U.S. patent application number 12/494789 was filed with the patent office on 2010-01-21 for differentiation of pluripotent stem cells.
Invention is credited to Pascal Ghislain Andre Bonnet, Janet Davis, Jiajian Liu, Christine Parmenter.
Application Number | 20100015711 12/494789 |
Document ID | / |
Family ID | 41100882 |
Filed Date | 2010-01-21 |
United States Patent
Application |
20100015711 |
Kind Code |
A1 |
Davis; Janet ; et
al. |
January 21, 2010 |
Differentiation of Pluripotent Stem Cells
Abstract
The present invention is directed to methods to differentiate
pluripotent stem cells. In particular, the present invention is
directed to methods and compositions to differentiate pluripotent
stem cells into cells expressing markers characteristic of the
definitive endoderm lineage comprising culturing the pluripotent
stem cells in medium comprising a sufficient amount of GDF-8 to
cause the differentiation of the pluripotent stem cells into cells
expressing markers characteristic of the definitive endoderm
lineage.
Inventors: |
Davis; Janet; (Skillman,
NJ) ; Liu; Jiajian; (Skillman, NJ) ;
Parmenter; Christine; (Skillman, NJ) ; Bonnet; Pascal
Ghislain Andre; (Beerse, BE) |
Correspondence
Address: |
PHILIP S. JOHNSON;JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
41100882 |
Appl. No.: |
12/494789 |
Filed: |
June 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61076900 |
Jun 30, 2008 |
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61076908 |
Jun 30, 2008 |
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61076915 |
Jun 30, 2008 |
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Current U.S.
Class: |
435/377 |
Current CPC
Class: |
C12N 5/0678 20130101;
C12N 2500/25 20130101; C12N 5/0606 20130101; C12N 2501/115
20130101; C12N 2506/02 20130101; C12N 2533/90 20130101; C12N
2501/16 20130101; C12N 2501/845 20130101; C12N 2501/42 20130101;
C12N 2531/00 20130101; C12N 2501/165 20130101; C12N 2501/999
20130101; C12N 5/0676 20130101; C12N 2501/19 20130101; C12N 2501/41
20130101; C12N 2501/155 20130101; C12N 2501/385 20130101; C12N
2501/727 20130101; C12N 2501/11 20130101; C12N 2533/50 20130101;
C12N 2501/135 20130101; C12N 2501/119 20130101; C12N 2501/15
20130101; C12N 2501/415 20130101 |
Class at
Publication: |
435/377 |
International
Class: |
C12N 5/00 20060101
C12N005/00 |
Claims
1. A method to differentiate pluripotent stem cells into cells
expressing markers characteristic of the definitive endoderm
lineage, comprising treating the pluripotent stem cells with a
medium lacking activin A, and containing a compound selected from
the group consisting of: EGF, FGF4, PDGF-A, PDGF-B, PDGF-C, PDGF-D,
VEGF, GDF-8, muscimol, PD98059, LY294002, U0124, U0126, and sodium
butyrate, for a period of time sufficient for the pluripotent stem
cells to differentiate into cells expressing markers characteristic
of the definitive endoderm lineage.
2. The method of claim 1, wherein the lacking activin A also
contains at least one other compound selected from the group
consisting of: an aniline-pyridinotriazine and a cyclic
aniline-pyridinotriazine.
Description
[0001] The present invention claims priority to application Ser.
No. 61/076,900, filed Jun. 30, 2008, application Ser. No.
61/076,908, filed Jun. 30, 2008, and application Ser. No.
61/076,915, filed Jun. 30, 2008.
FIELD OF THE INVENTION
[0002] The present invention is directed to methods to
differentiate pluripotent stem cells. In particular, the present
invention is directed to methods and compositions to differentiate
pluripotent stem cells into cells expressing markers characteristic
of the definitive endoderm lineage comprising culturing the
pluripotent stem cells in medium comprising a sufficient amount of
GDF-8 to cause the differentiation of the pluripotent stem cells
into cells expressing markers characteristic of the definitive
endoderm lineage.
BACKGROUND
[0003] Advances in cell-replacement therapy for Type I diabetes
mellitus and a shortage of transplantable islets of Langerhans have
focused interest on developing sources of insulin-producing cells,
or .beta. cells, appropriate for engraftment. One approach is the
generation of functional .beta. cells from pluripotent stem cells,
such as, for example, embryonic stem cells.
[0004] In vertebrate embryonic development, a pluripotent cell
gives rise to a group of cells comprising three germ layers
(ectoderm, mesoderm, and endoderm) in a process known as
gastrulation. Tissues such as, for example, thyroid, thymus,
pancreas, gut, and liver, will develop from the endoderm, via an
intermediate stage. The intermediate stage in this process is the
formation of definitive endoderm. Definitive endoderm cells express
a number of markers, such as, for example, HNF-3beta, GATA4, MIXL1,
CXCR4 and SOX17.
[0005] Formation of the pancreas arises from the differentiation of
definitive endoderm into pancreatic endoderm. Cells of the
pancreatic endoderm express the pancreatic-duodenal homeobox gene,
PDX1. In the absence of PDX1, the pancreas fails to develop beyond
the formation of ventral and dorsal buds. Thus, PDX1 expression
marks a critical step in pancreatic organogenesis. The mature
pancreas contains, among other cell types, exocrine tissue and
endocrine tissue. Exocrine and endocrine tissues arise from the
differentiation of pancreatic endoderm.
[0006] Cells bearing the features of islet cells have reportedly
been derived from embryonic cells of the mouse. For example,
Lumelsky et al. (Science 292:1389, 2001) report differentiation of
mouse embryonic stem cells to insulin-secreting structures similar
to pancreatic islets. Soria et al. (Diabetes 49: 157, 2000) report
that insulin-secreting cells derived from mouse embryonic stem
cells normalize glycemia in streptozotocin-induced diabetic
mice.
[0007] In one example, Hori et al. (PNAS 99: 16105, 2002) discloses
that treatment of mouse embryonic stem cells with inhibitors of
phosphoinositide 3-kinase (LY294002) produced cells that resembled
.beta. cells.
[0008] In another example, Blyszczuk et al. (PNAS 100:998, 2003)
reports the generation of insulin-producing cells from mouse
embryonic stem cells constitutively expressing Pax4.
[0009] Micallef et al. reports that retinoic acid can regulate the
commitment of embryonic stem cells to form Pdx1 positive pancreatic
endoderm. Retinoic acid is most effective at inducing Pdx1
expression when added to cultures at day 4 of embryonic stem cell
differentiation during a period corresponding to the end of
gastrulation in the embryo (Diabetes 54:301, 2005).
[0010] Miyazaki et al. reports a mouse embryonic stem cell line
over-expressing Pdx1. Their results show that exogenous Pdx1
expression clearly enhanced the expression of insulin,
somatostatin, glucokinase, neurogenin3, p48, Pax6, and HNF6 genes
in the resulting differentiated cells (Diabetes 53: 1030,
2004).
[0011] Skoudy et al. reports that activin A (a member of the
TGF-.beta. superfamily) up-regulates the expression of exocrine
pancreatic genes (p48 and amylase) and endocrine genes (Pdx1,
insulin, and glucagon) in mouse embryonic stem cells.
[0012] The maximal effect was observed using 1 nM activin A. They
also observed that the expression level of insulin and Pdx1 mRNA
was not affected by retinoic acid; however, 3 nM FGF7 treatment
resulted in an increased level of the transcript for Pdx1 (Biochem.
J. 379: 749, 2004).
[0013] Shiraki et al. studied the effects of growth factors that
specifically enhance differentiation of embryonic stem cells into
Pdx1 positive cells. They observed that TGF.beta.2 reproducibly
yielded a higher proportion of Pdx1 positive cells (Genes Cells.
June 2005; 10(6): 503-16).
[0014] Gordon et al. demonstrated the induction of brachyury
[positive]/HNF-3beta [positive] endoderm cells from mouse embryonic
stem cells in the absence of serum and in the presence of activin
along with an inhibitor of Wnt signaling (US 2006/0003446A1).
[0015] Gordon et al. (PNAS, Vol 103, page 16806, 2006) states: "Wnt
and TGF-beta/nodal/activin signaling simultaneously were required
for the generation of the anterior primitive streak."
[0016] However, the mouse model of embryonic stem cell development
may not exactly mimic the developmental program in higher mammals,
such as, for example, humans.
[0017] Thomson et al. isolated embryonic stem cells from human
blastocysts (Science 282:114, 1998). Concurrently, Gearhart and
coworkers derived human embryonic germ (hEG) cell lines from fetal
gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA
95:13726, 1998). Unlike mouse embryonic stem cells, which can be
prevented from differentiating simply by culturing with Leukemia
Inhibitory Factor (LIF), human embryonic stem cells must be
maintained under very special conditions (U.S. Pat. No. 6,200,806;
WO 99/20741; WO 01/51616).
[0018] D'Amour et al. describes the production of enriched cultures
of human embryonic stem cell-derived definitive endoderm in the
presence of a high concentration of activin and low serum (D'Amour
K A et al. 2005). Transplanting these cells under the kidney
capsule of mice resulted in differentiation into more mature cells
with characteristics of some endodermal organs. Human embryonic
stem cell-derived definitive endoderm cells can be further
differentiated into PDX1 positive cells after addition of FGF-10
(US 2005/0266554A1).
[0019] D'Amour et al. (Nature Biotechnology-24, 1392-1401 (2006))
states: "We have developed a differentiation process that converts
human embryonic stem (hES) cells to endocrine cells capable of
synthesizing the pancreatic hormones insulin, glucagon,
somatostatin, pancreatic polypeptide and ghrelin. This process
mimics in vivo pancreatic organogenesis by directing cells through
stages resembling definitive endoderm, gut-tube endoderm,
pancreatic endoderm and endocrine precursor en route to cells that
express endocrine hormones."
[0020] In another example, Fisk et al. reports a system for
producing pancreatic islet cells from human embryonic stem cells
(US2006/0040387A1). In this case, the differentiation pathway was
divided into three stages. Human embryonic stem cells were first
differentiated to endoderm using a combination of n-butyrate and
activin A. The cells were then cultured with TGF-.beta. antagonists
such as Noggin in combination with EGF or betacellulin to generate
PDX1 positive cells. The terminal differentiation was induced by
nicotinamide.
[0021] In one example, Benvenistry et al. states: "We conclude that
over-expression of PDX1 enhanced expression of pancreatic enriched
genes, induction of insulin expression may require additional
signals that are only present in vivo" (Benvenistry et al, Stem
Cells 2006; 24:1923-1930).
[0022] Activin A is a TGF-beta family member that exhibits a wide
range of biological activities including regulation of cellular
proliferation and differentiation, and promotion of neuronal
survival. Isolation and purification of activin A is often complex
and can often result in poor yields. For example, Pangas, S. A. and
Woodruff, T. K states: "Inhibin and activin are protein hormones
with diverse physiological roles including the regulation of
pituitary FSH secretion. Like other members of the transforming
growth factor-.beta. gene family, they undergo processing from
larger precursor molecules as well as assembly into functional
dimers. Isolation of inhibin and activin from natural sources can
only produce limited quantities of bioactive protein." (J.
Endocrinol. 172 (2002) 199-210).
[0023] In another example, Arai, K. Y. et al states: "Activins are
multifunctional growth factors belonging to the transforming growth
factor-.beta. superfamily. Isolation of activins from natural
sources requires many steps and only produces limited quantities.
Even though recombinant preparations have been used in recent
studies, purification of recombinant activins still requires
multiple steps." (Protein Expression and Purification 49 (2006)
78-82).
[0024] Therefore, there still remains a significant need for
alternatives for activin A to facilitate the differentiation of
pluripotent stem cells.
SUMMARY
[0025] In one embodiment, the present invention provides a method
to differentiate pluripotent stem cells into cells expressing
markers characteristic of the definitive endoderm lineage,
comprising culturing the pluripotent stem cells in medium
comprising a sufficient amount of GDF-8 to cause the
differentiation of the pluripotent stem cells into cells expressing
markers characteristic of the definitive endoderm lineage.
[0026] In one embodiment, the medium comprising a sufficient amount
of GDF-8 also contains at least one other compound. In one
embodiment, the at least one other compound is an
aniline-pyridinotriazine. In an alternate embodiment, the at least
one other compound is a cyclic aniline-pyridinotriazine.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIG. 1 shows the differentiation of HI human embryonic stem
cells into cells expressing markers characteristic of the
definitive endoderm lineage. Differentiation was determined by
measuring cell number (Panel A) and SOX17 intensity (Panel B) using
an IN Cell Analyzer 1000 (GE Healthcare). Human embryonic stem
cells were treated for a total of four days with medium containing
20 ng/ml Wnt3a plus activin A at the concentrations indicated
(black bars) or medium lacking Wnt3a but with activin A at the
concentrations indicated (white bars).
[0028] FIG. 2 shows the dose response relationship of activin A and
GDF8 used to differentiate cells of the human embryonic stem cell
line H1 toward cells expressing markers characteristic of the
definitive endoderm lineage. Cells were treated for a total of
three days with activin A or GDF8 at the concentrations shown in
combination with 20 ng/ml Wnt3a on the first day of assay.
Differentiation was determined by measuring SOX17 intensity using a
fluorescent antibody probe and high content analysis on a GE
Healthcare IN Cell Analyzer.
[0029] FIG. 3 shows the expression of CXCR4 in cells following the
first step of differentiation, according to the methods described
in Example 12. H1 cells were treated with 100 ng/ml activin A or
200 ng/ml GDF-8 for a total of three days in combination with 20
ng/ml Wnt3a for the first day or 2.5 .mu.M Compound 34 or 2.5 .mu.M
Compound 56 for all three days. CXCR4 expression was measured using
a fluorescent antibody probe and flow cytometry, yielding the
percentages of positive cells shown.
[0030] FIG. 4 shows the expression of SOX17 in cells after three
days differentiation to definitive endoderm according to the
methods described in Example 12. H1 cells were treated for a total
of three days with 100 ng/ml activin A or 200 ng/ml GDF-8 in
combination with 20 ng/ml Wnt3a for the first day or 2.5 .mu.M
Compound 34 or 2.5 .mu.M Compound 56 for all three days.
Differentiation was determined by measuring SOX17 intensity (black
bars) and resulting cell number (white bars) with fluorescent
antibody probes and high content analysis on a GE Healthcare IN
Cell Analyzer.
[0031] FIG. 5 shows the expression of PDX1 and CDX2 protein in
cells following the third step of differentiation, according to the
methods described in Example 12. H1 cells were treated for a total
of three days with 100 ng/ml activin A or 200 ng/ml GDF-8 in
combination with 20 ng/ml Wnt3a for the first day or 2.5 .mu.M
Compound 34 or 2.5 .mu.M Compound 56 for all three days followed by
subsequent differentiation through the second and third steps of
differentiation. Protein expression and cell numbers, as determined
with fluorescent antibody probes and high content analysis, are
depicted for each treatment group. For comparative purposes, values
are normalized relative to treatment with activin A/Wnt3a.
[0032] FIG. 6 shows the expression of PDX1 protein (white bars) and
cell number (black bars) in cells following the fourth step of
differentiation, according to the methods described in Example 12.
H1 cells were treated for a total of three days with 100 ng/ml
activin A or 200 ng/ml GDF-8 in combination with 20 ng/ml Wnt3a for
the first day or 2.5 .mu.M Compound 34 or 2.5 .mu.M Compound 56 for
all three days followed by subsequent differentiation through the
second, third, and fourth steps of differentiation. Protein
expression and cell numbers, as determined with fluorescent
antibody probes and high content analysis, are depicted for each
treatment group. For comparative purposes, values are normalized
relative to treatment with activin A/Wnt3a.
[0033] FIG. 7 shows the protein expression for insulin and glucagon
and cell number in cells differentiated according to the methods
described in Example 12. H1 cells were treated for a total of three
days with 100 ng/ml activin A or 200 ng/ml GDF-8 in combination
with 20 ng/ml Wnt3a for the first day or 2.5 .mu.M Compound 34 or
2.5 .mu.M Compound 56 for all three days followed by subsequent
differentiation through the second, third, fourth, and fifth steps
of differentiation. Protein expression and cell numbers, as
determined with fluorescent antibody probes and high content
analysis, are depicted for each treatment group. For comparative
purposes, values are normalized relative to treatment with activin
A/Wnt3a.
[0034] FIG. 8 shows SOX17 protein expression and cell number in
human embryonic stem cells after differentiation to definitive
endoderm, according to the methods described in Example 13. H1
cells were treated for a total of four days with 100 ng/ml of
activin A or 100 ng/ml of a GDF-growth factor in combination with
20 ng/ml Wnt3a for the first day or 2.5 .mu.M Compound 34 or 2.5
.mu.M Compound 56 for the first two days of assay. SOX17 protein
expression (black bars) and cell numbers (white bars), as
determined with fluorescent antibody probes and high content
analysis, are depicted for each treatment group. For comparative
purposes, values are normalized relative to treatment with activin
A/Wnt3a. Panel 8A shows a series of control conditions for
differentiation in the absence of any growth factors (NONE), or
with activin A/Wnt3a treatment (AA/Wnt3a) or with individual
reagents alone. Panel 8B shows differentiation with GDF-3, alone or
in multiple combinations with Wnt3a, Compound 34, or Compound 56.
Panel 8C shows differentiation with GDF-5, alone or in multiple
combinations with Wnt3a, Compound 34, or Compound 56. Panel 8D
shows differentiation with GDF-8, alone or in multiple combinations
with Wnt3a, Compound 34, or Compound 56. Panel 8E shows
differentiation with GDF-10, alone or in multiple combinations with
Wnt3a, Compound 34, or Compound 56. Panel 8F shows differentiation
with GDF-11, alone or in multiple combinations with Wnt3a, Compound
34, or Compound 56. Panel 8G shows differentiation with GDF-15,
alone or in multiple combinations with Wnt3a, Compound 34, or
Compound 56.
[0035] FIG. 9 shows SOX17 protein expression in human embryonic
stem cells after differentiation to definitive endoderm, according
to the methods described in Example 14. H1 cells were treated for a
total of three days with 100 ng/ml of activin A or various growth
factors at the concentrations shown in combination with 20 ng/ml
Wnt3a or 2.5 .mu.M Compound 34 for the first day of assay. SOX17
protein expression (black bars) and cell numbers (white bars), as
determined with fluorescent antibody probes and high content
analysis, are depicted for each treatment group. For comparative
purposes, values are normalized relative to treatment with activin
A/Wnt3a. Panel 9A shows a series of control conditions for
differentiation with Wnt3a alone or in the absence of any growth
factors (None) or with activin A/Wnt3a treatment (AA/Wnt3a). Panel
9B shows differentiation with GDF-8 (Vendor PeproTech), at the
concentrations shown, in combination with 20 ng/ml Wnt3a. Panel 9C
shows differentiation with GDF-8 (Vendor Shenendoah), at the
concentrations shown, in combination with 20 ng/ml Wnt3a. Panel 9D
shows differentiation with TGF.beta.1, at the concentrations shown,
in multiple combinations with Wnt3a or Compound 34. Panel 9E shows
differentiation with BMP2, at the concentrations shown, in multiple
combinations with Wnt3a or Compound 34. Panel 9F shows
differentiation with BMP3, at the concentrations shown, in multiple
combinations with Wnt3a or Compound 34. Panel 9G shows
differentiation with BMP4, at the concentrations shown, in multiple
combinations with Wnt3a or Compound 34.
[0036] FIG. 10 shows SOX17 protein expression in human embryonic
stem cells after differentiation to definitive endoderm, according
to the methods described in Example 15. H1 cells were treated for a
total of three days in various timed exposures with 100 ng/ml of
activin A or 100 ng/ml GDF-8 in combination with 20 ng/ml Wnt3a.
SOX17 protein expression, as determined with fluorescent antibody
probes and high content analysis, is shown as total intensity
values for each treatment group, testing control conditions for
differentiation with no growth factors added (no treatment), with
Wnt3a alone, with activin A or GDF-8 alone, or with activin A/Wnt3a
treatment or GDF-8/Wnt3a treatment, where Wnt3a was added only for
the first day of assay or for all three days of assay as shown.
[0037] FIG. 11 shows SOX17 protein expression in human embryonic
stem cells after differentiation to definitive endoderm, according
to the methods described in Example 15. H1 cells were treated for a
total of three days in various timed exposures with 100 ng/ml of
activin A in combination with test compound (Compound 181 (Panel
A), Compound 180 (Panel B), Compound 19(Panel C), Compound 202
(Panel D), Compound 40 (Panel E), Compound 34 (Panel F), or GSK3
inhibitor BIO (Panel G)) at the concentrations shown, where test
compound was added only on the first day of assay. Protein
expression for SOX17, as determined with fluorescent antibody
probes and high content analysis, is depicted by total intensity
values.
[0038] FIG. 12 shows SOX17 protein expression in human embryonic
stem cells after differentiation to definitive endoderm, according
to the methods described in Example 15. H1 cells were treated for a
total of three days in various timed exposures with 100 ng/ml of
activin A in combination with test compound (Compound 181 (Panel
A), Compound 180 (Panel B), Compound 19 (Panel C), Compound 202
(Panel D), Compound 40 (Panel E), Compound 34 (Panel F), or GSK3
inhibitor BIO (Panel G)) at the concentrations shown, where test
compound was added for all three days of assay. Protein expression
for SOX17, as determined with fluorescent antibody probes and high
content analysis, is depicted by total intensity values.
[0039] FIG. 13 shows SOX17 protein expression in human embryonic
stem cells after differentiation to definitive endoderm, according
to the methods described in Example 15. H1 cells were treated for a
total of three days in various timed exposures with 100 ng/ml of
GDF-8 in combination with test compound (Compound 181 (Panel A),
Compound 180 (Panel B), Compound 19 (Panel C), Compound 202 (Panel
D), Compound 40 (Panel E), Compound 34 (Panel F), or GSK3 inhibitor
BIO (Panel G)) at the concentrations shown, where test compound was
added only on the first day of assay. Protein expression for SOX17,
as determined with fluorescent antibody probes and high content
analysis, is depicted by total intensity values.
[0040] FIG. 14 shows SOX17 protein expression in human embryonic
stem cells after differentiation to definitive endoderm, according
to the methods described in Example 15. H1 cells were treated for a
total of three days in various timed exposures with 100 ng/ml of
GDF-8 in combination with test compound (Compound 181 (Panel A),
Compound 180 (Panel B), Compound 19 (Panel C), Compound 202 (Panel
D), Compound 40 (Panel E), Compound 34 (Panel F), or GSK3 inhibitor
BIO (Panel G)) at the concentrations shown, where test compound was
added for all three days of assay. Protein expression for SOX17, as
determined with fluorescent antibody probes and high content
analysis, is depicted by total intensity values.
[0041] FIG. 15 shows cell number yields after differentiation of
human embryonic stem cells to definitive endoderm, according to the
methods described in Example 15. H1 cells were treated for a total
of three days in various timed exposures with 100 ng/ml of activin
A or 100 ng/ml GDF-8 in combination with 20 ng/ml Wnt3a. Cell
numbers, as determined with a fluorescent nuclear probe and high
content analysis, are shown for each treatment group, testing
control conditions for differentiation with no growth factors added
(no treatment), with Wnt3a alone, with activin A or GDF-8 alone, or
with activin A/Wnt3a treatment or GDF-8/Wnt3a treatment, where
Wnt3a was added only for the first day of assay or for all three
days of assay as shown.
[0042] FIG. 16 shows cell number yields after differentiation of
human embryonic stem cells to definitive endoderm, according to the
methods described in Example 15. H1 cells were treated for a total
of three days in various timed exposures with 100 ng/ml of activin
A in combination with test compound (Compound 181 (Panel A),
Compound 180 (Panel B), Compound 19 (Panel C), Compound 202 (Panel
D), Compound 40 (Panel E), Compound 34 (Panel F), or GSK3 inhibitor
BIO (Panel G)) at the concentrations shown, where test compound was
added only on the first day of assay. Cell number yields, as
determined with a fluorescent nuclear probe and high content
analysis, are shown.
[0043] FIG. 17 shows cell number yields after differentiation of
human embryonic stem cells to definitive endoderm, according to the
methods described in Example 15. H1 cells were treated for a total
of three days in various timed exposures with 100 ng/ml of activin
A in combination with test compound (Compound 181 (Panel A),
Compound 180 (Panel B), Compound 19 (Panel C), Compound 202 (Panel
D), Compound 40 (Panel E), Compound 34 (Panel F), or GSK3 inhibitor
BIO (Panel G)) at the concentrations shown, where test compound was
added for all three days of assay. Cell number yields, as
determined with a fluorescent nuclear probe and high content
analysis, are shown.
[0044] FIG. 18 shows cell number yields after differentiation of
human embryonic stem cells to definitive endoderm, according to the
methods described in Example 15. H1 cells were treated for a total
of three days in various timed exposures with 100 ng/ml of GDF-8 in
combination with test compound (Compound 181 (Panel A), Compound
180 (Panel B), Compound 19 (Panel C), Compound 202 (Panel D),
Compound 40 (Panel E), Compound 34 (Panel F), or GSK3 inhibitor BIO
(Panel G)) at the concentrations shown, where test compound was
added only on the first day of assay. Cell number yields, as
determined with a fluorescent nuclear probe and high content
analysis, are shown.
[0045] FIG. 19 shows cell number yields after differentiation of
human embryonic stem cells to definitive endoderm, according to the
methods described in Example 15. H1 cells were treated for a total
of three days in various timed exposures with 100 ng/ml of GDF-8 in
combination with test compound (Compound 181 (Panel A), Compound
180 (Panel B), Compound 19 (Panel C), Compound 202 (Panel D),
Compound 40 (Panel E), Compound 34 (Panel F), or GSK3 inhibitor BIO
(Panel G)) at the concentrations shown, where test compound was
added for all three days of assay. Cell number yields, as
determined with a fluorescent nuclear probe and high content
analysis, are shown.
[0046] FIG. 20 shows the expression of various protein markers in
cells throughout multiple steps of differentiation according to the
methods described in Example 16. H1 cells were treated with 100
ng/ml activin A or 100 ng/ml GDF-8 for a total of three days in
combination with 20 ng/ml Wnt3a for the first day or 2.5 .mu.M
various compounds (Compound 19, Compound 202, Compound 40, or GSK3
inhibitor BIO) added only on the first day. FIG. 20, panel A shows
FACS analysis for the definitive endoderm marker, CXCR4, in cells
after the first step of differentiation. CXCR4 expression was
measured using a fluorescent antibody probe and flow cytometry,
yielding the percentages of positive cells as shown. FIG. 20, panel
B shows high content image analysis for normalized SOX17 protein
expression (black bars) and recovered cell numbers (white bars)
resulting from the first step of differentiation, testing the
corresponding treatments shown. FIG. 20, panel C shows high content
image analysis for relative cell numbers recovered from cultures
treated through differentiation step 5. FIG. 20, panel D shows high
content image analysis for glucagon protein expression from
cultures treated through differentiation step 5. FIG. 20, panel E
shows high content image analysis for insulin protein expression
from cultures treated through differentiation step 5. FIG. 20,
panel F shows the ratio of glucagon to insulin expression in cells
from cultures treated through differentiation step 5. For
comparison purposes, expression values in panels B, C, D, E, and F
are normalized to the control treatment with activin A and Wnt3a
during step 1.
[0047] FIG. 21 shows the expression of various protein and RT-PCR
markers in cells throughout multiple steps of differentiation
according to the methods described in Example 17. H1 cells were
treated with 100 ng/ml activin A or 100 ng/ml GDF-8 for a total of
three days in combination with 20 ng/ml Wnt3a for the first day or
various compounds at the following concentrations (Compound 181,
Compound 180, Compound 19, Compound 202, Compound 40, Compound 56,
or GSK3 inhibitor BIO) added only on the first day. FACS analysis
for the definitive endoderm marker, CXCR4, is shown in cells after
the first step of differentiation where treatment combined activin
A (Panel A) or GDF-8 (Panel B) with Wnt3a or various compounds.
CXCR4 expression was measured using a fluorescent antibody probe
and flow cytometry, yielding the percentages of positive cells as
shown. In subsequent panels of FIG. 21, normalized RT-PCR values
for various differentiation markers are shown with respective
treatments using activin A or GDF-8 during the first step of
differentiation as follows: markers at the end of step one of
differentiation for treatments combining activin A (Panel C) or
GDF-8 (Panel D); markers at the end of step three of
differentiation for treatments combining activin A (Panel E) or
GDF-8 (Panel F); markers at the end of step four of differentiation
for treatments combining activin A (Panel G) or GDF-8 (Panel H);
markers at the end of step five of differentiation for treatments
combining activin A (Panel I) or GDF-8 (Panel J). At the conclusion
of step five of differentiation, high content analysis was
performed to measure recovered cell numbers for corresponding
treatments during the first step of differentiation using activin A
(Panel K) or GDF-8 (Panel M). High content analysis was also used
to measure glucagon and insulin intensity in recovered cell
populations at the end of step five of differentiation,
corresponding to treatment with activin A (Panel L) or GDF-8 (Panel
N) during the first step of differentiation.
[0048] FIG. 22 shows the expression of various protein and RT-PCR
markers in cells treated according to the methods described in
Example 18. H1 cells were treated with 100 ng/ml activin A or 100
ng/ml GDF-8 for a total of three days in combination with 20 ng/ml
Wnt3a for the first day or 2.5 .mu.M Compound 40 or 2.5 .mu.M
Compound 202 only on the first day. FIG. 22, panel A shows FACS
analysis for the definitive endoderm marker, CXCR4, in cells after
the first step of differentiation. CXCR4 expression was measured
using a fluorescent antibody probe and flow cytometry, yielding the
percentages of positive cells as shown. In FIG. 22, panel B.
normalized RT-PCR values for various differentiation markers in
cells recovered after the fourth step of differentiation are shown
corresponding to respective treatments using activin A/Wnt3a or
GDF-8/Compound 40 or GDF-8/Compound 202 during the first step of
differentiation.
[0049] FIG. 23 shows the level of C-peptide detected in SCID-beige
mice that received cells at the end of step four of the
differentiation protocol as described in Example 18.
[0050] FIG. 24 panel A shows the expression of CXCR4, as determined
by FACS in cells at the end of step one of the differentiation
protocol described in Example 19. Panel B shows the expression of
various genes, as determined by RT-PCR in cells at the end of step
four of the differentiation protocol described in Example 19. Two
different experimental replicates are shown (Rep-1 and Rep-2), each
subjected to identical treatment protocols. Panel C shows the level
of C-peptide detected in SCID-beige mice that received cells at the
end of step four of the differentiation protocol as treated with
GDF-8 and Wnt3a during the first step of in vitro differentiation.
Panel D shows the level of C-peptide detected in SCID-beige mice
that received cells at the end of step four of the differentiation
protocol as treated with GDF-8 and Compound 28 during the first
step of in vitro differentiation.
[0051] FIG. 25 shows the cell number (panel A) and expression of
CXCR4 (panel B) from cells grown on microcarrier beads, treated
according to the methods of the present invention as described in
Example 22. Cells were grown on Cytodex3 beads without treatment
(undifferentiated) or with treatment combining 100 ng/ml activin A
with 20 ng/ml Wnt3a (AA/Wnt3a) or with various treatments combining
GDF-8 as shown: 50 ng/ml GDF-8with 2.5 .mu.M Compound 34 (Cmp
34+8); or 50 ng/ml GDF-8 with 2.5 .mu.M Compound 34 and 50 ng/ml
PDGF (Cmp 34+8+D); or 50 ng/ml GDF-8 with 2.5 .mu.M Compound 34 and
50 ng/ml PDGF and 50 ng/ml VEGF (Cmp 34+8+D+V); or 50 ng/ml GDF-8
with 2.5 .mu.M Compound 34 and 50 ng/ml PDGF and 50 ng/ml VEGF and
20 ng/ml muscimol (Cmp 34+8+D+V+M).
[0052] FIG. 26 shows the proliferation of cells following treatment
of the compounds of the present invention as described in Example
23. Panels B through I show assay results for treatment using a
compound in combination with GDF-8 and measuring MTS OD readings at
1 day, 2 days, and 3 days after initiating the differentiation
assay.
[0053] FIG. 27 shows the expression of various proteins and genes
from cells grown on microcarrier beads, treated according to the
methods of the present invention. Panel A shows the percent
positive expression of CXCR4, CD99, and CD9 as determined by FACS
in cells at the end of step one of the differentiation protocol
described in Example 24. Panel B shows cells recovered from
treatments as shown differentiated through step three of the
differentiation protocol. Panel C shows ddCT values for various
gene markers expressed in cells treated as shown in step and
differentiated through step three of the protocol.
DETAILED DESCRIPTION
[0054] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the following
subsections that describe or illustrate certain features,
embodiments, or applications of the present invention.
Definitions
[0055] Stem cells are undifferentiated cells defined by their
ability at the single cell level to both self-renew and
differentiate to produce progeny cells, including self-renewing
progenitors, non-renewing progenitors, and terminally
differentiated cells. Stem cells are also characterized by their
ability to differentiate in vitro into functional cells of various
cell lineages from multiple germ layers (endoderm, mesoderm and
ectoderm), as well as to give rise to tissues of multiple germ
layers following transplantation and to contribute substantially to
most, if not all, tissues following injection into blastocysts.
[0056] Stem cells are classified by their developmental potential
as: (1) totipotent, meaning able to give rise to all embryonic and
extraembryonic cell types; (2) pluripotent, meaning able to give
rise to all embryonic cell types; (3) multipotent, meaning able to
give rise to a subset of cell lineages but all within a particular
tissue, organ, or physiological system (for example, hematopoietic
stem cells (HSC) can produce progeny that include HSC (self-
renewal), blood cell restricted oligopotent progenitors, and all
cell types and elements (e.g., platelets) that are normal
components of the blood); (4) oligopotent, meaning able to give
rise to a more restricted subset of cell lineages than multipotent
stem cells; and (5) unipotent, meaning able to give rise to a
single cell lineage (e.g., spermatogenic stem cells).
[0057] Differentiation is the process by which an unspecialized
("uncommitted") or less specialized cell acquires the features of a
specialized cell such as, for example, a nerve cell or a muscle
cell. A differentiated or differentiation-induced cell is one that
has taken on a more specialized ("committed") position within the
lineage of a cell. The term "committed", when applied to the
process of differentiation, refers to a cell that has proceeded in
the differentiation pathway to a point where, under normal
circumstances, it will continue to differentiate into a specific
cell type or subset of cell types, and cannot, under normal
circumstances, differentiate into a different cell type or revert
to a less differentiated cell type. De-differentiation refers to
the process by which a cell reverts to a less specialized (or
committed) position within the lineage of a cell. As used herein,
the lineage of a cell defines the heredity of the cell, i.e., which
cells it came from and what cells it can give rise to. The lineage
of a cell places the cell within a hereditary scheme of development
and differentiation. A lineage-specific marker refers to a
characteristic specifically associated with the phenotype of cells
of a lineage of interest and can be used to assess the
differentiation of an uncommitted cell to the lineage of
interest.
[0058] ".beta.-cell lineage" refers to cells with positive gene
expression for the transcription factor PDX-1 and at least one of
the following transcription factors: NGN3, NKX2.2, NKX6.1, NEUROD,
ISL1, HNF-3 beta, MAFA, PAX4, or PAX6. Cells expressing markers
characteristic of the .beta. cell lineage include .beta. cells.
[0059] "Cells expressing markers characteristic of the definitive
endoderm lineage", or "Stage 1 cells", or "Stage 1", as used
herein, refers to cells expressing at least one of the following
markers: SOX17, GATA4, HNF-3 beta, GSC, CER1, Nodal, FGF8,
Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin
(EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, or OTX2. Cells
expressing markers characteristic of the definitive endoderm
lineage include primitive streak precursor cells, primitive streak
cells, mesendoderm cells and definitive endoderm cells.
[0060] "Cells expressing markers characteristic of the pancreatic
endoderm lineage", as used herein, refers to cells expressing at
least one of the following markers: PDX1, HNF-1 beta, PTF1 alpha,
HNF6, or HB9. Cells expressing markers characteristic of the
pancreatic endoderm lineage include pancreatic endoderm cells,
primitive gut tube cells, and posterior foregut cells.
[0061] "Cells expressing markers characteristic of the pancreatic
endocrine lineage", or "Stage 5 cells", or "Stage 5", as used
herein, refers to cells expressing at least one of the following
markers: NGN3, NEUROD, ISL1, PDX1, NKX6.1, PAX4, or PTF-1 alpha.
Cells expressing markers characteristic of the pancreatic endocrine
lineage include pancreatic endocrine cells, pancreatic hormone
expressing cells, and pancreatic hormone secreting cells, and cells
of the .beta.-cell lineage.
[0062] "Definitive endoderm", as used herein, refers to cells which
bear the characteristics of cells arising from the epiblast during
gastrulation and which form the gastrointestinal tract and its
derivatives. Definitive endoderm cells express the following
markers: HNF-3 beta, GATA4, SOX-17, Cerberus, OTX2, goosecoid,
C-Kit, CD99, or MIXL1.
[0063] "Extraembryonic endoderm", as used herein, refers to a
population of cells expressing at least one of the following
markers: SOX7, AFP, or SPARC.
[0064] "Markers", as used herein, are nucleic acid or polypeptide
molecules that are differentially expressed in a cell of interest.
In this context, differential expression means an increased level
for a positive marker and a decreased level for a negative marker.
The detectable level of the marker nucleic acid or polypeptide is
sufficiently higher or lower in the cells of interest compared to
other cells, such that the cell of interest can be identified and
distinguished from other cells using any of a variety of methods
known in the art.
[0065] "Mesendoderm cell", as used herein, refers to a cell
expressing at least one of the following markers: CD48,
eomesodermin (EOMES), SOX17, DKK4, HNF-3 beta, GSC, FGF17, or
GATA-6.
[0066] "Pancreatic endocrine cell", or "pancreatic hormone
expressing cell", as used herein, refers to a cell capable of
expressing at least one of the following hormones: insulin,
glucagon, somatostatin, and pancreatic polypeptide.
[0067] "Pancreatic endoderm cell", or "Stage 4 cells", or "Stage
4", as used herein, refers to a cell capable of expressing at least
one of the following markers: NGN3, NEUROD, ISL1, PDX1, PAX4, or
NKX2.2.
[0068] "Pancreatic hormone producing cell", as used herein, refers
to a cell capable of producing at least one of the following
hormones: insulin, glucagon, somatostatin, and pancreatic
polypeptide.
[0069] "Pancreatic hormone secreting cell", as used herein, refers
to a cell capable of secreting at least one of the following
hormones: insulin, glucagon, somatostatin, and pancreatic
polypeptide.
[0070] "Posterior foregut cell" or "Stage 3 cells", or "Stage 3",
as used herein, refers to a cell capable of secreting at least one
of the following markers: PDX1, HNF1, PTF-1 alpha, HNF6, HB-9, or
PROX-1.
[0071] "Pre-primitive streak cell", as used herein, refers to a
cell expressing at least one of the following markers: Nodal, or
FGF8.
[0072] "Primitive gut tube cell" or "Stage 2 cells", or "Stage2",
as used herein, refers to a cell capable of secreting at least one
of the following markers: HNF1, HNF-4 alpha.
[0073] "Primitive streak cell", as used herein, refers to a cell
expressing at least one of the following markers: Brachyury,
Mix-like homeobox protein, or FGF4.
Isolation, Expansion, and Culture of Pluripotent Stem Cells
Characterization of Pluripotent Stem Cells
[0074] The pluripotency of pluripotent stem cells can be confirmed,
for example, by injecting cells into severe combined
immunodeficient (SCID) mice, fixing the teratomas that form using
4% paraformaldehyde, and then examining them histologically for
evidence of cell types from the three germ layers. Alternatively,
pluripotency may be determined by the creation of embryoid bodies
and assessing the embryoid bodies for the presence of markers
associated with the three germinal layers.
[0075] Propagated pluripotent stem cell lines may be karyotyped
using a standard G-banding technique and compared to published
karyotypes of the corresponding primate species. It is desirable to
obtain cells that have a "normal karyotype," which means that the
cells are euploid, wherein all human chromosomes are present and
not noticeably altered.
Sources of Pluripotent Stem Cells
[0076] The types of pluripotent stem cells that may be used include
established lines of pluripotent cells derived from tissue formed
after gestation, including pre-embryonic tissue (such as, for
example, a blastocyst), embryonic tissue, or fetal tissue taken any
time during gestation, typically but not necessarily before
approximately 10 to 12 weeks gestation. Non-limiting examples are
established lines of human embryonic stem cells or human embryonic
germ cells, such as, for example, the human embryonic stem cell
lines H1, H7, and H9 (WiCell). Also contemplated is use of the
compositions of this disclosure during the initial establishment or
stabilization of such cells, in which case the source cells would
be primary pluripotent cells taken directly from the source
tissues. Also suitable are cells taken from a pluripotent stem cell
population already cultured in the absence of feeder cells. Also
suitable are mutant human embryonic stem cell lines, such as, for
example, BG01v (BresaGen, Athens, Ga.).
[0077] In one embodiment, human embryonic stem cells are prepared
as described by Thomson et al. (U.S. Pat. No. 5,843,780; Science
282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl.
Acad. Sci. U.S.A. 92:7844, 1995).
[0078] In one embodiment, pluripotent stem cells are prepared as
described by Takahashi et al. (Cell 131: 1-12, 2007).
Culture of Pluripotent Stem Cells
[0079] In one embodiment, pluripotent stem cells are typically
cultured on a layer of feeder cells that support the pluripotent
stem cells in various ways. Alternatively, pluripotent stem cells
are cultured in a culture system that is essentially free of feeder
cells but nonetheless supports proliferation of pluripotent stem
cells without undergoing substantial differentiation. The growth of
pluripotent stem cells in feeder-free culture without
differentiation is supported using a medium conditioned by
culturing previously with another cell type. Altematively, the
growth of pluripotent stem cells in feeder-free culture without
differentiation is supported using a chemically defined medium.
[0080] The pluripotent stem cells may be plated onto a suitable
culture substrate. In one embodiment, the suitable culture
substrate is an extracellular matrix component, such as, for
example, those derived from basement membrane or that may form part
of adhesion molecule receptor-ligand couplings. In one embodiment,
the suitable culture substrate is MATRIGEL.RTM. (Becton Dickenson).
MATRIGEL.RTM. is a soluble preparation from Engelbreth-Holm-Swarm
tumor cells that gels at room temperature to form a reconstituted
basement membrane.
[0081] Other extracellular matrix components and component mixtures
are suitable as an alternative. Depending on the cell type being
proliferated, this may include laminin, fibronectin, proteoglycan,
entactin, heparan sulfate, and the like, alone or in various
combinations.
[0082] The pluripotent stem cells may be plated onto the substrate
in a suitable distribution and in the presence of a medium that
promotes cell survival, propagation, and retention of the desirable
characteristics. All these characteristics benefit from careful
attention to the seeding distribution and can readily be determined
by one of skill in the art.
[0083] Suitable culture media may be made from the following
components, such as, for example, Dulbecco's modified Eagle's
medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified
Eagle's medium (KO DMEM), Gibco #10829-018; Ham's F12/50% DMEM
basal medium; 200 mM L-glutamine, Gibco #15039-027; non-essential
amino acid solution, Gibco 11140-050; .beta.-mercaptoethanol, Sigma
#M7522; human recombinant basic fibroblast growth factor (bFGF),
Gibco #13256-029.
Formation of Pancreatic Hormone Producing Cells from Pluripotent
Stem Cells
[0084] In one embodiment, the present invention provides a method
for producing pancreatic hormone producing cells from pluripotent
stem cells, comprising the steps of: [0085] a. Culturing
pluripotent stem cells, [0086] b. Differentiating the pluripotent
stem cells into cells expressing markers characteristic of the
definitive endoderm lineage, [0087] c. Differentiating the cells
expressing markers characteristic of the definitive endoderm
lineage into cells expressing markers characteristic of the
pancreatic endoderm lineage, and [0088] d. Differentiating the
cells expressing markers characteristic of the pancreatic endoderm
lineage into cells expressing markers characteristic of the
pancreatic endocrine lineage.
[0089] In one aspect of the present invention, the pancreatic
endocrine cell is a pancreatic hormone producing cell. In an
alternate aspect, the pancreatic endocrine cell is a cell
expressing markers characteristic of the .beta.-cell lineage. A
cell expressing markers characteristic of the .beta.-cell lineage
expresses PDX1 and at least one of the following transcription
factors: NGN3, NKX2.2, NKX6.1, NEUROD, ISL1, HNF-3 beta, MAFA,
PAX4, or Pax6. In one aspect of the present invention, a cell
expressing markers characteristic of the .beta.-cell lineage is a
.beta.-cell.
[0090] Pluripotent stem cells suitable for use in the present
invention include, for example, the human embryonic stem cell line
H9 (NIH code: WA09), the human embryonic stem cell line H1 (NIH
code: WA01), the human embryonic stem cell line H7 (NIH code:
WA07), and the human embryonic stem cell line SA002 (Cellartis,
Sweden). Also suitable for use in the present invention are cells
that express at least one of the following markers characteristic
of pluripotent cells: ABCG2, cripto, CD9, FOXD3, Connexin43,
Connexin45, OCT4, SOX2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4,
Tral-60, or Tral-81.
[0091] The pluripotent stem cells may be cultured on a feeder cell
layer. Alternatively, the pluripotent stem cells may be cultured on
an extracellular matrix. The extracellular matrix may be a
solubilized basement membrane preparation extracted from mouse
sarcoma cells (as sold by BD Biosciences under the trade name
MATRIGEL.TM.). Alternatively, the extracellular matrix may be
growth factor-reduced MATRIGEL.TM.. Alternatively, the
extracellular matrix may be fibronectin. In an alternate
embodiment, the pluripotent stem cells are cultured and
differentiated on tissue culture substrate coated with human
serum.
[0092] The extracellular matrix may be diluted prior to coating the
tissue culture substrate. Examples of suitable methods for diluting
the extracellular matrix and for coating the tissue culture
substrate may be found in Kleinman, H. K., et al., Biochemistry
25:312 (1986), and Hadley, M. A., et al., J. Cell. Biol. 101:1511
(1985).
[0093] In one embodiment, the extracellular matrix is MATRIGEL.TM..
In one embodiment, the tissue culture substrate is coated with
MATRIGEL.TM. at a 1:10 dilution. In an alternate embodiment, the
tissue culture substrate is coated with MATRIGEL.TM. at a 1:15
dilution. In an alternate embodiment, the tissue culture substrate
is coated with MATRIGEL.TM. at a 1:30 dilution. In an alternate
embodiment, the tissue culture substrate is coated with
MATRIGEL.TM. at a 1:60 dilution.
[0094] In one embodiment, the extracellular matrix is growth
factor-reduced MATRIGEL.TM.. In one embodiment, the tissue culture
substrate is coated with growth factor-reduced MATRIGEL.TM. at a
1:10 dilution. In an alternate embodiment, the tissue culture
substrate is coated with growth factor-reduced MATRIGEL.TM. at a
1:15 dilution. In an alternate embodiment, the tissue culture
substrate is coated with growth factor-reduced MATRIGEL.TM. at a
1:30 dilution. In an alternate embodiment, the tissue culture
substrate is coated with growth factor-reduced MATRIGEL.TM. at a
1:60 dilution.
[0095] Markers characteristic of the definitive endoderm lineage
are selected from the group consisting of SOX17, GATA4, HNF-3 beta,
GSC, CER1, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4
CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99,
and OTX2. Suitable for use in the present invention is a cell that
expresses at least one of the markers characteristic of the
definitive endoderm lineage. In one aspect of the present
invention, a cell expressing markers characteristic of the
definitive endoderm lineage is a primitive streak precursor cell.
In an alternate aspect, a cell expressing markers characteristic of
the definitive endoderm lineage is a mesendoderm cell. In an
alternate aspect, a cell expressing markers characteristic of the
definitive endoderm lineage is a definitive endoderm cell.
[0096] Markers characteristic of the pancreatic endoderm lineage
are selected from the group consisting of PDX1, HNF-1 beta, PTF1
alpha, HNF6, HB9 and PROX1. Suitable for use in the present
invention is a cell that expresses at least one of the markers
characteristic of the pancreatic endoderm lineage. In one aspect of
the present invention, a cell expressing markers characteristic of
the pancreatic endoderm lineage is a pancreatic endoderm cell.
[0097] Markers characteristic of the pancreatic endocrine lineage
are selected from the group consisting of NGN3, NEUROD, ISL1, PDX1,
NKX6.1, PAX4, and PTF-1 alpha. In one embodiment, a pancreatic
endocrine cell is capable of expressing at least one of the
following hormones: insulin, glucagon, somatostatin, and pancreatic
polypeptide. Suitable for use in the present invention is a cell
that expresses at least one of the markers characteristic of the
pancreatic endocrine lineage. In one aspect of the present
invention, a cell expressing markers characteristic of the
pancreatic endocrine lineage is a pancreatic endocrine cell. The
pancreatic endocrine cell may be a pancreatic hormone expressing
cell. Alternatively, the pancreatic endocrine cell may be a
pancreatic hormone secreting cell.
Formation of Cells Expressing Markers Characteristic of the
Definitive Endoderm Lineage
[0098] In one aspect of the present invention, pluripotent stem
cells may be differentiated into cells expressing markers
characteristic of the definitive endoderm lineage by culturing the
pluripotent stem cells in medium comprising a sufficient amount of
GDF-8 to cause the differentiation of the pluripotent stem cells
into cells expressing markers characteristic of the definitive
endoderm lineage.
[0099] The pluripotent stem cells may be cultured in the medium
containing a sufficient amount of GDF-8 for about one day to about
seven days. Alternatively, the pluripotent stem cells may be
cultured in the medium containing a sufficient amount of GDF-8 for
about one day to about six days. Alternatively, the pluripotent
stem cells may be cultured in the medium containing a sufficient
amount of GDF-8 for about one day to about five days.
Alternatively, the pluripotent stem cells may be cultured in the
medium containing a sufficient amount of GDF-8 for about one day to
about four days. Alternatively, the pluripotent stem cells may be
cultured in the medium containing a sufficient amount of GDF-8 for
about one day to about three days. Alternatively, the pluripotent
stem cells may be cultured in the medium containing a sufficient
amount of GDF-8 for about one day to about two days. Alternatively,
the pluripotent stem cells may be cultured in the medium containing
a sufficient amount of GDF-8 for about one day.
[0100] In one embodiment, the GDF-8 is used at a concentration from
about 5 ng/ml to about 500 ng/ml. In an alternate embodiment, the
GDF-8 is used at a concentration from about 5 ng/ml to about 50
ng/ml. In an alternate embodiment, the GDF-8 is used at a
concentration from about 5 ng/ml to about 25 ng/ml. In an alternate
embodiment, the GDF-8 is used at a concentration of about 25
ng/ml.
[0101] In one embodiment, the medium comprising a sufficient amount
of GDF-8 also contains at least one other factor. In one
embodiment, the at least one other factor is selected from the
group consisting of: EGF, FGF4, PDGF-A, PDGF-B, PDGF-C, PDGF-D,
VEGF, muscimol, PD98059, LY294002, U0124, U0126, and sodium
butyrate.
[0102] In one embodiment, the EGF is used at a concentration from
about 5 ng/ml to about 500 ng/ml. In an alternate embodiment, the
EGF is used at a concentration from about 5 ng/ml to about 50
ng/ml. In an alternate embodiment, the EGF is used at a
concentration of about 50 ng/ml.
[0103] In one embodiment, the FGF4 is used at a concentration from
about 5 ng/ml to about 500 ng/ml. In an alternate embodiment, the
FGF4 is used at a concentration from about 5 ng/ml to about 50
ng/ml. In an alternate embodiment, the FGF4 is used at a
concentration of about 50 ng/ml.
[0104] In one embodiment, the PDGF-A is used at a concentration
from about 5 ng/ml to about 500 ng/ml. In an alternate embodiment,
the PDGF-A is used at a concentration from about 5 ng/ml to about
50 ng/ml. In an alternate embodiment, the PDGF-A is used at a
concentration of about 50 ng/ml.
[0105] In one embodiment, the PDGF-B is used at a concentration
from about 5 ng/ml to about 500 ng/ml. In an alternate embodiment,
the PDGF-B is used at a concentration from about 5 ng/ml to about
50 ng/ml. In an alternate embodiment, the PDGF-B is used at a
concentration of about 50 ng/ml.
[0106] In one embodiment, the PDGF-C is used at a concentration
from about 5 ng/ml to about 500 ng/ml. In an alternate embodiment,
the PDGF-C is used at a concentration from about 5 ng/ml to about
50 ng/ml. In an alternate embodiment, the PDGF-C is used at a
concentration of about 50 ng/ml.
[0107] In one embodiment, the PDGF-D is used at a concentration
from about 5 ng/ml to about 500 ng/ml. In an alternate embodiment,
the PDGF-D is used at a concentration from about 5 ng/ml to about
50 ng/ml. In an alternate embodiment, the PDGF-D is used at a
concentration of about 50 ng/ml.
[0108] In one embodiment, the VEGF is used at a concentration from
about 5 ng/ml to about 500 ng/ml. In an alternate embodiment, the
VEGF is used at a concentration from about 5 ng/ml to about 50
ng/ml. In an alternate embodiment, the VEGF is used at a
concentration of about 50 ng/ml.
[0109] In one embodiment, the muscimol is used at a concentration
from about 1 .mu.M to about 200 .mu.M. In an alternate embodiment,
the muscimol is used at a concentration from about 1 .mu.M to about
20 .mu.M. In an alternate embodiment, the muscimol is used at a
concentration of about 20 .mu.M.
[0110] In one embodiment, the PD98059 is used at a concentration
from about 0.1 .mu.M to about 10 .mu.M. In an alternate embodiment,
the PD98059 is used at a concentration from about 0.1 .mu.M to
about 1 .mu.M. In an alternate embodiment, the PD98059 is used at a
concentration of about 1 .mu.M.
[0111] In one embodiment, the LY294002 is used at a concentration
from about 0.25 .mu.M to about 25 .mu.M. In an alternate
embodiment, the LY294002 is used at a concentration from about 0.25
.mu.M to about 2.5 .mu.M. In an alternate embodiment, the LY294002
is used at a concentration of about 2.5 .mu.M.
[0112] In one embodiment, the U0124 is used at a concentration from
about 0.1 .mu.M to about 10 .mu.M. In an alternate embodiment, the
U0124 is used at a concentration from about 0.1 .mu.M to about 1
.mu.M. In an alternate embodiment, the U0124 is used at a
concentration of about 1 .mu.M.
[0113] In one embodiment, the U0126 is used at a concentration from
about 0.1 .mu.M to about 10 .mu.M. In an alternate embodiment, the
U0126 is used at a concentration from about 0.1 .mu.M to about 1
.mu.M. In an alternate embodiment, the U0126 is used at a
concentration of about 1 .mu.M.
[0114] In one embodiment, the sodium butyrate is used at a
concentration from about 0.05 .mu.M to about 5 .mu.M. In an
alternate embodiment, the sodium butyrate is used at a
concentration from about 0.05 .mu.M to about 0.5 .mu.M. In an
alternate embodiment, the sodium butyrate is used at a
concentration of about 0.5 .mu.M.
[0115] In an alternate embodiment, the at least one other factor is
selected from the group consisting of: an aniline-pyridinotriazine,
a cyclic aniline-pyridinotriazine,
N-{[1-(Phenylmethyl)azepan-4-yl]methyl}-2-pyridin-3-ylacetamide,
4-{[4-(4-{[2-(Pyridin-2-ylamino)ethyl]amino}-1,3,5-triazin-2-yl)pyridin-2-
-yl]oxy}butan-1-ol
3-({3-[4-({2-[Methyl(pyridin-2-yl)amino]ethyl}amino)-1,3,5-triazin-2-yl]p-
yridin-2-yl}amino)propan-1-ol,
N.about.4.about.-[2-(3-Fluorophenyl)ethyl]-N.about.2.about.-[3-(4-methylp-
iperazin-1-yl)propyl]pyrido[2,3-d]pyrimidine-2,4-diamine,
1-Methyl-N-[(4-pyridin-3-yl-2-{[3-(trifluoromethyl)phenyl]amino}-1,3-thia-
zol-5-yl)methyl]piperidine-4-carboxamide,
1,1-Dimethylethyl{2-[4-({5-[3-(3-hydroxypropyl)phenyl]-4H-1,2,4-triazol-3-
-yl}amino)phenyl]ethyl}carbamate,
1,1-Dimethylethyl{[3-({5-[5-(3-hydroxypropyl)-2-(methyloxy)phenyl]-1,3-ox-
azol-2-yl}amino)phenyl]methyl}carbamate,
1-({5-[6-({4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}amino)pyrazin-2-yl]-
thiophen-2-yl}methyl)piperidin-4-ol,
1-({4-[6-({4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}amino)pyrazin-2-yl]-
thiophen-2-yl}methyl)piperidine-4-carboxamide, and
2-{[4-(1-Methylethyl)phenyl]amino}-N-(2-thiophen-2-ylethyl)-7,8-dihydropy-
rido[4,3-d]pyrimidine-6(5H)-carboxamide.
The Compounds of the Present Invention
[0116] The present invention provides compounds that are capable of
differentiating pluripotent stem cells into cells expressing
markers characteristic of the definitive endoderm lineage.
[0117] In one embodiment, the compound that is capable of
differentiating pluripotent stem cells into cells expressing
markers characteristic of the definitive endoderm lineage is an
aniline-pyridinotriazine of the Formula (1):
##STR00001##
[0118] The N-oxide forms, the pharmaceutically acceptable addition
salts and the stereochemically isomeric forms thereof, wherein:
[0119] m represents an integer from 1 to 4; n represents an integer
from 1 to 4; Z represents N or C;
[0120] R.sup.1 and R.sup.8 each independently represent hydrogen,
Het.sup.14, cyano, halo, hydroxy, C.sub.1-6alkoxy-,
C.sub.1-6alkyl-, mono- or di(C.sub.1-4alkyl)amino-carbonyl-, mono-
or di(C.sub.1-4alkyl)amino-sulfonyl, C.sub.1-6alkoxy-substituted
with halo or R.sup.1 represents C.sub.1-6alkyl substituted with one
or where possible two or more substituents selected from hydroxy or
halo;
[0121] R.sup.2 and R.sup.9 each independently represents hydrogen,
C.sub.1-4alkyl, C.sub.2-4alkenyl, Het.sup.3,
Het.sup.4-C.sub.1-4alkyl-, Het.sup.5-C.sub.1-4alkylcarbonyl-, mono-
or di(C.sub.1-4alkyl)amino-C.sub.1-4alkyl-carbonyl- or phenyl
optionally substituted with one or where possible two or more
substituents selected from hydrogen, hydroxy, amino or
C.sub.1-4alkyloxy-;
[0122] R.sup.3 and R.sup.7 each independently represent hydrogen,
C.sub.1-4alkyl, Het.sup.6, Het.sup.7-C.sub.1-4alkyl-,
C.sub.2-4alkenylcarbonyl-optionally substituted with
Het.sup.8-C.sub.1-4alkylaminocarbonyl-, C.sub.2-4alkenylsulfonyl-,
C.sub.1-4alkyloxyC.sub.1-4alkyl- or phenyl optionally substituted
with one or where possible two or more substituents selected from
hydrogen, hydroxy, amino or C.sub.1-4alkyloxy-;
[0123] R4, R5, R6 and R.sup.10 each independently represent
hydrogen or C.sub.1-4alkyl optionally substituted with hydroxy,
Het.sup.9 or C.sub.1-4alkyloxy;
[0124] Het.sup.1 and Het.sup.2 each independently represent a
heterocycle selected from pyrrolidinyl, piperidinyl, piperazinyl,
pyridinyl, pyrimidinyl, pyrazinyl, imidazolidinyl or pyrazolidinyl
wherein said Het.sup.1 and Het.sup.2 are optionally substituted
with amino, hydroxy, C.sub.1-4alkyl, hydroxy-C.sub.1-4allcyl-,
phenyl, phenyl-C.sub.1-4alkyl-,
C.sub.1-4alkyl-oxy-C.sub.1-4alkyl-mono- or di(C.sub.1-4alkyl)
amino- or amino-carbonyl-;
[0125] Het.sup.3 and Het.sup.6 each independently represent,
heterocycle selected from pyrrolidinyl or piperidinyl wherein said
Het.sup.3 and Het.sup.6 are optionally substituted with one or
where possible two or more substituents selected from
C.sub.1-4alkyl, C.sub.3-6cycloalkyl, hydroxy-C.sub.1-4alkyl-,
C.sub.1-4alkyloxyC.sub.1-4alkyl or polyhydroxy-C.sub.1-4alkyl-;
[0126] Het.sup.4, Het.sup.7 and Het.sup.9 each independently
represent a heterocycle selected from morpholinyl, pyrrolidinyl,
piperazinyl or piperidinyl wherein said Het.sup.4, Het.sup.7 and
Het.sup.9 are optionally substituted with one or where possible two
or more substituents selected from C.sub.1-4alkyl,
C.sub.3-6cycloalkyl, hydroxy-C.sub.1-4alkyl-,
C.sub.1-4alkyloxyC.sub.1-4alkyl or polyhydroxy-C.sub.1-4alkyl-;
[0127] Het.sup.5 represents a heterocycle selected from
morpholinyl, pyrrolidinyl, piperazinyl or pipendinyl wherein said
Het.sup.5 is optionally substituted with one or where possible two
or more substituents selected from C.sub.1-4alkyl,
C.sub.3-6cycloalkyl, hydroxy-C.sub.1-4alkyl-,
C.sub.1-4alkyloxyC.sub.1-4alkyl or polyhydroxy-C.sub.1-4alkyl-;
[0128] Het.sup.10, Het.sup.11 and Het.sup.13 each independently
represent a heterocycle selected from pyrrolidinyl, piperidinyl,
piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, imidazolidinyl or
pyrazolidinyl wherein said Het.sup.10, Het.sup.11 and Het.sup.13
are optionally substituted with amino, hydroxy, C.sub.1-4alkyl,
hydroxy-C.sub.1-4alkyl-, phenyl, phenyl-C.sub.1-4alkyl-,
C.sub.1-4alkyl-oxy-C.sub.1-4alkyl-, amino-carbonyl- or mono- or
di(C.sub.1-4alkyl)amino-;
[0129] Het.sup.12 represents a heterocycle selected from
pyrrolidinyl, piperidinyl, piperazinyl, pyridinyl, pyrimidinyl,
pyrazinyl, imidazolidinyl or pyrazolidinyl wherein said Het.sup.12
is optionally substituted with amino, hydroxy, C.sub.1-4alkyl,
hydroxy-C.sub.1-4alkyl-, phenyl, phenyl-C.sub.1-4alkyl-,
C.sub.1-4alkyl-oxy-C.sub.1-4alkyl-; mono- or
di(C.sub.1-4alkyl)amino- or amino-carbonyl-;
[0130] Het.sup.14 represents a heterocycle selected from
morpholinyl; pyrrolidinyl; piperazinyl; imidazolyl; pyrrolyl;
2,3,4-triazapyrrolyl; 1,2,3-triazolyl; pyrazolyl; or piperidinyl
wherein said Het.sup.14 is optionally substituted with one or where
possible two or more substituents selected from C.sub.1-4alkyl,
C.sub.3-6cycloalkyl, hydroxy-C.sub.1-4alkyl-,
C.sub.1-4alkyloxyC.sub.1-4alkyl or polyhydroxy-C.sub.1-4alkyl-; in
particular Het.sup.14 represents a heterocycle selected from
morpholinyl; pyrrolidinyl; pyrrolyl; 2,3,4-triazapyrrolyl;
piperazinyl or piperidinyl wherein said Het.sup.14 is optionally
substituted with one or where possible two or more substituents
selected from C.sub.1-4alkyl, C.sub.3-6cycloalkyl,
hydroxy-C.sub.1-4alkyl-, C.sub.1-4alkyloxyC.sub.1-4alkyl or
polyhydroxy-C.sub.1-4alkyl-; more particular Het.sup.14 represents
a heterocycle selected from morpholinyl; pyrrolidinyl; piperazinyl
or piperidinyl wherein said Het.sup.14 is optionally substituted
with one or where possible two or more substituents selected from
C.sub.1-4alkyl, C.sub.3-6cycloalkyl, hydroxy-C.sub.1-4alkyl-,
C.sub.1-4alkyloxyC.sub.1-4alkyl or polyhydroxy-C.sub.1-4alkyl-.
[0131] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (1).
[0132] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (2).
##STR00002##
Formula (2):
3-{3-[(4-Pyridin-3-yl-1,3,5-triazin-2-yl)amino]phenyl}propanoic
acid. Referred to herein as "Compound 1".
[0133] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (3).
##STR00003##
Formula (3):
2-{3-[(4-Pyridin-3-yl-1,3,5-triazin-2-yl)amino]phenyl}ethanol.
Referred to herein as "Compound 2".
[0134] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (4).
##STR00004##
Formula (4):
1,1-Dimethylethyl{2-[3-({4-[2-(3-hydroxyprop-1-yn-1-yl)pyridin-4-yl]-1,3,-
5-triazin-2-yl}amino)phenyl]ethyl}carbamate. Referred to herein as
"Compound 3".
[0135] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (5).
##STR00005##
Formula (5):
1,1-Dimethylethyl{4-[4-(4-{[3-(hydroxymethyl)phenyl]amino
}-1,3,5-triazin-2-yl)pyridin-2-yl]butyl}carbamate. Referred to
herein as "Compound 4".
[0136] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (6).
##STR00006##
Formula (6):
1,1-Dimethylethyl{3-[{[5-(2-{[3-bromo-5-(hydroxymethyl)phenyl]amino}pyrim-
idin-4-yl)-2-(methyloxy)phenyl]methyl}(methyl)amino]propyl}carbamate.
Referred to herein as "Compound 5".
[0137] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (7).
##STR00007##
Formula (7):
4-{[3-(3-Fluorophenyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl]amino}benz-
oic acid. Referred to herein as "Compound 6".
[0138] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (8).
##STR00008##
Formula (8):
2-Fluoro-5-[(3-phenyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)amino]benzo-
ic acid. Referred to herein as "Compound 7".
[0139] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (9).
##STR00009##
Formula (9):
N-{[3-(5-{[3-(2-Aminopyrimidin-4-yl)phenyl]amino}-3H-[1,2,3]triazolo[4,5--
d]pyrimidin-3-yl)phenyl]methyl}cyclopropanecarboxamide. Referred to
herein as "Compound 8".
[0140] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (10).
##STR00010##
[0141] Formula (10):
4-[(1-Cyclohexyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino]-N-[3-(methyloxy)-
propyl]benzenesulfonamide. Referred to herein as "Compound 9".
[0142] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (11).
##STR00011##
Formula (11):
4-Chloro-2-[(6-{[3-(chloromethyl)-4-methoxyphenyl]amino}pyrimidin-4-yl)am-
ino]phenol. Referred to herein as "Compound 10".
[0143] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (12).
##STR00012##
Formula (12):
4-{[4-(4-Methyl-3,4-dihydroquinoxalin-1(2H)-yl)pyrimidin-2-yl]amino}-N-(1-
-methylpiperidin-4-yl)benzamide. Referred to herein as "Compound
11".
[0144] In one embodiment, the aniline-pyridinotriazine is a
compound of the Formula (13).
##STR00013##
Formula (13):
N-(2-Methoxy-4-{[(3-methoxypropyl)amino]methyl}phenyl)-4-(
1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine. Referred to herein
as "Compound 12".
[0145] In one embodiment, the compound that is capable of
differentiating pluripotent stem cells into cells expressing
markers characteristic of the definitive endoderm lineage is a
cyclic aniline-pyridinotriazine of the Formula (14):
##STR00014##
[0146] The N-oxide forms, the pharmaceutically acceptable addition
salts and the stereochemically isomeric forms thereof, wherein:
[0147] m represents an integer from 1 to 4; n represents an integer
from 1 to 4; Z represents N or C;
[0148] Y represents --NR.sup.2--C.sub.1-6alkyl-CO--NR.sup.4--,
--C.sub.1-4alkyl-NR.sup.9--C.sub.1-4alkyl-,
C.sub.1-6alkyl-CO-Het.sup.10-, -Het.sup.11-CO--C.sub.1-6alkyl-,
-Het.sup.12-C.sub.1-6alkyl-, --CO-Het.sup.13-C.sub.1-6alkyl-,
--CO--NR.sup.10--C.sub.1-6alkyl-,
-Het.sup.1-C.sub.1-6alkyl-CO--NR.sup.5--, or
-Het.sup.2-CO--NR.sup.6-- wherein the-C.sub.1-6alkyl-linker in
--NR.sup.2--C.sup.1-6alkyl-CO--NR.sup.4-- or
-Het.sup.1-C.sub.1-6alkyl-CO--NR.sup.5-- is optionally substituted
with one or where possible two or more substituents selected from
hydroxy, methoxy, aminocarbonyl, halo, phenyl, indolyl,
methylsulfide, thiol, hydroxyphenyl, cyanophenyl, amino and
hydroxycarbonyl;
[0149] X.sup.1 represents a direct bond, C.sub.1-4alkyl,
C.sub.1-4alkyloxy-, C.sub.1-4alkyl-CO--, C.sub.2-4alkenyl,
C.sub.2-4alkynyl, or C.sub.1-4alkyl-NR.sup.3--, wherein said
C.sub.1-4alkyl or C.sub.2-4alkenyl is optionally substituted with
one or where possible two or more halo substituents;
[0150] X.sup.2 represents a direct bond, C.sub.1-4alkyl,
C.sub.1-4alkyloxy-, C.sub.1-4alkyl-CO--, C.sub.2-4alkenyl,
C.sub.2-4alkynyl, or C.sub.1-4alkyl-NR.sup.7--, wherein said
C.sub.1-4alkyl or C.sub.2-4alkenyl is optionally substituted with
one or where possible two or more halo substituents;
[0151] R.sup.1 and R.sup.8 each independently represent hydrogen,
Het.sup.14, cyano, halo, hydroxy, C.sub.1-6alkoxy-,
C.sub.1-6alkyl-, mono- or di(C.sub.1-4alkyl)amino-carbonyl-, mono-
or di(C.sub.1-4alkyl)amino-sulfonyl, C.sub.1-6alkoxy-substituted
with halo or R.sup.1 represents C.sub.1-6alkyl substituted with one
or where possible two or more substituents selected from hydroxy or
halo;
[0152] R.sup.2 and R.sup.9 each independently represents hydrogen,
C.sub.1-4alkyl, C.sub.2-4alkenyl, Het.sup.3,
Het.sup.4-C.sub.1-4alkyl-, Het.sup.5-C.sub.1-4alkylcarbonyl-, mono-
or di(C.sub.1-4alkyl)amino-C.sub.1-4alkyl-carbonyl- or phenyl
optionally substituted with one or where possible two or more
substituents selected from hydrogen, hydroxy, amino or
C.sub.1-4alkyloxy-;
[0153] R.sup.3 and R.sup.7 each independently represent hydrogen,
C.sub.1-4alkyl, Het.sup.6, Het.sup.7-C.sub.1-4alkyl-,
C.sub.2-4alkenylcarbonyl-optionally substituted with
Het.sup.8-C.sub.1-4alkylaminocarbonyl-, C.sub.2-4alkenylsulfonyl-,
C.sub.1-4alkyloxyC.sub.1-4alkyl- or phenyl optionally substituted
with one or where possible two or more substituents selected from
hydrogen, hydroxy, amino or C.sub.1-4alkyloxy-;
[0154] R4, R5, R6 and R.sup.10 each independently represent
hydrogen or C.sub.1-4alkyl optionally substituted with hydroxy,
Het.sup.9 or C.sub.1-4alkyloxy;
[0155] Het.sup.1 and Het.sup.2 each independently represent a
heterocycle selected from pyrrolidinyl, piperidinyl, piperazinyl,
pyridinyl, pyrimidinyl, pyrazinyl, imidazolidinyl or pyrazolidinyl
wherein said Het.sup.1 and Het.sup.2 are optionally substituted
with amino, hydroxy, C.sub.1-4alkyl, hydroxy-C.sub.1-4allcyl-,
phenyl, phenyl-C.sub.1-4alkyl-,
C.sub.1-4alkyl-oxy-C.sub.1-4alkyl-mono- or di(C.sub.1-4alkyl)
amino- or amino-carbonyl-;
[0156] Het.sup.3 and Het.sup.6 each independently represent,
heterocycle selected from pyrrolidinyl or piperidinyl wherein said
Het.sup.3 and Het.sup.6 are optionally substituted with one or
where possible two or more substituents selected from
C.sub.1-4alkyl, C.sub.3-6cycloalkyl, hydroxy-C.sub.1-4alkyl-,
C.sub.1-4alkyloxyC.sub.1-4alkyl or polyhydroxy-C.sub.1-4alkyl-;
[0157] Het.sup.4, Het.sup.7 and Het.sup.9 each independently
represent a heterocycle selected from morpholinyl, pyrrolidinyl,
piperazinyl or piperidinyl wherein said Het.sup.4, Het.sup.7 and
Het.sup.9 are optionally substituted with one or where possible two
or more substituents selected from C.sub.1-4alkyl,
C.sub.3-6cycloalkyl, hydroxy-C.sub.1-4alkyl-,
C.sub.1-4alkyloxyC.sub.1-4alkyl or polyhydroxy-C.sub.1-4alkyl-;
[0158] Het.sup.5 represents a heterocycle selected from
morpholinyl, pyrrolidinyl, piperazinyl or pipendinyl wherein said
Het.sup.5 is optionally substituted with one or where possible two
or more substituents selected from C.sub.1-4alkyl,
C.sub.3-6cycloalkyl, hydroxy-C.sub.1-4alkyl-,
C.sub.1-4alkyloxyC.sub.1-4alkyl or polyhydroxy-C.sub.1-4alkyl-;
[0159] Het.sup.10, Het.sup.11 and Het.sup.13 each independently
represent a heterocycle selected from pyrrolidinyl, piperidinyl,
piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, imidazolidinyl or
pyrazolidinyl wherein said Het.sup.10, Het.sup.11 and Het.sup.13
are optionally substituted with amino, hydroxy, C.sub.1-4alkyl,
hydroxy-C.sub.1-4alkyl-, phenyl, phenyl-C.sub.1-4alkyl-,
C.sub.1-4alkyl-oxy-C.sub.1-4alkyl-, amino-carbonyl- or mono- or
di(C.sub.1-4alkyl)amino-;
[0160] Het.sup.12 represents a heterocycle selected from
pyrrolidinyl, piperidinyl, piperazinyl, pyridinyl, pyrimidinyl,
pyrazinyl, imidazolidinyl or pyrazolidinyl wherein said Het.sup.12
is optionally substituted with amino, hydroxy, C.sub.1-4alkyl,
hydroxy-C.sub.1-4alkyl-, phenyl, phenyl-C.sub.1-4alkyl-,
C.sub.1-4alkyl-oxy-C.sub.1-4alkyl-; mono- or
di(C.sub.1-4alkyl)amino- or amino-carbonyl-;
[0161] Het.sup.14 represents a heterocycle selected from
morpholinyl; pyrrolidinyl; piperazinyl; imidazolyl; pyrrolyl;
2,3,4-triazapyrrolyl; 1,2,3-triazolyl; pyrazolyl; or piperidinyl
wherein said Het.sup.14 is optionally substituted with one or where
possible two or more substituents selected from C.sub.1-4alkyl,
C.sub.3-6cycloalkyl, hydroxy-C.sub.1-4alkyl-,
C.sub.1-4alkyloxyC.sub.1-4alkyl or polyhydroxy-C.sub.1-4alkyl-; in
particular Het.sup.14 represents a heterocycle selected from
morpholinyl; pyrrolidinyl; pyrrolyl; 2,3,4-triazapyrrolyl;
piperazinyl or piperidinyl wherein said Het.sup.14 is optionally
substituted with one or where possible two or more substituents
selected from C.sub.1-4alkyl, C.sub.3-6cycloalkyl,
hydroxy-C.sub.1-4alkyl-, C.sub.1-4alkyloxyC.sub.1-4alkyl or
polyhydroxy-C.sub.1-4alkyl-; more particular Het.sup.14 represents
a heterocycle selected from morpholinyl; pyrrolidinyl; piperazinyl
or piperidinyl wherein said Het.sup.14 is optionally substituted
with one or where possible two or more substituents selected from
C.sub.1-4alkyl, C.sub.3-6cycloalkyl, hydroxy-C.sub.1-4alkyl-,
C.sub.1-4alkyloxyC.sub.1-4alkyl or polyhydroxy-C.sub.1-4alkyl-.
[0162] Compounds of Formula (7) are disclosed in WO2007/003525,
assigned to Janssen Pharmaceutica N.V.
[0163] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (14).
[0164] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (15).
##STR00015##
Formula (15):
1,8,10,12,17,19,23,27,33-Nonaazapentacyclo[25.2.2.1.about.3,7.about..1.ab-
out.9,13.about..1.about.14,18.about.]tetratriaconta-3(34),4,6,9(33),10,12,-
14(32),15,17-nonaen-24-one. Referred to herein as "Compound
13".
[0165] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (16).
##STR00016##
Formula (16):
10-Chloro-14-ethyl-3,5,7,14,17,22,27-heptaazatetracyclo[19.3.1.1.about.2,-
6.about..1.about.8,12.about.]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-no-
naen-16-one. Referred to herein as "Compound 14".
[0166] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (17).
##STR00017##
Formula (17):
14-Ethyl-3,5,7,14,17,27-hexaazatetracyclo[19.3.1.1.about.2,6.about..1.abo-
ut.8,12.about.]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one.
Referred to herein as "Compound 15".
[0167] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (18).
##STR00018##
Formula (18):
10-Chloro-14-ethyl-3,5,7,14,17,27-hexaazatetracyclo[19.3.1.1.about.2,6.ab-
out..1.about.8,12.about.]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-
-16-one. Referred to herein as "Compound 16".
[0168] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (19).
##STR00019##
Formula (19):
3,5,7,14,20,26,31-Heptaazapentacyclo[22.3.1.1.about.2,6.about..1.about.8,-
12.about..1.about.14,18.about.]hentriaconta-1(28),2(31),3,5,8(30),9,11,24,-
26-nonaen-19-one. Referred to herein as "Compound 17".
[0169] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (20).
##STR00020##
Formula (20):
(18S)-3,5,7,14,20,26,30-Heptaazapentacyclo[22.3.1.1.about.2,6.about..1.ab-
out.8,12.about..0.about.14,18.about.]triaconta-1(28),2(30),3,5,8(29),9,11,-
24,26-nonaen-19-one. Referred to herein as "Compound 18".
[0170] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (21).
##STR00021##
Formula (21):
14-Methyl-3,5,7,14,18,24,28-heptaazatetracyclo[20.3.1.1.about.2,6.about..-
1.about.8,12.about.]octacosa-1(26),2(28),3,5,8(27),9,11,22,24-nonaen-17-on-
e. Referred to herein as "Compound 19".
[0171] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (22).
##STR00022##
Formula (22):
14-Methyl-3,5,7,14,19,25,29-heptaazatetracyclo[21.3.1.1.about.2,6.about..-
1.about.8,12.about.]nonacosa-1(27),2(29),3,5,8(28),9,11,23,25-nonaen-18-on-
e. Referred to herein as "Compound 20".
[0172] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (23).
##STR00023##
Formula (23):
14-Methyl-3,5,7,14,18,22,29-heptaazatetracyclo[21.3.1.1.about.2,6.about..-
1.about.8,12.about.]nonacosa-1(27),2(29),3,5,8(28),9,11,23,25-nonaen-17-on-
e. Referred to herein as "Compound 21".
[0173] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (24).
##STR00024##
Formula (24):
1,8,10,12,16,22,30-Heptaazapentacyclo[22.2.2.1.about.3,7.1.about.9,13.abo-
ut..1.about.14,18.about.]hentriaconta-3(31),4,6,9(30),10,12,14(29),15,17-n-
onaen-23-one. Referred to herein as "Compound 22".
[0174] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (25).
##STR00025##
Formula (25):
1,8,10,12,16,22,26,32-Octaazapentacyclo[24.2.2.1.about.3,7.about..1.about-
.9,13.about..1.about.14,18.about.]tritriaconta-3(33),4,6,9(32),10,12,14(31-
),15,17-nonaen-23-one. Referred to herein as "Compound 23".
[0175] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (26).
##STR00026##
Formula (26):
5-Chloro-17-fluoro-1,8,10,12,22,26,32-heptaazapentacyclo[24.2.2.1.about.3-
,7.about..1.about.9,13.about..1.about.14,18.about.]tritriaconta-3(33),4,6,-
9(32),10,12,14(31),15,17-nonaen-23-one. Referred to herein as
"Compound 24".
[0176] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (27).
##STR00027##
Formula (27):
10-Chloro-14-ethyl-22-fluoro-3,5,7,14,17,27-hexaazatetracyclo[19.3.1.1.ab-
out.2,6.about..1.about.8,12.about.]heptacosa-1(25),2(27),3,5,8(26),9,11,21-
,23-nonaen-16-one. Referred to herein as "Compound 25".
[0177] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (28).
##STR00028##
Formula (28):
10-Chloro-25-fluoro-3,5,7,14,20,31-hexaazapentacyclo[22.3.1.1.about.2,6.a-
bout..1.about.8,12.about..1.about.14,18.about.]hentriaconta-1(28),2(31),3,-
5,8(30),9,11,24,26-nonaen-19-one. Referred to herein as "Compound
26".
[0178] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (29).
##STR00029##
Formula (29):
4-Chloro-1,8,10,12,17,22,26,32-octaazapentacyclo[24.2.2.1.about.3,7.about-
..1.about.9,13.about..1.about.14,18.about.]tritriaconta-3(33),4,6,9(32),10-
,12,14(31),15,17-nonaen-23-one. Referred to herein as "Compound
27".
[0179] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (30).
##STR00030##
Formula (30):
18-Methyl-3,5,7,15,18,28-hexaazatetracyclo[20.3.1.1.about.2,6.about..1.ab-
out.8,12.about.]octacosa-1(26),2(28),3,5,8(27),9,11,22,24-nonaen-16-one.
Referred to herein as "Compound 28".
[0180] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (31).
##STR00031##
Formula (31):
18-Ethyl-3,5,7,15,18,28-hexaazatetracyclo[20.3.1.1.about.2,6.about..1.abo-
ut.8,12.about.]octacosa-1(26),2(28),3,5,8(27),9,11,22,24-nonaen-16-one.
Referred to herein as "Compound 29".
[0181] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (32).
##STR00032##
Formula (32):
1,8,10,12,17,19,23,27,33-Nonaazapentacyclo[25.2.2.1.about.3,7.about..1.ab-
out.9,13.about..1.about.14,18.about.]tetratriaconta-3(34),4,6,9(33),10,12,-
14(32),15,17-nonaen-24-one. Referred to herein as "Compound
30".
[0182] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (33).
##STR00033##
Formula (33):
1,11,13,15,23,31-Hexaazapentacyclo[23.2.2.1.about.5,9.about..1.about.10,1-
4.about..1.about.16,20.about.]dotriaconta-5(32),6,8,10(31),11,13,16(30),17-
,19-nonaen-24-one. Referred to herein as "Compound 31".
[0183] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (34).
##STR00034##
Formula (34):
15-Ethyl-13,14,15,16,18,19-hexahydro-1H-6,2-(azeno)-7,11-(metheno)-1,3,5,-
15,18-benzopentaazacyclohenicosin-17(12H)-one. Referred to herein
as "Compound 32".
[0184] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (35).
##STR00035##
Formula (35):
20-Methyl-3,5,7,15,20,30-hexaazatetracyclo[22.3.1.1.about.2,6.about..1.ab-
out.8,12.about.]triaconta-1(28),2(30),3,5,8(29),9,11,24,26-nonaen-16-one.
Referred to herein as "Compound 33".
[0185] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (36).
##STR00036##
Formula (36):
5-Chloro-1,8,10,12,16,22,26,32-octaazapentacyclo[24.2.2.1.about.3,7.about-
..1.about.9,13.about..1.about.14,18.about.]tritriaconta-3(33),4,6,9(32),10-
,12,14(31),15,17-nonaen-23-one. Referred to herein as "Compound
34".
[0186] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (37).
##STR00037##
Formula (37):
10-Chloro-14-ethyl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1.about.2,-
6.about..1.about.8,12.about.]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-no-
naen-16-one. Referred to herein as "Compound 35".
[0187] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (38).
##STR00038##
Formula (38):
(18S)-10-Chloro-3,5,7,14,20,26,30-heptaazapentacyclo[22.3.1.1.about.2,6.a-
bout..1.about.8,12.about..0.about.14,18.about.]triaconta-1(28),2(30),3,5,8-
(29),9,11,24,26-nonaen-19-one. Referred to herein as "Compound
36".
[0188] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (39).
##STR00039##
Formula (39):
10-Chloro-3,5,7,14,20,26,31-heptaazapentacyclo[22.3.1.1.about.2,6.about..-
1.about.8,12.about..1.about.14,18.about.]hentriaconta-1(28),2(31),3,5,8(30-
),9,11,24,26-nonaen-19-one. Referred to herein as "Compound
37".
[0189] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (40).
##STR00040##
Formula (40):
5-Chloro-1,8,10,12,16,22,30-heptaazapentacyclo[22.2.2.2.1.about.3,7.about-
..1.about.9,13.about..1.about.14,18.about.]hentriaconta-3(31),4,6,9(30),10-
,12,14(29),15,17-nonaen-23-one. Referred to herein as "Compound
38".
[0190] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (41).
##STR00041##
Formula (41):
9-Methyl-2,3,4,5,7,8,9,10-octahydro-16H-17,21-(azeno)-11,15-(metheno)pyri-
do[3,2-g][1,3,5,9,13,17]hexaazacyclotricosin-6(1H)-one. Referred to
herein as "Compound 39".
[0191] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (42).
##STR00042##
Formula (42):
14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1.about.2,6-
.about..1.about.8,12.about.]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-non-
aen-16-one. Referred to herein as "Compound 40".
[0192] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (43).
##STR00043##
Formula (43):
18-Oxo-14-oxa-2,4,8,17,25-pentaazatetracyclo[17.3.1.1.about.3,7.about..1.-
about.9,13.about.]pentacosa-1(23),3(25),4,6,9(24),10,12,19,21-nonaene-6-ca-
rbonitrile. Referred to herein as "Compound 41".
[0193] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (44).
##STR00044##
Formula (44):
14,21-Dioxa-2,4,8,18,28-pentaazatetracyclo[20.3.1.1.about.3,7.about..1.ab-
out.9,13.about.]octacosa-1(26),3(28),4,6,9(27),10,12,22,24-nonaen-19-one.
Referred to herein as "Compound 42".
[0194] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (45).
##STR00045##
Formula (45):
21-Methyl-1,8,10,11,21,24,30-heptaazapentacyclo[22.2.2.2.1.about.3,7.abou-
t..1.about.9,12.about..1.about.13,17.about.]hentriaconta-3(31),4,6,9,11,13-
(29),14,16-octaen-23-one. Referred to herein as "Compound 43".
[0195] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (46).
##STR00046##
Formula (46):
(18S)-11-(Morpholin-4-ylcarbonyl)-5,7,14,20,28-pentaazapentacyclo[20.3.1.-
1.about.2,6.about..1.about.8,12.about..0.about.14,18.about.]octacosa-1(26)-
,2(28),3,5,8(27),9,11,22,24-nonaen-19-one. Referred to herein as
"Compound 44".
[0196] In one embodiment, the cyclic aniline-pyridinotriazine is a
compound of the Formula (47).
##STR00047##
Formula (47):
10-Methoxy-17-methyl-2,14,15,17,18,19,20,22-octahydro-6H-19,21-methano-7,-
11-(metheno)-12-oxa-2,3,5,6,17,21-hexaazacycloicosa[1,2,3-cd]inden-16(13H)-
-one. Referred to herein as "Compound 45".
[0197] In one embodiment, the at least one other factor is a
compound of the Formula (48):
##STR00048##
Formula (48).
N-{[1-(Phenylmethyl)azepan-4-yl]methyl}-2-pyridin-3-ylacetamide.
Referred herein as "Compound 46".
[0198] In one embodiment, the at least one other factor is a
compound of the Formula (49):
##STR00049##
Formula (49).
4-{[4-(4-{[2-(Pyridin-2-ylamino)ethyl]amino}-1,3,5-triazin-2-yl)pyridin-2-
-yl]oxy}butan-1-ol. Referred herein as "Compound 47".
[0199] In one embodiment, the at least one other factor is a
compound of the Formula (50):
##STR00050##
Formula (50).
3-({3-[4-({2-[Methyl(pyridin-2-yl)amino]ethyl}amino)-1,3,5-triazin-2-yl]p-
yridin-2-yl}amino)propan-1-ol. Referred herein as "Compound
48".
[0200] In one embodiment, the at least one other factor is a
compound of the Formula (51):
##STR00051##
Formula (51).
N.about.4.about.-[2-(3-Fluorophenyl)ethyl]-N.about.2.about.-[3-(4-methylp-
iperazin-1-yl)propyl]pyrido[2,3-d]pyrimidine-2,4-diamine. Referred
herein as "Compound 49".
[0201] In one embodiment, the at least one other factor is a
compound of the Formula (52):
##STR00052##
Formula (52).
1-Methyl-N-[(4-pyridin-3-yl-2-{[3-(trifluoromethyl)phenyl]amino}-1,3-thia-
zol-5-yl)methyl]piperidine-4-carboxamide. Referred herein as
"Compound 50".
[0202] In one embodiment, the at least one other factor is a
compound of the Formula (53):
##STR00053##
Formula (53).
1,1-Dimethylethyl{2-[4-({5-[3-(3-hydroxypropyl)phenyl]-4H-1,2,4-triazol-3-
-yl}amino)phenyl]ethyl}carbamate. Referred herein as "Compound
51".
[0203] In one embodiment, the at least one other factor is a
compound of the Formula (54):
##STR00054##
Formula (54).
1,1-Dimethylethyl{[3-({5-[5-(3-hydroxypropyl)-2-(methyloxy)phenyl]-1,3-ox-
azol-2-yl}amino)phenyl]methyl}carbamate. Referred herein as
"Compound 52".
[0204] In one embodiment, the at least one other factor is a
compound of the Formula (55):
##STR00055##
Formula (55).
1-({5-[6-({4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}amino)pyrazin-2-yl]-
thiophen-2-yl}methyl)piperidin-4-ol. Referred herein as "Compound
53".
[0205] In one embodiment, the at least one other factor is a
compound of the Formula (56):
##STR00056##
Formula (56).
1-({4-[6-({4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}amino)pyrazin-2-yl]-
thiophen-2-yl}methyl)piperidine-4-carboxamide. Referred herein as
"Compound 54".
[0206] In one embodiment, the at least one other factor is a
compound of the Formula (57):
##STR00057##
Formula (57).
2-{[4-(1-Methylethyl)phenyl]amino}-N-(2-thiophen-2-ylethyl)-7,8-dihydropy-
rido[4,3-d]pyrimidine-6(5H)-carboxamide. Referred herein as
"Compound 55".
[0207] In one embodiment, the at least one other factor is a
compound of the Formula (58):
##STR00058##
Formula (58).
6-[(2-{[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-y-
l]amino}ethyl)amino]pyridine-3-carbonitrile. Referred herein as
"Compound 56".
[0208] In one embodiment, the at least one other factor is a
compound of the Formula (59):
##STR00059##
Formula (59).
4-(6-{[(3-Chlorophenyl)methyl]amino}imidazo[1,2-b]pyridazin-3-yl)-N-[2-(d-
imethylamino)ethyl]benzamide. Referred herein as "Compound 57".
Detection of Cells Expressing Markers Characteristic of the
Definitive Endoderm Lineage
[0209] Formation of cells expressing markers characteristic of the
definitive endoderm lineage may be determined by testing for the
presence of the markers before and after following a particular
protocol. Pluripotent stem cells typically do not express such
markers. Thus, differentiation of pluripotent cells is detected
when cells begin to express them.
[0210] The efficiency of differentiation may be determined by
exposing a treated cell population to an agent (such as an
antibody) that specifically recognizes a protein marker expressed
by cells expressing markers characteristic of the definitive
endoderm lineage.
[0211] Methods for assessing expression of protein and nucleic acid
markers in cultured or isolated cells are standard in the art.
These include quantitative reverse transcriptase polymerase chain
reaction (RT-PCR), Northern blots, in situ hybridization (see,
e.g., Current Protocols in Molecular Biology (Ausubel et al., eds.
2001 supplement)), and immunoassays such as immunohistochemical
analysis of sectioned material, Western blotting, and for markers
that are accessible in intact cells, flow cytometry analysis (FACS)
(see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual,
New York: Cold Spring Harbor Laboratory Press (1998)).
[0212] For example, characteristics of pluripotent stem cells are
well known to those skilled in the art, and additional
characteristics of pluripotent stem cells continue to be
identified. Pluripotent stem cell markers include, for example, the
expression of one or more of the following: ABCG2, cripto, FOXD3,
Connexin43, Connexin45, OCT4, SOX2, Nanog, hTERT, UTF-1, ZFP42,
SSEA-3, SSEA-4, Tral-60, or Tral-81.
[0213] After treating pluripotent stem cells with the methods of
the present invention, the differentiated cells may be purified by
exposing a treated cell population to an agent (such as an
antibody) that specifically recognizes a protein marker, such as
CXCR4, expressed by cells expressing markers characteristic of the
definitive endoderm lineage.
Formation of Cells Expressing Markers Characteristic of the
Pancreatic Endoderm Lineage
[0214] Cells expressing markers characteristic of the definitive
endoderm lineage may be differentiated into cells expressing
markers characteristic of the pancreatic endoderm lineage by any
method in the art or by any method proposed in this invention.
[0215] For example, cells expressing markers characteristic of the
definitive endoderm lineage may be differentiated into cells
expressing markers characteristic of the pancreatic endoderm
lineage according to the methods disclosed in D=Amour et al, Nature
Biotechnology 24, 1392-1401 (2006).
[0216] For example, cells expressing markers characteristic of the
definitive endoderm lineage are further differentiated into cells
expressing markers characteristic of the pancreatic endoderm
lineage, by treating the cells expressing markers characteristic of
the definitive endoderm lineage with a fibroblast growth factor and
the hedgehog signaling pathway inhibitor KAAD-cyclopamine, then
removing the medium containing the fibroblast growth factor and
KAAD-cyclopamine and subsequently culturing the cells in medium
containing retinoic acid, a fibroblast growth factor and
KAAD-cyclopamine. An example of this method is disclosed in Nature
Biotechnology 24, 1392-1401 (2006).
[0217] In one aspect of the present invention, cells expressing
markers characteristic of the definitive endoderm lineage are
further differentiated into cells expressing markers characteristic
of the pancreatic endoderm lineage, by treating the cells
expressing markers characteristic of the definitive endoderm
lineage with retinoic acid and at least one fibroblast growth
factor for a period of time, according to the methods disclosed in
U.S. patent application Ser. No. 11/736,908, assigned to LifeScan,
Inc.
[0218] In one aspect of the present invention, cells expressing
markers characteristic of the definitive endoderm lineage are
further differentiated into cells expressing markers characteristic
of the pancreatic endoderm lineage, by treating the cells
expressing markers characteristic of the definitive endoderm
lineage with retinoic acid and at least one fibroblast growth
factor for a period of time, according to the methods disclosed in
U.S. patent application Ser. No. 11/779,311, assigned to LifeScan,
Inc.
[0219] In one aspect of the present invention, cells expressing
markers characteristic of the definitive endoderm lineage are
further differentiated into cells expressing markers characteristic
of the pancreatic endoderm lineage, by treating the cells
expressing markers characteristic of the definitive endoderm
lineage according to the methods disclosed in U.S. patent
application Ser. No. 60/990,529.
[0220] Cells expressing markers characteristic of the definitive
endoderm lineage may be treated with at least one other additional
factor that may enhance the formation of cells expressing markers
characteristic of the pancreatic endoderm lineage. Alternatively,
the at least one other additional factor may enhance the
proliferation of the cells expressing markers characteristic of the
pancreatic endoderm lineage formed by the methods of the present
invention. Further, the at least one other additional factor may
enhance the ability of the cells expressing markers characteristic
of the pancreatic endoderm lineage formed by the methods of the
present invention to form other cell types, or improve the
efficiency of any other additional differentiation steps.
[0221] The at least one additional factor may be, for example,
nicotinamide, members of TGF-.beta. family, including TGF-.beta.1,
2, and 3, serum albumin, members of the fibroblast growth factor
family, platelet-derived growth factor-AA, and -BB, platelet rich
plasma, insulin growth factor (IGF-I, II), growth differentiation
factor (such as, for example, GDF-5, -6, -8, -10, -11), glucagon
like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody,
Exendin-4, retinoic acid, parathyroid hormone, insulin,
progesterone, aprotinin, hydrocortisone, ethanolamine, beta
mercaptoethanol, epidermal growth factor (EGF), gastrin I and II,
copper chelators such as, for example, triethylene pentamine,
forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite
growth factor, nodal, valproic acid, trichostatin A, sodium
butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF,
MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid
alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte
growth factor (KGF), Dickkopf protein family, bovine pituitary
extract, islet neogenesis-associated protein (INGAP), Indian
hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway
inhibitors, sonic hedgehog inhibitors, or combinations thereof.
[0222] The at least one other additional factor may be supplied by
conditioned media obtained from pancreatic cells lines such as, for
example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79),
BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic
cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and
intestinal cell lines such as, for example, FHs 74 (ATCC No:
CCL-241).
Detection of Cells Expressing Markers Characteristic of the
Pancreatic Endoderm Linage
[0223] Markers characteristic of the pancreatic endoderm lineage
are well known to those skilled in the art, and additional markers
characteristic of the pancreatic endoderm lineage continue to be
identified. These markers can be used to confirm that the cells
treated in accordance with the present invention have
differentiated to acquire the properties characteristic of the
pancreatic endoderm lineage. Pancreatic endoderm lineage specific
markers include the expression of one or more transcription factors
such as, for example, Hlxb9, PTF-1a, PDX-1, HNF-6, HNF-1beta.
[0224] The efficiency of differentiation may be determined by
exposing a treated cell population to an agent (such as an
antibody) that specifically recognizes a protein marker expressed
by cells expressing markers characteristic of the pancreatic
endoderm lineage.
[0225] Methods for assessing expression of protein and nucleic acid
markers in cultured or isolated cells are standard in the art.
These include quantitative reverse transcriptase polymerase chain
reaction (RT-PCR), Northern blots, in situ hybridization (see,
e.g., Current Protocols in Molecular Biology (Ausubel et al., eds.
2001 supplement)), and immunoassays such as immunohistochemical
analysis of sectioned material, Western blotting, and for markers
that are accessible in intact cells, flow cytometry analysis (FACS)
(see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual,
New York: Cold Spring Harbor Laboratory Press (1998)).
Formation of Cells Expressing Markers of the Pancreatic Endocrine
Lineage
[0226] Cells expressing markers characteristic of the pancreatic
endoderm lineage may be differentiated into cells expressing
markers characteristic of the pancreatic endocrine lineage by any
method in the art or by any method disclosed in this invention.
[0227] For example, cells expressing markers characteristic of the
pancreatic endoderm lineage may be differentiated into cells
expressing markers characteristic of the pancreatic endocrine
lineage according to the methods disclosed in D'Amour et al, Nature
Biotechnology 24, 1392-1401 (2006).
[0228] For example, cells expressing markers characteristic of the
pancreatic endoderm lineage are further differentiated into cells
expressing markers characteristic of the pancreatic endocrine
lineage, by culturing the cells expressing markers characteristic
of the pancreatic endoderm lineage in medium containing DAPT and
exendin 4, then removing the medium containing DAPT and exendin 4
and subsequently culturing the cells in medium containing exendin
1, IGF-1 and HGF. An example of this method is disclosed in Nature
Biotechnology 24, 1392-1401 (2006).
[0229] For example, cells expressing markers characteristic of the
pancreatic endoderm lineage are further differentiated into cells
expressing markers characteristic of the pancreatic endocrine
lineage, by culturing the cells expressing markers characteristic
of the pancreatic endoderm lineage in medium containing exendin 4,
then removing the medium containing exendin 4 and subsequently
culturing the cells in medium containing exendin 1, IGF-1 and HGF.
An example of this method is disclosed in D'Amour et al, Nature
Biotechnology, 2006.
[0230] For example, cells expressing markers characteristic of the
pancreatic endoderm lineage are further differentiated into cells
expressing markers characteristic of the pancreatic endocrine
lineage, by culturing the cells expressing markers characteristic
of the pancreatic endoderm lineage in medium containing DAPT and
exendin 4. An example of this method is disclosed in D'Amour et al,
Nature Biotechnology, 2006.
[0231] For example, cells expressing markers characteristic of the
pancreatic endoderm lineage are further differentiated into cells
expressing markers characteristic of the pancreatic endocrine
lineage, by culturing the cells expressing markers characteristic
of the pancreatic endoderm lineage in medium containing exendin 4.
An example of this method is disclosed in D'Amour et al, Nature
Biotechnology, 2006.
[0232] In one aspect of the present invention, cells expressing
markers characteristic of the pancreatic endoderm lineage are
further differentiated into cells expressing markers characteristic
of the pancreatic endocrine lineage, by treating the cells
expressing markers characteristic of the pancreatic endoderm
lineage with a factor that inhibits the Notch signaling pathway,
according to the methods disclosed in U.S. patent application Ser.
No. 11/736,908, assigned to LifeScan, Inc.
[0233] In one aspect of the present invention, cells expressing
markers characteristic of the pancreatic endoderm lineage are
further differentiated into cells expressing markers characteristic
of the pancreatic endocrine lineage, by treating the cells
expressing markers characteristic of the pancreatic endoderm
lineage with a factor that inhibits the Notch signaling pathway,
according to the methods disclosed in U.S. patent application Ser.
No. 11/779,311, assigned to LifeScan, Inc.
[0234] In one aspect of the present invention, cells expressing
markers characteristic of the pancreatic endoderm lineage are
further differentiated into cells expressing markers characteristic
of the pancreatic endocrine lineage, by treating the cells
expressing markers characteristic of the pancreatic endoderm
lineage with a factor that inhibits the Notch signaling pathway,
according to the methods disclosed in U.S. patent application Ser.
No. 60/953,178, assigned to LifeScan, Inc.
[0235] In one aspect of the present invention, cells expressing
markers characteristic of the pancreatic endoderm lineage are
further differentiated into cells expressing markers characteristic
of the pancreatic endocrine lineage, by treating the cells
expressing markers characteristic of the pancreatic endoderm
lineage according to the methods disclosed in U.S. patent
application Ser. No. 60/990,529.
[0236] Cells expressing markers characteristic of the pancreatic
endoderm lineage may be treated with at least one other additional
factor that may enhance the formation of cells expressing markers
characteristic of the pancreatic endocrine lineage.
[0237] Alternatively, the at least one other additional factor may
enhance the proliferation of the cells expressing markers
characteristic of the pancreatic endocrine lineage formed by the
methods of the present invention. Further, the at least one other
additional factor may enhance the ability of the cells expressing
markers characteristic of the pancreatic endocrine lineage formed
by the methods of the present invention to form other cell types or
improve the efficiency of any other additional differentiation
steps.
[0238] The at least one additional factor may be, for example,
nicotinamide, members of TGF-.beta. family, including TGF-.beta.1,
2, and 3, serum albumin, members of the fibroblast growth factor
family, platelet-derived growth factor-AA, and -BB, platelet rich
plasma, insulin growth factor (IGF-I, II), growth differentiation
factor (such as, for example, GDF-5, -6, -8, -10, -11), glucagon
like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody,
Exendin-4, retinoic acid, parathyroid hormone, insulin,
progesterone, aprotinin, hydrocortisone, ethanolamine, beta
mercaptoethanol, epidermal growth factor (EGF), gastrin I and II,
copper chelators such as, for example, triethylene pentamine,
forskolin, Na-Butyrate, activin, betacellulin, ITS, noggin, neurite
growth factor, nodal, valproic acid, trichostatin A, sodium
butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF,
MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid
alkaloid such as, for example, cyclopamine (EMD, CA), keratinocyte
growth factor (KGF), Dickkopf protein family, bovine pituitary
extract, islet neogenesis-associated protein (INGAP), Indian
hedgehog, sonic hedgehog, proteasome inhibitors, notch pathway
inhibitors, sonic hedgehog inhibitors, or combinations thereof.
[0239] The at least one other additional factor may be supplied by
conditioned media obtained from pancreatic cells lines such as, for
example, PANC-1 (ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79),
BxPC-3 (ATCC No: CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic
cell lines such as, for example, HepG2 (ATCC No: HTB-8065), and
intestinal cell lines such as, for example, FHs 74 (ATCC No:
CCL-241).
Detection of Cells Expressing Markers Characteristic of the
Pancreatic Endocrine Lineage
[0240] Markers characteristic of cells of the pancreatic endocrine
lineage are well known to those skilled in the art, and additional
markers characteristic of the pancreatic endocrine lineage continue
to be identified. These markers can be used to confirm that the
cells treated in accordance with the present invention have
differentiated to acquire the properties characteristic of the
pancreatic endocrine lineage. Pancreatic endocrine lineage specific
markers include the expression of one or more transcription factors
such as, for example, NGN3, NEURO, or ISL1.
[0241] Markers characteristic of cells of the .beta. cell lineage
are well known to those skilled in the art, and additional markers
characteristic of the .beta. cell lineage continue to be
identified. These markers can be used to confirm that the cells
treated in accordance with the present invention have
differentiated to acquire the properties characteristic of the
.beta.-cell lineage. .beta. cell lineage specific characteristics
include the expression of one or more transcription factors such
as, for example, PDX1, NKX2.2, NKX6.1, ISL1, PAX6, PAX4, NEUROD,
HNF1 beta, HNF6, HNF3 beta, or MAFA, among others. These
transcription factors are well established in the art for
identification of endocrine cells. See, e.g., Edlund (Nature
Reviews Genetics 3: 524-632 (2002)).
[0242] The efficiency of differentiation may be determined by
exposing a treated cell population to an agent (such as an
antibody) that specifically recognizes a protein marker expressed
by cells expressing markers characteristic of the pancreatic
endocrine lineage. Alternatively, the efficiency of differentiation
may be determined by exposing a treated cell population to an agent
(such as an antibody) that specifically recognizes a protein marker
expressed by cells expressing markers characteristic of the .beta.
cell lineage.
[0243] Methods for assessing expression of protein and nucleic acid
markers in cultured or isolated cells are standard in the art.
These include quantitative reverse transcriptase polymerase chain
reaction (RT-PCR), Northern blots, in situ hybridization (see,
e.g., Current Protocols in Molecular Biology (Ausubel et al., eds.
2001 supplement)), and immunoassays such as immunohistochemical
analysis of sectioned material, Western blotting, and for markers
that are accessible in intact cells, flow cytometry analysis (FACS)
(see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual,
New York: Cold Spring Harbor Laboratory Press (1998)).
[0244] In one aspect of the present invention, the efficiency of
differentiation is determined by measuring the percentage of
insulin positive cells in a given cell culture following treatment.
In one embodiment, the methods of the present invention produce
about 100% insulin positive cells in a given culture. In an
alternate embodiment, the methods of the present invention produce
about 90% insulin positive cells in a given culture. In an
alternate embodiment, the methods of the present invention produce
about 80% insulin positive cells in a given culture. In an
alternate embodiment, the methods of the present invention produce
about 70% insulin positive cells in a given culture. In an
alternate embodiment, the methods of the present invention produce
about 60% insulin positive cells in a given culture. In an
alternate embodiment, the methods of the present invention produce
about 50% insulin positive cells in a given culture. In an
alternate embodiment, the methods of the present invention produce
about 40% insulin positive cells in a given culture. In an
alternate embodiment, the methods of the present invention produce
about 30% insulin positive cells in a given culture. In an
alternate embodiment, the methods of the present invention produce
about 20% insulin positive cells in a given culture. In an
alternate embodiment, the methods of the present invention produce
about 10% insulin positive cells in a given culture. In an
alternate embodiment, the methods of the present invention produce
about 5% insulin positive cells in a given culture.
[0245] In one aspect of the present invention, the efficiency of
differentiation is determined by measuring glucose-stimulated
insulin secretion, as detected by measuring the amount of C-peptide
released by the cells. In one embodiment, cells produced by the
methods of the present invention produce about 1000 ng C-peptide/pg
DNA. In an alternate embodiment, cells produced by the methods of
the present invention produce about 900 ng C-peptide/pg DNA. In an
alternate embodiment, cells produced by the methods of the present
invention produce about 800 ng C-peptide/pg DNA. In an alternate
embodiment, cells produced by the methods of the present invention
produce about 700 ng C-peptide/pg DNA. In an alternate embodiment,
cells produced by the methods of the present invention produce
about 600 ng C-peptide/pg DNA. In an alternate embodiment, cells
produced by the methods of the present invention produce about 500
ng C-peptide/pg DNA. In an alternate embodiment, cells produced by
the methods of the present invention produce about 400 ng
C-peptide/pg DNA. In an alternate embodiment, cells produced by the
methods of the present invention produce about 500 ng C-peptide/pg
DNA. In an alternate embodiment, cells produced by the methods of
the present invention produce about 400 ng C-peptide/pg DNA. In an
alternate embodiment, cells produced by the methods of the present
invention produce about 300 ng C-peptide/pg DNA. In an alternate
embodiment, cells produced by the methods of the present invention
produce about 200 ng C-peptide/pg DNA. In an alternate embodiment,
cells produced by the methods of the present invention produce
about 100 ng C-peptide/pg DNA. In an alternate embodiment, cells
produced by the methods of the present invention produce about 90
ng C-peptide/pg DNA. In an alternate embodiment, cells produced by
the methods of the present invention produce about 80 ng
C-peptide/pg DNA. In an alternate embodiment, cells produced by the
methods of the present invention produce about 70 ng C-peptide/pg
DNA. In an alternate embodiment, cells produced by the methods of
the present invention produce about 60 ng C-peptide/pg DNA. In an
alternate embodiment, cells produced by the methods of the present
invention produce about 50 ng C-peptide/pg DNA. In an alternate
embodiment, cells produced by the methods of the present invention
produce about 40 ng C-peptide/pg DNA. In an alternate embodiment,
cells produced by the methods of the present invention produce
about 30 ng C-peptide/pg DNA. In an alternate embodiment, cells
produced by the methods of the present invention produce about 20
ng C-peptide/pg DNA. In an alternate embodiment, cells produced by
the methods of the present invention produce about 10 ng
C-peptide/pg DNA.
Therapies
[0246] In one aspect, the present invention provides a method for
treating a patient suffering from, or at risk of developing, Type1
diabetes. This method involves culturing pluripotent stem cells,
differentiating the pluripotent stem cells in vitro into a
.beta.-cell lineage, and implanting the cells of a .beta.-cell
lineage into a patient.
[0247] In yet another aspect, this invention provides a method for
treating a patient suffering from, or at risk of developing, Type 2
diabetes. This method involves culturing pluripotent stem cells,
differentiating the cultured cells in vitro into a .beta.-cell
lineage, and implanting the cells of a .beta.-cell lineage into the
patient.
[0248] If appropriate, the patient can be further treated with
pharmaceutical agents or bioactives that facilitate the survival
and function of the transplanted cells. These agents may include,
for example, insulin, members of the TGF-.beta. family, including
TGF-.beta.1, 2, and 3, bone morphogenic proteins (BMP-2, -3, -4,
-5, -6, -7, -11, -12, and -13), fibroblast growth factors-1 and -2,
platelet-derived growth factor-AA, and -BB, platelet rich plasma,
insulin growth factor (IGF-I, II) growth differentiation factor
(such as, for example, GDF-5, -6, -7, -8, -10, -15), vascular
endothelial cell-derived growth factor (VEGF), pleiotrophin,
endothelin, among others. Other pharmaceutical compounds can
include, for example, nicotinamide, glucagon like peptide-I (GLP-1)
and II, GLP-1 and -2 mimetibody, Exendin-4, retinoic acid,
parathyroid hormone, MAPK inhibitors, such as, for example,
compounds disclosed in U.S. Published Application 2004/0209901 and
U.S. Published Application 2004/0132729.
[0249] The pluripotent stem cells may be differentiated into an
insulin-producing cell prior to transplantation into a recipient.
In a specific embodiment, the pluripotent stem cells are fully
differentiated into .beta.-cells prior to transplantation into a
recipient. Alternatively, the pluripotent stem cells may be
transplanted into a recipient in an undifferentiated or partially
differentiated state. Further differentiation may take place in the
recipient.
[0250] Definitive endoderm cells or, alternatively, pancreatic
endoderm cells, or, alternatively, .beta. cells, may be implanted
as dispersed cells or formed into clusters that may be infused into
the hepatic portal vein. Alternatively, cells may be provided in
biocompatible degradable polymeric supports, porous non-degradable
devices or encapsulated to protect from host immune response. Cells
may be implanted into an appropriate site in a recipient. The
implantation sites include, for example, the liver, natural
pancreas, renal subcapsular space, omentum, peritoneum, subserosal
space, intestine, stomach, or a subcutaneous pocket.
[0251] To enhance further differentiation, survival or activity of
the implanted cells, additional factors, such as growth factors,
antioxidants or anti-inflammatory agents, can be administered
before, simultaneously with, or after the administration of the
cells. In certain embodiments, growth factors are utilized to
differentiate the administered cells in vivo. These factors can be
secreted by endogenous cells and exposed to the administered cells
in situ. Implanted cells can be induced to differentiate by any
combination of endogenous and exogenously administered growth
factors known in the art.
[0252] The amount of cells used in implantation depends on a number
of various factors including the patient's condition and response
to the therapy, and can be determined by one skilled in the
art.
[0253] In one aspect, this invention provides a method for treating
a patient suffering from, or at risk of developing diabetes. This
method involves culturing pluripotent stem cells, differentiating
the cultured cells in vitro into a .beta.-cell lineage, and
incorporating the cells into a three-dimensional support. The cells
can be maintained in vitro on this support prior to implantation
into the patient. Alternatively, the support containing the cells
can be directly implanted in the patient without additional in
vitro culturing. The support can optionally be incorporated with at
least one pharmaceutical agent that facilitates the survival and
function of the transplanted cells.
[0254] Support materials suitable for use for purposes of the
present invention include tissue templates, conduits, barriers, and
reservoirs useful for tissue repair. In particular, synthetic and
natural materials in the form of foams, sponges, gels, hydrogels,
textiles, and nonwoven structures, which have been used in vitro
and in vivo to reconstruct or regenerate biological tissue, as well
as to deliver chemotactic agents for inducing tissue growth, are
suitable for use in practicing the methods of the present
invention. See, for example, the materials disclosed in U.S. Pat.
No. 5,770,417, U.S. Pat. No. 6,022,743, U.S. Pat. No. 5,567,612,
U.S. Pat. No. 5,759,830, U.S. Pat. No. 6,626,950, U.S. Pat. No.
6,534,084, U.S. Pat. No. 6,306,424, U.S. Pat. No. 6,365,149, U.S.
Pat. No. 6,599,323, U.S. Pat. No. 6,656,488, U.S. Published
Application 2004/0062753 A1, U.S. Pat. No. 4,557,264 and U.S. Pat.
No. 6,333,029.
[0255] To form a support incorporated with a pharmaceutical agent,
the pharmaceutical agent can be mixed with the polymer solution
prior to forming the support. Alternatively, a pharmaceutical agent
could be coated onto a fabricated support, preferably in the
presence of a pharmaceutical carrier. The pharmaceutical agent may
be present as a liquid, a finely divided solid, or any other
appropriate physical form. Alternatively, excipients may be added
to the support to alter the release rate of the pharmaceutical
agent. In an alternate embodiment, the support is incorporated with
at least one pharmaceutical compound that is an anti-inflammatory
compound, such as, for example, compounds disclosed in U.S. Pat.
No. 6,509,369.
[0256] The support may be incorporated with at least one
pharmaceutical compound that is an anti-apoptotic compound, such
as, for example, compounds disclosed in U.S. Pat. No.
6,793,945.
[0257] The support may also be incorporated with at least one
pharmaceutical compound that is an inhibitor of fibrosis, such as,
for example, compounds disclosed in U.S. Pat. No. 6,331,298.
[0258] The support may also be incorporated with at least one
pharmaceutical compound that is capable of enhancing angiogenesis,
such as, for example, compounds disclosed in U.S. Published
Application 2004/0220393 and U.S. Published Application
2004/0209901.
[0259] The support may also be incorporated with at least one
pharmaceutical compound that is an immunosuppressive compound, such
as, for example, compounds disclosed in U.S. Published Application
2004/0171623.
[0260] The support may also be incorporated with at least one
pharmaceutical compound that is a growth factor, such as, for
example, members of the TGF-.beta. family, including TGF-.beta.1,
2, and 3, bone morphogenic proteins (BMP-2, -3, -4, -5, -6, -7,
-11, -12, and -13), fibroblast growth factors-1 and -2,
platelet-derived growth factor-AA, and -BB, platelet rich plasma,
insulin growth factor (IGF-I, II) growth differentiation factor
(such as, for example, GDF-5, -6, -8, -10, -15), vascular
endothelial cell-derived growth factor (VEGF), pleiotrophin,
endothelin, among others. Other pharmaceutical compounds can
include, for example, nicotinamide, hypoxia inducible factor
1-alpha, glucagon like peptide-I (GLP-1), GLP-1 and GLP-2
mimetibody, and II, Exendin-4, nodal, noggin, NGF, retinoic acid,
parathyroid hormone, tenascin-C, tropoelastin, thrombin-derived
peptides, cathelicidins, defensins, laminin, biological peptides
containing cell- and heparin-binding domains of adhesive
extracellular matrix proteins such as fibronectin and vitronectin,
MAPK inhibitors, such as, for example, compounds disclosed in U.S.
Published Application 2004/0209901 and U.S. Published Application
2004/0132729.
[0261] The incorporation of the cells of the present invention into
a scaffold can be achieved by the simple depositing of cells onto
the scaffold. Cells can enter into the scaffold by simple diffusion
(J. Pediatr. Surg. 23 (1 Pt 2): 3-9 (1988)). Several other
approaches have been developed to enhance the efficiency of cell
seeding. For example, spinner flasks have been used in seeding of
chondrocytes onto polyglycolic acid scaffolds (Biotechnol. Prog.
14(2): 193-202 (1998)). Another approach for seeding cells is the
use of centrifugation, which yields minimum stress to the seeded
cells and enhances seeding efficiency. For example, Yang et al.
developed a cell seeding method (J. Biomed. Mater. Res. 55(3):
379-86 (2001)), referred to as Centrifugational Cell Immobilization
(CCI).
[0262] The present invention is further illustrated, but not
limited by, the following examples.
Examples
[0263] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the following
subsections that describe or illustrate certain features,
embodiments, or applications of the present invention.
Example 1
Human Embryonic Stem Cell Culture
[0264] The human embryonic stem cell lines H1, H7, and H9 were
obtained from WiCell Research Institute, Inc., (Madison, Wis.) and
cultured according to instructions provided by the source
institute. The human embryonic stem cells were also seeded on
plates coated with a 1:30 dilution of reduced growth factor
MATRIGEL.TM. (BD Biosciences; Cat #356231) and cultured in
MEF-conditioned medium supplemented with 8 ng/ml bFGF (R&D
Systems; Cat #233-FB). The cells cultured on MATRIGEL.TM. were
routinely passaged as clusters using collagenase IV
(Invitrogen/GIBCO; Cat #17104-019), Dispase (Invitrogen; Cat
#17105-041), or Liberase CI enzyme (Roche; Cat #11814435001). In
some instances, the cells were passaged as single cells using
ACCUTASE (Sigma; Cat #A6964).
[0265] Human embryonic stem cells used in these examples were
maintained in an undifferentiated, pluripotent state with passage
on average every four-days. Passage was performed by exposing cell
cultures to a solution of collagenase (1 or 10 mg/ml;
Sigma-Aldrich) for 10 to 30 minutes at 37.degree. C. followed by
gentle scraping with a pipette tip to recover cell clusters.
Clusters were allowed to sediment by gravity, followed by washing
to remove residual collagenase. Cell clusters were split at a 1:3
ratio for routine maintenance culture or a 1:1 ratio for later
assay. All human ES cell lines were maintained at passage numbers
less than 50 and routinely evaluated for normal karyotypic
phenotype and absence of mycoplasma contamination.
Example 2
Bioassay for the Formation of Cells Expressing Markers
Characteristic of the Definitive Endoderm Lineage
[0266] Activin A is an important mediator of differentiation in a
broad range of cell types, including differentiation of embryonic
stem cells to definitive endoderm. When human embryonic stem cells
are treated with a combination of activin A and Wnt3a, various
genes representative of definitive endoderm are up-regulated. A
bioassay that measures this differentiation in human embryonic stem
cells was adapted in miniaturized format to 96-well plates for
screening purposes. Validation was completed using treatment with
commercial sources of activin A and Wnt3a recombinant proteins and
measuring protein expression of the transcription factor SOX17,
considered to be a representative marker of definitive
endoderm.
[0267] Live Cell Assay: Briefly, clusters of H1 human embryonic
stem cells were grown on reduced growth factor MATRIGEL.TM.
(Invitrogen; Cat #356231)-coated tissue culture plastic. Cells were
passaged using collagenase (Invitrogen; Cat #17104-019) treatment
and gentle scraping, washed to remove residual enzyme, and plated
in a ratio of 1:1 (surface area) on reduced growth factor
MATRIGEL.TM.-coated 96-well black plates (Packard ViewPlates;
Perkin Elmer; Cat #6005182). Cells were allowed to attach as
clusters and then recover log phase growth over a 1 to 3 day
period, feeding daily with 100 .mu.l per well mouse embryonic
fibroblast (MEF) conditioned medium supplemented with 8 ng/ml bFGF
(R&D Systems; Cat #233-FB).
[0268] The assay was initiated by washing the wells of each plate
twice in PBS (Invitrogen; Cat #14190), followed by adding an
aliquot (100 .mu.l) of test sample in DMEM:F12 basal medium
(Invitrogen; Cat #11330-032) to each well. Test conditions were
performed in triplicate, feeding on alternate days by aspirating
and replacing the medium from each well with test samples over a
total four-day assay period. On the first and second day of assay,
test samples added to the assay wells were diluted in DMEM:F12 with
0.5% FCS (HyClone; Cat #SH30070.03) and 20 ng/ml Wnt3a (R&D
Systems; Cat #1324-WN). On the third and fourth day of assay, test
samples added to the assay wells were diluted in DMEM:F12 with 2%
FCS, without any Wnt3a. Positive control samples consisted of
recombinant human activin A (PeproTech; Cat #120-14) added at a
concentration of 100 ng/ml throughout assay plus Wnt3a (20 ng/ml)
on days 1 and 2. Negative control samples omitted treatment with
both activin A and Wnt3a.
[0269] High Content Analysis: At the conclusion of four-days of
culture, assay plates were washed twice with PBS (Invitrogen; Cat
#14190), fixed with 4% paraformaldehyde (Alexis Biochemical; Cat
#ALX-350-011) at room temperature for 20 minutes, then washed three
times with PBS and permeabilized with 0.5% Triton X-100 (Sigma; Cat
#T8760-2) for 20 minutes at room temperature. Cells were washed
again three times with PBS and blocked with 4% chicken serum
(Invitrogen; Cat #16110082) in PBS for 30 minutes at room
temperature. Primary antibody (goat anti-human SOX17; R&D
Systems; Cat #AF1924) was diluted 1:100 in 4% chicken serum and
added to each well for one hour at room temperature. Alexa Fluor
488 conjugated secondary antibody (chicken anti-goat IgG; Molecular
Probes; Cat #AZ1467) was diluted 1:200 in PBS and added to each
sample well after washing three times with PBS. To counter stain
nuclei, 4 .mu.g/ml Hoechst 33342 (Invitrogen; Cat #H3570) was added
for ten minutes at room temperature. Plates were washed once with
PBS and left in 100 .mu.l/well PBS for imaging.
[0270] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized
from positive control wells and from untreated negative control
wells stained with secondary antibody alone. Images from 15 fields
per well were acquired to compensate for any cell loss during the
bioassay and subsequent staining procedures. Measurements for total
cell number and total SOX17 intensity were obtained from each well
using IN Cell Developer Toolbox 1.7 (GE Healthcare) software.
Segmentation for the nuclei was determined based on gray-scale
levels (baseline range 100-300) and nuclear size. Averages and
standard deviations were calculated for each replicate data set.
Total SOX17 protein expression was reported as total intensity or
integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria of gray-scale ranges between 200 to 3500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control. Normalized data were calculated for averages and standard
deviations for each replicate set.
[0271] FIG. 1 shows validation of the screening assay, testing a
two-fold dilution curve of a commercial source of activin A
(PeproTech) and measuring both cell number (FIG. 1A) and SOX17
intensity (FIG. 1B). Optimal activin A effects for induction of
SOX17 expression were generally observed in the 100-200 ng/ml range
with an EC.sub.50 of 30-50 ng/ml. Omitting Wnt3a from treatment on
days 1 and 2 of assay failed to produce measurable SOX17 expression
(FIG. 1B, white bars). Absence of activin A also failed to yield
SOX17 expression (FIG. 1B).
Example 3
Primary Screening: Effects of the Compounds of the Present
Invention on the Differentiation of Human Embryonic Stem Cells into
Cells Expressing Markers Characteristic of the Definitive Endoderm
Lineage in the Absence of Activin A
[0272] Differentiation of pluripotent stem cells into cells
expressing markers characteristic of the definitive endoderm
lineage is mediated through a series of receptor-ligand
interactions that in turn activate receptor kinases leading to
phosphorylation and nuclear translocation of downstream substrates,
eventually regulating expression of specific target genes. Optimal
activation of these signaling cascades in some cell types may
require inhibition of opposing default pathways. In other cases,
redundant pathways involving alternative members of a larger kinase
family may substitute in part for one or more signaling molecules.
In other cases, canonical and non-canonical pathways may diverge
with different initiating stimuli but may lead to a similar
functional outcome.
[0273] Cell-based functional screens are one approach to identify
novel targets and methods that can impact specific cellular
responses. One very powerful approach involves a series of
iterative screens whereby leads or hits from one screen are
integrated into a subsequent screen. Alternatively, a series of
different variables are integrated in a combinatorial fashion (for
example, growth factors with kinase inhibitors) to identify novel
effects on cellular differentiation. In this case, a library of
small molecules comprising aniline-pyridinotriazines, cyclic
aniline-pyridinotriazines and intermediate structures in their
synthesis was tested for properties important during definitive
endoderm differentiation of human embryonic stem cells,
specifically for effects to retain or enhance cell number at the
conclusion of a `first` differentiation step in low serum and in
the absence of the growth factor activin A.
Screening Assay
[0274] Cell assay seeding: Briefly, clusters of H1 human embryonic
stem cells were grown on reduced growth factor MATRIGEL.TM.
(Invitrogen; Cat #356231)-coated tissue culture plastic. Cells were
passaged using collagenase (Invitrogen; Cat #17104-019) treatment
and gentle scraping, washed to remove residual enzyme, and plated
with even dispersal at a ratio of 1:1 (surface area) on reduced
growth factor MATRIGEL.TM.-coated 96-well black plates (Packard
ViewPlates; PerkinElmer; Cat #6005182) using volumes of 100
.mu.l/well. Cells were allowed to attach as clusters and then
recover log phase growth over a 1 to 3 day period, feeding daily
with MEF conditioned medium supplemented with 8 ng/ml bFGF (R&D
Systems; Cat #233-FB). Plates were maintained at 37.degree. C., 5%
CO.sub.2 in a humidified box throughout the duration of assay.
[0275] Preparation of compounds and assay: The compounds tested
were made available as 5 mM stocks in 96-well plate format,
solubilized in 100% DMSO (Sigma; Cat #D2650) and stored at
-80.degree. C. The library compounds were further diluted to an
intermediate concentration of 0.2 mM in 50 mM HEPES (Invitrogen;
Cat #15630-080), 20% DMSO and stored at 4.degree. C. Test
conditions were performed in triplicate, feeding on alternate days
over a four-day assay period. Primary screening assays were
initiated by aspirating culture medium from each well followed by
three washes in PBS (Invitrogen; Cat #14190) to remove residual
growth factors and serum. On the first day of assay, test volumes
of 200 .mu.l per well were added back containing DMEM:F12 base
medium (Invitrogen; Cat #11330-032) supplemented with 0.5% FCS
(HyClone; Cat #SH30070.03) and 20 ng/ml Wnt3a (R&D Systems; Cat
#1324-WN) plus 2.5 .mu.M test compound. On the third day of assay,
test volumes of 200 .mu.l per well were added back containing
DMEM:F12 base medium supplemented with 2% FCS plus 2.5 .mu.M test
compound, without Wnt3a. Positive control samples contained the
same base medium supplemented with FCS, substituting 100 ng/ml
recombinant human activin A (PeproTech; Cat #120-14) for the test
compound throughout the four-day assay along with Wnt3a (20 ng/ml)
added only on days 1 and 2. Negative control samples contained
DMEM:F12 base medium supplemented with FCS, adding Wnt3a on days 1
and 2 but omitting activin A.
[0276] High Content Analysis: At the conclusion of four-days of
culture, assay plates were washed twice with PBS (Invitrogen; Cat
#14190), fixed with 4% paraformaldehyde (Alexis Biochemical; Cat
#ALX-350-011) at room temperature for 20 minutes, then washed three
times with PBS and permeabilized with 0.5% Triton X-100 (Sigma; Cat
#T8760-2) for 20 minutes at room temperature. Cells were washed
again three times with PBS and blocked with 4% chicken serum
(Invitrogen; Cat #16110082) in PBS for 30 minutes at room
temperature. Primary antibody (goat anti-human SOX17; R&D
Systems; Cat #AF1924) was diluted 1:100 in 4% chicken serum and
added to each well for one hour at room temperature. Alexa Fluor
488 conjugated secondary antibody (chicken anti-goat IgG; Molecular
Probes; Cat #AZ1467) was diluted 1:200 in PBS and added to each
sample well after washing three times with PBS. To counter stain
nuclei, 4 .mu.g/ml Hoechst 33342 (Invitrogen; Cat #H3570) was added
for ten minutes at room temperature. Plates were washed once with
PBS and left in 100 .mu.l/well PBS for imaging.
[0277] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized
from positive control wells and from untreated negative control
wells stained with secondary antibody alone. Images from 15 fields
per well were acquired to compensate for any cell loss during the
bioassay and subsequent staining procedures. Measurements for total
cell number and total SOX17 intensity were obtained from each well
using IN Cell Developer Toolbox 1.7 (GE Healthcare) software.
Segmentation for the nuclei was determined based on gray-scale
levels (baseline range 100-300) and nuclear size. Averages and
standard deviations were calculated for each replicate data set.
Total SOX17 protein expression was reported as total intensity or
integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria of gray-scale ranges between 200 to 3500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control. Normalized data were calculated for averages and standard
deviations for each replicate set.
[0278] Table 1 shows results of primary screening for the compounds
tested, showing their effects on the differentiation of human
embryonic stem cells to cells expressing markers characteristic of
the definitive endoderm lineage in the absence of activin A. The
results include quantitative measures of both cell number and SOX17
intensity, where respective data points were averaged from
triplicate wells and analyzed for each parameter using identical
fields in each well. Expression of the transcription factor SOX17
is considered indicative of definitive endoderm differentiation.
Primary screening results were captured from eight 96-well
screening plates. Plate to plate variability was reduced with
inclusion of individual positive and negative controls on each
plate. Results are normalized and expressed as a percentage of the
positive control. Emphasis was placed on retention or amplification
of cell number at the conclusion of assay.
[0279] Table 2 lists a subset of 27 compounds and their analyzed
results from the primary screening, where these hits appeared to
retain cell number at a level equivalent to or better than the
positive control despite the absence of activin A in the screening
assay.
[0280] In some cases, SOX17 expression was induced in the absence
of activin A (for example, the cyclic aniline-pyridinotriazines
Compound 35 and Compound 22.
[0281] The compounds shown in Table 2 were selected for further
evaluation for effects on the differentiation of human embryonic
stem cells to cells expressing markers characteristic of the
definitive endoderm lineage in the absence of activin A.
Example 4
Secondary Screening: Effects of the Compounds of the Present
Invention on the Differentiation of Human Embryonic Stem Cells into
Cells Expressing Markers Characteristic of the Definitive Endoderm
Lineage with EGF/FGF4 in the Absence of Activin A
[0282] A titration curve for activin A with a constant amount of
Wnt3a showed at least two effects during DE differentiation: 1)
maintaining cell numbers or preventing cell loss; and 2) inducing a
marker of DE, for example, SOX17 expression (Example 2). Primary
screening from Example 3 identified compounds that could maintain
similar or improved cell numbers in assay relative to addition of
activin A/Wnt3a alone. A secondary screening assay was conducted to
evaluate the effect of combinations of the identified compounds
with other growth factors, specifically EGF and FGF4, on the
generation of definitive endoderm.
[0283] Cell assay seeding: Clusters of H1 human embryonic stem
cells were grown on reduced growth factor MATRIGEL.TM. (Invitrogen;
Cat #356231)-coated tissue culture plastic. Cells were passaged
using collagenase (Invitrogen; Cat # Cat #17104-019) treatment and
gentle scraping, washed to remove residual enzyme, and plated with
even dispersal at a ratio of 1:1 (surface area) on reduced growth
factor MATRIGEL.TM.-coated 96-well black plates (Packard
ViewPlates; PerkinElmer; Cat #6005182) using volumes of 100
.mu.l/well. Cells were allowed to attach as clusters and then
recover log phase growth over a 1 to 3 day period, feeding daily
with MEF conditioned medium supplemented with 8 ng/ml bFGF (R&D
Systems; Cat #233-FB). Plates were maintained at 37.degree. C., 5%
CO.sub.2 in a humidified box throughout the duration of assay.
[0284] Preparation of compounds and growth factors: Stock
concentrations for EGF (R&D Systems; Cat #236-EG) and FGF4
(R&D Systems; Cat #235-F4) were 250 ng/ml, each solubilized in
PBS with 0.1% BSA (Sigma; Cat #A7888). Compounds were available as
5 mM stocks in 96-well plate format, solubilized in 100% DMSO
(Sigma; Cat #D2650) and stored at -80.degree. C. The compounds were
further diluted to an intermediate concentration of 0.2 mM in 50 mM
HEPES (Invitrogen; Cat #15630-080), 20% DMSO and stored at
4.degree. C. All growth factors and inhibitors were prepared in a
deep well, 96-well polypropylene plate, diluted to 5.times.
intermediate stocks in DMEM:F12 base medium at the beginning of
assay and stored at 4.degree. C.
[0285] A secondary screening assay was conducted, testing in
triplicate and feeding on alternate days over the four-day assay
timeframe. Assays were initiated by aspirating culture medium from
each well followed by three washes in PBS to remove residual growth
factors and serum. Test volumes of 80 .mu.l per well were added
back containing DMEM:F12 base medium (Invitrogen; Cat #11330-032)
supplemented with 0.625% FCS (HyClone; Cat #SH30070.03), 25 ng/ml
Wnt3a (R&D Systems), and 3.125 .mu.M compound plus 20 .mu.l
5.times. stock of growth factors to yield a final concentration of
0.5% FCS, 20 ng/ml Wnt3a, and 2.5 .mu.M compound plus 50 ng/ml EGF
and 50 ng/ml FGF4 in the assay. Positive control wells (100
.mu.l/well) contained the same base medium supplemented with 0.5%
FCS, 20 ng/ml Wnt3a and 100 ng/ml activin A. Negative control wells
(100 .mu.l/well) contained the same base medium with 0.5% FCS and
20 ng/ml Wnt3a, omitting activin A.
[0286] On day 3, wells were aspirated and fed with 80 .mu.l
DMEM:F12 base medium supplemented with 2.5% FCS (HyClone) and 3.125
.mu.M compound plus 20 .mu.l 5.times. stock of growth factors per
well to yield a final concentration of 2% FCS and 2.5 .mu.M
compound (omitting Wnt3a) plus 50 ng/ml EGF and FGF4 in the assay.
Positive control wells (100 .mu.l/well) contained the same base
medium supplemented with 2% FCS and 100 ng/ml activin A, omitting
Wnt3a. Negative control wells (100 .mu.l/well) contained the same
base medium with 2% FCS, omitting both activin A and Wnt3a.
[0287] High Content Analysis: At the conclusion of four-days of
culture, assay plates were washed twice with PBS, fixed with 4%
paraformaldehyde (Alexis Biochemical; Cat #ALX-350-011) at room
temperature for 20 minutes, then washed three times with PBS and
permeabilized with 0.5% Triton X-100 (Sigma; Cat #T8760-2) for 20
minutes at room temperature. Cells were washed again three times
with PBS and blocked with 4% chicken serum (Invitrogen; Cat
#16110082) in PBS for 30 minutes at room temperature. Primary
antibody (goat anti-human SOX17; R&D Systems; cat #AF1924) was
diluted 1:100 in 4% chicken serum and added to each well for one
hour at room temperature. Alexa Fluor 488 conjugated secondary
antibody (chicken anti-goat IgG; Molecular Probes; Cat #AZ1467) was
diluted 1:200 in PBS and added to each sample well after washing
three times with PBS. To counterstain nuclei, 4 .mu.g/ml Hoechst
33342 (Invitrogen; Cat #H3570) was added for ten minutes at room
temperature. Plates were washed once with PBS and left in 100
.mu.l/well PBS for imaging.
[0288] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized
from positive control wells and from untreated negative control
wells stained with secondary antibody alone. Images from 15 fields
per well were acquired to compensate for any cell loss during the
bioassay and subsequent staining procedures. Measurements for total
cell number and total SOX17 intensity were obtained from each well
using IN Cell Developer Toolbox 1.7 (GE Healthcare) software.
Segmentation for the nuclei was determined based on gray-scale
levels (baseline range 100-300) and nuclear size. Averages and
standard deviations were calculated for each replicate data set.
Total SOX17 protein expression was reported as total intensity or
integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria of gray-scale ranges between 200 to 3500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control. Normalized data were calculated for averages and standard
deviations for each replicate set.
[0289] Table 3A shows the results for two growth factors, EGF and
FGF 4 (50 ng/ml each) tested in combination with the
aniline-pyridinotriazine compounds shown in Table 2 for their
effects on the differentiation of human embryonic stem cells into
cells expressing markers characteristic of the definitive endoderm
lineage in the absence of activin A. Results are ranked in
descending order for best effects on SOX17 expression. Although the
effects of these compounds on SOX17 expression were considered weak
relative to the activin A/Wnt3a positive control, the responses for
some of these compounds were considered significant. For example a
selection of the compounds appear to have unique properties with
respect to retaining high cell numbers per well during assay,
presumably either by preventing apoptosis or by modulating cell
cycle. In addition, these compounds appear to synergize with EGF
and FGF4 to promote modest definitive endoderm differentiation, as
measured by SOX17 expression. The most potent compounds are listed
in Table 3B. Other compounds tested in combination with EGF and
FGF4 in this assay were ineffective at inducing SOX17 expression
but could retain cell numbers in assay (e.g. Compound 90: 85% cell
number; 2% SOX17 expression).
Example 5
Effects of Compounds of the Present Invention in Combination with
Other Factors on the Differentiation of Human Embryonic Stem Cells
to Cells Expressing Markers Characteristic of the Definitive
Endoderm Lineage in the Absence of Activin A
[0290] A secondary assay was conducted to evaluate the effect of
the compounds of the present invention with combinations of other
individual growth factors or compounds known from the literature to
regulate definitive endoderm differentiation.
[0291] Cell assay seeding: Clusters of H1 human embryonic stem
cells were grown on reduced growth factor MATRIGEL.TM. (Invitrogen;
Cat #356231)-coated tissue culture plastic. Cells were passaged
using collagenase (Invitrogen; Cat # Cat #17104-019) treatment and
gentle scraping, washed to remove residual enzyme, and plated with
even dispersal at a ratio of 1:1 (surface area) on reduced growth
factor MATRIGEL.TM.-coated 96-well black plates (Packard
ViewPlates; PerkinElmer; Cat #6005182) using volumes of 100
.mu.l/well. Cells were allowed to attach as clusters and then
recover log phase growth over a 1 to 3 day period, feeding daily
with MEF conditioned medium supplemented with 8 ng/ml bFGF (R&D
Systems; Cat #233-FB). Plates were maintained at 37.degree. C., 5%
CO.sub.2 in a humidified box throughout the duration of assay.
[0292] Preparation of compounds and growth factors: Stocks of
growth factors purchased from R&D Systems were EGF (Cat
#236-EG), FGF4 (Cat #235-F4), PDGF-A (Cat #221-AA), PDGF-B (Cat
#220-BB), PDGF-C (Cat #1687-CC), PDGF-D (Cat #1159-SB), PDGF-A/B
(Cat #222-AB), VEGF (Cat #293-VE), BMP-1 (Cat #1927-ZN) BMP-2 (Cat
#355-BM), BMP-4 (Cat #314-BP), BMP-6 (Cat #507-BP), BMP-7 (Cat
#222-AB), BMP-2/7 (Cat #3229-BM). Other agents tested were
purchased as follows: BMP-7 (Sigma; Cat #B1434), LY294002 (Cayman;
Cat 70920), PD98059, U0126, U0124 (EMD Biosciences; Cat #453710),
muscimol (Tocris; Cat #0289), biuculline (Tocris; Cat #0130),
sodium butyrate (Sigma; Cat #B5887). All growth factors were
solubilized in PBS with 0.1% BSA (Sigma; Cat #A7888) and stored
frozen at -80.degree. C. Small molecules were solubilized in 100%
DMSO (Sigma; Cat #D2650) and stored frozen at -80.degree. C. The
compounds were available as 5 mM stocks in 96-well plate format,
solubilized in 100% DMSO and stored at -80.degree. C. The compounds
of the present invention were further diluted to an intermediate
concentration of 0.2 mM in 50 mM HEPES (Invitrogen; Cat
#15630-080), 20% DMSO and stored at 4.degree. C. All growth factors
and inhibitors were prepared in a deep well, 96-well polypropylene
plate, diluted to 5.times. intermediate stocks in DMEM:F12 base
medium at the beginning of assay and stored at 4.degree. C.
[0293] A secondary screening assay was conducted, testing in
triplicate and feeding on alternate days over the four-day assay
timeframe. Assays were initiated by aspirating culture medium from
each well followed by three washes in PBS to remove residual growth
factors and serum. Test volumes of 80 .mu.l per well were added
back containing DMEM:F12 base medium (Invitrogen; Cat #11330-032)
supplemented with 0.625% FCS (HyClone; Cat #SH30070.03), 25 ng/ml
Wnt3a (R&D Systems), and 3.125 .mu.M compound plus 20 .mu.l
5.times. stock of growth factor or small molecule to yield a final
concentration of 0.5% FCS, 20 ng/ml Wnt3a, and 2.5 .mu.M compound.
All remaining growth factors were tested at a final assay
concentration of 50 ng/ml (EGF, FGF4, PDGF-A, PDGF-B, PDGF-C,
PDGF-D, PDGF-A/B, VEGF, BMP-1, BMP-2, BMP-4, BMP-6, BMP-7,
BMP-2/7). Final assay concentrations of small molecules tested were
as follows: muscimol (20 .mu.M), PD98059 (1 .mu.M), LY294002 (2.5
.mu.M), U0124 (1 .mu.M), U0126 (1 .mu.M), sodium butyrate (0.5 mM).
Positive control wells (100 .mu.l/well) contained the same base
medium supplemented with 0.5% FCS, 20 ng/ml Wnt3a and 100 ng/ml
activin A. Negative control wells (100 .mu.l/well) contained the
same base medium with 0.5% FCS and 20 ng/ml Wnt3a, omitting activin
A.
[0294] On day 3, wells were aspirated and fed with 80 .mu.l
DMEM:F12 base medium supplemented with 2.5% FCS (HyClone) and 3.125
.mu.M cyclic aniline-pyridinotriazine compound plus 20 .mu.l
5.times. stock of growth factors or small molecules per well to
yield a final concentration of 2% FCS and 2.5 .mu.M compound
(omitting Wnt3a) and as denoted on day one for all remaining growth
factors or small molecules. Positive control wells (100 .mu.l/well)
contained the same base medium supplemented with 2% FCS and 100
ng/ml activin A, omitting Wnt3a. Negative control wells (100
.mu.l/well) contained the same base medium with 2% FCS, omitting
both activin A and Wnt3a.
[0295] High Content Analysis: At the conclusion of four-days of
culture, assay plates were washed twice with PBS, fixed with 4%
paraformaldehyde (Alexis Biochemical; Cat #ALX-350-011) at room
temperature for 20 minutes, then washed three times with PBS and
permeabilized with 0.5% Triton X-100 (Sigma; Cat #T8760-2) for 20
minutes at room temperature. Cells were washed again three times
with PBS and blocked with 4% chicken serum (Invitrogen; Cat
#16110082) in PBS for 30 minutes at room temperature. Primary
antibody (goat anti-human SOX17; R&D Systems; cat #AF1924) was
diluted 1:100 in 4% chicken serum and added to each well for one
hour at room temperature. Alexa Fluor 488 conjugated secondary
antibody (chicken anti-goat IgG; Molecular Probes; Cat #AZ1467) was
diluted 1:200 in PBS and added to each sample well after washing
three times with PBS. To counterstain nuclei, 4 .mu.g/ml Hoechst
33342 (Invitrogen; Cat #H3570) was added for ten minutes at room
temperature. Plates were washed once with PBS and left in 100
.mu.l/well PBS for imaging.
[0296] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized
from positive control wells and from untreated negative control
wells stained with secondary antibody alone. Images from 15 fields
per well were acquired to compensate for any cell loss during the
bioassay and subsequent staining procedures. Measurements for total
cell number and total SOX17 intensity were obtained from each well
using IN Cell Developer Toolbox 1.7 (GE Healthcare) software.
Segmentation for the nuclei was determined based on gray-scale
levels (baseline range 100-300) and nuclear size. Averages and
standard deviations were calculated for each replicate data set.
Total SOX17 protein expression was reported as total intensity or
integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria of gray-scale ranges between 200 to 3500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control. Normalized data were calculated for averages and standard
deviations for each replicate set.
[0297] Table 4 shows the results for the differentiation of human
embryonic stem cells into cells expressing markers characteristic
of the definitive endoderm lineage following treatment with the
compounds of the present invention in combination with individual
growth factors or other small molecules. In general, members of the
BMP family (BMP-1, BMP-2, BMP-4, BMP-6, BMP-7, BMP-2/7) inhibited
or had negligible effects on SOX17 expression. The same was true
for most of the small molecule enzyme inhibitors tested in this
assay (LY294002, PD98059, U0126, U0124, sodium butyrate). However,
some members of the PDGF family (PDGF-A, -AB, -C, and -D) provided
an increase in SOX17 expression (10-25% of the activin A/Wnt3a
control). Other growth factors showing similar increases in SOX17
expression included EGF (34%), VEGF (18%), and FGF4 (17%), although
FGF4 was not able to support retention of cell numbers. The small
molecule muscimol (GABA.sub.A receptor agonist) tested in
combination with Compound 35 also provided a modest increase in
SOX17 expression; the GABA.sub.A antagonist bicuculline had no
effect on SOX17 expression. EGF, FGF4, PDGF-A, PDGF-B, PDGF-AB,
PDGF-C, and PDGF-D and muscimol were selected for additional
evaluation during definitive endoderm differentiation.
Example 6
Effects of the Compounds of the Present Invention in Combination
with Other Factors on the Differentiation of Human Embryonic Stem
Cells into Cells Expressing Markers Characteristic of the
Definitive Endoderm Lineage in the Absence of Activin A
[0298] A secondary assay was conducted to evaluate the effects of
combinations of different compounds with other individual agents on
definitive endoderm differentiation. The other agents selected for
this screen had previously shown a modest increase in definitive
endoderm formation, as tested with Compound 17 and as denoted in
Table 5. In this screen, a broader panel of compounds was evaluated
in with these agents, either in single pair-wise comparisons or
pooled combinations.
[0299] Cell assay seeding: Clusters of H1 human embryonic stem
cells were grown on reduced growth factor MATRIGEL.TM. (Invitrogen;
Cat #356231)-coated tissue culture plastic. Cells were passaged
using collagenase (Invitrogen; Cat #17104-019) treatment and gentle
scraping, washed to remove residual enzyme, and plated with even
dispersal at a ratio of 1:1 (surface area) on reduced growth factor
MATRIGEL.TM.-coated 96-well black plates (Packard ViewPlates;
PerkinElmer; Cat #6005182) using volumes of 100 .mu.l/well. Cells
were allowed to attach as clusters and then recover log phase
growth over a 1 to 3 day period, feeding daily with MEF-conditioned
medium supplemented with 8 ng/ml bFGF (R&D Systems; Cat
#233-FB). Plates were maintained at 37.degree. C., 5% CO.sub.2 in a
humidified box throughout the duration of the assay.
[0300] Preparation of compounds and growth factors: Stocks of
growth factors purchased from R&D Systems were EGF (Cat
#236-EG), FGF4 (Cat #235-F4), PDGF-A (Cat #221-AA), PDGF-D (Cat
#1159-SB), PDGF-A/B (Cat #222-AB), and VEGF (Cat #293-VE). Muscimol
was purchased from Tocris (Cat #0289). All growth factors were
solubilized in PBS with 0.1% BSA (Sigma; Cat #A7888) and stored
frozen at -80.degree. C. Muscimol was solubilized in 100% DMSO
(Sigma; Cat #D2650) and stored frozen at -80.degree. C. Compounds
were available as 5 mM stocks in 96-well plate format, solubilized
in 100% DMSO and stored at -80.degree. C. Compounds were further
diluted to an intermediate concentration of 0.2 mM in 50 mM HEPES
(Invitrogen; Cat #15630-080), 20% DMSO and stored at 4.degree. C.
All growth factors and inhibitors were prepared in a deep well,
96-well polypropylene plate, diluted to 5.times. intermediate
stocks in DMEM:F12 base medium at the beginning of assay and stored
at 4.degree. C.
[0301] A secondary screening assay was conducted, testing in
triplicate and feeding on alternate days over the four-day assay
timeframe. Assays were initiated by aspirating culture medium from
each well followed by three washes in PBS to remove residual growth
factors and serum. Test volumes of 80 .mu.l per well were added
back containing DMEM:F12 base medium (Invitrogen; Cat #11330-032)
supplemented with 0.625% FCS (HyClone; Cat #SH30070.03), 25 ng/ml
Wnt3a (R&D Systems), and 3.125 .mu.M compound plus 20 .mu.l
5.times. stock of growth factor or small molecule to yield a final
concentration of 0.5% FCS, 20 ng/ml Wnt3a, and 2.5 .mu.M. All
remaining growth factors were tested at a final assay concentration
of 50 ng/ml (EGF, FGF4, PDGF-A, PDGF-A/B, VEGF). Final assay
concentration of muscimol was 20 .mu.M. Positive control wells (100
.mu.l/well) contained the same base medium supplemented with 0.5%
FCS, 20 ng/ml Wnt3a and 100 ng/ml activin A. Negative control wells
(100 .mu.l/well) contained the same base medium with 0.5% FCS and
20 ng/ml Wnt3a, omitting activin A.
[0302] On day 3, wells were aspirated and fed with 80 .mu.l
DMEM:F12 base medium supplemented with 2.5% FCS (HyClone) and 3.125
.mu.M compound plus 20 .mu.l 5.times. stock of growth factors or
small molecules per well to yield a final concentration of 2% FCS
and 2.5 .mu.M compound (omitting Wnt3a) and as denoted on day one
for all remaining growth factors or small molecules. Positive
control wells (100 .mu.l/well) contained the same base medium
supplemented with 2% FCS and 100 ng/ml activin A, omitting Wnt3a.
Negative control wells (100 .mu.l/well) contained the same base
medium with 2% FCS, omitting both activin A and Wnt3a.
[0303] High Content Analysis: At the conclusion of four-days of
culture, assay plates were washed twice with PBS, fixed with 4%
paraformaldehyde (Alexis Biochemical; Cat #ALX-350-011) at room
temperature for 20 minutes, then washed three times with PBS and
permeabilized with 0.5% Triton X-100 (Sigma; Cat #T8760-2) for 20
minutes at room temperature. Cells were washed again three times
with PBS and blocked with 4% chicken serum (Invitrogen; Cat
#16110082) in PBS for 30 minutes at room temperature. Primary
antibody (goat anti-human SOX17; R&D Systems; cat #AF1924) was
diluted 1:100 in 4% chicken serum and added to each well for one
hour at room temperature. Alexa Fluor 488 conjugated secondary
antibody (chicken anti-goat IgG; Molecular Probes; Cat #AZ1467) was
diluted 1:200 in PBS and added to each sample well after washing
three times with PBS. To counterstain nuclei, 4 .mu.g/ml Hoechst
33342 (Invitrogen; Cat #H3570) was added for ten minutes at room
temperature. Plates were washed once with PBS and left in 100
.mu.l/well PBS for imaging.
[0304] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized
from positive control wells and from untreated negative control
wells stained with secondary antibody alone. Images from 15 fields
per well were acquired to compensate for any cell loss during the
bioassay and subsequent staining procedures. Measurements for total
cell number and total SOX17 intensity were obtained from each well
using IN Cell Developer Toolbox 1.7 (GE Healthcare) software.
Segmentation for the nuclei was determined based on grayscale
levels (baseline range 100-300) and nuclear size. Averages and
standard deviations were calculated for each replicate data set.
Total SOX17 protein expression was reported as total intensity or
integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria of gray-scale ranges between 200 to 3500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control. Normalized data were calculated for averages and standard
deviations for each replicate set.
[0305] Table 5 shows compounds previously identified as hits (Table
2) tested in a definitive endoderm bioassay in various combinations
with growth factors and muscimol, without activin A. Some compounds
had minimal or weak effects on SOX17 expression with all growth
factor combinations tested. However, some compounds were able to
induce significant SOX17 expression with some but not all growth
factor combinations. One compound in particular, Compound 34, had
significant synergistic responses with all growth factors tested
and mediated increases in both cell numbers as well as SOX17
expression in this assay: Compound 39 with 1) EGF+FGF4=77% of
positive control response; or 2) EGF+FGF4+PDGF-AB=68% of positive
control response; or 3) EGF+FGF4+PDGF-A+VEGF=31% of positive
control response.
Example 7
Effects of Compound 34 in Combination with Other Factors on the
Differentiation of Human Embryonic Stem Cells into Cells Expressing
Markers Characteristic of the Definitive Endoderm Lineage in the
Absence of Activin A
[0306] In this example, an effort was made to analyze the minimum
number of growth factors required in combination with the best
cyclic aniline-pyridinotriazine compound, Compound 34 to yield a
robust SOX17 response in the absence of activin A. Also in this
example, a new growth factor, GDF-8, was added for evaluation.
GDF-8, also known as myostatin, is a member of the TGF-.beta.
family and has been shown to use the activin type II and TGF-.beta.
type I receptors (ALK4/5) to induce SMAD 2/3 phosphorylation.
[0307] Cell assay seeding: Clusters of H1 human embryonic stem
cells were grown on reduced growth factor MATRIGEL.TM. (Invitrogen;
Cat #356231)-coated tissue culture plastic. Cells were passaged
using collagenase (Invitrogen; Cat #17104-019) treatment and gentle
scraping, washed to remove residual enzyme, and plated with even
dispersal at a ratio of 1:1 (surface area) on reduced growth factor
MATRIGEL.TM.-coated 96-well black plates (Packard ViewPlates;
PerkinElmer; Cat #6005182) using volumes of 100 .mu.l/well. Cells
were allowed to attach as clusters and then recover log phase
growth over a 1 to 3 day period, feeding daily with MEF conditioned
medium supplemented with 8 ng/ml bFGF (R&D Systems; Cat
#233-FB). Plates were maintained at 37.degree. C., 5% CO.sub.2 in a
humidified box throughout the duration of assay.
[0308] Preparation of compounds and growth factors: Stocks of
growth factors purchased from R&D Systems were EGF (Cat
#236-EG), FGF4 (Cat #235-F4), PDGF-A (Cat #221-AA), PDGF-D (Cat
#1159-SB), PDGF-A/B (Cat #222-AB), VEGF (Cat #293-VE), and GDF-8
(Cat #788-G8). Muscimol was purchased from Tocris (Cat #0289). All
growth factors were solubilized in PBS with 0.1% BSA (Sigma; Cat
#A7888) and stored frozen at -80.degree. C. Muscimol was
solubilized in 100% DMSO (Sigma; Cat #D2650) and stored frozen at
-80.degree. C. Cyclic aniline-pyridinotriazine compounds were
available as 5 mM stocks in 96-well plate format, solubilized in
100% DMSO and stored at -80.degree. C. Compound 34 was further
diluted to an intermediate concentration of 0.2 mM in 50 mM HEPES
(Invitrogen; Cat #15630-080), 20% DMSO and stored at 4.degree. C.
All growth factors and inhibitors were prepared in a deep well,
96-well polypropylene plate, diluted to 5.times. intermediate
stocks in DMEM:F12 base medium at the beginning of assay and stored
at 4.degree. C.
[0309] A secondary screening assay was conducted, testing in
triplicate and feeding on alternate days over the four-day assay
timeframe. Assays were initiated by aspirating culture medium from
each well followed by three washes in PBS to remove residual growth
factors and serum. Test volumes of 80 .mu.l per well were added
back containing DMEM:F12 base medium (Invitrogen; Cat #11330-032)
supplemented with 0.625% FCS (HyClone; Cat #SH30070.03), 25 ng/ml
Wnt3a (R&D Systems), and 3.125 .mu.M Compound 27 plus 20 .mu.l
5.times. stock of growth factor or small molecule to yield a final
concentration of 0.5% FCS, 20 ng/ml Wnt3a, and 2.5 .mu.M Compound
34. All remaining growth factors were tested at a final assay
concentration of 50 ng/ml (EGF, FGF4, PDGF-A, PDGF-A/B, VEGF) with
the exception of GDF-8 tested at 25 ng/ml. Final assay
concentration of muscimol was 20 .mu.M. Positive control wells (100
.mu.l/well) contained the same base medium supplemented with 0.5%
FCS, 20 ng/ml Wnt3a and 100 ng/ml activin A. Negative control wells
(100 .mu.l/well) contained the same base medium with 0.5% FCS and
20 ng/ml Wnt3a, omitting activin A.
[0310] On day 3, wells were aspirated and fed with 80 .mu.l
DMEM:F12 base medium supplemented with 2.5% FCS (HyClone) and 3.125
.mu.M Compound 34 plus 20 .mu.l 5.times. stock of growth factors or
small molecules per well to yield a final concentration of 2% FCS
and 2.5 .mu.M Compound 34 (omitting Wnt3a) and as denoted on day
one for all remaining growth factors or small molecules. Positive
control wells (100 .mu.l/well) contained the same base medium
supplemented with 2% FCS and 100 ng/ml activin A, omitting Wnt3a.
Negative control wells (100 .mu.l/well) contained the same base
medium with 2% FCS, omitting both activin A and Wnt3a.
[0311] High Content Analysis: At the conclusion of four-days of
culture, assay plates were washed twice with PBS, fixed with 4%
paraformaldehyde (Alexis Biochemical; Cat #ALX-350-011) at room
temperature for 20 minutes, then washed three times with PBS and
permeabilized with 0.5% Triton X-100 (Sigma; Cat #T8760-2) for 20
minutes at room temperature. Cells were washed again three times
with PBS and blocked with 4% chicken serum (Invitrogen; Cat
#16110082) in PBS for 30 minutes at room temperature. Primary
antibody (goat anti-human SOX17; R&D Systems; cat #AF1924) was
diluted 1:100 in 4% chicken serum and added to each well for one
hour at room temperature. Alexa Fluor 488 conjugated secondary
antibody (chicken anti-goat IgG; Molecular Probes; Cat #AZ1467) was
diluted 1:200 in PBS and added to each sample well after washing
three times with PBS. To counterstain nuclei, 4 .mu.g/ml Hoechst
33342 (Invitrogen; Cat #H3570) was added for ten minutes at room
temperature. Plates were washed once with PBS and left in 100
.mu.l/well PBS for imaging.
[0312] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized
from positive control wells and from untreated negative control
wells stained with secondary antibody alone. Images from 15 fields
per well were acquired to compensate for any cell loss during the
bioassay and subsequent staining procedures. Measurements for total
cell number and total SOX17 intensity were obtained from each well
using IN Cell Developer Toolbox 1.7 (GE Healthcare) software.
Segmentation for the nuclei was determined based on gray-scale
levels (baseline range 100-300) and nuclear size. Averages and
standard deviations were calculated for each replicate data set.
Total SOX17 protein expression was reported as total intensity or
integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria of gray-scale ranges between 200 to 3500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control. Normalized data were calculated for averages and standard
deviations for each replicate set.
[0313] Table 6 shows results of this assay. Where GDF-8 was present
in any combination with the Compound 34, a substantial increase in
SOX17 expression was observed. Furthermore, GDF-8 and Wnt3a with
Compound 34 were sufficient to yield SOX17 expression (88% of
control) in a range similar to that seen with 100 ng/ml activin
A/Wnt3a treatment. It appears that the growth factor GDF-8 can
serve as a replacement for activin A during definitive endoderm
differentiation of human embryonic stem cells.
Example 8
Additional Screening for Compounds Capable of Differentiating
Pluripotent Stem Cells into Cells Expressing Markers Characteristic
of the Definitive Endoderm Lineage
[0314] Based on the compound structures for hits identified thus
far, an analog search was conducted to find additional related
compounds to test in the definitive endoderm bioassay. The
substructure search yielded compounds for screening. Screening
parameters for this assay were designed with the combination of
factors that had yielded optimal results in previous assays,
specifically combining EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
and GDF-8 with the small molecule compound.
[0315] Cell assay seeding: Briefly, clusters of H1 human embryonic
stem cells were grown on reduced growth factor MATRIGEL.TM.
(Invitrogen; Cat #356231)-coated tissue culture plastic. Cells were
passaged using collagenase (Invitrogen; Cat #17104-019) treatment
and gentle scraping, washed to remove residual enzyme, and plated
with even dispersal at a ratio of 1:1 (surface area) on reduced
growth factor MATRIGEL.TM.-coated 96-well black plates (Packard
ViewPlates; PerkinElmer; Cat #6005182) using volumes of 100
.mu.l/well. Cells were allowed to attach as clusters and then
recover log phase growth over a 1 to 3 day period, feeding daily
with MEF conditioned medium supplemented with 8 ng/ml bFGF (R&D
Systems; Cat #233-FB). Plates were maintained at 37.degree. C., 5%
CO.sub.2 in a humidified box throughout the duration of assay.
[0316] Preparation of compounds and assay: Growth factors purchased
from R&D Systems were EGF (Cat #236-EG), FGF4 (Cat #235-F4),
PDGF-A (Cat #221-AA), PDGF-D (Cat #1159-SB), PDGF-A/B (Cat
#222-AB), VEGF (Cat #293-VE), and GDF-8 (Cat #788-G8). Muscimol was
purchased from Tocris (Cat #0289). Screening was conducted using a
library of compounds that were made available as 5 mM stocks in
96-well plate format, solubilized in 100% DMSO (Sigma; Cat #D2650)
and stored at -80.degree. C. The compounds were further diluted to
an intermediate concentration of 0.2 mM in 50 mM HEPES (Invitrogen;
Cat #15630-080), 20% DMSO and stored at 4.degree. C. Test
conditions were performed in single wells, feeding on alternate
days over a four-day assay period. Primary screening assays were
initiated by aspirating culture medium from each well followed by
three washes in PBS (Invitrogen; Cat #14190) to remove residual
growth factors and serum. On the first day of assay, test volumes
of 200 .mu.l per well were added back containing DMEM:F12 base
medium (Invitrogen; Cat #11330-032) supplemented with 0.5% FCS
(HyClone; Cat #SH30070.03) and 20 ng/ml Wnt3a (R&D Systems; Cat
#1324-WN) plus 2.5 .mu.M compound. All remaining growth factors
were tested at a final assay concentration of 50 ng/ml (EGF, FGF4,
PDGF-A, PDGF-A/B, VEGF) with the exception of GDF-8 tested at 25
ng/ml. Final assay concentration of muscimol was 20 .mu.M. Positive
control samples contained the same base medium supplemented with
0.5% FCS plus 20 ng/ml Wnt3a and 100 ng/ml recombinant human
activin A (PeproTech; Cat #120-14). Negative control samples
contained DMEM:F12 base medium supplemented with 0.5% FCS and 20
ng/ml Wnt3a. On the third day of assay, test volumes of 200 .mu.l
per well were added back containing DMEM:F12 base medium
supplemented with 2% FCS plus 2.5 .mu.M compound, without Wnt3a.
All remaining growth factors were tested at a final assay
concentration of 50 ng/ml (EGF, FGF4, PDGF-A, PDGF-A/B, VEGF) with
the exception of GDF-8 tested at 25 ng/ml. Final assay
concentration of muscimol was 20 .mu.M. Positive control samples
contained the same base medium supplemented with 2% FCS and 100
ng/ml recombinant human activin A (PeproTech; Cat #120-14).
Negative control samples contained DMEM:F12 base medium
supplemented with 2% FCS. Positive control samples contained the
same base medium supplemented with FCS, substituting 100 ng/ml
recombinant human activin A (PeproTech; Cat #120-14) for the
aniline-pyridinotriazine compound throughout the four-day assay
along with Wnt3a (20 ng/ml) on days 1 and 2. Negative control
samples contained DMEM:F 12 base medium supplemented with FCS,
adding Wnt3a on days 1 and 2 but omitting treatment with activin
A.
[0317] High Content Analysis: At the conclusion of four-days of
culture, assay plates were washed twice with PBS (Invitrogen; Cat
#14190), fixed with 4% paraformaldehyde (Alexis Biochemical; Cat
#ALX-350-011) at room temperature for 20 minutes, then washed three
times with PBS and permeabilized with 0.5% Triton X-100 (Sigma; Cat
#T8760-2) for 20 minutes at room temperature. Cells were washed
again three times with PBS and blocked with 4% chicken serum
(Invitrogen; Cat #16110082) in PBS for 30 minutes at room
temperature. Primary antibody (goat anti-human SOX17; R&D
Systems; Cat #AF1924) was diluted 1:100 in 4% chicken serum and
added to each well for one hour at room temperature. Alexa Fluor
488 conjugated secondary antibody (chicken anti-goat IgG; Molecular
Probes; Cat #AZ1467) was diluted 1:200 in PBS and added to each
sample well after washing three times with PBS. To counterstain
nuclei, 4 .mu.g/ml Hoechst 33342 (Invitrogen; Cat #H3570) was added
for ten minutes at room temperature. Plates were washed once with
PBS and left in 100 .mu.l/well PBS for imaging.
[0318] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized
from positive control wells and from untreated negative control
wells stained with secondary antibody alone. Images from 15 fields
per well were acquired to compensate for any cell loss during the
bioassay and subsequent staining procedures. Measurements for total
cell number and total SOX17 intensity were obtained from each well
using IN Cell Developer Toolbox 1.7 (GE Healthcare) software.
Segmentation for the nuclei was determined based on gray-scale
levels (baseline range 100-300) and nuclear size. Averages and
standard deviations were calculated for each replicate data set.
Total SOX17 protein expression was reported as total intensity or
integrated intensity, defined as total fluorescence of the cell
times area of the cell. Background was eliminated based on
acceptance criteria of gray-scale ranges between 200 to 3500. Total
intensity data were normalized by dividing total intensities for
each well by the average total intensity for the positive control.
Normalized data were calculated for averages and standard
deviations for each replicate set.
[0319] In Table 7, GDF-8 and a combination of growth
factors/agonists (EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol) were
tested with a new set of aniline-pyridinotriazine compounds.
Results from two assay plates in this single experiment are ranked
with respect to SOX17 responses (as a percentage of the positive
control treatment with activin A and Wnt3a). Additional compounds
were identified that show significant synergistic activity with the
growth factor/agonist pool. These compounds were effective in both
retaining assay cell number and yielding SOX17 expression during
human embryonic stem cell differentiation in the absence of activin
A. A list of these hits with greater than 25% activity of the
positive control is shown in Table 8.
[0320] Of note, four hits from the initial primary screening (Table
2) were duplicated in the analog library. Two of these compounds
repeated as hits with the analog screening (Compound 34 and
Compound 35; shown boxed in Table 8); one was a weak hit in the
analog screening, and one compound did not repeat.
Example 9
Effects of the Compounds of the Present Invention on the
Differentiation of Human Embryonic Stem Cells to Cells Expressing
Markers Characteristic of the Definitive Endoderm Lineage in the
Presence of Low Concentrations of Activin A
[0321] It was important to determine if the compounds that had been
identified as hits in the definitive endoderm bioassays above could
also show synergistic activity with very low doses of activin A. An
initial evaluation was performed using the short hit list of cyclic
aniline-pyridinotriazine compounds denoted in Table 3B.
[0322] Cell assay seeding: Clusters of H1 human embryonic stem
cells were grown on reduced growth factor MATRIGEL.TM. (Invitrogen;
Cat #356231)-coated tissue culture plastic. Cells were passaged
using collagenase (Invitrogen; Cat #17104-019) treatment and gentle
scraping, washed to remove residual enzyme, and plated with even
dispersal at a ratio of 1:1 (surface area) on reduced growth factor
MATRIGEL.TM.-coated 96-well black plates (Packard ViewPlates;
PerkinElmer; Cat #6005182) using volumes of 100 .mu.l/well. Cells
were allowed to attach as clusters and then recover log phase
growth over a 1 to 3 day period, feeding daily with MEF conditioned
medium supplemented with 8 ng/ml bFGF (R&D Systems; Cat
#233-FB). Plates were maintained at 37.degree. C., 5% CO.sub.2 in a
humidified box throughout the duration of assay.
[0323] Preparation of compounds and growth factors: Stocks of
growth factors purchased from R&D Systems were EGF (Cat
#236-EG), FGF4 (Cat #235-F4), PDGF-A (Cat #221-AA), PDGF-D (Cat
#1159-SB), PDGF-A/B (Cat #222-AB), VEGF (Cat #293-VE), and GDF-8
(Cat #788-G8). Activin A was purchased from PeproTech (Cat #).
Muscimol was purchased from Tocris (Cat #0289). All growth factors
were solubilized in PBS with 0.1% BSA (Sigma; Cat #A7888) and
stored frozen at -80.degree. C. Muscimol was solubilized in 100%
DMSO (Sigma; Cat #D2650) and stored frozen at -80.degree. C. The
compounds were available as 5 mM stocks in 96-well plate format,
solubilized in 100% DMSO and stored at -80.degree. C. The compounds
were further diluted to an intermediate concentration of 0.2 mM in
50 mM HEPES (Invitrogen; Cat #15630-080), 20% DMSO and stored at
4.degree. C. All growth factors and inhibitors were prepared in a
deep well, 96-well polypropylene plate, diluted to 5.times.
intermediate stocks in DMEM:F12 base medium at the beginning of
assay and stored at 4.degree. C.
[0324] A secondary screening assay was conducted, testing in
triplicate and feeding on alternate days over the four-day assay
timeframe. Assays were initiated by aspirating culture medium from
each well followed by three washes in PBS to remove residual growth
factors and serum. Test volumes of 80 .mu.l per well were added
back containing DMEM:F12 base medium (Invitrogen; Cat #11330-032)
supplemented with 0.625% FCS (HyClone; Cat #SH30070.03), 25 ng/ml
Wnt3a (R&D Systems), 12.5 ng/ml activin A, and 3.125 .mu.M
compound plus 20 .mu.l 5.times. stock of growth factor or small
molecule to yield a final concentration of 0.5% FCS, 20 ng/ml
Wnt3a, 10 ng/ml activin A, and 2.5 .mu.M compound. All remaining
growth factors were tested at a final assay concentration of 50
ng/ml (EGF, FGF4, PDGF-A, PDGF-A/B, VEGF), with the exception of
GDF-8 used at 25 ng/ml. Final assay concentration of muscimol was
20 .mu.M. Positive control wells (100 .mu.l/well) contained the
same base medium supplemented with 0.5% FCS, 20 ng/ml Wnt3a and 10
ng/ml (low dose) or 100 ng/ml (high dose) activin A. Negative
control wells (100 .mu.l/well) contained the same base medium with
0.5% FCS and 20 ng/ml Wnt3a, omitting activin A.
[0325] On day 3, wells were aspirated and fed with 80 .mu.l
DMEM:F12 base medium supplemented with 2.5% FCS (HyClone), 12.5
ng/ml activin A, and 3.125 .mu.M compound plus 20 .mu.l 5.times.
stock of growth factors or small molecules per well to yield a
final concentration of 2% FCS, 10 ng/ml activin A, and 2.5 .mu.M
compound (omitting Wnt3a) and as denoted on day one for all
remaining growth factors or small molecules. Positive control wells
(100 .mu.l/well) contained the same base medium supplemented with
2% FCS and 10 ng/ml or 100 ng/ml activin A, omitting Wnt3a.
Negative control wells (100 .mu.l/well) contained the same base
medium with 2% FCS, omitting both activin A and Wnt3a.
[0326] High Content Analysis: At the conclusion of four-days of
culture, assay plates were washed twice with PBS, fixed with 4%
paraformaldehyde (Alexis Biochemical; Cat #ALX-350-011) at room
temperature for 20 minutes, then washed three times with PBS and
permeabilized with 0.5% Triton X-100 (Sigma; Cat #T8760-2) for 20
minutes at room temperature. Cells were washed again three times
with PBS and blocked with 4% chicken serum (Invitrogen; Cat
#16110082) in PBS for 30 minutes at room temperature. Primary
antibody (goat anti-human SOX17; R&D Systems; cat #AF1924) was
diluted 1:100 in 4% chicken serum and added to each well for one
hour at room temperature. Alexa Fluor 488 conjugated secondary
antibody (chicken anti-goat IgG; Molecular Probes; Cat #AZ1467) was
diluted 1:200 in PBS and added to each sample well after washing
three times with PBS. To counterstain nuclei, 4 .mu.g/ml Hoechst
33342 (Invitrogen; Cat #H3570) was added for ten minutes at room
temperature. Plates were washed once with PBS and left in 100
.mu.l/well PBS for imaging.
[0327] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized
from positive control wells and from untreated negative control
wells stained with secondary antibody alone. Images from 15 fields
per well were acquired to compensate for any cell loss during the
bioassay and subsequent staining procedures. Measurements for total
cell number and total SOX17 intensity were obtained from each well
using IN Cell Developer Toolbox 1.7 (GE Healthcare) software.
Segmentation for the nuclei was determined based on gray-scale
levels (baseline range 100-300) and nuclear size. Averages and
standard deviations were calculated for each replicate data set.
Total SOX17 protein expression was reported as total intensity or
integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria of gray-scale ranges between 200 to 3500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control. Normalized data were calculated for averages and standard
deviations for each replicate set.
[0328] Table 9 shows results from assay of various compounds and
different combinations of growth factors with low doses of activin
A. Some compounds showed robust synergistic responses with various
growth factors. In other cases, the synergistic effects were more
modest but significant relative to a low dose activin A control.
Other compounds had no activity relative to the low dose activin A
control.
Example 10
Effects of the Compounds of the Present Invention on the
Differentiation of Single Human Embryonic Stem Cells to Cells
Expressing Markers of the Definitive Endoderm Lineage in the
Absence of Activin A
[0329] Cyclic aniline-pyridinotriazine compounds were also tested
in a screening format using cells dispersed through enzymatic
treatment to single cells and plated in monolayer for assay. The
assay also made changes to eliminate serum that can provide growth
factors even at low doses. To that end the basal medium was changed
and serum was replaced with fatty acid free BSA. The assay was
shortened from four days to three days to provide a more narrow
timeframe to measure results. Finally, the assay included two
growth factors, EGF and FGF4 that had previously shown significant
but sub-optimal effects on definitive endoderm differentiation in
the absence of activin A.
Screening Assay
[0330] Cell assay seeding: Briefly, clusters of H1 human embryonic
stem cells were grown on reduced growth factor MATRIGEL.TM.
(Invitrogen; Cat #356231)-coated tissue culture plastic. Cultures
were treated with Accutase (Sigma; Cat #A6964), using equivalent
volumes of 10 ml per 10 cm.sup.2 surface area for 5 minutes at
37.degree. C., then gently resuspended, pelleted by centrifugation,
and resuspended in MEF conditioned medium for counting. For assay
seeding, cells were plated at 50,000 cells/cm.sup.2 on TM reduced
growth factor MATRIGEL.TM.-coated 96-well black plates (Packard
ViewPlates; Cat #6005182) using volumes of 100 .mu.l/well. Cells
were allowed to attach and recover log phase growth over a 3 to 5
day period, feeding daily with MEF conditioned medium supplemented
with 8 ng/ml bFGF (R&D Systems; Cat #233-FB). Plates were
maintained at 37.degree. C., 5% CO.sub.2 in a humidified box
throughout the duration of assay.
[0331] Preparation of compounds and assay: Stocks of EGF and FGF4
were prepared in a 96-well polypropylene plate (Corning, Inc.; Cat
#3960). Compound 22 was available as a 5 mM stock solubilized in
100% DMSO (Sigma; Cat #D2650) and stored at -80.degree. C. Assays
were initiated by aspirating culture medium from each well followed
by three washes in PBS to remove residual growth factors and serum.
Test volumes of 80 .mu.l per well were added back containing RPMI
1640 base medium (Invitrogen; Cat #22400-089) supplemented with
2.5% fatty acid free BSA (MP Biomedicals LLC; Cat #152401), 10
ng/ml bFGF (PeproTech Inc; Cat #100-18B), 25 ng/ml Wnt3a (R&D
Systems; Cat #1324-WN) and 3.125 .mu.M Compound 22 plus 20 .mu.l
5.times. stock of growth factors to yield a final concentration of
2% fatty acid free BSA, 8 ng/ml bFGF (PeproTech Inc; Cat #100-18B),
20 ng/ml Wnt3a, and 2.5 .mu.M Compound 22 in assay. Positive
control wells contained the same base medium supplemented with 2%
fatty acid free BSA, 8 ng/ml bFGF, 20 ng/ml Wnt3a, and 100 ng/ml
recombinant human activin A (PeproTech; Cat #120-14). Negative
control wells contained the same base medium supplemented with 2%
fatty acid free BSA, 8 ng/ml bFGF, 20 ng/ml Wnt3a but omitted
treatment with activin A.
[0332] On the second day of assay, wells were again aspirated and
fed with 80 .mu.l per well were added back containing RPMI 1640
base medium supplemented with 2.5% fatty acid free BSA, 10 ng/ml
bFGF, and 3.125 .mu.M Compound 22 plus 20 .mu.l 5.times. stock of
growth factors to yield a final concentration of 2% fatty acid free
BSA, 8 ng/ml bFGF and 2.5 .mu.M Compound 22 in assay. Positive
control wells contained the same base medium supplemented with 2%
fatty acid free BSA, 8 ng/ml bFGF and 100 ng/ml recombinant human
activin A. Negative control samples contained the same base medium
supplemented with 2% fatty acid free BSA and 8 ng/ml bFGF but
omitted treatment with activin A.
[0333] High Content Analysis: At the conclusion of four-days of
culture, assay plates were washed twice with PBS, fixed with 4%
paraformaldehyde (Alexis Biochemical; Cat #ALX-350-011) at room
temperature for 20 minutes, then washed three times with PBS and
permeabilized with 0.5% Triton X-100 (Sigma; Cat #T8760-2) for 20
minutes at room temperature. Cells were washed again three times
with PBS and blocked with 4% chicken serum (Invitrogen; Cat
#16110082) in PBS for 30 minutes at room temperature. Primary
antibody (goat anti-human SOX17; R&D Systems; cat #AF1924) was
diluted 1:100 in 4% chicken serum and added to each well for one
hour at room temperature. Alexa Fluor 488 conjugated secondary
antibody (chicken anti-goat IgG; Molecular Probes; Cat #AZ1467) was
diluted 1:200 in PBS and added to each sample well after washing
three times with PBS. To counterstain nuclei, 4 .mu.g/ml Hoechst
33342 (Invitrogen; Cat #H3570) was added for ten minutes at room
temperature. Plates were washed once with PBS and left in 100
.mu.l/well PBS for imaging.
[0334] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized
from positive control wells and from untreated negative control
wells stained with secondary antibody alone. Images from 15 fields
per well were acquired to compensate for any cell loss during the
bioassay and subsequent staining procedures. Measurements for total
cell number and total SOX17 intensity were obtained from each well
using IN Cell Developer Toolbox 1.7 (GE Healthcare) software.
Segmentation for the nuclei was determined based on gray-scale
levels (baseline range 100-300) and nuclear size. Averages and
standard deviations were calculated for each replicate data set.
Total SOX17 protein expression was reported as total intensity or
integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria of gray-scale ranges between 200 to 3500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control. Normalized data were calculated for averages and standard
deviations for each replicate set.
[0335] Table 10 shows results of this assay with Compound 34.
Control samples with EGF and/or FGF4 alone without the Compound 34
had low SOX17 expression. Addition of Compound 34 added significant
enhancement of SOX17 expression.
Example 11
A Comparison of the Ability of Activin A and GDF-8 to Differentiate
Human Embryonic Stem Cells to Cells Expressing Markers
Characteristic of the Definitive Endoderm Lineage
[0336] A previous example showed that GDF-8 is able to replace
activin A to differentiate human embryonic stem cells to cells
expressing markers characteristic of the definitive endoderm
lineage. It was important to know the relative potencies of
GDF-8GDF-8 and activin A with respect their ability to
differentiate human embryonic stem cells to cells expressing
markers characteristic of the definitive endoderm lineage. A dose
response assay was conducted using equivalent concentrations of
each growth factor to compare results during embryonic stem cell
differentiation.
[0337] Preparation of cells for assay: Stock cultures of human
embryonic stem cells (H1 human embryonic stem cell line) were
maintained in an undifferentiated, pluripotent state on reduced
growth factor MATRIGEL-coated dishes in MEF conditioned medium with
passage on average every four days. Passage was performed by
exposing cell cultures to a solution of 1 mg/ml dispase
(Invitrogen, Cat #17105-041) for 5 to 7 minutes at 37.degree. C.
followed by rinsing the monolayer with MEF conditioned culture
medium and gentle scraping to recover cell clusters. Clusters were
centrifuged at low speed to collect a cell pellet and remove
residual dispase. Cell clusters were split at a 1:3 or 1:4 ratio
for routine maintenance culture or a 1:1 ratio for immediate assay.
All human embryonic stem cell lines were maintained at passage
numbers less than 50 and routinely evaluated for normal karyotypic
phenotype and for absence of mycoplasma contamination.
[0338] Cell clusters used in the assay were evenly resuspended in
MEF conditioned medium supplemented with 8 ng/ml bFGF and seeded
onto reduced growth factor MATRIGEL.TM.-coated 96-well Packard
VIEWPLATES (PerkinElmer; Cat #6005182) in volumes of 100
.mu.l/well. MEF conditioned medium supplemented with 8 ng/ml bFGF
was used for initial plating and expansion. Daily feeding was
conducted by aspirating spent culture medium from each well and
replacing with an equal volume of fresh medium. Plates were
maintained at 37.degree. C., 5% CO.sub.2 in a humidified box
throughout the duration of assay.
[0339] Assay: The assay was initiated by aspirating the culture
medium from each well and adding back an aliquot (100 .mu.l) of
test medium. Test conditions were performed in quadruplicate over a
total three-day assay period, feeding on day 1 and day 2 by
aspirating and replacing the medium from each well with fresh test
medium. Two 12-channel polypropylene basins (Argos technologies,
Inc, Cat #B3135) were used to make the test media containing
different concentrations of Activin A (PeproTech; Cat #120-14) or
GDF-8 (R&D Systems, Cat #788-G8). Channels numbered 2 through
12 of each basin contained 1 ml assay medium composed of RPMI-1640
medium (Invitrogen; Cat #22400) supplemented with 2% Albumin Bovine
Fraction V, Fatty Acid Free (FAF BSA) (MP Biomedicals, Inc; Cat
#152401) and 8 ng/ml bFGF (PeproTech Inc.; Cat #100-18B), and with
20 ng/ml Wnt3a (R&D Systems; Cat #1324-WN/CF) added on day 1,
omitted on day 2 and 3. Channel number 1 of each basin contained
1600 ng/ml Activin A or 1600 ng/ml GDF-8, diluted into the same
assay medium. One ml of medium was transferred from channel number
1 to channel number 2 and mixed well. A fresh pipette tip was used
to transfer one ml of medium from channel number 2 to channel
number 3, followed by thorough mixing. The same procedure was
repeated in sequence through channel number 11 for each respective
basin. Channel number 12 of each basin contained medium without
Activin A or GDF-8. By doing this, a series of two-fold test
dilutions was created, containing Activin A or GDF-8 at
concentrations ranging from 1.6 ng/ml to 1600 ng/ml, for addition
to the respective assay wells.
[0340] High Content Analysis: At the conclusion of three days of
culture, assay plates were washed once with PBS (Invitrogen; Cat
#14190), fixed with 4% paraformaldehyde (Alexis Biochemical; Cat
#ALX-350-011) at room temperature for 20 minutes, then washed three
times with PBS and permeabilized with 0.5% Triton X-100 (Sigma; Cat
#T8760-2) for 20 minutes at room temperature. Cells were washed
again three times with PBS and blocked with 4% chicken serum
(Invitrogen; Cat #16110082) in PBS for 30 minutes at room
temperature. Primary antibody (goat anti-human SOX17; R&D
Systems; Cat #AF1924) was diluted 1:100 in 4% chicken serum and
added to each well for two hours at room temperature. After washing
three times with PBS, Alexa Fluor 488 conjugated secondary antibody
(chicken anti-goat IgG; Invitrogen; Cat #A21467) diluted 1:200 in
PBS was added to each well. To counterstain nuclei, 5 .mu.g/ml
Hoechst 33342 (Invitrogen; Cat #H3570) was added for fifteen
minutes at room temperature. Plates were washed once with PBS and
left in 100 .mu.l/well PBS for imaging.
[0341] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Images were acquired from 25
fields per well. Measurements for total SOX17 intensity in each
well were obtained using IN Cell Developer Toolbox 1.7 (GE
Healthcare) software. Segmentation for the nuclei was determined
based on gray-scale levels (baseline range 100-300) and nuclear
size. Averages and standard deviations were calculated for each
quadruplicate data set. Total SOX17 protein expression was reported
as total intensity or integrated intensity, defined as total
fluorescence of the cell multiplied by area of the cell. Background
was eliminated based on acceptance criteria for gray-scale ranges
between 200 to 4500. Total SOX17 intensity data were calculated
using GraphPad Prism 4.02 (GraphPad Software, Inc., Lo Jolla,
Calif.). Data were normalized to define the smallest and largest
values in each data set as 0% and 100%, respectively. Table 11
shows the normalized values for each of the activin A and GDF-8
data sets. Two sigmoidal dose-response curves are shown in FIG. 2
as generated using the normalized values shown in Table 11. The
R.sup.2 values, indicating curve fit, were calculated using
GraphPad Prism and determined to be 0.9944 for activin A and 0.9964
for GDF-8. Using GraphPad Prism, EC.sub.50 values for each growth
factor were calculated and determined to be 13.9 ng/ml for activin
A and 184.8 ng/ml for GDF-8. These data indicate that GDF-8 is less
potent than activin A with respect to inducing human embryonic stem
cells to differentiate to cells expressing markers characteristic
of the definitive endoderm lineage. Nonetheless, GDF-8 can
substitute for activin A and at specific concentrations, can induce
an equivalent population of definitive endoderm cells, as denoted
by SOX17 expression.
Example 12
Cells Expressing Markers Characteristic of the Definitive Endoderm
Lineage that were Formed According to the Methods of the Present
Invention are able to Further Differentiate into Cells Expressing
Markers Characteristic of the Pancreatic Endocrine Lineage
[0342] Parallel populations of human embryonic stem cells were
differentiated to cells expressing markers characteristic of the
definitive endoderm lineage using GDF-8 in combination with either
Compound 34 or Compound 56. Thereafter, a step-wise differentiation
protocol was applied to treated cells to promote differentiation
toward pancreatic endoderm and endocrine lineages. A parallel
control consisting of cells treated with Activin A and Wnt3a was
maintained for comparison purposes throughout the step-wise
differentiation process. Samples were taken at every stage of the
differentiation to determine the appearance of proteins and mRNA
biomarkers representative of the various stages of
differentiation.
[0343] Preparation of cells for assay: Stock cultures of human
embryonic stem cells (H1 human embryonic stem cell line) were
maintained in an undifferentiated, pluripotent state on reduced
growth factor MATRIGEL.TM.-coated dishes in MEF conditioned medium
with passage on average every four days. Passage was performed by
exposing cell cultures to a solution of 1 mg/ml dispase
(Invitrogen; Cat #17105-041) for 5 to 7 minutes at 37.degree. C.
followed by rinsing the monolayer with MEF conditioned culture
medium and gentle scraping to recover cell clusters. Clusters were
centrifuged at low speed to collect a cell pellet and remove
residual dispase. Cell clusters were split at a 1:3 or 1:4 ratio
for routine maintenance culture or a 1:1 ratio for immediate assay.
All human ES cell lines were maintained at passage numbers less
than 50 and routinely evaluated for normal karyotype and absence of
mycoplasma.
[0344] Cell clusters were evenly resuspended in MEF conditioned
medium supplemented with 8 ng/ml bFGF and seeded onto reduced
growth factor MATRIGEL.TM.-coated 24-well, black wall culture
plates (Arctic White; Cat #AWLS-303012) in volumes of 0.5 ml/well.
Daily feeding was conducted by aspirating spent culture medium from
each well and replacing with an equal volume of fresh medium.
Plates were maintained at 37.degree. C., 5% CO.sub.2 throughout the
duration of assay.
[0345] Assay: The assay was initiated by aspirating the culture
medium from each well and adding back an aliquot (0.5 ml) of test
medium. Test conditions for the first step of differentiation were
conducted over a three-day period, feeding daily by aspirating and
replacing the medium from each well with fresh test medium. On the
first day of assay, 100 ng/ml activin A (PeproTech; Cat #120-14) or
200 ng/ml GDF-8 (R&D Systems, Cat #788-G8) was added to
respective assay wells where each growth factor was diluted into
RPMI-1640 medium (Invitrogen; Cat #22400) with 1% Albumin Bovine
Fraction V, Fatty Acid Free (FAF BSA) (MP Biomedicals, Inc; Cat
#152401), 1% Probumin (Millipore; Cat #81-068-3) and 20 ng/ml Wnt3a
(R&D Systems; Cat #1324-WN/CF). On the second day of assay, 100
ng/ml activin A or 200 ng/ml GDF-8 was diluted into RPMI-1640
medium supplemented with 2% FAF BSA without Wnt3a. In some test
samples using GDF-8, Wnt3a was replaced with a either Compound 34
or Compound 56 at a concentration of 2.5 .mu.M, and either Compound
34 or Compound 56 was added daily during all three days of
definitive endoderm differentiation. At the conclusion of the first
step of differentiation, cells from some wells were harvested for
flow cytometry analysis to evaluate levels of CXCR4, a marker of
definitive endoderm formation. Additional wells were harvested for
RT-PCR analysis to measure other markers of differentiation.
[0346] At the conclusion of the first step of differentiation,
replicate sets of parallel wells from each treatment group were
subjected to further step-wise differentiation. It is important to
note that after the first differentiation step, all wells
undergoing continuing culture and differentiation received the same
treatment. The protocol for this continuing differentiation is
described below.
[0347] Step 2 of the differentiation protocol was carried out over
two days. Cells were fed daily by aspirating the medium from each
well and replacing with a fresh aliquot (0.5 ml) of DMEM:F12 medium
(Invitrogen; Cat #11330-032) containing 2% Albumin Bovine Fraction
V, Fatty Acid Free (FAF BSA) (MP Biomedicals, Inc; Cat #152401), 50
ng/ml FGF7 (PeproTech; Cat #100-19), and 250 nM cyclopamine
(Calbiochem; Cat #239804).
[0348] Step 3 of the differentiation protocol was carried out over
four days. Cells were fed daily by aspirating medium from each well
and replacing with a fresh aliquot (0.5 ml) of DMEM-high glucose
(Invitrogen; Cat #10569) supplemented with 1% B27 (Invitrogen; Cat
#17504-044), 50 ng/ml FGF7, 100 ng/ml Noggin (R&D Systems; Cat
#3344-NG), 250 nM KAAD-cyclopamine (Calbiochem; Cat #239804), and 2
.mu.M all-trans retinoic acid (RA) (Sigma-Aldrich; Cat #R2625). At
the conclusion of the third step of differentiation, cells from
some wells were harvested for analysis by RT-PCR to measure markers
of differentiation. Other culture wells were subjected to high
content image analysis for protein expression levels of Pdx1, a
transcription factor associated with pancreatic endoderm, and Cdx2,
a transcription factor associated with intestinal endoderm.
[0349] Step 4 of the differentiation protocol was carried out over
three days. Cells were fed daily by aspirating the medium from each
well and replacing with a fresh aliquot (0.5 ml) of DMEM-high
glucose supplemented with 1% B27, 100 ng/ml Noggin, 100 ng/ml
Netrin-4, 1 .mu.M DAPT (EMD Biosciences; Cat #565770), and 1 .mu.M
Alk 5 inhibitor (Axxora; Cat #ALX-270-445). At the conclusion of
the fourth step of differentiation, cells from some wells were
harvested for analysis by RT-PCR to measure markers of
differentiation. Other culture wells were subjected to high content
image analysis for protein expression levels of PDX1.
[0350] Step 5 of the differentiation protocol was carried out over
seven days in DMEM-high glucose with 1% B27, and 1 .mu.M Alk 5
inhibitor. Medium in each well was aspirated and replaced with a
fresh aliquot (0.5 ml) on all days. At the conclusion of the fifth
step of differentiation, cells from some wells were harvested for
analysis by RT-PCR to measure markers of differentiation. Other
culture wells were subjected to high content image analysis for
protein expression levels of insulin and glucagon.
[0351] Step 6 of the differentiation protocol was carried out over
seven days in DMEM-high glucose with 1% B27. Medium in each well
was aspirated and replaced with a fresh aliquot (0.5 ml) on
alternating days. At the conclusion of the sixth step of
differentiation, cells from some wells were harvested for analysis
by RT-PCR to measure markers of differentiation.
[0352] FACS Analysis: Cells for FACS analysis were blocked in a 1:5
solution of 0.5% human gamma-globulin (Sigma; Cat #G-4386) in PBS
(Invitrogen; Cat #14040-133): BD FACS staining buffer--BSA (BD; Cat
#554657) for 15 minutes at 4.degree. C. Cells were then stained
with antibodies for CD9 PE (BD; Cat #555372), CD99 PE (Caltag; Cat
#MHCD9904) and CXCR4 APC (R&D Systems; Cat #FAB173A) for 30
minutes at 4.degree. C. After a series of washes in BD FACS
staining buffer, the cells were stained for viability with 7-AAD
(BD; Cat #559925) and run on a BD FACSArray. A mouse IgG1K Isotype
control antibody for both PE and APC was used to gate percent
positive cells.
[0353] RT-PCR Analysis: RNA samples were purified by binding to a
silica-gel membrane (Rneasy Mini Kit, Qiagen, Calif.) in the
presence of an ethanol-containing, high-salt buffer followed by
washing to remove contaminants. The RNA was further purified using
a TURBO DNA-free kit (Ambion, INC), and high-quality RNA was then
eluted in water. Yield and purity were assessed by A260 and A280
readings on a spectrophotometer. CDNA copies were made from
purified RNA using an ABI (ABI, CA) high capacity cDNA archive
kit.
[0354] Unless otherwise stated, all reagents were purchased from
Applied Biosystems. Real-time PCR reactions were performed using
the ABI PRISM.RTM. 7900 Sequence Detection System. TAQMAN.RTM.
UNIVERSAL PCR MASTER MIX.RTM. (ABI, CA) was used with 20 ng of
reverse transcribed RNA in a total reaction volume of 20 .mu.l.
Each cDNA sample was run in duplicate to correct for pipetting
errors. Primers and FAM-labeled TAQMAN.RTM. probes were used at
concentrations of 200 nM. The level of expression for each target
gene was normalized using a human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) endogenous control previously developed by
Applied Biosystems. Primer and probe sets are listed in Table 12.
After an initial incubation at 50.degree. C. for 2 min followed by
95.degree. C. for 10 min, samples were cycled 40 times in two
stages--a denaturation step at 95.degree. C. for 15 sec followed by
an annealing/extension step at 60.degree. C. for 1 min. Data
analysis was carried out using GENEAMP.RTM.7000 Sequence Detection
System software. For each primer/probe set, a Ct value was
determined as the cycle number at which the fluorescence intensity
reached a specific value in the middle of the exponential region of
amplification. Relative gene expression levels were calculated
using the comparative Ct method. Briefly, for each cDNA sample, the
endogenous control Ct value was subtracted from the gene of
interest Ct to give the delta Ct value (.DELTA.Ct). The normalized
amount of target was calculated as 2-.DELTA.Ct, assuming
amplification to be 100% efficiency. Final data were expressed
relative to a calibrator sample.
[0355] High Content Analysis: At the conclusion of culture, assay
plates were washed once with PBS (Invitrogen; Cat #14190), fixed
with 4% paraformaldehyde (Alexis Biochemical; Cat #ALX-350-011) at
room temperature for 20 minutes, then washed three times with PBS
and permeabilized with 0.5% Triton X-100 (Sigma; Cat #T8760-2) for
20 minutes at room temperature. Cells were washed again three times
with PBS and blocked with 4% chicken serum (Invitrogen; Cat
#16110082) in PBS for 30 minutes at room temperature. Primary
antibody (goat anti-human SOX17; R&D Systems; Cat #AF1924) was
diluted 1:100 in 4% chicken serum and added to each well for two
hours at room temperature. After washing three times with PBS,
Alexa Fluor 488 conjugated secondary antibody (chicken anti-goat
IgG; Invitrogen; Cat #A21467) diluted 1:200 in PBS was added to
each well. To counterstain nuclei, 5 .mu.g/ml Hoechst 33342
(Invitrogen; Cat #H3570) was added for fifteen minutes at room
temperature. Plates were washed once with PBS and left in 100
.mu.l/well PBS for imaging. Other primary antibodies used for
analysis included 1:100 dilution mouse anti-human CDX2 (Invitrogen;
Cat #397800), 1:100 dilution goat anti-human Pdx1 (Santa Cruz
Biotechnology; Cat #SC-14664), 1:200 dilution rabbit anti-human
insulin (Cell Signaling; Cat #C27C9), and 1:1500 dilution mouse
anti-human glucagon (Sigma-Aldrich; Cat #G2654). Secondary
antibodies used for analysis included 1:400 dilution Alexa Fluor
647 chicken anti-mouse IgG (Invitrogen; Cat #A-21463), 1:200
dilution Alexa Fluor 488 donkey anti-goat IgG (Invitrogen; Cat
#A11055), 1:1000 dilution Alexa Fluor 647 chicken anti-rabbit IgG
(Invitrogen; Cat #A21443), and 1:1000 dilution Alexa Fluor 488
chicken anti-mouse IgG (Invitrogen; Cat #A21200).
[0356] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Images were acquired from 25
fields per well. Measurements for total intensity were obtained
from each well using IN Cell Developer Toolbox 1.7 (GE Healthcare)
software. Segmentation for the nuclei was determined based on
gray-scale levels (baseline range 100-300) and nuclear size.
Averages and standard deviations were calculated for each replicate
data set. Total protein expression was reported as total intensity
or integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria for gray-scale ranges between 200 and 4500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control.
[0357] PCR results for representative differentiation markers are
shown in Table 13 for cells harvested from each step of
differentiation. Samples treated with GDF-8 and Wnt3a or with GDF-8
and either Compound 34 or Compound 56 showed similar, or in some
instances, improved expression levels of expression markers
associated with endodermal and endocrine differentiation.
[0358] FIG. 3 shows the results of the FACS analysis, showing the
expression of the definitive endoderm marker, CXCR4, after the
first step of differentiation. Treatment of human embryonic stem
cells with GDF-8 and Wnt3a yielded an equivalent percentage of
CXCR4 positive cells compared to treatment with activin A and
Wnt3a. Similarly, treatment of human embryonic stem cells with
GDF-8 and a small molecule (Compound 34 or Compound 56) also
yielded an equivalent or slightly higher percentage of CXC4
positive cells. FIG. 4 shows high content image analysis for
normalized SOX17 protein expression in human embryonic stem cells
after three days differentiation to definitive endoderm. Levels of
expression for treatment groups using GDF-8 with Wnt3a or GDF-8
with a small molecule are similar to treatment with Activin A and
Wnt3a.
[0359] FIG. 5 shows high content image analysis for normalized Pdx1
and Cdx2 protein expression in human embryonic stem cells after the
third step of differentiation to pancreatic endoderm. Levels of
expression for treatment groups using GDF-8 with Wnt3a or GDF-8
with Wnt3a or GDF-8 with a compound of the present invention show
equivalent levels of PDX1 and CDX2. In some treatment groups the
cell number retained after differentiation decreased thereby
increasing the ratio of PDX1 expressing cells. Similar results were
obtained showing equivalent normalized PDX1 expression in all
treatment groups after the fourth step of differentiation as shown
in FIG. 6. In FIG. 7, normalized protein levels of insulin and
glucagon are shown, demonstrating equivalent expression between the
Activin A and GDF-8 treatment groups.
[0360] These collective results demonstrate that GDF-8, in
combination with Wnt3a or Compound 34 or Compound 56, can
substitute for activin A during definitive endoderm differentiation
and subsequent pancreatic endoderm and endocrine
differentiation.
Example 13
Formation of Cells Expressing Markers Characteristic of the
Definitive Endoderm Lineage with Other Members of the GDF Family of
Proteins
[0361] It was important to determine if treating human embryonic
stem cells with other GDF family members could the formation of
cells expressing markers characteristic of the definitive endoderm
lineage. Wnt3a in combination with either Compound 34 or Compound
56 were tested on human embryonic stem cells in combination with
six different GDF growth factors [GDF-3, GDF-5, GDF-8, GDF-10,
GDF-11, and GDF-15] to determine the ability of members of the GDF
family of proteins to differentiate human embryonic stem cells
toward cells expressing markers characteristic of the definitive
endoderm lineage. A parallel control of cells treated with activin
A and Wnt3a was maintained for comparison purposes.
[0362] Preparation of cells for assay: Stock cultures of human
embryonic stem cells (H1 human embryonic stem cell line) were
maintained in an undifferentiated, pluripotent state on reduced
growth factor MATRIGEL.TM. (BD Biosciences; Cat #356231)-coated
dishes in MEF conditioned medium with passage on average every four
days. Passage was performed by exposing cell cultures to a solution
of 1 mg/ml dispase (Invitrogen; Cat #17105-041) for 5 to 7 minutes
at 37.degree. C. followed by rinsing the monolayer with MEF
conditioned culture medium and gentle scraping to recover cell
clusters. Clusters were centrifuged at low speed to collect a cell
pellet and remove residual dispase. Cell clusters were split at a
1:3 or 1:4 ratio for routine maintenance culture or a 1:1 ratio for
immediate assay. All human ES cell lines were maintained at passage
numbers less than 50 and routinely evaluated for normal karyotype
and absence of mycoplasma.
[0363] Cell clusters were evenly resuspended in MEF conditioned
medium supplemented with 8 ng/ml bFGF and seeded onto reduced
growth factor MATRIGEL.TM.-coated 96-well Packard VIEWPLATES
(PerkinElmer; Cat #6005182) in volumes of 0.1 ml/well. Daily
feeding was conducted by aspirating spent culture medium from each
well and replacing with an equal volume of fresh medium. Plates
were maintained at 37.degree. C., 5% CO.sub.2 throughout the
duration of assay.
[0364] Assay: The assay was initiated by aspirating the culture
medium from each well and adding back aliquots (100 .mu.l) of test
medium. Test conditions were performed in triplicate over a total
four-day assay period, feeding on day 1 and day 3 by aspirating and
replacing the medium from each well with fresh test medium. Various
members of the GDF family of proteins were obtained for testing as
follows: GDF-3 (PeproTech; Cat #120-22); GDF-5 (DePuy Orthopaedics,
Inc., a Johnson & Johnson company); GDF-8 (R&D Systems; Cat
#788-G8); GDF-10 (R&D Systems; Cat #1543-BP); GDF11 (PeproTech;
Cat #120-11); GDF-15 (R&D Systems; Cat #957-GD). On the first
day of assay, all wells received an aliquot (80 .mu.l) of basal
medium DMEM:F12 medium (Invitrogen; Cat #11330-032) supplemented
with 0.5% fetal bovine serum (Hyclone; Cat #SH30070.03). A series
of five different control or experimental test samples was created
to evaluate activin A or various GDFs in combination with Wnt3a or
Compound 34 or Compound 56. These test samples were added in 20
.mu.l aliquots (5.times. concentrated) to appropriately matched
assay wells to yield a final assay volume of 100 .mu.l in each well
at the final assay conditions indicated. In the first set of
control samples, the following conditions were tested: 1) no
additive (i.e. no supplementary growth factor or small molecule);
2) 100 ng/ml activin A (PeproTech; Cat #120-14) in combination with
20 ng/ml Wnt3a (R&D Systems; Cat #1324-WN/CF); 3) 20 ng/ml
Wnt3a alone; 4) Compound 34 alone (2.5 .mu.M) without any growth
factor or small molecule; 5) Compound 56 alone (2.5 .mu.M) without
any growth factor or small molecule. In the second set of test
samples, the following conditions were tested in combination with
100 ng/ml GDF3: 1) no additive (i.e. GDF-3 alone); 2) 20 ng/ml
Wnt3a; 3) 20 ng/ml Wnt3a with Compound 34 (2.5 .mu.M); 4) Compound
34 (2.5 .mu.M); 5) Compound 56 (2.5 .mu.M); and 6) 20 ng/ml Wnt3a
with Compound 56 (2.5 .mu.M). In the third set of test samples,
each of the six conditions was combined with 100 ng/ml GDF-5. In
the fourth set of test samples, each of the six conditions was
combined with 100 ng/ml GDF-8. In the fifth set of test samples,
each of the six conditions was combined with 100 ng/ml GDF-10. In
the sixth set of test samples, each of the six conditions was
combined with 100 ng/ml GDF-11. In the seventh set of test samples,
each of the six conditions was combined with 100 ng/ml GDF-15. On
the third day of assay, all wells for all test samples, received
100 ng/ml Activin A or 100 ng/ml respective GDF growth factor,
without Wnt3a or Compound 34 or Compound 56, diluted into DMEM:F12
medium supplemented with 2% FBS.
[0365] High Content Analysis: At the conclusion of culture, assay
plates were washed once with PBS (Invitrogen; Cat #14190), fixed
with 4% paraformaldehyde (Alexis Biochemical; Cat #ALX-350-011) at
room temperature for 20 minutes, then washed three times with PBS
and permeabilized with 0.5% Triton X-100 (Sigma; Cat #T8760-2) for
20 minutes at room temperature. Cells were washed again three times
with PBS and blocked with 4% chicken serum (Invitrogen; Cat
#16110082) in PBS for 30 minutes at room temperature. Primary
antibody (goat anti-human SOX17; R&D Systems; Cat #AF1924) was
diluted 1:100 in 4% chicken serum and added to each well for two
hours at room temperature. After washing three times with PBS,
Alexa Fluor 488 conjugated secondary antibody (chicken anti-goat
IgG; Invitrogen; Cat #A21467) diluted 1:200 in PBS was added to
each well. To counterstain nuclei, 5 .mu.g/ml Hoechst 33342
(Invitrogen; Cat #H3570) was added for fifteen minutes at room
temperature. Plates were washed once with PBS and left in 100
.mu.l/well PBS for imaging.
[0366] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Images were acquired from 25
fields per well. Measurements for total intensity were obtained
from each well using IN Cell Developer Toolbox 1.7 (GE Healthcare)
software. Segmentation for the nuclei was determined based on
gray-scale levels (baseline range 100-300) and nuclear size.
Averages and standard deviations were calculated for each replicate
data set. Total protein expression was reported as total intensity
or integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria for gray-scale ranges between 200 and 4500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control.
[0367] FIG. 8 shows high content image analysis for SOX17 protein
expression in human embryonic stem cells after four days
differentiation to definitive endoderm. In each case, results are
normalized to the positive control treatment with activin A and
Wnt3a. In FIG. 8A, only the positive control treatment yielded
significant expression of SOX17; treatment with Wnt3a alone or
either Compound 34 or Compound 56 alone failed to induce SOX17
expression. In FIG. 8, panels B through G, normalized SOX17
expression levels are shown for each GDF growth factor substituting
for activin A in the respective treatments. GDF-3 (FIG. 8B) and
GDF-5 (FIG. 8C) induced weak expression of SOX17 and only in test
samples where one of the compounds of the present invention was
present. GDF10 (FIG. 8D), GDF11 (FIG. 8E) and GDF15 (FIG. 8G)
induced significant levels of SOX17 expression, more than observed
with GDF3 or 5 treatments but less than observed that observed with
activin A and Wnt3a treatment. In general, SOX17 expression was
negligible when GDF-10, GDF-11, or GDF-15 was combined with Wnt3a,
but improved in combination with one of the compounds of the
present invention; in particular when combined with Compound 34.
FIG. 8D shows results for treatment groups using GDF-8 where GDF-8
in combination with either Compound 34 or Compound 56 caused a
robust induction of SOX17, exceeding results seen with the activin
A/Wnt3a positive control. In some of these examples, the presence
of Compound 34 or Compound 56 combined with a GDF growth factor
also caused an increase in cell number during differentiation.
[0368] These collective results demonstrate that GDF-8 was superior
to all other GDF family members tested when used in combination
with Compound 34 or Compound 56, and could substitute for activin A
during definitive endoderm differentiation.
Example 14
Formation of Cells Expressing Markers Characteristic of the
Definitive Endoderm Lineage with Other Members of the TGF
Superfamily of Proteins
[0369] It was important to determine if treating human embryonic
stem cells with other TGF superfamily members could facilitate the
formation of cells expressing markers characteristic of the
definitive endoderm lineage. Compound 34 and Wnt3a were tested on
human embryonic stem cells in combination with either TGF.beta.-1,
BMP2, BMP3, or BMP4 to determine the ability of members of the TGF
superfamily members to differentiate human embryonic stem cells
toward cells expressing markers characteristic of the definitive
endoderm lineage. In parallel, two different commercial sources of
GDF-8 were tested with Wnt3a for their ability to differentiate
human embryonic stem cells toward cells expressing markers
characteristic of the definitive endoderm lineage. A positive
control using activin A with Wnt3a was maintained for comparison
purposes.
[0370] Preparation of cells for assay: Stock cultures of human
embryonic stem cells (H1 human embryonic stem cell line) were
maintained in an undifferentiated, pluripotent state on reduced
growth factor MATRIGEL.TM.-(BD Biosciences; Cat #356231)-coated
dishes in MEF conditioned medium with passage on average every four
days. Passage was performed by exposing cell cultures to a solution
of 1 mg/ml dispase (Invitrogen; Cat #17105-041) for 5 to 7 minutes
at 37.degree. C. followed by rinsing the monolayer with MEF
conditioned culture medium and gentle scraping to recover cell
clusters. Clusters were centrifuged at low speed to collect a cell
pellet and remove residual dispase. Cell clusters were split at a
1:3 or 1:4 ratio for routine maintenance culture or a 1:1 ratio for
immediate assay. All human embryonic stem cell lines were
maintained at passage numbers less than 50 and routinely evaluated
for normal karyotype and absence of mycoplasma.
[0371] Cell clusters were evenly resuspended in MEF conditioned
medium supplemented with 8 ng/ml bFGF and seeded onto reduced
growth factor MATRIGEL.TM.-coated 96-well Packard VIEWPLATES
(PerkinElmer; Cat #6005182) in volumes of 0.1 ml/well. Daily
feeding was conducted by aspirating spent culture medium from each
well and replacing with an equal volume of fresh medium. Plates
were maintained at 37.degree. C., 5% CO.sub.2 throughout assay.
[0372] Assay: The assay was initiated by aspirating the culture
medium from each well and adding back aliquots (100 .mu.l) of test
medium. Test conditions were performed in triplicate over a total
three day assay period, feeding on day 1 and day 2 by aspirating
and replacing the medium from each well with fresh test medium.
Various growth factor proteins were obtained for testing as
follows: BMP-2 (R&D Systems; Cat #355-BM); BMP-3 (R&D
Systems; Cat #113-BP); BMP-4 (R&D Systems; Cat #314-BP);
TGF.beta.-1 (R&D Systems; Cat #240-B); GDF-8 (PeproTech; Cat
#120-00); GDF-8 (Shenandoah; Cat #100-22); and activin A
(PeproTech; Cat #120-14). On the first day of assay, each well was
treated with 80 .mu.l of growth medium [RPMI-1640 (Invitrogen; Cat
#22400) containing 2.5% Albumin Bovine Fraction V, Fatty Acid Free
(FAF BSA) (MP Biomedicals, Inc; Cat #152401), and 10 ng/ml bFGF].
In some wells, 25 ng/ml Wnt3a (R&D Systems; Cat #1324-WN/CF)
was added to the growth medium to yield a final assay concentration
of 20 ng/ml. In some wells, activin A was added to the growth
medium to yield a final assay concentration of 100 ng/ml. In some
wells, 3.125 .mu.M Compound 34 was added to the growth medium to
yield a final assay concentration of 2.5 .mu.M. A dose titration of
additional growth factors (5.times. concentrated, diluted in
RPMI-1640) was also added to respective test wells to yield a final
assay volume of 100 .mu.l in each well for all treatment
conditions. On the second day of assay, Wnt3a and Compound 34 were
omitted from assay. All wells received 80 .mu.l of growth medium
[RPMI-1640 containing 2.5% FAF BSA, and 10 ng/ml bFGF] and 20 .mu.l
of respective growth factor dilution (5.times. concentrated,
diluted in RPMI-1640). Comparative controls for this assay
included: 1) no added growth factors; 2) Wnt3a alone; and 3)
activin A with Wnt3a. Each commercial source of GDF-8 was tested in
combination with Wnt3a. Each of the BMP growth factors, as well as
TGF.beta.-1, was tested in combination with Wnt3a, with Compound
34, and with both Wnt3a in combination with Compound 34.
[0373] High Content Analysis: At the conclusion of culture, assay
plates were washed once with PBS (Invitrogen; Cat #14190), fixed
with 4% paraformaldehyde (Alexis Biochemical; Cat #ALX-350-011) at
room temperature for 20 minutes, then washed three times with PBS
and permeabilized with 0.5% Triton X-100 (Sigma; Cat #T8760-2) for
20 minutes at room temperature. Cells were washed again three times
with PBS and blocked with 4% chicken serum (Invitrogen; Cat
#16110082) in PBS for 30 minutes at room temperature. Primary
antibody (goat anti-human SOX17; R&D Systems; Cat #AF1924) was
diluted 1:100 in 4% chicken serum and added to each well for two
hours at room temperature. After washing three times with PBS,
Alexa Fluor 488 conjugated secondary antibody (chicken anti-goat
IgG; Invitrogen; Cat #A21467) diluted 1:200 in PBS was added to
each well. To counterstain nuclei, 5 .mu.g/ml Hoechst 33342
(Invitrogen; Cat #H3570) was added for fifteen minutes at room
temperature. Plates were washed once with PBS and left in 100
.mu.l/well PBS for imaging.
[0374] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Images were acquired from 25
fields per well. Measurements for total intensity were obtained
from each well using IN Cell Developer Toolbox 1.7 (GE Healthcare)
software. Segmentation for the nuclei was determined based on
gray-scale levels (baseline range 100-300) and nuclear size.
Averages and standard deviations were calculated for each replicate
data set. Total protein expression was reported as total intensity
or integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria for gray-scale ranges between 200 and 4500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control.
[0375] FIG. 9 shows high content image analysis for SOX17 protein
expression in human embryonic stem cells after three days
differentiation to definitive endoderm. In each case, results are
normalized to the positive control treatment for activin A with
Wnt3a. The results in FIG. 9A, show that treatment with growth
medium alone, or Wnt3a alone failed to induce SOX17 expression;
only the addition of activin A caused a robust expression of SOX17.
In FIG. 9, panels B and C, results for each of the commercial
sources of GDF-8 are depicted, showing differences in potency
between the two vendors. Although less potent than activin A, there
was significant induction of SOX17 expression in cells treated with
GDF-8 in combination with Wnt3a. In FIG. 9, panels D, E, F and G,
results are shown for definitive endoderm differentiation using
BMP2, BMP3, BMP4, and TGF.beta.-1, incorporating a dose titration
for each growth factor in combination with Wnt3a, or Compound 34,
or both Wnt3a with Compound 34. Although some treatments had a
significant effect on cell numbers at the conclusion of assay (e.g.
BMP2 and BMP4), induction of SOX17 expression resulting from any of
these growth factors and treatment combinations was weak or
negligible compared to the Wnt3a treatment alone.
Example 15
Dose Ranging Studies for Formation of Cells Expressing Markers
Characteristic of the Definitive Endoderm Lineage with a Selection
of the Compounds of the Present Invention
[0376] It was important to know the optimal working concentrations
for Compound 181, Compound 180, Compound 19, Compound 202, Compound
40, and Compound 34 that would mediate the formation of cells
expressing markers characteristic of the definitive endoderm
lineage. In conjunction, side-by-side comparisons were performed
for titrations of each compound in combination with activin A or
GDF-8 in the definitive endoderm assay. Finally, the duration of
exposure for each compound was tested in assay, also in combination
with activin A or GDF-8, adding compound only on the first day of
assay or throughout all three days of definitive endoderm
formation.
[0377] Preparation of cells for assay: Stock cultures of human
embryonic stem cells (H1 human embryonic stem cell line) were
maintained in an undifferentiated, pluripotent state on reduced
growth factor MATRIGEL.TM. (BD Biosciences; Cat #356231)-coated
dishes in MEF conditioned medium supplemented with 8 ng/ml bFGF
(PeproTech Inc.; Cat #100-18B) with passage on average every four
days. Passage was performed by exposing cell cultures to a solution
of 1 mg/ml dispase (Invitrogen; Cat #17105-041) for 5 to 7 minutes
at 37.degree. C. followed by rinsing the monolayer with MEF
conditioned culture medium and gentle scraping to recover cell
clusters. Clusters were centrifuged at low speed to collect a cell
pellet and remove residual dispase. Cell clusters were split at a
1:3 or 1:4 ratio for routine maintenance culture or a 1:1 ratio for
immediate assay. All human embryonic stem cell lines were
maintained at passage numbers less than 50 and routinely evaluated
for normal karyotype and absence of mycoplasma.
[0378] Cell clusters were evenly resuspended in MEF conditioned
medium supplemented with 8 ng/ml bFGF and seeded onto reduced
growth factor MATRIGEL.TM.-coated 96-well Packard VIEWPLATES
(PerkinElmer; Cat #6005182) in volumes of 0.1 ml/well. Daily
feeding was conducted by aspirating spent culture medium from each
well and replacing with an equal volume of fresh medium. Plates
were maintained at 37.degree. C., 5% CO.sub.2 throughout the
duration of assay.
[0379] Assay: Assay was initiated by aspirating the culture medium
from each well and adding back aliquots (100 .mu.l) of test medium.
Test conditions were performed in quadruplicate over a total
four-day assay period, feeding daily by aspirating and replacing
the medium from each well with fresh test medium. Each well was
treated with 80 .mu.l of growth medium [RPMI-1640 (Invitrogen; Cat
#22400) containing 2.5% Albumin Bovine Fraction V, Fatty Acid Free
(FAF BSA) (MP Biomedicals, Inc; Cat #152401), 10 ng/ml bFGF, and
additional growth factors (1.25.times. concentrated)] and 20 .mu.l
of test compound (5.times. concentrated diluted in RPMI-1640) to
yield a final assay volume of 100 ul in each well. Test compounds
in this assay included six of the compounds of the present
invention: Compound 181, Compound 180, Compound 19, Compound 202,
Compound 40, and Compound 34, and a commercial GSK3i inhibitor BIO
(EMD Chemicals, Inc.; Cat #361550). On the first day of assay,
wells were treated with various control or experimental conditions.
Control conditions, with final assay concentrations as indicated,
were as follows: 1) growth medium alone; 2) 20 ng/ml Wnt3a only
R&D Systems; Cat #1324-WN/CF); 3) 100 ng/ml activin A
(PeproTech; Cat #120-14); 4) 100 ng/ml activin A and 20 ng/ml
Wnt3a; 5) 100 ng/ml GDF-8 (R&D Systems, Cat #788-G8); 6) 100
ng/ml GDF-8 and 20 ng/ml Wnt3a. Test compounds were diluted
two-fold in series to yield a concentration range from 78 nM to 10
.mu.M in the final assay. Experimental test samples combined each
individual compound dilution series with 100 ng/ml activin A or 100
ng/ml GDF-8, both treatment sets in the absence of Wnt3a. On the
second and third day of assay, some wells continued to be treated
with 20 ng/ml Wnt3a or diluted test compound in combination with
either activin A or GDF-8. In other wells, activin A or GDF-8
treatment continued on the second and third day of assay, but Wnt3a
or diluted test compound was removed.
[0380] High Content Analysis: At the conclusion of culture, assay
plates were washed once with PBS (Invitrogen; Cat #14190), fixed
with 4% paraformaldehyde (Alexis Biochemical; Cat #ALX-350-011) at
room temperature for 20 minutes, then washed three times with PBS
and permeabilized with 0.5% Triton X-100 (Sigma; Cat #T8760-2) for
20 minutes at room temperature. Cells were washed again three times
with PBS and blocked with 4% chicken serum (Invitrogen; Cat
#16110082) in PBS for 30 minutes at room temperature. Primary
antibody (goat anti-human SOX17; R&D Systems; Cat #AF1924) was
diluted 1:100 in 4% chicken serum and added to each well for two
hours at room temperature. After washing three times with PBS,
Alexa Fluor 488 conjugated secondary antibody (chicken anti-goat
IgG; Invitrogen; Cat #A21467) diluted 1:200 in PBS was added to
each well. To counterstain nuclei, 5 .mu.g/ml Hoechst 33342
(Invitrogen; Cat #H3570) was added for fifteen minutes at room
temperature. Plates were washed once with PBS and left in 100
.mu.l/well PBS for imaging.
[0381] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Images were acquired from 25
fields per well. Measurements for total intensity were obtained
from each well using IN Cell Developer Toolbox 1.7 (GE Healthcare)
software. Segmentation for the nuclei was determined based on
gray-scale levels (baseline range 100-300) and nuclear size.
Averages and standard deviations were calculated for each replicate
data set. Total protein expression was reported as total intensity
or integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria for gray-scale ranges between 200 and 4500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control.
[0382] Results: High content analysis results are shown for SOX17
expression in FIGS. 10-14 and resulting cell number at the
conclusion of assay in FIGS. 15-19. In FIG. 10, results are shown
for SOX17 expression resulting from control treatments using
activin A or GDF-8, either alone or in combination with Wnt3a.
Activin A treatments resulted in significantly higher SOX17
expression than was observed with GDF-8 treatment. Similarly, as
seen in FIG. 15, activin A treatment resulted in a higher number of
cells at the conclusion of assay than was seen with GDF-8
treatment, regardless of whether Wnt3a was present for one or three
days during assay. Adding any of Compound 181, Compound 180,
Compound 19, Compound 202, Compound 40, or Compound 34 with activin
A treatment did not enhance SOX 17 expression (FIGS. 11-12) or
increase cell numbers (FIGS. 17-18), regardless of whether the
compound was present for one day at the initiation of assay or
three days throughout the duration of assay. However, treatment
with either Compound 181, Compound 180, Compound 19, Compound 202,
Compound 40, or Compound 34 in combination with GDF-8 significantly
improved SOX17 expression (FIGS. 13-14) and also enhanced cell
numbers at the end of assay (FIGS. 18-19). When either Compound
181, Compound 180, Compound 19, Compound 202, Compound 40, or
Compound 34and GDF-8 were used in combination, the improvements to
SOX17 expression and cell number in many cases were equivalent to
results observed with activin A treatment. Improved differentiation
in combination with GDF-8 was apparent in a dose titration effect
for many of the compounds, although toxicity was sometimes observed
at the highest concentrations. In most cases, optimal beneficial
effects from treatment with the compound and GDF-8 were apparent
with only one day of compound exposure at the initiation of assay.
In some cases, presence of the compound throughout the duration of
assay had no detrimental effect or had a slight beneficial effect.
From these collective results an optimal working concentration
range for each compound in combination with GDF8 treatment was
determined. Results were compound specific, generally in the 1-10
.mu.M range as tested in this assay.
Example 16
Cells Expressing Markers Characteristic of the Definitive Endoderm
Lineage that were Formed Without According to the Methods of the
Present Invention are able to Further Differentiate into Cells
Expressing Markers Characteristic of the Pancreatic Endocrine
Lineage
[0383] Additional small molecules were tested in combination with
GDF-8 for definitive endoderm differentiation. These included a
commercial inhibitor of GSK3 as well as compounds of the present
invention. A step-wise differentiation protocol was applied to
cells treated with GDF-8 in combination with various small
molecules. The efficacy of differentiation was determined by gene
expression for biomarkers representative the pancreatic endoderm,
or pancreatic endocrine lineages. A parallel control sample of
cells treated with activin A and Wnt3a was maintained for
comparison purposes throughout the step-wise differentiation
process.
[0384] Preparation of cells for assay: Stock cultures of human
embryonic stem cells (H1 human embryonic stem cell line) were
maintained in an undifferentiated, pluripotent state on reduced
growth factor MATRIGEL.TM. (BD Biosciences; Cat #356231)-coated
dishes in MEF conditioned medium with passage on average every four
days. Passage was performed by exposing cell cultures to a solution
of 1 mg/ml dispase (Invitrogen, Cat #17105-041) for 5 to 7 minutes
at 37.degree. C. followed by rinsing the monolayer with MEF
conditioned culture medium and gentle scraping to recover cell
clusters. Clusters were centrifuged at low speed to collect a cell
pellet and remove residual dispase. Cell clusters were split at a
1:3 or 1:4 ratio for routine maintenance culture or a 1:1 ratio for
immediate assay. All human embryonic stem cell lines were
maintained at passage numbers less than 50 and routinely evaluated
for normal karyotype and absence of mycoplasma.
[0385] Cell clusters were evenly resuspended in MEF conditioned
medium supplemented with 8 ng/ml bFGF and plated onto reduced
growth factor MATRIGEL-coated 24-well, black wall culture plates
(Arctic White; Cat #AWLS-303012) in volumes of 0.5 ml/well. Daily
feeding was conducted by aspirating spent culture medium from each
well and replacing with an equal volume of fresh medium. Plates
were maintained at 37.degree. C., 5% CO.sub.2 throughout the
duration of assay.
[0386] Assay: The assay was initiated by aspirating the culture
medium from each well and adding back an aliquot (0.5 ml) of test
medium. Test conditions for the first step of differentiation were
conducted over a three-day period, feeding daily by aspirating and
replacing the medium from each well with fresh test medium. On the
first day of assay, 100 ng/ml activin A (PeproTech; Cat #120-14) or
100 ng/ml GDF-8 (R&D Systems, Cat #788-G8) was added to
respective assay wells where each growth factor was diluted into
RPMI-1640 medium (Invitrogen; Cat #22400) with 2% Albumin Bovine
Fraction V, Fatty Acid Free (FAF BSA) (Proliant Inc. Cat #: SKU
68700), and 20 ng/ml Wnt3a (R&D Systems; Cat #1324-WN/CF). On
the second day of assay, 100 ng/ml activin A or 100 ng/ml GDF-8 was
diluted into RPMI-1640 medium supplemented with 2% FAF BSA without
Wnt3a. In some test samples using GDF-8, Wnt3a was replaced with a
small molecule compound, added only on the first day of definitive
endoderm differentiation. These small molecules included Compound
19 (2.5 .mu.M in assay), Compound 202 (2.5 .mu.M in assay),
Compound 40 (2.5 .mu.M in assay), or a commercially available GSK3
inhibitor BIO (0.5 .mu.M in assay) (EMD Chemicals, Inc.; Cat
#361550). At the conclusion of the first step of differentiation,
cells from some wells were harvested for flow cytometry analysis to
evaluate levels of CXCR4, a marker of definitive endoderm
formation. Additional wells were harvested for RT-PCR analysis to
measure other markers of differentiation.
[0387] At the conclusion of the first step of definitive endoderm
differentiation, replicate sets of parallel wells from each
treatment group were subjected to further step-wise
differentiation. It is important to note that after the first
differentiation step, all wells undergoing subsequent culture and
differentiation received the same treatment. The protocol for this
continuing differentiation is described below.
[0388] Step 2 of the differentiation protocol was carried out over
two days. Cells were fed daily by aspirating the medium from each
well and replacing with a fresh aliquot (0.5 ml) of DMEM:F12 medium
(Invitrogen; Cat #11330-032) containing 2% FAF BSA, 50 ng/ml FGF7
(PeproTech; Cat #100-19), and 250 nM cyclopamine-KAAD (Calbiochem;
Cat #239804).
[0389] Step 3 of the differentiation protocol was carried out over
seven days. Cells were fed daily by aspirating medium from each
well and replacing with a fresh aliquot (0.5 ml) of DMEM-high
glucose (Invitrogen; Cat #10569) supplemented with 0.1% Albumax
(Invitrogen; Cat #11020-021), 0.5.times.
Insulin-Transferrin-Selenium (ITS-X; Invitrogen; Cat #51500056), 50
ng/ml FGF7, 100 ng/ml Noggin (R&D Systems; Cat #3344-NG), 250
nM KAAD-cyclopamine, and 2 .mu.M all-trans retinoic acid (RA)
(Sigma-Aldrich; Cat #R2625). At the conclusion of the third step of
differentiation, cells from some wells were harvested for analysis
by RT-PCR to measure markers of differentiation. Other culture
wells were subjected to high content image analysis for protein
expression levels of Pdx1, and Cdx2.
[0390] Step 4 of the differentiation protocol was carried out over
three days. Cells were fed daily by aspirating the medium from each
well and replacing with a fresh aliquot (0.5 ml) of DMEM-high
glucose supplemented with 0.1% Albumax, 0.5.times.
Insulin-Transferrin-Selenium, 100 ng/ml Noggin, and 1 .mu.M Alk 5
inhibitor (Axxora; Cat #ALX-270-445). At the conclusion of the
fourth step of differentiation, cells from some wells were
harvested for analysis by RT-PCR to measure markers of
differentiation. Other culture wells were subjected to high content
image analysis for protein expression levels of Pdx1.
[0391] Step 5 of the differentiation protocol was carried out over
seven days in DMEM-high glucose with 0.1% Albumax, 0.5.times.
Insulin-Transferrin-Selenium, and 1 .mu.M Alk 5 inhibitor. Medium
in each well was aspirated and replaced with a fresh aliquot (0.5
ml) on all days. At the conclusion of the fifth step of
differentiation, cells from some wells were harvested for analysis
by RT-PCR to measure markers of differentiation. Other culture
wells were subjected to high content image analysis for protein
expression levels of insulin and glucagon.
[0392] FACS Analysis: Cells for FACS analysis were blocked in a 1:5
solution of 0.5% human gamma-globulin (Sigma; Cat #G-4386) in PBS
(Invitrogen; Cat #14040-133): BD FACS staining buffer--BSA (BD; Cat
#554657) for 15 minutes at 4.degree. C. Cells were then stained
with antibodies for CD9 PE (BD; Cat #555372), CD99 PE (Caltag; Cat
#MHCD9904) and CXCR4 APC (R&D Systems; Cat #FAB173A) for 30
minutes at 4.degree. C. After a series of washes in BD FACS
staining buffer, the cells were stained for viability with 7-AAD
(BD; Cat #559925) and run on a BD FACSArray. A mouse IgG1K Isotype
control antibody for both PE and APC was used to gate percent
positive cells.
[0393] RT-PCR Analysis: RNA samples were purified by binding to a
silica-gel membrane (Rneasy Mini Kit, Qiagen, Calif.) in the
presence of an ethanol-containing, high-salt buffer followed by
washing to remove contaminants. The RNA was further purified using
a TURBO DNA-free kit (Ambion, INC), and high-quality RNA was then
eluted in water. Yield and purity were assessed by A260 and A280
readings on a spectrophotometer. CDNA copies were made from
purified RNA using an ABI (ABI, CA) high capacity cDNA archive
kit.
[0394] Unless otherwise stated, all reagents were purchased from
Applied Biosystems. Real-time PCR reactions were performed using
the ABI PRISM.RTM. 7900 Sequence Detection System. TAQMAN.RTM.
UNIVERSAL PCR MASTER MIX.RTM. (ABI, CA) was used with 20 ng of
reverse transcribed RNA in a total reaction volume of 20 .mu.l.
Each cDNA sample was run in duplicate to correct for pipetting
errors. Primers and FAM-labeled TAQMAN.RTM. probes were used at
concentrations of 200 nM. The level of expression for each target
gene was normalized using a human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) endogenous control previously developed by
Applied Biosystems. Primer and probe sets are listed in Table 12.
After an initial incubation at 50.degree. C. for 2 min followed by
95.degree. C. for 10 min, samples were cycled 40 times in two
stages--a denaturation step at 95.degree. C. for 15 sec followed by
an annealing/extension step at 60.degree. C. for 1 min. Data
analysis was carried out using GENEAMP.RTM.7000 Sequence Detection
System software. For each primer/probe set, a Ct value was
determined as the cycle number at which the fluorescence intensity
reached a specific value in the middle of the exponential region of
amplification. Relative gene expression levels were calculated
using the comparative Ct method. Briefly, for each cDNA sample, the
endogenous control Ct value was subtracted from the gene of
interest Ct to give the delta Ct value (.DELTA.Ct). The normalized
amount of target was calculated as 2-.DELTA.Ct, assuming
amplification to be 100% efficiency. Final data were expressed
relative to a calibrator sample.
[0395] High Content Analysis: At the conclusion of culture, assay
plates were washed once with PBS (Invitrogen; Cat #14190), fixed
with 4% paraformaldehyde (Alexis Biochemical; Cat #ALX-350-011) at
room temperature for 20 minutes, then washed three times with PBS
and permeabilized with 0.5% Triton X-100 (Sigma; Cat #T8760-2) for
20 minutes at room temperature. Cells were washed again three times
with PBS and blocked with 4% chicken serum (Invitrogen; Cat
#16110082) in PBS for 30 minutes at room temperature. Primary
antibody (goat anti-human SOX17; R&D Systems; Cat #AF1924) was
diluted 1:100 in 4% chicken serum and added to each well for two
hours at room temperature. After washing three times with PBS,
Alexa Fluor 488 conjugated secondary antibody (chicken anti-goat
IgG; Invitrogen; Cat #A21467) diluted 1:200 in PBS was added to
each well. To counterstain nuclei, 5 .mu.g/ml Hoechst 33342
(Invitrogen; Cat #H3570) was added for fifteen minutes at room
temperature. Plates were washed once with PBS and left in 100
.mu.l/well PBS for imaging. Other primary antibodies used for
analysis included 1:200 dilution rabbit anti-human insulin (Cell
Signaling; Cat #C27C9), and 1:1500 dilution mouse anti-human
glucagon (Sigma-Aldrich; Cat #G2654). Secondary antibodies used for
analysis included 1:1000 dilution Alexa Fluor 647 chicken
anti-rabbit IgG (Invitrogen; Cat #A21443), and 1:1000 dilution
Alexa Fluor 488 chicken anti-mouse IgG (Invitrogen; Cat
#A21200).
[0396] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Images were acquired from 25
fields per well. Measurements for total intensity were obtained
from each well using IN Cell Developer Toolbox 1.7 (GE Healthcare)
software. Segmentation for the nuclei was determined based on
gray-scale levels (baseline range 100-300) and nuclear size.
Averages and standard deviations were calculated for each replicate
data set. Total protein expression was reported as total intensity
or integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria for gray-scale ranges between 200 and 4500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control.
[0397] PCR results for representative differentiation markers are
shown in Table 14 for cells harvested from each step of
differentiation. Samples treated with GDF-8 and Wnt3a or with GDF-8
and a small molecule showed similar expression levels of markers
associated with endodermal and endocrine differentiation.
[0398] FIG. 20, panel A shows FACS analysis for the definitive
endoderm marker, CXCR4, after the first step of differentiation.
Treatment of human embryonic stem cells with GDF-8 and Wnt3a
yielded a similar percentage of CXCR4 positive cells compared to
treatment with activin A and Wnt3a. Treatment of human embryonic
stem cells with GDF-8 and a compound of the present invention
(Compound 19, Compound 202, Compound 40, or GSK3 inhibitor 1.times.
BIO) also yielded an equivalent or slightly higher percentage of
CXC4 positive cells. FIG. 20, panel B shows high content image
analysis for normalized SOX17 protein expression in human embryonic
stem cells after three days differentiation to definitive endoderm.
In some cases, treatment with GDF-8 resulted in a lower cell number
at the conclusion of the first step of differentiation. However,
GDF-8 treatment in combination with Wnt3a or with the small
molecule inhibitors clearly induced expression of SOX17, a marker
of definitive endoderm. In one instance, treatment with GDF-8 and
Compound 40 yielded cell numbers in culture and SOX17 expression
equivalent to treatment with activin A and Wnt3a.
[0399] FIG. 20, panel C shows high content image analysis for
relative cell numbers recovered from cultures treated through
differentiation step 5. As observed earlier at the end of step 1,
some treatments caused a drop in cell recovery relative to
treatment with activin A and Wnt3a. This decrease in cell number
was seen specifically with treatment groups using GDF-8 with GSK3
inhibitor BIO and also using GDF-8 with Compound 19. Additional
GDF-8 treatment groups had cell recoveries similar to treatment
with activin A and Wnt3a. In FIG. 20, panels D-F, normalized
protein levels of insulin and glucagon are shown, along with their
respective ratio for each treatment group. Similar levels of
insulin and glucagon could be obtained with each of the GDF-8
treatments relative to treatment with activin A and Wnt3a,
demonstrating that GDF-8, in combination with Wnt3a or a small
molecule, can substitute for activin A during definitive endoderm
differentiation and subsequent pancreatic endoderm and endocrine
differentiation.
Example 17
Cells Expressing Markers Characteristic of the Definitive Endoderm
Lineage that were Formed Using GDF-8 and a Compound of the Present
Invention are able to Further Differentiate into Cells Expressing
Markers Characteristic of the Pancreatic Endocrine Lineage
[0400] Additional small molecules were tested in combination with
GDF-8 and activin A for definitive endoderm differentiation. These
included a commercial inhibitor of GSK3 as well as the compounds of
the present invention. A step-wise differentiation protocol was
applied to cells treated with GDF-8 in combination with various
small molecules. The efficacy of differentiation was determined by
gene expression for biomarkers representative of the pancreatic
endoderm and pancreatic endocrine lineages. A parallel control
sample of cells treated with activin A and Wnt3a was maintained for
comparison purposes throughout the step-wise differentiation
process.
[0401] Preparation of cells for assay. Stock cultures of human
embryonic stem cells (Hi human embryonic stem cell line) were
maintained in an undifferentiated, pluripotent state on reduced
growth factor MATRIGEL.TM. (BD Biosciences; Cat #356231)-coated
dishes in MEF conditioned medium supplemented with 8 ng/ml bFGF
(PeproTech Inc.; Cat #100-18B) with passage on average every four
days. Passage was performed by exposing cell cultures to a solution
of 1 mg/ml dispase (Invitrogen; Cat #17105-041) for 5 to 7 minutes
at 37.degree. C. followed by rinsing the monolayer with MEF
conditioned culture medium and gentle scraping to recover cell
clusters. Clusters were centrifuged at low speed to collect a cell
pellet and remove residual dispase. Cell clusters were split at a
1:3 or 1:4 ratio for routine maintenance culture or a 1:1 ratio for
immediate assay. All human ES cell lines were maintained at passage
numbers less than 50 and routinely evaluated for normal karyotype
and absence of mycoplasma.
[0402] Cell clusters were evenly resuspended in MEF conditioned
medium supplemented with 8 ng/ml bFGF and plated onto reduced
growth factor MATRIGEL.TM. coated 24-well, black wall culture
plates (Arctic White; Cat #AWLS-303012) in volumes of 0.5 ml/well.
Daily feeding was conducted by aspirating spent culture medium from
each well and replacing with an equal volume of fresh medium.
Plates were maintained at 37.degree. C., 5% CO.sub.2 throughout
assay.
[0403] Assay. The assay was initiated by aspirating the culture
medium from each well and adding back an aliquot (0.5 ml) of test
medium. Test conditions for the first step of differentiation were
conducted over a three-day period, feeding daily by aspirating and
replacing the medium from each well with fresh test medium. On the
first day of assay, 100 ng/ml activin A (PeproTech; Cat #120-14) or
100 ng/ml GDF-8 (R&D Systems, Cat #788-G8) was added to
respective assay wells where each growth factor was diluted into
RPMI-1640 medium (Invitrogen; Cat #: 22400) with 2% Albumin Bovine
Fraction V, Fatty Acid Free (FAF BSA) (MP Biomedicals, Inc; Cat
#152401). In some samples, 20 ng/ml Wnt3a (R&D Systems; Cat
#1324-WN/CF) was also included. On the second day of assay, 100
ng/ml activin A or 100 ng/ml GDF-8 was diluted into RPMI-1640
medium supplemented with 2% FAF BSA, omitting Wnt3a from all
samples. In some test samples using GDF-8, Wnt3a was replaced with
a given concentration of small molecule compound, added only on the
first day of definitive endoderm differentiation. These small
molecules included: Compound 181 (1.25 .mu.M in assay), Compound
180 (2.5 .mu.M in assay), Compound 19 (10 .mu.M in assay), Compound
202 (2.5 .mu.M in assay), Compound 40 (5 .mu.M in assay), Compound
34 (2.5 .mu.M in assay), Compound 206 (2.5 .mu.M in assay), and a
commercially available GSK3 inhibitor 1.times. BIO (10 .mu.M in
assay) (EMD Chemicals, Inc.; Cat #361550). At the conclusion of the
first step of differentiation, cells from some wells were harvested
for flow cytometry analysis to evaluate levels of CXCR4, a marker
of definitive endoderm formation. Additional wells were harvested
for RT-PCR analysis to measure other markers of
differentiation.
[0404] At the conclusion of the first step of definitive endoderm
differentiation, replicate sets of parallel wells from each
treatment group were subjected to further step-wise
differentiation. It is important to note that after the first
differentiation step, all wells undergoing subsequent culture and
differentiation received the same treatment. The protocol for this
continuing differentiation is described below.
[0405] Step 2 of the differentiation protocol was carried out over
two days. Cells were fed daily by aspirating the medium from each
well and replacing with a fresh aliquot (0.5 ml) of DMEM:F12 medium
(Invitrogen; Cat #11330-032) containing 2% FAF BSA, 50 ng/ml FGF7
(PeproTech; Cat #100-19), and 250 nM cyclopamine-KAAD (Calbiochem;
Cat #239804).
[0406] Step 3 of the differentiation protocol was carried out over
four days. Cells were fed daily by aspirating medium from each well
and replacing with a fresh aliquot (0.5 ml) of DMEM-high glucose
(Invitrogen; Cat #10569) supplemented with 0.1% Albumax
(Invitrogen; Cat #: 11020-021), 0.5x Insulin-Transferrin-Selenium
(ITS-X; Invitrogen; Cat #51500056), 50 ng/ml FGF7, 100 ng/ml Noggin
(R&D Systems; Cat #3344-NG), 250 nM KAAD-cyclopamine, and 2
.mu.M all-trans retinoic acid (RA) (Sigma-Aldrich; Cat #R2625). At
the conclusion of the third step of differentiation, cells from
some wells were harvested for analysis by RT-PCR to measure markers
of differentiation.
[0407] Step 4 of the differentiation protocol was carried out over
three days. Cells were fed daily by aspirating the medium from each
well and replacing with a fresh aliquot (0.5 ml) of DMEM-high
glucose supplemented with 0.1% Albumax, 0.5.times.
Insulin-Transferrin-Selenium, 100 ng/ml Noggin, and 1 .mu.M Alk 5
inhibitor (Axxora; Cat #ALX-270-445). At the conclusion of the
fourth step of differentiation, cells from some wells were
harvested for analysis by RT-PCR to measure markers of
differentiation.
[0408] Step 5 of the differentiation protocol was carried out over
seven days in DMEM-high glucose with 0.1% Albumax, 0.5.times.
Insulin-Transferrin-Selenium, and 1 .mu.M Alk 5 inhibitor. Medium
in each well was aspirated and replaced with a fresh aliquot (0.5
ml) on all days. At the conclusion of the fifth step of
differentiation, cells from some wells were harvested for analysis
by RT-PCR to measure markers of differentiation. Other culture
wells were subjected to high content image analysis for protein
expression levels of insulin and glucagon.
[0409] FACS Analysis: Cells for FACS analysis were blocked in a 1:5
solution of 0.5% human gamma-globulin (Sigma; Cat #G-4386) in PBS
(Invitrogen; Cat #14040-133): BD FACS staining buffer--BSA (BD; Cat
#554657) for 15 minutes at 4.degree. C. Cells were then stained
with antibodies for CD9 PE (BD; Cat #555372), CD99 PE (Caltag; Cat
#MHCD9904) and CXCR4 APC (R&D Systems; Cat #FAB173A) for 30
minutes at 4.degree. C. After a series of washes in BD FACS
staining buffer, the cells were stained for viability with 7-AAD
(BD; Cat #559925) and run on a BD FACSArray. A mouse IgG1K Isotype
control antibody for both PE and APC was used to gate percent
positive cells.
[0410] RT-PCR Analysis: RNA samples were purified by binding to a
silica-gel membrane (Rneasy Mini Kit, Qiagen, Calif.) in the
presence of an ethanol-containing, high-salt buffer followed by
washing to remove contaminants. The RNA was further purified using
a TURBO DNA-free kit (Ambion, INC), and high-quality RNA was then
eluted in water. Yield and purity were assessed by A260 and A280
readings on a spectrophotometer. CDNA copies were made from
purified RNA using an ABI (ABI, CA) high capacity cDNA archive
kit.
[0411] Unless otherwise stated, all reagents were purchased from
Applied Biosystems. Real-time PCR reactions were performed using
the ABI PRISM.RTM. 7900 Sequence Detection System. TAQMAN.RTM.
UNIVERSAL PCR MASTER MIX.RTM. (ABI, CA) was used with 20 ng of
reverse transcribed RNA in a total reaction volume of 20 .mu.l.
Each cDNA sample was run in duplicate to correct for pipetting
errors. Primers and FAM-labeled TAQMAN.RTM. probes were used at
concentrations of 200 nM. The level of expression for each target
gene was normalized using a human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) endogenous control previously developed by
Applied Biosystems. Primer and probe sets are listed in Table 12.
After an initial incubation at 50.degree. C. for 2 min followed by
95.degree. C. for 10 min, samples were cycled 40 times in two
stages--a denaturation step at 95.degree. C. for 15 sec followed by
an annealing/extension step at 60.degree. C. for 1 min. Data
analysis was carried out using GENEAMP.RTM.7000 Sequence Detection
System software. For each primer/probe set, a Ct value was
determined as the cycle number at which the fluorescence intensity
reached a specific value in the middle of the exponential region of
amplification. Relative gene expression levels were calculated
using the comparative Ct method. Briefly, for each cDNA sample, the
endogenous control Ct value was subtracted from the gene of
interest Ct to give the delta Ct value (.DELTA.Ct). The normalized
amount of target was calculated as 2-.DELTA.Ct, assuming
amplification to be 100% efficiency. Final data were expressed
relative to a calibrator sample.
[0412] High Content Analysis. At the conclusion of culture, assay
plates were washed once with PBS (Invitrogen; Cat #14190), fixed
with 4% paraformaldehyde (Alexis Biochemical; Cat #ALX-350-011) at
room temperature for 20 minutes, then washed three times with PBS
and permeabilized with 0.5% Triton X-100 (Sigma; Cat #T8760-2) for
20 minutes at room temperature. Cells were washed again three times
with PBS and blocked with 4% chicken serum (Invitrogen; Cat
#16110082) in PBS for 30 minutes at room temperature. Primary
antibody (goat anti-human SOX17; R&D Systems; Cat #AF1924) was
diluted 1:100 in 4% chicken serum and added to each well for two
hours at room temperature. After washing three times with PBS,
Alexa Fluor 488 conjugated secondary antibody (chicken anti-goat
IgG; Invitrogen; Cat #A21467) diluted 1:200 in PBS was added to
each well. To counterstain nuclei, 5 .mu.g/ml Hoechst 33342
(Invitrogen; Cat #H3570) was added for fifteen minutes at room
temperature. Plates were washed once with PBS and left in 100
.mu.l/well PBS for imaging. Other primary antibodies used for
analysis included 1:100 dilution mouse anti-human CDX2 (Invitrogen;
Cat #397800), 1:100 dilution goat anti-human Pdx1 (Santa Cruz
Biotechnology; Cat #SC-14664), 1:200 dilution rabbit anti-human
insulin (Cell Signaling; Cat #C27C9), and 1:1500 dilution mouse
anti-human glucagon (Sigma-Aldrich; Cat #G2654). Secondary
antibodies used for analysis included 1:400 dilution Alexa Fluor
647 chicken anti-mouse IgG (Invitrogen; Cat #A-21463), 1:200
dilution Alexa Fluor 488 donkey anti-goat IgG (Invitrogen; Cat #A
11055), 1:1000 dilution Alexa Fluor 647 chicken anti-rabbit IgG
(Invitrogen; Cat #A21443), and 1:1000 dilution Alexa Fluor 488
chicken anti-mouse IgG (Invitrogen; Cat #A21200).
[0413] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Images were acquired from 25
fields per well. Measurements for total intensity were obtained
from each well using IN Cell Developer Toolbox 1.7 (GE Healthcare)
software. Segmentation for the nuclei was determined based on
gray-scale levels (baseline range 100-300) and nuclear size.
Averages and standard deviations were calculated for each replicate
data set. Total protein expression was reported as total intensity
or integrated intensity, defined as total fluorescence of the cell
multiplied by the area of the cell. Background was eliminated based
on acceptance criteria for gray-scale ranges between 200 and 4500.
Total intensity data were normalized by dividing total intensities
for each well by the average total intensity for the positive
control.
[0414] Results. Results for representative differentiation markers
are shown in FIG. 21 and Table 15 for cells harvested from each
step of differentiation. In FIGS. 21A and B, flow cytometric
results for CXCR4 are shown for various treatments during the first
step of definitive endoderm differentiation. FIG. 21A shows the
effects on CXCR4 expression from treatment with various compounds
in combination with activin A. FIG. 21B shows the effects on CXCR4
from treatment with various compounds in combination with GDF-8.
Compounds of the present invention in combination with activin A
did not enhance CXCR4 expression. However, all of the compounds of
the present invention tested in this Example enhanced CXCR4
expression in combination with GDF-8.
[0415] In FIGS. 21C and 21D, normalized RT-PCR values for various
differentiation markers at the end of step one of differentiation
are shown for treatments applied during step one of the protocol,
using selected compounds of the present invention in combination
with activin A (FIG. 21C) or in combination with GDF-8 (FIG. 21D).
Similar normalized RT-PCR values were evaluated at the conclusion
of step three of the differentiation protocol (FIGS. 21E and 21F)
and at the end of step four of the differentiation protocol (FIGS.
21G and 21H) and at the end of step 5 of the differentiation
protocol (FIGS. 21I and 21J). Treatments during differentiation
step 1, which combined a compound of the present invention with
GDF-8, had improved expression of various endoderm and pancreatic
markers relative to GDF-8 treatment alone (FIGS. 21F and 21H and
21J). Treatments combining compounds of the present invention with
activin A had minimal or no improvement in expression markers
relative to treatment with activin A alone or activin A with Wnt3a
(FIGS. 21E, and 21G and 21I). Table 15 summarizes comparative CT
values for additional gene markers at the end of each
differentiation step, comparing treatments during step one that
combined activin A or GDF-8 with or without a compound of the
present invention. At the conclusion of step five of
differentiation, high content analysis was performed to measure
cell numbers (FIG. 21K and 21M) and protein expression of insulin
and glucagon (FIGS. 21L and 21N). Treatment with GDF-8 during the
first step of differentiation, alone or in combination with a
compound of the present invention, resulted in insulin and glucagon
expression at the conclusion of step five of differentiation,
demonstrating that GDF-8 was able to substitute for activin A
during the initiation of definitive endoderm formation and
subsequently led to pancreatic hormonal cells. Collectively, these
data show that addition of any of the respective small molecules
had minimal effects on differentiation markers for treatments in
combination with activin A. However, addition of a small molecule
in combination with GDF8 treatment had significant improved effects
on immediate definitive endoderm differentiation at the conclusion
of step 1 differentiation and also on downstream differentiation
markers at the conclusion of steps 3, 4, and 5. Variability was
observed within the panel of small molecules, perhaps attributable
to the concentration of compound used in assay and/or mechanism of
action.
Example 18
Cells Expressing Markers Characteristic of the Definitive Endoderm
Lineage that were Formed Using GDF-8 and a Compound of the Present
Invention are able to Release C-Peptide Following Transplantation
into a Rodent
[0416] It was important to determine whether cells expressing
markers characteristic of the pancreatic endoderm lineage generated
in vitro by treatment with GDF-8 and a small molecule could produce
functional endocrine cells in vivo. An in vivo transplant study was
done to compare cells differentiated by treatment with activin A
and Wnt3a versus treatment with GDF-8 and small molecule
compounds.
[0417] Preparation of cells. Clusters of H1 human embryonic stem
cells were grown on reduced growth factor MATRIGEL.TM. (Invitrogen;
Cat #356231)-coated tissue culture plastic with passage on average
every four days. MEF conditioned medium supplemented with 8 ng/ml
bFGF was used for initial seeding and expansion. All human ES cell
lines were maintained at passage numbers less than 50 and routinely
evaluated for normal karyotype and absence of mycoplasma
contamination.
[0418] Cell passage was performed by exposing cell cultures to a
solution of 1 mg/ml dispase (Invitrogen; Cat #17105-041) for 5 to 7
minutes at 37.degree. C. followed by rinsing the cell monolayer
with MEF conditioned medium and gentle scraping to recover cell
clusters. Cell clusters were centrifuged at low speed in MEF
conditioned medium to remove residual dispase and then evenly
resuspended in MEF conditioned medium supplemented with 8 ng/ml
bFGF (PeproTech Inc.; Cat #100-18B) for seeding on reduced growth
factor MATRIGEL (BD Biosciences; Cat #356231)-coated 6-well plates
(Nunc; Cat #140685) at a 1:3 ratio using volumes of 2.5 ml/well.
Daily feeding was conducted by aspirating spent culture medium from
each well and replacing with an equal volume of fresh medium.
Plates were maintained at 37.degree. C., 5% CO.sub.2 throughout the
time in culture.
[0419] Cell differentiation. The differentiation process was
started three days after the cells were seeded onto 6-well plates
coated with reduced growth factor MATRIGEL.TM.. A four-step
protocol was used for the in vitro differentiation of H1 human
embryonic stem cells to cells expressing markers characteristic of
the pancreatic endoderm lineage. Step 1 was conducted over three
days to generate definitive endoderm cells. On the first day of
step 1, differentiation was initiated by aspirating spent culture
medium and adding an equal volume of RPMI-1640 basal medium
(Invitrogen; Cat #22400) with 2% Albumin Bovine Fraction V, Fatty
Acid Free (FAF BSA) (Proliant Biologicals; Cat #SKU 68700) and 8
ng/ml bFGF. In one treatment group, cells were exposed to 100 ng/ml
activin A (PeproTech; Cat #120-14) with 20 ng/ml Wnt3a (R&D
Systems; Cat #1324-WN/CF). In a second treatment group, cells were
exposed to 100 ng/ml GDF-8 (R&D Systems; Cat #788-G8) with 2.5
.mu.M Compound 40. In a third treatment group, cells were exposed
to 100 ng/ml GDF-8 (R&D Systems; Cat #788-G8) with 2.5 .mu.M
Compound 202. On the second and third day of step 1 of
differentiation, cells in all treatment groups were fed with
RPMI-1640 containing 2% FAF BSA, 8 ng/ml bFGF and either 100 ng/ml
activin A (treatment group 1) or 100 ng/ml GDF-8 (treatment groups
2 and 3), without the addition of Wnt3a or a compound of the
present invention. At the end of the third day of culture, one well
from each treatment group was collected for FACS analysis.
[0420] Step 2 of the differentiation protocol was conducted over
three days. Cells for all treatment groups were fed daily with
DMEM:F12 (Invitrogen; Cat #11330-032) supplemented with 2% FAF BSA
and 50 ng/ml FGF7 (PeproTech; Cat #100-19).
[0421] Step 3 of the differentiation protocol was conducted over
four days. Cells for all treatment groups were fed daily with
DMEM-high glucose (Invitrogen; Cat #10569) supplemented with 1% B27
(Invitrogen; Cat #: 17504-044), 50 ng/ml FGF7, 100 ng/ml Noggin
(R&D Systems; Cat #3344-NG), 250 nM KAAD-cyclopamine
(Calbiochem; Cat #239804), and 2 .mu.M all-trans retinoic acid (RA)
(Sigma-Aldrich; Cat #R2625).
[0422] Step 4 of the differentiation protocol was conducted over
three days. Cells for all treatment groups were fed daily for the
first two days with DMEM-high glucose supplemented with 1% B27, 100
ng/ml Noggin, and 1 .mu.M ALK5 inhibitor (Axxora; Cat
#ALX-270-445). On the third day, cells were lifted from the
substratum by using a 20 .mu.l tip (Rainin; Cat #RT-L10F) and a
cell scraper (Corning; Cat #3008), then transferred to a 50 ml
tube. The cells were allowed to sediment by gravity, and the
supernatant was aspirated without disturbing the cell pellet. Cells
were resuspended in DMEM-high glucose supplemented with 1% B27, 100
ng/ml Noggin and 1 .mu.M ALK5 inhibitor, then cultured overnight in
six-well Costar Ultra Low Attachment Microplates (Corning Inc., Cat
#3471). On the following day, cells in suspension culture were
collected and counted. Aliquots of 10.times.10.sup.6 cells/mouse
were used for transplantation. Aliquots of 0.5.times.10.sup.6 cells
were collected for RT-PCR analysis.
[0423] FIG. 22, panel A shows flow cytometric results for
definitive endoderm cells generated at the end of step 1 for each
of the respective treatment groups. Treatment with activin A and
Wnt3a or treatment with GDF-8 and a compound of the present
invention resulted in cells expressing similar levels of CXCR4
(greater than 85%) at the end of step 1, suggesting that an
equivalent definitive endoderm population of cells was derived from
each treatment group.
[0424] Results for RT-PCR analysis for cells from each treatment
group at the conclusion of step 4 of the differentiation protocol
are shown in FIG. 22, panel B. Cells differentiated to pancreatic
endoderm (PE) using Activin A and Wnt3a or using GDF-8 and Compound
40 or using GDF-8 and Compound 202 expressed equivalent levels of
PE markers: CDX2, MAFA, NGN3, NKX6.1, PDX1 and Ptf1 alpha. These
results suggest that the differentiation protocol utilizing GDF-8
and a small molecule was equally effective in creating a pancreatic
endoderm precursor population of cells.
[0425] Transplantation of Human Embryonic Stem Cells Treated
According to the Methods of the Present Invention into Mice. Five
to six-week-old male scid-beige mice
(C.B-Igh-1b/GbmsTac-Prkdc.sup.scid-Lyst.sup.bg N7) were purchased
from Taconic Farms. Mice were housed in microisolator cages with
free access to sterilized food and water. In preparation for
surgery, mice were identified by ear tagging, their body weight was
measured, and their blood glucose was determined using a hand held
glucometer (LifeScan; One Touch). On the day of surgery, mice were
anesthetized with a mixture of isolflurane and oxygen, and the
surgical site was shaved with small animal clippers. Mice were
dosed with 0.1 mg.kg Buprenex subcutaneously pre-operatively. The
surgical site was prepared with successive washes of 70% isopropyl
alcohol, 10% povidone-iodide, and 70% isopropyl alcohol, and a left
lateral incision was made through the skin and muscle layers. The
left kidney was externalized and kept moist with 0.9% sodium
chloride. A 24G.times.3/4'' I.V. catheter was used to penetrate the
kidney capsule, and the needle was removed. The catheter was then
advanced under the kidney capsule to the distal pole of the kidney.
During preoperative preparation of the mice, cells for transplant
were centrifuged in a 1.5 mL microfuge tube, and most of the
supernatant was removed, leaving sufficient medium to collect the
pellet of cells. The cells were collected into a Rainin Pos-D
positive displacement pipette tip, and the pipette was inverted to
allow the cells to settle by gravity. Excess medium was dispensed
leaving a packed cell preparation for transplant. For
transplantation, the Pos-D pipette tip was placed firmly in the hub
of the catheter, and the cells were dispensed from the pipette
through the catheter under the kidney capsule for delivery to the
distal pole of the kidney. The lumen of the catheter was flushed
with a small volume of culture medium to deliver any remaining
cells, and the catheter was withdrawn. The kidney capsule was
sealed with a low temperature cautery, and the kidney was returned
to its original anatomical position. The muscle was closed with
continuous sutures using 5-0 VICRYL sutures, and the skin was
closed with wound clips. The mouse was removed from anesthesia and
allowed to fully recover. Mice were dosed with 1.0 mg.kg Metacam
subcutaneously post-operatively.
[0426] Following transplantation, mice were weighed once per week
and blood glucose was measured twice per week. At various intervals
following transplantation, mice were dosed with 3 g/kg glucose IP,
and blood was drawn 60 minutes following glucose injection via the
retro-orbital sinus into microfuge tubes containing a small amount
of heparin. The blood was centrifuged, and the plasma was placed
into a second microfuge tube, frozen on dry ice, for storage at
-80.degree. C. until the human C-peptide assay was performed. Human
C-peptide levels were determined using the Mercodia/ALPCO
Diagnotics Ultrasensitive C-peptide ELISA according to the
manufacturer's instructions.
[0427] ELISA results for human C-peptide are shown in FIG. 23 for
mice transplanted with cells from each of the respective treatment
groups. No circulating human C-peptide was detected at four weeks
post-transplant for any mice receiving cells from any of the
treatment groups. At 8-weeks post-transplant, detectable C-peptide
was found in one of two mice receiving cells treated with activin A
and Wnt3a; one of three mice receiving cells treated with GDF-8 and
Compound 40; and two of three mice receiving cells treated with
GDF-8 and Compound 202. These results suggest that an equivalent
endocrine precursor cell population could be derived from the
differentiation protocol with GDF-8 and a small molecule and that
the cells further matured in vivo to a glucose responsive, insulin
secreting cell.
Example 19
Cells Expressing Markers Characteristic of the Definitive Endoderm
Lineage that were Formed Using GDF-8 are able to Release C-Peptide
Following Transplantation into a Rodent
[0428] It was important to demonstrate that cells differentiated
with GDF-8 in the absence of activin A could also be further
differentiated to an endocrine cell population capable of secreting
human C-peptide in an in vivo rodent transplant model.
[0429] Preparation of cells. Clusters of H1 human embryonic stem
cells were grown on reduced growth factor MATRIGEL.TM. (Invitrogen;
Cat #356231) -coated tissue culture plastic with passage on average
every four days. MEF conditioned medium supplemented with 8 ng/ml
bFGF was used for initial seeding and expansion. All human ES cell
lines were maintained at passage numbers less than 50 and routinely
evaluated for normal karyotype and absence of mycoplasma
contamination.
[0430] Cell passage was performed by exposing cell cultures to a
solution of 1 mg/ml dispase (Invitrogen; Cat #17105-041) for 5 to 7
minutes at 37.degree. C. followed by rinsing the cell monolayer
with MEF conditioned medium and gentle scraping to recover cell
clusters. Cell clusters were centrifuged at low speed in MEF
conditioned medium to remove residual dispase and then evenly
resuspended in MEF conditioned medium supplemented with 8 ng/ml
bFGF (PeproTech Inc.; Cat #100-18B) for seeding on reduced growth
factor MATRIGEL.TM. (BD Biosciences; Cat #356231)-coated 6-well
plates (Nunc; Cat #140685) at a 1:3 ratio using volumes of 2.5
ml/well. Daily feeding was conducted by aspirating spent culture
medium from each well and replacing with an equal volume of fresh
medium. Plates were maintained at 37.degree. C., 5% CO.sub.2
throughout culture.
[0431] Cell differentiation. The differentiation process was
started three days after the cells were seeded into 6-well plates.
A four-step protocol was used for the in vitro differentiation of
H1 human embryonic stem cells to cells expressing markers
characteristic of the pancreatic endoderm lineage. Step 1 was
conducted over three days to generate cells expressing markers
characteristic of the definitive endoderm lineage. On the first day
of step 1, differentiation was initiated by aspirating spent
culture medium and adding an equal volume of RPMI-1640 basal medium
(Invitrogen; Cat #22400) with 2% Albumin Bovine Fraction V, Fatty
Acid Free (FAF BSA) (Proliant Biologicals; Cat #SKU 68700) and 8
ng/ml bFGF. In one treatment group, duplicate sets of cells were
treated with 100 ng/ml GDF-8 (R&D Systems; Cat #788-G8) and 20
ng/ml Wnt3a (R&D Systems; Cat #1324-WN/CF). In a second
treatment group, duplicate sets of cells were treated with 100
ng/ml GDF-8 and 2.5 .mu.M Compound 40. On the second and third day
of step 1 differentiation, cells in all treatment groups were fed
with RPMI-1640 containing 2% FAF BSA, 8 ng/ml bFGF and 100 ng/ml
GDF-8 but without the addition of Wnt3a or Compound 40. At the end
of the third day of culture, one well from each treatment group was
collected for FACS analysis.
[0432] Step 2 of the differentiation protocol was carried out over
three days. Cells for all treatment groups were fed daily with
DMEM:F12 (Invitrogen; Cat #11330-032) supplemented with 2% FAF BSA
and 50 ng/ml FGF7 (PeproTech; Cat #100-19).
[0433] Step 3 of the differentiation protocol was carried out over
four days. Cells for all treatment groups were fed daily with
DMEM-high glucose (Invitrogen; Cat #10569) supplemented with 1% B27
(Invitrogen; Cat #: 17504-044), 50 ng/ml FGF7, 100 ng/ml Noggin
(R&D Systems; Cat #3344-NG), 250 nM KAAD-cyclopamine
(Calbiochem; Cat #239804), and 2 .mu.M all-trans retinoic acid (RA)
(Sigma-Aldrich; Cat #R2625).
[0434] Step 4 of the differentiation protocol was carried out over
three days. Cells for all treatment groups were fed daily with
DMEM-high glucose supplemented with 1% B27, 100 ng/ml Noggin and 1
.mu.M ALK5 inhibitor (Axxora; Cat #ALX-270-445), and 100 ng/ml
GDF-8 (R&D Systems; Cat #788-G8) during the first two days. On
the third day of step 4, cells were harvested from the 6-well
plates using a 20 .mu.l tip (Rainin; Cat #RT-L10F) and a cell
scraper (Corning; Cat #3008) and transferred to a 50 ml tube. Cells
were allowed to sediment by gravity, and the supernatant was
aspirated without disturbing the cell pellet. Cells were
resuspended in DMEM-high glucose supplemented with 1% B27, 100
ng/ml Noggin, and 1 .mu.M ALK5 inhibitor, then cultured overnight
in six-well Costar Ultra Low Attachment Microplates (Corning Inc.,
Cat #3471). On the following day, cells in suspension culture were
collected and counted. Aliquots of 10.times.10.sup.6 cells/mouse
were used for transplantation. Aliquots of 0.5.times.10.sup.6 cells
were collected for RT-PCR analysis.
[0435] FIG. 24A shows flow cytometric results for definitive
endoderm cells generated at the end of step 1 for each of the
respective treatment groups. Results for treatment with GDF-8 and
Wnt3a or treatment with GDF-8 and Compound 40 expressed similar
levels of CXCR4 at the end of step 1, suggesting that an equivalent
and robust definitive endoderm population of cells resulted from
each treatment group. Duplicate treatment sets were in strong
agreement. Results prior to transplant for RT-PCR analysis at the
conclusion of step 4 of the differentiation protocol are shown in
FIG. 24B. Cells differentiated to pancreatic endoderm (PE) using
GDF-8 and Wnt3a or GDF-8 and Compound 40 expressed equivalent
levels of markers characteristic of the pancreatic endoderm
lineage, such as: CDX2, MafA, Ngn3, NKX6.1, Pdx-1 and Ptf1A. These
results demonstrate that the differentiation protocol utilizing
GDF-8 and Wnt3a or GDF-8 and a compound of the present invention
was effective in creating a pancreatic endoderm precursor
population of cells. The differentiation protocol was conducted in
two independent but identical treatment sets. Results from
duplicate treatment sets were in strong agreement as shown by
RT-PCR analysis.
[0436] Human embryonic stem cell Transplantation into Mice. Five to
six-week-old male scid-beige mice
(C.B-Igh-1bGbmsTac-Prkdc.sup.scid-Lyst.sup.bg N7) were purchased
from Taconic Farms. Mice were housed in microisolator cages with
free access to sterilized food and water. In preparation for
surgery, mice were identified by ear tagging, their body weight was
measured, and their blood glucose was determined using a hand held
glucometer (LifeScan; One Touch). On the day of surgery, mice were
anesthetized with a mixture of isolflurane and oxygen, and the
surgical site was shaved with small animal clippers. Mice were
dosed with 0.1 mg.kg Buprenex subcutaneously pre-operatively. The
surgical site was prepared with successive washes of 70% isopropyl
alcohol, 10% povidone-iodide, and 70% isopropyl alcohol, and a left
lateral incision was made through the skin and muscle layers. The
left kidney was externalized and kept moist with 0.9% sodium
chloride. A 24G.times.3/4'' I.V. catheter was used to penetrate the
kidney capsule, and the needle was removed. The catheter was then
advanced under the kidney capsule to the distal pole of the kidney.
During preoperative preparation of the mice, cells for transplant
were centrifuged in a 1.5 mL microfuge tube, and most of the
supernatant was removed, leaving sufficient medium to collect the
pellet of cells. The cells were collected into a Rainin Pos-D
positive displacement pipette tip, and the pipette was inverted to
allow the cells to settle by gravity. Excess medium was dispensed
leaving a packed cell preparation for transplant. For
transplantation, the Pos-D pipette tip was placed firmly in the hub
of the catheter, and the cells were dispensed from the pipette
through the catheter under the kidney capsule for delivery to the
distal pole of the kidney. The lumen of the catheter was flushed
with a small volume of culture medium to deliver any remaining
cells, and the catheter was withdrawn. The kidney capsule was
sealed with a low temperature cautery, and the kidney was returned
to its original anatomical position. The muscle was closed with
continuous sutures using 5-0 vicryl, and the skin was closed with
wound clips. The mouse was removed from anesthesia and allowed to
fully recover. Mice were dosed with 1.0 mg.kg Metacam
subcutaneously post-operatively.
[0437] Following transplantation, mice were weighed once per week
and blood glucose was measured twice per week. At various intervals
following transplantation, mice were dosed with 3 g/kg glucose IP,
and blood was drawn 60 minutes following glucose injection via the
retro-orbital sinus into microfuge tubes containing a small amount
of heparin. The blood was centrifuged, and the plasma was placed
into a second microfuge tube, frozen on dry ice, for storage at
-80.degree. C. until the human C-peptide assay was performed. Human
C-peptide levels were determined using the Mercodia/ALPCO
Diagnotics Ultrasensitive C-peptide ELISA according to the
manufacturer's instructions. ELISA results for human C-peptide are
shown in FIGS. 29C and D for mice transplanted with cells from each
of the respective treatment groups. Similar levels of human
C-peptide were detectable at 8 weeks post-transplant for each
treatment category, indicating that an equivalent endocrine
precursor cell population could be derived from the differentiation
protocol using GDF-8 and Wnt3a or GDF-8 and a compound of the
present invention.
Example 20
Evaluation of the Potential of Inhibitors of CDK, GSK3, and TRK to
Differentiate Human Embryonic Stem Cells into Cells Expressing
Markers Characteristic of the Definitive Endoderm Lineage
[0438] A subset of 14 proprietary small molecules, known to have
specificity for the CDK, GSK3, and/or TRK signaling pathways were
evaluated for their potential to differentiate human embryonic stem
cells to cells expressing markers characteristic of the definitive
endoderm lineage.
[0439] Cell assay seeding. Briefly, clusters of Hi human embryonic
stem cells were grown on reduced growth factor Matrigel.TM.
(Invitrogen; Cat #356231) coated tissue culture plastic. Cells were
passaged using collagenase (Invitrogen; Cat #17104-019) treatment
and gentle scraping, washed to remove residual enzyme, and plated
with even dispersal at a ratio of 1:1 (surface area) on reduced
growth factor MATRIGEL.TM. (BD Biosciences; Cat #356231)-coated
96-well black plates (Packard ViewPlates; PerkinElmer; Cat
#6005182) using volumes of 100 .mu.l/well. Cells were allowed to
attach and then recover log phase growth over a 1 to 3 day time
period, feeding daily with MEF conditioned medium supplemented with
8 ng/ml bFGF (R&D Systems; Cat #233-FB). Plates were maintained
at 37.degree. C., 5% CO.sub.2 in a humidified box throughout the
duration of assay.
[0440] Preparation of compounds and assay. Screening was conducted
using the compounds described in Table 16. In addition Compound 34
was included as a positive control, as demonstrated in previous
examples. Compounds were made available as 5 mM stocks in 96-well
plate format, solubilized in 100% DMSO (Sigma; Cat #D2650) and
stored at -80.degree. C. The library compounds were further diluted
to an intermediate concentration of 0.2 mM in 50 mM HEPES
(Invitrogen; Cat #15630-080), 20% DMSO and stored at 4.degree. C.
Test conditions were performed in triplicate, feeding on
alternating days over a four-day assay period. Assay was initiated
by aspirating culture medium from each well followed by three
washes in PBS (Invitrogen; Cat #14190) to remove residual growth
factors. On the first day of assay, test volumes of 200 .mu.l per
well were added to each well using DMEM:F12 base medium
(Invitrogen; Cat #11330-032) supplemented with 0.5% FCS (HyClone;
Cat #SH30070.03) and 100 ng/ml GDF-8 (R&D Systems, Cat #788-G8)
plus 2.5 .mu.M compound. A parallel set of test samples were
treated in an identical manner but omitting GDF-8 from the medium.
On the third day of assay, test volumes of 100 .mu.l per well were
added to each well using DMEM:F12 base medium supplemented with 2%
FCS plus 100 ng/ml GDF-8 (R&D Systems, Cat #788-G8). GDF-8 was
omitted from test samples that did not get treated with GDF-8 on
the first day of assay. Positive control samples contained the same
base medium supplemented with FCS and 100 ng/ml recombinant human
activin A (PeproTech; Cat #120-14) throughout the four day assay
along with Wnt3a (20 ng/ml) addition on days 1 and 2. Negative
control samples contained DMEM:F12 base medium supplemented with
FCS.
[0441] High Content Analysis. At the conclusion of four-days of
culture, assay plates were washed twice with PBS (Invitrogen; Cat
#14190), fixed with 4% paraformaldehyde (Alexis Biochemical; Cat
#ALX-350-011) at room temperature for 20 minutes, then washed three
times with PBS and permeabilized with 0.5% Triton X-100 (Sigma; Cat
#T8760-2) for 20 minutes at room temperature. Cells were washed
again three times with PBS and blocked with 4% chicken serum
(Invitrogen; Cat #16110082) in PBS for 30 minutes at room
temperature. Primary antibody (goat anti-human SOX17; R&D
Systems; Cat #AF 1924) was diluted 1:100 in 4% chicken serum and
added to each well for one hour at room temperature. Alexa Fluor
488 conjugated secondary antibody (chicken anti-goat IgG; Molecular
Probes; Cat #AZ1467) was diluted 1:200 in PBS and added to each
sample well after washing three times with PBS. To counterstain
nuclei, 4 .mu.g/ml Hoechst 33342 (Invitrogen; Cat #H3570) was added
for ten minutes at room temperature. Plates were washed once with
PBS and left in 100 .mu.l/well PBS for imaging.
[0442] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized
from positive control wells and from untreated negative control
wells stained with secondary antibody alone. Images from 15 fields
per well were acquired to compensate for any cell loss during the
bioassay and subsequent staining procedures. Measurements for total
cell number and total SOX17 intensity were obtained from each well
using IN Cell Developer Toolbox 1.7 (GE Healthcare) software.
Segmentation for the nuclei was determined based on gray-scale
levels (baseline range 100-300) and nuclear size. Averages and
standard deviations were calculated for each replicate data set.
Total SOX17 protein expression was reported as total intensity or
integrated intensity, defined as total fluorescence of the cell
multiplied by area of the cell. Background was eliminated based on
acceptance criteria of gray-scale ranges between 200 and 3500.
Average data from triplicate wells were collected. The percentage
of treated wells relative to the positive control was
calculated.
[0443] Results for this screen are shown in Table 17. None of the
small molecules induced significant SOX17 expression in the absence
of GDF-8 during the four day differentiation process. Compound 34
served as an experimental control and induced significant SOX17
expression in the presence of GDF-8, equivalent to levels observed
with the positive control using activin A and Wnt3a. The remaining
compounds of the present invention tested in this example showed a
range of activities with weak to moderate induction of SOX17
expression. Of note, differentiation activity in this subset of
compounds was observed in association with selectivity for all
three enzymatic signal pathways, making it difficult to
conclusively determine a clear mechanism of action.
Example 21
Screening for Analogues of the Compounds of the Present Invention
that are Capable of Mediating the Formation of Cells Expressing
Markers Characteristic of the Definitive Endoderm Lineage
[0444] Based on the structures for the compounds of the present
invention, an analog search was conducted and 118 analogues were
found. Initial screening determined that some analogues were able
to induce definitive endoderm differentiation in the absence of
activin A in combination with other growth factors. It was
important to determine if these analogues could also induce
definitive endoderm differentiation in combination with only
GDF-8.
[0445] Cell assay seeding. Briefly, clusters of H1 human embryonic
stem cells were grown on reduced growth factor Matrigel.TM.
(Invitrogen; Cat #356231)-coated tissue culture plastic. Cells were
passaged using collagenase (Invitrogen; Cat #17104-019) treatment
and gentle scraping, washed to remove residual enzyme, and plated
with even dispersal at a ratio of 1:1 (surface area) on reduced
growth factor MATRIGEL.TM. (BD Biosciences; Cat #356231)-coated
96-well black plates (Packard ViewPlates; PerkinElmer; Cat
#6005182) using volumes of 100 .mu.l/well. Cells were allowed to
attach and then recover log phase growth over a 1 to 3 day time
period, feeding daily with MEF conditioned medium supplemented with
8 ng/ml bFGF (R&D Systems; Cat #233-FB). Plates were maintained
at 37.degree. C., 5% CO.sub.2 in a humidified box throughout the
duration of assay.
[0446] Preparation of compounds and assay. Screening was conducted
using a library of the analogue compounds. Compounds from this
library were made available as 5 mM stocks in 96-well plate format,
solubilized in 100% DMSO (Sigma; Cat #D2650) and stored at
-80.degree. C. The library compounds were further diluted to an
intermediate concentration of 0.2 mM in 50 mM HEPES (Invitrogen;
Cat #15630-080), 20% DMSO and stored at 4.degree. C. Test
conditions were performed in triplicate, feeding on alternating
days over a four-day assay period. Assays were initiated by
aspirating culture medium from each well followed by three washes
in PBS (Invitrogen; Cat #14190) to remove residual growth factors.
On the first day of assay, test volumes of 200 .mu.l per well were
added to each well using DMEM:F12 base medium (Invitrogen; Cat
#11330-032) supplemented with 0.5% FCS (HyClone; Cat #SH30070.03)
and 200 ng/ml GDF-8 (R&D Systems, Cat #788-G8) plus 2.5 .mu.M
compound. On the third day of assay, test volumes of 100 .mu.l per
well were added to each well using DMEM:F12 base medium
supplemented with 2% FCS plus 200 ng/ml GDF-8 (R&D Systems, Cat
#788-G8). Positive control samples contained the same base medium
supplemented with FCS and 100 ng/ml recombinant human activin A
(PeproTech; Cat #120-14) throughout the four-day assay along with
Wnt3a (20 ng/ml) on days 1 and 2. Negative control samples
contained DMEM:F12 base medium supplemented with FCS, adding Wnt3a
on days 1 and 2 but omitting treatment with activin A.
[0447] High Content Analysis. At the conclusion of four-days of
culture, assay plates were washed twice with PBS (Invitrogen; Cat
#14190), fixed with 4% paraformaldehyde (Alexis Biochemical; Cat
#ALX-350-011) at room temperature for 20 minutes, then washed three
times with PBS and permeabilized with 0.5% Triton X-100 (Sigma; Cat
#T8760-2) for 20 minutes at room temperature. Cells were washed
again three times with PBS and blocked with 4% chicken serum
(Invitrogen; Cat #16110082) in PBS for 30 minutes at room
temperature. Primary antibody (goat anti-human SOX17; R&D
Systems; Cat #AF 1924) was diluted 1:100 in 4% chicken serum and
added to each well for one hour at room temperature. Alexa Fluor
488 conjugated secondary antibody (chicken anti-goat IgG; Molecular
Probes; Cat #AZ1467) was diluted 1:200 in PBS and added to each
sample well after washing three times with PBS. To counterstain
nuclei, 4 .mu.g/ml Hoechst 33342 (Invitrogen; Cat #H3570) was added
for ten minutes at room temperature. Plates were washed once with
PBS and left in 100 .mu.l/well PBS for imaging.
[0448] Imaging was performed using an IN Cell Analyzer 1000 (GE
Healthcare) utilizing the 51008bs dichroic for cells stained with
Hoechst 33342 and Alexa Fluor 488. Exposure times were optimized
from positive control wells and from untreated negative control
wells stained with secondary antibody alone. Images from 15 fields
per well were acquired to compensate for any cell loss during the
bioassay and subsequent staining procedures. Measurements for total
cell number and total SOX17 intensity were obtained from each well
using IN Cell Developer Toolbox 1.7 (GE Healthcare) software.
Segmentation for the nuclei was determined based on gray-scale
levels (baseline range 100-300) and nuclear size. Averages and
standard deviations were calculated for each replicate data set.
Total SOX17 protein expression was reported as total intensity or
integrated intensity, defined as total fluorescence of the cell
times area of the cell. Background was eliminated based on
acceptance criteria of gray-scale ranges between 200 to 3500. Total
intensity data were normalized by dividing total intensities for
each well by the average total intensity for the positive control.
Normalized data were calculated for averages and standard
deviations for each replicate set.
[0449] Screening results are shown in Table 18 from four assay
plates in this single experiment. Compounds are ranked with respect
to SOX17 expression as a percentage of the positive control
treatment with activin A and Wnt3a. This assay identified a list of
12 new analogue hits as shown in Table 19.
Example 22
Human Embryonic Stem Cells Grown on Microcarriers can be
Differentiated into Cells Expressing Markers Characteristic of the
Definitive Endoderm Lineage According to the Methods of the Present
Invention
[0450] For purposes of differentiation and production of large
numbers of endocrine cells under scalable conditions, it was
important to show that human embryonic stem cells could be grown
and differentiated to definitive endoderm on microcarrier beads
using the methods of the present invention.
[0451] Preparation of cells for assay and differentiation. H1 p49C3
cells were routinely grown on Cytodex3 beads (GE Healthcare Life
Sciences, NJ) in a 125 ml spinner flask, according to the methods
described in U.S. Patent Application No. 61/116,447. After seven
days, cells and beads were transferred to a 6 well plate (Vendor;
Cat #XXX) at a ratio of 30 cm.sup.2 bead surface area per well, and
the plate was placed on a rocking platform. Cells on beads in the
positive control treatment well (designated AA/Wnt3a) were
differentiated with addition of 100 ng/ml activin A (PeproTech; Cat
#120-14) and 20 ng/ml Wnt3a (R&D Systems; Cat #1324-WN/CF) for
two days followed by 100 ng/ml activin A and 8 ng/ml bFGF
(PeproTech Inc.; Cat #: 100-18B) for one day in RPMI-1640
(Invitrogen; Cat #: 22400) with 2% Fatty Acid Free BSA (MP
Biomedicals, Inc; Cat #152401) using volumes of 2 ml/well. Compound
34, at a final concentration of 2.5 .mu.M was added to a negative
control treatment well (designated CMP alone) in RPMI-1640 with 2%
Fatty Acid Free BSA (2 ml/well) for three days in the absence of
any other growth factor treatment. A third treatment well
(designated CMP+8) received Compound 34at 2.5 .mu.M plus 50 ng/ml
GDF-8 (R&D Systems, Cat #788-G8) in RPMI-1640 with 2% Fatty
Acid Free BSA (2 ml/well) for three days. A fourth treatment well
(designated CMP+8+D) received Compound 34 at 2.5 .mu.M with 50
ng/ml GDF-8 and 50 ng/ml PDGF-D in RPMI-1640 with 2% Fatty Acid
Free BSA (2 ml/well) for three days. A fifth treatment well
(designated CMP+8+D+V) received Compound 34at 2.5 .mu.M with 50
ng/ml GDF-8, 50 ng/ml PDGF-D, and 50 ng/ml VEGF in RPMI-1640 with
2% Fatty Acid Free BSA (2 ml/well) for three days. A sixth
treatment well (designated CMP+8+D+V+M) received Compound 34at 2.5
.mu.M with 50 ng/ml GDF-8, 50 ng/ml PDGF-D, 50 ng/ml VEGF, and 20
ng/ml Muscimol in RPMI-1640 with 2% Fatty Acid Free BSA (2 ml/well)
for three days. All media and treatments were exchanged daily.
[0452] At the conclusion of treatment and culture, cells were
harvested from the beads, according to the methods described in
U.S. Patent Application No. 61/116,447. The harvested cells were
counted and analyzed by flow cytometry, according to the methods
described above.
[0453] Results are shown in FIG. 25. As shown in panel A, similar
numbers of cells were recovered for all treatment groups undergoing
differentiation. As shown in panel B, cells treated with the
Compound 34 alone did not differentiate into CXCR4 positive cells.
The positive control treatment, adding activin A and Wnt3a during
differentiation, induced expression of CXCR4 in 68% of the
resulting cell population. Compound 34 added with the various
growth factor combinations induced CXCR4 expression in 50% of the
cells, on average. Of note, equivalent levels of CXCR4 expression
were observed during treatment with Compound 34 in combination with
a single growth factor, GDF-8, or in combination with multiple
growth factors that included GDF-8. This proves that Compound 34 in
combination with at least GDF-8 can substitute for activin A and
Wnt3a to promote definitive endoderm differentiation. This example
also shows that the treatment procedure is effective for cells
grown and differentiated on microcarrier beads.
Example 23
The Compounds of the Present Invention, Together with GDF-8 Enhance
Cell Proliferation
[0454] A previous example showed that GDF-8 is able to replace
activin A to differentiate human embryonic stem cells to cells
expressing markers characteristic of the definitive endoderm
lineage. It was important to know the relative potencies of GDF-8
and activin A with respect to definitive endoderm formation. A dose
response assay was conducted using equivalent concentrations of
each growth factor to compare results during human embryonic stem
cell differentiation.
[0455] The compounds of the present invention used in combination
with GDF-8 during definitive endoderm differentiation were
evaluated for their ability to induce cell proliferation. Results
were compared to treatment with activin A or GDF-8 alone.
[0456] Preparation of cells for assay. Stock cultures of human
embryonic stem cells (Hi human embryonic stem cell line) were
maintained in an undifferentiated, pluripotent state on reduced
growth factor MATRIGEL.TM. (BD Biosciences; Cat #356231)-coated
dishes in MEF conditioned medium with passage on average every four
days. Passage was performed by exposing cell cultures to a solution
of 1 mg/ml dispase (Invitrogen, Cat #: 17105-041) for 5 to 7
minutes at 37.degree. C. followed by rinsing the monolayer with MEF
conditioned culture medium and gentle scraping to recover cell
clusters. Clusters were centrifuged at low speed to collect a cell
pellet and remove residual dispase. Cell clusters were split at a
1:3 or 1:4 ratio for routine maintenance culture or a 1:1 ratio for
immediate assay. All human embryonic stem cell lines were
maintained at passage numbers less than 50 and routinely evaluated
for normal karyotypic phenotype and for absence of mycoplasma
contamination.
[0457] Cell clusters used in the assay were evenly resuspended in
MEF conditioned medium supplemented with 8 ng/ml bFGF and seeded
onto reduced growth factor MATRIGEL.TM.-coated 96-well Packard
VIEWPLATES (PerkinElmer; Cat #6005182) in volumes of 100
.mu.l/well. MEF conditioned medium supplemented with 8 ng/ml bFGF
was used for initial seeding and expansion. Daily feeding was
conducted by aspirating spent culture medium from each well and
replacing with an equal volume of fresh medium. A background set of
wells in each assay plate was not seeded with cells but was treated
throughout assay with basal media conditions. Plates were
maintained at 37.degree. C., 5% CO.sub.2 in a humidified box
throughout the duration of assay.
[0458] Assay. The assay was initiated by aspirating the culture
medium from each well and adding back a final aliquot (100 .mu.l)
of test medium. Test conditions were performed in triplicate over a
total three-day assay period, feeding daily by aspirating and
replacing the medium from each well with fresh test medium.
Identical assays were set up simultaneously in parallel for
evaluation at the end of 24, 48, and 72 hours.
[0459] On the first day of assay, all wells containing cells
received an aliquot (80 .mu.l) of RPMI-1640 medium (Invitrogen; Cat
#: 22400) supplemented with 2.5% Albumin Bovine Fraction V, Fatty
Acid Free (FAF BSA; 2% in final assay) (Proliant Inc. Cat #: SKU
68700). Various control and test samples were created at 5.times.
concentration to be added to appropriate wells (20 .mu.l per well).
Control conditions included the following, with final growth factor
concentrations as indicated: 1) basal medium with 2% FAF BSA; 2)
100 ng/ml activin A (PeproTech; Cat #120-14) with 8 ng/ml bFGF
(PeproTech; Cat #100-18B); 3) 100 ng/ml activin A with 8 ng/ml bFGF
and 20 ng/ml Wnt3a (R&D Systems; Cat #1324-WN/CF); 4) 100 ng/ml
GDF-8 (R&D Systems, Cat #788-G8) with 8 ng/ml bFGF; 5) GDF-8
with 8 ng/ml bFGF and 20 ng/ml Wnt3a. Cells in an additional set of
control wells were treated with MEF conditioned medium throughout
the assay. In some control samples using GDF-8, Wnt3a was replaced
with a compound of the present invention. For experimental test
samples, eight different compounds were diluted two-fold in series
to create three different dose concentrations then combined with
100 ng/ml GDF-8 and 8 ng/ml bFGF. These small molecules included
proprietary compounds Compound 181, Compound 180, Compound 19,
Compound 202, Compound 40, Compound 34, Compound 56, and a
commercially available GSK3 inhibitor BIO (EMD Chemicals, Inc.; Cat
#361550). On the second and third day of assay, all wells for
control and experimental samples were aspirated and fed again using
identical treatment conditions except that Wnt3a was removed from
some control wells.
[0460] MTS Assay: At the conclusion of 24, 48, or 72 hours of
culture, one set of assay plates was subjected to a MTS assay
(Promega; Cat #G3581), following the manufacturer's instructions.
In brief, 20 .mu.l of MTS was added to each well, and assay plates
were incubated at 37.degree. C., 5% CO.sub.2 for four hours prior
to taking OD490 readings. Statistical measures were calculated
minus background (i.e. treatment wells without cells) to determine
mean values for each triplicate set in addition to a standard error
of the mean.
[0461] The MTS assay is a measure of cellular metabolic activity in
the enzymatic reduction of a tetrazolium compound to a formazan
product. At a single time point, the MTS assay can be used as a
comparative indicator of cell viability. MTS assays evaluated in
parallel at sequential time points can add additional information
regarding increases in cellular metabolic activity which in turn
can be correlated with cell proliferation for each treatment
condition. FIG. 26, panel A shows OD490 readings for all control
treatments over the three day assay period. Cells treated with
conditioned medium showed little change in OD490 over three days,
indicating that cell numbers in this treatment group remained
static. In contrast, cells cultured in basal medium without growth
factors (no treatment), showed a steady decline in OD490 correlated
with a loss in cell number over time. Activin A treatments during
the differentiation process, with and without Wnt3a, showed
incremental increases in OD490, indicating significant expansion of
the cell population over time. GDF-8 treatment in the absence of
Wnt3a resulted in a decrease in OD490 relative to activin A
treatment; this was noticeable on the first day and sustained
throughout all three days of culture. Addition of Wnt3a to the
GDF-8 treatment group resulted in a recovery and increase in OD490
by the third day of culture.
[0462] FIG. 26, panel B through FIG. 26, panel I show MTS assay
results for treatment with a small molecule inhibitor in
combination with GDF-8. OD490 readings from treatments with a
compound of the present invention and GDF-8 were equivalent to or
exceeded results from treatment with activin A. In all cases, an
optimal concentration of each small molecule combined with GDF-8
resulted in improved OD490 readings over the three day assay
relative to treatment with GDF-8 alone. This suggests that the
compounds of the present invention are important for inducing
proliferation and expansion of a cell population during definitive
endoderm differentiation.
Example 24
Human Embryonic Stem Cells Grown on Microcarriers can be
Differentiated into Endocrine Progenitor Cells According to the
Methods of the Present Invention
[0463] For purposes of differentiation and production of large
numbers of endocrine cells under industrial conditions, it was
important to show that human embryonic stem cells could be grown
and differentiated to endocrine progenitor cells on microcarrier
beads using a protocol without activin A.
[0464] Preparation of cells for assay and differentiation. H1 p45
cells were grown on Cytodex3 beads (GE Healthcare; Cat #17-0485-01)
in a 6 well ultra low attachment plate (Costar; Cat #3471) placed
on a rocking platform at about 1 rotation every 10 seconds (Vari
Mix, Thermo Scientific, Cat #M79735). MEF conditioned media was
changed daily for six days. Then the media was changed to the
following treatments to initiate endoderm differentiation. Cells on
beads in the positive control treatment well (designated AA+Wnt)
were differentiated with addition of 100 ng/ml activin A
(PeproTech; Cat #120-14), 8 ng/ml bFGF (PeproTech Inc.; Cat #:
100-18B), and 20 ng/ml Wnt3a (R&D Systems; Cat #1324-WN/CF) for
one day followed by 100 ng/ml activin A and 8 ng/ml bFGF (PeproTech
Inc.; Cat #: 100-18B) for two days in RPMI-1640 (Invitrogen; Cat #:
22400) with 2% Fatty Acid Free BSA (Proliant Biomedicals, Inc; SKU
#68700) using volumes of 2 ml/well. A second treatment well
(designated GDF-8+MCX) received Compound 202 at 2.5 .mu.M plus 200
ng/ml GDF-8 (R&D Systems, Cat #788-G8) and 8 ng/ml bFGF for one
day followed by two days with 200 ng/ml GDF-8 and 8 ng/ml bFGF in
RPMI-1640 with 2% Fatty Acid Free BSA (2 ml/well) media. A third
treatment well (designated GDF-8+Wnt) received 200 ng/ml GDF-8 with
20 ng/ml Wnt3a and 8 ng/ml bFGF for one day followed by two days
with 200 ng/ml GDF-8 and 8 ng/ml bFGF in RPMI-1640 with 2% Fatty
Acid Free BSA (2 ml/well) media. All media and treatments were
exchanged daily.
[0465] At the conclusion of treatment and culture, cells were
harvested and counted to determine cell recovery and undergo flow
cytometric analysis. High levels of CXCR4 and CD99 was seen
following all three treatment regiments (FIG. 27A). Cell number
varied between samples (FIG. 27B). A lower cell number was observed
in samples treated with GDF-8 and at the definitive endoderm and
fourth stage than the other treatment groups. This suggests that
the compounds of the present invention may increase proliferation
of the cells during differentiation.
[0466] At the end of stage 3 the endodermal genes PDX1, HNF4 alpha,
and CDX2 are expressed in the cells (FIGS. 27C, D). Treatment of
the cells with GDF-8 and a compound of the present invention during
stage one of differentiation resulted in better expression of Pdx1
than the control differentiation treatment. At the end of stage 4,
endodermal genes were up regulated further (FIGS. 27E, F). These
results conclude that GDF-8 plus Compound 202 can replace activin A
and Wnt3a for definitive endoderm differentiation resulting in
pancreatic endoderm formation.
[0467] Publications cited throughout this document are hereby
incorporated by reference in their entirety. Although the various
aspects of the invention have been illustrated above by reference
to examples and preferred embodiments, it will be appreciated that
the scope of the invention is defined not by the foregoing
description but by the following claims properly construed under
the principles of patent law.
TABLE-US-00001 TABLE 1 Cell Number Sox17 Expression % of Average %
of Average Total positive Total positive Plate # Compound # Cell
Number control Intensity control plate 5 no Activin A (with Wnt3a)
7159 67.42 8.12E+06 2.51 plate 5 Activin A/Wnt3a 10619 100.00
3.23E+08 100.00 plate 5 Compound 58 4848 45.66 -1.60E+06 -0.49
plate 5 Compound 59 20 0.19 -4.62E+06 -1.43 plate 5 Compound 60
3348 31.52 -2.33E+05 -0.07 plate 5 Compound 61 2931 27.60 -3.05E+06
-0.94 plate 5 Compound 62 7171 67.53 -2.04E+06 -0.63 plate 5
Compound 3 14211 133.82 -2.34E+06 -0.73 plate 6 no Activin A (with
Wnt3a) 3264 32.97 2.52E+06 0.80 plate 6 Activin A/Wnt3a 9902 100.00
3.14E+08 100.00 plate 6 Compound 63 1917 19.36 4.75E+05 0.15 plate
6 Compound 26 5434 54.88 -6.33E+05 -0.20 plate 6 Compound 27 6288
63.50 -1.13E+06 -0.36 plate 6 Compound 28 4121 41.62 -1.89E+06
-0.60 plate 6 Compound 29 5164 52.15 -1.66E+06 -0.53 plate 6
Compound 30 4726 47.73 -1.23E+06 -0.39 plate 7 no Activin A (with
Wnt3a) 9545 47.57 -4.87E+06 -0.99 plate 7 Activin A/Wnt3a 20064
100.00 4.92E+08 100.00 plate 7 Compound 31 7230 36.03 -3.45E+06
-0.70 plate 7 Compound 32 14655 73.04 -3.03E+06 -0.62 plate 7
Compound 33 13891 69.23 -8.11E+06 -1.65 plate 7 Compound 34 11674
58.18 -2.24E+06 -0.46 plate 7 Compound 35 15379 76.65 -7.30E+06
-1.48 plate 7 Compound 36 8356 41.65 -4.57E+06 -0.93 plate 8 no
Activin A (with Wnt3a) 6868 36.97 -2.31E+06 -0.52 plate 8 Activin
A/Wnt3a 18575 100.00 4.47E+08 100.00 plate 8 Compound 37 9048 48.71
-3.51E+06 -0.79 plate 8 Compound 38 11361 61.16 -4.31E+06 -0.96
plate 8 Compound 39 7054 37.98 -3.83E+06 -0.86 plate 8 Compound 40
8104 43.63 -4.59E+06 -1.03 plate 1 no Activin A (with Wnt3a) 2972
27.98 1.64E+07 19.74 plate 1 Activin A/Wnt3a 3126 29.44 8.33E+07
100.00 plate 1 Compound 64 2201 20.72 1.71E+07 20.52 plate 1
Compound 65 3030 28.53 2.83E+07 33.95 plate 1 Compound 66 1990
18.74 2.36E+07 28.30 plate 1 Compound 67 2074 19.53 2.63E+07 31.55
plate 1 Compound 68 1432 13.48 1.03E+07 12.39 plate 1 Compound 69
2593 24.42 2.62E+07 31.43 plate 1 Compound 70 2236 21.05 2.59E+07
31.11 plate 1 Compound 71 2996 28.22 3.07E+07 36.92 plate 1
Compound 72 2179 20.52 1.21E+07 14.50 plate 1 Compound 73 2817
26.53 2.93E+07 35.25 plate 1 Compound 74 2853 26.86 2.25E+07 27.01
plate 1 Compound 75 1689 15.91 1.42E+07 17.05 plate 1 Compound 76
2324 21.89 1.48E+07 17.81 plate 1 Compound 77 2306 21.71 2.04E+07
24.55 plate 1 Compound 78 3298 31.06 2.58E+07 31.00 plate 1
Compound 79 2855 26.88 2.79E+07 33.47 plate 1 Compound 80 3603
33.93 3.22E+07 38.62 plate 1 Compound 81 2263 21.31 1.07E+07 12.91
plate 1 Compound 82 1210 11.39 1.36E+07 16.33 plate 1 Compound 83
1805 17.00 1.82E+07 21.87 plate 1 Compound 84 2024 19.06 2.48E+07
29.80 plate 1 Compound 85 2840 26.74 3.45E+07 41.44 plate 1
Compound 86 1447 13.63 8.43E+06 10.13 plate 1 Compound 87 5336
50.25 4.20E+07 50.38 plate 2 no Activin A (with Wnt3a) 4033 35.50
2.14E+07 21.70 plate 2 Activin A/Wnt3a 4292 37.78 9.86E+07 100.00
plate 2 Compound 88 3416 30.06 4.17E+07 42.28 plate 2 Compound 89
4751 41.82 2.11E+07 21.40 plate 2 Compound 90 4542 39.98 3.03E+07
30.70 plate 2 Compound 91 1401 12.33 1.29E+06 1.31 plate 2 Compound
92 4210 37.06 2.95E+07 29.90 plate 2 Compound 93 4157 36.59
2.29E+07 23.26 plate 2 Compound 94 4046 35.61 2.85E+07 28.91 plate
2 Compound 95 8368 73.66 4.02E+07 40.72 plate 2 Compound 96 3695
32.53 2.92E+07 29.57 plate 2 Compound 97 3437 30.26 2.41E+07 24.44
plate 2 Compound 98 4178 36.77 3.75E+07 38.07 plate 2 Compound 99
3739 32.91 2.10E+07 21.29 plate 2 Compound 100 2275 20.02 1.27E+07
12.86 plate 2 Compound 101 3496 30.77 2.98E+07 30.17 plate 2
Compound 102 4874 42.90 2.10E+07 21.32 plate 2 Compound 103 4228
37.22 2.69E+07 27.32 plate 2 Compound 104 6115 53.82 4.93E+07 49.99
plate 2 Compound 105 6484 57.07 5.03E+07 50.95 plate 2 Compound 106
4211 37.06 3.94E+07 40.00 plate 2 Compound 107 2853 25.11 1.78E+07
18.04 plate 2 Compound 108 3779 33.27 2.39E+07 24.26 plate 2
Compound 108 2869 25.26 2.04E+07 20.71 plate 2 Compound 110 4398
38.71 2.53E+07 25.65 plate 3 no Activin A (with Wnt3a) 2589 91.17
1.17E+07 5.89 plate 3 Activin A/Wnt3a 6933 244.13 1.98E+08 100.00
plate 3 Compound 111 6816 240.04 5.33E+07 26.90 plate 3 Compound
112 5357 188.66 3.52E+07 17.74 plate 3 Compound 113 6002 211.37
8.55E+07 43.11 plate 3 Compound 114 3308 116.49 3.85E+07 19.44
plate 3 Compound 115 5007 176.31 3.96E+07 19.95 plate 3 Compound
116 3802 133.89 3.12E+07 15.75 plate 3 Compound 117 6521 229.64
4.16E+07 20.97 plate 3 Compound 118 6128 215.81 5.53E+07 27.91
plate 3 Compound 119 4184 147.35 3.41E+07 17.21 plate 3 Compound
120 2489 87.66 2.87E+07 14.49 plate 3 Compound 121 4985 175.54
3.94E+07 19.87 plate 3 Compound 25 4151 146.17 4.03E+07 20.32 plate
3 Compound 122 6407 225.61 4.15E+07 20.95 plate 3 Compound 123 4465
157.24 5.35E+07 26.99 plate 3 Compound 124 4417 155.53 4.67E+07
23.55 plate 3 Compound 125 6367 224.23 5.73E+07 28.93 plate 3
Compound 126 6157 216.82 7.47E+07 37.70 plate 3 Compound 127 5593
196.97 5.61E+07 28.28 plate 3 Compound 128 4160 146.50 4.91E+07
24.77 plate 3 Compound 129 3778 133.03 3.54E+07 17.88 plate 3
Compound 130 4357 153.43 4.15E+07 20.92 plate 3 Compound 131 6135
216.05 4.28E+07 21.61 plate 3 Compound 132 4421 155.69 4.58E+07
23.12 plate 3 Compound 133 7069 248.94 6.52E+07 32.88 plate 4 no
Activin A (with Wnt3a) 3274 86.62 1.25E+07 12.79 plate 4 Activin
A/Wnt3a 4158 110.03 9.79E+07 100.00 plate 4 Compound 134 5277
139.62 3.43E+07 35.04 plate 4 Compound 64 5657 149.67 3.38E+07
34.48 plate 4 Compound 135 2790 73.83 1.63E+07 16.63 plate 4
Compound 34 4774 126.33 4.35E+07 44.47 plate 4 Compound 136 4881
129.16 3.20E+07 32.73 plate 4 Compound 137 1740 46.05 9.16E+06 9.35
plate 4 Compound 30 6367 168.46 4.22E+07 43.13 plate 4 Compound 37
5377 142.27 2.85E+07 29.14 plate 4 Compound 138 7722 204.32
3.07E+07 31.37 plate 4 Compound 139 3574 94.56 1.30E+07 13.32 plate
4 Compound 140 3893 103.00 1.12E+07 11.46 plate 4 Compound 39 6114
161.77 3.45E+07 35.22 plate 4 Compound 141 4310 114.04 1.61E+07
16.48 plate 4 Compound 142 5091 134.71 3.74E+07 38.22 plate 4
Compound 35 6601 174.65 8.50E+07 86.77 plate 4 Compound 143 3582
94.79 2.17E+07 22.14 plate 4 Compound 144 6787 179.57 5.45E+07
55.69 plate 4 Compound 145 3752 99.29 2.23E+07 22.81 plate 4
Compound 146 2554 67.59 1.83E+07 18.71 plate 4 Compound 112 3289
87.03 1.48E+07 15.11 plate 4 Compound 113 3819 101.06 2.34E+07
23.93 plate 4 Compound 114 1259 33.32 1.34E+07 13.67 plate 4
Compound 22 5517 145.98 7.09E+07 72.39 plate 4 Compound 150 5104
135.04 3.34E+07 34.11 plate 5 no Activin A (with Wnt3a) 7159 116.70
8.12E+06 2.51 plate 5 Activin A/Wnt3a 10619 173.09 3.23E+08 100.00
plate 5 Compound 151 2785 45.39 -1.03E+06 -0.32 plate 5 Compound
152 4693 76.50 -3.08E+06 -0.95 plate 5 Compound 153 9718 158.40
-1.20E+06 -0.37 plate 5 Compound 154 3479 56.70 -1.97E+06 -0.61
plate 5 Compound 155 9343 152.28 -3.45E+06 -1.07 plate 5 Compound
156 3813 62.16 -2.58E+05 -0.08 plate 6 no Activin A (with Wnt3a)
3264 68.37 2.52E+06 0.80 plate 6 Activin A/Wnt3a 9902 207.40
3.14E+08 100.00 plate 6 Compound 157 2480 51.94 -1.22E+06 -0.39
plate 6 Compound 158 5271 110.41 -1.30E+06 -0.41 plate 6 Compound
159 6478 135.68 -1.84E+06 -0.59 plate 6 Compound 160 4212 88.21
1.30E+05 0.04 plate 6 Compound 161 2439 51.09 -9.20E+05 -0.29 plate
6 Compound 162 1260 26.39 -1.35E+06 -0.43 plate 7 no Activin A
(with Wnt3a) 9545 156.12 -4.87E+06 -0.99 plate 7 Activin A/Wnt3a
20064 328.17 4.92E+08 100.00 plate 7 Compound 163 16557 270.81
-7.31E+06 -1.49 plate 7 Compound 164 16472 269.42 -7.37E+06 -1.50
plate 7 Compound 165 3015 49.32 -7.34E+06 -1.49 plate 7 Compound
166 13845 226.45 -7.98E+06 -1.62 plate 7 Compound 167 10325 168.87
-7.35E+06 -1.49 plate 7 Compound 168 14139 231.26 -6.49E+06 -1.32
plate 7 Compound 169 4468 73.08 -6.38E+06 -1.30 plate 8 no Activin
A (with Wnt3a) 6868 179.83 -2.31E+06 -0.52 plate 8 Activin A/Wnt3a
18575 486.35 4.47E+08 100.00 plate 8 Compound 170 13140 344.04
-4.13E+06 -0.93 plate 8 Compound 171 10894 285.22 -2.61E+06 -0.58
plate 8 Compound 172 3416 89.44 -4.72E+06 -1.06 plate 8 Compound
173 8815 230.81 -4.25E+06 -0.95 plate 8 Compound 174 11760 307.91
-3.33E+06 -0.75 plate 8 Compound 175 5 0.13 -4.91E+06 -1.10 plate 8
Compound 176 10139 265.47 -4.73E+06 -1.06 plate 8 Compound 177 9994
261.68 -2.95E+06 -0.66 plate 8 Compound 178 8998 235.58 -3.74E+06
-0.84 plate 5 no Activin A (with Wnt3a) 7159 67.42 8.12E+06 2.51
plate 5 Activin A/Wnt3a 10619 100.00 3.23E+08 100.00 plate 5
Compound 21 4719 44.44 -1.96E+06 -0.61 plate 5 Compound 22 2036
19.18 -1.79E+06 -0.55 plate 5 Compound 23 2563 24.13 -1.56E+06
-0.48 plate 5 Compound 24 4470 42.09 -7.05E+05 -0.22 plate 5
Compound 24 6085 57.30 -3.08E+06 -0.95 plate 5 Compound 26 7276
68.52 -2.38E+06 -0.74 plate 5 Compound 27 4588 43.20 -5.63E+05
-0.17 plate 5 Compound 28 2682 25.26 -1.37E+06 -0.43 plate 5
Compound 29 5778 54.41 -1.94E+06 -0.60 plate 5 Compound 30 620 5.84
-5.05E+06 -1.56 plate 5 Compound 31 3419 32.19 -1.42E+06 -0.44
plate 6 no Activin A (with Wnt3a) 3264 69.07 2.52E+06 0.80 plate 6
Activin A/Wnt3a 9902 209.51 3.14E+08 100.00 plate 6 Compound 32
2142 45.32 -1.33E+06 -0.42 plate 6 Compound 33 5564 117.73
-8.63E+05 -0.27 plate 6 Compound 34 5927 125.41 -2.01E+06 -0.64
plate 6 Compound 35 10068 213.01 -2.15E+06 -0.68 plate 6 Compound
36 5170 109.39 -1.22E+06 -0.39 plate 6 Compound 37 3098 65.55
1.91E+06 0.61 plate 6 Compound 38 1537 32.52 4.48E+04 0.01 plate 6
Compound 39 3650 77.23 -2.01E+06 -0.64 plate 6 Compound 40 5817
123.07 4.91E+05 0.16 plate 6 Compound 64 4359 92.23 -1.07E+05 -0.03
plate 6 Compound 30 4035 85.38 2.09E+06 0.66 plate 6 Compound 65
3279 69.37 -5.63E+05 -0.18 plate 6 Compound 67 2698 57.08 -1.95E+06
-0.62 plate 7 no Activin A (with Wnt3a) 9545 321.22 -4.87E+06 -0.99
plate 7 Activin A/Wnt3a 20064 675.20 4.92E+08 100.00 plate 7
Compound 68 10894 366.62 -5.15E+06 -1.05 plate 7 Compound 69 9734
327.58 -3.97E+06 -0.81 plate 7 Compound 70 16736 563.21 -6.51E+06
-1.32 plate 7 Compound 71 17999 605.71 -7.38E+06 -1.50 plate 7
Compound 72 7309 245.96 -6.47E+06 -1.32 plate 7 Compound 73 8888
299.10 -3.03E+06 -0.62 plate 7 Compound 74 11496 386.85 -2.67E+06
-0.54 plate 7 Compound 75 9739 327.74 -7.75E+06 -1.57 plate 7
Compound 76 14439 485.89 -4.19E+06 -0.85 plate 7 Compound 77 12331
414.95 -6.03E+06 -1.22 plate 7 Compound 78 9702 326.49 -6.57E+06
-1.33 plate 7 Compound 79 8535 287.22 -6.92E+06 -1.41 plate 8 no
Activin A (with Wnt3a) 6868 295.49 -2.31E+06 -0.52 plate 8 Activin
A/Wnt3a 18575 799.17 4.47E+08 100.00 plate 8 Compound 80 13939
599.68 -4.23E+06 -0.95 plate 8 Compound 81 10466 450.29 -4.91E+06
-1.10 plate 8 Compound 82 10323 444.14 -4.90E+06 -1.10 plate 8
Compound 83 14619 628.95 1.48E+06 0.33 plate 8 Compound 84 14105
606.84 -4.44E+06 -0.99 plate 8 Compound 85 12172 523.66 -3.48E+06
-0.78 plate 8 Compound 86 7218 310.54 -4.22E+06 -0.94 plate 8
Compound 87 5383 231.58 -4.07E+06 -0.91 plate 8 Compound 88 10419
448.27 -4.27E+06 -0.96 plate 8 Compound 89 11780 506.83 -3.94E+06
-0.88 plate 8 Compound 90 7002 301.25 -1.54E+06 -0.35 plate 8
Compound 91 6224 267.78 -4.53E+06 -1.01
TABLE-US-00002 TABLE 2 Cell Number Sox17 Intensity % of positive %
of positive Compound # control control Compound 17 133.8 -0.7
Compound 95 195.0 40.7 Compound 138 185.7 31.4 Compound 87 170.7
50.4 Compound 144 163.2 55.7 Compound 35 158.7 86.8 Compound 30
153.1 43.1 Compound 105 151.0 51.0 Compound 39 147.0 35.2 Compound
104 142.5 50.0 Compound 29 136.0 34.5 Compound 22 132.7 72.4
Compound 37 129.3 29.1 Compound 134 126.9 35.0 Compound 150 122.7
34.1 Compound 142 122.4 38.2 Compound 136 117.4 32.7 Compound 80
115.2 38.6 Compound 34 114.8 44.5 Compound 102 113.5 21.3 Compound
89 110.7 21.4 Compound 105 105.8 30.7 Compound 78 105.5 31.0
Compound 141 103.6 16.5 Compound 110 102.5 25.7 Compound 133 102.0
32.9
TABLE-US-00003 TABLE 3 Cell Number Sox17 Expression Average Total %
of positive % of positive Compound # Treatments Cell Number control
Average Total Intensity control none no Activin A, with Wnt3a 23253
124.16 1.97E+07 10.59 none Activin A/Wnt3a 18728 100.00 1.86E+08
100.00 Compound 17 no AA (with Wnt3a) EGF + FGF4 21445 114.51
3.43E+07 18.48 none no Activin A, with Wnt3a 23253 124.16 1.97E+07
10.59 none Activin A/Wnt3a 18728 100.00 1.86E+08 100.00 Compound 22
no AA (with Wnt3a) EGF + FGF4 18336 97.91 3.72E+07 20.05 Compound
34 no AA (with Wnt3a) EGF + FGF4 18891 100.87 3.26E+07 17.55
Compound 29 no AA (with Wnt3a) EGF + FGF4 20221 107.97 2.83E+07
15.27 Compound 39 no AA (with Wnt3a) EGF + FGF4 17095 91.28
2.82E+07 15.19 Compound 37 no AA (with Wnt3a) EGF + FGF4 15605
83.32 2.67E+07 14.37 Compound 35 no AA (with Wnt3a) EGF + FGF4
23823 127.20 2.54E+07 13.69 Compound 80 no AA (with Wnt3a) EGF +
FGF4 19864 106.07 2.33E+07 12.54 Compound 141 no AA (with Wnt3a)
EGF + FGF4 17719 94.61 2.24E+07 12.04 Compound 30 no AA (with
Wnt3a) EGF + FGF4 18063 96.45 2.18E+07 11.73 Compound 150 no AA
(with Wnt3a) EGF + FGF4 16833 89.88 2.16E+07 11.63 Compound 144 no
AA (with Wnt3a) EGF + FGF4 17100 91.31 2.04E+07 11.01 Compound 104
no AA (with Wnt3a) EGF + FGF4 17863 95.38 1.89E+07 10.19 Compound
142 no AA (with Wnt3a) EGF + FGF4 18955 101.21 1.84E+07 9.90
Compound 110 no AA (with Wnt3a) EGF + FGF4 17534 93.62 1.76E+07
9.45 Compound 78 no AA (with Wnt3a) EGF + FGF4 17703 94.52 1.71E+07
9.23 Compound 133 no AA (with Wnt3a) EGF + FGF4 16521 88.22
1.67E+07 8.97 Compound 87 no AA (with Wnt3a) EGF + FGF4 16495 88.07
1.55E+07 8.33 Compound 95 no AA (with Wnt3a) EGF + FGF4 16900 90.24
1.43E+07 7.72 Compound 136 no AA (with Wnt3a) EGF + FGF4 19167
102.34 7.91E+06 4.26 Compound 105 no AA (with Wnt3a) EGF + FGF4
15217 81.25 7.45E+06 4.01 Compound 134 no AA (with Wnt3a) EGF +
FGF4 17208 91.88 7.40E+06 3.99 Compound 138 no AA (with Wnt3a) EGF
+ FGF4 16695 89.14 6.65E+06 3.58 Compound 89 no AA (with Wnt3a) EGF
+ FGF4 14652 78.24 3.89E+06 2.10 Compound 90 no AA (with Wnt3a) EGF
+ FGF4 15903 84.92 3.53E+06 1.90 Compound 102 no AA (with Wnt3a)
EGF + FGF4 12943 69.11 2.85E+05 0.15 none no Activin A, with Wnt3a
23253 124.16 1.97E+07 10.59 none Activin A/Wnt3a 18728 100.00
1.86E+08 100.00 Compound 35 no AA (with Wnt3a) EGF + FGF4 18294
97.68 1.99E+07 10.70
TABLE-US-00004 TABLE 3B Cell Number Sox17 Intensity % of positive %
of positive Compound # control control Compound 22 97.91 20.05
Compound 34 100.87 17.55 Compound 29 107.97 15.27 Compound 39 91.28
15.19 Compound 37 83.32 14.37 Compound 35 127.20 13.69
TABLE-US-00005 TABLE 4 Sox17 Expression Cell Number Average Average
Total % of positive Total % of positive Compound # Treatments Cell
Number control Intensity control none no Activin A (with Wnt3a)
7107 67.96 -1.27E+07 -7.94 none Activin A/Wnt3a 10459 100.00
1.60E+08 100.00 Compound 17 no AA (with Wnt3a) EGF 6942 73.43
1.27E+06 0.74 Compound 17 no AA (with Wnt3a) EGF + FGF4 5738 60.69
3.14E+06 1.83 Compound 17 no AA (with Wnt3a) EGF + FGF4 + PDGF-AB
4453 47.10 9.30E+05 0.54 Compound 17 no AA (with Wnt3a) EGF + FGF4
+ PDGF-AB + Muscimol 10391 109.91 8.92E+06 5.20 Compound 17 no AA
(with Wnt3a) EGF + PDGF-A + VEGF 5728 60.59 2.14E+06 1.24 Compound
17 no AA (with Wnt3a) FGF4 + PDGF-A + VEGF 13198 139.59 1.29E+07
7.54 Compound 17 no AA (with Wnt3a) EGF + FGF4 + PDGF-A + VEGF
10480 110.85 8.97E+06 5.23 Compound 17 no AA (with Wnt3a) EGF +
FGF4 + PDGF-A + Muscimol 13649 144.37 1.45E+07 8.43 none no Activin
A (with Wnt3a) 3117 34.86 -1.41E+06 -0.72 none Activin A/Wnt3a 8942
100.00 1.95E+08 100.00 Compound 35 no AA (with Wnt3a) EGF 19334
216.23 6.62E+07 33.86 Compound 35 no AA (with Wnt3a) PDGF-AB 16662
186.34 4.95E+07 25.33 Compound 35 no AA (with Wnt3a) PDGF-A 16885
188.84 4.48E+07 22.94 Compound 35 no AA (with Wnt3a) VEGF 18263
204.25 3.51E+07 17.98 Compound 35 no AA (with Wnt3a) FGF4 4410
49.32 3.33E+07 17.04 Compound 35 no AA (with Wnt3a) Muscimol 18867
211.00 2.61E+07 13.35 Compound 35 no AA (with Wnt3a) PDGF-C 16642
186.12 1.85E+07 9.46 Compound 35 no AA (with Wnt3a) PDGF-D 17618
197.03 1.84E+07 9.41 Compound 35 no AA (with Wnt3a) PDGF-B 14168
158.46 1.52E+07 7.76 Compound 35 no AA (with Wnt3a) PD98059 18877
211.11 1.30E+07 6.64 Compound 35 no AA (with Wnt3a) BMP1 18849
210.81 1.29E+07 6.59 Compound 35 no AA (with Wnt3a) LY294002 18374
205.49 1.03E+07 5.28 Compound 35 no AA (with Wnt3a) BMP4 16748
187.31 8.97E+06 4.59 Compound 35 no AA (with Wnt3a) BMP2 16218
181.38 8.89E+06 4.55 Compound 35 no AA (with Wnt3a) BMP7 20111
224.91 8.05E+06 4.12 Compound 35 no AA (with Wnt3a) U0124 16539
184.97 7.54E+06 3.86 Compound 35 no AA (with Wnt3a) BMP6 17838
199.50 7.32E+06 3.75 Compound 35 no AA (with Wnt3a) BMP2/7 12042
134.67 7.08E+06 3.62 Compound 35 no AA (with Wnt3a) bicuculline
19312 215.98 1.95E+06 1.00 Compound 35 no AA (with Wnt3a) U0126
19961 223.24 -5.75E+05 -0.29 Compound 35 no AA (with Wnt3a)
Butyrate 14238 159.24 -1.85E+06 -0.94 none no Activin A (with
Wnt3a) 6049 45.2 -1.31E+07 -5.2 none Activin A/Wnt3a 13392 100.0
2.50E+08 100.0 Compound 20 EGF, FGF, PDGF-A, VEGF, PDGF- 9434 70.4
1.48E+08 59.1 D, muscimol, GDF8 Compound 17 EGF, FGF, PDGF-A, VEGF,
PDGF- 7988 59.6 1.13E+08 45.0 D, muscimol, GDF8 Compound 16 EGF,
FGF, PDGF-A, VEGF, PDGF- 8303 62.0 9.20E+07 36.7 D, muscimol, GDF8
Compound 13 EGF, FGF, PDGF-A, VEGF, PDGF- 7045 52.6 7.22E+07 28.8
D, muscimol, GDF8 Compound 19 EGF, FGF, PDGF-A, VEGF, PDGF- 7799
58.2 6.82E+07 27.2 D, muscimol, GDF8 Compound 92 EGF, FGF, PDGF-A,
VEGF, PDGF- 5886 44.0 5.63E+07 22.5 D, muscimol, GDF8 Compound 93
EGF, FGF, PDGF-A, VEGF, PDGF- 5463 40.8 4.38E+07 17.5 D, muscimol,
GDF8 Compound 94 EGF, FGF, PDGF-A, VEGF, PDGF- 5100 38.1 4.18E+07
16.7 D, muscimol, GDF8 Compound 95 EGF, FGF, PDGF-A, VEGF, PDGF-
4510 33.7 3.32E+07 13.3 D, muscimol, GDF8 Compound 96 EGF, FGF,
PDGF-A, VEGF, PDGF- 4570 34.1 3.09E+07 12.3 D, muscimol, GDF8
Compound 97 EGF, FGF, PDGF-A, VEGF, PDGF- 4561 34.1 2.15E+07 8.6 D,
muscimol, GDF8 Compound 98 EGF, FGF, PDGF-A, VEGF, PDGF- 3176 23.7
9.86E+06 3.9 D, muscimol, GDF8 Compound 99 EGF, FGF, PDGF-A, VEGF,
PDGF- 1209 9.0 -1.56E+07 -6.2 D, muscimol, GDF8 none no Activin A
(with Wnt3a) 15494 98.0 -1.25E+07 -4.4 none Activin A/Wnt3a 15807
100.0 2.86E+08 100.0 Compound 18 EGF, FGF, PDGF-A, VEGF, PDGF- 8742
55.3 1.01E+08 35.4 D, muscimol, GDF8 Compound 14 EGF, FGF, PDGF-A,
VEGF, PDGF- 8464 53.5 8.33E+07 29.1 D, muscimol, GDF8 Compound 15
EGF, FGF, PDGF-A, VEGF, PDGF- 7234 45.8 7.95E+07 27.8 D, muscimol,
GDF8 Compound 100 EGF, FGF, PDGF-A, VEGF, PDGF- 6805 43.0 5.88E+07
20.6 D, muscimol, GDF8 Compound 101 EGF, FGF, PDGF-A, VEGF, PDGF-
5668 35.9 5.34E+07 18.7 D, muscimol, GDF8 Compound 102 EGF, FGF,
PDGF-A, VEGF, PDGF- 6195 39.2 5.29E+07 18.5 D, muscimol, GDF8
Compound 103 EGF, FGF, PDGF-A, VEGF, PDGF- 7545 47.7 5.13E+07 18.0
D, muscimol, GDF8 Compound 104 EGF, FGF, PDGF-A, VEGF, PDGF- 4757
30.1 4.58E+07 16.0 D, muscimol, GDF8 Compound 105 EGF, FGF, PDGF-A,
VEGF, PDGF- 6285 39.8 4.29E+07 15.0 D, muscimol, GDF8 Compound 106
EGF, FGF, PDGF-A, VEGF, PDGF- 5622 35.6 2.86E+07 10.0 D, muscimol,
GDF8 Compound 107 EGF, FGF, PDGF-A, VEGF, PDGF- 3951 25.0 1.72E+07
6.0 D, muscimol, GDF8 Compound 108 EGF, FGF, PDGF-A, VEGF, PDGF-
3226 20.4 1.58E+07 5.5 D, muscimol, GDF8 Compound 109 EGF, FGF,
PDGF-A, VEGF, PDGF- 3473 22.0 1.46E+07 5.1 D, muscimol, GDF8
Compound 110 EGF, FGF, PDGF-A, VEGF, PDGF- 3703 23.4 1.32E+07 4.6
D, muscimol, GDF8 Compound 111 EGF, FGF, PDGF-A, VEGF, PDGF- 2918
18.5 1.22E+07 4.3 D, muscimol, GDF8 Compound 112 EGF, FGF, PDGF-A,
VEGF, PDGF- 2975 18.8 1.04E+07 3.6 D, muscimol, GDF8 Compound 113
EGF, FGF, PDGF-A, VEGF, PDGF- 2910 18.4 9.18E+06 3.2 D, muscimol,
GDF8 Compound 114 EGF, FGF, PDGF-A, VEGF, PDGF- 2734 17.3 6.13E+06
2.1 D, muscimol, GDF8 Compound 115 EGF, FGF, PDGF-A, VEGF, PDGF-
2169 13.7 3.77E+06 1.3 D, muscimol, GDF8 Compound 116 EGF, FGF,
PDGF-A, VEGF, PDGF- 3107 19.7 3.52E+06 1.2 D, muscimol, GDF8
Compound 117 EGF, FGF, PDGF-A, VEGF, PDGF- 3343 21.1 5.35E+05 0.2
D, muscimol, GDF8 Compound 118 EGF, FGF, PDGF-A, VEGF, PDGF- 3034
19.2 2.37E+05 0.1 D, muscimol, GDF8 Compound 119 EGF, FGF, PDGF-A,
VEGF, PDGF- 2263 14.3 -1.66E+06 -0.6 D, muscimol, GDF8 Compound 120
EGF, FGF, PDGF-A, VEGF, PDGF- 1771 11.2 -5.57E+06 -2.0 D, muscimol,
GDF8 Compound 121 EGF, FGF, PDGF-A, VEGF, PDGF- 1136 7.2 -1.79E+07
-6.3 D, muscimol, GDF8 Compound 122 EGF, FGF, PDGF-A, VEGF, PDGF-
2021 12.8 -2.09E+07 -7.3 D, muscimol, GDF8
TABLE-US-00006 TABLE 5 Sox17 Expression Cell Number Average Average
Total % of positive Total % of positive Compound # Treatments Cell
Number control Intensity control none no Activin A (with Wnt3a)
7107 67.96 -1.27E+07 -7.94 none Activin A/Wnt3a 10459 100.00
1.60E+08 100.00 Compound 17 no AA (with Wnt3a) EGF 6942 73.43
1.27E+06 0.74 Compound 17 no AA (with Wnt3a) EGF + FGF4 5738 60.69
3.14E+06 1.83 Compound 17 no AA (with Wnt3a) EGF + FGF4 + PDGF-AB
4453 47.10 9.30E+05 0.54 Compound 17 no AA (with Wnt3a) EGF + FGF4
+ PDGF-AB + Muscimol 10391 109.91 8.92E+06 5.20 Compound 17 no AA
(with Wnt3a) EGF + PDGF-A + VEGF 5728 60.59 2.14E+06 1.24 Compound
17 no AA (with Wnt3a) FGF4 + PDGF-A + VEGF 13198 139.59 1.29E+07
7.54 Compound 17 no AA (with Wnt3a) EGF + FGF4 + PDGF-A + VEGF
10480 110.85 8.97E+06 5.23 Compound 17 no AA (with Wnt3a) EGF +
FGF4 + PDGF-A + Muscimol 13649 144.37 1.45E+07 8.43 none no Activin
A (with Wnt3a) 7107 67.96 -1.27E+07 -7.94 none Activin A/Wnt3a
10459 100.00 1.60E+08 100.00 Compound 35 no AA (with Wnt3a) EGF
23887 228.40 -1.01E+07 -6.32 Compound 35 no AA (with Wnt3a) EGF +
FGF4 21268 203.36 1.36E+06 0.85 Compound 35 no AA (with Wnt3a) EGF
+ FGF4 + PDGF-AB 17611 168.39 1.28E+07 8.03 Compound 35 no AA (with
Wnt3a) EGF + FGF4 + PDGF-AB + Muscimol 17949 171.62 1.54E+06 0.96
Compound 35 no AA (with Wnt3a) EGF + PDGF-A + VEGF 23242 222.23
1.23E+07 7.72 Compound 35 no AA (with Wnt3a) FGF4 + PDGF-A + VEGF
16068 153.63 3.92E+07 24.57 Compound 35 no AA (with Wnt3a) EGF +
FGF4 + PDGF-A + VEGF 16132 154.25 9.11E+07 57.04 Compound 35 no AA
(with Wnt3a) EGF + FGF4 + PDGF-A + Muscimol 15457 147.80 6.89E+07
43.15 Compound 29 no AA (with Wnt3a) EGF 1971 18.84 -1.44E+07 -9.00
Compound 29 no AA (with Wnt3a) EGF + FGF4 7436 71.10 -4.35E+06
-2.72 Compound 29 no AA (with Wnt3a) EGF + FGF4 + PDGF-AB 6535
62.48 -7.52E+06 -4.71 Compound 29 no AA (with Wnt3a) EGF + FGF4 +
PDGF-AB + Muscimol 1376 13.15 -1.42E+07 -8.91 Compound 29 no AA
(with Wnt3a) EGF + PDGF-A + VEGF 8880 84.91 -8.53E+06 -5.34
Compound 29 no AA (with Wnt3a) FGF4 + PDGF-A + VEGF 8146 77.89
-4.82E+06 -3.02 Compound 29 no AA (with Wnt3a) EGF + FGF4 + PDGF-A
+ VEGF 8858 84.70 -7.15E+06 -4.48 Compound 29 no AA (with Wnt3a)
EGF + FGF4 + PDGF-A + Muscimol 10071 96.30 2.95E+06 1.85 Compound
37 no AA (with Wnt3a) EGF 7966 76.17 -1.19E+07 -7.42 Compound 37 no
AA (with Wnt3a) EGF + FGF4 6932 66.28 -4.62E+06 -2.89 Compound 37
no AA (with Wnt3a) EGF + FGF4 + PDGF-AB 7473 71.46 -2.61E+06 -1.63
Compound 37 no AA (with Wnt3a) EGF + FGF4 + PDGF-AB + Muscimol 7914
75.67 -1.91E+06 -1.20 Compound 37 no AA (with Wnt3a) EGF + PDGF-A +
VEGF 12956 123.88 -1.25E+07 -7.82 Compound 37 no AA (with Wnt3a)
FGF4 + PDGF-A + VEGF 6731 64.36 -1.10E+07 -6.89 Compound 37 no AA
(with Wnt3a) EGF + FGF4 + PDGF-A + VEGF 8778 83.93 1.39E+05 0.09
Compound 37 no AA (with Wnt3a) EGF + FGF4 + PDGF-A + Muscimol 5821
55.66 -1.22E+07 -7.64 Compound 34 no AA (with Wnt3a) EGF 13062
124.89 2.78E+07 17.39 Compound 34 no AA (with Wnt3a) EGF + FGF4
13133 125.58 1.23E+08 76.85 Compound 34 no AA (with Wnt3a) EGF +
FGF4 + PDGF-AB 12532 119.83 1.09E+08 68.41 Compound 34 no AA (with
Wnt3a) EGF + FGF4 + PDGF-AB + Muscimol 15811 151.18 6.90E+06 4.32
Compound 34 no AA (with Wnt3a) EGF + PDGF-A + VEGF 11801 112.84
4.04E+06 2.53 Compound 34 no AA (with Wnt3a) FGF4 + PDGF-A + VEGF
15262 145.93 1.15E+07 7.18 Compound 34 no AA (with Wnt3a) EGF +
FGF4 + PDGF-A + VEGF 12901 123.36 5.01E+07 31.35 Compound 34 no AA
(with Wnt3a) EGF + FGF4 + PDGF-A + Muscimol 12208 116.72 5.56E+07
34.83 none no Activin A (with Wnt3a) 10224 108.14 7.36E+05 0.43
none Activin A/Wnt3a 9455 100.00 1.72E+08 100.00 Compound 39 no AA
(with Wnt3a) EGF 11615 122.85 1.49E+05 0.09 Compound 39 no AA (with
Wnt3a) EGF + FGF4 10456 110.59 5.11E+06 2.98 Compound 39 no AA
(with Wnt3a) EGF + FGF4 + PDGF-AB 9972 105.47 1.62E+06 0.94
Compound 39 no AA (with Wnt3a) EGF + FGF4 + PDGF-AB + Muscimol
10540 111.48 2.22E+06 1.29 Compound 39 no AA (with Wnt3a) EGF +
PDGF-A + VEGF 17050 180.34 4.84E+06 2.82 Compound 39 no AA (with
Wnt3a) FGF4 + PDGF-A + VEGF 8856 93.67 7.01E+05 0.41 Compound 39 no
AA (with Wnt3a) EGF + FGF4 + PDGF-A + VEGF 7973 84.33 5.30E+06 3.09
Compound 39 no AA (with Wnt3a) EGF + FGF4 + PDGF-A + Muscimol 9103
96.28 7.32E+05 0.43 Compound 22 no AA (with Wnt3a) EGF 14105 149.19
1.75E+06 1.02 Compound 22 no AA (with Wnt3a) EGF + FGF4 12971
137.19 1.04E+07 6.05 Compound 22 no AA (with Wnt3a) EGF + FGF4 +
PDGF-AB 16580 175.36 8.60E+06 5.01 Compound 22 no AA (with Wnt3a)
EGF + FGF4 + PDGF-AB + Muscimol 14676 155.23 5.61E+06 3.27 Compound
22 no AA (with Wnt3a) EGF + PDGF-A + VEGF 20372 215.48 4.99E+06
2.91 Compound 22 no AA (with Wnt3a) FGF4 + PDGF-A + VEGF 12277
129.85 4.90E+06 2.86 Compound 22 no AA (with Wnt3a) EGF + FGF4 +
PDGF-A + VEGF 12522 132.44 7.88E+06 4.59 Compound 22 no AA (with
Wnt3a) EGF + FGF4 + PDGF-A + Muscimol 11610 122.80 1.33E+07
7.77
TABLE-US-00007 TABLE 6 Cell Number Sox17 Expression Average Total %
of positive Average Total % of positive Compound # Treatments Cell
Number control Intensity control none no Activin A (with Wnt3a) 477
6.64 7.4E+04 0.09 none Activn A/Wnt3a 7185 100.00 8.0E+07 100.00
Compound no AA (with Wnt3a) none 4611 64.18 1.4E+07 17.21 34
Compound no AA (with Wnt3a) EGF 6145 85.53 1.5E+07 19.18 34
Compound no AA (with Wnt3a) FGF4 5323 74.09 2.7E+07 33.75 34
Compound no AA (with Wnt3a) PDGF-D 5017 69.84 1.5E+07 18.76 34
Compound no AA (with Wnt3a) PDGF-A 4175 58.11 1.1E+07 13.43 34
Compound no AA (with Wnt3a) VEGF 4713 65.60 1.0E+07 12.49 34
Compound no AA (with Wnt3a) GDF8 6354 88.44 7.1E+07 88.59 34
Compound no AA (with Wnt3a) Muscimol 7286 101.41 3.1E+07 38.38 34
Compound no AA (with Wnt3a) PDGF-D + VEGF 5030 70.01 1.2E+07 14.58
34 Compound no AA (with Wnt3a) VEGF + Muscimol 776 10.81 1.3E+06
1.56 34 Compound no AA (with Wnt3a) PDGF-D + Muscimol 3490 48.57
6.5E+06 8.02 34 Compound no AA (with Wnt3a) GDF8 + PDGF-D 6889
95.88 5.8E+07 72.59 34 Compound no AA (with Wnt3a) PDGF-D +
Muscimol + VEGF 2133 29.68 2.7E+06 3.32 34 Compound no AA (with
Wnt3a) GDF8 + PDGF-D + VEGF 5585 77.74 6.6E+07 81.75 34 Compound no
AA (with Wnt3a) GDF8 + VEGF + Muscimol 6083 84.67 5.6E+07 69.62 34
Compound no AA (with Wnt3a) GDF8 + PDGF- 9455 131.60 9.6E+07 119.24
34 D + VEGF + Muscimol Compound no AA, no Wnt3a EGF + FGF4 + PDGF-
4757 66.21 3.9E+07 48.77 34 A + VEGF + PDGF- D + Muscimol + GDF8
Compound no AA (with Wnt3a) EGF + FGF4 + PDGF- 6028 83.90 7.0E+07
87.44 34 A + VEGF + PDGF- D + Muscimol + GDF8
TABLE-US-00008 TABLE 7 Cell Number Sox17 Expression Average Average
Total Cell % of positive Total % of positive Plate Treatment
Compound # Number control Intensity control 1 no Activin A (with
Wnt3a) none 6049 45.2 -1.31E+07 -5.2 1 Activin A/Wnt3a none 13392
100.0 2.50E+08 100.0 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 18 13037 97.3 1.63E+08 65.2 GDF8 1 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 14 9344 69.8 1.23E+08 49.0 GDF8 1 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 15 8448 63.1 8.64E+07
34.5 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 16
5498 41.1 6.56E+07 26.2 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D,
muscimol, Compound 64 5063 37.8 5.88E+07 23.5 GDF8 1 EGF, FGF,
PDGF-A, VEGF, PDGF-D, muscimol, Compound 65 4788 35.8 4.57E+07 18.2
GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 66 8129
60.7 3.53E+07 14.1 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 67 6791 50.7 3.18E+07 12.7 GDF8 1 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 68 3456 25.8 2.30E+07 9.2 GDF8 1 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 69 3995 29.8 1.69E+07
6.8 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 70
474 3.5 -1.80E+07 -7.2 GDF8 2 no Activin A (with Wnt3a) none 15494
98.0 -1.25E+07 -4.4 2 Activin A/Wnt3a none 15807 100.0 2.86E+08
100.0 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 19 8425
53.3 1.19E+08 41.6 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 13 9123 57.7 1.13E+08 39.7 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 71 6048 38.3 5.51E+07 19.3 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 72 6060 38.3 5.46E+07
19.1 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 73
5545 35.1 3.99E+07 14.0 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D,
muscimol, Compound 74 10898 68.9 3.91E+07 13.7 GDF8 2 EGF, FGF,
PDGF-A, VEGF, PDGF-D, muscimol, Compound 75 4117 26.0 3.01E+07 10.5
GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 76 3825
24.2 2.74E+07 9.6 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 77 5928 37.5 2.44E+07 8.5 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 78 3303 20.9 2.03E+07 7.1 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 79 4767 30.2 1.85E+07
6.5 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 80
2194 13.9 1.22E+07 4.3 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D,
muscimol, Compound 81 2920 18.5 9.16E+05 0.3 GDF8 2 EGF, FGF,
PDGF-A, VEGF, PDGF-D, muscimol, Compound 82 1819 11.5 -1.05E+07
-3.7 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 83
2153 13.6 -1.19E+07 -4.2 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D,
muscimol, Compound 84 58 0.4 -2.94E+07 -10.3 GDF8 2 EGF, FGF,
PDGF-A, VEGF, PDGF-D, muscimol, Compound 85 57 0.4 -3.03E+07 -10.6
GDF8 1 no Activin A (with Wnt3a) none 6049 45.2 -1.31E+07 -5.2 1
Activin A/Wnt3a none 13392 100.0 2.50E+08 100.0 1 EGF, FGF, PDGF-A,
VEGF, PDGF-D, muscimol, Compound 20 9434 70.4 1.48E+08 59.1 GDF8 1
EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 17 7988 59.6
1.13E+08 45.0 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 16 8303 62.0 9.20E+07 36.7 GDF8 1 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 13 7045 52.6 7.22E+07 28.8 GDF8 1 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 19 7799 58.2 6.82E+07
27.2 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 92
5886 44.0 5.63E+07 22.5 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D,
muscimol, Compound 93 5463 40.8 4.38E+07 17.5 GDF8 1 EGF, FGF,
PDGF-A, VEGF, PDGF-D, muscimol, Compound 94 5100 38.1 4.18E+07 16.7
GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 95 4510
33.7 3.32E+07 13.3 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 96 4570 34.1 3.09E+07 12.3 GDF8 1 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 97 4561 34.1 2.15E+07 8.6 GDF8 1 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 98 3176 23.7 9.86E+06
3.9 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 99
1209 9.0 -1.56E+07 -6.2 GDF8 2 no Activin A (with Wnt3a) none 15494
98.0 -1.25E+07 -4.4 2 Activin A/Wnt3a none 15807 100.0 2.86E+08
100.0 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 18 8742
55.3 1.01E+08 35.4 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 14 8464 53.5 8.33E+07 29.1 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 15 7234 45.8 7.95E+07 27.8 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 100 6805 43.0
5.88E+07 20.6 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 101 5668 35.9 5.34E+07 18.7 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 102 6195 39.2 5.29E+07 18.5 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 103 7545 47.7
5.13E+07 18.0 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 104 4757 30.1 4.58E+07 16.0 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 105 6285 39.8 4.29E+07 15.0 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 106 5622 35.6
2.86E+07 10.0 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 107 3951 25.0 1.72E+07 6.0 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 108 3226 20.4 1.58E+07 5.5 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 109 3473 22.0
1.46E+07 5.1 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 110 3703 23.4 1.32E+07 4.6 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 111 2918 18.5 1.22E+07 4.3 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 112 2975 18.8
1.04E+07 3.6 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 113 2910 18.4 9.18E+06 3.2 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 114 2734 17.3 6.13E+06 2.1 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 115 2169 13.7
3.77E+06 1.3 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 116 3107 19.7 3.52E+06 1.2 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 117 3343 21.1 5.35E+05 0.2 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 118 3034 19.2
2.37E+05 0.1 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 119 2263 14.3 -1.66E+06 -0.6 GDF8 2 EGF, FGF, PDGF-A,
VEGF, PDGF-D, muscimol, Compound 120 1771 11.2 -5.57E+06 -2.0 GDF8
2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 121 1136 7.2
-1.79E+07 -6.3 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 122 2021 12.8 -2.09E+07 -7.3 GDF8 1 no Activin A (with
Wnt3a) none 6049 45.2 -1.31E+07 -5.2 1 Activin A/Wnt3a none 13392
100.0 2.50E+08 100.0 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 19 15878 118.6 2.67E+08 106.5 GDF8 1 EGF, FGF, PDGF-A,
VEGF, PDGF-D, muscimol, Compound 24 12714 94.9 2.46E+08 98.2 GDF8 1
EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 23 12165 90.8
2.15E+08 86.0 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 21 12640 94.4 1.65E+08 65.9 GDF8 1 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 13 11491 85.8 1.61E+08 64.3 GDF8 1 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 30 11396 85.1
1.34E+08 53.4 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 36 7964 59.5 9.47E+07 37.8 GDF8 1 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 32 8066 60.2 9.29E+07 37.1 GDF8 1 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 26 7415 55.4 8.30E+07
33.1 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 17
6994 52.2 7.76E+07 31.0 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D,
muscimol, Compound 31 6957 51.9 6.59E+07 26.3
GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 179 3573
26.7 2.43E+07 9.7 GDF8 1 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 180 922 6.9 -2.20E+07 -8.8 GDF8 1 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 181 8 0.1 -2.68E+07 -10.7 GDF8 2 no
Activin A (with Wnt3a) none 15494 98.0 -1.25E+07 -4.4 2 Activin
A/Wnt3a none 15807 100.0 2.86E+08 100.0 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 18 21102 133.5 4.18E+08 146.3 GDF8 2
EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 15 15373 97.3
3.74E+08 130.8 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 14 9008 57.0 2.62E+08 91.6 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 38 9650 61.0 2.46E+08 86.2 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 35 10461 66.2
1.59E+08 55.7 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 16 9064 57.3 1.48E+08 51.8 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 34 8907 56.3 9.99E+07 35.0 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 20 7346 46.5 8.90E+07
31.2 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 27
8044 50.9 8.81E+07 30.8 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D,
muscimol, Compound 28 7591 48.0 8.77E+07 30.7 GDF8 2 EGF, FGF,
PDGF-A, VEGF, PDGF-D, muscimol, Compound 40 4049 25.6 8.23E+07 28.8
GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 33 7485
47.4 8.10E+07 28.3 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 25 6571 41.6 7.60E+07 26.6 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 182 7631 48.3 6.74E+07 23.6 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 183 6777 42.9
5.93E+07 20.8 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 184 5475 34.6 5.44E+07 19.0 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 185 4093 25.9 4.92E+07 17.2 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 186 5274 33.4
4.63E+07 16.2 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 187 5342 33.8 4.02E+07 14.1 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 188 5533 35.0 3.98E+07 13.9 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 189 5928 37.5
3.96E+07 13.9 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 190 4822 30.5 3.90E+07 13.7 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 191 4249 26.9 3.81E+07 13.3 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 192 5616 35.5
3.54E+07 12.4 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 193 4158 26.3 3.23E+07 11.3 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 194 3470 22.0 2.96E+07 10.4 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 195 3800 24.0
2.95E+07 10.3 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 196 4619 29.2 2.78E+07 9.7 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 197 4011 25.4 2.45E+07 8.6 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 198 4367 27.6
1.92E+07 6.7 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 199 3162 20.0 1.20E+07 4.2 GDF8 2 EGF, FGF, PDGF-A, VEGF,
PDGF-D, muscimol, Compound 200 2087 13.2 4.43E+06 1.6 GDF8 2 EGF,
FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 201 1568 9.9
-6.17E+06 -2.2 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 202 5213 33.0 -1.41E+07 -4.9 GDF8 2 EGF, FGF, PDGF-A,
VEGF, PDGF-D, muscimol, Compound 203 7 0.0 -3.04E+07 -10.6 GDF8 2
EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol, Compound 204 11 0.1
-3.18E+07 -11.1 GDF8 2 EGF, FGF, PDGF-A, VEGF, PDGF-D, muscimol,
Compound 205 10 0.1 -3.20E+07 -11.2 GDF8
TABLE-US-00009 TABLE 8 Cell Number Sox17 Expression Average % of
Average % of Total positive Total positive Compound # Cell Number
control Intensity control Compound 18 21102 133.5 4.18E+08 146.3
Compound 15 15373 97.3 3.74E+08 130.8 Compound 19 15878 118.6
2.67E+08 106.5 Compound 24 12714 94.9 2.46E+08 98.2 Compound 14
9008 57.0 2.62E+08 91.6 Compound 38 9650 61.0 2.46E+08 86.2
Compound 23 12165 90.8 2.15E+08 86.0 Compound 21 12640 94.4
1.65E+08 65.9 Compound 13 11491 85.8 1.61E+08 64.3 Compound 35
10461 66.2 1.59E+08 55.7 Compound 30 11396 85.1 1.34E+08 53.4
Compound 16 9064 57.3 1.48E+08 51.8 Compound 36 7964 59.5 9.47E+07
37.8 Compound 32 8066 60.2 9.29E+07 37.1 Compound 34 8907 56.3
9.99E+07 35.0 Compound 26 7415 55.4 8.30E+07 33.1 Compound 20 7346
46.5 8.90E+07 31.2 Compound 17 6994 52.2 7.76E+07 31.0 Compound 27
8044 50.9 8.81E+07 30.8 Compound 28 7591 48.0 8.77E+07 30.7
Compound 40 4049 25.6 8.23E+07 28.8 Compound 33 7485 47.4 8.10E+07
28.3 Compound 25 6571 41.6 7.60E+07 26.6 Compound 31 6957 51.9
6.59E+07 26.3 Compound 20 9434 70.4 1.48E+08 59.1 Compound 17 7988
59.6 1.13E+08 45.0 Compound 16 8303 62.0 9.20E+07 36.7 Compound 18
8742 55.3 1.01E+08 35.4 Compound 14 8464 53.5 8.33E+07 29.1
Compound 13 7045 52.6 7.22E+07 28.8 Compound 15 7234 45.8 7.95E+07
27.8 Compound 19 7799 58.2 6.82E+07 27.2
TABLE-US-00010 TABLE 9 Cell Number Sox17 Expression Average % of
Average % of Treatments Total Cell positive Total positive Plate #
Activin A Compound # Growth Factors Number control Intensity
control 1 none none none 9164 149.46 -5.91E+06 -5.17 1 10 ng/ml AA
none none 6132 100.00 1.52E+06 1.33 1 100 ng/ml AA none none 9658
157.51 1.14E+08 100.00 1 10 ng/ml AA Compound 22 EGF + FGF4 +
PDGF-A + VEGF + PDGF- 8556 139.53 8.78E+07 76.82 D + Muscimol +
GDF8 + Wnt3a 1 10 ng/ml AA Compound 22 EGF + FGF4 + PDGF-AB + VEGF
+ Wnt3a 7657 124.87 4.70E+07 41.09 1 10 ng/ml AA Compound 22 EGF +
FGF4 + PDGF-A + Muscimol + Wnt3a 8100 132.10 4.42E+07 38.65 1 10
ng/ml AA Compound 22 EGF + FGF4 + PDGF-AB + Wnt3a 7975 130.06
3.43E+07 30.03 1 10 ng/ml AA Compound 22 EGF + FGF4 + Wnt3a 9800
159.83 4.59E+07 40.13 1 10 ng/ml AA Compound 22 FGF4 + Wnt3a 6490
105.84 4.28E+07 37.43 1 10 ng/ml AA Compound 22 EGF + Wnt3a 5001
81.55 2.80E+07 24.45 1 10 ng/ml AA Compound 22 Wnt3a 4543 74.09
3.05E+07 26.65 1 10 ng/ml AA Compound 35 EGF + FGF4 + PDGF-A + VEGF
+ PDGF- 2522 41.14 -4.86E+06 -4.25 D + Muscimol + GDF8 + Wnt3a 1 10
ng/ml AA Compound 35 EGF + FGF4 + PDGF-AB + VEGF + Wnt3a 3479 56.74
-3.96E+06 -3.46 1 10 ng/ml AA Compound 35 EGF + FGF4 + PDGF-A +
Muscimol + Wnt3a 3820 62.29 -1.67E+06 -1.46 1 10 ng/ml AA Compound
35 EGF + FGF4 + PDGF-AB + Wnt3a 3263 53.21 -4.56E+06 -3.99 1 10
ng/ml AA Compound 35 EGF + FGF4 + Wnt3a 2704 44.10 -4.17E+06 -3.65
1 10 ng/ml AA Compound 35 FGF4 + Wnt3a 284 4.64 -7.54E+06 -6.59 1
10 ng/ml AA Compound 35 EGF + Wnt3a 155 2.53 -7.82E+06 -6.84 1 10
ng/ml AA Compound 35 Wnt3a 173 2.83 -7.61E+06 -6.66 1 10 ng/ml AA
Compound 29 EGF + FGF4 + PDGF-A + VEGF + PDGF- 2737 44.63 2.41E+07
21.10 D + Muscimol + GDF8 + Wnt3a 1 10 ng/ml AA Compound 29 EGF +
FGF4 + PDGF-AB + VEGF + Wnt3a 2283 37.23 5.59E+06 4.88 1 10 ng/ml
AA Compound 29 EGF + FGF4 + PDGF-A + Muscimol + Wnt3a 4676 76.26
2.41E+07 21.11 1 10 ng/ml AA Compound 29 EGF + FGF4 + PDGF-AB +
Wnt3a 3964 64.65 2.27E+07 19.89 1 10 ng/ml AA Compound 29 EGF +
FGF4 + Wnt3a 1736 28.31 1.98E+06 1.73 1 10 ng/ml AA Compound 29
FGF4 + Wnt3a 2139 34.89 6.98E+06 6.10 1 10 ng/ml AA Compound 29 EGF
+ Wnt3a 365 5.96 -4.86E+06 -4.25 1 10 ng/ml AA Compound 29 Wnt3a
2090 34.09 4.89E+06 4.28 2 none none none 9325 121.89 -3.35E+06
-3.01 2 10 ng/ml AA none none 5177 67.67 3.89E+06 3.49 2 100 ng/ml
AA none none 7650 100.00 1.11E+08 100.00 2 10 ng/ml AA Compound 34
EGF + FGF4 + PDGF-A + VEGF + PDGF- 18362 240.02 3.45E+08 309.74 D +
Muscimol + GDF8 + Wnt3a 2 10 ng/ml AA Compound 34 EGF + FGF4 +
PDGF-AB + VEGF + Wnt3a 15574 203.58 2.59E+08 232.70 2 10 ng/ml AA
Compound 34 EGF + FGF4 + PDGF-A + Muscimol + Wnt3a 17890 233.85
2.88E+08 258.30 2 10 ng/ml AA Compound 34 EGF + FGF4 + PDGF-AB +
Wnt3a 17875 233.65 2.68E+08 241.07 2 10 ng/ml AA Compound 34 EGF +
FGF4 + Wnt3a 14158 185.07 2.40E+08 215.35 2 10 ng/ml AA Compound 34
FGF4 + Wnt3a 13323 174.15 2.19E+08 196.86 2 10 ng/ml AA Compound 34
EGF + Wnt3a 14527 189.89 2.28E+08 204.84 2 10 ng/ml AA Compound 34
Wnt3a 3589 46.91 7.02E+07 63.08 2 10 ng/ml AA Compound 39 EGF +
FGF4 + PDGF-A + VEGF + PDGF- 5738 75.00 2.14E+07 19.24 D + Muscimol
+ GDF8 + Wnt3a 2 10 ng/ml AA Compound 39 EGF + FGF4 + PDGF-AB +
VEGF + Wnt3a 2531 33.08 2.82E+06 2.53 2 10 ng/ml AA Compound 39 EGF
+ FGF4 + PDGF-A + Muscimol + Wnt3a 2879 37.64 3.61E+06 3.24 2 10
ng/ml AA Compound 39 EGF + FGF4 + PDGF-AB + Wnt3a 2989 39.07
-1.78E+04 -0.02 2 10 ng/ml AA Compound 39 EGF + FGF4 + Wnt3a 734
9.59 -3.93E+06 -3.53 2 10 ng/ml AA Compound 39 FGF4 + Wnt3a 521
6.81 -4.46E+06 -4.01 2 10 ng/ml AA Compound 39 EGF + Wnt3a 211 2.75
-4.54E+06 -4.08 2 10 ng/ml AA Compound 39 Wnt3a 518 6.78 -2.37E+06
-2.13 2 10 ng/ml AA Compound 37 EGF + FGF4 + PDGF-A + VEGF + PDGF-
5711 74.65 1.21E+07 10.82 D + Muscimol + GDF8 + Wnt3a 2 10 ng/ml AA
Compound 37 EGF + FGF4 + PDGF-AB + VEGF + Wnt3a 4767 62.31
-5.16E+05 -0.46 2 10 ng/ml AA Compound 37 EGF + FGF4 + PDGF-A +
Muscimol + Wnt3a 4540 59.34 9.23E+05 0.83 2 10 ng/ml AA Compound 37
EGF + FGF4 + PDGF-AB + Wnt3a 4223 55.20 -6.15E+05 -0.55 2 10 ng/ml
AA Compound 37 EGF + FGF4 + Wnt3a 3501 45.77 5.60E+05 0.50 2 10
ng/ml AA Compound 37 FGF4 + Wnt3a 3930 51.37 -1.88E+06 -1.69 2 10
ng/ml AA Compound 37 EGF + Wnt3a 1431 18.70 -2.75E+06 -2.47 2 10
ng/ml AA Compound 37 Wnt3a 791 10.34 -2.99E+06 -2.68
TABLE-US-00011 TABLE 10 Cell Number Sox17 Expression Average
Average Total Cell % of positive Total % of positive Compound #
Treatments Number control Intensity control none No Activin A (with
Wnt3a) 4273 33.70 4.75E+07 17.49 none Activin A (with Wnt3a) 12676
100.00 2.72E+08 100.00 Compound 34 No AA (without Wnt3a) FGF (50
ng/ml) EGF (50 ng/ml) 13317 105.06 2.01E+08 74.08 Compound 34 No AA
(without Wnt3a) FGF (50 ng/ml) EGF (100 ng/ml) 14189 111.93
2.01E+08 73.90 Compound 34 No AA (without Wnt3a) FGF (100 ng/ml)
EGF (50 ng/ml) 12616 99.52 1.80E+08 66.21 Compound 34 No AA
(without Wnt3a) FGF (100 ng/ml) EGF (100 ng/ml) 8269 65.23 1.13E+08
41.73 Compound 34 No AA (with Wnt3a) none none 11711 92.38 1.65E+08
60.68 Compound 34 No AA (with Wnt3a) none EGF (25 ng/ml) 16052
126.63 2.14E+08 78.82 Compound 34 No AA (with Wnt3a) none EGF (50
ng/ml) 13593 107.23 1.94E+08 71.52 Compound 34 No AA (with Wnt3a)
none EGF (100 ng/ml) 13170 103.90 1.93E+08 71.04 Compound 34 No AA
(with Wnt3a) FGF (25 ng/ml) none 18433 145.41 2.49E+08 91.72
Compound 34 No AA (with Wnt3a) FGF (25 ng/ml) EGF (25 ng/ml) 18841
148.63 2.60E+08 95.72 Compound 34 No AA (with Wnt3a) FGF (25 ng/ml)
EGF (50 ng/ml) 16232 128.05 2.30E+08 84.79 Compound 34 No AA (with
Wnt3a) FGF (25 ng/ml) EGF (100 ng/ml) 9309 73.44 1.39E+08 51.00
Compound 34 No AA (with Wnt3a) FGF (50 ng/ml) none 12757 100.64
1.66E+08 61.10 Compound 34 No AA (with Wnt3a) FGF (50 ng/ml) EGF
(25 ng/ml) 17720 139.79 2.31E+08 85.01 Compound 34 No AA (with
Wnt3a) FGF (50 ng/ml) EGF (50 ng/ml) 16331 128.83 2.26E+08 83.11
Compound 34 No AA (with Wnt3a) FGF (50 ng/ml) EGF (100 ng/ml) 16336
128.87 2.32E+08 85.24 Compound 34 No AA (with Wnt3a) FGF (100
ng/ml) none 19853 156.61 2.59E+08 95.45 Compound 34 No AA (with
Wnt3a) FGF (100 ng/ml) EGF (25 ng/ml) 19880 156.83 2.59E+08 95.47
Compound 34 No AA (with Wnt3a) FGF (100 ng/ml) EGF (50 ng/ml) 18166
143.30 2.35E+08 86.30 Compound 34 No AA (with Wnt3a) FGF (100
ng/ml) EGF (100 ng/ml) 11241 88.68 1.55E+08 57.10 none No AA (with
Wnt3a) none EGF (50 ng/ml) 5558 43.85 5.01E+07 18.44 none No AA
(with Wnt3a) none EGF (100 ng/ml) 6818 53.79 6.42E+07 23.62 none No
AA (with Wnt3a) FGF (50 ng/ml) none 8494 67.01 6.62E+07 24.35 none
No AA (with Wnt3a) FGF (50 ng/ml) EGF (50 ng/ml) 10138 79.98
7.30E+07 26.87 none No AA (with Wnt3a) FGF (50 ng/ml) EGF (100
ng/ml) 10219 80.62 7.75E+07 28.51 none No AA (with Wnt3a) FGF (100
ng/ml) none 9944 78.45 6.68E+07 24.59 none No AA (with Wnt3a) FGF
(100 ng/ml) EGF (50 ng/ml) 11046 87.14 8.17E+07 30.07 none No AA
(with Wnt3a) FGF (100 ng/ml) EGF (100 ng/ml) 7695 60.71 6.87E+07
25.28
TABLE-US-00012 TABLE 11 Normalized SOX17 Intensity Activin A GDF8
ng/ml Average SD Average SD 1600 100.00 9.20 100.00 9.00 800 100.00
6.60 84.90 6.30 400 100.00 3.30 72.20 7.50 200 100.00 1.90 51.30
5.30 100 90.70 8.70 32.70 5.10 50 85.20 4.70 17.60 4.80 25 73.10
2.80 5.10 3.60 12.50 50.90 6.20 0.90 0.80 6.25 18.40 4.80 0.70 1.40
3.13 3.00 1.90 0.10 0.20 1.56 0.10 0.00 0.00 0.20 0.00 0.00 0.20
0.30 0.30
TABLE-US-00013 TABLE 12 Marker name Catalog #* AFP Hs00173490_m1
CD99 Hs00365982_m1 CD9 Hs00233521_m1 CDH1 Hs00170423_m1 CDH2
Hs00169953_m1 CDX2 Hs00230919_m1 CER1 Hs00193796_m1 CXCR4
Hs00237052_m1 FGF17 Hs00182599_m1 FGF4 Hs00173564_m1 FOXA2
Hs00232764_m1 GAPDH Hs99999905_m1 GATA4 Hs00171403_m1 GATA6
Hs00232018_m1 GSC Hs00418279_m1 HLXB9 Hs00232128_m1 KIT
Hs00174029_m1 MIXL1 Hs00430824_g1 NANOG Hs02387400_g1 OTX2
Hs00222238_m1 POU5F1 Hs00742896_s1 SOX17 Hs00751752_s1 SOX7
Hs00846731_s1 T Hs00610080_m1 ALB Hs00609411_m1 AMY2A Hs00420710_g1
ARX Hs00292465_m1 CDX2 Hs00230919_m1 GAPDH Hs99999905_m1 GCG
Hs00174967_m1 HNF4A Hs00230853_m1 INS Hs00355773_m1 ISL1
Hs00158126_m1 MAFA Hs01651425_s1 MAFB Hs00534343_s1 NEUROD1
Hs00159598_m1 NEUROG3 Hs00360700_g1 NKX2-2 Hs00159616_m1 NKX2-5
Hs00231763_m1 NKX6-1 Hs00232355_m1 PAX4 Hs00173014_m1 PAX6
Hs00240871_m1 PDX1 Hs00236830_m1 PECAM1 Hs00169777_m1 POU3F4
Hs00264887_s1 PTF1A Hs00603586_g1 SST Hs00356144_m1 ZIC1
Hs00602749_m1
TABLE-US-00014 TABLE 13 Differentiation Step 1 CT Values Treatment
GAPDH AFP CD9 CD99 CDH1 CDH2 CDX2 CER1 CXCR4 FGF17 FGF4 FOXA2
AA/Wnt3a 19.5 34.7 23.8 24.1 24.5 21.5 36.8 18.4 22.7 20 33.5 24.7
GDF8/Wnt3a 18.7 36.1 23 23.5 23.3 21 36.2 17.8 21.9 19.9 33.1 23.8
GDF8/Compound 34 18.5 33 23 23.1 23.6 20.9 35.3 17.9 21.3 19.7 32.6
24 GDF8/Compound 56 17 31.2 20.8 20.9 21.2 18.4 35.3 15.5 19.4 17.2
29.7 21.1 Differentiation Step 1 CT Values Treatment GATA4 GATA6
GSC KIT MIXL1 MNX1 NANOG OTX2 POU5F1 SOX17 SOX7 T AA/Wnt3a 23.7
22.1 22.3 25 23.4 28 23.8 22.6 31.4 23.5 32.2 32.3 GDF8/Wnt3a 23.7
21.9 22.1 23.9 23.1 28.6 23 21.9 29.6 23.4 31.9 32.2 GDF8/Compound
34 23.2 21.7 21.9 24 23 27.5 23.2 21.5 30.1 23.2 31.7 32.1
GDF8/Compound 56 20.8 19.6 20 21.5 21.5 25 21.1 19.3 27.9 21.8 31.1
30.3 Differentiation Step 3 CT Values Treatment\CTs GAPDH ALB AMY2A
ARX CDX2 GCG HNF4 INS ISL1 MAFA MAFB NEUROD1 AA/Wnt3a 18.7 23.1
30.2 30.8 22.4 34.1 21.1 34.9 27.9 35.1 26.8 30.6 GDF8/Wnt3a 18.4
23.1 29.7 30.9 22.5 34.5 21.1 34.7 27.4 34.6 26.9 30.5
GDF8/Compound 34 18.4 23.3 29.7 34.7 22.5 36.6 21.1 38.1 27.5 34.2
26.8 33.3 GDF8/Compound 56 18.2 23.5 29.7 31.6 22.5 36.3 21.2 35.7
27.3 34.4 27 30.6 CT Values Differentiation Step 3 NKX2- NKX2-
Treatment\CTs NEUROG3 2 5 NKX6-1 PAX4 PAX6 PDX1 PECAM1 POU3F4 PTF1A
SST ZIC1 AA/Wnt3a 28 29.3 33.1 38.5 30.6 36 25.6 28.3 29.5 38.4
30.3 32.7 GDF8/Wnt3a 28 30 36.4 36 31.1 33.5 25.4 30.4 30.1 38.5
27.9 32.4 GDF8/Compound 34 31 32.2 33.8 37.8 33.2 36.5 26.3 28.1
31.1 36.4 27.6 33.2 GDF8/Compound 56 27.9 30 33.8 38.2 30.8 32.7 25
28.9 30.4 34.8 27.5 32.7 Differentiation Step 4 CT Values
Treatment\CTs GAPDH ALB AMY2A ARX CDX2 GCG HNF4 INS ISL1 MAFA MAFB
NEUROD1 AA/Wnt3a 18.3 18.3 27.1 23.8 21.7 19.5 20.6 20.5 23.3 31
23.8 23 GDF8/Wnt3a 18.9 19.3 27.7 24.1 22.2 19.9 21.2 20.7 23.7
31.2 24.2 23.5 GDF8/Compound 34 18.9 18.9 27.1 25 22.3 21.5 21 22.2
24.4 31.3 24.6 24.6 GDF8/Compound 56 18.3 18.8 27.1 22.3 22.6 17.6
21.3 18.6 22.4 29.9 22.9 22.7 CT Values Differentiation Step 4
NKX2- NKX2- Treatment\CTs NEUROG3 2 5 NKX6-1 PAX4 PAX6 PDX1 PECAM1
POU3F4 PTF1A SST ZIC1 AA/Wnt3a 27.9 24.6 31.5 31.5 27.5 26.4 24.1
26.9 27.6 40 25.2 31.4 GDF8/Wnt3a 27.8 25 35.4 31.5 27.7 26.3 24.5
29.5 29.3 38.2 24.9 31.4 GDF8/Compound 34 29.7 25.8 31.6 29.6 27.9
26.4 23.9 27.5 28.1 37.1 24.7 29.5 GDF8/Compound 56 25.5 23.2 32
27.9 25.5 23.8 23.2 27.8 27.5 30.9 22.8 32.1 Differentiation Step 5
CT Values Treatment\CTs GAPDH ALB AMY2A ARX CDX2 GCG HNF4 INS ISL1
MAFA MAFB NEUROD1 AA/Wnt3a 18.3 18.5 27.8 24 22.3 18.2 21.4 16.5
23.4 31.3 23.7 24.1 GDF8/Wnt3a 19.3 19.7 28.5 24.2 23.1 18.2 22
16.7 23.4 31.1 23.4 24.6 GDF8/Compound 34 19.9 20.6 29 24.1 23.7
17.8 22.4 16.3 23.7 31.3 24 24.4 GDF8/Compound 56 20 21.1 29 25.1
24.8 18.3 23.2 17.1 24.6 32.1 24.9 24.8 CT Values Differentiation
Step 5 NKX2- NKX2- Treatment\CTs NEUROG3 2 5 NKX6-1 PAX4 PAX6 PDX1
PECAM1 POU3F4 PTF1A SST ZIC1 AA/Wnt3a 32.5 25.4 29.7 32 28 25.1
24.2 26.9 29 37.5 22.5 29.9 GDF8/Wnt3a 32.3 25.6 29.9 30.5 28.1
25.4 24.6 29.9 29.9 33.4 22.1 32.1 GDF8/Compound 34 32.4 25.8 31.3
32 28.4 25.6 24.6 29.7 30.7 34.5 21.6 35 GDF8/Compound 56 33.8 26.3
34 30.2 29.7 27.3 25.9 29.7 31.5 34.7 22.1 33.3 Differentiation
Step 6 CT Values Treatment\CTs GAPDH ALB AMY2A ARX CDX2 GCG HNF4
INS ISL1 MAFA MAFB NEUROD1 AA/Wnt3a 20.4 24.3 30.7 27.6 25.7 19.4
24.7 20.5 26.4 34.6 27.2 27.1 GDF8/Wnt3a 20.7 23.5 30.4 26.8 25.2
18.4 24.3 19.3 26.2 35.2 26.3 26.5 GDF8/Compound 34 21.3 24.6 31.3
27.1 26 18.4 24.7 20.1 26.3 34.8 26.4 27 GDF8/Compound 56 21.2 25
30.9 26 25.9 17.4 24.4 19.6 25.7 34.7 25.9 26.1 CT Values
Differentiation Step 6 NKX2- NKX2- Treatment\CTs NEUROG3 2 5 NKX6-1
PAX4 PAX6 PDX1 PECAM1 POU3F4 PTF1A SST ZIC1 AA/Wnt3a 40 29.6 30.7
33.4 30.9 28.6 29.2 31.3 32.8 38.4 22.2 34.8 GDF8/Wnt3a 35 29 32.3
30.5 30.7 27.8 28.5 32.3 31.5 33.9 22.4 27.4 GDF8/Compound 34 34.5
29.2 31.6 33.1 30.4 28.1 29 32.9 33.9 37.7 22.1 34.5 GDF8/Compound
56 34.3 28.1 33.8 30.7 29.3 27.2 27.9 33.6 33.2 35.2 21 34.9
TABLE-US-00015 TABLE 14 Differentiation Step 1 Ct Value Treatment
GAPDH AFP CD9 CD99 CDH1 CDH CDX2 CER1 CXCR4 FGF17 FGF4 FOXA2
AA/Wnt3a 20 35.6 24.1 24.2 26 20.9 40 17.5 22.7 19.8 35.8 24.7
GDF8/Wnt3a 20.1 34 23.8 24.5 24.6 21.6 40 19.5 23.3 21 34.8 25.1
GDF8/GSK3 inh BIO 19 34.4 23.7 24.1 24.3 21.3 36 18.7 23 20.1 33.5
24.2 GDF8/Compound 19 19.8 34.8 23.8 24 24.6 20.7 37.7 18.8 22.3 20
34.4 24.2 GDF8/Compound 202 19.8 40 24.5 23.5 25.9 20.8 40 18.8
22.2 20.3 36.5 24.4 GDF8/Compound 40 19.8 36.1 24.3 22.9 26.2 21.6
33.3 18.8 22.5 20.3 38.1 25.3 Differentiation Step 1 Ct Value
Treatment GATA4 GATA6 GSC HLXB9 KIT MIXL1 NANOG OTX2 POU5F1 SOX17
SOX7 T AA/Wnt3a 23.8 22.1 21.6 23.4 23.2 28.1 24.5 22 32.6 23.2 33
36.8 GDF8/Wnt3a 24.5 23.3 23.3 17.6 25.5 28.3 24.9 23 31 23.7 33.3
34.2 GDF8/GSK3 inh BIO 24.2 22.4 21.8 23.4 24.2 28.4 23.7 21.8 30.8
23 33.7 33.1 GDF8/Compound 19 23.6 22.5 21.9 23.1 24.3 28 24.3 21.8
31.3 22.3 33 32.9 GDF8/Compound 202 23.4 22.3 22.3 24 24.8 27.3 26
21.9 33.3 22.7 32.6 32.1 GDF8/Compound 40 23.4 23 23 25 25.7 27.8
26 22.3 32.8 23.2 27.2 29.3 Differentiation Step 3 Ct Value
Treatment GAPDH ALB AMY2A ARX CDX2 GCG HNF4A INS ISL1 MAFA MAFB
NEUROD1 AA/Wnt3a 17.9 25.4 29.5 28.4 23.3 34.1 21.8 29.2 29.4 34 27
25.8 GDF8/Wnt3a 18.5 26.5 30.4 29.4 23.9 34.2 22.6 29 29 34.4 27.1
27.2 GDF8/GSK3 inh BIO 18.5 25.2 30.3 29.4 23.6 32.8 22.6 28.8 29.3
34.7 27.6 26.8 GDF8/Compound 19 18.4 26.1 30.2 29.1 24 33.1 22.5
28.5 30 34.4 27.3 26.6 GDF8/Compound 202 18.7 26.7 31.1 29.6 24
34.9 22.7 30.3 31.6 34.2 27.8 27.2 GDF8/Compound 40 18.6 25.8 30.5
29.6 23.8 37.6 22.5 30 31.1 34.5 27.9 27.2 Ct Value Differentiation
Step 3 NKX2- NKX2- Treatment NEUROG3 2 5 NKX6-1 PAX4 PAX6 PDX1
PECAM POU3F4 PTF1A SST ZIC1 AA/Wnt3a 25.2 27.3 34.1 28.3 27.8 35.2
22.7 28.3 28.6 30.8 32.2 37.4 GDF8/Wnt3a 26.4 27.9 37.8 29 29.2
31.4 23.3 32.2 30.1 30.7 31 30.1 GDF8/GSK3 inh BIO 26.2 27.6 35
28.8 28.7 32.9 23.2 32.2 29.5 30.6 31.3 31.1 GDF8/Compound 19 25.9
27.5 37.6 27.8 28.3 33.8 22.9 31.7 29.7 30 32.4 33.4 GDF8/Compound
202 27 28 40 30 29 36.2 23.7 30.9 30.2 32.4 32.4 34.6 GDF8/Compound
40 26.2 27.8 37.2 29.5 29 37.1 23.2 31.5 30.2 31.5 32.4 35.5
Differentiation Step 4 Ct Value Treatment GAPDH ALB AMY2A ARX CDX2
GCG HNF4A INS ISL1 MAFA MAFB NEUROD1 AA/Wnt3a 18.9 21.3 28.8 24.6
23.4 21.7 21.9 21.6 25.2 32.4 24.9 23.7 GDF8/Wnt3a 18.3 21.3 28.5
25.3 23.1 22.6 21.9 21.9 25.7 33.1 24.9 24.3 GDF8/GSK3 inh BIO 19
21.1 28.7 25.3 23.3 22.3 21.9 22 25.7 32.5 25.4 24 GDF8/Compound 19
18.9 21.7 28.9 25.2 23.5 22.4 22.2 22 25.6 34 25.4 24.1
GDF8/Compound 202 19 20.9 29.2 25.1 23.6 22.4 22.1 22 25.5 33.3
25.5 23.9 GDF8/Compound 40 19.2 21.1 29.4 25.5 23.7 22.8 22.3 22.3
26 33.4 25.8 24.2 Ct Value Differentiation Step 4 NKX2- NKX2-
Treatment NEUROG3 2 5 NKX6-1 PAX4 PAX6 PDX1 PECAM POU3F4 PTF1A SST
ZIC1 AA/Wnt3a 23.8 24.2 33.9 25.6 25.6 27 23 29.2 27 28.1 25.3 32.6
GDF8/Wnt3a 24.2 24.7 35.4 25.8 26.2 27.3 23.2 31.2 27 28.7 24.7
24.6 GDF8/GSK3 inh BIO 24 24.7 35.4 26.1 26 27.7 23.2 30.7 27.4
28.5 25.6 31.5 GDF8/Compound 19 23.9 24.6 35.9 25.7 25.7 27.8 23.1
31.4 27.1 28.6 25.5 31.4 GDF8/Compound 202 24.1 24.5 35.7 26 25.8
27.6 23.4 30.2 27.5 28.8 26.1 35.7 GDF8/Compound 40 24.2 24.6 37.3
25.9 25.9 28.4 23.1 30.4 27.6 28.4 26.3 34.4 Differentiation Step 5
Ct Value Treatment GAPDH ALB AMY2A ARX CDX2 GCG HNF4A INS ISL1 MAFA
MAFB NEUROD1 AA/Wnt3a 19.1 19.5 28.6 23.1 23.9 16.2 21.9 16.9 23.4
33.7 23.3 21.7 GDF8/Wnt3a 18.4 19.9 28.4 23.8 23.8 17.2 22.3 17.4
24 32.6 23.9 22.6 GDF8/GSK3 inh BIO 19.1 19.2 29.1 24 24.2 17.2
22.4 17.6 24 33.5 23.8 22.9 GDF8/Compound 19 19 20 28.8 23.4 24.2
17 22.6 17.1 23.8 33.2 23.8 22.8 GDF8/Compound 202 19.2 20 29 23
23.9 16.7 22.2 16.8 23.2 32.7 23.8 22.3 GDF8/Compound 40 19.6 19.5
29 23.7 24.2 16.9 22.2 17.1 23.9 33.2 23.9 22.5 Ct Value
Differentiation Step 5 NKX2- NKX2- Treatment NEUROG3 2 5 NKX6-1
PAX4 PAX6 PDX1 PECAM POU3F4 PTF1A SST ZIC1 AA/Wnt3a 27.4 24 33.1 25
26.4 24.7 22.6 27.1 28.4 27.5 22 34.1 GDF8/Wnt3a 28.6 24.2 33.1
25.8 27.2 25.6 23.6 29.2 28.9 29.1 22.7 25.6 GDF8/GSK3 inh BIO 28.4
24.4 40 25.1 27.3 25.6 23.6 29.2 28.1 28.7 23 26.3 GDF8/Compound 19
28.6 24.1 34.6 25 26.8 25.6 23.4 29.8 28.2 28.2 22.9 28.8
GDF8/Compound 202 28.2 23.2 40 26 27.2 25.8 23.4 29.9 29.1 28.4
22.6 33.8 GDF8/Compound 40 28.1 23.5 34.8 25 27.3 26.4 23.4 29.9
29.2 27.5 22.3 34.8
TABLE-US-00016 TABLE 15 Step 1 RT-PCR CT Values Treatment GAPDH AFP
CD9 CD99 CDH1 CDH CDX2 CER1 CXCR4 FGF17 FGF4 FOXA2 AA 19.4 32.8
25.2 24.0 26.1 21.8 36.0 18.3 20.4 20.7 34.0 25.1 AA + Wnt3a 18.2
40.0 23.5 22.0 24.1 20.9 40.0 17.1 22.0 18.7 33.4 22.9 AA +
Compound 181 20.1 40.0 24.5 23.3 26.0 20.7 35.9 18.2 22.0 20.1 35.1
25.6 AA + Compound 180 18.4 34.2 23.6 21.6 25.9 21.4 35.2 17.2 22.0
18.9 34.0 24.0 AA + Compound 19 20.1 35.4 24.4 24.9 26.3 20.8 40.0
17.9 17.7 20.5 32.8 25.6 AA + Compound 202 20.3 40.0 25.1 23.7 25.9
21.4 40.0 18.3 22.0 20.4 36.0 25.7 AA + Compound 40 19.9 40.0 24.6
23.6 25.8 20.4 40.0 17.7 22.7 20.2 35.1 25.5 AA + GSK3 inhib BIO
20.2 35.0 25.4 23.7 27.2 21.9 35.5 18.5 22.2 20.9 36.0 25.8 AA +
Compound 206 19.8 40.0 24.9 23.7 25.5 21.1 40.0 18.3 19.6 20.7 36.2
24.4 GDF8 21.6 40.0 25.5 25.9 25.2 22.3 40.0 20.1 24.6 22.1 34.9
25.9 GDF8 + Wnt3a 21.2 40.0 25.0 25.8 25.1 22.6 40.0 19.7 23.6 22.0
34.8 25.7 GDF8 + Compound 181 20.7 40.0 25.1 23.6 25.5 22.4 40.0
20.0 23.0 21.5 36.3 25.2 GDF8 + Compound 180 20.9 40.0 25.6 24.0
26.9 22.1 34.7 19.9 22.7 21.1 36.6 25.0 GDF8 + Compound 19 19.6
40.0 23.9 23.7 24.6 20.7 40.0 18.0 21.8 20.4 33.2 24.2 GDF8 +
Compound 202 18.5 30.6 22.1 20.2 22.8 19.7 35.8 18.5 22.4 19.9 34.3
23.6 GDF8 + Compound 40 19.7 40.0 23.0 22.6 24.7 20.5 33.8 18.2
22.4 20.5 35.4 24.9 GDF8 + GSK3 inhib 19.6 30.1 23.1 21.8 24.3 20.0
33.4 17.7 23.3 20.1 34.8 24.7 BIO GDF8 + Compound 206 19.7 40.0
22.7 22.5 23.2 21.0 29.9 18.4 23.0 20.3 34.5 25.3 Step 1 RT-PCR CT
Values Treatment GATA4 GATA6 GSC HLXB9 KIT MIXL1 NANOG OTX2 POU5F1
SOX17 SOX7 T AA 25.0 22.9 22.4 24.8 22.7 28.9 24.1 22.5 32.2 21.6
32.3 36.1 AA + Wnt3a 23.1 21.8 22.8 23.3 22.9 27.7 22.7 19.9 31.0
21.2 31.2 34.1 AA + Compound 181 24.7 22.9 20.8 25.5 19.7 27.8 24.8
22.3 32.8 22.2 33.0 33.8 AA + Compound 180 23.1 22.6 22.5 24.8 22.6
27.9 23.7 20.3 32.1 21.4 32.0 32.7 AA + Compound 19 25.1 22.7 21.3
26.0 22.6 29.5 24.1 22.9 32.1 22.3 33.0 29.6 AA + Compound 202 24.5
23.0 21.8 25.2 23.9 27.7 24.5 22.5 32.7 22.6 32.1 33.5 AA +
Compound 40 24.6 22.5 20.9 25.5 22.9 28.0 23.8 22.0 33.6 22.3 32.5
32.8 AA + GSK3 inhib BIO 25.0 23.2 22.2 25.2 23.7 28.5 24.9 22.8
34.3 23.4 32.7 33.5 AA + Compound 206 24.8 22.7 22.6 24.1 23.7 27.6
23.9 22.0 33.2 23.3 32.4 34.6 GDF8 27.5 24.8 24.2 25.3 25.4 30.6
25.0 24.3 29.5 25.2 29.8 32.9 GDF8 + Wnt3a 27.3 24.4 23.8 25.0 25.1
30.5 24.8 24.4 31.1 25.0 34.5 32.6 GDF8 + Compound 181 25.4 23.9
23.6 24.3 26.3 28.1 25.4 23.3 31.8 23.7 32.8 31.9 GDF8 + Compound
180 25.2 23.9 23.6 24.2 25.8 28.3 26.3 23.0 33.2 24.2 32.8 32.3
GDF8 + Compound 19 25.0 22.8 22.1 24.0 22.6 28.5 23.1 22.7 30.3
22.6 32.1 29.3 GDF8 + Compound 202 22.7 22.5 23.5 23.1 25.9 27.7
25.1 21.5 30.0 22.3 32.0 32.5 GDF8 + Compound 40 24.2 22.6 23.4
23.4 25.4 28.5 24.0 22.2 31.6 23.7 31.2 32.7 GDF8 + GSK3 inhib 24.5
22.4 22.7 24.1 25.0 29.3 24.8 21.8 31.7 23.3 33.7 34.6 BIO GDF8 +
Compound 206 24.9 22.9 23.6 23.8 25.7 29.7 24.7 22.2 30.7 24.4 32.8
33.8 Step 3 RT-PCR CT Values Treatment GAPDH ALB AMY2A ARX CDX2 GCG
HNF4A INS ISL1 MAFA MAFB NEUROD1 AA 18.5 26.9 30.6 32.0 22.9 34.1
22.4 34.8 29.3 36.4 27.4 30.1 AA + Wnt3a 18.4 27.3 30.2 33.0 23.0
34.7 22.4 40.0 28.8 33.9 27.7 30.4 AA + Compound 181 18.6 26.0 30.1
34.2 22.3 40.0 22.3 40.0 30.4 34.6 28.5 31.4 AA + Compound 180 18.8
25.5 30.0 33.3 22.5 35.1 22.5 40.0 29.4 34.3 28.6 32.6 AA +
Compound 19 34.1 40.0 40.0 40.0 40.0 40.0 37.4 40.0 40.0 40.0 34.9
40.0 AA + Compound 202 18.5 26.2 30.7 33.0 22.5 34.8 22.6 35.1 29.8
35.1 28.2 30.0 AA + Compound 40 18.5 25.8 30.1 34.9 22.2 40.0 22.3
40.0 29.7 34.1 28.1 30.8 AA + GSK3 inhib BIO 18.5 24.9 30.1 34.6
22.0 40.0 21.5 40.0 30.2 34.9 27.8 34.0 AA + Compound 206 18.3 27.0
30.3 33.7 22.7 35.7 22.5 40.0 28.4 34.4 27.8 30.7 GDF8 18.0 28.7
30.4 35.3 23.8 40.0 23.6 40.0 28.5 33.6 27.2 30.3 GDF8 + Wnt3a 17.4
27.0 29.5 33.8 23.1 35.1 22.4 40.0 26.3 30.5 26.1 30.1 GDF8 +
Compound 181 18.8 27.8 30.2 31.3 23.4 40.0 22.7 34.5 28.8 35.5 27.3
28.9 GDF8 + Compound 180 18.8 27.3 30.6 32.4 22.8 34.9 22.7 40.0
29.5 35.9 27.7 30.0 GDF8 + Compound 19 18.3 24.9 29.7 33.4 22.0
40.0 22.2 40.0 29.7 34.5 28.4 33.3 GDF8 + Compound 202 18.7 27.8
30.4 32.8 23.8 40.0 22.9 34.6 28.5 34.2 27.6 29.6 GDF8 + Compound
40 18.4 27.7 30.1 32.5 23.0 35.1 22.4 40.0 29.1 34.3 27.5 30.0 GDF8
+ GSK3 inhib 18.4 24.9 30.3 31.5 22.2 34.7 21.7 34.5 29.9 35.3 27.6
29.2 BIO GDF8 + Compound 206 18.2 27.6 30.2 33.5 23.8 40.0 22.9
40.0 27.9 35.1 27.1 29.9 RT-PCR CT Values Step 3 NKX2- NKX2- NKX6-
Treatment NEUROG3 2 5 1 PAX4 PAX6 PDX1 PECAM POU3F4 PTF1A SST ZIC1
AA 28.0 29.3 34.6 32.2 31.4 40.0 23.3 31.0 30.5 34.6 33.2 34.8 AA +
Wnt3a 28.1 29.0 40.0 34.9 31.9 38.1 23.8 30.9 30.7 40.0 32.8 40.0
AA + Compound 181 29.0 30.3 33.9 35.8 33.3 40.0 25.0 30.0 30.8 36.0
34.6 34.8 AA + Compound 180 30.3 31.1 32.5 35.3 35.3 36.2 26.3 28.9
31.3 40.0 34.7 34.4 AA + Compound 19 40.0 40.0 40.0 40.0 40.0 40.0
40.0 40.0 40.0 40.0 40.0 40.0 AA + Compound 202 28.5 29.5 33.6 33.9
32.0 36.6 24.1 29.7 31.9 40.0 34.2 40.0 AA + Compound 40 29.1 30.3
33.6 34.4 32.9 40.0 24.8 30.3 31.3 35.6 35.3 35.4 AA + GSK3 inhib
BIO 31.0 31.8 32.6 40.0 34.9 40.0 26.1 31.0 32.4 40.0 35.2 34.9 AA
+ Compound 206 28.6 29.8 32.8 35.0 31.5 30.4 23.9 29.7 31.2 35.0
31.9 40.0 GDF8 28.7 30.3 34.4 35.0 32.4 24.8 25.5 30.0 30.8 34.2
29.5 26.1 GDF8 + Wnt3a 27.5 29.1 40.0 32.9 31.5 25.8 23.1 32.2 29.8
34.0 27.7 28.2 GDF8 + Compound 181 27.0 28.0 35.4 32.5 30.5 35.4
23.5 29.4 30.5 33.9 32.4 34.5 GDF8 + Compound 180 27.5 29.0 35.4
32.6 31.2 40.0 23.7 31.6 30.8 34.6 34.4 40.0 GDF8 + Compound 19
31.5 32.2 34.4 35.2 34.4 35.0 25.0 29.4 31.6 40.0 34.1 33.5 GDF8 +
Compound 202 27.1 28.4 35.7 30.5 30.4 31.8 23.0 30.1 30.2 31.6 30.9
34.0 GDF8 + Compound 40 27.3 28.8 35.3 32.9 31.5 40.0 23.2 29.2
30.5 36.3 33.7 40.0 GDF8 + GSK3 inhib 26.8 27.9 34.6 33.9 30.6 35.1
24.0 30.4 30.0 34.8 33.6 40.0 BIO GDF8 + Compound 206 27.7 29.3
40.0 32.3 31.3 27.4 23.3 32.7 30.8 33.0 29.2 30.3 Step 4 RT-PCR CT
Values Treatment GAPDH ALB AMY2A ARX CDX2 GCG HNF4A INS ISL1 MAFA
MAFB NEUROD1 AA 19.0 22.4 28.6 23.9 23.7 22.8 22.1 23.6 24.1 30.6
23.5 23.7 AA + Wnt3a 19.5 23.5 29.3 24.6 24.1 25.1 22.7 25.2 25.1
31.1 24.4 24.4 AA + Compound 181 18.0 21.0 27.7 24.0 22.1 24.5 21.0
24.6 24.5 31.6 24.0 24.2 AA + Compound 180 19.4 21.0 27.3 26.3 21.6
25.4 20.6 26.7 26.1 34.7 24.6 25.5 AA + Compound 19 (insufficient
RNA sample) AA + Compound 202 19.2 20.7 29.1 24.0 23.3 21.8 21.9
22.7 24.4 31.1 23.9 24.0 AA + Compound 40 19.2 20.8 29.4 24.7 22.9
22.2 21.8 23.5 25.3 31.5 24.7 24.7 AA + GSK3 inhib BIO 19.0 19.1
29.2 26.7 22.7 25.2 21.1 26.3 26.5 33.0 25.4 26.3 AA + Compound 206
18.8 20.9 28.4 23.3 22.9 21.2 21.7 22.8 24.0 30.4 23.5 23.3 GDF8
18.0 25.5 29.1 29.8 24.6 31.3 23.8 30.9 27.6 32.8 24.1 29.2 GDF8 +
Wnt3a 19.0 24.3 29.0 25.4 24.4 27.7 23.0 25.5 25.4 32.7 25.1 25.4
GDF8 + Compound 181 18.0 22.8 28.1 23.5 23.1 24.6 21.4 22.7 23.8
30.6 23.0 22.9 GDF8 + Compound 180 19.5 24.0 29.3 24.4 23.9 25.7
22.5 24.5 24.7 31.4 24.4 24.3 GDF8 + Compound 19 19.1 22.6 28.7
25.5 22.9 26.7 22.1 27.2 25.8 32.6 25.5 26.2 GDF8 + Compound 202
19.0 22.0 28.9 23.7 24.5 21.8 22.3 21.7 24.3 30.3 23.5 22.9 GDF8 +
Compound 40 19.0 21.4 29.0 23.4 23.6 21.0 22.0 21.5 23.8 30.2 23.3
23.2 GDF8 + GSK3 inhib 19.1 19.4 29.1 24.3 23.0 21.5 21.4 21.8 24.6
31.0 24.0 23.8 BIO GDF8 + Compound 206 18.9 21.6 28.9 24.4 24.0
22.6 22.3 22.7 24.9 30.9 24.2 23.7 RT-PCR CT Values Step 4 NKX2-
NKX2- NKX6- Treatment NEUROG3 2 5 1 PAX4 PAX6 PDX1 PECAM POU3F4
PTF1A SST ZIC1 AA 23.3 24.0 31.9 27.4 25.7 27.1 23.7 29.8 26.5 29.6
24.3 32.5 AA + Wnt3a 24.1 24.5 31.8 27.5 26.1 27.9 24.1 30.4 27.2
31.1 25.9 32.4 AA + Compound 181 24.6 24.6 30.9 28.1 26.3 28.0 24.0
27.9 27.5 31.0 25.5 31.2 AA + Compound 180 25.8 25.0 29.4 28.9 27.7
28.9 24.0 26.7 28.5 32.6 27.1 31.2 AA + Compound 19 AA + Compound
202 24.1 24.4 32.1 27.4 26.3 26.9 24.3 29.6 27.7 29.5 24.0 35.1 AA
+ Compound 40 25.3 25.0 32.2 28.7 27.1 28.2 25.1 30.3 28.5 31.2
25.6 32.7 AA + GSK3 inhib BIO 27.7 26.2 31.5 32.9 29.4 30.0 26.5
29.6 30.5 33.7 27.2 32.4 AA + Compound 206 23.3 23.8 31.9 27.2 25.5
26.3 23.6 29.7 26.6 29.3 23.6 32.9 GDF8 28.7 29.0 32.3 31.2 31.1
27.9 26.5 29.4 29.4 40.0 26.0 22.8 GDF8 + Wnt3a 24.0 25.0 33.3 28.0
26.7 28.8 23.8 33.4 27.7 30.5 24.4 29.0 GDF8 + Compound 181 21.8
23.2 32.1 25.7 24.2 27.0 22.3 27.6 25.4 28.6 24.2 31.6 GDF8 +
Compound 180 23.7 24.4 33.5 27.7 26.0 28.3 23.8 30.6 26.9 30.7 26.3
33.4 GDF8 + Compound 19 26.4 25.6 34.0 30.2 28.2 30.0 25.1 30.2
29.2 32.6 27.9 31.1 GDF8 + Compound 202 22.2 23.4 33.4 25.9 24.8
26.5 23.0 29.5 26.1 27.9 22.4 34.1 GDF8 + Compound 40 22.7 23.7
33.2 26.6 24.8 26.1 23.4 29.3 26.4 28.4 22.7 32.6 GDF8 + GSK3 inhib
23.8 24.2 33.3 27.9 26.1 27.0 24.2 30.0 27.8 29.5 23.4 32.7 BIO
GDF8 + Compound 206 23.0 24.0 35.3 26.3 25.5 27.2 23.5 31.4 26.9
27.7 24.0 28.9 Step 5 RT-PCR CT Values Treatment GAPDH ALB AMY2A
ARX CDX2 GCG HNF4A INS ISL1 MAFA MAFB NEUROD1 AA 18.3 20.2 27.6
22.0 23.4 14.1 21.3 14.9 22.4 31.8 22.2 22.8 AA + Wnt3a 18.0 20.0
27.7 21.9 23.1 14.0 20.9 14.6 22.3 31.6 22.0 21.6 AA + Compound 181
18.0 18.8 27.6 22.0 22.9 14.3 20.9 14.5 22.1 31.4 22.2 21.5 AA +
Compound 180 18.0 18.8 27.6 22.4 22.9 14.9 21.0 14.7 22.4 31.9 22.6
21.7 AA + Compound 19 17.9 23.6 28.6 28.2 25.4 27.0 24.2 26.9 26.2
32.0 24.4 27.2 AA + Compound 202 18.6 19.2 28.0 22.6 23.4 14.9 21.3
15.0 22.7 31.8 22.6 21.9 AA + Compound 40 18.3 18.9 27.9 22.3 23.0
14.6 21.1 14.7 22.5 31.5 22.4 21.6 AA + GSK3 inhib BIO 18.3 17.1
28.0 23.0 22.6 15.1 20.5 15.1 22.8 31.8 22.8 22.1 AA + Compound 206
18.2 19.5 27.9 22.0 23.4 14.4 21.3 14.8 22.5 31.1 22.4 21.7 GDF8
17.4 20.5 28.2 25.2 24.4 18.1 22.9 17.7 24.3 31.8 23.3 24.2 GDF8 +
Wnt3a 17.8 20.6 28.2 24.8 24.3 17.7 22.9 17.5 24.2 31.9 23.5 24.0
GDF8 + Compound 181 18.0 19.1 27.6 22.4 23.4 14.5 21.2 14.8 22.5
31.5 22.6 21.7 GDF8 + Compound 180 18.0 18.0 27.3 22.2 22.9 14.2
20.9 14.4 22.2 31.4 22.1 21.2 GDF8 + Compound 19 18.3 18.5 27.8
23.4 23.0 16.2 21.2 15.6 23.2 32.2 23.2
22.6 GDF8 + Compound 202 18.7 19.6 28.3 23.1 24.0 15.7 21.9 15.8
23.5 32.8 23.3 22.3 GDF8 + Compound 40 18.1 18.7 27.9 22.3 23.0
14.8 21.1 14.8 22.6 31.5 22.5 21.5 GDF8 + GSK3 inhib 18.4 17.1 27.6
23.2 22.8 15.2 20.6 15.5 23.1 32.6 22.6 22.1 BIO GDF8 + Compound
206 18.0 20.0 27.9 23.4 24.0 16.0 22.1 16.0 23.6 32.1 22.9 22.8
RT-PCR CT Values Step 5 NKX2- NKX2- NKX6- Treatment NEUROG3 2 5 1
PAX4 PAX6 PDX1 PECAM POU3F4 PTF1A SST ZIC1 AA 28.1 23.7 34.1 25.5
27.3 24.0 23.2 30.1 28.8 27.3 19.6 34.4 AA + Wnt3a 28.0 23.4 34.8
26.1 27.3 23.7 23.3 29.6 28.8 27.5 19.4 32.5 AA + Compound 181 28.9
23.3 32.2 26.1 26.8 24.0 23.1 27.5 28.8 28.0 18.8 31.2 AA +
Compound 180 29.5 23.8 30.2 26.5 27.2 24.3 23.2 26.7 29.0 28.7 18.7
30.3 AA + Compound 19 31.2 28.0 30.1 25.8 35.1 28.6 29.4 31.5 28.2
32.4 23.1 24.0 AA + Compound 202 28.6 23.7 29.9 25.8 26.9 24.7 23.6
27.7 29.2 27.9 19.4 32.8 AA + Compound 40 29.0 23.5 32.9 26.1 27.1
24.4 23.2 28.1 29.2 28.1 19.1 31.9 AA + GSK3 inhib BIO 29.5 24.2
33.8 27.5 27.4 24.9 23.8 28.3 29.9 29.7 19.5 32.0 AA + Compound 206
28.0 23.6 35.8 25.9 27.1 24.1 23.3 29.0 28.7 27.5 19.7 32.7 GDF8
30.1 25.9 31.4 26.6 29.4 26.6 25.6 29.7 27.8 29.5 21.1 22.5 GDF8 +
Wnt3a 30.2 25.6 31.9 27.0 29.3 26.5 25.6 29.8 28.1 30.7 21.5 22.8
GDF8 + Compound 181 27.4 23.4 33.8 25.0 26.8 24.4 23.0 27.5 28.8
27.3 19.5 31.9 GDF8 + Compound 180 27.9 23.2 40.0 25.1 26.1 23.8
22.9 29.6 28.7 27.3 18.8 31.5 GDF8 + Compound 19 31.2 24.3 33.9
28.6 27.3 25.2 24.2 29.0 30.1 31.0 20.5 31.9 GDF8 + Compound 202
27.8 23.8 31.3 24.8 27.1 25.2 23.2 29.0 29.5 27.1 20.8 30.9 GDF8 +
Compound 40 27.3 23.2 33.2 25.0 26.6 24.1 22.9 27.3 28.9 27.4 20.4
32.0 GDF8 + GSK3 inhib 28.2 24.2 35.0 26.7 27.3 24.8 24.0 28.1 30.1
28.5 19.3 32.4 BIO GDF8 + Compound 206 28.5 24.4 30.5 25.9 27.5
25.4 24.0 29.7 28.3 28.0 20.8 23.9
TABLE-US-00017 TABLE 16 Compound # Primary Selectivity Compound 6
Selective for GSK Compound 7 Selective for GSK Compound 8 Selective
for GSK Compound 9 Selective for CDK Compound 57 Selective for Trk
Compound 41 Selective for GSK Compound 42 Selective for CDK
Compound 10 Selective for CDK Compound 34 Positive Control Compound
11 Selective for CDK Compound 43 Selective for Trk Compound 44
Selective for GSK Compound 12 Selective for CDK Compound 45
Selective for Trk
TABLE-US-00018 TABLE 17 Cell Number Sox17 Expression Average % of
Average Compound Total positive Total % of positive Plate Treatment
Compound # Selectivity Cell Number control Intensity control 1 no
Activin A none n/a 9809 67.8 -4.0E+05 -0.2 1 Activin none n/a 14476
100.0 2.3E+08 100.0 A/Wnt3a 1 No GDF8 Compound 11 Selective for CDK
565 3.9 -1.1E+06 -0.5 1 No GDF8 Compound 44 Selective for GSK 14
0.1 -1.1E+06 -0.5 1 No GDF8 Compound 43 Selective for Trk 8610 59.5
-2.1E+05 -0.1 1 No GDF8 Compound 42 Selective for CDK 8700 60.1
-2.4E+05 -0.1 1 No GDF8 Compound 57 Selective for Trk 1222 8.4
-7.1E+05 -0.3 1 No GDF8 Compound 10 Selective for CDK 7011 48.4
-6.6E+05 -0.3 1 No GDF8 Compound 41 Selective for GSK 9995 69.0
5.9E+04 0.0 1 No GDF8 Compound 7 Selective for CDK 3 0.0 -1.4E+06
-0.6 1 No GDF8 Compound 45 Selective for Trk 8857 61.2 -4.5E+05
-0.2 1 No GDF8 Compound 6 Selective for GSK 14827 102.4 -1.8E+05
-0.1 1 No GDF8 Compound 9 Selective for CDK 7156 49.4 -4.2E+04 0.0
1 No GDF8 Compound 12 Selective for GSK 13124 90.7 -2.3E+05 -0.1 1
No GDF8 Compound 8 Selective for GSK 13235 91.4 3.8E+05 0.2 1 GDF8
Compound 34 Positive Control 13926 96.2 2.6E+08 111.8 1 GDF8
Compound 45 Selective for Trk 9540 65.9 1.1E+08 47.9 1 GDF8
Compound 7 Selective for GSK 5296 36.6 7.0E+07 30.4 1 GDF8 Compound
10 Selective for CDK 4627 32.0 6.6E+07 28.6 1 GDF8 Compound 6
Selective for GSK 5118 35.4 5.8E+07 25.2 1 GDF8 Compound 43
Selective for Trk 6682 46.2 5.4E+07 23.4 1 GDF8 Compound 42
Selective for CDK 5686 39.3 4.9E+07 21.2 1 GDF8 Compound 8
Selective for GSK 5018 34.7 4.7E+07 20.4 1 GDF8 Compound 9
Selective for CDK 4816 33.3 4.5E+07 19.4 1 GDF8 Compound 41
Selective for GSK 4455 30.8 3.4E+07 14.8 1 GDF8 n/a n/a 2856 19.7
2.2E+07 9.4 1 GDF8 Compound 57 Selective for Trk 2110 14.6 1.1E+07
4.8 1 GDF8 Compound 11 Selective for CDK 210 1.4 -4.9E+05 -0.2 1
GDF8 Compound 44 Selective for GSK 226 1.6 -9.5E+05 -0.4 1 GDF8
Compound 12 Selective for CDK 31 0.2 -1.3E+06 -0.6
TABLE-US-00019 TABLE 18 Cell Number Sox17 Expression Average % of
Average % of Total Cell positive Total positive Plate Treatment
Compound # Number SD CV % control Intensity SD CV % control 1 no
Activin A (with Wnt3a) none 15489 0 0.00 103.2 3.75E+07 0.00E+00
0.00 10.9 1 ActivinA/Wnt3a none 15007 1991 13.27 100.0 3.45E+08
7.16E+07 20.75 100.0 1 GDF8 Compound 206 20568 1683 8.18 137.1
5.19E+08 4.41E+07 8.51 150.3 1 GDF8 Compound 207 19224 1091 5.68
128.1 2.54E+08 5.69E+07 22.41 73.6 1 GDF8 Compound 19 12569 1524
12.13 83.8 2.40E+08 6.34E+07 26.44 69.5 1 GDF8 Compound 23 8758 474
5.41 58.4 1.16E+08 9.07E+06 7.80 33.7 1 GDF8 Compound 170 6460 2305
35.68 43.0 9.44E+07 6.98E+07 73.93 27.4 1 GDF8 Compound 208 4848
1225 25.27 32.3 2.26E+07 2.15E+07 94.96 23.6 1 GDF8 Compound 209
4831 1243 25.74 32.2 3.97E+07 1.61E+07 40.56 11.5 1 GDF8 Compound
32 4338 1520 35.04 28.9 3.63E+07 3.27E+07 90.14 10.5 1 GDF8
Compound 30 4679 435 9.29 31.2 3.47E+07 1.04E+07 30.03 10.1 1 GDF8
Compound 223 3704 1077 29.08 24.7 3.45E+07 2.74E+07 79.43 10.0 1
GDF8 Compound 2 4538 632 13.93 30.2 2.95E+07 2.81E+06 9.50 8.6 1
GDF8 Compound 210 2645 817 30.88 17.6 2.90E+07 2.45E+07 84.73 8.4 1
GDF8 Compound 24 5012 1263 25.21 33.4 2.64E+07 1.66E+07 62.95 7.7 1
GDF8 Compound 211 5165 796 15.41 34.4 2.61E+07 5.02E+06 19.23 7.6 1
GDF8 Compound 212 5476 1445 26.39 36.5 2.54E+07 1.18E+07 46.53 7.4
1 GDF8 Compound 224 5188 761 14.67 34.6 2.46E+07 8.26E+06 33.56 7.1
1 GDF8 Compound 225 4431 1149 25.92 29.5 2.45E+07 2.65E+07 108.19
7.1 1 GDF8 Compound 13 3123 1508 48.27 20.8 2.44E+07 2.30E+07 94.13
7.1 1 GDF8 Compound 213 1261 1028 81.49 8.4 2.07E+07 1.97E+07 95.03
6.0 1 GDF8 Compound 52 4932 386 7.82 32.9 1.99E+07 6.90E+06 34.67
5.8 1 GDF8 Compound 214 3345 335 10.01 22.3 1.93E+07 1.39E+07 72.18
5.6 1 GDF8 Compound 51 4289 940 21.91 28.6 1.70E+07 1.10E+07 64.86
4.9 1 GDF8 Compound 26 4896 545 11.14 32.6 1.65E+07 5.93E+06 36.02
4.8 1 GDF8 Compound 226 3617 577 15.94 24.1 1.59E+07 4.96E+06 31.21
4.6 1 GDF8 Compound 215 4326 165 3.81 28.8 1.45E+07 2.69E+06 18.53
4.2 1 GDF8 Compound 31 3619 1011 27.92 24.1 1.36E+07 4.63E+06 34.15
3.9 1 GDF8 Compound 216 3364 629 18.70 22.4 8.75E+06 2.30E+06 26.32
2.5 1 GDF8 Compound 217 2859 544 19.03 19.1 8.75E+06 1.94E+06 22.16
2.5 1 GDF8 Compound 218 1327 118 8.92 8.8 6.44E+06 9.70E+05 15.05
1.9 1 GDF8 Compound 219 368 168 45.67 2.5 1.79E+06 1.29E+06 72.17
0.5 2 no Activin A (with Wnt3a) none 15778 0 0.00 103.2 2.24E+07
0.00E+00 0.00 6.7 2 Activin A/Wnt3a none 15290 1119 7.32 100.0
3.37E+08 2.84E+07 8.44 100.0 2 GDF8 Compound 202 20177 987 4.89
132.0 4.85E+08 1.94E+07 4.00 144.0 2 GDF8 Compound 227 2911 4619
158.69 19.0 3.89E+07 6.69E+07 172.00 11.5 2 GDF8 Compound 15 4383
1775 40.49 28.7 3.57E+07 3.57E+07 100.03 10.6 2 GDF8 Compound 228
4043 1253 30.98 26.4 3.10E+07 2.53E+07 81.62 9.2 2 GDF8 Compound
229 3451 892 25.85 22.6 1.80E+07 1.46E+07 81.07 5.4 2 GDF8 Compound
4 3163 805 25.44 20.7 1.58E+07 3.54E+06 22.32 4.7 2 GDF8 Compound
220 2791 1453 52.05 18.3 1.40E+07 9.00E+06 64.28 4.2 2 GDF8
Compound 5 3137 1172 37.34 20.5 1.30E+07 7.52E+06 57.85 3.9 2 GDF8
Compound 230 2624 248 9.46 17.2 1.24E+07 1.55E+07 124.73 3.7 2 GDF8
Compound 231 4773 2651 55.55 31.2 1.22E+07 6.51E+06 53.37 3.6 2
GDF8 Compound 232 3273 1290 39.41 21.4 1.18E+07 1.51E+07 127.98 3.5
2 GDF8 Compound 221 1950 361 18.52 12.8 1.18E+07 1.54E+07 131.11
3.5 2 GDF8 Compound 233 3041 180 5.93 19.9 1.12E+07 1.09E+07 97.44
3.3 2 GDF8 Compound 147 3434 1199 34.91 22.5 1.12E+07 9.80E+06
87.75 3.3 2 GDF8 Compound 234 2835 623 21.98 18.5 9.47E+06 5.67E+06
59.84 2.8 2 GDF8 Compound 235 3391 2269 66.91 22.2 9.10E+06
6.51E+06 71.52 2.7 2 GDF8 Compound 236 2868 561 19.57 18.8 6.73E+06
6.32E+06 93.82 2.0 2 GDF8 Compound 33 2362 511 21.66 15.4 6.60E+06
2.45E+06 37.20 2.0 2 GDF8 Compound 1 3213 166 5.16 21.0 6.48E+06
3.09E+06 47.67 1.9 2 GDF8 Compound 53 2783 441 15.86 18.2 6.36E+06
2.89E+06 45.36 1.9 2 GDF8 Compound 237 2973 292 9.83 19.4 6.02E+06
3.00E+06 49.79 1.8 2 GDF8 Compound 238 2739 485 17.70 17.9 5.97E+06
6.10E+06 102.07 1.8 2 GDF8 Compound 239 3156 667 21.15 20.6
5.60E+06 2.42E+06 43.24 1.7 2 GDF8 Compound 240 3002 287 9.55 19.6
4.68E+06 3.13E+06 66.80 1.4 2 GDF8 Compound 200 2308 209 9.04 15.1
4.39E+06 1.88E+06 42.83 1.3 2 GDF8 Compound 222 1776 719 40.47 11.6
3.33E+06 2.52E+06 75.78 1.0 2 GDF8 Compound 241 2949 446 15.14 19.3
3.29E+06 1.55E+06 47.03 1.0 2 GDF8 Compound 242 385 184 47.83 2.5
1.08E+06 8.85E+05 81.61 0.3 2 GDF8 Compound 243 249 55 22.21 1.6
2.53E+05 3.07E+05 121.25 0.1 2 GDF8 Compound 204 250 21 8.38 1.6
1.36E+05 2.27E+04 16.66 0.0 3 no Activin A (with Wnt3a) none 15796
0 0.00 99.6 2.82E+07 0.00E+00 0.00 8.0 3 Activin A/Wnt3a none 15867
785 4.95 100.0 3.54E+08 2.40E+07 6.77 100.0 3 GDF8 Compound 34 6974
3723 53.38 44.0 2.07E+08 9.51E+07 45.85 58.6 3 GDF8 Compound 185
10892 1552 14.24 68.6 1.53E+08 4.08E+07 26.72 43.1 3 GDF8 Compound
35 7746 1873 24.17 48.8 1.35E+08 4.86E+07 36.08 38.0 3 GDF8
Compound 22 6727 1927 28.64 42.4 1.06E+08 5.04E+07 47.73 29.8 3
GDF8 Compound 34 4889 1152 23.57 30.8 4.31E+07 2.11E+07 48.95 12.2
3 GDF8 Compound 184 4173 1758 42.14 26.3 3.94E+07 2.24E+07 56.78
11.1 3 GDF8 Compound 223 4234 1604 37.88 26.7 3.55E+07 2.51E+07
70.56 10.0 3 GDF8 Compound 37 4187 338 8.06 26.4 3.11E+07 1.56E+07
50.18 8.8 3 GDF8 Compound 244 4479 1229 27.43 28.2 2.73E+07
1.52E+07 55.71 7.7 3 GDF8 Compound 245 4725 99 2.09 29.8 2.59E+07
1.03E+07 39.90 7.3 3 GDF8 Compound 246 3820 1091 28.57 24.1
2.30E+07 2.69E+07 117.08 6.5 3 GDF8 Compound 247 3730 966 25.90
23.5 2.14E+07 1.04E+07 48.63 6.1 3 GDF8 Compound 248 3875 445 11.48
24.4 2.13E+07 9.45E+06 44.45 6.0 3 GDF8 Compound 25 3879 658 16.95
24.4 1.76E+07 1.21E+07 69.04 5.0 3 GDF8 Compound 195 3703 405 10.94
23.3 1.61E+07 3.27E+06 20.34 4.5 3 GDF8 Compound 227 2904 397 13.68
18.3 1.43E+07 1.35E+07 94.25 4.0 3 GDF8 Compound 183 3306 969 29.32
20.8 1.35E+07 1.14E+07 84.25 3.8 3 GDF8 Compound 187 2768 1426
51.51 17.4 1.35E+07 9.02E+06 66.67 3.8 3 GDF8 Compound 201 3213
1114 34.66 20.3 1.35E+07 1.69E+07 125.02 3.8 3 GDF8 Compound 197
3268 211 6.46 20.6 1.30E+07 5.25E+06 40.51 3.7 3 GDF8 Compound 249
3840 348 9.06 24.2 1.29E+07 6.79E+06 52.72 3.6 3 GDF8 Compound 141
2404 213 8.86 15.1 1.12E+07 4.95E+06 44.30 3.2 3 GDF8 Compound 194
3177 354 11.14 20.0 9.75E+06 2.11E+06 21.63 2.8 3 GDF8 Compound 250
3683 420 11.40 23.2 9.14E+06 4.78E+06 52.32 2.6 3 GDF8 Compound 251
3021 668 22.10 19.0 8.41E+06 4.59E+06 54.60 2.4 3 GDF8 Compound 20
2793 205 7.35 17.6 6.77E+06 1.86E+06 27.45 1.9 3 GDF8 Compound 252
2580 135 5.24 16.3 6.20E+06 2.31E+05 3.72 1.8 3 GDF8 Compound 253
2485 820 32.98 15.7 5.83E+06 1.47E+06 25.20 1.6 3 GDF8 Compound 202
2095 518 24.71 13.2 5.75E+06 2.62E+06 45.66 1.6 3 GDF8 Compound 21
371 294 79.19 2.3 2.36E+06 3.07E+06 129.78 0.7 4 no Activin A (with
Wnt3a) none 16629 0 0.00 119.3 2.42E+07 0.00E+00 0.00 7.8 4 Activin
A/Wnt3a none 13945 1535 11.01 100.0 3.09E+08 4.77E+07 15.46 100.0 4
GDF8 Compound 34 7416 6482 87.41 53.2 2.10E+08 1.82E+08 86.70 68.0
4 GDF8 Compound 240 11283 2023 17.93 80.9 1.61E+08 4.41E+07 27.34
52.2 4 GDF8 Compound 28 5236 1787 34.12 37.5 4.03E+07 3.08E+07
76.36 13.1 4 GDF8 Compound 198 3985 2674 67.10 28.6 3.89E+07
5.55E+07 142.91 12.6 4 GDF8 Compound 196 4861 1501 30.87 34.9
3.03E+07 1.98E+07 65.37 9.8 4 GDF8 Compound 18 1921 1759 91.56 13.8
2.94E+07 3.65E+07 123.90 9.5 4 GDF8 Compound 186 3486 425 12.19
25.0 2.34E+07 1.42E+07 60.78 7.6 4 GDF8 Compound 254 3960 1521
38.42 28.4 2.31E+07 2.27E+07 98.10 7.5 4 GDF8 Compound 168 3460 324
9.36 24.8 2.28E+07 7.13E+06 31.23 7.4 4 GDF8 Compound 190 3402 1318
38.74 24.4 1.87E+07 1.58E+07 84.61 6.1 4 GDF8 Compound 255 4006
1625 40.57 28.7 1.52E+07 1.05E+07 68.91 4.9 4 GDF8 Compound 50 2666
743 27.86 19.1 1.48E+07 8.30E+06 56.15 4.8 4 GDF8 Compound 27 3721
721 19.37 26.7 1.19E+07 9.69E+06 81.29 3.9 4 GDF8 Compound 256 2922
1275 43.64 21.0 9.41E+06 8.65E+06 92.01 3.0 4 GDF8 Compound 257
3182 705 22.14 22.8 8.06E+06 4.49E+06 55.75 2.6 4 GDF8 Compound 258
2731 472 17.29 19.6 7.89E+06 7.24E+06 91.70 2.6 4 GDF8 Compound 189
2350 1625 69.16 16.9 7.72E+06 5.36E+06 69.41 2.5 4 GDF8 Compound
259 2195 955 43.49 15.7 6.92E+06 2.58E+06 37.29 2.2 4 GDF8 Compound
260 2468 741 30.04 17.7 6.64E+06 3.33E+06 50.18 2.2 4 GDF8 Compound
261 2965 456 15.38 21.3 6.23E+06 2.10E+06 33.61 2.0 4 GDF8 Compound
192 2377 572 24.08 17.0 6.17E+06 2.76E+06 44.65 2.0 4 GDF8 Compound
262 2894 399 13.78 20.8 5.75E+06 3.00E+06 52.20 1.9 4 GDF8 Compound
188 3005 759 25.26 21.6 5.02E+06 3.97E+06 79.06 1.6 4 GDF8 Compound
263 2129 230 10.79 15.3 4.77E+06 1.14E+06 23.93 1.5 4 GDF8 Compound
264 2630 342 13.00 18.9 4.28E+06 2.17E+06 50.73 1.4 4 GDF8 Compound
265 2636 1372 52.04 18.9 4.27E+06 1.15E+06 26.86 1.4 4 GDF8
Compound 14 274 14 5.02 2.0 1.56E+05 9.51E+04 60.91 0.1 4 GDF8
Compound 205 241 3 1.20 1.7 1.36E+05 6.83E+04 50.42 0.0 4 GDF8
Compound 266 271 7 2.67 1.9 1.18E+05 3.34E+04 28.43 0.0 4 GDF8
Compound 203 253 4 1.49 1.8 1.09E+05 3.49E+04 32.09 0.0
TABLE-US-00020 TABLE 19 Sox17 Expression % of Compound # positive
control Compound 181 150.3 Compound 202 144.0 Compound 180 73.6
Compound 19 69.5 Compound 34 68.0 Compound 40 52.2 Compound 185
43.1 Compound 185 38.0 Compound 35 33.7 Compound 23 29.8 Compound
22 27.4 Compound 17 23.6
* * * * *