U.S. patent application number 16/540657 was filed with the patent office on 2019-12-05 for encapsulated intestinal midgut endoderm cells.
This patent application is currently assigned to Janssen Biotech, Inc.. The applicant listed for this patent is Janssen Biotech, Inc.. Invention is credited to Alireza Rezania, Sebastian Rieck.
Application Number | 20190365823 16/540657 |
Document ID | / |
Family ID | 60039713 |
Filed Date | 2019-12-05 |
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United States Patent
Application |
20190365823 |
Kind Code |
A1 |
Rieck; Sebastian ; et
al. |
December 5, 2019 |
ENCAPSULATED INTESTINAL MIDGUT ENDODERM CELLS
Abstract
Cell populations of intestinal midgut endoderm cells and methods
of generating the cells expressing markers characteristic of
intestinal endoderm lineage are disclosed. Methods of treating
conditions such as diabetes are also disclosed.
Inventors: |
Rieck; Sebastian; (San
Diego, CA) ; Rezania; Alireza; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janssen Biotech, Inc. |
Horsham |
PA |
US |
|
|
Assignee: |
Janssen Biotech, Inc.
Horsham
PA
|
Family ID: |
60039713 |
Appl. No.: |
16/540657 |
Filed: |
August 14, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15478881 |
Apr 4, 2017 |
10420803 |
|
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16540657 |
|
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62322636 |
Apr 14, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/385 20130101;
A61K 35/545 20130101; A61K 35/38 20130101; A61P 3/10 20180101; C12N
2501/119 20130101; C12N 2501/19 20130101; C12N 2501/727 20130101;
C12N 2500/38 20130101; C12N 2501/415 20130101; C12N 2501/155
20130101; C12N 5/0679 20130101; C12N 2506/02 20130101 |
International
Class: |
A61K 35/38 20060101
A61K035/38; A61K 35/545 20060101 A61K035/545; C12N 5/071 20060101
C12N005/071 |
Claims
1. A population of substantially intestinal midgut endoderm cells,
wherein the population of cells is encapsulated.
2. The population of substantially intestinal midgut endoderm cells
of claim 1, wherein the population of cells is
micro-encapsulated.
3. The population of substantially intestinal midgut endoderm cells
of claim 1, wherein the population cells is macro-encapsulated.
4. The population of substantially intestinal midgut endoderm cells
of claim 1, wherein the intestinal midgut endoderm cells express
CDX2 and FOXA2.
5. The population of substantially intestinal midgut endoderm cells
of claim 1, wherein the intestinal midgut endoderm cells express
transcription factors selected from the group consisting of SOX9,
PDX1, KLF5 and HOXC5.
6. The population of substantially intestinal midgut endoderm cells
of claim 1, wherein the intestinal midgut endoderm cells do not
express transcription factors selected from the group consisting of
SOX2, ALB, PTF1A, HOXA13 and LGR5.
7. The population of substantially intestinal midgut endoderm cells
of claim 1, wherein the intestinal midgut endoderm cells do not
express HAND1.
8. The population of substantially intestinal midgut endoderm cells
of claim 1, wherein the population comprises greater than 70%
intestinal midgut endoderm cells.
9. The population of substantially intestinal midgut endoderm cells
of claim 8, wherein the population comprises greater than 80%
intestinal midgut endoderm cells.
10. The population of substantially intestinal midgut endoderm
cells of claim 8, wherein the population comprises greater than 90%
intestinal midgut endoderm cells.
11. The population of substantially intestinal midgut endoderm
cells of claim 1, wherein the population comprises less than 20%
mesenchymal cells.
12. The population of substantially intestinal midgut endoderm
cells of claim 11, wherein the population comprises less than 10%
mesenchymal cells.
13. The population of substantially intestinal midgut endoderm
cells of claim 11, wherein the population comprises less than 1%
mesenchymal cells.
14. The population of substantially intestinal midgut endoderm
cells of claim 1, wherein the population does not include
mesenchymal cells.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/478,881, filed Apr. 4, 2017, which claims the benefit of
U.S. Provisional Application No. 62/322,636, filed Apr. 14, 2016.
The above-listed applications are herein incorporated by reference
in their entirety.
FIELD OF INVENTION
[0002] The invention relates to the field of cell-based therapy for
conditions such as diabetes. In particular, the invention relates
to cell differentiation, including directing differentiation of
human pluripotent stem cells to generate a population of intestinal
midgut endoderm cells. The invention provides cells or a cell
population and methods of producing the cells that express markers
characteristic of intestinal midgut endoderm.
BACKGROUND
[0003] Advances in the knowledge of incretin hormone mechanism of
action coupled with advancements in the understanding of intestinal
differentiation, both at the stem cell and endocrine cell stages,
have led to interest in developing sources of incretin hormone
producing cells, appropriate for engraftment. One approach is the
generation of functional enteroendocrine L- or K-cells from
pluripotent stem cells, such as human embryonic stem cells ("hESC")
or induced pluripotent stem cells ("iPS").
[0004] The production/secretion of glucagon-like peptide 1 (GLP-1)
from intestinal L-cells or glucose-dependent insulinotropic
polypeptide (GIP) from intestinal K-cells has beneficial effects
for the treatment of diabetes mellitus. Incretin hormones have
systemic effects beneficial for the treatment of diabetes mellitus
(Type 1 and Type 2) (Unger, J., Curr Diab Rep., 2013;
13(5):663-668). Benefits may include augmentation of many aspects
of beta (.beta.) cell function and number, suppression of glucagon
secretion, increases in the insulin sensitivity of peripheral
metabolic tissues, reduction of hepatic gluconeogenesis, and
reduction of appetite. Two classes of incretin-based therapeutic
agents have been identified for the treatment of diabetes mellitus
(GLP-1 receptor agonists and dipeptidyl peptidase 4 (DPP-4)
inhibitors). However, there is currently no incretin-based cell
therapy option that would encompass an endogenous and cellular
barometer for improved and efficient GLP1-based diabetes treatment.
Furthermore, current incretin-based therapies are not regulated by
circulating blood glucose levels and thus provide
non-physiologically regulated GLP production.
[0005] 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. The mesenchyme tissue is derived from the mesoderm,
and is marked by the genes heart and neural crest derivatives
expressed 1 (HAND1), and forkhead box F1 (FOXF1), among others.
Tissues such as, 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 the
definitive endoderm. By the end of gastrulation, the endoderm is
partitioned into anterior-posterior domains that can be recognized
by the expression of a panel of factors that uniquely mark anterior
foregut, posterior foregut, midgut, and hindgut regions of the
endoderm.
[0006] The level of expression of specific transcription factors
("TFs") may be used to designate the identity of a tissue, as
described in Grapin-Botten et al., Trends Genet, 2000;
16(3):124-130. FOXA2 marks the entire endoderm along the
anterior-posterior axis. During transformation of the definitive
endoderm into a primitive gut tube, the gut tube becomes
regionalized into broad domains that can be observed at the
molecular level by restricted gene expression patterns. The
anterior foregut is marked broadly by the high expression of SOX2,
and encompasses organ domains such as the thyroid, lung, and
esophagus. The midgut (includes the duodenum, ileum, jejunum) and
hindgut (includes the colon) are marked by high expression of
caudal type homeobox 2 (CDX2). The SOX2-CDX2 boundary occurs within
the posterior foregut, within which additional TFs mark specific
organ domains. The regionalized pancreas domain within the
posterior foregut shows a very high expression of PDX1 and very low
expression of CDX2 and SOX2. PTF1A is highly expressed in
pancreatic tissue. Low PDX1 expression, together with high CDX2
expression marks the duodenum domain. The intestinal endoderm is
patterned by specific homeobox (HOX) genes. For example, HOXC5 is
preferentially expressed in midgut endoderm cells. In addition, the
expression of HOXA13 and HOXD13 are restricted to hindgut endoderm
cells. The ALB gene, or albumin 1 protein, marks the earliest liver
progenitors in the posterior foregut endoderm (Zaret et al., Curr
Top Dev Biol, 2016; 117:647-669).
[0007] Strides have been made in improving protocols to generate
intestinal endoderm cells from human pluripotent stem cells. For
example, the following publications (Spence et al., Nature, 2011;
470(7332):105-109; Watson et al., Nature Medicine, 2014;
20(11):1310-1314; and Kauffman et al., Front Pharmacol, 2013;
4(79):1-18) outline differentiation protocols using either
fibroblast growth factor (FGF)-4, Wingless-type MMTV integration
site family, member 3A (WNT3A), Chiron 99021, or retinoic acid (RA)
and FGF7 starting at the definitive endoderm stage, that generate
mid-/hindgut spheroids, containing not only a
CDX2.sup.+/FOXA2.sup.+ endodermal population, but also a
significant mesenchymal CDX2.sup.+ cell population. The process of
differentiating enteroendocrine cells from these hESC-derived
mid-/hindgut spheroids is very inefficient, requiring a long time
period, and is directed non-discriminately towards the generation
of all intestinal cell types of the intervillus and villus regions.
A need still exists for technology to generate intestinal midgut
endoderm cells, without substantial contaminating mesenchyme, so as
to be able to produce with high efficiency intestinal
enteroendocrine cells for cell therapeutics.
SUMMARY OF THE INVENTION
[0008] As embodied and fully described, the invention provides
cells, cell populations and methods of generating the cells by
differentiating human pluripotent stem cells. In particular, the
invention features methods of directed differentiation of human
pluripotent stem cells, to generate intestinal midgut endoderm
cells, more particularly an endodermal monolayer of intestinal
midgut endoderm cells.
[0009] One aspect of the invention is a method of producing a
population of intestinal midgut endoderm cells comprising culturing
human pluripotent stem cells in culture media. In embodiments, the
method comprises inducing differentiation of human pluripotent stem
cells to intestinal midgut endoderm cells. In some embodiments, a
population of intestinal midgut endoderm cells is produced. In some
embodiments, a population of substantially intestinal midgut
endoderm cells is produced. In embodiments of the invention, the
intestinal midgut endoderm cells form and are stable as a monolayer
in culture. In embodiments, greater than 50% of the differentiated
cells express markers characteristic of intestinal midgut endoderm,
preferably greater than 60% of the differentiated cells express
markers characteristic of intestinal midgut endoderm, more
preferably greater than 70%, greater than 80%, and greater than 90%
express markers characteristic of intestinal midgut endoderm. In
embodiments, differentiated cells express markers characteristic of
intestinal midgut endoderm are intestinal midgut endoderm cells. In
embodiments, the intestinal midgut endoderm cells express CDX2 and
FOXA2. In all embodiments, the intestinal midgut endoderm cells
express transcription factors selected from SOX9, PDX1, KLF5 and
HOXC5. In embodiments, the intestinal midgut endoderm cells do not
express transcription factors selected from SOX2, ALB, PTF1A,
HOXA13 and LGR5.
[0010] In embodiments of the invention, human pluripotent stem
cells are differentiated to intestinal midgut endoderm cells by
steps including: a) culturing the human pluripotent stem cells in a
first culture media containing GDF-8 and a GSK3.beta. inhibitor,
such as MCX compound, to induce differentiation into definitive
endoderm cells; b) culturing the definitive endoderm cells in a
second culture media containing ascorbic acid and FGF7 to induce
differentiation into primitive gut tube cells; and c) culturing the
primitive gut tube cells in a third culture media containing
retinoic acid and BMP2 or BMP4 in acidic conditions to induce
differentiation into intestinal midgut endoderm cells. In
particular embodiments, acidic conditions is culture with BLAR
medium. The pH of the acidic culture can range from 6.8 to 7.2. In
the embodiments of the invention, the intestinal midgut endoderm
cells form a monolayer in culture. In embodiments, the monolayer of
intestinal midgut endoderm cells is maintained in culture.
[0011] Another embodiment of the invention is a method of treating
a patient suffering from or at risk of developing diabetes
comprising differentiating human pluripotent stem cells to
intestinal midgut endoderm cells, and administering the
differentiated intestinal midgut endoderm cells in a patient with
diabetes. In embodiments, diabetes is Type 1 or Type 2. In
embodiments, administering the cells may be via implantation,
injection or otherwise administration directly or indirectly to the
site of treatment. In some embodiments, the intestinal midgut
endoderm cells are implanted in the body, such as in subcutaneous
space, omentum, liver, kidney, etc. Further embodiments encompass
encapsulated delivery of the cells including encapsulation of
macro- or micro-encapsulation devices.
[0012] A further embodiments of the invention is a method of
producing intestinal midgut endoderm cells comprising inducing
differentiation of definitive endoderm cells in culture to
primitive gut tube cells. In embodiments, the definitive endoderm
cells are cultured in culture media containing ascorbic acid and
FGF7. In further embodiments, the primitive gut tube cells are
cultured in culture media containing retinoic acid and BMP2 or
BMP4. The primitive gut tube cells are differentiated to intestinal
midgut endoderm cells. In some embodiments, primitive gut tube
cells are differentiated to intestinal midgut endoderm cells in
acidic conditions (acidic culture media). In particular
embodiments, acidic conditions is culture with BLAR medium. The pH
of the acidic culture can range from 6.8 to 7.2. In the
embodiments, the intestinal midgut endoderm cells form and maintain
a monolayer in culture.
[0013] In each of the embodiments discussed above, human
pluripotent stem cells are human embryonic stem cells or induced
pluripotent stem cells. In each of the embodiments above, the
intestinal midgut endoderm cells express CDX2 and FOXA2. In all
embodiments, the intestinal midgut endoderm cells express
transcription factors selected from SOX9, PDX1, KLF5 and HOXC5. In
the embodiments, the intestinal midgut endoderm cells do not
express transcription factors selected from SOX2, ALB, PTF1A,
HOXA13 and LGR5. In the embodiments above, the intestinal midgut
endoderm cells express CDX2, FOXA2, SOX9, PDX1, KLF5 and HOXC5. In
the embodiments above, the intestinal midgut endoderm cells do not
express SOX2, ALB, PTF1A, HOXA13 and LGR5. In embodiments, greater
than 50% of the differentiated cells express markers characteristic
of intestinal midgut endoderm, preferably greater than 60% of the
differentiated cells express markers characteristic of intestinal
midgut endoderm, more preferably greater than 70%, greater than
80%, and greater than 90% express markers characteristic of
intestinal midgut endoderm. In embodiments, differentiated cells
express markers characteristic of intestinal midgut endoderm are
intestinal midgut endoderm cells. In embodiments, the intestinal
midgut endoderm cells express CDX2 and FOXA2. In embodiments, the
intestinal midgut endoderm cells express transcription factors
selected from SOX9, PDX1, KLF5 and HOXC5. In embodiments, the
intestinal midgut endoderm cells do not express transcription
factors selected from SOX2, ALB, PTF1A, HOXA13 and LGR5. In the
embodiments, intestinal midgut endoderm cells do not express
HAND1.
[0014] In the embodiments discussed above, the population of
intestinal midgut endoderm cells is substantially intestinal midgut
endoderm cells. In some embodiments, the population of intestinal
midgut endoderm cells comprises greater than 70% intestinal midgut
endoderm cells, preferably greater than 80%, greater than 90%, and
greater than 95% of intestinal midgut endoderm cells. In some
embodiments, the population of intestinal midgut endoderm cells
comprises less than 20% mesenchymal cells, preferably less than
15%, more preferably less than 10%, less than 5%, less than 2%,
less than 1%, less than 0.5%. In embodiments, intestinal midgut
endoderm cells lack expression of HAND1.
[0015] In some embodiments of the invention described above,
differentiation is induced in vitro. In other embodiments,
intestinal midgut endoderm cells differentiate further in vivo.
Another embodiment relates to the intestinal midgut endoderm cells
further differentiating into enteroendocrine cells in vivo. The
enteroendocrine cells express or secrete incretin hormones. In
embodiments, the incretin hormones are GLP1 and GIP.
[0016] In a further embodiment, intestinal midgut endoderm cells
serve as starting material for the identification of small
molecules that promote at high efficiency the in vitro
differentiation of intestinal midgut endoderm cells into, first,
enteroendocrine precursors, and ultimately, incretin expressing or
secreting enteroendocrine cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGS. 1A-1D depict a differentiation method for intestinal
midgut endoderm cells. FIG. 1A is a summary of the differentiation
method, including medium components, growth factors and small
molecules added to each stage, and key stage-specific markers of
the differentiating intestinal midgut endoderm cells (FOXA2,
forkhead box A2; CDX2, caudal type homeobox 2; KLF5, Kruppel-like
factor 5; SOX9, SRY (sex determining region Y)-box 9; PDX1,
pancreatic and duodenal homeobox 1; LO, low expression and protein
presence). Compared to the neutral pH noted at S2D2 (7.35.+-.0.04),
cells were exposed to slightly acidic conditions in BLAR medium
(used interchangeably with "BLAR acidic medium"), during stage 3
(pH; S3D1, 6.98.+-.0.05; S3D2, 7.02.+-.0.04; S3D5, 7.18.+-.0.03)
(FIG. 1B), and as a result of lower sodium bicarbonate levels in
BLAR medium. FIG. 1C depicts representative phase-contrast images
of a S3D5 monolayer (left), and human epithelial colon
adenocarcinoma cell line ("Caco-2") (right), which was used as a
benchmark for characterization of differentiation. A uniform
morphology at S3D5 was consistently observed. Characterization of
cell number using the Nucleocounter.RTM. NC-100 (Chemometec,
Alleroed, Denmark , Catalog No. 900-004) shows that one hESC
differentiated into 4.56.+-.2.60 S3D5 intestinal midgut endoderm
cells (FIG. 1D).
[0018] FIGS. 2A-2D demonstrate the differentiation method,
utilizing Bone morphogenic protein-4 (BMP4), generates intestinal
midgut endoderm cells in monolayer, comprising both CDX2 and FOXA2
on transcript and protein levels. FIG. 2A (bottom) shows that
90.0.+-.5.85 percent of S3D5 cells were co-present for both CDX2
and FOXA2 protein, similar to percentage seen in Caco-2 cells
(86.0.+-.6.67). Conversely, definitive endoderm (DE--S1D3) cells
were devoid of CDX2 and FOXA2 co-presence (2.3.+-.1.2). Gene
expression analysis shows that CDX2 was induced (FIG. 2B), and
FOXA2 maintained (FIG. 2C) during Stage 3. FIG. 2D shows that the
induction of CDX2 protein levels and CDX2/FOXA2 protein co-presence
after the establishment of the FOXA2-positive primitive gut
endoderm stage, S2D2 (FIG. 2D-i), progressively increased through
S3D2 (FIG. 2D-ii), and at S3D5 (FIG. 2D-iii) reached similar levels
as seen in Caco-2 cells (FIG. 2D-iv). CDX2 protein is depicted on
the bottom row, and FOXA2 protein is depicted on the top row. Each
image was taken using the same parameters to allow for quantitative
analysis. Protein expression was assessed by FACS; gene expression
was assessed by qPCR.
[0019] FIGS. 3A-3Q demonstrate the induction by S3D5 of transcript
and protein levels of additional transcription factors (TF) that
constitute robust intestinal midgut endoderm induction; proper
intestinal midgut endoderm was achieved. In addition to CDX2 and
FOXA2 co-expression, S3D5 cells also exhibited co-expression of
SOX9, PDX1, KLF5, HOXC5 (homeobox C5), but not SOX2 (SRY-box 2),
ALB (albumin), PTF1A (pancreas specific transcription factor, 1a),
and LGR5 (leucine rich repeat containing G protein coupled receptor
5). The protein presence of all TFs is depicted in separate single
channel images. FIG. 3A (bottom) demonstrates that 98.7.+-.0.25
percent of cells were co-present for both CDX2 and SOX9 at S3D5.
Strong induction of SOX9 gene expression to levels seen in Caco-2
cells (FIG. 3B), and protein presence as assessed by
immunofluorescence (IF)-analysis were observed (FIG. 3C).
69.4.+-.14.2 percent of cells were co-positive for both CDX2 and
PDX1 (FIG. 3D--bottom). PDX1 gene expression was induced at low
levels, when compared to pancreas-biased S4D3 cells (See, e.g.,
US2014/0242693) (FIG. 3E), and low to absent protein levels was
reflected in the IF-analysis (FIG. 3F). Anterior endoderm TF SOX2
was not observed in S3D5 cells, as 1.45.+-.0.15 of S3D5 cells
exhibited SOX2 and CDX2 co-presence (FIG. 3G--bottom; FIG. 3I), and
gene expression was below levels seen in hESC and Caco-2 cells
(FIG. 3H). The gene expression of KLF5, essential for proper
development of intestinal mid-/hindgut endoderm, was upregulated at
S3D5 (FIG. 3J). Protein co-presence of KLF5 within CDX2-positive
cells at S3D5 was observed (FIG. 3K). ALB gene expression (FIG.
3L), and protein presence (FIG. 3M) was not observed in S3D5 cells.
The gene expression of pancreas lineage allocating TF, PTF1A, was
not induced in S3D5 cells, unlike pancreas-biased S4D3 cells (FIG.
3N). The homeobox gene, HOXC5, present in the embryonic midgut
endoderm was induced in S3D5 cells (FIG. 3O). FIG. 3P demonstrates
that LGR5, a marker of embryonic intestinal endoderm beginning at
mid-gestation in the mouse, was not induced in S3D5 cells. FIG. 3Q
demonstrates that HOXA13, a marker of the intestinal hindgut
endoderm, was not induced in S3D5 cells (FIG. 3P). Gene expression
was assessed by qPCR.
[0020] FIGS. 4A-4B characterize the proliferative profile of
differentiating S3D5 cells. FIG. 4A depicts Caco-2 cells, where
most of CDX2-protein positive cells were in active cell cycle (as
indicated by the co-expression with KI67 protein) (left), and the
proliferative index of the H1-hESC-derived cells during Stage 3
that decreased over time (S3D2--middle; S3D5--right). CDX2 (top
row) and KI67 (bottom row) protein levels are depicted as single
channel images. The percentage KI67-protein positive cells of total
S3D5 cells (total cells are >90% CDX2-positive), assessed by
FACS, was 16.8.+-.3.12, in contrast with percentage seen at S1D3
(97.3.+-.1.3), and in Caco-2 cells (99.2.+-.0.2) (FIG. 4B).
[0021] FIGS. 5A-5C demonstrate use of BMP2 as an alternative to
BMP4, during Stage 3 to achieve a monolayer of intestinal midgut
endoderm cells with CDX2 and FOXA2 protein co-presence. FIG. 5A
summarizes the differentiation method, including the medium
components, growth factors and small molecules that were added to
each stage, and stage-specific markers of the differentiating
intestinal midgut endoderm cells (FOXA2, CDX2, KLF5, SOX9, and
PDX1.sup.LO). Compared to the neutral pH noted at S2D2
(7.35.+-.0.04), cells were exposed to slightly acidic conditions in
BLAR medium, during the entirety of stage 3 (pH; S3D1, 6.92; S3D2,
7.01; S3D5, 7.22) (FIG. 5B), and as a result of lower sodium
bicarbonate levels in BLAR medium. FIG. 5C depicts representative
phase-contrast images of a S3D5 monolayer (left), and Caco-2 cells
(right); a uniform morphology at S3D5 was observed.
[0022] FIGS. 6A-6U demonstrate generation of proper intestinal
midgut endoderm cells in monolayer, each comprising of CDX2, FOXA2,
KLF5, SOX9, PDX1.sup.LO and HOXC5 on transcript and protein levels.
For IF-images, all TF protein levels are depicted as single channel
images. FIG. 6A (bottom) shows that 94 percent of S3D5 cells were
co-present for both CDX2 and FOXA2 protein, similar to or greater
than the percentage seen in Caco-2 cells (86.0.+-.6.67). Gene
expression analysis shows CDX2 induced (FIG. 6B), and FOXA2
maintained (FIG. 6C) during Stage 3. FIG. 6D shows CDX2 protein
levels and complete CDX2/FOXA2 protein co-presence induced at S3D5,
reaching similar levels as seen in Caco-2 cells (FIG. 2D-iv). FIG.
6E (bottom) demonstrates that 99.8 percent of cells were co-present
for both CDX2 and SOX9 at S3D5. Strong induction of SOX9 gene
expression to levels seen in Caco-2 cells (FIG. 6F), and protein
presence as assessed by IF-analysis were observed (FIG. 6G). 45.5
percent of cells were co-positive for both CDX2 and PDX1 (FIG.
6H--bottom). PDX1 gene expression was induced at low levels
compared to pancreas-biased S4D3 cells (FIG. 6I); low to absent
protein levels was reflected in the IF-analysis (FIG. 6J). Anterior
endoderm TF SOX2 was not observed in S3D5 cells, as 0.8 percent of
S3D5 cells exhibited SOX2 and CDX2 co-presence (FIG. 6K--bottom;
FIG. 6M), and gene expression was below levels seen in hESC and
Caco-2 cells (FIG. 6L). Gene expression of KLF5, essential for
proper development of intestinal mid-/hindgut endoderm, was
upregulated at S3D5 (FIG. 6N). Protein co-presence of KLFS within
CDX2-positive cells was observed at S3D5 (FIG. 6O). ALB gene
expression (FIG. 6P), and protein presence (FIG. 6Q) was not
observed in S3D5 cells. Gene expression of pancreas lineage
allocating TF, PTF1A, was not induced in S3D5 cells, unlike
pancreas-biased S4D3 cells (FIG. 6R). The homeobox gene HOXC5,
present in the embryonic intestinal midgut endoderm, was induced in
S3D5 cells (FIG. 6S). FIG. 6T demonstrates that LGR5, a marker of
embryonic intestinal endoderm beginning at mid-gestation, was not
induced in S3D5 cells. FIG. 6U demonstrates that HOXA13, a marker
of the intestinal hindgut endoderm, was not induced in S3D5 cells
(FIG. 6U).
[0023] FIG. 7 characterizes the proliferative profile of
differentiating S3D5 cells. Compared to Caco-2 cells, where most of
CDX2-protein positive cells were in active cell cycle (as indicated
by the co-expression with KI67 protein) (left), the proliferative
index of the H1-hESC-derived cells during Stage 3 was lower
(S3D5--right). CDX2 (top row) and KI67 (bottom row) protein levels
are depicted as single channel images. The percentage KI67-protein
positive cells of total S3D5 cells (total cells are >90%
CDX2-positive), assessed by FACS, was 14.1 percent, in contrast
with percentage seen at S1D3 (97.3.+-.1.3), and in Caco-2 cells
(99.2.+-.0.2).
[0024] FIGS. 8A-8F demonstrate the induction of a heterogeneous
population of CDX2.sup.+ cells. FIG. 8A is a summary of the
differentiation methods, including medium components, growth
factors and small molecules added to each stage, and key
stage-specific markers of the differentiating intestinal
mid-/hindgut endoderm cells (HAND1). FIG. 8B shows phase-contrast
images of H1-hESC cells (top row, left), post-Stage 1 cells
conditioned two days with 500 ng/ml FGF4 and 3 .mu.M Chiron99021
(top row, middle), post-Stage 1 cells conditioned two days with 500
ng/ml FGF4 and 500 ng/ml WNT3A (top row, right), a S3D5 monolayer
conditioned by RA/BMP4 (bottom row, left), and a S3D5 monolayer
conditioned by RA/BMP2 (bottom row, right). The induction of gene
expression after two days of conditioning is shown for CDX2 at low
levels (FIG. 8C), is maintained for the endoderm marker FOXA2 (FIG.
8D), and is induced for the mesoderm/mesenchyme marker HAND1 (FIG.
8F). KLF5 was not induced (FIG. 8E).
DETAILED DESCRIPTION OF THE INVENTION
[0025] It is to be understood that this invention is not limited to
particular methods, reagents, compounds, compositions or biological
systems, which can, of course, vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to be
limiting.
[0026] The present invention pertains to the generation of
intestinal midgut endoderm cells. The cells were generated using a
specific culturing sequence. Accordingly, the present invention
provides an in vitro cell culture for differentiating cells derived
from pluripotent stem cells into cells expressing markers
characteristic of the intestinal midgut endoderm cell lineage, such
as expression of CDX2 and FOXA2. The invention further provides a
method for obtaining and maintaining such cells in a monolayer via
an in vitro cell culture. In certain embodiments, the invention is
based on the discovery that the inclusion of retinoic acid and BMP4
or BMP2 or analogues thereof, act to induce CDX2 and maintain FOXA2
protein expression in differentiating cells to facilitate
differentiation towards intestinal midgut endoderm cells. CDX2 is
not expressed at the protein level at definitive endoderm (Stage 1)
or primitive gut tube (Stage 2). Accordingly, the present invention
provides methods of differentiating pluripotent stem cells to
generate intestinal midgut endoderm cells that express CDX2 and
FOXA2.
Definitions
[0027] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice for testing of the present
invention, the preferred materials and methods are described
herein. In describing and claiming the present invention, the
following terminology will be used.
[0028] Stem cells are undifferentiated cells defined by the ability
of a single cell both to self-renew, and to 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.
[0029] Stem cells are classified according to their developmental
potential as: (1) totipotent; (2) pluripotent; (3) multipotent; (4)
oligopotent; and (5) unipotent. Totipotent cells are able to give
rise to all embryonic and extraembryonic cell types. Pluripotent
cells are able to give rise to all embryonic cell types.
Multipotent cells include those 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). Cells that are oligopotent can give rise to a more
restricted subset of cell lineages than multipotent stem cells; and
cells that are unipotent are able to give rise to a single cell
lineage (e.g., spermatogenic stem cells).
[0030] Stem cells are also categorized on the basis of the source
from which they may be obtained. An adult stem cell is generally a
multipotent undifferentiated cell found in tissue comprising
multiple differentiated cell types. The adult stem cell can renew
itself. Under normal circumstances, it can also differentiate to
yield the specialized cell types of the tissue from which it
originated, and possibly other tissue types. Induced pluripotent
stem cells (iPS cells) are adult cells that are converted into
pluripotent stem cells. (Takahashi et al., Cell, 2006;
126(4):663-676; Takahashi et al., Cell, 2007; 131:1-12). An
embryonic stem cell is a pluripotent cell from the inner cell mass
of a blastocyst-stage embryo. A fetal stem cell is one that
originates from fetal tissues or membranes.
[0031] Embryonic tissue is typically defined as tissue originating
from the embryo (which in humans refers to the period from
fertilization to about six weeks of development). Fetal tissue
refers to tissue originating from the fetus, which in humans refers
to the period from about six weeks of development to parturition.
Extraembryonic tissue is tissue associated with, but not
originating from, the embryo or fetus. Extraembryonic tissues
include extraembryonic membranes (chorion, amnion, yolk sac and
allantois), umbilical cord and placenta (which itself forms from
the chorion and the maternal decidua basalis).
[0032] Differentiation is the process by which an unspecialized
("uncommitted") or less specialized cell acquires the features of a
specialized cell, such as an intestinal cell or pancreatic cell,
for example. A differentiated 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.
[0033] In a broad sense, a progenitor cell is a cell that has the
capacity to create progeny that are more differentiated than
itself, and yet retains the capacity to replenish the pool of
progenitors. By that definition, stem cells themselves are also
progenitor cells, as are the more immediate precursors to
terminally differentiated cells. In a narrower sense, a progenitor
cell is often defined as a cell that is intermediate in the
differentiation pathway, i.e., it arises from a stem cell and is
intermediate in the production of a mature cell type or subset of
cell types. This type of progenitor cell is generally not able to
self-renew. Accordingly, if this type of cell is referred to
herein, it will be referred to as a non-renewing progenitor cell or
as an intermediate progenitor or precursor cell.
[0034] "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
as compared to an undifferentiated cell. 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.
[0035] As used herein, a cell is "positive for" a specific marker
or "positive" when the specific marker is sufficiently detected in
the cell. Similarly, the cell is "negative for" a specific marker,
or "negative" when the specific marker is not sufficiently detected
in the cell. In particular, positive by fluorescence-activated flow
cytometry (FACS) is usually greater than 2%, whereas the negative
threshold by FACS is usually less than 1%.
[0036] As used herein, positive by real-time PCR (RT-PCR) had less
than 28 cycles (Cts), and using Taqman.RTM. Low Density Array
(TLDA) had less than 33 Cts; whereas negative by Open Array.RTM. is
more than 28.5 cycles and negative by TLDA is more than 33.5
Cts.
[0037] To differentiate pluripotent stem cells into functional
intestinal midgut endoderm cells in static in vitro cell culture,
the differentiation process is often viewed as progressing through
consecutive stages. Here, the differentiation process to intestinal
midgut endoderm occurs through three stages In this step-wise
progression, "Stage 1" refers to the first step in the
differentiation process, the differentiation of pluripotent stem
cells into cells expressing markers characteristic of definitive
endoderm cells (hereinafter referred to alternatively as "Stage 1
cells"). "Stage 2" refers to the second step, the differentiation
of cells expressing markers characteristic of definitive endoderm
cells into cells expressing markers characteristic of primitive gut
tube cells (hereinafter referred to alternatively as "Stage 2
cells"). "Stage 3" refers to the third step, the differentiation of
cells expressing markers characteristic of gut tube cells into
cells expressing markers characteristic of intestinal midgut
endoderm cells (hereinafter referred to alternatively as "Stage 3
cells").
[0038] However, it should be noted that not all cells in a
particular population progress through these stages at the same
rate. Consequently, it is not uncommon in in vitro cell cultures to
detect the presence of cells that have progressed less, or more,
down the differentiation pathway than the majority of cells present
in the population, particularly at the later differentiation
stages. For purposes of illustrating the present invention,
characteristics of the various cell types associated with the
above-identified stages are described herein.
[0039] "Definitive endoderm cells," 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 at least one of
the following markers: FOXA2 (also known as hepatocyte nuclear
factor 3-.beta. ("HNF3.beta.")), GATA4, SOX17, CXCR4, Brachyury,
Cerberus, OTX2, goosecoid, C-Kit, CD99, and MIXL1. Markers
characteristic of the definitive endoderm cells include CXCR4,
FOXA2 and SOX17. Thus, definitive endoderm cells may be
characterized by their expression of CXCR4, FOXA2 and SOX17. In
addition, depending on the length of time cells are allowed to
remain in Stage 1, an increase in HNF4.alpha. may be observed.
[0040] "Primitive gut tube cells," as used herein, refers to
endoderm cells derived from definitive endoderm that can give rise
to all endodermal organs, such as lungs, liver, pancreas, stomach,
and intestine. Primitive gut tube cells may be characterized by
their substantially increased expression of HNF4.alpha. over that
expressed by definitive endoderm cells.
[0041] "Foregut endoderm cells," as used herein, refers to endoderm
cells that give rise to the esophagus, lungs, stomach, liver,
pancreas, gall bladder, and the most anterior portion of the
duodenum. Foregut endoderm cells may be characterized by their
expression of SOX2, PDX1, ALB, SOX17 and FOXA2, among others.
[0042] "Intestinal midgut endoderm cell," as used herein, refers to
endoderm cells that give rise to small intestine. Intestinal midgut
endoderm cells may be characterized by their expression of CDX2,
FOXA2, and low expression of PDX1 (PDX1.sup.LO). The expression of
certain HOX genes can distinguish between midgut and hindgut
endoderm. For example, HOXC5 is preferentially expressed in midgut
endoderm cells.
[0043] "Hindgut endodermal cell" as used herein, refers to endoderm
cells that give rise to large intestine. Hindgut endoderm cells may
be characterized by their expression of CDX2, FOXA2, HOXA13 and
HOXD13.
[0044] "Mesenchyme cell," as used herein, refers to mesoderm cells
that give rise to connective tissues, such as bones, cartilage,
lymphatic, and circulatory systems. Expression of HAND1 and FOXF1
define mesenchyme cells.
[0045] The term "patient" or "subject" or "host" refers to animals,
including mammals, preferably humans, who are treated with
compositions or pharmaceutical compositions, or in accordance with
the methods described herein.
[0046] The term "effective amount" or equivalents thereof refers to
an amount of an agent or compound including but not limited to a
growth factor, which is sufficient to promote and differentiate
human pluripotent stem cells to a differentiated cell population,
for example, to a definitive endoderm, foregut endoderm, intestinal
midgut endoderm, hindgut endoderm, pancreatic endoderm and the
like.
[0047] The terms "administering" and "administration" are used
interchangeably herein and mean the cells may be implanted,
injected, transplanted or otherwise administered directly or
indirectly to the site of treatment. When cells are administered in
semi-solid or solid devices, implantation is a suitable means of
delivery, particularly surgical implantation into a precise
location in the body, such as into subcutaneous space, omentum,
liver, kidney (kidney capsule). Liquid or fluid pharmaceutical
compositions may be administered to a more general location.
[0048] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural referents unless
the content clearly dictates otherwise. Thus, for example,
reference to "a cell" includes a combination of two or more cells,
and the like.
[0049] As used herein, the term "about" when referring to a
measurable value such as an amount, a temporal duration, and the
like, is meant to encompass variations of between .+-.20% and
.+-.0.1%, preferably .+-.20% or .+-.10%, more preferably .+-.5%,
even more preferably .+-.1%, and still more preferably .+-.0.5%,
.+-.0.1%. 0.05% or 0.01% of the specified value, as such variations
are appropriate to perform the disclosed methods.
[0050] The following abbreviations may appear throughout the
specification and claims: [0051] ABCG2--ATP-Binding Cassette,
Sub-Family G, Member 2; [0052] ALB--albumin; [0053] BMP--bone
morphogenic protein; [0054] CDX2--caudal type homeobox 2; [0055]
CXCR4--C-X-C chemokine receptor type 4; [0056] FAF-BSA--Fatty-acid
Free Bovine Serum Albumin [0057] FGF--fibroblast growth factor;
[0058] FOXA2--Forkhead Box A2; [0059] GATA4--GATA binding protein
4; [0060] GDF--growth differentiation factor; [0061]
GIP--glucose-dependent insulinotropic polypeptide; [0062]
GLP-1--glucagon-like peptide 1; [0063] GSK3B--Glycogen synthase
kinase 3 beta; [0064] HAND1--heart and neural crest derivatives
expressed 1; [0065] HOX--homeobox; [0066] hTERT--human telomerase
reverse transcriptase; [0067] KLF--Kruppel-like factor; [0068]
LGR5--leucine rich repeat containing G protein coupled receptor 5;
[0069] MIXL1--Mix Paired-Like Homeobox-1; [0070]
OCT4--octamer-binding transcription factor 4; [0071]
OTX2--orthodenticle homeobox 2; [0072] PDX1--pancreatic and
duodenal homeobox 1; [0073] PTF1A--pancreas specific transcription
factor, 1a; [0074] SOX--sex determining region Y (SRY)-box; [0075]
TRA1-60--T cell receptor alpha-1-60; [0076] UTF1--Undifferentiated
embryonic cell transcription factor 1; [0077] WNT3A--wingless-type
MMTV integration site family, member 3A; and [0078] ZFP42--zinc
finger protein 42.
DETAILED DESCRIPTION
[0079] Pluripotent stem cells have the potential to differentiate
into cells of all three germinal layers: endoderm, mesoderm, and
ectoderm tissues. Exemplary types of pluripotent stem cells that
may be used include established lines of pluripotent cells,
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 Research Institute, Madison, Wis.,
USA). Cells taken from a pluripotent stem cell population already
cultured in the absence of feeder cells are also suitable. iPS, or
reprogrammed pluripotent cells, derived from adult somatic cells
using forced expression of a number of pluripotent related
transcription factors, such as OCT4, NANOG, SOX2, KLF4, and ZFP42
(Annu Rev Genomics Hum Genet 2011, 12:165-185; see also iPS, Cell,
126(4): 663-676) may also be used. The human embryonic stem cells
used in the methods of the invention may also be prepared as
described by Thomson et al. (U.S. Pat. No. 5,843,780; Science,
1998, 282:1145-1147; Curr Top Dev Biol 1998, 38:133-165; Proc Natl
Acad Sci U.S.A. 1995, 92:7844-7848). Mutant human embryonic stem
cell lines, such as, BG01v (BresaGen, Athens, Ga.), or cells
derived from adult human somatic cells, such as, cells disclosed in
Takahashi et al., Cell 131: 1-12 (2007) may also be used. In
certain embodiments, pluripotent stem cells suitable for use in the
present invention may be derived according to the methods described
in: Li et al. (Cell Stem Cell 4: 16-19, 2009); Maherali et al.
(Cell Stem Cell 1: 55-70, 2007); Stadtfeld et al. (Cell Stem Cell
2: 230-240); Nakagawa et al. (Nature Biotechnol 26: 101-106, 2008);
Takahashi et al. (Cell 131: 861-872, 2007); and U.S. Patent App.
Pub. No. 2011/0104805. In certain embodiments, the pluripotent stem
cells may be of non-embryonic origins. All of these references,
patents, and patent applications are herein incorporated by
reference in their entirety, in particular, as they pertain to the
isolation, culture, expansion and differentiation of pluripotent
cells.
[0080] Pluripotent stem cells differentiate through various stages
each of which may be characterized by the presence or absence of
particular markers. Differentiation of the cells into these stages
is achieved by the specific culturing conditions, including the
presence or lack of certain factors added to the culture media. In
general, this differentiation may involve differentiation of
pluripotent stem cells into definitive endoderm cells, referred to
herein as Stage 1. These definitive endoderm cells may then be
further differentiated into primitive gut tube cells, referred to
herein as Stage 2. Primitive gut tube cells in turn may then be
differentiated into intestinal midgut endoderm cells, referred to
herein as Stage 3.
Differentiation of Pluripotent Stem Cells into Cells Expressing
Markers Characteristic of Intestinal Midgut Endoderm Cells
[0081] 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; UTF1; ZFP42; SSEA-3; SSEA-4; TRA-1-60; and
TRA-1-81.
[0082] Exemplary pluripotent stem cells include the human embryonic
stem cell line H1 (NIH code: WA01), the human embryonic stem cell
line H9 (NIH code: WA09), the human embryonic stem cell line H7
(NIH code: WA07), and the human embryonic stem cell line SA002
(Cellartis, Sweden). Also suitable 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, UTF1, ZFP42, SSEA-3, SSEA-4, TRA-1-60, and
TRA-1-81.
[0083] Also 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 embodiment of the invention, a
cell expressing markers characteristic of the definitive endoderm
lineage is a primitive streak precursor cell. In an alternate
embodiment, a cell expressing markers characteristic of the
definitive endoderm lineage is a mesendoderm cell. In an alternate
embodiment, a cell expressing markers characteristic of the
definitive endoderm lineage is a definitive endoderm cell.
[0084] Also suitable for use in the present invention is a cell
that expresses at least one of the markers characteristic of the
intestinal midgut endoderm lineage. In one embodiment of the
present invention, a cell expressing markers characteristic of the
intestinal endoderm lineage is an intestinal midgut endoderm cell
in which the cell expresses FOXA2 and CDX2. In some embodiments,
the cell does not express SOX2, ALB, PTF1A, HOXA13 or LGR5. In
embodiments, a cell expressing markers characteristic of the
intestinal endoderm lineage is an intestinal midgut endoderm cell
in which the cell expresses each of FOXA2, CDX2, SOX9, PDX1, KLF5
and HOXC5. In embodiments, a cell expressing markers characteristic
of the intestinal endoderm lineage is an intestinal midgut endoderm
cell in which the cell does not express any of SOX2, ALB, PTF1A,
HOXA13 and LGR5.
[0085] The invention provides for staged, directed differentiation
of pluripotent stem cells toward intestinal midgut endoderm cells
using cell culture conditions and media. In embodiments of the
invention, to arrive at a cell expressing markers characteristic of
intestinal midgut endoderm cell, a protocol starting with
pluripotent stem cells, such as embryonic stem cells and induced
pluripotent cells, is employed. This protocol includes the
following stages. [0086] Stage 1: Pluripotent stem cells, such as
embryonic stem cells obtained for cell culture lines, are treated
with appropriate factors to induce differentiation into cells
expressing markers characteristic of definitive endoderm cells.
[0087] Stage 2: Cells resulting from Stage 1 are treated with
appropriate factors to induce further differentiation into cells
expressing markers characteristic of primitive gut tube cells.
[0088] Stage 3: Cells resulting from Stage 2 are treated with
appropriate factors to induce further differentiation into cells
expressing markers characteristic of intestinal midgut endoderm
cells.
[0089] Methods for assessing expression of protein and nucleic acid
markers in cultured or isolated cells are standard in the art.
These methods include 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, FACS
(see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual,
New York: Cold Spring Harbor Laboratory Press (1998)). 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 cell type of interest.
[0090] 1. Differentiation of Pluripotent Stem Cells into Cells
Expressing Markers Characteristic of Definitive Endoderm Cells
[0091] Pluripotent stem cells may be differentiated into cells
expressing markers characteristic of definitive endoderm cells by
any suitable method known in the art, or by any method proposed in
this invention. In one embodiment of the invention, pluripotent
stem cells are treated with a medium, such as MCDB-131 medium (Life
Technologies, Carlsbad, Calif.) supplemented with factors including
GDF8 and a GSK3.beta. inhibitor (such as the cyclic
aniline-pyridinotriazine compounds disclosed in U.S. Patent App.
Pub. No. 2010/0015711; incorporated herein by reference in its
entirety) to induce differentiation into cells expressing markers
characteristic of definitive endoderm cells. There is a broad range
of GSK3.beta. inhibitors, such as staurosporine, and the preferred
GSK3.beta. inhibitor
(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-nonaen-16-one, referred to herein as "MCX
Compound". Treatment may involve contacting pluripotent stem cells
with a medium supplemented with about 10 ng/ml to 1000 ng/ml,
preferably 50 ng/ml to about 150 ng/ml, alternatively about 75
ng/ml to about 125 ng/ml, alternatively about 100 ng/ml of GDF8.
The treatment may also involve contacting the cells with about 0.1
to 10 .mu.M, preferably about 0.1 to 5 .mu.M, alternatively about
0.5 to about 2.5 .mu.M, preferable about 1.5 .mu.M or about 1.0
.mu.M of MCX compound. Other components of the medium may include:
Sodium bicarbonate at about 2.7 g/1000 ml to 3.6 g/1000 ml,
preferably 2.7 g/1000 ml; FAF-BSA at about 0.1% to 2.0%, preferably
about 0.5%; GlutaMAX.TM. (Life Technologies Corporation, Carlsbad,
Calif.) at 1:100 dilution ("1.times. concentration"); and D-Glucose
at a concentration range of about 2 mM to 20 mM, preferably 4.5 mM
to obtain a concentration of 10 mM D-Glucose.
[0092] The pluripotent cells may be cultured for approximately two
to five days, preferably about three to four days, to facilitate
their differentiation into definitive endoderm cells. In one
embodiment, the pluripotent cells are cultured in the presence of
an effective amount of TGF.beta. signaling molecule and/or
GSK3.beta. inhibitor, for example, an effective amount of GDF8 and
MCX compound for one day, followed by culturing in the presence of
GDF8 and a lower concentration of MCX compound for one day,
followed by culturing in the presence of GDF8 for one day in the
absence of the MCX compound. In particular, the cells may be
cultured in the presence of GDF8 and about 1.5 .mu.M of MCX
compound for one day, followed by culturing in the presence of GDF8
and about 0.1 .mu.M MCX compound for one day, followed by culturing
in the presence of GDF8 for one day in the absence of the MCX
compound. In an alternate embodiment, the cells may be cultured in
the presence of GDF8 and about 1.5 .mu.M of MCX compound for one
day, followed by culturing in the presence of GDF8 and about 0.1
.mu.M MCX compound for one day.
[0093] Generation of cells expressing markers characteristic of
definitive endoderm cells 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 can be detected
when the cells begin to express markers characteristic of
definitive endoderm cells, such as CXCR4, FOXA2 and SOX17. In the
embodiments, cells expressing markers characteristic of definitive
endoderm cells are definitive endoderm cells.
[0094] 2. Differentiation of Cells Expressing Markers
Characteristic of Definitive Endoderm Cells into Cells Expressing
Markers Characteristic of Primitive Gut Tube Cells
[0095] The cells expressing markers characteristic of definitive
endoderm cells may be further differentiated into cells expressing
markers characteristic of primitive gut tube cells. In one
embodiment, the formation of cells expressing markers
characteristic of primitive gut tube cells includes culturing cells
expressing markers characteristic of definitive endoderm cells with
a medium, such as MCDB-131, containing FGF7 to differentiate these
cells. For example, the culture medium may include from about 10
ng/ml to 100 ng/ml, preferably about 25 ng/ml to about 75 ng/ml,
alternatively from about 30 ng/ml to about 60 ng/ml, alternatively
about 50 ng/ml of FGF7. The cells may be cultured under these
conditions for about two to three days, preferably about two
days.
[0096] In another embodiment, differentiation into cells expressing
markers characteristic of primitive gut tube cells includes
culturing cells expressing markers characteristic of definitive
endoderm cells with FGF7 and ascorbic acid (vitamin C). The culture
medium, such as MCDB-131, may include from about 0.1 mM to about
1.0 mM ascorbic acid, preferably about 0.1 mM to about 1.0 mM,
alternatively from about 0.2 mM to about 0.4 mM, alternatively
about 0.25 mM of ascorbic acid. The culture medium may also include
from about 10 ng/ml to 100 ng/ml, preferably about 10 ng/ml to
about 50 ng/ml, alternatively from about 15 ng/ml to about 30
ng/ml, alternatively about 50 ng/ml or about 25 ng/ml of FGF7. For
example, the culture medium may include about 0.25 mM ascorbic acid
and about 50 ng/ml FGF7. Other components of the medium may
include: sodium bicarbonate at about 2.7 g/1000 ml to 3.6 g/1000
ml, preferably 2.7 g/1000 ml; FAF-BSA at about 0.1% to 2.0%,
preferably about 0.5%; GlutaMAX.TM. at 1:100 dilution ("1.times.
concentration"); and D-Glucose at a concentration range of about 2
mM to 20 mM, preferably 4.5 mM to obtain a concentration of 10 mM
D-Glucose. In one embodiment, cells expressing markers
characteristic of definitive endoderm cells are treated for 2 days
with FGF7 and ascorbic acid. Differentiation of definitive endoderm
cells can be detected when the cells begin to express markers
characteristic of primitive gut tube cells, such as expression of
FOXA2 and increased expression of HNF4.alpha.. In the embodiments,
cells expressing markers characteristic of primitive gut tube cells
are primitive gut tube cells.
[0097] 3. Differentiation of Cells Expressing Markers
Characteristic of Primitive Gut Tube Cells into Cells Expressing
Markers Characteristic of Intestinal Midgut Endoderm Cells
[0098] Cells expressing markers characteristic of primitive gut
tube cells may be further differentiated into cells expressing
markers characteristic of intestinal midgut endoderm cells. In one
embodiment, primitive gut tube cells are further differentiated
into intestinal midgut endoderm cells by culturing the primitive
gut tube cells in a culture medium, such as BLAR medium (Life
Technologies, Corporation, Carlsbad, Calif.), supplemented with
retinoic acid and a BMP4 or BMP2. In a preferred embodiment, the
medium is supplemented with from about 0.5 .mu.M to about 5 .mu.M
of retinoic acid, preferably about 1 .mu.M, and from about 10 ng/ml
to about 100 ng/ml BMP4 or BMP2, preferably about 50 ng/ml of BMP4
or BMP2. Other supplements to the medium may include: FAF-BSA at
about 0.1% to 2.0%, preferably about 0.5%; GlutaMAX.TM.; and
D-Glucose at a concentration range of about 2 mM to 20 mM,
preferably 4.5 mM to obtain a concentration of 10 mM D-Glucose. In
one embodiment, cells expressing markers characteristic of
primitive gut cells are treated for 3 to 5 days, preferably for 5
days with BMP4 or BMP2 and retinoic acid. The pH of the culture can
range from 6.8 to 7.2 during the 5-day Stage 3 conditioning period
(compared to normal pH at S2D2 being 7.3 or more).
[0099] The invention relates to a method of producing a population
of intestinal midgut endoderm cells by culturing human pluripotent
stem cells in selected culture media for generating intestinal
midgut endoderm cells. In embodiments, the method induces
differentiation of human pluripotent stem cell to intestinal midgut
endoderm cells in a staged process. In embodiments, a population of
intestinal midgut endoderm cells is produced. In some embodiments,
a population of substantially intestinal midgut endoderm cells is
produced. In the embodiments, the intestinal midgut endoderm cells
form and maintain a monolayer on planar culture. In embodiments,
the intestinal midgut endoderm cells are stable as a monolayer in
culture. Cells stable as a monolayer, or remain stable as a
monolayer, herein refers to a monolayer of cells that do not form
spheroids in culture.
[0100] In embodiments, greater than 50% of the differentiated cells
express markers characteristic of intestinal midgut endoderm. In
embodiments, greater than 60%, greater than 70%, greater than 80%,
greater than 90% or greater than 95% of the differentiated cells
express markers characteristic of intestinal midgut endoderm. In
embodiments, differentiated cells express markers characteristic of
intestinal midgut endoderm are intestinal midgut endoderm cells. In
embodiments, the intestinal midgut endoderm cells express CDX2 and
FOXA2 as determined by FACS analysis and qPCR. In some embodiments,
intestinal midgut endoderm cells express transcription factors
selected from SOX9, PDX1, KLF5 and HOXC5 as determined by IF
analysis and qPCR. In embodiments, the intestinal midgut endoderm
cells do not express transcription factors selected from SOX2, ALB,
PTF1A as determined by IF analysis and qPCR, and HOXA13 and LGR5 as
determined by qPCR.
[0101] A further embodiment of the invention is a method of
producing intestinal midgut endoderm cells comprising inducing
differentiation of definitive endoderm cells in culture to
primitive gut tube cells. In embodiments, the definitive endoderm
cells are cultured in culture media containing ascorbic acid and
FGF7. In further embodiments, the primitive gut tube cells are
cultured in culture media containing retinoic acid and BMP2 or
BMP4. The primitive gut tube cells are differentiated to intestinal
midgut endoderm cells. In some embodiments, primitive gut tube
cells are differentiated to intestinal midgut endoderm cells in
acidic conditions (acidic culture media). In particular
embodiments, acidic conditions is culture in BLAR media. The pH of
the acidic culture can range from 6.8 to 7.2 during the 5-day
differentiation conditioning period from primitive gut tube cells
to intestinal midgut endoderm cells (compared to normal pH at S2D2
being 7.3 or more). In the embodiments, the intestinal midgut
endoderm cells form and maintain a monolayer in culture.
[0102] In each of the embodiments discussed herein, human
pluripotent stem cells are human hESC or iPS cells. In each of the
embodiments above and herein, the intestinal midgut endoderm cells
express CDX2 and FOXA2 as determined by FACS analysis and qPCR. In
all embodiments, the intestinal midgut endoderm cells express
transcription factors selected from SOX9, PDX1, KLF5 and HOXC5 as
determined by IF analysis and qPCR. In the embodiments above and
herein, the intestinal midgut endoderm cells do not express
transcription factors selected from SOX2, ALB, PTF1A as determined
by IF analysis and qPCR, and HOXA13 and LGR5 as determined by qPCR.
In the embodiments above and herein, the intestinal midgut endoderm
cells express CDX2, FOXA2, SOX9, PDX1, KLF5 and HOXC5 by IF
analysis and qPCR. In each of the embodiments, the intestinal
midgut endoderm cells do not express SOX2, ALB and PTF1A as
determined by IF analysis and qPCR, and HOXA13 and LGR5 as
determined by qPCR.
[0103] As a result of the differentiation protocol described above
and herein, using specific culture components and culture
conditions, in particular acidic culture condition, such as culture
in BLAR medium, a culture of cells expressing markers for
intestinal midgut endoderm cells is generated; the cells lack
expression of HAND1 as determined by qPCR, a marker of
mesoderm/mesenchymal lineage. Varying the differentiation protocol
to induce pluripotent stem cells to midgut/hindgut endoderm
lineage, such as inducing stem cells at definitive endoderm stage 1
rather than primitive gut tube cell stage 2, results in a mixed
population of endoderm-mesenchyme CDX2+ mid-/hindgut cells that
express HAND1 as determined by qPCR.
[0104] In certain embodiments, the population of intestinal midgut
endoderm cells is substantially intestinal midgut endoderm cells.
In some embodiments, the population of intestinal midgut endoderm
cells comprises greater than 70% intestinal midgut endoderm cells,
preferably greater than 80%, greater than 90%, and greater than 95%
of intestinal midgut endoderm cells. In some embodiments, the
population of intestinal midgut endoderm cells comprises less than
20% mesenchymal cells, preferably less than 15%, more preferably
less than 10%, less than 5%, less than 2%, less than 1%, less than
0.5%. In embodiments, intestinal midgut endoderm cells lack
expression of HAND1.
Use of Differentiated Intestinal Midgut Endoderm Cells
[0105] In another embodiment of the invention, the differentiated
intestinal midgut endoderm cells may be used for treating a patient
suffering from or at risk of developing diabetes alone or in
combination with differentiated or mature endocrine cells, for
example, enteroendocrine cells. In such embodiments, differentiated
intestinal midgut endoderm cells, or mixtures thereof, may be
administered to a patient having diabetes, for example Type 1 or
Type 2 diabetes. In embodiments, intestinal midgut endoderm cells
differentiate and mature to enteroendocrine cells. In embodiments,
intestinal midgut endoderm cells differentiate and mature to
enteroendocrine cells, and the enteroendocrine cells express or
secrete incretin type hormones. In embodiments, incretin hormones
include GLP1 and GIP. Administration of the cells may be via
implantation or injection in the body, in particular implantation
into subcutaneous space, omentum, liver, kidney, etc.
[0106] In some embodiments of the invention described above,
differentiation of intestinal midgut endoderm cells is induced in
vitro. In other embodiments, intestinal midgut endoderm cells
further differentiate and mature in vivo. Another embodiment
relates to the intestinal midgut endoderm cells further
differentiating into enteroendocrine cells in vivo or in a mixture
with enteroendocrine cells in vivo. Such enteroendocrine cells
express or secrete incretin hormones. In embodiments, the
enteroendocrine cell-secreted incretin hormones include GLP1 and
GIP.
[0107] In a further embodiment, intestinal midgut endoderm cells
serve as starting material for the identification of small
molecules that promote at high efficiency the in vitro
differentiation of intestinal midgut endoderm cell type into,
first, enteroendocrine precursors, and then to incretin expressing
or secreting enteroendocrine cells.
[0108] Cells and cell populations and mixtures such as those
described herein may be micro- or macro-encapsulated and
subsequently transplanted into a mammalian host. Encapsulated cells
or cells alone may be transplanted (administered) subcutaneously or
anywhere else in the body whereby the cells may be vascularized and
differentiate and mature in vivo.
EXAMPLES
[0109] The invention can be further understood in view of the
following non-limiting examples.
Example 1
Method of Producing an Intestinal Midgut Endoderm Cell Population
with CDX2 and FOXA2 Co-Presence/Co-Expression
[0110] The following example describes a directed-based method to
generate intestinal midgut endoderm cells from human embryonic stem
cell ("hESC"). "Intestinal midgut endoderm" refers to a
corresponding in vivo or in situ cell type which is CDX2-positive
and FOXA2-positive endoderm cells present at about embryonic day
8.5 ("E8.5") during mouse development, or at about the 3-4 week
time point during human embryonic development.
Materials and Methods
[0111] Cell culture: Cells of the human embryonic stem cell line H1
("H1-hESC") (WA01 cells, WiCell Research Institute, Madison, Wis.)
cultured with EZ8 media (Cat #A1516901 Gibco, Thermo Fisher
Scientific) at passage 28 were seeded as single cells at
0.094.times.10.sup.6 cells/cm.sup.2 on MATRIGEL.TM., at a 1:30
dilution, (Corning Incorporated, Corning, N.Y., Catalog #356231)
coated dishes in a media of Dulbecco's Modified Eagle's Medium
Nutrient mixture F-12 ("DMEM-F12") (Life Technologies Corporation,
Carlsbad, Calif., Catalog No. 11330-032), with the following:
TABLE-US-00001 GlutaMAX .TM. 1:100 dilution (Life Technologies
Corporation, Carlsbad, ("1.times. concentration") California,
Catalog No. 35050-079) Ascorbic acid 0.25 mM (Sigma Aldrich Co.
LLC, St. Louis, Missouri, Catalog No. A4544) FGF2 100 ng/ml (R
& D Systems Inc., Minneapolis, Minnesota, Catalog No.
233-FB-025) Transforming Growth Factor beta ("TGF.beta.") 1 ng/ml
(R & D Systems Inc., Minneapolis, Minnesota, Catalog No.
240-B-002) insulin-transferrin-selenium-ethanolamine 1:200 dilution
("ITS-X") (Life Technologies, Carlsbad, California, Catalog No.
51500056) at a 1:100 dilution FAF-BSA 2% (Proliant, Inc., Boone,
Idaho, Catalog No. 68700) Insulin-like Growth Factor-1 ("IGF-1") 20
ng/ml (R & D Systems Inc., Minneapolis, Minnesota, Catalog No.
291-G1-200) Rock Inhibitor Y-27632 ("Y-compound") 10 .mu.M (Sigma
Aldrich Co. LLC, St. Louis, Missouri, Catalog No. Y-0503)
[0112] About forty-eight hours post-seeding, the cultures were
washed in incomplete PBS (phosphate buffered saline without
magnesium or calcium) (Life Technologies, Carlsbad, Calif., Catalog
No. 14190). Rock Inhibitor Y-27632 (Y compound) was used only for
the first 24 hours of culture.
[0113] Differentiation: The cultures were differentiated using the
following protocol. During Stages 1 through 3 of the protocol,
cultures were maintained on planar adherent cultures. Others,
however, have described differentiation using suspension culture
including US US2014/0242693, which is incorporated by reference in
its entirety; the protocol described herein can be modified and
performed in suspension, which provides for scalability of
manufacturing. The following nomenclature, S#D#, specifies exact
time during Stages 1 through 3. For example, S1D3 is stage 1 day 3.
Briefly, each stage defines differentiation towards definitive
endoderm (stage 1), primitive gut tube (stage 2), and intestinal
midgut endoderm (stage 3).
[0114] a. Stage 1 (3 days): Cells were cultured for one day in the
following Stage 1 media:
TABLE-US-00002 MCDB-131 medium (Life Technologies, Carlsbad,
California, Catalog No. ME120219L2) Sodium bicarbonate 2.7 g/1000
ml (Sigma-Aldrich Co. LLC, St. Louis, Missouri, Catalog No. 5761)
FAF-BSA 0.5% GlutaMAX .TM. 1:100 dilution ("1.times.
concentration") D-Glucose 4.5 mM to obtain a (Sigma-Aldrich Co.
LLC, St. Louis, concentration of 10 mM Missouri, Catalog No. G8769)
D-Glucose GDF8 100 ng/ml (Peprotech, Rocky Hill, New Jersey,
Catalog No. 120-00) 14-Prop-2-en-1-yl-3,5,7,14,17,23,27- 1.5 .mu.M
heptaazatetracyclo [19.3.1.1~2,6~.1~8,12~]heptacosa-
1(25),2(27),3,5,8(26),9,11,21,23-nonaen- 16-one ("MCX
compound").
[0115] Cells were then cultured for an additional day in the
following media:
TABLE-US-00003 [0115] MCDB-131 medium Sodium bicarbonate 2.7 g/1000
ml FAF-BSA 0.5% GlutaMAX .TM. 1.times. concentration D-Glucose 4.5
mM to obtain a concentration of 10 mM D-Glucose GDF8 100 ng/ml MCX
compound 0.1 .mu.M
[0116] Cells were then cultured for an additional day in the same
media as day 2 above but without MCX compound.
[0117] b. Stage 2 (2 days): Cells were treated for two days with
the following medium:
TABLE-US-00004 MCDB-131 medium Sodium bicarbonate 2.7 g/1000 ml
FAF-BSA 0.5% GlutaMAX .TM. 1.times. concentration D-Glucose 4.5 mM
to obtain a concentration of 10 mM D-Glucose Ascorbic Acid 0.25 mM
FGF7 50 ng/ml (R & D Systems, Inc., Minneapolis, Minnesota,
Catalog No. 251-K
[0118] c. Stage 3 (5 days): Cells were treated for five day with
BLAR 001 custom medium:
TABLE-US-00005 BLAR medium (custom manufactured by Life
Technologies, Corporation, Carlsbad, California, Catalog No.
ME120123L2, components listed on Table I) FAF-BSA 0.5% GlutaMAX
.TM. 1.times. concentration D-Glucose 4.5 mM to obtain a
concentration of 10 mM D-Glucose Retinoic Acid 1 .mu.M (Sigma
Aldrich, St. Louis, Missouri, Catalog No. R2625) BMP4 compound 50
ng/ml (R & D Systems, Inc., Minneapolis, Minnesota, Catalog No.
314-BP OR BMP2 compound (R & D Systems, Inc., Minneapolis,
Minnesota, Catalog No. 355-BM),
[0119] Either BMP4 or BMP2 can be used during Stage 3 in this
method to achieve intestinal midgut endoderm cells in monolayer
with CDX2 and FOXA2 protein co-presence, as illustrated in FIGS.
1A-4B (BMP4-based), and FIGS. 5A-7 (BMP2-based).
TABLE-US-00006 TABLE I List of components of BLAR medium.
Concentration (mM) Amino Acids Glycine 3.00E-02 Alanine 3.00E-02
Arginine 3.00E-01 Asparagine 1.00E-01 Aspartic Acid 1.00E-01
Cysteine 1.99E-01 Glutamic acid 3.00E-02 Histidine 1.10E-01
Isoleucine 1.00E-02 Leucine 9.00E-02 Lysine hydrochloride 1.50E-01
Methionine 3.00E-02 Phenylalanine 3.00E-02 Proline 1.00E-01 Serine
1.00E-01 Threonine 3.00E-02 Tryptophan 2.00E-03 Tyrosine disodium
1.00E-02 Valine 3.00E-02 Vitamins Biotin 3.00E-05 Choline chloride
5.00E-03 D-Calcium pantothenate 1.50E-03 Folinic Acid Calcium salt
2.30E-03 Niacinamide 4.90E-03 Pyridoxine hydrochloride 9.70E-04
Riboflavin 1.00E-05 Thiamine hydrochloride 3.00E-03 Vitamin B12
3.70E-06 i-Inositol 2.80E-03 Salts/Minerals Calcium Chloride
(CaCl.sub.2--2H.sub.2O) 3.00E-01 Cupric sulfate
(CuSO.sub.4--5H.sub.2O) 4.80E-06 Ferric sulfate
(FeSO.sub.4--7H.sub.2O) 1.00E-03 Magnesium Sulfate
(MgSO.sub.4--7H.sub.2O) 4.10E-01 Potassium Chloride (KCl) 3.80E+00
Sodium Bicarbonate (NaHCO.sub.3) 1.40E+01 Sodium Chloride (NaCl)
1.10E+02 Sodium Phosphate dibasic 5.00E-01
(Na.sub.2HPO.sub.4--7H.sub.2O) Zinc Sulfate (ZnSO4--H2O) 1.00E-04
Other Adenine 1.00E-03 D-Glucose (Dextrose) 5.00E+00 Lipoic Acid
1.20E-05 Phenol Red 1.00E-02 Sodium Pyruvate 1.00E+00 Thymidine
9.80E-05
[0120] Quantification of differentiated cells: For quantification
of protein presence co-localization, S3D5 cells were harvested and
analyzed by FACS. FACS staining was conducted as described in
Nature Biotechnology, 2014 (32) 11, 1121-1133, incorporated herein
by reference in its entirety, and using the antibodies listed in
Table II. In brief, differentiated cells were incubated in
TrypLE.TM. Express (Life Technologies, Carlsbad, Calif., Catalog
No. 12604) for 5-10 minutes at 37.degree. C., released into a
single-cell suspension, after which they were washed twice with a
staining buffer of PBS containing 0.2% BSA (BD Biosciences, San
Jose, Calif., Catalog No. 554657). Intracellular antibody staining
was accomplished by utilizing the LIVE/DEAD Violet Fluorescent
reactive dye (Life Technologies, Carlsbad, Calif., Catalog No.
L34955) at 4.degree. C. for 30 minutes followed by a single wash in
cold PBS. Fixing of cells was in 300 .mu.l of Cytofix/Cytoperm
Buffer (BD Biosciences, San Jose, Calif., Catalog No. 554723)
followed by two washes in Perm/Wash Buffer (BD Biosciences, San
Jose, Calif., Catalog No. 554722). Cells were then incubated with
the appropriate antibodies at 4.degree. C. for 30 minutes (for
unconjugated antibodies) or 1 hour (for conjugated antibodies), and
then washed twice prior to analysis on the BD FACS Canto II using
BD FACS Diva Software with at least 30,000 events being acquired.
Non-viable cells were excluded during FACS analysis, and gating was
determined by using isotype antibodies ("IgG"). IgG FACS data is
shown as the top panel for each FACS experiment presented.
Antibodies were tested for specificity using positive controls,
such as Caco-2 cells, or negative controls, such as S1D3 definitive
endoderm ("DE") cells.
TABLE-US-00007 TABLE II List of antibodies used for FACS analysis.
Source/Catalog Antigen Species Number Dilution PE IgG1, .kappa.
Isotype control Mouse BD Cat# 555749 1:5 Alexa Fluor 647 IgG1,
.kappa. Mouse BD Cat# 557732 Neat Isotype control PE anti-Human
Mouse BD Cat# 561589 Neat FOXA2 PE Anti-human CDX2 Mouse BD Cat#
563428 Neat Alexa Fluor 647 anti- Mouse BD Cat# 560395 Neat CDX2
Alexa Fluor 647 Mouse BD Cat# 561126 Neat anti-KI67 PE anti-PDX1
Mouse BD Cat# 562161 Neat PE anti-SOX2 Mouse BD Cat# 560291 Neat
Anti-Mouse IgG(H + L) Goat Life technology 1:4000 Secondary
Antibody, Cat# A21235 Alexa Fluor 647 conjugate F(ab')2 anti-rabbit
Goat Life technology Neat IgG(H + L) Secondary Cat# A10542
Antibody, RPE conjugate Anti-SOX9 Rabbit Millipore Cat# 1:10 AB5535
Anti-CDX2 Mouse BioGenex Cat# 1:10 [CDX2-88] MU392A-UC
[0121] For quantification of protein co-localization at various
stages, Caco-2, S2D2, S3D2 and S3D5 cells were harvested as a
monolayer and analyzed by immunofluorescence ("IF"). Note that the
morphology seen in IF images was caused by the method of cell
scraping from the monolayer of adherent cultures. H1-hESC-derived
cells were prepared and stained as described in Nature
Biotechnology, 2014 (32) 11, 1121-1133, and using the antibodies
listed in Table III. For cryosectioning, cells were rinsed with PBS
followed by overnight fixation in 4% PFA (Sigma Aldrich, St. Louis,
Mo., Catalog No. 158127) at 4.degree. C. Following fixation, 4% PFA
was removed, cells rinsed twice with PBS, and incubated overnight
at 4.degree. C. in 30% sucrose solution (Amresco, Solon, Ohio,
Catalog No. 0335). The samples were cryopreserved in OCT solution
(Sakura Finetek USA Inc., Torrance, Calif., Catalog No. 4583), and
5 .mu.m sections placed on Superfrost plus slides (VWR
International, LLC, Radnor, Pa., Catalog No. 48311-703). For
IF-staining, primary antibodies were added at appropriate dilutions
overnight at 4.degree. C., while secondary antibodies were added
for 30 min at room temperature followed by rinsing with PBS and
adding Vectastain mounting reagent with DAPI (Vector Laboratories
Inc., Burlingame, California, Catalog No. H-1200). The sections
were visualized using a Nikon Ti fluorescence microscope (Nikon
Instruments, Inc., Melville, N.Y.).
TABLE-US-00008 TABLE III List of antibodies used for IF analysis.
Antigen Species Source Dilution CDX2 Mouse BioGenex 1:50 (Catalog
No. MU392A-UC) FOXA2 Rabbit Seven Hills 1:500 (Catalog No. WRAB
1200) SOX9 Rabbit Millipore EMD 1:100 (Catalog No. AB5535) PDX1
Goat R & D Systems 1:33 (Catalog No. AF2419) SOX2 Goat Sigma
1:50 (Catalog No. sc-17320) KLF5 Rabbit Abcam 1:150 (Catalog No.
ab24331) (Tyramide amplification- Perkin Elmer, Waltham, MA,
NEL744001KT) ALB Rabbit Sigma 1:250 (Catalog No. A0433) KI67 Rabbit
Abcam 1:100 (Catalog No. ab16667) Donkey anti-mouse IgG Mouse Life
Technologies 1:100 (H + L) Secondary (Catalog No. A21202) antibody,
Alexa Fluor 488 Donkey anti-rabbit IgG Rabbit Life Technologies
1:200 (H + L) Secondary (Catalog No. A10040) Antibody, Alexa Fluor
546 Donkey anti-goat IgG Goat Life Technologies 1:50-1:100 (H + L)
Secondary (Catalog No. A11056) Antibody, Alexa Fluor 546 Goat
anti-rabbit IgG, Rabbit Perkin Elmer, Waltham, MA 1:250 HRP-Labeled
(Catalog No. NEF812001)
[0122] For quantification of gene expression at various stages,
Caco-2, H1-hESC, S1D3, S2D2, S3D2 and S3D5 cells were harvested, as
described in Nature Biotechnology, 2014 (32) 11, 1121-1133.
Briefly, gene expression was assessed in cells using custom
Taqman.RTM. Arrays (Applied Biosystems, Foster City, Calif.); Open
Array.RTM. (OA) was used for CDX2, FOXA2, SOX2, SOX9, PDX1, ALB,
PTF1A, and Taqman.RTM. Low Density Array (TLDA) was used for KLF5,
HOXC5, and LGR5, with housekeeping gene GAPDH used for both tests.
Data were analyzed using Sequence Detection Software (Applied
Biosystems, Foster City, Calif.), and normalized using GAPDH as a
housekeeping gene to undifferentiated H1-hESC using the
.DELTA..DELTA.Ct method. Primer details are outlined in Table
IV.
TABLE-US-00009 TABLE IV List of RT-qPCR primers. Gene Assay ID 1
ALB Hs00609411_m1 2 CDX2 Hs00230919_m1 3 FOXA2 Hs00232764_m1 4
GAPDH Hs99999905_m1 5 PDX1 Hs00236830_m1 6 PTF1A Hs00603586_g1 7
SOX2 Hs01053049_s1 8 SOX9 Hs00165814_m1 9 HOXC5 Hs00232747_m1 10
KLF5 Hs00156145_m1 11 LGR5 Hs00969422_m1 12 HOXA13 Hs00426284_m1 13
HAND1 Hs00231848_m1
Results
[0123] A summary of a differentiation method, including the
important medium components, growth factors and small molecules
that were added to each stage, and key stage-specific markers of
the differentiating intestinal midgut endoderm cells (FOXA2; CDX2;
KLF5; SOX9; PDX1.sup.LO) is depicted in FIG. 1A. As compared to the
neutral pH noted for S2D2 (7.35.+-.0.04), cells were exposed to
slightly acidic conditions in the BLAR medium, during the entirety
of stage 3 (pH; S3D1, 6.98.+-.0.05; S3D2, 7.02.+-.0.04; S3D5,
7.18.+-.0.03) (FIG. 1B), and as a result of lower sodium
bicarbonate levels in BLAR medium. The pH of the culture can range
from about 6.8 to 7.2 during the five days of stage 3. FIG. 1C
depicts representative phase-contrast images of a S3D5 monolayer
(left), and human epithelial colon adenocarcinoma cell line
("Caco-2") (right), which was used as a benchmark for
characterization of differentiation. A uniform morphology at S3D5
was observed. Characterization of cell number using the
Nucleocounter.RTM. NC-100 (Chemometec, Alleroed, Denmark , Catalog
No. 900-004) shows that one hESC differentiated into 4.56.+-.2.60
S3D5 hindgut endoderm cells (FIG. 1D).
[0124] The differentiation method, utilizing BMP4, efficiently
generates and maintains intestinal midgut endoderm cells in
monolayer, each comprising both CDX2 and FOXA2 on transcript and
protein levels. FIG. 2A (bottom) shows that 90.0.+-.5.85 percent of
S3D5 cells were co-present for both CDX2 and FOXA2 protein, similar
to percentage observed in Caco-2 cells (86.0.+-.6.67). Conversely,
definitive endoderm (DE--S1D3) cells were devoid of CDX2 and FOXA2
co-presence (2.3.+-.1.2). Gene expression analysis shows CDX2
induced (FIG. 2B), and FOXA2 maintained (FIG. 2C) during Stage 3.
FIG. 2D shows that the induction of CDX2 protein levels and
CDX2/FOXA2 protein co-presence after the establishment of the
FOXA2-positive primitive gut endoderm stage, S2D2 (FIG. 2D-i),
progressively increased through S3D2 (FIG. 2D-ii), and at S3D5
(FIG. 2D-iii) reached similar levels as seen in Caco-2 cells (FIG.
2D-iv). CDX2 protein is depicted on the bottom row, and FOXA2
protein is depicted on the top row.
[0125] Transcript and protein levels of additional TFs are found at
S3D5, which constitute robust intestinal midgut endoderm induction.
FIGS. 3A-3P show that proper intestinal midgut endoderm was
achieved. In addition to CDX2 and FOXA2 co-presence, S3D5 cells
also exhibited co-presence of SOX9, PDX1, KLF5, HOXC5, but did not
express SOX2, ALB, PTF1A, and LGR5. The protein presence of all TFs
is depicted in separate single channel images. FIG. 3A (bottom)
shows that 98.7.+-.0.25 percent of cells were co-present for both
CDX2 and SOX9 at S3D5. Strong induction of SOX9 gene expression was
comparable to levels observed in Caco-2 cells (FIG. 3B), and
protein presence as assessed by IF-analysis were observed (FIG.
3C). 69.4.+-.14.2 percent of cells were co-positive for both CDX2
and PDX1 (FIG. 3D--bottom). PDX1 gene expression was induced at low
levels, as compared to pancreas-biased S4D3 cells (See, e.g.,
US2014/0242693) (FIG. 3E), and this was reflected low to absent
protein levels in the IF-analysis (FIG. 3F).
[0126] S3D5 cells did not express the anterior endoderm TF SOX2,
and only 1.45.+-.0.15 of S3D5 cells exhibited SOX2 and CDX2
co-presence (FIG. 3G--bottom; 3I), and gene expression was below
levels seen in hESC and Caco-2 cells (FIG. 3H). Gene expression of
KLF5, essential for proper development of hindgut endoderm, was
upregulated at S3D5 (FIG. 3J). Protein co-presence of KLF5 within
CDX2-positive cells at S3D5 was observed (FIG. 3K). ALB gene
expression (FIG. 3L), and protein presence (FIG. 3M) was not
observed in S3D5 cells. Similarly, the gene expression of PTF1A, a
pancreas lineage marker, was not induced in S3D5 cells, as compared
to pancreas-biased S4D3 cells (FIG. 3N). The homeobox gene, HOXC5,
present in the embryonic intestinal midgut endoderm was strongly
induced in S3D5 cells (FIG. 3O). FIG. 3P shows that LGR5, a marker
of embryonic intestinal endoderm beginning at mid-gestation in the
mouse, was not induced in S3D5 cells (FIG. 3P). FIG. 3Q shows that
HOXA13, a marker of the intestinal hindgut endoderm, was not
induced in S3D5 cells (FIG. 3P).
[0127] FIGS. 4A-4B illustrate the proliferative profile of
differentiating S3D5 cells. FIG. 4A depicts Caco-2 cells, where
most of CDX2-protein positive cells were in active cell cycle (as
indicated by the co-expression with KI67 protein) (left), and the
proliferative index of the H1-hESC-derived cells during Stage 3
that decreased over time (S3D2--middle; S3D5--right). CDX2 (top
row) and KI67 (bottom row) protein levels are depicted as single
channel images. The percentage KI67-protein positive cells of total
S3D5 cells (total cells are >90% CDX2-positive), assessed by
FACS, was 16.8.+-.3.12, in contrast with percentage seen at S1D3
(97.3.+-.1.3), and in Caco-2 cells (99.2.+-.0.2) (FIG. 4B).
[0128] BMP2 can be used as an alternative to BMP4, during Stage 3
in this method to produce a monolayer of intestinal midgut endoderm
cells with CDX2 and FOXA2 protein co-presence. FIG. 5A depicts a
summary of a differentiation method, including the medium
components, growth factors and small molecules that were added to
each stage, and key stage-specific markers of the differentiating
intestinal midgut endoderm cells (FOXA2, CDX2, KLF5, SOX9, and
PDX1.sup.LO). Compared to the neutral pH noted at S2D2
(7.35.+-.0.04), cells were exposed to slightly acidic conditions in
BLAR medium, during the entirety of stage 3 (pH; S3D1, 6.92; S3D2,
7.01; S3D5, 7.22) (FIG. 5B), and as a result of lower sodium
bicarbonate levels in BLAR acidic medium. FIG. 5C depicts
representative phase-contrast images of a S3D5 monolayer (left),
and Caco-2 cells (right). A uniform morphology at S3D5 was
observed.
[0129] The differentiation method generates and maintains proper
intestinal midgut endoderm cells in monolayer, each comprising of
CDX2, FOXA2, KLF5, SOX9, PDX1.sup.LO and HOXC5 on transcript and
protein levels. All TF protein levels are depicted as single
channel IF images. FIG. 6A (bottom) shows that 94 percent of S3D5
cells were co-present for both CDX2 and FOXA2 protein, similar to
percentage seen in Caco-2 cells (86.0.+-.6.67). Gene expression
analysis shows that CDX2 was induced (FIG. 6B), and FOXA2
maintained (FIG. 6C) during Stage 3. FIG. 6D shows that CDX2
protein levels and complete CDX2/FOXA2 protein co-presence were
induced at S3D5 (FIG. 6D), which is comparable to levels observed
in Caco-2 cells (FIG. 2D-iv). FIG. 6E (bottom) shows that 99.8
percent of cells were co-present for both CDX2 and SOX9 at S3D5.
Strong induction of SOX9 gene expression was observed similar to
levels in Caco-2 cells (FIG. 6F), and protein presence as assessed
by IF-analysis was observed (FIG. 6G). 45.5 percent of cells were
co-positive for both CDX2 and PDX1 (FIG. 6H--bottom). PDX1 gene
expression was induced at low levels, when compared to
pancreas-biased S4D3 cells (FIG. 6I), and low to absent protein
levels was reflected in the IF-analysis (FIG. 6J). Anterior
endoderm TF SOX2 was not observed in S3D5 cells, as 0.8 percent of
S3D5 cells exhibited SOX2 and CDX2 co-presence (FIG. 6K--bottom;
6M), and gene expression was below levels seen in hESC and Caco-2
cells (FIG. 6L). The gene expression of KLF5, an essential marker
for demonstrating proper development of hindgut endoderm, was
strongly upregulated at S3D5 (FIG. 6N). Protein co-presence of KLF5
within CDX2-positive cells at S3D5 was observed (FIG. 6O). ALB gene
expression (FIG. 6P), and protein presence (FIG. 6Q) was not
observed in S3D5 cells. The gene expression of pancreas lineage
allocating TF, PTF1A, was not induced in S3D5 cells, as compared to
pancreas-biased S4D3 cells (FIG. 6R). The homeobox gene HOXC5,
present in the embryonic intestinal midgut endoderm, was strongly
induced in S3D5 cells (FIG. 6S). FIG. 6T demonstrates that LGR5, a
marker of embryonic intestinal endoderm beginning at mid-gestation,
was not induced in S3D5 cells. FIG. 6U demonstrates that HOXA13, a
marker of the intestinal hindgut endoderm, was not induced in S3D5
cells (FIG. 6U).
[0130] FIG. 7 characterizes the proliferative profile of
differentiating S3D5 cells, showing Caco-2 cells, where most of
CDX2-protein positive cells were in active cell cycle (as indicated
by the co-expression with KI67 protein) (left), compared to the
proliferative index of the H1-hESC-derived cells during Stage 3
that was lower (S3D5--right). CDX2 (top row) and KI67 (bottom row)
protein levels are depicted as single channel images. The
percentage KI67-protein positive cells of total S3D5 cells (total
cells are >90% CDX2-positive), as assessed by FACS, was 14.1
percent, in contrast with percentage seen at S1D3 (97.3.+-.1.3),
compared to Caco-2 cells (99.2.+-.0.2) (FIG. 7; 4B).
EXAMPLE 2
Intestinal Culturing Starting from the Definitive Endoderm, and
Using FGF4 and WNT-Agonists, Generates an Endoderm-Mesenchyme
Mixture of CDX2+ Mid-/Hindgut Cells
[0131] This example demonstrates the endoderm-mesenchyme-mixed
quality of the CDX2+ mid-/hindgut cells generated from intestinal
culturing beginning at the definitive endoderm stage using FGF4 and
WNT-agonists (Spence et al., Nature, 2011; 470:105-109; Watson et
al. Nature Med, 2014; 11:1310-1314). To examine the induction to
midgut/hindgut endoderm cells described in Spence et al., infra,
hESCs were differentiated using the protocol below. Note that the
differentiation conditions outlined in this Example differ from
Example 1 by the following: (i) intestinal condition starting point
begins at the definitive endoderm stage; (ii) different growth
factors and small molecules are used than RA and BMP4 or BMP2; and
(iii) acidic culture conditions are not used.
Materials and Methods
[0132] Cell culture: H1-hESC cells were cultured and maintained as
described in Example 1.
[0133] Differentiation: The cultures were differentiated using the
following protocol. [0134] Stage 1--Mimic (3 days): Cells were
cultured for one day in the following Stage 1 medium:
TABLE-US-00010 [0134] RMPI 1640 medium (Thermo Fisher Scientific,
Catalog No. 11875) Penicillin-Streptomycin 1.times. concentration
(1:100 dilution (Thermo Fisher Scientific, Catalog No. from stock
concentration) 15140122) L-Glutamine 2 mM (Thermo Fisher
Scientific, Catalog No. 25030081) Activin A 100 ng/ml (R & D
Systems, Inc., Minneapolis, Minnesota, Catalog No. 338-AC)
[0135] Cells were then cultured for an additional day in the
following media:
TABLE-US-00011 RMPI 1640 medium Penicillin-Streptomycin 1.times.
concentration (1:100 dilution from stock concentration) L-Glutamine
2 mM Activin A 100 ng/ml Defined Fetal Bovine Serum (FBS) 0.2%
(Hyclone, Catalog No. SH30070.02)
[0136] Cells were then cultured for an additional day in the
following media:
TABLE-US-00012 RMPI 1640 medium Penicillin-Streptomycin 1.times.
concentration (1:100 dilution from stock concentration) L-Glutamine
2 mM Activin A 100 ng/ml Defined FBS 2.0%
[0137] Post-Stage 1 (2 days): For example, pS1d1 is post-Stage 1
day 1 and pS1d2 is post-Stage 1 day 2. Cells were cultured for two
days in the following Post-Stage 1 medium:
TABLE-US-00013 RMPI 1640 medium Penicillin-Streptomycin 1.times.
concentration (1:100 dilution from stock concentration) L-Glutamine
2 mM FGF4 500 ng/ml (R & D Systems, Inc., Minneapolis,
Minnesota, Catalog No. 235-F4) Defined Fetal Bovine Serum (FBS)
2.0% WNT3A 500 ng/ml (R & D Systems, Inc., Minneapolis,
Minnesota, Catalog No. 5036-WN) OR Chiron99201 3 .mu.M (Stemgent,
Catalog No. 0400041)
[0138] Quantification: Phase contrast imaging and quantification of
gene expression followed the procedures in Example 1.
Results
[0139] A summary of the differentiation methods, including medium
components, growth factors and small molecules added to each stage,
and signature or key stage-specific markers of the differentiating
intestinal mid-/hindgut endoderm cells (HAND1) is depicted in FIG.
8A. Intestinal conditioning (post-Stage 1), started at the
definitive endoderm stage, with 500 ng/ml FGF4, and either 3 .mu.M
Chiron99021 (Watson et al.), or 500 ng/ml Wnt3A (Spence et al.).
The term "Stage 1-mimic" refers to the definitive endoderm
differentiation protocol in this example that differs from the
"S1D3-Original" that refers to Stage 1 conditioning described in
Example 1. FIG. 8B shows phase-contrast images of H1-hESC cells
(top row, left), post-Stage 1 cells conditioned two days with 500
ng/ml FGF4 and 3 .mu.M Chiron99021 (top row, middle), post-Stage 1
cells conditioned two days with 500 ng/ml FGF4 and 500 ng/ml Wnt3A
(top row, right), along with a S3D5 monolayer conditioned by
RA/BMP4 (bottom row, left), and a S3D5 monolayer conditioned by
RA/BMP2 (bottom row, right) (from Example 1).
[0140] Induction of CDX2 gene expression was achieved after two
days of conditioning, but at much lower levels compared to RA/BMP2
or RA/BMP4 S3D5 (FIG. 8C). However, the gene expression of the
endoderm marker FOXA2 was maintained (FIG. 8D), and
mesoderm/mesenchyme marker HAND1 was strongly induced (FIG. 8F).
Also, KLF5 was not induced at the two day time point unlike by
RA/BMP4 and RA/BMP2 conditioning (FIG. 8E). As conclusively shown
in FIG. 8F, this gene expression pattern is reflective of the
heterogeneous cell population seen in Watson et al. and Spence et
al. containing not only a CDX2.sup.+FOXA2.sup.+ endodermal
population, but also a significant mesenchymal CDX2.sup.+ cell
population. In contrast, RA/BMP4 or RA/BMP2 post-Stage 2 (primitive
gut tube cell) conditioning did not induce the mesoderm/mesenchyme
marker HAND1; only endodermal CDX2.sup.+FOXA2.sup.+ population was
induced.
[0141] In describing the present invention and its various
embodiments, specific terminology is employed for the sake of
clarity. However, the invention is not intended to be limited to
the specific terminology so selected. A person skilled in the
relevant art will recognize that other equivalent components can be
employed and other methods developed without departing from the
broad concepts of the current invention. All references cited
anywhere in this specification are incorporated by reference as if
each had been individually incorporated.
* * * * *