U.S. patent application number 15/144752 was filed with the patent office on 2016-11-24 for definitive endoderm cells and human pluripotent stem cells.
This patent application is currently assigned to ViaCyte, Inc.. The applicant listed for this patent is ViaCyte, Inc.. Invention is credited to Alan D. Agulnick, Emmanuel E. Baetge, Kevin Allen D'Amour, Susan Eliazer.
Application Number | 20160340645 15/144752 |
Document ID | / |
Family ID | 46322165 |
Filed Date | 2016-11-24 |
United States Patent
Application |
20160340645 |
Kind Code |
A1 |
D'Amour; Kevin Allen ; et
al. |
November 24, 2016 |
DEFINITIVE ENDODERM CELLS AND HUMAN PLURIPOTENT STEM CELLS
Abstract
Disclosed herein are methods of identifying one or more
differentiation factors that are useful for differentiating cells
in a cell population comprising definitive endoderm cells into
cells which are capable of forming tissues and/or organs that are
derived from the gut tube.
Inventors: |
D'Amour; Kevin Allen; (San
Diego, CA) ; Agulnick; Alan D.; (San Diego, CA)
; Eliazer; Susan; (Dallas, TX) ; Baetge; Emmanuel
E.; (Encinitas, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ViaCyte, Inc. |
San Diego |
CA |
US |
|
|
Assignee: |
ViaCyte, Inc.
San Diego
CA
|
Family ID: |
46322165 |
Appl. No.: |
15/144752 |
Filed: |
May 2, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13544870 |
Jul 9, 2012 |
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15144752 |
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12476570 |
Jun 2, 2009 |
8216836 |
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13544870 |
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11165305 |
Jun 23, 2005 |
7541185 |
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12476570 |
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11115868 |
Apr 26, 2005 |
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11165305 |
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11021618 |
Dec 23, 2004 |
7510876 |
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11165305 |
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60566293 |
Apr 27, 2004 |
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60587942 |
Jul 14, 2004 |
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60586566 |
Jul 9, 2004 |
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60587942 |
Jul 14, 2004 |
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60586566 |
Jul 9, 2004 |
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60532004 |
Dec 23, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/16 20130101;
C12N 2501/415 20130101; C12N 2503/00 20130101; C12N 5/0018
20130101; C12N 2501/15 20130101; C12N 2501/155 20130101; C12N
5/0606 20130101; C12Q 1/6881 20130101; C12N 2510/00 20130101; C12N
2500/90 20130101; C12N 15/1086 20130101; C12N 2502/02 20130101;
C12N 2501/385 20130101; G01N 33/56966 20130101; C12Q 2600/158
20130101; C12N 5/0679 20130101; C12N 5/0676 20130101; G01N 33/5023
20130101; C12N 2501/119 20130101; C12N 5/0603 20130101; A61K 35/12
20130101; C12N 2501/115 20130101; G01N 33/5073 20130101; C12N
2506/02 20130101; C12N 2502/13 20130101 |
International
Class: |
C12N 5/0735 20060101
C12N005/0735; C12N 5/00 20060101 C12N005/00 |
Claims
1.-20. (canceled)
21. An in vitro method of producing human definitive endoderm
cells, comprising: obtaining a cell population comprising
pluripotent human stem cells; and providing said cell population
with a TGF.beta. superfamily growth factor selected from the group
consisting of activn A, activin B, BMP4, nodal, and combinations
thereof, thereby producing human definitive endoderm cells.
22. The method of claim 21, further comprising removing TGF.beta.
superfamily growth factor from the cell population.
23. The method of claim 21, wherein at least 15% of the pluripotent
human stem cells differentiate into definitive endoderm cells.
24. The method of claim 21, wherein the TGF.beta. superfamily
growth factor is activin A.
25. The method of claim 21, further comprising providing a Wnt
family member to the cell population.
26. The method of claim 25, wherein the Wnt family member is
Wnt3a.
27. The method of claim 21, wherein at least 10 ng/ml activin A is
provided to the cell population.
28. The method of claim 21, wherein at least 100 ng/ml activin A is
provided to the cell population.
29. The method of claim 21, further comprising providing serum to
the cell population.
30. The method of claim 21, wherein the pluripotent human stem
cells comprise embryonic stem cells.
31. The method of claim 29, wherein the embryonic stem cells are
derived from tissue selected from the group consisting of the
morula and the inner cell mass (ICM) of the embryo.
32. The method of claim 29, wherein providing serum to the cell
population comprises providing increasing concentrations of serum
to the cell population.
33. An in vitro cell culture, comprising: human pluripotent cells;
human definitive endoderm cells; and a medium comprising an
effective amount of a TGF.beta. superfamily member and an effective
amount of a TGF.beta. superfamily growth factor selected from the
group consisting of activn A, activin B, BMP4, nodal and
combinations thereof, wherein the effective amount of a TGF.beta.
superfamily growth factor selected from the group consisting of
activn A, activin B, BMP4, nodal and combinations thereof promote
differentiation of pluripotent cells to definitive endoderm
cells.
34. The in vitro cell culture of claim 33, wherein at least 15% of
said pluripotent cells differentiate into definitive endoderm
cells.
35. The in vitro cell culture of claim 33, further comprising a Wnt
family member.
36. The in vitro cell culture of claim 35, wherein said Wnt family
member is Wnt3a.
37. The in vitro cell culture of claim 33, further comprising serum
in the medium.
38. The in vitro cell culture of claim 37, wherein the medium
comprises less than 2% serum.
39. An in vitro method of producing human definitive endoderm
cells, comprising: obtaining a cell population comprising
pluripotent human stem cells; and providing said cell population
with activn A, activin B and BMP4, thereby generating human
definitive endoderm cells.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
13/544,870, filed Jul. 9, 2012, which is a continuation of U.S.
patent application Ser. No. 12/476,570, filed Jun. 2, 2009, issued
as U.S. Pat. No. 8,216,836, which is a continuation of U.S. patent
application Ser. No. 11/165,305, filed Jun. 23, 2005, issued as
U.S. Pat. No. 7,541,185, which is a continuation-in-part of U.S.
patent application Ser. No. 11/115,868, filed Apr. 26, 2005, which
claims the benefit of U.S. Provisional Application No. 60/566,293,
filed Apr. 27, 2004, U.S. Provisional Application No. 60/587,942,
filed Jul. 14, 2004, and U.S. Provisional Application No.
60/586,566, filed Jul. 9, 2004. U.S. patent application Ser. No.
11/165,305, filed Jun. 23, 2005, is also a continuation-in-part of
U.S. patent application Ser. No. 11/021,618, filed Dec. 23, 2004,
issued as U.S. Pat. No. 7,510,876, which claims the benefit of U.S.
Provisional Application No. 60/587,942, filed Jul. 14, 2004, U.S.
Provisional Application No. 60/586,566, filed Jul. 9, 2004 and U.S.
Provisional Application No. 60/532,004, filed Dec. 23, 2003. The
disclosure of each of the foregoing applications is incorporated
herein by reference in its entirety.
SEQUENCE LISTING
[0002] The Sequence Listing is submitted as an ASCII text file
[9511-96316-09_Sequence_Listing.txt, May 2, 2016, 5.62 KB], which
is incorporated by reference herein.
FIELD OF THE INVENTION
[0003] The present invention relates to the fields of medicine and
cell biology. In particular, the present invention relates the
identification of factors that are useful for differentiating
definitive endoderm cells into other cell types.
BACKGROUND
[0004] Human pluripotent stem cells, such as embryonic stem (ES)
cells and embryonic germ (EG) cells, were first isolated in culture
without fibroblast feeders in 1994 (Bongso et al., 1994) and with
fibroblast feeders (Hogan, 1997). Later, Thomson, Reubinoff and
Shamblott established continuous cultures of human ES and EG cells
using mitotically inactivated mouse feeder layers (Reubinoff et
al., 2000; Shamblott et al., 1998; Thomson et al., 1998).
[0005] Human ES and EG cells (hESCs) offer unique opportunities for
investigating early stages of human development as well as for
therapeutic intervention in several disease states, such as
diabetes mellitus and Parkinson's disease. For example, the use of
insulin-producing .beta.-cells derived from hESCs would offer a
vast improvement over current cell therapy procedures that utilize
cells from donor pancreases for the treatment of diabetes. However,
presently it is not known how to generate an insulin-producing
.beta.-cell from hESCs. As such, current cell therapy treatments
for diabetes mellitus, which utilize islet cells from donor
pancreases, are limited by the scarcity of high quality islet cells
needed for transplant. Cell therapy for a single Type I diabetic
patient requires a transplant of approximately 8.times.10.sup.8
pancreatic islet cells. (Shapiro et al., 2000; Shapiro et al.,
2001a; Shapiro et al., 2001b). As such, at least two healthy donor
organs are required to obtain sufficient islet cells for a
successful transplant. Human embryonic stem cells offer a source of
starting material from which to develop substantial quantities of
high quality differentiated cells for human cell therapies.
[0006] Two properties that make hESCs uniquely suited to cell
therapy applications are pluripotence and the ability to maintain
these cells in culture for prolonged periods. Pluripotency is
defined by the ability of hESCs to differentiate to derivatives of
all 3 primary germ layers (endoderm, mesoderm, ectoderm) which, in
turn, form all somatic cell types of the mature organism in
addition to extraembryonic tissues (e.g. placenta) and germ cells.
Although pluripotency imparts extraordinary utility upon hESCs,
this property also poses unique challenges for the study and
manipulation of these cells and their derivatives. Owing to the
large variety of cell types that may arise in differentiating hESC
cultures, the vast majority of cell types are produced at very low
efficiencies. Additionally, success in evaluating production of any
given cell type depends critically on defining appropriate markers.
Achieving efficient, directed differentiation is of great
importance for therapeutic application of hESCs.
[0007] In order to use hESCs as a starting material to generate
cells that are useful in cell therapy applications, it would be
advantageous to overcome the foregoing problems. Additionally, it
would be beneficial to identify factors which promote the
differentiation of precursor cells derived from hESCs to cell types
useful for cell therapies.
SUMMARY OF THE INVENTION
[0008] Embodiments of the present invention relate to methods of
identifying one or more differentiation factors that are useful for
differentiating cells in a cell population comprising PDX1-positive
(PDX1-expressing) endoderm cells and/or PDX1-negative endoderm
cells (endoderm cells which do not significantly express PDX1),
such as definitive endoderm cells, into cells that are useful for
cell therapy. For example, some embodiments of the methods
described herein relate to methods of identifying factors capable
of promoting the differentiation of definitive endoderm cells into
cells which are precursors for tissues and/or organs which include,
but are not limited to, pancreas, liver, lungs, stomach, intestine,
thyroid, thymus, pharynx, gallbladder and urinary bladder. In some
embodiments, such precursor cells are PDX1-positive endoderm cells.
In other embodiments, such precursor cells are endoderm cells that
do not significantly express PDX1.
[0009] In some embodiments of the methods described herein, cell
cultures or cell populations of definitive endoderm cells are
contacted or otherwise provided with a candidate (test)
differentiation factor. In preferred embodiments, the definitive
endoderm cells are human definitive endoderm cells. In more
preferred embodiments, the human definitive endoderm cells are
multipotent cells that can differentiate into cells of the gut tube
or organs derived therefrom.
[0010] In other embodiments of the methods described herein, cell
cultures or cell populations of PDX1-positive endoderm cells are
contacted or otherwise provided with a candidate differentiation
factor. In preferred embodiments, the PDX1-positive endoderm cells
are human PDX1-positive endoderm cells. In certain embodiments, the
human PDX1-positive endoderm cells are PDX1-positive foregut/midgut
endoderm cells. In more preferred embodiments, the human
PDX1-positive endoderm cells are PDX1-positive foregut endoderm
cells. In other preferred embodiments, the human PDX1-positive
endoderm cells are PDX1-positive endoderm cells of the posterior
portion of the foregut. In especially preferred embodiments, the
human PDX1-positive foregut endoderm cells are multipotent cells
that can differentiate into cells, tissues or organs derived from
the anterior portion of the gut tube.
[0011] As related to the methods described herein, the candidate
differentiation factor may be one that is known to cause cell
differentiation or one that is not known to cause cell
differentiation. In certain embodiments, the candidate
differentiation factor can be a polypeptide, such as a growth
factor. In some embodiments, the growth factor includes, but is not
limited to, FGF10, FGF4, FGF2, Wnt3A or Wnt3B. In other
embodiments, the candidate differentiation factor can be a small
molecule. In particular embodiments, the small molecule is a
retinoid compound, such as retinoic acid. Alternatively, in some
embodiments, the candidate differentiation factor is not a
retinoid, is not a foregut differentiation factor or is not a
member of the TGF.beta. superfamily. In other embodiments, the
candidate differentiation factor is any molecule other than a
retinoid compound, a foregut differentiation factor, or a member of
the TGF.beta. superfamily of growth factors, such as activins A and
B. In still other embodiments, the candidate differentiation factor
is a factor that was not previously known to cause the
differentiation of definitive endoderm cells.
[0012] Additional embodiments of the methods described herein
relate to testing candidate differentiation factors at a plurality
of concentrations. For example, a candidate differentiation factor
may cause the differentiation of definitive endoderm cells and/or
PDX1-positive endoderm cells only at concentrations above a certain
threshold. Additionally, a candidate differentiation factor can
cause the same cell to differentiate into a first cell type when
provided at a low concentration and a second cell type when
provided at a higher concentration. In some embodiments, the
candidate differentiation factor is provided at one or more
concentrations ranging from about 0.1 ng/ml to about 10 mg/ml.
[0013] Prior to or at approximately the same time as contacting or
otherwise providing the cell culture or cell population comprising
definitive endoderm cells and/or PDX1-positive endoderm cells with
the candidate differentiation factor, at least one marker is
selected and evaluated so as to determine its expression. This step
may be referred to as the first marker evaluation step.
Alternatively, this step may be referred to as determining
expression of a marker at a first time point. The marker can be any
marker that is useful for monitoring cell differentiation, however,
preferred markers include, but are not limited to, sex determining
region Y-box 17 (SOX17), pancreatic-duodenal homeobox factor-1
(PDX1), albumin, hepatocyte specific antigen (HAS),
prospero-related homeobox 1 (PROX1), thyroid transcription factor 1
(TITF1), villin, alpha fetoprotein (AFP), cytochrome P450 7A
(CYP7A), tyrosine aminotransferase (TAT), hepatocyte nuclear factor
4a (HNF4.alpha.), CXC-type chemokine receptor 4 (CXCR4), von
Willebrand factor (VWF), vascular cell adhesion molecule-1 (VACM1),
apolipoprotein A1 (APOA1), glucose transporter-2 (GLUT2),
alpha-1-antitrypsin (AAT), glukokinase (GLUKO), and human
hematopoietically expressed homeobox (hHEX) and CDX2.
[0014] After sufficient time has passed since contacting or
otherwise providing cell culture or cell population comprising
definitive endoderm cells and/or PDX1-positive endoderm cells with
the candidate differentiation factor, the expression of the at
least one marker in the cell culture or cell population is again
evaluated. This step may be referred to as the second marker
evaluation step. Alternatively, this step may be referred to as
determining expression of a marker at a second time point. In
preferred embodiments, the marker evaluated at the first and second
time points is the same marker.
[0015] In some embodiments of the methods described herein, it is
further determined whether the expression of the at least one
marker at the second time point has increased or decreased as
compared to the expression of this marker at the first time point.
An increase or decrease in the expression of the at least one
marker indicates that the candidate differentiation factor is
capable of promoting the differentiation of the definitive endoderm
cells and/or the PDX1-positive endoderm cells. Sufficient time
between contacting or otherwise providing a cell culture or cell
population comprising definitive endoderm cells and/or
PDX1-positive endoderm cells with the candidate differentiation
factor and determining expression of the at least one marker at the
second time point can be as little as from about 1 hour to as much
as about 10 days. In some embodiments, the expression of the at
least one marker is evaluated multiple times subsequent to
contacting or otherwise providing the cell culture or cell
population comprising definitive endoderm cells and/or
PDX1-positive endoderm cells with the candidate differentiation
factor. In certain embodiments, marker expression is evaluated by
Q-PCR. In other embodiments, marker expression is evaluated by
immunocytochemistry.
[0016] Additional embodiments of the present invention relate to a
method of identifying a differentiation factor capable of promoting
the differentiation of PDX1-negative definitive endoderm cells to
PDX1-positive foregut endoderm cells. In such methods,
PDX1-negative definitive endoderm cells are contacted with a
candidate differentiation factor and it is determined whether PDX1
expression in the cell population after contact with the candidate
differentiation factor has increased as compared to PDX1 expression
in the cell population before contact with the candidate
differentiation factor. An increase in the PDX1 expression in the
cell population indicates that the candidate differentiation factor
is capable of promoting the differentiation of PDX1-negative
definitive endoderm cells to PDX1-positive foregut endoderm cells.
In some embodiments, PDX1 expression is determined by quantitative
polymerase chain reaction (Q-PCR). Some embodiments of the
foregoing method further comprise the step of determining
expression of the HOXA13 and/or the HOXC6 gene in the cell
population before and after contact with the candidate
differentiation factor. In some embodiments, the candidate
differentiation factor is a small molecule, for example, a
retinoid, such as RA. In others, the candidate differentiation
factor is a polypeptide, for example, a growth factor, such as
FGF-10.
[0017] Still other embodiments of the present invention relate to a
method of identifying a differentiation factor capable of promoting
the differentiation of PDX1-positive foregut endoderm cells. In
such methods, PDX1-positive foregut endoderm cells are contacted
with a candidate differentiation factor and it is determined
whether expression of a marker in the population is increased or
decreased after contact with the candidate differentiation factor
as compared to the expression of the same marker in the population
before contact with the candidate differentiation factor. An
increase or decrease in the expression of the marker indicates that
the candidate differentiation factor is capable of promoting the
differentiation of PDX1-positive foregut endoderm cells. In some
embodiments, marker expression is determined by Q-PCR. In some
embodiments, the candidate differentiation factor is a small
molecule, for example, a retinoid, such as RA. In others, the
candidate differentiation factor is a polypeptide, for example, a
growth factor, such as FGF-10.
[0018] Yet other embodiments of the present invention relate to
cells differentiated by the methods described herein. Such cells
include but are not limited to precursors of the pancreas, liver,
lungs, stomach, intestine, thyroid, thymus, pharynx, gallbladder
and urinary bladder. In some embodiments, the cells may be
terminally differentiated. Other embodiments described herein
relate to cell cultures and/or cell populations comprising the
above-described cells.
[0019] In certain jurisdictions, there may not be any generally
accepted definition of the term "comprising." As used herein, the
term "comprising" is intended to represent "open" language which
permits the inclusion of any additional elements. With this in
mind, additional embodiments of the present inventions are
described with reference to the numbered paragraphs below:
[0020] 1. A method of identifying a differentiation factor capable
of promoting the differentiation of human definitive endoderm cells
in a cell population comprising human cells, said method comprising
the steps of: (a) obtaining a cell population comprising human
definitive endoderm cells; (b) providing a candidate
differentiation factor to said cell population; (c) determining
expression of a marker in said cell population at a first time
point; (d) determining expression of the same marker in said cell
population at a second time point, wherein said second time point
is subsequent to said first time point and wherein said second time
point is subsequent to providing said cell population with said
candidate differentiation factor; and (e) determining if expression
of the marker in said cell population at said second time point is
increased or decreased as compared to the expression of the marker
in said cell population at said first time point, wherein an
increase or decrease in expression of said marker in said cell
population indicates that said candidate differentiation factor is
capable of promoting the differentiation of said human definitive
endoderm cells.
[0021] 2. The method of paragraph 1, wherein said human definitive
endoderm cells comprise at least about 10% of the human cells in
said cell population.
[0022] 3. The method of paragraph 1, wherein human feeder cells are
present in said cell population and wherein at least about 10% of
the human cells other than said feeder cells are definitive
endoderm cells.
[0023] 4. The method of paragraph 1, wherein said human definitive
endoderm cells comprise at least about 90% of the human cells in
said cell population.
[0024] 5. The method of paragraph 1, wherein said human feeder
cells are present in said cell population and wherein at least
about 90% of the human cells other than said feeder cells are
definitive endoderm cells.
[0025] 6. The method of paragraph 1, wherein said human definitive
endoderm cells differentiate into cells, tissues or organs derived
from the gut tube in response to said candidate differentiation
factor.
[0026] 7. The method of paragraph 1, wherein said human definitive
endoderm cells differentiate into pancreatic precursor cells in
response to said candidate differentiation factor.
[0027] 8. The method of paragraph 7, wherein said marker is
selected from the group consisting of pancreatic-duodenal homeobox
factor-1 (PDX1), homeobox A13 (HOXA13) and homeobox C6 (HOXC6).
[0028] 9. The method of paragraph 1, wherein said human definitive
endoderm cells differentiate into liver precursor cells in response
to said candidate differentiation factor.
[0029] 10. The method of paragraph 9, wherein said marker is
selected from the group consisting of albumin, prospero-related
homeobox 1 (PROX1) and hepatocyte specific antigen (HSA).
[0030] 11. The method of paragraph 1, wherein said human definitive
endoderm cells differentiate into lung precursor cells in response
to said candidate differentiation factor.
[0031] 12. The method of paragraph 11, wherein said marker is
thyroid transcription factor 1 (TITF1).
[0032] 13. The method of paragraph 1, wherein said human definitive
endoderm cells differentiate into intestinal precursor cells in
response to said candidate differentiation factor.
[0033] 14. The method of paragraph 13, wherein said marker is
selected from the group consisting of villin and caudal type
homeobox transcription factor 2 (CDX2).
[0034] 15. The method of paragraph 1, wherein said first time point
is prior to providing said candidate differentiation factor to said
cell population.
[0035] 16. The method of paragraph 1, wherein said first time point
is at approximately the same time as providing said candidate
differentiation factor to said cell population.
[0036] 17. The method of paragraph 1, wherein said first time point
is subsequent to providing said candidate differentiation factor to
said cell population.
[0037] 18. The method of paragraph 1, wherein expression of said
marker is increased.
[0038] 19. The method of paragraph 1, wherein expression of said
marker is decreased.
[0039] 20. The method of paragraph 1, wherein expression of said
marker is determined by quantitative polymerase chain reaction
(Q-PCR).
[0040] 21. The method of paragraph 1, wherein expression of said
marker is determined by immunocytochemistry.
[0041] 22. The method of paragraph 1, wherein said marker is
selected from the group consisting of pancreatic-duodenal homeobox
factor-1 (PDX1), homeobox A13 (HOXA13) and homeobox C6 (HOXC6).
[0042] 23. The method of paragraph 1, wherein said marker is
selected from the group consisting of albumin, prospero-related
homeobox 1 (PROX1) and hepatocyte specific antigen (HSA).
[0043] 24. The method of paragraph 1, wherein said marker is
selected from the group consisting of villin and caudal type
homeobox transcription factor 2 (CDX2).
[0044] 25. The method of paragraph 1, wherein said marker is
thyroid transcription factor 1 (TITF1).
[0045] 26. The method of paragraph 1, wherein said differentiation
factor comprises a foregut differentiation factor.
[0046] 27. The method of paragraph 1, wherein said differentiation
factor comprises a small molecule.
[0047] 28. The method of paragraph 1, wherein said differentiation
factor comprises a retinoid.
[0048] 29. The method of paragraph 1, wherein said differentiation
factor comprises retinoic acid.
[0049] 30. The method of paragraph 1, wherein said differentiation
factor comprises a polypeptide.
[0050] 31. The method of paragraph 1, wherein said differentiation
factor comprises a growth factor.
[0051] 32. The method of paragraph 1, wherein said differentiation
factor comprises FGF-10.
[0052] 33. The method of paragraph 1, wherein said differentiation
factor comprises FGF-2.
[0053] 34. The method of paragraph 1, wherein said differentiation
factor comprises Wnt3B.
[0054] 35. The method of paragraph 1, wherein said differentiation
factor is not a foregut differentiation factor.
[0055] 36. The method of paragraph 1, wherein said differentiation
factor is not a retinoid.
[0056] 37. The method of paragraph 1, wherein said differentiation
factor is not retinoic acid.
[0057] 38. The method of paragraph 1, wherein said differentiation
factor is provided to said cell population at a concentration of
between about 0.1 ng/ml to about 10 mg/ml.
[0058] 39. The method of paragraph 1, wherein said differentiation
factor is provided to said cell population at a concentration of
between about 1 ng/ml to about 1 mg/ml
[0059] 40. The method of paragraph 1, wherein said differentiation
factor is provided to said cell population at a concentration of
between about 10 ng/ml to about 100 .mu.g/ml.
[0060] 41. The method of paragraph 1, wherein said differentiation
factor is provided to said cell population at a concentration of
between about 100 ng/ml to about 10 .mu.g/ml.
[0061] 42. The method of paragraph 1, wherein said differentiation
factor is provided to said cell population at a concentration of
about 1 .mu.g/ml.
[0062] 43. The method of paragraph 1, wherein said differentiation
factor is provided to said cell population at a concentration of
about 100 ng/ml.
[0063] It will be appreciated that the methods and compositions
described above relate to cells cultured in vitro. However, the
above-described in vitro differentiated cell compositions may be
used for in vivo applications.
[0064] Additional embodiments of the present invention may also be
found in U.S. Provisional Patent Application No. 60/532,004,
entitled DEFINITIVE ENDODERM, filed Dec. 23, 2003; U.S. Provisional
Patent Application No. 60/566,293, entitled PDX1 EXPRESSING
ENDODERM, filed Apr. 27, 2004; U.S. Provisional Patent Application
No. 60/586,566, entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE
ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9, 2004; U.S.
Provisional Patent Application No. 60/587,942, entitled CHEMOKINE
CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM,
filed Jul. 14, 2004; U.S. patent application Ser. No. 11/021,618,
entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, and U.S. patent
application Ser. No. 11/115,868, entitled PDX1 EXPRESSING ENDODERM,
filed Apr. 26, 2005 the disclosures of which are incorporated
herein by reference in their entireties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] FIG. 1 is a schematic of a proposed differentiation pathway
for the production of beta-cells from hESCs. The first step in the
pathway commits the ES cell to the definitive endoderm lineage and
also represents the first step prior to further differentiation
events to pancreatic endoderm, endocrine endoderm, or
islet/beta-cells. The second step in the pathway shows the
conversion of SOX17-positive/PDX1-negative definitive endoderm to
PDX1-positive foregut endoderm. Some factors useful for mediating
these transitions are italicized. Relevant markers for defining the
target cells are underlined.
[0066] FIG. 2 is a diagram of the human SOX17 cDNA which displays
the positions of conserved motifs and highlights the region used
for the immunization procedure by GENOVAC.
[0067] FIG. 3 is a relational dendrogram illustrating that SOX17 is
most closely related to SOX7 and somewhat less to SOX18. The SOX17
proteins are more closely related among species homologs than to
other members of the SOX group F subfamily within the same
species.
[0068] FIG. 4 is a Western blot probed with the rat anti-SOX17
antibody. This blot demonstrates the specificity of this antibody
for human SOX17 protein over-expressed in fibroblasts (lane 1) and
a lack of immunoreactivity with EGFP (lane 2) or the most closely
related SOX family member, SOX7 (lane 3).
[0069] FIGS. 5A-5B are micrographs showing a cluster of SOX17.sup.+
cells that display a significant number of AFP co-labeled cells
(A). This is in striking contrast to other SOX17.sup.+ clusters (B)
where little or no AFP.sup.+ cells are observed.
[0070] FIGS. 6A-6C are micrographs showing parietal endoderm and
SOX17. (A) shows immunocytochemistry for human Thrombomodulin (TM)
protein located on the cell surface of parietal endoderm cells in
randomly differentiated cultures of hES cells. (B) is the identical
field shown in (A) double-labeled for TM and SOX17. (C) is the
phase contrast image of the same field with DAPI labeled nuclei.
Note the complete correlation of DAPI labeled nuclei and SOX17
labeling.
[0071] FIGS. 7A-7B are bar charts showing SOX17 gene expression by
quantitative PCR (Q-PCR) and anti-SOX17 positive cells by
SOX17-specific antibody. (A) shows that activin A increases SOX17
gene expression while retinoic acid (RA) strongly suppresses SOX17
expression relative to the undifferentiated control media (SR20).
(B) shows the identical pattern as well as a similar magnitude of
these changes is reflected in SOX17.sup.+ cell number, indicating
that Q-PCR measurement of SOX17 gene expression is very reflective
of changes at the single cell level.
[0072] FIG. 8A is a bar chart which shows that a culture of
differentiating hESCs in the presence of activin A maintains a low
level of AFP gene expression while cells allowed to randomly
differentiate in 10% fetal bovine serum (FBS) exhibit a strong
upregulation of AFP. The difference in expression levels is
approximately 7-fold.
[0073] FIGS. 8B-8C are images of two micrographs showing that the
suppression of AFP expression by activin A is also evident at the
single cell level as indicated by the very rare and small clusters
of AFP cells observed in activin A treatment conditions (bottom)
relative to 10% FBS alone (top).
[0074] FIGS. 9A-9B are comparative images showing the quantitation
of the AFP cell number using flow cytometry. This figure
demonstrates that the magnitude of change in AFP gene expression
(FIG. 8A) in the (A) presence and (B) absence of activin A exactly
corresponds to the number of AFP cells, further supporting the
utility of Q-PCR analyses to indicate changes occurring at the
individual cell level.
[0075] FIGS. 10A-10F are micrographs which show that exposure of
hESCs to nodal, activin A and activin B (NAA) yields a striking
increase in the number of SOX17.sup.+ cells over the period of 5
days (A-C). By comparing to the relative abundance of SOX17.sup.+
cells to the total number of cells present in each field, as
indicated by DAPI stained nuclei (D-F), it can be seen that
approximately 30-50% of all cells are immunoreactive for SOX17
after five days treatment with NAA.
[0076] FIG. 11 is a bar chart which demonstrates that activin A (0,
10, 30 or 100 ng/ml) dose-dependently increases SOX17 gene
expression in differentiating hESCs. Increased expression is
already robust after 3 days of treatment on adherent cultures and
continues through subsequent 1, 3 and 5 days of suspension culture
as well.
[0077] FIGS. 12A-12C are bar charts which demonstrate the effect of
activin A on the expression of MIXL1 (A), GATA4 (B) and HNF3b (C).
Activin A dose-dependent increases are also observed for three
other markers of definitive endoderm; MIXL1, GATA4 and HNF3b. The
magnitudes of increased expression in response to activin dose are
strikingly similar to those observed for SOX17, strongly indicating
that activin A is specifying a population of cells that co-express
all four genes (SOX17.sup.+, MIXL1.sup.+, GATA4.sup.+ and
HNF3b.sup.+).
[0078] FIGS. 13A-13C are bar charts which demonstrate the effect of
activin A on the expression of AFP (A), SOX7 (B) and SPARC (C).
There is an activin A dose-dependent decrease in expression of the
visceral endoderm marker AFP. Markers of primitive endoderm (SOX7)
and parietal endoderm (SPARC) remain either unchanged or exhibit
suppression at some time points indicating that activin A does not
act to specify these extra-embryonic endoderm cell types. This
further supports the fact that the increased expression of SOX17,
MIXL1, GATA4, and HNF3b are due to an increase in the number of
definitive endoderm cells in response to activin A.
[0079] FIGS. 14A-14B are bar charts showing the effect of activin A
on ZIC1 (A) and Brachyury expression (B) Consistent expression of
the neural marker ZIC1 demonstrates that there is not a
dose-dependent effect of activin A on neural differentiation. There
is a notable suppression of mesoderm differentiation mediated by
100 ng/ml of activin A treatment as indicated by the decreased
expression of brachyury. This is likely the result of the increased
specification of definitive endoderm from the mesendoderm
precursors. Lower levels of activin A treatment (10 and 30 ng/ml)
maintain the expression of brachyury at later time points of
differentiation relative to untreated control cultures.
[0080] FIGS. 15A-15B are micrographs showing decreased parietal
endoderm differentiation in response to treatment with activins.
Regions of TM.sup.hi parietal endoderm are found through the
culture (A) when differentiated in serum alone, while
differentiation to TM.sup.+ cells is scarce when activins are
included (B) and overall intensity of TM immunoreactivity is
lower.
[0081] FIGS. 16A-16D are micrographs which show marker expression
in response to treatment with activin A and activin B. hESCs were
treated for four consecutive days with activin A and activin B and
triple labeled with SOX17, AFP and TM antibodies. (A) SOX17; (B)
AFP; (C) TM; and (D) Phase/DAPI. Notice the numerous SOX17 positive
cells (A) associated with the complete absence of AFP (B) and TM
(C) immunoreactivity.
[0082] FIG. 17 is a micrograph showing the appearance of definitive
endoderm and visceral endoderm in vitro from hESCs. The regions of
visceral endoderm are identified by AFP.sup.hi/SOX17.sup.lo/- while
definitive endoderm displays the complete opposite profile,
SOX17.sup.hi/AFP.sup.lo/-. This field was selectively chosen due to
the proximity of these two regions to each other. However, there
are numerous times when SOX17.sup.hi/AFP.sup.lo/- regions are
observed in absolute isolation from any regions of AFP.sup.hi
cells, suggesting the separate origination of the definitive
endoderm cells from visceral endoderm cells.
[0083] FIG. 18 is a diagram depicting the TGF.beta. family of
ligands and receptors. Factors activating AR Smads and BR Smads are
useful in the production of definitive endoderm from human
embryonic stem cells (see, J Cell Physiol. 187:265-76).
[0084] FIG. 19 is a bar chart showing the induction of SOX17
expression over time as a result of treatment with individual and
combinations of TGF.beta. factors.
[0085] FIG. 20 is a bar chart showing the increase in SOX17.sup.+
cell number with time as a result of treatment with combinations of
TGF.beta. factors.
[0086] FIG. 21 is a bar chart showing induction of SOX17 expression
over time as a result of treatment with combinations of TGF.beta.
factors.
[0087] FIG. 22 is a bar chart showing that activin A induces a
dose-dependent increase in SOX17.sup.+ cell number.
[0088] FIG. 23 is a bar chart showing that addition of Wnt3a to
activin A and activin B treated cultures increases SOX17 expression
above the levels induced by activin A and activin B alone.
[0089] FIGS. 24A-24C are bar charts showing differentiation to
definitive endoderm is enhanced in low FBS conditions. Treatment of
hESCs with activins A and B in media containing 2% FBS (2AA) yields
a 2-3 times greater level of SOX17 expression as compared to the
same treatment in 10% FBS media (10AA) (A). Induction of the
definitive endoderm marker MIXL1 (B) is also affected in the same
way and the suppression of AFP (visceral endoderm) (C) is greater
in 2% FBS than in 10% FBS conditions.
[0090] FIGS. 25A-25D are micrographs which show SOX17.sup.+ cells
are dividing in culture. SOX17 immunoreactive cells are present at
the differentiating edge of an hESC colony (C, D) and are labeled
with proliferating cell nuclear antigen (PCNA) (B) yet are not
co-labeled with OCT4 (C). In addition, clear mitotic figures can be
seen by DAPI labeling of nuclei in both SOX17.sup.+ cells (arrows)
as well as OCT4+, undifferentiated hESCs (arrowheads) (D).
[0091] FIG. 26 is a bar chart showing the relative expression level
of CXCR4 in differentiating hESCs under various media
conditions.
[0092] FIGS. 27A-27D are bar charts that show how a panel of
definitive endoderm markers, (A) SOX17, (B) GSC, (C) HNF3B, and (D)
MIXL1, share a very similar pattern of expression to CXCR4 across
the same differentiation treatments displayed in FIG. 26.
[0093] FIGS. 28A-28E are bar charts showing how markers for
mesoderm (A, BRACHYURY, B, MOX1), ectoderm (C, SOX1, D, ZIC1) and
visceral endoderm (E, SOX7) exhibit an inverse relationship to
CXCR4 expression across the same treatments displayed in FIG.
26.
[0094] FIGS. 29A-29F are micrographs that show the relative
difference in SOX17 immunoreactive cells across three of the media
conditions displayed in FIGS. 26-28. (A&D) 10% NF (B&E) 10%
A100, (C&F) 0.5% A100A. (A-C) SOX17, (D-F) SOX17/DAPI.
[0095] FIGS. 30A-30C are flow cytometry dot plots that demonstrate
the increase in CXCR4.sup.+ cell number with increasing
concentration of activin A added to the differentiation media. (A)
0 ng/mL activin A, (B) 10 ng/ml activin A, (C) 100 ng/ml activin
A.
[0096] FIGS. 31A-31D are bar charts that show the CXCR4.sup.+ cells
isolated from the high dose activin A treatment (A100-CX+) are even
further enriched for definitive endoderm markers (A) SOX17, (B)
GSC, (C) HNF3B, and (D) MIXL1, than the parent population
(A100).
[0097] FIG. 32 is a bar chart showing gene expression from
CXCR4.sup.+ and CXCR4.sup.- cells isolated using
fluorescence-activated cell sorting (FACS) as well as gene
expression in the parent populations. This demonstrates that the
CXCR4.sup.+ cells contain essentially all the CXCR4 gene expression
present in each parent population and the CXCR4.sup.- populations
contain very little or no CXCR4 gene expression.
[0098] FIGS. 33A-33D are bar charts that demonstrate the depletion
of mesoderm (A, BRACHYURY, B, MOX1), ectoderm (C, ZIC1) and
visceral endoderm (D, SOX7) gene expression in the CXCR4.sup.+
cells isolated from the high dose activin A treatment which is
already suppressed in expression of these non-definitive endoderm
markers.
[0099] FIGS. 34A-34M are bar charts showing the expression patterns
of marker genes that can be used to identify definitive endoderm
cells. The expression analysis of definitive endoderm markers,
FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is shown in G-L,
respectively. The expression analysis of previously described
lineage marking genes, SOX17, SOX7, SOX17/SOX7, TM, ZIC1, and MOX1
is shown in A-F, respectively. (M) shows the expression analysis of
CXCR4. With respect to each of A-M, the column labeled hESC
indicates gene expression from purified human embryonic stem cells;
2NF indicates cells treated with 2% FBS, no activin addition;
0.1A100 indicates cells treated with 0.1% FBS, 100 ng/ml activin A;
1A100 indicates cells treated with 1% FBS, 100 ng/ml activin A; and
2A100 indicates cells treated with 2% FBS, 100 ng/ml activin A.
[0100] FIG. 35 is a chart which shows the relative expression of
the PDX1 gene in a culture of hESCs after 4 days and 6 days with
and without activin in the presence of retinoic acid (RA) and
fibroblast growth factor-10 (FGF-10) added on day 4.
[0101] FIGS. 36A-36F are charts which show the relative expression
of marker genes in a culture of hESCs after 4 days and 6 days with
and without activin in the presence of retinoic acid (RA) and
fibroblast growth factor-10 (FGF-10) added on day 4. The panels
show the relative levels of expression of the following marker
genes: (A) SOX17; (B) SOX7; (C) AFP; (D) SOX1; (E) ZIC1; and (F)
NFM.
[0102] FIGS. 37A-37C are charts which show the relative expression
of marker genes in a culture of hESCs after 4 days and 8 days with
and without activin in the presence or absence of combinations of
retinoic acid (RA), fibroblast growth factor-10 (FGF-10) and
fibroblast growth factor-4 (FGF-4) added on day 4. The panels show
the relative levels of expression of the following marker genes:
(A) PDX1; (B) SOX7; and (C) NFM.
[0103] FIGS. 38A-38G are charts which show the relative expression
of marker genes in a culture of definitive endoderm cells contacted
with 50 ng/ml FGF-10 in combination with either 1 .mu.M, 0.2 .mu.M
or 0.04 .mu.M retinoic acid (RA) added on day 4. The panels show
the relative levels of expression of the following marker genes:
(A) PDX1; (B) HOXA3; (C) HOXC6; (D) HOXA13; (E) CDX1; (F) SOX1; and
(G) NFM.
[0104] FIGS. 39A-39E are charts which show the relative expression
of marker genes in a culture of hESCs after 4 days and 8 days with
and without activin in the presence of combinations of retinoic
acid (RA), fibroblast growth factor-10 (FGF-10) and one of the
following: serum replacement (SR), fetal bovine serum (FBS) or B27.
The panels show the relative levels of expression of the following
marker genes: (A) PDX1; (B) SOX7; (C) AFP; (D) ZIC1; and (E)
NFM.
[0105] FIGS. 40A-40B are charts which show the relative expression
of marker genes for pancreas (PDX1, HNF6) and liver (HNF6) in a
culture of hESCs after 6 days (just prior to addition of RA) and at
9 days (three days after exposure to RA). Various conditions were
included to compare the addition of activin B at doses of 10 ng/ml
(a10), 25 ng/ml (a25) or 50 ng/ml (a50) in the presence of either
25 ng/ml (A25) or 50 ng/ml (A50) activin A. The condition without
any activin A or activin B (NF) serves as the negative control for
definitive endoderm and PDX1-positive endoderm production. The
panels show the relative levels of expression of the following
marker genes: (A) PDX1 and (B) HNF6.
[0106] FIGS. 41A-41C are charts which show the relative expression
of marker genes in a culture of hESCs with 100 ng/ml (A100), 50
ng/ml (A50) or without (NF) activin A at 5 days (just prior to
retinoic acid addition) and at 2, 4, and 6 days after RA exposure
(day 7, 9, and 11, respectively). The percentage label directly
under each bar indicates the FBS dose during days 3-5 of
differentiation. Starting at day 7, cells treated with RA (R) were
grown in RPMI medium comprising 0.5% FBS. The RA concentration was
2 .mu.M on day 7, 1 .mu.M on day 9 and 0.2 .mu.M on day 11. The
panels show the relative levels of expression of the following
marker genes: (A) PDX1; (B) ZIC1; (C) SOX7.
[0107] FIGS. 42A-42B are charts which show the relative expression
of marker genes in a culture of hESCs treated first with activin A
in low FBS to induce definitive endoderm (day 5) and then with
fresh (A25R) medium comprising 25 ng/ml activin A and RA or various
conditioned media (MEFCM, CM#2, CM#3 and CM#4) and RA to induce
PDX1-expressing endoderm. Marker expression was determined on days
5, 6, 7, 8 and 9. The panels show the relative levels of expression
of the following marker genes: (A) PDX1; (B) CDX1.
[0108] FIG. 43 is a chart which shows the relative expression of
PDX1 in a culture of hESCs treated first with activin A in low FBS
to induce definitive endoderm and followed by fresh media
comprising activin A and retinoic acid (A25R) or varying amounts of
RA in conditioned media diluted into fresh media. Total volume of
media is 5 ml in all cases.
[0109] FIG. 44 is a Western blot showing PDX1 immunoprecipitated
from RA-treated definitive endoderm cells 3 days (d8) and 4 days
(d9) after the addition of RA and 50 ng/ml activin A.
[0110] FIG. 45 is a summary chart displaying the results of a
fluorescence-activated cell sort (FACs) of PDX1-positive foregut
endoderm cells genetically tagged with a EGFP reporter under
control of the PDX1 promoter.
[0111] FIG. 46 is a chart showing relative PDX1 expression levels
normalized to housekeeping genes for sorted populations of live
cells (Live), EGFP-negative cells (Neg) and EGFP-positive cells
(GFP+).
[0112] FIG. 47 is a chart showing relative PDX1 expression levels
normalized to housekeeping genes for sorted populations of live
cells (Live), EGFP-negative cells (Neg), the half of the
EGFP-positive cell population that has the lowest EGFP signal
intensity (Lo) and the half of the EGFP-positive cell population
that has the highest EGFP signal intensity (Hi).
[0113] FIGS. 48A-48E are a charts showing the relative expression
levels normalized to housekeeping genes of five pancreatic endoderm
markers in sorted populations of live cells (Live), EGFP-negative
cells (Neg) and EGFP-positive cells (GFP+). (A) NKX2.2; (B) GLUT2;
(C) HNF3.beta.; (D) KRT19 and (E) HNF4.alpha..
[0114] FIGS. 49A-49B are a charts showing the relative expression
levels normalized to housekeeping genes of two non-pancreatic
endoderm markers in sorted populations of live cells (Live),
EGFP-negative cells (Neg) and EGFP-positive cells (GFP+). (A) ZIC1
and (B) GFAP.
[0115] FIGS. 50A-50D show the in vivo differentiation of definitive
endoderm cells that are transplanted under the kidney capsule of
immunocompromised mice. (A)--hetatoxylin-eosin staining showing
gut-tube-like structures; (B)--antibody immunoreactivity against
hepatocyte specific antigen (liver); (C)--antibody immunoreactivity
against villin (intestine); and (D)--antibody immunoreactivity
against CDX2 (intestine).
[0116] FIGS. 51A-51C are charts showing the normalized relative
expression levels of markers for liver (albumin and PROX1) and lung
(TITF1) tissues in cells contacted with Wnt3B at 20 ng/ml, FGF2 at
5 ng/ml or FGF2 at 100 ng/ml on days 5-10. DE refers to definitive
endoderm. (A) albumin, (B) PROX1, and (C) TITF1.
[0117] FIGS. 52A-52L are charts showing the normalized relative
expression levels of markers for liver (AFP, AAT, hHEX, GLUT2,
APOA1 and VCAM1) and lung (VWF and CXR4) tissues in cells contacted
with Wnt3A at 20-50 ng/ml, FGF2 at 5 ng/ml or FGF2 at 100 ng/ml on
days 5-10 and BMP4 on days 9 and 10. DE refers to definitive
endoderm. (A) AFP, (B) AAT, (C) GLUKO, (D) hHEX, (E) TAT, (F)
hNF4a, (G) CYP7A, (H) GLUT2, (I) APOA1, (J) VCAM1, (K) VWF, and (L)
CXCR4.
DETAILED DESCRIPTION
[0118] A crucial stage in early human development termed
gastrulation occurs 2-3 weeks after fertilization. Gastrulation is
extremely significant because it is at this time that the three
primary germ layers are first specified and organized (Lu et al.,
2001; Schoenwolf and Smith, 2000). The ectoderm is responsible for
the eventual formation of the outer coverings of the body and the
entire nervous system whereas the heart, blood, bone, skeletal
muscle and other connective tissues are derived from the mesoderm.
Definitive endoderm is defined as the germ layer that is
responsible for formation of the entire gut tube which includes the
esophagus, stomach and small and large intestines, and the organs
which derive from the gut tube such as the lungs, liver, thymus,
parathyroid and thyroid glands, gall bladder and pancreas
(Grapin-Botton and Melton, 2000; Kimelman and Griffin, 2000;
Tremblay et al., 2000; Wells and Melton, 1999; Wells and Melton,
2000). A very important distinction should be made between the
definitive endoderm and the completely separate lineage of cells
termed primitive endoderm. The primitive endoderm is primarily
responsible for formation of extra-embryonic tissues, mainly the
parietal and visceral endoderm portions of the placental yolk sac
and the extracellular matrix material of Reichert's membrane.
[0119] During gastrulation, the process of definitive endoderm
formation begins with a cellular migration event in which
mesendoderm cells (cells competent to form mesoderm or endoderm)
migrate through a structure called the primitive streak. Definitive
endoderm is derived from cells, which migrate through the anterior
portion of the streak and through the node (a specialized structure
at the anterior-most region of the streak). As migration occurs,
definitive endoderm populates first the most anterior gut tube and
culminates with the formation of the posterior end of the gut
tube.
[0120] Definitive endoderm and endoderm cells derived therefrom
represent important multipotent starting points for the derivation
of cells which make up terminally differentiated tissues and/or
organs derived from the definitive endoderm lineage. Such cells,
tissues and/or organs are extremely useful in cell therapies. As
such, the methods described herein for identifying differentiation
factors capable of causing the differentiation of definitive
endoderm cells and/or PDX1 expressing endoderm cells to other cells
types derived from the definitive endoderm cell lineage are
beneficial for the advancement of cell therapy.
[0121] In particular, some embodiments of the present invention
relate to methods of identifying one or more differentiation
factors that are useful for differentiating cells in a cell
population comprising PDX1-positive endoderm cells and/or
definitive endoderm cells into cells that are capable of promoting
the differentiation of definitive endoderm cells into cells which
are precursors for tissues and/or organs which include, but are not
limited to, pancreas, liver, lungs, stomach, intestine, thyroid,
thymus, pharynx, gallbladder and urinary bladder.
[0122] Additional aspects which relate to compositions of
definitive endoderm cells, PDX1-positive endoderm as well as
methods and compositions useful for producing such cells are also
described herein.
DEFINITIONS
[0123] Certain terms and phrases as used throughout this
application have the meanings provided as follows:
[0124] As used herein, "embryonic" refers to a range of
developmental stages of an organism beginning with a single zygote
and ending with a multicellular structure that no longer comprises
pluripotent or totipotent cells other than developed gametic cells.
In addition to embryos derived by gamete fusion, the term
"embryonic" refers to embryos derived by somatic cell nuclear
transfer.
[0125] As used herein, "multipotent" or "multipotent cell" refers
to a cell type that can give rise to a limited number of other
particular cell types.
[0126] As used herein, "expression" refers to the production of a
material or substance as well as the level or amount of production
of a material or substance. Thus, determining the expression of a
specific marker refers to detecting either the relative or absolute
amount of the marker that is expressed or simply detecting the
presence or absence of the marker.
[0127] As used herein, "marker" refers to any molecule that can be
observed or detected. For example, a marker can include, but is not
limited to, a nucleic acid, such as a transcript of a specific
gene, a polypeptide product of a gene, a non-gene product
polypeptide, a glycoprotein, a carbohydrate, a glycolipd, a lipid,
a lipoprotein or a small molecule (for example, molecules having a
molecular weight of less than 10,000 amu)
[0128] When used in connection with cell cultures and/or cell
populations, the term "portion" means any non-zero amount of the
cell culture or cell population, which ranges from a single cell to
the entirety of the cell culture or cells population.
[0129] With respect to cells in cell cultures or in cell
populations, the phrase "substantially free of" means that the
specified cell type of which the cell culture or cell population is
free, is present in an amount of less than about 5% of the total
number of cells present in the cell culture or cell population.
[0130] As used herein, "retinoid" refers to retinol, retinal or
retinoic acid as well as derivatives of any of these compounds.
[0131] By "conditioned medium" is meant, a medium that is altered
as compared to a base medium.
[0132] As used herein, "foregut/midgut" refers to cells of the
anterior portion of the gut tube as well as cells of the middle
portion of the gut tube, including cells of the foregut/midgut
junction.
Definitive Endoderm Cells and Processes Related Thereto
[0133] Embodiments described herein relate to novel, defined
processes for the production of definitive endoderm cells in
culture by differentiating pluripotent cells, such as stem cells
into multipotent definitive endoderm cells. As described above,
definitive endoderm cells do not differentiate into tissues
produced from ectoderm or mesoderm, but rather, differentiate into
the gut tube as well as organs that are derived from the gut tube.
In certain preferred embodiments, the definitive endoderm cells are
derived from hESCs. Such processes can provide the basis for
efficient production of human endodermal derived tissues such as
pancreas, liver, lung, stomach, intestine, thyroid and thymus. For
example, production of definitive endoderm may be the first step in
differentiation of a stem cell to a functional insulin-producing
.beta.-cell. To obtain useful quantities of insulin-producing
.beta.-cells, high efficiency of differentiation is desirable for
each of the differentiation steps that occur prior to reaching the
pancreatic islet/.beta.-cell fate. Since differentiation of stem
cells to definitive endoderm cells represents perhaps the earliest
step towards the production of functional pancreatic
islet/.beta.-cells (as shown in FIG. 1), high efficiency of
differentiation at this step is particularly desirable.
[0134] In view of the desirability of efficient differentiation of
pluripotent cells to definitive endoderm cells, some aspects of the
differentiation processes described herein relate to in vitro
methodology that results in approximately 50-80% conversion of
pluripotent cells to definitive endoderm cells. Typically, such
methods encompass the application of culture and growth factor
conditions in a defined and temporally specified fashion. Further
enrichment of the cell population for definitive endoderm cells can
be achieved by isolation and/or purification of the definitive
endoderm cells from other cells in the population by using a
reagent that specifically binds to definitive endoderm cells. As
such, some embodiments described herein relate to definitive
endoderm cells as well as methods for producing and isolating
and/or purifying such cells.
[0135] In order to determine the amount of definitive endoderm
cells in a cell culture or cell population, a method of
distinguishing this cell type from the other cells in the culture
or in the population is desirable. Accordingly, certain embodiments
described herein relate to cell markers whose presence, absence
and/or relative expression levels are specific for definitive
endoderm and methods for detecting and determining the expression
of such markers.
[0136] In some embodiments described herein, the presence, absence
and/or level of expression of a marker is determined by
quantitative PCR (Q-PCR). For example, the amount of transcript
produced by certain genetic markers, such as SOX17, CXCR4, OCT4,
AFP, TM, SPARC, SOX7, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR,
FOXQ1, CMKOR1, CRIP1 and other markers described herein is
determined by quantitative Q-PCR. In other embodiments,
immunohistochemistry is used to detect the proteins expressed by
the above-mentioned genes. In still other embodiments, Q-PCR and
immunohistochemical techniques are both used to identify and
determine the amount or relative proportions of such markers.
[0137] By using methods, such as those described above, to
determine the expression of one or more appropriate markers, it is
possible to identify definitive endoderm cells, as well as
determine the proportion of definitive endoderm cells in a cell
culture or cell population. For example, in some embodiments of the
present invention, the definitive endoderm cells or cell
populations that are produced express the SOX17 and/or the CXCR4
gene at a level of about 2 orders of magnitude greater than
non-definitive endoderm cell types or cell populations. In other
embodiments, the definitive endoderm cells or cell populations that
are produced express the SOX17 and/or the CXCR4 gene at a level of
more than 2 orders of magnitude greater than non-definitive
endoderm cell types or cell populations. In still other
embodiments, the definitive endoderm cells or cell populations that
are produced express one or more of the markers selected from the
group consisting of SOX17, CXCR4, GSC, FGF17, VWF, CALCR, FOXQ1,
CMKOR1 and CRIP1 at a level of about 2 or more than 2 orders of
magnitude greater than non-definitive endoderm cell types or cell
populations. In some embodiments described herein, definitive
endoderm cells do not substantially express PDX1.
[0138] Embodiments described herein also relate to definitive
endoderm compositions. For example, some embodiments relate to cell
cultures comprising definitive endoderm, whereas others relate to
cell populations enriched in definitive endoderm cells. Some
preferred embodiments relate to cell cultures which comprise
definitive endoderm cells, wherein at least about 50-80% of the
cells in culture are definitive endoderm cells. An especially
preferred embodiment relates to cells cultures comprising human
cells, wherein at least about 50-80% of the human cells in culture
are definitive endoderm cells. Because the efficiency of the
differentiation procedure can be adjusted by modifying certain
parameters, which include but are not limited to, cell growth
conditions, growth factor concentrations and the timing of culture
steps, the differentiation procedures described herein can result
in about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 95%, or greater than about 95% conversion of pluripotent
cells to definitive endoderm. In other preferred embodiments,
conversion of a pluripotent cell population, such as a stem cell
population, to substantially pure definitive endoderm cell
population is contemplated.
[0139] The compositions and methods described herein have several
useful features. For example, the cell cultures and cell
populations comprising definitive endoderm as well as the methods
for producing such cell cultures and cell populations are useful
for modeling the early stages of human development. Furthermore,
the compositions and methods described herein can also serve for
therapeutic intervention in disease states, such as diabetes
mellitus. For example, since definitive endoderm serves as the
source for only a limited number of tissues, it can be used in the
development of pure tissue or cell types.
Production of Definitive Endoderm from Pluripotent Cells
[0140] Processes for differentiating pluripotent cells to produce
cell cultures and enriched cell populations comprising definitive
endoderm is described below and in U.S. patent Ser. No. 11/021,618,
entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, the disclosure
of which is incorporated herein by reference in its entirety. In
some of these processes, the pluripotent cells used as starting
material are stem cells. In certain processes, definitive endoderm
cell cultures and enriched cell populations comprising definitive
endoderm cells are produced from embryonic stem cells. A preferred
method for deriving definitive endoderm cells utilizes human
embryonic stem cells as the starting material for definitive
endoderm production. Such pluripotent cells can be cells that
originate from the morula, embryonic inner cell mass or those
obtained from embryonic gonadal ridges. Human embryonic stem cells
can be maintained in culture in a pluripotent state without
substantial differentiation using methods that are known in the
art. Such methods are described, for example, in U.S. Pat. Nos.
5,453,357, 5,670,372, 5,690,926 5,843,780, 6,200,806 and 6,251,671
the disclosures of which are incorporated herein by reference in
their entireties.
[0141] In some processes for producing definitive endoderm cells,
hESCs are maintained on a feeder layer. In such processes, any
feeder layer which allows hESCs to be maintained in a pluripotent
state can be used. One commonly used feeder layer for the
cultivation of human embryonic stem cells is a layer of mouse
fibroblasts. More recently, human fibroblast feeder layers have
been developed for use in the cultivation of hESCs (see US Patent
Application No. 2002/0072117, the disclosure of which is
incorporated herein by reference in its entirety). Alternative
processes for producing definitive endoderm permit the maintenance
of pluripotent hESC without the use of a feeder layer. Methods of
maintaining pluripotent hESCs under feeder-free conditions have
been described in US Patent Application No. 2003/0175956, the
disclosure of which is incorporated herein by reference in its
entirety.
[0142] The human embryonic stem cells used herein can be maintained
in culture either with or without serum. In some embryonic stem
cell maintenance procedures, serum replacement is used. In others,
serum free culture techniques, such as those described in US Patent
Application No. 2003/0190748, the disclosure of which is
incorporated herein by reference in its entirety, are used.
[0143] Stem cells are maintained in culture in a pluripotent state
by routine passage until it is desired that they be differentiated
into definitive endoderm. In some processes, differentiation to
definitive endoderm is achieved by providing to the stem cell
culture a growth factor of the TGF.beta. superfamily in an amount
sufficient to promote differentiation to definitive endoderm.
Growth factors of the TGF.beta. superfamily which are useful for
the production of definitive endoderm are selected from the
Nodal/Activin or BMP subgroups. In some preferred differentiation
processes, the growth factor is selected from the group consisting
of Nodal, activin A, activin B and BMP4. Additionally, the growth
factor Wnt3a and other Wnt family members are useful for the
production of definitive endoderm cells. In certain differentiation
processes, combinations of any of the above-mentioned growth
factors can be used.
[0144] With respect to some of the processes for the
differentiation of pluripotent stem cells to definitive endoderm
cells, the above-mentioned growth factors are provided to the cells
so that the growth factors are present in the cultures at
concentrations sufficient to promote differentiation of at least a
portion of the stem cells to definitive endoderm cells. In some
processes, the above-mentioned growth factors are present in the
cell culture at a concentration of at least about 5 ng/ml, at least
about 10 ng/ml, at least about 25 ng/ml, at least about 50 ng/ml,
at least about 75 ng/ml, at least about 100 ng/ml, at least about
200 ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, at
least about 500 ng/ml, at least about 1000 ng/ml, at least about
2000 ng/ml, at least about 3000 ng/ml, at least about 4000 ng/ml,
at least about 5000 ng/ml or more than about 5000 ng/ml.
[0145] In certain processes for the differentiation of pluripotent
stem cells to definitive endoderm cells, the above-mentioned growth
factors are removed from the cell culture subsequent to their
addition. For example, the growth factors can be removed within
about one day, about two days, about three days, about four days,
about five days, about six days, about seven days, about eight
days, about nine days or about ten days after their addition. In a
preferred processes, the growth factors are removed about four days
after their addition.
[0146] Cultures of definitive endoderm cells can be grown in medium
containing reduced serum or no serum. Under certain culture
conditions, serum concentrations can range from about 0.05% v/v to
about 20% v/v. For example, in some differentiation processes, the
serum concentration of the medium can be less than about 0.05%
(v/v), less than about 0.1% (v/v), less than about 0.2% (v/v), less
than about 0.3% (v/v), less than about 0.4% (v/v), less than about
0.5% (v/v), less than about 0.6% (v/v), less than about 0.7% (v/v),
less than about 0.8% (v/v), less than about 0.9% (v/v), less than
about 1% (v/v), less than about 2% (v/v), less than about 3% (v/v),
less than about 4% (v/v), less than about 5% (v/v), less than about
6% (v/v), less than about 7% (v/v), less than about 8% (v/v), less
than about 9% (v/v), less than about 10% (v/v), less than about 15%
(v/v) or less than about 20% (v/v). In some processes, definitive
endoderm cells are grown without serum or with serum replacement.
In still other processes, definitive endoderm cells are grown in
the presence of B27. In such processes, the concentration of B27
supplement can range from about 0.1% v/v to about 20% v/v.
Monitoring the Differentiation of Pluripotent Cells to Definitive
Endoderm
[0147] The progression of the hESC culture to definitive endoderm
can be monitored by determining the expression of markers
characteristic of definitive endoderm. In some processes, the
expression of certain markers is determined by detecting the
presence or absence of the marker. Alternatively, the expression of
certain markers can be determined by measuring the level at which
the marker is present in the cells of the cell culture or cell
population. In such processes, the measurement of marker expression
can be qualitative or quantitative. One method of quantitating the
expression of markers that are produced by marker genes is through
the use of quantitative PCR (Q-PCR). Methods of performing Q-PCR
are well known in the art. Other methods which are known in the art
can also be used to quantitate marker gene expression. For example,
the expression of a marker gene product can be detected by using
antibodies specific for the marker gene product of interest. In
certain processes, the expression of marker genes characteristic of
definitive endoderm as well as the lack of significant expression
of marker genes characteristic of hESCs and other cell types is
determined.
[0148] As described further in the Examples below, a reliable
marker of definitive endoderm is the SOX17 gene. As such, the
definitive endoderm cells produced by the processes described
herein express the SOX17 marker gene, thereby producing the SOX17
gene product. Other markers of definitive endoderm are MIXL1,
GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
Since definitive endoderm cells express the SOX17 marker gene at a
level higher than that of the SOX7 marker gene, which is
characteristic of primitive and visceral endoderm (see Table 1), in
some processes, the expression of both SOX17 and SOX7 is monitored.
In other processes, expression of the both the SOX17 marker gene
and the OCT4 marker gene, which is characteristic of hESCs, is
monitored. Additionally, because definitive endoderm cells express
the SOX17 marker gene at a level higher than that of the AFP, SPARC
or Thrombomodulin (TM) marker genes, the expression of these genes
can also be monitored.
[0149] Another marker of definitive endoderm is the CXCR4 gene. The
CXCR4 gene encodes a cell surface chemokine receptor whose ligand
is the chemoattractant SDF-1. The principal roles of the CXCR4
receptor-bearing cells in the adult are believed to be the
migration of hematopoetic cells to the bone marrow, lymphocyte
trafficking and the differentiation of various B cell and
macrophage blood cell lineages [Kim, C., and Broxmeyer, H. J.
Leukocyte Biol. 65, 6-15 (1999)]. The CXCR4 receptor also functions
as a coreceptor for the entry of HIV-1 into T-cells [Feng, Y., et
al. Science, 272, 872-877 (1996)]. In an extensive series of
studies carried out by [McGrath, K. E. et al. Dev. Biology 213,
442-456 (1999)], the expression of the chemokine receptor CXCR4 and
its unique ligand, SDF-1 [Kim, C., and Broxmyer, H., J. Leukocyte
Biol. 65, 6-15 (1999)], were delineated during early development
and adult life in the mouse. The CXCR4/SDF1 interaction in
development became apparent when it was demonstrated that if either
gene was disrupted in transgenic mice [Nagasawa et al. Nature, 382,
635-638 (1996)], Ma, Q., et al Immunity, 10, 463-471 (1999)] it
resulted in late embryonic lethality. McGrath et al. demonstrated
that CXCR4 is the most abundant chemokine receptor messenger RNA
detected during early gastrulating embryos (E7.5) using a
combination of RNase protection and in situ hybridization
methodologies. In the gastrulating embryo, CXCR4/SDF-1 signaling
appears to be mainly involved in inducing migration of
primitive-streak germlayer cells and is expressed on definitive
endoderm, mesoderm and extraembryonic mesoderm present at this
time. In E7.2-7.8 mouse embryos, CXCR4 and alpha-fetoprotein are
mutually exclusive indicating a lack of expression in visceral
endoderm [McGrath, K. E. et al. Dev. Biology 213, 442-456
(1999)].
[0150] Since definitive endoderm cells produced by differentiating
pluripotent cells express the CXCR4 marker gene, expression of
CXCR4 can be monitored in order to track the production of
definitive endoderm cells. Additionally, definitive endoderm cells
produced by the methods described herein express other markers of
definitive endoderm including, but not limited to, SOX17, MIXL1,
GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
Since definitive endoderm cells express the CXCR4 marker gene at a
level higher than that of the SOX7 marker gene, the expression of
both CXCR4 and SOX7 can be monitored. In other processes,
expression of both the CXCR4 marker gene and the OCT4 marker gene,
is monitored. Additionally, because definitive endoderm cells
express the CXCR4 marker gene at a level higher than that of the
AFP, SPARC or Thrombomodulin (TM) marker genes, the expression of
these genes can also be monitored.
[0151] It will be appreciated that expression of CXCR4 in
endodermal cells does not preclude the expression of SOX17. As
such, definitive endoderm cells produced by the processes described
herein will substantially express SOX17 and CXCR4 but will not
substantially express AFP, TM, SPARC or PDX1.
[0152] It will be appreciated that SOX17 and/or CXCR4 marker
expression is induced over a range of different levels in
definitive endoderm cells depending on the differentiation
conditions. As such, in some embodiments described herein, the
expression of the SOX17 marker and/or the CXCR4 marker in
definitive endoderm cells or cell populations is at least about
2-fold higher to at least about 10,000-fold higher than the
expression of the SOX17 marker and/or the CXCR4 marker in
non-definitive endoderm cells or cell populations, for example
pluripotent stem cells. In other embodiments, the expression of the
SOX17 marker and/or the CXCR4 marker in definitive endoderm cells
or cell populations is at least about 4-fold higher, at least about
6-fold higher, at least about 8-fold higher, at least about 10-fold
higher, at least about 15-fold higher, at least about 20-fold
higher, at least about 40-fold higher, at least about 80-fold
higher, at least about 100-fold higher, at least about 150-fold
higher, at least about 200-fold higher, at least about 500-fold
higher, at least about 750-fold higher, at least about 1000-fold
higher, at least about 2500-fold higher, at least about 5000-fold
higher, at least about 7500-fold higher or at least about
10,000-fold higher than the expression of the SOX17 marker and/or
the CXCR4 marker in non-definitive endoderm cells or cell
populations, for example pluripotent stem cells. In some
embodiments, the expression of the SOX17 marker and/or CXCR4 marker
in definitive endoderm cells or cell populations is infinitely
higher than the expression of the SOX17 marker and/or the CXCR4
marker in non-definitive endoderm cells or cell populations, for
example pluripotent stem cells.
[0153] It will also be appreciated that in some embodiments
described herein, the expression of markers selected from the group
consisting of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1,
CMKOR1 and CRIP1 in definitive endoderm cells or cell populations
is increased as compared to the expression of GATA4, MIXL1, HNF3b,
GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 in non-definitive
endoderm cells or cell populations.
[0154] Additionally, it will be appreciated that there is a range
of differences between the expression level of the SOX17 marker and
the expression levels of the OCT4, SPARC, AFP, TM and/or SOX7
markers in definitive endoderm cells. Similarly, there exists a
range of differences between the expression level of the CXCR4
marker and the expression levels of the OCT4, SPARC, AFP, TM and/or
SOX7 markers in definitive endoderm cells. As such, in some
embodiments described herein, the expression of the SOX17 marker or
the CXCR4 marker is at least about 2-fold higher to at least about
10,000-fold higher than the expression of OCT4, SPARC, AFP, TM
and/or SOX7 markers. In other embodiments, the expression of the
SOX17 marker or the CXCR4 marker is at least about 4-fold higher,
at least about 6-fold higher, at least about 8-fold higher, at
least about 10-fold higher, at least about 15-fold higher, at least
about 20-fold higher, at least about 40-fold higher, at least about
80-fold higher, at least about 100-fold higher, at least about
150-fold higher, at least about 200-fold higher, at least about
500-fold higher, at least about 750-fold higher, at least about
1000-fold higher, at least about 2500-fold higher, at least about
5000-fold higher, at least about 7500-fold higher or at least about
10,000-fold higher than the expression of OCT4, SPARC, AFP, TM
and/or SOX7 markers. In some embodiments, OCT4, SPARC, AFP, TM
and/or SOX7 markers are not significantly expressed in definitive
endoderm cells.
[0155] It will also be appreciated that in some embodiments
described herein, the expression of markers selected from the group
consisting of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1,
CMKOR1 and CRIP1 in definitive endoderm cells is increased as
compared to the expression of OCT4, SPARC, AFP, TM and/or SOX7 in
definitive endoderm cells.
Enrichment, Isolation and/or Purification of Definitive
Endoderm
[0156] Definitive endoderm cells produced by any of the
above-described processes can be enriched, isolated and/or purified
by using an affinity tag that is specific for such cells. Examples
of affinity tags specific for definitive endoderm cells are
antibodies, ligands or other binding agents that are specific to a
marker molecule, such as a polypeptide, that is present on the cell
surface of definitive endoderm cells but which is not substantially
present on other cell types that would be found in a cell culture
produced by the methods described herein. In some processes, an
antibody which binds to CXCR4 is used as an affinity tag for the
enrichment, isolation or purification of definitive endoderm cells.
In other processes, the chemokine SDF-1 or other molecules based on
SDF-1 can also be used as affinity tags. Such molecules include,
but not limited to, SDF-1 fragments, SDF-1 fusions or SDF-1
mimetics.
[0157] Methods for making antibodies and using them for cell
isolation are known in the art and such methods can be implemented
for use with the antibodies and definitive endoderm cells described
herein. In one process, an antibody which binds to CXCR4 is
attached to a magnetic bead and then allowed to bind to definitive
endoderm cells in a cell culture which has been enzymatically
treated to reduce intercellular and substrate adhesion. The
cell/antibody/bead complexes are then exposed to a movable magnetic
field which is used to separate bead-bound definitive endoderm
cells from unbound cells. Once the definitive endoderm cells are
physically separated from other cells in culture, the antibody
binding is disrupted and the cells are replated in appropriate
tissue culture medium.
[0158] Additional methods for obtaining enriched, isolated or
purified definitive endoderm cell cultures or populations can also
be used. For example, in some embodiments, the CXCR4 antibody is
incubated with a definitive endoderm-containing cell culture that
has been treated to reduce intercellular and substrate adhesion.
The cells are then washed, centrifuged and resuspended. The cell
suspension is then incubated with a secondary antibody, such as an
FITC-conjugated antibody that is capable of binding to the primary
antibody. The cells are then washed, centrifuged and resuspended in
buffer. The cell suspension is then analyzed and sorted using a
fluorescence activated cell sorter (FACS). CXCR4-positive cells are
collected separately from CXCR4-negative cells, thereby resulting
in the isolation of such cell types. If desired, the isolated cell
compositions can be further purified by using an alternate
affinity-based method or by additional rounds of sorting using the
same or different markers that are specific for definitive
endoderm.
[0159] In still other processes, definitive endoderm cells are
enriched, isolated and/or purified using a ligand or other molecule
that binds to CXCR4. In some processes, the molecule is SDF-1 or a
fragment, fusion or mimetic thereof.
[0160] In preferred processes, definitive endoderm cells are
enriched, isolated and/or purified from other non-definitive
endoderm cells after the stem cell cultures are induced to
differentiate towards the definitive endoderm lineage. It will be
appreciated that the above-described enrichment, isolation and
purification procedures can be used with such cultures at any stage
of differentiation.
[0161] In addition to the procedures just described, definitive
endoderm cells may also be isolated by other techniques for cell
isolation. Additionally, definitive endoderm cells may also be
enriched or isolated by methods of serial subculture in growth
conditions which promote the selective survival or selective
expansion of the definitive endoderm cells.
[0162] Using the methods described herein, enriched, isolated
and/or purified populations of definitive endoderm cells and or
tissues can be produced in vitro from pluripotent cell cultures or
cell populations, such as stem cell cultures or populations, which
have undergone at least some differentiation. In some methods, the
cells undergo random differentiation. In a preferred method,
however, the cells are directed to differentiate primarily into
definitive endoderm. Some preferred enrichment, isolation and/or
purification methods relate to the in vitro production of
definitive endoderm from human embryonic stem cells. Using the
methods described herein, cell populations or cell cultures can be
enriched in definitive endoderm content by at least about 2- to
about 1000-fold as compared to untreated cell populations or cell
cultures. In some embodiments, definitive endoderm cells can be
enriched by at least about 5- to about 500-fold as compared to
untreated cell populations or cell cultures. In other embodiments,
definitive endoderm cells can be enriched from at least about 10-
to about 200-fold as compared to untreated cell populations or cell
cultures. In still other embodiments, definitive endoderm cells can
be enriched from at least about 20- to about 100-fold as compared
to untreated cell populations or cell cultures. In yet other
embodiments, definitive endoderm cells can be enriched from at
least about 40- to about 80-fold as compared to untreated cell
populations or cell cultures. In certain embodiments, definitive
endoderm cells can be enriched from at least about 2- to about
20-fold as compared to untreated cell populations or cell
cultures.
Compositions Comprising Definitive Endoderm
[0163] Cell compositions produced by the above-described methods
include cell cultures comprising definitive endoderm and cell
populations enriched in definitive endoderm. For example, cell
cultures which comprise definitive endoderm cells, wherein at least
about 50-80% of the cells in culture are definitive endoderm cells,
can be produced. Because the efficiency of the differentiation
process can be adjusted by modifying certain parameters, which
include but are not limited to, cell growth conditions, growth
factor concentrations and the timing of culture steps, the
differentiation procedures described herein can result in about 5%,
about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,
about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,
about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,
or greater than about 95% conversion of pluripotent cells to
definitive endoderm. In processes in which isolation of definitive
endoderm cells is employed, for example, by using an affinity
reagent that binds to the CXCR4 receptor, a substantially pure
definitive endoderm cell population can be recovered.
[0164] Some embodiments described herein relate to compositions,
such as cell populations and cell cultures, that comprise both
pluripotent cells, such as stem cells, and definitive endoderm
cells. For example, using the methods described herein,
compositions comprising mixtures of hESCs and definitive endoderm
cells can be produced. In some embodiments, compositions comprising
at least about 5 definitive endoderm cells for about every 95
pluripotent cells are produced. In other embodiments, compositions
comprising at least about 95 definitive endoderm cells for about
every 5 pluripotent cells are produced. Additionally, compositions
comprising other ratios of definitive endoderm cells to pluripotent
cells are contemplated. For example, compositions comprising at
least about 1 definitive endoderm cell for about every 1,000,000
pluripotent cells, at least about 1 definitive endoderm cell for
about every 100,000 pluripotent cells, at least about 1 definitive
endoderm cell for about every 10,000 pluripotent cells, at least
about 1 definitive endoderm cell for about every 1000 pluripotent
cells, at least about 1 definitive endoderm cell for about every
500 pluripotent cells, at least about 1 definitive endoderm cell
for about every 100 pluripotent cells, at least about 1 definitive
endoderm cell for about every 10 pluripotent cells, at least about
1 definitive endoderm cell for about every 5 pluripotent cells, at
least about 1 definitive endoderm cell for about every 2
pluripotent cells, at least about 2 definitive endoderm cells for
about every 1 pluripotent cell, at least about 5 definitive
endoderm cells for about every 1 pluripotent cell, at least about
10 definitive endoderm cells for about every 1 pluripotent cell, at
least about 20 definitive endoderm cells for about every 1
pluripotent cell, at least about 50 definitive endoderm cells for
about every 1 pluripotent cell, at least about 100 definitive
endoderm cells for about every 1 pluripotent cell, at least about
1000 definitive endoderm cells for about every 1 pluripotent cell,
at least about 10,000 definitive endoderm cells for about every 1
pluripotent cell, at least about 100,000 definitive endoderm cells
for about every 1 pluripotent cell and at least about 1,000,000
definitive endoderm cells for about every 1 pluripotent cell are
contemplated. In some embodiments, the pluripotent cells are human
pluripotent stem cells. In certain embodiments the stem cells are
derived from a morula, the inner cell mass of an embryo or the
gonadal ridges of an embryo. In certain other embodiments, the
pluripotent cells are derived from the gondal or germ tissues of a
multicellular structure that has developed past the embryonic
stage.
[0165] Some embodiments described herein relate to cell cultures or
cell populations comprising from at least about 5% definitive
endoderm cells to at least about 95% definitive endoderm cells. In
some embodiments the cell cultures or cell populations comprise
mammalian cells. In preferred embodiments, the cell cultures or
cell populations comprise human cells. For example, certain
specific embodiments relate to cell cultures comprising human
cells, wherein from at least about 5% to at least about 95% of the
human cells are definitive endoderm cells. Other embodiments relate
to cell cultures comprising human cells, wherein at least about 5%,
at least about 10%, at least about 15%, at least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90% or greater than 90% of the human cells are definitive
endoderm cells. In embodiments where the cell cultures or cell
populations comprise human feeder cells, the above percentages are
calculated without respect to the human feeder cells in the cell
cultures or cell populations.
[0166] Further embodiments described herein relate to compositions,
such as cell cultures or cell populations, comprising human cells,
such as human definitive endoderm cells, wherein the expression of
either the SOX17 or the CXCR4 marker is greater than the expression
of the OCT4, SPARC, alpha-fetoprotein (AFP), Thrombomodulin (TM)
and/or SOX7 marker in at least about 5% of the human cells. In
other embodiments, the expression of either the SOX17 or the CXCR4
marker is greater than the expression of the OCT4, SPARC, AFP, TM
and/or SOX7 marker in at least about 10% of the human cells, in at
least about 15% of the human cells, in at least about 20% of the
human cells, in at least about 25% of the human cells, in at least
about 30% of the human cells, in at least about 35% of the human
cells, in at least about 40% of the human cells, in at least about
45% of the human cells, in at least about 50% of the human cells,
in at least about 55% of the human cells, in at least about 60% of
the human cells, in at least about 65% of the human cells, in at
least about 70% of the human cells, in at least about 75% of the
human cells, in at least about 80% of the human cells, in at least
about 85% of the human cells, in at least about 90% of the human
cells, in at least about 95% of the human cells or in greater than
95% of the human cells. In embodiments where the cell cultures or
cell populations comprise human feeder cells, the above percentages
are calculated without respect to the human feeder cells in the
cell cultures or cell populations.
[0167] It will be appreciated that some embodiments described
herein relate to compositions, such as cell cultures or cell
populations, comprising human cells, such as human definitive
endoderm cells, wherein the expression of one or more markers
selected from the group consisting of GATA4, MIXL1, HNF3b, GSC,
FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is greater than the
expression of the OCT4, SPARC, AFP, TM and/or SOX7 markers in from
at least about 5% to greater than at least about 95% of the human
cells. In embodiments where the cell cultures or cell populations
comprise human feeder cells, the above percentages are calculated
without respect to the human feeder cells in the cell cultures or
cell populations.
[0168] Still other embodiments described herein relate to
compositions, such as cell cultures or cell populations, comprising
human cells, such as human definitive endoderm cells, wherein the
expression both the SOX17 and the CXCR4 marker is greater than the
expression of the OCT4, SPARC, AFP, TM and/or SOX7 marker in at
least about 5% of the human cells. In other embodiments, the
expression of both the SOX17 and the CXCR4 marker is greater than
the expression of the OCT4, SPARC, AFP, TM and/or SOX7 marker in at
least about 10% of the human cells, in at least about 15% of the
human cells, in at least about 20% of the human cells, in at least
about 25% of the human cells, in at least about 30% of the human
cells, in at least about 35% of the human cells, in at least about
40% of the human cells, in at least about 45% of the human cells,
in at least about 50% of the human cells, in at least about 55% of
the human cells, in at least about 60% of the human cells, in at
least about 65% of the human cells, in at least about 70% of the
human cells, in at least about 75% of the human cells, in at least
about 80% of the human cells, in at least about 85% of the human
cells, in at least about 90% of the human cells, in at least about
95% of the human cells or in greater than 95% of the human cells.
In embodiments where the cell cultures or cell populations comprise
human feeder cells, the above percentages are calculated without
respect to the human feeder cells in the cell cultures or cell
populations.
[0169] It will be appreciated that some embodiments described
herein relate to compositions, such as cell cultures or cell
populations, comprising human cells, such as human definitive
endoderm cells, wherein the expression of the GATA4, MIXL1, HNF3b,
GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 markers is greater
than the expression of the OCT4, SPARC, AFP, TM and/or SOX7 markers
in from at least about 5% to greater than at least about 95% of the
human cells. In embodiments where the cell cultures or cell
populations comprise human feeder cells, the above percentages are
calculated without respect to the human feeder cells in the cell
cultures or cell populations.
[0170] Additional embodiments described herein relate to
compositions, such as cell cultures or cell populations, comprising
mammalian endodermal cells, such as human endoderm cells, wherein
the expression of either the SOX17 or the CXCR4 marker is greater
than the expression of the OCT4, SPARC, AFP, TM and/or SOX7 marker
in at least about 5% of the endodermal cells. In other embodiments,
the expression of either the SOX17 or the CXCR4 marker is greater
than the expression of the OCT4, SPARC, AFP, TM and/or SOX7 marker
in at least about 10% of the endodermal cells, in at least about
15% of the endodermal cells, in at least about 20% of the
endodermal cells, in at least about 25% of the endodermal cells, in
at least about 30% of the endodermal cells, in at least about 35%
of the endodermal cells, in at least about 40% of the endodermal
cells, in at least about 45% of the endodermal cells, in at least
about 50% of the endodermal cells, in at least about 55% of the
endodermal cells, in at least about 60% of the endodermal cells, in
at least about 65% of the endodermal cells, in at least about 70%
of the endodermal cells, in at least about 75% of the endodermal
cells, in at least about 80% of the endodermal cells, in at least
about 85% of the endodermal cells, in at least about 90% of the
endodermal cells, in at least about 95% of the endodermal cells or
in greater than 95% of the endodermal cells.
[0171] It will be appreciated that some embodiments described
herein relate to compositions, such as cell cultures or cell
populations comprising mammalian endodermal cells, wherein the
expression of one or more markers selected from the group
consisting of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1,
CMKOR1 and CRIP1 is greater than the expression of the OCT4, SPARC,
AFP, TM and/or SOX7 markers in from at least about 5% to greater
than at least about 95% of the endodermal cells.
[0172] Still other embodiments described herein relate to
compositions, such as cell cultures or cell populations, comprising
mammalian endodermal cells, such as human endodermal cells, wherein
the expression of both the SOX17 and the CXCR4 marker is greater
than the expression of the OCT4, SPARC, AFP, TM and/or SOX7 marker
in at least about 5% of the endodermal cells. In other embodiments,
the expression of both the SOX17 and the CXCR4 marker is greater
than the expression of the OCT4, SPARC, AFP, TM and/or SOX7 marker
in at least about 10% of the endodermal cells, in at least about
15% of the endodermal cells, in at least about 20% of the
endodermal cells, in at least about 25% of the endodermal cells, in
at least about 30% of the endodermal cells, in at least about 35%
of the endodermal cells, in at least about 40% of the endodermal
cells, in at least about 45% of the endodermal cells, in at least
about 50% of the endodermal cells, in at least about 55% of the
endodermal cells, in at least about 60% of the endodermal cells, in
at least about 65% of the endodermal cells, in at least about 70%
of the endodermal cells, in at least about 75% of the endodermal
cells, in at least about 80% of the endodermal cells, in at least
about 85% of the endodermal cells, in at least about 90% of the
endodermal cells, in at least about 95% of the endodermal cells or
in greater than 95% of the endodermal cells.
[0173] It will be appreciated that some embodiments described
herein relate to compositions, such as cell cultures or cell
populations comprising mammalian endodermal cells, wherein the
expression of the GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR,
FOXQ1, CMKOR1 and CRIP1 markers is greater than the expression of
the OCT4, SPARC, AFP, TM and/or SOX7 markers in from at least about
5% to greater than at least about 95% of the endodermal cells.
[0174] Using the methods described herein, compositions comprising
definitive endoderm cells substantially free of other cell types
can be produced. In some embodiments described herein, the
definitive endoderm cell populations or cell cultures produced by
the methods described herein are substantially free of cells that
significantly express the OCT4, SOX7, AFP, SPARC, TM, ZIC1 or BRACH
marker genes.
[0175] In one embodiment, a description of a definitive endoderm
cell based on the expression of marker genes is, SOX17 high, MIXL1
high, AFP low, SPARC low, Thrombomodulin low, SOX7 low, CXCR4
high.
The PDX1 Gene Expression During Development
[0176] PDX1 (also called STF-1, IDX-1 and IPF-1) is a transcription
factor that is necessary for development of the pancreas and
rostral duodenum. PDX1 is first expressed in the pancreatic
endoderm, which arises from posterior foregut endoderm and will
produce both the exocrine and endocrine cells, starting at E8.5 in
the mouse. Later, PDX1 becomes restricted to beta-cells and some
delta-cells of the endocrine pancreas. This expression pattern is
maintained in the adult. PDX1 is also expressed in duodenal
endoderm early in development, which is adjacent to the forming
pancreas, then in the duodenal enterocytes and enteroendocrine
cells, antral stomach and in the common bile, cystic and biliary
ducts. This region of expression also becomes limited, at the time
that pancreatic expression becomes restricted, to predominantly the
rostral duodenum.
PDX1-Positive Cells and Processes Related Thereto
[0177] Embodiments of other differentiation processes described
herein relate to novel, defined processes for the production of
PDX1-positive endoderm cells, wherein the PDX1-positive endoderm
cells are multipotent cells that can differentiate into cells,
tissues or organs derived from the foregut/midgut region of the gut
tube (PDX1-positive foregut/midgut endoderm). Some preferred
embodiments relate to processes for the production of PDX1-positive
foregut endoderm cells. In some embodiments, these PDX1-positive
foregut endoderm cells are multipotent cells that can differentiate
into cells, tissues or organs derived from the anterior portion of
the gut tube (PDX1-positive foregut endoderm). Additional preferred
embodiments relate to processes for the production of PDX1-positive
endoderm cells of the posterior portion of the foregut. In some
embodiments, these PDX1-positive endoderm cells are multipotent
cells that can differentiate into cells, tissues or organs derived
from the posterior portion of the foregut region of the gut
tube.
[0178] The PDX1-positive foregut endoderm cells, such as those
produced according to the methods described herein, can be used to
produce fully differentiated insulin-producing .beta.-cells. In
some embodiments, PDX1-positive foregut endoderm cells are produced
by differentiating definitive endoderm cells that do not
substantially express PDX1 (PDX1-negative definitive endoderm
cells; also referred to herein as definitive endoderm) so as to
form PDX1-positive foregut endoderm cells. PDX1-negative definitive
endoderm cells can be prepared by differentiating pluripotent
cells, such as embryonic stem cells, as described herein or by any
other known methods. A convenient and highly efficient method for
producing PDX1-negative definitive endoderm from pluripotent cells
is described previously herein and in U.S. patent Ser. No.
11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, the
disclosure of which is incorporated herein by reference in its
entirety.
[0179] Processes of producing PDX1-positive foregut endoderm cells
provide a basis for efficient production of pancreatic tissues such
as acinar cells, ductal cells and islet cells from pluripotent
cells. In certain preferred embodiments, human PDX1-positive
foregut endoderm cells are derived from human PDX1-negative
definitive endoderm cells, which in turn, are derived from hESCs.
These human PDX1-positive foregut endoderm cells can then be used
to produce functional insulin-producing .beta.-cells. To obtain
useful quantities of insulin-producing .beta.-cells, high
efficiency of differentiation is desirable for each of the
differentiation steps that occur prior to reaching the pancreatic
islet/.beta.-cell fate. Because differentiation of PDX1-negative
definitive endoderm cells to PDX1-positive foregut endoderm cells
represents an early step towards the production of functional
pancreatic islet/.beta.-cells (as shown in FIG. 1), high efficiency
of differentiation at this step is particularly desirable.
[0180] In view of the desirability of efficient differentiation of
PDX1-negative definitive endoderm cells to PDX1-positive foregut
endoderm cells, some aspects of the processes described herein
relate to in vitro methodology that results in approximately 2-25%
conversion of PDX1-negative definitive endoderm cells to
PDX1-positive foregut endoderm cells. Typically, such methods
encompass the application of culture and growth factor conditions
in a defined and temporally specified fashion. Further enrichment
of the cell population for PDX1-positive foregut endoderm cells can
be achieved by isolation and/or purification of the PDX1-positive
foregut endoderm cells from other cells in the population by using
a reagent that specifically binds to the PDX1-positive foregut
endoderm cells. As an alternative, PDX1-positive foregut endoderm
cells can be labeled with a reporter gene, such as green
fluorescent protein (GFP), so as to enable the detection of PDX1
expression. Such fluorescently labeled cells can then be purified
by fluorescent activated cell sorting (FACS). Further aspects of
the present invention relate to cell cultures and enriched cell
populations comprising PDX1-positive foregut endoderm cells as well
as methods for identifying factors useful in the differentiation to
and from PDX1-positive foregut endoderm.
[0181] In order to determine the amount of PDX1-positive foregut
endoderm cells in a cell culture or cell population, a method of
distinguishing this cell type from the other cells in the culture
or in the population is desirable. Accordingly, certain embodiments
described herein relate to cell markers whose presence, absence
and/or relative expression levels are indicative of PDX1-positive
foregut endoderm cells as well as methods for detecting and
determining the expression of such markers.
[0182] In some embodiments described herein, the presence, absence
and/or level of expression of a marker is determined by
quantitative PCR (Q-PCR). For example, the amount of transcript
produced by certain genetic markers, such as PDX1, SOX17, SOX7,
SOX1, ZIC1, NFM, alpha-fetoprotein (AFP), homeobox A13 (HOXA13),
homeobox C6 (HOXC6), and/or other markers described herein is
determined by Q-PCR. In other embodiments, immunohistochemistry is
used to detect the proteins expressed by the above-mentioned genes.
In still other embodiments, Q-PCR and immunohistochemical
techniques are both used to identify and determine the amount or
relative proportions of such markers.
[0183] By using the differentiation and detection methods described
herein, it is possible to identify PDX1-positive foregut endoderm
cells, as well as determine the proportion of PDX1-positive foregut
endoderm cells in a cell culture or cell population. For example,
in some embodiments, the PDX1-positive foregut endoderm cells or
cell populations that are produced express the PDX1 gene at a level
of at least about 2 orders of magnitude greater than PDX1-negative
cells or cell populations. In other embodiments, the PDX1-positive
foregut endoderm cells and cell populations that are produced
express the PDX1 gene at a level of more than 2 orders of magnitude
greater than PDX1-negative cells or cell populations. In still
other embodiments, the PDX1-positive foregut endoderm cells or cell
populations that are produced express one or more of the markers
selected from the group consisting of PDX1, SOX17, HOXA13 and HOXC6
at a level of about 2 or more than 2 orders of magnitude greater
than PDX1-negative definitive endoderm cells or cell
populations.
[0184] The compositions and methods described herein have several
useful features. For example, the cell cultures and cell
populations comprising PDX1-positive endoderm, as well as the
methods for producing such cell cultures and cell populations, are
useful for modeling the early stages of human development.
Furthermore, the compositions and methods described herein can also
serve for therapeutic intervention in disease states, such as
diabetes mellitus. For example, since PDX1-positive foregut
endoderm serves as the source for only a limited number of tissues,
it can be used in the development of pure tissue or cell types.
Production of PDX1-Positive Foregut Endoderm from PDX1-Negative
Definitive Endoderm
[0185] The PDX1-positive foregut endoderm cell cultures and
populations comprising PDX1-positive foregut endoderm cells that
are described herein are produced from PDX1-negative definitive
endoderm, which is generated from pluripotent cells as described
above. A preferred method utilizes human embryonic stem cells as
the starting material. In one embodiment, hESCs are first converted
to PDX1-negative definitive endoderm cells, which are then
converted to PDX1-positive foregut endoderm cells. It will be
appreciated, however, that the starting materials for the
production of PDX1-positive foregut endoderm is not limited to
definitive endoderm cells produced using pluripotent cell
differentiation methods. Rather, any PDX1-negative definitive
endoderm cells can be used in the methods described herein
regardless of their origin.
[0186] In some embodiments described herein, cell cultures or cell
populations comprising PDX1-negative definitive endoderm cells can
be used for further differentiation to cell cultures and/or
enriched cell populations comprising PDX1-positive foregut endoderm
cells. For example, a cell culture or cell population comprising
human PDX1-negative, SOX17-positive definitive endoderm cells can
be used. In some embodiments, the cell culture or cell population
may also comprise differentiation factors, such as activins, nodals
and/or BMPs, remaining from the previous differentiation step (that
is, the step of differentiating pluripotent cells to definitive
endoderm cells). In other embodiments, factors utilized in the
previous differentiation step are removed from the cell culture or
cell population prior to the addition of factors used for the
differentiation of the PDX1-negative, SOX17-positive definitive
endoderm cells to PDX1-positive foregut endoderm cells. In other
embodiments, cell populations enriched for PDX1-negative,
SOX17-positive definitive endoderm cells are used as a source for
the production of PDX1-positive foregut endoderm cells.
[0187] PDX1-negative definitive endoderm cells in culture are
differentiated to PDX1-positive endoderm cells by providing to a
cell culture comprising PDX1-negative, SOX17-positive definitive
endoderm cells a differentiation factor that promotes
differentiation of the cells to PDX1-positive foregut endoderm
cells (foregut differentiation factor). In some embodiments of the
present invention, the foregut differentiation factor is retinoid,
such as retinoic acid (RA). In some embodiments, the retinoid is
used in conjunction with a fibroblast growth factor, such as FGF-4
or FGF-10. In other embodiments, the retinoid is used in
conjunction with a member of the TGF.beta. superfamily of growth
factors and/or a conditioned medium.
[0188] As defined above, the phrase "conditioned medium" refers to
a medium that is altered as compared to a base medium. For example,
the conditioning of a medium may cause molecules, such as nutrients
and/or growth factors, to be added to or depleted from the original
levels found in the base medium. In some embodiments, a medium is
conditioned by allowing cells of certain types to be grown or
maintained in the medium under certain conditions for a certain
period of time. For example, a medium can be conditioned by
allowing hESCs to be expanded, differentiated or maintained in a
medium of defined composition at a defined temperature for a
defined number of hours. As will be appreciated by those of skill
in the art, numerous combinations of cells, media types, durations
and environmental conditions can be used to produce nearly an
infinite array of conditioned media. In some embodiments of the
present invention, a medium is conditioned by allowing
differentiated pluripotent cells to be grown or maintained in a
medium comprising about 1% to about 20% serum concentration. In
other embodiments, a medium is conditioned by allowing
differentiated pluripotent cells to be grown or maintained in a
medium comprising about 1 ng/ml to about 1000 ng/ml activin A. In
still other embodiments, a medium is conditioned allowing
differentiated pluripotent cells to be grown or maintained in a
medium comprising about 1 ng/ml to about 1000 ng/ml BMP4. In a
preferred embodiment, a conditioned medium is prepared by allowing
differentiated hESCs to be grown or maintained for 24 hours in a
medium, such as RPMI, comprising about 25 ng/ml activin A and about
2 .mu.M RA.
[0189] In some embodiments described herein, the cells used to
condition the medium, which is used to enhance the differentiation
of PDX1-negative definitive endoderm to PDX1-positive foregut
endoderm, are cells that are differentiated from pluripotent cells,
such as hESCs, over about a 5 day time period in a medium such as
RPMI comprising about 0% to about 20% serum and/or one or more
growth/differentiation factors of the TGF.beta. superfamily
Differentiation factors, such as activin A and BMP4 are supplied at
concentrations ranging from about 1 ng/ml to about 1000 ng/ml. In
certain embodiments of the present invention, the cells used to
condition the medium are differentiated from hESCs over about a 5
day period in low serum RPMI. According to some embodiments, low
serum RPMI refers to a low serum containing medium, wherein the
serum concentration is gradually increased over a defined time
period. For example, in one embodiment, low serum RPMI comprises a
concentration of about 0.2% fetal bovine serum (FBS) on the first
day of cell growth, about 0.5% FBS on the second day of cell growth
and about 2% FBS on the third through fifth day of cell growth. In
another embodiment, low serum RPMI comprises a concentration of
about 0% on day one, about 0.2% on day two and about 2% on days
3-6. In certain preferred embodiments, low serum RPMI is
supplemented with one or more differentiation factors, such as
activin A and BMP4. In addition to its use in preparing cells used
to condition media, low serum RPMI can be used as a medium for the
differentiation of PDX1-positive foregut endoderm cells from
PDX1-negative definitive endoderm cells.
[0190] It will be appreciated by those of ordinary skill in the art
that conditioned media can be prepared from media other than RPMI
provided that such media do not interfere with the growth or
maintenance of PDX1-positive foregut endoderm cells. It will also
be appreciated that the cells used to condition the medium can be
of various types. In embodiments where freshly differentiated cells
are used to condition a medium, such cells can be differentiated in
a medium other than RPMI provided that the medium does not inhibit
the growth or maintenance of such cells. Furthermore, a skilled
artisan will appreciate that neither the duration of conditioning
nor the duration of preparation of cells used for conditioning is
required to be 24 hours or 5 days, respectively, as other time
periods will be sufficient to achieve the effects reported
herein.
[0191] In general, the use of a retinoid in combination with a
fibroblast growth factor, a member of the TGF.beta. superfamily of
growth factors, a conditioned medium or a combination of any of
these foregut differentiation factors causes greater
differentiation of PDX1-negative definitive endoderm to
PDX1-positive foregut endoderm than the use of a retinoid alone. In
a preferred embodiment, RA and FGF-10 are both provided to the
PDX1-negative definitive endoderm cell culture. In another
preferred embodiment, PDX1-negative definitive endoderm cells are
differentiated in a culture comprising a conditioned medium,
activin A, activin B and RA.
[0192] With respect to some of the embodiments of differentiation
processes described herein, the above-mentioned foregut
differentiation factors are provided to the cells so that these
factors are present in the cell culture or cell population at
concentrations sufficient to promote differentiation of at least a
portion of the PDX1-negative definitive endoderm cell culture or
cell population to PDX1-positive foregut endoderm cells. As defined
previously, when used in connection with cell cultures and/or cell
populations, the term "portion" means any non-zero amount of the
cell culture or cell population, which ranges from a single cell to
the entirety of the cell culture or cells population.
[0193] In some embodiments of processes described herein, a
retinoid is provided to the cells of a cell culture such that it is
present at a concentration of at least about 0.01 .mu.M, at least
about 0.02 .mu.M, at least about 0.04 .mu.M, at least about 0.08
.mu.M, at least about 0.1 .mu.M, at least about 0.2 .mu.M, at least
about 0.3 .mu.M, at least about 0.4 .mu.M, at least about 0.5
.mu.M, at least about 0.6 .mu.M, at least about 0.7 .mu.M, at least
about 0.8 .mu.M, at least about 0.9 .mu.M, at least about 1 .mu.M,
at least about 1.1 .mu.M, at least about 1.2 .mu.M, at least about
1.3 .mu.M, at least about 1.4 .mu.M, at least about 1.5 .mu.M, at
least about 1.6 .mu.M, at least about 1.7 .mu.M, at least about 1.8
.mu.M, at least about 1.9 .mu.M, at least about 2 .mu.M, at least
about 2.1 .mu.M, at least about 2.2 .mu.M, at least about 2.3
.mu.M, at least about 2.4 .mu.M, at least about 2.5 .mu.M, at least
about 2.6 .mu.M, at least about 2.7 .mu.M, at least about 2.8
.mu.M, at least about 2.9 .mu.M, at least about 3 .mu.M, at least
about 3.5 .mu.M, at least about 4 .mu.M, at least about 4.5 .mu.M,
at least about 5 .mu.M, at least about 10 .mu.M, at least about 20
.mu.M, at least about 30 .mu.M, at least about 40 .mu.M or at least
about 50 .mu.M. In a preferred embodiment, the retinoid is retinoic
acid.
[0194] In other embodiments of the processes described herein, one
or more differentiation factors of the fibroblast growth factor
family are present in the cell culture. For example, in some
embodiments, FGF-4 can be present in the cell culture at a
concentration of at least about 10 ng/ml, at least about 25 ng/ml,
at least about 50 ng/ml, at least about 75 ng/ml, at least about
100 ng/ml, at least about 200 ng/ml, at least about 300 ng/ml, at
least about 400 ng/ml, at least about 500 ng/ml, or at least about
1000 ng/ml. In further embodiments, FGF-10 is present in the cell
culture at a concentration of at least about 10 ng/ml, at least
about 25 ng/ml, at least about 50 ng/ml, at least about 75 ng/ml,
at least about 100 ng/ml, at least about 200 ng/ml, at least about
300 ng/ml, at least about 400 ng/ml, at least about 500 ng/ml, or
at least about 1000 ng/ml. In some embodiments, either FGF-4 or
FGF-10, but not both, is provided to the cell culture along with
RA. In a preferred embodiment, RA is present in the cell culture at
1 .mu.M and FGF-10 is present at a concentration of 50 ng/ml.
[0195] In some embodiments of the processes described herein,
growth factors of the TGF.beta. superfamily and/or a conditioned
medium are present in the cell culture. These differentiation
factors can be used in combination with RA and/or other mid-foregut
differentiation factors including, but not limited to, FGF-4 and
FGF-10. For example, in some embodiments, activin A and/or activin
B can be present in the cell culture at a concentration of at least
about 5 ng/ml, at least about 10 ng/ml, at least about 25 ng/ml, at
least about 50 ng/ml, at least about 75 ng/ml, at least about 100
ng/ml, at least about 200 ng/ml, at least about 300 ng/ml, at least
about 400 ng/ml, at least about 500 ng/ml, or at least about 1000
ng/ml. In further embodiments, a conditioned medium is present in
the cell culture at a concentration of at least about 1%, at least
about 5%, at least about 10%, at least about 20%, at least about
30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, or at
least about 100% of the total medium. In some embodiments, activin
A, activin B and a conditioned medium are provided to the cell
culture along with RA. In a preferred embodiment, PDX1-negative
definitive endoderm cells are differentiated to PDX1-positive
foregut endoderm cells in cultures comprising about 1 .mu.M RA,
about 25 ng/ml activin A and low serum RPMI medium that has been
conditioned for about 24 hours by differentiated hESCs, wherein the
differentiated hESCs have been differentiated for about 5 days in
low serum RPMI comprising about 100 ng/ml activin A. In another
preferred embodiment, activin B and/or FGF-10 are also present in
the culture at 25 ng/ml and 50 ng/ml, respectively.
[0196] In certain embodiments of the processes described herein,
the above-mentioned foregut differentiation factors are removed
from the cell culture subsequent to their addition. For example,
the foregut differentiation factors can be removed within about one
day, about two days, about three days, about four days, about five
days, about six days, about seven days, about eight days, about
nine days or about ten days after their addition.
[0197] Cultures of PDX1-positive foregut endoderm cells can be
grown in a medium containing reduced serum. Serum concentrations
can range from about 0.05% (v/v) to about 20% (v/v). In some
embodiments, PDX1-positive foregut endoderm cells are grown with
serum replacement. For example, in certain embodiments, the serum
concentration of the medium can be less than about 0.05% (v/v),
less than about 0.1% (v/v), less than about 0.2% (v/v), less than
about 0.3% (v/v), less than about 0.4% (v/v), less than about 0.5%
(v/v), less than about 0.6% (v/v), less than about 0.7% (v/v), less
than about 0.8% (v/v), less than about 0.9% (v/v), less than about
1% (v/v), less than about 2% (v/v), less than about 3% (v/v), less
than about 4% (v/v), less than about 5% (v/v), less than about 6%
(v/v), less than about 7% (v/v), less than about 8% (v/v), less
than about 9% (v/v), less than about 10% (v/v), less than about 15%
(v/v) or less than about 20% (v/v). In some embodiments,
PDX1-positive foregut endoderm cells are grown without serum. In
other embodiments, PDX1-positive foregut endoderm cells are grown
with serum replacement.
[0198] In still other embodiments, PDX1-positive foregut endoderm
cells are grown in the presence of B27. In such embodiments, B27
can be provided to the culture medium in concentrations ranging
from about 0.1% (v/v) to about 20% (v/v) or in concentrations
greater than about 20% (v/v). In certain embodiments, the
concentration of B27 in the medium is about 0.1% (v/v), about 0.2%
(v/v), about 0.3% (v/v), about 0.4% (v/v), about 0.5% (v/v), about
0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about 0.9% (v/v),
about 1% (v/v), about 2% (v/v), about 3% (v/v), about 4% (v/v),
about 5% (v/v), about 6% (v/v), about 7% (v/v), about 8% (v/v),
about 9% (v/v), about 10% (v/v), about 15% (v/v) or about 20%
(v/v). Alternatively, the concentration of the added B27 supplement
can be measured in terms of multiples of the strength of a
commercially available B27 stock solution. For example, B27 is
available from Invitrogen (Carlsbad, Calif.) as a SOX stock
solution. Addition of a sufficient amount of this stock solution to
a sufficient volume of growth medium produces a medium supplemented
with the desired amount of B27. For example, the addition of 10 ml
of SOX B27 stock solution to 90 ml of growth medium would produce a
growth medium supplemented with 5.times.B27. The concentration of
B27 supplement in the medium can be about 0.1.times., about
0.2.times., about 0.3.times., about 0.4.times., about 0.5.times.,
about 0.6.times., about 0.7.times., about 0.8.times., about
0.9.times., about 1.times., about 1.1.times., about 1.2.times.,
about 1.3.times., about 1.4.times., about 1.5.times., about
1.6.times., about 1.7.times., about 1.8.times., about 1.9.times.,
about 2.times., about 2.5.times., about 3.times., about 3.5.times.,
about 4.times., about 4.5.times., about 5.times., about 6.times.,
about 7.times., about 8.times., about 9.times., about 10.times.,
about 11.times., about 12.times., about 13.times., about 14.times.,
about 15.times., about 16.times., about 17.times., about 18.times.,
about 19.times., about 20.times. and greater than about
20.times..
Monitoring the Differentiation of PDX1-Negative Definitive Endoderm
to PDX1-Positive Endoderm
[0199] As with the differentiation of definitive endoderm cells
from pluripotent cells, the progression of differentiation from
PDX1-negative, SOX17-positive definitive endoderm to PDX1-positive
foregut endoderm can be monitored by determining the expression of
markers characteristic of these cell types. Such monitoring permits
one to determine the amount of time that is sufficient for the
production of a desired amount of PDX1-positive foregut endoderm
under various conditions, for example, one or more differentiation
factor concentrations and environmental conditions. In preferred
embodiments, the amount of time that is sufficient for the
production of a desired amount of PDX1-positive foregut endoderm is
determined by detecting the expression of PDX1. In some
embodiments, the expression of certain markers is determined by
detecting the presence or absence of the marker. Alternatively, the
expression of certain markers can be determined by measuring the
level at which the marker is present in the cells of the cell
culture or cell population. In such embodiments, the measurement of
marker expression can be qualitative or quantitative. As described
above, a preferred method of quantitating the expression markers
that are produced by marker genes is through the use of Q-PCR. In
particular embodiments, Q-PCR is used to monitor the progression of
cells of the PDX1-negative, SOX17-positive definitive endoderm
culture to PDX1-positive foregut endoderm cells by quantitating
expression of marker genes characteristic of PDX1-positive foregut
endoderm and the lack of expression of marker genes characteristic
of other cell types. Other methods which are known in the art can
also be used to quantitate marker gene expression. For example, the
expression of a marker gene product can be detected by using
antibodies specific for the marker gene product of interest. In
some embodiments, the expression of marker genes characteristic of
PDX1-positive foregut endoderm as well as the lack of significant
expression of marker genes characteristic of PDX1-negative
definitive endoderm, hESCs and other cell types is determined.
[0200] As described further in the Examples below, PDX1 is a marker
gene that is associated with PDX1-positive foregut endoderm. As
such, in some embodiments of the processes described herein, the
expression of PDX1 is determined. In other embodiments, the
expression of other markers, which are expressed in PDX1-positive
foregut endoderm, including, but not limited to, SOX17, HOXA13
and/or HOXC6 is also determined Since PDX1 can also be expressed by
certain other cell types (that is, visceral endoderm and certain
neural ectoderm), some embodiments described herein relate to
demonstrating the absence or substantial absence of marker gene
expression that is associated with visceral endoderm and/or neural
ectoderm. For example, in some embodiments, the expression of
markers, which are expressed in visceral endoderm and/or neural
cells, including, but not limited to, SOX7, AFP, SOX1, ZIC1 and/or
NFM is determined.
[0201] In some embodiments, PDX1-positive foregut endoderm cell
cultures produced by the methods described herein are substantially
free of cells expressing the SOX7, AFP, SOX1, ZIC1 or NFM marker
genes. In certain embodiments, the PDX1-positive foregut endoderm
cell cultures produced by the processes described herein are
substantially free of visceral endoderm, parietal endoderm and/or
neural cells.
Enrichment, Isolation and/or Purification of PDX1-Positive Foregut
Endoderm
[0202] With respect to additional aspects of the processes
described herein, PDX1-positive foregut endoderm cells can be
enriched, isolated and/or purified. In some embodiments, cell
populations enriched for PDX1-positive foregut endoderm cells are
produced by isolating such cells from cell cultures.
[0203] In some embodiments of the processes described herein,
PDX1-positive foregut endoderm cells are fluorescently labeled then
isolated from non-labeled cells by using a fluorescence activated
cell sorter (FACS). In such embodiments, a nucleic acid encoding
green fluorescent protein (GFP) or another nucleic acid encoding an
expressible fluorescent marker gene is used to label PDX1-positive
cells. For example, in some embodiments, at least one copy of a
nucleic acid encoding GFP or a biologically active fragment thereof
is introduced into a pluripotent cell, preferably a human embryonic
stem cell, downstream of the PDX1 promoter such that the expression
of the GFP gene product or biologically active fragment thereof is
under control of the PDX1 promoter. In some embodiments, the entire
coding region of the nucleic acid, which encodes PDX1, is replaced
by a nucleic acid encoding GFP or a biologically active fragment
thereof. In other embodiments, the nucleic acid encoding GFP or a
biologically active fragment thereof is fused in frame with at
least a portion of the nucleic acid encoding PDX1, thereby
generating a fusion protein. In such embodiments, the fusion
protein retains a fluorescent activity similar to GFP.
[0204] Fluorescently marked cells, such as the above-described
pluripotent cells, are differentiated to definitive endoderm and
then to PDX1-positive foregut endoderm as described previously
above. Because PDX1-positive foregut endoderm cells express the
fluorescent marker gene, whereas PDX1-negative cells do not, these
two cell types can be separated. In some embodiments, cell
suspensions comprising a mixture of fluorescently-labeled
PDX1-positive cells and unlabeled PDX1-negative cells are sorted
using a FACS. PDX1-positive cells are collected separately from
PDX1-negative cells, thereby resulting in the isolation of such
cell types. If desired, the isolated cell compositions can be
further purified by additional rounds of sorting using the same or
different markers that are specific for PDX1-positive foregut
endoderm.
[0205] In addition to the procedures just described, PDX1-positive
foregut endoderm cells may also be isolated by other techniques for
cell isolation. Additionally, PDX1-positive foregut endoderm cells
may also be enriched or isolated by methods of serial subculture in
growth conditions which promote the selective survival or selective
expansion of said PDX1-positive foregut endoderm cells.
[0206] It will be appreciated that the above-described enrichment,
isolation and purification procedures can be used with such
cultures at any stage of differentiation.
[0207] Using the methods described herein, enriched, isolated
and/or purified populations of PDX1-positive foregut endoderm cells
and/or tissues can be produced in vitro from PDX1-negative,
SOX17-positive definitive endoderm cell cultures or cell
populations which have undergone at least some differentiation. In
some embodiments, the cells undergo random differentiation. In a
preferred embodiment, however, the cells are directed to
differentiate primarily into PDX1-positive foregut endoderm cells.
Some preferred enrichment, isolation and/or purification methods
relate to the in vitro production of PDX1-positive foregut endoderm
cells from human embryonic stem cells.
[0208] Using the methods described herein, cell populations or cell
cultures can be enriched in PDX1-positive foregut endoderm cell
content by at least about 2- to about 1000-fold as compared to
untreated cell populations or cell cultures. In some embodiments,
PDX1-positive foregut endoderm cells can be enriched by at least
about 5- to about 500-fold as compared to untreated cell
populations or cell cultures. In other embodiments, PDX1-positive
foregut endoderm cells can be enriched from at least about 10- to
about 200-fold as compared to untreated cell populations or cell
cultures. In still other embodiments, PDX1-positive foregut
endoderm cells can be enriched from at least about 20- to about
100-fold as compared to untreated cell populations or cell
cultures. In yet other embodiments, PDX1-positive foregut endoderm
cells can be enriched from at least about 40- to about 80-fold as
compared to untreated cell populations or cell cultures. In certain
embodiments, PDX1-positive foregut endoderm cells can be enriched
from at least about 2- to about 20-fold as compared to untreated
cell populations or cell cultures.
Compositions Comprising PDX1-Positive Foregut Endoderm
[0209] Some embodiments described herein relate to cell
compositions, such as cell cultures or cell populations, comprising
PDX1-positive endoderm cells, wherein the PDX1-positive endoderm
cells are multipotent cells that can differentiate into cells,
tissues or organs derived from the anterior portion of the gut tube
(PDX1-positive foregut endoderm). In accordance with certain
embodiments, the PDX1-positive foregut endoderm are mammalian
cells, and in a preferred embodiment, these cells are human
cells.
[0210] Other embodiments described herein relate to compositions,
such as cell cultures or cell populations, comprising cells of one
or more cell types selected from the group consisting of hESCs,
PDX1-negative definitive endoderm cells, PDX1-positive foregut
endoderm cells and mesoderm cells. In some embodiments, hESCs
comprise less than about 5%, less than about 4%, less than about
3%, less than about 2% or less than about 1% of the total cells in
the culture. In other embodiments, PDX1-negative definitive
endoderm cells comprise less than about 90%, less than about 85%,
less than about 80%, less than about 75%, less than about 70%, less
than about 65%, less than about 60%, less than about 55%, less than
about 50%, less than about 45%, less than about 40%, less than
about 35%, less than about 30%, less than about 25%, less than
about 20%, less than about 15%, less than about 12%, less than
about 10%, less than about 8%, less than about 6%, less than about
5%, less than about 4%, less than about 3%, less than about 2% or
less than about 1% of the total cells in the culture. In yet other
embodiments, mesoderm cells comprise less than about 90%, less than
about 85%, less than about 80%, less than about 75%, less than
about 70%, less than about 65%, less than about 60%, less than
about 55%, less than about 50%, less than about 45%, less than
about 40%, less than about 35%, less than about 30%, less than
about 25%, less than about 20%, less than about 15%, less than
about 12%, less than about 10%, less than about 8%, less than about
6%, less than about 5%, less than about 4%, less than about 3%,
less than about 2% or less than about 1% of the total cells in the
culture.
[0211] Additional embodiments described herein relate to
compositions, such as cell cultures or cell populations, produced
by the processes described herein, which comprise PDX1-positive
foregut endoderm as the majority cell type. In some embodiments,
the processes described herein produce cell cultures and/or cell
populations comprising at least about 99%, at least about 98%, at
least about 97%, at least about 96%, at least about 95%, at least
about 94%, at least about 93%, at least about 92%, at least about
91%, at least about 90%, at least about 85%, at least about 80%, at
least about 75%, at least about 70%, at least about 65%, at least
about 60%, at least about 55%, at least about 54%, at least about
53%, at least about 52% or at least about 51% PDX1-positive foregut
endoderm cells. In preferred embodiments the cells of the cell
cultures or cell populations comprise human cells. In other
embodiments, the processes described herein produce cell cultures
or cell populations comprising at least about 50%, at least about
45%, at least about 40%, at least about 35%, at least about 30%, at
least about 25%, at least about 24%, at least about 23%, at least
about 22%, at least about 21%, at least about 20%, at least about
19%, at least about 18%, at least about 17%, at least about 16%, at
least about 15%, at least about 14%, at least about 13%, at least
about 12%, at least about 11%, at least about 10%, at least about
9%, at least about 8%, at least about 7%, at least about 6%, at
least about 5%, at least about 4%, at least about 3%, at least
about 2% or at least about 1% PDX1-positive foregut endoderm cells.
In preferred embodiments, the cells of the cell cultures or cell
populations comprise human cells. In some embodiments, the
percentage of PDX1-positive foregut endoderm cells in the cell
cultures or populations is calculated without regard to the feeder
cells remaining in the culture.
[0212] Still other embodiments described herein relate to
compositions, such as cell cultures or cell populations, comprising
mixtures of PDX1-positive foregut endoderm cells and PDX1-negative
definitive endoderm cells. For example, cell cultures or cell
populations comprising at least about 5 PDX1-positive foregut
endoderm cells for about every 95 PDX1-negative definitive endoderm
cells can be produced. In other embodiments, cell cultures or cell
populations comprising at least about 95 PDX1-positive foregut
endoderm cells for about every 5 PDX1-negative definitive endoderm
cells can be produced. Additionally, cell cultures or cell
populations comprising other ratios of PDX1-positive foregut
endoderm cells to PDX1-negative definitive endoderm cells are
contemplated. For example, compositions comprising at least about 1
PDX1-positive foregut endoderm cell for about every 1,000,000
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 100,000
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 10,000
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 1000
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 500
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 100
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 10
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 5 PDX1-negative
definitive endoderm cells, at least about 1 PDX1-positive foregut
endoderm cell for about every 4 PDX1-negative definitive endoderm
cells, at least about 1 PDX1-positive foregut endoderm cell for
about every 2 PDX1-negative definitive endoderm cells, at least
about 1 PDX-1 positive foregut endoderm cell for about every 1
PDX1-negative definitive endoderm cell, at least about 2
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 4
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 5
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 10
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 20
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 50
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 100
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 1000
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 10,000
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 100,000
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell and at least about 1,000,000
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell are contemplated.
[0213] In some embodiments described herein, the PDX1-negative
definitive endoderm cells from which PDX1-positive foregut endoderm
cells are produced are derived from human pluripotent cells, such
as human pluripotent stem cells. In certain embodiments, the human
pluripotent cells are derived from a morula, the inner cell mass of
an embryo or the gonadal ridges of an embryo. In certain other
embodiments, the human pluripotent cells are derived from the
gonadal or germ tissues of a multicellular structure that has
developed past the embryonic stage.
[0214] Further embodiments described herein relate to compositions,
such as cell cultures or cell populations, comprising human cells,
including human PDX1-positive foregut endoderm, wherein the
expression of the PDX1 marker is greater than the expression of the
AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at least about 2% of the
human cells. In other embodiments, the expression of the PDX1
marker is greater than the expression of the AFP, SOX7, SOX1, ZIC1
and/or NFM marker in at least about 5% of the human cells, in at
least about 10% of the human cells, in at least about 15% of the
human cells, in at least about 20% of the human cells, in at least
about 25% of the human cells, in at least about 30% of the human
cells, in at least about 35% of the human cells, in at least about
40% of the human cells, in at least about 45% of the human cells,
in at least about 50% of the human cells, in at least about 55% of
the human cells, in at least about 60% of the human cells, in at
least about 65% of the human cells, in at least about 70% of the
human cells, in at least about 75% of the human cells, in at least
about 80% of the human cells, in at least about 85% of the human
cells, in at least about 90% of the human cells, in at least about
95% of the human cells or in at least about 98% of the human cells.
In some embodiments, the percentage of human cells in the cell
cultures or populations, wherein the expression of PDX1 is greater
than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker,
is calculated without regard to feeder cells.
[0215] It will be appreciated that some embodiments described
herein relate to compositions, such as cell cultures or cell
populations, comprising human PDX1-positive foregut endoderm cells,
wherein the expression of one or more markers selected from the
group consisting of SOX17, HOXA13 and HOXC6 is greater than the
expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in from
at least about 2% to greater than at least about 98% of the human
cells. In some embodiments, the expression of one or more markers
selected from the group consisting of SOX17, HOXA13 and HOXC6 is
greater than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM
marker in at least about 5% of the human cells, in at least about
10% of the human cells, in at least about 15% of the human cells,
in at least about 20% of the human cells, in at least about 25% of
the human cells, in at least about 30% of the human cells, in at
least about 35% of the human cells, in at least about 40% of the
human cells, in at least about 45% of the human cells, in at least
about 50% of the human cells, in at least about 55% of the human
cells, in at least about 60% of the human cells, in at least about
65% of the human cells, in at least about 70% of the human cells,
in at least about 75% of the human cells, in at least about 80% of
the human cells, in at least about 85% of the human cells, in at
least about 90% of the human cells, in at least about 95% of the
human cells or in at least about 98% of the human cells. In some
embodiments, the percentage of human cells in the cell cultures or
populations, wherein the expression of one or more markers selected
from the group consisting of SOX17, HOXA13 and HOXC6 is greater
than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker,
is calculated without regard to feeder cells.
[0216] Additional embodiments described herein relate to
compositions, such as cell cultures or cell populations, comprising
mammalian endodermal cells, such as human endoderm cells, wherein
the expression of the PDX1 marker is greater than the expression of
the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at least about 2% of
the endodermal cells. In other embodiments, the expression of the
PDX1 marker is greater than the expression of the AFP, SOX7, SOX1,
ZIC1 and/or NFM marker in at least about 5% of the endodermal
cells, in at least about 10% of the endodermal cells, in at least
about 15% of the endodermal cells, in at least about 20% of the
endodermal cells, in at least about 25% of the endodermal cells, in
at least about 30% of the endodermal cells, in at least about 35%
of the endodermal cells, in at least about 40% of the endodermal
cells, in at least about 45% of the endodermal cells, in at least
about 50% of the endodermal cells, in at least about 55% of the
endodermal cells, in at least about 60% of the endodermal cells, in
at least about 65% of the endodermal cells, in at least about 70%
of the endodermal cells, in at least about 75% of the endodermal
cells, in at least about 80% of the endodermal cells, in at least
about 85% of the endodermal cells, in at least about 90% of the
endodermal cells, in at least about 95% of the endodermal cells or
in at least about 98% of the endodermal cells.
[0217] Still other embodiments described herein relate to
compositions, such as cell cultures or cell populations, comprising
mammalian endodermal cells, such as human endodermal cells, wherein
the expression of one or more markers selected from the group
consisting of SOX17, HOXA13 and HOXC6 is greater than the
expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at
least about 2% of the endodermal cells. In other embodiments, the
expression of one or more markers selected from the group
consisting of SOX17, HOXA13 and HOXC6 is greater than the
expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at
least about 5% of the endodermal cells, in at least about 10% of
the endodermal cells, in at least about 15% of the endodermal
cells, in at least about 20% of the endodermal cells, in at least
about 25% of the endodermal cells, in at least about 30% of the
endodermal cells, in at least about 35% of the endodermal cells, in
at least about 40% of the endodermal cells, in at least about 45%
of the endodermal cells, in at least about 50% of the endodermal
cells, in at least about 55% of the endodermal cells, in at least
about 60% of the endodermal cells, in at least about 65% of the
endodermal cells, in at least about 70% of the endodermal cells, in
at least about 75% of the endodermal cells, in at least about 80%
of the endodermal cells, in at least about 85% of the endodermal
cells, in at least about 90% of the endodermal cells, in at least
about 95% of the endodermal cells or at least about 98% of the
endodermal cells.
[0218] Using the processes described herein, compositions
comprising PDX1-positive foregut endoderm cells substantially free
of other cell types can be produced. In some embodiments of the
present invention, the PDX1-positive foregut endoderm cell
populations or cell cultures produced by the methods described
herein are substantially free of cells that significantly express
the AFP, SOX7, SOX1, ZIC1 and/or NFM marker genes.
[0219] In one embodiment, a description of a PDX1-positive foregut
endoderm cell based on the expression of marker genes is, PDX1
high, AFP low, SOX7 low, SOX1 low, ZIC1 low and NFM low.
Increasing Expression of PDX1 in a SOX17-Positive Definitive
Endoderm Cell
[0220] Some aspects of the processes described herein are related
to methods of increasing the expression of the PDX1 gene product in
cell cultures or cell populations comprising SOX17-positive
definitive endoderm cells. In such embodiments, the SOX17-positive
definitive endoderm cells are contacted with a differentiation
factor in an amount that is sufficient to increase the expression
of the PDX1 gene product. The SOX17-positive definitive endoderm
cells that are contacted with the differentiation factor can be
either PDX1-negative or PDX1-positive. In some embodiments, the
differentiation factor can be a retinoid. In certain embodiments,
SOX17-positive definitive endoderm cells are contacted with a
retinoid at a concentration ranging from about 0.01 .mu.M to about
50 .mu.M. In a preferred embodiment, the retinoid is RA.
[0221] In other embodiments of the processes described herein, the
expression of the PDX1 gene product in cell cultures or cell
populations comprising SOX17-positive definitive endoderm cells is
increased by contacting the SOX17-positive cells with a
differentiation factor of the fibroblast growth factor family Such
differentiation factors can either be used alone or in conjunction
with RA. In some embodiments, the SOX17-positive definitive
endoderm cells are contacted with a fibroblast growth factor at a
concentration ranging from about 10 ng/ml to about 1000 ng/ml. In a
preferred embodiment, the FGF growth factor is FGF-10.
[0222] In some embodiments of the processes described herein, the
expression of the PDX1 gene product in cell cultures or cell
populations comprising SOX17-positive definitive endoderm cells is
increased by contacting the SOX17-positive cells with B27. This
differentiation factor can either be used alone or in conjunction
with one or both of retinoid and FGF family differentiation
factors. In some embodiments, the SOX17-positive definitive
endoderm cells are contacted with B27 at a concentration ranging
from about 0.1% (v/v) to about 20% (v/v). In a preferred
embodiment, the SOX17-positive definitive endoderm cells are
contacted with RA, FGF-10 and B27.
[0223] Methods for increasing the expression of the PDX1 gene
product in cell cultures or cell populations comprising
SOX17-positive definitive endoderm cells can be carried out in
growth medium containing reduced or no serum. In some embodiments,
serum concentrations range from about 0.05% (v/v) to about 20%
(v/v). In some embodiments, the SOX17-positive cells are grown with
serum replacement.
Identification of Factors Capable of Promoting the Differentiation
of Definitive Endoderm Cells
[0224] Certain screening methods described herein relate to methods
for identifying at least one differentiation factor that is capable
of promoting the differentiation of definitive endoderm cells. In
some embodiments of these methods, cell populations comprising
definitive endoderm cells, such as human definitive endoderm cells,
are obtained. The cell population is then provided with a candidate
differentiation factor. At a first time point, which is prior to or
at approximately the same time as providing the candidate
differentiation factor, expression of a marker is determined.
Alternatively, expression of the marker can be determined after
providing the candidate differentiation factor. At a second time
point, which is subsequent to the first time point and subsequent
to the step of providing the candidate differentiation factor to
the cell population, expression of the same marker is again
determined. Whether the candidate differentiation factor is capable
of promoting the differentiation of the definitive endoderm cells
is determined by comparing expression of the marker at the first
time point with the expression of the marker at the second time
point. If expression of the marker at the second time point is
increased or decreased as compared to expression of the marker at
the first time point, then the candidate differentiation factor is
capable of promoting the differentiation of definitive endoderm
cells.
[0225] Some embodiments of the screening methods described herein
utilize cell populations or cell cultures which comprise human
definitive endoderm cells. For example, the cell population can be
a substantially purified population of human definitive endoderm
cells. Alternatively, the cell population can be an enriched
population of human definitive endoderm cells, wherein at least
about 90%, at least about 91%, at least about 92%, at least about
93%, at least about 94%, at least about 95%, at least about 96%, at
least about 97% or greater than at least about 97% of the human
cells in the cell population are human definitive endoderm cells.
In other embodiments described herein, the cell population
comprises human cells wherein at least about 10%, at least about
15%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85% or greater than at least about 85% of the human
cells are human definitive endoderm cells. In some embodiments, the
cell population includes non-human cells such as non-human feeder
cells. In other embodiments, the cell population includes human
feeder cells. In such embodiments, at least about 10%, at least
about 15%, at least about 20%, at least about 25%, at least about
30%, at least about 35%, at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95% or
greater than at least about 95% of the human cells, other than said
feeder cells, are human definitive endoderm cells. In some
embodiments of the screening methods described herein, the cell
populations further comprise PDX1-positive endoderm cells
including, but not limited to, PDX1-positive foregut endoderm
cells.
[0226] In embodiments of the screening methods described herein,
the cell population is contacted or otherwise provided with a
candidate (test) differentiation factor. The candidate
differentiation factor can comprise any molecule that may have the
potential to promote the differentiation of human definitive
endoderm cells. In some embodiments described herein, the candidate
differentiation factor comprises a molecule that is known to be a
differentiation factor for one or more types of cells. In alternate
embodiments, the candidate differentiation factor comprises a
molecule that in not known to promote cell differentiation. In
preferred embodiments, the candidate differentiation factor
comprises molecule that is not known to promote the differentiation
of human definitive endoderm cells.
[0227] In some embodiments of the screening methods described
herein, the candidate differentiation factor comprises a small
molecule. In preferred embodiments, a small molecule is a molecule
having a molecular mass of about 10,000 amu or less. In some
embodiments, the small molecule comprises a retinoid. In some
embodiments, the small molecule comprises retinoic acid.
[0228] In other embodiments described herein, the candidate
differentiation factor comprises a polypeptide. The polypeptide can
be any polypeptide including, but not limited to, a glycoprotein, a
lipoprotein, an extracellular matrix protein, a cytokine, a
chemokine, a peptide hormone, an interleukin or a growth factor.
Preferred polypeptides include growth factors. In some preferred
embodiments, the candidate differentiation factors comprises one or
more growth factors selected from the group consisting of FGF10,
FGF4, FGF2 and Wnt3B.
[0229] In some embodiments of the screening methods described
herein, the candidate differentiation factors comprise one or more
growth factors selected from the group consisting of Amphiregulin,
B-lymphocyte stimulator, IL-16, Thymopoietin, TRAIL/Apo-2, Pre B
cell colony enhancing factor, Endothelial differentiation-related
factor 1 (EDF1), Endothelial monocyte activating polypeptide II,
Macrophage migration inhibitory factor (MIF), Natural killer cell
enhancing factor (NKEFA), Bone mophogenetic protein 2, Bone
mophogenetic protein 8 (osteogeneic protein 2), Bone morphogenic
protein 6, Bone morphogenic protein 7, Connective tissue growth
factor (CTGF), CGI-149 protein (neuroendocrine differentiation
factor), Cytokine A3 (macrophage inflammatory protein 1-alpha),
Gliablastoma cell differentiation-related protein (GBDR1),
Hepatoma-derived growth factor, Neuromedin U-25 precursor, Vascular
endothelial growth factor (VEGF), Vascular endothelial growth
factor B (VEGF-B), T-cell specific RANTES precursor, thymic
dendritic cell-derived factor 1, Transferrin, Interleukin-1 (IL 1),
Interleukin-2 (IL 2), Interleukin-3 (IL 3), Interleukin-4 (IL 4),
Interleukin-5 (IL 5), Interleukin-6 (IL 6), Interleukin-7 (IL 7),
Interleukin-8 (IL 8), Interleukin-9 (IL 9), Interleukin-10 (IL 10),
Interleukin-11 (IL 11), Interleukin-12 (IL 12), Interleukin-13 (IL
13), Granulocyte-colony stimulating factor (G-CSF), Granulocyte
macrophage colony stimulating factor (GM-CSF), Macrophage colony
stimulating factor (M-CSF), Erythropoietin, Thrombopoietin, Vitamin
D3, Epidermal growth factor (EGF), Brain-derived neurotrophic
factor, Leukemia inhibitory factor, Thyroid hormone, Basic
fibroblast growth factor (bFGF), aFGF, FGF-4, FGF-6, Keratinocyte
growth factor (KGF), Platelet-derived growth factor (PDGF),
Platelet-derived growth factor-BB, beta nerve growth factor,
activin A, Transforming growth factor beta 1 (TGF-.beta.1),
Interferon-.alpha., Interferon-.beta., Interferon-.gamma., Tumor
necrosis factor-.alpha., Tumor necrosis factor-.beta., Burst
promoting activity (BPA), Erythroid promoting activity (EPA), PGE2,
insulin growth factor-1 (IGF-1), IGF-II, Neutrophin growth factor
(NGF), Neutrophin-3, Neutrophin 4/5, Ciliary neurotrophic factor,
Glial-derived nexin, Dexamethasone, .beta.-mercaptoethanol,
Retinoic acid, Butylated hydroxyanisole, 5-azacytidine,
Amphotericin B, Ascorbic acid, Ascrorbate, isobutylxanthine,
indomethacin, .beta.-glycerolphosphate, nicotinamide, DMSO,
Thiazolidinediones, TWS119, oxytocin, vasopressin,
melanocyte-stimulating hormone, corticortropin, lipotropin,
thyrotropin, growth hormone, prolactin, luteinizing hormone, human
chorionic gonadotropin, follicle stimulating hormone,
corticotropin-releasing factor, gonadotropin-releasing factor,
prolactin-releasing factor, prolactin-inhibiting factor,
growth-hormone releasing factor, somatostatin,
thyrotropin-releasing factor, calcitonin gene-related peptide,
parathyroid hormone, glucagon-like peptide 1, glucose-dependent
insulinotropic polypeptide, gastrin, secretin, cholecystokinin,
motilin, vasoactive intestinal peptide, substance P, pancreatic
polypeptide, peptide tyrosine tyrosine, neuropeptide tyrosine,
insulin, glucagon, placental lactogen, relaxin, angiotensin II,
calctriol, atrial natriuretic peptide, and melatonin. thyroxine,
triiodothyronine, calcitonin, estradiol, estrone, progesterone,
testosterone, cortisol, corticosterone, aldosterone, epinephrine,
norepinepherine, androstiene, calcitriol, collagen, Dexamethasone,
.beta.-mercaptoethanol, Retinoic acid, Butylated hydroxyanisole,
5-azacytidine, Amphotericin B, Ascorbic acid, Ascrorbate,
isobutylxanthine, indomethacin, .beta.-glycerolphosphate,
nicotinamide, DMSO, Thiazolidinediones, and TWS119.
[0230] In some embodiments of the screening methods described
herein, the candidate differentiation factor is provided to the
cell population in one or more concentrations. In some embodiments,
the candidate differentiation factor is provided to the cell
population so that the concentration of the candidate
differentiation factor in the medium surrounding the cells ranges
from about 0.1 ng/ml to about 10 mg/ml. In some embodiments, the
concentration of the candidate differentiation factor in the medium
surrounding the cells ranges from about 1 ng/ml to about 1 mg/ml.
In other embodiments, the concentration of the candidate
differentiation factor in the medium surrounding the cells ranges
from about 10 ng/ml to about 100 .mu.g/ml. In still other
embodiments, the concentration of the candidate differentiation
factor in the medium surrounding the cells ranges from about 100
ng/ml to about 10 .mu.g/ml. In preferred embodiments, the
concentration of the candidate differentiation factor in the medium
surrounding the cells is about 5 ng/ml, about 25 ng/ml, about 50
ng/ml, about 75 ng/ml, about 100 ng/ml, about 125 ng/ml, about 150
ng/ml, about 175 ng/ml, about 200 ng/ml, about 225 ng/ml, about 250
ng/ml, about 275 ng/ml, about 300 ng/ml, about 325 ng/ml, about 350
ng/ml, about 375 ng/ml, about 400 ng/ml, about 425 ng/ml, about 450
ng/ml, about 475 ng/ml, about 500 ng/ml, about 525 ng/ml, about 550
ng/ml, about 575 ng/ml, about 600 ng/ml, about 625 ng/ml, about 650
ng/ml, about 675 ng/ml, about 700 ng/ml, about 725 ng/ml, about 750
ng/ml, about 775 ng/ml, about 800 ng/ml, about 825 ng/ml, about 850
ng/ml, about 875 ng/ml, about 900 ng/ml, about 925 ng/ml, about 950
ng/ml, about 975 ng/ml, about 1 .mu.g/ml, about 2 .mu.g/ml, about 3
.mu.g/ml, about 4 .mu.g/ml, about 5 .mu.g/ml, about 6 .mu.g/ml,
about 7 .mu.g/ml, about 8 .mu.g/ml, about 9 .mu.g/ml, about 10
.mu.g/ml, about 11 .mu.g/ml, about 12 .mu.g/ml, about 13 .mu.g/ml,
about 14 .mu.g/ml, about 15 .mu.g/ml, about 16 .mu.g/ml, about 17
.mu.g/ml, about 18 .mu.g/ml, about 19 .mu.g/ml, about 20 .mu.g/ml,
about 25 .mu.g/ml, about 50 .mu.g/ml, about 75 .mu.g/ml, about 100
.mu.g/ml, about 125 .mu.g/ml, about 150 .mu.g/ml, about 175
.mu.g/ml, about 200 .mu.g/ml, about 250 .mu.g/ml, about 300
.mu.g/ml, about 350 .mu.g/ml, about 400 .mu.g/ml, about 450
.mu.g/ml, about 500 .mu.g/ml, about 550 .mu.g/ml, about 600
.mu.g/ml, about 650 .mu.g/ml, about 700 .mu.g/ml, about 750
.mu.g/ml, about 800 .mu.g/ml, about 850 .mu.g/ml, about 900
.mu.g/ml, about 950 .mu.g/ml, about 1000 .mu.g/ml or greater than
about 1000 .mu.g/ml.
[0231] In certain embodiments of the screening methods described
herein, the cell population is provided with a candidate
differentiation factor which comprises any molecule other than
foregut differentiation factor. For example, in some embodiments,
the cell population is provided with a candidate differentiation
factor which comprises any molecule other than a retinoid, a member
of the TGF.beta. superfamily of growth factors, FGF10 or FGF4. In
some embodiments, the cell population is provided with a candidate
differentiation factor which comprises any molecule other than
retinoic acid.
[0232] In some embodiments, steps of the screening methods
described herein comprise determining expression of at least one
marker at a first time point and a second time point. In some of
these embodiments, the first time point can be prior to or at
approximately the same time as providing the cell population with
the candidate differentiation factor. Alternatively, in some
embodiments, the first time point is subsequent to providing the
cell population with the candidate differentiation factor. In some
embodiments, expression of a plurality of markers is determined at
a first time point.
[0233] In addition to determining expression of at least one marker
at a first time point, some embodiments of the screening methods
described herein contemplate determining expression of at least one
marker at a second time point, which is subsequent to the first
time point and which is subsequent to providing the cell population
with the candidate differentiation factor. In such embodiments,
expression of the same marker is determined at both the first and
second time points. In some embodiments, expression of a plurality
of markers is determined at both the first and second time points.
In such embodiments, expression of the same plurality of markers is
determined at both the first and second time points. In some
embodiments, marker expression is determined at a plurality of time
points, each of which is subsequent to the first time point, and
each of which is subsequent to providing the cell population with
the candidate differentiation factor. In certain embodiments,
marker expression is determined by Q-PCR. In other embodiments,
marker expression is determined by immunocytochemistry.
[0234] In certain embodiments of the screening methods described
herein, the marker having its expression is determined at the first
and second time points is a marker that is associated with the
differentiation of human definitive endoderm cells to cells which
are the precursors of cells which make up tissues and/or organs
that are derived from the gut tube. In some embodiments, the
tissues and/or organs that are derived from the gut tube comprise
terminally differentiated cells. In some embodiments, the marker is
indicative of pancreatic cells or pancreatic precursor cells. In
preferred embodiments, the marker is pancreatic-duodenal homeobox
factor-1 (PDX1). In other embodiments, the marker is homeobox A13
(HOXA13) or homeobox C6 (HOXC6). Additionally, in other
embodiments, the marker is indicative of liver cells or liver
precursor cells. In certain preferred embodiments, the marker is
albumin, hepatocyte specific antigen (HSA) or prospero-related
homeobox 1 (PROX1). In other embodiments, the marker is indicative
of lung or lung precursor cells. In some preferred embodiments, the
marker is thyroid transcription factor 1 (TITF1). In yet other
embodiments, the marker is indicative of intestinal or intestinal
precursor cells. In additional preferred embodiments, the marker is
villin, glucose transporter-2 (GLUT2), apolipoprotein A1 (APOA1),
vascular cell adhesion molecule-1 (VACM1), von Willebrand factor
(VWF), CXC-type chemokine receptor 4 (CXCR4) or caudal type
homeobox transcription factor 2 (CDX2). In still other embodiments,
the marker is indicative of stomach or stomach precursor cells. In
additional preferred embodiments, the marker is VCAM1, VWF or
CXCR4. In other embodiments, the marker is indicative of thyroid or
thyroid precursor cells. In such embodiments, the marker is TITF1.
In still other embodiments, the marker is indicative of thymus or
thymus precursor cells.
[0235] In some embodiments of the screening methods described
herein, sufficient time is allowed to pass between providing the
cell population with the candidate differentiation factor and
determining marker expression at the second time point. Sufficient
time between providing the cell population with the candidate
differentiation factor and determining expression of the marker at
the second time point can be as little as from about 1 hour to as
much as about 10 days. In some embodiments, the expression of at
least one marker is determined multiple times subsequent to
providing the cell population with the candidate differentiation
factor. In some embodiments, sufficient time is at least about 1
hour, at least about 6 hours, at least about 12 hours, at least
about 18 hours, at least about 24 hours, at least about 30 hours,
at least about 36 hours, at least about 42 hours, at least about 48
hours, at least about 54 hours, at least about 60 hours, at least
about 66 hours, at least about 72 hours, at least about 78 hours,
at least about 84 hours, at least about 90 hours, at least about 96
hours, at least about 102 hours, at least about 108 hours, at least
about 114 hours, at least about 120 hours, at least about 126
hours, at least about 132 hours, at least about 138 hours, at least
about 144 hours, at least about 150 hours, at least about 156
hours, at least about 162 hours, at least about 168 hours, at least
about 174 hours, at least about 180 hours, at least about 186
hours, at least about 192 hours, at least about 198 hours, at least
about 204 hours, at least about 210 hours, at least about 216
hours, at least about 222 hours, at least about 228 hours, at least
about 234 hours or at least about 240 hours.
[0236] In some embodiments of the methods described herein, it is
further determined whether the expression of the marker at the
second time point has increased or decreased as compared to the
expression of this marker at the first time point. An increase or
decrease in the expression of the at least one marker indicates
that the candidate differentiation factor is capable of promoting
the differentiation of the definitive endoderm cells. Similarly, if
expression of a plurality of markers is determined, it is further
determined whether the expression of the plurality of markers at
the second time point has increased or decreased as compared to the
expression of this plurality of markers at the first time point. An
increase or decrease in marker expression can be determined by
measuring or otherwise evaluating the amount, level or activity of
the marker in the cell population at the first and second time
points. Such determination can be relative to other markers, for
example housekeeping gene expression, or absolute. In certain
embodiments, wherein marker expression is increased at the second
time point as compared with the first time point, the amount of
increase is at least about 2-fold, at least about 5-fold, at least
about 10-fold, at least about 20-fold, at least about 30-fold, at
least about 40-fold, at least about 50-fold, at least about
60-fold, at least about 70-fold, at least about 80-fold, at least
about 90-fold, at least about 100-fold or more than at least about
100-fold. In some embodiments, the amount of increase is less than
2-fold. In embodiments where marker expression is decreased at the
second time point as compared with the first time point, the amount
of decrease is at least about 2-fold, at least about 5-fold, at
least about 10-fold, at least about 20-fold, at least about
30-fold, at least about 40-fold, at least about 50-fold, at least
about 60-fold, at least about 70-fold, at least about 80-fold, at
least about 90-fold, at least about 100-fold or more than at least
about 100-fold. In some embodiments, the amount of decrease is less
than 2-fold.
[0237] In some embodiments of the screening methods described
herein, after providing the cell population with a candidate
differentiation factor, the human definitive endoderm cells
differentiate into one or more cell types of the definitive
endoderm lineage. In some embodiments, after providing the cell
population with a candidate differentiation factor, the human
definitive endoderm cells differentiate into cells that are derived
from the gut tube. Such cells include, but are not limited to,
cells of the pancreas, liver, lungs, stomach, intestine, thyroid,
thymus, pharynx, gallbladder and urinary bladder as well as
precursors of such cells. Additionally, these cells can further
develop into higher order structures such as tissues and/or
organs.
[0238] It will be appreciated that screening methods similar to
those described above can be used to identify one or more
differentiation factors capable of promoting the differentiation of
human PDX1-positive endoderm cells in a cell population which
comprises human PDX1-positive endoderm cells. In certain
embodiments, the human PDX1-positive endoderm cells are
PDX1-positive foregut/midgut endoderm cells. In preferred
embodiments, the human PDX1-positive endoderm cells are
PDX1-positive foregut endoderm cells. In other preferred
embodiments, the human PDX1-positive endoderm cells are
PDX1-positive endoderm cells of the posterior portion of the
foregut. In especially preferred embodiments, the human
PDX1-positive foregut endoderm cells are multipotent cells that can
differentiate into cells, tissues or organs derived from the
anterior portion of the gut tube.
Identification of Factors Capable of Promoting the Differentiation
of PDX1-Negative Definitive Endoderm Cells to PDX1-Positive Foregut
Endoderm Cells
[0239] Aspects of the screening methods described herein relate to
methods of identifying one or more differentiation factors capable
of promoting the differentiation of PDX1-negative definitive
endoderm cells to PDX1-positive foregut endoderm cells. In such
methods, a cell culture or cell population comprising PDX1-negative
definitive endoderm cells is obtained and the expression of PDX1 in
the cell culture or cell population is determined After determining
the expression of PDX1, the cells of the cell culture or cell
population are contacted with a candidate differentiation factor.
In some embodiments, the expression of PDX1 is determined at the
time of contacting or shortly after contacting the cells with a
candidate differentiation factor. PDX1 expression is then
determined at one or more times after contacting the cells with the
candidate differentiation factor. If the expression of PDX1 has
increased after contact with the candidate differentiation factor
as compared to PDX1 expression prior to contact with the candidate
differentiation factor, the candidate differentiation factor is
identified as capable of promoting the differentiation of
PDX1-negative definitive endoderm cells to PDX1-positive foregut
endoderm cells.
[0240] In some embodiments, the above-described methods of
identifying factors capable of promoting the differentiation of
PDX1-negative definitive endoderm cells to PDX1-positive foregut
endoderm cells also include determining the expression of the
HOXA13 gene and/or the HOXC6 gene in the cell culture or cell
population. In such embodiments, the expression of HOXA13 and/or
HOXC6 is determined both before and after the cells are contacted
with the candidate differentiation factor. If the expression of
PDX1 and HOXA13 has increased after contact with the candidate
differentiation factor as compared to PDX1 and HOXA13 expression
prior to contact with the candidate differentiation factor, the
candidate differentiation factor is identified as capable of
promoting the differentiation of PDX1-negative definitive endoderm
cells to PDX1-positive foregut endoderm cells. Similarly, if the
expression of PDX1 and HOXC6 has increased after contact with the
candidate differentiation factor as compared to PDX1 and HOXC6
expression prior to contact with the candidate differentiation
factor, the candidate differentiation factor is identified as
capable of promoting the differentiation of PDX1-negative
definitive endoderm cells to PDX1-positive foregut endoderm cells.
In a preferred embodiment, a candidate differentiation factor is
identified as being capable of promoting the differentiation of
PDX1-negative definitive endoderm cells to PDX1-positive foregut
endoderm cells by determining the expression of PDX1, HOXA13 and
HOXC6 both before and after contacting the cells of the cell
culture or cell population with the candidate differentiation
factor. In preferred embodiments, the expression of PDX1, HOXA13
and/or HOXC6 is determined Q-PCR.
[0241] It will be appreciated that in some embodiments, the
expression of one or more of PDX1, HOXA13 and HOXC6 can be
determined at the time of contacting or shortly after contacting
the cells of the cell cultures or cell populations with a candidate
differentiation factor rather than prior to contacting the cells
with a candidate differentiation factor. In such embodiments, the
expression of one or more of PDX1, HOXA13 and HOXC6 at the time of
contacting or shortly after contacting the cells with a candidate
differentiation factor is compared to the expression of one or more
of PDX1, HOXA13 and HOXC6 at one or more times after contacting the
cells with a candidate differentiation factor.
[0242] In some embodiments of the above-described methods, the one
or more times at which PDX1 expression is determined after
contacting the cells with the candidate differentiation factor can
range from about 1 hour to about 10 days. For example, PDX1
expression can be determined about 1 hour after contacting the
cells with the candidate differentiation factor, about 2 hours
after contacting the cells with the candidate differentiation
factor, about 4 hours after contacting the cells with the candidate
differentiation factor, about 6 hours after contacting the cells
with the candidate differentiation factor, about 8 hours after
contacting the cells with the candidate differentiation factor,
about 10 hours after contacting the cells with the candidate
differentiation factor, about 12 hours after contacting the cells
with the candidate differentiation factor, about 16 hours after
contacting the cells with the candidate differentiation factor,
about 24 hours after contacting the cells with the candidate
differentiation factor, about 2 days after contacting the cells
with the candidate differentiation factor, about 3 days after
contacting the cells with the candidate differentiation factor,
about 4 days after contacting the cells with the candidate
differentiation factor, about 5 days after contacting the cells
with the candidate differentiation factor, about 6 days after
contacting the cells with the candidate differentiation factor,
about 7 days after contacting the cells with the candidate
differentiation factor, about 8 days after contacting the cells
with the candidate differentiation factor, about 9 days after
contacting the cells with the candidate differentiation factor,
about 10 days after contacting the cells with the candidate
differentiation factor or more than 10 days after contacting the
cells with the candidate differentiation factor.
[0243] Candidate differentiation factors for use in the methods
described herein can be selected from compounds, such as
polypeptides and small molecules. For example, candidate
polypeptides can include, but are not limited to, growth factors,
cytokines, chemokines, extracellular matrix proteins, and synthetic
peptides. In a preferred embodiment, the growth factor is from the
FGF family, for example FGF-10. Candidate small molecules include,
but are not limited to, compounds generated from combinatorial
chemical synthesis and natural products, such as steroids,
isoprenoids, terpenoids, phenylpropanoids, alkaloids and
flavinoids. It will be appreciated by those of ordinary skill in
the art that thousands of classes of natural and synthetic small
molecules are available and that the small molecules contemplated
for use in the methods described herein are not limited to the
classes exemplified above. Typically, small molecules will have a
molecular weight less than 10,000 amu. In a preferred embodiment,
the small molecule is a retinoid, for example RA.
Identification of Factors Capable of Promoting the Differentiation
of PDX1-Positive Foregut Endoderm Cells
[0244] Other aspects of the screening methods described herein
relate to methods of identifying one or more differentiation
factors capable of promoting the differentiation of PDX1-positive
foregut endoderm cells. In such methods, a cell culture or cell
population comprising PDX1-positive foregut endoderm cells is
obtained and the expression of a marker in the cell culture or cell
population is determined. After determining the expression of the
marker, the cells of the cell culture or cell population are
contacted with a candidate differentiation factor. In some
embodiments, the expression of the marker is determined at the time
of contacting or shortly after contacting the cells with a
candidate differentiation factor. The expression of the same marker
is then determined at one or more times after contacting the cells
with the candidate differentiation factor. If the expression of the
marker has increased or decreased after contact with the candidate
differentiation factor as compared to the marker expression prior
to contact with the candidate differentiation factor, the candidate
differentiation factor is identified as capable of promoting the
differentiation of PDX1-positive foregut endoderm cells. In
preferred embodiments, expression of the marker is determined by
Q-PCR.
[0245] In some embodiments of the above-described methods, the one
or more times at which the marker expression is determined after
contacting the cells with the candidate differentiation factor can
range from about 1 hour to about 10 days. For example, marker
expression can be determined about 1 hour after contacting the
cells with the candidate differentiation factor, about 2 hours
after contacting the cells with the candidate differentiation
factor, about 4 hours after contacting the cells with the candidate
differentiation factor, about 6 hours after contacting the cells
with the candidate differentiation factor, about 8 hours after
contacting the cells with the candidate differentiation factor,
about 10 hours after contacting the cells with the candidate
differentiation factor, about 12 hours after contacting the cells
with the candidate differentiation factor, about 16 hours after
contacting the cells with the candidate differentiation factor,
about 24 hours after contacting the cells with the candidate
differentiation factor, about 2 days after contacting the cells
with the candidate differentiation factor, about 3 days after
contacting the cells with the candidate differentiation factor,
about 4 days after contacting the cells with the candidate
differentiation factor, about 5 days after contacting the cells
with the candidate differentiation factor, about 6 days after
contacting the cells with the candidate differentiation factor,
about 7 days after contacting the cells with the candidate
differentiation factor, about 8 days after contacting the cells
with the candidate differentiation factor, about 9 days after
contacting the cells with the candidate differentiation factor,
about 10 days after contacting the cells with the candidate
differentiation factor or more than 10 days after contacting the
cells with the candidate differentiation factor.
[0246] As described previously, candidate differentiation factors
for use in the methods described herein can be selected from
compounds such as polypeptides and small molecules.
[0247] Although each of the methods disclosed herein have been
described with respect to PDX1-positive foregut endoderm cells, it
will be appreciated that in certain embodiments, these methods can
be used to produce compositions comprising the PDX1-positive
foregut/midgut endoderm cells that are described herein and/or the
PDX1-positive endoderm cells of the posterior portion of the
foregut that are described herein. Furthermore, any of the
PDX1-positive endoderm cell types disclosed in this specification
can be utilized in the screening methods described herein.
[0248] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of illustration
only, and are not intended to be limiting.
EXAMPLES
[0249] Many of the examples below describe the use of pluripotent
human cells. Methods of producing pluripotent human cells are well
known in the art and have been described numerous scientific
publications, including U.S. Pat. Nos. 5,453,357, 5,670,372,
5,690,926, 6,090,622, 6,200,806 and 6,251,671 as well as U.S.
Patent Application Publication No. 2004/0229350, the disclosures of
which are incorporated herein by reference in their entireties.
Example 1
Human ES Cells
[0250] For our studies of endoderm development we employed human
embryonic stem cells, which are pluripotent and can divide
seemingly indefinitely in culture while maintaining a normal
karyotype. ES cells were derived from the 5-day-old embryo inner
cell mass using either immunological or mechanical methods for
isolation. In particular, the human embryonic stem cell line
hESCyt-25 was derived from a supernumerary frozen embryo from an in
vitro fertilization cycle following informed consent by the
patient. Upon thawing the hatched blastocyst was plated on mouse
embryonic fibroblasts (MEF), in ES medium (DMEM, 20% FBS, non
essential amino acids, beta-mercaptoethanol, ITS supplement). The
embryo adhered to the culture dish and after approximately two
weeks, regions of undifferentiated hESCs were transferred to new
dishes with MEFs. Transfer was accomplished with mechanical cutting
and a brief digestion with dispase, followed by mechanical removal
of the cell clusters, washing and re-plating. Since derivation,
hESCyt-25 has been serially passaged over 100 times. We employed
the hESCyt-25 human embryonic stem cell line as our starting
material for the production of definitive endoderm.
[0251] It will be appreciated by those of skill in the art that
stem cells or other pluripotent cells can also be used as starting
material for the differentiation procedures described herein. For
example, cells obtained from embryonic gonadal ridges, which can be
isolated by methods known in the art, can be used as pluripotent
cellular starting material.
Example 2
hESCyt-25 Characterization
[0252] The human embryonic stem cell line, hESCyt-25 has maintained
a normal morphology, karyotype, growth and self-renewal properties
over 18 months in culture. This cell line displays strong
immunoreactivity for the OCT4, SSEA-4 and TRA-1-60 antigens, all of
which, are characteristic of undifferentiated hESCs and displays
alkaline phosphatase activity as well as a morphology identical to
other established hESC lines. Furthermore, the human stem cell
line, hESCyt-25, also readily forms embryoid bodies (EBs) when
cultured in suspension. As a demonstration of its pluripotent
nature, hESCyT-25 differentiates into various cell types that
represent the three principal germ layers. Ectoderm production was
demonstrated by Q-PCR for ZIC1 as well as immunocytochemistry (ICC)
for nestin and more mature neuronal markers. Immunocytochemical
staining for .beta.-111 tubulin was observed in clusters of
elongated cells, characteristic of early neurons. Previously, we
treated EBs in suspension with retinoic acid, to induce
differentiation of pluripotent stem cells to visceral endoderm
(VE), an extra-embryonic lineage. Treated cells expressed high
levels of .alpha.-fetoprotein (AFP) and SOX7, two markers of VE, by
54 hours of treatment. Cells differentiated in monolayer expressed
AFP in sporadic patches as demonstrated by immunocytochemical
staining. As will be described below, the hESCyT-25 cell line was
also capable of forming definitive endoderm, as validated by
real-time quantitative polymerase chain reaction (Q-PCR) and
immunocytochemistry for SOX17, in the absence of AFP expression. To
demonstrate differentiation to mesoderm, differentiating EBs were
analyzed for Brachyury gene expression at several time points.
Brachyury expression increased progressively over the course of the
experiment. In view of the foregoing, the hESCyT-25 line is
pluripotent as shown by the ability to form cells representing the
three germ layers.
Example 3
Production of SOX17 Antibody
[0253] A primary obstacle to the identification of definitive
endoderm in hESC cultures is the lack of appropriate tools. We
therefore undertook the production of an antibody raised against
human SOX17 protein.
[0254] The marker SOX17 is expressed throughout the definitive
endoderm as it forms during gastrulation and its expression is
maintained in the gut tube (although levels of expression vary
along the A-P axis) until around the onset of organogenesis. SOX17
is also expressed in a subset of extra-embryonic endoderm cells. No
expression of this protein has been observed in mesoderm or
ectoderm. It has now been discovered that SOX17 is an appropriate
marker for the definitive endoderm lineage when used in conjunction
with markers to exclude extra-embryonic lineages.
[0255] As described in detail herein, the SOX17 antibody was
utilized to specifically examine effects of various treatments and
differentiation procedures aimed at the production of SOX17
positive definitive endoderm cells. Other antibodies reactive to
AFP, SPARC and Thrombomodulin were also employed to rule out the
production of visceral and parietal endoderm (extra-embryonic
endoderm).
[0256] In order to produce an antibody against SOX17, a portion of
the human SOX17 cDNA (SEQ ID NO: 1) corresponding to amino acids
172-414 (SEQ ID NO: 2) in the carboxyterminal end of the SOX17
protein (FIG. 2) was used for genetic immunization in rats at the
antibody production company, GENOVAC (Freiberg, Germany), according
to procedures developed there. Procedures for genetic immunization
can be found in U.S. Pat. Nos. 5,830,876, 5,817,637, 6,165,993 and
6,261,281 as well as International Patent Application Publication
Nos. WO00/29442 and WO99/13915, the disclosures of which are
incorporated herein by reference in their entireties.
[0257] Other suitable methods for genetic immunization are also
described in the non-patent literature. For example, Barry et al.
describe the production of monoclonal antibodies by genetic
immunization in Biotechniques 16: 616-620, 1994, the disclosure of
which is incorporated herein by reference in its entirety. Specific
examples of genetic immunization methods to produce antibodies
against specific proteins can be found, for example, in Costaglia
et al., (1998) Genetic immunization against the human thyrotropin
receptor causes thyroiditis and allows production of monoclonal
antibodies recognizing the native receptor, J. Immunol. 160:
1458-1465; Kilpatrick et al (1998) Gene gun delivered DNA-based
immunizations mediate rapid production of murine monoclonal
antibodies to the Flt-3 receptor, Hybridoma 17: 569-576; Schmolke
et al., (1998) Identification of hepatitis G virus particles in
human serum by E2-specific monoclonal antibodies generated by DNA
immunization, J. Virol. 72: 4541-4545; Krasemann et al., (1999)
Generation of monoclonal antibodies against proteins with an
unconventional nucleic acid-based immunization strategy, J.
Biotechnol. 73: 119-129; and Ulivieri et al., (1996) Generation of
a monoclonal antibody to a defined portion of the Heliobacter
pylori vacuolating cytotoxin by DNA immunization, J. Biotechnol.
51: 191-194, the disclosures of which are incorporated herein by
reference in their entireties.
[0258] SOX7 and SOX18 are the closest Sox family relatives to SOX17
as depicted in the relational dendrogram shown in FIG. 3. We
employed the human SOX7 polypeptide as a negative control to
demonstrate that the SOX17 antibody produced by genetic
immunization is specific for SOX17 and does not react with its
closest family member. In particular, SOX7 and other proteins were
expressed in human fibroblasts, and then, analyzed for cross
reactivity with the SOX17 antibody by Western blot and ICC. For
example, the following methods were utilized for the production of
the SOX17, SOX7 and EGFP expression vectors, their transfection
into human fibroblasts and analysis by Western blot. Expression
vectors employed for the production of SOX17, SOX7, and EGFP were
pCMV6 (OriGene Technologies, Inc., Rockville, Md.), pCMV-SPORT6
(Invitrogen, Carlsbad, Calif.) and pEGFP-N1 (Clonetech, Palo Alto,
Calif.), respectively. For protein production, telomerase
immortalized MDX human fibroblasts were transiently transfected
with supercoiled DNA in the presence of Lipofectamine 2000
(Invitrogen, Carlsbad, Calif.). Total cellular lysates were
collected 36 hours post-transfection in 50 mM TRIS-HCl (pH 8), 150
mM NaCl, 0.1% SDS, 0.5% deoxycholate, containing a cocktail of
protease inhibitors (Roche Diagnostics Corporation, Indianapolis,
Ind.). Western blot analysis of 100 .mu.g of cellular proteins,
separated by SDS-PAGE on NuPAGE (4-12% gradient polyacrylamide,
Invitrogen, Carlsbad, Calif.), and transferred by electro-blotting
onto PDVF membranes (Hercules, Calif.), were probed with a 1/1000
dilution of the rat SOX17 anti-serum in 10 mM TRIS-HCl (pH 8), 150
mM NaCl, 10% BSA, 0.05% Tween-20 (Sigma, St. Louis, Mo.), followed
by Alkaline Phosphatase conjugated anti-rat IgG (Jackson
ImmunoResearch Laboratories, West Grove, Pa.), and revealed through
Vector Black Alkaline Phosphatase staining (Vector Laboratories,
Burlingame, Calif.). The proteins size standard used was wide range
color markers (Sigma, St. Louis, Mo.).
[0259] In FIG. 4, protein extracts made from human fibroblast cells
that were transiently transfected with SOX17, SOX7 or EGFP cDNA's
were probed on Western blots with the SOX17 antibody. Only the
protein extract from hSOX17 transfected cells produced a band of
.about.51 Kda which closely matched the predicted 46 Kda molecular
weight of the human SOX17 protein. There was no reactivity of the
SOX17 antibody to extracts made from either human SOX7 or EGFP
transfected cells. Furthermore, the SOX17 antibody clearly labeled
the nuclei of human fibroblast cells transfected with the hSOX17
expression construct but did not label cells transfected with EGFP
alone. As such, the SOX17 antibody exhibits specificity by ICC.
Example 4
Validation of SOX17 Antibody as a Marker of Definitive Endoderm
[0260] Partially differentiated hESCs were co-labeled with SOX17
and AFP antibodies to demonstrate that the SOX17 antibody is
specific for human SOX17 protein and furthermore marks definitive
endoderm. It has been demonstrated that SOX17, SOX7 (which is a
closely related member of the SOX gene family subgroup F (FIG. 3))
and AFP are each expressed in visceral endoderm. However, AFP and
SOX7 are not expressed in definitive endoderm cells at levels
detectable by ICC, and thus, they can be employed as negative
markers for bonifide definitive endoderm cells. It was shown that
SOX17 antibody labels populations of cells that exist as discrete
groupings of cells or are intermingled with AFP positive cells. In
particular, FIG. 5A shows that small numbers of SOX17 cells were
co-labeled with AFP; however, regions were also found where there
were little or no AFP cells in the field of SOX17.sup.+ cells (FIG.
5B). Similarly, since parietal endoderm has been reported to
express SOX17, antibody co-labeling with SOX17 together with the
parietal markers SPARC and/or Thrombomodulin (TM) can be used to
identify the SOX17.sup.+ cells that are parietal endoderm. As shown
in FIGS. 6A-6C, Thrombomodulin and SOX17 co-labeled parietal
endoderm cells were produced by random differentiation of hES
cells.
[0261] In view of the above cell labeling experiments, the identity
of a definitive endoderm cell can be established by the marker
profile SOX17.sup.hi/AFP.sup.lo/[TM.sup.lo or SPARC.sup.lo]. In
other words, the expression of the SOX17 marker is greater than the
expression of the AFP marker, which is characteristic of visceral
endoderm, and the TM or SPARC markers, which are characteristic of
parietal endoderm. Accordingly, those cells positive for SOX17 but
negative for AFP and negative for TM or SPARC are definitive
endoderm.
[0262] As a further evidence of the specificity of the
SOX17.sup.hi/AFP.sup.lo/TM.sup.lo/SPARC.sup.lo marker profile as
predictive of definitive endoderm, SOX17 and AFP gene expression
was quantitatively compared to the relative number of antibody
labeled cells. As shown in FIG. 7A, hESCs treated with retinoic
acid (visceral endoderm inducer), or activin A (definitive endoderm
inducer), resulted in a 10-fold difference in the level of SOX17
mRNA expression. This result mirrored the 10-fold difference in
SOX17 antibody-labeled cell number (FIG. 7B). Furthermore, as shown
in FIG. 8A, activin A treatment of hESCs suppressed AFP gene
expression by 6.8-fold in comparison to no treatment. This was
visually reflected by a dramatic decrease in the number of AFP
labeled cells in these cultures as shown in FIGS. 8B-8C. To
quantify this further, it was demonstrated that this approximately
7-fold decrease in AFP gene expression was the result of a similar
7-fold decrease in AFP antibody-labeled cell number as measured by
flow cytometry (FIGS. 9A-B). This result is extremely significant
in that it indicates that quantitative changes in gene expression
as seen by Q-PCR mirror changes in cell type specification as
observed by antibody staining.
[0263] Incubation of hESCs in the presence of Nodal family members
(Nodal, activin A and activin B--NAA) resulted in a significant
increase in SOX17 antibody-labeled cells over time. By 5 days of
continuous activin treatment greater than 50% of the cells were
labeled with SOX17 (FIGS. 10A-10F). There were few or no cells
labeled with AFP after 5 days of activin treatment.
[0264] In summary, the antibody produced against the
carboxy-terminal 242 amino acids of the human SOX17 protein
identified human SOX17 protein on Western blots but did not
recognize SOX7, it's closest Sox family relative. The SOX17
antibody recognized a subset of cells in differentiating hESC
cultures that were primarily SOX17.sup.+/AFP.sup.lo/- (greater than
95% of labeled cells) as well as a small percentage (<5%) of
cells that co-label for SOX17 and AFP (visceral endoderm).
Treatment of hESC cultures with activins resulted in a marked
elevation of SOX17 gene expression as well as SOX17 labeled cells
and dramatically suppressed the expression of AFP mRNA and the
number of cells labeled with AFP antibody.
Example 5
Q-PCR Gene Expression Assay
[0265] In the following experiments, real-time quantitative RT-PCR
(Q-PCR) was the primary assay used for screening the effects of
various treatments on hESC differentiation. In particular,
real-time measurements of gene expression were analyzed for
multiple marker genes at multiple time points by Q-PCR. Marker
genes characteristic of the desired as well as undesired cell types
were evaluated to gain a better understanding of the overall
dynamics of the cellular populations. The strength of Q-PCR
analysis includes its extreme sensitivity and relative ease of
developing the necessary markers, as the genome sequence is readily
available. Furthermore, the extremely high sensitivity of Q-PCR
permits detection of gene expression from a relatively small number
of cells within a much larger population. In addition, the ability
to detect very low levels of gene expression provides indications
for "differentiation bias" within the population. The bias towards
a particular differentiation pathway, prior to the overt
differentiation of those cellular phenotypes, is unrecognizable
using immunocytochemical techniques. For this reason, Q-PCR
provides a method of analysis that is at least complementary and
potentially much superior to immunocytochemical techniques for
screening the success of differentiation treatments. Additionally,
Q-PCR provides a mechanism by which to evaluate the success of a
differentiation protocol in a quantitative format at semi-high
throughput scales of analysis.
[0266] The approach taken here was to perform relative quantitation
using SYBR Green chemistry on a Rotor Gene 3000 instrument (Corbett
Research) and a two-step RT-PCR format. Such an approach allowed
for the banking of cDNA samples for analysis of additional marker
genes in the future, thus avoiding variability in the reverse
transcription efficiency between samples.
[0267] Primers were designed to lie over exon-exon boundaries or
span introns of at least 800 bp when possible, as this has been
empirically determined to eliminate amplification from
contaminating genomic DNA. When marker genes were employed that do
not contain introns or they possess pseudogenes, DNase I treatment
of RNA samples was performed.
[0268] We routinely used Q-PCR to measure the gene expression of
multiple markers of target and non-target cell types in order to
provide a broad profile description of gene expression in cell
samples. The markers relevant for the early phases of hESC
differentiation (specifically ectoderm, mesoderm, definitive
endoderm and extra-embryonic endoderm) and for which validated
primer sets are available are provided below in Table 1. The human
specificity of these primer sets has also been demonstrated. This
is an important fact since the hESCs were often grown on mouse
feeder layers. Most typically, triplicate samples were taken for
each condition and independently analyzed in duplicate to assess
the biological variability associated with each quantitative
determination.
[0269] To generate PCR template, total RNA was isolated using
RNeasy (Qiagen) and quantitated using RiboGreen (Molecular Probes).
Reverse transcription from 350-500 ng of total RNA was carried out
using the iScript reverse transcriptase kit (BioRad), which
contains a mix of oligo-dT and random primers. Each 20 .mu.L
reaction was subsequently diluted up to 100 .mu.L total volume and
3 .mu.L was used in each 10 .mu.L Q-PCR reaction containing 400 nM
forward and reverse primers and 5 .mu.L 2.times.SYBR Green master
mix (Qiagen). Two step cycling parameters were used employing a 5
second denature at 85-94.degree. C. (specifically selected
according to the melting temp of the amplicon for each primer set)
followed by a 45 second anneal/extend at 60.degree. C. Fluorescence
data was collected during the last 15 seconds of each extension
phase. A three point, 10-fold dilution series was used to generate
the standard curve for each run and cycle thresholds (Ct's) were
converted to quantitative values based on this standard curve. The
quantitated values for each sample were normalized to housekeeping
gene performance and then average and standard deviations were
calculated for triplicate samples. At the conclusion of PCR
cycling, a melt curve analysis was performed to ascertain the
specificity of the reaction. A single specific product was
indicated by a single peak at the T.sub.m appropriate for that PCR
amplicon. In addition, reactions performed without reverse
transcriptase served as the negative control and do not
amplify.
[0270] A first step in establishing the Q-PCR methodology was
validation of appropriate housekeeping genes (HGs) in the
experimental system. Since the HG was used to normalize across
samples for the RNA input, RNA integrity and RT efficiency, it was
of value that the HG exhibited a constant level of expression over
time in all sample types in order for the normalization to be
meaningful. We measured the expression levels of Cyclophilin G,
hypoxanthine phosphoribosyltransferase 1 (HPRT),
beta-2-microglobulin, hydroxymethylbiane synthase (HMBS),
TATA-binding protein (TBP), and glucoronidase beta (GUS) in
differentiating hESCs. Our results indicated that beta-2-micro
globulin expression levels increased over the course of
differentiation and therefore we excluded the use of this gene for
normalization. The other genes exhibited consistent expression
levels over time as well as across treatments. We routinely used
both Cyclophilin G and GUS to calculate a normalization factor for
all samples. The use of multiple HGs simultaneously reduces the
variability inherent to the normalization process and increases the
reliability of the relative gene expression values.
[0271] After obtaining genes for use in normalization, Q-PCR was
then utilized to determine the relative gene expression levels of
many marker genes across samples receiving different experimental
treatments. The marker genes employed have been chosen because they
exhibit enrichment in specific populations representative of the
early germ layers and in particular have focused on sets of genes
that are differentially expressed in definitive endoderm and
extra-embryonic endoderm. These genes as well as their relative
enrichment profiles are highlighted in Table 1.
TABLE-US-00001 TABLE 1 Germ Layer Gene Expression Domains Endoderm
SOX17 definitive, visceral and parietal endoderm MIXL1 endoderm and
mesoderm GATA4 definitive and primitive endoderm HNF3b definitive
endoderm and primitive endoderm, mesoderm, neural plate GSC
endoderm and mesoderm Extra- SOX7 visceral endoderm embryonic AFP
visceral endoderm, liver SPARC parietal endoderm TM parietal
endoderm/trophectoderm Ectoderm ZIC1 neural tube, neural
progenitors Mesoderm BRACH nascent mesoderm
[0272] Since many genes are expressed in more than one germ layer
it is useful to quantitatively compare expression levels of many
genes within the same experiment. SOX17 is expressed in definitive
endoderm and to a smaller extent in visceral and parietal endoderm.
SOX7 and AFP are expressed in visceral endoderm at this early
developmental time point. SPARC and TM are expressed in parietal
endoderm and Brachyury is expressed in early mesoderm.
[0273] Definitive endoderm cells were predicted to express high
levels of SOX17 mRNA and low levels of AFP and SOX7 (visceral
endoderm), SPARC (parietal endoderm) and Brachyury (mesoderm). In
addition, ZIC1 was used here to further rule out induction of early
ectoderm. Finally, GATA4 and HNF3b were expressed in both
definitive and extra-embryonic endoderm, and thus, correlate with
SOX17 expression in definitive endoderm (Table 1). A representative
experiment is shown in FIGS. 11-14 which demonstrates how the
marker genes described in Table 1 correlate with each other among
the various samples, thus highlighting specific patterns of
differentiation to definitive endoderm and extra-embryonic endoderm
as well as to mesodermal and neural cell types.
[0274] In view of the above data it is clear that increasing doses
of activin resulted in increasing SOX17 gene expression. Further
this SOX17 expression predominantly represented definitive endoderm
as opposed to extra-embryonic endoderm. This conclusion stems from
the observation that SOX17 gene expression was inversely correlated
with AFP, SOX7, and SPARC gene expression.
Example 6
Directed Differentiation of Human ES Cells to Definitive
Endoderm
[0275] Human ES cell cultures randomly differentiate if cultured
under conditions that do not actively maintain their
undifferentiated state. This heterogeneous differentiation results
in production of extra-embryonic endoderm cells comprised of both
parietal and visceral endoderm (AFP, SPARC and SOX7 expression) as
well as early ectodermal and mesodermal derivatives as marked by
ZIC1 and Nestin (ectoderm) and Brachyury (mesoderm) expression.
Definitive endoderm cell appearance has not been examined or
specified for lack of specific antibody markers in ES cell
cultures. As such, and by default, early definitive endoderm
production in ES cell cultures has not been well studied. Since
satisfactory antibody reagents for definitive endoderm cells have
been unavailable, most of the characterization has focused on
ectoderm and extra-embryonic endoderm. Overall, there are
significantly greater numbers of extra-embryonic and neurectodermal
cell types in comparison to SOX17.sup.hi definitive endoderm cells
in randomly differentiated ES cell cultures.
[0276] As undifferentiated hESC colonies expand on a bed of
fibroblast feeders, the cells at the edges of the colony take on
alternative morphologies that are distinct from those cells
residing within the interior of the colony. Many of these outer
edge cells can be distinguished by their less uniform, larger cell
body morphology and by the expression of higher levels of OCT4. It
has been described that as ES cells begin to differentiate they
alter the levels of OCT4 expression up or down relative to
undifferentiated ES cells. Alteration of OCT4 levels above or below
the undifferentiated threshold may signify the initial stages of
differentiation away from the pluripotent state.
[0277] When undifferentiated colonies were examined by SOX17
immunocytochemistry, occasionally small 10-15-cell clusters of
SOX17-positive cells were detected at random locations on the
periphery and at the junctions between undifferentiated hESC
colonies. As noted above, these scattered pockets of outer colony
edges appeared to be some of the first cells to differentiate away
from the classical ES cell morphology as the colony expanded in
size and became more crowded. Younger, smaller fully
undifferentiated colonies (<1 mm; 4-5 days old) showed no SOX17
positive cells within or at the edges of the colonies while older,
larger colonies (1-2 mm diameter, >5 days old) had sporadic
isolated patches of SOX17 positive, AFP negative cells at the
periphery of some colonies or in regions interior to the edge that
did not display the classical hESC morphology described previously.
Given that this was the first development of an effective SOX17
antibody, definitive endoderm cells generated in such early
"undifferentiated" ES cell cultures have never been previously
demonstrated.
[0278] Based on negative correlations of SOX17 and SPARC gene
expression levels by Q-PCR, the vast majority of these SOX17
positive, AFP negative cells will be negative for parietal endoderm
markers by antibody co-labeling. This was specifically demonstrated
for TM-expressing parietal endoderm cells as shown in FIGS. 15A-B.
Exposure to Nodal factors activin A and B resulted in a dramatic
decrease in the intensity of TM expression and the number of TM
positive cells. By triple labeling using SOX17, AFP and TM
antibodies on an activin treated culture, clusters of SOX17
positive cells that were also negative for AFP and TM were observed
(FIGS. 16A-D). These are the first cellular demonstrations of SOX17
positive definitive endoderm cells in differentiating hESC cultures
(FIGS. 16A-D and 17).
[0279] With the SOX17 antibody and Q-PCR tools described above we
have explored a number of procedures capable of efficiently
programming hESCs to become SOX17.sup.hi/AFP.sup.lo/SPARC/TM.sup.lo
definitive endoderm cells. We applied a variety of differentiation
protocols aimed at increasing the number and proliferative capacity
of these cells as measured at the population level by Q-PCR for
SOX17 gene expression and at the level of individual cells by
antibody labeling of SOX17 protein.
[0280] We were the first to analyze and describe the effect of
TGF.beta. family growth factors, such as Nodal/activin/BMP, for use
in creating definitive endoderm cells from embryonic stem cells in
in vitro cell cultures. In typical experiments, activin A, activin
B, BMP or combinations of these growth factors were added to
cultures of undifferentiated human stem cell line hESCyt-25 to
begin the differentiation process.
[0281] As shown in FIG. 19, addition of activin A at 100 ng/ml
resulted in a 19-fold induction of SOX17 gene expression vs.
undifferentiated hESCs by day 4 of differentiation. Adding activin
B, a second member of the activin family, together with activin A,
resulted in a 37-fold induction over undifferentiated hESCs by day
4 of combined activin treatment. Finally, adding a third member of
the TGF.beta. family from the Nodal/Activin and BMP subgroups,
BMP4, together with activin A and activin B, increased the fold
induction to 57 times that of undifferentiated hESCs (FIG. 19).
When SOX17 induction with activins and BMP was compared to no
factor medium controls 5-, 10-, and 15-fold inductions resulted at
the 4-day time point. By five days of triple treatment with
activins A, B and BMP, SOX17 was induced more than 70 times higher
than hESCs. These data indicate that higher doses and longer
treatment times of the Nodal/activin TGF.beta. family members
results in increased expression of SOX17.
[0282] Nodal and related molecules activin A, B and BMP facilitate
the expression of SOX17 and definitive endoderm formation in vivo
or in vitro. Furthermore, addition of BMP results in an improved
SOX17 induction possibly through the further induction of Cripto,
the Nodal co-receptor.
[0283] We have demonstrated that the combination of activins A and
B together with BMP4 result in additive increases in SOX17
induction and hence definitive endoderm formation. BMP4 addition
for prolonged periods (>4 days), in combination with activin A
and B may induce SOX17 in parietal and visceral endoderm as well as
definitive endoderm. In some embodiments of the present invention,
it is therefore valuable to remove BMP4 from the treatment within 4
days of addition.
[0284] To determine the effect of TGF.beta. factor treatment at the
individual cell level, a time course of TGF.beta. factor addition
was examined using SOX17 antibody labeling. As previously shown in
FIGS. 10A-F, there was a dramatic increase in the relative number
of SOX17 labeled cells over time. The relative quantification (FIG.
20) shows more than a 20-fold increase in SOX17-labeled cells. This
result indicates that both the numbers of cells as well SOX17 gene
expression level are increasing with time of TGF.beta. factor
exposure. As shown in FIG. 21, after four days of exposure to
Nodal, activin A, activin B and BMP4, the level of SOX17 induction
reached 168-fold over undifferentiated hESCs. FIG. 22 shows that
the relative number of SOX17-positive cells was also dose
responsive. activin A doses of 100 ng/ml or more were capable of
potently inducing SOX17 gene expression and cell number.
[0285] In addition to the TGF.beta. family members, the Wnt family
of molecules may play a role in specification and/or maintenance of
definitive endoderm. The use of Wnt molecules was also beneficial
for the differentiation of hESCs to definitive endoderm as
indicated by the increased SOX17 gene expression in samples that
were treated with activins plus Wnt3a over that of activins alone
(FIG. 23).
[0286] All of the experiments described above were performed using
a tissue culture medium containing 10% serum with added factors.
Surprisingly, we discovered that the concentration of serum had an
effect on the level of SOX17 expression in the presence of added
activins as shown in FIGS. 24A-C. When serum levels were reduced
from 10% to 2%, SOX17 expression tripled in the presence of
activins A and B.
[0287] Finally, we demonstrated that activin induced SOX17.sup.+
cells divide in culture as depicted in FIGS. 25A-D. The arrows show
cells labeled with SOX17/PCNA/DAPI that are in mitosis as evidenced
by the PCNA/DAPI-labeled mitotic plate pattern and the phase
contrast mitotic profile.
Example 7
Chemokine Receptor 4 (CXCR4) Expression Correlates with Markers for
Definitive Endoderm and not Markers for Mesoderm, Ectoderm or
Visceral Endoderm
[0288] As described above, hESCs can be induced to differentiate to
the definitive endoderm germ layer by the application of cytokines
of the TGF.beta. family and more specifically of the activin/nodal
subfamily Additionally, we have shown that the proportion of fetal
bovine serum (FBS) in the differentiation culture medium effects
the efficiency of definitive endoderm differentiation from hESCs.
This effect is such that at a given concentration of activin A in
the medium, higher levels of FBS will inhibit maximal
differentiation to definitive endoderm. In the absence of exogenous
activin A, differentiation of hESCs to the definitive endoderm
lineage is very inefficient and the FBS concentration has much
milder effects on the differentiation process of hESCs.
[0289] In these experiments, hESCs were differentiated by growing
in RPMI medium (Invitrogen, Carlsbad, Calif.; cat#61870-036)
supplemented with 0.5%, 2.0% or 10% FBS and either with or without
100 ng/ml activin A for 6 days. In addition, a gradient of FBS
ranging from 0.5% to 2.0% over the first three days of
differentiation was also used in conjunction with 100 ng/ml of
activin A. After the 6 days, replicate samples were collected from
each culture condition and analyzed for relative gene expression by
real-time quantitative PCR. The remaining cells were fixed for
immunofluorescent detection of SOX17 protein.
[0290] The expression levels of CXCR4 varied dramatically across
the 7 culture conditions used (FIG. 26). In general, CXCR4
expression was high in activin A treated cultures (A100) and low in
those which did not receive exogenous activin A (NF). In addition,
among the A100 treated cultures, CXCR4 expression was highest when
FBS concentration was lowest. There was a remarkable decrease in
CXCR4 level in the 10% FBS condition such that the relative
expression was more in line with the conditions that did not
receive activin A (NF).
[0291] As described above, expression of the SOX17, GSC, MIXL1, and
HNF3.beta. genes is consistent with the characterization of a cell
as definitive endoderm. The relative expression of these four genes
across the 7 differentiation conditions mirrors that of CXCR4
(FIGS. 27A-27D). This demonstrates that CXCR4 is also a marker of
definitive endoderm.
[0292] Ectoderm and mesoderm lineages can be distinguished from
definitive endoderm by their expression of various markers. Early
mesoderm expresses the genes Brachyury and MOX1 while nascent
neuro-ectoderm expresses SOX1 and ZIC1. FIGS. 28A-28D demonstrate
that the cultures which did not receive exogenous activin A were
preferentially enriched for mesoderm and ectoderm gene expression
and that among the activin A treated cultures, the 10% FBS
condition also had increased levels of mesoderm and ectoderm marker
expression. These patterns of expression were inverse to that of
CXCR4 and indicated that CXCR4 was not highly expressed in mesoderm
or ectoderm derived from hESCs at this developmental time
period.
[0293] Early during mammalian development, differentiation to
extra-embryonic lineages also occurs. Of particular relevance here
is the differentiation of visceral endoderm that shares the
expression of many genes in common with definitive endoderm,
including SOX17. To distinguish definitive endoderm from
extra-embryonic visceral endoderm one should examine a marker that
is distinct between these two. SOX7 represents a marker that is
expressed in the visceral endoderm but not in the definitive
endoderm lineage. Thus, culture conditions that exhibit robust
SOX17 gene expression in the absence of SOX7 expression are likely
to contain definitive and not visceral endoderm. It is shown in
FIG. 28E that SOX7 was highly expressed in cultures that did not
receive activin A, SOX7 also exhibited increased expression even in
the presence of activin A when FBS was included at 10%. This
pattern is the inverse of the CXCR4 expression pattern and suggests
that CXCR4 is not highly expressed in visceral endoderm.
[0294] The relative number of SOX17 immunoreactive (SOX17.sup.+)
cells present in each of the differentiation conditions mentioned
above was also determined. When hESCs were differentiated in the
presence of high dose activin A and low FBS concentration
(0.5%-2.0%) SOX17.sup.+ cells were ubiquitously distributed
throughout the culture. When high dose activin A was used but FBS
was included at 10% (v/v), the SOX17.sup.+ cells appeared at much
lower frequency and always appeared in isolated clusters rather
than evenly distributed throughout the culture (FIGS. 29A and 29C
as well as 29B and 29E). A further decrease in SOX17.sup.+ cells
was seen when no exogenous activin A was used. Under these
conditions the SOX17.sup.+ cells also appeared in clusters and
these clusters were smaller and much more rare than those found in
the high activin A, low FBS treatment (FIGS. 29 C and 29F). These
results demonstrate that the CXCR4 expression patterns not only
correspond to definitive endoderm gene expression but also to the
number of definitive endoderm cells in each condition.
Example 8
Differentiation Conditions that Enrich for Definitive Endoderm
Increase the Proportion of CXCR4 Positive Cells
[0295] The dose of activin A also effects the efficiency at which
definitive endoderm can be derived from hESCs. This example
demonstrates that increasing the dose of activin A increases the
proportion of CXCR4.sup.+ cells in the culture.
[0296] hESCs were differentiated in RPMI media supplemented with
0.5%-2% FBS (increased from 0.5% to 1.0% to 2.0% over the first 3
days of differentiation) and either 0, 10, or 100 ng/ml of activin
A. After 7 days of differentiation the cells were dissociated in
PBS without Ca.sup.2+/Mg.sup.2+ containing 2% FBS and 2 mM (EDTA)
for 5 minutes at room temperature. The cells were filtered through
35 .mu.m nylon filters, counted and pelleted. Pellets were
resuspended in a small volume of 50% human serum/50% normal donkey
serum and incubated for 2 minutes on ice to block non-specific
antibody binding sites. To this, 1 .mu.l of mouse anti-CXCR4
antibody (Abcam, cat# ab10403-100) was added per 50 .mu.l
(containing approximately 10.sup.5 cells) and labeling proceeded
for 45 minutes on ice. Cells were washed by adding 5 ml of PBS
containing 2% human serum (buffer) and pelleted. A second wash with
5 ml of buffer was completed then cells were resuspended in 50
.mu.l buffer per 10.sup.5 cells. Secondary antibody (FITC
conjugated donkey anti-mouse; Jackson ImmunoResearch,
cat#715-096-151) was added at 5 .mu.g/ml final concentration and
allowed to label for 30 minutes followed by two washes in buffer as
above. Cells were resuspended at 5.times.10.sup.6 cells/ml in
buffer and analyzed and sorted using a FACS Vantage (Beckton
Dickenson) by the staff at the flow cytometry core facility (The
Scripps Research Institute). Cells were collected directly into RLT
lysis buffer (Qiagen) for subsequent isolation of total RNA for
gene expression analysis by real-time quantitative PCR.
[0297] The number of CXCR4.sup.+ cells as determined by flow
cytometry were observed to increase dramatically as the dose of
activin A was increased in the differentiation culture media (FIGS.
30A-30C). The CXCR4.sup.+ cells were those falling within the R4
gate and this gate was set using a secondary antibody-only control
for which 0.2% of events were located in the R4 gate. The
dramatically increased numbers of CXCR4.sup.+ cells correlates with
a robust increase in definitive endoderm gene expression as activin
A dose is increased (FIGS. 31A-31D).
Example 9
Isolation of CXCR4 Positive Cells Enriches for Definitive Endoderm
Gene Expression and Depletes Cells Expressing Markers of Mesoderm,
Ectoderm and Visceral Endoderm
[0298] The CXCR4.sup.+ and CXCR4.sup.- cells identified in Example
8 above were collected and analyzed for relative gene expression
and the gene expression of the parent populations was determined
simultaneously.
[0299] The relative levels of CXCR4 gene expression was
dramatically increased with increasing dose of activin A (FIG. 32).
This correlated very well with the activin A dose-dependent
increase of CXCR4.sup.+ cells (FIGS. 30A-30C). It is also clear
that isolation of the CXCR4.sup.+ cells from each population
accounted for nearly all of the CXCR4 gene expression in that
population. This demonstrates the efficiency of the FACS method for
collecting these cells.
[0300] Gene expression analysis revealed that the CXCR4.sup.+ cells
contain not only the majority of the CXCR4 gene expression, but
they also contained gene expression for other markers of definitive
endoderm. As shown in FIGS. 31A-31D, the CXCR4.sup.+ cells were
further enriched over the parent A100 population for SOX17, GSC,
HNF3B, and MIXL1. In addition, the CXCR4.sup.- fraction contained
very little gene expression for these definitive endoderm markers.
Moreover, the CXCR4.sup.+ and CXCR4.sup.- populations displayed the
inverse pattern of gene expression for markers of mesoderm,
ectoderm and extra-embryonic endoderm. FIGS. 33A-33D shows that the
CXCR4.sup.+ cells were depleted for gene expression of Brachyury,
MOX1, ZIC1, and SOX7 relative to the A100 parent population. This
A100 parent population was already low in expression of these
markers relative to the low dose or no activin A conditions. These
results show that the isolation of CXCR4.sup.+ cells from hESCs
differentiated in the presence of high activin A yields a
population that is highly enriched for and substantially pure
definitive endoderm.
Example 10
Quantitation of Definitive Endoderm Cells in a Cell Population
Using CXCR4
[0301] To confirm the quantitation of the proportion of definitive
endoderm cells present in a cell culture or cell population as
determined previously herein and as determined in U.S. Provisional
Patent Application No. 60/532,004, entitled DEFINITIVE ENDODERM,
filed Dec. 23, 2003, the disclosure of which is incorporated herein
by reference in its entirety, cells expressing CXCR4 and other
markers of definitive endoderm were analyzed by FACS.
[0302] Using the methods such as those described in the above
Examples, hESCs were differentiated to produce definitive endoderm.
In particular, to increase the yield and purity in differentiating
cell cultures, the serum concentration of the medium was controlled
as follows: 0.2% FBS on day 1, 1.0% FBS on day 2 and 2.0% FBS on
days 3-6. Differentiated cultures were sorted by FACS using three
cell surface epitopes, E-Cadherin, CXCR4, and Thrombomodulin.
Sorted cell populations were then analyzed by Q-PCR to determine
relative expression levels of markers for definitive and
extraembryonic-endoderm as well as other cell types. CXCR4 sorted
cells taken from optimally differentiated cultures resulted in the
isolation of definitive endoderm cells that were >98% pure.
[0303] Table 2 shows the results of a marker analysis for a
definitive endoderm culture that was differentiated from hESCs
using the methods described herein.
TABLE-US-00002 TABLE 2 Composition of Definitive Endoderm Cultures
Percent Percent Percent Percent of Definitive Extraembryonic hES
Marker(s) culture Endoderm endoderm cells SOX17 70-80 100
Thrombomodulin <2 0 75 AFP <1 0 25 CXCR4 70-80 100 0 ECAD 10
0 100 other (ECAD neg.) 10-20 Total 100 100 100 100
[0304] In particular, Table 2 indicates that CXCR4 and SOX17
positive cells (endoderm) comprised from 70%-80% of the cells in
the cell culture. Of these SOX17-expressing cells, less than 2%
expressed TM (parietal endoderm) and less than 1% expressed AFP
(visceral endoderm). After subtracting the proportion of
TM-positive and AFP-positive cells (combined parietal and visceral
endoderm; 3% total) from the proportion of SOX17/CXCR4 positive
cells, it can be seen that about 67% to about 77% of the cell
culture was definitive endoderm. Approximately 10% of the cells
were positive for E-Cadherin (ECAD), which is a marker for hESCs,
and about 10-20% of the cells were of other cell types.
[0305] We have discovered that the purity of definitive endoderm in
the differentiating cell cultures that are obtained prior to FACS
separation can be improved as compared to the above-described low
serum procedure by maintaining the FBS concentration at <0.5%
throughout the 5-6 day differentiation procedure. However,
maintaining the cell culture at <0.5% throughout the 5-6 day
differentiation procedure also results in a reduced number of total
definitive endoderm cells that are produced.
[0306] Definitive endoderm cells produced by methods described
herein have been maintained and expanded in culture in the presence
of activin for greater than 50 days without appreciable
differentiation. In such cases, SOX17, CXCR4, MIXL1, GATA4, HNF313
expression is maintained over the culture period. Additionally, TM,
SPARC, OCT4, AFP, SOX7, ZIC1 and BRACH were not detected in these
cultures. It is likely that such cells can be maintained and
expanded in culture for substantially longer than 50 days without
appreciable differentiation.
Example 11
Additional Markers of Definitive Endoderm Cells
[0307] In the following experiment, RNA was isolated from purified
definitive endoderm and human embryonic stem cell populations. Gene
expression was then analyzed by gene chip analysis of the RNA from
each purified population. Q-PCR was also performed to further
investigate the potential of genes expressed in definitive
endoderm, but not in embryonic stem cells, as a marker for
definitive endoderm.
[0308] Human embryonic stem cells (hESCs) were maintained in
DMEM/F12 media supplemented with 20% KnockOut Serum Replacement, 4
ng/ml recombinant human basic fibroblast growth factor (bFGF), 0.1
mM 2-mercaptoethanol, L-glutamine, non-essential amino acids and
penicillin/streptomycin. hESCs were differentiated to definitive
endoderm by culturing for 5 days in RPMI media supplemented with
100 ng/ml of recombinant human activin A, fetal bovine serum (FBS),
and penicillin/streptomycin. The concentration of FBS was varied
each day as follows: 0.1% (first day), 0.2% (second day), 2% (days
3-5).
[0309] Cells were isolated by fluorescence activated cell sorting
(FACS) in order to obtain purified populations of hESCs and
definitive endoderm for gene expression analysis
Immuno-purification was achieved for hESCs using SSEA4 antigen
(R&D Systems, cat# FAB1435P) and for definitive endoderm using
CXCR4 (R&D Systems, cat# FAB170P). Cells were dissociated using
trypsin/EDTA (Invitrogen, cat#25300-054), washed in phosphate
buffered saline (PBS) containing 2% human serum and resuspended in
100% human serum on ice for 10 minutes to block non-specific
binding. Staining was carried out for 30 minutes on ice by adding
200 .mu.l of phycoerythrin-conjugated antibody to 5.times.10.sup.6
cells in 800 .mu.l human serum. Cells were washed twice with 8 ml
of PBS buffer and resuspended in 1 ml of the same. FACS isolation
was carried out by the core facility of The Scripps Research
Institute using a FACS Vantage (BD Biosciences). Cells were
collected directly into RLT lysis buffer and RNA was isolated by
RNeasy according to the manufacturers instructions (Qiagen).
[0310] Purified RNA was submitted in duplicate to Expression
Analysis (Durham, N.C.) for generation of the expression profile
data using the Affymetrix platform and U133 Plus 2.0 high-density
oligonucleotide arrays. Data presented is a group comparison that
identifies genes differentially expressed between the two
populations, hESCs and definitive endoderm. Genes that exhibited a
robust upward change in expression level over that found in hESCs
were selected as new candidate markers that are highly
characteristic of definitive endoderm. Select genes were assayed by
Q-PCR, as described above, to verify the gene expression changes
found on the gene chip and also to investigate the expression
pattern of these genes during a time course of hESC
differentiation.
[0311] FIGS. 34A-34M show the gene expression results for certain
markers. Results are displayed for cell cultures analyzed 1, 3 and
5 days after the addition of 100 ng/ml activin A, CXCR4-expressing
definitive endoderm cells purified at the end of the five day
differentiation procedure (CXDE), and in purified hESCs. A
comparison of FIGS. 34C and 34G-34M demonstrates that the six
marker genes, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1, exhibit
an expression pattern that is almost identical to each other and
which is also identical to the pattern of expression of CXCR4 and
the ratio of SOX17/SOX7. As described previously, SOX17 is
expressed in both the definitive endoderm as well as in the
SOX7-expressing extra-embryonic endoderm. Since SOX7 is not
expressed in the definitive endoderm, the ratio of SOX17/SOX7
provides a reliable estimate of definitive endoderm contribution to
the SOX17 expression witnessed in the population as a whole. The
similarity of panels G-L and M to panel C indicates that FGF17,
VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 are likely markers of
definitive endoderm and that they are not significantly expressed
in extra-embryonic endoderm cells.
[0312] It will be appreciated that the Q-PCR results described
herein can be further confirmed by ICC.
Example 12
Retinoic Acid and FGF-10 Induces PDX1 Specifically in Definitive
Endoderm Cultures
[0313] The following experiment demonstrates that RA and FGF-10
induces the expression of PDX1 in definitive endoderm cells.
[0314] Human embryonic stem cells were cultured with or without
activins for four days. On day four, 1 .mu.M RA and 50 ng/ml FGF-10
were added to the cell culture. Forty-eight hours after the
RA/FGF-10 addition, the expression of the PDX1 marker gene and
other marker genes not specific to foregut endoderm were
quantitated by Q-PCR.
[0315] The application of RA to definitive endoderm cells caused a
robust increase in PDX1 gene expression (see FIG. 35) without
increasing the expression of visceral endoderm (SOX7, AFP), neural
(SOX1, ZIC1), or neuronal (NFM) gene expression markers (see FIGS.
36A-36F). PDX1 gene expression was induced to levels approximately
500-fold higher than observed in definitive endoderm after 48 hours
exposure to 1 .mu.M RA and 50 ng/ml FGF-10. Furthermore, these
results show that substantial PDX1 induction occurred only in cell
cultures which had been previously differentiated to definitive
endoderm (SOX17) as indicated by the 160-fold higher PDX1
expression found in the activin treated cell cultures relative to
those cultures that received no activin prior to RA
application.
Example 13
FGF-10 Provides Additional Increase in PDX1 Expression Over RA
Alone
[0316] This Example shows that the combination of RA and FGF-10
induces PDX1 expression to a greater extent than RA alone.
[0317] As in the previous Example, hESCs were cultured with or
without activins for four days. On day four, the cells were treated
with one of the following: 1 .mu.M RA alone; 1 .mu.M RA in
combination with either FGF-4 or FGF-10; or 1 .mu.M RA in
combination with both FGF-4 and FGF-10. The expression of PDX1,
SOX7 and NFM were quantitated by Q-PCR ninety six hours after RA or
RA/FGF.
[0318] The treatment of hESC cultures with activin followed by
retinoic acid induced a 60-fold increase in PDX1 gene expression.
The addition of FGF-4 to the RA treatment induced slightly more
PDX1 (approximately 3-fold over RA alone). However, by adding
FGF-10 and retinoic acid together, the induction of PDX1 was
further enhanced 60-fold over RA alone (see FIG. 37A). This very
robust PDX1 induction was greater than 1400-fold higher than with
no activin or RA/FGF treatment. Interestingly, addition of FGF-4
and FGF-10 simultaneously abolished the beneficial effect of the
FGF-10, producing only the modest PDX1 increase attributed to FGF-4
addition.
[0319] Addition of RA/FGF-4 or RA/FGF-10 combinations did not
increase the expression of marker genes not associated with foregut
endoderm when compared to cells not exposed to RA/FGF combinations
(see FIGS. 37B-37C).
Example 14
Retinoic Acid Dose Affects Anterior-Posterior (A-P) Position In
Vitro
[0320] To determine whether the dose of RA affects A-P position in
in vitro cell cultures, the following experiment was performed.
[0321] Human embryonic stem cells were cultured with or without
activins for four days. On day four, FGF-10 at 50 ng/ml was added
to the culture in combination with RA at 0.04 .mu.M, 0.2 .mu.M or
1.0 .mu.M. The expression of the PDX1 marker gene as well as other
markers not specific for foregut endoderm were quantitated by
Q-PCR.
[0322] The addition of retinoic acid at various doses, in
combination with FGF-10 at 50 ng/ml, induced differential gene
expression patterns that correlate with specific anterior-posterior
positional patterns. The highest dose of RA (1 .mu.M)
preferentially induced expression of anterior endoderm marker
(HOXA3) and also produced the most robust increase in PDX1 (FIGS.
38A38-B). The middle dose of RA (0.2 .mu.M) induced midgut endoderm
markers (CDX1, HOXC6) (see FIGS. 38C and 41E), while the lowest
dose of RA (0.04 .mu.M) preferentially induced a marker of hindgut
endoderm (HOXA13) (see FIG. 38D). The RA dose had essentially no
effect on the relative expression of either neural (SOX1) or
neuronal (NFM) markers (see FIGS. 38F-38G). This example highlights
the use of RA as a morphogen in vitro and in particular as a
morphogen of endoderm derivatives of differentiating hESCs.
Example 15
Use of B27 Supplement Enhances Expression of PDX1
[0323] PDX1 expression in definitive endoderm can be influenced by
the use of a number of factors and cell growth/differentiation
conditions. In the following experiment, we show that the use of
B27 supplement enhances the expression of PDX1 in definitive
endoderm cells.
[0324] Human embryonic stem cells were induced to differentiate to
definitive endoderm by treatment of undifferentiated hES cells
grown on mouse embryonic fibroblast feeders with high dose activin
A (100-200 ng/ml in 0.5-2% FBS/DMEM/F12) for 4 days. The no activin
A control received 0.5-2% 1-BS/DMEM/F12 with no added activin A. At
four days, cultures received either no activin A in 2% FBS (none),
and in 2% serum replacement (SR), or 50 ng/ml activin A together
with 2 .mu.M RA and 50 ng/ml FGF-10 in 2% FBS/DMEM/F12 (none, +FBS,
+B27) and similarly in 2% Serum replacement (SR). B27 supplement,
(Gibco/BRL), was added as a 1/50 dilution directly into 2%
FBS/DMEM/F12 (+B27). Duplicate cell samples where taken for each
point, and total RNA was isolated and subjected to Q-PCR as
previously described.
[0325] FIGS. 39A-39E shows that serum-free supplement B27 provided
an additional benefit for induction of PDX1 gene expression without
inducing an increase in the expression of markers genes not
specific for foregut endoderm as compared to such marker gene
expression in cells grown without serum.
Example 16
Use of Activin B to Enhance Induction of PDX1
[0326] This Example shows that the use of activin B enhances the
differentiation of PDX1-negative cells to PDX1-positive cells in in
vitro cell culture.
[0327] Human embryonic stem cells were induced to differentiate to
definitive endoderm by treatment of undifferentiated hESCs grown on
mouse embryonic fibroblast feeders with high dose activin A (50
ng/ml) in low serum/RPMI for 6 days. The FBS dose was 0% on day
one, 0.2% on day two and 2% on days 3-6. The negative control for
definitive endoderm production (NF) received 2% FBS/RPMI with no
added activin A. In order to induce PDX1 expression, each of the
cultures received retinoic acid at 2 .mu.M in 2% FBS/RPMI on day 6.
The cultures treated with activin A on days one through five were
provided with different dosing combinations of activin A and
activin B or remained in activin A alone at 50 ng/ml. The no
activin A control culture (NF) was provided neither activin A nor
activin B. This RA/activin treatment was carried out for 3 days at
which time PDX1 gene expression was measured by Q-PCR from
duplicate cell samples.
[0328] FIG. 40A shows that the addition of activin B at doses
ranging from 10-50 ng/ml (a10, a25 and a50) in the presence of 25
ng/ml (A25) or 50 ng/ml (A50) of activin A increased the PDX1
expression at least 2-fold over the culture that received only
activin A at 50 ng/ml. The increase in PDX1 as a result of activin
B addition was without increase in HNF6 expression (see FIG. 40B),
which is a marker for liver as well as pancreas at this time in
development. This result suggests that the proportion of cells
differentiating to pancreas had been increased relative to
liver.
Example 17
Use of Serum Dose to Enhance Induction of PDX1
[0329] The expression of PDX1 in definitive endoderm cells is
influenced by the amount of serum present in the cell culture
throughout the differentiation process. The following experiment
shows that the level of serum in a culture during the
differentiation of hESCs to PDX1-negative definitive endoderm has
an effect on the expression of PDX1 during further differentiation
of these cells to PDX1-positive endoderm.
[0330] Human embryonic stem cells were induced to differentiate to
definitive endoderm by treatment of undifferentiated hESCs grown on
mouse embryonic fibroblast feeders with high dose activin A (100
ng/ml) in low serum/RPMI for 5 days. The FBS dose was 0.1% on day
one, 0.5% on day two and either 0.5%, 2% or 10% on days 3-5. The no
activin A control (NF) received the same daily FBS/RPMI dosing, but
with no added activin A. PDX1 expression was induced beginning at
day 6 by the addition of RA. During days 6-7, cultures received
retinoic acid at 2 .mu.M in 0.5% FBS/RPMI, 1 .mu.M on day 8 and 0.2
.mu.M on day 9-11. The activin A was lowered to 50 ng/ml during
retinoic acid treatment and was left absent from the no activin A
control (NF).
[0331] FIG. 41A shows that the FBS dosing during the 3 day period
of definitive endoderm induction (days 3, 4 and 5) had a lasting
ability to change the induction of PDX1 gene expression during the
retinoic acid treatment. This was without significant alteration in
the expression pattern of ZIC1 (FIG. 41B) or SOX7 (FIG. 41C) gene
expression.
Example 18
Use of Conditioned Medium to Enhance Induction of PDX1
[0332] Other factors and growth conditions which influence the
expression of PDX1 in definitive endoderm cells were also studied.
The following experiment shows the effect of conditioned media on
the differentiation of PDX1-negative definitive endoderm cells to
PDX1-positive endoderm cells.
[0333] Human embryonic stem cells were induced to differentiate to
definitive endoderm by treatment of undifferentiated hESCs grown on
mouse embryonic fibroblast feeders with high dose activin A (100
ng/ml) in low serum/RPMI for 5 days. The FBS dose was 0.2% on day
one, 0.5% on day two and 2% on days 3-5.
[0334] The definitive endoderm cultures generated by 5 days of
activin A treatment were then induced to differentiate to PDX1
expressing endoderm by the addition of RA in 2% FBS/RPMI containing
activin A at 25 ng/ml for four days. The RA was 2 .mu.M for the
first two days of addition, 1 .mu.M on the third day and 0.5 .mu.M
on the fourth day. This base medium for PDX1 induction was provided
fresh (2A25R) or after conditioning for 24 hours by one of four
different cell populations. Conditioned media (CM) were generated
from either mouse embryonic fibroblasts (MEFCM) or from hESCs that
were first differentiated for 5 days by one of three conditions; i)
3% FBS/RPMI (CM2), or ii) activin A (CM3) or iii) bone morphogenic
protein 4 (BMP4) (CM4). Activin A or BMP4 factors were provided at
100 ng/ml under the same FBS dosing regimen described above (0.2%,
0.5%, 2%). These three different differentiation paradigms yield
three very different populations of human cells by which the PDX1
induction media can be conditioned. The 3% FBS without added growth
factor (NF) yields a heterogeneous population composed in large
part of extraembryonic endoderm, ectoderm and mesoderm cells. The
activin A treated culture (A100) yields a large proportion of
definitive endoderm and the BMP4 treated culture (B100) yields
primarily trophectoderm and some extraembryonic endoderm.
[0335] FIG. 42A shows that PDX1 was induced equivalently in fresh
and conditioned media over the first two days of RA treatment.
However, by the third day PDX1 expression had started to decrease
in fresh media and MEF conditioned media treatments. The
differentiated hESCs produced conditioned media that resulted in
maintenance or further increases in the PDX1 gene expression at
levels 3 to 4-fold greater than fresh media. The effect of
maintaining high PDX1 expression in hESC-conditioned media was
further amplified on day four of RA treatment achieving levels 6 to
7-fold higher than in fresh media. FIG. 42B shows that the
conditioned media treatments resulted in much lower levels of CDX1
gene expression, a gene not expressed in the region of PDX1
expressing endoderm. This indicates that the overall purity of
PDX1-expressing endoderm was much enhanced by treating definitive
endoderm with conditioned media generated from differentiated hESC
cultures.
[0336] FIG. 43 shows that PDX1 gene expression exhibited a positive
dose response to the amount of conditioned media applied to the
definitive endoderm cells. Total volume of media added to each
plate was 5 ml and the indicated volume (see FIG. 43) of
conditioned media was diluted into fresh media (A25R). It is of
note that just 1 ml of conditioned media added into 4 ml of fresh
media was still able to induce and maintain higher PDX1 expression
levels than 5 ml of fresh media alone. This suggests that the
beneficial effect of conditioned media for induction of PDX1
expressing endoderm is dependent on the release of some substance
or substances from the cells into the conditioned media and that
this substance(s) dose dependently enhances production of
PDX1-expressing endoderm.
Example 19
Validation of Antibodies which Bind to PDX1
[0337] Antibodies that bind to PDX1 are useful tools for monitoring
the induction of PDX1 expression in a cell population. This Example
shows that rabbit polyclonal and IgY antibodies to PDX1 can be used
to detect the presence of this protein.
[0338] In a first experiment, IgY anti-PDX1 (IgY .alpha.-PDX1)
antibody binding to PDX1 in cell lysates was validated by Western
blot analysis. In this analysis, the binding of IgY .alpha.-PDX1
antibody to 50 .mu.g of total cell lysate from MDX12 human
fibroblasts or MDX12 cells transfected 24 hrs previously with a
PDX1 expression vector was compared. The cell lysates separated by
SDS-PAGE, transferred to a membrane by electroblotting, and then
probed with the IgY .alpha.-PDX1 primary antiserum followed by
alkaline phosphatase conjugated rabbit anti-IgY (Rb .alpha.-IgY)
secondary antibodies. Different dilutions of primary and secondary
antibodies were applied to separate strips of the membrane in the
following combinations: A (500.times. dilution of primary,
10,000.times. dilution of secondary), B (2,000.times.,
10,000.times.), C (500.times., 40,000.times.), D (2,000.times.,
40,000), E (8,000.times., 40,000.times.).
[0339] Binding was detected in cells transfected with the PDX1
expression vector (PDX1-positive) at all of the tested antibody
combinations. Binding was only observed in untransfected
(PDX1-negative) fibroblasts when using the highest concentrations
of both primary and secondary antibody together (combination A).
Such non-specific binding was characterized by the detection of an
additional band at a molecular weight slightly higher than PDX1 in
both the transfected and untransfected fibroblasts.
[0340] In a second experiment, the binding of polyclonal rabbit
anti-PDX1 (Rb .alpha.-PDX1) antibody to PDX1 was tested by
immunocytochemistry. To produce a PDX1 expressing cell for such
experiments, MS1-V cells (ATCC # CRL-2460) were transiently
transfected with an expression vector of PDX1-EGFP (constructed
using pEGFP-N1, Clontech). Transfected cells were then labeled with
Rb .alpha.-PDX1 and .alpha.-EGFP antisera. Transfected cells were
visualized by both EGFP fluorescence as well as .alpha.-EGFP
immunocytochemistry through the use of a Cy5 conjugated secondary
antibody. PDX1 immunofluorescence was visualized through the use of
an .alpha.-Rb Cy3-conjugated secondary antibody.
[0341] Binding of the Rb .alpha.-PDX1 and the .alpha.-EGPF
antibodies co-localized with GPF expression.
Example 20
Immunocytochemistry of Human Pancreatic Tissue
[0342] This Example shows that antibodies having specificity for
PDX1 can be used to identify human PDX1-positive cells by
immunocytochemistry.
[0343] In a first experiment, paraffin embedded sections of human
pancreas were stained for insulin with guinea pig anti-insulin (Gp
.alpha.-Ins) primary antibody at a 1/200 dilution followed by dog
anti-guinea pig (D .alpha.-Gp) secondary antibody conjugated to Cy2
at a 1/100 dilution. In a second experiment, the same paraffin
embedded sections of human pancreas were stained for PDX1 with IgY
.alpha.-PDX1 primary antibody at a 1/4000 dilution followed Rb
.alpha.-IgY secondary antibody conjugated to AF555 at a 1/300
dilution. The images collected from the first and second
experiments where then merged. In a third experiment, cells that
were stained with IgY .alpha.-PDX1 antibodies were also stained
with DAPI.
[0344] Analysis of the human pancreatic sections revealed the
presence of strong staining of islets of Langerhans. Although the
strongest PDX1 signal appeared in islets (insulin-positive), weak
staining was also seen in acinar tissue (insulin-negative). DAPI
and PDX1 co-staining shows that PDX1 was mostly but not exclusively
localized to the nucleus.
Example 21
Immunoprecipitation of PDX1 from Retinoic Acid Treated Cells
[0345] To further confirm PDX1 expression in definitive endoderm
cells that have been differentiated in the presence of RA and the
lack of PDX1 in definitive endoderm cells that have not been
differentiated with RA, a rabbit anti-PDX1 (Rb .alpha.-PDX1)
antibody was used to immunoprecipitate PDX1 from both RA
differentiated and undifferentiated definitive endoderm cells
Immunoprecipitated RA was detected by Western blot analysis using
IgY .alpha.-PDX1 antibody.
[0346] To obtain undifferentiated and differentiated definitive
endoderm cell lysates for immunoprecipitation, hESCs were treated
for 5 days with activin A at 100 ng/ml in low serum (definitive
endoderm) followed by treatment with activin A at 50 ng/ml and 2
.mu.M all-trans RA for two days, 1 .mu.M for one day and 0.2 .mu.M
for one day (PDX1-positive foregut endoderm). As a positive control
cell lysates were also prepared from MS1-V cells (ATCC # CRL-2460)
transfected with a PDX1 expression vector. PDX1 was
immunoprecipitated by adding Rb .alpha.-PDX1 and rabbit-specific
secondary antibodies to each lysate. The precipitate was harvested
by centrifugation. Immunoprecipitates were dissolved in
SDS-containing buffer then loaded onto a polyacrylamide gel. After
separation, the proteins were transferred to a membrane by
electroblotting, and then probed with the IgY .alpha.-PDX1 primary
antibody followed by labeled Rb .alpha.-IgY secondary
antibodies.
[0347] Immunoprecipitates collected from the MS1-V positive control
cells as well as those from day 8 (lane d8, three days after the
start of RA treatment) and day 9 (lane d9, four days after the
start of RA) cells were positive for PDX1 protein (FIG. 44).
Precipitates obtained from undifferentiated definitive endoderm
cells (that is, day 5 cells treated with activin A--designated (A)
in FIG. 44) and undifferentiated hESCs (that is, untreated day 5
cells--designated as (NF) in FIG. 44) were negative for PDX1.
Example 22
Generation of PDX1 Promoter-EGFP Transgenic hESC Lines
[0348] In order to use the PDX1 marker for cell isolation, we
genetically tagged PDX1-positive foregut endoderm cells with an
expressible reporter gene. This Example describes the construction
of a vector comprising a reporter cassette which comprises a
reporter gene under the control of the PDX1 regulatory region. This
Example also describes the preparation of a cell, such as a human
embryonic stem cell, transfected with this vector as well as a cell
having this reporter cassette integrated into its genome.
[0349] PDX1-expressing definitive endoderm cell lines genetically
tagged with a reporter gene were constructed by placing a GFP
reporter gene under the control of the regulatory region (promoter)
of the PDX1 gene. First, a plasmid construct in which EGFP
expression is driven by the human PDX1 gene promoter was generated
by replacing the CMV promoter of vector pEGFP-N1 (Clontech) with
the human PDX1 control region (Genbank Accession No. AF192496, the
disclosure of which is incorporated herein by reference in its
entirety), which comprises a nucleotide sequence ranging from about
4.4 kilobase pairs (kb) upstream to about 85 base pairs (bp)
downstream of the PDX1 transcription start site. This region
contains the characterized regulatory elements of the PDX1 gene,
and it is sufficient to confer the normal PDX1 expression pattern
in transgenic mice. In the resulting vector, expression of EFGP is
driven by the PDX1 promoter. In some experiments, this vector can
be transfected into hESCs.
[0350] The PDX1 promoter/EGFP cassette was excised from the above
vector, and then subcloned into a selection vector containing the
neomycin phosphotransferase gene under control of the
phosphoglycerate kinase-1 promoter. The selection cassette was
flanked by flp recombinase recognition sites to allow removal of
the cassette. This selection vector was linearized, and then
introduced into hESCs using standard lipofection methods. Following
10-14 days of selection in G418, undifferentiated transgenic hESC
clones were isolated and expanded.
Example 23
Isolation of PDX1-Positive Foregut Endoderm
[0351] The following Example demonstrates that hESCs comprising the
PDX1 promoter/EGFP cassette can be differentiated into
PDX1-positive endoderm cells and then subsequently isolated by
fluorescence-activated cell sorting (FACS).
[0352] PDX1 promoter/EGFP transgenic hESCs were differentiated for
5 days in activin A-containing media followed by two days in media
comprising activin A and RA. The differentiated cells were then
harvested by trypsin digestion and sorted on a Becton Dickinson
FACS Diva directly into RNA lysis buffer or PBS. A sample of single
live cells was taken without gating for EGFP (Live) and single live
cells were gated into EGFP positive (GFP) and GFP negative (Neg)
populations. In one experiment, the EGFP positive fraction was
separated into two equally sized populations according to
fluorescence intensity (Hi and Lo).
[0353] Following sorting, cell populations were analyzed by both
Q-PCR and immunocytochemistry. For Q-PCR analysis, RNA was prepared
using Qiagen RNeasy columns and then converted to cDNA. Q-PCR was
conducted as described previously. For immunocytochemistry
analysis, cells were sorted into PBS, fixed for 10 minutes in 4%
paraformaldehyde, and adhered to glass slides using a Cytospin
centrifuge. Primary antibodies to Cytokeratin19 (KRT19) were from
Chemicon; to Hepatocyte nuclear factor 3 beta (HNF3.beta.) from
Santa Cruz; to Glucose Transporter 2 (GLUT2) from R&D systems.
Appropriate secondary antibodies conjugated to FITC (green) or
Rhodamine (Red) were used to detect binding of the primary
antibodies.
[0354] A typical FACS sort of differentiated cells is shown in FIG.
45. The percent isolated PDX1-positive cells in this example was
approximately 7%, which varied depending on the differentiation
efficiency from about 1% to about 20%.
[0355] Sorted cells were further subjected to Q-PCR analysis.
Differentiated cells showed a correlation of EGFP fluorescence with
endogenous PDX1 gene expression. Compared to non-fluorescing cells,
the EGFP positive cells showed a greater than 20-fold increase in
PDX1 expression levels (FIG. 46). The separation of high and low
EGFP intensity cells indicated that EGFP expression level
correlated with PDX1 expression level (FIG. 47). In addition to
PDX1 marker analysis, sorted cells were subjected to Q-PCR analysis
of several genes that are expressed in pancreatic endoderm.
Products of each of these marker genes (NKX2.2, GLUT2, KRT19,
HNF4.alpha. and HNF3.beta.) were all enriched in the EGFP positive
fraction (FIGS. 48A-48E). In contrast, the neural markers ZIC1 and
GFAP were not enriched in sorted EGFP expressing cells (FIGS. 49A
and 49B).
[0356] By immunocytochemistry, virtually all the isolated
PDX1-positive cells were seen to express KRT19 and GLUT2. This
result is expected for cells of the pancreatic endoderm lineage.
Many of these cells were also HNF3.beta. positive by antibody
staining.
Example 24
Transplantation of Human Definitive Endoderm Cells Under Mouse
Kidney Capsule
[0357] To demonstrate that the human definitive endoderm cells
produced using the methods described herein are capable of
responding to differentiation factors so as to produce cells that
are derived from the gut tube, such human definitive endoderm cells
were subjected to an in vivo differentiation protocol.
[0358] Human definitive endoderm cells were produced as described
in the foregoing Examples. Such cells were harvested and
transplanted under the kidney capsule of immunocompromised mice
using standard procedures. After three weeks, the mice were
sacrificed and the transplanted tissue was removed, sectioned and
subjected to histological and immunocytochemical analysis.
[0359] FIGS. 50A-50D show that after three weeks
post-transplantation, the human definitive endoderm cells
differentiated into cells and cellular structures derived from the
gut tube. In particular, FIG. 50A shows hematoxylin and eosin
stained sections of transplanted human definitive endoderm tissue
that has differentiated into gut-tube-like structures. FIG. 50B
shows a transplanted human definitive endoderm section
immunostained with antibody to hepatocyte specific antigen (HSA).
This result indicates that the human definitive endoderm cells are
capable of differentiating into liver or liver precursor cells.
FIGS. 50C and 50D show a transplanted human definitive endoderm
section immunostained with antibody to villin and antibody to
caudal type homeobox transcription factor 2 (CDX2), respectively.
These results indicate that the human definitive endoderm cells are
capable of differentiating into intestinal cells or intestinal cell
precursors.
Example 25
Identification of Differentiation Factors Capable of Promoting the
Differentiation of Human Definitive Endoderm Cells In Vitro
[0360] To exemplify the differentiation factor screening methods
described herein, populations of human definitive endoderm cells
produced using the methods described herein were separately
provided with several candidate differentiation factors while
determining the normalized expression levels of certain marker gene
products at various time points.
[0361] Human definitive endoderm cells were produced as described
in the foregoing Examples. In brief, hESCs cells were grown in the
presence of 100 ng/ml activin A in low serum RPMI medium for four
days, wherein the fetal bovine serum (FBS) concentration on day 1
was 0%, on day 2 was 0.2% and on days 3-4 was 2%. After formation
of definitive endoderm, beginning on day 5 and ending on day 10,
cell populations maintained in individual plates in RPMI containing
0.2% FBS were treated with one of: Wnt3B at 20 ng/ml, FGF2 at 5
ng/ml or FGF2 at 100 ng/ml. The expression of marker gene products
for albumin, PROX1 and TITF1 were quantitated using Q-PCR.
[0362] FIG. 51A shows that expression of the albumin gene product
(a marker for liver precursors and liver cells) substantially
increased on days 9 and 10 in response to FGF2 at 5 ng/ml as
compared to expression in definitive endoderm cells on day 4 prior
to treatment with this differentiation factor. Expression of the
albumin gene product was also increased in response to 20 ng/ml
Wnt3B on days 9 and 10 as compared to expression in untreated
definitive endoderm cells, however, the increase was not as large
as that observed for the 5 ng/ml FGF2 treatment. Of particular
significance is the observation that the expression of the albumin
gene product was not increased on days 9 and 10 in response to FGF2
at 100 ng/ml as compared to expression in definitive endoderm cells
on day 4. Similar results were seen with the PROX1 marker (a second
marker for liver precursors and liver cells) as shown in FIG. 51B.
FIG. 51C shows that in cell populations provided with 100 ng/ml
FGF2, expression of the TITF1 marker gene substantially increased
on days 7, 9 and 10 as compared to expression in definitive
endoderm cells on day 4 prior to treatment with this
differentiation factor, but FGF2 at 5 ng/ml had very little effect
on expression of this gene product as compared to untreated
definitive endoderm. Taken together, the results shown in FIGS.
51A-51C indicate that the concentration at which the candidate
differentiation factor is provided to the cell population can
affect the differentiation fate of definitive endoderm cells in
vitro.
Example 26
Marker Upregulation and Downregulation in Response to Candidate
Differentiation Factors
[0363] To further exemplify the differentiation factor screening
methods described herein, populations of human definitive endoderm
cells were screened with candidate differentiation factors using
procedures similar to those described in Example 25.
[0364] Human definitive endoderm cells were produced as described
in the foregoing Examples. In brief, hESCs cells were grown in the
presence of 100 ng/ml activin A in low serum RPMI medium for four
days, wherein the fetal bovine serum (FBS) concentration on day 1
was 0%, on day 2 was 0.2% and on days 3-4 was 2%. After formation
of definitive endoderm, beginning on day 5 and ending on day 10,
cell populations maintained in individual plates in RPMI containing
0.2% FBS were treated with one of: Wnt3A at 20-50 ng/ml, FGF2 at 5
ng/ml or FGF2 at 100 ng/ml. On day 5 post definitive endoderm
formation (day 9 after the start of the differentiation from
hESCs), BMP4 was added to all the cultures at a concentration of 50
ng/ml. The expression of marker gene products (mRNAs) for alpha
fetoprotein (AFP), cytochrome P450 7A (CYP7A), tyrosine
aminotransferase (TAT), hepatocyte nuclear factor 4a (HNF4.alpha.),
CXC-type chemokine receptor 4 (CXCR4), von Willebrand factor (VWF),
vascular cell adhesion molecule-1 (VACM1), apolipoprotein A1
(APOA1), glucose transporter-2 (GLUT2), alpha-1-antitrypsin (AAT),
glukokinase (GLUKO), and human hematopoietically expressed homeobox
(hHEX) were quantitated using Q-PCR.
[0365] FIGS. 52A-52B show that expression of the AFP gene product
(a marker for liver precursors and liver cells) and AAT
substantially increased on days 9 and 10 in response to FGF2 at 5
ng/ml and BMP4 at 50 ng/ml as compared to expression in definitive
endoderm cells on day 4. Expression of AFP and AAT mRNAs was not
substantially increased by higher concentration of FGF2 (100 ng/ml)
even in the presence of BMP4 (FIGS. 51A-51B days 9 and 10). In
contrast to the above results, the expression of GLUKO, hHEX and
TAT mRNAs was substantially upregulated in the presence of FGF2 at
100 ng/ml and BMP4 at 50 ng/ml on days 9 and 10 as compared to
expression in definitive endoderm cells on day 4. In the case of
GLUKO, neither Wnt3A nor FGF2 at 5 ng/ml with or without BMP4
caused an increase in the expression of this marker (FIG. 52C).
FGF2 at 5 ng/ml did, however, cause an increase in expression of
hHEX in the presence of BMP to an extent greater than or equal to
the increase caused by FGF2 at 100 ng/ml in the presence of BMP
(FIG. 52D). Expression of TAT on days 9 and 10 as compared to
expression in definitive endoderm cells was increased by each of
the factors tested (FIG. 52E). Additionally, certain cell markers
were expressed at an increased level as compared to definitive
endoderm cells in the presence of Wnt3A, but not in response to
FGF/BMP combinations. In particular, the expression of hNF4a mRNA
significantly increased on days 9 and 10 in response to the
combination of Wnt3A and BMP4 (FIG. 52F). Furthermore, CYP7A showed
a marginal increase on response to Wnt3A/BMP4 on day 10 (FIG.
52G).
[0366] Several markers that are known to be expressed in a number
of different cells types were also observed. Specifically the
markers APOA1, GLUT2, VCAM1, VWF and CXCR4 were examined.
Previously the expression of each of these markers has been
correlated with specific cell types as follows: The markers APOA1
and GLUT2 are highly expressed in the liver and moderately
expressed in the duodenum and small intestine. The marker VCAM1 is
expressed at a high level in the liver, expressed at a moderate
level in the stomach, duodenum, and small intestine, and expressed
at lower but significant levels in the lung and pancreas. In
contrast, the markers VWF and CXCR4 are expressed at high levels in
the lung but only at low levels in liver. Both VWF and CXCR4 are
also expressed at moderate to high levels in the stomach, pancreas,
duodenum, and small intestine.
[0367] Expression of each of the above-described markers was
monitored in definitive endoderm cell cultures contacted with
combinations of Wnt3A, FGF2 and BMP4. Consistent with the above
results, FIGS. 52H-52J show that GLUT2, APOA1 and VCAM1 mRNA
expression was increased in response to the combination of FGF2 at
5 ng/ml and BMP4 on days 9 and 10 as compared to the expression in
definitive endoderm. The mRNA expression for these markers was not
substantially increased in response to the combination of FGF2 at
100 ng/ml an BMP4. In the case of the APOA1 and VCAM1 marker mRNAs,
the largest increase in expression on days 9 and 10 was mediated by
the combination of Wnt3A and BMP4 (FIGS. 52I-52J).
[0368] In addition to the foregoing, the expression of certain
mRNAs was decreased as compared to the expression in definitive
endoderm. For example, as compared to the expression in definitive
endoderm, both VWF and CXCR4 mRNA expression was decreased after
contact with Wnt3A in the presence and in the absence of BMP4 as
well as after contact with FGF2 at 5 ng/ml in the presence and in
the absence of BMP4 (FIGS. 52K-52L). Contact with FGF2 at 100
ng/ml, both in the absence and in the presence of BMP4, greatly
slowed the rate of decrease of these two markers (FIGS. 52K-52L).
In fact, expression of CXCR4 was substantially maintained even on
day 10 (FIG. 52L).
[0369] The methods, compositions, and devices described herein are
presently representative of preferred embodiments and are exemplary
and are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention and
are defined by the scope of the disclosure. Accordingly, it will be
apparent to one skilled in the art that varying substitutions and
modifications may be made to the invention disclosed herein without
departing from the scope and spirit of the invention.
[0370] As used in the claims below and throughout this disclosure,
by the phrase "consisting essentially of" is meant including any
elements listed after the phrase, and limited to other elements
that do not interfere with or contribute to the activity or action
specified in the disclosure for the listed elements. Thus, the
phrase "consisting essentially of" indicates that the listed
elements are required or mandatory, but that other elements are
optional and may or may not be present depending upon whether or
not they affect the activity or action of the listed elements.
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Sequence CWU 1
1
211245DNAHomo sapiens 1atgagcagcc cggatgcggg atacgccagt gacgaccaga
gccagaccca gagcgcgctg 60cccgcggtga tggccgggct gggcccctgc ccctgggccg
agtcgctgag ccccatcggg 120gacatgaagg tgaagggcga ggcgccggcg
aacagcggag caccggccgg ggccgcgggc 180cgagccaagg gcgagtcccg
tatccggcgg ccgatgaacg ctttcatggt gtgggctaag 240gacgagcgca
agcggctggc gcagcagaat ccagacctgc acaacgccga gttgagcaag
300atgctgggca agtcgtggaa ggcgctgacg ctggcggaga agcggccctt
cgtggaggag 360gcagagcggc tgcgcgtgca gcacatgcag gaccacccca
actacaagta ccggccgcgg 420cggcgcaagc aggtgaagcg gctgaagcgg
gtggagggcg gcttcctgca cggcctggct 480gagccgcagg cggccgcgct
gggccccgag ggcggccgcg tggccatgga cggcctgggc 540ctccagttcc
ccgagcaggg cttccccgcc ggcccgccgc tgctgcctcc gcacatgggc
600ggccactacc gcgactgcca gagtctgggc gcgcctccgc tcgacggcta
cccgttgccc 660acgcccgaca cgtccccgct ggacggcgtg gaccccgacc
cggctttctt cgccgccccg 720atgcccgggg actgcccggc ggccggcacc
tacagctacg cgcaggtctc ggactacgct 780ggccccccgg agcctcccgc
cggtcccatg cacccccgac tcggcccaga gcccgcgggt 840ccctcgattc
cgggcctcct ggcgccaccc agcgcccttc acgtgtacta cggcgcgatg
900ggctcgcccg gggcgggcgg cgggcgcggc ttccagatgc agccgcaaca
ccagcaccag 960caccagcacc agcaccaccc cccgggcccc ggacagccgt
cgccccctcc ggaggcactg 1020ccctgccggg acggcacgga ccccagtcag
cccgccgagc tcctcgggga ggtggaccgc 1080acggaatttg aacagtatct
gcacttcgtg tgcaagcctg agatgggcct cccctaccag 1140gggcatgact
ccggtgtgaa tctccccgac agccacgggg ccatttcctc ggtggtgtcc
1200gacgccagct ccgcggtata ttactgcaac tatcctgacg tgtga
12452414PRTHomo sapiens 2Met Ser Ser Pro Asp Ala Gly Tyr Ala Ser
Asp Asp Gln Ser Gln Thr1 5 10 15 Gln Ser Ala Leu Pro Ala Val Met
Ala Gly Leu Gly Pro Cys Pro Trp 20 25 30 Ala Glu Ser Leu Ser Pro
Ile Gly Asp Met Lys Val Lys Gly Glu Ala 35 40 45 Pro Ala Asn Ser
Gly Ala Pro Ala Gly Ala Ala Gly Arg Ala Lys Gly 50 55 60 Glu Ser
Arg Ile Arg Arg Pro Met Asn Ala Phe Met Val Trp Ala Lys65 70 75 80
Asp Glu Arg Lys Arg Leu Ala Gln Gln Asn Pro Asp Leu His Asn Ala 85
90 95 Glu Leu Ser Lys Met Leu Gly Lys Ser Trp Lys Ala Leu Thr Leu
Ala 100 105 110 Glu Lys Arg Pro Phe Val Glu Glu Ala Glu Arg Leu Arg
Val Gln His 115 120 125 Met Gln Asp His Pro Asn Tyr Lys Tyr Arg Pro
Arg Arg Arg Lys Gln 130 135 140 Val Lys Arg Leu Lys Arg Val Glu Gly
Gly Phe Leu His Gly Leu Ala145 150 155 160 Glu Pro Gln Ala Ala Ala
Leu Gly Pro Glu Gly Gly Arg Val Ala Met 165 170 175 Asp Gly Leu Gly
Leu Gln Phe Pro Glu Gln Gly Phe Pro Ala Gly Pro 180 185 190 Pro Leu
Leu Pro Pro His Met Gly Gly His Tyr Arg Asp Cys Gln Ser 195 200 205
Leu Gly Ala Pro Pro Leu Asp Gly Tyr Pro Leu Pro Thr Pro Asp Thr 210
215 220 Ser Pro Leu Asp Gly Val Asp Pro Asp Pro Ala Phe Phe Ala Ala
Pro225 230 235 240 Met Pro Gly Asp Cys Pro Ala Ala Gly Thr Tyr Ser
Tyr Ala Gln Val 245 250 255 Ser Asp Tyr Ala Gly Pro Pro Glu Pro Pro
Ala Gly Pro Met His Pro 260 265 270 Arg Leu Gly Pro Glu Pro Ala Gly
Pro Ser Ile Pro Gly Leu Leu Ala 275 280 285 Pro Pro Ser Ala Leu His
Val Tyr Tyr Gly Ala Met Gly Ser Pro Gly 290 295 300 Ala Gly Gly Gly
Arg Gly Phe Gln Met Gln Pro Gln His Gln His Gln305 310 315 320 His
Gln His Gln His His Pro Pro Gly Pro Gly Gln Pro Ser Pro Pro 325 330
335 Pro Glu Ala Leu Pro Cys Arg Asp Gly Thr Asp Pro Ser Gln Pro Ala
340 345 350 Glu Leu Leu Gly Glu Val Asp Arg Thr Glu Phe Glu Gln Tyr
Leu His 355 360 365 Phe Val Cys Lys Pro Glu Met Gly Leu Pro Tyr Gln
Gly His Asp Ser 370 375 380 Gly Val Asn Leu Pro Asp Ser His Gly Ala
Ile Ser Ser Val Val Ser385 390 395 400 Asp Ala Ser Ser Ala Val Tyr
Tyr Cys Asn Tyr Pro Asp Val 405 410
* * * * *