U.S. patent application number 11/588693 was filed with the patent office on 2007-05-31 for pdx1-expressing dorsal and ventral foregut endoderm.
Invention is credited to Alan D. Agulnick, E. Edward Baetge, Kevin Allen D'Amour, Susan Eliazer.
Application Number | 20070122905 11/588693 |
Document ID | / |
Family ID | 37769419 |
Filed Date | 2007-05-31 |
United States Patent
Application |
20070122905 |
Kind Code |
A1 |
D'Amour; Kevin Allen ; et
al. |
May 31, 2007 |
PDX1-expressing dorsal and ventral foregut endoderm
Abstract
Disclosed herein are cell cultures comprising dorsal and/or
ventral PDX1-positive foregut endoderm cells and methods of
producing the same. Also disclosed herein are cell populations
comprising substantially purified dorsal and/or ventral
PDX1-positive foregut endoderm cells as well as methods for
enriching, isolating and purifying dorsal and/or ventral
PDX1-positive foregut endoderm cells from other cell types. Methods
of identifying differentiation factors capable of promoting the
differentiation of dorsal and/or ventral PDX1-positive foregut
endoderm cells, are also disclosed.
Inventors: |
D'Amour; Kevin Allen; (San
Diego, CA) ; Agulnick; Alan D.; (San Diego, CA)
; Eliazer; Susan; (San Diego, CA) ; Baetge; E.
Edward; (Encinitas, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
37769419 |
Appl. No.: |
11/588693 |
Filed: |
October 27, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60730917 |
Oct 27, 2005 |
|
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Current U.S.
Class: |
435/366 |
Current CPC
Class: |
C12N 2501/117 20130101;
C12N 5/068 20130101; C12N 2501/119 20130101; C12N 5/0678 20130101;
C12N 2501/16 20130101; C12N 2510/00 20130101; C12N 5/0603 20130101;
C12N 2501/415 20130101; C12N 2501/41 20130101; C12N 2506/02
20130101; C12N 2501/405 20130101; C12N 5/0676 20130101; C12N
2501/155 20130101; C07K 16/18 20130101; C12N 2502/13 20130101; C12N
2501/385 20130101; C12N 5/0606 20130101; C12N 2500/38 20130101 |
Class at
Publication: |
435/366 |
International
Class: |
C12N 5/08 20060101
C12N005/08 |
Claims
1. A cell culture comprising human cells wherein at least about 26%
of said human cells are pancreatic-duodenal homoebox factor-1
(PDX1) positive, dorsally-biased, foregut endoderm cells that
express at least one marker selected from the group consisting of
CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2,
SLC27A2, SERPINF2, DUSP9, CDH6 and SOX9, said PDX1-positive,
dorsally-biased, foregut endoderm cells being multipotent cells
that can differentiate into cells of the dorsal pancreatic bud.
2. The cell culture of claim 1, wherein said marker is selected
from the group consisting of SERPINF2, DUSP9, CDH6 and SOX9.
3. The cell culture of claim 1, wherein said marker is a cell
surface marker selected from the group consisting of CDH6, GABRA2,
GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.
4. The cell culture of claim 1, wherein said PDX1-positive,
dorsally-biased, foregut endoderm cells express at least one marker
selected from the group consisting of ADORA2A, CD47, EPB41L1, MAG,
SFRP5, SLC16AI0, SLC16A2, SLCIA3, SLC3OA4, SLICK, SLITRK4, XPR1,
HOXA1, PDEI IA, FAM49A and WNT5A.
5. The cell culture of claim 4, wherein said marker is selected
from the group consisting of HOXAI, PDEI IA, FAM49A and WNT5A.
6. The cell culture of claim 4, wherein said marker is a cell
surface marker selected from the group consisting of ADORA2A, CD47,
EPB41L1, MAG, SFRP5, SLC16A10, SLC16A2, SLCIA3, SLC3OA4, SLICK,
SLITRK4 and XPRI.
7. The cell culture of claim 1, wherein at least about 30% of said
human cells are PDX1-positive, dorsally-biased, foregut endoderm
cells.
8. The cell culture of claim 1, wherein at least about 40% of said
human cells are PDX1-positive, dorsally-biased, foregut endoderm
cells.
9. The cell culture of claim 1, wherein at least about 50% of said
human cells are PDX1-positive, dorsally-biased, foregut endoderm
cells.
10. The cell culture of claim 1, wherein at least about 60% of said
human cells are PDX1-positive, dorsally-biased, foregut endoderm
cells.
11. The cell culture of claim 1, wherein at least about 75% of said
human cells are PDX1-positive, dorsally-biased, foregut endoderm
cells.
12. The cell culture of claim I, wherein human feeder cells are
present in said culture, and wherein at least about 2% of human
cells other than said human feeder cells are PDX1-positive,
dorsally biased, foregut endoderm cells.
13. The cell culture of claim 1, wherein the expression of PDX1 is
greater than the expression of a marker selected from the group
consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in
said PDX1-positive, dorsally biased, foregut endoderm cells.
14. The cell culture of claim 1, wherein said cell culture is
substantially free of cells selected from the group consisting of
visceral endodermal cells, parietal endodermal cells and neural
cells.
15. The cell culture of claim I further comprising a retinoid.
16. The cell culture of claim 15, wherein said retinoid is retinoic
acid (RA).
17. The cell culture of claim 16 further comprising B27.
18. A cell culture comprising human cells wherein at least about 2%
of said human cells are pancreatic-duodenal homoebox factor-.beta.
(PDX1) positive, ventrally-biased, foregut endoderm cells that
express at least one marker is selected from the group consisting
of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG,
SERPINF2, SLC27A2, SERPINF2, DUSP9, CDH6 and SOX9, said
PDX1-positive, ventrally-biased, foregut endoderm cells being
multipotent cells that can differentiate into cells of the ventral
pancreatic bud.
19. The cell culture of claim 18, wherein said marker is selected
from the group consisting of SERPINF2, DUSP9, CDH6 and SOX9.
20. The cell culture of claim 18, wherein said marker is a cell
surface marker selected from the group consisting of CDH6, GABRA2,
GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPFNF2 and SLC27A2.
21. The cell culture of claim 18, wherein said PDX1-positive,
ventrally-biased, foregut endoderm cells do not substantially
express one or more markers selected from the group consisting of
ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16AI0, SLC16A2, SLCIA3,
SLC3OA4, SLICK, SLITRK4, XPRI, HOXAI, PDEI IA, FAM49A and
WNT5A.
22. The cell culture of claim 18, wherein at least about 5% of said
human cells are PDX1-positive, ventrally-biased, foregut endoderm
cells.
23. The cell culture of claim 18, wherein at least about 10% of
said human cells are PDX1-positive, ventrally-biased, foregut
endoderm cells.
24. The cell culture of claim 18, wherein at least about 25% of
said human cells are PDX1-positive, ventrally-biased, foregut
endoderm cells.
25. The cell culture of claim 18, wherein at least about 50% of
said human cells are PDX1-positive, ventrally-biased, foregut
endoderm cells.
26. The cell culture of claim 18, wherein at least about 75% of
said human cells are PDX1-positive, ventrally-biased, foregut
endoderm cells.
27. The cell culture of claim 18, wherein human feeder cells are
present in said culture, and wherein at least about 2% of human
cells other than said human feeder cells are PDX1-positive,
ventrally-biased, foregut endoderm cells.
28. The cell culture of claim 18, wherein the expression of PDX1 is
greater than the expression of a marker selected from the group
consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in
said PDX1-positive, ventrally-biased, foregut endoderm cells.
29. The cell culture of claim 18, wherein said cell culture is
substantially free of cells selected from the group consisting of
visceral endodermal cells, parietal endodermal cells and neural
cells.
30. The cell culture of claim 18 further comprising a retinoid.
31. The cell culture of claim 30, wherein said retinoid is retinoic
acid (RA).
32. The cell culture of claim 31 further comprising B27.
33. A cell population comprising cells wherein at least about 90%
of said cells are human PDX1-positive, dorsally-biased, foregut
endoderm cells that express at least one marker selected from the
group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3,
LGALS3/GALIG, SERPINF2, SLC27A2, SERPINF2, DUSP9, CDH6 and SOX9,
said PDX1-positive, dorsally-biased, foregut endoderm cells being
multipotent cells that can differentiate into cells of the dorsal
pancreatic bud.
34. The cell population of claim 33, wherein said marker is
selected from the group consisting of SERPINF2, DUSP9, CDH6 and
SOX9.
35. The cell population of claim 33, wherein said marker is a cell
surface marker selected from the group consisting of CDH6, GABRA2,
GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.
36. The cell population of claim 33, wherein said PDX1-positive,
dorsally-biased, foregut endoderm cells express at least one marker
selected from the group consisting of ADORA2A, CD47, EPB41LI, MAG,
SFRP5, SLC16A10, SLC16A2, SLCIA3, SLC3OA4, SLICK, SLITRK4, XPRI,
HOXAI, PDEI IA, FAM49A and WNT5A.
37. The cell population of claim 36, wherein said marker is
selected from the group consisting of HOXA I, PDE I IA, FAM49A and
WNT5A.
38. The cell population of claim 36, wherein said marker is a cell
surface marker selected from the group consisting of ADORA2A, CD47,
EPB41L1, MAG, SFRP5, SLC16A10, SLCI6A2, SLCIA3, SLC3OA4, SLICK,
SLITRK4 and XPRI.
39. The cell population of claim 33, wherein at least about 95% of
said cells are PDX1-positive, dorsally-biased, foregut endoderm
cells.
40. The cell population of claim 33, wherein at least about 98% of
said cells are PDX1-positive, dorsally-biased, foregut endoderm
cells.
41. The cell population of claim 33, wherein the expression of PDX1
is greater than the expression of a marker selected from the group
consisting of AFP, SOX7, SOX1, ZIC1 and NFM in said PDX1-positive,
dorsally-biased, foregut endoderm cells.
42. A cell population comprising cells wherein at least about
90%.of said cells are human PDX1-positive, ventrally-biased,
foregut endoderm cells that express at least one marker selected
from the group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2,
LGALS3, LGALS3/GALIG, SERPINF2, SLC27A2, SERPINF2, DUSP9, CDH6 and
SOX9, said PDX1-positive, ventrally-biased, foregut endoderm cells
being multipotent cells that can differentiate into cells of the
ventral pancreatic bud.
43. The cell population of claim 42, wherein said marker is
selected from the group consisting of SERPINF2, DUSP9, CDH6 and
SOX9.
44. The cell population of claim 42, wherein said marker is a cell
surface marker selected from the group consisting of CDH6, GABRA2,
GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.
45. The cell population of claim 42, wherein said PDX1-positive,
ventrally-biased, foregut endoderm cells do not substantially
express one or more markers selected from the group consisting of
ADORA2A, CD47, EPB41LI, MAG, SFRP5, SLC16AI0, SLC16A2, SLCIA3,
SLC3OA4, SLICK, SLITRK4, XPRI, HOXA1, PDEI IA, FAM49A and
WNT5A.
46. The cell population of claim 42, wherein at least about 95% of
said cells are PDX1-positive, ventrally-biased, foregut endoderm
cells.
47. The cell population of claim 42, wherein at least about 98% of
said cells are PDX1-positive, ventrally-biased, foregut endoderm
cells.
48. The cell population of claim 42, wherein the expression of PDX1
is greater than the expression of a marker selected from the group
consisting of AFP, SOX7, SOX I, ZIC1 and NFM in said PDX1-positive,
ventrally-biased, foregut endoderm cells.
Description
RELATED APPLICATIONS
[0001] This application is a nonprovisional application of and
claims priority to U.S. Provisional Patent Application No.
60/730,917, entitled PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT
ENDODERM, filed Oct. 27, 2005, the disclosure of which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the fields of medicine and
cell biology. In particular, the present invention relates to
compositions comprising mammalian foregut endoderm cells and
compositions comprising dorsal and/or ventral PDX1-positive foregut
endoderm cells and methods of making, isolating and using such
cells.
BACKGROUND
[0003] 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).
[0004] 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 x 108 pancreatic
islet cells. (Shapiro et al., 2000; Shapiro et al., 2001 a; 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.
[0005] 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, ectoderrn) 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.
[0006] 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. For example, in
order to achieve the level of cellular material required for islet
cell transplantation therapy, it would be advantageous to
efficiently direct hESCs toward the pancreatic islet/.beta.-cell
lineage at the very earliest stages of differentiation.
[0007] In addition to efficient direction of the differentiation
process, it would also be beneficial to isolate and characterize
intermediate cell types along the differentiation pathway towards
the pancreatic islet/.beta.-cell lineage and to use such cells as
appropriate lineage precursors for further steps in the
differentiation.
SUMMARY OF THE INVENTION
[0008] Embodiments of the present invention relate to cell cultures
of PDX1-negative foregut endoderm cells (foregut endoderm cells).
In some embodiments, the foregut endoderm expresses the HNF1b and
FOXA1 markers but does not substantially express PDX1. Other
embodiments of the present invention relate to cell cultures of
PDX1-positive, dorsally-biased, foregut endoderm cells (dorsal
PDX1-positive foregut endoderm cells). In some embodiments, the
PDX1-positive, dorsally-biased, foregut endoderm cells express one
or more markers selected from Table 3 and/or one or more markers
selected from Table 4. Additional embodiments, relate to cell
cultures of PDX1-positive, ventrally-biased, foregut endoderm cells
(ventral PDX1-positive foregut endoderm cells). In some
embodiments, the PDX1-positive, ventrally-biased, foregut endoderm
cells express one or more markers selected from Table 3 but do not
substantially express a marker selected from Table 4 as compared to
the expression of the same marker in PDX1-positive,
dorsally-biased, foregut endoderm cells.
[0009] Additional embodiments of the present invention relate to
enriched, isolated and/or purified cell populations comprising
PDX1-negative foregut endoderm cells. Other embodiments relate to
PDX1-positive, dorsally-biased, foregut endoderm cells. Still other
embodiments relate to enriched, isolated and/or purified cell
populations comprising PDX1-positive, ventrally-biased, foregut
endoderm cells.
[0010] Aspects of the present invention also relate to methods or
processes for the production of cell cultures of PDX1-negative
foregut endoderm cells from definitive endoderm cells. Such
processes include reducing or eliminating TGF.beta. superfamily
growth factor signaling in a cell culture or cell population of
definitive endoderm cells. In some embodiments, reducing or
eliminating TGF.beta. superfamily growth factor signaling is
mediated by diluting or removing an exogenously added TGF.beta.
superfamily growth factor, such as activin A, from the cell culture
or cell population of definitive endoderm. In some embodiments,
differentiation of definitive endoderm cells to foregut endoderm
cells is enhanced by providing the definitive endoderm cell culture
or cell population with an FGF-family growth factor and/or a
hedgehog pathway inhibitor. In some embodiments, the definitive
endoderm cells are derived from stem cells. Preferably, the stem
cells are embryonic stem cells. Even more preferably, the stem
cells are human embryonic stem cells (hESCs). In some embodiments,
the PDX1-negative foregut endoderm cells are differentiated
to-PDX1-positive endoderm cells (pancreatic endoderm cells) by the
addition of a retinoid, such as retinoic acid. Other aspects relate
to methods or processes for the production of cell cultures of
PDX1-positive, dorsally-biased, foregut endoderm cells. Such
processes include providing definitive endoderm cells with retinoic
acid. In some embodiments, the definitive endoderm cells are
derived from stem cells. Preferably, the stem cells are embryonic
stem cells. Even more preferably, the stem cells are human
embryonic stem cells (hESCs). Further aspects of the present
invention relate to methods or processes for the production of cell
cultures of PDX1-positive, ventrally-biased, foregut endoderm
cells. Such processes include providing. definitive endoderm cells
with an FGF-family growth factor. In some embodiments, the
definitive endoderm cells are derived from stem cells. Preferably,
the stem cells are embryonic stem cells. Even more preferably, the
stem cells are hESCs.
[0011] Additional embodiments of the present invention relate to
methods of enriching, isolating and/or purifying PDX1-negative
foregut endoderm cells. In such embodiments, PDX1-negative foregut
endoderm cells are separated from other cells in the cell
population by using an antibody, ligand or other molecule that
binds to a molecule that is expressed on the cell surface of
PDX1-negative foregut endoderm cells, such as a cell surface
molecule. Other embodiments of the present invention relate to
methods of enriching, isolating and/or purifying PDX1-positive,
dorsally-biased, foregut endoderm cells. In such embodiments,
PDX1-positive, dorsally-biased, foregut endoderm cells are
separated from other cells in the cell population by using an
antibody, ligand or other molecule that binds to a molecule that is
expressed on the cell surface of PDX1-positive, dorsally-biased,
foregut endoderm cells, such as a cell surface molecule selected
from Table 3 or a cell surface molecule selected from Table 4.
Still other embodiments of the present invention relate to methods
of enriching, isolating and/or purifying PDX1-positive,
ventrally-biased, foregut endoderm cells. In such embodiments,
PDX1-positive, ventrally-biased, foregut endoderm cells are
separated from other cells in the cell population by using an
antibody, ligand or other molecule that binds to a molecule that is
expressed on the cell surface of PDX1-positive, ventrally-biased,
foregut endoderm cells, such as a cell surface molecule selected
from Table 3.
[0012] Embodiments of the present invention relate to additional
methods of enriching, isolating and/or purifying PDX1-negative
foregut endoderm cells. In such embodiments, pluripotent or
multipotent cells that are precursors to PDX1-negative foregut
endoderm cells are engineered to contain a fluorescent reporter
gene under control of a promoter that endogenously controls the
expression of a marker gene such as HNF1b or FOXA1. The
fluorescently-tagged PDX1-negative foregut endoderm cells are then
separated from other cells in the cell population by fluorescence
activated cell sorting (FACS). Still other embodiments of the
present invention relate to additional methods of enriching,
isolating and/or purifying PDX1-positive, dorsally-biased, foregut
endoderm cells. In such embodiments, pluripotent or multipotent
PDX1-negative cells that are precursors to PDX1-positive cells are
engineered to contain a fluorescent reporter gene under control of
a promoter that endogenously controls the expression of a marker
gene selected from Table 3 or Table 4. The fluorescently-tagged
PDX1-positive, dorsally-biased, foregut endoderm cells are then
separated from other cells in the cell population by fluorescence
activated cell sorting (FACS). Yet other embodiments of the present
invention relate to additional methods of enriching, isolating
and/or purifying PDX1-positive, ventrally-biased, foregut endoderm
cells. In such embodiments, pluripotent or multipotent
PDX1-negative cells that are precursors to PDX1-positive cells are
engineered to contain a fluorescent reporter gene under control of
a promoter that endogenously controls the expression of a marker
gene selected from Table 3. The fluorescently-tagged PDX1-positive,
ventrally-biased, foregut endoderm cells are then separated from
other cells in the cell population by FACS.
[0013] Further embodiments of the present invention relate to
methods of identifying a differentiation factor capable of
promoting the differentiation of human PDX1-negative foregut
endoderm cells in a cell population comprising human cells. The
method includes the steps of obtaining a cell population comprising
human PDX1-negative foregut endoderm cells, providing a candidate
differentiation factor to the cell population, determining
expression of a marker, such as HNF1b, FOXA1 or PDX1, in the cell
population at a first time point and determining expression of the
same marker in the cell population at a second time point. In such
embodiments, the second time point is subsequent to the first time
point and the second time point is subsequent to providing the cell
population with the candidate differentiation factor. If expression
of the marker in the cell population at the second time point is
increased or decreased as compared to the expression of the marker
in the cell population at the first time point, then the candidate
differentiation factor is capable of promoting the differentiation
of the human PDX1-negative foregut endoderm cells. Still further
embodiments of the present invention relate to methods of
identifying a differentiation factor capable of promoting the
differentiation of human PDX1-positive, dorsally-biased, foregut
endoderm cells in a cell population comprising human cells. The
method includes the steps of obtaining a cell population comprising
human PDX1-positive, dorsally-biased, foregut endoderm cells,
providing a candidate differentiation factor to the cell
population, determining expression of a marker, such as a marker
selected from Table 3 or a marker selected from Table 4, in the
cell population at a first time point and determining expression of
the same marker in the cell population at a second time point. In
such embodiments, the second time point is subsequent to the first
time point and the second time point is subsequent to providing the
cell population with the candidate differentiation factor. If
expression of the marker in the cell population at the second time
point is increased or decreased as compared to the expression of
the marker in the cell population at the first time point, then the
candidate differentiation factor is capable of promoting the
differentiation of the human PDX1-positive, dorsally-biased,
foregut endoderm cells. Yet further embodiments of the present
invention relate to methods of identifying a differentiation factor
capable of promoting the differentiation of human PDX1-positive,
ventrally-biased, foregut endoderm cells in a cell population
comprising human cells. The method includes the steps of obtaining
a cell population comprising human PDX1-positive, ventrally-biased,
foregut endoderm cells, providing a candidate differentiation
factor to the cell population, determining expression of a marker,
such as a marker selected from Table 3, in the cell population at a
first time point and determining expression of the same marker in
the cell population at a second time point. In such embodiments,
the second time point is subsequent to the first time point and the
second time point is subsequent to providing the cell population
with the candidate differentiation factor. If expression of the
marker in the cell population at the second time point is increased
or decreased as compared to the expression of the marker in the
cell population at the first time point, then the candidate
differentiation factor is capable of promoting the differentiation
of the human PDX1-positive, ventrally-biased, foregut endoderm
cells.
[0014] 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:
[0015] 1. A cell culture comprising human cells wherein at least
about 26% of said human cells are pancreatic-duodenal homoebox
factor-1 (PDX1) positive, dorsally-biased, foregut endoderm cells
that express at least one marker selected from Table 3, said
PDX1-positive, dorsally-biased, foregut endoderm cells being
multipotent cells that can differentiate into cells of the dorsal
pancreatic bud.
[0016] 2. The cell culture of paragraph 1, wherein said marker
selected from Table 3 is a marker expressed on the cell
surface.
[0017] 3. The cell culture of paragraph 2, wherein said marker
expressed on the cell surface is selected from the group consisting
of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2
and SLC27A2.
[0018] 4. The cell culture of paragraph 1, wherein said
PDX1-positive, dorsally-biased, foregut endoderm cells express at
least one marker selected from Table 4.
[0019] 5. The cell culture of paragraph 4, wherein said marker
selected from Table 4 is a marker expressed on the cell
surface.
[0020] 6. The cell culture of paragraph 5, wherein said marker
expressed on the cell surface is selected from the group consisting
of ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16A10, SLC16A2, SLC1A3,
SLC30A4, SLICK, SLITRK4 and XPR1.
[0021] 7. The cell culture of paragraph 1, wherein at least about
30% of said human cells are PDX1-positive, dorsally-biased, foregut
endoderm cells.
[0022] 8. The cell culture of paragraph 1, wherein at least about
40% of said human cells are PDX1-positive, dorsally-biased, foregut
endoderm cells.
[0023] 9. The cell culture of paragraph 1, wherein at least about
50% of said human cells are PDX1-positive, dorsally-biased, foregut
endoderm cells.
[0024] 10. The cell culture of paragraph 1, wherein at least about
60% of said human cells are PDX1-positive, dorsally-biased, foregut
endoderm cells.
[0025] 11. The cell culture of paragraph 1, wherein at least about
75% of said human cells are PDX1-positive, dorsally-biased, foregut
endoderm cells.
[0026] 12. The cell culture of paragraph 1, wherein human feeder
cells are present in said culture, and wherein at least about 2% of
human cells other than said human feeder cells are PDX1-positive,
dorsally biased, foregut endoderm cells.
[0027] 13. The cell culture of paragraph 1, wherein the expression
of PDX1 is greater than the expression of a marker selected from
the group consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1
and NFM in said PDX1-positive, dorsally biased, foregut endoderm
cells.
[0028] 14. The cell culture of paragraph 1, wherein said cell
culture is substantially free of cells selected from the group
consisting of visceral endodermal cells, parietal endodermal cells
and neural cells.
[0029] 15. The cell culture of paragraph 1 further comprising a
retinoid.
[0030] 16. The cell culture of paragraph 15, wherein said retinoid
is retinoic acid (RA).
[0031] 17. The cell culture of paragraph 16 further comprising
B27.
[0032] 18. A cell culture comprising human cells wherein at least
about 2% of said human cells are pancreatic-duodenal homoebox
factor-1 (PDX1) positive, ventrally-biased, foregut endoderm cells
that express at least one marker selected from Table 3, said
PDX1-positive, ventrally-biased, foregut endoderm cells being
multipotent cells that can differentiate into cells of the ventral
pancreatic bud.
[0033] 19. The cell culture of paragraph 18, wherein said marker
selected from Table 3 is a marker expressed on the cell
surface.
[0034] 20. The cell culture of paragraph 19, wherein said marker
expressed on the cell surface is selected from the group consisting
of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2
and SLC27A2.
[0035] 21. The cell culture of paragraph 18, wherein said
PDX1-positive, ventrally-biased, foregut endoderm cells do not
substantially express one or more markers selected from Table
4.
[0036] 22. The cell culture of paragraph 18, wherein at least about
5% of said human cells are PDX1-positive, ventrally-biased, foregut
endoderm cells.
[0037] 23. The cell culture of paragraph 18, wherein at least about
10% of said human cells are PDX1-positive, ventrally-biased,
foregut endoderm cells.
[0038] 24. The cell culture of paragraph 18, wherein at least about
25% of said human cells are PDX1-positive, ventrally-biased,
foregut endoderm cells.
[0039] 25. The cell culture of paragraph 18, wherein at least about
50% of said human cells are PDX1-positive, ventrally-biased,
foregut endoderm cells.
[0040] 26. The cell culture of paragraph 18, wherein at least about
75% of said human cells are PDX1-positive, ventrally-biased,
foregut endoderm cells.
[0041] 27. The cell culture of paragraph 18, wherein human feeder
cells are present in said culture, and wherein at least about 2% of
human cells other than said human feeder cells are PDX1-positive,
ventrally-biased, foregut endoderm cells.
[0042] 28. The cell culture of paragraph 18, wherein the expression
of PDX1 is greater than the expression of a marker selected from
the group consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1
and NFM in said PDX1-positive, ventrally-biased, foregut endoderm
cells.
[0043] 29. The cell culture of paragraph 18, wherein said cell
culture is substantially free of cells selected from the group
consisting of visceral endodermal cells, parietal endodermal cells
and neural cells.
[0044] 30. The cell culture of paragraph 18 further comprising a
retinoid.
[0045] 31. The cell culture of paragraph 30, wherein said retinoid
is retinoic acid (RA).
[0046] 32. The cell culture of paragraph 31 further comprising
B27.
[0047] 33. A cell population comprising cells wherein at least
about 90% of said cells are human PDX1-positive, dorsally-biased,
foregut endoderm cells that express at least one marker selected
from Table 3, said PDX1-positive, dorsally-biased, foregut endoderm
cells being multipotent cells that can differentiate into cells of
the dorsal pancreatic bud.
[0048] 34. The cell population of paragraph 33, wherein said marker
selected from Table 3 is a marker expressed on the cell
surface.
[0049] 35. The cell population of paragraph 34, wherein said marker
expressed on the cell surface is selected from the group consisting
of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2
and SLC27A2.
[0050] 36. The cell population of paragraph 33, wherein said
PDX1-positive, dorsally-biased, foregut endoderm cells express at
least one marker selected from Table 4.
[0051] 37. The cell population of paragraph 36, wherein said marker
selected from Table 4 is a marker expressed on the cell
surface.
[0052] 38. The cell population of paragraph 37, wherein said marker
expressed on the cell surface is selected from the group consisting
of ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16A10, SLC16A2, SLC1A3,
SLC30A4, SLICK, SLITRK4 and XPR1.
[0053] 39. The cell population of paragraph 33, wherein at least
about 95% of said cells are PDX1-positive, dorsally-biased, foregut
endoderm cells.
[0054] 40. The cell population of paragraph 33, wherein at least
about 98% of said cells are PDX1-positive, dorsally-biased, foregut
endoderm cells.
[0055] 41. The cell population of paragraph 33, wherein the
expression of PDX1 is greater than the expression of a marker
selected from the group consisting of AFP, SOX7, SOX1, ZIC1 and NFM
in said PDX1-positive, dorsally-biased, foregut endoderm cells.
[0056] 42. A cell population comprising cells wherein at least
about 90% of said cells are human PDX1-positive, ventrally-biased,
foregut endoderm cells that express at least one marker selected
from Table 3, said PDX1-positive, ventrally-biased, foregut
endoderm cells being multipotent cells that can differentiate into
cells of the ventral pancreatic bud.
[0057] 43. The cell population of paragraph 42, wherein said marker
selected from Table 3 is a marker expressed on the cell
surface.
[0058] 44. The cell population of paragraph 43, wherein said marker
expressed on the cell surface is selected from the group consisting
of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2
and SLC27A2.
[0059] 45. The cell population of paragraph 42, wherein said
PDX1-positive, ventrally-biased, foregut endoderm cells do not
substantially express one or more markers selected from Table
4.
[0060] 46. The cell population of paragraph 42, wherein at least
about 95% of said cells are PDX1-positive, ventrally-biased,
foregut endoderm cells.
[0061] 47. The cell population of paragraph 42, wherein at least
about 98% of said cells are PDX1-positive, ventrally-biased,
foregut endoderm cells.
[0062] 48. The cell population of paragraph 42, wherein the
expression of PDX1 is greater than the expression of a marker
selected from the group consisting of AFP, SOX7, SOX1, ZIC1 and NFM
in said PDX1-positive, ventrally-biased, foregut endoderm
cells.
[0063] 49. A method of producing PDX1-positive, dorsally-biased,
foregut endoderm cells, said method comprising the steps of
obtaining a cell population comprising PDX1-negative definitive
endoderm cells and providing said cell population with a retinoid
in an amount sufficient to promote differentiation of at least 26%
of said PDX1-negative definitive endoderm cell population to
PDX1-positive, dorsally-biased, foregut endoderm cells that express
at least one marker selected from Table 3, wherein said
PDX1-positive, dorsally-biased, foregut endoderm cells are
multipotent cells that can differentiate into cells of the dorsal
pancreatic bud.
[0064] 50. The method of paragraph 49, wherein said PDX1-positive,
dorsally-biased, foregut endoderm cells also express at least one
marker selected from Table 4.
[0065] 51. The method of paragraph 50 further comprising the step
of allowing sufficient time for PDX1-positive, dorsally-biased,
foregut endoderm cells to form, wherein said sufficient time for
PDX1-positive, dorsally-biased, foregut endoderm cells to form has
been determined by detecting the presence of a marker from Table 4
in dorsally-biased foregut endoderm cells in said cell
population.
[0066] 52. The method of paragraph 50, wherein the expression of
said marker selected from Table 3 or Table 4 is determined by
quantitative polymerase chain reaction (Q-PCR).
[0067] 53. The method of paragraph 50, wherein the expression of
said marker selected from Table 3 or Table 4 is determined by
immunocytochemistry.
[0068] 54. The method of paragraph 49, wherein the expression of
PDX1 is greater than the expression of a marker selected from the
group consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and
NFM in said PDX1-positive, dorsally-biased, foregut endoderm
cells.
[0069] 55. The method of paragraph 49, wherein said retinoid is
RA.
[0070] 56. The method of paragraph 55, wherein RA is provided in a
concentration ranging from about 0.5 .mu.M to about 50 .mu.M.
[0071] 57. The method of paragraph 56, wherein RA is provided in a
concentration ranging from about 1 .mu.M to about 20 .mu.M.
[0072] 58. The method of paragraph 57, wherein RA is provided in a
concentration of about 2 .mu.M.
[0073] 59. The method of paragraph 55, wherein RA is provided when
said culture is about 5-days-old.
[0074] 60. The method of paragraph 49 further comprising providing
B27 to said culture.
[0075] 61. The method of paragraph 60, wherein said B27 is provided
in a concentration ranging from about 0.1% to about 20% of the
total medium.
[0076] 62. The method of paragraph 61, wherein B27 is provided in a
concentration ranging from about 0.5% to about 2% of the total
medium.
[0077] 63. The method of paragraph 62, wherein B27 is provided in a
concentration of about 0.5% of the total medium.
[0078] 64. The method of paragraph 60, wherein B27 is provided at
approximately the same time as said retinoid.
[0079] 65. The method of paragraph 49 further comprising providing
activin A to said culture.
[0080] 66. The method of paragraph 65, wherein activin A is
provided in a concentration ranging from about 10 ng/ml to about
200 ng/ml.
[0081] 67. The method of paragraph 66, wherein activin A is
provided in a concentration ranging from about 20 ng/ml to about
100 ng/ml.
[0082] 68. The method of paragraph 67, wherein activin A is
provided in a concentration of about 25 ng/ml.
[0083] 69. The method of paragraph 49, wherein said PDX1-positive,
dorsally-biased, foregut endoderm cells are grown in CMRL
medium.
[0084] 70. The method of paragraph 69, wherein said CMRL medium
comprises RA at about 2 .mu.M, activin A at about 25 ng/ml and B27
at about 0.5% of the total medium.
[0085] 71. The method of paragraph 49, wherein said step of
obtaining a cell population comprising PDX1-negative definitive
endoderm cells comprises obtaining a cell population comprising
pluripotent human cells, providing said cell population with at
least one growth factor of the TGF.beta. superfamily in an amount
sufficient to promote differentiation of said pluripotent cells to
definitive endoderm cells and allowing sufficient time for
definitive endoderm cells to form, wherein said sufficient time for
definitive endoderm cells to form has been determined by detecting
the presence of definitive endoderm cells in said cell
population.
[0086] 72. A PDX1-positive, dorsally-biased, foregut endoderm cell
produced by the method of paragraph 49.
[0087] 73. A method of producing PDX1-positive, ventrally-biased,
foregut endoderm cells, said method comprising the steps of
obtaining a cell population comprising PDX1-negative definitive
endoderm cells and providing said cell population with an
FGF-family growth factor in an amount sufficient to promote
differentiation of at least a portion of said PDX1-negative
definitive endoderm cell population to PDX1-positive,
ventrally-biased, foregut endoderm cells that express at least one
marker selected from Table 3, wherein said PDX1-positive,
ventrally-biased, foregut endoderm cells are multipotent cells that
can differentiate into cells of the ventral pancreatic bud.
[0088] 74. The method of paragraph 73, wherein said cell population
is differentiated in the absence of RA.
[0089] 75. The method of paragraph 73, wherein said PDX1-positive,
ventrally-biased, foregut endoderm cells do not express one or more
markers selected from Table 4.
[0090] 76. The method of paragraph 75 further comprising the step
of allowing sufficient time for PDX1-positive, ventrally-biased,
foregut endoderm cells to form, wherein said sufficient time for
PDX1-positive, ventrally-biased, foregut endoderm cells to form has
been determined by detecting the presence of a marker from Table 3
in ventrally-biased foregut endoderm cells in said cell
population.
[0091] 77. The method of paragraph 76, wherein the expression of
said marker selected from Table 3 is determined by quantitative
polymerase chain reaction (Q-PCR).
[0092] 78. The method of paragraph 76, wherein the expression of
said marker selected from Table 3 is determined by
immunocytochemistry.
[0093] 79. The method of paragraph 73, wherein the expression of
PDX1 is greater than the expression of a marker selected from the
group consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and
NFM in said PDX1-positive, ventrally-biased, foregut endoderm
cells.
[0094] 80. The method of paragraph 73, wherein said FGF-family
growth factor is FGF-10, said FGF-10 being provided in a
concentration ranging from about 5 ng/ml to about 500 ng/ml.
[0095] 81. The method of paragraph 80, wherein FGF-10 is provided
in a concentration ranging from about 10 ng/ml to about 100
ng/ml.
[0096] 82. The method of paragraph 81, wherein FGF-10 is provided
in a concentration of about 50 ng/ml.
[0097] 83. The method of paragraph 73, wherein said FGF-family
growth factor is provided when said culture is about
3-days-old.
[0098] 84. The method of paragraph 73 further comprising providing
B27 to said culture.
[0099] 85. The method of paragraph 84, wherein said B27 is provided
in a concentration ranging from about 0.1% to about 20% of the
total medium.
[0100] 86. The method of paragraph 85, wherein B27 is provided in a
concentration ranging from about 0.5% to about 2% of the total
medium.
[0101] 87. The method of paragraph 86, wherein B27 is provided in a
concentration of about 0.5% of the total medium.
[0102] 88. The method of paragraph 84, wherein B27 is provided at
approximately the same time as said FGF-family growth factor.
[0103] 89. The method of paragraph 73 further comprising providing
a hedgehog inhibitor to said culture
[0104] 90. The method of paragraph 89, wherein said hedgehog
inhibitor is KAAD-cyclopamine, said KAAD-cyclopamine being provided
in a concentration of about 0.1 .mu.M to about 50 .mu.M.
[0105] 91. The method of paragraph 90, wherein KAAD-cyclopamine is
provided in a concentration ranging from about 0.5 .mu.M to about
10 .mu.M.
[0106] 92. The method of paragraph 91, wherein KAAD-cyclopamine is
provided in a concentration of about 0.5 .mu.M.
[0107] 93. The method of paragraph 73, wherein said PDX1-positive,
ventrally-biased, foregut endoderm cells are grown in CMRL
medium.
[0108] 94. The method of paragraph 93, wherein said CMRL medium
comprises FGF-10 at about 50 ng/ml, KAAD-cyclopamine at about 0.5
.mu.M, and B27 at about 0.5% of the total medium.
[0109] 95. The method of paragraph 94, wherein said CMRL medium
lacks RA.
[0110] 96. The method of paragraph 73, wherein said step of
obtaining a cell population comprising PDX1-negative definitive
endoderm cells comprises obtaining a cell population comprising
pluripotent human cells, providing said cell population with at
least one growth factor of the TGF.beta. superfamily in an amount
sufficient to promote differentiation of said pluripotent cells to
definitive endoderm cells and allowing sufficient time for
definitive endoderm cells to form, wherein said sufficient time for
definitive endoderm cells to form has been determined by detecting
the presence of definitive endoderm cells in said cell
population.
[0111] 97. A PDX1-positive, ventrally-biased, foregut endoderm cell
produced by the method of paragraph 73.
[0112] 98. A method of producing a cell population enriched in
PDX1-positive, dorsally-biased, foregut endoderm cells, said method
comprising the steps of differentiating cells in a population of
PDX1-negative definitive endoderm cells so as to produce
PDX1-positive, dorsally-biased, foregut endoderm cells, said
PDX1-positive, dorsally-biased, foregut endoderm cells being
multipotent cells that can differentiate into cells of the dorsal
pancreatic bud, providing to said cell population a reagent which
binds to a marker expressed in said PDX1-positive, dorsally-biased,
foregut endoderm cells but which is not substantially expressed in
other cell types present in said cell population and separating
said PDX1-positive, dorsally-biased, foregut endoderm cells bound
to said reagent from said other cell types present in said cell
population, thereby producing a cell population enriched in
PDX1-positive, dorsally-biased, foregut endoderm cells.
[0113] 99. The method for paragraph 98, wherein said marker is
selected from the group consisting of ADORA2A, CD47, EPB41L1, MAG,
SFRP5, SLC16A10, SLC16A2, SLC1A3, SLC30A4, SLICK, SLITRK4 and
XPR1.
[0114] 100. The method for paragraph 98, wherein said marker is
selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R,
KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.
[0115] 101. The method of paragraph 98, wherein the differentiating
step further comprises obtaining a cell population comprising
PDX1-negative definitive endoderm cells, providing said cell
population with a retinoid in an amount sufficient to promote
differentiation of said PDX1-negative definitive endoderm cells to
PDX1-positive, dorsally-biased, foregut endoderm cells, said
PDX1-positive, dorsally-biased, foregut endoderm cells being
multipotent cells that can differentiate into cells of the dorsal
pancreatic bud, and allowing sufficient time for PDX1-positive,
dorsally-biased, foregut endoderm cells to form, wherein said
sufficient time for PDX1-positive, dorsally-biased, foregut
endoderm cells to form has been determined by detecting the
presence of PDX1-positive, dorsally-biased, foregut endoderm cells
in said cell population.
[0116] 102. The method of paragraph 101, wherein the providing step
further comprises providing B27.
[0117] 103. The method of paragraph 101, wherein detecting
comprises detecting the expression of at least one marker selected
from Table 4.
[0118] 104. The method of paragraph 98, wherein at least about 95%
of said cells are PDX1-positive, dorsally-biased, foregut endoderm
cells.
[0119] 105. The method of paragraph 98, wherein at least about 98%
of said cells are PDX1 -positive, dorsally-biased, foregut endoderm
cells.
[0120] 106. The method of paragraph 98, wherein said reagent is an
antibody
[0121] 107. The method of paragraph 106, wherein said antibody has
affinity for a cell surface polypeptide selected from the group
consisting of ADORA2A, CD47, EPB41L1, MAG, SFRP5, SLC16A10,
SLC16A2, SLC1A3, SLC30A4, SLICK, SLITRK4, XPR1, CDH6, GABRA2,
GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.
[0122] 108. An enriched population of PDX1-positive,
dorsally-biased, foregut endoderm cells produced by the method of
paragraph 98.
[0123] 109. A method of producing a cell population enriched in
PDX1-positive, ventrally-biased, foregut endoderm cells, said
method comprising the steps of differentiating cells in a
population of PDX1-negative definitive endoderm cells so as to
produce PDX1-positive, ventrally-biased, foregut endoderm cells,
said PDX1-positive, ventrally-biased, foregut endoderm cells being
multipotent cells that can differentiate into cells of the ventral
pancreatic bud, providing to said cell population a reagent which
binds to a marker expressed in said PDX1-positive,
ventrally-biased, foregut endoderm cells but which is not
substantially expressed in other cell types present in said cell
population and separating said PDX1-positive, ventrally-biased,
foregut endoderm cells bound to said reagent from said other cell
types present in said cell population, thereby producing a cell
population enriched in PDX1-positive, ventrally-biased, foregut
endoderm cells.
[0124] 110. The method for paragraph 109, wherein said marker is
selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R,
KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2.
[0125] 111. The method of paragraph 109, wherein the
differentiating step further comprises obtaining a cell population
comprising PDX1-negative definitive endoderm cells, providing said
cell population with an FGF-family growth factor in an amount
sufficient to promote differentiation of said PDX1-negative
definitive endoderm cells to PDX1-positive, ventrally-biased,
foregut endoderm cells, said PDX1-positive, ventrally-biased,
foregut endoderm cells being multipotent cells that can
differentiate into cells of the ventral pancreatic bud, and
allowing sufficient time for PDX1-positive, ventrally-biased,
foregut endoderm cells to form, wherein said sufficient time for
PDX1-positive, ventrally-biased, foregut endoderm cells to form has
been determined by detecting the presence of PDX1-positive,
ventrally-biased, foregut endoderm cells in said cell
population.
[0126] 112. The method of paragraph 111, wherein the providing step
further comprises providing B27.
[0127] 113. The method of paragraph 111, wherein detecting
comprises detecting the expression of at least one marker selected
from Table 3.
[0128] 114. The method of paragraph 109, wherein at least about 95%
of said cells are PDX1-positive, ventrally-biased, foregut endoderm
cells.
[0129] 115. The method of paragraph 109, wherein at least about 98%
of said cells are PDX1-positive, ventrally-biased, foregut endoderm
cells.
[0130] 116. The method of paragraph 109, wherein said reagent is an
antibody
[0131] 117. The method of paragraph 116, wherein said antibody has
affinity for a cell surface polypeptide selected from the group
consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3,
LGALS3/GALIG, SERPINF2 and SLC27A2.
[0132] 118. An enriched population of PDX1-positive,
ventrally-biased, foregut endoderm cells produced by the method of
paragraph 109.
[0133] 119. A method of producing a cell population enriched in
PDX1-positive, dorsally-biased, foregut endoderm cells, said method
comprising the steps of obtaining a population of pluripotent
cells, wherein at least one cell of said pluripotent cell
population comprises at least one copy of a nucleic acid under the
control of the a promoter of any one of the marker genes selected
from Table 4, said nucleic acid comprising a sequence encoding a
fluorescent protein or a biologically active fragment thereof,
differentiating said pluripotent cells so as to produce
PDX1-positive, dorsally-biased foregut endoderm cells, said
PDX1-positive, dorsally-biased, foregut endoderm cells being
multipotent cells that can differentiate into cells of the dorsal
pancreatic bud, and separating said PDX1-positive, dorsally-biased,
foregut endoderm cells from other cell types present in the cell
population.
[0134] 120. The method of paragraph 119, wherein said enriched cell
population comprises at least about 95% PDX1-positive,
dorsally-biased, foregut endoderm cells.
[0135] 121. The method of paragraph 119, wherein said enriched cell
population comprises at least about 98% PDX1-positive,
dorsally-biased, foregut endoderm cells.
[0136] 122. The method of paragraph 119, wherein the
differentiating step further comprises, providing said pluripotent
cell population with at least one growth factor of the TGF.beta.
superfamily in an amount sufficient to promote differentiation of
said pluripotent cells to PDX1-negative definitive endoderm cells,
and providing said PDX1-negative definitive endoderm cells with a
retinoid in an amount sufficient to promote differentiation of said
PDX1-negative definitive endoderm cells to PDX1-positive,
dorsally-biased, foregut endoderm cells.
[0137] 123. The method of paragraph 122, wherein said retinoid is
RA.
[0138] 124. The method of paragraph 123, wherein the providing step
further comprises providing B27.
[0139] 125. The method of paragraph 119, wherein said fluorescent
protein is green fluorescent protein (GFP).
[0140] 126. The method of paragraph 119, wherein said
PDX1-positive, dorsally-biased, foregut endoderm cells are
separated from other cell types present in the cell population by
fluorescence activated cell sorting (FACS).
[0141] 127. An enriched population of PDX1-positive,
dorsally-biased, foregut endoderm cells produced by the method of
paragraph 119.
[0142] 128. A method of producing a cell population enriched in
PDX1-positive, ventrally-biased, foregut endoderm cells, said
method comprising the steps of obtaining a population of
pluripotent cells, wherein at least one cell of said pluripotent
cell population comprises at least one copy of a nucleic acid under
the control of the a promoter of any one of the marker genes
selected from Table 3, said nucleic acid comprising a sequence
encoding a fluorescent protein or a biologically active fragment
thereof, differentiating said pluripotent cells so as to produce
PDX1-positive, ventrally-biased foregut endoderm cells, said
PDX1-positive, ventrally-biased, foregut endoderm cells being
multipotent cells that can differentiate into cells of the ventral
pancreatic bud and separating said PDX1-positive, ventrally-biased,
foregut endoderm cells from non-ventrally-biased foregut endoderm
cells.
[0143] 129. The method of paragraph 128, wherein said enriched cell
population comprises at least about 95% PDX1-positive,
ventrally-biased, foregut endoderm cells.
[0144] 130. The method of paragraph 128, wherein said enriched cell
population comprises at least about 98% PDX1-positive,
ventrally-biased, foregut endoderm cells.
[0145] 131. The method of paragraph 128, wherein the
differentiating step further comprises, providing said pluripotent
cell population with at least one growth factor of the TGF.beta.
superfamily in an amount sufficient to promote differentiation of
said pluripotent cells to PDX1-negative definitive endoderm cells,
and providing said PDX1-negative definitive endoderm cells with an
FGF-family growth factor in an amount sufficient to promote
differentiation of said PDX1-negative definitive endoderm cells to
PDX1-positive, ventrally-biased, foregut endoderm cells.
[0146] 132. The method of paragraph 128, wherein the providing step
further comprises providing B27.
[0147] 133. The method of paragraph 128, wherein said fluorescent
protein is green fluorescent protein (GFP).
[0148] 134. The method of paragraph 128, wherein said
PDX1-positive, dorsally-biased, foregut endoderm cells are
separated from other cell types present in the cell population by
fluorescence activated cell sorting (FACS).
[0149] 135. An enriched population of PDX1-positive,
dorsally-biased, foregut endoderm cells produced by the method of
paragraph 128.
[0150] 136. A method of identifying a differentiation factor
capable of promoting the differentiation of human PDX1-positive,
dorsally-biased, foregut endoderm cells in a cell population
comprising human cells, said method comprising the steps of
obtaining a cell population comprising human PDX1-positive,
dorsally-biased, foregut endoderm cells, providing a candidate
differentiation factor to said cell population, determining
expression of a marker in said cell population at a first time
point, 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 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
PDX1-positive, dorsally-biased, foregut endoderm cells.
[0151] 137. The method of paragraph 136, wherein said marker is
selected from Table 4.
[0152] 138. The method of paragraph 136, wherein said human
PDX1-positive, dorsally-biased, foregut endoderm cells comprise at
least about 10% of the human cells in said cell population.
[0153] 139. The method of paragraph 136, 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 PDX1-positive,
dorsally-biased, foregut endoderm cells.
[0154] 140. The method of paragraph 136, wherein said human
PDX1-positive, dorsally-biased, foregut endoderm cells comprise at
least about 90% of the human cells in said cell population.
[0155] 141. The method of paragraph 136, 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
PDX1-positive, dorsally-biased, foregut endoderm cells.
[0156] 142. The method of paragraph 136, wherein said human
PDX1-positive, dorsally-biased, foregut endoderm cells can
differentiate into cells of the dorsal pancreatic bud.
[0157] 143. The method of paragraph 136, wherein said human
definitive endoderm cells differentiate into pancreatic precursor
cells in response to said candidate differentiation factor.
[0158] 144. The method of paragraph 136, wherein said first time
point is prior to providing said candidate differentiation factor
to said cell population.
[0159] 145. The method of paragraph 136, wherein said first time
point is at approximately the same time as providing said candidate
differentiation factor to said cell population.
[0160] 146. The method of paragraph 136, wherein said first time
point is subsequent to providing said candidate differentiation
factor to said cell population.
[0161] 147. The method of paragraph 136, wherein expression of said
marker is increased.
[0162] 148. The method of paragraph 136, wherein expression of said
marker is decreased.
[0163] 149. The method of paragraph 136, wherein expression of said
marker is determined by quantitative polymerase chain reaction
(Q-PCR).
[0164] 150. The method of paragraph 136, wherein expression of said
marker is determined by immunocytochemistry.
[0165] 151. The method of paragraph 136, wherein said
differentiation factor comprises a small molecule.
[0166] 152. The method of paragraph 136, wherein said
differentiation factor comprises a polypeptide.
[0167] 153. The method of paragraph 136, wherein said
differentiation factor comprises a growth factor.
[0168] 154. A method of identifying a differentiation factor
capable of promoting the differentiation of human PDX1-positive,
ventrally-biased, foregut endoderm cells in a cell population
comprising human cells, said method comprising the steps of
obtaining a cell population comprising human PDX1-positive,
ventrally-biased, foregut endoderm cells, providing a candidate
differentiation factor to said cell population, determining
expression of a marker in said cell population at a first time
point, 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 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
PDX1-positive, ventrally-biased, foregut endoderm cells.
[0169] 155. The method of paragraph 154, wherein said marker is
selected from Table 3.
[0170] 156. The method of paragraph 154, wherein said human
PDX1-positive, ventrally-biased, foregut endoderm cells comprise at
least about 10% of the human cells in said cell population.
[0171] 157. The method of paragraph 154, 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 PDX1-positive,
ventrally-biased, foregut endoderm cells.
[0172] 158. The method of paragraph 154, wherein said human
PDX1-positive, ventrally-biased, foregut endoderm cells comprise at
least about 90% of the human cells in said cell population.
[0173] 159. The method of paragraph 154, 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
PDX1-positive, ventrally-biased, foregut endoderm cells.
[0174] 160. The method of paragraph 154, wherein said human
PDX1-positive, ventrally-biased, foregut endoderm cells can
differentiate into cells of the ventral pancreatic bud.
[0175] 161. The method of paragraph 154, wherein said human
definitive endoderm cells differentiate into pancreatic precursor
cells in response to said candidate differentiation factor.
[0176] 162. The method of paragraph 154, wherein said first time
point is prior to providing said candidate differentiation factor
to said cell population.
[0177] 163. The method of paragraph 154, wherein said first time
point is at approximately the same time as providing said candidate
differentiation factor to said cell population.
[0178] 164. The method of paragraph 154, wherein said first time
point is subsequent to providing said candidate differentiation
factor to said cell population.
[0179] 165. The method of paragraph 154, wherein expression of said
marker is increased.
[0180] 166. The method of paragraph 154, wherein expression of said
marker is decreased.
[0181] 167. The method of paragraph 154, wherein expression of said
marker is determined by quantitative polymerase chain reaction
(Q-PCR).
[0182] 168. The method of paragraph 154, wherein expression of said
marker is determined by immunocytochemistry.
[0183] 169. The method of paragraph 154, wherein said
differentiation factor comprises a small molecule.
[0184] 170. The method of paragraph 154, wherein said
differentiation factor comprises a polypeptide.
[0185] 171. The method of paragraph 154, wherein said
differentiation factor comprises a growth factor.
[0186] 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.
[0187] 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; U.S. patent application Ser. No. 11/165,305,
entitled METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIATING
DEFINITIVE ENDODERM, filed Jun. 23, 2005, the disclosures of which
are incorporated herein by reference in their entireties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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).
[0192] FIGS. 5A-B 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 SOX 17+clusters (B)
where little or no AFP+cells are observed.
[0193] FIGS. 6A-C are micrographs showing parietal endoderm and
SOX17. Panel A shows immunocytochemistry for human Thrombomodulin
(TM) protein located on the cell surface of parietal endoderm cells
in randomly differentiated cultures of hES cells. Panel B is the
identical field shown in A double-labeled for TM and SOX17. Panel 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.
[0194] FIGS. 7A-B are bar charts showing SOX17 gene expression by
quantitative PCR (Q-PCR) and anti-SOX17 positive cells by
SOX17-specific antibody. Panel A shows that activin A increases SOX
17 gene expression while retinoic acid (RA) strongly suppresses SOX
17 expression relative to the undifferentiated control media
(SR20). Panel 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.
[0195] 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.
[0196] FIGS. 8B-C 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.sup.+ cells observed in activin A treatment conditions
(bottom) relative to 10% FBS alone (top).
[0197] FIGS. 9A-B are comparative images showing the quantitation
of the AFP.sup.+ cell number using flow cytometry. This figure
demonstrates that the magnitude of change in AFP gene expression
(FIG. 8A) in the presence (right panel) and absence (left panel) of
activin A exactly corresponds to the number of AFP.sup.+ cells,
further supporting the utility of Q-PCR analyses to indicate
changes occurring at the individual cell level.
[0198] FIGS. 10A-F 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.
[0199] 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.
[0200] FIGS. 12A-C are bar charts which demonstrate the effect of
activin A on the expression of MIXLI (panel A), GATA4 (panel B) and
HNF3b (panel C). Activin A dose-dependent increases are also
observed for three other markers of definitive endoderm; MIXLI,
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+, MIXLI+, GATA4+and
HNF3b+).
[0201] FIGS. 13A-C are bar charts which demonstrate the effect of
activin A on the expression of AFP (panel A), SOX7 (panel B) and
SPARC (panel C). There is an activin A dose-dependent decrease in
expression of the visceral endoderm marker AFP. Markers of
primitive endoderm (SOX7) and parietal endodenn (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, MIXLI, GATA4, and HNF3b are
due to an increase in the number of definitive endoderm cells in
response to activin A.
[0202] FIGS. 14A-B are bar charts showing the effect of activin A
on ZIC1 (panel A) and Brachyury expression (panel 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.
[0203] FIGS. 15A-B are micrographs showing decreased parietal
endoderm differentiation in response to treatment with activins.
Regions of TMhi parietal endoderm are found through the culture (A)
when differentiated in serum alone, while differentiation to
TM+cells is scarce when activins are included (B) and overall
intensity of TM immunoreactivity is lower.
[0204] FIGS. 16A-D 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. Panel A--SOX17;
Panel B--AFP; Panel C--TM; and Panel D--Phase/DAPI. Notice the
numerous SOX 17 positive cells (A) associated with the complete
absence of AFP (B) and TM (C) immunoreactivity.
[0205] 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 AFPhi cells,
suggesting the separate origination of the definitive endoderm
cells from visceral endoderm cells.
[0206] 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).
[0207] 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.
[0208] FIG. 20 is a bar chart showing the increase in SOX17+cell
number with time as a result of treatment with combinations of
TGF.beta. factors.
[0209] FIG. 21 is a bar chart showing induction of SOX 17
expression over time as a result of treatment with combinations of
TGF.beta. factors.
[0210] FIG. 22 is a bar chart showing that activin A induces a
dose-dependent increase in SOX17+cell number.
[0211] 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.
[0212] FIGS. 24A-C 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 (IOAA) (panel A). Induction of the
definitive endoderm marker MIXLI (panel B) is also affected in the
same way and the suppression of AFP (visceral endoderm) (panel C)
is greater in 2% FBS than in 10% FBS conditions.
[0213] FIGS. 25A-D 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) (panel B) yet are not
co-labeled with OCT4 (panel C). In addition, clear mitotic figures
can be seen by DAPI labeling of nuclei in both SOX17+cells (arrows)
as well as OCT4+, undifferentiated hESCs (arrowheads) (D).
[0214] FIG. 26 is a bar chart showing the relative expression level
of CXCR4 in differentiating hESCs under various media
conditions.
[0215] FIGS. 27A-D are bar charts that show how a panel of
definitive endoderm markers share a very similar pattern of
expression to CXCR4 across the same differentiation treatments
displayed in FIG. 26.
[0216] FIGS. 28A-E are bar charts showing how markers for mesoderm
(BRACHYURY, MOXI), ectoderm (SOX1, ZIC1) and visceral endoderm
(SOX7) exhibit an inverse relationship to CXCR4 expression across
the same treatments displayed in FIG. 26.
[0217] FIGS. 29A-F are micrographs that show the relative
difference in SOX17 immunoreactive cells across three of the media
conditions displayed in FIGS. 26-28.
[0218] FIGS. 30A-C are flow cytometry dot plots that demonstrate
the increase in CXCR4+cell number with increasing concentration of
activin A added to the differentiation media.
[0219] FIGS. 31A-D are bar charts that show the CXCR4+cells
isolated from the high dose activin A treatment (A100-CX+) are even
further enriched for definitive endoderm markers than the parent
population (A100).
[0220] FIG. 32 is a bar chart showing gene expression from
CXCR4+and CXCR4-cells isolated using fluorescence-activated cell
sorting (FACS) as well as gene expression in the parent
populations. This demonstrates that the CXCR4+cells contain
essentially all the CXCR4 gene expression present in each parent
population and the CXCR4- populations contain very little or no
CXCR4 gene expression.
[0221] FIGS. 33A-D are bar charts that demonstrate the depletion of
mesoderm (BRACHYURY, MOXI), ectoderm (ZIC1) and visceral endoderm
(SOX7) gene expression in the CXCR4+cells isolated from the high
dose activin A treatment which is already suppressed in expression
of these non-definitive endoderm markers.
[0222] FIGS. 34A-M 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, FOXQI, CMKORI and CRIPI is shown in panels G-L,
respectively. The expression analysis of previously described
lineage marking genes, SOX17, SOX7, SOX17/SOX7, TM, ZIC1, and MOXI
is shown in panels A-F, respectively. Panel M shows the expression
analysis of CXCR4. With respect to each of panels 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.IAIOO indicates cells treated with 0.1% FBS,
100 ng/ml activin A; IAIOO indicates cells treated with 1% FBS, 100
ng/ml activin A; and 2A 100 indicates cells treated with 2% FBS,
100 ng/ml activin A. 102231 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 (FGF-10) added on
day 4.
[0223] FIGS. 36A-F 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 (FGF-10) added on day 4. The panels show
the relative levels of expression of the following marker genes:
(A) SOX I 7; (B) SOX7; (C) AFP; (D) SOX1; (E) ZIC1; and (F)
NFM.
[0224] FIGS. 37A-C 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 (FGF-10) and
fibroblast growth factor (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.
[0225] FIGS. 38A-G 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) CDXI; (F) SOX1; and
(G) NFM.
[0226] FIGS. 39A-E 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 (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.
[0227] FIGS. 40A-B 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
(alO), 25 ng/ml (a25) or 50 ng/ml (a5O) 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.
[0228] FIGS. 41A-C 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 Oust prior to retinoic
acid addition) and at 2, 4, and 6 days after RA exposure (day 7, 9,
and I1, 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
liM on day 9 and 0.2 .mu.M on day I1. The panels show the relative
levels of expression of the following marker genes: (A) PDX1; (B)
ZIC I; (C) SOX7.
[0229] FIGS. 42A-B 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) CDXI.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] 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+).
[0234] 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).
[0235] FIGS. 48A-E 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+). Panels: A--NKX2.2;
B--GLUT2; C--HNF3.beta.; D--RT19 and E--HNF4.alpha..
[0236] FIG. 49 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+). Panels: A--ZIC1 and
B--GFAP.
[0237] FIGS. 50A-D are charts showing the relative expression of
marker genes in a culture of hESCs at the start of differentiation
prior to addition of any factor (O d), and either in the presence
of 100 ng/ml activin A (A1OO) for 3 days (3 d) followed by 3 ng/ml
BMP4 and 50 ng/ml FGF-10 (B3F50) on days 4-11 (7d, 9d and lid) or
in the presence of 100 ng/ml activin A (A100) for 5 days (5d)
followed by 25 ng/ml activin A and 2 .mu.M RA (A25R) on day 6 and 7
(7 d). The panels show the relative levels of expression of the
following marker genes: (A) ALB; (B) PDX1; (C) HB9 and (D)
HHEX.
[0238] FIGS. 51 A-D are charts showing the relative expression of
marker genes in a culture of hESCs at the start of differentiation
prior to addition of any factor (O d), and after four days of
differentiation under one of the four following conditions: (a) 100
ng/ml BMP4 and 5 .mu.M SU5402 (B/SU); (b) no factor (NF); (c) 15
ng/ml activin A (A15); or (d) 100 ng/ml activin A. This
differentiation is followed by incubation in the presence of 3
ng/ml BMP4, 50 ng/ml FGF-10 and 0.5 .mu.M KAAD-cyclopamine
(B3F50KO.5) on days 5-12 (6d, 8d, lOd and 12d). The panels show the
relative levels of expression of the following marker genes: (A)
CER; (B) SOX17; (C) PDX1 and (D) ALB.
[0239] FIGS. 52A-B are charts showing the relative expression of
marker genes in a culture of hESCs at the start of differentiation
prior to addition of any factor (O d); in the presence of 100 ng/ml
activin A (A1OO) for 3 days (3 d); in the presence of no factor
(NF), 50 ng/ml FGF-10 and 0.5 .mu.M KAAD-cyclopamine (FK) or 3
ng/ml BMP4, 50 ng/ml FGF-10 and 0.5 .mu.M KAAD-cyclopamine (BFK) on
days 4 and 5 (5d); and in the presence of no factor in RPMI medium
(2R), no factor in RPMI medium supplemented with B27 (ORB), no
factor in CMRL medium supplemented with B27 (OCB), 50 ng/ml FGF-10
and 0.5 .mu.M KAAD-cyclopamine in CMRL medium supplemented with B27
(FK) or 3 ng/ml BMP4, 50 ng/ml FGF-10 and 0.5 .mu.M
KAAD-cyclopamine in CMRL medium supplemented with B27 (BFK) on days
6-13 (7d, 9d, lI d and 13d). The panels show the relative levels of
expression of the following marker genes: (A) PDX1 and (B) ALB.
[0240] FIGS. 53A-E are charts showing the relative expression of
marker genes in a culture of hESCs at the start of differentiation
prior to addition of any factor (O d), and either in the presence
of 100 ng/ml activin A (AIOO) for 3 days (3 d) followed by 3 ng/ml
BMP4 and 50 ng/ml FGF-I0 (B3F50) on days 4-11 (7d, 9d and lid) or
in the presence of 100 ng/ml activin A (A100) for 5 days (5d)
followed by 25 ng/ml activin A and 2 .mu.M RA (A25R) on day 6 and 7
(7 d). The panels show the relative levels of expression of the
following marker genes: (A) PDX1; (B) SERPINF2; (C) DUSP9; (D) CDH6
and (E) SOX9.
[0241] FIGS. 54A-D are charts showing the relative expression of
marker genes in a culture of hESCs at the start of differentiation
prior to addition of any factor (0 d), and either in the presence
of 100 ng/ml activin A (A100) for 3 days (3 d) followed by 3 ng/ml
BMP4 and 50 ng/ml FGF-10 (B3F50) on days 4-11 (7d, 9d and lI d) or
in the presence of 100 ng/ml activin A (AIOO) for 5 days (5d)
followed by 25 ng/ml activin A and 2 .mu.M RA (A25R) on day 6 and 7
(7 d). The panels show the relative levels of expression of the
following marker genes: (A) HOXAI; (B) PDEI IA; (C) FAM49A and (D)
WNT5A.
DETAILED DESCRIPTION
[0242] 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.
[0243] 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.
The PDX1 Gene Expression During Development
[0244] PDX1 (also called STF-1, IDX-1, IPF-1, IUF-I and GSF) 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. This expression pattem 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.
[0245] Targeted disruption of the PDX1 gene in mouse and man leads
to pancreatic agenesis (Jonsson, J., et al., Nature, 606-609, 1994;
Offield, MF, et al. Devel. 983-995, 1996; Stoffers, D. A., et al.
Nature Genetics, 106-110, 1997). PDX1 is also required during
terminal differentiation of the insulin and somatostatin pancreatic
endocrine cells and functional disruption of a single allele in
humans is associated with severe pancreatic dysfunction of MODY
type 4 (maturity onset diabetes of the young) and late onset type
1I diabetes (Stoffers D. A., et al. Nature Genetics 138-139,
1997).
[0246] During embryogenesis of the pancreas a budding of the
prospective pancreatic tissue occurs on both the dorsal and ventral
side of the primitive gut endoderm. These protrusions occur in a
regionally defined manner at the most posterior end of the foregut
endoderm. In mice this occurs at approximately 8.5-9.5 days post
conception (dpc) and in humans at 30 dpc. By 35 dpc in the human,
the ventral and dorsal buds have grown, developed a branched ductal
system and fused to form the definitive organ. PDX1 protein is
required for the early pancreatic buds to expand and differentiate
into the principal cells comprising the pancreas which include
duct, acinar and endocrine cells.
[0247] Due to their locations on opposite sides of the gut tube and
their respective associations with notochord and cardiac mesoderm
the developmental programs for the dorsal and ventral pancreatic
structures are distinctly different. With regard to the unique
developmental programs controlling specification of the dorsal and
ventral pancreatic anlaga we have developed two separate
methodologies for producing dorsally-biased and ventrally-biased
PDX1-expressing foregut endoderm from definitive endoderm (DE)
cultures generated from human embryonic stem cells (hESCs). These
PDX1-expressing (PDX1-positive) foregut endoderm cells are
competent to develop into pancreatic and duodenal epithelium as
well as endocrine cells of the anterior gastric mucosa. 102491
Aspects of the present invention relate to the discovery that
definitive endoderm cells can be differentiated into at least two
distinguishable types of PDX1-expressing (PDX1-positive) foregut
endoderm cells. We have also discovered that prior to the
expression of PDX1, definitive endoderm cells can be differentiated
into a PDX1-negative foregut endoderm cell. Providing these
PDX1-negative cells with a retinoid compound, such as retinoic
acid, induces the expression of PDX1. In another aspect of the
present invention, definitive endoderm cells are differentiated to
form dorsal PDX1-positive foregut endoderm cells. As used herein,
with respect to PDX1-positive foregut endoderm, "dorsal" or
"dorsally-biased" means that the PDX1-positive foregut endoderm
cells are those that can give rise to tissues derived from the
dorsal side of the posterior portion of the foregut, such as the
dorsal pancreatic bud. Once a PDX1-positive foregut endoderm cell
becomes "dorsally-biased" it does not typically develop into
tissues derived from the ventral side of the posterior portion of
the foregut. In another aspect, definitive endoderm cells are
differentiated to form ventral PDX1-positive foregut endoderm
cells. As used herein, with respect to PDX1-positive foregut
endoderm, "ventral" or "ventrally-biased" means that the
PDX1-positive foregut endoderm cells are those that can give rise
to tissues derived from the ventral side of the posterior portion
of the foregut, such as the liver and the ventral pancreatic bud.
Once a PDX1-positive foregut endoderm cell becomes
"ventrally-biased" it does not typically develop into tissues
derived from the dorsal side of the posterior portion of the
foregut.
[0248] In view of the foregoing discovery, embodiments of the
present invention relate to compositions of PDX1-negative foregut
endoerm cells, dorsally-biased PDX1-positive foregut endoderm
cells, ventrally-biased PDX1-positive foregut endoderm cells and/or
compositions comprising mixtures of dorsally-biased and
ventrally-biased PDX1-positive foregut endoderm cells as well as
methods for the production of such compositions. Other embodiments
of the present invention relate to screening of PDX1-negative
foregut endoderm cells for factors that promote the differentiation
of such cells. Still other embodiments relate to screening dorsal,
ventral or mixed populations of PDX1-positive foregut endoderm
cells for factors that promote the differentiation of such cells.
By "mixed populations" is meant a cell population comprising
significant amounts of both dorsal PDX1-positive foregut endoderm
cells and ventral PDX1-positive foregut endoderm cells.
[0249] As used herein, FGF-family growth factor includes, but is
not limited to, FGF-family growth factor selected from the group
consisting of FGFI, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9,
FGF10, FGFII, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF 18, FGF
19, FGF20, FGF21, FGF22 and/or FGF23.
[0250] As used herein, hedgehog inhibitor includes, but is not
limited to, KKAD-cyclopamine, KKAD-cyclopamine analogs, jervine,
jervine analogs, hedgehog pathway blocking antibodies and any other
inhibitors of hedgehog pathway function known to those of ordinary
skill in the art.
PDX1-Negative Foregut Endoderm Cells and Processes Related
Thereto
[0251] Embodiments of the present invention relate to novel,
defined processes for the production of PDX1-negative endoderm
cells, wherein the PDX1-negative endoderm cells are multipotent
cells that can differentiate into cells, tissues or organs derived
from the foregut/midgut region of the gut tube (PDX1-negative
foregut/midgut endoderm). 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 but which cannot give
rise to all three primary embryonic cells lineages (endoderm,
ectoderm and mesoderm). 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.
[0252] Some preferred embodiments of the present invention relate
to processes for the production of PDX1-negative foregut endoderm
cells. In some embodiments, these PDX1-negative foregut endoderm
cells are multipotent cells that can differentiate into cells,
tissues or organs derived from the anterior portion of the gut tube
(PDX1-negative foregut endoderm).
Dorsal, Ventral and Mixed Populations of PDX1-Positive Foregut
Endoderm Cells and Processes Related Thereto
[0253] Embodiments of the present invention relate to novel,
defined processes for the production of PDX1-negative 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). Other embodiments of the present
invention 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). 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 but
which cannot give rise to all three primary embryonic cells
lineages (endoderm, ectoderm and mesoderm). 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. In some
embodiments, the PDX1 positive endoderm cells are dorsal foregut
endoderm cells. In other embodiments, the PDX1 positive endoderm
cells are ventral foregut endoderm cells.
[0254] Some preferred embodiments of the present invention 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). In some embodiments, the PDX1
positive endoderm cells are dorsal foregut endoderm cells. In other
embodiments, the PDX1 positive endoderm cells are ventral foregut
endoderm cells.
[0255] 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. In some embodiments, the PDX1 positive
endoderm cells are dorsal endoderm cells that can differentiate
into cells, tissues or organs derived from the posterior portion of
the foregut, such as cells of the dorsal pancreatic bud. In other
embodiments, the PDX1 positive endoderm cells are ventral foregut
endoderm cells that can differentiate into cells, tissues or organs
derived from the posterior portion of the foregut, such as cells of
the ventral pancreatic bud.
[0256] The dorsal and/or ventral 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 of the present
invention, positive dorsal and/or ventral PDX1- 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 positive dorsal and/or ventral 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 in U.S.
Pat. No. 11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23,
2004, the disclosure of which is incorporated herein by reference
in its entirety.
[0257] Processes of producing PDX1-positive foregut endoderm cells,
including dorsal and/or ventral 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 dorsal
and/or ventral PDX1-positive foregut endoderm cells are derived
from human PDX1-negative definitive endoderm cells, which in turn,
are derived from hESCs. These human dorsal and/or ventral
PDX1-positive foregut endoderm cells can then be used to produce
functional insulin-producing .beta.-cells. To obtain useful
quantities of insulin-producing P-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 dorsal and/or ventral PDX1-positive foregut endoderm cells
represents an early step towards the production of functional
pancreatic islet/p-cells (as shown in FIG. 1), high efficiency of
differentiation at this step is particularly desirable.
[0258] In view of the desirability of efficient differentiation of
PDX1-negative definitive endoderm cells to PDX1-positive foregut
endoderm cells, some aspects of the present invention relate to in
vitro methodology that results in approximately 2-25% conversion of
PDX1-negative definitive endoderm cells to PDX1-positive foregut
endoderm cells. Some aspects of the present invention relate to in
vitro methodology that results in approximately 26% to at least
approximately 75% conversion of PDX1-negative definitive endoderm
cells to dorsal PDX1-positive foregut endoderm cells. Other aspects
of the present invention relate to in vitro methodology that
results in approximately 26% to at least approximately 75%
conversion of PDX1-negative definitive endoderm cells to ventral
PDX1-positive foregut endoderm cells. Typically, the
above-described 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, including dorsal and/or
ventral 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, including
dorsal and/or ventral 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. Additional aspects of the present
invention relate to cell cultures and enriched cell populations
comprising dorsal PDX1-positive foregut endoderm cells as well as
methods for identifying factors useful in the differentiation to
and from dorsal PDX1-positive foregut endoderm. Still other aspects
of the present invention relate to cell cultures and enriched cell
populations comprising ventral PDX1-positive foregut endoderm cells
as well as methods for identifying factors useful in the
differentiation to and from ventral PDX1-positive foregut
endoderm.
[0259] 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
of the present invention relate to cell markers whose presence,
absence and/or relative expression levels are indicative of
PDX1-positive foregut endoderm cells, including dorsal and/or
ventral PDX1-positive foregut endoderm cells, as well as methods
for detecting and determining the expression of such markers. 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. 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).
[0260] In some embodiments of the present invention, 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, SOX 17, 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. In some embodiments, markers
that are common to both dorsal and ventral PDX1-positive foregut
endoderm cells, such as PDX1 and/or one or more markers selected
from Table 3, are detected by Q-PCR and/or immunohistochemistry. In
other embodiments, markers that are preferentially, specifically or
uniquely expressed in dorsal PDX1-positive foregut endoderm cells,
such as one or more markers selected from Table 4, are detected by
Q-PCR and/or immunohistochemistry. 102631 By using the
differentiation and detection methods described herein, it is
possible to identify PDX1-positive foregut endoderm cells,
including dorsal and/or ventral PDX1-positive foregut endoderm
cells, as well as determine the proportion of dorsal and/or ventral
PDX1-positive foregut endoderm cells in a cell culture or cell
population. For example, in some embodiments of the present
invention, the dorsal and/or ventral 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
dorsal and/or ventral 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 dorsal and/or
ventral 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. In yet
other embodiments, the dorsal and/or ventral PDX1-positive foregut
endoderm cells or cell populations that are produced express one or
more of the markers selected from Table 3 at a level of about 2 or
more than 2 orders of magnitude greater than PDX1-negative
definitive endoderm cells or cell populations. In further
embodiments, the dorsal PDX1-positive foregut endoderm cells or
cell populations that are produced express one or more of the
markers selected from Table 4 at a level of about 2 or more than 2
orders of magnitude greater than PDX1-negative definitive endoderm
cells or cell populations.
[0261] The compositions and methods described herein have several
useful features. For example, the cell cultures and cell
populations comprising PDX1-positive endoderm, including dorsal
and/or ventral PDX1-positive foregut 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-Negative Definitive Endoderm Endoderm
(Definitive Endoderm) from Pluripotent Cells
[0262] Cell cultures and/or cell populations comprising
PDX1-positive foregut endoderm cells are produced from pluripotent
cells by first producing PDX1-negative definitive endoderm (also
referred to as "definitive endoderm"). Processes for
differentiating pluripotent cells to produce cell cultures and
enriched cell populations comprising definitive endoderm is
described briefly below and in detail in U.S. Pat. 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. 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. 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.
[0263] 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 U.S. Pat.
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.
[0264] 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.
[0265] 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.
[0266] 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.
[0267] 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.
[0268] 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 Differntiation of Pluripotent Cells to PDX1-Negative
Definitive Endoderm (Definitive Endoderm)
[0269] 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.
[0270] 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 MIXLI,
GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQI, CMKORI and CRIPI.
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 SOX 17 marker gene
and the OCT4 marker gene, which is characteristic of hESCs, is
monitored. Additionally, because definitive endoderm cells express
the SOX 17 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.
[0271] 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-I [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/SDFI 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-I 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)].
[0272] 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, MIXLI,
GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQI, CMKORI and CRIPI.
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 the 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. 102761 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.
Enrichment, Isolation and/or Purification of Definitive
Endoderm
[0273] 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-I or other molecules based on
SDF-I can also be used as affinity tags. Such molecules include,
but not limited to, SDF-I fragments, SDF-I fusions or SDF-I
mimetics.
[0274] 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.
[0275] 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.
[0276] 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-I or a
fragment, fusion or mimetic thereof.
[0277] 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.
[0278] 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.
[0279] 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.
Compositions Comprising PDX1-Negative Definitive Endoderm
(Definitive Endoderm)
[0280] 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.
Productio of PDX1-Negative Foregut Endoderm
[0281] Definitive endoderm cells can be specified toward pancreatic
differentiation by further differentiation of these cells to
produce PDX1-negative foregut endoderm cells. In some of the
differentiation processes described herein, cell cultures as well
as enriched or purified cell populations comprising definitive
endoderm cells can be used for further differentiation to cell
cultures and/or enriched cell populations comprising PDX1-negative
foregut endoderm cells.
[0282] Typically, definitive endoderm cells are differentiated to
PDX1-negative foregut endoderm cells by reducing or eliminating
TGF.beta. superfamily growth factor signaling in a cell culture or
cell population of SOX17-positive definitive endoderm cells. In
some embodiments, reducing or eliminating TGFP superfamily growth
factor signaling is mediated by diluting or removing an exogenously
added TGF.beta. superfamily growth factor, such as activin A, from
the cell culture or cell population of definitive endoderm. In
other embodiments, TGF.beta. superfamily growth factor signaling is
reduced or eliminated by providing the definitive endoderm cells
with a compound that blocks TGF.beta. superfamily growth factor
signaling, such as follistatin and/or noggin. In some embodiments,
TGF.beta. superfamily growth factor signaling can be reduced or
eliminated for 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, about ten days or greater than
about ten days subsequent to the differentiation of the human
pluripotent cells to definitive endoderm cells.
[0283] In some embodiments, differentiation of definitive endoderm
cells to foregut endoderm cells is enhanced by providing the
definitive endoderm cell culture or cell population with an
FGF-family growth factor and/or a hedgehog pathway inhibitor. In
such embodiments the FGF-family growth factor and/or hedgehog
pathway inhibitor is provided at 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, about ten days
or greater than about ten days subsequent to reducing or
eliminating TGF.beta. superfamily growth factor signaling in the
definitive endoderm cell culture. In a preferred embodiment, the
FGF-family growth factor and/or hedgehog pathway inhibitor is
provided at about the same time as reducing or eliminating TGFP
superfamily growth factor signaling in the definitive endoderm cell
culture.
[0284] In a preferred embodiment, the FGF-family growth factor
provided to the definitive endoderm cell culture or cell population
is FGFIO and/or FGF7. However, it will be appreciated that other
FGF-family growth factors or FGF-family growth factor analogs or
mimetics may be provided instead of or in addition to FGFIO and/or
FGF7. For example, an FGF-family growth factor selected from the
group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8,
FGF9, FGFIO, FGFII, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17,
FGF18, FGFI9, FGF20, FGF21, FGF22 and/or FGF23 may be provided. In
such embodiments, the FGF-family growth factor and/or the
FGF-family growth factor analog or mimetic is provided to the cells
of a cell culture such that it is present 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.
[0285] In other preferred embodiments, the hedgehog inhibitor is
KAAD-cyclopamine. However, it will be appreciated that other
hedgehog inhibitors can be used. Such inhibitors include, but are
not limited to, KKAD-cyclopamine analogs, jervine, jervine analogs,
hedgehog pathway blocking antibodies and any other inhibitors of
hedgehog pathway function known to those of ordinary skill in the
art. When used alone or in conjunction with FGF-family growth
factor, the hedgehog inhibitor can be provided 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.
[0286] In a preferred process for the production of a population of
PDX1-negative foregut endoderm cells from definitive endoderm
cells, TGF.beta. superfamily growth factor signaling is reduced or
eliminated for about two day subsequent to the differentiation of a
substantial portion of human pluripotent cells to definitive
endoderm (for example, after a three day, four or five day
differentiation protocol as described in the examples below). At
about the same time, the cell culture or cell population of
definitive endoderm cells is provided with 50 ng/ml of FGF-10 and
0.2 .mu.M KAAD-cyclopamine.
[0287] Cultures of PDX1-negative foregut endoderm cells can be
differentiated and further grown in a medium containing reduced or
no serum. Serum concentrations can range from about 0.05% (v/v) to
about 20% (v/v). In some processes, PDX1-negative foregut endoderm
cells are grown with serum replacement. For example, in certain
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 certain
processes described herein, the differentiation medium does not
include serum, serum replacement or any supplement comprising
insulin or insulin-like growth factors.
[0288] In certain processes, PDX1-negative foregut endoderm cells
are grown in the presence of B27. In such differentiation
processes, 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
processes, 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, CA) as a 5OX 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 5OX B27 stock solution to 90 ml of growth medium would produce a
growth medium supplemented with 5X B27. The concentration of B27
supplement in the medium can be about O.IX, about 0.2X, about 0.3X,
about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about
0.9X, about 1 X, about 1.1 X, about 1.2X, about 1.3X, about 1.4X,
about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, about
2X, about 2.5X, about 3X, about 3.5X, about 4X, about 4.5X, about
5X, about 6X, about 7X, about 8X, about 9X, about IOX, about I IX,
about 12X, about 13X, about 14X, about 15X, about 16X, about 17X,
about 18X, about 19X, about 20X and greater than about 20X.
[0289] In some embodiments, the PDX1-negative foregut endoderm
cells can be further differentiated to PDX1-positive foregut
endoderm cells by contacting the cells with a medium comprising, or
otherwise providing to the cells, a retinoid, such as retinoic acid
(RA). In some embodiments, the retinoid is provided to the cells of
a cell culture such that it is present at a concentration of at
least about 1 nM, at least about 0.01 .mu.M, at least about 0.02
.mu.M, at least about 0.04 PM, at least about 0.08 .mu.M, at least
about 0.1 PM, at least about 0.2 .mu.M, at least about 0.3 .mu.M,
at least about 0.4 PM, at least about 0.5 .mu.M, at least about 0.6
PM, at least about 0.7 PM, at least about 0.8 .mu.M, at least about
0.9 PM, at least about 1 .mu.M, at least about 1.1 .mu.M, at least
about 1.2 PM, 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 PM, 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 PM, at least
about 40 .mu.M or at least about 50 .mu.M. In such embodiments, the
retinoid is provided to the cells at 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, about ten days
or greater than about ten days subsequent to reducing or
eliminating TGF.beta. superfamily growth factor signaling in the
definitive endoderm cell culture. In a preferred embodiment, from
about 0.05 .mu.M RA to about 2 .mu.M RA is provided to the PDX-1
negative foregut endoderm cell culture about 2 to 3 days subsequent
to reducing or eliminating TGFP superfamily growth factor
signaling.
[0290] In some of the differentiation processes described herein,
the above-mentioned differentiation factors are removed from the
cell culture subsequent to their addition. For example, the
above-mentioned 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.
Monitoring the Differentiation of PDX1-Negative Definitive Endoderm
to PDX1-Negative Foregut Endoderm
[0291] Expression of HNF1b and/or FOXA1 and the lack of expression
of PDX1 can be detected and/or quantitated using the
above-described methods, such as Q-PCR and/or immunocytochemistry,
to monitor the differentiation of PDX1-negative definitive endoderm
to PDX1-negative foregut endoderm. In addition to the
above-described markers, in some embodiments of the present
invention, the expression of SOX17 is also determined.
[0292] In some embodiments, PDX1-negative 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-negative foregut endoderm
cell cultures produced by the processes described herein are
substantially free of visceral endoderm, parietal endoderm and/or
neural cells.
Compositions Comprising PDX1-Negative Foregut Endoderm
[0293] Some embodiments of the present invention relate to cell
compositions, such as cell cultures or cell populations, comprising
PDX1-negative foregut endoderm cells, wherein the PDX1-negtive
foregut endoderm cells are multipotent cells that can differentiate
into cells, tissues or organs derived from the anterior portion of
the gut tube. In accordance with certain embodiments, the
PDX1-negative foregut endoderm cells are mammalian cells, and in a
preferred embodiment, such cells are human cells.
[0294] Other embodiments of the present invention 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-negative
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.
[0295] Additional embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, produced
by the processes described herein comprise PDX1-negative 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 89%, at least about 88%, at
least about 87%, at least about 86%, at least about 85%, at least
about 84%, at least about 83%, at least about 82%, at least about
81%, at least about 80%, at least about 79%, at least about 78%, at
least about 77%, at least about 76%, at least about 75%, at least
about 74%, at least about 73%, at least about 72%, at least about
71%, at least about 70%, at least about 69%, at least about 68%, at
least about 67%, at least about 66%, at least about 65%, at least
about 64%, at least about 63%, at least about 62%, at least about
61%, at least about 60%, at least about 59%, at least about 58%, at
least about 57%, at least about 56%, at least about 55%, at least
about 54%, at least about 53%, at least about 52%, at least about
51% or at least about 50% PDX1-negative 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 .sup.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-negative 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-negative foregut endoderm cells in the cell
cultures or populations is calculated Without regard to the feeder
cells remaining in the culture.
[0296] Still other embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
PDX1-negative foregut endoderm cells and PDX1-negative definitive
endoderm cells. For example, cell cultures or cell populations
comprising at least about 5 PDX1-negative 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-negative 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-negative foregut endoderm cells to
PDX1-negative definitive endoderm cells are contemplated. For
example, compositions comprising at least about I PDX1-negative
foregut endoderm cell for about every 1,000,000 PDX1-negative
definitive endoderm cells, at least about I PDX1-negative foregut
endoderm cell for about every 100,000 PDX1-negative definitive
endoderm cells, at least about I PDX1-negative foregut endoderm
cell for about every 10,000 PDX1-negative definitive endoderm
cells, at least about I PDX1-negative foregut endoderm cell for
about every 1000 PDX1-negative definitive endoderm cells, at least
about I PDX1-negative foregut endoderm cell for about every 500
PDX1-negative definitive endoderm cells, at least about I
PDX1-negative foregut endoderm cell for about every 100
PDX1-negative definitive endoderm cells, at least about I
PDX1-negative foregut endoderm cell for about every 10
PDX1-negative definitive endoderm cells, at least about I
PDX1-negative foregut endoderm cell for about every 5 PDX1-negative
definitive endoderm cells, at least about I PDX1-negative foregut
endoderm cell for about every 4 PDX1-negative definitive endoderm
cells, at least about I PDX1-negative foregut endoderm cell for
about every 2 PDX1-negative definitive endoderm cells, at least
about I PDX1-negative foregut endoderm cell for about every I
PDX1-negative definitive endoderm cell, at least about 2
PDX1-negative foregut endoderm cells for about every I
PDX1-negative definitive endoderm cell, at least about 4
PDX1-negative foregut endoderm cells for about every I
PDX1-negative definitive endoderm cell, at least about 5
PDX1-negative foregut endoderm cells for about every I
PDX1-negative definitive endoderm cell, at least about 10
PDX1-negative foregut endoderm cells for about every I
PDX1-negative definitive endoderm cell, at least about 20
PDX1-negative foregut endoderm cells for about every I
PDX1-negative definitive endoderm cell, at least about 50
PDX1-negative foregut endoderm cells for about every I
PDX1-negative definitive endoderm cell, at least about 100
PDX1-negative foregut endoderm cells for about every I
PDX1-negative definitive endoderm cell, at least about 1000
PDX1-negative foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 10,000
PDX1-negative foregut endoderm cells for about every I
PDX1-negative definitive endoderm cell, at least about 100,000
PDX1-negative foregut endoderm cells for about every I
PDX1-negative definitive endoderm cell and at least about 1,000,000
PDX1-negative foregut endoderm cells for about every I
PDX1-negative definitive endoderm cell are contemplated.
[0297] In some embodiments of the present invention, the
PDX1-negative definitive endoderm cells from which PDX1-negative
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.
[0298] Further embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
human cells, including human PDX1-negative foregut endoderm cells,
wherein the expression of the SOX 17, HNF I b and/or FOXA I marker
is greater than the expression of the AFP, SOX7, SOX , ZIC I and/or
NFM marker in at least about 2% of the human cells. In other
embodiments, the expression of the SOX17, HNF1b and/or FOXA1 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 SOX17, HNFIb
and/or FOXA1 is greater than the expression of the AFP, SOX7, SOX
1, ZIC I and/or NFM marker, is calculated without regard to feeder
cells.
[0299] It will be appreciated that some embodiments of the present
invention relate to compositions, such as cell cultures or cell
populations, comprising human PDX1-negative foregut endoderm cells,
wherein the expression of one or more markers selected from the
group consisting of SOX17, HNF1b and/or FOXA1 is greater than the
expression of the PDX1 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, HNF1b and/or FOXA1 is greater than the
expression of the PDX1 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, HNFIb
and/or FOXA1 is greater than the expression of the PDX1 marker, is
calculated without regard to feeder cells.
[0300] Using the processes described herein, compositions
comprising PDX1-negative foregut endoderm cells substantially free
of other cell types can be produced. With respect to cells in cell
cultures or in cell populations, the term 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. In some embodiments of the present invention, the
PDX1-negative 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.
[0301] In one embodiment of the present invention, a description of
a PDX1-negative foregut endoderm cell based on the expression of
marker genes is, SOX17 high, HNF1b high, FOXA1 high, PDX1 low, AFP
low, SOX7 low, SOX1 low, ZIC1 low and NFM low.
Production of PDX1-Positive Foregut Endoderm Directly from
PDX1-Negative Definitive Endoderm
[0302] 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.
[0303] In some embodiments of the present invention, 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, SOX I 7-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.
[0304] 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-.alpha. 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.
[0305] By "conditioned medium" is meant, 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 I 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 I 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.
[0306] In some embodiments of the present invention, 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 I 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.
[0307] 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.
[0308] 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-.beta.0 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.
[0309] 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. 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.
[0310] In some embodiments of the present invention, 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 I PM, 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 PM,
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.
As used herein, "retinoid" refers to retinol, retinal or retinoic
acid as well as derivatives of any of these compounds. In a
preferred embodiment, the retinoid is retinoic acid.
[0311] In other embodiments of the present invention, 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 of the present invention, 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.
[0312] In some embodiments of the present invention, 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 of the present invention, 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.
[0313] In certain embodiments of the present invention, 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.
[0314] 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.
[0315] 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, CA) as a 5OX 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 5OX B27 stock solution to 90 ml of growth medium would produce a
growth medium supplemented with 5X B27. The concentration of B27
supplement in the medium can be about O.IX, about 0.2X, about 0.3X,
about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about
0.9X, about IX, about 1.1X, about 1.2X, about 1.3X, about 1.4X,
about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, about
2X, about 2.5X, about 3X, about 3.5X, about 4X, about 4.5X, about
5X, about 6X, about 7X, about 8X, about 9X, about 10X, about I IX,
about 12X, about 13X, about 14X, about 1 5X, about 1 6X, about 1
7X, about 1 8X, about 1 9X, about 20X and greater than about
2OX.
Production of Dorsal PDX1-Positive Foregut Endoderm from
PDX1-Negative Definitive Endoderm
[0316] The dorsal PDX1-positive foregut endoderm cell cultures and
populations comprising dorsal 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. Furthermore, 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 dorsal PDX1-positive foregut endoderm cells. It will be
appreciated, however, that the starting materials for the
production of dorsal 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.
[0317] As described in connection with the production of mixed
populations of PDX1-positive foregut endoderm cells, in some
embodiments of the present invention, 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 dorsal 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 dorsal
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 dorsal
PDX1-positive foregut endoderm cells.
[0318] PDX1-negative definitive endoderm cells in culture are
differentiated to dorsal PDX1-positive endoderm cells by providing
to a cell culture comprising PDX1-negative, SOX17-positive
definitive endoderm cells a retinoid, such as retinoic acid (RA).
In some embodiments, the retinoid is used in conjunction with a
member of the TGFD superfamily of growth factors and/or Connaught
Medical Research Labs niedium (CRML medium) (Invitrogen, Carlsbad,
Calif.).
[0319] With respect to some of the embodiments of differentiation
processes described herein, the RA or a combination of the
above-mentioned 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 dorsal PDX1-positive foregut endoderm
cells. 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 cell population. In preferred
embodiments, the term "portion" means at least .sup.5%, at least
.sup.6%, at least 7%, at least 8%, at least 9%, at least 10%, at
least 11%, at least 12%, at least 13%, at least 14%, at least 15%,
at least 16%, at least 17%, at least 18%, at least 19%, at least
20%, at least 21%, at least 22%, at least .sup.23%, at least 24%,
at least 25%, at least .sup.26%, at least .sup.27%, at least 28%,
at least 29%, at least 30%, at least 3 1%, at least 32%, at least
33%, at least 34%, at least 35%, at least 36%, at least 37%, at
least 38%, at least 39%, at least 40%, at least 41%, at least 42%,
at least 43%, at least 44%, at least 45%, at least 46%, at least
47%, at least 48%, at least 49%, at least 50%, at least 51%, at
least 52%, at least 53%, at least 54%, at least 55%, at least 56%,
at least 57%, at least 58%, at least 59%, at least 60%, at least
61%, at least 62%, at least 63%, at least 64%, at least 65%, at
least 66%, at least 67%, at least 68%, at least 69%, at least 70%,
at least 71%, at least 72%, at least 73%, at least 74% or at least
75% of the cell culture or cell population.
[0320] In some embodiments of the present invention, 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
PM, 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 I PM, at least
about 1.1 .mu.M, at least about 1.2 .mu.M, at least about 1.3 PM,
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 PM, 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.
[0321] In preferred embodiments of the present invention, a
population of dorsally-biased PDX1-positive foregut endoderm cells
is produced by providing retinoic acid in the absence of exogenous
FGF-10 or other FGF family growth factor. In such embodiments, RA
is provided at a concentration of about 2 .mu.M. In a preferred
embodiment, RA is provided at a concentration of about 2 .mu.M in
CMRL medium.
[0322] In some embodiments, activin A and/or activin B are provided
to the cell culture along with RA. For example, in some
embodiments, RA is provided to the cell culture at a concentration
of about 2 .mu.M and activin A and/or activin B is provided to 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.
[0323] In some embodiments, the differentiation factors and/or CRML
medium is provided to the PDX1-negative definitive endoderm cells
at about three days, at about four days, at about five days, at
about six days, at about seven days, at about eight days, at about
nine days, at about ten days or at about greater than ten days
subsequent to the initiation of differentiation from hESCs. In
preferred embodiments, differentiation factors and/or CRML medium
is provided to the PDX1-negative definitive endoderm cells at about
five days subsequent to the initiation of differentiation from
hESCs.
[0324] In certain embodiments of the present invention, the
above-mentioned differentiation factors are removed from the cell
culture subsequent to their addition. For example, the
above-mentioned 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.
[0325] Cultures of dorsal 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, dorsal 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, dorsal
PDX1-positive foregut endoderm cells are grown without serum. In
other embodiments, dorsal PDX1-positive foregut endoderm cells are
grown with serum replacement.
[0326] In still other embodiments, dorsal 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, CA) as a 5OX 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 5OX B27 stock solution to 90 ml of growth medium would produce a
growth medium supplemented with 5X B27. The concentration of B27
supplement in the medium can be about 0.1X, about 0.2X, about 0.3X,
about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about
0.9X, about 1X, about 1X, about 1.2X, about 1.3X, about 1.4X, about
1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, about 2X,
about 2.5X, about 3X, about 3.5X, about 4X, about 4.5X, about 5X,
about 6X, about 7X, about 8X, about 9X, about 10X, about I IX,
about 12X, about 13X, about 14X, about 15X, about 16X, about 17X,
about 18X, about 19X, about 20X and greater than about 20X.
Production of Ventral PDX1-Positive Foregut Endoderm from
PDX1-Negative Definitive Endoderm
[0327] The ventral PDX1-positive foregut endoderm cell cultures and
populations comprising ventral 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. Furthermore, 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 ventral PDX1-positive foregut endoderm cells. It will be
appreciated, however, that the starting materials for the
production of ventral 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.
[0328] As described in connection with the production of mixed
populations of PDX1-positive foregut endoderm cells, in some
embodiments of the present invention, 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 ventral 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 ventral
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 ventral
PDX1-positive foregut endoderm cells.
[0329] PDX1-negative definitive endoderm cells in culture are
differentiated to ventral PDX1-positive endoderm cells by providing
to a cell culture comprising PDX1-negative, SOX 17-positive
definitive endoderm cells an FGF-family growth factor or FGF-family
growth factor analog or mimetic. In some embodiments, the
FGF-family growth factor or FGF-family growth factor analog or
mimetic is used in conjunction with a hedgehog inhibitor and/or
Connaught Medical Research Labs medium (CRML medium) (Invitrogen,
Carlsbad, CA). In especially preferred embodiments, FGF-10 and/or
KAAD-cyclopamine is provided to a cell culture comprising
PDX1-negative definitive endoderm cells in the absence of RA or
other retinoid. In certain embodiments, BMP4 may be included in
FGF-10 and/or KAAD-cyclopamine in the absence of RA or other
retinoid. After about one day to about ten days subsequent to the
addition of the FGF-family growth factor analog or mimetic and/or
hedgehog inhibitor, a retinoid, such as RA, or a retinoid
containing supplement, such as B27, is provided to induce the
expression of PDX1. In a preferred embodiments, RA is provided at
about 2 days, about 3 day, about 4 day or about 5 days subsequent
to the addition of the FGF-family growth factor analog or mimetic
and/or hedgehog inhibitor. In other embodiments, B27 is provided at
about the same time as providing the FGF-family growth factor
analog or mimetic and/or hedgehog inhibitor.
[0330] With respect to some of the embodiments of differentiation
processes described herein, a retinoid and a combination of the
above-mentioned 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 ventral PDX1-positive foregut
endoderm cells. 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 cell population. In
preferred embodiments, the term "portion" means at least 5%, at
least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at
least I 1%, at least 12%, at least 13%, at least 14%, at least 15%,
at least 16%, at least 17%, at least 18%, at least 19%, at least
20%, at least 21%, at least 22%, at least 23%, at least 24%, at
least 25%, at least 26%, at least 27%, at least 28%, at least 29%,
at least 30%, at least 31%, at least 32%, at least 33%, at least
34%, at least 35%, at least 36%, at least 37%, at least 38%, at
least 39%, at least .sup.40%, at least 41%, at least 42%, at least
43%, at least 44%, at least 45%, at least 46%, at least 47%, at
least 48%, at least 49%, at least 50%, at least 51%, at least 52%,
at least 53%, at least 54%, at least 55%, at least 56%, at least
57%, at least 58%, at least 59%, at least 60%, at least 61%, at
least 62%, at least 63%, at least 64%, at least 65%, at least 66%,
at least 67%, at least 68%, at least 69%, at least 70%, at least
71%, at least 72%, at least 73%, at least 74% or at least 75% of
the cell culture or cell population.
[0331] In some embodiments of the present invention, the FGF-family
growth factor or FGF-family growth factor analog or mimetic is
provided to the cells of a cell culture such that it is present 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 other embodiments, when used alone or in
conjunction with FGF-10, KAAD-cyclopamine can be provided 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 .parallel.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.
[0332] In preferred embodiments of the present invention, a
population of ventrally-biased PDX1-positive foregut endoderm cells
is produced by providing a population of PDX1-negative definitive
endoderm with 50 ng/ml of FGF-10 and 0.5 .mu.M KAAD-cyclopamine in
CMRL medium in the absence of RA. About two days subsequent to the
addition of FGF-10 and KAAD-cyclopamine 2 .mu.M RA is added to
complete the differentiation of the cells to PDX1-positive
cells.
[0333] In some embodiments, the differentiation factors and/or CRML
medium is provided to the PDX1-negative definitive endoderm cells
at about three days, at about four days, at about five days, at
about six days, at about seven days, at about eight days, at about
nine days, at about ten days or at about greater than ten days
subsequent to the initiation of differentiation from hESCs. In
preferred embodiments, differentiation factors and/or CRML medium
is provided to the PDX1-negative definitive endoderm cells at about
three days subsequent to the initiation of differentiation from
hESCs.
[0334] In certain embodiments of the present invention, the
above-mentioned differentiation factors are removed from the cell
culture subsequent to their addition. For example, the
above-mentioned 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.
[0335] Cultures of ventral 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, ventral 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, ventral
PDX1-positive foregut endoderm cells are grown without serum. In
other embodiments, ventral PDX1-positive foregut endoderm cells are
grown with serum replacement.
[0336] In still other embodiments, ventral 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, CA) as a 5OX 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 5OX B27 stock solution to 90 ml of growth medium would produce a
growth medium supplemented with 5X B27. The concentration of B27
supplement in the medium can be about 0.1X, about 0.2X, about 0.3X,
about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about
0.9X, about IX, about 1.1 X, about 1.2X, about 1.3X, about 1.4X,
about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, about
2X, about 2.5X, about 3X, about 3.5X, about 4X, about 4.5X, about
5X, about 6X, about 7X, about 8X, about 9X, about 10X, about 11X,
about 12X, about 13X, about 14X, about 15X, about 16X, about 17X,
about 18X, about 19X, about 20X and greater than about 20X. In some
embodiments where B27 is provided, a retinoid is not provided to
complete the differentiation of the PDX1-negative cells to ventral
PDX1-positive foregut endoderm. Monitoring the Differentiation of
PDX1-Negative Definitive Endoderm to PDX1-Positive Foregut
Endoderm
[0337] As with the differentiation of definitive endoderm cells
from pluripotent cells, the progression of differentiation from
PDX1-negative, SOX 17-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
of the present invention, 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 of the
present invention, 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.
[0338] 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 present invention, 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 of the present invention 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.
[0339] 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.
Monitoring the Differentiation of PDX1-Negative Definitive Endoderm
to Dorsal PDX1-Positive Foregut Endoderm
[0340] Expression of one or more of the markers described in Table
3 and/or Table 4, in the Examples below, can be detected and/or
quantitated using the above-described methods, such as Q-PCR and/or
immunocytochemistry, to monitor the differentiation of
PDX1-negative definitive endoderm to dorsal PDX1-positive endoderm.
Markers associated with both dorsally-biased and ventrally-biased
PDX1-positive foregut endoderm cells are described in Table 3. Of
these markers, the markers selected from the group consisting of
CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2
and SLC27A2 are cell surface markers. Some preferred markers listed
in Table 3 for monitoring the production of dorsal PDX1-positive
foregut endoderm are selected from the group consisting of
SERPINF2, DUSP9, CDH6 and SOX9. Markers associated with
dorsally-biased foregut endoderm are described in Table 4. Each of
the Table 4 markers is expressed preferentially, specifically or
uniquely in dorsal PDX1-positive foregut endoderm cells as compared
to other PDX1-positive cells. Of these markers, the markers
selected from the group consisting of ADORA2A, CD47, EPB41L1, MAG,
SFRP5, SLC16A10, SLC16A2, SLCIA3, SLC3OA4, SLICK, SLITRK4 and XPRI
are cell surface markers. Some preferred markers listed in Table 4
for monitoring the production of dorsal PDX1-positive foregut
endoderm are selected from the group consisting of HOXAI, PDEI IA,
FAM49A and WNT5A.
[0341] In addition to the above-described markers, in some
embodiments of the present invention, 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 of the present invention 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, PDX1 ZIC1 and/or NFM is determined.
[0342] In some embodiments, dorsal 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 dorsal PDX1-positive
foregut endoderm cell cultures produced by the processes described
herein are substantially free of visceral endoderm, parietal
endoderm and/or neural cells.
Monitoring the Differentiation of PDX1-Negative Definitive Endoderm
to Ventral PDX1-Positive Foregut Endoderm
[0343] As described in the previous section, markers associated
with both dorsally-biased and ventrally-biased PDX1-positive
foregut endoderm cells are described in Table 3. As such,
expression of one or more of the markers described in Table 3 can
be detected and/or quantitated using the above-described methods,
such as Q-PCR and/or immunocytochemistry, to monitor the
differentiation of PDX1-negative definitive endoderm to ventral
PDX1-positive endoderm. Of the markers described in Table 3, the
markers selected from the group consisting of CDH6, GABRA2, GRIA3,
IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2 and SLC27A2 are cell
surface markers. Some preferred markers listed in Table 3 for
monitoring the production of ventral PDX1-positive foregut endoderm
are selected from the group consisting of SERPINF2, DUSP9, CDH6 and
SOX9. Furthermore, because the markers described in Table 4 are
preferentially, specifically or uniquely expressed in
dorsally-biased foregut endoderm, detecting the lack of expression,
or reduced expression relative to the expression in dorsal
PDX1-positive foregut endoderm, of one or more of these markers is
also useful for monitoring the differentiation of PDX1-negative
definitive endoderm to ventral PDX1-positive foregut endoderm. Of
the Table 4 markers, the markers selected from the group consisting
of ADORA2A, CD47, EPB41LI, MAG, SFRP5, SLC16AI0, SLC16A2, SLCIA3,
SLC3OA4, SLICK, SLITRK4 and XPRI are cell surface markers. Some
preferred markers listed in Table 4 for monitoring the production
of dorsal PDX1-positive foregut endoderm are selected from the
group consisting of HOXAI, PDEIIA, FAM49A and WNT5A. As such, the
absence, or insubstantial expression, of these markers in
PDX1-positive cells expressing one or more markers selected from
Table 3, is indicative of ventral PDX1-positive
[0344] In addition to the above-described markers, in some
embodiments of the present invention, 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 of the present invention 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.
[0345] In some embodiments, ventral 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 ventral 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 Dorsal and/or Ventral
PDX1-Positive Foregut Endoderm
[0346] PDX1-positive foregut endoderm cells, including dorsal
and/or ventral PDX1-positive foregut 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 dorsal and/or ventral
PDX1-positive foregut 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 dorsal
and/or ventral PDX1-positive foregut 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 a cell surface marker
selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R,
KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2, SLC27A2, ADORA2A, CD47,
EPB41L1, MAG, SFRP5, SLC16AI0, SLC16A2, SLCIA3, SLC3OA4, SLICK,
SLITRK4 and XPRI is used as an affinity tag for the enrichment,
isolation or purification of dorsal and/or ventral PDX1-positive
foregut endoderm cells.
[0347] 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 dorsal and/or ventral PDXl-positive
foregut endoderm cells described herein. In one process, an
antibody which binds to a marker selected from CDH6, GABRA2, GRIA3,
IL6R, KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2, SLC27A2, ADORA2A,
CD47, EPB41LI, MAG, SFRP5, SLC16A10, SLC16A2, SLCIA3, SLC3OA4,
SLICK, SLITRK4 and XPRI is attached to a magnetic bead and then
allowed to bind to dorsal and/or ventral PDX1-positive foregut
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 dorsal and/or ventral
PDX1-positive foregut 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.
[0348] Additional methods for obtaining enriched, isolated or
purified dorsal and/or ventral PDX1-positive foregut endoderm cell
cultures or populations can also be used. For example, in some
embodiments, an antibody that binds to a marker selected from the
group consisting of CDH6, GABRA2, GRIA3, IL6R, KCNJ2, LGALS3,
LGALS3/GALIG, SERPINF2, SLC27A2, ADORA2A, CD47, EPB41LI, MAG,
SFRP5, SLC16AI0, SLC16A2, SLCIA3, SLC3OA4, SLICK, SLITRK4 and XPRI
is incubated with a dorsal and/or ventral PDX1-positive foregut
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). The marker-positive cells are collected separately
from marker-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 dorsal and/or ventral PDX1-positive foregut
endoderm cells.
[0349] In still other processes, dorsal and/or ventral
PDX1-positive foregut endoderm cells are enriched, isolated and/or
purified using a ligand or other molecule that binds to a marker
selected from the group consisting of CDH6, GABRA2, GRIA3, IL6R,
KCNJ2, LGALS3, LGALS3/GALIG, SERPINF2, SLC27A2, ADORA2A, CD47,
EPB41LI, MAG, SFRP5, SLC16A10, SLC16A2, SLCIA3, SLC30A4, SLICK,
SLITRK4 and XPRI.
[0350] In preferred processes, dorsal and/or ventral PDX1-positive
foregut endoderm cells are enriched, isolated and/or purified from
other cells after the PDX1-negative definitive endoderm cell
cultures are induced to differentiate towards the dorsal and/or
ventral PDX1-positive foregut 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.
[0351] In addition to the procedures just described, dorsal and/or
ventral PDX1-positive foregut endoderm cells may also be isolated
by other techniques for cell isolation. Additionally, dorsal and/or
ventral 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 the
dorsal and/or ventral PDX1-positive foregut endoderm cells.
[0352] Using the methods described herein, enriched, isolated
and/or purified populations of dorsal and/or ventral PDX1-positive
foregut endoderm cells and or tissues can be produced in vitro from
PDX1-negative definitive endoderm cell cultures or cell
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 dorsal and/or ventral PDX1-positive foregut endoderm
cells. Some preferred enrichment, isolation and/or purification
methods relate to the in vitro production of dorsal and/or ventral
PDX1-positive foregut endoderm cells from human PDX1-negative
definitive endoderm cells. Using the methods described herein, cell
populations or cell cultures can be enriched in dorsal and/or
ventral PDX1-positive foregut endoderm content by at least about 2-
to about 1000-fold as compared to untreated cell populations or
cell cultures. In some embodiments, dorsal and/or ventral
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, dorsal and/or
ventral 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, dorsal
and/or ventral 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, dorsal
and/or ventral 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, dorsal
and/or ventral 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.
Enrichment, Isolation and/or Purification of Dorsal and/or Ventral
PDX1-Positive Foregut Endoderm
[0353] With respect to additional aspects of the present invention,
dorsal and/or ventral PDX1-positive foregut endoderm cells can be
enriched, isolated and/or purified. In some embodiments of the
present invention, cell populations enriched for dorsal and/or
ventral PDX1-positive foregut endoderm cells are produced by
isolating such cells from cell cultures.
[0354] In some embodiments of the present invention, dorsal and/or
ventral 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 fluorescent protein (GFP) or another nucleic
acid encoding an expressible fluorescent marker gene, such as the
gene encoding luciferase, 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 promoter of a gene selected from
Table 3 or Table 4 such that the expression of the GFP gene product
or biologically active fragment thereof is under control of the
such promoter. In some embodiments, the entire coding region of the
nucleic acid, which encodes the marker selected from Table 3 or
Table 4, 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 the marker selected from Table 3 or Table 4, thereby
generating a fusion protein. In such embodiments, the fusion
protein retains a fluorescent activity similar to GFP.
[0355] Fluorescently marked cells, such as the above-described
pluripotent cells, are differentiated to definitive endoderm and
then to dorsal and/or ventral PDX1-positive foregut endoderm cells
as described previously above. Because dorsal and/or ventral
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 dorsal and/or ventral
PDX1-positive foregut endoderm cells and unlabeled PDX1-negative
cells are sorted using a FACS. Dorsal and/or ventral PDX1-positive
foregut endoderm 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 dorsal and/or ventral PDX1-positive foregut
endoderm.
[0356] It will be appreciated that the above-described enrichment,
isolation and purification procedures can be used with such
cultures at any stage of differentiation.
[0357] Using the methods described herein, enriched, isolated
and/or purified populations of dorsal and/or ventral 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 dorsal and/or ventral
PDX1-positive foregut endoderm cells. Some preferred enrichment,
isolation and/or purification methods relate to the in vitro
production of dorsal and/or ventral PDX1-positive foregut endoderm
cells from human embryonic stem cells.
[0358] Using the methods described herein, cell populations or cell
cultures can be enriched in dorsal and/or ventral 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, dorsal and/or ventral 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, dorsal and/or ventral 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, dorsal and/or ventral
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, dorsal
and/or ventral 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, dorsal
and/or ventral 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.
Compistion Comprising Dorsal and/or Ventral PDX1-Positive Foregut
Endoderm
[0359] Some embodiments of the present invention relate to cell
compositions, such as cell cultures or cell populations, comprising
dorsal and/or ventral PDX1-positive foregut endoderm cells, wherein
the dorsal and/or ventral 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, such as
the dorsal pancreatic bud and/or the ventral pancreatic bud. In
accordance with certain embodiments, the dorsal and/or ventral
PDX1-positive foregut endoderm cells are mammalian cells, and in a
preferred embodiment, such cells are human cells.
[0360] Other embodiments of the present invention 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, dorsal and/or
ventral 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.
[0361] Additional embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, produced
by the processes described herein comprise dorsal and/or ventral
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 89%, at least
about 88%, at least about 87%, at least about 86%, at least about
85%, at least about 84%, at least about 83%, at least about 82%, at
least about 81%, at least about 80%, at least about 79%, at least
about 78%, at least about 77%, at least about 76%, at least about
75%, at least about 74%, at least about 73%, at least about 72%, at
least about 71%. at least about 70%, at least about 69%, at least
about 68%, at least about 67%, at least about 66%, at least about
65%, at least about 64%, at least about 63%, at least about 62%, at
least about 61%, at least about 60%, at least about 59%, at least
about 58%, at least about 57%, at least about 56%, at least about
55%, at least about 54%, at least about 53%, at least about 52%, at
least about 51% or at least about 50% dorsal and/or ventral
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% dorsal and/or ventral
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 dorsal and/or ventral
PDX1-positive foregut endoderm cells in the cell cultures or
populations is calculated without regard to the feeder cells
remaining in the culture.
[0362] Still other embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
mixtures of dorsal and/or ventral PDX1-positive foregut endoderm
cells and PDX1-negative definitive endoderm cells. For example,
cell cultures or cell populations comprising at least about 5
dorsal and/or ventral 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 dorsal and/or ventral 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 dorsal and/or ventral
PDX1-positive foregut endoderm cells to PDX1-negative definitive
endoderm cells are contemplated. For example, compositions
comprising at least about 1 dorsal or ventral PDX1-positive foregut
endoderm cell for about every 1,000,000 PDX1-negative definitive
endoderm cells, at least about I dorsal or ventral PDX1-positive
foregut endoderm cell for about every 100,000 PDX1-negative
definitive endoderm cells, at least about I dorsal or ventral
PDX1-positive foregut endoderm cell for about every 10,000
PDX1-negative definitive endoderm cells, at least about I dorsal or
ventral PDX1-positive foregut endoderm cell for about every 1000
PDX1-negative definitive endoderm cells, at least about I dorsal or
ventral PDX1-positive foregut endoderm cell for about every 500
PDX1-negative definitive endoderm cells, at least about I dorsal or
ventral PDX1-positive foregut endoderm cell for about every 100
PDX1-negative definitive endoderm cells, at least about I dorsal or
ventral PDX1-positive foregut endoderm cell for about every 10
PDX1-negative definitive endoderm cells, at least about I dorsal or
ventral PDX1-positive foregut endoderm cell for about every 5
PDX1-negative definitive endoderm cells, at least about I dorsal or
ventral PDX1-positive foregut endoderm cell for about every 4
PDX1-negative definitive endoderm cells, at least about I dorsal or
ventral PDX1-positive foregut endoderm cell for about every 2
PDX1-negative definitive endoderm cells, at least about I dorsal or
ventral PDX1-positive foregut endoderm cell for about every I
PDX1-negative definitive endoderm cell, at least about 2 dorsal
and/or ventral PDX1-positive foregut endoderm cells for about every
1 PDX1-negative definitive endoderm cell, at least about 4 dorsal
and/or ventral PDX1-positive foregut endoderm cells for about every
I PDX1-negative definitive endoderm cell, at least about 5 dorsal
and/or ventral PDX1-positive foregut endoderm cells for about every
I PDX1-negative definitive endoderm cell, at least about 10 dorsal
and/or ventral PDX1-positive foregut endoderm cells for about every
I PDX1-negative definitive endoderm cell, at least about 20 dorsal
and/or ventral PDX1-positive foregut endoderm cells for about every
I PDX1-negative definitive endoderm cell, at least about 50 dorsal
and/or ventral PDX1-positive foregut endoderm cells for about every
I PDX1-negative definitive endoderm cell, at least about 100 dorsal
and/or ventral PDX1-positive foregut endoderm cells for about every
I PDX1-negative definitive endoderm cell, at least about 1000
dorsal and/or ventral PDX1-positive foregut endoderm cells for
about every I PDX1-negative definitive endoderm cell, at least
about 10,000 dorsal and/or ventral PDX1-positive foregut endoderm
cells for about every I PDX1-negative definitive endoderm cell, at
least about 100,000 dorsal and/or ventral PDX1-positive foregut
endoderm cells for about every I PDX1-negative definitive endoderm
cell and at least about 1,000,000 dorsal and/or ventral
PDX1-positive foregut endoderm cells for about every I
PDX1-negative definitive endoderm cell are contemplated.
[0363] In some embodiments of the present invention, the
PDX1-negative definitive endoderm cells from which dorsal and/or
ventral 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.
[0364] Further embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
human cells, including human dorsal and/or ventral PDX1-positive
foregut endoderm cells, wherein the expression of the PDX1 marker
is greater than the expression of the AFP, SOX7, SOX I, ZIC I
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, SOX I, ZIC I and/or NFM marker, is calculated
without regard to feeder cells.
[0365] It will be appreciated that some embodiments of the present
invention relate to compositions, such as cell cultures or cell
populations, comprising human dorsal and/or ventral PDX1-positive
foregut endoderm cells, wherein the expression of one or more
markers selected from the group consisting of SOX 17, HOXA 13 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 SOX7, HOXA 13 and HOXC6 is greater than the
expression of the AFP, SOX7, SOX 1, ZIC I and/or NFM marker, is
calculated without regard to feeder cells.
[0366] Other embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
human cells, including human dorsal and/or ventral PDX1-positive
foregut endoderm cells, wherein the expression of one or more
markers selected from Table 3 is greater than the expression of the
AFP, SOX7, SOX I, ZIC I and/or NFM marker in at least about 2% of
the human cells. In other embodiments, the expression of the one or
more markers selected from Table 3 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 cell 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 Table 3 is greater
than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker,
is calculated without regard to feeder cells.
[0367] Other embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
human cells, including human dorsal PDX1-positive foregut endoderm
cells, wherein the expression of one or more markers selected from
Table 4 is greater than the expression of the AFP, SOX7, SOX I, ZIC
I and/or NFM marker in at least about 2% of the human cells. In
other embodiments, the expression of the one or more markers
selected from Table 4 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 Table 4 is greater than the
expression of the AFP, SOX7, SOX I, ZIC I and/or NFM marker, is
calculated without regard to feeder cells.
[0368] Other embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
human cells, including human ventral PDX1-positive foregut endoderm
cells, wherein the expression of a marker selected from Table 3 is
greater than the expression of the AFP, SOX7, SOX1, ZIC I and/or
NFM marker in at least about 2% of the human cells, and wherein a
marker selected from Table 4 is not substantially expressed as
compared to the expression of the same marker in dorsal
PDX1-positive foregut endoderm cells. In other embodiments, the
expression of the marker selected from Table 3 is greater than the
expression of the AFP, SOX7, SOX 1, ZIC I 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 .sup.30% of the human cells, in at least
about .sup.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 such
embodiments, a marker selected from Table 4 is not substantially
expressed as compared to the expression of the same marker in
dorsal PDX1-positive foregut endoderm cells. In some embodiments,
the percentage of human cells in the cell cultures or populations,
wherein the expression of a marker selected from Table 3 is greater
than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker,
and wherein a marker selected from Table 4 is not substantially
expressed as compared to the expression of the same marker in
dorsal PDX1-positive foregut endoderm cells, is calculated without
regard to feeder cells.
[0369] Additional embodiments of the present invention 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 and the expression of one or more
markers selected from Table 3 or Table 4 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 and the expression of one or more
markers selected from Table 3 or Table 4 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.
[0370] Still other embodiments of the present invention 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 SOX 17, HOXA 13 and HOXC6 and the expression of one
or more markers selected from Table 3 or Table 4 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 and the expression of one or
more markers selected from Table 3 or Table 4 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.
[0371] Using the processes described herein, compositions
comprising dorsal and/or ventral PDX1-positive foregut endoderm
cells substantially free of other cell types can be produced. With
respect to cells in cell cultures or in cell populations, the term
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. In some embodiments of the
present invention, the dorsal and/or ventral 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, SOX I, ZIC I and/or NFM marker genes.
[0372] In one embodiment of the present invention, a description of
a dorsal PDX1-positive foregut endoderm cell based on the
expression of marker genes is, PDX1 high, a marker selected from
Table 3 high, a marker selected from Table 4 high, AFP low, SOX7
low, SOX I low, ZIC1 low and NFM low.
[0373] In one embodiment of the present invention, a description of
a ventral PDX1-positive foregut endoderm cell based on the
expression of marker genes is, PDX1 high, a marker selected from
Table 3 high, a marker selected from Table 4 low as compared to the
expression of the same marker in dorsal PDX1-positive foregut
endoderm, AFP low, SOX7 low, SOX1 low, ZIC1 low and NFM low.
Increasing Expression of PDX in a SOX17-Positive Definitive
Endoderm Cell
[0374] Some aspects of the present invention 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.
[0375] In other embodiments of the present invention, the
expression of the PDX1 gene product in cell cultures or cell
populations comprising SOX I 7-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.
[0376] In some embodiments of the present invention, 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.
[0377] 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.
[0378] It will be appreciated that the above described methods can
also be used to increase the expression of one or more markers
selected from Table 3 and/or Table 4 in dorsal PDX1-positive
foregut endoderm cells. Similarly, such methods can be used to
increase the expression of one or more markers selected from Table
3 in ventral PDX1-positive foregut endoderm cells.
Identification of Factors Capable of Promoting the Differentiation
of PDX1-Negative Definitive Endoderm Cells to PDX1-Positive Foregut
Endoderm Cells
[0379] Additional aspects of the present invention relate to
methods of identifying one or more differentiation factors capable
of promoting the differentiation of PDX1-negative definitive
endoderm cells to PDXl-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.
[0380] 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
HOXAI3 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 HOC16 is determined Q-PCR.
[0381] 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.
[0382] 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 I 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.
[0383] 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.
[0384] It will be appreciated that the above-described methods can
be used to identify factors capable of promoting the
differentiation of PDX1-negative definitive endoderm cells to
dorsal PDX1-positive foregut endoderm cells by monitoring the
expression of one or more markers expression from Table 4. In some
embodiments, the expression of one or more markers selected from
Table 3 and one or more markers selected from Table 4 is
monitored.
[0385] Similarly, it will also be appreciated that the
above-described methods can be used to identify factors capable of
promoting the differentiation of PDX1-negative definitive endoderm
cells to ventral PDX1-positive foregut endoderm cells by monitoring
the expression of one or more markers expression from Table 3. In
some embodiments, the expression of one or more markers selected
from Table 3 and one or more markers selected from Table 4 is
monitored.
Identification of Factors Capable of Promoting the Differentiation
of Dorsal and/or Ventral PDX1-Positive Foregut Endoderm Cells
[0386] Certain screening methods described herein relate to methods
for identifying at least one differentiation factor that is capable
of promoting the differentiation of dorsal and/or ventral
PDX1-positive foregut endoderm cells. In some embodiments of these
methods, cell populations comprising dorsal and/or ventral
PDX1-positive foregut endoderm cells, such as human dorsal and/or
ventral PDX1-positive foregut 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 dorsal and/or ventral
PDX1-positive foregut 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 dorsal and/or ventral PDX1-positive foregut
endoderm cells. In preferred embodiments, expression of the marker
is determined by Q-PCR.
[0387] Some embodiments of the screening methods described herein
utilize cell populations or cell cultures which comprise human
dorsal and/or ventral PDX1-positive foregut endoderm cells. For
example, the cell population can be a substantially purified
population of human dorsal and/or ventral PDX1-positive foregut
endoderm cells. Alternatively, the cell population can be an
enriched population of human dorsal and/or ventral PDX1-positive
foregut 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 dorsal and/or ventral PDX1-positive foregut 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 dorsal and/or ventral PDX1-positive foregut
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 dorsal and/or ventral PDX1-positive foregut endoderm
cells.
[0388] 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 dorsal and/or
ventral PDX1-positive foregut 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 dorsal and/or ventral
PDX1-positive foregut endoderm cells.
[0389] 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.
[0390] 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.
[0391] 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 I (EDFI), Endothelial monocyte activating polypeptide 11,
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
.beta.-alpha), Gliablastoma cell differentiation-related protein
(GBDRI), 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-I (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-1 I (IL 11), Interleukin-12 (IL 12), lnterleukin-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
D.sub.3, 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 I (TGF-pI),
Interferon-a, Interferon-.beta., Interferon-.gamma., Tumor necrosis
factor-.alpha., Tumor necrosis factor-.beta., Burst promoting
activity (BPA), Erythroid promoting activity (EPA), PGE.sub.2,
insulin growth factor-1 (IGF-I), IGF-11, Neutrophin growth factor
(NGF), Neutrophin-3, Neutrophin 4/5, Ciliary neurotrophic factor,
Glial-derived nexin, Dexamethasone, P-mercaptoethanol, Retinoic
acid, Butylated hydroxyanisole, 5-azacytidine, Amphotericin B,
Ascorbic acid, Ascrorbate, isobutylxanthine, indomethacin,
.alpha.-glycerolphosphate, nicotinamide, DMSO, Thiazolidinediones,
TWS 119, 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 11, 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 TWSI 19.
[0392] 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/mi. In some embodiments, the
concentration of the candidate differentiation factor in the medium
surrounding the cells ranges from about I ng/ml to about I mg/mi.
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/mI, 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/mi, 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 I pg/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
pg/ml, about 11 .mu.g/ml, about 12 pg/ml, about 13 .mu.g/ml, about
14 pg/ml, about 15 .mu.g/ml, about 16 .mu.g/ml, about 17 pg/ml,
about 18 .mu.g/ml, about 19 pg/ml, about 20 pg/ml, about 25
.mu.g/ml, about 50 .mu.g/ml, about 75 pg/mI, 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 pg/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.
[0393] 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 a
retinoid, FGF-10, FGF-4, BMP-4, activin A, activin B or any other
foregut differentiation factor. In some embodiments, the cell
population is provided with a candidate differentiation factor
which comprises any molecule other than retinoic acid.
[0394] 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.
[0395] 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.
[0396] 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 dorsal and/or ventral PDX1-positive
foregut endoderm cells to cells which are the precursors of cells
which make up tissues and/or organs that are derived from the
posterior portion of the foregut. In some embodiments, the tissues
and/or organs that are derived from the posterior portion of the
foregut comprise terminally differentiated cells. In some
embodiments, the marker is indicative of pancreatic cells or
pancreatic precursor cells. In some embodiments, the marker is a
marker that is selected from Table 3 or Table 4.
[0397] 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 I 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.
[0398] 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 dorsal and/or ventral PDX1-positive
foregut 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.
[0399] Although each of the methods disclosed herein have been
described with respect to dorsal and/or ventral PDX1-positive
foregut endoderm cells, it will be appreciated that in certain
embodiments, these methods can be used to produce compositions
comprising the dorsal and/or ventral PDX1-positive foregut/midgut
endoderm cells that are described herein and/or the dorsal and/or
ventral 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.
[0400] 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
[0401] 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
[0402] 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.
[0403] 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
[0404] 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.-III 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
[0405] 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.
[0406] 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.
[0407] 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).
[0408] 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. WOOO/29442 and WO99/13915, the disclosures of which are
incorporated herein by reference in their entireties.
[0409] 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.
[0410] 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 SOX 17 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, CA) and pEGFP-NI (Clonetech, Palo Alto, CA),
respectively. For protein production, telomerase immortalized MDX
human fibroblasts were transiently transfected with supercoiled DNA
in the presence of Lipofectamine 2000 (Invitrogen, Carlsbad, CA).
Total cellular lysates were collected 36 hours post-transfection in
50 mM TRIS-HCI (pH 8), 150 mM NaCI, 0.1% SDS, 0.5% deoxycholate,
containing a cocktail of protease inhibitors (Roche Diagnostics
Corporation, Indianapolis, IN). Western blot analysis of 100 .mu.g
of cellular proteins, separated by SDS-PAGE on NuPAGE (4-12%
gradient polyacrylamide, Invitrogen, Carlsbad, CA), and transferred
by electro-blotting onto PDVF membranes (Hercules, CA), were probed
with a 1/1000 dilution of the rat SOX17 anti-serum in 10 mM
TRIS-HCI (pH 8), 150 mM NaCI, 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.).
[0411] 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.15 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 SOX 17 Antibody as a Marker of Definitive
Endoderm
[0412] 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 SOX 17, 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+cells that are parietal endoderm. As shown in
FIGS. 6A-C, Thrombomodulin and SOX17 co-labeled parietal endoderm
cells were produced by random differentiation of hES cells.
[0413] 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.
[0414] As a further evidence of the specificity of the
SOX17hi/AFPO/TMIO/SPARCo 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-C. 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.
[0415] 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-F). There were few or no cells
labeled with AFP after 5 days of activin treatment.
[0416] 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+/AFP.sup.10/- (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
[0417] 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.
[0418] 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.
[0419] 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.
[0420] 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.
[0421] 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 pL 2X 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 Tm appropriate for that PCR
amplicon. In addition, reactions performed without reverse
transcriptase served as the negative control and do not
amplify.
[0422] 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 I (HPRT),
beta-2-microglobulin, hydroxymethylbiane synthase (HMBS),
TATA-binding protein (TBP), and glucoronidase beta (GUS) in
differentiating hESCs. Our results indicated that
beta-2-microglobulin 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.
[0423] 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 endodern 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
[0424] 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. SOX 17 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.
[0425] 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 I 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.
[0426] 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
[0427] 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.
[0428] 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.
[0429] 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 (<lmm; 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, >5days 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.
[0430] Based on negative correlations of SOX 17 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).
[0431] With the SOX17 antibody and Q-PCR tools described above we
have explored a number of procedures capable of efficiently
programming hESCs to become SOX17hi/AFP/SPARC/TM.sup.10 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 SOX 17 protein.
[0432] 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.
[0433] 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.
[0434] 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.
[0435] 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.
[0436] 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 ofSOX17-positive cells was also dose
responsive. activin A doses of 100 ng/ml or more were capable of
potently inducing SOX 17 gene expression and cell number.
[0437] 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).
[0438] 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%, SOX 17 expression tripled in the presence of
activins A and B.
[0439] Finally, we demonstrated that activin induced SOX17+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
[0440] 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.
[0441] In these experiments, hESCs were differentiated by growing
in RPMI medium (Invitrogen, Carlsbad, CA; 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. 104461 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 AIOO 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).
[0442] As described above, expression of the SOX17, GSC, MIXLI, and
HNF3, 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-D). This demonstrates that CXCR4 is also a marker of
definitive endoderm.
[0443] Ectoderm and mesoderm lineages can be distinguished from.
definitive endoderm by their expression of various markers. Early
mesoderm expresses the genes Brachyury and MOXI while nascent
neuro-ectoderm expresses SOX1 and ZIC1. FIGS. 28A-D 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.
[0444] 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.
[0445] The relative number of SOX17 immunoreactive (SOX17+) 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+cells were ubiquitously distributed throughout the culture.
When high dose activin A was used but FBS was included at 10%
(v/v), the SOX17+cells appeared at much lower frequency and always
appeared in isolated clusters rather than evenly distributed
throughout the culture (FIGS. 29A and C as well as B and E). A
further decrease in SOX17+cells was seen when no exogenous activin
A was used. Under these conditions the SOX17+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 (FIG. 29 C and
F). 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
[0446] 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+cells in the culture.
[0447] 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 105 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 105 cells. Secondary antibody (FITC conjugated donkey
anti-mouse; Jackson ImmunoResearch, cat# 715-096-151) was added at
5 .mu.Lg/ml final concentration and allowed to label for 30 minutes
followed by two washes in buffer as above. Cells were resuspended
at 5x10.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.
[0448] The number of CXCR4+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-C). The
CXCR4+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+cells correlates with a robust increase in
definitive endoderm gene expression as activin A dose is increased
(FIGS. 31 A-D).
Example 9
Isolation of CXCR4 Positive Cells Enriches for Definitive Endoderm
Gene Expression and Depletes Cells Expressing Markers of Mesoderm
Ectoderm and Visceral Endoderm
[0449] The CXCR4+and CXCR4- 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.
[0450] 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+cells (FIGS. 30A-C). It is also clear that
isolation of the CXCR4+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.
[0451] Gene expression analysis revealed that the CXCR4+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-D, the CXCR4+cells were further
enriched over the parent A100 population for SOX17, GSC, HNF3B, and
MIXLI. In addition, the CXCR4- fraction contained very little gene
expression for these definitive endoderm markers. Moreover, the
CXCR4+and CXCR4- populations displayed the inverse pattern of gene
expression for markers of mesoderm, ectoderm and extra-embryonic
endoderm. FIGS. 33A-D shows that the CXCR4+cells were depleted for
gene expression of Brachyury, MOXI, 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+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
[0452] 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.
[0453] 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 dayl, 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.
[0454] 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.) 10-20 000
000 000 Total 100 100 100 100
[0455] 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.
[0456] 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.
[0457] 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, MIXLI, GATA4, HNF3p
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 Marker of Definitive Endoderm Cells
[0458] 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.
[0459] 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).
[0460] 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 x 106 cells in
800 .mu.l human serum. Cells were washed twice with 8 ml of PBS
buffer and resuspended in I 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).
[0461] Purified RNA was submitted in duplicate to Expression
Analysis (Durham, NC) for generation of the expression profile data
using the Affymetrix platform and U 133 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.
[0462] FIGS. 34A-M 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 G-M demonstrates that the six marker
genes, FGF17, VWF, CALCR, FOXQI, CMKORI 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,
FOXQI, CMKORI and CRIPI are likely markers of definitive endoderm
and that they are not significantly expressed in extra-embryonic
endoderm cells.
[0463] 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
[0464] The following experiment demonstrates that RA and FGF-10
induces the expression of PDX1 in definitive endoderm cells.
[0465] 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.
[0466] 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 FIG.
36A-F). 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
[0467] This Example shows that the combination of RA and FGF-10
induces PDX1 expression to a greater extent than RA alone.
[0468] 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
RAIFGF.
[0469] 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.
[0470] 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 RAIFGF combinations
(see FIG. 37B-C).
Example 14
Retinoic Acid Dose Affects Anterior-Posterior (A-P) Position In
Vitro
[0471] To determine whether the dose of RA affects A-P position in
in vitro cell cultures, the following experiment was performed.
[0472] 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.
[0473] 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 (FIG.
38A-B). The middle dose of RA (0.2 .mu.M) induced midgut endoderm
markers (CDXI, HOXC6) (see FIG. 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 FIG. 38F-G). 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
[0474] 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.
[0475] 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% FBS/DMEMIF12 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.
[0476] FIG. 39A-E 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
[0477] 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.
[0478] 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 50ng/mI. 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.
[0479] FIG. 40A shows that the addition of activin B at doses
ranging from 10-50 ng/ml (a10, a25 and a5O) 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
[0480] 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.
[0481] 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 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).
[0482] 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 ZIC I (FIG. 41 B) or SOX7 (FIG. 41 C)
gene expression.
Example 18
Use of Conditioned Medium to Enhance Induction of PDX1
[0483] 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.
[0484] 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.
[0485] 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.
[0486] 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 CDXI
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. 104921 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 I 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
[0487] 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.
[0488] In a first experiment, IgY anti-PDX1 (IgY cc-PDX1) antibody
binding to PDX1 in cell lysates was validated by Western blot
analysis. In this analysis, the binding of IgY (X-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 (500x dilution of primary, l0,OOOx dilution of
secondary), B (2,000x, 10,000x), C (500x, 40,000x), D (2,000x,
40,000), E (8,000x, 40,000x).
[0489] 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.
[0490] In a second experiment, the binding of polyclonal rabbit
anti-PDX1 (Rb .alpha.X-PDX1) antibody to PDX1 was tested by
immunocytochemistry. To produce a PDX1 expressing cell for such
experiments, MSI-V cells (ATCC # CRL-2460) were transiently
transfected with an expression vector of PDX1-EGFP (constructed
using pEGFP-NI, Clontech). Transfected cells were then labeled with
Rb A-PDX1 and .mu.-EGFP antisera. Transfected cells were visualized
by both EGFP fluorescence as well as .mu.-EGFP immunocytochemistry
through the use of a Cy5 conjugated secondary antibody. PDX1
immunofluorescence was visualized through the use of an .mu.-Rb
Cy3-conjugated secondary antibody.
[0491] Binding ofthe Rb A-PDX1 and the .mu.-EGPF antibodies
co-localized with GPF expression.
Example 20
Immunocvtochemistry of Human Pancreatic Tissue
[0492] This Example shows that antibodies having specificity for
PDX1 can be used to identify human PDX1-positive cells by
immunocytochemistry.
[0493] In a first experiment, paraffin embedded sections of human
pancreas were stained for insulin with guinea pig anti-insulin (Gp
a-Ins) primary antibody at a 1/200 dilution followed by dog
anti-guinea pig (D a-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 a-PDX1
primary antibody at a 1/4000 dilution followed Rb a-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 a-PDX1
antibodies were also stained with DAPI.
[0494] 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
[0495] 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 a-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 a-PDX1
antibody.
[0496] 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 MSI-V cells (ATCC # CRL-2460)
transfected with a.PDX1 expression vector. PDX1 was
immunoprecipitated by adding Rb a-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 A-PDX1 primary
antibody followed by labeled Rb a-lgY secondary antibodies.
[0497] Immunoprecipitates collected from the MSI-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 transzenic hESC lines
[0498] 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.
[0499] 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-NI (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.
[0500] 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-l 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 G41 8, undifferentiated transgenic hESC
clones were isolated and expanded.
Example 23
Isolation of PDX1-Positive Foregut Endoderm
[0501] 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).
[0502] 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).
[0503] 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 Cytokeratinl9 (KRTI9) were from
Chemicon; to Hepatocyte nuclear factor 3 beta (HNF.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.
[0504] 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%.
[0505] 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, KRTI9, HNF4a
and HNF3) were all enriched in the EGFP positive fraction (FIGS.
48A-E). In contrast, the neural markers ZIC1 and GFAP were not
enriched in sorted EGFP expressing cells (FIGS. 49A and B).
[0506] 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 HNF3positive by antibody
staining.
Example 24
Production of PDX1-positive Dorsal and Ventral Foregut Endoderm
[0507] This Example describes the production of PDX1-positive,
dorsally-biased, foregut endoderm as well as the production of
PDX1-positive, ventrally-biased, foregut endoderm.
[0508] Definitive endoderm was produced from undifferentiated hESCs
using a three or five day protocol in which activin A was provided
to the culture medium at a concentration of 100 ng/ml each day. For
both dorsal and ventral differentiation, the medium composition for
the first five days was as follows: Dayl - RPMI +0% fetal bovine
serum (FBS), Day 2 - RPMI +0.2% FBS, Day 3 - RPMI +2.0% FBS, Day 4
- RPMI +2.0% FBS and Day 5 - RPMI +2.0% FBS. For the ventral
differentiation, definitive endoderm was produced for 3 days in
activin A at 100 ng/ml and then exposed to BMP4 at 3 ng/ml and
FGFIO at 50 ng/ml. BMP4/FGF10 addition was carried out in RPMI +2%
FBS for the first 2 days and then subsequently in Connaught Medical
Research Labs (CMRL) medium (Invitrogen, Carlsbad, CA) (see, Parker
R.C., et al. 1957. N.Y. Academy of Sciences 5:303, the disclosure
of which is incorporated herein by reference in its entirety)
containing B27 supplement (I part B27 to 200 parts medium by volume
- (1:200)) (Invitrogen, Carlsbad, CA). For the dorsal
differentiation procedure, definitive endoderm was produced for 5
days in activin A at 100 ng/ml and then exposed to retinoic acid
(RA) at 2 .mu.M and activin A at 25 ng/ml in CMRL medium containing
B27 supplement (1:200). 105151 In the RA-based, dorsal
differentiation procedure, there was a strong induction of PDX1 and
a maintenance of HB9 expression with no induced expression of HHEX
or albumin, which are ventral liver markers (FIGS. 50A-D). In the
ventral differentiation protocol, which does not use RA but instead
uses FGFIO and BMP, PDX1 gene expression was also strongly induced.
In contrast to the RA treatment, HB9 (dorsal endoderm marker)
expression was not maintained and ventral liver markers, such as
albumin and HHEX, were strongly induced along with PDX1 (FIGS.
50A-D). These data indicated that in the presence of RA, the
foregut PDX1-expressing endoderm was devoid of liver (a ventral
organ) markers and expressed dorsal markers like HB9. In the
absence of RA, the PDX1 expression was not accompanied by high HB9
expression levels. Furthermore, the expression of classical liver
markers, such as albumin and HUEX, indicated that the definitive
endoderm was preferentially executing a ventral differentiation
program since liver is exclusively derived from the ventral
endoderm.
Example 25
Production of PDX1-positive Ventral Foregut Endoderm Cells is
Dependent on Definitive Endoderm Formation
[0509] This Example describes the production of PDX1-positive,
ventrally-biased, foregut endoderm from cultures comprising varying
amounts of definitive endoderm cells. Cultures with no definitive
endoderm show very little production of PDX1-positive, ventrally
biased, foregut endoderm. As the initial amount of definitive
endoderm cells increase, so does the production of
ventrally-biased, foregut endoderm.
[0510] Four separate conditions were used to treat hESCs that
result in varying proportions of differentiation to definitive
endoderm. All four conditions utilized RPMI supplemented with 0%
FBS on the first day, 0.2% FBS on second day, and 2% FBS on days 3
and 4. The four conditions were as follows: (a) BMP4 at lOOng/mL
with SU5402 at 5 PM; (b) no exogenous growth factors; (c) activin A
at 15ng/mL; and (d) activin A at 100 ng/ml. After the first four
days of differentiation, the relative levels of definitive endoderm
produced were indicated by cerberus (CER) and SOX17 expression
levels, whereby definitive endoderm was essentially absent under
condition (a), minimal under condition (b), present under condition
(c) and highly present under condition (d). All cultures were then
incubated for 2 days with BMP4 at 3ng/mL, FGFIO at 50 ng/ml and
KAAD-cyclopamine at 0.5 .mu.M in a base medium of 2% FBS in RPMI
followed by 6 days with BMP4 at 3ng/mL, FGFI0 at 50 ng/ml and
KAAD-cyclopamine at 0.5 .mu.M in a base medium composed of CMRL
with 1:200 dilution of B27 extract. 105181 In the presence of
SU5402 and BMP4, conditions under which no definitive endoderm was
produced as demonstrated by lack of CER and SOX17 gene expression
(FIGS. 5IA and 51B), there was no induction of PDX1 or albumin gene
expression after treatment with BMP4/FGF1O (ventral endoderm
condition) (FIGS. 51C and 51D). This was similarly true for the no
growth factor condition (condition (b)), in which very minimal
levels of definitive endoderm were formed as indicated by the low
levels of CER and SOX17 (FIGS. 51A and 51B). Although PDX1 and
albumin gene expression was very low under the no growth factor
condition (FIGS. 51C and 51D), the amount of gene expression was
significantly greater than that produced from condition (a). The
hESCs treated with intermediate (15 ng/ml) and high (100 ng/ml)
doses of activin A yielded robust definitive endoderm
differentiation, indicated by high SOX17 gene expression levels
(FIG. 51B). The high dose activin treatment produced definitive
endoderm primarily of anterior character as indicated by very high
CER expression levels. Both the condition (c) and condition (d)
treatments exhibited robust ventral endoderm differentiation as
indicated by high level PDX1 and albumin gene expression (FIGS. 51C
and 51D). The levels of PDX1 and albumin expression were greatest
in the most anterior endoderm because anterior endoderm remains
competent to differentiate to more posterior endoderm fates while
posterior endoderm cells have lost competence to acquire more
anterior fates. These data strongly indicated that the production
of ventral PDX1-expressing foregut endoderm and liver were
dependent upon efficient production of definitive endoderm.
Example 26
BMP4 is not Necessary for PDX1-positive Ventral Foregut
Endoderm
[0511] This Example describes the production of PDX1-positive,
ventrally-biased, foregut endoderm in the absence of BMP4.
[0512] Definitive endoderm was produced by exposing
undifferentiated hESCs to activin at 100 ng/mL in RPMI base medium
supplemented with 0%, 0.2%, and 2% FBS on days I through 3,
respectively. After 3 days of activin A treatment, the cultures
were switched to a base medium composed of RPMI containing 2% FBS
and maintained under one of the following conditions: (a) BMP4 at 3
ng/ml with FGFIO at 50 ng/ml and KAAD-cyclopamine at 0.5 uM; (b)
FGFIO at 50 ng/ml and KAAD-cyclopamine at 0.5 1M; or (c-e) no
exogenous factors. After two days, the base medium was changed to
CMRL plus B27 supplement (1:200) and cells were maintained
according to conditions (a-c) above. Alternatively, cells were
maintained with no exogenous factors in RPMI with B27 supplement
(1:200) (condition (d)) or RPMI with 2% FBS (condition (e)). The
same factor treatment conditions were maintained for 8 more days of
differentiation.
[0513] BMP4 was not needed to produce either PDX1-positive ventral
foregut or liver endoderm cells as indicated by the robust
induction of PDX1 and albumin expression in the absence of BMP4
(FIGS. 52A and 52B). BMP4 addition appeared to be less favorable
for the production of PDX1-positive ventral foregut endoderm but
the addition of BMP4 to FGFIO and KAAD-cyclopamine treatment does
not decrease ventral foregut liver endoderm gene expression (FIGS.
52A and 52B). The use of CMRL with B27 supplement had some ability
to induce PDX1 expression in the absence of added factors
(condition (c)) while RPMI with B27 (condition (d)) and RPMI with
2% FBS (condition (e)) did not exhibit any induction of PDX1
expression (FIG. 52A). There did not appear to be a significant
effect of base media on the induction of liver gene expression. In
summary, FGF10 and KAAD-cyclopamine are sufficient to produce
PDX1-positive ventral foregut endoderm.
Example 27
Markers for the Identification of PDX1-positive Dorsal and Ventral
Foregut Endoderm
[0514] This Example describes markers useful for, among other
things, the identification, detection, enrichment, isolation,
purification, targeting and/or validation of PDX1-positive dorsal
and ventral foregut endoderm.
[0515] Cell cultures differentiated as described in Example 24 were
subjected to gene chip analysis to globally monitor the gene
expression dynamics occurring during differentiation of hESC to
definitive endoderm and further on to more mature dorsal and
ventral endoderm phenotypes. Duplicate samples were isolated at the
times indicated in Example 24. Gene expression profiles were
determined using Affymetrix U133 plus 2.0 high density
oligonucleotide arrays by Expression Analysis (Durham, NC)
according to their internal standard operating procedures. We have
evaluated the patterns of gene expression across these 7
conditions/time points through manual inspection as well as through
hierarchical clustering analyses. We have looked for patterns of
gene expression that match the temporal pattern of PDX1 expression
(dorsal and ventral) to find novel genes that are expressed in both
ventral and dorsal differentiation paradigms.
[0516] Provided are genes that have a significant similarity in
expression pattern to PDX1, and thus, may be co-expressed in
PDX1-expressing foregut endoderm cells The genes listed in Table 3
are expressed in both the dorsal and ventral PDX1 differentiation.
The genes in Table 4 are dorsally biased and are preferentially
expressed in the dorsal PDX1 pattern.
[0517] Table 3 lists 39 markers that are expressed in both dorsal
and ventral PDX1-positive foregut endoderm. Column I provides the
commonly known gene symbol for each marker. Columns 2 through 4
provide the Unigene, LocusLink, and OMIM accession numbers,
respectively. Column 5 described the Genebank accession number for
a nucleic acid sequence which includes the marker described in
column 1. Finally, column 6 provides a description of the
functional activity of the polypeptide marker that is encoded by
the listed genetic marker.
[0518] It will be appreciated that the accession numbers listed in
Table 3 can be used by those of ordinary skill in the art to
retrieve specific information about each sequence described in the
table, including both the primary nucleic acid and polypeptide
sequence of each of these marker. TABLE-US-00003 TABLE 3 Markers
expressed in both dorsal and ventral PDX1-positive foregut endoderm
Gene Symbol Unigene LocusLink OMIM SeqDerivedFrom Gene Descriptor
ANXA4 Hs.422986 307 106491 NM_001153 annexin A4 ASCL1 Hs.524672 429
100790 BC001638 achaete-scute complex-like 1 (Drosophila) BNC1
Hs.459153 646 601930 NM_001717 basonuclin 1 C10orf30 Hs.498740
222389 AW195407 Chromosome 10 open reading frame 30 C2orf23
Hs.368884 65055 609139 BE535746 chromosome 2 open reading frame 23
C9orf150 Hs.445356 286343 AI972386 chromosome 9 open reading frame
150 CDH6 Hs.171054 1004 603007 BC000019 cadherin 6, type 2,
K-cadherin (fetal kidney) DACH1 Hs.129452 1602 603803 AI650353
dachshund homolog 1 (Drosophila) DUSP9 Hs.144879 1852 300134
NM_001395 dual specificity phosphatase 9 ELMOD1 Hs.495779 55531
AL359601 ELMO domain containing 1 FLJ21462 fis Hs.24321 AW236803
CDNA clone IMAGE: 5273964, partial cds FLJ22761 Hs.522988 80201
W81116 hypothetical protein FLJ22761 GABRA2 Hs.116250 2555 137140
NM_000807 gamma-aminobutyric acid (GABA) A receptor, alpha 2 GRIA3
Hs.377070 2892 305915 BC032004 glutamate receptor, ionotrophic,
AMPA 3 HNF4G Hs.241529 3174 605966 AI916600 hepatocyte nuclear
factor 4, gamma IDH2 Hs.513141 3418 147650 U52144 isocitrate
dehydrogenase 2 (NADP+), mitochondrial IL6R Hs.135087 3570 147880
AV700030 interleukin 6 receptor KCNJ2 Hs.1547 3759 170390 AF153820
potassium inwardly-rectifying channel, subfamily J, member 2 KLF3
Hs.298658 51274 AA130132 Kruppel-like factor 3 (basic) LGALS3
Hs.531081 3958 153619 AW085690 Lectin, galactoside-binding,
soluble, 3 (galectin 3) LGALS3///GALIG Hs.531081 3958/// 153619
BC001120 lectin, galactoside-binding, soluble, 3 (galectin
3)///galectin-3 internal gene LIPC Hs.188630 3990 151670 NM_000236
lipase, hepatic MEIS1 Hs.526754 4211 601739 NM_002398 Meisl,
myeloid ecotropic viral integration site 1 homolog (mouse) NR2F1
Hs.519445 7025 132890 AI951185 Nuclear receptor subfamily 2, group
F, member 1 ONECUT2 Hs.194725 9480 604894 NM_004852 one cut domain,
family member 2 PAPPA Hs.494928 5069 176385 AA148534
pregnancy-associated plasma protein A, pappalysin 1 PDE3B Hs.445711
5140 602047 NM_000753 phosphodiesterase 3B, cGMP-inhibited PGPEP1
Hs.131776 54858 NM_017712 pyroglutamyl-peptidase 1 PMS2LI Hs.520575
5379 605038 D38503 postmeiotic segregation increased 2-like 1
SERPINF2 Hs.159509 5345 262850 NM_000934 serine (or cysteine)
proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment
epithelium derived factor), member 2 SLC27A2 Hs.11729 11001 603247
NM_003645 solute carrier family 27 (fatty acid transporter), member
2 SLN Hs.334629 6588 602203 NM_003063 sarcolipin SOX9 Hs.2316 6662
114290 NM_000346 SRY (sex determining region Y)-box 9 (campomelic
dysplasia, autosomal sex-reversal) SULT2A1 Hs.515835 6822 125263
U08024 sulfotransferase family, cytosolic, 2A,
dehydroepiandrosterone (DHEA)-preferring, member 1 TFPI Hs.516578
7035 152310 BF511231 Tissue factor pathway inhibitor
(lipoprotein-associated coagulation inhibitor) ZHX1 Hs.521264 11244
604764 AI123518 zinc fingers and homeoboxes 1 ZNF467 Hs.112158
168544 BE549732 zinc finger protein 467 ZNF503 Hs.195710 84858
AA603467 zinc finger protein 503 Hs.142869 AI935586 Transcribed
locus
[0519] FIGS. 53A-E further illustrate the commonality of expression
profile betveen PDX1 and markers selected from Table 3. In
particular, FIGS. 53A-E provide examples of genes that displayed
nearly identical gene expression patterns to that of PDX1 across
the 7 conditions/time monitored in this experiment. Pattern
recognition to this degree of similarity most likely reflects
co-expression of these genes in the same cells that express PDX1,
thus making these markers excellent novel candidate markers for
PDX1-positive foregut endoderm from both dorsal and ventral
endoderm origins.
[0520] Table 4 lists 50 markers that are specifically and/or
preferentially expressed in dorsal PDX1-positive foregut endoderm.
Column I provides the commonly known gene symbol for each marker.
Columns 2 through 4 provide the Unigene, LocusLink, and OMIM
accession numbers, respectively. Column 5 described the Genebank
accession number for a nucleic acid sequence which includes the
marker described in column 1. Finally, column 6 provides a
description of the functional activity of the polypeptide marker
that is encoded by the listed genetic marker.
[0521] It will be appreciated that the accession numbers listed in
Table 4 can be used by those of ordinary skill in the art to
retrieve specific information about each sequence described in the
table, including both the primary nucleic acid and polypeptide
sequence of each of these marker. TABLE-US-00004 TABLE 4 Markers
expressed in dorsally-biased PDX1-positive foregut endoderm Gene
Symbol Unigene LocusLink OMIM SeqDerivedFrom Gene Descriptor
ADORA2A Hs.197029 135 102776 NM_000675 adenosine A2a receptor
AMSH-LP Hs.16229 57559 AI638611 associated molecule with the SH3
domain of STAM (AMSH) like protein BAIAP2L1 Hs.489237 55971
AA628400 BAI1-associated protein 2-like 1 CD47 Hs.446414 961 601028
BG230614 CD47 antigen (Rh-related antigen, integrin-associated
signal transducer) CHN2 Hs.203663 1124 602857 AK026415 Chimerin
(chimaerin) 2 CLDN3 Hs.25640 1365 602910 BE791251 claudin 3 CPVL
Hs.233389 54504 NM_031311 carboxypeptidase,
vitellogenic-like///carboxypeptidase, vitellogenic-like CREB3L1
Hs.405961 90993 AF055009 cAMP responsive element binding protein
3-like 1 DACT1 Hs.48950 51339 607861 NM_016651 dapper homolog 1,
antagonist of beta-catenin (xenopus) DPP6 Hs.490684 1804 126141
AW071705 Dipeptidylpeptidase 6 ELF3 Hs.67928 1999 602191 AF017307
E74-like factor 3 (ets domain transcription factor,
epithelial-specific) ENPP2 Hs.190977 5168 601060 L35594
ectonucleotide pyrophosphatase/phosphodiesterase 2 (autotaxin)
EPB41L1 Hs.437422 2036 602879 AA912711 erythrocyte membrane protein
band 4.1-like 1 FAM46C Hs.356216 54855 AL046017 family with
sequence similarity 46, member C FAM49A Hs.467769 81553 NM_030797
family with sequence similarity 49, member A///family with sequence
similarity 49, member A FLJ30596 Hs.81907 133686 AI453203
hypothetical protein FLJ30596 HOXA1 Hs.67397 3198 142955 S79910
homeo box A1 HOXA3 Hs.533357 3200 142954 AW137982 homeo box A3
HOXB2 Hs.514289 3212 142967 NM_002145 homeo box B2 LAF4 Hs.444414
3899 601464 AW085505 Lymphoid nuclear protein related to AF4
LOC283658 Hs.87194 283658 AA233912 hypothetical protein LOC283658
MAF Hs.134859 4094 177075 AF055376 v-maf musculoaponeurotic
fibrosarcoma oncogene homolog (avian) MAG Hs.515354 4099 159460
X98405 myelin associated glycoprotein MYCPBP Hs.513817 10260 600382
BE268538 c-myc promoter binding protein NR4A2 Hs.165258 4929
168600/ NM_006186 nuclear receptor subfamily 4, group A, member 2
NRXN3 Hs.368307 9369 600567 AI129949 neurexin 3 NSE1 Hs.260855
151354 AI601101 NSE1 PCGF5 Hs.500512 84333 AL045882 polycomb group
ring finger 5 PDE11A Hs.130312 50940 604961 AB038041
phosphodiesterase 11A PDE5A Hs.370661 8654 603310 BF221547
Phosphodiesterase 5A, cGMP-specific PGA3 5220 169710 AI570199
pepsinogen 3, group I (pepsinogen A) PLN Hs.170839 5350 115200
NM_002667 phospholamban PTGIS Hs.302085 5740 145500 NM_000961
prostaglandin 12 (prostacyclin) synthase///prostaglandin 12
(prostacyclin) synthase RARB Hs.436538 5915 180220 NM_000965
retinoic acid receptor, beta RGN Hs.77854 9104 300212 D31815
regucalcin (senescence marker protein-30) RND1 Hs.124940 27289
609038 U69563 Rho family GTPase 1 SFRP5 Hs.279565 6425 604158
NM_003015 secreted frizzled-related protein 5 SGKL Hs.380877 23678
607591 AV690866 serum/glucocorticoid regulated kinase-like SLC16A10
Hs.520321 117247 607550 N30257 solute carrier family 16
(monocarboxylic acid transporters), member 10 SLC16A2 Hs.75317 6567
300095 NM_006517 solute carrier family 16 (monocarboxylic acid
transporters), member 2 SLC1A3 Hs.481918 6507 600111 NM_004172
solute carrier family 1 (glial high affinity glutamate
transporter), member 3 SLC30A4 Hs.162989 7782 602095 NM_013309
solute carrier family 30 (zinc transporter), member 4 SLICK
Hs.420016 343450 AI732637 sodium- and chloride-activated
ATP-sensitive potassium channel SLITRK4 Hs.272284 139065 AL080239
SLIT and NTRK-like family, member 4 ST8SIA3 Hs.298923 51046
NM_015879 ST8 alpha-N-acetyl-neuraminide
alpha-2,8-sialyltransferase 3 wingless-type MMTV integration site
family, member 5A///wingless-type WNT5A Hs.152213 7474 164975
AI968085 MMTV integration site family, member 5A XPR1 Hs.227656
9213 605237 AF089744 xenotropic and polytropic retrovirus receptor
Hs.535688 AK001582 CDNA FLJ10720 fis, clone NT2RP3001116 Hs.127009
AI935541 Transcribed locus Hs.4749 AL137310 CDNA FLJ31660 fis,
clone NT2RI2004410
[0522] FIG. 54A-D provide examples of genes that display patterns
of gene expression that indicate specific (HOXAI and PDEI IA) or
preferential (FAM49A and WNT5A) expression in the dorsal endoderm
condition (RA treatment). These markers are novel candidate genes
for identification of PDX1-positive, dorsally-biased, foregut
endoderm.
Example 28
Production of PDX1-Negative Foregut Endoderm
[0523] This Example describes the production of PDX1-negative
foregut endoderm.
[0524] Human embryonic stem cells were differentiated for 7 days
via a 2-step protocol to achieve PDX1 cells. The first step
comprised 5 days differentiation in activin A (100 ng/ml) to
robustly produce DE (D'Amour, K., et al., Nature Biotechnology 23,
1534-1541, (2005)). Step 2 comprised 2 days differentiation in
fresh RPMI with 2% FBS containing FGFIO (50 ng/ml) and
KAAD-cyclopamine (0.5 .mu.M).
[0525] The addition of FGFIO (5-500 ng/ml) was beneficial together
with the addition of KAAD-cyclopamine (0.1-2 .mu.M, sonic hedgehog
inhibitor), which further specified definitive endoderm cells into
the foregut endoderm domain.
[0526] 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.
[0527] 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.
REFERENCES
[0528] Numerous literature and patent references have been cited in
the present patent application. Each and every reference that is
cited in this patent application is incorporated by reference
herein in its entirety.
[0529] For some references, the complete citation is in the body of
the text. For other references the citation in the body of the text
is by author and year, the complete citation being as follows:
[0530] Alexander, J., Rothenberg, M., Henry, G. L., and Stainier,
D. Y. (1999). Casanova plays an early and essential role in
endoderm formation in zebrafish. Dev Biol 215, 343-357.
[0531] Alexander, J., and Stainier, D. Y. (1999). A molecular
pathway leading to endoderm formation in zebrafish. Curr Biol 9,
1147-1157.
[0532] Aoki, T. O., Mathieu, J., Saint-Etienne, L., Rebagliati, M.
R., Peyrieras, N., and Rosa, F. M. (2002). Regulation of nodal
signalling and mesendoderm formation by TARAM-A, a TGFbeta-related
type I receptor. Dev Biol 241, 273-288.
[0533] Beck, S., Le Good, J. A., Guzman, M., Ben Haim, N., Roy, K.,
Beermann, F., and Constam, D. B. (2002). Extra-embryonic proteases
regulate Nodal signalling during gastrulation. Nat Cell Biol 4,
981-985.
[0534] Beddington, R. S., Rashbass, P., and Wilson, V. (1992).
Brachyury--a gene affecting mouse gastrulation and early
organogenesis. Dev Suppl, 157-165.
[0535] Bongso, A., Fong, C. Y., Ng, S. C., and Ratnam, S. (1994).
Isolation and culture of inner cell mass cells from human
blastocysts. Hum Reprod 9, 2110-2117.
[0536] Chang, H., Brown, C. W., and Matzuk, M. M. (2002). Genetic
analysis of the mammalian transforming growth factor-beta
superfamily. Endocr Rev 23, 787-823.
[0537] Conlon, F. L., Lyons, K. M., Takaesu, N., Barth, K. S.,
Kispert, A., Herrmann, B., and Robertson, E. J. (1994). A primary
requirement for nodal in the formation and maintenance of the
primitive streak in the mouse. Development 120, 1919-1928. 105461
Dougan, S. T., Warga, R. M., Kane, D. A., Schier, A. F., and
Talbot, W. S. (2003). The role of the zebrafish nodal-related genes
squint and cyclops in patterning of mesendoderm. Development 130,
1837-1851.
[0538] Feldman, B., Gates, M. A., Egan, E. S., Dougan, S. T.,
Rennebeck, G., Sirotkin, H. I., Schier, A. F., and Talbot, W. S.
(1998). Zebrafish organizer development and germ-layer formation
require nodal-related signals. Nature 395, 181-185.
[0539] Feng, Y., Broder, C. C., Kennedy, P. E., and Berger, E. A.
(1996). HIV-1 entry cofactor: functional cDNA cloning of a
seven-transmembrane, G protein-coupled receptor. Science 272,
872-877.
[0540] Futaki, S., Hayashi, Y., Yamashita, M., Yagi, K., Bono, H.,
Hayashizaki, Y., Okazaki, Y., and Sekiguchi, K. (2003). Molecular
basis of constitutive production of basement membrane components:
Gene expression profiles of engelbreth-holm-swarm tumor and F9
embryonal carcinoma cells. J Biol Chem.
[0541] Grapin-Botton, A., and Melton, D. A. (2000). Endoderm
development: from patterning to organogenesis. Trends Genet 16,
124-130.
[0542] Harris, T. M., and Childs, G. (2002). Global gene expression
patterns during differentiation of F9 embryonal carcinoma cells
into parietal endoderm. Funct lntegr Genomics 2, 105-119.
[0543] Hogan, B. L. (1996). Bone morphogenetic proteins in
development. Curr Opin Genet Dev 6, 432-438.
[0544] Hogan, B. L. (1997). Pluripotent embryonic cells and methods
of making same (U.S.A., Vanderbilt University).
[0545] Howe, C. C., Overton, G. C., Sawicki, J., Solter, D., Stein,
P., and Strickland, S. (1988). Expression of SPARC/osteonectin
transcript in murine embryos and gonads. Differentiation 37,
20-25.
[0546] Hudson, C., Clements, D., Friday, R. V., Stott, D., and
Woodland, H. R. (1997). Xsoxl 7alpha and -beta mediate endoderm
formation in Xenopus. Cell 91, 397-405.
[0547] Imada, M., Imada, S., Iwasaki, H., Kume, A., Yamaguchi, H.,
and Moore, E. E. (1987). Fetomodulin: marker surface protein of
fetal development which is modulatable by cyclic AMP. Dev Biol 122,
483-491.
[0548] Kanai-Azuma, M., Kanai, Y., Gad, J. M., Tajima, Y., Taya,
C., Kurohmaru, M., Sanai, Y., Yonekawa, H., Yazaki, K., Tam, P. P.,
and Hayashi, Y. (2002). Depletion of definitive gut endoderm in
Soxl 7-null mutant mice. Development 129, 2367-2379.
[0549] Katoh, M. (2002). Expression of human SOX7 in normal tissues
and tumors. Int J Mol Med 9, 363-368.
[0550] Kikuchi, Y., Agathon, A., Alexander, J., Thisse, C.,
Waldron, S., Yelon, D., Thisse, B., and Stainier, D. Y. (2001).
casanova encodes a novel Sox-related protein necessary and
sufficient for early endoderm formation in zebrafish. Genes Dev 15,
1493-1505.
[0551] Kim, C. H., and Broxmeyer, H. E. (1999). Chemokines: signal
lamps for trafficking of T and B cells for development and effector
function. J Leukoc Biol 65, 6-15.
[0552] Kimelman, D., and Griffin, K. J. (2000). Vertebrate
mesendoderm induction and patterning. Curr Opin Genet Dev 10,
350-356.
[0553] Kubo A, Shinozaki K, Shannon JM, Kouskoff V, Kennedy M, Woo
S, Fehling H J, Keller G. (2004) Development of definitive endoderm
from embryonic stem cells in culture. Development. 131,1651-62.
[0554] Kumar, A., Novoselov, V., Celeste, A. J., Wolfman, N. M.,
ten Dijke, P., and Kuehn, M. R. (2001). Nodal signaling uses
activin and transforming growth factor-beta receptor-regulated
Smads. J Biol Chem 276, 656-661.
[0555] Labosky, P. A., Barlow, D. P., and Hogan, B. L. (1994a).
Embryonic germ cell lines and their derivation from mouse
primordial germ cells. Ciba Found Symp 182, 157-168; discussion
168-178.
[0556] Labosky, P. A., Barlow, D. P., and Hogan, B. L. (1994b).
Mouse embryonic germ (EG) cell lines: transmission through the
germline and differences in the methylation imprint of insulin-like
growth factor 2 receptor (Igf2r) gene compared with embryonic stem
(ES) cell lines. Development 120, 3197-3204.
[0557] Lickert, H., Kutsch, S., Kanzler, B., Tamai, Y., Taketo, M.
M., and Kemler, R. (2002). Formation of multiple hearts in mice
following deletion of beta-catenin in the embryonic endoderm. Dev
Cell 3, 171-181.
[0558] Lu, C. C., Brennan, J., and Robertson, E. J. (2001). From
fertilization to gastrulation: axis formation in the mouse embryo.
Curr Opin Genet Dev 11, 384-392.
[0559] Ma, Q., Jones, D., and Springer, T. A. (1999). The chemokine
receptor CXCR4 is required for the retention of B lineage and
granulocytic precursors within the bone marrow microenvironment.
Immunity 10, 463-471.
[0560] McGrath KE, Koniski AD, Maltby KM, McGann JK, Palis J.
(1999) Embryonic expression and function of the chemokine SDF-I and
its receptor, CXCR4. Dev Biol. 213, 442-56.
[0561] Miyazono, K., Kusanagi, K., and Inoue, H. (2001). Divergence
and convergence of TGF-beta/BMP signaling. J Cell Physiol 187,
265-276.
[0562] Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N.,
Nishikawa, S., Kitamura, Y., Yoshida, N., Kikutani, H., and
Kishimoto, T. (1996). Defects of B-cell lymphopoiesis and
bone-marrow myelopoiesis in mice lacking the CXC chemokine
PBSF/SDF-1. Nature 382, 635-638.
[0563] Niwa, H. (2001). Molecular mechanism to maintain stem cell
renewal of ES cells. Cell Struct Funct 26, 137-148. 105731 Ogura,
H., Aruga, J., and Mikoshiba, K. (2001). Behavioral abnormalities
of Zicl and Zic2 mutant mice: implications as models for human
neurological disorders. Behav Genet 31, 317-324.
[0564] Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A.,
and Bongso, A. (2000). Embryonic stem cell lines from human
blastocysts: somatic differentiation in vitro. Nat Biotechnol 18,
399-404.
[0565] Rodaway, A., and Patient, R. (2001). Mesendoderm. an ancient
germ layer? Cell 105, 169-172.
[0566] Rodaway, A., Takeda, H., Koshida, S., Broadbent, J., Price,
B., Smith, J. C., Patient, R., and Holder, N. (1999). Induction of
the mesendoderm in the zebrafish germ ring by yolk cell-derived
TGF-beta family signals and discrimination of mesoderm and endoderm
by FGF. Development 126,3067-3078.
[0567] Rohr, K. B., Schulte-Merker, S., and Tautz, D. (1999).
Zebrafish zicl expression in brain and somites is affected by BMP
and hedgehog signalling. Mech Dev 85, 147-159.
[0568] Schier, A. F. (2003). Nodal signaling in vertebrate
development. Annu Rev Cell Dev Biol 19, 589-621.
[0569] Schoenwolf, G. C., and Smith, J. L. (2000). Gastrulation and
early mesodermal patterning in vertebrates. Methods Mol Biol 135,
113-125.
[0570] Shamblott, M. J., Axelman, J., Wang, S., Bugg, E. M.,
Littlefield, J. W., Donovan, P. J., Blumenthal, P. D., Huggins, G.
R., and Gearhart, J. D. (1998). Derivation of pluripotent stem
cells from cultured human primordial germ cells. Proc Natl Acad Sci
U S A 95, 13726-13731.
[0571] Shapiro, A. M., Lakey, J. R., Ryan, E. A., Korbutt, G. S.,
Toth, E., Warnock, G. L., Kneteman, N. M., and Rajotte, R. V.
(2000). Islet transplantation in seven patients with type I
diabetes mellitus using a glucocorticoid-free immunosuppressive
regimen. N EngI J Med 343, 230-238.
[0572] Shapiro, A. M., Ryan, E. A., and Lakey, J. R. (2001a).
Pancreatic islet transplantation in the treatment of diabetes
mellitus. Best Pract Res Clin Endocrinol Metab 15, 241-264.
[0573] Shapiro, J., Ryan, E., Warnock, G. L., Kneteman, N. M.,
Lakey, J., Korbutt, G. S., and Rajotte, R. V. (2001b). Could fewer
islet cells be transplanted in type I diabetes? Insulin
independence should be dominant force in islet transplantation. Bmj
322, 861.
[0574] Shiozawa, M., Hiraoka, Y., Komatsu, N., Ogawa, M., Sakai,
Y., and Aiso, S. (1996). Cloning and characterization of Xenopus
laevis xSox7 cDNA. Biochim Biophys Acta 1309, 73-76.
[0575] Smith, J. (1997). Brachyury and the T-box genes. Curr Opin
Genet Dev 7, 474-480.
[0576] Smith, J. C., Armes, N. A., Conlon, F. L., Tada, M.,
Umbhauer, M., and Weston, K. M. (1997). Upstream and downstream
from Brachyury, a gene required for vertebrate mesoderm formation.
Cold Spring Harb Symp Quant Biol 62, 337-346.
[0577] Takash, W., Canizares, J., Bonneaud, N., Poulat, F., Mattei,
M. G., Jay, P., and Berta, P. (2001). SOX7 transcription factor:
sequence, chromosomal localisation, expression, transactivation and
interference with Wnt signalling. Nucleic Acids Res 29,
4274-4283.
[0578] Taniguchi, K., Hiraoka, Y., Ogawa, M., Sakai, Y., Kido, S.,
and Aiso, S. (1999). Isolation and characterization of a mouse
SRY-related cDNA, mSox7. Biochim Biophys Acta 1445, 225-231.
[0579] Technau, U. (2001). Brachyury, the blastopore and the
evolution of the mesoderm. Bioessays 23, 788-794.
[0580] Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S.,
Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., and Jones, J. M.
(1998). Embryonic stem cell lines derived from human blastocysts.
Science 282, 1145-1147.
[0581] Tremblay, K. D., Hoodless, P. A., Bikoff, E. K., and
Robertson, E. J. (2000). Formation of the definitive endoderm in
mouse is a Smad2-dependent process. Development 127, 3079-3090.
[0582] Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van
Roy, N., De Paepe, A., and Speleman, F. (2002). Accurate
normalization of real-time quantitative RT-PCR data by geometric
averaging of multiple internal control genes. Genome Biol 3,
RESEARCH0034.
[0583] Varlet, I., Collignon, J., and Robertson, E. J. (1997).
nodal expression in the primitive endoderm is required for
specification of the anterior axis during mouse gastrulation.
Development 124, 1033-1044.
[0584] Vincent, S. D., Dunn, N. R., Hayashi, S., Norris, D. P., and
Robertson, E. J. (2003). Cell fate decisions within the mouse
organizer are governed by graded Nodal signals. Genes Dev 17,
1646-1662.
[0585] Weiler-Guettler, H., Aird, W. C., Rayburn, H., Husain, M.,
and Rosenberg, R. D. (1996). Developmentally regulated gene
expression of thrombomodulin in postimplantation mouse embryos.
Development 122, 2271-2281.
[0586] Weiler-Guettler, H., Yu, K., Soff, G., Gudas, L. J., and
Rosenberg, R. D. (1992). Thrombomodulin gene regulation by cAMP and
retinoic acid in F9 embryonal carcinoma cells. Proceedings Of The
National Academy Of Sciences Of The United States Of America 89,
2155-2159.
[0587] Wells, J. M., and Melton, D. A. (1999). Vertebrate endoderm
development. Annu Rev Cell Dev Biol 15, 393-410.
[0588] Wells, J. M., and Melton, D. A. (2000). Early mouse endoderm
is patterned by soluble factors from adjacent germ layers.
Development 127, 1563-1572.
[0589] Willison, K. (1990). The mouse Brachyury gene and mesoderm
formation. Trends Genet 6, 104-105.
[0590] Zhao, G. Q. (2003). Consequences of knocking out BMP
signaling in the mouse. Genesis 35, 43-56.
[0591] Zhou, X., Sasaki, H., Lowe, L., Hogan, B. L., and Kuehn, M.
R. (1993). Nodal is a novel TGF-beta-like gene expressed in the
mouse node during gastrulation. Nature 361, 543-547.
[0592] Jonsson, J., et al., Nature, 606-609, 1994
[0593] Offield, M F, et al. Devel. 983-995, 1996
[0594] Stoffers, D. A., et al. Nature Genetics, 106-110 1997
[0595] Stoffers D. A., et al. Nature Genetics 138-139, 1997
[0596] Stafford et al. Genes Evol. 432-441, 2004
[0597] Chen et al., Dev. Biol. 144-160, 2004
[0598] Martin, M., et al. Dev. Biol. 2005
[0599] Molotkov, A., et al. Dev. Dyn. 950-957, 2005
[0600] Harrison, K. A., et al. Nature Genetics 71-75 1999
[0601] Li, H., et al. Nature Genetics 67-70, 1999
[0602] Kawahira, H., et al. Dev. Biology 280, 111-121, 2005
Sequence CWU 1
1
2 1 1245 DNA Homo sapiens 1 atgagcagcc cggatgcggg atacgccagt
gacgaccaga gccagaccca gagcgcgctg 60 cccgcggtga tggccgggct
gggcccctgc ccctgggccg agtcgctgag ccccatcggg 120 gacatgaagg
tgaagggcga ggcgccggcg aacagcggag caccggccgg ggccgcgggc 180
cgagccaagg gcgagtcccg tatccggcgg ccgatgaacg ctttcatggt gtgggctaag
240 gacgagcgca agcggctggc gcagcagaat ccagacctgc acaacgccga
gttgagcaag 300 atgctgggca agtcgtggaa ggcgctgacg ctggcggaga
agcggccctt cgtggaggag 360 gcagagcggc tgcgcgtgca gcacatgcag
gaccacccca actacaagta ccggccgcgg 420 cggcgcaagc aggtgaagcg
gctgaagcgg gtggagggcg gcttcctgca cggcctggct 480 gagccgcagg
cggccgcgct gggccccgag ggcggccgcg tggccatgga cggcctgggc 540
ctccagttcc ccgagcaggg cttccccgcc ggcccgccgc tgctgcctcc gcacatgggc
600 ggccactacc gcgactgcca gagtctgggc gcgcctccgc tcgacggcta
cccgttgccc 660 acgcccgaca cgtccccgct ggacggcgtg gaccccgacc
cggctttctt cgccgccccg 720 atgcccgggg actgcccggc ggccggcacc
tacagctacg cgcaggtctc ggactacgct 780 ggccccccgg agcctcccgc
cggtcccatg cacccccgac tcggcccaga gcccgcgggt 840 ccctcgattc
cgggcctcct ggcgccaccc agcgcccttc acgtgtacta cggcgcgatg 900
ggctcgcccg gggcgggcgg cgggcgcggc ttccagatgc agccgcaaca ccagcaccag
960 caccagcacc agcaccaccc cccgggcccc ggacagccgt cgccccctcc
ggaggcactg 1020 ccctgccggg acggcacgga ccccagtcag cccgccgagc
tcctcgggga ggtggaccgc 1080 acggaatttg aacagtatct gcacttcgtg
tgcaagcctg agatgggcct cccctaccag 1140 gggcatgact ccggtgtgaa
tctccccgac agccacgggg ccatttcctc ggtggtgtcc 1200 gacgccagct
ccgcggtata ttactgcaac tatcctgacg tgtga 1245 2 414 PRT Homo sapiens
2 Met Ser Ser Pro Asp Ala Gly Tyr Ala Ser Asp Asp Gln Ser Gln Thr 1
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 Lys 65 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 Ala
145 150 155 160 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 Pro 225 230 235 240 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 Gln 305 310 315 320 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 Ser 385
390 395 400 Asp Ala Ser Ser Ala Val Tyr Tyr Cys Asn Tyr Pro Asp Val
405 410
* * * * *