U.S. patent application number 12/710300 was filed with the patent office on 2010-06-17 for definitive endoderm.
Invention is credited to Alan D. Agulnick, Emmanuel E. Baetge, Kevin Allen D'Amour.
Application Number | 20100151568 12/710300 |
Document ID | / |
Family ID | 34743694 |
Filed Date | 2010-06-17 |
United States Patent
Application |
20100151568 |
Kind Code |
A1 |
D'Amour; Kevin Allen ; et
al. |
June 17, 2010 |
DEFINITIVE ENDODERM
Abstract
Disclosed herein are cell cultures comprising definitive
endoderm cells and methods of producing the same. Also disclosed
herein are cell populations comprising substantially purified
definitive endoderm cells as well as methods for enriching,
isolating and purifying definitive endoderm cells from other cell
types.
Inventors: |
D'Amour; Kevin Allen; (San
Diego, CA) ; Agulnick; Alan D.; (San Diego, CA)
; Baetge; Emmanuel E.; (Encinitas, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
34743694 |
Appl. No.: |
12/710300 |
Filed: |
February 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10584338 |
Jan 9, 2007 |
7704738 |
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PCT/US04/43696 |
Dec 23, 2004 |
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12710300 |
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60587942 |
Jul 14, 2004 |
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60586566 |
Jul 9, 2004 |
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60532004 |
Dec 23, 2003 |
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Current U.S.
Class: |
435/347 |
Current CPC
Class: |
C12N 2501/415 20130101;
C12N 5/068 20130101; C12N 5/0606 20130101; C12N 5/0603 20130101;
C12N 2506/02 20130101; C12N 2501/15 20130101; C12N 2501/155
20130101; C12N 2502/13 20130101; C12N 2501/16 20130101 |
Class at
Publication: |
435/347 |
International
Class: |
C12N 5/073 20100101
C12N005/073 |
Claims
1. An in vitro cell culture comprising human ectoderm cells and
human definitive endoderm cells, wherein said definitive endoderm
cells are multipotent cells that can differentiate into cells of
the gut tube or organs derived therefrom, and wherein said ectoderm
cells comprise less than 5% of the cells in said cell culture.
2. The cell culture of claim 1, wherein said ectoderm cells and
said definitive endoderm cells together comprise greater than 10%
of the cells in said cell culture.
3. The cell culture of claim 1, wherein said ectoderm cells and
said definitive endoderm cells together comprise greater than 15%
of the cells in said cell culture.
4. The cell culture of claim 1 further comprising a human
pluripotent stem cell.
5. The cell culture of claim 1 further comprising an extraembryonic
endoderm cell.
6. The cell culture of claim 5 further comprising a human
pluripotent stem cell.
7. The cell culture of claim 6, wherein said human pluripotent stem
cell comprises an embryonic stem cell.
8. The cell culture of claim 7, wherein said embryonic stem cell is
derived from a tissue selected from the group consisting of the
morula and the inner cell mass (ICM) of an embryo.
9. The cell culture of claim 7, wherein said human pluripotent cell
is derived from a gondal ridge of an embryo.
10. The cell culture of claim 7 further comprising a medium.
11. The cell culture of claim 10, wherein said medium lacks
B27.
12. The cell culture of claim 10, wherein said medium lacks serum
replacement.
13. The cell culture of claim 10, wherein said medium comprises a
growth factor of the Nodal/Activin subgroup of the TGF.beta.
superfamily.
14. The cell culture of claim 10, further comprising a growth
factor selected from the group consisting of Nodal, Activin A,
Activin B and combinations thereof.
15. The cell culture of claim 14, wherein said medium comprises a
Wnt family member.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority
under 35 U.S.C. .sctn.120 to U.S. patent application Ser. No.
10/584,338, entitled DEFINITIVE ENDODERM, filed Jan. 9, 2007, which
is a U.S. national phase application under 35 U.S.C. .sctn.371 of
PCT Application No. PCT/US2004/043696, entitled DEFINITIVE
ENDODERM, filed Dec. 23, 2004, which was published in English as
PCT Application Publication Number WO2005/063971 on Jul. 14, 2005,
and which is a nonprovisional application of and claims priority to
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. Provisional Patent Application
No. 60/586,566, entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE
ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9, 2004, and U.S.
Provisional Patent Application No. 60/532,004, entitled DEFINITIVE
ENDODERM, filed Dec. 23, 2003. The disclosure of each of the
above-listed priority applications 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 definitive 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 which utilize
cells from donor pancreases. However, presently it is not known how
to generate an insulin-producing .beta.-cell from hESCs. As such,
current cell therapy treatments for diabetes mellitus, which
utilize islet cells from donor pancreases, are limited by the
scarcity of high quality islet cells needed for transplant. Cell
therapy for a single Type I diabetic patient requires a transplant
of approximately 8.times.10.sup.8 pancreatic islet cells. (Shapiro
et al., 2000; Shapiro et al., 2001a; Shapiro et al., 2001b). As
such, at least two healthy donor organs are required to obtain
sufficient islet cells for a successful transplant. HESCs 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 without accumulation
of genetic changes. Pluripotency is defined by the ability of hESCs
to differentiate to derivatives of all 3 primary germ layers
(endoderm, mesoderm, ectoderm) which, in turn, form all cell
somatic 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] Some embodiments of the present invention relate to cell
cultures comprising definitive endoderm cells, wherein the
definitive endoderm cells are multipotent cells that can
differentiate into cells of the gut tube or organs derived from the
gut tube. In accordance with certain embodiments, the definitive
endoderm cells are mammalian cells, and in a preferred embodiment,
the definitive endoderm cells are human cells. In some embodiments
of the present invention, definitive endoderm cells express or fail
to significantly express certain markers. In some embodiments, one
or more markers selected from SOX17, CXCR4, MIXL1, GATA4, HNF3b,
GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 are expressed in
definitive endoderm cells. In other embodiments, one or more
markers selected from OCT4, alpha-fetoprotein (AFP), Thrombomodulin
(TM), SPARC and SOX7 are not significantly expressed in definitive
endoderm cells.
[0009] In accordance with other embodiments of the present
invention, methods of producing definitive endoderm from
pluripotent cells are described. In some embodiments, pluripotent
cells are derived from a morula. In some embodiments, pluripotent
stem cells are stem cells. Stem cells used in these methods can
include, but are not limited to, embryonic stem cells. Embryonic
stem cells can be derived from the embryonic inner cell mass or
from the embryonic gonadal ridges. Embryonic stem cells can
originate from a variety of animal species including, but not
limited to, various mammalian species including humans. In a
preferred embodiment, human embryonic stem cells are used to
produce definitive endoderm.
[0010] In some embodiments of the present invention, one or more
growth factors are used in the differentiation process from
pluripotent cell to definitive endoderm cell. The one or more
growth factors used in the differentiation process can include
growth factors from the TGF.beta. superfamily. In such embodiments,
the one or more growth factors comprise the Nodal/Activin and/or
the BMP subgroups of the TGF.beta. superfamily of growth factors.
In some embodiments, the one or more growth factors are selected
from the group consisting of Nodal, Activin A, Activin B, BMP4,
Wnt3a or combinations of any of these growth factors.
[0011] Embodiments of the present invention also relate to
populations of cells enriched in definitive endoderm cells. In
certain embodiments, the definitive endoderm cells are isolated or
substantially purified. In some embodiments, the isolated or
substantially purified definitive endoderm cells express the SOX17
and/or the CXRC4 marker to a greater extent than the OCT4, AFP, TM,
SPARC and/or SOX7 markers.
[0012] Methods for enriching a cell population with definitive
endoderm are also contemplated. In some embodiments, definitive
endoderm cells can be isolated or substantially purified from a
mixed cell population by contacting the cells with a reagent that
binds to a molecule that is present on the surface of definitive
endoderm cells but which is not present on the surface of other
cells in the mixed cell population, and then isolating the cells
bound to the reagent. In certain embodiments, the molecule that is
present on the surface of definitive endoderm cells is CXCR4.
[0013] Still other embodiments of the present invention relate to
CXCR4 antibodies, SDF-1 ligands or other ligands for CXCR4 can be
used to obtain definitive endoderm cells in an enriched, isolated
or substantially purified form. For example, a CXCR4 antibody, an
SDF-1 ligand or another ligand for CXCR4 can be used as a reagent
in a method, such as affinity-based separation or magnetic-based
separation, to enrich, isolate or substantially purify preparations
of definitive endoderm cells which bind to the reagent.
[0014] Other embodiments of the invention described herein relate
to compositions, such as cell cultures, which comprise pluripotent
cells and definitive endoderm cells. In certain embodiments, the
cell cultures comprise both stem cells and definitive endoderm
cells. The number of stem cells present in such cultures can be
greater than, equal to or less than the number of definitive
endoderm cells in the culture. In some embodiments, the stem cells
are human embryonic stem cells. In certain embodiments the hESCs
are maintained on a feeder layer. In such embodiments, the feeder
layer cells can be cells, such as fibroblasts, which are obtained
from humans, mice or any other suitable organism.
[0015] In some embodiments of the present invention, the
compositions comprising definitive endoderm cells and hESCs also
includes one or more growth factors. Such growth factors can
include growth factors from the TGF.beta. superfamily. In such
embodiments, the one or more growth factors comprise the
Nodal/Activin and/or the BMP subgroups of the TGF.beta. superfamily
of growth factors. In some embodiments, the one or more growth
factors are selected from the group consisting of Nodal, Activin A,
Activin B, BMP4, Wnt3a or combinations of any of these growth
factors.
[0016] Other embodiments of the present inventions are described
with reference to the numbered paragraphs below:
[0017] 1. A cell culture comprising human cells wherein at least
about 10% of said human cells are definitive endoderm cells, said
definitive endoderm cells being multipotent cells that can
differentiate into cells of the gut tube or organs derived
therefrom.
[0018] 2. The cell culture of paragraph 1, wherein at least about
50% of said human cells are definitive endoderm cells.
[0019] 3. The cell culture of paragraph 1, wherein at least about
80% of said human cells are definitive endoderm cells.
[0020] 4. The cell culture of paragraph 1, wherein said definitive
endoderm cells express a marker selected from the group consisting
of SOX17 and CXCR4.
[0021] 5. The cell culture of paragraph 4, wherein the expression
of a marker selected from the group consisting of SOX17 and CXCR4
is greater than the expression of a marker selected from the group
consisting of OCT4, alpha-fetoprotein (AFP), Thrombomodulin (TM),
SPARC and SOX7 in said definitive endoderm cells.
[0022] 6. The cell culture of paragraph 4, wherein said definitive
endoderm cells do not express a marker selected from the group
consisting of OCT4, AFP, TM, SPARC and SOX7.
[0023] 7. The cell culture of paragraph 4, wherein said definitive
endoderm cells express a marker selected from the group consisting
of MIXL1, GATA4 and HNF3b.
[0024] 8. The cell culture of paragraph 4, wherein said definitive
endoderm cells express a marker selected from the group consisting
of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
[0025] 9. The cell culture of paragraph 1, wherein said definitive
endoderm cells express SOX17 and CXCR4.
[0026] 10. The cell culture of paragraph 9, wherein the expression
of SOX17 and CXCR4 is greater than the expression of OCT4, AFP, TM,
SPARC and SOX7 in said definitive endoderm cells.
[0027] 11. The cell culture of paragraph 9, wherein said definitive
endoderm cells do not express OCT4, AFP, TM, SPARC and SOX7.
[0028] 12. The cell culture of paragraph 9, wherein said definitive
endoderm cells express MIXL1, GATA4 and HNF3b.
[0029] 13. The cell culture of paragraph 9, wherein said definitive
endoderm cells express a marker selected from the group consisting
of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
[0030] 14. The cell culture of paragraph 1, wherein at least about
2 definitive endoderm cells are present for about every 1
pluripotent cell in said cell culture.
[0031] 15. The cell culture of paragraph 14, wherein said
pluripotent cell comprises an embryonic stem cell.
[0032] 16. The cell culture of paragraph 15, wherein said embryonic
stem cell is derived from a tissue selected from the group
consisting of the morula, the inner cell mass (ICM) of an embryo
and the gonadal ridges of an embryo.
[0033] 17. The cell culture of paragraph 1 further comprising a
medium which comprises less than about 10% serum.
[0034] 18. The cell culture of paragraph 1 further comprising a
growth factor of the Nodal/Activin subgroup of the TGF.beta.
superfamily.
[0035] 19. The cell culture of paragraph 1, further comprising a
growth factor selected from the group consisting of Nodal, Activin
A, Activin B and combinations thereof.
[0036] 20. A cell population comprising cells wherein at least
about 90% of said cells are human definitive endoderm cells, said
human definitive endoderm cells being multipotent cells that can
differentiate into cells of the gut tube or organs derived
therefrom.
[0037] 21. The cell population of paragraph 20, wherein at least
about 95% of said cells are human definitive endoderm cells.
[0038] 22. The cell population of paragraph 20, wherein at least
about 98% of said cells are human definitive endoderm cells.
[0039] 23. The cell population of paragraph 20, wherein said human
definitive endoderm cells express a marker selected from the group
consisting of SOX17 and CXCR4.
[0040] 24. The cell population of paragraph 23, wherein the
expression of a marker selected from the group consisting of SOX17
and CXCR4 is greater than the expression of a marker selected from
the group consisting of OCT4, AFP, TM, SPARC and SOX7 in said human
definitive endoderm cells.
[0041] 25. The cell population of paragraph 23, wherein said human
definitive endoderm cells do not express a marker selected from the
group consisting of OCT4, AFP, TM, SPARC and SOX7.
[0042] 26. The cell population of paragraph 23, wherein said human
definitive endoderm cells express a marker selected from the group
consisting of MIXL1, GATA4 and HNF3b.
[0043] 27. The cell population of paragraph 23, wherein said
definitive endoderm cells express a marker selected from the group
consisting of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
[0044] 28. The cell population of paragraph 20, wherein said human
definitive endoderm cells express SOX17 and CXCR4.
[0045] 29. The cell population of paragraph 28, wherein the
expression of SOX17 and CXCR4 is greater than the expression of
OCT4, AFP, TM, SPARC and SOX7 in said human definitive endoderm
cells.
[0046] 30. The cell population of paragraph 28, wherein said human
definitive endoderm cells do not express OCT4, AFP, TM, SPARC and
SOX7.
[0047] 31. The cell population of paragraph 28, wherein said human
definitive endoderm cells express MIXL1, GATA4 and HNF3b.
[0048] 32. The cell population of paragraph 28, wherein said
definitive endoderm cells express a marker selected from the group
consisting of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
[0049] 33. The cell population of paragraph 20, wherein at least
about 2 human definitive endoderm cells are present for about every
1 pluripotent cell in said cell population.
[0050] 34. The cell population of paragraph 33, wherein said
pluripotent cell comprises an embryonic stem cell.
[0051] 35. The cell population of paragraph 34, wherein said
embryonic stem cell is derived from a tissue selected from the
morula, the ICM of an embryo and the gonadal ridges of an
embryo.
[0052] 36. A method of producing definitive endoderm cells, said
method comprising the steps of:
[0053] obtaining a cell population comprising pluripotent human
cells;
[0054] 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, said definitive endoderm cells being multipotent
cells that can differentiate into cells of the gut tube or organs
derived therefrom; and
[0055] 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.
[0056] 37. The method of paragraph 36, wherein at least about 10%
of said pluripotent cells differentiate into definitive endoderm
cells.
[0057] 38. The method of paragraph 36, wherein at least about 50%
of said pluripotent cells differentiate into definitive endoderm
cells.
[0058] 39. The method of paragraph 36, wherein at least about 70%
of said pluripotent cells differentiate into definitive endoderm
cells.
[0059] 40. The method of paragraph 36, wherein at least about 80%
of said pluripotent cells differentiate into definitive endoderm
cells.
[0060] 41. The method of paragraph 36, wherein detecting the
presence of definitive endoderm cells in said cell population
comprises detecting the expression of at least one marker selected
from the group consisting of SOX17 and CXCR4 and at least one
marker from the group consisting of OCT4, AFP, TM, SPARC and SOX7
in cells of said cell population, wherein the expression of a
marker selected from the group consisting of SOX17 and CXCR4 is
greater than the expression of a marker selected from the group
consisting of OCT4, AFP, TM, SPARC and SOX7 in said definitive
endoderm cells.
[0061] 42. The method of paragraph 36, wherein detecting the
presence of definitive endoderm cells in said cell population
comprises detecting the expression of at least one marker selected
from the group consisting of SOX17 and CXCR4 and at least one
marker from the group consisting of AFP, TM, and SOX7 in cells of
said cell population, wherein the expression of a marker selected
from the group consisting of SOX17 and CXCR4 is greater than the
expression of a marker selected from the group consisting of AFP,
TM, and SOX7 in said definitive endoderm cells.
[0062] 43. The method of paragraph 42, wherein the expression of at
least one of said markers is determined by Q-PCR.
[0063] 44. The method of paragraph 42, wherein the expression of at
least one of said markers is determined by immunocytochemistry.
[0064] 45. The method of paragraph 36, wherein detecting the
presence of definitive endoderm cells in said cell population
comprises detecting the expression of at least one marker selected
from the group consisting of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and
CRIP1 and at least one marker from the group consisting of OCT4,
AFP, TM, SPARC and SOX7 in cells of said cell population, wherein
the expression of a marker selected from the group consisting of
FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is greater than the
expression of a marker selected from the group consisting of OCT4,
AFP, TM, SPARC and SOX7 in said definitive endoderm cells.
[0065] 46. The method of paragraph 36, wherein said at least one
growth factor is of the Nodal/Activin subgroup of the TGF.beta.
superfamily.
[0066] 47. The method of paragraph 46, wherein said at least one
growth factor is selected from the group consisting of Nodal
Activin A, Activin B and combinations thereof.
[0067] 48. The method of paragraph 47, wherein said at least one
growth factor is
[0068] Nodal.
[0069] 49. The method of paragraph 47, wherein said at least one
growth factor is Activin A.
[0070] 50. The method of paragraph 47, wherein said at least one
growth factor is Activin B.
[0071] 51. The method of paragraph 36, wherein a plurality of
growth factors of the TGF.beta. superfamily is provided.
[0072] 52. The method of paragraph 51, wherein said plurality of
growth factors comprises Nodal Activin A and Activin B.
[0073] 53. The method of paragraph 36, wherein said at least one
growth factor is provided in a concentration of at least about 10
ng/ml.
[0074] 54. The method of paragraph 36, wherein said at least one
growth factor is provided in a concentration of at least about 100
ng/ml.
[0075] 55. The method of paragraph 36, wherein said at least one
growth factor is provided in a concentration of at least about 500
ng/ml.
[0076] 56. The method of paragraph 36, wherein said at least one
growth factor is provided in a concentration of at least about 1000
ng/ml.
[0077] 57. The method of paragraph 36, wherein said at least one
growth factor is provided in a concentration of at least about 5000
ng/ml.
[0078] 58. The method of paragraph 36, wherein said cell population
is grown in a medium comprising less than about 10% serum.
[0079] 59. The method of paragraph 36, wherein said pluripotent
cells comprise stem cells.
[0080] 60. The method of paragraph 59, wherein said pluripotent
cells comprise embryonic stem cells.
[0081] 61. The method of paragraph 60, wherein said embryonic stem
cells are derived from a tissue selected from the group consisting
of the morula, the ICM of an embryo and the gonadal ridges of an
embryo.
[0082] 62. A definitive endoderm cell produced by the method of
paragraph 36.
[0083] 63. A method of producing a cell population enriched in
definitive endoderm cells, said method comprising the steps of:
[0084] differentiating cells in a population of pluripotent human
cells so as to produce definitive endoderm cells, said definitive
endoderm cells being multipotent cells that can differentiate into
cells of the gut tube or organs derived therefrom;
[0085] providing to said cell population a reagent which binds to a
marker expressed in said definitive endoderm cells but which is not
substantially expressed in other cell types present in said cell
population; and
[0086] separating said definitive endoderm cells bound to said
reagent from said other cell types present in said cell population,
thereby producing a cell population enriched in definitive endoderm
cells.
[0087] 64. The method of paragraph 63, wherein the differentiating
step further 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, said definitive endoderm cells being
multipotent cells that can differentiate into cells of the gut tube
or organs derived therefrom, 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.
[0088] 65. The method of paragraph 63, wherein detecting comprises
detecting the expression of at least one marker selected from the
group consisting of SOX17 and CXCR4 and at least one marker from
the group consisting of OCT4, AFP, TM, SPARC and SOX7 in cells of
said cell population, wherein the expression of a marker selected
from the group consisting of SOX17 and CXCR4 is greater than the
expression of a marker selected from the group consisting of OCT4,
AFP, TM, SPARC and SOX7 in said definitive endoderm cells.
[0089] 66. The method of paragraph 63, wherein detecting comprises
detecting the expression of at least one marker selected from the
group consisting of SOX17 and CXCR4 and at least one marker from
the group consisting of AFP, TM, and SOX7 in cells of said cell
population, wherein the expression of a marker selected from the
group consisting of SOX17 and CXCR4 is greater than the expression
of a marker selected from the group consisting of AFP, TM, and SOX7
in said definitive endoderm cells.
[0090] 67. The method of paragraph 63, wherein detecting comprises
detecting the expression of at least one marker selected from the
group consisting of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 and
at least one marker from the group consisting of OCT4, AFP, TM,
SPARC and SOX7 in cells of said cell population, wherein the
expression of a marker selected from the group consisting of FGF17,
VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is greater than the expression
of a marker selected from the group consisting of OCT4, AFP, TM,
SPARC and SOX7 in said definitive endoderm cells.
[0091] 68. The method of paragraph 63, wherein at least about 95%
of said cells are definitive endoderm cells.
[0092] 69. The method of paragraph 63, wherein at least about 98%
of said cells are definitive endoderm cells.
[0093] 70. The method of paragraph 63, wherein said marker is
CXCR4.
[0094] 71. The method of paragraph 63, wherein said reagent is an
antibody
[0095] 72. The method of paragraph 71, wherein said antibody has
affinity for CXCR4.
[0096] 73. An enriched population of definitive endoderm cells
produced by the method of paragraph 63.
[0097] 74. The cell culture of any one of paragraphs 4 or 9,
wherein said definitive endoderm cells do not significantly express
a marker selected from the group consisting of OCT4, AFP, TM, SPARC
and SOX7.
[0098] 75. The cell population of any one of paragraphs 23 or 28,
wherein said definitive endoderm cells do not significantly express
a marker selected from the group consisting of OCT4, AFP, TM, SPARC
and SOX7.
[0099] 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.
[0100] Additional embodiments of the present inventions 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. 60/586,566, entitled CHEMOKINE CELL SURFACE
RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9,
2004; and U.S. Provisional Patent Application No. 60/587,942,
entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF
DEFINITIVE ENDODERM, filed Jul. 14, 2004, the disclosures of which
are incorporated herein by reference in their entireties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0101] 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
represents one of the earliest known steps in the further
differentiation of ES cells to pancreatic endoderm, endocrine
endoderm, or islet/beta-cell. Some factors useful for mediating
this transition are members of the TGF.beta. family which include,
but are not limited to, activins, nodals and BMPs. Exemplary
markers for defining the definitive endoderm target cell are SOX17,
GATA4, HNF3b, MIX1 and CXCR4.
[0102] 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.
[0103] 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.
[0104] 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).
[0105] FIGS. 5A-B are micrographs showing a cluster of SOX17.sup.+
cells that display a significant number of AFP.sup.+ co-labeled
cells (A). This is in striking contrast to other SOX17.sup.+
clusters (B) where little or no AFP.sup.+ cells are observed.
[0106] 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.
[0107] 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
SOX17 gene expression while retinoic acid (RA) strongly suppresses
SOX17 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.
[0108] 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.
[0109] 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).
[0110] 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.
[0111] 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.
[0112] 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.
[0113] FIGS. 12A-C are bar charts which demonstrate the effect of
Activin A on the expression of MIXL1 (panel A), GATA4 (panel B) and
HNF3b (panel C). Activin A dose-dependent increases are also
observed for three other markers of definitive endoderm; MIXL1,
GATA4 and HNF3b. The magnitudes of increased expression in response
to activin dose are strikingly similar to those observed for SOX17,
strongly indicating that Activin A is specifying a population of
cells that co-express all four genes (SOX17.sup.+, MIXL1.sup.+,
GATA4.sup.+ and HNF3b.sup.+).
[0114] 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 endoderm (SPARC) remain
either unchanged or exhibit suppression at some time points
indicating that Activin A does not act to specify these
extra-embryonic endoderm cell types. This further supports the fact
that the increased expression of SOX17, MIXL1, GATA4, and HNF3b are
due to an increase in the number of definitive endoderm cells in
response to Activin A.
[0115] 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.
[0116] FIGS. 15A-B are micrographs showing decreased parietal
endoderm differentiation in response to treatment with activins.
Regions of TM.sup.hi parietal endoderm are found through the
culture (A) when differentiated in serum alone, while
differentiation to TM.sup.+ cells is scarce when activins are
included (B) and overall intensity of TM immunoreactivity is
lower.
[0117] 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 SOX17 positive cells (A) associated with the complete
absence of AFP (B) and TM (C) immunoreactivity.
[0118] FIG. 17 is a micrograph showing the appearance of definitive
endoderm and visceral endoderm in vitro from hESCs. The regions of
visceral endoderm are identified by AFP.sup.hi/SOX17.sup.lo/- while
definitive endoderm displays the complete opposite profile,
SOX17.sup.hi/AFP.sup.lo/-. This field was selectively chosen due to
the proximity of these two regions to each other. However, there
are numerous times when SOX17.sup.hi/AFP.sup.lo/- regions are
observed in absolute isolation from any regions of AFP.sup.hi
cells, suggesting the separate origination of the definitive
endoderm cells from visceral endoderm cells.
[0119] 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).
[0120] 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.
[0121] FIG. 20 is a bar chart showing the increase in SOX17.sup.+
cell number with time as a result of treatment with combinations of
TGF.beta. factors.
[0122] FIG. 21 is a bar chart showing induction of SOX17 expression
over time as a result of treatment with combinations of TGF.beta.
factors.
[0123] FIG. 22 is a bar chart showing that Activin A induces a
dose-dependent increase in SOX17.sup.+ cell number.
[0124] 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.
[0125] 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 (10AA) (panel A). Induction of the
definitive endoderm marker MIXL1 (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.
[0126] 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.sup.+ cells
(arrows) as well as OCT4.sup.+, undifferentiated hESCs (arrowheads)
(D).
[0127] FIG. 26 is a bar chart showing the relative expression level
of CXCR4 in differentiating hESCs under various media
conditions.
[0128] 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.
[0129] FIGS. 28A-E are bar charts showing how markers for mesoderm
(BRACHYURY, MOX1), ectoderm (SOX1, ZIC1) and visceral endoderm
(SOX7) exhibit an inverse relationship to CXCR4 expression across
the same treatments displayed in FIG. 26.
[0130] 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.
[0131] FIGS. 30A-C are flow cytometry dot plots that demonstrate
the increase in CXCR4.sup.+ cell number with increasing
concentration of activin A added to the differentiation media.
[0132] FIGS. 31A-D are bar charts that show the CXCR4.sup.+ cells
isolated from the high dose activin A treatment (A100-CX+) are even
further enriched for definitive endoderm markers than the parent
population (A100).
[0133] FIG. 32 is a bar chart showing gene expression from
CXCR4.sup.+ and CXCR4.sup.- cells isolated using
fluorescence-activated cell sorting (FACS) as well as gene
expression in the parent populations. This demonstrates that the
CXCR4.sup.+ cells contain essentially all the CXCR4 gene expression
present in each parent population and the CXCR4.sup.- populations
contain very little or no CXCR4 gene expression.
[0134] FIGS. 33A-D are bar charts that demonstrate the depletion of
mesoderm (BRACHYURY, MOX1), 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.
[0135] 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, FOXQ1, CMKOR1 and CRIP1 is shown in panels G-L,
respectively. The expression analysis of previously described
lineage marking genes, SOX17, SOX7, SOX17/SOX7, TM, ZIC1, and MOX1
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; 0.1A100 indicates cells treated with 0.1% FBS,
100 ng/ml Activin A; 1A100 indicates cells treated with 1% FBS, 100
ng/ml Activin A; and 2A100 indicates cells treated with 2% FBS, 100
ng/ml Activin A.
DETAILED DESCRIPTION
[0136] 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.
[0137] 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.
[0138] In vivo analyses of the formation of definitive endoderm,
such as the studies in Zebrafish and Xenopus by Conlon et al.,
1994; Feldman et al., 1998; Zhou et al., 1993; Aoki et al., 2002;
Dougan et al., 2003; Tremblay et al., 2000; Vincent et al., 2003;
Alexander et al., 1999; Alexander and Stainier, 1999; Kikuchi et
al., 2001; Hudson et al., 1997 and in mouse by Kanai-Azuma et al.,
2002 lay a foundation for how one might attempt to approach the
development of a specific germ layer cell type in the culture dish
using human embryonic stem cells. There are two aspects associated
with in vitro ESC culture that pose major obstacles in the attempt
to recapitulate development in the culture dish. First, organized
germ layer or organ structures are not produced. The majority of
germ layer and organ specific genetic markers will be expressed in
a heterogeneous fashion in the differentiating hESC culture system.
Therefore it is difficult to evaluate formation of a specific
tissue or cell type due to this lack of organ specific boundaries.
Almost all genes expressed in one cell type within a particular
germ layer or tissue type are expressed in other cells of different
germ layer or tissue types as well. Without specific boundaries
there is considerably less means to assign gene expression
specificity with a small sample of 1-3 genes. Therefore one must
examine considerably more genes, some of which should be present as
well as some that should not be expressed in the particular cell
type of the organ or tissue of interest. Second, the timing of gene
expression patterns is crucial to movement down a specific
developmental pathway.
[0139] To further complicate matters, it should be noted that stem
cell differentiation in vitro is rather asynchronous, likely
considerably more so than in vivo. As such, one group of cells may
be expressing genes associated with gastrulation, while another
group may be starting final differentiation. Furthermore,
manipulation of hESC monolayers or embryoid bodies (EBs) with or
without exogenous factor application may result in profound
differences with respect to overall gene expression pattern and
state of differentiation. For these reasons, the application of
exogenous factors must be timed according to gene expression
patterns within a heterogeneous cell mixture in order to
efficiently move the culture down a specific differentiation
pathway. It is also beneficial to consider the morphological
association of the cells in the culture vessel. The ability to
uniformly influence hESCs when formed into so called embryoid
bodies may be less optimal than hESCs grown and differentiated as
monolayers and or hESC colonies in the culture vessel.
[0140] As an effective way to deal with the above-mentioned
problems of heterogeneity and asynchrony, some embodiments of the
present invention contemplate combining a method for
differentiating cells with a method for the enrichment, isolation
and/or purification of intermediate cell types in the
differentiation pathway.
[0141] Embodiments of the present invention relate to novel,
defined processes for the production of definitive endoderm cells
in culture by differentiating pluripotent cells, such as stem cells
into multipotent definitive endoderm cells. As used herein,
"multipotent" or "multipotent cell" refers to a cell type that can
give rise to a limited number of other particular cell types. As
described above, definitive endoderm cells do not differentiate
into tissues produced from ectoderm or mesoderm, but rather,
differentiate into the gut tube as well as organs that are derived
from the gut tube. In certain preferred embodiments, the definitive
endoderm cells are derived from hESCs. Such processes can provide
the basis for efficient production of human endodermal derived
tissues such as pancreas, liver, lung, stomach, intestine and
thyroid. For example, production of definitive endoderm may be the
first step in differentiation of a stem cell to a functional
insulin-producing .beta.-cell. To obtain useful quantities of
insulin-producing .beta.-cells, high efficiency of differentiation
is desirable for each of the differentiation steps that occur prior
to reaching the pancreatic islet/.beta.-cell fate. Since
differentiation of stem cells to definitive endoderm cells
represents perhaps the earliest step towards the production of
functional pancreatic islet/.beta.-cells (as shown in FIG. 1), high
efficiency of differentiation at this step is particularly
desirable.
[0142] In view of the desirability of efficient differentiation of
pluripotent cells to definitive endoderm cells, some aspects of the
present invention relate to in vitro methodology that results in
approximately 50-80% conversion of pluripotent cells to definitive
endoderm cells. Typically, such methods encompass the application
of culture and growth factor conditions in a defined and temporally
specified fashion. Further enrichment of the cell population for
definitive endoderm cells can be achieved by isolation and/or
purification of the definitive endoderm cells from other cells in
the population by using a reagent that specifically binds to
definitive endoderm cells. As such, aspects of the present
invention relate to definitive endoderm cells as well as methods
for producing and isolating and/or purifying such cells.
[0143] In order to determine the amount of definitive endoderm
cells in a cell culture or cell population, a method of
distinguishing this cell type from the other cells in the culture
or in the population is desirable. Accordingly, certain embodiments
of the present invention relate to cell markers whose presence,
absence and/or relative expression levels are specific for
definitive endoderm and methods for detecting and determining the
expression of such markers. 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.
[0144] 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 SOX17, CXCR4, OCT4,
AFP, TM, SPARC, SOX7, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR,
FOXQ1, CMKOR1, CRIP1 and other markers described herein is
determined by quantitative Q-PCR. In other embodiments,
immunohistochemistry is used to detect the proteins expressed by
the above-mentioned genes. In still other embodiments, Q-PCR and
immunohistochemical techniques are both used to identify and
determine the amount or relative proportions of such markers.
[0145] By using methods, such as those described above, to
determine the expression of one or more appropriate markers, it is
possible to identify definitive endoderm cells, as well as
determine the proportion of definitive endoderm cells in a cell
culture or cell population. For example, in some embodiments of the
present invention, the definitive endoderm cells or cell
populations that are produced express the SOX17 and/or the CXCR4
gene at a level of about 2 orders of magnitude greater than
non-definitive endoderm cell types or cell populations. In other
embodiments, the definitive endoderm cells or cell populations that
are produced express the SOX17 and/or the CXCR4 gene at a level of
more than 2 orders of magnitude greater than non-definitive
endoderm cell types or cell populations. In still other
embodiments, the definitive endoderm cells or cell populations that
are produced express one or more of the markers selected from the
group consisting of SOX17, CXCR4, GSC, FGF17, VWF, CALCR, FOXQ1,
CMKOR1 and CRIP1 at a level of about 2 or more than 2 orders of
magnitude greater than non-definitive endoderm cell types or cell
populations.
[0146] Further aspects of the present invention relate to cell
cultures comprising definitive endoderm as well as cell populations
enriched in definitive endoderm cells. As such, certain embodiments
relate to cell cultures which comprise definitive endoderm cells,
wherein at least about 50-80% of the cells in culture are
definitive endoderm cells. A preferred embodiment relates to cells
cultures comprising human cells, wherein at least about 50-80% of
the human cells in culture are definitive endoderm cells. Because
the efficiency of the differentiation procedure can be adjusted by
modifying certain parameters, which include but are not limited to,
cell growth conditions, growth factor concentrations and the timing
of culture steps, the differentiation procedures described herein
can result in about 5%, about 10%, about 15%, about 20%, about 25%,
about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,
about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,
about 90%, about 95%, or greater than about 95% conversion of
pluripotent cells to definitive endoderm. In other embodiments of
the present invention, conversion of a pluripotent cell population,
such as a stem cell population, to substantially pure definitive
endoderm cell population is contemplated.
[0147] The compositions and methods described herein have several
useful features. For example, the cell cultures and cell
populations comprising definitive endoderm as well as the methods
for producing such cell cultures and cell populations are useful
for modeling the early stages of human development. Furthermore,
the compositions and methods described herein can also serve for
therapeutic intervention in disease states, such as diabetes
mellitus. For example, since definitive endoderm serves as the
source for only a limited number of tissues, it can be used in the
development of pure tissue or cell types.
Production of Definitive Endoderm from Pluripotent Cells
[0148] The definitive endoderm cell cultures and compositions
comprising definitive endoderm cells that are described herein can
be produced from pluripotent cells, such as 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 (hESC) as the starting material for definitive
endoderm production. The embryonic stem cells used in this method
can be cells that originate from the morula, embryonic inner cell
mass or those obtained from embryonic gonadal ridges. Human 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.
[0149] In some embodiments of the methods described herein, hESCs
are maintained on a feeder layer. In such embodiments, any feeder
layer which allows hESCs to be maintained in a pluripotent state
can be used in the methods described herein. One commonly used
feeder layer for the cultivation of human embryonic stem cells is a
layer of mouse fibroblasts. More recently, human fibroblast feeder
layers have been developed for use in the cultivation of hESCs (see
US Patent Application No. 2002/0072117, the disclosure of which is
incorporated herein by reference in its entirety). Alternative
embodiments of the methods described herein permit the maintenance
of pluripotent hESC without the use of a feeder layer. Such methods
have been described in US Patent Application No. 2003/0175956, the
disclosure of which is incorporated herein by reference in its
entirety.
[0150] The human embryonic stem cells used herein can be maintained
in culture either with or without serum. In some embodiments, serum
replacement is used. In other embodiments, 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.
[0151] 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 embodiments, 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 embodiments of the
differentiation methods described herein, 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 embodiments of the present invention,
combinations of any of the above-mentioned growth factors can be
used.
[0152] With respect to some of the embodiments of differentiation
methods described herein, 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. In
some embodiments of the present invention, 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.
[0153] In certain embodiments of the present invention, 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 embodiment, the growth factors are removed
about four days after their addition.
[0154] Cultures of definitive endoderm cells can be grown in medium
containing reduced serum or no serum. In certain embodiments of the
present invention, serum concentrations can range from about 0.05%
v/v to about 20% v/v. 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, definitive
endoderm cells are grown without serum. In other embodiments,
definitive endoderm cells are grown with serum replacement. In
still other embodiments, definitive endoderm cells are grown in the
presence of B27. In such embodiments, the concentration of B27
supplement can range from about 0.2% v/v to about 20% v/v.
[0155] The progression of the hESC culture to definitive endoderm
can be monitored by determining the expression of markers
characteristic of definitive endoderm. In some embodiments, the
expression of certain markers are determined by detecting the
presence or absence of the marker. Alternatively, the expression of
certain markers can 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. One method of
quantitating the expression 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 some embodiments of the present invention,
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
[0156] 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 methods described herein
express the SOX17 marker gene, thereby producing the SOX17 gene
product. Other markers of definitive endoderm are MIXL1, GATA4,
HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1. In some
embodiments of the present invention, 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). Additionally, in some embodiments,
expression of the SOX17 marker gene is higher than the expression
of the OCT4 marker gene, which is characteristic of hESCs. In other
embodiments of the present invention, definitive endoderm cells
express the SOX17 marker gene at a level higher than that of the
AFP, SPARC or Thrombomodulin (TM) marker genes. In certain
embodiments of the present invention, the SOX17-expressing
definitive endoderm cells produced by the methods described herein
do not express significant levels or amounts of PDX1
(PDX1-negative).
[0157] Another marker of definitive endoderm is the CXCR4 gene. The
CXCR4 gene encodes a cell surface chemokine receptor whose ligand
is the chemoattractant SDF-1. The principal roles of the CXCR4
receptor-bearing cells in the adult are believed to be the
migration of hematopoetic cells to the bone marrow, lymphocyte
trafficking and the differentiation of various B cell and
macrophage blood cell lineages [Kim, C., and Broxmeyer, H. J.
Leukocyte Biol. 65, 6-15 (1999)]. The CXCR4 receptor also functions
as a coreceptor for the entry of HIV-1 into T-cells [Feng, Y., et
al. Science, 272, 872-877 (1996)]. In an extensive series of
studies carried out by [McGrath, K. E. et al. Dev. Biology 213,
442-456 (1999)], the expression of the chemokine receptor CXCR4 and
its unique ligand, SDF-1 [Kim, C., and Broxmyer, H., J. Leukocyte
Biol. 65, 6-15 (1999)], were delineated during early development
and adult life in the mouse. The CXCR4/SDF1 interaction in
development became apparent when it was demonstrated that if either
gene was disrupted in transgenic mice [Nagasawa et al. Nature, 382,
635-638 (1996)], Ma, Q., et al Immunity, 10, 463-471 (1999)] it
resulted in late embryonic lethality. McGrath et al. demonstrated
that CXCR4 is the most abundant chemokine receptor messenger RNA
detected during early gastrulating embryos (E7.5) using a
combination of RNase protection and in situ hybridization
methodologies. In the gastrulating embryo, CXCR4/SDF-1 signaling
appears to be mainly involved in inducing migration of
primitive-streak germlayer cells and is expressed on definitive
endoderm, mesoderm and extraembryonic mesoderm present at this
time. In E7.2-7.8 mouse embryos, CXCR4 and alpha-fetoprotein are
mutually exclusive indicating a lack of expression in visceral
endoderm [McGrath, K. E. et al. Dev. Biology 213, 442-456
(1999)].
[0158] In some embodiments of the present invention, the definitive
endoderm cells produced by the methods described herein express the
CXCR4 marker gene. In other embodiments, the definitive endoderm
cells produced by the methods described herein express the CXCR4
marker gene as well as other markers of definitive endoderm
including, but not limited to, SOX17, MIXL1, GATA4, HNF3b, GSC,
FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1. In some embodiments of
the present invention, definitive endoderm cells express the CXCR4
marker gene at a level higher than that of the SOX7 marker gene.
Additionally, in some embodiments, expression of the CXCR4 marker
gene is higher than the expression of the OCT4 marker gene. In
other embodiments of the present invention, definitive endoderm
cells express the CXCR4 marker gene at a level higher than that of
the AFP, SPARC or Thrombomodulin (TM) marker genes. In certain
embodiments of the present invention, the CXCR4-expressing
definitive endoderm cells produced by the methods described herein
do not express significant levels or amounts of PDX1
(PDX1-negative).
[0159] It will be appreciated that expression of CXCR4 in
endodermal cells does not preclude the expression of SOX17.
Accordingly, in some embodiments of the present invention,
definitive endoderm cells are those that express both the SOX17 and
CXCR4 marker genes at a level higher than that of the SOX7 marker
gene. Additionally, in some embodiments, the expression of both the
SOX17 and CXCR4 marker genes is higher than the expression of the
OCT4 marker gene. In other embodiments of the present invention,
definitive endoderm cells express both the SOX17 and the CXCR4
marker genes at a level higher than that of the AFP, SPARC or
Thrombomodulin (TM) marker genes. In certain embodiments of the
present invention, the SOX17/CXCR4-expressing definitive endoderm
cells produced by the methods described herein do not express
significant levels or amounts of PDX1 (PDX1-negative).
[0160] It will be appreciated that SOX17 and/or CXCR4 marker
expression is induced over a range of different levels in
definitive endoderm cells depending on the differentiation
conditions. As such, in some embodiments of the present invention,
the expression of the SOX17 marker and/or the CXCR4 marker in
definitive endoderm cells or cell populations is at least about
2-fold higher to at least about 10,000-fold higher than the
expression of the SOX17 marker and/or the CXCR4 marker in
non-definitive endoderm cells or cell populations, for example
pluripotent stem cells. In other embodiments of the present
invention, the expression of the SOX17 marker and/or the CXCR4
marker in definitive endoderm cells or cell populations is at least
about 4-fold higher, at least about 6-fold higher, at least about
8-fold higher, at least about 10-fold higher, at least about
15-fold higher, at least about 20-fold higher, at least about
40-fold higher, at least about 80-fold higher, at least about
100-fold higher, at least about 150-fold higher, at least about
200-fold higher, at least about 500-fold higher, at least about
750-fold higher, at least about 1000-fold higher, at least about
2500-fold higher, at least about 5000-fold higher, at least about
7500-fold higher or at least about 10,000-fold higher than the
expression of the SOX17 marker and/or the CXCR4 marker in
non-definitive endoderm cells or cell populations, for example
pluripotent stem cells. In some embodiments, the expression of the
SOX17 marker and/or CXCR4 marker in definitive endoderm cells or
cell populations is infinitely higher than the expression of the
SOX17 marker and/or the CXCR4 marker in non-definitive endoderm
cells or cell populations, for example pluripotent stem cells.
[0161] It will be appreciated that in some embodiments of the
present invention, the expression of markers selected from the
group consisting of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR,
FOXQ1, CMKOR1 and CRIP1 in definitive endoderm cells or cell
populations is increased as compared to the expression of GATA4,
MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 in
non-definitive endoderm cells or cell populations.
[0162] It will also be appreciated that there is a range of
differences between the expression level of the SOX17 marker and
the expression levels of the OCT4, SPARC, AFP, TM and/or SOX7
markers in definitive endoderm cells. Similarly, there exists a
range of differences between the expression level of the CXCR4
marker and the expression levels of the OCT4, SPARC, AFP, TM and/or
SOX7 markers in definitive endoderm cells. As such, in some
embodiments of the present invention, the expression of the SOX17
marker or the CXCR4 marker is at least about 2-fold higher to at
least about 10,000-fold higher than the expression of OCT4, SPARC,
AFP, TM and/or SOX7 markers. In other embodiments of the present
invention, the expression of the SOX17 marker or the CXCR4 marker
is at least about 4-fold higher, at least about 6-fold higher, at
least about 8-fold higher, at least about 10-fold higher, at least
about 15-fold higher, at least about 20-fold higher, at least about
40-fold higher, at least about 80-fold higher, at least about
100-fold higher, at least about 150-fold higher, at least about
200-fold higher, at least about 500-fold higher, at least about
750-fold higher, at least about 1000-fold higher, at least about
2500-fold higher, at least about 5000-fold higher, at least about
7500-fold higher or at least about 10,000-fold higher than the
expression of OCT4, SPARC, AFP, TM and/or SOX7 markers. In some
embodiments, OCT4, SPARC, AFP, TM and/or SOX7 markers are not
significantly expressed in definitive endoderm cells.
[0163] It will be appreciated that in some embodiments of the
present invention, the expression of markers selected from the
group consisting of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR,
FOXQ1, CMKOR1 and CRIP1 in definitive endoderm cells is increased
as compared to the expression of OCT4, SPARC, AFP, TM and/or SOX7
in definitive endoderm cells.
Compositions Comprising Definitive Endoderm
[0164] Some aspects of the present invention relate to
compositions, such as cell populations and cell cultures, that
comprise both pluripotent cells, such as stem cells, and definitive
endoderm cells. For example, using the methods described herein,
compositions comprising mixtures of hESCs and definitive endoderm
cells can be produced. In some embodiments, compositions comprising
at least about 5 definitive endoderm cells for about every 95
pluripotent cells are produced. In other embodiments, compositions
comprising at least about 95 definitive endoderm cells for about
every 5 pluripotent cells are produced. Additionally, compositions
comprising other ratios of definitive endoderm cells to pluripotent
cells are contemplated. For example, compositions comprising at
least about 1 definitive endoderm cell for about every 1,000,000
pluripotent cells, at least about 1 definitive endoderm cell for
about every 100,000 pluripotent cells, at least about 1 definitive
endoderm cell for about every 10,000 pluripotent cells, at least
about 1 definitive endoderm cell for about every 1000 pluripotent
cells, at least about 1 definitive endoderm cell for about every
500 pluripotent cells, at least about 1 definitive endoderm cell
for about every 100 pluripotent cells, at least about 1 definitive
endoderm cell for about every 10 pluripotent cells, at least about
1 definitive endoderm cell for about every 5 pluripotent cells, at
least about 1 definitive endoderm cell for about every 2
pluripotent cells, at least about 2 definitive endoderm cells for
about every 1 pluripotent cell, at least about 5 definitive
endoderm cells for about every 1 pluripotent cell, at least about
10 definitive endoderm cells for about every 1 pluripotent cell, at
least about 20 definitive endoderm cells for about every 1
pluripotent cell, at least about 50 definitive endoderm cells for
about every 1 pluripotent cell, at least about 100 definitive
endoderm cells for about every 1 pluripotent cell, at least about
1000 definitive endoderm cells for about every 1 pluripotent cell,
at least about 10,000 definitive endoderm cells for about every 1
pluripotent cell, at least about 100,000 definitive endoderm cells
for about every 1 pluripotent cell and at least about 1,000,000
definitive endoderm cells for about every 1 pluripotent cell are
contemplated. In some embodiments of the present invention, the
pluripotent cells are human pluripotent stem cells. In certain
embodiments the stem cells are derived from a morula, the inner
cell mass of an embryo or the gonadal ridges of an embryo. In
certain other embodiments, the pluripotent cells are derived from
the gondal or germ tissues of a multicellular structure that has
developed past the embryonic stage.
[0165] Some aspects of the present invention relate to cell
cultures or cell populations comprising from at least about 5%
definitive endoderm cells to at least about 95% definitive endoderm
cells. In some embodiments the cell cultures or cell populations
comprise mammalian cells. In preferred embodiments, the cell
cultures or cell populations comprise human cells. For example,
certain specific embodiments relate to cell cultures comprising
human cells, wherein from at least about 5% to at least about 95%
of the human cells are definitive endoderm cells. Other embodiments
of the present invention relate to cell cultures comprising human
cells, wherein at least about 5%, at least about 10%, at least
about 15%, at least about 20%, at least about 25%, at least about
30%, at least about 35%, at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90% or greater than 90% of
the human cells are definitive endoderm cells.
[0166] Further embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
human cells, such as human definitive endoderm cells, wherein the
expression of either the SOX17 or the CXCR4 marker is greater than
the expression of the OCT 4, SPARC, alpha-fetoprotein (AFP),
Thrombomodulin (TM) and/or SOX7 marker in at least about 5% of the
human cells. In other embodiments, the expression of either the
SOX17 or the CXCR4 marker is greater than the expression of the
OCT4, SPARC, AFP, TM and/or SOX7 marker in at least about 10% of
the human cells, in at least about 15% of the human cells, in at
least about 20% of the human cells, in at least about 25% of the
human cells, in at least about 30% of the human cells, in at least
about 35% of the human cells, in at least about 40% of the human
cells, in at least about 45% of the human cells, in at least about
50% of the human cells, in at least about 55% of the human cells,
in at least about 60% of the human cells, in at least about 65% of
the human cells, in at least about 70% of the human cells, in at
least about 75% of the human cells, in at least about 80% of the
human cells, in at least about 85% of the human cells, in at least
about 90% of the human cells, in at least about 95% of the human
cells or in greater than 95% of the human cells.
[0167] It will be appreciated that some embodiments of the present
invention relate to compositions, such as cell cultures or cell
populations, comprising human cells, such as human definitive
endoderm cells, wherein the expression of one or more markers
selected from the group consisting of GATA4, MIXL1, HNF3b, GSC,
FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is greater than the
expression of the OCT4, SPARC, AFP, TM and/or SOX7 markers in from
at least about 5% to greater than at least about 95% of the human
cells.
[0168] Still other embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
human cells, such as human definitive endoderm cells, wherein the
expression both the SOX17 and the CXCR4 marker is greater than the
expression of the OCT4, SPARC, AFP, TM and/or SOX7 marker in at
least about 5% of the human cells. In other embodiments, the
expression of both the SOX17 and the CXCR4 marker is greater than
the expression of the OCT4, SPARC, AFP, TM and/or SOX7 marker in at
least about 10% of the human cells, in at least about 15% of the
human cells, in at least about 20% of the human cells, in at least
about 25% of the human cells, in at least about 30% of the human
cells, in at least about 35% of the human cells, in at least about
40% of the human cells, in at least about 45% of the human cells,
in at least about 50% of the human cells, in at least about 55% of
the human cells, in at least about 60% of the human cells, in at
least about 65% of the human cells, in at least about 70% of the
human cells, in at least about 75% of the human cells, in at least
about 80% of the human cells, in at least about 85% of the human
cells, in at least about 90% of the human cells, in at least about
95% of the human cells or in greater than 95% of the human
cells.
[0169] It will be appreciated that some embodiments of the present
invention relate to compositions, such as cell cultures or cell
populations, comprising human cells, such as human definitive
endoderm cells, wherein the expression of the GATA4, MIXL1, HNF3b,
GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 markers is greater
than the expression of the OCT4, SPARC, AFP, TM and/or SOX7 markers
in from at least about 5% to greater than at least about 95% of the
human cells.
[0170] 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 either the SOX17 or the CXCR4 marker is greater
than the expression of the OCT4, SPARC, AFP, TM and/or SOX7 marker
in at least about 5% of the endodermal cells. In other embodiments,
the expression of either the SOX17 or the CXCR4 marker is greater
than the expression of the OCT4, SPARC, AFP, TM and/or SOX7 marker
in at least about 10% of the endodermal cells, in at least about
15% of the endodermal cells, in at least about 20% of the
endodermal cells, in at least about 25% of the endodermal cells, in
at least about 30% of the endodermal cells, in at least about 35%
of the endodermal cells, in at least about 40% of the endodermal
cells, in at least about 45% of the endodermal cells, in at least
about 50% of the endodermal cells, in at least about 55% of the
endodermal cells, in at least about 60% of the endodermal cells, in
at least about 65% of the endodermal cells, in at least about 70%
of the endodermal cells, in at least about 75% of the endodermal
cells, in at least about 80% of the endodermal cells, in at least
about 85% of the endodermal cells, in at least about 90% of the
endodermal cells, in at least about 95% of the endodermal cells or
in greater than 95% of the endodermal cells.
[0171] It will be appreciated that some embodiments of the present
invention relate to compositions, such as cell cultures or cell
populations comprising mammalian endodermal cells, wherein the
expression of one or more markers selected from the group
consisting of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1,
CMKOR1 and CRIP1 is greater than the expression of the OCT4, SPARC,
AFP, TM and/or SOX7 markers in from at least about 5% to greater
than at least about 95% of the endodermal cells.
[0172] Still other embodiments 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 both the SOX17 and the CXCR4 marker is greater than
the expression of the OCT4, SPARC, AFP, TM and/or SOX7 marker in at
least about 5% of the endodermal cells. In other embodiments, the
expression of both the SOX17 and the CXCR4 marker is greater than
the expression of the OCT4, SPARC, AFP, TM and/or SOX7 marker in at
least about 10% of the endodermal cells, in at least about 15% of
the endodermal cells, in at least about 20% of the endodermal
cells, in at least about 25% of the endodermal cells, in at least
about 30% of the endodermal cells, in at least about 35% of the
endodermal cells, in at least about 40% of the endodermal cells, in
at least about 45% of the endodermal cells, in at least about 50%
of the endodermal cells, in at least about 55% of the endodermal
cells, in at least about 60% of the endodermal cells, in at least
about 65% of the endodermal cells, in at least about 70% of the
endodermal cells, in at least about 75% of the endodermal cells, in
at least about 80% of the endodermal cells, in at least about 85%
of the endodermal cells, in at least about 90% of the endodermal
cells, in at least about 95% of the endodermal cells or in greater
than 95% of the endodermal cells.
[0173] It will be appreciated that some embodiments of the present
invention relate to compositions, such as cell cultures or cell
populations comprising mammalian endodermal cells, wherein the
expression of the GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR,
FOXQ1, CMKOR1 and CRIP1 markers is greater than the expression of
the OCT4, SPARC, AFP, TM and/or SOX7 markers in from at least about
5% to greater than at least about 95% of the endodermal cells.
[0174] Using the methods described herein, compositions comprising
definitive endoderm cells substantially free of other cell types
can be produced. 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 definitive endoderm
cell populations or cell cultures produced by the methods described
herein are substantially free of cells that significantly express
the OCT4, SOX7, AFP, SPARC, TM, ZIC1 or BRACH marker genes.
[0175] In one embodiment of the present invention, a description of
a definitive endoderm cell based on the expression of marker genes
is, SOX17 high, MIXL1 high, AFP low, SPARC low, Thrombomodulin low,
SOX7 low, CXCR4 high.
Enrichment, Isolation and/or Purification of Definitive
Endoderm
[0176] With respect to additional aspects of the present invention,
definitive endoderm cells 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 embodiments, an
antibody which binds to CXCR4 is used as an affinity tag for the
enrichment, isolation or purification of definitive endoderm cells.
In other embodiments, the chemokine SDF-1 or other molecules based
on SDF-1 can also be used as affinity tags. Such molecules include,
but not limited to, SDF-1 fragments, SDF-1 fusions or SDF-1
mimetics.
[0177] 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 cells described herein. In one
embodiment, an antibody which binds to CXCR4 is attached to a
magnetic bead 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.
[0178] Embodiments of the present invention contemplate additional
methods for obtaining enriched, isolated or purified definitive
endoderm cell cultures or populations. For example, in some
embodiments, the CXCR4 antibody is incubated with 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.
[0179] In other embodiments of the present invention, definitive
endoderm is enriched, isolated and/or purified using a ligand or
other molecule that binds to CXCR4. In some embodiments, the
molecule is SDF-1 or a fragment, fusion or mimetic thereof.
[0180] In preferred embodiments, 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.
[0181] 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 said definitive endoderm cells.
[0182] 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 embodiments,
the cells undergo random differentiation. In a preferred
embodiment, however, the cells are directed to differentiate
primarily into definitive endoderm. Some preferred enrichment,
isolation and/or purification methods relate to the in vitro
production of definitive endoderm from human embryonic stem cells.
Using the methods described herein, cell populations or cell
cultures can be enriched in definitive endoderm content by at least
about 2- to about 1000-fold as compared to untreated cell
populations or cell cultures. In some embodiments, definitive
endoderm cells can be enriched by at least about 5- to about
500-fold as compared to untreated cell populations or cell
cultures. In other embodiments, definitive endoderm cells can be
enriched from at least about 10- to about 200-fold as compared to
untreated cell populations or cell cultures. In still other
embodiments, definitive endoderm cells can be enriched from at
least about 20- to about 100-fold as compared to untreated cell
populations or cell cultures. In yet other embodiments, definitive
endoderm cells can be enriched from at least about 40- to about
80-fold as compared to untreated cell populations or cell cultures.
In certain embodiments, definitive endoderm cells can be enriched
from at least about 2- to about 20-fold as compared to untreated
cell populations or cell cultures.
[0183] 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
[0184] 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
[0185] 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.
[0186] 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
[0187] 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
[0188] 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.
[0189] 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.
[0190] 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).
[0191] In order to produce an antibody against SOX17, a portion of
the human SOX17 cDNA (SEQ ID NO: 1) corresponding to amino acids
172-414 (SEQ ID NO: 2) in the carboxyterminal end of the SOX17
protein (FIG. 2) was used for genetic immunization in rats at the
antibody production company, GENOVAC (Freiberg, Germany), according
to procedures developed there. Procedures for genetic immunization
can be found in U.S. Pat. Nos. 5,830,876, 5,817,637, 6,165,993 and
6,261,281 as well as International Patent Application Publication
Nos. WO00/29442 and WO99/13915, the disclosures of which are
incorporated herein by reference in their entireties.
[0192] 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.
[0193] SOX7 and SOX18 are the closest Sox family relatives to SOX17
as depicted in the relational dendrogram shown in FIG. 3. We
employed the human SOX7 polypeptide as a negative control to
demonstrate that the SOX17 antibody is specific for SOX17 and does
not react with its closest family member. In particular, to
demonstrate that the antibody produced by genetic immunization is
specific for SOX17, SOX7 and other proteins were expressed in human
fibroblasts, and then, analyzed for cross reactivity with the SOX17
antibody by Western blot and ICC. For example, the following
methods were utilized for the production of the SOX17, SOX7 and
EGFP expression vectors, their transfection into human fibroblasts
and analysis by Western blot. Expression vectors employed for the
production of SOX17, SOX7, and EGFP were pCMV6 (OriGene
Technologies, Inc., Rockville, Md.), pCMV-SPORT6 (Invitrogen,
Carlsbad, Calif.) and pEGFP-N1 (Clonetech, Palo Alto, Calif.),
respectively. For protein production, telomerase immortalized MDX
human fibroblasts were transiently transfected with supercoiled DNA
in the presence of Lipofectamine 2000 (Invitrogen, Carlsbad,
Calif.). Total cellular lysates were collected 36 hours
post-transfection in 50 mM TRIS-HCl (pH 8), 150 mM NaCl, 0.1% SDS,
0.5% deoxycholate, containing a cocktail of protease inhibitors
(Roche Diagnostics Corporation, Indianapolis, Ind.). Western blot
analysis of 100 .mu.g of cellular proteins, separated by SDS-PAGE
on NuPAGE (4-12% gradient polyacrylamide, Invitrogen, Carlsbad,
Calif.), and transferred by electro-blotting onto PDVF membranes
(Hercules, Calif.), were probed with a 1/1000 dilution of the rat
SOX17 anti-serum in 10 mM TRIS-HCl (pH 8), 150 mM NaCl, 10% BSA,
0.05% Tween-20 (Sigma, St. Louis, Mo.), followed by Alkaline
Phosphatase conjugated anti-rat IgG (Jackson ImmunoResearch
Laboratories, West Grove, Pa.), and revealed through Vector Black
Alkaline Phosphatase staining (Vector Laboratories, Burlingame,
Calif.). The proteins size standard used was wide range color
markers (Sigma, St. Louis, Mo.).
[0194] In FIG. 4, protein extracts made from human fibroblast cells
that were transiently transfected with SOX17, SOX7 or EGFP cDNA's
were probed on Western blots with the SOX17 antibody. Only the
protein extract from hSOX17 transfected cells produced a band of
.about.51 Kda which closely matched the predicted 46 Kda molecular
weight of the human SOX17 protein. There was no reactivity of the
SOX17 antibody to extracts made from either human SOX7 or EGFP
transfected cells. Furthermore, the SOX17 antibody clearly labeled
the nuclei of human fibroblast cells transfected with the hSOX17
expression construct but did not label cells transfected with EGFP
alone. As such, the SOX17 antibody exhibits specificity by ICC.
Example 4
Validation of SOX17 Antibody as a Marker of Definitive Endoderm
[0195] As evidence that the SOX17 antibody is specific for human
SOX17 protein and furthermore marks definitive endoderm, partially
differentiated hESCs were co-labeled with SOX17 and AFP antibodies.
It has been demonstrated that SOX17, SOX7, which is a closely
related member of the SOX gene family subgroup F (FIG. 3), and AFP
are each expressed in visceral endoderm. However, AFP and SOX7 are
not expressed in definitive endoderm cells at levels detectable by
ICC, and thus, they be can 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.sup.+ cells in the field of SOX17.sup.+ cells
(FIG. 5B). Similarly, since parietal endoderm has also been
reported to express SOX17, antibody co-labeling with SOX17 together
with the parietal markers SPARC and/or Thrombomodulin (TM) can be
used to identify the SOX17.sup.+ cells which are parietal endoderm.
As shown in FIGS. 6A-C, Thrombomodulin and SOX17 co-labelled
parietal endoderm cells were produced by random differentiation of
hES cells.
[0196] In view of the above cell labelling 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.
[0197] As a further evidence of the specificity of the
SOX17.sup.hi/AFP.sup.lo/TM.sup.lo/SPARC.sup.lo marker profile as
predictive of definitive endoderm, SOX17 and AFP gene expression
was quantitatively compared to the relative number of antibody
labeled cells. As shown in FIG. 7A, hESCs treated with retinoic
acid (visceral endoderm inducer), or Activin A (definitive endoderm
inducer), resulted in a 10-fold difference in the level of SOX17
mRNA expression. This result mirrored the 10-fold difference in
SOX17 antibody-labeled cell number (FIG. 7B). Furthermore, as shown
in FIG. 8A, Activin A treatment of hESCs suppressed AFP gene
expression by 6.8-fold in comparison to no treatment. This was
visually reflected by a dramatic decrease in the number of AFP
labeled cells in these cultures as shown in FIGS. 8B-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
[0198] 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.
[0199] In summary, the antibody produced against the
carboxy-terminal 242 amino acids of the human SOX17 protein
identified human SOX17 protein on Western blots but did not
recognize SOX7, it's closest Sox family relative. The SOX17
antibody recognized a subset of cells in differentiating hESC
cultures that were primarily SOX17.sup.+/AFP.sup.lo/- (greater than
95% of labeled cells) as well as a small percentage (<5%) of
cells that co-label for SOX17 and AFP (visceral endoderm).
Treatment of hESC cultures with activins resulted in a marked
elevation of SOX17 gene expression as well as SOX17 labeled cells
and dramatically suppressed the expression of AFP mRNA and the
number of cells labeled with AFP antibody.
Example 5
Q-PCR Gene Expression Assay
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] To generate PCR template, total RNA was isolated using
RNeasy (Qiagen) and quantitated using RiboGreen (Molecular Probes).
Reverse transcription from 350-500 ng of total RNA was carried out
using the iScript reverse transcriptase kit (BioRad), which
contains a mix of oligo-dT and random primers. Each 20 .mu.L
reaction was subsequently diluted up to 100 .mu.L total volume and
3 .mu.L was used in each 10 .mu.L Q-PCR reaction containing 400 nM
forward and reverse primers and 5 .mu.L 2.times. SYBR Green master
mix (Qiagen). Two step cycling parameters were used employing a 5
second denature at 85-94.degree. C. (specifically selected
according to the melting temp of the amplicon for each primer set)
followed by a 45 second anneal/extend at 60.degree. C. Fluorescence
data was collected during the last 15 seconds of each extension
phase. A three point, 10-fold dilution series was used to generate
the standard curve for each run and cycle thresholds (Ct's) were
converted to quantitative values based on this standard curve. The
quantitated values for each sample were normalized to housekeeping
gene performance and then average and standard deviations were
calculated for triplicate samples. At the conclusion of PCR
cycling, a melt curve analysis was performed to ascertain the
specificity of the reaction. A single specific product was
indicated by a single peak at the T.sub.m appropriate for that PCR
amplicon. In addition, reactions performed without reverse
transcriptase served as the negative control and do not
amplify.
[0205] A first step in establishing the Q-PCR methodology was
validation of appropriate housekeeping genes (HGs) in the
experimental system. Since the HG was used to normalize across
samples for the RNA input, RNA integrity and RT efficiency, it was
of value that the HG exhibited a constant level of expression over
time in all sample types in order for the normalization to be
meaningful. We measured the expression levels of Cyclophilin G,
hypoxanthine phosphoribosyltransferase 1 (HPRT),
beta-2-microglobulin, hydroxymethylbiane synthase (HMBS),
TATA-binding protein (TBP), and glucoronidase beta (GUS) in
differentiating hESCs. Our results indicated that
beta-2-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.
[0206] After obtaining genes for use in normalization, Q-PCR was
then utilized to determine the relative gene expression levels of
many marker genes across samples receiving different experimental
treatments. The marker genes employed have been chosen because they
exhibit enrichment in specific populations representative of the
early germ layers and in particular have focused on sets of genes
that are differentially expressed in definitive endoderm and
extra-embryonic endoderm. These genes as well as their relative
enrichment profiles are highlighted in Table 1.
TABLE-US-00001 TABLE 1 Germ Layer Gene Expression Domains Endoderm
SOX17 definitive, visceral and parietal endoderm MIXL1 endoderm and
mesoderm GATA4 definitive and primitive endoderm HNF3b definitive
endoderm and primitive endoderm, mesoderm, neural plate GSC
endoderm and mesoderm Extra- SOX7 visceral endoderm embryonic AFP
visceral endoderm, liver SPARC parietal endoderm TM parietal
endoderm/trophectoderm Ectoderm ZIC1 neural tube, neural
progenitors Mesoderm BRACH nascent mesoderm
[0207] Since many genes are expressed in more than one germ layer
it is useful to quantitatively compare expression levels of many
genes within the same experiment. SOX17 is expressed in definitive
endoderm and to a smaller extent in visceral and parietal endoderm.
SOX7 and AFP are expressed in visceral endoderm at this early
developmental time point. SPARC and TM are expressed in parietal
endoderm and Brachyury is expressed in early mesoderm.
[0208] Definitive endoderm cells were predicted to express high
levels of SOX17 mRNA and low levels of AFP and SOX7 (visceral
endoderm), SPARC (parietal endoderm) and Brachyury (mesoderm). In
addition, ZIC1 was used here to further rule out induction of early
ectoderm. Finally, GATA4 and HNF3b were expressed in both
definitive and extra-embryonic endoderm, and thus, correlate with
SOX17 expression in definitive endoderm (Table 1). A representative
experiment is shown in FIGS. 11-14 which demonstrates how the
marker genes described in Table 1 correlate with each other among
the various samples, thus highlighting specific patterns of
differentiation to definitive endoderm and extra-embryonic endoderm
as well as to mesodermal and neural cell types.
[0209] 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
[0210] Human ES cell cultures will randomly differentiate if they
are 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 traditionally 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 no
good antibody reagents for definitive endoderm cells have been
available, 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.
[0211] As undifferentiated hESC colonies expand on a bed of
fibroblast feeders 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.
[0212] 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 ESC
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 ESC morphology as the colony expanded in size
and became more crowded. Younger, smaller fully undifferentiated
colonies (<1 mm; 4-5 days old) showed no SOX17 positive cells
within or at the edges of the colonies while older, larger colonies
(1-2 mm diameter, >5 days old) had sporadic isolated patches of
SOX17 positive, AFP negative cells at the periphery of some
colonies or in regions interior to the edge that were
differentiated away from 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" ESC cultures have never been
previously demonstrated.
[0213] Based on negative correlations of SOX17 and SPARC gene
expression levels by Q-PCR, the vast majority of these SOX17
positive, AFP negative cells will be negative for parietal 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 to 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 which 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 ESC
cultures (FIGS. 16A-D and 17).
[0214] With the SOX17 antibody and Q-PCR tools described above we
have explored a number of procedures capable of efficiently
programming ESCs to become SOX17.sup.hi/AFP.sup.lo/SPARC/TM.sup.lo
definitive endoderm cells. We applied a variety of differentiation
protocols aimed at increasing the number and proliferative capacity
of these cells as measured at the population level by Q-PCR for
SOX17 gene expression and at the level of individual cells by
antibody labeling of SOX17 protein.
[0215] 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.
[0216] 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.
[0217] 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.
[0218] 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.
[0219] To determine the effect of TGF.beta. factor treatment at the
individual cell level, a time course of TGF.beta. factor addition
was examined using SOX17 antibody labeling. As previously shown in
FIGS. 10A-F, there was a dramatic increase in the relative number
of SOX17 labeled cells over time. The relative quantification (FIG.
20) shows more than a 20-fold increase in SOX17-labeled cells. This
result indicates that both the numbers of cells as well SOX17 gene
expression level are increasing with time of TGF.beta. factor
exposure. As shown in FIG. 21, after four days of exposure to
Nodal, Activin A, Activin B and BMP4, the level of SOX17 induction
reached 168-fold over undifferentiated hESCs. FIG. 22 shows that
the relative number of SOX17-positive cells was also dose
responsive. Activin A doses of 100 ng/mL or more were capable of
potently inducing SOX17 gene expression and cell number.
[0220] 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 indicted
by the increased SOX17 gene expression in samples that were treated
with activins plus Wnt3a over that of activins alone (FIG. 23).
[0221] All of the experiments described above were performed using
tissue culture medium containing 10% serum with added factors.
Surprisingly, we discovered that the concentration of serum had an
effect on the level of SOX17 expression in the presence of added
activins as shown in FIGS. 24A-C. When serum levels were reduced
from 10% to 2%, SOX17 expression tripled in the presence of
Activins A and B.
[0222] Finally, we demonstrated that activin induced SOX17.sup.+
cells divide in culture as depicted in FIGS. 25A-D. The arrows show
cells labeled with SOX17/PCNA/DAPI that are in mitosis as evidenced
by the PCNA/DAPI-labeled mitotic plate pattern and the phase
contrast mitotic profile.
Example 7
Chemokine Receptor 4 (CXCR4) Expression Correlates with Markers for
Definitive Endoderm and not Markers for Mesoderm, Ectoderm or
Visceral Endoderm
[0223] As described above, ESCs 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 ESCs.
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 ESCs to the definitive endoderm
lineage is very inefficient and the FBS concentration has much
milder effects on the differentiation process of ESCs.
[0224] In these experiments, hESCs were differentiated by growing
in RPMI medium (Invitrogen, Carlsbad, Calif.; cat# 61870-036)
supplemented with 0.5%, 2.0% or 10% FBS and either with or without
100 ng/mL activin A for 6 days. In addition, a gradient of FBS
ranging from 0.5% to 2.0% over the first three days of
differentiation was also used in conjunction with 100 ng/mL of
activin A. After the 6 days, replicate samples were collected from
each culture condition and analyzed for relative gene expression by
real-time quantitative PCR. The remaining cells were fixed for
immunofluorescent detection of SOX17 protein.
[0225] The expression levels of CXCR4 varied dramatically across
the 7 culture conditions used (FIG. 26). In general, CXCR4
expression was high in activin A treated cultures (A100) and low in
those which did not receive exogenous activin A (NF). In addition,
among the A100 treated cultures, CXCR4 expression was highest when
FBS concentration was lowest. There was a remarkable decrease in
CXCR4 level in the 10% FBS condition such that the relative
expression was more in line with the conditions that did not
receive activin A (NF).
[0226] As described above, expression of the SOX17, GSC, MIXL1, and
HNF3.beta. genes is consistent with the characterization of a cell
as definitive endoderm. The relative expression of these four genes
across the 7 differentiation conditions mirrors that of CXCR4
(FIGS. 27A-D). This demonstrates that CXCR4 is also a marker of
definitive endoderm.
[0227] Ectoderm and mesoderm lineages can be distinguished from
definitive endoderm by their expression of various markers. Early
mesoderm expresses the genes Brachyury and MOX1 while nascent
neuro-ectoderm expresses SOX1 and ZIC1. FIGS. 28A-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 ESCs at this developmental time
period.
[0228] 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.
[0229] The relative number of SOX17 immunoreactive (SOX17.sup.+)
cells present in each of the differentiation conditions mentioned
above was also determined. When hESCs were differentiated in the
presence of high dose activin A and low FBS concentration (0.5%
-2.0%) SOX17.sup.+ cells were ubiquitously distributed throughout
the culture. When high dose activin A was used but FBS was included
at 10% (v/v), the SOX17.sup.+ cells appeared at much lower
frequency and always appeared in isolated clusters rather than
evenly distributed throughout the culture (FIGS. 29A and C as well
as B and E). A further decrease in SOX17.sup.+ cells was seen when
no exogenous activin A was used. Under these conditions the
SOX17.sup.+ cells also appeared in clusters and these clusters were
smaller and much more rare than those found in the high activin A,
low FBS treatment (FIGS. 29 C and 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
[0230] The dose of activin A also effects the efficiency at which
definitive endoderm can be derived from ESCs. This example
demonstrates that increasing the dose of activin A increases the
proportion of CXCR4.sup.+ cells in the culture.
[0231] 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 um 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 uL of mouse anti-CXCR4 antibody (Abeam,
cat# ab10403-100) was added per 50 uL (containing approximately
10.sup.5 cells) and labeling proceeded for 45 minutes on ice. Cells
were washed by adding 5 mL of PBS containing 2% human serum
(buffer) and pelleted. A second wash with 5 mL of buffer was
completed then cells were resuspended in 50 uL buffer per 10.sup.5
cells. Secondary antibody (FITC conjugated donkey anti-mouse;
Jackson ImmunoResearch, cat# 715-096-151) was added at 5 ug/mL
final concentration and allowed to label for 30 minutes followed by
two washes in buffer as above. Cells were resuspended at
5.times.10.sup.6 cells/mL in buffer and analyzed and sorted using a
FACS Vantage (Beckton Dickenson) by the staff at the flow cytometry
core facility (The Scripps Research Institute). Cells were
collected directly into RLT lysis buffer (Qiagen) for subsequent
isolation of total RNA for gene expression analysis by real-time
quantitative PCR.
[0232] The number of CXCR4.sup.+ cells as determined by flow
cytometry were observed to increase dramatically as the dose of
activin A was increased in the differentiation culture media (FIGS.
30A-C). The CXCR4.sup.+ cells were those falling within the R4 gate
and this gate was set using a secondary antibody-only control for
which 0.2% of events were located in the R4 gate. The dramatically
increased numbers of CXCR4.sup.+ cells correlates with a robust
increase in definitive endoderm gene expression as activin A dose
is increased (FIGS. 31A-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
[0233] The CXCR4.sup.+ and CXCR4.sup.- cells identified in Example
8 above were collected and analyzed for relative gene expression
and the gene expression of the parent populations was determined
simultaneously.
[0234] The relative levels of CXCR4 gene expression was
dramatically increased with increasing dose of activin A (FIG. 32).
This correlated very well with the activin A dose-dependent
increase of CXCR4.sup.+ cells (FIGS. 30A-C). It is also clear that
isolation of the CXCR4.sup.+ cells from each population accounted
for nearly all of the CXCR4 gene expression in that population.
This demonstrates the efficiency of the FACS method for collecting
these cells.
[0235] Gene expression analysis revealed that the CXCR4.sup.+ cells
contain not only the majority of the CXCR4 gene expression, but
they also contained other gene expression for markers of definitive
endoderm. As shown in FIGS. 31A-D, the CXCR4.sup.+ cells were
further enriched over the parent A100 population for SOX17, GSC,
HNF3B, and MIXL1. In addition, the CXCR4.sup.- fraction contained
very little gene expression for these definitive endoderm markers.
Moreover, the CXCR4.sup.+ and CXCR4.sup.- populations displayed the
inverse pattern of gene expression for markers of mesoderm,
ectoderm and extra-embryonic endoderm. FIGS. 33A-D shows that the
CXCR4.sup.+ cells were depleted for gene expression of Brachyury,
MOX1, ZIC1, and SOX7 relative to the A100 parent population. This
A100 parent population was already low in expression of these
markers relative to the low dose or no activin A conditions. These
results show that the isolation of CXCR4.sup.+ cells from hESCs
differentiated in the presence of high activin A yields a
population that is highly enriched for and substantially pure
definitive endoderm.
Example 10
Quantitation of Definitive Endoderm Cells in a Cell Population
Using CXCR4
[0236] 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.
[0237] Using the methods such as those described in the above
Examples, hESCs were differentiated to produce definitive endoderm.
In particular, increase yield and purity expressed in
differentiating cell cultures, the serum concentration of the
medium was controlled as follows: 0.2% FBS on day 1, 1.0% FBS on
day 2 and 2.0% FBS on days 3-6. Differentiated cultures were sorted
by FACS using three cell surface epitopes, E-Cadherin, CXCR4, and
Thrombomodulin. Sorted cell populations were then analyzed by Q-PCR
to determine relative expression levels of markers for definitive
and extraembryonic-endoderm as well as other cell types. CXCR4
sorted cells taken from optimally differentiated cultures resulted
in the isolation of definitive endoderm cells that were >98%
pure.
[0238] 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 endododerm cells SOX17 70-80 100
Thrombomodulin <2 0 75 AFP <1 0 25 CXCR4 70-80 100 0 ECAD 10
0 100 other (ECAD neg.) 10-20 Total 100 100 100 100
[0239] 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.
[0240] 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
.ltoreq.0.5% throughout the 5-6 day differentiation procedure.
However, maintaining the cell culture at .ltoreq.0.5% throughout
the 5-6 day differentiation procedure also results in a reduced
number of total definitive endoderm cells that are produced.
[0241] Definitive endoderm cells produced by methods described
herein have been maintained and expanded in culture in the presence
of activin for greater than 50 days without appreciable
differentiation. In such cases, SOX17, CXCR4, MIXL1, GATA4,
HNF3.beta. 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
[0242] 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.
[0243] 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).
[0244] 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 uL of phycoerythrin-conjugated antibody to 5.times.10.sup.6
cells in 800 uL human serum. Cells were washed twice with 8 mL of
PBS buffer and resuspended in 1 mL of the same. FACS isolation was
carried out by the core facility of The Scripps Research Institute
using a FACS Vantage (BD Biosciences). Cells were collected
directly into RLT lysis buffer and RNA was isolated by RNeasy
according to the manufacturers instructions (Qiagen).
[0245] Purified RNA was submitted in duplicate to Expression
Analysis (Durham, N.C.) for generation of the expression profile
data using the Affymetrix platform and U133 Plus 2.0 high-density
oligonucleotide arrays. Data presented is a group comparison that
identifies genes differentially expressed between the two
populations, hESCs and definitive endoderm. Genes that exhibited a
robust upward change in expression level over that found in hESCs
were selected as new candidate markers that are highly
characteristic of definitive endoderm. Select genes were assayed by
Q-PCR, as described above, to verify the gene expression changes
found on the gene chip and also to investigate the expression
pattern of these genes during a time course of hESC
differentiation.
[0246] 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 human embryonic
stem cells (HESC). A comparison of FIGS. 34C and G-M demonstrates
that the six marker genes, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and
CRIP1, exhibit an expression pattern that is almost identical to
each other and which is also identical to the pattern of expression
of CXCR4 and SOX17/SOX7. As described previously, SOX17 is
expressed in both the definitive endoderm as well as in the
SOX7-expressing extra-embryonic endoderm. Since SOX7 is not
expressed in the definitive endoderm, the ratio of SOX17/SOX7
provides a reliable estimate of definitive endoderm contribution to
the SOX17 expression witnessed in the population as a whole. The
similarity of panels G-L and M to panel C indicates that FGF17,
VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 are likely markers of
definitive endoderm and that they are not significantly expressed
in extra-embryonic endoderm cells.
[0247] It will be appreciated that the Q-PCR results described
herein can be further confirmed by ICC.
[0248] 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.
[0249] 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
[0250] Numerous literature and patent references have been cited in
the present patent application. Each and every reference that cited
in this patent application is incorporated by reference herein in
its entirety.
[0251] 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:
[0252] 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.
[0253] Alexander, J., and Stainier, D. Y. (1999). A molecular
pathway leading to endoderm formation in zebrafish. Curr Biol 9,
1147-1157.
[0254] 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.
[0255] 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.
[0256] Beddington, R. S., Rashbass, P., and Wilson, V. (1992).
Brachyury--a gene affecting mouse gastrulation and early
organogenesis. Dev Suppl, 157-165.
[0257] 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.
[0258] 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.
[0259] 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.
[0260] 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.
[0261] 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.
[0262] 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.
[0263] 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.
[0264] Grapin-Botton, A., and Melton, D. A. (2000). Endoderm
development: from patterning to organogenesis. Trends Genet 16,
124-130.
[0265] Harris, T. M., and Childs, G. (2002). Global gene expression
patterns during differentiation of F9 embryonal carcinoma cells
into parietal endoderm. Funct Integr Genomics 2, 105-119.
[0266] Hogan, B. L. (1996). Bone morphogenetic proteins in
development. Curr Opin Genet Dev 6, 432-438.
[0267] Hogan, B. L. (1997). Pluripotent embryonic cells and methods
of making same (U.S.A., Vanderbilt University).
[0268] Howe, C. C., Overton, G. C., Sawicki, J., Softer, D., Stein,
P., and Strickland, S. (1988). Expression of SPARC/osteonectin
transcript in murine embryos and gonads. Differentiation 37,
20-25.
[0269] Hudson, C., Clements, D., Friday, R. V., Stott, D., and
Woodland, H. R. (1997). Xsox17alpha and -beta mediate endoderm
formation in Xenopus. Cell 91, 397-405.
[0270] 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.
[0271] 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
Sox17-null mutant mice. Development 129, 2367-2379.
[0272] Katoh, M. (2002). Expression of human SOX7 in normal tissues
and tumors. Int J Mol Med 9, 363-368.
[0273] 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.
[0274] 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.
[0275] Kimelman, D., and Griffin, K. J. (2000). Vertebrate
mesendoderm induction and patterning. Curr Opin Genet Dev 10,
350-356.
[0276] Kubo A, Shinozaki K, Shannon J M, 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.
[0277] 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.
[0278] 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.
[0279] 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.
[0280] 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.
[0281] 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.
[0282] 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.
[0283] McGrath K E, Koniski A D, Maltby K M, McGann J K, Palis J.
(1999) Embryonic expression and function of the chemokine SDF-1 and
its receptor, CXCR4. Dev Biol. 213, 442-56.
[0284] Miyazono, K., Kusanagi, K., and Inoue, H. (2001). Divergence
and convergence of TGF-beta/BMP signaling. J Cell Physiol 187,
265-276.
[0285] 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.
[0286] Niwa, H. (2001). Molecular mechanism to maintain stem cell
renewal of ES cells. Cell Struct Funct 26, 137-148.
[0287] Ogura, H., Aruga, J., and Mikoshiba, K. (2001). Behavioral
abnormalities of Zic1 and Zic2 mutant mice: implications as models
for human neurological disorders. Behav Genet 31, 317-324.
[0288] 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.
[0289] Rodaway, A., and Patient, R. (2001). Mesendoderm. an ancient
germ layer? Cell 105, 169-172.
[0290] 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.
[0291] Rohr, K. B., Schulte-Merker, S., and Tautz, D. (1999).
Zebrafish zic1 expression in brain and somites is affected by BMP
and hedgehog signalling. Mech Dev 85, 147-159.
[0292] Schier, A. F. (2003). Nodal signaling in vertebrate
development. Annu Rev Cell Dev Biol 19, 589-621.
[0293] Schoenwolf, G. C., and Smith, J. L. (2000). Gastrulation and
early mesodermal patterning in vertebrates. Methods Mol Biol 135,
113-125.
[0294] 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
USA 95, 13726-13731.
[0295] 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 1
diabetes mellitus using a glucocorticoid-free immunosuppressive
regimen. N Engl J Med 343, 230-238.
[0296] 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.
[0297] 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 1 diabetes? Insulin
independence should be dominant force in islet transplantation. Bmj
322, 861.
[0298] 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.
[0299] Smith, J. (1997). Brachyury and the T-box genes. Curr Opin
Genet Dev 7, 474-480.
[0300] 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.
[0301] 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.
[0302] 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.
[0303] Technau, U. (2001). Brachyury, the blastopore and the
evolution of the mesoderm. Bioessays 23, 788-794.
[0304] 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.
[0305] 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.
[0306] 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.
[0307] 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.
[0308] 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.
[0309] 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.
[0310] 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.
[0311] Wells, J. M., and Melton, D. A. (1999). Vertebrate endoderm
development. Annu Rev Cell Dev Biol 15, 393-410.
[0312] Wells, J. M., and Melton, D. A. (2000). Early mouse endoderm
is patterned by soluble factors from adjacent germ layers.
Development 127, 1563-1572.
[0313] Willison, K. (1990). The mouse Brachyury gene and mesoderm
formation. Trends Genet 6, 104-105.
[0314] Zhao, G. Q. (2003). Consequences of knocking out BMP
signaling in the mouse. Genesis 35, 43-56.
[0315] 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.
Sequence CWU 1
1
211245DNAHomo sapiens 1atgagcagcc cggatgcggg atacgccagt gacgaccaga
gccagaccca gagcgcgctg 60cccgcggtga tggccgggct gggcccctgc ccctgggccg
agtcgctgag ccccatcggg 120gacatgaagg tgaagggcga ggcgccggcg
aacagcggag caccggccgg ggccgcgggc 180cgagccaagg gcgagtcccg
tatccggcgg ccgatgaacg ctttcatggt gtgggctaag 240gacgagcgca
agcggctggc gcagcagaat ccagacctgc acaacgccga gttgagcaag
300atgctgggca agtcgtggaa ggcgctgacg ctggcggaga agcggccctt
cgtggaggag 360gcagagcggc tgcgcgtgca gcacatgcag gaccacccca
actacaagta ccggccgcgg 420cggcgcaagc aggtgaagcg gctgaagcgg
gtggagggcg gcttcctgca cggcctggct 480gagccgcagg cggccgcgct
gggccccgag ggcggccgcg tggccatgga cggcctgggc 540ctccagttcc
ccgagcaggg cttccccgcc ggcccgccgc tgctgcctcc gcacatgggc
600ggccactacc gcgactgcca gagtctgggc gcgcctccgc tcgacggcta
cccgttgccc 660acgcccgaca cgtccccgct ggacggcgtg gaccccgacc
cggctttctt cgccgccccg 720atgcccgggg actgcccggc ggccggcacc
tacagctacg cgcaggtctc ggactacgct 780ggccccccgg agcctcccgc
cggtcccatg cacccccgac tcggcccaga gcccgcgggt 840ccctcgattc
cgggcctcct ggcgccaccc agcgcccttc acgtgtacta cggcgcgatg
900ggctcgcccg gggcgggcgg cgggcgcggc ttccagatgc agccgcaaca
ccagcaccag 960caccagcacc agcaccaccc cccgggcccc ggacagccgt
cgccccctcc ggaggcactg 1020ccctgccggg acggcacgga ccccagtcag
cccgccgagc tcctcgggga ggtggaccgc 1080acggaatttg aacagtatct
gcacttcgtg tgcaagcctg agatgggcct cccctaccag 1140gggcatgact
ccggtgtgaa tctccccgac agccacgggg ccatttcctc ggtggtgtcc
1200gacgccagct ccgcggtata ttactgcaac tatcctgacg tgtga
12452414PRTHomo sapiens 2Met Ser Ser Pro Asp Ala Gly Tyr Ala Ser
Asp Asp Gln Ser Gln Thr1 5 10 15Gln Ser Ala Leu Pro Ala Val Met Ala
Gly Leu Gly Pro Cys Pro Trp 20 25 30Ala Glu Ser Leu Ser Pro Ile Gly
Asp Met Lys Val Lys Gly Glu Ala 35 40 45Pro Ala Asn Ser Gly Ala Pro
Ala Gly Ala Ala Gly Arg Ala Lys Gly 50 55 60Glu Ser Arg Ile Arg Arg
Pro Met Asn Ala Phe Met Val Trp Ala Lys65 70 75 80Asp Glu Arg Lys
Arg Leu Ala Gln Gln Asn Pro Asp Leu His Asn Ala 85 90 95Glu Leu Ser
Lys Met Leu Gly Lys Ser Trp Lys Ala Leu Thr Leu Ala 100 105 110Glu
Lys Arg Pro Phe Val Glu Glu Ala Glu Arg Leu Arg Val Gln His 115 120
125Met Gln Asp His Pro Asn Tyr Lys Tyr Arg Pro Arg Arg Arg Lys Gln
130 135 140Val Lys Arg Leu Lys Arg Val Glu Gly Gly Phe Leu His Gly
Leu Ala145 150 155 160Glu Pro Gln Ala Ala Ala Leu Gly Pro Glu Gly
Gly Arg Val Ala Met 165 170 175Asp Gly Leu Gly Leu Gln Phe Pro Glu
Gln Gly Phe Pro Ala Gly Pro 180 185 190Pro Leu Leu Pro Pro His Met
Gly Gly His Tyr Arg Asp Cys Gln Ser 195 200 205Leu Gly Ala Pro Pro
Leu Asp Gly Tyr Pro Leu Pro Thr Pro Asp Thr 210 215 220Ser Pro Leu
Asp Gly Val Asp Pro Asp Pro Ala Phe Phe Ala Ala Pro225 230 235
240Met Pro Gly Asp Cys Pro Ala Ala Gly Thr Tyr Ser Tyr Ala Gln Val
245 250 255Ser Asp Tyr Ala Gly Pro Pro Glu Pro Pro Ala Gly Pro Met
His Pro 260 265 270Arg Leu Gly Pro Glu Pro Ala Gly Pro Ser Ile Pro
Gly Leu Leu Ala 275 280 285Pro Pro Ser Ala Leu His Val Tyr Tyr Gly
Ala Met Gly Ser Pro Gly 290 295 300Ala Gly Gly Gly Arg Gly Phe Gln
Met Gln Pro Gln His Gln His Gln305 310 315 320His Gln His Gln His
His Pro Pro Gly Pro Gly Gln Pro Ser Pro Pro 325 330 335Pro Glu Ala
Leu Pro Cys Arg Asp Gly Thr Asp Pro Ser Gln Pro Ala 340 345 350Glu
Leu Leu Gly Glu Val Asp Arg Thr Glu Phe Glu Gln Tyr Leu His 355 360
365Phe Val Cys Lys Pro Glu Met Gly Leu Pro Tyr Gln Gly His Asp Ser
370 375 380Gly Val Asn Leu Pro Asp Ser His Gly Ala Ile Ser Ser Val
Val Ser385 390 395 400Asp Ala Ser Ser Ala Val Tyr Tyr Cys Asn Tyr
Pro Asp Val 405 410
* * * * *