U.S. patent application number 11/115868 was filed with the patent office on 2005-12-01 for pdx1 expressing endoderm.
Invention is credited to Agulnick, Alan D., Baetge, Emmanuel E., D'Amour, Kevin Allen, Eliazer, Susan.
Application Number | 20050266554 11/115868 |
Document ID | / |
Family ID | 35057079 |
Filed Date | 2005-12-01 |
United States Patent
Application |
20050266554 |
Kind Code |
A1 |
D'Amour, Kevin Allen ; et
al. |
December 1, 2005 |
PDX1 expressing endoderm
Abstract
Disclosed herein are cell cultures comprising PDX1-positive
endoderm cells and methods of producing the same. Also disclosed
herein are cell populations comprising substantially purified
PDX1-positive endoderm cells as well as methods for enriching,
isolating and purifying PDX1-positive endoderm cells from other
cell types. Methods of identifying differentiation factors capable
of promoting the differentiation of endoderm cells, such as
PDX1-positive foregut endoderm cells and PDX1-negative definitive
endoderm cells, are also disclosed.
Inventors: |
D'Amour, Kevin Allen; (San
Diego, CA) ; Agulnick, Alan D.; (San Marcos, CA)
; Eliazer, Susan; (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: |
35057079 |
Appl. No.: |
11/115868 |
Filed: |
April 26, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11115868 |
Apr 26, 2005 |
|
|
|
11021618 |
Dec 23, 2004 |
|
|
|
60566293 |
Apr 27, 2004 |
|
|
|
60587942 |
Jul 14, 2004 |
|
|
|
60586566 |
Jul 9, 2004 |
|
|
|
Current U.S.
Class: |
435/366 |
Current CPC
Class: |
C12N 2500/90 20130101;
C12N 2501/119 20130101; C12N 2501/16 20130101; C12N 2510/00
20130101; C12N 2502/13 20130101; A61K 35/12 20130101; C12N 15/1086
20130101; C12N 2501/115 20130101; C12N 2506/02 20130101; C12N
5/0679 20130101; C12N 5/0603 20130101; C12Q 2600/158 20130101; C12N
5/0608 20130101; C12N 2501/415 20130101; C12N 5/0606 20130101; C12N
5/0018 20130101; C12N 2503/00 20130101; C12N 5/0676 20130101; C12N
2502/02 20130101; C12N 2501/155 20130101; C12N 2501/385 20130101;
C12Q 1/6881 20130101 |
Class at
Publication: |
435/366 |
International
Class: |
C12N 005/08 |
Claims
What is claimed is:
1. A cell culture comprising human cells, wherein at least about 2%
of said human cells are pancreatic-duodenal homoebox factor-1
(PDX1) positive foregut endoderm cells, said PDX1-positive foregut
endoderm cells being multipotent cells that can differentiate into
cells, tissues or organs derived from the anterior portion of the
gut tube.
2. The cell culture of claim 1, wherein at least about 5% of said
human cells are PDX1-positive foregut endoderm cells.
3. The cell culture of claim 1, wherein at least about 10% of said
human cells are PDX1-positive foregut endoderm cells.
4. The cell culture of claim 1, wherein at least about 25% of said
human cells are PDX1-positive foregut endoderm cells.
5. The cell culture of claim 1, wherein human feeder cells are
present in said culture, and wherein at least about 2% of human
cells other than said human feeder cells are PDX1-positive foregut
endoderm cells.
6. The cell culture of claim 1, wherein said PDX1-positive foregut
endoderm cells express the homeobox A13 (HOXA13) gene.
7. The cell culture of claim 1, wherein said PDX1-positive foregut
endoderm cells express the homeobox C6 (HOXC6) gene.
8. The cell culture of claim 1, wherein said PDX1-positive foregut
endoderm cells express SOX17.
9. The cell culture of claim 1, wherein the expression of PDX1 is
greater than the expression of a marker selected from the group
consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in
said PDX1-positive foregut endoderm cells.
10. The cell culture of claim 1, wherein said cell culture is
substantially free of cells selected from the group consisting of
visceral endodermal cells, parietal endodermal cells and neural
cells.
11. The cell culture of claim 1, wherein at least about 1
PDX1-positive foregut endoderm cell is present for about every 10
PDX1-negative definitive endoderm cells in said cell culture.
12. The cell culture of claim 1, wherein at least about 1
PDX1-positive foregut endoderm cell is present for about every 5
PDX1-negative definitive endoderm cells in said cell culture.
13. The cell culture of claim 1, wherein at least about 1
PDX1-positive foregut endoderm cell is present for about every 4
PDX1-negative definitive endoderm cells in said cell culture.
14. The cell culture of claim 1 further comprising an embryonic
stem cell.
15. The cell culture of claim 14, 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.
16. The cell culture of claim 1 further comprising a retinoid.
17. The cell culture of claim 16, wherein said retinoid is retinoic
acid (RA).
18. The cell culture of claim 1 further comprising FGF-10.
19. The cell culture of claim 1 further comprising B27.
20. The cell culture of claim 1 further comprising both RA and
FGF-10.
21. The cell culture of claim 20 further comprising B27.
22. A cell population comprising cells wherein at least about 90%
of said cells are human PDX1-positive foregut endoderm cells, said
PDX1-positive foregut endoderm cells being multipotent cells that
can differentiate into cells, tissues or organs derived from the
anterior portion of the gut tube.
23. The cell population of claim 22, wherein at least about 95% of
said cells are PDX1-positive foregut endoderm cells.
24. The cell population of claim 22, wherein at least about 98% of
said cells are PDX1-positive foregut endoderm cells.
25. The cell population of claim 22, wherein said PDX1-positive
foregut endoderm cells express the HOXA13 gene.
26. The cell population of claim 22, wherein said PDX1-positive
foregut endoderm cells express the HOXC6 gene.
27. The cell population of claim 22, wherein said PDX1-positive
foregut endoderm cells express SOX17.
28. The cell population of claim 22, wherein the expression of PDX1
is greater than the expression of a marker selected from the group
consisting of AFP, SOX7, SOX1, ZIC1 and NFM in said PDX1-positive
foregut endoderm cells.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 11/021,618, entitled DEFINITIVE ENDODERM,
filed Dec. 23, 2004, which claims priority under 35 U.S.C. .sctn.
119(e) to the following three provisional patent applications: U.S.
Provisional Patent Application No. 60/566,293, entitled PDX1
EXPRESSING ENDODERM, filed Apr. 27, 2004; U.S. Provisional Patent
Application No. 60/587,942, entitled CHEMOKINE CELL SURFACE
RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 14,
2004; and U.S. Provisional Patent Application No. 60/586,566,
entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF
DEFINITIVE ENDODERM, filed Jul. 9, 2004. The disclosure of each of
the foregoing 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 PDX1-positive endoderm cells and
methods of making, isolating and using such cells.
BACKGROUND
[0003] Human pluripotent stem cells, such as embryonic stem (ES)
cells and embryonic germ (EG) cells, were first isolated in culture
without fibroblast feeders in 1994 (Bongso et al., 1994) and with
fibroblast feeders (Hogan, 1997). Later, Thomson, Reubinoff and
Shamblott established continuous cultures of human ES and EG cells
using mitotically inactivated mouse feeder layers (Reubinoff et
al., 2000; Shamblott et al., 1998; Thomson et al., 1998).
[0004] Human ES and EG cells (hESCs) offer unique opportunities for
investigating early stages of human development as well as for
therapeutic intervention in several disease states, such as
diabetes mellitus and Parkinson's disease. For example, the use of
insulin-producing .beta.-cells derived from hESCs would offer a
vast improvement over current cell therapy procedures that utilize
cells from donor pancreases for the treatment of diabetes. However,
presently it is not known how to generate an insulin-producing
.beta.-cell from hESCs. As such, current cell therapy treatments
for diabetes mellitus, which utilize islet cells from donor
pancreases, are limited by the scarcity of high quality islet cells
needed for transplant. Cell therapy for a single Type I diabetic
patient requires a transplant of approximately 8.times.10.sup.8
pancreatic islet cells. (Shapiro et al., 2000; Shapiro et al.,
2001a; Shapiro et al., 2001b). As such, at least two healthy donor
organs are required to obtain sufficient islet cells for a
successful transplant. Human embryonic stem cells offer a source of
starting material from which to develop substantial quantities of
high quality differentiated cells for human cell therapies.
[0005] Two properties that make hESCs uniquely suited to cell
therapy applications are pluripotence and the ability to maintain
these cells in culture for prolonged periods. Pluripotency is
defined by the ability of hESCs to differentiate to derivatives of
all 3 primary germ layers (endoderm, mesoderm, ectoderm) which, in
turn, form all somatic cell types of the mature organism in
addition to extraembryonic tissues (e.g. placenta) and germ cells.
Although pluripotency imparts extraordinary utility upon hESCs,
this property also poses unique challenges for the study and
manipulation of these cells and their derivatives. Owing to the
large variety of cell types that may arise in differentiating hESC
cultures, the vast majority of cell types are produced at very low
efficiencies. Additionally, success in evaluating production of any
given cell type depends critically on defining appropriate markers.
Achieving efficient, directed differentiation is of great
importance for therapeutic application of hESCs.
[0006] In order to use hESCs as a starting material to generate
cells that are useful in cell therapy applications, it would be
advantageous to overcome the foregoing problems. For example, in
order to achieve the level of cellular material required for islet
cell transplantation therapy, it would be advantageous to
efficiently direct hESCs toward the pancreatic islet/.beta.-cell
lineage at the very earliest stages of differentiation.
[0007] In addition to efficient direction of the differentiation
process, it would also be beneficial to isolate and characterize
intermediate cell types along the differentiation pathway towards
the pancreatic islet/.beta.-cell lineage and to use such cells as
appropriate lineage precursors for further steps in the
differentiation.
SUMMARY OF THE INVENTION
[0008] Embodiments of the present invention relate to compositions
comprising PDX1-expressing (PDX1-positive) endoderm cells as well
as methods for producing the same. Additional embodiments relate to
cell populations enriched in PDX1-positive endoderm and methods for
the production of such cell populations. Other embodiments relate
to methods of increasing the expression of PDX1 in endoderm cells
as well as identifying factors useful for further differentiating
PDX1-negative and/or PDX1-positive endoderm. In some embodiments of
the compositions and methods described throughout this application,
the PDX1-positive endoderm cells are PDX1-positive foregut/midgut
endoderm cells. In certain preferred embodiments of the
compositions and methods described throughout this application, the
PDX1-positive endoderm cells are PDX1-positive foregut endoderm
cells. In other preferred embodiments, the PDX1-positive endoderm
cells are PDX1-positive endoderm cells of the posterior portion of
the foregut.
[0009] Some embodiments of the present invention relate to cell
cultures comprising PDX1-positive foregut endoderm cells, wherein
the PDX1-positive foregut endoderm cells are multipotent cells that
can differentiate into cells, tissues or organs derived from the
anterior portion of the gut tube. In some embodiments, the cell
cultures comprise human cells. In such human cell cultures,
PDX1-positive foregut endoderm can comprise at least about 2%, at
least about 5%, at least about 10% or at least about 25% of the
human cells in the culture. In some embodiments, the at least about
2%, the at least about 5%, the at least about 10% or the at least
about 25% is calculated without respect to any feeder cells present
in said culture. The PDX1-positive foregut endoderm cells in
certain embodiments of the cell cultures described herein can
express a marker selected from the group consisting of the homeobox
A13 (HOXA13) gene, the homeobox C6 (HOXC6) gene and SOX17. In other
embodiments, cell cultures comprising PDX1-positive foregut
endoderm cells are substantially free of visceral endoderm cells,
parietal endoderm cells and/or neural cells. In some embodiments,
the cell cultures further comprise one of more of the following: a
retinoid compound, such as retinoic acid (RA), FGF-10 or B27.
[0010] Additional embodiments of the present invention relate to
enriched, isolated or substantially purified PDX1-positive foregut
endoderm cell populations, wherein the PDX1-positive foregut
endoderm cells are multipotent cells that can differentiate into
cells, tissues or organs derived from the anterior portion of the
gut tube. In some embodiments, the PDX1-positive foregut endoderm
cells are derived from pluripotent cells, such as human embryonic
stem cells. Other embodiments of the present invention, relate to a
cell population which comprises cells, wherein at least about 90%
of the cells are PDX1-positive foregut endoderm cells, and wherein
the PDX1-positive foregut endoderm cells are multipotent cells that
can differentiate into cells, tissues or organs derived from the
anterior portion of the gut tube. In preferred embodiments, the
PDX-1 positive foregut endoderm cells comprise at least about 95%
of the cells in the cell population. In even more preferred
embodiments, the PDX1-positive foregut endoderm cells comprise at
least about 98% of the cells in the cell population.
[0011] Further embodiments described herein relate to methods of
producing PDX1-positive foregut endoderm cells by providing a cell
culture or cell population comprising definitive endoderm cells
which do not substantially express PDX1 (PDX1-negative definitive
endoderm cells) with a foregut differentiation factor, such as a
retinoid. The retinoid, for example RA, can be supplied in a
concentration ranging from about 0.01 .mu.M to about 50 .mu.M. In
some embodiments, the differentiation of PDX1-negative definitive
endoderm to PDX1 positive foregut endoderm is increased by
providing the cell culture or cell population with FGF-10 and/or
B27. FGF-10 can be supplied in a concentration ranging from about 5
ng/ml to about 1000 ng/ml. In some embodiments, B27 is supplied to
the cell culture or cell population at a concentration ranging from
about 0.1% to about 20%. FGF-10 and/or B27 can be added to the cell
culture or cell population at about the same time as the retinoid
or each of the factors may be added separately with up to several
hours between each addition. In certain embodiments, the retinoid
is added to an approximately 4-day-old PDX1-negative definitive
endoderm culture. In some embodiments, the retinoid is added to an
approximately 5-day-old PDX1-negative definitive endoderm
culture.
[0012] Still other embodiments relate to methods of using a foregut
differentiation factor to further increase the production of
PDX1-positive foregut endoderm cells in a cell culture or cell
population that has been contacted with a retinoid, such as RA. In
such embodiments, the differentiation of PDX1-negative definitive
endoderm to PDX1 positive foregut endoderm is increased by
providing the cell culture or cell population with activin A and/or
activin B. Activin A and/or activin B can be supplied in a
concentration ranging from about 5 ng/ml to about 1000 ng/ml. Other
embodiments relate to methods of increasing the production of
PDX1-positive foregut endoderm cells in a cell culture or cell
population by differentiating PDX1-negative cells in a medium
comprising a retinoid, wherein the medium has been previously
conditioned by the maintenance or growth of certain cell types.
Such cell types include, but are not limited to, embryonic stem
cells or other pluripotent cells that have been differentiated in
medium comprising serum or members of the TGF.beta. superfamily of
growth factors, such as activin A, activin B, Nodal and/or bone
morphogenic protein (BMP). In some embodiments, conditioned medium
is supplied to the cell culture or cell population at a
concentration ranging from about 1% to about 100% of the entire
growth medium. TGF.beta. superfamily growth factors and/or
conditioned medium can be added to the cell culture or cell
population at about the same time as the retinoid or each of the
factors may be added separately with up to several hours between
each addition.
[0013] Embodiments of the present invention also relate to methods
of producing a cell population enriched in PDX1-positive foregut
endoderm cells. In certain embodiments, these methods comprise the
step of obtaining a population of pluripotent cells, wherein at
least one cell of the pluripotent cell population comprises at
least one copy of a nucleic acid that is under the control of the
PDX1 promoter. In some embodiments, the nucleic acid comprises a
sequence encoding green fluorescent protein (GFP) or a biologically
active fragment thereof. In other embodiments, additional method
steps include, differentiating the pluripotent cells so as to
produce PDX1-positive foregut endoderm cells, wherein the
PDX1-positive foregut endoderm cells are multipotent cells that can
differentiate into cells, tissues or organs derived from the
anterior portion of the gut tube, and separating PDX1-positive
cells from PDX1-negative cells. In some embodiments of the methods
described herein, the differentiation step further comprises
providing the pluripotent cell population with at least one growth
factor of the TGF.beta. superfamily in an amount sufficient to
promote differentiation of the pluripotent cells to PDX1-negative
definitive endoderm cells, and providing the PDX1-negative
definitive endoderm cells with a foregut differentiation factor in
an amount sufficient to promote differentiation of the
PDX1-negative definitive endoderm cells to PDX1-positive endoderm
cells of the foregut.
[0014] Some embodiments of the present invention relate to a method
of increasing the expression of the PDX1 gene product in
SOX17-expressing (SOX17-positive) definitive endoderm cells by
contacting such cells with a differentiation factor in an amount
that is sufficient to increase the expression of the PDX1 gene
product. In some embodiments, the differentiation factor is
selected from the group consisting of RA, FGF-10 and B27.
[0015] Additional embodiments of the present invention relate to a
method of identifying a differentiation factor capable of promoting
the differentiation of PDX1-negative definitive endoderm cells to
PDX1-positive foregut endoderm cells. In such methods,
PDX1-negative definitive endoderm cells are contacted with a
candidate differentiation factor and it is determined whether PDX1
expression in the cell population after contact with the candidate
differentiation factor has increased as compared to PDX1 expression
in the cell population before contact with the candidate
differentiation factor. An increase in the PDX1 expression in the
cell population indicates that the candidate differentiation factor
is capable of promoting the differentiation of PDX1-negative
definitive endoderm cells to PDX1-positive foregut endoderm cells.
In some embodiments, PDX1 expression is determined by quantitative
polymerase chain reaction (Q-PCR). Some embodiments of the
foregoing method further comprise the step of determining
expression of the HOXA13 and/or the HOXC6 gene in the cell
population before and after contact with the candidate
differentiation factor. In some embodiments, the candidate
differentiation factor is a small molecule, for example, a
retinoid, such as RA. In others, the candidate differentiation
factor is a polypeptide, for example, a growth factor, such as
FGF-10.
[0016] Still other embodiments of the present invention relate to a
method of identifying a differentiation factor capable of promoting
the differentiation of PDX1-positive foregut endoderm cells. In
such methods, PDX1-positive foregut endoderm cells are contacted
with a candidate differentiation factor and it is determined
whether expression of a marker in the population is increased or
decreased after contact with the candidate differentiation factor
as compared to the expression of the same marker in the population
before contact with the candidate differentiation factor. An
increase or decrease in the expression of the marker indicates that
the candidate differentiation factor is capable of promoting the
differentiation of PDX1-positive foregut endoderm cells. In some
embodiments, marker expression is determined by Q-PCR. In some
embodiments, the candidate differentiation factor is a small
molecule, for example, a retinoid, such as RA. In others, the
candidate differentiation factor is a polypeptide, for example, a
growth factor, such as FGF-10.
[0017] In certain jurisdictions, there may not be any generally
accepted definition of the term "comprising." As used herein, the
term "comprising" is intended to represent "open" language which
permits the inclusion of any additional elements. With this in
mind, additional embodiments of the present inventions are
described with reference to the numbered paragraphs below:
[0018] 1. A cell culture comprising human cells wherein at least
about 2% of said human cells are pancreatic-duodenal homoebox
factor-1 (PDX1) positive foregut endoderm cells, said PDX1-positive
foregut endoderm cells being multipotent cells that can
differentiate into cells, tissues or organs derived from the
anterior portion of the gut tube.
[0019] 2. The cell culture of paragraph 1, wherein at least about
5% of said human cells are PDX1-positive foregut endoderm
cells.
[0020] 3. The cell culture of paragraph 1, wherein at least about
10% of said human cells are PDX1-positive foregut endoderm
cells.
[0021] 4. The cell culture of paragraph 1, wherein at least about
25% of said human cells are PDX1-positive foregut endoderm
cells.
[0022] 5. The cell culture of paragraph 1, wherein human feeder
cells are present in said culture, and wherein at least about 2% of
human cells other than said human feeder cells are PDX1-positive
foregut endoderm cells.
[0023] 6. The cell culture of paragraph 1, wherein said
PDX1-positive foregut endoderm cells express the homeobox A13
(HOXA13) gene.
[0024] 7. The cell culture of paragraph 1, wherein said
PDX1-positive foregut endoderm cells express the homeobox C6
(HOXC6) gene.
[0025] 8. The cell culture of paragraph 1, wherein said
PDX1-positive foregut endoderm cells express SOX17.
[0026] 9. The cell culture of paragraph 1, wherein the expression
of PDX1 is greater than the expression of a marker selected from
the group consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1
and NFM in said PDX1-positive foregut endoderm cells.
[0027] 10. The cell culture of paragraph 1, wherein said cell
culture is substantially free of cells selected from the group
consisting of visceral endodermal cells, parietal endodermal cells
and neural cells.
[0028] 11. The cell culture of paragraph 1, wherein at least about
1 PDX1-positive foregut endoderm cell is present for about every 10
PDX1-negative definitive endoderm cells in said cell culture.
[0029] 12. The cell culture of paragraph 1, wherein at least about
1 PDX1-positive foregut endoderm cell is present for about every 5
PDX1-negative definitive endoderm cells in said cell culture.
[0030] 13. The cell culture of paragraph 1, wherein at least about
1 PDX1-positive foregut endoderm cell is present for about every 4
PDX1-negative definitive endoderm cells in said cell culture.
[0031] 14. The cell culture of paragraph 1 further comprising an
embryonic stem cell.
[0032] 15. The cell culture of paragraph 14, 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] 16. The cell culture of paragraph 1 further comprising a
retinoid.
[0034] 17. The cell culture of paragraph 16, wherein said retinoid
is retinoic acid (RA).
[0035] 18. The cell culture of paragraph 1 further comprising
FGF-10.
[0036] 19. The cell culture of paragraph 1 further comprising
B27.
[0037] 20. The cell culture of paragraph 1 further comprising both
RA and FGF-10.
[0038] 21. The cell culture of paragraph 20 further comprising
B27.
[0039] 22. A cell population comprising cells wherein at least
about 90% of said cells are human PDX1-positive foregut endoderm
cells, said PDX1-positive foregut endoderm cells being multipotent
cells that can differentiate into cells, tissues or organs derived
from the anterior portion of the gut tube.
[0040] 23. The cell population of paragraph 22, wherein at least
about 95% of said cells are PDX1-positive foregut endoderm
cells.
[0041] 24. The cell population of paragraph 22, wherein at least
about 98% of said cells are PDX1-positive foregut endoderm
cells.
[0042] 25. The cell population of paragraph 22, wherein said
PDX1-positive foregut endoderm cells express the HOXA13 gene.
[0043] 26. The cell population of paragraph 22, wherein said
PDX1-positive fore gut endoderm cells express the HOXC6 gene.
[0044] 27. The cell population of paragraph 22, wherein said
PDX1-positive foregut endoderm cells express SOX17.
[0045] 28. The cell population of paragraph 22, wherein the
expression of PDX1 is greater than the expression of a marker
selected from the group consisting of AFP, SOX7, SOX1, ZIC1 and NFM
in said PDX1-positive foregut endoderm cells.
[0046] 29. A method of producing PDX1-positive foregut endoderm
cells, said method comprising the steps of obtaining a cell
population comprising PDX I-negative definitive endoderm cells and
providing said cell population with a retinoid in an amount
sufficient to promote differentiation of at least a portion of said
PDX1-negative definitive endoderm cells to PDX1-positive foregut
endoderm cells, wherein said PDX1-positive foregut endoderm cells
are multipotent cells that can differentiate into cells, tissues or
organs derived from the anterior portion of the gut tube.
[0047] 30. The method of paragraph 29 further comprising the step
of allowing sufficient time for PDX1-positive foregut endoderm
cells to form, wherein said sufficient time for PDX1-positive
foregut endoderm cells to form has been determined by detecting the
presence of PDX1-positive foregut endoderm cells in said cell
population.
[0048] 31. The method of paragraph 29, wherein at least about 2% of
said PDX1-negative definitive endoderm cells differentiate into
PDX1-positive foregut endoderm cells.
[0049] 32. The method of paragraph 29, wherein at least about 5% of
said PDX1-negative definitive endoderm cells differentiate into
PDX1-positive foregut endoderm cells.
[0050] 33. The method of paragraph 29, wherein at least about 10%
of said PDX1-negative definitive endoderm cells differentiate into
PDX1-positive foregut endoderm cells.
[0051] 34. The method of paragraph 29, wherein at least about 25%
of said PDX1-negative definitive endoderm cells differentiate into
PDX1-positive foregut endoderm cells.
[0052] 35. The method of paragraph 29, wherein detecting the
presence of PDX1-positive foregut endoderm cells in said cell
population comprises detecting the expression of PDX1.
[0053] 36. The method of paragraph 35, wherein the expression of
PDX1 is greater than the expression of a marker selected from the
group consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and
NFM in said PDX1-positive foregut endoderm cells.
[0054] 37. The method of paragraph 35, wherein the expression of
said PDX1 is determined by quantitative polymerase chain reaction
(Q-PCR).
[0055] 38. The method of paragraph 35, wherein the expression of
PDX1 is determined by immunocytochemistry.
[0056] 39. The method of paragraph 29, wherein said retinoid is
RA.
[0057] 40. The method of paragraph 39, wherein RA is provided in a
concentration ranging from about 0.01 .mu.M to about 50 .mu.M.
[0058] 41. The method of paragraph 39, wherein RA is provided in a
concentration ranging from about 0.04 .mu.M to about 20 .mu.M.
[0059] 42. The method of paragraph 39, wherein RA is provided in a
concentration ranging from about 0.1 .mu.M to about 10 .mu.M.
[0060] 43. The method of paragraph 39, wherein RA is provided in a
concentration ranging from about 0.2 .mu.M to about 2.5 .mu.M.
[0061] 44. The method of paragraph 39, wherein RA is provided in a
concentration ranging from about 0.5 .mu.M to about 1.5 .mu.M.
[0062] 45. The method of paragraph 39, wherein RA is provided in a
concentration of about 1 .mu.M.
[0063] 46. The method of paragraph 39, wherein RA is provided when
said culture is about 4-days-old.
[0064] 47. The method of paragraph 29 further comprising providing
to said culture a factor selected from the group consisting of
FGF-10, FGF-4, activin A, activin B, B27, conditioned medium, and
combinations of said factors in an amount sufficient to enhance the
production of PDX1-positive foregut endoderm cells.
[0065] 48. The method of paragraph 47, wherein said factor is
selected from the group consisting of FGF-10, FGF-4, activin A and
activin B.
[0066] 49. The method of paragraph 48, wherein said factor is
provided in a concentration ranging from about 10 ng/ml to about
500 ng/ml.
[0067] 50. The method of paragraph 48, wherein said factor is
provided in a concentration ranging from about 20 ng/ml to about
200 ng/ml.
[0068] 51. The method of paragraph 48, wherein said factor is
provided in a concentration ranging from about 25 ng/ml to about 75
ng/ml.
[0069] 52. The method of paragraph 48, wherein said factor is
provided in a concentration about 50 ng/ml.
[0070] 53. The method of paragraph 48, wherein said factor is
provided at approximately the same time as said retinoid.
[0071] 54. The method of paragraph 47 wherein said factor is
B27.
[0072] 55. The method of paragraph 54, wherein B27 is provided in a
concentration ranging from about 0.1% to about 20% of the total
medium.
[0073] 56. The method of paragraph 54, wherein B27 is provided in a
concentration ranging from about 0.2% to about 5% of the total
medium.
[0074] 57. The method of paragraph 54, wherein B27 is provided in a
concentration ranging from 0.5% to about 2% of the total
medium.
[0075] 58. The method of paragraph 54, wherein B27 is provided in a
concentration of about 1% of the total medium.
[0076] 59. The method of paragraph 54, wherein B27 is provided at
approximately the same time as said retinoid.
[0077] 60. The method of paragraph 47 wherein said factor is
conditioned medium.
[0078] 61. The method of paragraph 60, wherein conditioned medium
is provided in a concentration ranging from about 10% to about 100%
of the total medium.
[0079] 62. The method of paragraph 60, wherein conditioned medium
is provided in a concentration ranging from about 20% to about 80%
of the total medium.
[0080] 63. The method of paragraph 60, wherein conditioned medium
is provided in a concentration ranging from about 40% to about 60%
of the total medium.
[0081] 64. The method of paragraph 60, wherein conditioned medium
is provided in a concentration of about 50% of the total
medium.
[0082] 65. The method of paragraph 60, wherein conditioned medium
is provided at approximately the same time as said retinoid.
[0083] 66. The method of paragraph 60, wherein conditioned medium
is prepared by contacting differentiated human embryonic stem cells
(hESCs) with a cell culture medium for about 24 hours.
[0084] 67. The method of paragraph 66, wherein said hESCs are
differentiated for about 5 days in a cell culture medium selected
from the group consisting of RPMI supplemented with 3% serum, low
serum RPMI supplemented with activin A and low serum RPMI
supplemented with BMP4.
[0085] 68. A PDX1-positive foregut endoderm cell produced by the
method of paragraph 29.
[0086] 69. A method of producing a cell population enriched in
PDX1-positive foregut endoderm cells, said method comprising the
steps of obtaining a population of pluripotent cells, wherein at
least one cell of said pluripotent cell population comprises at
least one copy of a nucleic acid under the control of the PDX1
promoter, said nucleic acid comprising a sequence encoding green
fluorescent protein (GFP) or a biologically active fragment
thereof, differentiating said pluripotent cells so as to produce
PDX1-positive foregut endoderm cells, said PDX1-positive foregut
endoderm cells being multipotent cells that can differentiate into
cells, tissues or organs derived from the anterior portion of the
gut tube and separating said PDX1-positive foregut endoderm cells
from PDX1-negative cells.
[0087] 70. The method of paragraph 69, wherein said enriched cell
population comprises at least about 95% PDX1-positive foregut
endoderm cells.
[0088] 71. The method of paragraph 69, wherein said enriched cell
population comprises at least about 98% PDX1-positive foregut
endoderm cells.
[0089] 72. The method of paragraph 69, wherein the differentiating
step further comprises, providing said pluripotent cell population
with at least one growth factor of the TGF.beta. superfamily in an
amount sufficient to promote differentiation of said pluripotent
cells to PDX1-negative definitive endoderm cells, and providing
said PDX1-negative definitive endoderm cells with a retinoid in an
amount sufficient to promote differentiation of said PDX1-negative
definitive endoderm cells to PDX1-positive foregut endoderm
cells.
[0090] 73. The method of paragraph 72, wherein said retinoid is
RA.
[0091] 74. An enriched population of PDX1-positive foregut endoderm
cells produced by the method of paragraph 69.
[0092] 75. A method of increasing the expression of the PDX1 gene
product in a SOX17 expressing definitive endoderm cell, said method
comprising contacting said definitive endoderm cell with a
differentiation factor in an amount sufficient to increase
expression of the PDX1 gene product.
[0093] 76. The method of paragraph 75, wherein said differentiation
factor is a retinoid.
[0094] 77. The method of paragraph 76, wherein said differentiation
factor is RA.
[0095] 78. The method of paragraph 75, wherein said differentiation
factor is selected from the group consisting of FGF-10, FGF-4,
activin A, activin B, B27, conditioned medium and combinations of
said factors.
[0096] 79. A method of identifying a differentiation factor capable
of promoting the differentiation of PDX1-negative definitive
endoderm cells to PDX1-positive foregut endoderm cells, said method
comprising the steps of obtaining a population comprising
PDX1-negative definitive endoderm cells, contacting said population
comprising PDX1-negative definitive endoderm cells with a candidate
differentiation factor and determining if PDX1 expression in said
cell population after contact with said candidate differentiation
factor has increased as compared to PDX1 expression in said cell
population before contact with said candidate differentiation
factor, wherein an increase in said PDX1 expression in said cell
population indicates that said candidate differentiation factor is
capable of promoting the differentiation of PDX1-negative
definitive endoderm cells to PDX1-positive foregut endoderm cells,
said PDX1-positive foregut endoderm cells being multipotent cells
that can differentiate into cells, tissues or organs derived from
the anterior portion of the gut tube.
[0097] 80. The method of paragraph 79, wherein said PDX1 expression
is determined by Q-PCR.
[0098] 81. The method of paragraph 79 further comprising the step
of determining the expression of the HOXA13 gene in said cell
population before and after contact with said candidate
differentiation factor.
[0099] 82. The method of paragraph 79 further comprising the step
of determining the expression of the HOXC6 gene in said cell
population before and after contact with said candidate
differentiation factor.
[0100] 83. The method of paragraph 79, wherein said candidate
differentiation factor is a small molecule.
[0101] 84. The method of paragraph 83, wherein said small molecule
is a retinoid.
[0102] 85. The method of paragraph 84, wherein said retinoid is
RA.
[0103] 86. The method of paragraph 79, wherein said candidate
differentiation factor is a polypeptide.
[0104] 87. The method of paragraph 79, wherein said candidate
differentiation factor is a growth factor.
[0105] 88. The method of paragraph 79, wherein said candidate
differentiation factor is FGF-10.
[0106] 89. A method of identifying a differentiation factor capable
of promoting the differentiation of PDX1-positive foregut endoderm
cells, said method comprising the steps of obtaining a population
comprising PDX1-positive foregut endoderm cells, contacting said
population comprising PDX1-positive foregut endoderm cells with a
candidate differentiation factor and determining if expression of a
marker in said population is increased or decreased after contact
with said candidate differentiation factor, as compared to
expression of the same marker in said population before contact
with said candidate differentiation factor, wherein an increase or
decrease in expression of said marker in said population indicates
that said candidate differentiation factor is capable of promoting
the differentiation of PDX1-positive foregut endoderm cells.
[0107] 90. The method of paragraph 81, wherein said marker
expression is determined by Q-PCR.
[0108] 91. The method of paragraph 81, wherein said candidate
differentiation factor is a small molecule.
[0109] 92. The method of paragraph 81, wherein said candidate
differentiation factor is a polypeptide.
[0110] 93. The method of paragraph 81, wherein said candidate
differentiation factor is a growth factor.
[0111] 94. A vector comprising a reporter gene operably linked to a
PDX1 control region.
[0112] 95. The vector of paragraph 94, wherein said reporter gene
is EGFP.
[0113] 96. A cell comprising the vector of paragraph 94.
[0114] 97. A cell comprising a reporter gene operably linked to a
PDX1 control region.
[0115] 98. The cell of paragraph 97, wherein said reporter gene
operably linked to said PDX1 control region is integrated into a
chromosome.
[0116] 99. The cell of paragraph 97, wherein said reporter gene is
EGFP.
[0117] 100. The cell of paragraph 97, wherein said cell is
pluripotent.
[0118] 101. The cell of paragraph 100, wherein said cell is a
hESC.
[0119] 102. The cell of paragraph 97, wherein said cell is a
definitive endoderm cell.
[0120] 103. The cell of paragraph 97, wherein said cell is a
PDX1-positive foregut endoderm cell.
[0121] 104. A conditioned medium prepared by the steps of
contacting fresh cell culture medium with a population of
differentiated hESCs for about 24 hours, wherein said hESCs have
been differentiated for about 5 days in a cell culture medium
selected from the group consisting of RPMI supplemented with 3%
serum, low serum RPMI supplemented with activin A and low serum
RPMI supplemented with BMP4 and removing said population of
differentiated hESCs from the medium.
[0122] 105. The conditioned medium of paragraph 104, wherein said
fresh cell culture medium is RPMI.
[0123] 106. The conditioned medium of paragraph 105, wherein said
RPMI is low serum RPMI.
[0124] 107. A method for conditioning medium, said method
comprising the steps of contacting fresh cell culture medium with a
population of differentiated hESCs for about 24 hours, wherein said
hESCs have been differentiated for about 5 days in a cell culture
medium selected from the group consisting of RPMI supplemented with
3% serum, low serum RPMI supplemented with activin A and low serum
RPMI supplemented with BMP4 and removing said population of
differentiated hESCs from the medium.
[0125] 108. The method of paragraph 107, wherein said fresh cell
culture medium is RPMI.
[0126] 109. The method of paragraph 108, wherein said RPMI is low
serum RPMI.
[0127] 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.
[0128] Additional embodiments of the present invention may also be
found in U.S. Provisional Patent Application No. 60/532,004,
entitled DEFINITIVE ENDODERM, filed Dec. 23, 2003; U.S. Provisional
Patent Application No. 60/566,293, entitled PDX1 EXPRESSING
ENDODERM, filed Apr. 27, 2004; U.S. Provisional Patent Application
No. 60/586,566, entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE
ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9, 2004; U.S.
Provisional Patent Application No. 60/587,942, entitled CHEMOKINE
CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM,
filed Jul. 14, 2004; and U.S. patent application Ser. No.
11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, the
disclosures of which are incorporated herein by reference in their
entireties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0129] FIG. 1 is a schematic of a proposed differentiation pathway
for the production of beta-cells from hESCs. The first step in the
pathway commits the ES cell to the definitive endoderm lineage and
also represents the first step prior to further differentiation
events to pancreatic endoderm, endocrine endoderm, or
islet/beta-cells. The second step in the pathway shows the
conversion of SOX17-positive/PDX1-negative definitive endoderm to
PDX1-positive foregut endoderm. Some factors useful for mediating
these transitions are italicized. Relevant markers for defining the
target cells are underlined.
[0130] 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.
[0131] 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.
[0132] 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).
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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).
[0138] 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.
[0139] 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.
[0140] 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.
[0141] 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.+).
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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).
[0148] 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.
[0149] 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.
[0150] FIG. 21 is a bar chart showing induction of SOX17 expression
over time as a result of treatment with combinations of TGF.beta.
factors.
[0151] FIG. 22 is a bar chart showing that activin A induces a
dose-dependent increase in SOX17.sup.+ cell number.
[0152] 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.
[0153] 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.
[0154] 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).
[0155] FIG. 26 is a bar chart showing the relative expression level
of CXCR4 in differentiating hESCs under various media
conditions.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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.
[0160] 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 (A1100).
[0161] FIG. 32 is a bar chart showing gene expression from
CXCR4.sup.+ and CXCR4-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.
[0162] 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.
[0163] 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.
[0164] FIG. 35 is a chart which shows the relative expression of
the PDX1 gene in a culture of hESCs after 4 days and 6 days with
and without activin in the presence of retinoic acid (RA) and
fibroblast growth factor (FGF-10) added on day 4.
[0165] FIGS. 36A-F are charts which show the relative expression of
marker genes in a culture of hESCs after 4 days and 6 days with and
without activin in the presence of retinoic acid (RA) and
fibroblast growth factor (FGF-10) added on day 4. The panels show
the relative levels of expression of the following marker genes:
(A) SOX17; (B) SOX7; (C) AFP; (D) SOX1; (E) ZIC1; and (F) NFM.
[0166] FIGS. 37A-C are charts which show the relative expression of
marker genes in a culture of hESCs after 4 days and 8 days with and
without activin in the presence or absence of combinations of
retinoic acid (RA), fibroblast growth factor (FGF-10) and
fibroblast growth factor (FGF-4) added on day 4. The panels show
the relative levels of expression of the following marker genes:
(A) PDX1; (B) SOX7; and (C)NFM.
[0167] FIGS. 38A-G are charts which show the relative expression of
marker genes in a culture of definitive endoderm cells contacted
with 50 ng/ml FGF-10 in combination with either 1 .mu.M, 0.2 .mu.M
or 0.04 .mu.M retinoic acid (RA) added on day 4. The panels show
the relative levels of expression of the following marker genes:
(A) PDX1; (B) HOXA3; (C) HOXC6; (D) HOXA13; (E) CDX1; (F) SOX1; and
(G) NFM.
[0168] FIGS. 39A-E are charts which show the relative expression of
marker genes in a culture of hESCs after 4 days and 8 days with and
without activin in the presence of combinations of retinoic acid
(RA), fibroblast growth factor (FGF-10) and one of the following:
serum replacement (SR), fetal bovine serum (FBS) or B27. The panels
show the relative levels of expression of the following marker
genes: (A) PDX1; (B) SOX7; (C) AFP; (D) ZIC1; and (E) NFM.
[0169] FIGS. 40A-B are charts which show the relative expression of
marker genes for pancreas (PDX1, HNF6) and liver (HNF6) in a
culture of hESCs after 6 days Oust prior to addition of RA) and at
9 days (three days after exposure to RA). Various conditions were
included to compare the addition of activin B at doses of 10 ng/ml
(a10), 25 ng/ml (a25) or 50 ng/ml (a50) in the presence of either
25 ng/ml (A25) or 50 ng/ml (A50) activin A. The condition without
any activin A or activin B (NF) serves as the negative control for
definitive endoderm and PDX1-positive endoderm production. The
panels show the relative levels of expression of the following
marker genes: (A) PDX1 and (B) HNF6.
[0170] FIGS. 41A-C are charts which show the relative expression of
marker genes in a culture of hESCs with 100 ng/ml (A100), 50 ng/ml
(A50) or without (NF) activin A at 5 days Oust prior to retinoic
acid addition) and at 2, 4, and 6 days after RA exposure (day 7, 9,
and 11, respectively). The percentage label directly under each bar
indicates the FBS dose during days 3-5 of differentiation. Starting
at day 7, cells treated with RA (R) were grown in RPMI medium
comprising 0.5% FBS. The RA concentration was 2 .mu.M on day 7, 1
.mu.M on day 9 and 0.2 .mu.M on day 11. The panels show the
relative levels of expression of the following marker genes: (A)
PDX1; (B) ZIC1; (C)SOX7.
[0171] FIGS. 42A-B are charts which show the relative expression of
marker genes in a culture of hESCs treated first with activin A in
low FBS to induce definitive endoderm (day 5) and then with fresh
(A25R) medium comprising 25 ng/ml activin A and RA or various
conditioned media (MEFCM, CM#2, CM#3 and CM#4) and RA to induce
PDX1-expressing endoderm. Marker expression was determined on days
5, 6, 7, 8 and 9. The panels show the relative levels of expression
of the following marker genes: (A) PDX1; (B) CDX1.
[0172] FIG. 43 is a chart which shows the relative expression of
PDX1 in a culture of hESCs treated first with activin A in low FBS
to induce definitive endoderm and followed by fresh media
comprising activin A and retinoic acid (A25R) or varying amounts of
RA in conditioned media diluted into fresh media. Total volume of
media is 5 ml in all cases.
[0173] FIG. 44 is a Western blot showing PDX1 immunoprecipitated
from RA-treated definitive endoderm cells 3 days (d8) and 4 days
(d9) after the addition of RA and 50 ng/ml activin A.
[0174] FIG. 45 is a summary chart displaying the results of a
fluorescence-activated cell sort (FACs) of PDX1-positive foregut
endoderm cells genetically tagged with a EGFP reporter under
control of the PDX1 promoter.
[0175] FIG. 46 is a chart showing relative PDX1 expression levels
normalized to housekeeping genes for sorted populations of live
cells (Live), EGFP-negative cells (Neg) and EGFP-positive cells
(GFP+).
[0176] FIG. 47 is a chart showing relative PDX1 expression levels
normalized to housekeeping genes for sorted populations of live
cells (Live), EGFP-negative cells (Neg), the half of the
EGFP-positive cell population that has the lowest EGFP signal
intensity (Lo) and the half of the EGFP-positive cell population
that has the highest EGFP signal intensity (Hi).
[0177] FIGS. 48A-E are a charts showing the relative expression
levels normalized to housekeeping genes of five pancreatic endoderm
markers in sorted populations of live cells (Live), EGFP-negative
cells (Neg) and EGFP-positive cells (GFP+). Panels: A--NKX2.2;
B--GLUT2; C--HNF3.beta.; D--KRT19 and E--HNF4.alpha..
[0178] FIG. 49 are a charts showing the relative expression levels
normalized to housekeeping genes of two non-pancreatic endoderm
markers in sorted populations of live cells (Live), EGFP-negative
cells (Neg) and EGFP-positive cells (GFP+). Panels: A--ZIC1 and
B--GFAP.
DETAILED DESCRIPTION
[0179] 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.
[0180] 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.
[0181] The PDX1 Gene Expression During Development
[0182] PDX1 (also called STF-1, IDX-1 and IPF-1) is a transcription
factor that is necessary for development of the pancreas and
rostral duodenum. PDX1 is first expressed in the pancreatic
endoderm, which arises from posterior foregut endoderm and will
produce both the exocrine and endocrine cells, starting at E8.5 in
the mouse. Later, PDX1 becomes restricted to beta-cells and some
delta-cells. This expression pattern is maintained in the adult.
PDX1 is also expressed in duodenal endoderm early in development,
which is adjacent to the forming pancreas, then in the duodenal
enterocytes and enteroendocrine cells, antral stomach and in the
common bile, cystic and biliary ducts. This region of expression
also becomes limited, at the time that pancreatic expression
becomes restricted, to predominantly the rostral duodenum.
[0183] PDX1-Positive Cells and Processes Related Thereto
[0184] Embodiments of the present invention relate to novel,
defined processes for the production of PDX1-positive endoderm
cells, wherein the PDX1-positive endoderm cells are multipotent
cells that can differentiate into cells, tissues or organs derived
from the foregut/midgut region of the gut tube (PDX1-positive
foregut/midgut endoderm). As used herein, "multipotent" or
"multipotent cell" refers to a cell type that can give rise to a
limited number of other particular cell types. As used herein,
"foregut/midgut" refers to cells of the anterior portion of the gut
tube as well as cells of the middle portion of the gut tube,
including cells of the foregut/midgut junction.
[0185] Some preferred embodiments of the present invention relate
to processes for the production of PDX1-positive foregut endoderm
cells. In some embodiments, these PDX1-positive foregut endoderm
cells are multipotent cells that can differentiate into cells,
tissues or organs derived from the anterior portion of the gut tube
(PDX1-positive foregut endoderm).
[0186] Additional preferred embodiments relate to processes for the
production of PDX1-positive endoderm cells of the posterior portion
of the foregut. In some embodiments, these PDX1-positive endoderm
cells are multipotent cells that can differentiate into cells,
tissues or organs derived from the posterior portion of the foregut
region of the gut tube.
[0187] The PDX1-positive foregut endoderm cells, such as those
produced according to the methods described herein, can be used to
produce fully differentiated insulin-producing .beta.-cells. In
some embodiments of the present invention, PDX1-positive foregut
endoderm cells are produced by differentiating definitive endoderm
cells that do not substantially express PDX1 (PDX1-negative
definitive endoderm cells; also referred to herein as definitive
endoderm) so as to form PDX1-positive foregut endoderm cells.
PDX1-negative definitive endoderm cells can be prepared by
differentiating pluripotent cells, such as embryonic stem cells, as
described herein or by any other known methods. A convenient and
highly efficient method for producing PDX1-negative definitive
endoderm from pluripotent cells is described in U.S. patent Ser.
No. 11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004,
the disclosure of which is incorporated herein by reference in its
entirety.
[0188] Processes of producing PDX1-positive foregut endoderm cells
provide a basis for efficient production of pancreatic tissues such
as acinar cells, ductal cells and islet cells from pluripotent
cells. In certain preferred embodiments, human PDX1-positive
foregut endoderm cells are derived from human PDX1-negative
definitive endoderm cells, which in turn, are derived from hESCs.
These human PDX1-positive foregut endoderm cells can then be used
to produce functional insulin-producing .beta.-cells. To obtain
useful quantities of insulin-producing .beta.-cells, high
efficiency of differentiation is desirable for each of the
differentiation steps that occur prior to reaching the pancreatic
islet/.beta.-cell fate. Because differentiation of PDX1-negative
definitive endoderm cells to PDX1-positive foregut endoderm cells
represents an early step towards the production of functional
pancreatic islet/.beta.-cells (as shown in FIG. 1), high efficiency
of differentiation at this step is particularly desirable.
[0189] In view of the desirability of efficient differentiation of
PDX1-negative definitive endoderm cells to PDX1-positive foregut
endoderm cells, some aspects of the present invention relate to in
vitro methodology that results in approximately 2-25% conversion of
PDX1-negative definitive endoderm cells to PDX1-positive foregut
endoderm cells. Typically, such methods encompass the application
of culture and growth factor conditions in a defined and temporally
specified fashion. Further enrichment of the cell population for
PDX1-positive foregut endoderm cells can be achieved by isolation
and/or purification of the PDX1-positive foregut endoderm cells
from other cells in the population by using a reagent that
specifically binds to the PDX1-positive foregut endoderm cells. As
an alternative, PDX1-positive foregut endoderm cells can be labeled
with a reporter gene, such as green fluorescent protein (GFP), so
as to enable the detection of PDX1 expression. Such fluorescently
labeled cells can then be purified by fluorescent activated cell
sorting (FACS). Further aspects of the present invention relate to
cell cultures and enriched cell populations comprising
PDX1-positive foregut endoderm cells as well as methods for
identifying factors useful in the differentiation to and from
PDX1-positive foregut endoderm.
[0190] In order to determine the amount of PDX1-positive foregut
endoderm cells in a cell culture or cell population, a method of
distinguishing this cell type from the other cells in the culture
or in the population is desirable. Accordingly, certain embodiments
of the present invention relate to cell markers whose presence,
absence and/or relative expression levels are indicative of
PDX1-positive foregut endoderm cells as well as methods for
detecting and determining the expression of such markers. As used
herein, "expression" refers to the production of a material or
substance as well as the level or amount of production of a
material or substance. Thus, determining the expression of a
specific marker refers to detecting either the relative or absolute
amount of the marker that is expressed or simply detecting the
presence or absence of the marker. As used herein, "marker" refers
to any molecule that can be observed or detected. For example, a
marker can include, but is not limited to, a nucleic acid, such as
a transcript of a specific gene, a polypeptide product of a gene, a
non-gene product polypeptide, a glycoprotein, a carbohydrate, a
glycolipd, a lipid, a lipoprotein or a small molecule (for example,
molecules having a molecular weight of less than 10,000 amu).
[0191] In some embodiments of the present invention, the presence,
absence and/or level of expression of a marker is determined by
quantitative PCR (Q-PCR). For example, the amount of transcript
produced by certain genetic markers, such as PDX1, SOX17, SOX7,
SOX1, ZIC1, NFM, alpha-fetoprotein (AFP), homeobox A13 (HOXA13),
homeobox C6 (HOXC6), and/or other markers described herein is
determined by Q-PCR. In other embodiments, immunohistochemistry is
used to detect the proteins expressed by the above-mentioned genes.
In still other embodiments, Q-PCR and immunohistochemical
techniques are both used to identify and determine the amount or
relative proportions of such markers.
[0192] By using the differentiation and detection methods described
herein, it is possible to identify PDX1-positive foregut endoderm
cells, as well as determine the proportion of PDX1-positive foregut
endoderm cells in a cell culture or cell population. For example,
in some embodiments of the present invention, the PDX1-positive
foregut endoderm cells or cell populations that are produced
express the PDX1 gene at a level of at least about 2 orders of
magnitude greater than PDX1-negative cells or cell populations. In
other embodiments, the PDX1-positive foregut endoderm cells and
cell populations that are produced express the PDX1 gene at a level
of more than 2 orders of magnitude greater than PDX1-negative cells
or cell populations. In still other embodiments, the PDX1-positive
foregut endoderm cells or cell populations that are produced
express one or more of the markers selected from the group
consisting of PDX1, SOX17, HOXA13 and HOXC6 at a level of about 2
or more than 2 orders of magnitude greater than PDX1-negative
definitive endoderm cells or cell populations.
[0193] The compositions and methods described herein have several
useful features. For example, the cell cultures and cell
populations comprising PDX1-positive endoderm, as well as the
methods for producing such cell cultures and cell populations, are
useful for modeling the early stages of human development.
Furthermore, the compositions and methods described herein can also
serve for therapeutic intervention in disease states, such as
diabetes mellitus. For example, since PDX1-positive foregut
endoderm serves as the source for only a limited number of tissues,
it can be used in the development of pure tissue or cell types.
[0194] Production of PDX1-Negative Definitive Endoderm (Definitive
Endoderm) from Pluripotent Cells
[0195] Cell cultures and/or cell populations comprising
PDX1-positive foregut endoderm cells are produced from pluripotent
cells by first producing PDX1-negative definitive endoderm (also
referred to as "definitive endoderm"). Processes for
differentiating pluripotent cells to produce cell cultures and
enriched cell populations comprising definitive endoderm is
described briefly below and in detail in U.S. patent Ser. No.
11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, the
disclosure of which is incorporated herein by reference in its
entirety. In some of these processes, the pluripotent cells used as
starting material are stem cells. In certain processes, definitive
endoderm cell cultures and enriched cell populations comprising
definitive endoderm cells are produced from embryonic stem cells.
As used herein, "embryonic" refers to a range of developmental
stages of an organism beginning with a single zygote and ending
with a multicellular structure that no longer comprises pluripotent
or totipotent cells other than developed gametic cells. In addition
to embryos derived by gamete fusion, the term "embryonic" refers to
embryos derived by somatic cell nuclear transfer. A preferred
method for deriving definitive endoderm cells utilizes human
embryonic stem cells as the starting material for definitive
endoderm production. Such pluripotent cells can be cells that
originate from the morula, embryonic inner cell mass or those
obtained from embryonic gonadal ridges. Human embryonic stem cells
can be maintained in culture in a pluripotent state without
substantial differentiation using methods that are known in the
art. Such methods are described, for example, in U.S. Pat. Nos.
5,453,357, 5,670,372, 5,690,926 5,843,780, 6,200,806 and 6,251,671
the disclosures of which are incorporated herein by reference in
their entireties.
[0196] In some processes for producing definitive endoderm cells,
hESCs are maintained on a feeder layer. In such processes, any
feeder layer which allows hESCs to be maintained in a pluripotent
state can be used. One commonly used feeder layer for the
cultivation of human embryonic stem cells is a layer of mouse
fibroblasts. More recently, human fibroblast feeder layers have
been developed for use in the cultivation of hESCs (see U.S. Patent
Application No. 2002/0072117, the disclosure of which is
incorporated herein by reference in its entirety). Alternative
processes for producing definitive endoderm permit the maintenance
of pluripotent hESC without the use of a feeder layer. Methods of
maintaining pluripotent hESCs under feeder-free conditions have
been described in U.S. Patent Application No. 2003/0175956, the
disclosure of which is incorporated herein by reference in its
entirety.
[0197] The human embryonic stem cells used herein can be maintained
in culture either with or without serum. In some embryonic stem
cell maintenance procedures, serum replacement is used. In others,
serum free culture techniques, such as those described in U.S.
Patent Application No. 2003/0190748, the disclosure of which is
incorporated herein by reference in its entirety, are used.
[0198] Stem cells are maintained in culture in a pluripotent state
by routine passage until it is desired that they be differentiated
into definitive endoderm. In some processes, differentiation to
definitive endoderm is achieved by providing to the stem cell
culture a growth factor of the TGF.beta. superfamily in an amount
sufficient to promote differentiation to definitive endoderm.
Growth factors of the TGF.beta. superfamily which are useful for
the production of definitive endoderm are selected from the
Nodal/Activin or BMP subgroups. In some preferred differentiation
processes, the growth factor is selected from the group consisting
of Nodal, activin A, activin B and BMP4. Additionally, the growth
factor Wnt3a and other Wnt family members are useful for the
production of definitive endoderm cells. In certain differentiation
processes, combinations of any of the above-mentioned growth
factors can be used.
[0199] With respect to some of the processes for the
differentiation of pluripotent stem cells to definitive endoderm
cells, the above-mentioned growth factors are provided to the cells
so that the growth factors are present in the cultures at
concentrations sufficient to promote differentiation of at least a
portion of the stem cells to definitive endoderm cells. In some
processes, the above-mentioned growth factors are present in the
cell culture at a concentration of at least about 5 ng/ml, at least
about 10 ng/ml, at least about 25 ng/ml, at least about 50 ng/ml,
at least about 75 ng/ml, at least about 100 ng/ml, at least about
200 ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, at
least about 500 ng/ml, at least about 1000 ng/ml, at least about
2000 ng/ml, at least about 3000 ng/ml, at least about 4000 ng/ml,
at least about 5000 ng/ml or more than about 5000 ng/ml.
[0200] In certain processes for the differentiation of pluripotent
stem cells to definitive endoderm cells, the above-mentioned growth
factors are removed from the cell culture subsequent to their
addition. For example, the growth factors can be removed within
about one day, about two days, about three days, about four days,
about five days, about six days, about seven days, about eight
days, about nine days or about ten days after their addition. In a
preferred processes, the growth factors are removed about four days
after their addition.
[0201] Cultures of definitive endoderm cells can be grown in medium
containing reduced serum or no serum. Under certain culture
conditions, serum concentrations can range from about 0.05% v/v to
about 20% v/v. For example, in some differentiation processes, the
serum concentration of the medium can be less than about 0.05%
(v/v), less than about 0.1% (v/v), less than about 0.2% (v/v), less
than about 0.3% (v/v), less than about 0.4% (v/v), less than about
0.5% (v/v), less than about 0.6% (v/v), less than about 0.7% (v/v),
less than about 0.8% (v/v), less than about 0.9% (v/v), less than
about 1% (v/v), less than about 2% (v/v), less than about 3% (v/v),
less than about 4% (v/v), less than about 5% (v/v), less than about
6% (v/v), less than about 7% (v/v), less than about 8% (v/v), less
than about 9% (v/v), less than about 10% (v/v), less than about 15%
(v/v) or less than about 20% (v/v). In some processes, definitive
endoderm cells are grown without serum or with serum replacement.
In still other processes, definitive endoderm cells are grown in
the presence of B27. In such processes, the concentration of B27
supplement can range from about 0.1% v/v to about 20% v/v.
[0202] Monitoring the Differentiation of Pluripotent Cells to
PDX1-Negative Definitive Endoderm (Definitive Endoderm)
[0203] The progression of the hESC culture to definitive endoderm
can be monitored by determining the expression of markers
characteristic of definitive endoderm. In some processes, the
expression of certain markers is determined by detecting the
presence or absence of the marker. Alternatively, the expression of
certain markers can be determined by measuring the level at which
the marker is present in the cells of the cell culture or cell
population. In such processes, the measurement of marker expression
can be qualitative or quantitative. One method of quantitating the
expression of markers that are produced by marker genes is through
the use of quantitative PCR (Q-PCR). Methods of performing Q-PCR
are well known in the art. Other methods which are known in the art
can also be used to quantitate marker gene expression. For example,
the expression of a marker gene product can be detected by using
antibodies specific for the marker gene product of interest. In
certain processes, the expression of marker genes characteristic of
definitive endoderm as well as the lack of significant expression
of marker genes characteristic of hESCs and other cell types is
determined.
[0204] As described further in the Examples below, a reliable
marker of definitive endoderm is the SOX17 gene. As such, the
definitive endoderm cells produced by the processes described
herein express the SOX17 marker gene, thereby producing the SOX17
gene product. Other markers of definitive endoderm are MIXL1,
GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
Since definitive endoderm cells express the SOX17 marker gene at a
level higher than that of the SOX7 marker gene, which is
characteristic of primitive and visceral endoderm (see Table 1), in
some processes, the expression of both SOX17 and SOX7 is monitored.
In other processes, expression of the both the SOX17 marker gene
and the OCT4 marker gene, which is characteristic of hESCs, is
monitored. Additionally, because definitive endoderm cells express
the SOX17 marker gene at a level higher than that of the AFP, SPARC
or Thrombomodulin (TM) marker genes, the expression of these genes
can also be monitored.
[0205] 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)].
[0206] Since definitive endoderm cells produced by differentiating
pluripotent cells express the CXCR4 marker gene, expression of
CXCR4 can be monitored in order to track the production of
definitive endoderm cells. Additionally, definitive endoderm cells
produced by the methods described herein express other markers of
definitive endoderm including, but not limited to, SOX17, MIXL1,
GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.
Since definitive endoderm cells express the CXCR4 marker gene at a
level higher than that of the SOX7 marker gene, the expression of
both CXCR4 and SOX7 can be monitored. In other processes,
expression of the both the CXCR4 marker gene and the OCT4 marker
gene, is monitored. Additionally, because definitive endoderm cells
express the CXCR4 marker gene at a level higher than that of the
AFP, SPARC or Thrombomodulin (TM) marker genes, the expression of
these genes can also be monitored.
[0207] It will be appreciated that expression of CXCR4 in
endodermal cells does not preclude the expression of SOX17. As
such, definitive endoderm cells produced by the processes described
herein will substantially express SOX 17 and CXCR4 but will not
substantially express AFP, TM, SPARC or PDX1.
[0208] Enrichment, Isolation and/or Purification of Definitive
Endoderm
[0209] Definitive endoderm cells produced by any of the
above-described processes can be enriched, isolated and/or purified
by using an affinity tag that is specific for such cells. Examples
of affinity tags specific for definitive endoderm cells are
antibodies, ligands or other binding agents that are specific to a
marker molecule, such as a polypeptide, that is present on the cell
surface of definitive endoderm cells but which is not substantially
present on other cell types that would be found in a cell culture
produced by the methods described herein. In some processes, an
antibody which binds to CXCR4 is used as an affinity tag for the
enrichment, isolation or purification of definitive endoderm cells.
In other processes, the chemokine SDF-1 or other molecules based on
SDF-1 can also be used as affinity tags. Such molecules include,
but not limited to, SDF-1 fragments, SDF-1 fusions or SDF-1
mimetics.
[0210] Methods for making antibodies and using them for cell
isolation are known in the art and such methods can be implemented
for use with the antibodies and definitive endoderm cells described
herein. In one process, an antibody which binds to CXCR4 is
attached to a magnetic bead and then allowed to bind to definitive
endoderm cells in a cell culture which has been enzymatically
treated to reduce intercellular and substrate adhesion. The
cell/antibody/bead complexes are then exposed to a movable magnetic
field which is used to separate bead-bound definitive endoderm
cells from unbound cells. Once the definitive endoderm cells are
physically separated from other cells in culture, the antibody
binding is disrupted and the cells are replated in appropriate
tissue culture medium.
[0211] Additional methods for obtaining enriched, isolated or
purified definitive endoderm cell cultures or populations can also
be used. For example, in some embodiments, the CXCR4 antibody is
incubated with a definitive endoderm-containing cell culture that
has been treated to reduce intercellular and substrate adhesion.
The cells are then washed, centrifuged and resuspended. The cell
suspension is then incubated with a secondary antibody, such as an
FITC-conjugated antibody that is capable of binding to the primary
antibody. The cells are then washed, centrifuged and resuspended in
buffer. The cell suspension is then analyzed and sorted using a
fluorescence activated cell sorter (FACS). CXCR4-positive cells are
collected separately from CXCR4-negative cells, thereby resulting
in the isolation of such cell types. If desired, the isolated cell
compositions can be further purified by using an alternate
affinity-based method or by additional rounds of sorting using the
same or different markers that are specific for definitive
endoderm.
[0212] In still other processes, definitive endoderm cells are
enriched, isolated and/or purified using a ligand or other molecule
that binds to CXCR4. In some processes, the molecule is SDF-1 or a
fragment, fusion or mimetic thereof.
[0213] In preferred processes, definitive endoderm cells are
enriched, isolated and/or purified from other non-definitive
endoderm cells after the stem cell cultures are induced to
differentiate towards the definitive endoderm lineage. It will be
appreciated that the above-described enrichment, isolation and
purification procedures can be used with such cultures at any stage
of differentiation.
[0214] In addition to the procedures just described, definitive
endoderm cells may also be isolated by other techniques for cell
isolation. Additionally, definitive endoderm cells may also be
enriched or isolated by methods of serial subculture in growth
conditions which promote the selective survival or selective
expansion of the definitive endoderm cells.
[0215] Using the methods described herein, enriched, isolated
and/or purified populations of definitive endoderm cells and or
tissues can be produced in vitro from pluripotent cell cultures or
cell populations, such as stem cell cultures or populations, which
have undergone at least some differentiation. In some methods, the
cells undergo random differentiation. In a preferred method,
however, the cells are directed to differentiate primarily into
definitive endoderm. Some preferred enrichment, isolation and/or
purification methods relate to the in vitro production of
definitive endoderm from human embryonic stem cells. Using the
methods described herein, cell populations or cell cultures can be
enriched in definitive endoderm content by at least about 2- to
about 1000-fold as compared to untreated cell populations or cell
cultures.
[0216] Compositions Comprising PDX1-Negative Definitive Endoderm
(Definitive Endoderm)
[0217] Cell compositions produced by the above-described methods
include cell cultures comprising definitive endoderm and cell
populations enriched in definitive endoderm. For example, cell
cultures which comprise definitive endoderm cells, wherein at least
about 50-80% of the cells in culture are definitive endoderm cells,
can be produced. Because the efficiency of the differentiation
process can be adjusted by modifying certain parameters, which
include but are not limited to, cell growth conditions, growth
factor concentrations and the timing of culture steps, the
differentiation procedures described herein can result in about 5%,
about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,
about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,
about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,
or greater than about 95% conversion of pluripotent cells to
definitive endoderm. In processes in which isolation of definitive
endoderm cells is employed, for example, by using an affinity
reagent that binds to the CXCR4 receptor, a substantially pure
definitive endoderm cell population can be recovered.
[0218] Production of PDX1-Positve Foregut Endoderm from
PDX1-Negative Definitive Endoderm
[0219] The PDX1-positive foregut endoderm cell cultures and
populations comprising PDX1-positive foregut endoderm cells that
are described herein are produced from PDX1-negative definitive
endoderm, which is generated from pluripotent cells as described
above. A preferred method utilizes human embryonic stem cells as
the starting material. In one embodiment, hESCs are first converted
to PDX1-negative definitive endoderm cells, which are then
converted to PDX1-positive foregut endoderm cells. It will be
appreciated, however, that the starting materials for the
production of PDX1-positive foregut endoderm is not limited to
definitive endoderm cells produced using pluripotent cell
differentiation methods. Rather, any PDX1-negative definitive
endoderm cells can be used in the methods described herein
regardless of their origin.
[0220] In some embodiments of the present invention, cell cultures
or cell populations comprising PDX1-negative definitive endoderm
cells can be used for further differentiation to cell cultures
and/or enriched cell populations comprising PDX1-positive foregut
endoderm cells. For example, a cell culture or cell population
comprising human PDX1-negative, SOX17-positive definitive endoderm
cells can be used. In some embodiments, the cell culture or cell
population may also comprise differentiation factors, such as
activins, nodals and/or BMPs, remaining from the previous
differentiation step (that is, the step of differentiating
pluripotent cells to definitive endoderm cells). In other
embodiments, factors utilized in the previous differentiation step
are removed from the cell culture or cell population prior to the
addition of factors used for the differentiation of the
PDX1-negative, SOX17-positive definitive endoderm cells to
PDX1-positive foregut endoderm cells. In other embodiments, cell
populations enriched for PDX1-negative, SOX17-positive definitive
endoderm cells are used as a source for the production of
PDX1-positive foregut endoderm cells.
[0221] PDX1-negative definitive endoderm cells in culture are
differentiated to PDX1-positive endoderm cells by providing to a
cell culture comprising PDX1-negative, SOX17-positive definitive
endoderm cells a differentiation factor that promotes
differentiation of the cells to PDX1-positive foregut endoderm
cells (foregut differentiation factor). In some embodiments of the
present invention, the foregut differentiation factor is retinoid,
such as retinoic acid (RA). In some embodiments, the retinoid is
used in conjunction with a fibroblast growth factor, such as FGF-4
or FGF-10. In other embodiments, the retinoid is used in
conjunction with a member of the TGF.beta. superfamily of growth
factors and/or a conditioned medium.
[0222] By "conditioned medium" is meant, a medium that is altered
as compared to a base medium. For example, the conditioning of a
medium may cause molecules, such as nutrients and/or growth
factors, to be added to or depleted from the original levels found
in the base medium. In some embodiments, a medium is conditioned by
allowing cells of certain types to be grown or maintained in the
medium under certain conditions for a certain period of time. For
example, a medium can be conditioned by allowing hESCs to be
expanded, differentiated or maintained in a medium of defined
composition at a defined temperature for a defined number of hours.
As will be appreciated by those of skill in the art, numerous
combinations of cells, media types, durations and environmental
conditions can be used to produce nearly an infinite array of
conditioned media. In some embodiments of the present invention, a
medium is conditioned by allowing differentiated pluripotent cells
to be grown or maintained in a medium comprising about 1% to about
20% serum concentration. In other embodiments, a medium is
conditioned by allowing differentiated pluripotent cells to be
grown or maintained in a medium comprising about 1 ng/ml to about
1000 ng/ml activin A. In still other embodiments, a medium is
conditioned allowing differentiated pluripotent cells to be grown
or maintained in a medium comprising about 1 ng/ml to about 1000
ng/ml BMP4. In a preferred embodiment, a conditioned medium is
prepared by allowing differentiated hESCs to be grown or maintained
for 24 hours in a medium, such as RPMI, comprising about 25 ng/ml
activin A and about 2 .mu.M RA.
[0223] In some embodiments of the present invention, the cells used
to condition the medium, which is used to enhance the
differentiation of PDX1-negative definitive endoderm to
PDX1-positive foregut endoderm, are cells that are differentiated
from pluripotent cells, such as hESCs, over about a 5 day time
period in a medium such as RPMI comprising about 0% to about 20%
serum and/or one or more growth/differentiation factors of the
TGF.beta. superfamily. Differentiation factors, such as activin A
and BMP4 are supplied at concentrations ranging from about 1 ng/ml
to about 1000 ng/ml. In certain embodiments of the present
invention, the cells used to condition the medium are
differentiated from hESCs over about a 5 day period in low serum
RPMI. According to some embodiments, low serum RPMI refers to a low
serum containing medium, wherein the serum concentration is
gradually increased over a defined time period. For example, in one
embodiment, low serum RPMI comprises a concentration of about 0.2%
fetal bovine serum (FBS) on the first day of cell growth, about
0.5% FBS on the second day of cell growth and about 2% FBS on the
third through fifth day of cell growth. In another embodiment, low
serum RPMI comprises a concentration of about 0% on day one, about
0.2% on day two and about 2% on days 3-6. In certain preferred
embodiments, low serum RPMI is supplemented with one or more
differentiation factors, such as activin A and BMP4. In addition to
its use in preparing cells used to condition media, low serum RPMI
can be used as a medium for the differentiation of PDX1-positive
foregut endoderm cells from PDX1-negative definitive endoderm
cells.
[0224] It will be appreciated by those of ordinary skill in the art
that conditioned media can be prepared from media other than RPMI
provided that such media do not interfere with the growth or
maintenance of PDX1-positive foregut endoderm cells. It will also
be appreciated that the cells used to condition the medium can be
of various types. In embodiments where freshly differentiated cells
are used to condition a medium, such cells can be differentiated in
a medium other than RPMI provided that the medium does not inhibit
the growth or maintenance of such cells. Furthermore, a skilled
artisan will appreciate that neither the duration of conditioning
nor the duration of preparation of cells used for conditioning is
required to be 24 hours or 5 days, respectively, as other time
periods will be sufficient to achieve the effects reported
herein.
[0225] In general, the use of a retinoid in combination with a
fibroblast growth factor, a member of the TGF.beta. superfamily of
growth factors, a conditioned medium or a combination of any of
these foregut differentiation factors causes greater
differentiation of PDX1-negative definitive endoderm to
PDX1-positive foregut endoderm than the use of a retinoid alone. In
a preferred embodiment, RA and FGF-10 are both provided to the
PDX1-negative definitive endoderm cell culture. In another
preferred embodiment, PDX1-negative definitive endoderm cells are
differentiated in a culture comprising a conditioned medium,
activin A, activin B and RA.
[0226] With respect to some of the embodiments of differentiation
processes described herein, the above-mentioned foregut
differentiation factors are provided to the cells so that these
factors are present in the cell culture or cell population at
concentrations sufficient to promote differentiation of at least a
portion of the PDX1-negative definitive endoderm cell culture or
cell population to PDX1-positive foregut endoderm cells. When used
in connection with cell cultures and/or cell populations, the term
"portion" means any non-zero amount of the cell culture or cell
population, which ranges from a single cell to the entirety of the
cell culture or cells population.
[0227] In some embodiments of the present invention, a retinoid is
provided to the cells of a cell culture such that it is present at
a concentration of at least about 0.01 .mu.M, at least about 0.02
.mu.M, at least about 0.04 .mu.M, at least about 0.08 .mu.M, at
least about 0.1 .mu.M, at least about 0.2 .mu.M, at least about 0.3
.mu.M, at least about 0.4 .mu.M, at least about 0.5 .mu.M, at least
about 0.6 .mu.M, at least about 0.7 .mu.M, at least about 0.8
.mu.M, at least about 0.9 .mu.M, at least about 1 .mu.M, at least
about 1.1 .mu.M, at least about 1.2 .mu.M, at least about 1.3
.mu.M, at least about 1.4 .mu.M, at least about 1.5 .mu.M, at least
about 1.6 .mu.M, at least about 1.7 .mu.M, at least about 1.8
.mu.M, at least about 1.9 .mu.M, at least about 2 .mu.M, at least
about 2.1 .mu.M, at least about 2.2 .mu.M, at least about 2.3
.mu.M, at least about 2.4 .mu.M, at least about 2.5 .mu.M, at least
about 2.6 .mu.M, at least about 2.7 .mu.M, at least about 2.8
.mu.M, at least about 2.9 .mu.M, at least about 3 .mu.M, at least
about 3.5 .mu.M, at least about 4 .mu.M, at least about 4.5 .mu.M,
at least about 5 .mu.M, at least about 10 .mu.M, at least about 20
.mu.M, at least about 30 .mu.M, at least about 40 .mu.M or at least
about 50 .mu.M. As used herein, "retinoid" refers to retinol,
retinal or retinoic acid as well as derivatives of any of these
compounds. In a preferred embodiment, the retinoid is retinoic
acid.
[0228] In other embodiments of the present invention, one or more
differentiation factors of the fibroblast growth factor family are
present in the cell culture. For example, in some embodiments,
FGF-4 can be present in the cell culture at a concentration of at
least about 10 ng/ml, at least about 25 ng/ml, at least about 50
ng/ml, at least about 75 ng/ml, at least about 100 ng/ml, at least
about 200 ng/ml, at least about 300 ng/ml, at least about 400
ng/ml, at least about 500 ng/ml, or at least about 1000 ng/ml. In
further embodiments of the present invention, FGF-10 is present in
the cell culture at a concentration of at least about 10 ng/ml, at
least about 25 ng/ml, at least about 50 ng/ml, at least about 75
ng/ml, at least about 100 ng/ml, at least about 200 ng/ml, at least
about 300 ng/ml, at least about 400 ng/ml, at least about 500
ng/ml, or at least about 1000 ng/ml. In some embodiments, either
FGF-4 or FGF-10, but not both, is provided to the cell culture
along with RA. In a preferred embodiment, RA is present in the cell
culture at 1 .mu.M and FGF-10 is present at a concentration of 50
ng/ml.
[0229] In some embodiments of the present invention, growth factors
of the TGF.beta. superfamily and/or a conditioned medium are
present in the cell culture. These differentiation factors can be
used in combination with RA and/or other mid-foregut
differentiation factors including, but not limited to, FGF-4 and
FGF-10. For example, in some embodiments, activin A and/or activin
B can be present in the cell culture at a concentration of at least
about 5 ng/ml, at least about 10 ng/ml, at least about 25 ng/ml, at
least about 50 ng/ml, at least about 75 ng/ml, at least about 100
ng/ml, at least about 200 ng/ml, at least about 300 ng/ml, at least
about 400 ng/ml, at least about 500 ng/ml, or at least about 1000
ng/ml. In further embodiments of the present invention, a
conditioned medium is present in the cell culture at a
concentration of at least about 1%, at least about 5%, at least
about 10%, at least about 20%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, or at least about 100% of the
total medium. In some embodiments, activin A, activin B and a
conditioned medium are provided to the cell culture along with RA.
In a preferred embodiment, PDX1-negative definitive endoderm cells
are differentiated to PDX1-positive foregut endoderm cells in
cultures comprising about 1 .mu.M RA, about 25 ng/ml activin A and
low serum RPMI medium that has been conditioned for about 24 hours
by differentiated hESCs, wherein the differentiated hESCs have been
differentiated for about 5 days in low serum RPMI comprising about
100 ng/ml activin A. In another preferred embodiment, activin B
and/or FGF-10 are also present in the culture at 25 ng/ml and 50
ng/ml, respectively.
[0230] In certain embodiments of the present invention, the
above-mentioned foregut differentiation factors are removed from
the cell culture subsequent to their addition. For example, the
foregut differentiation factors can be removed within about one
day, about two days, about three days, about four days, about five
days, about six days, about seven days, about eight days, about
nine days or about ten days after their addition.
[0231] Cultures of PDX I-positive foregut endoderm cells can be
grown in a medium containing reduced serum. Serum concentrations
can range from about 0.05% (v/v) to about 20% (v/v). In some
embodiments, PDX1-positive foregut endoderm cells are grown with
serum replacement. For example, in certain embodiments, the serum
concentration of the medium can be less than about 0.05% (v/v),
less than about 0.1% (v/v), less than about 0.2% (v/v), less than
about 0.3% (v/v), less than about 0.4% (v/v), less than about 0.5%
(v/v), less than about 0.6% (v/v), less than about 0.7% (v/v), less
than about 0.8% (v/v), less than about 0.9% (v/v), less than about
1% (v/v), less than about 2% (v/v), less than about 3% (v/v), less
than about 4% (v/v), less than about 5% (v/v), less than about 6%
(v/v), less than about 7% (v/v), less than about 8% (v/v), less
than about 9% (v/v), less than about 10% (v/v), less than about 15%
(v/v) or less than about 20% (v/v). In some embodiments,
PDX1-positive foregut endoderm cells are grown without serum. In
other embodiments, PDX1-positive foregut endoderm cells are grown
with serum replacement.
[0232] In still other embodiments, PDX1-positive foregut endoderm
cells are grown in the presence of B27. In such embodiments, B27
can be provided to the culture medium in concentrations ranging
from about 0.1% (v/v) to about 20% (v/v) or in concentrations
greater than about 20% (v/v). In certain embodiments, the
concentration of B27 in the medium is about 0.1% (v/v), about 0.2%
(v/v), about 0.3% (v/v), about 0.4% (v/v), about 0.5% (v/v), about
0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about 0.9% (v/v),
about 1% (v/v), about 2% (v/v), about 3% (v/v), about 4% (v/v),
about 5% (v/v), about 6% (v/v), about 7% (v/v), about 8% (v/v),
about 9% (v/v), about 10% (v/v), about 15% (v/v) or about 20%
(v/v). Alternatively, the concentration of the added B27 supplement
can be measured in terms of multiples of the strength of a
commercially available B27 stock solution. For example, B27 is
available from Invitrogen (Carlsbad, Calif.) as a 50.times. stock
solution. Addition of a sufficient amount of this stock solution to
a sufficient volume of growth medium produces a medium supplemented
with the desired amount of B27. For example, the addition of 10 ml
of 50.times. B27 stock solution to 90 ml of growth medium would
produce a growth medium supplemented with 5.times. B27. The
concentration of B27 supplement in the medium can be about
0.1.times., about 0.2.times., about 0.3.times., about 0.4.times.,
about 0.5.times., about 0.6.times., about 0.7.times., about
0.8.times., about 0.9.times., about 1.times., about 1.1.times.,
about 1.2.times., about 1.3.times., about 1.4.times., about
1.5.times., about 1.6.times., about 1.7.times., about 1.8.times.,
about 1.9.times., about 2.times., about 2.5.times., about 3.times.,
about 3.5.times., about 4.times., about 4.5.times., about 5.times.,
about 6.times., about 7.times., about 8.times., about 9.times.,
about 10.times., about 11.times., about 12.times., about 13.times.,
about 14.times., about 15.times., about 16.times., about 17.times.,
about 18.times., about 19.times., about 20.times. and greater than
about 20.times..
[0233] Monitoring the Differentiation of PDX1-Negative Definitive
Endoderm to PDX1-Positive Endoderm
[0234] As with the differentiation of definitive endoderm cells
from pluripotent cells, the progression of differentiation from
PDX1-negative, SOX17-positive definitive endoderm to PDX1-positive
foregut endoderm can be monitored by determining the expression of
markers characteristic of these cell types. Such monitoring permits
one to determine the amount of time that is sufficient for the
production of a desired amount of PDX1-positive foregut endoderm
under various conditions, for example, one or more differentiation
factor concentrations and environmental conditions. In preferred
embodiments, the amount of time that is sufficient for the
production of a desired amount of PDX1-positive foregut endoderm is
determined by detecting the expression of PDX1. In some embodiments
of the present invention, the expression of certain markers is
determined by detecting the presence or absence of the marker.
Alternatively, the expression of certain markers can be determined
by measuring the level at which the marker is present in the cells
of the cell culture or cell population. In such embodiments, the
measurement of marker expression can be qualitative or
quantitative. As described above, a preferred method of
quantitating the expression markers that are produced by marker
genes is through the use of Q-PCR. In particular embodiments, Q-PCR
is used to monitor the progression of cells of the PDX1-negative,
SOX17-positive definitive endoderm culture to PDX1-positive foregut
endoderm cells by quantitating expression of marker genes
characteristic of PDX1-positive foregut endoderm and the lack of
expression of marker genes characteristic of other cell types.
Other methods which are known in the art can also be used to
quantitate marker gene expression. For example, the expression of a
marker gene product can be detected by using antibodies specific
for the marker gene product of interest. In some embodiments of the
present invention, the expression of marker genes characteristic of
PDX1-positive foregut endoderm as well as the lack of significant
expression of marker genes characteristic of PDX1-negative
definitive endoderm, hESCs and other cell types is determined.
[0235] As described further in the Examples below, PDX1 is a marker
gene that is associated with PDX1-positive foregut endoderm. As
such, in some embodiments of the present invention, the expression
of PDX1 is determined. In other embodiments, the expression of
other markers, which are expressed in PDX1-positive foregut
endoderm, including, but not limited to, SOX17, HOXA13 and/or HOXC6
is also determined. Since PDX1 can also be expressed by certain
other cell types (that is, visceral endoderm and certain neural
ectoderm), some embodiments of the present invention relate to
demonstrating the absence or substantial absence of marker gene
expression that is associated with visceral endoderm and/or neural
ectoderm. For example, in some embodiments, the expression of
markers, which are expressed in visceral endoderm and/or neural
cells, including, but not limited to, SOX7, AFP, SOX1, ZIC1 and/or
NFM is determined.
[0236] In some embodiments, PDX1-positive foregut endoderm cell
cultures produced by the methods described herein are substantially
free of cells expressing the SOX7, AFP, SOX1, ZIC1 or NFM marker
genes. In certain embodiments, the PDX1-positive foregut endoderm
cell cultures produced by the processes described herein are
substantially free of visceral endoderm, parietal endoderm and/or
neural cells.
[0237] Enrichment Isolation and/or Purification of PDX1-Positive
Foregut Endoderm
[0238] With respect to additional aspects of the present invention,
PDX1-positive foregut endoderm cells can be enriched, isolated
and/or purified. In some embodiments of the present invention, cell
populations enriched for PDX1-positive foregut endoderm cells are
produced by isolating such cells from cell cultures.
[0239] In some embodiments of the present invention, PDX1-positive
foregut endoderm cells are fluorescently labeled then isolated from
non-labeled cells by using a fluorescence activated cell sorter
(FACS). In such embodiments, a nucleic acid encoding green
fluorescent protein (GFP) or another nucleic acid encoding an
expressible fluorescent marker gene is used to label PDX1-positive
cells. For example, in some embodiments, at least one copy of a
nucleic acid encoding GFP or a biologically active fragment thereof
is introduced into a pluripotent cell, preferably a human embryonic
stem cell, downstream of the PDX1 promoter such that the expression
of the GFP gene product or biologically active fragment thereof is
under control of the PDX1 promoter. In some embodiments, the entire
coding region of the nucleic acid, which encodes PDX1, is replaced
by a nucleic acid encoding GFP or a biologically active fragment
thereof. In other embodiments, the nucleic acid encoding GFP or a
biologically active fragment thereof is fused in frame with at
least a portion of the nucleic acid encoding PDX1, thereby
generating a fusion protein. In such embodiments, the fusion
protein retains a fluorescent activity similar to GFP.
[0240] Fluorescently marked cells, such as the above-described
pluripotent cells, are differentiated to definitive endoderm and
then to PDX I-positive foregut endoderm as described previously
above. Because PDX1-positive foregut endoderm cells express the
fluorescent marker gene, whereas PDX1-negative cells do not, these
two cell types can be separated. In some embodiments, cell
suspensions comprising a mixture of fluorescently-labeled
PDX1-positive cells and unlabeled PDX1-negative cells are sorted
using a FACS. PDX1-positive cells are collected separately from
PDX1-negative cells, thereby resulting in the isolation of such
cell types. If desired, the isolated cell compositions can be
further purified by additional rounds of sorting using the same or
different markers that are specific for PDX1-positve foregut
endoderm.
[0241] In addition to the procedures just described, PDX 1-positive
foregut endoderm cells may also be isolated by other techniques for
cell isolation. Additionally, PDX1-positive foregut endoderm cells
may also be enriched or isolated by methods of serial subculture in
growth conditions which promote the selective survival or selective
expansion of said PDX1-positive foregut endoderm cells.
[0242] It will be appreciated that the above-described enrichment,
isolation and purification procedures can be used with such
cultures at any stage of differentiation.
[0243] Using the methods described herein, enriched, isolated
and/or purified populations of PDX1-positive foregut endoderm cells
and/or tissues can be produced in vitro from PDX1-negative,
SOX17-positive definitive endoderm cell cultures or cell
populations which have undergone at least some differentiation. In
some embodiments, the cells undergo random differentiation. In a
preferred embodiment, however, the cells are directed to
differentiate primarily into PDX1-positive foregut endoderm cells.
Some preferred enrichment, isolation and/or purification methods
relate to the in vitro production of PDX1-positive foregut endoderm
cells from human embryonic stem cells.
[0244] Using the methods described herein, cell populations or cell
cultures can be enriched in PDX1-positive foregut endoderm cell
content by at least about 2- to about 1000-fold as compared to
untreated cell populations or cell cultures. In some embodiments,
PDX1-positive foregut endoderm cells can be enriched by at least
about 5- to about 500-fold as compared to untreated cell
populations or cell cultures. In other embodiments, PDX1-positive
foregut endoderm cells can be enriched from at least about 10- to
about 200-fold as compared to untreated cell populations or cell
cultures. In still other embodiments, PDX1-positive foregut
endoderm cells can be enriched from at least about 20- to about
100-fold as compared to untreated cell populations or cell
cultures. In yet other embodiments, PDX1-positive foregut endoderm
cells can be enriched from at least about 40- to about 80-fold as
compared to untreated cell populations or cell cultures. In certain
embodiments, PDX1-positive foregut endoderm cells can be enriched
from at least about 2- to about 20-fold as compared to untreated
cell populations or cell cultures.
[0245] Compositions Comprising PDX1-Positive Foregut Endoderm
[0246] Some embodiments of the present invention relate to cell
compositions, such as cell cultures or cell populations, comprising
PDX1-positive endoderm cells, wherein the PDX1-positive endoderm
cells are multipotent cells that can differentiate into cells,
tissues or organs derived from the anterior portion of the gut tube
(PDX1-positive foregut endoderm). In accordance with certain
embodiments, the PDX1-positive foregut endoderm are mammalian
cells, and in a preferred embodiment, the definitive endoderm cells
are human cells.
[0247] Other embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
cells of one or more cell types selected from the group consisting
of hESCs, PDX1-negative definitive endoderm cells, PDX1-positive
foregut endoderm cells and mesoderm cells. In some embodiments,
hESCs comprise less than about 5%, less than about 4%, less than
about 3%, less than about 2% or less than about 1% of the total
cells in the culture. In other embodiments, PDX1-negative
definitive endoderm cells comprise less than about 90%, less than
about 85%, less than about 80%, less than about 75%, less than
about 70%, less than about 65%, less than about 60%, less than
about 55%, less than about 50%, less than about 45%, less than
about 40%, less than about 35%, less than about 30%, less than
about 25%, less than about 20%, less than about 15%, less than
about 12%, less than about 10%, less than about 8%, less than about
6%, less than about 5%, less than about 4%, less than about 3%,
less than about 2% or less than about 1% of the total cells in the
culture. In yet other embodiments, mesoderm cells comprise less
than about 90%, less than about 85%, less than about 80%, less than
about 75%, less than about 70%, less than about 65%, less than
about 60%, less than about 55%, less than about 50%, less than
about 45%, less than about 40%, less than about 35%, less than
about 30%, less than about 25%, less than about 20%, less than
about 15%, less than about 12%, less than about 10%, less than
about 8%, less than about 6%, less than about 5%, less than about
4%, less than about 3%, less than about 2% or less than about 1% of
the total cells in the culture.
[0248] Additional embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, produced
by the processes described herein comprise PDX1-positive foregut
endoderm as the majority cell type. In some embodiments, the
processes described herein produce cell cultures and/or cell
populations comprising at least about 99%, at least about 98%, at
least about 97%, at least about 96%, at least about 95%, at least
about 94%, at least about 93%, at least about 92%, at least about
91%, at least about 90%, at least about 85%, at least about 80%, at
least about 75%, at least about 70%, at least about 65%, at least
about 60%, at least about 55%, at least about 54%, at least about
53%, at least about 52% or at least about 51% PDX1-positive foregut
endoderm cells. In preferred embodiments the cells of the cell
cultures or cell populations comprise human cells. In other
embodiments, the processes described herein produce cell cultures
or cell populations comprising at least about 50%, at least about
45%, at least about 40%, at least about 35%, at least about 30%, at
least about 25%, at least about 24%, at least about 23%, at least
about 22%, at least about 21%, at least about 20%, at least about
19%, at least about 18%, at least about 17%, at least about 16%, at
least about 15%, at least about 14%, at least about 13%, at least
about 12%, at least about 11%, at least about 10%, at least about
9%, at least about 8%, at least about 7%, at least about 6%, at
least about 5%, at least about 4%, at least about 3%, at least
about 2% or at least about 1% PDX1-positive foregut endoderm cells.
In preferred embodiments, the cells of the cell cultures or cell
populations comprise human cells. In some embodiments, the
percentage of PDX1-positive foregut endoderm cells in the cell
cultures or populations is calculated without regard to the feeder
cells remaining in the culture.
[0249] Still other embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
mixtures of PDX1-positive foregut endoderm cells and PDX1-negative
definitive endoderm cells. For example, cell cultures or cell
populations comprising at least about 5 PDX1-positive foregut
endoderm cells for about every 95 PDX1-negative definitive endoderm
cells can be produced. In other embodiments, cell cultures or cell
populations comprising at least about 95 PDX1-positive foregut
endoderm cells for about every 5 PDX1-negative definitive endoderm
cells can be produced. Additionally, cell cultures or cell
populations comprising other ratios of PDX1-positive foregut
endoderm cells to PDX1-negative definitive endoderm cells are
contemplated. For example, compositions comprising at least about 1
PDX1-positive foregut endoderm cell for about every 1,000,000
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 100,000
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 10,000
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 1000
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 500
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 100
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 10
PDX1-negative definitive endoderm cells, at least about 1
PDX1-positive foregut endoderm cell for about every 5 PDX1-negative
definitive endoderm cells, at least about 1 PDX1-positive foregut
endoderm cell for about every 4 PDX1-negative definitive endoderm
cells, at least about 1 PDX1-positive foregut endoderm cell for
about every 2 PDX1-negative definitive endoderm cells, at least
about 1 PDX1-positive foregut endoderm cell for about every 1
PDX1-negative definitive endoderm cell, at least about 2
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 4
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 5
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 10
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 20
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 50
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 100
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 1000
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 10,000
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell, at least about 100,000
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell and at least about 1,000,000
PDX1-positive foregut endoderm cells for about every 1
PDX1-negative definitive endoderm cell are contemplated.
[0250] In some embodiments of the present invention, the
PDX1-negative definitive endoderm cells from which PDX1-positive
foregut endoderm cells are produced are derived from human
pluripotent cells, such as human pluripotent stem cells. In certain
embodiments, the human pluripotent cells are derived from a morula,
the inner cell mass of an embryo or the gonadal ridges of an
embryo. In certain other embodiments, the human pluripotent cells
are derived from the gondal or germ tissues of a multicellular
structure that has developed past the embryonic stage.
[0251] Further embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
human cells, including human PDX1-positive foregut endoderm,
wherein the expression of the PDX1 marker is greater than the
expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at
least about 2% of the human cells. In other embodiments, the
expression of the PDX1 marker is greater than the expression of the
AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at least about 5% of the
human cells, in at least about 10% of the human cells, in at least
about 15% of the human cells, in at least about 20% of the human
cells, in at least about 25% of the human cells, in at least about
30% of the human cells, in at least about 35% of the human cells,
in at least about 40% of the human cells, in at least about 45% of
the human cells, in at least about 50% of the human cells, in at
least about 55% of the human cells, in at least about 60% of the
human cells, in at least about 65% of the human cells, in at least
about 70% of the human cells, in at least about 75% of the human
cells, in at least about 80% of the human cells, in at least about
85% of the human cells, in at least about 90% of the human cells,
in at least about 95% of the human cells or in at least about 98%
of the human cells. In some embodiments, the percentage of human
cells in the cell cultures or populations, wherein the expression
of PDX1 is greater than the expression of the AFP, SOX7, SOX1, ZIC1
and/or NFM marker, is calculated without regard to feeder
cells.
[0252] It will be appreciated that some embodiments of the present
invention relate to compositions, such as cell cultures or cell
populations, comprising human PDX1-positive foregut endoderm cells,
wherein the expression of one or more markers selected from the
group consisting of SOX17, HOXA13 and HOXC6 is greater than the
expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in from
at least about 2% to greater than at least about 98% of the human
cells. In some embodiments, the expression of one or more markers
selected from the group consisting of SOX17, HOXA13 and HOXC6 is
greater than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM
marker in at least about 5% of the human cells, in at least about
10% of the human cells, in at least about 15% of the human cells,
in at least about 20% of the human cells, in at least about 25% of
the human cells, in at least about 30% of the human cells, in at
least about 35% of the human cells, in at least about 40% of the
human cells, in at least about 45% of the human cells, in at least
about 50% of the human cells, in at least about 55% of the human
cells, in at least about 60% of the human cells, in at least about
65% of the human cells, in at least about 70% of the human cells,
in at least about 75% of the human cells, in at least about 80% of
the human cells, in at least about 85% of the human cells, in at
least about 90% of the human cells, in at least about 95% of the
human cells or in at least about 98% of the human cells. In some
embodiments, the percentage of human cells in the cell cultures or
populations, wherein the expression of one or more markers selected
from the group consisting of SOX17, HOXA13 and HOXC6 is greater
than the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker,
is calculated without regard to feeder cells.
[0253] Additional embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
mammalian endodermal cells, such as human endoderm cells, wherein
the expression of the PDX1 marker is greater than the expression of
the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at least about 2% of
the endodermal cells. In other embodiments, the expression of the
PDX1 marker is greater than the expression of the AFP, SOX7, SOX1,
ZIC1 and/or NFM marker in at least about 5% of the endodermal
cells, in at least about 10% of the endodermal cells, in at least
about 15% of the endodermal cells, in at least about 20% of the
endodermal cells, in at least about 25% of the endodermal cells, in
at least about 30% of the endodermal cells, in at least about 35%
of the endodermal cells, in at least about 40% of the endodermal
cells, in at least about 45% of the endodermal cells, in at least
about 50% of the endodermal cells, in at least about 55% of the
endodermal cells, in at least about 60% of the endodermal cells, in
at least about 65% of the endodermal cells, in at least about 70%
of the endodermal cells, in at least about 75% of the endodermal
cells, in at least about 80% of the endodermal cells, in at least
about 85% of the endodermal cells, in at least about 90% of the
endodermal cells, in at least about 95% of the endodermal cells or
in at least about 98% of the endodermal cells.
[0254] Still other embodiments of the present invention relate to
compositions, such as cell cultures or cell populations, comprising
mammalian endodermal cells, such as human endodermal cells, wherein
the expression of one or more markers selected from the group
consisting of SOX17, HOXA13 and HOXC6 is greater than the
expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at
least about 2% of the endodermal cells. In other embodiments, the
expression of one or more markers selected from the group
consisting of SOX17, HOXA13 and HOXC6 is greater than the
expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at
least about 5% of the endodermal cells, in at least about 10% of
the endodermal cells, in at least about 15% of the endodermal
cells, in at least about 20% of the endodermal cells, in at least
about 25% of the endodermal cells, in at least about 30% of the
endodermal cells, in at least about 35% of the endodermal cells, in
at least about 40% of the endodermal cells, in at least about 45%
of the endodermal cells, in at least about 50% of the endodermal
cells, in at least about 55% of the endodermal cells, in at least
about 60% of the endodermal cells, in at least about 65% of the
endodermal cells, in at least about 70% of the endodermal cells, in
at least about 75% of the endodermal cells, in at least about 80%
of the endodermal cells, in at least about 85% of the endodermal
cells, in at least about 90% of the endodermal cells, in at least
about 95% of the endodermal cells or at least about 98% of the
endodermal cells.
[0255] Using the processes described herein, compositions
comprising PDX1-positive foregut endoderm cells substantially free
of other cell types can be produced. With respect to cells in cell
cultures or in cell populations, the term "substantially free of"
means that the specified cell type of which the cell culture or
cell population is free, is present in an amount of less than about
5% of the total number of cells present in the cell culture or cell
population. In some embodiments of the present invention, the
PDX1-positive foregut endoderm cell populations or cell cultures
produced by the methods described herein are substantially free of
cells that significantly express the AFP, SOX7, SOX1, ZIC1 and/or
NFM marker genes.
[0256] In one embodiment of the present invention, a description of
a PDX1-positive foregut endoderm cell based on the expression of
marker genes is, PDX1 high, AFP low, SOX7 low, SOX1 low, ZIC1 low
and NFM low.
[0257] Increasing Expression of PDX1 In a SOX17-Positive Definitive
Endoderm Cell
[0258] Some aspects of the present invention are related to methods
of increasing the expression of the PDX1 gene product in cell
cultures or cell populations comprising SOX17-positive definitive
endoderm cells. In such embodiments, the SOX17-positive definitive
endoderm cells are contacted with a differentiation factor in an
amount that is sufficient to increase the expression of the PDX1
gene product. The SOX17-positive definitive endoderm cells that are
contacted with the differentiation factor can be either
PDX1-negative or PDX1-positive. In some embodiments, the
differentiation factor can be a retinoid. In certain embodiments,
SOX17-positive definitive endoderm cells are contacted with a
retinoid at a concentration ranging from about 0.01 .mu.M to about
50 .mu.M. In a preferred embodiment, the retinoid is RA.
[0259] In other embodiments of the present invention, the
expression of the PDX1 gene product in cell cultures or cell
populations comprising SOX17-positive definitive endoderm cells is
increased by contacting the SOX17-positive cells with a
differentiation factor of the fibroblast growth factor family. Such
differentiation factors can either be used alone or in conjunction
with RA. In some embodiments, the SOX17-positive definitive
endoderm cells are contacted with a fibroblast growth factor at a
concentration ranging from about 10 ng/ml to about 1000 ng/ml. In a
preferred embodiment, the FGF growth factor is FGF-10.
[0260] In some embodiments of the present invention, the expression
of the PDX1 gene product in cell cultures or cell populations
comprising SOX17-positive definitive endoderm cells is increased by
contacting the SOX17-positive cells with B27. This differentiation
factor can either be used alone or in conjunction with one or both
of retinoid and FGF family differentiation factors. In some
embodiments, the SOX17-positive definitive endoderm cells are
contacted with B27 at a concentration ranging from about 0.1% (v/v)
to about 20% (v/v). In a preferred embodiment, the SOX17-positive
definitive endoderm cells are contacted with RA, FGF-10 and
B27.
[0261] Methods for increasing the expression of the PDX1 gene
product in cell L cultures or cell populations comprising
SOX17-positive definitive endoderm cells can be carried out in
growth medium containing reduced or no serum. In some embodiments,
serum concentrations range from about 0.05% (v/v) to about 20%
(v/v). In some embodiments, the SOX17-positive cells are grown with
serum replacement.
[0262] Identification of Factors Capable of Promoting the
Differentiation of PDX1-Negative Definitive Endoderm Cells to
PDX1-Positive Foregut Endoderm Cells
[0263] Additional aspects of the present invention relate to
methods of identifying one or more differentiation factors capable
of promoting the differentiation of PDX1-negative definitive
endoderm cells to PDX1-positive foregut endoderm cells. In such
methods, a cell culture or cell population comprising PDX1-negative
definitive endoderm cells is obtained and the expression of PDX1 in
the cell culture or cell population is determined. After
determining the expression of PDX1, the cells of the cell culture
or cell population are contacted with a candidate differentiation
factor. In some embodiments, the expression of PDX1 is determined
at the time of contacting or shortly after contacting the cells
with a candidate differentiation factor. PDX1 expression is then
determined at one or more times after contacting the cells with the
candidate differentiation factor. If the expression of PDX1 has
increased after contact with the candidate differentiation factor
as compared to PDX1 expression prior to contact with the candidate
differentiation factor, the candidate differentiation factor is
identified as capable of promoting the differentiation of
PDX1-negative definitive endoderm cells to PDX1-positive foregut
endoderm cells.
[0264] In some embodiments, the above-described methods of
identifying factors capable of promoting the differentiation of
PDX1-negative definitive endoderm cells to PDX1-positive foregut
endoderm cells also include determining the expression of the
HOXA13 gene and/or the HOXC6 gene in the cell culture or cell
population. In such embodiments, the expression of HOXA13 and/or
HOXC6 is determined both before and after the cells are contacted
with the candidate differentiation factor. If the expression of
PDX1 and HOXA13 has increased after contact with the candidate
differentiation factor as compared to PDX1 and HOXA13 expression
prior to contact with the candidate differentiation factor, the
candidate differentiation factor is identified as capable of
promoting the differentiation of PDX1-negative definitive endoderm
cells to PDX1-positive foregut endoderm cells. Similarly, if the
expression of PDX1 and HOXC6 has increased after contact with the
candidate differentiation factor as compared to PDX1 and HOXC6
expression prior to contact with the candidate differentiation
factor, the candidate differentiation factor is identified as
capable of promoting the differentiation of PDX1-negative
definitive endoderm cells to PDX1-positive foregut endoderm cells.
In a preferred embodiment, a candidate differentiation factor is
identified as being capable of promoting the differentiation of
PDX1-negative definitive endoderm cells to PDX1-positive foregut
endoderm cells by determining the expression of PDX1, HOXA13 and
HOXC6 both before and after contacting the cells of the cell
culture or cell population with the candidate differentiation
factor. In preferred embodiments, the expression of PDX1, HOXA13
and/or HOXC6 is determined Q-PCR.
[0265] It will be appreciated that in some embodiments, the
expression of one or more of PDX1, HOXA13 and HOXC6 can be
determined at the time of contacting or shortly after contacting
the cells of the cell cultures or cell populations with a candidate
differentiation factor rather than prior to contacting the cells
with a candidate differentiation factor. In such embodiments, the
expression of one or more of PDX1, HOXA13 and HOXC6 at the time of
contacting or shortly after contacting the cells with a candidate
differentiation factor is compared to the expression of one or more
of PDX1, HOXA13 and HOXC6 at one or more times after contacting the
cells with a candidate differentiation factor.
[0266] In some embodiments of the above-described methods, the one
or more times at which PDX1 expression is determined after
contacting the cells with the candidate differentiation factor can
range from about 1 hour to about 10 days. For example, PDX1
expression can be determined about 1 hour after contacting the
cells with the candidate differentiation factor, about 2 hours
after contacting the cells with the candidate differentiation
factor, about 4 hours after contacting the cells with the candidate
differentiation factor, about 6 hours after contacting the cells
with the candidate differentiation factor, about 8 hours after
contacting the cells with the candidate differentiation factor,
about 10 hours after contacting the cells with the candidate
differentiation factor, about 12 hours after contacting the cells
with the candidate differentiation factor, about 16 hours after
contacting the cells with the candidate differentiation factor,
about 24 hours after contacting the cells with the candidate
differentiation factor, about 2 days after contacting the cells
with the candidate differentiation factor, about 3-days after
contacting the cells with the candidate differentiation factor,
about 4 days after contacting the cells with the candidate
differentiation factor, about 5 days after contacting the cells
with the candidate differentiation factor, about 6 days after
contacting the cells with the candidate differentiation factor,
about 7 days after contacting the cells with the candidate
differentiation factor, about 8 days after contacting the cells
with the candidate differentiation factor, about 9 days after
contacting the cells with the candidate differentiation factor,
about 10 days after contacting the cells with the candidate
differentiation factor or more than 10 days after contacting the
cells with the candidate differentiation factor.
[0267] Candidate differentiation factors for use in the methods
described herein can be selected from compounds, such as
polypeptides and small molecules. For example, candidate
polypeptides can include, but are not limited to, growth factors,
cytokines, chemokines, extracellular matrix proteins, and synthetic
peptides. In a preferred embodiment, the growth factor is from the
FGF family, for example FGF-10. Candidate small molecules include,
but are not limited to, compounds generated from combinatorial
chemical synthesis and natural products, such as steroids,
isoprenoids, terpenoids, phenylpropanoids, alkaloids and
flavinoids. It will be appreciated by those of ordinary skill in
the art that thousands of classes of natural and synthetic small
molecules are available and that the small molecules contemplated
for use in the methods described herein are not limited to the
classes exemplified above. Typically, small molecules will have a
molecular weight less than 10,000 amu. In a preferred embodiment,
the small molecule is a retinoid, for example RA.
[0268] Identification of Factors Capable of Promoting the
Differentiation of PDX1-Positive Foregut Endoderm Cells
[0269] Other aspects of the present invention relate to methods of
identifying one or more differentiation factors capable of
promoting the differentiation of PDX1-positive foregut endoderm
cells. In such methods, a cell culture or cell population
comprising PDX1-positive foregut endoderm cells is obtained and the
expression of a marker in the cell culture or cell population is
determined. After determining the expression of the marker, the
cells of the cell culture or cell population are contacted with a
candidate differentiation factor. In some embodiments, the
expression of the marker is determined at the time of contacting or
shortly after contacting the cells with a candidate differentiation
factor. The expression of the same marker is then determined at one
or more times after contacting the cells with the candidate
differentiation factor. If the expression of the marker has
increased or decreased after contact with the candidate
differentiation factor as compared to the marker expression prior
to contact with the candidate differentiation factor, the candidate
differentiation factor is identified as capable of promoting the
differentiation of PDX1-positive foregut endoderm cells. In
preferred embodiments, expression of the marker is determined by
Q-PCR.
[0270] In some embodiments of the above-described methods, the one
or more times at which the marker expression is determined after
contacting the cells with the candidate differentiation factor can
range from about 1 hour to about 10 days. For example, marker
expression can be determined about 1 hour after contacting the
cells with the candidate differentiation factor, about 2 hours
after contacting the cells with the candidate differentiation
factor, about 4 hours after contacting the cells with the candidate
differentiation factor, about 6 hours after contacting the cells
with the candidate differentiation factor, about 8 hours after
contacting the cells with the candidate differentiation factor,
about 10 hours after contacting the cells with the candidate
differentiation factor, about 12 hours after contacting the cells
with the candidate differentiation factor, about 16 hours after
contacting the cells with the candidate differentiation factor,
about 24 hours after contacting the cells with the candidate
differentiation factor, about 2 days after contacting the cells
with the candidate differentiation factor, about 3 days after
contacting the cells with the candidate differentiation factor,
about 4 days after contacting the cells with the candidate
differentiation factor, about 5 days after contacting the cells
with the candidate differentiation factor, about 6 days after
contacting the cells with the candidate differentiation factor,
about 7 days after contacting the cells with the candidate
differentiation factor, about 8 days after contacting the cells
with the candidate differentiation factor, about 9 days after
contacting the cells with the candidate differentiation factor,
about 10 days after contacting the cells with the candidate
differentiation factor or more than 10 days after contacting the
cells with the candidate differentiation factor.
[0271] As described previously, candidate differentiation factors
for use in the methods described herein can be selected from
compounds such as polypeptides and small molecules.
[0272] Although each of the methods disclosed herein have been
described with respect to PDX1-positive foregut endoderm cells, it
will be appreciated that in certain embodiments, these methods can
be used to produce compositions comprising the PDX1-positive
foregut/midgut endoderm cells that are described herein and/or the
PDX1-positive endoderm cells of the posterior portion of the
foregut that are described herein. Furthermore, any of the
PDX1-positive endoderm cell types disclosed in this specification
can be utilized in the screening methods described herein.
[0273] 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
[0274] 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
[0275] 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.
[0276] 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
[0277] 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
[0278] 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.
[0279] 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.
[0280] 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).
[0281] 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.
[0282] 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.
[0283] SOX7 and SOX18 are the closest Sox family relatives to SOX17
as depicted in the relational dendrogram shown in FIG. 3. We
employed the human SOX7 polypeptide as a negative control to
demonstrate that the SOX17 antibody produced by genetic
immunization is specific for SOX17 and does not react with its
closest family member. In particular, SOX7 and other proteins were
expressed in human fibroblasts, and then, analyzed for cross
reactivity with the SOX17 antibody by Western blot and ICC. For
example, the following methods were utilized for the production of
the SOX17, SOX7 and EGFP expression vectors, their transfection
into human fibroblasts and analysis by Western blot. Expression
vectors employed for the production of SOX17, SOX7, and EGFP were
pCMV6 (OriGene Technologies, Inc., Rockville, Md.), pCMV-SPORT6
(Invitrogen, Carlsbad, Calif.) and pEGFP-N1 (Clonetech, Palo Alto,
Calif.), respectively. For protein production, telomerase
immortalized MDX human fibroblasts were transiently transfected
with supercoiled DNA in the presence of Lipofectamine 2000
(Invitrogen, Carlsbad, Calif.). Total cellular lysates were
collected 36 hours post-transfection in 50 mM TRIS-HCl (pH 8), 150
mM NaCl, 0.1% SDS, 0.5% deoxycholate, containing a cocktail of
protease inhibitors (Roche Diagnostics Corporation, Indianapolis,
Ind.). Western blot analysis of 100 .mu.g of cellular proteins,
separated by SDS-PAGE on NuPAGE (4-12% gradient polyacrylamide,
Invitrogen, Carlsbad, Calif.), and transferred by electro-blotting
onto PDVF membranes (Hercules, Calif.), were probed with a
{fraction (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.).
[0284] 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 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
[0285] Partially differentiated hESCs were co-labeled with SOX17
and AFP antibodies to demonstrate that the SOX17 antibody is
specific for human SOX17 protein and furthermore marks definitive
endoderm. It has been demonstrated that SOX17, SOX7 (which is a
closely related member of the SOX gene family subgroup F (FIG. 3))
and AFP are each expressed in visceral endoderm. However, AFP and
SOX7 are not expressed in definitive endoderm cells at levels
detectable by ICC, and thus, they can be employed as negative
markers for bonifide definitive endoderm cells. It was shown that
SOX17 antibody labels populations of cells that exist as discrete
groupings of cells or are intermingled with AFP positive cells. In
particular, FIG. 5A shows that small numbers of SOX17 cells were
co-labeled with AFP; however, regions were also found where there
were little or no AFP.sup.+ cells in the field of SOX17.sup.+ cells
(FIG. 5B). Similarly, since parietal endoderm has been reported to
express SOX17, antibody co-labeling with SOX17 together with the
parietal markers SPARC and/or Thrombomodulin (TM) can be used to
identify the SOX17.sup.+ cells that are parietal endoderm. As shown
in FIGS. 6A-C, Thrombomodulin and SOX17 co-labeled parietal
endoderm cells were produced by random differentiation of hES
cells.
[0286] In view of the above cell labeling experiments, the identity
of a definitive endoderm cell can be established by the marker
profile SOX17.sup.hi/AFP.sup.lo/[TM.sup.lo or SPARC.sup.lo]. In
other words, the expression of the SOX17 marker is greater than the
expression of the AFP marker, which is characteristic of visceral
endoderm, and the TM or SPARC markers, which are characteristic of
parietal endoderm. Accordingly, those cells positive for SOX17 but
negative for AFP and negative for TM or SPARC are definitive
endoderm.
[0287] 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.
[0288] 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.
[0289] 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
[0290] 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.
[0291] 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.
[0292] 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.
[0293] 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.
[0294] 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.
[0295] 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.
[0296] 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.
1TABLE 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
[0297] 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.
[0298] 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.
[0299] 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
[0300] Human ES cell cultures randomly differentiate if cultured
under conditions that do not actively maintain their
undifferentiated state. This heterogeneous differentiation results
in production of extra-embryonic endoderm cells comprised of both
parietal and visceral endoderm (AFP, SPARC and SOX7 expression) as
well as early ectodermal and mesodermal derivatives as marked by
ZIC1 and Nestin (ectoderm) and Brachyury (mesoderm) expression.
Definitive endoderm cell appearance has not been examined or
specified for lack of specific antibody markers in ES cell
cultures. As such, and by default, early definitive endoderm
production in ES cell cultures has not been well studied. Since
satisfactory antibody reagents for definitive endoderm cells have
been unavailable, most of the characterization has focused on
ectoderm and extra-embryonic endoderm. Overall, there are
significantly greater numbers of extra-embryonic and neurectodermal
cell types in comparison to SOX17.sup.hi definitive endoderm cells
in randomly differentiated ES cell cultures.
[0301] As undifferentiated hESC colonies expand on a bed of
fibroblast feeders, the cells at the edges of the colony take on
alternative morphologies that are distinct from those cells
residing within the interior of the colony. Many of these outer
edge cells can be distinguished by their less uniform, larger cell
body morphology and by the expression of higher levels of OCT4. It
has been described that as ES cells begin to differentiate they
alter the levels of OCT4 expression up or down relative to
undifferentiated ES cells. Alteration of OCT4 levels above or below
the undifferentiated threshold may signify the initial stages of
differentiation away from the pluripotent state.
[0302] When undifferentiated colonies were examined by SOX17
immunocytochemistry, occasionally small 10-15-cell clusters of
SOX17-positive cells were detected at random locations on the
periphery and at the junctions between undifferentiated hESC
colonies. As noted above, these scattered pockets of outer colony
edges appeared to be some of the first cells to differentiate away
from the classical ES cell morphology as the colony expanded in
size and became more crowded. Younger, smaller fully
undifferentiated colonies (<1 mm; 4-5 days old) showed no SOX17
positive cells within or at the edges of the colonies while older,
larger colonies (1-2 mm diameter, >5 days old) had sporadic
isolated patches of SOX17 positive, AFP negative cells at the
periphery of some colonies or in regions interior to the edge (that
did not display the classical hESC morphology described previously.
Given that this was the first development of an effective SOX17
antibody, definitive endoderm cells generated in such early
"undifferentiated" ES cell cultures have never been previously
demonstrated.
[0303] Based on negative correlations of SOX17 and SPARC gene
expression levels by Q-PCR, the vast majority of these SOX17
positive, AFP negative cells will be negative for parietal endoderm
markers by antibody co-labeling. This was specifically demonstrated
for TM-expressing parietal endoderm cells as shown in FIGS. 15A-B.
Exposure to Nodal factors activin A and B resulted in a dramatic
decrease in the intensity of TM expression and the number of TM
positive cells. By triple labeling using SOX17, AFP and TM
antibodies on an activin treated culture, clusters of SOX17
positive cells that were also negative for AFP and TM were observed
(FIGS. 16A-D). These are the first cellular demonstrations of SOX17
positive definitive endoderm cells in differentiating hESC cultures
(FIGS. 16A-D and 17).
[0304] With the SOX17 antibody and Q-PCR tools described above we
have explored a number of procedures capable of efficiently
programming hESCs to become SOX17.sup.hi/AFP.sup.lo/SPARC/TM.sup.lo
definitive endoderm cells. We applied a variety of differentiation
protocols aimed at increasing the number and proliferative capacity
of these cells as measured at the population level by Q-PCR for
SOX17 gene expression and at the level of individual cells by
antibody labeling of SOX17 protein.
[0305] 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.
[0306] 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.
[0307] 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.
[0308] 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.
[0309] 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.
[0310] In addition to the TGF.beta. family members, the Wnt family
of molecules may play a role in specification and/or maintenance of
definitive endoderm. The use of Wnt molecules was also beneficial
for the differentiation of hESCs to definitive endoderm as
indicated by the increased SOX17 gene expression in samples that
were treated with activins plus Wnt3a over that of activins alone
(FIG. 23).
[0311] All of the experiments described above were performed using
a tissue culture medium containing 10% serum with added factors.
Surprisingly, we discovered that the concentration of serum had an
effect on the level of SOX17 expression in the presence of added
activins as shown in FIGS. 24A-C. When serum levels were reduced
from 10% to 2%, SOX17 expression tripled in the presence of
activins A and B.
[0312] 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
[0313] As described above, hESCs can be induced to differentiate to
the definitive endoderm germ layer by the application of cytokines
of the TGF.beta. family and more specifically of the activin/nodal
subfamily. Additionally, we have shown that the proportion of fetal
bovine serum (FBS) in the differentiation culture medium effects
the efficiency of definitive endoderm differentiation from hESCs.
This effect is such that at a given concentration of activin A in
the medium, higher levels of FBS will inhibit maximal
differentiation to definitive endoderm. In the absence of exogenous
activin A, differentiation of hESCs to the definitive endoderm
lineage is very inefficient and the FBS concentration has much
milder effects on the differentiation process of hESCs.
[0314] 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.
[0315] 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).
[0316] 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.
[0317] 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 hESCs at this developmental time
period.
[0318] 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.
[0319] 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 (FIG. 29 C and F). These results demonstrate that
the CXCR4 expression patterns not only correspond to definitive
endoderm gene expression but also to the number of definitive
endoderm cells in each condition.
Example 8
Differentiation Conditions that Enrich for Definitive Endoderm
Increase the Proportion of CXCR4 Positive Cells
[0320] The dose of activin A also effects the efficiency at which
definitive endoderm can be derived from hESCs. This example
demonstrates that increasing the dose of activin A increases the
proportion of CXCR4.sup.+ cells in the culture.
[0321] hESCs were differentiated in RPMI media supplemented with
0.5%-2% FBS (increased from 0.5% to 1.0% to 2.0% over the first 3
days of differentiation) and either 0, 10, or 100 ng/ml of activin
A. After 7 days of differentiation the cells were dissociated in
PBS without Ca.sup.2+/Mg.sup.2+ containing 2% FBS and 2 mM (EDTA)
for 5 minutes at room temperature. The cells were filtered through
35 .mu.m nylon filters, counted and pelleted. Pellets were
resuspended in a small volume of 50% human serum/50% normal donkey
serum and incubated for 2 minutes on ice to block non-specific
antibody binding sites. To this, 1 .mu.l of mouse anti-CXCR4
antibody (Abcam, cat# ab10403-100) was added per 50 .mu.l
(containing approximately 10.sup.5 cells) and labeling proceeded
for 45 minutes on ice. Cells were washed by adding 5 ml of PBS
containing 2% human serum (buffer) and pelleted. A second wash with
5 ml of buffer was completed then cells were resuspended in 50
.mu.l buffer per 10.sup.5 cells. Secondary antibody (FITC
conjugated donkey anti-mouse; Jackson ImmunoResearch, cat#
715-096-151) was added at 5 .mu.g/ml final concentration and
allowed to label for 30 minutes followed by two washes in buffer as
above. Cells were resuspended at 5.times.10.sup.6 cells/ml in
buffer and analyzed and sorted using a FACS Vantage (Beckton
Dickenson) by the staff at the flow cytometry core facility (The
Scripps Research Institute). Cells were collected directly into RLT
lysis buffer (Qiagen) for subsequent isolation of total RNA for
gene expression analysis by real-time quantitative PCR.
[0322] 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
[0323] 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.
[0324] 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.
[0325] Gene expression analysis revealed that the CXCR4.sup.+ cells
contain not only the majority of the CXCR4 gene expression, but
they also contained gene expression for other markers of definitive
endoderm. As shown in FIGS. 31A-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-- 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
[0326] 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.
[0327] Using the methods such as those described in the above
Examples, hESCs were differentiated to produce definitive endoderm.
In particular, to increase the yield and purity in differentiating
cell cultures, the serum concentration of the medium was controlled
as follows: 0.2% FBS on day 1, 1.0% FBS on day 2 and 2.0% FBS on
days 3-6. Differentiated cultures were sorted by FACS using three
cell surface epitopes, E-Cadherin, CXCR4, and Thrombomodulin.
Sorted cell populations were then analyzed by Q-PCR to determine
relative expression levels of markers for definitive and
extraembryonic-endoderm as well as other cell types. CXCR4 sorted
cells taken from optimally differentiated cultures resulted in the
isolation of definitive endoderm cells that were >98% pure.
[0328] Table 2 shows the results of a marker analysis for a
definitive endoderm culture that was differentiated from hESCs
using the methods described herein.
2TABLE 2 Composition of Definitive Endoderm Cultures Percent
Percent Percent Percent of Definitive Extraembryonic hES Marker(s)
culture Endoderm endoderm cells SOX17 70-80 100 Thrombomodulin
<2 0 75 AFP <1 0 25 CXCR4 70-80 100 0 ECAD 10 0 100 other
(ECAD neg.) 10-20 Total 100 100 100 100
[0329] 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.
[0330] We have discovered that the purity of definitive endoderm in
the differentiating cell cultures that are obtained prior to FACS
separation can be improved as compared to the above-described low
serum procedure by maintaining the FBS concentration at <0.5%
throughout the 5-6 day differentiation procedure. However,
maintaining the cell culture at <0.5% throughout the 5-6 day
differentiation procedure also results in a reduced number of total
definitive endoderm cells that are produced.
[0331] 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
[0332] 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.
[0333] Human embryonic stem cells (hESCs) were maintained in DMEM/F
12 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).
[0334] Cells were isolated by fluorescence activated cell sorting
(FACS) in order to obtain purified populations of hESCs and
definitive endoderm for gene expression analysis.
Immuno-purification was achieved for hESCs using SSEA4 antigen
(R&D Systems, cat# FAB1435P) and for definitive endoderm using
CXCR4 (R&D Systems, cat# FAB170P). Cells were dissociated using
trypsin/EDTA (Invitrogen, cat# 25300-054), washed in phosphate
buffered saline (PBS) containing 2% human serum and resuspended in
100% human serum on ice for 10 minutes to block non-specific
binding. Staining was carried out for 30 minutes on ice by adding
200 .mu.l of phycoerythrin-conjugated antibody to 5.times.10.sup.6
cells in 800 .mu.l human serum. Cells were washed twice with 8 ml
of PBS buffer and resuspended in 1 ml of the same. FACS isolation
was carried out by the core facility of The Scripps Research
Institute using a FACS Vantage (BD Biosciences). Cells were
collected directly into RLT lysis buffer and RNA was isolated by
RNeasy according to the manufacturers instructions (Qiagen).
[0335] 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.
[0336] FIGS. 34A-M show the gene expression results for certain
markers. Results are displayed for cell cultures analyzed 1, 3 and
5 days after the addition of 100 ng/ml activin A, CXCR4-expressing
definitive endoderm cells purified at the end of the five day
differentiation procedure (CXDE), and in purified hESCs. A
comparison of FIGS. 34C and G-M demonstrates that the six marker
genes, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1, exhibit an
expression pattern that is almost identical to each other and which
is also identical to the pattern of expression of CXCR4 and the
ratio of SOX17/SOX7. As described previously, SOX17 is expressed in
both the definitive endoderm as well as in the SOX7-expressing
extra-embryonic endoderm. Since SOX7 is not expressed in the
definitive endoderm, the ratio of SOX17/SOX7 provides a reliable
estimate of definitive endoderm contribution to the SOX17
expression witnessed in the population as a whole. The similarity
of panels G-L and M to panel C indicates that FGF17, VWF, CALCR,
FOXQ1, CMKOR1 and CRIP1 are likely markers of definitive endoderm
and that they are not significantly expressed in extra-embryonic
endoderm cells.
[0337] It will be appreciated that the Q-PCR results described
herein can be further confirmed by ICC.
Example 12
Retinoic Acid and FGF-10 Induces PDX1 Specifically in Definitive
Endoderm Cultures
[0338] The following experiment demonstrates that RA and FGF-10
induces the expression of PDX1 in definitive endoderm cells.
[0339] Human embryonic stem cells were cultured with or without
activins for four days. On day four, 1 .mu.M RA and 50 ng/ml FGF-10
were added to the cell culture. Forty-eight hours after the
RA/FGF-10 addition, the expression of the PDX1 marker gene and
other marker genes not specific to foregut endoderm were
quantitated by Q-PCR.
[0340] The application of RA to definitive endoderm cells caused a
robust increase in PDX1 gene expression (see FIG. 35) without
increasing the expression of visceral endoderm (SOX7, AFP), neural
(SOX1, ZIC1), or neuronal (NFM) gene expression markers (see FIG.
36A-F). PDX1 gene expression was induced to levels approximately
500-fold higher than observed in definitive endoderm after 48 hours
exposure to 1 .mu.M RA and 50 ng/ml FGF-10. Furthermore, these
results show that substantial PDX1 induction occurred only in cell
cultures which had been previously differentiated to definitive
endoderm (SOX17) as indicated by the 160-fold higher PDX1
expression found in the activin treated cell cultures relative to
those cultures that received no activin prior to RA
application.
Example 13
FGF-10 Provides Additional Increase in PDX1 Expression Over RA
Alone
[0341] This Example shows that the combination of RA and FGF-10
induces PDX1 expression to a greater extent than RA alone.
[0342] As in the previous Example, hESCs were cultured with or
without activins for four days. On day four, the cells were treated
with one of the following: 1 .mu.M RA alone; 1 .mu.M RA in
combination with either FGF-4 or FGF-10; or 1 .mu.M RA in
combination with both FGF-4 and FGF-10. The expression of PDX1,
SOX7 and NFM were quantitated by Q-PCR ninety six hours after RA or
RA/FGF.
[0343] The treatment of hESC cultures with activin followed by
retinoic acid induced a 60-fold increase in PDX1 gene expression.
The addition of FGF-4 to the RA treatment induced slightly more
PDX1 (approximately 3-fold over RA alone). However, by adding
FGF-10 and retinoic acid together, the induction of PDX1 was
further enhanced 60-fold over RA alone (see FIG. 37A). This very
robust PDX1 induction was greater than 1400-fold higher than with
no activin or RA/FGF treatment. Interestingly, addition of FGF-4
and FGF-10 simultaneously abolished the beneficial effect of the
FGF-10, producing only the modest PDX1 increase attributed to FGF-4
addition.
[0344] Addition of RA/FGF-4 or RA/FGF-10 combinations did not
increase the expression of marker genes not associated with foregut
endoderm when compared to cells not exposed to RA/FGF combinations
(see FIG. 37B-C).
Example 14
Retinoic Acid Dose Affects Anterior-Posterior (A-P) Position In
Vitro
[0345] To determine whether the dose of RA affects A-P position in
in vitro cell cultures, the following experiment was performed.
[0346] Human embryonic stem cells were cultured with or without
activins for four days. On day four, FGF-10 at 50 ng/ml was added
to the culture in combination with RA at 0.04 .mu.M, 0.2 .mu.M or
1.0 .mu.M. The expression of the PDX1 marker gene as well as other
markers not specific for foregut endoderm were quantitated by
Q-PCR.
[0347] The addition of retinoic acid at various doses, in
combination with FGF-10 at 50 ng/ml, induced differential gene
expression patterns that correlate with specific anterior-posterior
positional patterns. The highest dose of RA (1 .mu.M)
preferentially induced expression of anterior endoderm marker
(HOXA3) and also produced the most robust increase in PDX1 (FIG.
38A-B). The middle dose of RA (0.2 .mu.M) induced midgut endoderm
markers (CDX1, HOXC6) (see FIGS. 38C and 41E), while the lowest
dose of RA (0.04 .mu.M) preferentially induced a marker of hindgut
endoderm (HOXA13) (see FIG. 38D). The RA dose had essentially no
effect on the relative expression of either neural (SOX1) or
neuronal (NFM) markers (see FIG. 38F-G). This example highlights
the use of RA as a morphogen in vitro and in particular as a
morphogen of endoderm derivatives of differentiating hESCs.
Example 15
Use of B27 Supplement Enhances Expression of PDX1
[0348] PDX1 expression in definitive endoderm can be influenced by
the use of a number of factors and cell growth/differentiation
conditions. In the following experiment, we show that the use of
B27 supplement enhances the expression of PDX1 in definitive
endoderm cells.
[0349] Human embryonic stem cells were induced to differentiate to
definitive endoderm by treatment of undifferentiated hES cells
grown on mouse embryonic fibroblast feeders with high dose activin
A (100-200 ng/ml in 0.5-2% FBS/DMEM/F12) for 4 days. The no activin
A control received 0.5-2% FBS/DMEM/F12 with no added activin A. At
four days, cultures received either no activin A in 2% FBS (none),
and in 2% serum replacement (SR), or 50 ng/ml activin A together
with 2 .mu.M RA and 50 ng/ml FGF-10 in 2% FBS/DMEM/F12 (none, +FBS,
+B27) and similarly in 2% Serum replacement (SR). B27 supplement,
(Gibco/BRL), was added as a {fraction (1/50)} dilution directly
into 2% FBS/DMEM/F12 (+B27). Duplicate cell samples where taken for
each point, and total RNA was isolated and subjected to Q-PCR as
previously described.
[0350] FIG. 39A-E shows that serum-free supplement B27 provided an
additional benefit for induction of PDX1 gene expression without
inducing an increase in the expression of markers genes not
specific for foregut endoderm as compared to such marker gene
expression in cells grown without serum.
Example 16
Use of Activin B to Enhance Induction of PDX1
[0351] This Example shows that the use of activin B enhances the
differentiation of PDX1-negative cells to PDX1-positive cells in in
vitro cell culture.
[0352] Human embryonic stem cells were induced to differentiate to
definitive endoderm by treatment of undifferentiated hESCs grown on
mouse embryonic fibroblast feeders with high dose activin A (50
ng/ml) in low serum/RPMI for 6 days. The FBS dose was 0% on day
one, 0.2% on day two and 2% on days 3-6. The negative control for
definitive endoderm production (NF) received 2% FBS/RPMI with no
added activin A. In order to induce PDX1 expression, each of the
cultures received retinoic acid at 2 .mu.M in 2% FBS/RPMI on day 6.
The cultures treated with activin A on days one through five were
provided with different dosing combinations of activin A and
activin B or remained in activin A alone at 50 ng/ml. The no
activin A control culture (NF) was provided neither activin A nor
activin B. This RA/activin treatment was carried out for 3 days at
which time PDX1 gene expression was measured by Q-PCR from
duplicate cell samples.
[0353] FIG. 40A shows that the addition of activin B at doses
ranging from 10-50 ng/ml (a10, a25 and a50) in the presence of 25
ng/ml (A25) or 50 ng/ml (A50) of activin A increased the PDX1
expression at least 2-fold over the culture that received only
activin A at 50 ng/ml. The increase in PDX1 as a result of activin
B addition was without increase in HNF6 expression (see FIG. 40B),
which is a marker for liver as well as pancreas at this time in
development. This result suggests that the proportion of cells
differentiating to pancreas had been increased relative to
liver.
Example 17
Use of Serum Dose to Enhance Induction of PDX1
[0354] The expression of PDX1 in definitive endoderm cells is
influenced by the amount of serum present in the cell culture
throughout the differentiation process. The following experiment
shows that the level of serum in a culture during the
differentiation of hESCs to PDX1-negative definitive endoderm has
an effect on the expression of PDX1 during further differentiation
of these cells to PDX1-positive endoderm.
[0355] Human embryonic stem cells were induced to differentiate to
definitive endoderm by treatment of undifferentiated hESCs grown on
mouse embryonic fibroblast feeders with high dose activin A (100
ng/ml) in low serum/RPMI for 5 days. The FBS dose was 0.1% on day
one, 0.5% on day two and either 0.5%, 2% or 10% on days 3-5. The no
activin A control (NF) received the same daily FBS/RPMI dosing, but
with no added activin A. PDX1 expression was induced beginning at
day 6 by the addition of RA. During days 6-7, cultures received
retinoic acid at 2 .mu.M in 0.5% FBS/RPMI, 1 .mu.M on day 8 and 0.2
.mu.M on day 9-11. The activin A was lowered to 50 ng/ml during
retinoic acid treatment and was left absent from the no activin A
control (NF).
[0356] FIG. 41A shows that the FBS dosing during the 3 day period
of definitive endoderm induction (days 3, 4 and 5) had a lasting
ability to change the induction of PDX1 gene expression during the
retinoic acid treatment. This was without significant alteration in
the expression pattern of ZIC1 (FIG. 41B) or SOX7 (FIG. 41C) gene
expression.
Example 18
Use of Conditioned Medium to Enhance Induction of PDX1
[0357] Other factors and growth conditions which influence the
expression of PDX1 in definitive endoderm cells were also studied.
The following experiment shows the effect of conditioned media on
the differentiation of PDX1-negative definitive endoderm cells to
PDX1-positive endoderm cells.
[0358] Human embryonic stem cells were induced to differentiate to
definitive endoderm by treatment of undifferentiated hESCs grown on
mouse embryonic fibroblast feeders with high dose activin A (100
ng/ml) in low serum/RPMI for 5 days. The FBS dose was 0.2% on day
one, 0.5% on day two and 2% on days 3-5.
[0359] The definitive endoderm cultures generated by 5 days of
activin A treatment were then induced to differentiate to PDX1
expressing endoderm by the addition of RA in 2% FBS/RPMI containing
activin A at 25 ng/ml for four days. The RA was 2 .mu.M for the
first two days of addition, 1 .mu.M on the third day and 0.5 .mu.M
on the fourth day. This base medium for PDX1 induction was provided
fresh (2A25R) or after conditioning for 24 hours by one of four
different cell populations. Conditioned media (CM) were generated
from either mouse embryonic fibroblasts (MEFCM) or from hESCs that
were first differentiated for 5 days by one of three conditions; i)
3% FBS/RPMI (CM2), or ii) activin A (CM3) or iii) bone morphogenic
protein 4 (BMP4) (CM4). Activin A or BMP4 factors were provided at
100 ng/ml under the same FBS dosing regimen described above (0.2%,
0.5%, 2%). These three different differentiation paradigms yield
three very different populations of human cells by which the PDX1
induction media can be conditioned. The 3% FBS without added growth
factor (NF) yields a heterogeneous population composed in large
part of extraembryonic endoderm, ectoderm and mesoderm cells. The
activin A treated culture (A100) yields a large proportion of
definitive endoderm and the BMP4 treated culture (B100) yields
primarily trophectoderm and some extraembryonic endoderm.
[0360] FIG. 42A shows that PDX1 was induced equivalently in fresh
and conditioned media over the first two days of RA treatment.
However, by the third day PDX1 expression had started to decrease
in fresh media and MEF conditioned media treatments. The
differentiated hESCs produced conditioned media that resulted in
maintenance or further increases in the PDX1 gene expression at
levels 3 to 4-fold greater than fresh media. The effect of
maintaining high PDX1 expression in hESC-conditioned media was
further amplified on day four of RA treatment achieving levels 6 to
7-fold higher than in fresh media. FIG. 42B shows that the
conditioned media treatments resulted in much lower levels of CDX1
gene expression, a gene not expressed in the region of PDX1
expressing endoderm. This indicates that the overall purity of
PDX1-expressing endoderm was much enhanced by treating definitive
endoderm with conditioned media generated from differentiated hESC
cultures.
[0361] FIG. 43 shows that PDX1 gene expression exhibited a positive
dose response to the amount of conditioned media applied to the
definitive endoderm cells. Total volume of media added to each
plate was 5 ml and the indicated volume (see FIG. 43) of
conditioned media was diluted into fresh media (A25R). It is of
note that just 1 ml of conditioned media added into 4 ml of fresh
media was still able to induce and maintain higher PDX1 expression
levels than 5 ml of fresh media alone. This suggests that the
beneficial effect of conditioned media for induction of PDX1
expressing endoderm is dependent on the release of some substance
or substances from the cells into the conditioned media and that
this substance(s) dose dependently enhances production of
PDX1-expressing endoderm.
Example 19
Validation of Antibodies Which Bind to PDX1
[0362] Antibodies that bind to PDX1 are useful tools for monitoring
the induction of PDX1 expression in a cell population. This Example
shows that rabbit polyclonal and IgY antibodies to PDX1 can be used
to detect the presence of this protein.
[0363] In a first experiment, IgY anti-PDX1 (IgY .alpha.-PDX1)
antibody binding to PDX1 in cell lysates was validated by Western
blot analysis. In this analysis, the binding of IgY .alpha.-PDX1
antibody to 50 .mu.g of total cell lysate from MDX12 human
fibroblasts or MDX12 cells transfected 24 hrs previously with a
PDX1 expression vector was compared. The cell lysates separated by
SDS-PAGE, transferred to a membrane by electroblotting, and then
probed with the IgY .alpha.-PDX1 primary antiserum followed by
alkaline phosphatase conjugated rabbit anti-IgY (Rb .alpha.-IgY)
secondary antibodies. Different dilutions of primary and secondary
antibodies were applied to separate strips of the membrane in the
following combinations: A (500.times. dilution of primary,
10,000.times. dilution of secondary), B (2,000.times.,
10,000.times.), C (500.times., 40,000.times.), D (2,000.times.,
40,000), E (8,000.times., 40,000.times.).
[0364] Binding was detected in cells transfected with the PDX1
expression vector (PDX1-positive) at all of the tested antibody
combinations. Binding was only observed in untransfected
(PDX1-negative) fibroblasts when using the highest concentrations
of both primary and secondary antibody together (combination A).
Such non-specific binding was characterized by the detection of an
additional band at a molecular weight slightly higher than PDX1 in
both the transfected and untransfected fibroblasts.
[0365] In a second experiment, the binding of polyclonal rabbit
anti-PDX1 (Rb .alpha.-PDX1) antibody to PDX1 was tested by
immunocytochemistry. To produce a PDX1 expressing cell for such
experiments, MS 1-V cells (ATCC # CRL-2460) were transiently
transfected with an expression vector of PDX1-EGFP (constructed
using pEGFP-N1, Clontech). Transfected cells were then labeled with
Rb .alpha.-PDX1 and .alpha.-EGFP antisera. Transfected cells were
visualized by both EGFP fluorescence as well as .alpha.-EGFP
immunocytochemistry through the use of a Cy5 conjugated secondary
antibody. PDX1 immunofluorescence was visualized through the use of
an .alpha.-Rb Cy3-conjugated secondary antibody.
[0366] Binding of the Rb .alpha.-PDX1 and the .alpha.-EGPF
antibodies co-localized with GPF expression.
Example 20
Immunocytochemistry of Human Pancreatic Tissue
[0367] This Example shows that antibodies having specificity for
PDX1 can be used to identify human PDX1-positive cells by
immunocytochemistry.
[0368] In a first experiment, paraffin embedded sections of human
pancreas were stained for insulin with guinea pig anti-insulin (Gp
a-Ins) primary antibody at a {fraction (1/200)} dilution followed
by dog anti-guinea pig (D a-Gp) secondary antibody conjugated to
Cy2 at a {fraction (1/100)} dilution. In a second experiment, the
same paraffin embedded sections of human pancreas were stained for
PDX1 with IgY a-PDX1 primary antibody at a {fraction (1/4000)}
dilution followed Rb a-IgY secondary antibody conjugated to AF555
at a {fraction (1/300)} dilution. The images collected from the
first and second experiments where then merged. In a third
experiment, cells that were stained with IgY a-PDX1 antibodies were
also stained with DAPI.
[0369] Analysis of the human pancreatic sections revealed the
presence of strong staining of islets of Langerhans. Although the
strongest PDX1 signal appeared in islets (insulin-positive), weak
staining was also seen in acinar tissue (insulin-negative). DAPI
and PDX1 co-staining shows that PDX1 was mostly but not exclusively
localized to the nucleus.
Example 21
Immunoprecipitation of PDX1 from Retinoic Acid Treated Cells
[0370] To further confirm PDX1 expression in definitive endoderm
cells that have been differentiated in the presence of RA and the
lack of PDX1 in definitive endoderm cells that have not been
differentiated with RA, a rabbit anti-PDX1 (Rb a-PDX1) antibody was
used to immunoprecipitate PDX1 from both RA differentiated and
undifferentiated definitive endoderm cells. Immunoprecipitated RA
was detected by Western blot analysis using IgY a-PDX1
antibody.
[0371] To obtain undifferentiated and differentiated definitive
endoderm cell lysates for immunoprecipitation, hESCs were treated
for 5 days with activin A at 100 ng/ml in low serum (definitive
endoderm) followed by treatment with activin A at 50 ng/ml and 2
.mu.M all-trans RA for two days, 1 .mu.M for one day and 0.2 .mu.M
for one day (PDX1-positive foregut endoderm). As a positive control
cell lysates were also prepared from MS1-V cells (ATCC # CRL-2460)
transfected with a PDX1 expression vector. PDX1 was
immunoprecipitated by adding Rb a- PDX1 and rabbit-specific
secondary antibodies to each lysate. The precipitate was harvested
by centrifugation. Immunoprecipitates were dissolved in
SDS-containing buffer then loaded onto a polyacrylamide gel. After
separation, the proteins were transferred to a membrane by
electroblotting, and then probed with the IgY .alpha.-PDX1 primary
antibody followed by labeled Rb .alpha.-IgY secondary
antibodies.
[0372] Immunoprecipitates collected from the MS1-V positive control
cells as well as those from day 8 (lane d8, three days after the
start of RA treatment) and day 9 (lane d9, four days after the
start of RA) cells were positive for PDX1 protein (FIG. 44).
Precipitates obtained from undifferentiated definitive endoderm
cells (that is, day 5 cells treated with activin A--designated (A)
in FIG. 44) and undifferentiated hESCs (that is, untreated day 5
cells--designated as (NF) in FIG. 44) were negative for PDX1.
Example 22
Generation of PDX1 Promoter-EGFP Transgenic hESC Lines
[0373] In order to use the PDX1 marker for cell isolation, we
genetically tagged PDX1-positive foregut endoderm cells with an
expressible reporter gene. This Example describes the construction
of a vector comprising a reporter cassette which comprises a
reporter gene under the control of the PDX1 regulatory region. This
Example also describes the preparation of a cell, such as a human
embryonic stem cell, transfected with this vector as well as a cell
having this reporter cassette integrated into its genome.
[0374] PDX1-expressing definitive endoderm cell lines genetically
tagged with a reporter gene were constructed by placing a GFP
reporter gene under the control of the regulatory region (promoter)
of the PDX1 gene. First, a plasmid construct in which EGFP
expression is driven by the human PDX1 gene promoter was generated
by replacing the CMV promoter of vector pEGFP-N1 (Clontech) with
the human PDX1 control region (Genbank Accession No. AF192496, the
disclosure of which is incorporated herein by reference in its
entirety), which comprises a nucleotide sequence ranging from about
4.4 kilobase pairs (kb) upstream to about 85 base pairs (bp)
downstream of the PDX1 transcription start site. This region
contains the characterized regulatory elements of the PDX1 gene,
and it is sufficient to confer the normal PDX1 expression pattern
in transgenic mice. In the resulting vector, expression of EFGP is
driven by the PDX1 promoter. In some experiments, this vector can
be transfected into hESCs.
[0375] The PDX1 promoter/EGFP cassette was excised from the above
vector, and then subcloned into a selection vector containing the
neomycin phosphotransferase gene under control of the
phosphoglycerate kinase-1 promoter. The selection cassette was
flanked by flp recombinase recognition sites to allow removal of
the cassette. This selection vector was linearized, and then
introduced into hESCs using standard lipofection methods. Following
10-14 days of selection in G418, undifferentiated transgenic hESC
clones were isolated and expanded.
Example 23
Isolation of PDX1-Positive Foregut Endoderm
[0376] The following Example demonstrates that hESCs comprising the
PDX1 promoter/EGFP cassette can be differentiated into
PDX1-positive endoderm cells and then subsequently isolated by
fluorescence-activated cell sorting (FACS).
[0377] PDX1 promoter/EGFP transgenic hESCs were differentiated for
5 days in activin A-containing media followed by two days in media
comprising activin A and RA. The differentiated cells were then
harvested by trypsin digestion and sorted on a Becton Dickinson
FACS Diva directly into RNA lysis buffer or PBS. A sample of single
live cells was taken without gating for EGFP (Live) and single live
cells were gated into EGFP positive (GFP) and GFP negative (Neg)
populations. In one experiment, the EGFP positive fraction was
separated into two equally sized populations according to
fluorescence intensity (Hi and Lo).
[0378] Following sorting, cell populations were analyzed by both
Q-PCR and immunocytochemistry. For Q-PCR analysis, RNA was prepared
using Qiagen RNeasy columns and then converted to cDNA. Q-PCR was
conducted as described previously. For immunocytochemistry
analysis, cells were sorted into PBS, fixed for 10 minutes in 4%
paraformaldehyde, and adhered to glass slides using a Cytospin
centrifuge. Primary antibodies to Cytokeratin19 (KRT19) were from
Chemicon; to Hepatocyte nuclear factor 3 beta (HNF3.beta.) from
Santa Cruz; to Glucose Transporter 2 (GLUT2) from R&D systems.
Appropriate secondary antibodies conjugated to FITC (green) or
Rhodamine (Red) were used to detect binding of the primary
antibodies.
[0379] A typical FACS sort of differentiated cells is shown in FIG.
45. The percent isolated PDX1-positive cells in this example was
approximately 7%, which varied depending on the differentiation
efficiency from about 1% to about 20%.
[0380] Sorted cells were further subjected to Q-PCR analysis.
Differentiated cells showed a correlation of EGFP fluorescence with
endogenous PDX1 gene expression. Compared to non-fluorescing cells,
the EGFP positive cells showed a greater than 20-fold increase in
PDX1 expression levels (FIG. 46). The separation of high and low
EGFP intensity cells indicated that EGFP expression level
correlated with PDX1 expression level (FIG. 47). In addition to
PDX1 marker analysis, sorted cells were subjected to Q-PCR analysis
of several genes that are expressed in pancreatic endoderm.
Products of each of these marker genes (NKX2.2, GLUT2, KRT19,
HNF4.alpha. and HNF3.beta.) were all enriched in the EGFP positive
fraction (FIGS. 48A-E). In contrast, the neural markers ZIC1 and
GFAP were not enriched in sorted EGFP expressing cells (FIGS. 49A
and B).
[0381] By immunocytochemistry, virtually all the isolated
PDX1-positive cells were seen to express KRT19 and GLUT2. This
result is expected for cells of the pancreatic endoderm lineage.
Many of these cells were also HNF3.beta. positive by antibody
staining.
[0382] 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.
[0383] 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
[0384] Numerous literature and patent references have been cited in
the present patent application. Each and every reference that is
cited in this patent application is incorporated by reference
herein in its entirety.
[0385] 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:
[0386] 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.
[0387] Alexander, J., and Stainier, D. Y. (1999). A molecular
pathway leading to endoderm formation in zebrafish. Curr Biol 9,
1147-1157.
[0388] 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.
[0389] 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.
[0390] Beddington, R. S., Rashbass, P., and Wilson, V. (1992).
Brachyury--a gene affecting mouse gastrulation and early
organogenesis. Dev Suppl, 157-165.
[0391] 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.
[0392] 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.
[0393] 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.
[0394] 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.
[0395] 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.
[0396] 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.
[0397] 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.
[0398] Grapin-Botton, A., and Melton, D. A. (2000). Endoderm
development: from patterning to organogenesis. Trends Genet 16,
124-130.
[0399] 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.
[0400] Hogan, B. L. (1996). Bone morphogenetic proteins in
development. Curr Opin Genet Dev 6, 432-438.
[0401] Hogan, B. L. (1997). Pluripotent embryonic cells and methods
of making same (U.S.A., Vanderbilt University).
[0402] Howe, C. C., Overton, G. C., Sawicki, J., Solter, D., Stein,
P., and Strickland, S. (1988). Expression of SPARC/osteonectin
transcript in murine embryos and gonads. Differentiation 37,
20-25.
[0403] 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.
[0404] 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.
[0405] 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.
[0406] Katoh, M. (2002). Expression of human SOX7 in normal tissues
and tumors. Int J Mol Med 9, 363-368.
[0407] 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.
[0408] 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.
[0409] Kimelman, D., and Griffin, K. J. (2000). Vertebrate
mesendoderm induction and patterning. Curr Opin Genet Dev 10,
350-356.
[0410] 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.
[0411] 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.
[0412] 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.
[0413] 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.
[0414] 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.
[0415] 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.
[0416] 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.
[0417] 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.
[0418] Miyazono, K., Kusanagi, K., and Inoue, H. (2001). Divergence
and convergence of TGF-beta/BMP signaling. J Cell Physiol 187,
265-276.
[0419] 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.
[0420] Niwa, H. (2001). Molecular mechanism to maintain stem cell
renewal of ES cells. Cell Struct Funct 26, 137-148.
[0421] 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.
[0422] 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.
[0423] Rodaway, A., and Patient, R. (2001). Mesendoderm. an ancient
germ layer? Cell 105, 169-172.
[0424] 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.
[0425] 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.
[0426] Schier, A. F. (2003). Nodal signaling in vertebrate
development. Annu Rev Cell Dev Biol 19, 589-621.
[0427] Schoenwolf, G. C., and Smith, J. L. (2000). Gastrulation and
early mesodermal patterning in vertebrates. Methods Mol Biol 135,
113-125.
[0428] 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.
[0429] Shapiro, A. M., Lakey, J. R., Ryan, E. A., Korbutt, G. S.,
Toth, E., Warnock, G. L., Kneteman, N. M., and Rajotte, R. V.
(2000). Islet transplantation in seven patients with type I
diabetes mellitus using a glucocorticoid-free immunosuppressive
regimen. N Engl J Med 343, 230-238.
[0430] 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.
[0431] 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.
[0432] 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.
[0433] Smith, J. (1997). Brachyury and the T-box genes. Curr Opin
Genet Dev 7, 474-480.
[0434] 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.
[0435] 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.
[0436] 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.
[0437] Technau, U. (2001). Brachyury, the blastopore and the
evolution of the mesoderm. Bioessays 23, 788-794.
[0438] 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.
[0439] 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.
[0440] 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.
[0441] 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.
[0442] 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.
[0443] 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.
[0444] 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.
[0445] Wells, J. M., and Melton, D. A. (1999). Vertebrate endoderm
development. Annu Rev Cell Dev Biol 15, 393-410.
[0446] Wells, J. M., and Melton, D. A. (2000). Early mouse endoderm
is patterned by soluble factors from adjacent germ layers.
Development 127, 1563-1572.
[0447] Willison, K. (1990). The mouse Brachyury gene and mesoderm
formation. Trends Genet 6, 104-105.
[0448] Zhao, G. Q. (2003). Consequences of knocking out BMP
signaling in the mouse. Genesis 35, 43-56.
[0449] 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
2 1 1245 DNA Homo sapiens 1 atgagcagcc cggatgcggg atacgccagt
gacgaccaga gccagaccca gagcgcgctg 60 cccgcggtga tggccgggct
gggcccctgc ccctgggccg agtcgctgag ccccatcggg 120 gacatgaagg
tgaagggcga ggcgccggcg aacagcggag caccggccgg ggccgcgggc 180
cgagccaagg gcgagtcccg tatccggcgg ccgatgaacg ctttcatggt gtgggctaag
240 gacgagcgca agcggctggc gcagcagaat ccagacctgc acaacgccga
gttgagcaag 300 atgctgggca agtcgtggaa ggcgctgacg ctggcggaga
agcggccctt cgtggaggag 360 gcagagcggc tgcgcgtgca gcacatgcag
gaccacccca actacaagta ccggccgcgg 420 cggcgcaagc aggtgaagcg
gctgaagcgg gtggagggcg gcttcctgca cggcctggct 480 gagccgcagg
cggccgcgct gggccccgag ggcggccgcg tggccatgga cggcctgggc 540
ctccagttcc ccgagcaggg cttccccgcc ggcccgccgc tgctgcctcc gcacatgggc
600 ggccactacc gcgactgcca gagtctgggc gcgcctccgc tcgacggcta
cccgttgccc 660 acgcccgaca cgtccccgct ggacggcgtg gaccccgacc
cggctttctt cgccgccccg 720 atgcccgggg actgcccggc ggccggcacc
tacagctacg cgcaggtctc ggactacgct 780 ggccccccgg agcctcccgc
cggtcccatg cacccccgac tcggcccaga gcccgcgggt 840 ccctcgattc
cgggcctcct ggcgccaccc agcgcccttc acgtgtacta cggcgcgatg 900
ggctcgcccg gggcgggcgg cgggcgcggc ttccagatgc agccgcaaca ccagcaccag
960 caccagcacc agcaccaccc cccgggcccc ggacagccgt cgccccctcc
ggaggcactg 1020 ccctgccggg acggcacgga ccccagtcag cccgccgagc
tcctcgggga ggtggaccgc 1080 acggaatttg aacagtatct gcacttcgtg
tgcaagcctg agatgggcct cccctaccag 1140 gggcatgact ccggtgtgaa
tctccccgac agccacgggg ccatttcctc ggtggtgtcc 1200 gacgccagct
ccgcggtata ttactgcaac tatcctgacg tgtga 1245 2 414 PRT Homo sapiens
2 Met Ser Ser Pro Asp Ala Gly Tyr Ala Ser Asp Asp Gln Ser Gln Thr 1
5 10 15 Gln Ser Ala Leu Pro Ala Val Met Ala Gly Leu Gly Pro Cys Pro
Trp 20 25 30 Ala Glu Ser Leu Ser Pro Ile Gly Asp Met Lys Val Lys
Gly Glu Ala 35 40 45 Pro Ala Asn Ser Gly Ala Pro Ala Gly Ala Ala
Gly Arg Ala Lys Gly 50 55 60 Glu Ser Arg Ile Arg Arg Pro Met Asn
Ala Phe Met Val Trp Ala Lys 65 70 75 80 Asp Glu Arg Lys Arg Leu Ala
Gln Gln Asn Pro Asp Leu His Asn Ala 85 90 95 Glu Leu Ser Lys Met
Leu Gly Lys Ser Trp Lys Ala Leu Thr Leu Ala 100 105 110 Glu Lys Arg
Pro Phe Val Glu Glu Ala Glu Arg Leu Arg Val Gln His 115 120 125 Met
Gln Asp His Pro Asn Tyr Lys Tyr Arg Pro Arg Arg Arg Lys Gln 130 135
140 Val Lys Arg Leu Lys Arg Val Glu Gly Gly Phe Leu His Gly Leu Ala
145 150 155 160 Glu Pro Gln Ala Ala Ala Leu Gly Pro Glu Gly Gly Arg
Val Ala Met 165 170 175 Asp Gly Leu Gly Leu Gln Phe Pro Glu Gln Gly
Phe Pro Ala Gly Pro 180 185 190 Pro Leu Leu Pro Pro His Met Gly Gly
His Tyr Arg Asp Cys Gln Ser 195 200 205 Leu Gly Ala Pro Pro Leu Asp
Gly Tyr Pro Leu Pro Thr Pro Asp Thr 210 215 220 Ser Pro Leu Asp Gly
Val Asp Pro Asp Pro Ala Phe Phe Ala Ala Pro 225 230 235 240 Met Pro
Gly Asp Cys Pro Ala Ala Gly Thr Tyr Ser Tyr Ala Gln Val 245 250 255
Ser Asp Tyr Ala Gly Pro Pro Glu Pro Pro Ala Gly Pro Met His Pro 260
265 270 Arg Leu Gly Pro Glu Pro Ala Gly Pro Ser Ile Pro Gly Leu Leu
Ala 275 280 285 Pro Pro Ser Ala Leu His Val Tyr Tyr Gly Ala Met Gly
Ser Pro Gly 290 295 300 Ala Gly Gly Gly Arg Gly Phe Gln Met Gln Pro
Gln His Gln His Gln 305 310 315 320 His Gln His Gln His His Pro Pro
Gly Pro Gly Gln Pro Ser Pro Pro 325 330 335 Pro Glu Ala Leu Pro Cys
Arg Asp Gly Thr Asp Pro Ser Gln Pro Ala 340 345 350 Glu Leu Leu Gly
Glu Val Asp Arg Thr Glu Phe Glu Gln Tyr Leu His 355 360 365 Phe Val
Cys Lys Pro Glu Met Gly Leu Pro Tyr Gln Gly His Asp Ser 370 375 380
Gly Val Asn Leu Pro Asp Ser His Gly Ala Ile Ser Ser Val Val Ser 385
390 395 400 Asp Ala Ser Ser Ala Val Tyr Tyr Cys Asn Tyr Pro Asp Val
405 410
* * * * *