U.S. patent application number 11/799659 was filed with the patent office on 2007-11-08 for method of differentiating stem cells into cells of the endoderm and pancreatic lineage.
Invention is credited to Jon Odorico, Xiaofang Xu.
Application Number | 20070259423 11/799659 |
Document ID | / |
Family ID | 38541928 |
Filed Date | 2007-11-08 |
United States Patent
Application |
20070259423 |
Kind Code |
A1 |
Odorico; Jon ; et
al. |
November 8, 2007 |
Method of differentiating stem cells into cells of the endoderm and
pancreatic lineage
Abstract
Methods are described to more efficiently produce cells of the
endoderm and pancreatic lineage from mammalian pluripotent stem
cells. These methods provide a simple, reproducible culture
protocol using defined media components to enable consistent,
large-scale production of pancreatic cell types for research or
therapeutic uses.
Inventors: |
Odorico; Jon; (Fitchburg,
WI) ; Xu; Xiaofang; (Madison, WI) |
Correspondence
Address: |
QUARLES & BRADY LLP
33 E. MAIN ST, SUITE 900
P.O. BOX 2113
MADISON
WI
53701-2113
US
|
Family ID: |
38541928 |
Appl. No.: |
11/799659 |
Filed: |
May 2, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60796662 |
May 2, 2006 |
|
|
|
Current U.S.
Class: |
435/366 |
Current CPC
Class: |
C12N 2506/02 20130101;
C12N 2501/117 20130101; C12N 2501/155 20130101; C12N 2500/38
20130101; C12N 2501/115 20130101; C12N 2500/25 20130101; C12N
5/0676 20130101; C12N 2501/335 20130101 |
Class at
Publication: |
435/366 |
International
Class: |
C12N 5/08 20060101
C12N005/08 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This disclosure was made with United States government
support awarded by the following agency: NIH DK042502. The United
States has certain rights in this disclosure.
Claims
1. A method of culturing human pluripotent stem cells to produce
cells of the pancreatic lineage, the method comprising the steps of
(a) culturing the stem cells under conditions that induce
differentiation in the direction of mesendoderm, wherein the
conditions include the presence of an effective amount of a bone
morphogenetic protein; (b) culturing the cells from step (a) under
conditions favoring the formation of intact embryoid bodies (EBs),
wherein the EBs are surrounded by a layer of visceral yolk sac; and
(c) culturing the cells from the EBs of step (b) under conditions
favoring terminal differentiation of the cells to the pancreatic
lineage.
2. The method of claim 1 wherein the culture conditions in step (c)
include culturing the EB cells in a serum-free medium containing
insulin, transferrin, selenium, FGF7, nicotinamide, and
exendin-4.
3. The method of claim 1 wherein the amount of bone morphogenetic
protein ranges from about 10 ng/ml to about 100 ng/ml.
4. The method of claim 1 wherein the bone morphogenetic protein is
BMP4.
5. The method of claim 4 wherein an effective amount of BMP4 is
about 50 ng/ml.
6. The method of claim 1 wherein the stem cells of step (a) are
also cultured in the presence of an effective amount of a
fibroblast growth factor to induce differentiation in the direction
of mesendoderm.
7. The method of claim 1 wherein the amount of fibroblast growth
factor ranges from about 10 ng/ml to about 200 ng/ml.
8. The method of claim 7 wherein the fibroblast growth factor is
bFGF.
9. The method of claim 8 wherein the effective amount of bFGF is
about 100 ng/ml.
10. The method of claim 1 further comprising the step of (d)
selecting the cells of step (c) that positively express the
epithelial cell adhesion marker (EpCAM) to retain cells of the
pancreatic lineage that exhibit a reduction in tumorigenicity.
11. The method of claim 10 wherein the selecting is performed by
magnetic activated cell sorting.
12. The method of claim 1 wherein the mesendoderm cells express
Oct4 and Brachyury (T).
13. The method of claim 1 wherein the EBs include definitive
endoderm cells with duct-like structures containing Foxa2+, Sox17+
and PDX1+ cells.
14. The method of claim 1 wherein the terminally differentiated
cells simultaneously express insulin, C-peptide and PDX1.
15. A method of sequentially enriching a culture derived from human
pluripotent stem cells for cells of endoderm and pancreatic
lineages, the method comprising the steps of (a) culturing the stem
cells under conditions that induce differentiation in the direction
of mesendoderm, wherein the conditions include the presence of an
effective amount of a bone morphogenetic protein and fibroblast
growth factor; (b) culturing the cells from step (a) under
conditions favoring the formation of intact embryoid bodies (EBs),
wherein the EBs are surrounded by a layer of visceral yolk sac; and
(c) culturing the cells from the EBs of step (b) under conditions
favoring terminal differentiation of the cells to the pancreatic
lineage.
16. The method of claim 15 wherein the culture conditions in step
(c) include culturing the EB cells in a serum-free medium
containing insulin, transferrin, selenium, FGF7, nicotinamide, and
exendin-4.
17. The method of claim 15 further comprising the step of (d)
selecting the cells of step (c) that positively express the
epithelial cell adhesion molecule (EpCAM) to retain cells of the
pancreatic lineage that exhibit a reduction in tumorigenicity.
18. The method of claim 17 wherein the selecting is performed by
magnetic activated cell sorting.
19. The method of claim 15 wherein an effective amount of a bone
morphogenetic protein ranges from about 10 ng/ml to about 100 ng/ml
and an effective amount of a fibroblast growth factor ranges from
about 20 ng/ml to about 200 ng/ml.
20. A method of culturing human pluripotent stem cells to prepare a
cell population of the pancreatic lineage, which does not have
tumorigenic capability, the method comprising the steps of: (a)
culturing the stem cells under conditions that induce
differentiation in the direction of mesendoderm, wherein the
conditions include the presence of an effective amount of a bone
morphogenetic protein; (b) culturing the cells from step (a) under
conditions favoring the formation of intact embryoid bodies (EBs),
wherein the EBs are surrounded by a layer of visceral yolk sac; (c)
culturing the cells from the EBs of step (b) under conditions
favoring terminal differentiation of the cells to the pancreatic
lineage; and (d) selecting for expression of a cell surface marker
indicative of a commitment to a particular differentiated lineage,
wherein the marker is EpCAM, the resulting cell culture not forming
teratomas when injected in immunocompromised mice.
21. An isolated cell population of claim 20, step (a), the
mesendoderm cells characterized by the expression of Oct4 and
Brachyury (T).
22. An isolated cell population of claim 20, step (b), the EBs
include definitive endoderm cells with duct-like structures
containing Foxa2+, Sox17+ and PDX1+ cells.
23. An isolated cell population of claim 20, step (c), the
terminally differentiated cells simultaneously express insulin,
C-peptide and PDX1.
24. An isolated cell population of claim 20, step (d), the
differentiated cells expressing EpCAM and not forming teratomas
when injected in immunocompromised mice.
25. An isolated cell population derived from human pluripotent stem
cells comprising terminally differentiated cells of the pancreatic
lineage, wherein the cells simultaneously express one or more of
insulin, C-peptide and PDX1.
26. The isolated population of claim 25 wherein at least 50% of the
cells express one or more of insulin, C-peptide and PDX1.
27. An isolated cell population derived from human pluripotent stem
cells comprising terminally differentiated cells of the pancreatic
lineage, wherein the cells simultaneously express one or more of
insulin, C-peptide, PDX1 and EpCAM, and wherein the cells do not
form teratomas when injected in immunocompromised mice.
28. The isolated population of claim 27 wherein at least 70% of the
cells express one or more of insulin, C-peptide, PDX1 and EpCAM.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/796,662 filed May 2, 2006. This application is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] Type I diabetes is an autoimmune disease of humans caused by
destruction of pancreatic islet beta cells. At present the disease
is irreversible, although its symptoms are controlled by the
administration of exogenous insulin. Type I diabetes is one of the
most common autoimmune diseases in human populations and is a major
public health concern.
[0004] It has previously been found that transplantation of a whole
pancreas or of isolated islet cells is an effective treatment for
Type I diabetes to restore insulin independence, when combined with
immunosuppressive therapy. The success of existing therapies with
isolated islets from human cadaver donors is a proof in principle
that a cell-based therapy for human diabetes can be successful.
However, the lack of available organs or islet cells has restricted
this therapy only to very selected patients. The amount of islet
cells which can be harvested from human cadavers is extremely
limited. Therefore, a technology that is capable of producing
significant quantities of islet cells would be highly desirable
with regard to potential therapies for this disease.
[0005] Primate and human embryonic stem cells (ESCs) have been
isolated and proliferated in culture. Embryonic stem cells are stem
cells that can be maintained indefinitely through self-renewal and
proliferation in culture, but which also retain the ability to
differentiate spontaneously into cells of many different lineages.
Under nonselective conditions, it has been previously demonstrated
that a wide variety of stem cells, including mouse and human ESCs,
will differentiate spontaneously into cells of many lineages
including the pancreatic lineage. It has been previously shown that
such differentiated cells can express the pancreatic duodenal
homeobox 1 (PDX 1) gene, a transcription factor specifying the
pancreatic lineage and can also express the insulin hormone.
However, without selective conditions, stem cells will
spontaneously differentiate into a wide variety of different
lineages and only a small proportion of the cells will be
differentiated towards any particular lineage.
[0006] Culture systems that allow the spontaneous differentiation
of hESCs into insulin-staining cells have been reported (Assady, S.
et al., Insulin production by human embryonic stem cells. Diabetes
50, 1691-1697 (2001); and Segev, H. et al., Differentiation of
human embryonic stem cells into insulin-producing clusters. Stem
Cells 22, 265-274 (2004)). However, these studies neither
investigated endoderm marker expression nor demonstrated
development of cells possessing stereotypical characteristics of
.beta. cells: simultaneous expression of C-peptide and pancreatic
duodenal homeobox 1 (PDX1), which is required for pancreas
formation and co-activates the insulin promoter (Jonsson, J. et
al., Insulin-promoter-factor 1 is required for pancreas development
in mice. Nature 371, 606-609 (1994)). Because non-.beta. cells such
as neuronal cells, may express insulin (Sipione, S. et al., Insulin
expressing cells from differentiated embryonic stem cells are not
beta cells. Diabetologia 47, 499-508 (2004)), and insulin present
in the culture media may be taken up into other cell types under
certain conditions in vitro (Rajagopal, J. et al., Insulin staining
of ES cell progeny from insulin uptake. Science 299, 363 (2003)),
it is important that the endoderm and pancreatic origin of
insulin-staining cells derived from hESCs be ascertained.
[0007] It was recently reported that spontaneous differentiation of
human ESCs produced PDX1+/FOXA2+ cells and co-transplantation of
these differentiated cells with mouse dorsal pancreas (E13.5)
resulted in PDX1+/insulin+ cells, and co-staining of insulin and
C-peptide was observed (Brolen, G. K. et al., Signals from the
embryonic mouse pancreas induce differentiation of human embryonic
stem cells into insulin-producing beta-cell-like cells. Diabetes
54, 2867-2874 (2005)). This report demonstrated that pancreatic
lineage cells can be induced from spontaneously-differentiating
human ESCs by signals emanating from the embryonic pancreas.
However, the experimental methods would be impractical to adopt
into a high-throughput culture protocol and the nature of the
molecular signals was not revealed by this study. In addition,
unselected stem cell populations are tumorigenic, meaning that they
will generate non-malignant tumors, known as teratomas, in
immunodeficient animals in the same way that undifferentiated ES
cells will.
[0008] Several studies have evaluated the effects of growth factors
on human ESC differentiation to endoderm (Schuldiner, M. et al.,
Effects of eight growth factors on the differentiation of cells
derived from human embryonic stem cells. Proc Natl Acad Sci USA 97,
11307-11312 (2000) and D'Amour, K. A. et al. Efficient
differentiation of human embryonic stem cells to definitive
endoderm. Nat. Biotechnol. 23, 1534-1541 (2005). Yet, the state of
the art is that reproducible, highly efficient differentiation to
pancreatic precursors and islet cells is not routinely achievable.
Furthermore, insulin producing cells generated using previously
reported methods are less responsive to glucose (i.e. less
functionally mature) than adult human beta cells and are believed
to possess a phenotype more like immature beta cells. Taken
together, these studies indicate that additional signals may be
necessary to convert endoderm into pancreatic progenitors and
insulin expressing cells into maturely functional beta cells.
Studies of growth factor regulation of pancreas development in
embryo models may provide important insights for directing hESC
differentiation towards the pancreatic lineage (Wells, J. M. &
Melton, D. A. Early mouse endoderm is patterned by soluble factors
from adjacent germ layers. Development 127, 1563-1572 (2000)). For
example, in a chick-quail chimera system, it was demonstrated that
BMP4 induces pdx1 expression in uncommitted endoderm and noggin
blocks pdx1 expression in committed endoderm (Kumar, M. et al.,
Signals from lateral plate mesoderm instruct endoderm toward a
pancreatic fate. Dev. Biol. 259, 109-122 (2003)).
[0009] Methods have been discussed in the patent literature for the
differentiation of human ESCs, or other human pluripotent cell
types, into pancreatic or pancreatic islet cells. However, as the
technology of stem cell culture moves ahead, improvements in the
techniques to culture various differentiated cell types are
critical to the ultimate commercial use of these differentiated
cells. The previous techniques reported for culturing human ESCs
into cells of the pancreatic lineage, while suitable for laboratory
scale investigations, can not be readily scaled up to reliably and
consistently produce large numbers of pancreatic cell types for
research or for therapeutic uses. Thus, a simple, reproducible
culture method utilizing defined components that promotes islet
differentiation from human pluripotent stem cells is a desirable
addition to the field.
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention is broadly summarized as novel methods
for direct in vitro differentiation of human pluripotent stem cells
to cells of the endoderm and pancreatic lineage. The method
involves culturing the stem cells with an effective amount of a
bone morphogenetic protein to induce differentiation in the
direction of mesendoderm. These mesendoderm cells are further
cultured to form embryoid bodies (EBs), which under defined
conditions terminally differentiate to cells of to the pancreatic
lineage. By utilizing defined components that promote pancreatic
islet differentiation from human pluripotent stem cells, the
methods of the invention provide a simple, reproducible culture
protocol enabling large-scale production of pancreatic cell types
for research or therapeutic uses.
[0011] In a related aspect, the method includes culturing human
pluripotent stem cells in an effective amount of a bone
morphogenetic protein and a fibroblast growth factor to induce
differentiation in the direction of mesendoderm. This initial
culture step is followed by further culturing the cells under
defined conditions to induce differentiation of pancreatic lineage
cells.
[0012] Another feature of the direct differentiation methods
described herein is the ability to isolate on a large scale
endoderm and pancreatic cells, such as beta cells.
[0013] The methods described herein also overcome one of the
largest hurdles to potential use of stem cell derived cells for
transplant, the tumorigenic character of undifferentiated stem
cells. These methods may be used for deriving populations of
pancreatic islet cells that do not form teratomas when transplanted
into hosts.
[0014] In one aspect, isolated cell populations derived from human
pluripotent stem cells that have committed to the mesendoderm and
endoderm lineages are disclosed. The cells of this lineage are
characterized by their ability to differentiate further into
pancreatic islet cells, suitably beta cells. In a related aspect,
at least 50% of the terminally differentiated cells of the
pancreatic lineage are characterized by their ability to
simultaneously express at least one or more of insulin, c-peptide,
and PDX1 markers.
[0015] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
suitable methods and materials for the practice or testing of the
present invention are described below, other methods and materials
similar or equivalent to those described herein, which are well
known in the art, can also be used.
[0016] Other object features and advantages of the present
invention will become apparent from the following
specification.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0017] FIG. 1 is a flowchart summarizing the general method of
preparing pancreatic lineage cells from human ES cells.
[0018] FIG. 2, comprising FIGS. 2(a-f), shows a time-course of the
expression of genes: (a) secreted hCG protein levels from cultures
of BMP4/bFGF-treated hESCs; (b) human chorionic gonadotropin (hCG),
(c) brachyury (T), (d) oct4, (e) sox17 and (f) foxa2.
[0019] FIG. 3, comprising FIGS. 3(a-t), shows that BMP4 initiated
mesendoderm differentiation characterized by expression of
Brachyury, Foxa2 and Sox17. FIGS. 3 (a-n) are photomicrographs of
BMP4-treated hESCs and FIGS. 3 (o-q) are photomicrographs of
untreated hESCs: (a) Homogeneous colonies of hESC grown on MEF for
4 days. (b) hESCs grown on MEF and treated with 50 ng/ml BMP4 for 4
days become heterogeneous and display an altered cellular
morphology (Stage 1). (c-e) BMP4-treated hESCs display clusters of
brachyury-(BRACH) positive cells intermingled with FOXA2-positive
cells. (f-h) FOXA2 cells co-stain with SOX17. (i-k) In
BRACH-positive cell clusters in BMP4-treated hESCs, many cells are
co-stained with OCT4. (l-n) No co-staining of FOXA2 and OCT4 is
observed. (o-q) Untreated hESCs are stained with OCT4, but not with
BRACH. FIGS. 3 (r-t): RT-PCR (r) and Q-PCR (s, t) indicate changes
in mRNA levels of brachyury (T) and Foxa2. Scale bar for (a-b), 100
.mu.m; for (c-q): 50 .mu.m.
[0020] FIG. 4, comprising FIGS. 4 (a-g), shows the effects of BMP4
and bFGF treatment of hESCs on endoderm- and pancreas-associated
gene expression and on EB morphology. FIGS. 4 (a) RT-PCR and (b)
Q-PCR analysis depicts changes in expression of endoderm-(sox17,
foxa2, pdx1) and pancreas-associated (pdx1, glut2, insulin) genes
at EB14 for hESCs either untreated, treated with 50 ng/ml BMP4, or
treated with both 50 ng/ml BMP4 and 300 ng/ml Noggin. FIG. 4 (c)
depict mRNA levels from either hESCs grown on MEF treated with 50
ng/ml BMP4 or hESCs grown on Matrigel.TM. treated with 50 ng/ml
BMP4 alone, or treated with both 50 ng/ml BMP4 and 100 ng/ml bFGF
and analyzed at the EB14 stage. FIGS. 4 (d-g) are representative
phase contrast images of 14 day EB suspension cultures from
untreated or treated hESCs as follows: (d) EBs from untreated hESCs
grown on MEF; (e) EBs from BMP4-treated hESCs grown on MEF; (f) EBs
from BMP4-treated hESCs grown on Matrigel.TM.0; (g) EBs from hESCs
grown on Matrigel.TM. treated with both BMP4 and bFGF. Scale bar,
100 .mu.m.
[0021] FIG. 5, comprising FIGS. 5 (a-c), characterize EBs or Stage
2 cells. FIGS. 5 (a-c) are graphs showing expression levels of
endoderm and pancreas associated genes in hESC-derived EB14s. FIGS.
5 (d-o) show immunostaining of EB14s, where bFGF was added during
the EB stage and more than 50% of cells are stained with PDX1.
[0022] FIG. 6 depicts a time-course of gene expression after
plating intact EBs and culturing for 14, 21, and 28 days in growth
factor-supplemented serum-free ITSFNE media (Stage 3). EBs were
from untreated (-) or BMP4-treated (+) hESCs grown on MEF. The
expression of genes marking early neural (sox1), mesendoderm (T),
undifferentiated ESCs (oct4), trophoblasts (human chorionic
gonadotrophin, hCG), definitive endoderm (foxa2, pdx1), liver
(tyrosine amino transferase, TAT), and islet endocrine cell types
(ngn3, insulin, glucagon, glut2) were analyzed by RT-PCR.
[0023] FIG. 7, comprising FIGS. 7 (a-p), are photomicrographs
showing that PDX1+/insulin+ cells are present in cultures at
Stage3, viz. a viz., following BMP4 treatment of undifferentiated
cells, a 14 day EB formation period, and further differentiation of
plated EBs. FIGS. 7 (a-c) show that EBs plated in ITSFNE media for
14 days show co-staining of PDX1 and insulin. FIGS. 7 (d-f) depict
most PDX1-positive cells no longer co-stain with KI67 at this
stage. FIGS. 7 (g-i) show that larger clusters of
PDX1+insulin+co-staining cells appearing by EB14+28. FIGS. 7 (j-l)
show that no PDX1 or insulin staining is observed in cultures
previously not treated with BMP4. FIGS. 7 (m-p) show that cells
co-stained with Insulin, C-peptide and PDX1 at EB14+28. This
pattern is indicative of normal beta cells. Scale bar, 50
.mu.m.
[0024] FIG. 8, comprising FIGS. 8 (a-h), are photomicrographs
showing that glucagon-positive and somatostatin-positive cells are
present at EB14+28 in cultures previously treated with BMP4. FIGS.
8 (a-d) show that glucagon-positive cells are not co-stained with
C-peptide and PDX1, as would be expected on normal adult alpha
cells. FIGS. 8 (e-h) show that somatostatin-positive cells are not
co-stained with C-peptide, but some appear to co-stain with PDX1 as
would be expected on normal adult delta cells. Scale bar, 50
.mu.m.
[0025] FIG. 9 is a diagram depicting how different concentrations
of growth factors (BMP4 and bFGF) lead to different lineages:
(left) high bFGF and low BMP4 (or with Noggin to inhibit endogenous
sources of BMPs) maintain hESCs in an undifferentiated state;
(center) high bFGF and medium BMP4 leads to differentiation into
endoderm and pancreatic lineage cells; and (right) low bFGF and
high BMP4 leads to trophoectoderm/trophoblast cells.
[0026] FIG. 10, comprising FIGS. 10 (a-c), shows that MACS-sorting
for EpCAM+ cells results in enrichment of cultures for EpCAM and
endoderm and pancreas-associated transcripts. (A) Differentiated
hESCs co-express EpCAM (green) and PDX1 (red). Nearly all PDX1+
cells co-express EpCAM; some EpCAM+ cells that do not co-express
PDX1 are also seen. (B) Fold change values from QPCR analysis of
differentiated EpCAM-sorted cells compared with unsorted cells from
two independent experiments. (C) FACS analysis of EpCAM expression
following differentiation of hESCs by the BMP4/bFGF protocol. Cells
were analyzed pre and post-MACS-sorting for EpCAM. The percent of
EpCAM+ cells increases from .about.35% to .about.95% following
MACS-sorting for EpCAM.
[0027] FIG. 11 is a table showing a list of Applied Biosystems
TaqMan Assays used for quantitative and non-quantitative PCR
analysis of gene expression.
[0028] FIG. 12 is a table listing primer sequences used for
quantitative and non-quantitative PCR analysis of gene
expression.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention broadly relates to novel methods for
direct in vitro differentiation of mammalian pluripotent stem cells
to cells of the pancreatic lineage. The methods involve culturing
the stem cells in the presence of an effective amount of a bone
morphogenetic protein to induce differentiation in the direction of
mesendoderm. These mesendoderm cells are further cultured to form
embryoid bodies (EBs) enriched for definitive endoderm committed
cells, which under defined conditions terminally differentiate to
cells of the pancreatic lineage. By utilizing defined media
components that promote pancreatic islet differentiation, the
described methods provide a simple, reproducible approach to enable
large-scale production of pancreatic cell types for research or
therapeutic uses.
[0030] In an effort to better understand the methods disclosed
herein and the scientific literature surrounding them, it is noted
that recent studies with human embryonic stem cells (hESCs) have
begun to focus on the differentiation of definitive endoderm as a
first step toward development of pancreatic lineage cells. Others
have reported on Activin A induction of definitive endoderm from
hESCs (see D'Amour, K. A., et al. (2005)). However, pancreatic
lineage cells were not induced by this protocol. Furthermore,
preliminary results testing Activin A (at 5 ng/ml, 50 ng/ml, or 100
ng/ml) in serum-free media suggest that this treatment alone cannot
induce pancreatic cell differentiation. This is not surprising
given that it has been demonstrated that, in the absence of feeder
cells, Activin A can maintain pluripotency of hESCs (Beattie, G. M.
et al., Activin A maintains pluripotency of human embryonic stem
cells in the absence of feeder layers. Stem Cells 23, 489-495
(2005)). Other hESC studies evaluating pancreatic differentiation
have either been inconclusive as to the origin of insulin staining
cells or required a period of in vivo growth in undefined
conditions (Brolen, G. K. et al., (2005)).
[0031] With the goal of identifying culture conditions that promote
efficient derivation of .beta. cells from hESCs, a series of pilot
experiments were conducted to test a number of growth factors,
cytokines and developmentally-relevant molecules, including FGF4
(10 or 50 ng/ml), retinoic acid (10.sup.-9 M), FGF10 (10 ng/ml),
activin A (5 ng/ml, 50 ng/ml or 100 ng/ml), cyclopamine (1 .mu.M or
10 .mu.M) and BMP4 (5 ng/ml, 50 ng/ml or 100 ng/ml) at different
stages of hESC differentiation. Of the factors tested, BMP4
treatment of hESCs grown on MEFs had by far the strongest enhancing
effect on pdx1 expression.
[0032] The intercellular signaling molecule BMP4 is known to play
an important role in fate determination and lineage development
during embryogenesis. Several studies in other vertebrates have
shown that BMP4 inhibits early neurogenesis in ESC cultures and
promotes pancreatic endoderm specification from uncommitted
endoderm (Kumar, et al., Signals from lateral plate mesoderm
instruct endoderm toward a pancreatic fate. Dev. Biol. 259, 109-122
(2003) and Finley, et al., BMP-4 inhibits neural differentiation of
murine embryonic stem cells. J. Neurobiol. 40, 271-287 (1999)).
Based on these studies applicants hypothesized that BMP4 might
enhance endoderm and pancreatic differentiation from hESCs.
[0033] Again, through experimentation, applicants have shown that
BMP4 treatment of hESCs promotes mesendoderm differentiation and
subsequently supports pancreatic differentiation. Applicants' data,
together with the data of Xu, et al., were used to identify a
dosage effect of BMP4 on hESCs. It was found that treatment with
100 ng/ml alone transforms nearly all hESCs into trophoblast cells,
whereas lower doses were able to guide differentiation of hESCs to
mesendoderm and definitive endoderm.
[0034] It is known that certain inductive events in vivo mediated
by mesoderm-derived embryonic tissues (i.e. notochord, dorsal
aortae, lateral plate mesoderm) play an important role in the
patterning of uncommitted foregut endoderm and the specification of
a pancreatic fate (Wells, J. M. & Melton, D. A. (2000); and
Kumar, et al. (2003)). Applicants believe that effects from other
cell types and germ layers may be necessary for the further
maturation of endoderm cells and production of PDX1+ cells from
hESCs. The production of pancreatic cell types from ESCs is
probably a multi-step process, likely involving the sequential
steps of definitive endoderm induction, endoderm patterning and
induction of pancreas epithelium, each of which requires proper
environmental cues, such as soluble growth factors and cytokines,
and perhaps direct contact with other cell types.
[0035] Others report that PDX1+/FoxA2+ cells were produced when
hESCs have direct contact with MEFs and insulin-producing cells
were produced from PDX1+/FoxA2+ cells after in vivo exposure to
murine dorsal pancreas signals (Brolen, et al., (2005)). On the
other hand, it was recently reported that highly purified murine
definitive endodermal cells failed to express pancreatic markers
after short-term culture (Tada, S. et al., Characterization of
mesendoderm: a diverging point of the definitive endoderm and
mesoderm in embryonic stem cell differentiation culture.
Development 132, 4363-4374 (2005)). Similarly, purified cultures of
Activin A-induced hESC-derived endodermal cells were not shown to
express pdx1 and other signals may be necessary (see D'Amour, et
al., (2005)).
[0036] By contrast, applicants discovered that use of an
intermediate level of BMP4 results in adoption of a mesendoderm
fate (brachyury+) by many cells. The endoderm marker FoxA2 arises
during the initial treatment of hESCs with BMP4 and MEF or with
BMP4 and bFGF. Further, FoxA2 expression gradually increases
throughout the entire differentiation process and is associated
with duct-like structures in EBs. Subsequent pdx1 gene expression
and the emergence of PDX1+ cells also occurred during the EB phase.
Although FoxA2 expression is not unique to definitive endoderm, the
(1) proximity of the cells to brachyury+ cells, (2) observed
co-staining with Sox17, (3) absence of neural markers, such as
sox1, (4) low abundance of trophoblast markers in these EBs, and
(5) simultaneous expression of PDX1 suggest that the FoxA2+ cells
are likely to represent pre-pancreatic endoderm or endoderm-derived
cell populations. These results suggest that by employing a
multi-step procedure involving early BMP4 and bFGF induction of
mesendoderm with defined culture conditions illustrated in FIG. 1,
definitive endoderm and pancreatic lineage cells may be
sequentially derived from hESCs.
[0037] Accordingly, in a broad embodiment of the invention, the
method provides a method of culturing human pluripotent stem cells
in a medium containing an effective amount of bone morphogenetic
protein to induce differentiation into a mesendoderm direction. As
used herein, mammalian pluripotent stem cells include primate and
preferably human embryonic stem cells (hESCs) described by Thomson
et al. (Science 282:1145, 1998). These cells are characterized as
being capable under appropriate conditions of producing several
different cell types that are derivatives of all of the three
germinal layers (endoderm, mesoderm, and ectoderm). According to a
standard art-accepted test, these pluripotent cells also have the
ability to form a teratoma in 8-12 week old SCID mice.
[0038] In some aspects of this embodiment, the pluripotent stem
cells are cultured on a gelatin-coated tissue culture surface with
irradiated mouse embryonic fibroblast (MEF) as feeder cells. Feeder
cells are cells of one type that are co-cultured with cells of
another type, to provide an environment in which the cells of the
second type can grow. The length of culture time for this step
ranges from about 1 day to about 10 days, preferably 4 days.
Inducers of the bone morphogenetic signaling pathway include but
are not limited to bone morphogenetic proteins (BMPs), suitably
BMP2 and BMP7 and most suitably, BMP4.
[0039] As used herein, an effective amount of a BMP encompasses a
concentration ranging from about 10 ng/ml to about 100 ng/ml. It is
noted that while larger dosages of BMP4 have been shown to induce
human pluripotent stem cell differentiation into trophectoderm
lineages, it is disclosed here that culturing stem cell colonies
with low to intermediate amounts of BMP4, suitably 50 ng/ml, works
synergistically with MEFs or high concentrations of bFGF, such as
100 ng/ml, promoting the differentiation of the stem cells in the
mesendoderm direction.
[0040] As used herein, mesendoderm cells are defined by and are not
limited to the expression of Brachyury and Oct 4 nuclear
transcription factor markers (see Stage 1 in FIG. 1). Mesendoderm
cells may also be characterized by expression of Wnt3 and FGF4
(D'Amour et al. 2005) and/or goosecoid (Gsc), Ecadherin, Mixl1, and
FoxA2, and possibly Sox17 (Yasunaga et al. Nat. Biotechnol.
2005).
[0041] In the second phase of this embodiment, the cultures are
induced to form embryoid bodies (EBs) from small pluripotent stem
cell colonies in MEF conditioned media. Suitably, the EBs are
intact and are surrounded by a layer of visceral yolk sac (VYS),
which express stage specific embryonic antigen-3 (SSEA-3). The
length of culture time for this step ranges from about 1 day to
about 4 weeks, preferably 14 days. As used herein, EBs are three
dimensional structures of groups of cells which interact in such a
way to induce further differentiation within the cells of the EB
(see Stage 2 in FIG. 1). Suitable EBs include definitive endoderm
cells with duct-like structures, which include cells expressing
Foxa2, Sox17 and PDX1. It is believed that these endoderm cells
give rise to cells of the pancreatic lineage. As used herein,
pancreatic lineage cells include, for example, cells co-expressing
PDX1 and Nkx6.1, which are well known to represent either
pancreatic epithelial progenitor cells or beta cells. These cells
are the only two cell types in the body expressing this combination
of markers or PDX1, insulin, and C-peptide, which are well known to
be simultaneously expressed in normal beta cells; or cells
expressing somatostatin generally understood to represent delta
cells.
[0042] Cells within EBs derived from BMP4 treated hESCs were
characterized by immunostaining and Quantitative PCR as containing
a significant subset co-expressing both PDX1 and Nkx6.1 markers
(see FIG. 4). Some of the cells also appear to express the Ki67
marker indicating they are proliferating. Another subset appears to
be capable of committing to a terminally differentiated endocrine
lineage.
[0043] In the third phase of this embodiment, the cells from the
EBs are plated onto tissue culture plates in a serum-free medium
(without fetal bovine serum (FBS)) intended to induce terminal
differentiation into cells of the endocrine lineage. In this step,
the length of time the cells are cultured for varies from about 7
day to about 56 days, suitably 28 days. Suitable terminally
differentiated cells are characterized by the simultaneous
expression of insulin, C-peptide and PDX1. Other cell types of the
endocrine lineage, such as glucagon-expressing cells and
somatostatin-expressing cells also appear in this context and in
these regions of the cultures. A significant proportion of PDX1+
cells at these stages (stage 2 and stage 3) are found to co-express
the cell surface marker epithelial cell adhesion molecule
(EpCAM)
[0044] As used herein, serum-free medium means serum-free DMEM/F12
(17.5 mM glucose) medium with ITS supplement (BD, 5 .mu.g/ml
insulin+5 .mu.g/ml transferrin+5 ng/ml selenous acid), 10 ng/ml
FGF7 (R&D), 10 mM nicotinamide (Sigma), 10 nM exendin-4
(Sigma), and 2 g/L BSA (Sigma). Hence, the serum-free medium is
termed ITSFNE for
insulin-transferrin-selenium-FGF7-nicotiamide-exendin-4 (see Stage
3 in FIG. 1). A suitable concentration for extendin-4 ranges from
about 0.1 mM to about 1 mM. Also, the concentration of nicotinamide
may range from about 1 to about 25 mM.
[0045] Alternatively, in a related and more suitable embodiment,
the human pluripotent stem cells may be initially cultured without
feeder cells, suitably on Matrigel.TM.. When culturing hESCs on
Matrigel, applicants discovered that in addition to inducers of the
bone morphogenetic pathway, an effective amount of fibroblast
growth factors (FGFs) was required to induce the cells to
differentiate in the mesendoderm direction. The concentration of
FGF ranges from about 10 to about 200 ng/ml. A suitable bFGF
concentration is 100 ng/ml. At this stage applicants also
discovered that MEFs and the factors they elaborate or produce may
replace the function of FGFs in this induction protocol.
Supplementing bFGF to the cultures at Stage 2 leads to
significantly more cells of endoderm and pancreatic lineages, which
express the appropriate markers. The medium may be supplemented
with other suitable fibroblast growth factors, such as FGF2. To
characterize the cells of these three developmental stages, an RNA
expression time-course for a variety of endoderm- and
pancreas-associated genes was performed and described below.
[0046] In some embodiments, the invention is also directed toward
methods for deriving pancreatic cell enriched populations of cells,
suitably beta cells that do not form teratomas when transplanted
into hosts. Accordingly, it is contemplated that the stage 3 cells
of pancreatic lineage can be rendered non-tumorigenic by sorting
cells on the basis of cell surface markers as described in U.S.
Published Application No: 20050260749 to the applicants.
Specifically, the differentiated cells are sorted based on the
positive expression of the epithelial cell adhesion molecule
(EpCAM), a cell surface marker the expression of which can be used
for positive selection to identify human cells committed to the
endodermal, pancreatic or foregut lineages. For performing this
selection, any instrument capable of sorting single cells such as a
fluorescence-activated cell sorter (FACS) or magnetic activated
cell sorting (MACS) should be adaptable for use in this kind of
cell sorting procedure. Using this EpCAM/MACS selection protocol to
remove undifferentiated cells remaining in the later stage culture,
but also using antibodies to cell surface markers of
undifferentiated stem cells such as SSEA3 or SSEA4, applicants
discovered that the tumorigenic tendency of stem cell cultures can
be reduced effectively.
[0047] It is clearly evident from the stem cell literature that
when injected into immunocompromised mice, undifferentiated ES
cells will form teratomas, which are non-malignant growths or
tumors made up of many different tissue types in a poorly organized
structure. While the generation of teratomas is not thought to be
life-threatening to the host, the teratomas can grow to large size,
be unsightly and wasteful of metabolic energy to the host. A
characterization of the teratomas formed by human ES cells is found
in Gertow et al., Stem Cells and Development, 13:421-435 (2004). If
human ES cells are to be used ultimately for transplantation of
cells or tissues into human patients, the cells which are so
introduced would presumably be preferred to be free of tumorigenic
capacity. In the art, the main techniques which have been taught to
eliminate this capability are based on inserting exogenous gene
constructs into ES cells and then selecting for differentiated
cells based on expression characteristics of the introduced genes.
However, the use of exogenous genes inserted into human ES cell
cultures carries another set of safety concerns that are best
avoided.
[0048] By combining cell surface sorting technology with the direct
differentiation methods described herein, applicants reasonably
expect at a practical level to produce a stem cell derived cell
culture that is not tumorigenic and does not form teratomas. At the
risk of redundancy, and to avoid misunderstanding, the use of the
phrase tumorigenic is intended to apply to the teratoma-forming
characteristics of undifferentiated human undifferentiated stem
cells, such as ES cells and is not intended to imply malignancy of
any kind. This is because ES cells do not produce frank
malignancies when injected into mice. The removal of the
tumorigenic trait simply by direct differentiation and selection is
another important step in the progression of stem cell derivative
from laboratory model to useful human therapy.
[0049] In another embodiment, the invention provides an isolated
cell population derived from human pluripotent stem cells having
terminally differentiated cells of the pancreatic lineage. At least
50% of this cell population simultaneously express one or more of
insulin, C-peptide and PDX1.
[0050] In still another embodiment, the invention provides an
isolated non-tumorigenic cell population derived from human
pluripotent stem cells. This cell population has terminally
differentiated cells of the pancreatic lineage, wherein at least
70% of the cells simultaneously express one or more of insulin,
C-peptide, PDX1, NKX6.1 and EpCAM, and wherein the cells do not
form teratomas when injected in immunocompromised mice. Since these
cells, suitably Beta cells, develop from cultures that harbor cells
having a phenotype of endoderm (FoxA2 and Sox17) and pancreatic
progenitors (PDX1+, NKX6.1+, and insulin-proliferative), it is
expected that they express other markers, such as, NKX2.2, NeurOD,
Pax4 and IS11.
[0051] The following examples are provided as further non-limiting
illustrations of particular embodiments of the invention.
EXAMPLES
Example 1
Cell Culture and Differentiation
[0052] The general differentiation method is illustrated in
simplistic fashion in FIG. 1. NIH approved HESC lines, H1 (WA01)
and H9 (WA09) were used between passage 24 to 40. Media for
undifferentiated ESCs was comprised of 80% DMEM/F12 and 20%
Knockout serum replacement supplemented with 1 mM L-glutamine, 1%
nonessential amino acids, 0.1 mM 2-mercaptoethanol and 4 ng/ml bFGF
(all from Invitrogen). hESCs were cultured in 6-well plates on a
feeder layer of irradiated mouse embryonic fibroblasts (MEFs) in
either ESC media (control group), ESC media plus 50 ng/ml BMP4
(BMP4 group; R&D systems), or ESC media plus 50 ng/ml BMP4 plus
300 ng/ml noggin (noggin group; R&D systems) for 4 days.
[0053] In experiments with Matrigel.TM., hESCs were grown on
growth-factor depleted Matrigel.TM. (BD Biosciences) instead of MEF
and culture media was MEF-conditioned media (CM, control group), CM
plus 50 ng/ml BMP4 or CM plus 50 ng/ml BMP4 plus 100 ng/ml bFGF.
Colonies were transferred by incubating with 2 mg/ml dispase
(Invitrogen), after which cells were rinsed off the plates,
pipetted into small pieces, and filtered through a 70 .mu.m cell
strainer. The filtrates that contained small pieces of colonies
were put into 100-mm non-treated suspension culture dishes with CM
on a shaker for 14 days to form embryoid bodies (EBs). EBs were
then replated in serum-free DMEM/F-12 (17.5 mM glucose) medium with
ITS supplement (BD, 5 .mu.g/ml insulin+5 .mu.g/ml transferrin+5
ng/ml selenous acid), 10 .mu.M nicotinamide (Sigma), 10 ng/ml FGF7
(R&D), 10 nM exendin-4 (Sigma), and 2 g/L BSA (Sigma) for 14,
21 or 28 days, then harvested. A fraction of each culture was used
for RT-PCR and Q-PCR and the remaining cells were either embedded
in OCT (EB14; Tissue-Tek) or fixed on coverslips (EB14+14, EB14+21
and EB14+28) for immunostaining.
Example 2
Quantitative PCR and RT-PCR
[0054] Total cellular RNA was extracted with TriZol (Invitrogen).
cDNA was synthesized from 1 .mu.g total RNA using a SuperScript
First-Strand Synthesis kit (Invitrogen). Quantitative real time
RT-PCR (Q-PCR) was performed using Assays-on-demand agents (Applied
Biosystems) on an ABI PRISM 7700 Sequence Detection System (Applied
Biosystems) for the following transcripts: foxa2, sox17, brachyury,
ngn3, pdx1, insulin, glucagon, glut2 and an endogenous control,
.beta.-actin. Q-PCR was performed according to equipment
manufacturer's instructions. Relative quantification was carried
out using the comparative cycle threshold (C.sub.T) methods
recommended by the supplier. Fold change was calculated as:
2.sup.-.DELTA..DELTA.CT Mean .DELTA..DELTA.C.sub.T values from
Q-PCR analyses were compared using the unpaired, two-tailed
Student's t-test. P values <0.05 were considered
significant.
[0055] For non-quantitative RT-PCR, oligonucleotide primer pairs
were generated against human transcripts using Genebank sequences
(see FIG. 12). Primers were selected from two different exons and
spanned at least one intronic sequence. PCR was performed using
HotStarTaq DNA polymerase (Qiagen,) and reaction conditions were as
follows: initial denaturation at 95.degree. C. for 15 min, then
cycles of 94.degree. C. for 30 sec, 30 sec at annealing
temperature, 1 min at 72.degree. C., and a final 10 min at
72.degree. C. Primers were annealed at 53.degree. C. except for
pdx1 (56.degree. C.), sox17 (55.degree. C.) and foxa2 (50.degree.
C.; with Qiagen's Q-solution). A control sample without reverse
transcriptase (--RT) was amplified with GAPDH primers in all cases,
and human adult pancreas RNA was used as a positive control.
Example 3
Immunofluorescence Staining
[0056] Immunofluorescence staining of coverslips was carried out as
previously described (Kahan, B. W. et al., Pancreatic precursors
and differentiated islet cell types from murine embryonic stem
cells: an in vitro model to study islet differentiation. Diabetes
52, 2016-2024 (2003)). The following primary antibodies were used
at the listed dilutions: PDX1 rabbit anti-mouse serum 1:4000 (gift
of C. Wright); insulin mouse monoclonal 10 .mu.g/ml (ATCC No:
HB124); glucagon mouse monoclonal 1:2000 (Sigma); somatostatin
mouse monoclonal 1:2000 (Novonordisk); Ki-67 mouse monoclonal 1:25
(BD Pharmingen); C-peptide rat monoclonal 1:3000 (BCBC 1921);
Brachyury goat anti-human 1:20 (R&D); OCT4 goat anti mouse
1:100 (Santa Cruz); Sox17 goat anti-human 1:40 (R&D); FOXA2
rabbit anti-rat1:4000 (Gift of R. Costa). Secondary antibodies
(Goat anti-mouse IgG Alexa Fluor 488, 1:2000; Goat anti-rabbit
Alexa Fluor 568, 1:4000; Goat anti-rat Alexa Fluor 488, 1:2000;
Goat anti-rabbit, Alexa Fluor 647, 1:4000; Goat anti-mouse 568,
1:2000; Donkey anti-goat Alexa Fluor 568, 1:2000; Donkey anti-mouse
Alexa Fluor 488, 1:2000) were obtained from Molecular probes
(Eugene, Oreg.).
Example 4
Mesendoderm Induction by Treating hESCs with BMP4 on MEFs
[0057] The process of hESCs differentiation was initiated by
treating hESCs with BMP4 on MEFs, as shown overall in FIG. 1.
Notably a prior study showed that treatment of hESCs on
Matrigel.TM. with 100 ng/ml BMP4 for 7 days causes nearly 100% of
the cells to differentiate into human chorionic gonadotrophin
(hCG)-expressing trophoblast cells (Xu, R. H. et al. BMP4 initiates
human embryonic stem cell differentiation to trophoblast. Nature
Biotechnology. 20, 1261-1264 (2002)).
[0058] These results were also reproduced as shown in FIG. 2, where
after 7 days treatment with BMP4 100 ng/ml alone on Matrigel, the
cell types produced abundantly expressed hCG and secreted hCG
protein (FIGS. 2a and b). Specifically, undifferentiated hESCs
grown on Matrigel (MG) were divided into 7 groups and treated with:
(1) 10 ng/ml BMP4 (B10); (2) 50 ng/ml BMP4 (B50); (3) 100 ng/ml
BMP4 (B100); (4) 10 ng/ml BMP4+100 ng/ml bFGF (B10F); (5) 50
ng/mlBMP4+100 ng/ml bFGF (B50F); (6) 100 ng/mlBMP4+100 ng/mlbFGF
(B100F); (7) control(ctrl): without any added growth factors--for 1
day, 4 days and 7 days. As indicated above, hCG secretion and gene
expression were greatly increased in B100 group (FIG. 2a-b). By day
7, average hCG secretion was 21000 mIU/ml in culture medium and hCG
gene expression exhibited a 13000-fold increase over the control
group (statistical analysis in FIG. 2 are made with values compared
to first day control of the same group). Lower concentration of
BMP4 (10 ng/ml, 50 ng/ml) in fact had the same degree of
hCG-induction effect. On the other hand, bFGF obviously inhibits
the hCG-induction effect of BMP4. For example, the average hCG
secretion at day 7 in B10 group was 29000 mIU/ml and in B10F group
was 51 mIU/ml (FIG. 2a). At same time, T expression was greatly
induced in BMP4/bFGF-treated hESCs in a time-dependent manner (FIG.
2c). The induction peaked at day 4 with B10F group with a 1000-fold
increase and B50F about 200-fold increase. Sox17 and foxa2
expressions were also increased in BMP4/bFGF-treated hESCs (FIG.
2e-f). Oct4 expression, on the other hand, was significantly
decreased, especially in only BMP4-treated groups at day 4 (FIG.
2d).
[0059] Applicants also treated undifferentiated hESCs with 50 ng/ml
BMP4 for 4 days in the presence of either MEFs or bFGF (100 ng/ml)
This treatment morphologically altered the cells, with untreated
cells maintaining a homogeneous appearance, and treated cells
becoming heterogeneous and clearly changing their morphology (FIG.
3 a-b). BMP4 treatment results in some cells differentiating to
Brachyury+ cells, which are intermingled with relatively fewer
FoxA2+ cells in the same cell cluster (FIG. 3c-e). Applicants found
an average of 10 such clusters per 15 mm coverslip in BMP4-treated
cultures, whereas none were detected in untreated hESCs. Brachyury+
cells are also OCT4+, at least transiently (FIG. 3i-k). In
contrast, FoxA2+ cells are not OCT4+ (FIG. 3 l-n), and nearly all
FoxA2+ cells are co-stained with Sox17 (FIG. 3f-h).
[0060] FIG. 2 shows that in contrast to cells treated with BMP4
alone, cells treated with low and intermediate doses of BMP4 (10
ng/ml and 50 ng/ml) for 4 days did not express hCG or produce hCG
protein; instead, cells expressed significantly greater amounts of
brachyury and sox17 transcripts while expressing significantly
lower levels of Oct4 transcripts compared to control cells at the
same time point (FIG. 2 c-e).
[0061] In cultures not treated with BMP4, cells were OCT4+ but did
not express Brachyury (FIG. 3o-q). These OCT4 staining data are in
concert with recent reports showing that decreased OCT4 expression
induces loss of pluripotency and dedifferentiation to trophectoderm
whereas, increased OCT4 levels cause differentiation into primitive
endoderm and mesoderm (Niwa, H. et al., Quantitative expression of
Oct-3/4 defines differentiation, dedifferentiation or self-renewal
of ES cells. Nat. Genet. 24, 372-376 (2000)). Transcript levels of
Brachyury and Foxa2 are supportive of the staining data. Both
non-quantitative (FIG. 3r) and quantitative RT-PCR (FIGS. 3s and t)
show an increase in brachyury and Foxa2 transcripts when cultures
on MEFs are treated with BMP4. Applicants found an approximately
170-fold increase in brachyury gene expression (FIG. 3s) and a
roughly 5-fold increase in foxa2 expression with BMP4 treatment
(FIG. 3t). Together these data predict that a sequential series of
embryonic stages emerge from hESCs. First, brachyury+, and
brachyury and Oct4 co-expressing cells (possibly reflecting a
transition state) arise, representing mesendoderm. Then, cells go
through another rapid transition within a few days to co-express
FoxA2 and Sox17, while losing brachyury expression.
Example 5
Several Additional Essential Components for Early Pancreatic Cell
Lineage Differentiation from hESCs In Vitro are Identified
[0062] To further define the culture conditions and media additives
that promote development of pancreatic cells from hESCs in vitro,
applicants evaluated the requirement for MEFs during the
BMP4-treatment period. In BMP4-treated cells grown on Matrigel.TM.,
transcripts were not detected for any of the genes examined,
including brachyury, foxa2, pdx1 and glut2 (FIG. 4c), whereas they
were easily detected in BMP4-treated cells grown in MEFs. In
addition, hEBs derived from cells grown on MEFs vs. Matrigel.TM.
were morphologically different (FIG. 4d-g). Cells grown on
Matrigel.TM. resulted in EBs that were smaller, more compact, and
more opaque than EBs derived from BMP4-treated cells grown in the
presence of MEFs. Thus, MEFs play an important role in pancreatic
lineage specification in this protocol.
[0063] As a high concentration of bFGF has been recently reported
to have a MEF-like effect on the growth of hESCs (Ludwig, T. E., et
al., Derivation of human embryonic stem cells in defined
conditions. Nat. Biotechnol. 24, 185-187 (2006) and Levenstein, M.
E., et al., Basic FGF Support of Human Embryonic Stem Cell
Self-Renewal. Stem Cells (2005)), applicants examined whether bFGF
could replace MEFs in the culture protocol. To determine this, 50
ng/ml BMP4 and 100 ng/ml bFGF were added to hESCs grown on
Matrigel.TM. for 4 days; cells were then harvested for gene
expression analysis. Compared to untreated hESCs, brachyury gene
expression was .about.1000 fold greater in BMP4- and bFGF-treated
hESCs. EBs derived from BMP4- and bFGF-treated cells showed similar
morphologies and comparable levels of brachyury, foxa2, pdx1, and
glut2 gene expression to EBs from hESCs grown on MEFs and treated
with BMP4 (FIGS. 4c and 4e, 4g), suggesting that bFGF plays an
early, essential role in endoderm specification and/or pancreatic
lineage cell differentiation.
[0064] To determine whether the inductive effect of BMP4 on
mesendoderm formation from hESCs was dependent on canonical BMP
signaling, applicants tested whether the addition of soluble noggin
to the culture medium was able to reverse the effect of BMP4
treatment. Gene expression was examined at EB14 by RT-PCR (FIG. 4a)
and Q-PCR (FIG. 4b) after treatment of hESCs for 4 days with either
50 ng/ml BMP4 alone, or BMP4 plus 300 ng/ml noggin. The
simultaneous addition of noggin and BMP4 is capable of completely
blocking gene expression of brachyury, sox17, foxa2, pdx1 and glut2
in BMP4-treated HESC cultures. The effect of noggin was dose
dependent (data not shown). It is notable that transcript levels of
foxa2 and glut2 in noggin-treated cells were even lower than
non-BMP4 treated cells, suggesting that there exists some minimal
BMP4-like effect in HESC media or elaborated from hESCs
themselves.
Example 6
Embryoid Body (EB) Suspension Culture of BMP4-Treated hESCs
Promotes Endoderm and Pancreatic Lineage Cell Differentiation
[0065] Based on the defined role of BMP4 in pancreas specification
in chicken embryos, applicants aimed to determine whether BMP4
treatment would promote differentiation of mesendoderm and
pancreatic cells from hESCs. EBs were therefore formed from either
untreated hESCs, or hESCs treated with 50 ng/ml BMP4 for 4 days.
Applicants' previous experiments showed that differentiation
through an EB stage, in which inductive tissue interactions may
occur in three dimensions among the early embryonic germ layers,
positively influences development of pancreatic lineage cells,
compared to differentiation under two-dimensional conditions (Xu et
al., Endoderm and pancreatic lineage differentiation from human
embryonic stem cells, Cloning and Stem Cells, 8, 96-107,
(2006))).
[0066] Transcript analysis showed no detectable pdx1 expression in
untreated hESCs. After the cells were grown as EBs for 14 days,
pdx1 mRNA began to appear in the control group. Compared to the
untreated group at EB14, BMP4 treatment induced significant
up-regulation of genes including: sox17 (11 fold), foxa2 (12 fold),
tat (7 fold), pdx1 (22 fold), brachyury (8 fold) and glut2 (19
fold). Although brachyury induction by BMP4 was still significant
when cells were analyzed at EB14 (14 fold), the magnitude of
increase was much less than at the ESC stage (170 fold, treated
hESCs vs. untreated hESCs). Applicants reproducibly observed the
BMP4 effect in numerous (more than 15) independent experiments and
with two different hESC lines (H1 and H9).
[0067] In this regard, FIGS. 5 (a-c) show expression levels of
endoderm and pancreas associated genes in hESC-derived EB14s. FIG.
5 (b-c) show that BMP4/bFGF treatment induces significant
expression of endoderm and pancreas-associated genes in
hESC-derived EBs after 14 days of growth (EB14+BF vs. EB14, ctrl).
Also, the addition of 100 ng/ml bFGF during the EB stage (+F) led
to a reduction in brachyury (T) transcripts (a) on one hand and on
the other, greatly enhanced foxa2 and pdx1 expression (b and c); b)
foxa2 transcript levels increased from 219-fold greater than
undifferentiated hESCs (without bFGF during the EB phase) to
873-fold greater than undifferentiated hESCs (with bFGF during the
EB stage), and c) pdx1 transcript levels increased from 26-fold vs.
undifferentiated hESCs (without bFGF during the EB stage) to
312-fold greater than undifferentiated hESCs (with bFGF during the
EB stage). Notably, average delta Ct value of pdx1 from QPCR
testing was 7.7, comparable to the pdx1 expression level in human
islet preparations (approximately 50% pure).
[0068] Furthermore, FIGS. 5 (d-o) show immunostaining of EB14s,
where bFGF was added during the EB stage and more than 50% of cells
are stained with PDX1. The stainings are very strong and nuclear
and cells are often formed in clusters. PDX1+ cells are not
co-stained with the SOX17 antibody (FIG. 5d-f), yet some PDX1+
cells are co-stained with the NKX6.1 antibody (FIG. 5g-i). Some of
the PDX1+ cells are also Ki67+ (FIG. 5j-i), suggesting the
existence of proliferating PDX1+ cells.
[0069] The immunostaining of EB14 cells also revealed that hESCs
were capable of differentiating into mitotically-active pancreatic
progenitor cells, expressing PDX1 and Ki67 (FIG. 5j-l). Many PDX1+
cells were seen in the BMP4-treated group, most of which were also
Ki67+, suggesting that these cells were actively proliferating. In
addition, a significant proportion of cells expressing PDX1 also
co-expressed Nkx.times.6.1 (FIG. 5g-i), but the majority did not
express insulin at this stage (FIG. 5m-o). PDX1/insulin co-staining
began to appear at EB 14 (FIG. 5m-o). Notably, bFGF during the EB
stage appears to promote a transition from brachyury expressing
cells to the appearance of pancreas lineage committed cells.
[0070] The above-noted constellation of markers suggests the cells
represent a pancreatic progenitor stage similar to the
proliferating pancreatic epithelium that grows and expands during
mid-gestation prior to or around the time of the secondary
transition when most beta cells form. Furthermore, numerous FoxA2+
cells were seen in duct-like structures, and some cells were also
Ki67+. These structures formed during the EB stage may play an
important role in the induction of PDX1+ cells. In contrast, PDX1+
and FoxA2+ cells were not seen at these stages of hESC
differentiation without BMP4 treatment (data not shown).
[0071] Serum is known to have an inhibitory effect on the
differentiation of definitive endoderm from ESCs (D'Amour, K. A.,
et al. (2005)) and pancreatic lineage cell development (Gao, R., et
al., Characterization of endocrine progenitor cells and critical
factors for their differentiation in human adult pancreatic cell
culture. Diabetes 52, 2007-2015 (2003)). Cultures in which EBs are
plated in 15% serum-containing media show a gradual reduction in
pdx1 expression, as well as the expression of other endoderm and
pancreatic lineage associated genes (Xu et al., Cloning and Stem
Cells, 2006). In contrast, pdx1 transcript levels were maintained
and/or increased in growth factor-supplemented serum-free
media.
Example 7
Human ESCs Yield Cells with PDX1/Insulin/C-Peptide Co-Staining
[0072] To determine if BMP4-treated hESCs have the ability to
become fully differentiated, hormone-containing pancreatic islet
cells, applicants plated 14 day EBs onto gelatin-coated coverslips
in serum-free ITSFNE media for an additional 14, 21 and 28 days.
Transcriptional profiles over this time-course were determined by
RT-PCR (FIG. 6) and Q-PCR. After plating (EB14+14, EB14+21 and
EB14+28), endoderm and pancreas-associated gene transcripts,
including foxa2, pdx1, ngn3, insulin, glucagon and glut2, were
highly enriched in the BMP4-treated cells compared to untreated
cells. Q-PCR data demonstrated the increasing transcript levels of
foxa2 (18, 21 and 37 fold increase over the time-course EB14+14,
EB14+21 and EB14+28 respectively, compared to untreated EB14), pdx1
(179, 155 and 541 fold increase) and glut2 (47, 97 and 224 fold
increase). Importantly, insulin and glucagon transcripts were
undetectable in untreated EB14 cells.
[0073] In contrast, in BMP4-treated cultures, insulin and glucagon
mRNA levels increased dramatically over time. Q-PCR reaction cycles
for insulin mRNA change from 50 (undetectable) in EB14 untreated
cells to 28, 27 and 25 cycles in BMP4-treated cells over the
post-EB differentiation time-course. For glucagon, the change was
from 46 cycles to 22, 21 and 20 cycles; in all experiments,
.alpha.-actin cycle times were approximately 15 cycles. In contrast
to the increasing abundance of islet hormone transcripts, brachyury
mRNA continuously decreased over time during these late stages and
was not significantly different from the level in EB14 untreated
cells. Consistent with prior studies in hESCs, the trophoblast
marker hCG was expressed in the BMP4-treated cells at EB14 (Xu, R.
H., et al. (2002)), but not in EBs generated from hESCs that had
not been treated with BMP4. After EBs were plated in serum-free
media, hCG mRNA levels decreased dramatically (FIG. 6).
[0074] Also in concert with prior observations in embryos and
mESCs, the neuroectoderm marker soxI was not detected in
BMP4-treated cells, whereas it was induced in differentiation
cultures that had been initiated with untreated hESC colonies (FIG.
6). These data suggest that: 1) serum-free conditions favor islet
hormone cell differentiation over trophoblast differentiation
(e.g., retained islet hormone gene expression over time vs. lost
hCG gene expression over time under serum-free conditions); 2)
neural differentiation is likely inhibited by BMP4 treatment and/or
neural precursors are not selected by BMP4 treatment, 3) serum-free
conditions prevent Oct4-expressing undifferentiated cells from
surviving and/or expanding, and 4) TAT-expressing cells, such as
liver cells, are not supported or maintained by these serum-free
conditions.
[0075] Consistent with gene expression results, immunostaining data
show that BMP4-treated cultures contained large clusters of PDX1+
cells after plating. In contrast to PDX1+ cells found in EBs, most
of the cells no longer co-stained with Ki67 (FIG. 7d-f), suggesting
that the cells had begun terminal differentiation. Cells
co-staining for either PDX1 and insulin, or PDX1, insulin and
C-peptide appeared within the large clusters of PDX1+ cells. By 14
days after EB plating, 5 of 17 (29%) EBs showed insulin+ cells
which increased over time such that by 28 days after plating, 10 of
16 (63%) EBs contained insulin+ cells and the mean # of positive
cells per EB increased. The fact that applicants observed numerous
insulin/C-peptide immunostained cells, which always co-express PDX1
and are localized within PDX1+ clusters, as well as a significant
increase in insulin mRNA, suggests a phenotypic pattern
characteristic of bona fide .beta. cells. Glucagon and somatostatin
staining was also observed in BMP4-treated cultures (FIG. 8).
Glucagon+ and somatostatin+ cells did not co-stain for C-peptide as
would be expected for adult endocrine cell types. Cells staining
for either PDX1 or islet hormones were not observed in control
group cells.
Example 8
EpCAM Based MACS Sorting Further Enriched Cultures for Endoderm and
Pancreas Committed Cells
[0076] The applicant detected that when BMP4/bFGF treated cultures
are stained for EpCAM and PDX1, numerous cell clusters are seen
containing cells co-expressing both markers (FIG. 10a). This
detection provided an opportunity to preferentially select PDX1+
cells based on EpCAM expression and MACS sorting. To test this
hypothesis, cells differentiated according to the BMP4/bFGF
treatment protocol are labeled with an anti-EpCAM antibody followed
by labeling with an appropriate secondary antibody and sorted.
After EpCAM+ sorting of differentiated hESC progeny, transcript
levels of sox17 and foxa2 are increased, as are levels of pdx1 and
EpCAM (FIG. 10b). In this respect, MACS sorting for EpCAM can be
used to enrich or purify differentiated ESC cultures to eliminate
non-endodermal cells that arise as the result of non-selective
culture conditions. Sorting for EpCAM results in a population in
which .about.95% of the cells are EpCAM+ (FIG. 7c). Applicants
hypothesize that combining two or more sorts (selecting for EpCAM
and removing SSEA3 or 4+ cells) will further enrich the cultures
for endoderm/pancreatic progenitor cells and simultaneously remove
undifferentiated, teratoma-forming cells, from the cultures.
[0077] In summary, the methods of the invention are able to
facilitate reproducible differentiation of hESCs to cells highly
reminiscent of beta cells or beta-like cells. The differentiated
cells are a promising source of pancreatic beta cells, which can be
used to treat diabetic patients. However, little is known about
cellular and molecular events regulating pancreatic differentiation
in humans. As such, described herein are several components of the
direct differentiation process that results in reliable and robust
induction of PDX1-positive cells and generates cell clusters
co-expressing PDX1 and insulin/C-peptide. The entire
differentiation process is carried out in vitro in serum-free
media. It was found that mesendoderm, marked by early brachyury
expression, is induced when hESCs are treated with 50 ng/ml BMP4
and 100 ng/ml bFGF, and that endoderm and early proliferative
pancreatic lineage cells are specified in EBs made from these
cultures. Further differentiation occurs after cells are
transferred to serum-free media containing insulin, transferrin,
selenium, FGF7, nicotinamide and exendin-4. The in vitro-generated
pancreatic cells express PDX1 and C-peptide, characteristic of
.beta. cells. These inventive findings move the field a step closer
to the production of pancreatic .beta. cell populations suitable
for therapeutic purposes and facilitate the use of human
pluripotent stem cells as an in vitro model of pancreas
development.
[0078] The isolated populations of endodermal or pancreatic
progenitor populations could enhance adult therapy. For example,
these progenitor cells can be co-transplanted with human adult
islets or hepatocytes. These progenitor populations may also be
transplanted by themselves, without first going through the
terminal differentiation step. In this therapeutic approach, it is
expected that the progenitor cells will differentiate in the
patient to the functional cells or tissues of interest.
Regeneration of entire tissues is also envisioned starting from
such endodermal or pancreatic progenitor cells.
[0079] The disclosure of every patent, patent application, and
publication cited herein is hereby incorporated herein by reference
in its entirety.
[0080] It is understood that certain adaptations of the invention
described in this disclosure are a matter of routine optimization
for those skilled in the art, and can be implemented without
departing from the spirit of the invention, or the scope of the
appended claims.
Sequence CWU 1
1
24 1 25 DNA Artificial Synthetic polynucleotide 1 ggatgaagtc
taccaaagct cacgc 25 2 25 DNA Artificial SEQ ID NO3 2 ccagatcttg
atgtgtctct cggtc 25 3 24 DNA Artificial Synthetic polynucleotide 3
ggggttctat ttgggaaggt attc 24 4 23 DNA Artificial Synthetic
polynucleotide 4 attctccagg ttgcctctac atc 23 5 20 DNA Artificial
Synthetic polynucleotide 5 gggagcggtg aagatggaag 20 6 20 DNA
Artificial Synthetic polynucleotide 6 tcaggctggg actcaagtgc 20 7 22
DNA Artificial Synthetic polynucleotide 7 aagaacggca ggaggatgtt tc
22 8 24 DNA Artificial Synthetic polynucleotide 8 cccaactctc
actatgtgga ttcg 24 9 20 DNA Artificial Synthetic polynucleotide 9
gggatacgcc agtgacgacc 20 10 20 DNA Artificial Synthetic
polynucleotide 10 gctctgcctc ctccacgaag 20 11 23 DNA Artificial
Synthetic polynucleotide 11 gtagaaagga tgacgcctca acc 23 12 20 DNA
Artificial Synthetic polynucleotide 12 gctgcttgct cagtgccaac 20 13
21 DNA Artificial Synthetic polynucleotide 13 acacgagacc cactttttcc
g 21 14 23 DNA Artificial Synthetic polynucleotide 14 tgctggactt
gtgcttcttc aac 23 15 22 DNA Artificial Synthetic polynucleotide 15
ttctacgaca gcagcgacaa cc 22 16 22 DNA Artificial Synthetic
polynucleotide 16 cgtcacctcc atacctttct cg 22 17 24 DNA Artificial
Synthetic polynucleotide 17 atgctctggt ccctgtctgt atcc 24 18 24 DNA
Artificial Synthetic polynucleotide 18 tgactaataa gaatgcccgt gacg
24 19 20 DNA Artificial Synthetic polynucleotide 19 cagcctttgt
gaaccaacac 20 20 21 DNA Artificial Synthetic polynucleotide 20
gctttattcc atctctctcg g 21 21 21 DNA Artificial Synthetic
polynucleotide 21 tcagatgaac gaggacaagc g 21 22 21 DNA Artificial
Synthetic polynucleotide 22 cctggcggca agattatcaa g 21 23 20 DNA
Artificial Synthetic polynucleotide 23 cggatttggt cgtattgggc 20 24
22 DNA Artificial Synthetic polynucleotide 24 cagggatgat gttctggaga
gc 22
* * * * *