U.S. patent application number 12/726814 was filed with the patent office on 2010-10-14 for generation of clonal mesenchymal progenitors and mesenchymal stem cell lines under serum-free conditions.
Invention is credited to Igor I. Slukvin, Maksym A. Vodyanyk.
Application Number | 20100261274 12/726814 |
Document ID | / |
Family ID | 44009741 |
Filed Date | 2010-10-14 |
United States Patent
Application |
20100261274 |
Kind Code |
A1 |
Vodyanyk; Maksym A. ; et
al. |
October 14, 2010 |
GENERATION OF CLONAL MESENCHYMAL PROGENITORS AND MESENCHYMAL STEM
CELL LINES UNDER SERUM-FREE CONDITIONS
Abstract
Methods for obtaining multipotent Apelin receptor-positive
lateral plate mesoderm cells, mesenchymal stem cells, and
mesangioblasts under serum-free conditions are disclosed.
Inventors: |
Vodyanyk; Maksym A.;
(Madison, WI) ; Slukvin; Igor I.; (Verona,
WI) |
Correspondence
Address: |
QUARLES & BRADY LLP
411 E. WISCONSIN AVENUE, SUITE 2040
MILWAUKEE
WI
53202-4497
US
|
Family ID: |
44009741 |
Appl. No.: |
12/726814 |
Filed: |
March 18, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12554969 |
Sep 7, 2009 |
|
|
|
12726814 |
|
|
|
|
12024770 |
Feb 1, 2008 |
7615374 |
|
|
12554969 |
|
|
|
|
60974980 |
Sep 25, 2007 |
|
|
|
60989058 |
Nov 19, 2007 |
|
|
|
Current U.S.
Class: |
435/354 ;
435/325; 435/363; 435/366 |
Current CPC
Class: |
C12N 5/0662 20130101;
C12N 5/0647 20130101; C12N 2533/70 20130101; C12N 5/069 20130101;
C12N 2501/145 20130101; C12N 2501/155 20130101; C12N 2501/135
20130101; C12N 2501/23 20130101; C12N 5/0692 20130101; C12N
2501/165 20130101; C12N 2500/44 20130101; C12N 2502/1394 20130101;
C12N 2506/02 20130101; C12N 2533/78 20130101; C12N 2500/90
20130101; C12N 2506/45 20130101; C12N 2501/125 20130101; C12N
2500/38 20130101; C12N 2501/115 20130101; C12N 2533/54
20130101 |
Class at
Publication: |
435/354 ;
435/363; 435/366; 435/325 |
International
Class: |
C12N 5/0775 20100101
C12N005/0775 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with United States government
support awarded by the following agency: NIH RR052085 and NIH
HD044067. The United States government has certain rights in this
invention.
Claims
1. A method of generating a clonal population of primate
mesenchymal stem cells, the method comprising the steps of:
culturing a heterogeneous, single-cell suspension of primate cells
that contains mesenchymal progenitors in a serum-free, semi-solid
medium containing between about 5 and about 100 ng/ml bFGF until
independent colonies form; and culturing one of the independent
colonies in a serum-free, liquid medium containing between about 5
and about 100 ng/ml bFGF to obtain a substantially pure clonal
population of MSCs.
2. The method of claim 1, wherein the heterogeneous suspension is
obtained in a method comprising the steps of: co-culturing
pluripotent primate cells with bone marrow stromal cells in a
medium that supports differentiation for between two and five days
until differentiated cells are formed; and suspending the
differentiated cells.
3. The method of claim 2, wherein the pluripotent cells are
selected from the group consisting of embryonic stem cells (ESCs)
and induced pluripotent stem (iPS) cells.
4. The method of claim 2, further comprising the step of depleting
cells not derived by in vitro differentiation of pluripotent cells
from the heterogeneous suspension.
5. The method of claim 4, wherein the depleting step comprises the
steps of non-covalently binding the cells to be depleted to
paramagnetic monoclonal antibodies specific for epitopes on the
cells to be depleted; and segregating the antibody-bound cells with
a magnet.
6. The method of claim 2, wherein the bone marrow stromal cells are
mouse OP9 cells.
7. The method of claim 1, wherein the heterogeneous suspension is
obtained in a method comprising the steps of: dissociating an
embryoid body to single cells; and suspending the single cells.
8. The method of claim 1, wherein the single-cell suspension is
cultured for between ten to twenty days.
9. The method of claim 1, wherein the semi-solid medium contains
between about 20 and about 100 ng/ml bFGF.
10. The method of claim 1, wherein the semi-solid medium contains
about 5 ng/ml bFGF.
11. The method of claim 1, wherein the semi-solid medium contains
about 1% methylcellulose.
12. The method of claim 1, wherein the semi-solid medium contains
between about 10 ng/ml and about 20 ng/ml PDGF-BB.
13. The method of claim 1, wherein cells in the single-cell
suspension express MIXL1 and T.
14. The method of claim 1, wherein the primate cells are of human
origin.
15. The method of claim 1, wherein the MSCs are cultured in the
presence of an extracellular matrix protein.
16. The method of claim 15, wherein the extracellular matrix
protein is selected from the group consisting of Matrigel.RTM.,
collagen, gelatin and fibronectin.
17. The method of claim 1, wherein the mesenchymal colonies express
FOXF1, MSX1, MSX2, SNAI1, SNAI2, SOX9 and RUNX2.
18. The method of claim 1, wherein the mesenchymal colonies express
CD44, CD45, CD140a, CD146, and CD105, but do not express CD31,
CD43, CD45 and VE-cadherin.
19. The method of claim 1, wherein the method comprises the step
of: observing at least one mesenchymal characteristic of colonies
formed during culture in the serum-free, semi-solid medium, thereby
confirming identification of mesenchymal progenitors in the
suspension.
20. The method of claim 19 wherein the at least one mesenchymal
characteristic is selected from the group consisting of a
functional characteristic, a morphological characteristic and a
phenotypical characteristic.
21. The method of claim 20, wherein the functional characteristic
is selected from the group consisting of (1) growth stimulation by
factors that promote mesenchymal cell growth (e.g., PDGF-BB, EGF
and TGF-alpha) and growth suppression by factors involved in
mesodermal differentiation (e.g., VEGF, TGF-beta and Activin A) and
(2) differentiation into osteogenic, chondrogenic or adipogenic
cell lineages.
22. The method of claim 20, wherein the morphological
characteristic is selected from the group consisting of (1) a
tightly packed, round-shaped cell aggregate measuring 100-500 .mu.m
in diameter; and (2) lack of dense outer cell layer and irregular
inner structure.
23. The method of claim 20, wherein the phenotypical characteristic
is selected from the group consisting of (1) expression of CD44,
CD45, CD105, CD146, and CD140a, but no expression of CD31, CD43,
CD45 and VE-cadherin, (2) expression of FOXF1, MSX1, MSX2, SNAI1,
SNAI2, SOX9 and RUNX2 and (3) expression of vimentin, alpha smooth
muscle actin, and desmin.
24. The method of claim 19, wherein the method further comprises
counting colonies to estimate a number of mesenchymal progenitors
in the heterogeneous suspension.
25. A cell population comprising: a substantially pure line of
clonally-derived mesenchymal stem cells positive at least for CD44,
CD45, CD73, CD146, CD140a and CD105, but negative for CD31, CD43,
CD45 and VE cadherin.
26. The cell population of claim 25, wherein the population
comprises at least 99% mesenchymal stem cells.
27. A method of generating a population of primate Apelin
receptor-positive lateral plate mesoderm cells, the method
comprising the steps of: culturing primate pluripotent stem cells
in a medium that supports differentiation until Apelin receptor is
expressed isolating Apelin receptor-positive lateral plate mesoderm
cells.
28. The method of claim 27, wherein the primate pluripotent stem
cells are co-cultured with bone marrow stromal cells for about two
to about five days.
29. The method of claim 27 wherein about 1% to about 5% of the
Apelin receptor-positive cells have hemangioblast and mesangioblast
potential.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
U.S. patent application Ser. No. 12/554,696, which is a
continuation application of U.S. patent application Ser. No.
12/024,770, which claims the benefit of U.S. Provisional Patent
Application No. 60/974,980, filed Sep. 25, 2007; and U.S.
Provisional Patent Application No. 60/989,058, filed Nov. 19, 2007,
each of which is incorporated herein by reference as if set forth
in its entirety.
BACKGROUND
[0003] The invention relates generally to clonal primate
mesenchymal progenitors and to mesenchymal stem cell (MSC) lines
and methods for identifying and generating such cells, and more
particularly to methods for generating clonal mesenchymal
progenitors and MSC lines under serum-free conditions. The
invention further relates to a population of shared endothelial-
and mesenchymal cell precursors and methods for identifying and
generating such cells. The invention furthermore relates to a
population of cells comprising lateral plate mesoderm cells and
methods for their generation and isolation from cultured
pluripotent stem cells.
[0004] During embryonic development of animals, gastrulation forms
three germ layers, i.e., endoderm, ectoderm, and mesoderm, that
each give rise to distinct bodily cells. Mesoderm develops from
primitive streak, a transient embryonic structure formed at the
onset of gastrulation. Nascent mesoderm transitionally
differentiates into paraxial mesoderm, intermediate mesoderm, and
lateral plate mesoderm. Paraxial mesoderm gives rise to axial
skeleton, and skeletal muscles. Intermediate mesoderm forms the
urogenital system. Lateral plate mesoderm gives rise to the
circulatory system, including blood cells, vessels, and heart, and
forms the viscera and limbs Extraembryonic mesoderm is located
outside the developing embryo. Evidence suggests that
extraembryonic mesoderm is derived from the primitive streak during
gastrulation (Boucher and Pedersen, Reprod. Fertil. Dev. 8:765
(1996)). Extraembryonic mesoderm gives rise to several tissues that
provide the embryo with nutrients, a means of waste disposal, and
mechanical protection.
[0005] Both lateral plate and extraembryonic mesoderm can generate
endothelial and blood cells and express FOXF1, HAND1, HAND2,
GATA-2, BMP4, and WNT5a, expression of which is low or undetectable
in paraxial and intermediate mesoderm (Mahlapuu et al.,
Development. 128(2):155 (2001); Firulli et al., Nat. Genet.
18(3):266 (1998); Morikawa et al., Circ. Res. 103(12):1422 (2008);
Kelley et al., Dev. Biol. 165:193 (1994); Silver et al., Blood
89(4):1154 (1997); Fujiwara et al., Proc. Natl. Acad. Sci.
98(24):13739 (2001); Takada et al., Genes Dev. 8(2):174 (1994)).
Distinctive markers for lateral plate and extraembryonic mesoderm
remain to be elucidated. A recent finding by Bosse et al. suggests
that IRX3 is expressed in lateral plate mesoderm but not in
extraembryonic mesoderm (Bosse et al., Mech. Dev. 69(1-2):169
(1997)). For the purposes of this application, the term lateral
plate is used to describe both tissues.
[0006] Certain committed mesodermal progenitors can give rise to
cells of more than one lineage. Example of such progenitors
includes hemangioblasts, which can give rise to both hematopoietic-
and endothelial cells. Choi K, et al., "A common precursor of
hematopoietic and endothelial cells," Development 125:725
(1998).
[0007] MSCs can differentiate into at least three downstream
mesenchymal cell lineages (i.e., osteoblasts, chondroblasts, and
adipocytes). To date, no unique MSC marker has been identified. As
such, morphological and functional criteria are used to identify
these cells. See, Horwitz E, et al., "Clarification of the
nomenclature for MSC: the International Society for Cellular
Therapy position statement," Cytotherapy 7:393 (2005); and Dominici
M, et al., "Minimal criteria for defining multipotent mesenchymal
stromal cells. The International Society for Cellular Therapy
position statement," Cytotherapy 8:315 (2006). Because MSCs can
differentiate into many cell types, the art contemplates methods
for differentiating MSCs for cell-based therapies, for regenerative
medicine and for reconstructive medicine.
[0008] Typically, MSCs are isolated from adult bone marrow, fat,
cartilage and muscle. Pittenger F, et al., "Multilineage potential
of adult human mesenchymal stem cells," Science 284:143-147 (1999);
Zuk P, et al., "Multilineage cells from human adipose tissue:
implications for cell-based therapies," Tissue Eng. 7:211-228
(2001); and Young H, et al., "Human reserve pluripotent mesenchymal
stem cells are present in the connective tissues of skeletal muscle
and dermis derived from fetal, adult, and geriatric donors," Anat.
Rec. 264:51-62 (2001). MSCs have also been isolated from human
peripheral blood. Kassis I, et al., "Isolation of mesenchymal stem
cells from G-CSF-mobilized human peripheral blood using fibrin
microbeads," Bone Marrow Transplant. 37:967-976 (2006). MSCs can
also be isolated from human neonatal tissue, such as Wharton's
jelly (Wang H, et al., "Mesenchymal stem cells in the Wharton's
jelly of the human umbilical cord," Stem Cells 22:1330-1337
(2004)), human placenta (Fukuchi Y, et al., "Human placenta-derived
cells have mesenchymal stem/progenitor cell potential," Stem Cells
22:649-658 (2004)); and umbilical cord blood (Erices A, et al.,
"Mesenchymal progenitor cells in human umbilical cord blood," Br.
J. Haematol. 109:235-242 (2000)) and human fetal tissues
(Campagnoli C, et al., "Identification of mesenchymal
stem/progenitor cells in human first-trimester fetal blood, liver,
and bone marrow," Blood 98:2396-2402 (2001)).
[0009] The art is limited by an inability to isolate sufficient
MSCs for subsequent differentiation and use. Where suitable donors
are available, the invasive procedures required to isolate even a
limited number of cells present risks to donors. It also remains
difficult to maintain isolated MSCs in long-term culture and to
maintain such cultures free of bacterial or viral
contamination.
[0010] Efforts to devise methods for differentiating embryonic stem
cells (ESCs) including human ESCs (hESCs) to MSCs either have
required culturing the cells in a medium containing potentially
contaminating serum or have yielded cells that retain
characteristics of undifferentiated hESCs. For example, Barberi et
al. differentiated hESCs to MSCs on mitotically-inactivated mouse
stromal cell lines feeder cells) with 20% heat-inactivated fetal
bovine serum (FBS) in alpha MEM medium for 40 days. Barberi T, et
al. "Derivation of multipotent mesenchymal precursors from human
embryonic stem cells," PLoS Med. 2:e161 (2005). Cells were
harvested and assayed for CD73, and CD73.sup.+cells were then
plated in the absence of the feeder cells with 20% FBS in alpha MEM
for 7 to 10 days. Barberi et al. differentiated the MSCs into
adipogenic cells, chondrogenic cells, osteogenic cells and myogenic
cells.
[0011] Likewise, Olivier et al. differentiated hESCs to MSCs by
plating raclures (i.e., spontaneously differentiated cells that
appear in hESC culture in the center or at the edges of colonies)
with D10 medium (DMEM, 10% FBS, 1% penicillin/streptomycin and 1%
non-essential amino acids) changed weekly until a thick,
multi-layer epithelium developed. Olivier E, et al.,
"Differentiation of human embryonic stem cells into bipotent
mesenchymal stem cells," Stem Cells 24:1914-1922 (2006). After
approximately four weeks, MSCs were isolated by dissociating the
epithelium with a mixture of trypsin, collagenase type IV and
dispase for four to six hours, followed by re-plating in D10
medium. Olivier et al.'s MSCs grew robustly, had stable karyotypes,
were contact inhibited, senesced after twenty passages and
differentiated into adipogenic and osteogenic cells. Olivier et al.
did not report that the cells differentiated into chondroblasts.
Unlike Barberi et al., Olivier et al. did not require feeder cells
to support differentiation of hESC to MSCs. However, Olivier et
al.'s MSCs were SSEA-4 positive, suggesting that these MSCs
expressed cell surface markers characteristic of hESC.
[0012] Pike & Shevde differentiated hESCs to MSCs via embryoid
bodies (EBs) incubated for ten to twelve days in a
mesenchymal-specific medium (MesenCult.RTM. medium with 10% FBS;
alpha MEM with glutamine and nucleosides; or DMEM with glucose and
glutamine, replaced every two days). US Patent Publication No.
2006/0008902. The EBs were digested, and pre-mesenchymal cells were
cultured to 80% confluence. The cells were trypsinized and passaged
three times in mesenchymal-specific medium.
[0013] Meuleman et al. reported culturing MSCs in a serum-free
medium; however, it was later discovered that the medium did in
fact contain animal serum as a component. Meuleman N, et al.,
"Human marrow mesenchymal stem cell culture: serum-free medium
allows better expansion than classical alpha-minimal essential
medium (MEM)," Eur. J. Haematol. 76:309-316 (2006); and Meuleman N,
et al., "Human marrow mesenchymal stem cell culture: serum-free
medium allows better expansion than classical alpha-minimal
essential medium (MEM)," Eur. J. Haematol. 77:168 (2007); but see,
Korhonen M, "Culture of human mesenchymal stem cells in serum-free
conditions: no breakthroughs yet," Eur. J. Haematol. 77:167
(2007).
[0014] Those methods cultured and differentiated MSCs in
serum-containing medium. Serum-free conditions for culturing and
differentiating MSCs, if defined, would reduce variation among
batches and eliminate a risk of infection transmitted by xenogenic
by-products and pathogens. Sotiropoulou P, et al., "Cell culture
medium composition and translational adult bone marrow-derived stem
cell research," Stem Cells 24:1409-1410 (2006).
[0015] For the foregoing reasons, there is a need for new methods
for obtaining early mesenchymal progenitors and MSCs, especially
when derived under serum-free conditions.
[0016] Mesoderm and the neural crest can both give rise to
mesenchymal precursors during embryonic development. Dennis, J. E.,
and P. Charbord, "Origin and differentiation of human and murine
stroma," Stem Cells 20:205-214 (2002); Takashima, Y, et al.,
"Neuroepithelial cells supply an initial transient wave of MSC
differentiation," Cell 129:1377-1388 (2007). While conditions for
generating MSCs of neural crest origin from embryonic stem cells
have been described, Takashima et al., supra; Lee, G, et al.,
"Isolation and directed differentiation of neural crest stem cells
derived from human embryonic stem cells," Nat Biotechnol
25:1468-1475 (2007), it is not known how to generate MSCs from
mesoderm.
[0017] For the foregoing reasons, there is a need for new methods
for obtaining early mesenchymal progenitors and MSCs, particularly
under serum-free conditions. Further, there is a need to identify
and generate mesoderm-derived MSCs as well as early mesodermal
progenitors that can give rise to MSCs during differentiation of
pluripotent stem cells into MSCs.
BRIEF SUMMARY
[0018] The invention generally relates to a newly identified common
mesenchymal and endothelial cell precursor, i.e., mesangioblasts,
derived from in vitro-differentiated stem cells.
[0019] In a first aspect, the invention is summarized in that a
method of generating a clonal population of primate MSCs includes
the steps of culturing a heterogeneous, single-cell suspension of
primate cells that contains mesenchymal progenitors in a
serum-free, semi-solid medium containing between about 5 and about
100 ng/ml bFGF until independent colonies form, and culturing, one
of the independent colonies in a serum-free, liquid medium
containing between about 5 and about 100 ng/ml, or at about 5
ng/ml, or between about 20 and about 100 ng/ml, bFGF to obtain an
substantially pure clonal population of MSCs.
[0020] The heterogeneous suspension for use in the method can be
obtained, for example, by differentiating pluripotent cells from a
primate (e.g., human), such as ESCs or induced pluripotent stem
(iPS) cells, in culture until cells in the culture are mesenchymal
progenitors. This can be accomplished by co-culturing the
pluripotent cells with bone marrow stromal cells in a medium that
supports differentiation as described herein for at least two to
five days, or by dissociating EBs, which can themselves be obtained
by culture of pluripotent cells using well-known methods, and then
suspending the cells as a single cell suspension. The bone marrow
stromal cells can be mouse OP9 cells. A heterogeneous suspension
substantially free of some or all cells not derived by in vitro
differentiation of pluripotent cells (especially co-cultured bone
marrow cells) can be obtained by depleting those cells from the
suspension. These cells can be depleted from the suspension before
use, for example, by non-covalently binding the cells to be
depleted to paramagnetic monoclonal antibodies specific for the
epitopes on the cells to be depleted and then segregating the
antibody-bound cells with a magnet. Cells in a suspension obtained
from pluripotent cells can express at least MIXL1, and T
(BRACHYURY).
[0021] The medium can be rendered semi-solid by including about 1%
methylcellulose in the medium. The medium can optionally contain
between about 10 and about 20 ng/ml PDGF-BB. The suspension can be
cultured for between about ten to about twenty days or more to
produce the colonies.
[0022] Mesenchymal progenitors are identified as having been
present in the suspension if mesenchymal colonies form during
culture in the serum-free, semi-solid medium supplemented with
bFGF. An example of such bFGF-dependent colony-forming assay for
detecting mesenchymal progenitor is described in U.S. Pat. No.
7,615,374, incorporated herein as if set forth in its entirety. The
colonies obtained in the colony-forming assay can be identified as
mesenchymal by their expression of at least a plurality of FOXF1,
MSX1, MSX2, SNAI1, SNAI2, SOX9 and RUNX2. Characteristics of the
colonies include functional, morphological and phenotypical
characteristics and gene expression profile. Functional
characteristics of the colonies include (1) growth stimulation by
factors that promote mesenchymal cell growth (e.g., PDGF-BB, EGF
and TGF-alpha) and growth suppression by factors involved in
mesodermal differentiation (e.g., VEGF, TGF-beta and Activin A);
(2) differentiation into osteogenic, chondrogenic or adipogenic
cell lineages; and (3) differentiation into endothelial cells.
Morphological characteristics of the colonies include (1) tight
packing of cells to form round (i.e., spherical) aggregates
measuring 100-500 .mu.m in diameter; (2)colony formation through
establishing tightly packed structures (cores) that further develop
into compact spheroid colonies; and (3) even after prolonged
culture, lack of dense outer cell layer and irregular inner
structure, which are characteristics of EBs. Phenotypical
characteristics of the colonies include (1) expression of CD44,
CD45, CD105 and CD140a (PDGFRA), CD146, but not hematoendothelial
surface markers (i.e., CD31, CD43, CD45 and VE-cadherin); (2)
expression of FOXF1, MSX1, MSX2, SNAI1, SNAP, SOX9 and RUNX2; and
(3) expression of vimentin, alpha smooth muscle actin, and
desmin.
[0023] The mesenchymal colonies thus formed in the method can be
further cultured in the presence of an extracellular matrix
protein, such as Matrigel.RTM., collagen, gelatin or fibronectin,
as well as combinations thereof.
[0024] The invention is further summarized as a substantially pure
population of clonally-derived MSC lines produced from the methods
described above that are positive for at least CD44, CD45, CD 73,
CD105, CD140a, and CD146, but negative for CD31, CD43, CD45 and
VE-cadherin.
[0025] The described embodiments have many advantages, including
that mesenchymal progenitors and MSCs obtained in the methods may
be used to treat diseases associated with bone, cartilage and fat
cells.
[0026] It is also an advantage that a clonal population of MSCs can
be obtained from a single mesenchymal colony.
[0027] It is also an advantage that the cells obtained in the
methods can easily be selected for further expansion because the
mesenchymal progenitors have high proliferation potential and form
large colonies.
[0028] It is yet another advantage that cells obtained in the
methods can be tolerant or tolerogenic to allo- and auto-immune
response on transplantation.
[0029] It is still another advantage that the cells obtained in the
methods can differentiate into at least osteogenic, chondrogenic
and adipogenic lineages.
[0030] It is still another advantage that mesenchymal colonies
obtained in the methods possess angiogenic potential.
[0031] The invention is further summarized as a population of in
vitro-derived Apelin receptor-positive (APLNR.sup.+) lateral plate
mesoderm cells. These cells can be isolated from mixed populations
of differentiating pluripotent stem cells based on expression of
the Apelin receptor (APLNR). These cells can differentiate into
cells of the body wall and viscera and give rise to mesangiogenic
mesenchymal and hemangiogenic blast colonies in semisolid media
cultures in the presence of bFGF. The APLNR.sup.+ cells express
transcripts characteristic of mesoderm, specifically lateral plate
mesoderm.
[0032] It is still another advantage of the invention that MSCs
obtained by the claimed methods are of mesodermal origin and can be
derived from APLNR.sup.+ cells enriched in lateral plate mesoderm
cells.
[0033] These and other features, aspects and advantages of the
present invention will become better understood from the
description that follows. The description of preferred embodiments
is not intended to limit the invention to cover all modifications,
equivalents and alternatives. Reference should therefore be made to
the claims herein for interpreting the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1A-E illustrate the properties of two types of
hESC-derived colonies, i.e., mesenchymal colonies derived from
mesangioblasts (MB) and blast colonies derived from hemangioblasts
(HB). FIG. 1A depicts MB and HB colony morphologies following
growth in semisolid media for 3, 5, 7, and 12 days. FIG. 1B depicts
the kinetics of FGF-dependent colony formation. Bars represent
standard deviation of four independent experiments. Depending on
whether the hESC-derived single cells are initially co-cultured
with OP9 cells for 2 days or 3 days, they assume either MB or HB
potential. FIG. 1C illustrates that bFGF, but not PDGF or VEGF
alone, supports both MB and HB colony formation. The data are
represented as mean.+-.SD (n=4). The asterisk indicates statistical
significance (p<0.01) between cultures containing FGF alone and
FGF in combination with either PDGF or VEGF. FIG. 1D illustrates
the differentiation potential of MB-derived and HB-derived colonies
after coculture with OP9 cells for 4 days. Flow cytometry
demonstrated that MB colony-derived cells collected on day 12 of
clonogenic culture gave rise to CD146.sup.+ CD31.sup.- mesenchymal
and CD31.sup.+ CD43.sup.- endothelial cells, while HB
colony-derived cells gave rise to CD31.sup.+ CD43.sup.- endothelial
cells and CD43.sup.+ hematopoietic lineage cells. FIG. 1E
illustrates immunostaining analysis of cell clusters developed from
a single MB (top, scale bar, 100 .mu.m) and HB (bottom, scale bar,
50 .mu.m) colony collected on day 5 of clonogenic culture. Cells
were identified as CD144.sup.+ (also known as VE-cadherin)
CD43.sup.- endothelial, CD43.sup.+ hematopoietic, and
calponin.sup.+ CD144.sup.- mesenchymal. The scale bars represent
100 .mu.m. Colonies developed from cell clusters of a single MB
colony generate calponin.sup.+ CD144(VE-cadherin).sup.- mesenchymal
cells and CD144(VE-cadherin).sup.+calponin.sup.- endothelial cells
(upper panel). Colonies developed from cell clusters developed from
a single HB colony generate CD43.sup.+hematopoietic and
CD144(VE-cadherin).sup.+CD43.sup.-endothelial cells (lower
panel).
[0035] FIG. 2 illustrates microarray analysis of gene expression in
hESCs co-cultured with OP9 cells from day 0 (H1) to day 7. FIG. 2A
depicts a heat map for selected gene sets defining particular germ
layers and their subpopulations and derivatives. FIG. 2B depicts
relative gene expression of MIXL1, T, SNAI1, FOXF1, and SOX17 as
determined by quantitative PCR. FIG. 2C depicts the fold increase
in the number of hESC-derived cells after day 1-6 of OP9 co-culture
in relation to previous day. The data is represented as means.+-.SD
(n=3).
[0036] FIG. 3 illustrates analysis of APLNR.sup.+ cells. FIG. 3A
depicts dot plots of flow cytometry results. FIG. 3B depicts the
effect of inhibitors of mesoderm formation (SB431542 (5 .mu.g/ml)
and DKK1 (150 .mu.g/ml)) on generation of APLNR+cells from H1 cells
in OP9 cell co-cultures. FIG. 3C compares transcript expression
between APLNR.sup.+ and APLNR.sup.- cells. FIG. 3D depicts the
colony-forming potential of APLNR.sup.+ and APLNR.sup.- cells.
[0037] FIG. 4 illustrates the gene expression profiles of
APLNR.sup.+ cells, APLNR.sup.- cells, cores, colonies, and a
mesenchymal stem cell (MSC) line (at passages p1 and p5) obtained
from H1 hESCs differentiated for 2 (D2) or 3 (D3) days by coculture
with OP9 cells. FIG. 4A depicts heat maps for selected sets of
genes defining indicated germ layers and their
subpopulations/derivatives. Cores were collected on day 3 of
clonogenic cultures and fully developed colonies on day 12 of
clonogenic cultures. EMT is epithelial-mesenchymal transition. VSMC
is vascular smooth muscle cells. FIG. 4B shows lack of
SOX/neuroepithelium marker expression throughout all stages of
differentiation. Embryoid bodies derived from H1 hESCs
differentiated for 14 days were used as positive control. FIG. 4C
illustrates quantitative RT-PCR analysis of representative
transcripts in indicated cell subsets. Bars represent gene
expression in pooled samples from 3 experiments normalized to
RPL13.
[0038] FIG. 5 depicts a schematic diagram of mesodermal lineages
development and differentiation toward MSCs from pluripotent stem
cells.
[0039] FIG. 6 depicts a schematic diagram of the protocol used for
hESC differentiation, generation of MB colonies, and clonal MSC
lines.
[0040] FIG. 7 depicts a schematic diagram of the protocol used to
evaluate differentiation potential of mesenchymal and blast
colonies.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0041] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention belongs. Although
any methods and materials similar to or equivalent to those
described herein can be used in the practice or testing of the
present invention, the preferred methods and materials are
described herein. It is commonly understood by one of ordinary
skill in the art that "lack of expression" of a gene or the absence
of a certain marker on a cell refers to an inability to detect such
gene or marker expression using methods known in the art at the
time of filing It cannot be ruled out that more sensitive methods
could detect low levels of expression of such genes or markers.
[0042] In describing the embodiments and claiming the invention,
the following terminology is used in accordance with the
definitions set out below.
[0043] As used herein, "about" means within 5% of a stated
concentration.
[0044] As used herein, "clonal" means a population of cells
cultured from a single cell, not from an aggregate of cells. Cells
in a "clonal population" display a substantially uniform pattern of
cell surface markers and morphology and are substantially
genetically identical.
[0045] As used herein, an "embryoid body" or an "EB," is an
aggregate of cells derived from pluripotent cells, such as ESCs or
iPS cells, where cell aggregation can be initiated by hanging drop,
by plating upon non-tissue culture-treated plates or spinner flasks
(i.e., low attachment conditions); and any method that prevents the
cells from adhering to a surface to form typical colony growth. EBs
appear as rounded collections of cells and contain cell types
derived from all three germ layers (i.e., the ectoderm, mesoderm
and endoderm). Methods for generating EBs are well-known to one
having ordinary skill in the art. See, Itskovitz-Eldor J, et al.,
"Differentiation of human embryonic stem cells into embryoid bodies
compromising the three embryonic germ layers," Mol. Med. 6:88-95
(2000); Odorico J, et al., Stem Cells 19:193-204 (2001); and U.S.
Pat. No. 6,602,711, each of which is incorporated herein by
reference as if set forth in its entirety.
[0046] As used herein, "serum-free" means that neither the culture
nor the culture medium contains serum or plasma, although purified
or synthetic serum or plasma components (e.g., FGFs) can be
provided in the culture in reproducible amounts as described
below.
[0047] As used here, a "substantially pure population" means a
population of derived cells that contains at least 99% of the
desired cell type. Cell purification can be accomplished by any
means known to one of ordinary skill in the art. For example, a
substantially pure population of cells can be achieved by growth of
cells or by selection from a less pure population, as described
herein.
[0048] As used herein, "pluripotent cells" means a population of
cells capable of differentiating into all three germ layers and
becoming any cell type in the body. Pluripotent cells express a
variety of cell surface markers, have a cell morphology
characteristic of undifferentiated cells and form teratomas when
introduced into an immunocompromised animal, such as a SCID mouse.
Teratomas typically contain cells or tissues characteristic of all
three germ layers.
[0049] As used herein, "multipotent" cells are more differentiated
than pluripotent cells, but are not permanently committed to a
specific cell type. Pluripotent cells therefore have a higher
potency than multipotent cells.
[0050] As used herein, "induced pluripotent stem cells" or "iPS
cells" are cells that are differentiated, somatic cells
reprogrammed to pluripotency. The cells are substantially
genetically identical to their respective differentiated somatic
cell of origin and display characteristics similar to higher
potency cells, such as ES cells. See, Yu J, et al., "Induced
pluripotent stem cell lines derived from human somatic cells,"
Science 318:1917-1920 (2007), incorporated herein by reference as
if set forth in its entirety.
[0051] As used herein, a "mesenchymal stem cell" (MSC) is a cell
capable of differentiating into the skeletal cell lineages (i.e.,
osteoblasts, chondroblasts and adipocytes). As noted above, no
unique MSC marker has been identified. As such, morphological and
functional criteria well-known to those of ordinary skill in the
art are used to identify these cells. See, Horwitz et al., supra;
Dominici et al., supra; Trivedi P & Hematti P, "Derivation and
immunological characterization of mesenchymal stromal cells from
human embryonic stem cells," Exp. Hematol. Jan. 5, 2008 [Epub ahead
of print]; Trivedi P & Hematti P, "Simultaneous generation of
CD34+ primitive hematopoietic cells and CD56+ mesenchymal stem
cells from human embryonic stem cells cocultured with murine OP9
stromal cells," Exp. Hematol. 35:146-154 (2007); and US Published
Patent Application No. 2006/0008902, each of which is incorporated
herein by reference as if set forth in its entirety. MSCs produced
by the methods described herein can be characterized according to
phenotypic criteria. For example, MSCs can be recognized by their
characteristic mononuclear ovoid, stellate shape or spindle shape,
with a round to oval nucleus. The oval elongate nuclei typically
have prominent nucleoli and a mix of hetero- and euchromatin. These
cells have little cytoplasm, but many thin processes that appear to
extend from the nucleus. It is believed that MSCs will typically
stain for one, two, three or more of the following markers: CD106
(VCAM), CD73, CD146, CD166 (ALCAM), CD29, CD44 and alkaline
phosphatase, while being negative for hematopoietic lineage cell
markers (e.g., CD14 or CD45) and endothelial lineage cell markers
(e.g., CD31 and VE-cadherin). MSCs may also express STRO-1 as a
marker.
[0052] As used herein, a "mesangioblast" is a progenitor for MSCs
as well as endothelial cells.
[0053] As used herein, a "mesenchymal colony" is a colony composed
of mesenchymal cells originating from mesangioblasts.
[0054] As used herein, a "hemangioblast" is a precursor to blood
cells as well as endothelial cells.
[0055] As used herein, a "blast colony" is a colony composed of
predominantly hematopoietic cells originating from
hemangioblasts.
[0056] As used herein, "mesendoderm" is a tissue that gives rise to
mesoderm and endoderm.
[0057] As used herein, "mesoderm" is a cell subset that expresses
KDR and PDGFRa to much greater level than POU4F1, SOX1, and PAX6
(neural crest and neuroectoderm), LAMAS, KRT14, and KRT10 (surface
ectoderm), CGA and PLAC1 (trophectoderm) FOXA1, FOXA2, APOA1,
TMPRSS2, TTR1, and AFP (endoderm), and SOX2 and DPPA2
(undifferentiated hESCs).
[0058] As used herein, "lateral plate mesoderm" is a subset of
mesoderm that expresses at least FOXF1 and HAND1 but lacks
expression of MEOX1 and TCF15 (paraxial mesoderm), PAX2 and PAX8
(intermediate mesoderm), and is capable of at least endothelial and
hematopoietic differentiation.
[0059] It is contemplated that Matrigel.RTM., laminin, collagen
(especially collagen type I), fibronectin and glycosaminoglycans
may all be suitable as an extracellular matrix, by themselves or in
various combinations.
[0060] The invention will be more fully understood upon
consideration of the following non-limiting Examples.
EXAMPLES
Example 1
[0061] Generation of MSCs from Pluripotent Stem Cells Under
Serum-Free Conditions.
[0062] hESCs (H1; WiCell; Madison, Wis.) were maintained on
irradiated mouse embryonic fibroblasts in a serum-free medium, such
as DMEM/F12 medium supplemented with 20% Knockout.TM. serum
replacer, 2 mM L-glutamine, 1.times.(100 .mu.M) non-essential amino
acids, 100 .mu.M 2-mercaptoethanol and 4 ng/ml bFGF (all from
Gibco-Invitrogen; Carlsbad, Calif.). See Amit M, et al., "Clonally
derived human embryonic stem cell lines maintain pluripotency and
proliferative potential for prolonged periods of culture," Dev.
Biol. 227:271-278 (2000), incorporated herein by reference as if
set forth in its entirety. Mouse OP9 bone marrow stromal cells
(kindly provided by Dr. Toru Nakano and available from ATCC,
catalog # CRL-2749) were maintained by four-day subculture on
gelatin-coated dishes in alpha MEM medium (Gibco-Invitrogen) with
20% fetal calf serum (FCS; HyClone; Logan, Utah).
[0063] The hESCs were induced to differentiate by co-culture with
mouse OP9 bone marrow stromal cells, as previously described.
Vodyanik M, et al., "Human embryonic stem cell-derived CD34+ cells:
efficient production in the coculture with OP9 stromal cells and
analysis of lymphohematopoietic potential," Blood 105:617-626
(2005), incorporated herein by reference as if set forth in its
entirety. Briefly, small aggregates of hESCs were added to OP9
cells in alpha MEM supplemented with 10% FCS and 100 .mu.M MTG
(Sigma; St. Louis, Mo.). On the next day (day 1) of culture, the
medium was changed, and the cultures were harvested on the days
indicated below.
[0064] On day two of hESC (H1) co-culture with OP9 stromal cells,
peak expression of transcription factors for primitive streak
population (mesendoderm) (GSC, EOMES, MIXL1 and T (BRACHYURY)) and
early mesoderm (EVX1, LHX1 and TBX6) were detected with
NimbleGen.RTM. (Madison, Wis.) microarrays.
[0065] On days 3-5 of co-culture, the culture contained mesenchymal
progenitors, as well as cells expressing genes characteristic of
endoderm and mesoderm. Among the genes characteristic for mesoderm,
only genes characteristic of the lateral plate mesoderm, such as
FOXF1, HAND1, NKX2-5, and GATA2 were expressed consistently. In
contrast, genes characteristic for the axial (CHRD, SHH), paraxial
(MEOX1, TCF15), or intermediate (PAX2, PAX8) mesoderm were not
expressed consistently. Thus, hESCs co-cultured with OP9 cells for
3-5 days gave rise to cells expressing genes characteristic of the
lateral plate/extraembryonic mesoderm. On days 3-5 of hESC(H1)/OP9
co-culture, the cells were also characterized by maximal cell
proliferation and sustained expression of genes involved in
epithelial-mesenchymal transition (EMT, SNAI1, and SNAI2) and cell
expansion (HOXB2, HOXB3).
[0066] On days 5-7 of hESC(H1)/OP9 co-culture, differentiation into
specific mesodermal and endodermal lineages was observed, when
markers of developing endoderm (AFP and SERPINA1), mesenchymal
(SOX9, RUNX2, and PPARG2), and hematoendothelial (CDH5 and GATA1)
cells were detected. Neither muscle-inductive factors (MYOD1, MYF5,
and MYF6) nor neuroectoderm (SOX1, PAX6, and NEFL) or trophectoderm
(CGB and PLAC) markers were expressed throughout the seven days of
co-culture, indicating that OP9 cells provided an efficient
inductive environment for directed hESC differentiation toward the
mesendodermal pathway.
[0067] On day 2 of hESC(H1)/OP9 co-culture, a single-cell
suspension was harvested from the co-culture by successive
enzymatic treatment with collagenase IV (Gibco-Invitrogen) at 1
mg/ml in DMEM/F12 medium for 15 minutes at 37.degree. C. and 0.05%
Trypsin-0.5 mM EDTA (Gibco-Invitrogen) for 10 minutes at 37.degree.
C., Cells were washed 3 times with PBS-5% FBS, filtered through 70
.mu.M and 30 .mu.M cell strainers (BD Labware; Bedford, Mass.) and
labeled with anti-mouse CD29-PE (AbD Serotec; Raleigh, N.C.) and
anti-PE paramagnetic monoclonal antibodies (Miltenyi Biotec;
Auburn, Calif.). The cell suspension was purified with
magnet-activated cell sorting (MACS) by passing it through a LD
magnetic column attached to a Midi-MACS separation unit (Miltenyi
Biotech) to obtain a negative fraction of OP9-depleted,
hESC-derived cells. Purity was verified using pan anti-human
TRA-1-85 monoclonal antibodies (R&D Systems; Minneapolis,
Minn.).
[0068] The purified single-cell suspension was plated at density of
0.5-2.times.10.sup.4 cells/ml on a semisolid, serum-free medium
composed of StemLine.TM. serum-free medium (Sigma; St. Louis, Mo.)
supplemented with 5-100 ng/ml bFGF (PeproTech; Rocky Hill, N.J.)
and 1% methylcellulose (Stem Cell Technologies; Vancouver, Canada)
with or without 10-20 ng/ml PDGF-BB (PeproTech). PDGF-BB improved
growth of mesenchymal cells, but was not essential for colony
formation. Alternatively, single cell suspensions were plated in a
semisolid colony-forming serum-free medium containing 40% ES-Cult
M3120 methylcellulose, 25% serum-free expansion medium (SFEM, Stem
Cell Technologies), 25% endothelial serum-free medium (E-SFM,
Invotrogen), 10% BIT 9500 (Stem Cell Technologies), GlutaMAX
(diluted 1:100), Ex-Cyte (diluted 1:1000, Millipore), 100 .mu.M
monothioglycerol (MTG), 50 .mu.g/ml ascorbic acid and 20 ng/ml
bFGF.
[0069] After 10-20 days of culture, large, compact mesenchymal
colonies formed that resembled embryoid bodies (EBs). While these
mesenchymal colonies were detected as early as day 7, 10-20 days of
culture were required to reveal actively growing colonies.
Undifferentiated hESCs or cells harvested on day 1 or on day 6 of
co-culture did not form these mesenchymal colonies when cultured
under the same conditions.
[0070] Mesenchymal colonies, which resembled embryoid-like bodies,
were distinguished from EBs through several characteristics: (1)
formation and growth under serum-free conditions supplemented with
bFGF and stimulation by factors promoting mesenchymal cell growth
(e.g., PDGF-BB, EGF and TGF-.alpha.), but suppression by factors
involved in mesodermal differentiation (e.g., VEGF, TGF-.beta. and
Activin A) in mesenchymal colonies; (2) lack of a dense outer cell
layer and irregular cavitated structure characteristic of EBs, even
after prolonged culture in mesenchymal colonies; (3) presence of
morphological homogeneity in cells comprising the mesenchymal
colonies; and (4) formation of colonies through establishment of
tightly packed structures (cores) which further develop into
compact spheroid colonies.
[0071] To demonstrate that the single-cell suspensions did not form
aggregates upon plating in semi-solid medium, clonality of the
mesenchymal colonies obtained in the culture methods was tested and
confirmed using chimeric hESC lines established from cells
retrovirally marked with a reporter gene, e.g., either enhanced
green fluorescent protein (EGFP) or histone 2B-(H2BB) mOrange
fluorescent protein. Expression of a product of the reporter gene
indicated clonality. The chimeric hESC lines were generated from
two lentiviral constructs: (1) the EGFP protein expressed
constitutively from an elongation factor 1 alpha (EF1alpha)
promoter, and (2) the H2BB-mOrange protein expressed constitutively
from the EF1alpha promoter. Both constructs were packaged in 293FT
cells, and the lentiviruses were used to transduce H1 hESCs to
produce stable H1 hESC lines that expressed either green EGFP
protein or orange H2BB-mOrange protein. Mesenchymal colonies
derived from the described methods were of single colors, either
green or orange, thus indicating the clonal (i.e., single cell)
origin of the MSCs. In addition, prospective phenotypic analysis
demonstrated a positive correlation between mesenchymal-colony
forming cell (CFC) frequency and KDR (VEGFR2) expression, though
KDR.sup.highCD34+ population of the earliest hemangiogenic
precursors was devoid of mesenchymal-CFCs. Analysis of cells within
mesenchymal colonies revealed a homogeneous population of early
mesenchymal cells defined by high, CD90, CD140a and CD166
expression, low CD44, CD56 and CD105 expression and lack of CD24,
CD31, CD43, CD45, CD144 (VE-cadherin), and lack of SSEA4
expression. In addition, mesenchymal colonies expressed vimentin,
alpha smooth muscle actin, and desmin. Furthermore, mesenchymal
colonies expressed genes specific for MSC lineage, such as FOXF1,
MSX1, MSX2, SNAI1, SNAI2, SOX9, and RUNX2.
[0072] Individual mesenchymal colonies were transferred to wells of
a collagen- or fibronectin-coated, 96-well plate pre-filled with
0.2 ml/well StemLine.TM. serum-free medium supplemented with 5-100
ng/ml bFGF or serum-free expansion medium consisting of 50%
StemLine II serum-free HSC expansion medium (H-SFEM, Sigma), and
50% E-SFM supplemented with GlutaMAX (diluted 1:100), ExCyte
(diluted 1:2000), 100 .mu.M MTG, and 10 ng/ml bFGF. After 3-4 days
of culture, adherent cells from individual wells were harvested by
trypsin treatment and expanded on collagen- or fibronectin-coated
dishes in StemLine.TM. serum-free medium with 5-100 ng/ml bFGF or
serum-free expansion medium (M-SFEM) containing 50% StemLine.TM. II
serum-free HSC expansion medium (HSFEM; Sigma), 50% E-SFM,
GlutaMAX.TM. (1/100 dilution), Ex-Cyte.RTM. supplement (1/2000
dilution), 100 .mu.M MTG, and 5-100 ng of bFGF.
[0073] MSCs were expanded for many passages. When individual
colonies were plated on collagen- or fibronectin-coated plates,
immediate attachment and vigorous outgrowth of fibroblast-like
cells were observed. During subsequent passages, cells grew
intensively during the first 10 passages; however, growth rate was
attenuated at passages 10-15 and gradual senescence was observed
during passages 15-20. Cultures derived from single MB-CFC
accumulated up to 10.sup.22 total cells in the observed time
period. Because each colony is presumed to have originated from a
single cell, the number corresponds to the expansion potential of a
single hESC-derived mesenchymal precursor.
[0074] Cell lines established from individual colonies were
maintained in serum-free medium with bFGF for 10-15 passages at a
high proliferation rate. All cell lines displayed a mesenchymal
phenotype, characterized by expression of CD44, CD45, CD73, CD105,
CD146, and CD140a (PDGFRA) and lack of hematoendothelial markers
(i.e. CD31, CD43, CD45 and VE-cadherin). When tested in conditions
revealing mesenchymal differentiation potential, the cell lines
were capable of osteogenic, chondrogenic and adipogenic
differentiation. Interestingly, these cells resemble bone marrow
MSCs, but expand and proliferate better than bone marrow MSCs.
These expanded mesenchymal cells could be differentiated into cells
of the chondro-, osteo- and adipogenic lineage. However, these
cells could not give rise to hematopoietic or endothelial cells
when cultured with OP9 cells, or when cultured in feeder-free
cultures with hematoendothelial growth factors (VEGF, bFGF, SCF,
TPO, IL3, IL6), indicating a limited differentiation potential of
these mesenchymal cells.
[0075] Mesenchymal colonies were also generated from various
induced pluripotent stem (iPS) cells, such as iPS(IMR90)-1,
iPS(SK)-46, and iPS(FSK)-1 reprogrammed using a lentiviral vector
(Yu et al., Science 318:1917-1920 (2007)), or transgene-free iPS-5
4-3-7T and iPS-1 19-9-7T (Yu et al., Science 324:797-801 (2009)).
Mesenchymal colonies derived from transgene-containing iPS cells
displayed irregular or more loose morphology. Transgene-free iPSC
produced typical spheroid mesenchymal colonies.
Example 2
[0076] In vitro generation and characterization of
mesangioblasts.
[0077] To isolate and characterize a population of mesodermal
progenitors that can give rise to cells of the mesodermal lineage
with hematopoietic, endothelial, and mesenchymal stem cell
potentials, H1 hES cells were co-cultured with OP9 cells, as
described in Example 1. After two or three days of co-culture, when
genes representative of primitive streak population (mesendoderm)
(MIXL1, T, EOMES) were expressed, the hESC-derived cells depleted
of OP9 cells using anti-mouse CD29 antibody were plated in
semisolid, serum-free medium, essentially as described in Example
1, with 20 ng/ml bFGF (PeproTech; Rocky Hill, N.J.). The number of
colony-forming cells (CFCs) was calculated per 1000 plated
H1-derived TRA-1-85.sup.+ cells.
[0078] After 2-3 days in semisolid medium, the cells formed tightly
packed structures (cores). Cores derived from hESCs that were
differentiated in co-culture with OP9 cells for 2 days further grew
into spheroid mesenchymal colonies. Cores derived from hESCs that
were differentiated in co-culture with OP9 cells for 3 days further
grew into dispersed blast colonies with hematopoietic and
endothelial potential.
[0079] bFGF is necessary and sufficient for the formation of both
colonies from hESCs. bFGF supported both mesenchymal and blast
colony formation. In contrast, in the absence of bFGF, neither
VEGF, nor PDGF-BB (FIG. 1A), SCF, IGF1, or HGF (data not shown),
alone or in combination, supported formation of either colony.
While PDGF-BB (10 ng/ml) alone did not support colony formation,
PDGF-BB in combination with bFGF significantly increased the yield
and size of mesenchymal colonies compared to bFGF alone (FIG. 1A).
VEGF alone (20 ng/ml) did not support colony formation but its
addition to bFGF cultures slightly increased the number of blast
colonies, but inhibited formation of mesenchymal colonies (FIG.
1A). Cells that gave rise to each colony type constituted
approximately 2-3% of total hESC-derived cells (FIG. 1B).
[0080] To determine if cells within the mesenchymal colonies can
give rise to cells of the hermatovascular lineage, individual
mesenchymal colonies were picked from the methylcellulose on day
5-7 and plated onto OP9 cells in alpha-MEM medium with 10% FBS, and
the cytokines SCF (50 ng/ml), TPO (50 ng/ml), IL-3 (10 ng/ml), and
IL-6 (20 ng/ml). After 4 days of culture, cells were harvested and
analyzed by flow cytometry or stained in situ with rabbit
anti-human CD144 (VE-cadherin; 1 .mu.g/ml; eBioscience, San Diego,
Calif.) in combination with mouse anti-human CD43 (0.5 .mu.g/ml; BD
Bioscience) or mouse anti-human Calponin (0.5 .mu.g/ml; Thermo
Fisher Scientific) primary antibodies, followed by a mixture of
secondary cross-absorbed donkey anti-mouse IgG-DyLight 594 and
donkey anti-rabbit IgG-DyLight-488 (both at 2 .mu.g/ml; Jackson
ImmunoResearch Laboratories, Inc., West Grove, Pa.) antibodies.
[0081] The mesenchymal colonies originated from precursors that
gave rise to endothelial and mesenchymal cells, i.e.
mesangioblasts. As explained in Example 1, MSCs expanded from
mesenchymal colonies in adherent cultures did not give rise to
hematopoietic or endothelial cells when cocultured with OP9 cells.
In contrast, approximately 70% of mesenchymal colonies isolated
from day 5-7 colony-forming cultures in semisolid media gave rise
to CD31.sup.+CD144(VE-cadherin).sup.+ endothelial cells when
cocultured with OP9 cells. (FIG. 1D and E, upper panels). The
mesenchymal colonies, therefore, originated from common precursors
for endothelial and mesenchymal lineages, i.e., mesangioblasts. In
contrast, blast colonies contained CD31.sup.+CD43.sup.+
hematopoietic cells and could give rise to endothelial cells (FIG.
1D and E lower panels).
[0082] The endothelial potential of mesenchymal colonies could be
significantly enhanced with the addition of bone morphogenic
protein 4 (BMP4) to the clonogenic assay medium (3.2.+-.2.4%
CD31.sup.+CD43.sup.- cells without BMP4 vs. 11.6.+-.0.5 with 5
ng/ml BMP4).
Example 3
[0083] Generating and isolating a population of cells substantially
enriched in lateral plate/extraembryonic mesoderm cells.
[0084] Genetic profiling of H1 hESCs differentiated in OP9
cocultures demonstrated selective commitment toward mesodermal and
endodermal lineages with no detectable ectoderm (tropho-, neuro-,
or surface ectoderm) (FIG. 2). The cells became committed to
mesendoderm by day 2 of culture, when synchronous expression of
primitive streak genes (MIXL1, T, and EOMES) was detected. At
subsequent days of culture, mesoderm- and endoderm-specific genes
and, eventually, endoderm- and mesoderm derivative-specific genes
were expressed. Of the mesodermal genes, those characteristic of
the lateral plate/extraembryonic mesodermal subset (FOXF1, HAND1,
NKX2-5, GATA2) were expressed consistently, while expression of
genes of the axial (CHRD, SHH), paraxial (MEOX1, TCF15), or
intermediate (PAX2, PAX8) subsets was not consistent. Apelin
receptor (APLNR) expression is strongly induced and up-regulated on
days 2-3 of differentiation, concurrently with mesodermal
commitment.
[0085] To characterize APLNR expression and the cells that express
it, hESCs differentiated in OP9 co-cultures were stained with
monoclonal antibodies specific for Apelin receptor (APLNR) (R&D
Systems) in combination with antibodies against CD30, KDR, PDGFRA,
T, and FOXA2. Undifferentiated hESCs and hESC-derived cells on day
1 of OP9 co-culture were APLNR negative (FIG. 3A, Day 1 panels).
Expression of APLNR was strongly up-regulated in cells co-cultured
with OP9 cells for 2-3 days (FIG. 3A, Day 2 and 3). On day 2,
15-20% of cells were APLNR.sup.+ and by day 3, 60-70% of cells were
APLNR.sup.+. This upregulation coincided with mesodermal
commitment, as evidenced by the upregulation of mesodermal markers,
such as KDR (VEGFR2), T, and PDGFRA (FIG. 3A, Day 2 and 3 panels).
The number of APLNR.sup.+ cells gradually decreased on subsequent
days (FIG. 3B). Conversely, hESC markers (e.g. CD30) were
successively down-regulated.
[0086] While PDGFRA is expressed only at low levels in day 2
co-cultures, APLNR is expressed at high density as early as day 2
of co-culture allowing separation of APLNR positive from APLNR
negative cells. On days 2, 2.5, and 3 of H1/OP9 cell co-culture,
APLNR.sup.+ and APLNR.sup.- cells were separated by magnetic
sorting and gene expression was analyzed by microarray
analysis.
[0087] MIXL1, T, and EOMES, indicative of primitive streak cells
(mesendoderm), were all expressed in APLNR.sup.+ cells, while
transcripts associated with neural crest/neuroectoderm (POU4F1,
SOX1, SOX2, SOX3, SOX10) could not be detected (FIG. 4A and 4B). As
expected, APLNR.sup.+ cells were enriched in TCF21
mesoderm-specific transcripts, whereas transcripts marking
pan-endoderm (FOXA2, APOA1), definitive (FOXA1, TMPRSS2), and
visceral (TTR, AFP) endoderm were found in APLNR.sup.-cells (FIG.
4A and C).
[0088] Interestingly, APLNR.sup.+ cells expressed FOXF1, IRX3,
BMP4, WNT5A, NKX2.5, HAND1, and HAND2 representative of lateral
plate/extraembryonic mesoderm, but not markers of paraxial/myogenic
(MEOX1, TCF15, PAX3, PAX7) and intermediate (PAX2, PAX8) mesoderm
in the embryo. This data indicates that rather than being a total
population of cells committed to mesendodermal development,
APLNR.sup.+ cells represent mesoderm, or likely its subpopulation
reminiscent of lateral plate/extraembryonic mesoderm (FIG. 3C and
FIG. 4).
[0089] To further confirm mesodermal identity, APLNR.sup.+ cells
were analyzed for expression of T, a marker of early mesoderm, and
FOXA2, a marker of endoderm. As shown in FIG. 3A, APLNR.sup.+ cells
are T.sup.+ and maintain T expression until it subsides on day 4.
In contrast, FOXA2.sup.+ cells, which comprised less than 5% of
total cells in culture, did not express APLNR. Thus, APLNR.sup.+
cells are T.sup.+ FOXA2.sup.- mesodermal precursors on day 2-3 of
culture.
[0090] To further support the notion that APLNR.sup.+ cells are
mesodermal precursors, H1/OP9 cell co-cultures were supplemented
with inhibitors of mesoderm formation SB431542 (5 .mu.g/ml) or DKK1
(150 .mu.g/ml). APLNR.sup.+ cells could not be detected in cultures
that received the inhibitors of mesoderm formation (FIG. 3B),
confirming that APLNR.sup.+cells are mesodermal. Further,
mesenchymal and blast colony-forming potential was found
exclusively within the APLNR.sup.+ cell population (FIG. 3D),
further confirming that both mesangiogenic mesenchymal and
hemangiogenic blast colonies are formed by APLNR.sup.+ mesodermal
precursors.
Example 4
[0091] Enrichment of Mesangioblasts Derived from hESCs Under
Serum-Free Conditions through isolation of APLNR.sup.+lateral
plate/extraembryonic mesoderm cells.
[0092] To identify the origin of mesenchymal colonies and obtain a
population of cells enriched in mesangioblasts, pluripotent stem
cells were co-cultured with OP9 for 2-3 days to induce mesoderm
formation. After depletion of OP9 cells with mouse-specific CD29
antibodies, APLNR.sup.+ and APLNR.sup.- cells were isolated using
magnetic sorting. Colony formation assays in semisolid media in
presence of bFGF demonstrated that mesangioblast and hemangioblast
potential was confined solely to the APLNR.sup.+ fraction (FIG.
3D). Approximately 1 to 5% of cells within APLNR.sup.+fraction
possessed mesangioblast activity.
[0093] The invention has been described in connection with what are
presently considered to be the most practical and preferred
embodiments. However, the present invention has been presented by
way of illustration and is not intended to be limited to the
disclosed embodiments. Accordingly, those skilled in the art will
realize that the invention is intended to encompass all
modifications and alternative arrangements within the spirit and
scope of the invention as set forth in the appended claims.
* * * * *