U.S. patent application number 12/554696 was filed with the patent office on 2010-01-21 for generation of clonal mesenchymal progenitors and mesenchymal stem cell lines under serum-free conditions.
Invention is credited to James A. Thomson, Maksym A. Vodyanyk, Junying Yu.
Application Number | 20100015705 12/554696 |
Document ID | / |
Family ID | 40472086 |
Filed Date | 2010-01-21 |
United States Patent
Application |
20100015705 |
Kind Code |
A1 |
Vodyanyk; Maksym A. ; et
al. |
January 21, 2010 |
Generation of Clonal Mesenchymal Progenitors and Mesenchymal Stem
Cell Lines Under Serum-Free Conditions
Abstract
Methods for obtaining multipotent mesenchymal stem cells under
serum-free conditions and methods for identifying multipotent
mesenchymal progenitor cells are disclosed.
Inventors: |
Vodyanyk; Maksym A.;
(Madson, WI) ; Yu; Junying; (Madison, WI) ;
Thomson; James A.; (Madison, WI) |
Correspondence
Address: |
QUARLES & BRADY LLP
411 E. WISCONSIN AVENUE, SUITE 2040
MILWAUKEE
WI
53202-4497
US
|
Family ID: |
40472086 |
Appl. No.: |
12/554696 |
Filed: |
September 4, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12024770 |
Feb 1, 2008 |
7615374 |
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12554696 |
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60974980 |
Sep 25, 2007 |
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60989058 |
Nov 19, 2007 |
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Current U.S.
Class: |
435/354 ;
435/363; 435/366 |
Current CPC
Class: |
C12N 2502/1394 20130101;
C12N 2799/027 20130101; C12N 2501/115 20130101; C12N 2500/90
20130101; C12N 2506/02 20130101; C12N 2510/00 20130101; C12N 5/0662
20130101 |
Class at
Publication: |
435/354 ;
435/363; 435/366 |
International
Class: |
C12N 5/06 20060101
C12N005/06; C12N 5/00 20060101 C12N005/00; C12N 5/08 20060101
C12N005/08 |
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 an 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% methyl cellulose.
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 (BRACHYURY).
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, MEF2C, MSX1, MSX2, SNAI1, SNAI2, SOX9 and RUNX2.
18. The method of claim 1, wherein the mesenchymal colonies express
CD44, CD56 and CD105 expression, 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,
CD56, CD105 and CD140a, but no expression of CD31, CD43, CD45 and
VE-cadherin, (2) expression of FOXF1, MEF2C, MSX1, MSX2, SNAI1,
SNAI2, SOX9 and RUNX2 and (3) expression of vimentin and alpha
smooth muscle actin, but no expression of 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: an substantially pure line of
clonally-derived mesenchymal stem cells positive at least for CD44,
CD56, 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.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Utility patent
application Ser. No. 12/024,770, filed Feb. 1, 2008, 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.
[0004] 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.
[0005] 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)).
[0006] 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.
[0007] 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 (i.e., 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.
[0008] 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.
[0009] 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.
[0010] 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).
[0011] 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).
[0012] 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.
BRIEF SUMMARY
[0013] 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.
[0014] 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, to single
cells, 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).
[0015] 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.
[0016] 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. The colonies obtained in the method can be identified as
mesenchymal by their expression of at least a plurality of FOXF1,
MEF2C, 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);
and (2) differentiation into osteogenic, chondrogenic or adipogenic
cell lineages. Morphological characteristics of the colonies
include (1) tight packing of cells to form round aggregates
measuring 100-500 .mu.m in diameter and (2) 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,
CD56, CD105 and CD140a (PDGFRA), but not hematoendothelial surface
markers (i.e., CD31, CD43, CD45 and VE-cadherin); (2) expression of
FOXF1, MEF2C, MSX1, MSX2, SNAI1, SNAI2, SOX9 and RUNX2; and (3)
expression of vimentin and alpha smooth muscle actin, but no
expression of desmin.
[0017] 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.
[0018] The invention is further summarized as an substantially pure
population of clonally-derived MSC lines produced from the methods
described above that are positive for at least CD44, CD56, CD105
and CD140a, but negative for CD31, CD43, CD45 and VE-cadherin.
[0019] The described embodiments have many advantages, including
that mesenchymal progenitors or MSCs obtained in the methods may be
used to treat diseases associated with bone, cartilage and fat
cells.
[0020] It is also an advantage that a clonal population of MSCs can
be obtained from a single mesenchymal colony.
[0021] 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.
[0022] It is yet another advantage that cells obtained in the
methods can be non-immunoresponsive to allo- and auto-antigens on
transplantation.
[0023] It is still another advantage that the cells obtained in the
methods can differentiate into at least osteogenic, chondrogenic
and adipogenic lineages.
[0024] 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
[0025] Not applicable.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0026] 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.
[0027] In describing the embodiments and claiming the invention,
the following terminology is used in accordance with the
definitions set out below.
[0028] As used herein, "about" means within 5% of a stated
concentration.
[0029] 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.
[0030] 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); any method 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
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.
[0031] 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.
[0032] As used here, a "substantially pure population" means a
population of derived mesenchymal cells that contains at least 99%
mesenchymal cells. 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] As used herein, a "mesenchymal stem cell," "MSC," or
"mesenchymal progenitor" is a cell capable of differentiating into
the mesenchymal 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, mesenchymal progenitors or 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 mesenchymal progenitors or MSCs will typically stain
for one, two, three or more of the following markers: CD106 (VCAM),
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). Mesenchymal progenitors or MSCs may also express
STRO-1 as a marker.
[0037] 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.
[0038] The invention will be more fully understood upon
consideration of the following non-limiting Examples.
EXAMPLES
Example 1
Generation of MSCs from hESCs under Serum-Free Conditions
[0039] 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).
[0040] 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.
[0041] On day 2 of co-culture, mesodermal commitment was detected
by a peak expression of transcription factors for mesendoderm (GSC,
MIXL1 and T (BRACHYURY)) and early mesoderm (EVX1, LHX1 and TBX6)
with NimbleGen.RTM. (Madison, Wis.) microarrays. Mesenchymal
progenitors were still present in co-culture during days 3-5;
however, some specification to endoderm and mesodermal lineages was
also observed. This stage was accompanied with sustained expression
of genes involved in epithelial-mesenchymal transition (EMT; SNAI1
and SNAI2) and cell expansion (HOXB2-3). It also coincided with a
maximal cell proliferation rate in hESC/OP9 co-culture.
Differentiation of specific mesendodermal lineages was observed on
days 5-7 of co-culture, when markers of developing endoderm (AFP
and SERPINA1), mesenchymal (SOX9, RUNX2 and PPARG2) and
hematoendothelial (CDH5 and GATA1) cells were detected. However,
muscle-inductive factors (MYOD1, MYF5 and MYF6) were not expressed
throughout seven days of co-culture. Moreover, neuroectoderm (SOX1
and NEFL) or trophectoderm (CGB and PLAC) markers were not
detected, indicating that OP9 cells provided an efficient inductive
environment for directed hESC differentiation toward the
mesendodermal pathway.
[0042] Also on day 2 of 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.).
[0043] The purified single-cell suspension was plated at density of
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. After days 14-21 of culture, large, compact mesenchymal
colonies formed that resembled embryoid bodies (EBs). Mesenchymal
colonies were detected on day 7; however, 10-20 days were required
to reveal actively growing colonies. No mesenchymal colonies were
observed upon culture of undifferentiated hESCs or cells harvested
on day 1 or on day 6 of co-culture.
[0044] Mesenchymal colonies, which appeared as 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; and (3) presence of
morphological homogeneity in cells comprising the mesenchymal
colonies.
[0045] 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 (EF1 alpha)
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.
[0046] 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 CD73, CD90, CD140a and CD166 expression, low CD44,
CD56 and CD105 expression and lack of CD24, CD31, CD43, CD45,
CD144, and SSEA4 expression. In addition, mesenchymal colonies
expressed vimentin and alpha smooth muscle actin, but not desmin.
Furthermore, mesenchymal colonies expressed genes specific for MSC
lineage, such as FOXF1, MSX1/2, SNAI1/2, SOX9, RUNX2 and MEF2C.
[0047] 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. 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.
[0048] 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.
[0049] Cell lines established from individual colonies were
maintained in serum-free medium with bFGF for 10-15 passages at a
high proliferation rate. Phenotypically, all cell lines displayed a
mesenchymal phenotype as defined by expression of CD44, CD56, CD105
and CD140a (PDGFRA), but no expression 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.
[0050] 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.
* * * * *