U.S. patent application number 15/467201 was filed with the patent office on 2017-09-07 for mesenchymal stromal cells and uses related thereto.
This patent application is currently assigned to Astellas Institute for Regenerative Medicine. The applicant listed for this patent is Astellas Institute for Regenerative Medicine. Invention is credited to Jianlin Chu, Erin Anne Kimbrel, Nicholas Arthur Kouris, Robert P. Lanza.
Application Number | 20170252374 15/467201 |
Document ID | / |
Family ID | 48536141 |
Filed Date | 2017-09-07 |
United States Patent
Application |
20170252374 |
Kind Code |
A1 |
Kimbrel; Erin Anne ; et
al. |
September 7, 2017 |
MESENCHYMAL STROMAL CELLS AND USES RELATED THERETO
Abstract
The present invention generally relates to novel preparations of
mesenchymal stromal cells (MSCs) derived from hemangioblasts,
methods for obtaining such MSCs, and method sof treating a
pathology using such MSCs. The methods of the present invention
produce substantial numbers of MSCs having a potency-retaining
youthful phenotype, which are useful in the treatment of
pathologies.
Inventors: |
Kimbrel; Erin Anne;
(Sudbury, MA) ; Lanza; Robert P.; (Clinton,
MA) ; Chu; Jianlin; (Bedford, MA) ; Kouris;
Nicholas Arthur; (Hudson, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Astellas Institute for Regenerative Medicine |
Marlborough |
MA |
US |
|
|
Assignee: |
Astellas Institute for Regenerative
Medicine
Marlborough
MA
|
Family ID: |
48536141 |
Appl. No.: |
15/467201 |
Filed: |
March 23, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14504351 |
Oct 1, 2014 |
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15467201 |
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13691349 |
Nov 30, 2012 |
8962321 |
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14504351 |
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61565358 |
Nov 30, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/30 20130101;
A61K 35/407 20130101; A61P 13/12 20180101; A61P 35/02 20180101;
C12N 2501/155 20130101; A61P 1/04 20180101; A61P 17/02 20180101;
C12N 2501/115 20130101; A61K 35/34 20130101; A61P 27/16 20180101;
C12N 2533/54 20130101; A61P 35/00 20180101; A61P 11/02 20180101;
C12N 5/0647 20130101; A61P 25/16 20180101; A61P 37/06 20180101;
C12N 2502/1171 20130101; A61P 3/06 20180101; A61P 7/00 20180101;
A61P 1/16 20180101; A61P 11/00 20180101; A61P 17/00 20180101; A61P
19/02 20180101; A61P 37/00 20180101; A61P 9/00 20180101; A61P 27/02
20180101; A61P 1/02 20180101; A61P 43/00 20180101; C12N 2501/165
20130101; A61P 9/10 20180101; A61P 17/06 20180101; A61P 29/00
20180101; C12N 5/0668 20130101; A61P 37/08 20180101; C12N 2501/26
20130101; A61P 11/06 20180101; A61P 31/04 20180101; A61P 37/02
20180101; C12N 2501/145 20130101; A61K 35/39 20130101; A61P 19/08
20180101; C12N 2506/28 20130101; A61K 35/28 20130101; A61P 25/00
20180101; A61P 21/00 20180101; C12N 2501/125 20130101; A61P 3/10
20180101; C12N 2506/02 20130101; A61K 35/36 20130101; A61P 3/00
20180101; A61P 9/04 20180101; A61P 19/00 20180101; A61P 31/12
20180101; C12N 5/0692 20130101; C12N 2506/11 20130101; A61K 35/30
20130101; A61K 2300/00 20130101; A61K 35/34 20130101; A61K 2300/00
20130101; A61K 35/36 20130101; A61K 2300/00 20130101; A61K 35/39
20130101; A61K 2300/00 20130101; A61K 35/407 20130101; A61K 2300/00
20130101; A61K 35/28 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 35/28 20060101
A61K035/28; A61K 35/30 20060101 A61K035/30; A61K 35/34 20060101
A61K035/34; A61K 35/36 20060101 A61K035/36; C12N 5/0775 20060101
C12N005/0775; A61K 35/407 20060101 A61K035/407; C12N 5/077 20060101
C12N005/077; C12N 5/071 20060101 C12N005/071; C12N 5/0789 20060101
C12N005/0789; A61K 48/00 20060101 A61K048/00; A61K 35/39 20060101
A61K035/39 |
Claims
1. A pharmaceutical preparation, comprising at least 10.sup.6
mesenchymal stromal cells, wherein CD24 expression is upregulated
in mesenchymal stromal cells of the preparation, as compared to
mesenchymal stromal cells of bone marrow, and wherein mRNA encoding
interleukin-6 (IL-6) is expressed in mesenchymal stromal cells of
the preparation at a level that is less than ten percent of the
IL-6 mRNA level expressed by mesenchymal stromal cells of bone
marrow.
2-45. (canceled)
46. A method for generating mesenchymal stromal cells comprising
culturing embryonic stem cells under conditions that give rise to a
mesenchymal stromal cell population, wherein CD24 expression is
upregulated in mesenchymal stromal cells of the population, as
compared to mesenchymal stromal cells of bone marrow, and wherein
mRNA encoding interleukin-6 (IL-6) is expressed in mesenchymal
stromal cells of the population at a level that is less than ten
percent of the IL-6 mRNA level expressed by mesenchymal stromal
cells of bone marrow; and isolating the mesenchymal stromal cell
population.
47-102. (canceled)
103. A kit comprising the preparation of mesenchymal stromal cells
of claim 1.
104-125. (canceled)
126. The pharmaceutical preparation of claim 1 further comprising a
pharmaceutically acceptable carrier.
127. The pharmaceutical preparation of claim 1, wherein the
mesenchymal stromal cells are HLA-genotypically identical or
genomically identical.
128. The pharmaceutical preparation of claim 1, wherein at least
50% of the mesenchymal stromal cells of the preparation are
positive for CD24 expression.
129. The pharmaceutical preparation of claim 1, wherein the
preparation retains between 50% and 100% of its proliferative
capacity after ten population doublings.
130. The pharmaceutical preparation of claim 1, wherein the
preparation is pyrogen-free and/or pathogen-free.
131. The pharmaceutical preparation of claim 1, wherein the
mesenchymal stromal cells are generated in vitro from pluripotent
cells.
132. The pharmaceutical preparation of claim 131, wherein the
pluripotent cells are embryonic stem cells or induced pluripotent
stem cells.
133. The pharmaceutical preparation of claim 1, wherein the
mesenchymal stromal cells are isolated at early passage.
134. The pharmaceutical preparation of claim 133, wherein the
mesenchymal stromal cells have a replicative capacity to undergo at
least 10 population doublings in cell culture in less than 25
days.
135. The pharmaceutical preparation of claim 1, wherein the
mesenchymal stromal cells express lower Stro-1 expression levels,
relative to mesenchymal stromal cells derived from bone marrow.
136. The pharmaceutical preparation of claim 1, wherein the
preparation comprises less than 1% pluripotent stem cells.
137. The pharmaceutical preparation of claim 136, wherein the
preparation is devoid of pluripotent stem cells.
138. The pharmaceutical preparation of claim 1, wherein at least
90% of cells of the preparation are mesenchymal stromal cells.
139. The pharmaceutical preparation of claim 1, wherein the
pharmaceutical preparation comprises an effective amount of the
mesenchymal stromal cells to treat an autoimmune disease, an
inflammatory disease, pain, heat sensitivity or cold
sensitivity.
140. The pharmaceutical preparation of claim 139, wherein the
pharmaceutical preparation comprises an effective amount of the
mesenchymal stromal cells to treat an autoimmune disease selected
from multiple sclerosis, refractory systemic lupus erythematosus,
lupus nephritis and Crohn's disease.
141. The pharmaceutical preparation of claim 139, wherein the
pharmaceutical preparation comprises an effective amount of the
mesenchymal stromal cells to treat uveitis.
142. The pharmaceutical preparation of claim 139, wherein the
pharmaceutical preparation comprises an effective amount of the
mesenchymal stromal cells to treat pain.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Application Ser. No. 61/565,358, filed Nov. 30, 2011,
entitled "METHODS OF GENERATING MESENCHYMAL STROMAL CELLS USING
HEMANGIOBLASTS" (attorney docket no. 75820.210001) the contents of
which is incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the use of cell-based
therapies to reduce the manifestations of a pathology such as that
characterized by an inappropriate immune response in a subject, and
also to affect the origin of a pathology such that the abnormality
defining the pathology is returned to a normal posture. In
particular, the present invention relates to mesenchymal stromal
cells (MSCs) that retain a phenotype of "youthful" cells that
imparts a high potency in the reduction of a manifestation of a
pathology in a subject.
BACKGROUND OF THE INVENTION
[0003] Many pathologies manifest clinically through unwanted or
excessive immune responses within a host, e.g., transplant
rejection, inflammatory and autoimmune disorders. Immunosuppressive
therapies have been developed to treat the symptoms, but not the
underlying cause of pathologies characterized by excessive immune
responses. These therapies are effective at down-modulating immune
function and, as such, carry the potential for severe adverse
events, including cancer and opportunistic infection, as well as
side effects such as cataracts, hyperglycemia, bruising, and
nephrotoxicity from agents such as prednisone, cyclosporine, and
tacrolimus.
[0004] Although therapies that do not suppress the entire immune
system have been developed, there are limitations associated with
these regimens as well. These immunomodulatory treatments target a
narrower point of intervention within the immune system and, as
such, have different, sometimes less severe side effects. Examples
of such immunomodulatory therapies include the use of antibodies,
e.g., anti-CD3 or anti-IL2R. While successful at inducing a
heightened state of non-responsiveness, the withdrawal of these
immunomodulatory therapies results in a reversion to the unwanted
pathology.
[0005] Mesenchymal stem cells (MSC) are multipotent stem cells with
self-renewal capacity and the ability to differentiate into
osteoblasts, chondrocytes, and adipocytes, among other mesenchymal
cell lineages. In recent years, the intense research on the
multilineage differentiation potential and immunomodulatory
properties of human MSC have indicated that these cells can be used
to treat a range of clinical conditions, including immunological
disorders as well as degenerative diseases. Consequently, the
number of clinical studies with MSC has been steadily increasing
for a wide variety of conditions: graft-versus-host disease (GVHD),
myocardial infarction and inflammatory and autoimmune diseases and
disorders, among others. Cuurently, clinical programs utilizing
MSCs rely on isolation of these cells from adult sources and cord
blood. The high cell doses required for MSC clinical applications
(up to several million cells per kg of the patient) demands a
reliable, reproducible and efficient expansion protocol, capable of
generating a large number of cells from those isolated from the
donor source.
[0006] However, to reach the clinically meaningful cell numbers for
cellular therapy and tissue engineering applications, MSC ex-vivo
expansion is mandatory. As during aging in vivo, sequential ex-vivo
cell passaging of MSCs from a cord blood, fetal and adult sources
(such as bone marrow or adipost tissues) can cause replicative
stress, chromosomal abnormalities, or other stochastic cellular
defects, resulting in the progressive loss of the proliferative,
clonogenic and differentiation potential of the expanded MSCs,
which ultimately can jeopardize MSC clinical safety and efficacy.
The use of senescent MSCs in treatment should not be underestimated
since cells lose part of their differentiation potential and their
secretory profile is also altered. MSC senescence during culture
was found to induce cell growth arrest, with telomere shortening
and a continuous decrease in adipogenic differentiation potential
was reported for bone marrow (BM) MSC along increasing passages,
whereas the propensity for differentiation into the osteogenic
lineage increased.
[0007] Accordingly, some essential problems remain to be solved
before the clinical application of MSC. MSCs derived from ESCs can
be generated in sufficient quantities and in a highly controllable
manner, thus alleviating the problems with donor-dependent sources.
Since long-term engraftment of MSCs is not required, there is
basically no concern for mismatch of major histocompatibility (MHC)
[7, 8]. In the art, MSCs derived from ESCs have been obtained
through various methods including co-culture with murine OP9 cells
or handpicking procedures [9-13]. These methods, however, are
tedious and generate MSCs with a low yield, varying quality and a
lack of potency. Moreover, maximizing the potency of the injected
cells is desirable, both in terms of being able to provide a
cellular product with a better therapeutic index, ability to be
used at a reduce dosage (number of cells) relative to CB-derived,
BM-derived or adipost-derived MSCs, and/or the ability for the MSCs
to provide a tractable therapy for inflammatory and autoimmune
diseases for which CB-derived, BM-derived or adipost-derived MSCs
are not efficacious enough.
SUMMARY OF PREFERRED EMBODIMENTS
[0008] The present invention relates to mesenchymal stromal cells
(MSCs) and methods for generating MSCs. The methods of the present
invention produce substantial numbers of high quality mesenchymal
stromal cells, characterized by the phenotype of youthful cells
that imparts a high potency. In an embodiment of the invention, the
MSCs are derived from hemangioblasts. Preparations of the subject
MSCs are useful in the treatment of pathologies, including unwanted
immune responses, e.g., autoimmune diseases and disorders, as well
as inflammatory diseases and disorders.
[0009] In one aspect, the present invention comprises improved
preparations of MSCs generated from hemangioblasts using improved
methods for culturing the hemangioblasts. In exemplary embodiments,
mesenchymal stromal cells of the present invention retain higher
levels of potency and do not clump or clump substantially less than
mesenchymal stromal cells derived directly from embryonic stems
cells (ESCs). Mesenchymal stromal cells generated according to any
one or more of the processes of the present invention may retain
higher levels of potency, and may not clump or may clump
substantially less than mesenchymal stromal cells derived directly
from ESCs.
[0010] In one aspect, the invention provides pharmaceutical
preparations comprising mesenchymal stromal cells, wherein said
mesenchymal stromal cells are able to undergo at least 10
population doublings, e.g., at least 10 population doublings occur
within about 22-27 days. In another aspect, the invention provides
pharmaceutical preparations comprising mesenchymal stromal cells,
wherein said mesenchymal stromal cells are able to undergo at least
15 population doublings, e.g., at least 15 population doublings
occur within about 22-27 days. The pharmaceutical preparations may
be produced by in vitro differentiation of hemangioblasts. The
mesenchymal stromal cells may be primate cells, e.g., human cells.
The mesenchymal stromal cells may be able to undergo at least 15
population doublings. For example, the mesenchymal stromal cells
undergo at least 20, 25, 30, 35, 40, 45, 50 or more population
doublings. The preparation may comprise less than about 10%, 9%,
8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,
0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,
0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%,
0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%,
0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% pluripotent cells.
Preferably, the preparation is devoid of pluripotent cells. The
preparation may comprise at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% mesenchymal stromal cells.
[0011] In one aspect, at least 50% of said mesenchymal stromal
cells are positive for (i) at least one of CD10, CD24, IL-11,
AIRE-1, ANG-1, CXCL1, CD105, CD73 and CD90; (ii) at least one of
CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105,
CD13, CD29, CD 44, CD166, CD274, and HLA-ABC; or (iii) any
combination thereof. In another aspect, at least 50% of said
mesenchymal stromal cells are positive for (i) at least two of
CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73 and CD90; (ii)
all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90,
CD105, CD13, CD29,CD 44, CD166, CD274, and HLA-ABC. In yet another
aspect, at least 50% of said mesenchymal stromal cells are (i)
positive for all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105,
CD73, CD90, CD105, CD13, CD29,CD 44, CD166, CD274, and HLA-ABC and
(ii) do not express or express low levels of at least one of CD31,
34, 45, 133, FGFR2, CD271, Stro-1, CXCR4, TLR3. Additionally, at
least 60%, 70%, 80% or 90% of such mesenchymal stromal cells may be
positive for (i) one or more of CD10, CD24, IL-11, AIRE-1, ANG-1,
CXCL1, CD105, CD73 and CD90; or (ii) one or more of CD10, CD24,
IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13,
CD29,CD 44, CD166, CD274, and HLA-ABC.
[0012] In one aspect, the pharmaceutical preparation comprises an
amount of mesenchymal stromal cells effective to treat or prevent
an unwanted immune response in a subject in need thereof. The
pharmaceutical preparation may further comprise other cells,
tissues or organs for transplantation into a recipient in need
thereof. Exemplary other cells or tissues include RPE cells, skin
cells, corneal cells, pancreatic cells, liver cells, or cardiac
cells or tissue containing any of said cells.
[0013] In another aspect, the mesenchymal stromal cells are not
derived from bone marrow and the potency of the preparation in an
immune regulatory assay is greater than the potency of a
preparation of bone marrow derived mesenchymal stromal cells.
Potency may be assayed by an immune regulatory assay that
determines the EC50 dose.
[0014] In one aspect, the preparation retains between about 50 and
100% of its proliferative capacity after ten population
doublings.
[0015] In another aspect, the mesenchymal stromal cells of the
pharmaceutical preparation are not derived directly from
pluripotent cells and wherein said mesenchymal stromal cells (a) do
not clump or clump substantially less than mesenchymal stromal
cells derived directly from ESCs; (b) more easily disperse when
splitting compared to mesenchymal stromal cells derived directly
from ESCs; (c) are greater in number than mesenchymal stromal cells
derived directly from ESCs when starting with equivalent numbers of
ESCs; and/or (d) acquire characteristic mesenchymal cell surface
markers earlier than mesenchymal stromal cells derived directly
from ESCs.
[0016] The present invention further encompasses methods for
generating mesenchymal stromal cells comprising culturing
hemangioblast cells under conditions that give rise to mesenchymal
stem cells. The hemangioblasts may be cultured in feeder-free
conditions. Additonally, hemangioblasts may be plated on a matrix,
e.g., comprising transforming growth factor beta (TGF-beta),
epidermal growth factor (EGF), insulin-like growth factor 1, bovine
fibroblast growth factor (bFGF), and/or platelet-derived growth
factor (PDGF). The matrix may be selected from the group consisting
of: laminin, fibronectin, vitronectin, proteoglycan, entactin,
collagen, collagen I, collagen IV, heparan sulfate, Matrigel (a
soluble preparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma
cells), a human basement membrane extract, and any combination
thereof. The matrix may comprise a soluble preparation from
Engelbreth-Holm-Swarm mouse sarcoma cells.
[0017] In one aspect, the mesenchymal stromal cells are mammalian.
Preferably, the mesenchymal stromal cells are human, canine, or
equine.
[0018] In one aspect, the hemangioblasts may be cultured in a
medium comprising aMEM. In another aspect, the hemangioblasts may
be cultured in a medium comprising serum or a serum replacement.
For example, the hemangioblasts cells may be cultured in a medium
comprising, .alpha.MEM supplemented with 0%, 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, or
20% fetal calf serum. In additional exemplary embodiments the
medium may comprise higher percentages of fetal calf serum, e.g.,
more than 20%, e.g., at least 25%, at least 30%, at least 35%, at
least 40%, or even higher percentages of fetal calf serum. The
hemangioblasts may be cultured on said matrix for at least about
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or
30 days.
[0019] In one aspect, the hemangioblasts or hemangio-colony forming
cells are differentiated from pluripotent cells, e.g., iPS cells,
or blastomeres. The pluripotent cells may be derived from one or
more blastomeres without the destruction of a human embryo.
Additionally, the hemangioblasts may be differentiated from
pluripotent cells by a method comprising (a) culturing said
pluripotent cells to form clusters of cells. In one aspect, the
pluripotent cells are cultured in the presence of vascular
endothelial growth factor (VEGF) and/or bone morphogenic protein 4
(BMP-4). VEGF and BMP-4 may be added to the pluripotent cell
culture within 0-48 hours of initiation of said cell culture, and
said VEGF is optionally added at a concentration of 20-100 nm/mL
and said BMP-4 is optionally added at a concentration of 15-100
ng/mL.
[0020] In one aspect, the hemangioblasts are differentiated from
pluripotent cells by a method further comprising: (b) culturing
said single cells in the presence of at least one growth factor in
an amount sufficient to induce the differentiation of said clusters
of cells into hemangioblasts. The at least one growth factor added
in step (b) may comprise one or more of basic fibroblast growth
factor (bFGF), vascular endothelial growth factor (VEGF), bone
morphogenic protein 4 (BMP-4), stem cell factor (SCF), Flt 3L (FL),
thrombopoietin (TPO), EPO, and/or tPTD-HOXB4. The one or more of
said at least one growth factor added in step (b) may be added to
said culture within 36-60 hours from the start of step (a).
Preferably, the one or more of said at least one growth factor
added in step (b) is added to said culture within 40-48 hours from
the start of step (a). The at least one factor added in step (b)
may comprise one or more of bFGF, VEGF, BMP-4, SCF, FL and/or
tPTD-HOXB4. The concentration of said growth factors if added in
step (b) may range from about the following: bFGF is is about 20-25
ng/ml, VEGF is about 20-100 ng/ml, BMP-4 is about 15-100 ng/ml, SCF
is about 20-50 ng/ml, FL is about 10-50 ng/ml, TPO is about 20-50
ng/ml, and tPTD-HOXB4 is about 1.5-5 U/ml.
[0021] In another aspect, the method further comprises (c)
dissociating said clusters of cells, optionally into single cells.
In another aspect, the method further comprises (d) culturing said
hemangioblasts in a medium comprising at least one additional
growth factor, wherein said at least one additional growth factor
is in an amount sufficient to expand the hemangioblasts or
hemangio-colony forming cells. At least one additional growth
factors of (d) may comprise one or more of: insulin, transferrin,
granulocyte macrophage colony-stimulating factor (GM-CSF),
interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte
colony-stimulating factor (G-CSF), erythropoietin (EPO), stem cell
factor (SCF), vascular endothelial growth factor (VEGF), bone
morphogenic protein 4 (BMP-4), and/or tPTD-HOXB4. Exemplary
concentrations in step (d) include insulin about 10-100 .mu.g/ml,
transferrin about 200-2,000 .mu.g/ml, GM-CSF about 10-50 ng/ml,
IL-3 about 10-20 ng/ml, IL-6 about 10-1000 ng/ml, G-CSF about 10-50
ng/ml, EPO about 3-50 U/ml, SCF about 20-200 ng/ml, VEGF about
20-200 ng/ml, BMP-4 about 15-150 ng/ml, and/or tPTD-HOXB4 about
1.5-15U/ml. The medium in step (a), (b), (c) and/or (d) may be a
serum-free medium.
[0022] In one aspect, the method generates at least 80, 85, 90, 95,
100, 125, or 150 million mesenchymal stromal cells. The
hemangioblasts may be harvested after at least 10, 11, 12, 13, 14,
15, 16, 17 or 18 days of starting to induce differentiation of said
pluripotent cells. The mesenchymal stromal cells may be generated
within at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days of
starting to induce differentiation of said pluripotent cells. In
another aspect, the method results in at least 80, 85, 90, 95, 100,
125, or 150 million mesenchymal stromal cells being generated from
about 200,000 hemangioblasts within about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days
of culture. The mesenchymal stromal cells may be generated from
hemangioblasts and/or hemangio-colony forming cells in a ratio of
hemangioblasts to mesenchymal stromal cells of at least 1:200,
1:250, 1:300, 1:350, 1:400, 1:415, 1:425, 1:440; 1:450, 1:365,
1:475, 1:490 and 1:500 within about 26, 27, 28, 29, 30, 31, 32, 33,
34 or 35 days of culture. The cells may be human.
[0023] The present invention also contemplates mesenchymal stromal
cells derived from hemangioblasts obtained by the described
methods. In one aspect, the invention includes mesenchymal stromal
cells derived by in vitro differentiation of hemangioblasts. At
least 50% of said mesenchymal stromal cells may (i) be positive for
all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90,
CD105, CD13, CD29,CD 44, CD166, CD274, and HLA-ABC and (ii) not
express or express low levels of at least one of CD31, 34, 45, 133,
FGFR2, CD271, Stro-1, CXCR4, TLR3. Alternatively, at least 50% of
said mesenchymal stromal cells may be positive for (i) all of CD10,
CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73 and CD90; or (ii)
all of CD73, CD90, CD105, CD13, CD29, CD44, CD166, CD274, and
HLA-ABC. At least 60%, 70%, 80% or 90% of these mesencyhmal stromal
cells may be positive for (i) at least one of CD10, CD24, IL-11,
AIRE-1, ANG-1, CXCL1, CD105, CD73 and CD90; or (ii) at least one of
CD73, CD90, CD105, CD13, CD29,CD 44, CD166, CD274, and HLA-ABC.
Preferably, the mesenchymal stromal cells do not express or express
low levels of at least one of CD31, CD34, CD45, CD133, FGFR2,
CD271, Stro-1, CXCR4, TLR3.
[0024] In another aspect, the invention encompasses a preparation
of the mesenchymal stromal cells described herein. The preparation
may comprise less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,
0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%,
0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%,
0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,
0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or
0.0001% pluripotent cells. Preferably, the preparation is devoid of
pluripotent cells. The preparation may be substantially purified
and optionally comprises at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% human mesenchymal stromal cells. The
preparation may comprise substantially similar levels of p53 and
p21 protein or wherein the levels of p53 protein as compared to p21
protein are 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times greater. The
mesenchymal stromal cells may be capable of undergoing at least 5
population doublings in culture. Preferably, the mesenchymal
stromal cells are capable of undergoing at least 10, 15, 20, 25,
30, 35, 40, 45, 50, 55, 60 or more population doublings in
culture.
[0025] In one aspect, the mesenchymal stromal cells of the present
invention (a) do not clump or clump substantially less than
mesenchymal stromal cells derived directly from ESCs; (b) more
easily disperse when splitting compared to mesenchymal stromal
cells derived directly from ESCs; (c) are greater in number than
mesenchymal stromal cells derived directly from ESCs when starting
with equivalent numbers of ESCs; and/or (d) acquire characteristic
mesenchymal cell surface markers earlier than mesenchymal stromal
cells derived directly from ESCs. The invention contemplates a
pharmaceutical preparation comprising such mesenchymal stromal
cells, which comprises an amount of mesenchymal stromal cells
effective to treat an unwanted immune response. The preparation may
comprise an amount of mesenchymal stromal cells effective to treat
an unwanted immune response and further comprise other cells or
tissues for transplantation into a recipient in need thereof.
Exemplary other cells include allogeneic or syngeneic pancreatic,
neural, liver, RPE, or corneal cells or tissues containing any of
the foregoing. The pharmaceutical preparation may be useful in
treating an autoimmune disorder or an immune reaction against
allogeneic cells including, but not limited to, multiple sclerosis,
systemic sclerosis, hematological malignancies, myocardial
infarction, organ transplantation rejection, chronic allograft
nephropathy, cirrhosis, liver failure, heart failure, GvHD, tibial
fracture, left ventricular dysfunction, leukemia, myelodysplastic
syndrome, Crohn's disease, diabetes, chronic obstructive pulmonary
disease, osteogenesis imperfecta, homozygous familial
hypocholesterolemia, treatment following meniscectomy, adult
periodontitis, vasculogenesis in patients with severe myocardial
ischemia, spinal cord injury, osteodysplasia, critical limb
ischemia, diabetic foot disease, primary Sjogren's syndrome,
osteoarthritis, cartilage defects, laminitis, multisystem atrophy,
amyotropic lateral sclerosis, cardiac surgery, systemic lupus
erythematosis, living kidney allografts, nonmalignant red blood
cell disorders, thermal burn, radiation burn, Parkinson's disease,
microfractures, epidermolysis bullosa, severe coronary ischemia,
idiopathic dilated cardiomyopathy, osteonecrosis femoral head,
lupus nephritis, bone void defects, ischemic cerebral stroke, after
stroke, acute radiation syndrome, pulmonary disease, arthritis,
bone regeneration, uveitis or combinations thereof. The subject MSC
(including formulations or preparations thereof) may be used to
treat respiratory conditions, particularly those including
inflammatory components or acute injury, such as Adult Respiratory
Distress Syndrome, post-traumatic Adult Respiratory Distress
Syndrome, transplant lung disease, Chronic Obstructive Pulmonary
Disease, emphysema, chronic obstructive bronchitis, bronchitis, an
allergic reaction, damage due to bacterial or viral pneumonia,
asthma, exposure to irritants, and tobacco use. Additionally, the
subject MSC (including formulations or preparations thereof) may be
used to treat atopic dermatitis, allergic rhinitis, hearing loss
(particularly autoimmune hearing loss or noise-induced hearing
loss), psoriasis.
[0026] The invention further encompasses kits comprising the
mesenchymal stromal cells or preparation of mesenchymal stromal
cells described herein. The kits may comprise mesenchymal stromal
cells or preparations of mesenchymal stromal cells that are frozen
or cryopreserved. The mesenchymal stromal cells or preparation of
mesenchymal stromal cells comprised in the kit may be contained in
a cell delivery vehicle.
[0027] Moreover, the invention contemplates methods for treating a
disease or disorder, comprising administering an effective amount
of mesenchymal stromal cells or a preparation of mesenchymal
stromal cells described herein to a subject in need thereof. The
method may further comprise the transplantation of other cells or
tissues, e.g., retinal, RPE, corneal, neural, immune, bone marrow,
liver or pancreatic cells. Exemplary diseases or disorders treated
include, but are not limited to, multiple sclerosis, systemic
sclerosis, hematological malignancies, myocardial infarction, organ
transplantation rejection, chronic allograft nephropathy,
cirrhosis, liver failure, heart failure, GvHD, tibial fracture,
left ventricular dysfunction, leukemia, myelodysplastic syndrome,
Crohn's disease, diabetes, chronic obstructive pulmonary disease,
osteogenesis imperfecta, homozygous familial hypocholesterolemia,
treatment following meniscectomy, adult periodontitis,
vasculogenesis in patients with severe myocardial ischemia, spinal
cord injury, osteodysplasia, critical limb ischemia, diabetic foot
disease, primary Sjogren's syndrome, osteoarthritis, cartilage
defects, laminitis, multisystem atrophy, amyotropic lateral
sclerosis, cardiac surgery, refractory systemic lupus
erythematosis, living kidney allografts, nonmalignant red blood
cell disorders, thermal burn, radiation burn, Parkinson's disease,
microfractures, epidermolysis bullosa, severe coronary ischemia,
idiopathic dilated cardiomyopathy, osteonecrosis femoral head,
lupus nephritis, bone void defects, ischemic cerebral stroke, after
stroke, acute radiation syndrome, pulmonary disease, arthritis,
bone regeneration, or combinations thereof. In one aspect, the
disease or disorder is uveitis. In another aspect, the disease or
disorder is an autoimmune disorder, e.g., multiple sclerosis, or an
immune reaction against allogeneic cells.
[0028] The invention further encompasses methods of treating bone
loss or cartilage damage comprising administering an effective
amount of mesenchymal stromal cells or preparation of mesenchymal
stromal cells described herein to a subject in need thereof. The
mesenchymal stromal cells may be administered in combination with
an allogeneic or syngeneic transplanted cell or tissue, e.g.,
retinal pigment epithelium cell, retinal cell, corneal cell, or
muscle cell.
[0029] The present invention comprises methods of culturing
hemangioblasts that generate preparations MSCs, which retain
potency, despite increasing numbers of population doublings. The
pharmaceutical preparations of mesenchymal stromal cells of the
present invention demonstrate improved therapeutic properties when
administered to a mammalian host in need of such
administration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1. Generation of FM-MA09-MSC from pluripotent cells.
This figure shows a microscopic view of generating mesenchymal
stromal cells from ESCs via hemangioblasts.
[0031] FIG. 2. A phenotype of FM-MA09-MSC obtained from pluripotent
cell-derived hemangioblasts. This figure shows the percentage of
cells positive for MSC surface markers in the initial hemangioblast
population (left side of graph, day 7-11 hemangioblast) and after
culturing hemangioblasts on Matrigel coated plates (right side of
graph) and a microscopic view of the mesenchymal stromal cells
derived from the hemangioblasts (right panel photograph).
[0032] FIG. 3. Phenotypes of mesenchymal stromal cells derived from
different culture methods. This figure shows the percentage of
cells positive for MSC surface markers after culturing human
embryonic stem cells (ESC) on gelatin coated plates (left panel),
ESC on Matrigel coated plates (middle panel), and hemangioblasts on
Matrigel coated plates (right panel).
[0033] FIG. 4. Mesenchymal stromal cell yield from pluripotent
cells. This figure shows the yields of cells positive for MSC
surface markers obtained from culturing ESC on gelatin coated
plates (first column--no yield), ESC on Matrigel coated plates
(second column), and hemangioblasts on Matrigel coated plates
(third column).
[0034] FIG. 5. Acquisition of mesenchymal stromal cell markers.
This figure depicts the time for MSC surface markers to be acquired
using hemangioblasts (top line) and ESC (lower line).
[0035] FIG. 6. Phenotypes of mesenchymal stromal cells derived from
different culture methods. This figure shows the percentage of
cells positive for MSC markers and negative for hematopoiesis and
endothelial markers after culturing ESC on Matrigel coated plates
(left panel) and hemangioblasts on Matrigel coated plates (right
panel).
[0036] FIG. 7. FM-MA09-MSC display differentiation capabilities.
This figure depicts the differentiation capabilities of mesenchymal
stromal cells derived from hemangioblasts differentiated from MA09
ESC to form adipocytes and osteocytes.
[0037] FIG. 8. MSC chondrogenic differentiation. This figure
depicts chondrogenic differentiation of MA09 ESC
hemangioblast-derived mesenchymal stromal cells by mRNA expression
of Aggrecan (chondroitin proteoglycan sulfate 1) and Collagen
IIa.
[0038] FIG. 9. Transient expression of CD309 by FM-MA09-MSC. This
figure shows the transient expression of the cell surface marker
CD309.
[0039] FIG. 10A. T cell proliferation in response to mitogen is
suppressed by FM-MA09-MSC. This figure shows hemangioblast-derived
mesenchymal stromal cells suppression of T cell proliferation
caused by chemical stimulation (PMA/ionomycin).
[0040] FIG. 10B. T cell proliferation in response to antigen
presenting cells is suppressed by FM-MA09-MSC. This figure shows
hemangioblast-derived mesenchymal stromal cells suppression of T
cell proliferation caused by exposure to dendritic cells.
[0041] FIG. 11. T cell proliferation in response to antigen
presenting cells is suppressed by FM-MA09-MSC. FIG. 11A shows that
hemangioblast-derived mesenchymal stromal cells were able to
increase the percentage of CD4/CD25 double positive Tregs that are
induced in response to IL2 stimulus.
[0042] FIG. 11B shows that hemangioblast-derived mesenchymal
stromal cells inhibit Th1 secretion of IFN.gamma..
[0043] FIG. 12. Proinflammatory cytokine IFNg stimulates changes in
FM-MA09-MSC surface marker expression. This figure shows that
interferon gamma stimulates changes in MSC surface marker
expression and may enhance MSC immunosuppressive effects.
[0044] FIG. 13. Increased potency, greater inhibitory effects of
FM-MA09-MSCs as compared to BM-MSCs. FM-MA09-MSCs exert greater
inhibitory effects on T cell proliferation than do BM-MSCs. (A.)
Increasing the amount of MSCs in co-culture with PBMCs causes a
dose-dependent reduction in T cell proliferation in response to PMA
and ionomycin. Young (p4) FM-MA09-MSCs are the most potent of all
cell types tested. (B.) FM-MA09-MSCs inhibit T cell proliferation
to a greater degree than do BM-MSCs in response to PHA. A 5:1 ratio
of PBMCs:MSCs were co-cultured for 6 days. (C.) FM-MA09-MSCs
inhibit T cell proliferation in response to increasing amounts of
dendritic cells better than do BM-MSCs. In (A-C), percent T cell
proliferation was assessed by BrdU incorporation in the CD4+ and/or
CD8+ cell population.
[0045] FIG. 14. FM-MA09-MSCs enhance Treg induction: early passage
MSCs have greater effects than do late passage MSCs. Non-adherent
PBMCs (different donors) were cultured +/-IL2 for 4 days in the
absence or presence of FM-MA09-MSCs. The percentage of CD4/CD25
double positive Tregs was assessed by flow cytometry. Young (p6) or
old (p16-18) FM-MA09-MSCs were used. The black bars indicate the
average of 6 experiments. MSCs as a whole had a statistically
significant effect on induction of Tregs. (p=0.02).
[0046] FIG. 15. Enhanced Treg expansion by FM-MA09-MSCs as compared
to BM-MSCs. FM-MA09-MSCs induce Treg expansion better than do
BM-MSCs. (A.) Fold increase in CD4/CD25 double positive Tregs. The
minus IL2 condition was set to 1 and other groups are expressed as
fold induction over this level. MM=MA09-MSCs, BM=bone marrow MSCs.
"p"=passage number. (B.) FM MA09-MSCs (MM) induce CD4/CD25/FoxP3
triple positive Tregs better than do BM-MSCs. (C.) Percent of
responding PBMCs that are CD4+ are consistent among the different
treatment groups. (D.) Percent of responding PBMCs that are CD25+
vary among the different treatment groups. FM-MA09-MSCs induce
greater expression of CD25 than do BM-MSCs. This difference may
explain the difference in induction of Tregs.
[0047] FIG. 16. FM-MA09-MSCs have greater proliferative capacity
than BM-MSCs. FM-MA09-MSCs have a greater proliferative capacity
than do BM-MSCs. Cumulative population doublings are plotted
against the number of days in culture. After initial plating of
ESC-derived hemangioblasts or bone marrow-derived mononuclear
cells, adherent cells were considered p0 MSCs. Successive MSC
passages were replated at a density of 7000 cells/sq cm and
harvested when the cultures were approximately 70% confluent (every
3-5 days).
[0048] FIG. 17. Process of FM-MA09-MSC generation; Matrigel effect.
Removing cells from Matrigel at an early passage (i.e., p2) may
temporarily slow MSC growth as compared to those maintained on
Matrigel until p6.
[0049] FIG. 18. BM-MSCs and FM-MA09-MSCs undergo chondrogenesis.
Safranin O staining (indicative of cartilaginous matrix deposition)
was performed on paraffin-embedded pellet mass cultures after 21
days. Images are 40.times. magnification.
[0050] FIG. 19. In the basal state, FM-MA09-MSCs secrete less PGE2
than do BM-MSCs yet the fold increase upon IFN.gamma. or TNF.alpha.
stimulation is greater. (A.) The amount of prostaglandin E2
secretion (pg/ml) is shown for BM-MSCs versus FM-MA09-MSCs under
basal or various stimulation conditions. PGE2 amounts are
normalized to cell number. (B.) Basal PGE2 values are set to 1
(black line) and PGE2 secretion under various stimuli are expressed
as fold increase over basal level.
[0051] FIG. 20. FM-MA09-MSCs maintain phenotype over time. Flow
cytometry analysis of different MSC populations. (A.) Cell surface
marker expression of FM-MA09-MSCs is maintained on three different
substrates and compared to BM-MSCs. (B.) Cell surface marker
expression of FM-MA09-MSCs is evaluated over time (with successive
passages, as indicated).
[0052] FIG. 21. FM-MA09-MSCs express less Stro-1 and more CD10 as
compared to BM-MSCs. Flow cytometry analysis of different MSC
populations. Stro-1 expression is lower in FM-MA09-MSCs than in
BM-MSCs at the indicated passage number. CD10 expression is higher
in FM-MA09-MSCs than in BM-MSCs. Other markers are the same for
both MSC populations.
[0053] FIG. 22. Stro-1 and CD10 expression in 10 different lots of
early passage FM-MA09-MSCs consistently show low Stro-1 and
mid-range CD10 expression. Flow cytometry analysis of different MSC
populations. Ten different lots of FM-MA09-MSCs were evaluated at
the indicated passage number for expression of Stro-1 and CD10.
Stro-1 expression is consistently low in the different lots of
FM-MA09-MSCs (average of 5-10%). CD10 expression is consistently at
amid-range level in the different lots of FM-MA09-MSCs (average of
approximately 40%).
[0054] FIG. 23. FM-MA09-MSCs maintain their size as they age in
culture while BM-MSC cell size increases with age. Forward
scatter/side scatter dot plots on flow cytometry (shown on the
left) were used to capture the size of MSCs. The percentage of
cells in the upper right quadrant "large" cells were monitored and
are displayed in the bar graph.
[0055] FIG. 24. CD10 and CD24 are upregulated in FM-MA09-MSCs as
compared to BM-MSCs. Gene expression analysis is shown for BM-MSCs
and FM-MA09-MSCs in the basal state. Quantitative RT-PCR with
Taqman probes was used to assess the expression of the indicated
genes and normalized to two housekeeping genes. The average of
quadruplicate readings is shown +/- standard deviation.
[0056] FIG. 25. Aire-1 and IL-11 are upregulated in FM-MA09-MSCs as
compared to BM-MSCs. Gene expression analysis is shown for BM-MSCs
and FM-MA09-MSCs in the basal state. Quantitative RT-PCR with
Taqman probes was used to assess the expression of the indicated
genes and normalized to two housekeeping genes. The average of
quadruplicate readings is shown +/- standard deviation.
[0057] FIG. 26. Ang-1 and CXCL1 are upregulated in FM-MA09-MSCs as
compared to BM-MSCs. Gene expression analysis is shown for BM-MSCs
and FM-MA09-MSCs in the basal state. Quantitative RT-PCR with
Taqman probes was used to assess the expression of the indicated
genes and normalized to two housekeeping genes. The average of
quadruplicate readings is shown +/- standard deviation.
[0058] FIG. 27. IL6 and VEGF are downregulated in FM-MA09-MSCs as
compared to BM-MSCs. Gene expression analysis is shown for BM-MSCs
and FM-MA09-MSCs in the basal state. Quantitative RT-PCR with
Taqman probes was used to assess the expression of the indicated
genes and normalized to two housekeeping genes. The average of
quadruplicate readings is shown +/- standard deviation.
[0059] FIG. 28. FM-MA09-MSCs and BM-MSCs show increased indoleamine
2,3 deoxygenase (IDO) activity in response to 3 days of IFN.gamma.
stimulation. Comparison of MSCs stimulated with 50 ng/ml IFNg for 3
days, for their ability to convert tryptophan into kynurenine
(indicative of IDO activity). For each MSC population, 1 million
cells were lysed and used in the assay.
[0060] FIG. 29. Age-related changes in FM-MA09-MSC expression of
Aire-1 and Prion Protein (PrP): two proteins involved in immune
suppression and proliferation, respectively. Western blot analysis
of Aire-1 and PrP expression in FM-MA09-MSCs whole cell lysates at
different passage numbers (p). Actin expression is shown as loading
control. Differences in Aire-1 and PrP expression are noted by
referencing the actin loading controls.
[0061] FIG. 30. FM-MA09-MSCs secrete less IL6 than BM-MSCs do in
the basal state. Cytokine arrays showing positive controls for
normalization (4 dots on left) and IL6 (boxed) in MSC conditioned
medium. BM-MSCs from two different donors are compared to 4
different lots of FM-MA09-MSCs.
[0062] FIG. 31. FM-MA09-MSCs secrete less IL6 than BM-MSCs in the
basal and IFN.gamma.-stimulated state. Cytokine arrays showing
positive controls for normalization (4 dots on left) and IL6
(boxed) in MSC conditioned medium. Passage 7 BM-MSCs are compared
to p7 FM-MA09-MSCs after 48 hours +/- IFN.gamma. treatment.
[0063] FIG. 32. FM-MA09-MSCs secrete less VEGF than BM-MSCs in the
basal and IFN.gamma.-stimulated state. Cytokine arrays showing
positive controls for normalization (4 dots on left) and VEGF
(boxed) in MSC conditioned medium. Passage 7 BM-MSCs are compared
to p7 FM-MA09-MSCs after 48 hours +/- IFN.gamma. treatment.
DETAILED DESCRIPTION OF THE INVENTION
[0064] The instant invention relates to methods of generating
mesenchymal stromal cells, preparations of mesenchymal stromal
cells from culturing hemangioblasts, methods of culturing
hemangioblasts, and methods of treating a pathology using
mesenchymal stromal cells.
[0065] The methods of the instant invention, whereby hemangioblast
cultures produce increased yields of mesenchymal stromal cells,
compared to prior processes, are more efficient than previous
processes at producing substantially ESC-free mesenchymal stromal
cells. The hemangioblast-derived mesenchymal stromal cells of the
instant invention retain a novel, youthful phenotype as defined by
expression or lack thereof of specific markers.
[0066] In certain embodiments, the MSC preparation (such as
cultures having at least 10.sup.3, 10.sup.4, 10.sup.5 or even
10.sup.6 MSCs) may have, as an average, telomere lengths that are
at least 30 percent of the telomere length of an ESC and/or human
iPS cell (or the average of a population of ESC and/or human iPS
cells), and preferably at least 40, 50, 60, 70 80 or even 90
percent of the telomere length of an ESC and/or human iPS cell (or
of the average of a population of ESC and/or human iPS cells). For
example, said ESC and/or human iPS cell (or said population of ESC
and/or human iPS cells) may be a cell or cell population from which
said MSC cells were differentiated.
[0067] The MSC preparation may, as a population, have a mean
terminal restriction fragment length (TRF) that is longer than 4
kb, and preferably longer than 5, 6, 7, 8, 9, 10, 11, 12 or even 13
kb. In an exemplary embodiment, the MSCs of the preparation may
have an average TRF that is 10 kb or longer.
[0068] In certain embodiments, the MSC preparation (such as
cultures having at least 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6,
10.sup.7 or even 10.sup.8 MSCs) has a replicative lifespan that is
greater than the replicative lifespan of MSC preparations obtained
from other sources (e.g., cultures derived from donated human
tissue, such as fetal, infant, child, adolescent or adult tissue).
Replicative lifespan may be assessed by determining the number of
population doublings or passages in culture prior to replicative
senescence, i.e., where more than 10, 20, 30, 40 or even 50 percent
of the cells in culture senesce before the next doubling or
passage. For example, the subject MSC preparations may have a
replicative lifespan that is at least 10 doublings greater than
that of an MSC preparation derived from donated human tissue
(particularly derived from adult bone marrow or adult adipose
tissue), and preferably at least 20, 30, 40, 50, 60, 70 80, 90 or
even 100 population doublings. In certain embodiments, the MSC
preparations may have a replicative lifespan that permits at least
8 passages before more than 50 percent of the cells senesce and/or
differentiate into non-MSC cell types (such as fibroblasts), and
more preferably at least 10, 12, 14, 16, 18 or even 20 passages
before reaching that point. In certain embodiments, the MSC
preparation may have a replicative lifespan that permits at least 2
times as many doublings or passages relative to adult bone
marrow-derived MSC preparations and/or adipose-derived MSC
preparations (e.g., equivalent starting number of cells) before
more than 50 percent of the cells senesce and/or differentiate into
non-MSC cell types (such as fibroblasts), and more preferably at
least 4, 6, 8 or even 10 times as many doublings or passages.
[0069] In certain embodiments, the MSC preparation of the present
invention (such as cultures having at least 10.sup.3, 10.sup.4,
10.sup.5, 10.sup.6, 10.sup.7 or even 10.sup.8 MSCs) have a
statistically significant decreased content and/or enzymatic
activity of proteins involved in cell cycle regulation and aging
relative to passage 1 (P1), passage 2 (P2), passage 3 (P3), passage
4 (P4) and/or passage 5 (P5) MSC preparations derived from other
sources (e.g., cultures derived from donated human tissue, such as
fetal, infant, child, adolescent or adult tissue), and particularly
bone marrow-derived MSCs and adipose-derived MSCs. For example, the
subject MSC preparation has a proteasome 26S subunit, non-ATPase
regulatory subunit 11 (PSMD11) protein content that is less than 75
percent of the content in MSCs from donated human tissue
(particularly derived from adult bone marrow or adult adipose
tissue), and even more preferably less than 60, 50, 40, 30, 20 or
even 10 percent.
[0070] In certain embodiments, the MSC preparation of the present
invention (such as cultures having at least 10.sup.3, 10.sup.4,
10.sup.5, 10.sup.6, 10.sup.7 or even 10.sup.8 MSCs) have a
statistically significant decreased content and/or enzymatic
activity of proteins involved in energy and/or lipid metabolism of
the cell relative to passage 1 (P1), passage 2 (P2), passage 3
(P3), passage 4 (P4) and/or passage 5 (P5) MSC preparations derived
from other sources (e.g., cultures derived from donated human
tissue, such as fetal, infant, child, adolescent or adult tissue),
and particularly bone marrow-derived MSCs and adipose-derived MSCs.
To illustrate, the subject MSC preparation has a protein content
that is less than 90 percent of the content in MSCs from donated
human tissue (particularly derived from adult bone marrow or adult
adipose tissue), and even more preferably less than 60, 50, 40, 30,
20 or even 10 percent, for one or more proteins involved in
metabolic pathways for ATP or NADPH synthesis such as glycolysis
(such as fructose-biphosphate aldolase A, ALDOA; aldo-keto
reductase family 1, member A1, AKR1A1); glyceraldehyde-3-phosphate,
GAPDH), the tricarboxylic acid cycle (TCA cycle) (such as
isocitrate dehydrogenase 1, IDH1), the pentose phosphate pathway
(such as glucose-6-phosphate dehydrogenase, G6PD) and the
biosynthesis of UDP-glucose in the glucuronic acid biosynthetic
pathway (such as UDP-glucose 6-dehydrogenase, UGDH). To further
illustrate, the subject MSC preparation has a protein content that
is less than 90 percent of the content in MSCs from donated human
tissue (particularly derived from adult bone marrow or adult
adipose tissue), and even more preferably less than 60, 50, 40, 30,
20 or even 10 percent, for one or more proteins involved in lipid
metabolism, such as enoyl-CoA hydratase, short chain, 1 (ECHS1)
and/or acetyl-CoA acetyltransferase (ACAT2).
[0071] In certain embodiments, the MSC preparation of the present
invention (such as cultures having at least 10.sup.3, 10.sup.4,
10.sup.5, 10.sup.6, 10.sup.7 or even 10.sup.8 MSCs) have a
statistically significant decreased content and/or enzymatic
activity of proteins involved in apoptosis of the cell relative to
passage 1 (P1), passage 2 (P2), passage 3 (P3), passage 4 (P4)
and/or passage 5 (P5) MSC preparations derived from other sources
(e.g., cultures derived from donated human tissue, such as fetal,
infant, child, adolescent or adult tissue), and particularly bone
marrow-derived MSCs and adipose-derived MSCs. To illustrate, the
subject MSC preparation has a protein content that is less than 90
percent of the content in MSCs from donated human tissue
(particularly derived from adult bone marrow or adult adipose
tissue), and even more preferably less than 60, 50, 40, 30, 20 or
even 10 percent, for one or more proteins annexin A1 (ANXA1), A2
(ANXA2), A5 (ANXA5), the voltage-dependent anion-selective channel
protein 1 (VDAC1), and/or glyceraldehyde-3-phosphate dehydrogenase
(GAPDH).
[0072] Without being bound by theory, it is believed that the
statistically significant difference in content and/or enzymatic
activity of proteins involved in energy and/or lipid metabolism
and/or apotosis of the cell displayed by the hemangioblast-derived
MSCs of the present invention is attributable, at least in part, to
the homogeneous nature of the preparations. For example,
hemangioblast-derived MSCs of the present invention have
homogeneous MHC gene expression, i.e., completely MHC matched,
unlike adult derived MSC banks, in which the cells are derived from
multiple different donors, i.e., MHC mismatched. A therapeutic dose
of MSCs is about 2-8 million cells/kg (or about 130-500 million
cells per dose).
[0073] Definitions
[0074] "Pluripotent cells" and "pluripotent stem cells" as used
herein, refers broadly to a cell capable of prolonged or virtually
indefinite proliferation in vitro while retaining their
undifferentiated state, exhibiting a stable (preferably normal)
karyotype, and having the capacity to differentiate into all three
germ layers (i.e., ectoderm, mesoderm and endoderm) under the
appropriate conditions. Typically pluripotent cells (a) are capable
of inducing teratomas when transplanted in immunodeficient (SCID)
mice; (b) are capable of differentiating to cell types of all three
germ layers (e.g., ectodermal, mesodermal, and endodermal cell
types); and (c) express at least one hES cell marker (such as
Oct-4, alkaline phosphatase, SSEA 3 surface antigen, SSEA 4 surface
antigen, NANOG, TRA 1 60, TRA 1 81, SOX2, REX1). Exemplary
pluripotent cells may express Oct-4, alkaline phosphatase, SSEA 3
surface antigen, SSEA 4 surface antigen, TRA 1 60, and/or TRA 1 81.
Additional exemplary pluripotent cells include but are not limited
to embryonic stem cells, induced pluripotent cells (iPS) cells,
embryo-derived cells, pluripotent cells produced from embryonic
germ (EG) cells (e.g., by culturing in the presence of FGF-2, LIF
and SCF), parthenogenetic ES cells, ES cells produced from cultured
inner cell mass cells, ES cells produced from a blastomere, and ES
cells produced by nuclear transfer (e.g., a somatic cell nucleus
transferred into a recipient oocyte). Exemplary pluripotent cells
may be produced without destruction of an embryo. For example,
induced pluripotent cells may be produced from cells obtained
without embryo destruction. As a further example, pluripotent cells
may be produced from a biopsied blastomere (which can be
accomplished without harm to the remaining embryo); optionally, the
remaining embryo may be cryopreserved, cultured, and/or implanted
into a suitable host. Pluripotent cells (from whatever source) may
be genetically modified or otherwise modified to increase
longevity, potency, homing, or to deliver a desired factor in cells
that are differentiated from such pluripotent cells (for example,
MSCs, and hemangioblasts). As non-limiting examples thereof, the
pluripotent cells may be genetically modified to express Sirtl
(thereby increasing longevity), express one or more telomerase
subunit genes optionally under the control of an inducible or
repressible promoter, incorporate a fluorescent label, incorporate
iron oxide particles or other such reagent (which could be used for
cell tracking via in vivo imaging, MRI, etc., see Thu et al., Nat
Med. 2012 Feb. 26; 18(3):463-7), express bFGF which may improve
longevity (see Go et al., J. Biochem. 142, 741-748 (2007)), express
CXCR4 for homing (see Shi et al., Haematologica. 2007 July;
92(7):897-904), express recombinant TRAIL to induce
caspase-mediatedx apoptosis in cancer cells like Gliomas (see
Sasportas et al., Proc Natl Acad Sci USA. 2009 Mar. 24;
106(12):4822-7), etc.
[0075] "Embryo" or "embryonic," as used herein refers broadly to a
developing cell mass that has not implanted into the uterine
membrane of a maternal host. An "embryonic cell" is a cell isolated
from or contained in an embryo. This also includes blastomeres,
obtained as early as the two-cell stage, and aggregated
blastomeres.
[0076] "Embryonic stem cells" (ES cells or ESC) encompasses
pluripotent cells produced from embryonic cells (such as from
cultured inner cell mass cells or cultured blastomeres) as well as
induced pluripotent cells (further described below). Frequently
such cells are or have been serially passaged as cell lines.
Embryonic stem cells may be used as a pluripotent stem cell in the
processes of producing hemangioblasts as described herein. For
example, ES cells may be produced by methods known in the art
including derivation from an embryo produced by any method
(including by sexual or asexual means) such as fertilization of an
egg cell with sperm or sperm DNA, nuclear transfer (including
somatic cell nuclear transfer), or parthenogenesis. As a further
example, embryonic stem cells also include cells produced by
somatic cell nuclear transfer, even when non-embryonic cells are
used in the process. For example, ES cells may be derived from the
ICM of blastocyst stage embryos, as well as embryonic stem cells
derived from one or more blastomeres. Such embryonic stem cells can
be generated from embryonic material produced by fertilization or
by asexual means, including somatic cell nuclear transfer (SCNT),
parthenogenesis, and androgenesis. As further discussed above (see
"pluripotent cells), ES cells may be genetically modified or
otherwise modified to increase longevity, potency, homing, or to
deliver a desired factor in cells that are differentiated from such
pluripotent cells (for example, MSCs, and hemangioblasts).
[0077] ES cells may be generated with homozygosity or hemizygosity
in one or more HLA genes, e.g., through genetic manipulation,
screening for spontaneous loss of heterozygosity, etc. ES cells may
be genetically modified or otherwise modified to increase
longevity, potency, homing, or to deliver a desired factor in cells
that are differentiated from such pluripotent cells (for example,
MSCs and hemangioblasts). Embryonic stem cells, regardless of their
source or the particular method used to produce them, typically
possess one or more of the following attributes: (i) the ability to
differentiate into cells of all three germ layers, (ii) expression
of at least Oct-4 and alkaline phosphatase, and (iii) the ability
to produce teratomas when transplanted into immunocompromised
animals. Embryonic stem cells that may be used in embodiments of
the present invention include, but are not limited to, human ES
cells ("ESC" or "hES cells") such as MA01, MA09, ACT-4, No. 3, H1,
H7, H9, H14 and ACT30 embryonic stem cells. Additional exemplary
cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. See also
NIH Human Embryonic Stem Cell Registry. An exemplary human
embryonic stem cell line that may be used is MA09 cells. The
isolation and preparation of MA09 cells was previously described in
Klimanskaya, et al. (2006) "Human Embryonic Stem Cell lines Derived
from Single Blastomeres." Nature 444: 481-485. The human ES cells
used in accordance with exemplary embodiments of the present
invention may be derived and maintained in accordance with GMP
standards.
[0078] Exemplary hES cell markers include but are not limited to:
such as alkaline phosphatase, Oct-4, Nanog, Stage-specific
embryonic antigen-3 (SSEA-3), Stage-specific embryonic antigen-4
(SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49/6E, Sox2, growth and
differentiation factor 3 (GDF3), reduced expression 1 (REX1),
fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1
(ESG1), developmental pluripotency-associated 2 (DPPA2), DPPA4,
telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN,
Cluster designation 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis,
Germ cell nuclear factor, and Stem cell factor (SCF or c-Kit
ligand). As an addition example, embryonic stem cells may express
Oct-4, alkaline phosphatase, SSEA 3 surface antigen, SSEA 4 surface
antigen, TRA 1 60, and/or TRA 1 81.
[0079] The ESCs may be initially co-cultivated with murine
embryonic feeder cells (MEF) cells. The MEF cells may be
mitotically inactivated by exposure to mitomycin C prior to seeding
ESCs in co culture, and thus the MEFs do not propagate in culture.
Additionally, ESC cell cultures may be examined microscopically and
colonies containing non ESC cell morphology may be picked and
discarded, e.g., using a stem cell cutting tool, by laser ablation,
or other means. Typically, after the point of harvest of the ESCs
for seeding for embryoid body formation no additional MEF cells are
used.
[0080] "Embryo-derived cells" (EDC), as used herein, refers broadly
to pluripotent morula-derived cells, blastocyst-derived cells
including those of the inner cell mass, embryonic shield, or
epiblast, or other pluripotent stem cells of the early embryo,
including primitive endoderm, ectoderm, and mesoderm and their
derivatives. "EDC" also including blastomeres and cell masses from
aggregated single blastomeres or embryos from varying stages of
development, but excludes human embryonic stem cells that have been
passaged as cell lines.
[0081] Exemplary ESC cell markers include but are not limited to:
such as alkaline phosphatase, Oct-4, Nanog, Stage-specific
embryonic antigen-3 (SSEA-3), Stage-specific embryonic antigen-4
(SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49/6E, Sox2, growth and
differentiation factor 3 (GDF3), reduced expression 1 (REX1),
fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1
(ESG1), developmental pluripotency-associated 2 (DPPA2), DPPA4,
telomerase reverse transcriptase (hTERT), SALL4, E-CADHERIN,
Cluster designation 30 (CD30), Cripto (TDGF-1), GCTM-2, Genesis,
Germ cell nuclear factor, and Stem cell factor (SCF or c-Kit
ligand).
[0082] "Potency", as used herein, refers broadly to the
concentration, e.g., molar, of a reagent (such as
hemangioblast-derived MSCs) that produces a defined effect. Potency
may be defined in terms of effective concentration (EC50), which
does not involve measurements of maximal effect but, instead, the
effect at various locations along the concentration axis of dose
response curves. Potency may also be determined from either graded
(EC50) or quantal dose-response curves (ED50, TD50 and LD50);
however, potency is preferably measured by EC50. The term "EC50"
refers to the concentration of a drug, antibody or toxicant which
induces a response halfway between the baseline and maximum effect
after some specified exposure time. The EC50 of a graded dose
response curve therefore represents the concentration of a compound
where 50% of its maximal effect is observed. The EC50 of a quantal
dose response curve represents the concentration of a compound
where 50% of the population exhibit a response, after a specified
exposure duration. The EC50 may be determined using animal studies
in which a defined animal model demonstrates a measurable,
physiological change in response to application of the drug;
cell-based assays that use a specified cell system, which on
addition of the drug, demonstrate a measureable biological
response; and/or enzymatic reactions where the biological activity
of the drug can be measured by the accumulation of product
following the chemical reaction facilitated by the drug.
Preferably, an immune regulatory assay isused to determine EC50.
Non-limiting examples of such immune regulatory assays include
intracellular cytokine, cytotoxicity, regulatory capacity, cell
signaling capacity, proliferative capacity, apoptotic evaluations,
and other assays.
[0083] "Mesenchymal stem cells" (MSC) as used herein refers to
multipotent stem cells with self-renewal capacity and the ability
to differentiate into osteoblasts, chondrocytes, and adipocytes,
among other mesenchymal cell lineages. In addition to these
characteristics, MSCs may be identified by the expression of one or
more markers as further described herein. Such cells may be used to
treat a range of clinical conditions, including immunological
disorders as well as degenerative diseases such as
graft-versus-host disease (GVHD), myocardial infarction and
inflammatory and autoimmune diseases and disorders, among others.
Except where the context indicates otherwise, MSCs may include
cells from adult sources and cord blood. MSCs (or a cell from which
they are generated, such as a pluripotent cell) may be genetically
modified or otherwise modified to increase longevity, potency,
homing, or to deliver a desired factor in the MSCs or cells that
are differentiated from such MSCs. As non-limiting examples
thereof, the MSCs cells may be genetically modified to express
Sirt1 (thereby increasing longevity), express one or more
telomerase subunit genes optionally under the control of an
inducible or repressible promoter, incorporate a fluorescent label,
incorporate iron oxide particles or other such reagent (which could
be used for cell tracking via in vivo imaging, MRI, etc., see Thu
et al., Nat Med. 2012 Feb. 26; 18(3):463-7), express bFGF which may
improve longevity (see Go et al., J. Biochem. 142, 741-748 (2007)),
express CXCR4 for homing (see Shi et al., Haematologica. 2007 July;
92(7):897-904), express recombinant TRAIL to induce
caspase-mediatedx apoptosis in cancer cells like Gliomas (see
Sasportas et al., Proc Natl Acad Sci USA. 2009 Mar. 24;
106(12):4822-7), etc.
[0084] "Therapy," "therapeutic," "treating," "treat" or
"treatment", as used herein, refers broadly to treating a disease,
arresting or reducing the development of the disease or its
clinical symptoms, and/or relieving the disease, causing regression
of the disease or its clinical symptoms. Therapy encompasses
prophylaxis, prevention, treatment, cure, remedy, reduction,
alleviation, and/or providing relief from a disease, signs, and/or
symptoms of a disease. Therapy encompasses an alleviation of signs
and/or symptoms in patients with ongoing disease signs and/or
symptoms (e.g., muscle weakness, multiple sclerosis.) Therapy also
encompasses "prophylaxis" and "prevention". Prophylaxis includes
preventing disease occurring subsequent to treatment of a disease
in a patient or reducing the incidence or severity of the disease
in a patient. The term "reduced", for purpose of therapy, refers
broadly to the clinical significant reduction in signs and/or
symptoms. Therapy includes treating relapses or recurrent signs
and/or symptoms (e.g., retinal degeneration, loss of vision.)
Therapy encompasses but is not limited to precluding the appearance
of signs and/or symptoms anytime as well as reducing existing signs
and/or symptoms and eliminating existing signs and/or symptoms.
Therapy includes treating chronic disease ("maintenance") and acute
disease. For example, treatment includes treating or preventing
relapses or the recurrence of signs and/or symptoms (e.g., muscle
weakness, multiple sclerosis).
[0085] In order maintain regulatory compliance, MSC banks must
maintain a sufficient supply of cells, e.g., to provide a
sufficient number of cells to treat at least a few hundred to
10,000 patients, MSC banks must have at least 50 billion MSCs. The
present invention encompasses GMP-complaint and/or cryopreserved
MSC banks In one aspect, the MSC preparation of the present
invention comprise at least 10.sup.10 hemangioblast-derived MSCs.
In another aspect, the present invention provides a MSC preparation
comprising at least 10.sup.11, 10.sup.12, 10.sup.13, or 10.sup.14
hemangioblast-derived MSCs.
[0086] "Normalizing a pathology", as used herein, refers to
reverting the abnormal structure and/or function resulting from a
disease to a more normal state. Normalization suggests that by
correcting the abnormalities in structure and/or function of a
tissue, organ, cell type, etc. resulting from a disease, the
progression of the pathology can be controlled and improved. For
example, following treatment with the ESC-MSCs of the present
invention the abnormalities of the immune system as a result of
autoimmune disorders, e.g., MS, may be improved, corrected, and/or
reversed.
Induced Pluripotent Stem Cells
[0087] Further exemplary pluripotent stem cells include induced
pluripotent stem cells (iPS cells) generated by reprogramming a
somatic cell by expressing or inducing expression of a combination
of factors ("reprogramming factors"). iPS cells may be generated
using fetal, postnatal, newborn, juvenile, or adult somatic cells.
iPS cells may be obtained from a cell bank. Alternatively, iPS
cells may be newly generated (by processes known in the art) prior
to commencing differentiation to RPE cells or another cell type.
The making of iPS cells may be an initial step in the production of
differentiated cells. iPS cells may be specifically generated using
material from a particular patient or matched donor with the goal
of generating tissue-matched RPE cells. iPS cells can be produced
from cells that are not substantially immunogenic in an intended
recipient, e.g., produced from autologous cells or from cells
histocompatible to an intended recipient. As further discussed
above (see "pluripotent cells"), pluripotent cells including iPS
cells may be genetically modified or otherwise modified to increase
longevity, potency, homing, or to deliver a desired factor in cells
that are differentiated from such pluripotent cells (for example,
MSCs and hemangioblasts).
[0088] As a further example, induced pluripotent stem cells may be
generated by reprogramming a somatic or other cell by contacting
the cell with one or more reprogramming factors. For example, the
reprogramming factor(s) may be expressed by the cell, e.g., from an
exogenous nucleic acid added to the cell, or from an endogenous
gene in response to a factor such as a small molecule, microRNA, or
the like that promotes or induces expression of that gene (see Suh
and Blelloch, Development 138, 1653-1661 (2011); Miyosh et al.,
Cell Stem Cell (2011), doi:10.1016/j.stem.2011.05.001;
Sancho-Martinez et al., Journal of Molecular Cell Biology (2011)
1-3; Anokye-Danso et al., Cell Stem Cell 8, 376-388, Apr. 8, 2011;
Orkin and Hochedlinger, Cell 145, 835-850, Jun. 10, 2011, each of
which is incorporated by reference herein in its entirety).
Reprogramming factors may be provided from an exogenous source,
e.g., by being added to the culture media, and may be introduced
into cells by methods known in the art such as through coupling to
cell entry peptides, protein or nucleic acid transfection agents,
lipofection, electroporation, biolistic particle delivery system
(gene gun), microinjection, and the like. iPS cells can be
generated using fetal, postnatal, newborn, juvenile, or adult
somatic cells. In certain embodiments, factors that can be used to
reprogram somatic cells to pluripotent stem cells include, for
example, a combination of Oct4 (sometimes referred to as Oct 3/4),
Sox2, c-Myc, and Klf4. In other embodiments, factors that can be
used to reprogram somatic cells to pluripotent stem cells include,
for example, a combination of Oct-4, Sox2, Nanog, and Lin28. In
other embodiments, somatic cells are reprogrammed by expressing at
least 2 reprogramming factors, at least three reprogramming
factors, or four reprogramming factors. In other embodiments,
additional reprogramming factors are identified and used alone or
in combination with one or more known reprogramming factors to
reprogram a somatic cell to a pluripotent stem cell. iPS cells
typically can be identified by expression of the same markers as
embryonic stem cells, though a particular iPS cell line may vary in
its expression profile.
[0089] The induced pluripotent stem cell may be produced by
expressing or inducing the expression of one or more reprogramming
factors in a somatic cell. The somatic cell is a fibroblast, such
as a dermal fibroblast, synovial fibroblast, or lung fibroblast, or
a non-fibroblastic somatic cell. The somatic cell is reprogrammed
by expressing at least 1, 2, 3, 4, 5 reprogramming factors. The
reprogramming factors may be selected from Oct 3/4, Sox2, NANOG,
Lin28, c Myc, and Klf4. Expression of the reprogramming factors may
be induced by contacting the somatic cells with at least one agent,
such as a small organic molecule agents, that induce expression of
reprogramming factors.
[0090] The somatic cell may also be reprogrammed using a
combinatorial approach wherein the reprogramming factor is
expressed (e.g., using a viral vector, plasmid, and the like) and
the expression of the reprogramming factor is induced (e.g., using
a small organic molecule.) For example, reprogramming factors may
be expressed in the somatic cell by infection using a viral vector,
such as a retroviral vector or a lentiviral vector. Also,
reprogramming factors may be expressed in the somatic cell using a
non-integrative vector, such as an episomal plasmid. See, e.g., Yu
et al., Science. 2009 May 8; 324(5928):797-801, which is hereby
incorporated by reference in its entirety. When reprogramming
factors are expressed using non-integrative vectors, the factors
may be expressed in the cells using electroporation, transfection,
or transformation of the somatic cells with the vectors. For
example, in mouse cells, expression of four factors (Oct3/4, Sox2,
c myc, and Klf4) using integrative viral vectors is sufficient to
reprogram a somatic cell. In human cells, expression of four
factors (Oct3/4, Sox2, NANOG, and Lin28) using integrative viral
vectors is sufficient to reprogram a somatic cell.
[0091] Once the reprogramming factors are expressed in the cells,
the cells may be cultured. Over time, cells with ES characteristics
appear in the culture dish. The cells may be chosen and subcultured
based on, for example, ES morphology, or based on expression of a
selectable or detectable marker. The cells may be cultured to
produce a culture of cells that resemble ES cells--these are
putative iPS cells. iPS cells typically can be identified by
expression of the same markers as other embryonic stem cells,
though a particular iPS cell line may vary in its expression
profile. Exemplary iPS cells may express Oct-4, alkaline
phosphatase, SSEA 3 surface antigen, SSEA 4 surface antigen, TRA 1
60, and/or TRA 1 81.
[0092] To confirm the pluripotency of the iPS cells, the cells may
be tested in one or more assays of pluripotency. For example, the
cells may be tested for expression of ES cell markers; the cells
may be evaluated for ability to produce teratomas when transplanted
into SCID mice; the cells may be evaluated for ability to
differentiate to produce cell types of all three germ layers. Once
a pluripotent iPS cell is obtained it may be used to produce
hemangioblast and MSC cells.
[0093] Hemangioblasts
[0094] Hemangioblasts are multipotent and serve as the common
precursor to both hematopoietic and endothelial cell lineages.
During embryonic development, they are believed to arise as a
transitional cell type that emerges during early mesoderm
development and colonizes primitive blood islands (Choi et al.
Development 125 (4): 725-732 (1998). Once there, hemangioblasts are
capable of giving rise to both primitive and definitive
hematopoietic cells, HSCs, and endothelial cells (Mikkola et al, J.
Hematother. Stem Cell Res 11(1): 9-17 (2002).
[0095] Hemangioblasts may be derived in vitro from both mouse ESCs
(Kennedy et al, Nature (386): 488-493 (1997); Perlingeiro et al,
Stem Cells (21): 272-280 (2003)) and human ESCs (ref. 14, 15, Yu et
al., Blood 2010 116: 4786-4794). Other studies claim to have
isolated hemangioblasts from umbilical cord blood (Bordoni et al,
Hepatology 45 (5) 1218-1228), circulating CD34-lin-CD45-CD133-cells
from peripheral blood (Ciraci et al, Blood 118: 2105-2115), and
from mouse uterus (Sun et al, Blood 116 (16): 2932-2941 (2010)).
Both mouse and human ESC-derived hemangioblasts have been obtained
through the culture and differentiation of clusters of cells grown
in liquid culture followed by growth of the cells in semi-solid
medium containing various cytokines and growth factors (Kennedy,
Perlingeiro, ref 14, 15); see also, U.S. Pat. No. 8,017,393, which
is hereby incorporated by reference in its entirety. For the
purposes of this application, the term hemangioblasts also includes
the hemangio-colony forming cells described in U.S. Pat. No.
8,017,393, which in addition to being capable of differentiating
into hematopoietic and endothelial cell lineages, are capable of
becoming smooth muscle cells and which are not positive for CD34,
CD31, KDR, and CD133. Hemangioblasts useful in the methods
described herein may be derived or obtained from any of these known
methods. For example, embryoid bodies may be formed by culturing
pluripotent cells under non-attached conditions, e.g., on a
low-adherent substrate or in a "hanging drop." In these cultures,
ES cells can form clumps or clusters of cells denominated as
embryoid bodies. See Itskovitz-Eldor et al., Mol Med. 2000 Feb.;
6(2):88-95, which is hereby incorporated by reference in its
entirety. Typically, embryoid bodies initially form as solid clumps
or clusters of pluripotent cells, and over time some of the
embryoid bodies come to include fluid filled cavities, the latter
former being referred to in the literature as "simple" EBs and the
latter as "cystic" embryoid bodies. Id. The cells in these EBs
(both solid and cystic forms) can differentiate and over time
produce increasing numbers of cells. Optionally EBs may then be
cultured as adherent cultures and allowed to form outgrowths.
Likewise, pluripotent cells that are allowed to overgrow and form a
multilayer cell population can differentiate over time.
[0096] In one embodiment, hemangioblasts are generated by the steps
comprising (a) culturing an ESC line for 2, 3, 4, 5, 6 or 7 days to
form clusters of cells, and (b) inducing said clusters of cells to
differentiate into hemangioblasts. In a further embodiment, the
clusters of cells in step (b) of are cultured in a cytokine-rich
serum-free methylcellulose based medium (14, 15).
[0097] In one embodiment, hemangioblasts are generated by the steps
comprising (a) culturing an ESC line selected from the group
consisting of MA09, H7, H9, MA01, HuES3, and H1gfp for 2, 3, 4, 5,
6 or 7 days to form clusters of cells, and (b) inducing said
clusters of cells to differentiate into hemangioblasts by culturing
in a cytokine-rich, serum-free, methylcellulose based medium.
[0098] In another embodiment, hemangioblasts are generated by
inducing any pluripotent cell as described herein. In a further
embodiment, hemangioblasts are generated by inducing
differentiation of a pluripotent cell selected from the group
comprising blastocysts, plated ICMs, one or more blastomeres, or
other portions of a pre-implantation-stage embryo or embryo-like
structure, regardless of whether produced by fertilization, somatic
cell nuclear transfer (SCNT), parthenogenesis, androgenesis, or
other sexual or asexual means, and ESC derived through
reprogramming (e.g., iPS cells). In a still further embodiment,
hemangioblasts are generated from iPS cells, wherein the iPS cells
are generated using exogenously added factors or other methods
known in the art such as proteins or microRNA (see Zhou et al.,
Cell Stem Cell (4): 1-4, 2009; Miyoshi et al. Cell Stem Cell (8):
1-6, 2011; Danso et al., Cell Stem Cell (8): 376-388, 2011).
[0099] In another aspect, the disclosure provides preparations of
mesenchymal stromal cells (MSCs) and methods of generating MSCs
using hemangioblasts. The MSC may differ from pre-existing MSC in
one or more aspects, as further described herein. In one
embodiment, hemangioblasts are harvested after at least 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days in culture using
a serum free methylcellulose medium plus one or more ingredients
selected from the group comprising penicillin/streptomycin
(pen/strp), EX-CYTE.RTM. growth supplement (a water-soluble
concentrate comprising 9.0-11.0 g/L cholesterol and 13.0-18.0 g/L
lipoproteins and fatty acids at pH 7-8.4), Flt3-ligand (FL),
vascular endothelial growth factor (VEGF), thrombopoietin (TPO),
basic fibroblast growth factor (bFGF), stem cell derived factor
(SCF), granulocyte macrophage colony stimulating factor (GM-CSF),
interleukin 3 (IL3), and interleukin 6 (IL6), by inducing a
pluripotent cell selected from the group comprising blastocysts,
plated ICMs, one or more blastomeres, or other portions of a
pre-implantation-stage embryo or embryo-like structure, regardless
of whether produced by fertilization, somatic cell nuclear transfer
(SCNT), parthenogenesis, androgenesis, or other sexual or asexual
means, and cells derived through reprogramming (iPS cells). In a
preferred embodiment of the instant invention, hemangioblasts are
harvested between 6-14 days, of being cultured in, for example,
serum-free methylcellulose plus the ingredients of the previous
embodiment. In a preferred embodiment, the ingredients are present
in said medium at the following concentrations: Flt3-ligand (FL) at
50 ng/ml, vascular endothelial growth factor (VEGF) at 50 ng/ml,
thrombopoietin (TPO) at 50 ng/ml, and basic fibroblast growth
factor (bFGF) at 20 ng/ml, 50 ng/ml stem cell derived factor (SCF),
20 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF),
20 ng/ml interleukin 3 (IL3), 20 ng/ml interleukin 6 (IL6), 50
ng/ml FL, 50 ng/ml VEGF, 50 ng/ml TPO, and 30 ng/ml bFGF.
[0100] In another embodiment, a cluster of cells comprised
substantially of hemangioblasts are re-plated and cultured for at
least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 days forming
a preparation of mesenchymal stromal cells. In one embodiment,
mesenchymal stromal cells are generated by the steps comprising (a)
culturing ESCs for 8-12 days, (b) harvesting hemangioblasts that
form clusters of cells, (c) re-plating the hemangioblasts of step
(b), and (d) culturing the hemangioblasts of step (c) for between
14-30 days.
[0101] In one embodiment, the hemangioblasts are harvested,
re-plated and cultured in liquid medium under feeder-free
conditions wherein no feeder layer of cells such as mouse embryonic
fibroblasts, OP9 cells, or other cell types known to one of
ordinary skill in the art are contained in the culture. In a
preferred embodiment, hemangioblasts are cultured on an
extracellular matrix. In a further proferred embodiment,
hemangioblasts are cultured on an extracellular matrix, wherein
said matrix comprises a soluble preparation from
Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells that gels at room
temperature to form a reconstituted basement membrane (Matrigel).
In a still further preferred embodiment, hemangioblasts are
generated according to the steps comprising (a) culturing said
hemangioblasts on Matrigel for at least 7 days, (b) transferring
the hemangioblasts of step (a) to non-coated tissue culture plate
and further culturing said hemangioblasts of step (b) for between
about 7 to 14 days). The hemangioblasts may be cultured on a
substrate comprising one or more of the factors selected from the
group consisting of: transforming growth factor beta (TGF-beta),
epidermal growth factor (EGF), insulin-like growth factor 1, bovine
fibroblast growth factor (bFGF), and/or platelet-derived growth
factor (PDGF), Human Basement Membrane Extract (BME) (e.g., Cultrex
BME, Trevigen) or an EHS matrix, laminin, fibronectin, vitronectin,
proteoglycan, entactin, collagen (e.g., collagen I, collagen IV),
and heparan sulfate. Said matrix or matrix components may be of
mammalian, or more specifically human, origin. In one embodiment,
hemangioblasts are cultured in a liquid medium comprising serum on
a Matrigel-coated plate, wherein the culture medium may comprise
ingredients selected from .alpha.MEM (Sigma-Aldrich) supplemented
with 10-20% fetal calf serum (.alpha.MEM+20% FCS), .alpha.MEM
supplemented with 10-20% heat-inactivated human AB serum, and IMDM
supplemented with 10-20% heat inactivated AB human serum.
Mesenchymal Stromal Cells Generated by Culturing Hemangioblasts
[0102] An embodiment of the instant invention comprises improved
mesenchymal stromal cells. The mesenchymal stromal cells of the
instant invention may be generated from hemangioblasts using
improved processes of culturing hemangioblasts.
[0103] Mesenchymal stromal cells of the instant invention may
retain higher levels of potency and may not clump or may clump
substantially less than mesenchymal stromal cells derived directly
from ESCs. In an embodiment of the instant invention, a preparation
of mesenchymal stromal cells generated according to any one or more
of the processes of the instant invention retains higher levels of
potency, and do not clump or clump substantially less than
mesenchymal stromal cells derived directly from ESCs.
[0104] An embodiment of the instant invention provides a processes
of culturing hemangioblasts that generate preparations of
mesenchymal stromal cells, wherein said mesenchymal stromal cells
retain a youthful phenotype. The pharmaceutical preparations of
mesenchymal stromal cells of the instant invention may demonstrate
improved therapeutic properties when administered to a mammalian
host in need of treatment.
[0105] An embodiment of the instant invention provides a
preparation of mesenchymal stromal cells generated by culturing
human hemangioblasts. A further embodiment of the instant invention
provides a processes for generating a preparation of mesenchymal
stromal cells by culturing human hemangioblasts. An embodiment of a
process of the instant invention, wherein said human hemangioblasts
are cultured in feeder-free conditions then plated on a matrix. A
still further embodiment of the instant invention, wherein said
matrix is selected from the group comprising transforming growth
factor beta (TGF-beta), epidermal growth factor (EGF), insulin-like
growth factor 1, bovine fibroblast growth factor (bFGF),
platelet-derived growth factor (PDGF), laminin, fibronectin,
vitronectin, proteoglycan, entactin, collagen, collagen I, collagen
IV, heparan sulfate, a soluble preparation from
Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, Matrigel, and a
human basement membrane extract. In a still further embodiment,
said matrix may derive from mammalian or human origin.
[0106] In another embodiment, hemangioblasts are cultured in a
medium comprising serum or a serum replacment, such as .alpha.MEM
supplemented with 20% fetal calf serum. In a further embodiment,
hemangioblasts are cultured on a matrix for about 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29
or 30 days. In a still further embodiment of the instant invention,
a preparation of mesenchymal stromal cells are generated by the
steps comprising (a) culturing hemangioblasts on Matrigel for about
7 days, (b) transferring the hemangioblasts of step (a) off
Matrigel and growing the hemangioblasts on an uncoated tissue
culture dish for an additional 9-100 days, about 9, 10, 11, 12,
13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50,
60, 70, 80, 90 or 100 days.
[0107] In an embodiment of the instant invention, a preparation of
mesenchymal stromal cells is generated by culturing hemangioblasts
in a medium comprising serum or a serum replacement such as aMEM
supplemented with 20% fetal calf serum. In further embodiment of
the instant invention, said hemangioblasts are cultured on a matrix
for about 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, or 30 days.
[0108] In an embodiment of the instant invention hemangioblasts are
differentiated from ESCs. In a further embodiment of the instant
invention, the hemangioblasts of the previous embodiment are
differentiated from ESCs wherein, said ESCs are selected from the
group comprising iPS, MA09, H7, H9, MA01, HuES3, H1gfp, inner cell
mass cells and blastomeres.
[0109] An embodiment of the instant invention comprises a
preparation of mesenchymal stromal cells generated by a process
wherein hemangioblasts are differentiated from ESCs. In a further
embodiment of the instant invention, the hemangioblasts of the
previous embodiment are differentiated from ESCs wherein, said ESCs
are selected from the group comprising iPS, MA09, H7, H9, MA01,
HuES3, H1gfp, inner cell mass cells and blastomeres.
[0110] In an embodiment of the instant invention hemangioblasts are
differentiated from ESCs by following the steps comprising (a)
culturing ESCs in, for example, the presence of vascular
endothelial growth factor (VEGF) and/or bone morphogenic protein 4
(BMP-4) to form clusters of cells; (b) culturing said clusters of
cells in the presence of at least one growth factor (e.g., basic
fibroblast growth factor (bFGF), vascular endothelial growth factor
(VEGF), and bone morphogenic protein 4 (BMP-4), stem cell factor
(SCF), Flt 3L (FL), thrombopoietin (TPO), and/or tPTD-HOXB4) in an
amount sufficient to induce the differentiation of said clusters of
cells into hemangioblasts; and (c) culturing said hemangioblasts in
a medium comprising at least one additional growth factor (e.g.,
insulin, transferrin, granulocyte macrophage colony-stimulating
factor (GM-C SF), interleukin-3 (IL-3), interleukin-6 (IL-6),
granulocyte colony-stimulating factor (G-CSF), erythropoietin
(EPO), stem cell factor (SCF), vascular endothelial growth factor
(VEGF), bone morphogenic protein 4 (BMP-4), and tPTD-HOXB4),
wherein said at least one additional growth factor is provided in
an amount sufficient to expand said clusters of cells in said
culture, and wherein copper is optionally added to any of the steps
(a)-(c).
[0111] In an embodiment of the instant invention a preparation of
mesenchymal stromal cells is generated by culturing hemangioblasts,
wherein said hemangioblasts are differentiated from ESCs by
following the steps comprising (a) culturing ESCs in the presence
of vascular endothelial growth factor (VEGF) and bone morphogenic
protein 4 (BMP-4) within 0-48 hours of initiation of said culture
to form clusters of cells; (b) culturing said clusters of cells in
the presence of at least one growth factor selected from the group
comprising basic fibroblast growth factor (bFGF), vascular
endothelial growth factor (VEGF), bone morphogenic protein 4
(BMP-4), stem cell factor (SCF), Flt 3L (FL), thrombopoietin (TPO),
and tPTD-HOXB4 in an amount sufficient to induce the
differentiation of said clusters of cells into hemangioblasts; and
(c) culturing said hemangioblasts in a medium comprising at least
one additional growth factor selected from the group comprising
insulin, transferrin, granulocyte macrophage colony-stimulating
factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6),
granulocyte colony-stimulating factor (G-CSF), erythropoietin
(EPO), stem cell factor (SCF), vascular endothelial growth factor
(VEGF), bone morphogenic protein 4 (BMP-4), and tPTD-HOXB4, wherein
said at least one additional growth factor is provided in an amount
sufficient to expand human clusters of cells in said culture.
[0112] In another embodiment, a preparation of mesenchymal stem
cells is generated by the steps comprising (a) harvesting
hemangioblasts after at least 6, 7, 8, 9, 10, 11, 12, 13, or 14
days of inducing ESCs to differentiate into said hemangioblasts,
and (b) harvesting mesenchymal stromal cells that are generated
withinabout 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 days ofinducing
said hemangioblasts from step (a) to differentiate into said
mesenchymal cells.
[0113] In yet another embodiment, a preparation of at least 80, 85,
90, 95, 100, 125 or 125 million mesenchymal stromal cells are
generated from about 200,000 hemangioblasts within about 26, 27,
28, 29, 30, 31, 32, 33, 34, or 35 days of culturing the
hemangioblasts, wherein said preparation of mesenchymal stromal
cells comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,
2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,
0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%,
0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%,
0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%,
0.0003%, 0.0002%, or 0.0001% human mebryonic stem cells. In still
another embodiment, at least 80, 85, 90, 100, 125 or 150 million
mesenchymal stromal cells are generated from about 200,000
hemangioblasts within about 26, 27, 28, 29, 30, 31, 32, 33, 34, or
35 days of culturing the hemangioblasts.
[0114] In an embodiment of a process of the instant invention a
preparation of mesenchymal stromal cells are substantially purified
with respect to human embryonic stem cells. In a further embodiment
of a process of the instant invention a preparation of mesenchymal
stromal cells are substantially purified with respect to human
embryonic stem cells such that said preparation comprises at least
about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
mesenchymal stromal cells.
[0115] In another embodiment of the instant invention, a
preparation of mesenchymal stromal cells generated by any one or
more of the processes of the instant invention do not form
teratomas when introduced into a host.
[0116] In another embodiment of the instant invention, at least 50%
of a preparation of mesenchymal stromal cells are positive for
CD105 or CD73 within about 7-20 (e.g., 15) days of culture. I a
preferred embodiment of the instant invention, at least 50% of a
preparation of mesenchymal stromal cells generated according to any
one or more processes of the instant invention are positive for
CD105 or CD73 after about 7-15 days of culture. In a further
embodiment of the instant invention, at least 80% of a preparation
of mesenchymal stromal cells are positive for CD105 and CD73 within
about 20 days of culture. In still a further embodiment of the
instant invention, at least 80% of a preparation of mesenchymal
stromal cells generated according to any one or more of the
processes of the instant invention are positive for CD105 and CD73
within about 20 days of culture.
[0117] In an exemplary aspect, the present disclosure provides a
pharmaceutical preparation suitable for use in a mammalian patient,
comprising at least 10.sup.6 mesenchymal stromal cells and a
pharmaceutically acceptable carrier, wherein the mesenchymal
stromal cells have replicative capacity to undergo at least 10
population doublings in cell culture with less than 25 percent of
the cells undergoing cell death, senescing or differentiating into
non-MSC cells by the tenth doubling.
[0118] In an exemplary aspect, the present disclosure provides a
pharmaceutical preparation suitable for use in a mammalian patient
comprising at least 10.sup.6 mesenchymal stromal cells and a
pharmaceutically acceptable carrier, wherein the mesenchymal
stromal cells have replicative capacity to undergo at least 5
passages in cell culture with less than 25 percent of the cells
undergoing cell death, senescing or differentiating into
fibroblasts by the 5.sup.th passage.
[0119] In an exemplary aspect, the present disclosure provides a
pharmaceutical preparation comprising at least 10.sup.6 mesenchymal
stromal cells and a pharmaceutically acceptable carrier, wherein
the mesenchymal stromal cells are differentiated from a
hemangioblast cell.
[0120] In an exemplary aspect, the present disclosure provides a
cryogenic cell bank comprising at least 10.sup.8 mesenchymal
stromal cells, wherein the mesenchymal stromal cells have
replicative capacity to undergo at least 10 population doublings in
cell culture with less than 25 percent of the cells undergoing cell
death, senescing or differentiating into fibroblasts by the tenth
population doubling.
[0121] In an exemplary aspect, the present disclosure provides a
purified cellular preparation comprising at least 10.sup.6
mesenchymal stromal cells and less than one percent of any other
cell type, wherein the mesenchymal stromal cells have replicative
capacity to undergo at least 10 population doublings in cell
culture with less than 25 percent of the cells undergoing cell
death, senescing or differentiating into non-MSC cells by the tenth
population doubling.
[0122] The mesenchymal stromal cells may be differentiated from a
pluripotent stem cell source, such as an embryonic stem cell line
or induced pluripotent stem cell line. For example, all of the
mesenchymal stromal cells of the preparation or bank may be
differentiated from a common pluripotent stem cell source.
Additionally, the mesenchymal stromal cells may be differentiated
from a pluripotent stem cell source, passaged in culture to expand
the number of mesenchymal stromal cells, and isolated from culture
after less than twenty population doublings.
[0123] The mesenchymal stromal cells may be HLA-genotypically
identical. The mesenchymal stromal cells may be genomically
identical.
[0124] At least 30% of the mesenchymal stromal cells may be
positive for CD10. Additionally, at least 60% of the mesenchymal
stromal cells may be positive for markers CD73, CD90, CD105, CD13,
CD29, CD44, and CD166 and HLA-ABC. In an exemplary embodiment, less
than 30% of the mesenchymal stromal cells may be positive for
markers CD31, CD34, CD45, CD133, FGFR2, CD271, Stro-1, CXCR4 and
TLR3.
[0125] The mesenchymal stromal cells may have replicative rates to
undergo at least 10 population doublings in cell culture in less
than 25 days. The mesenchymal stromal cells may have a mean
terminal restriction fragment length (TRF) that may be longer than
8 kb. The mesenchymal stromal cells may have a statistically
significant decreased content and/or enzymatic activity, relative
to mesenchymal stromal cell preparations derived from bone marrow
that have undergone five population doublings, of proteins involved
in one or more of (i) cell cycle regulation and cellular aging,
(ii) cellular energy and/or lipid metabolism, and (iii) apoptosis.
The mesenchymal stromal cells may have a statistically significant
increased content and/or enzymatic activity of proteins involved in
cytoskeleton structure and cellular dynamics relating thereto,
relative to mesenchymal stromal cell preparations derived from bone
marrow. The mesenchymal stromal cells may not undergo more than a
75 percent increase in cells having a forward-scattered light
value, measured by flow cytometry, greater than 5,000,000 over 10
population doublings in culture. The mesenchymal stromal cells may
in a resting state, express mRNA encoding Interleukin-6 at a level
which may be less than ten percent of the IL-6 mRNA level expressed
by mesenchymal stromal cells preparations, in a resting state,
derived from bone marrow or adipose tissue.
[0126] The preparation may be suitable for administration to a
human patient. The preparation may be suitable for administration
to a non-human veterinarian mammal.
[0127] In an exemplary aspect, the disclosure provides a
pharmaceutical preparation comprising mesenchymal stromal cells,
wherein said mesenchymal stromal cells are able to undergo at least
10 population doublings and wherein the 10 population doublings
occur within about 27 days, more preferably less than about 26
days, preferably less than 25 days, more preferably less than about
24 days, still more preferably less than about 23 days, still more
preferably less than about 22 days, or lower.
[0128] In an exemplary aspect, the disclosure provides a
pharmaceutical preparation comprising mesenchymal stromal cells,
wherein said mesenchymal stromal cells are able to undergo at least
15 population doublings.
[0129] Said mesenchymal stromal cells may be able to undergo at
least 20, 25, 30, 35, 40, 45, 50 or more population doublings.
[0130] In an exemplary aspect, the disclosure provides a
pharmaceutical preparation comprising mesenchymal stromal cells,
wherein said mesenchymal stromal cells are able to undergo at least
15 population doublings, at least 20 population doublings, or at
least 25 population doublings in culture.
[0131] The mesenchymal stromal cells may be produced by in vitro
differentiation of hemangioblasts. The mesenchymal stromal cells
may be primate cells or other mammalian cells. The mesenchymal
stromal cells may be human cells.
[0132] Said population doublings occur within about 35 days, more
preferably within about 34 days, preferably within 33 days, more
preferably within 32 days, still more preferably within 31 days, or
still more preferably within about 30 days.
[0133] The preparation may comprise less than about 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,
0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,
0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%,
0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%,
0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% pluripotent
cells.
[0134] The preparation may be devoid of pluripotent cells.
[0135] The preparation may comprise at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% mesenchymal stromal
cells.
[0136] At least 50% of said mesenchymal stromal cells may be
positive for (i) at least one of CD10, CD24, IL-11, AIRE-1, ANG-1,
CXCL1, CD105, CD73 and CD90; (ii) at least one of CD10, CD24,
IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13,
CD29,CD 44, CD166, CD274, and HLA-ABC; (iii) CD105, CD73 and/or
CD90 or (iv) any combination thereof. At least 50% of said
mesenchymal stromal cells may be positive for (i) at least two of
CD105, CD73 and/or CD90 (ii) at least two of CD10, CD24, IL-11,
AIRE-1, ANG-1, CXCL1, CD105, CD73 and CD90; or (iii) all of CD10,
CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13,
CD29,CD 44, CD166, CD274, and HLA-ABC. At least 50% of said
mesenchymal stromal cells (i) may be positive for all of CD105,
CD73 and CD90; (ii) positive for all of CD10, CD24, IL-11, AIRE-1,
ANG-1, CXCL1, CD105, CD73, CD90, CD105, CD13, CD29,CD 44, CD166,
CD274, and HLA-ABC and/or (ii) may be negative for or less than 5%
or less than 10% of the cells express CD31, 34, 45, 133, FGFR2,
CD271, Stro-1, CXCR4, and/or TLR3. At least 60%, 70%, 80% or 90% of
said mesenchymal stromal cells may be positive for (i) one or more
of of CD105, CD73 and CD90 (ii) one or more of CD10, CD24, IL-11,
AIRE-1, ANG-1, CXCL1, CD105, CD73 and CD90; or (iii) one or more of
CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105, CD73, CD90, CD105,
CD13, CD29,CD 44, CD166,CD274, and HLA-ABC.
[0137] The pharmaceutical preparation may comprise an amount of
mesenchymal stromal cells effective to treat an unwanted immune
response in a subject in need thereof.
[0138] The pharmaceutical preparation may comprise other cells,
tissue or organ for transplantation into a recipient in need
thereof. The other cells or tissue may be RPE cells, skin cells,
corneal cells, pancreatic cells, liver cells, cardiac cells or
tissue containing any of said cells. Said mesenchymal stromal cells
may be not derived from bone marrow and the potency of the
preparation in an immune regulatory assay may be greater than the
potency of a preparation of bone marrow derived mesenchymal stromal
cells. Potency may be assayed by an immune regulatory assay that
determines the EC50 dose. The preparation may retain between about
50 and 100% of its proliferative capacity after ten population
doublings.
[0139] Said mesenchymal stromal cells may be not derived directly
from pluripotent cells and wherein said mesenchymal stromal cells
(a) do not clump or clump substantially less than mesenchymal
stromal cells derived directly from pluripotent cells; (b) more
easily disperse when splitting compared to mesenchymal stromal
cells derived directly from pluripotent cells; (c) may be greater
in number than mesenchymal stromal cells derived directly from
pluripotent cells when starting with equivalent numbers of
pluripotent cells; and/or (d) acquire characteristic mesenchymal
cell surface markers earlier than mesenchymal stromal cells derived
directly from pluripotent cells.
[0140] Said mesenchymal stromal cells may be mammalian. Said
mesenchymal stromal cells may be human, canine, bovine, non-human
primate, murine, feline, or equine
[0141] In an exemplary aspect, the present disclosure provides a
method for generating mesenchymal stromal cells comprising
culturing hemangioblasts under conditions that give rise to
mesenchymal stem cells. Said hemangioblasts may be cultured in
feeder-free conditions. Said hemangioblasts may be plated on a
matrix. Said matrix may comprise one or more of: transforming
growth factor beta (TGF-beta), epidermal growth factor (EGF),
insulin-like growth factor 1, bovine fibroblast growth factor
(bFGF), and/or platelet-derived growth factor (PDGF). Said matrix
may be selected from the group consisting of: laminin, fibronectin,
vitronectin, proteoglycan, entactin, collagen, collagen I, collagen
IV, heparan sulfate, Matrigel (a soluble preparation from
Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), a human basement
membrane extract, and any combination thereof. Said matrix may
comprise a soluble preparation from Engelbreth-Holm-Swarm mouse
sarcoma cells.
[0142] Said mesenchymal stromal cells may be mammalian. Said
mesenchymal stromal cells may be human, canine, bovine, non-human
primate, murine, feline, or equine.
[0143] Said hemangioblasts may be cultured in a medium comprising
.alpha.MEM. Said hemangioblasts may be cultured in a medium
comprising serum or a serum replacement. Said hemangioblasts may be
cultured in a medium comprising, .alpha.MEM supplemented with 0%,
0.1%-0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,
14%, 15%, 16%, 17%, 18%,19%, or 20% fetal calf serum. Said
hemangioblasts may be cultured on said matrix for at least about
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or
30 days.
[0144] Said hemangioblasts may be differentiated from pluripotent
cells.
[0145] Said pluripotent cells may be iPS cells or pluripotent cells
produced from blastomeres. Said pluripotent cells may be derived
from one or more blastomeres without the destruction of a human
embryo.
[0146] Said hemangioblasts may be differentiated from pluripotent
cells by a method comprising (a) culturing said pluripotent cells
to form clusters of cells. The pluripotent cells may be cultured in
the presence of vascular endothelial growth factor (VEGF) and/or
bone morphogenic protein 4 (BMP-4). In step (a), the pluripotent
cells may be cultured in the presence of vascular endothelial
growth factor (VEGF) and/or bone morphogenic protein 4 (BMP-4).
Said VEGF and BMP-4 may be added to the pluripotent cell culture
within 0-48 hours of initiation of said cell culture, and said VEGF
may be optionally added at a concentration of 20-100 nm/mL and said
BMP-4 may be optionally added at a concentration of 15-100 ng/mL.
Said VEGF and BMP-4 may be added to the cell culture of step (a)
within 0-48 hours of initiation of said cell culture, and said VEGF
may be optionally added at a concentration of 20-100 nm/mL and said
BMP-4 may be optionally added at a concentration of 15-100 ng/mL.
Said hemangioblasts may be differentiated from pluripotent cells by
a method which may further comprise: (b) culturing said clusters of
cells in the presence of at least one growth factor in an amount
sufficient to induce the differentiation of said clusters of cells
into hemangioblasts. Said at least one growth factor added in step
(b) may comprise one or more of basic fibroblast growth factor
(bFGF), vascular endothelial growth factor (VEGF), bone morphogenic
protein 4 (BMP-4), stem cell factor (SCF), Flt 3L (FL),
thrombopoietin (TPO), EPO, and/or tPTD-HOXB4.
[0147] Said at least one growth factor added in step (b) may
comprise one or more of: about 20-25 ng/ml basic fibroblast growth
factor (bFGF), about 20-100 ng/ml vascular endothelial growth
factor (VEGF), about 15-100 ng/ml bone morphogenic protein 4
(BMP-4), about 20-50 ng/ml stem cell factor (SCF), about 10-50
ng/ml Flt 3L (FL), about 20-50 ng/ml thrombopoietin (TPO), EPO,
and/or 1.5-5 U/ml tPTD-HOXB4.
[0148] One or more of said at least one growth factor optionally
added in step (b) may be added to said culture within 36-60 hours
or 40-48 hours from the start of step (a).
[0149] One or more of said at least one growth factor added in step
(b) may be added to said culture within 48-72 hours from the start
of step (a).
[0150] Said at least one factor added in step (b) may comprise one
or more of bFGF, VEGF, BMP-4, SCF and/or FL.
[0151] The method may further comprise (c) dissociating said
clusters of cells, optionally into single cells.
[0152] The method may further comprise (d) culturing said
hemangioblasts in a medium comprising at least one additional
growth factor, wherein said at least one additional growth factor
may be in an amount sufficient to expand the hemangioblasts.
[0153] In step (d), said at least one additional growth factor may
comprise one or more of: insulin, transferrin, granulocyte
macrophage colony-stimulating factor (GM-CSF), interleukin-3
(IL-3), interleukin-6 (IL-6), granulocyte colony-stimulating factor
(G-CSF), erythropoietin (EPO), stem cell factor (SCF), vascular
endothelial growth factor (VEGF), bone morphogenic protein 4
(BMP-4), and/or tPTD-HOXB4.
[0154] In step (d), said at least one additional growth factor may
comprise one or more of: about 10-100 .mu.g/ml insulin, about
200-2,000 .mu.g/ml transferrin, about 10-50 ng/ml granulocyte
macrophage colony-stimulating factor (GM-CSF), about 10-20 ng/ml
interleukin-3 (IL-3), about 10-1000 ng/ml interleukin-6 (IL-6),
about 10-50 ng/ml granulocyte colony-stimulating factor (G-CSF),
about 3-50 U/ml erythropoietin (EPO), about 20-200 ng/ml stem cell
factor (SCF), about 20-200 ng/ml vascular endothelial growth factor
(VEGF), about 15-150 ng/ml bone morphogenic protein 4 (BMP-4),
and/or about 1.5-15U/ml tPTD-HOXB4.
[0155] Said medium in step (a), (b), (c) and/or (d) may be a
serum-free medium.
[0156] The method as described above may further comprise (e)
mitotically inactivating the mesenchymal stromal cells.
[0157] At least 80, 85, 90, 95, 100, 125, or 150 million
mesenchymal stromal cells may be generated.
[0158] Said hemangioblasts may be harvested after at least 10, 11,
12, 13, 14, 15, 16, 17 or 18 days of starting to induce
differentiation of said pluripotent cells.
[0159] Said mesenchymal stromal cells may be generated within at
least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days of starting to
induce differentiation of said pluripotent cells.
[0160] The method may result in at least 80, 85, 90, 95, 100, 125,
or 150 million mesenchymal stromal cells being generated from about
200,000 hemangioblasts within about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days of
culture.
[0161] The mesenchymal stromal cells may be generated from
hemangioblasts in a ratio of hemangioblasts to mesenchymal stromal
cells of at least 1:200, 1:250, 1:300, 1:350,1:400, 1:415,1:425,
1:440; 1:450, 1:365, 1:475, 1:490 and 1:500 within about 26, 27,
28, 29, 30, 31, 32, 33, 34 or 35 days of culture as
hemangioblasts.
[0162] Said cells may be human.
[0163] In another aspect, the present disclosure provides
mesenchymal stromal cells derived from hemangioblasts obtained by
any of the methods described above.
[0164] In another aspect, the present disclosure provides
mesenchymal stromal cells derived by in vitro differentiation of
hemangioblasts.
[0165] At least 50% of said mesenchymal stromal cells (i) may be
positive for all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1, CD105,
CD73, CD90, CD105, CD13, CD29,CD 44, CD166, CD274, and HLA-ABC and
(ii) may be negative for or less than 5% or less than 10% of the
cells express CD31, 34, 45, 133, FGFR2, CD271, Stro-1, CXCR4 and/or
TLR3.
[0166] At least 50% of said mesenchymal stromal cells may be
positive for (i) all of CD10, CD24, IL-11, AIRE-1, ANG-1, CXCL1,
CD105, CD73 and CD90; or (ii) all of CD73, CD90, CD105, CD13, CD29,
CD44, CD166, CD274, and HLA-ABC.
[0167] At least 60%, 70%, 80% or 90% of said mesencyhmal stromal
cells may be positive for (i) at least one of CD10, CD24, IL-11,
AIRE-1, ANG-1, CXCL1, CD105, CD73 and CD90; or (ii) at least one of
CD73, CD90, CD105, CD13, CD29,CD 44, CD166, CD274, and HLA-ABC.
[0168] The mesenchymal stromal may not express or less than 5% or
less than 10% of the cells may express at least one of CD31, 34,
45, 133, FGFR2, CD271, Stro-1, CXCR4, or TLR3.
[0169] In another aspect, the present disclosure provides a
preparation of mesenchymal stromal cells as described above.
[0170] Said preparation may comprise less than 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%,
0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%,
0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%,
0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%,
0.0004%, 0.0003%, 0.0002%, or 0.0001% pluripotent cells.
[0171] The preparation may be devoid of pluripotent cells.
[0172] Said preparation may be substantially purified and
optionally may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% human mesenchymal stromal cells.
[0173] The preparation may comprise substantially similar levels of
p53 and p21 protein or wherein the levels of p53 protein as
compared to p21 protein may be 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10
times greater.
[0174] The mesenchymal stromal cells or the MSC in the preparation
may be capable of undergoing at least 5 population doublings in
culture, or may be capable of undergoing at least 10, 15, 20, 25,
30, 35, 40, 45, 50, 55, 60 or more population doublings in
culture.
[0175] Said mesenchymal stromal cells (a) may not clump or clump
substantially less than mesenchymal stromal cells derived directly
from pluripotent cells; (b) may more easily disperse when splitting
compared to mesenchymal stromal cells derived directly from
pluripotent cells; (c) may be greater in number than mesenchymal
stromal cells derived directly from pluripotent cells when starting
with equivalent numbers of pluripotent cellss; and/or (d) acquire
characteristic mesenchymal cell surface markers earlier than
mesenchymal stromal cells derived directly from pluripotent
cells.
[0176] In another aspect, the disclosure provides pharmaceutical
preparation comprising any mesenchymal stromal cells or preparation
of mesenchymal stromal cells as described above.
[0177] The pharmaceutical preparation may comprise an amount of
mesenchymal stromal cells effective to treat an unwanted immune
response.
[0178] The pharmaceutical preparation may comprise an amount of
mesenchymal stromal cells effective to treat an unwanted immune
response and may further comprise other cells or tissues for
transplantation into a recipient in need thereof.
[0179] Said other cells or tissues may be allogeneic or syngeneic
pancreatic, neural, liver, RPE, corneal cells or tissues containing
any of the foregoing.
[0180] The pharmaceutical preparation may be for use in treating an
autoimmune disorder or an immune reaction against allogeneic cells,
or for use in treating multiple sclerosis, systemic sclerosis,
hematological malignancies, myocardial infarction, organ
transplantation rejection, chronic allograft nephropathy,
cirrhosis, liver failure, heart failure, GvHD, tibial fracture,
left ventricular dysfunction, leukemia, myelodysplastic syndrome,
Crohn's disease, diabetes, chronic obstructive pulmonary disease,
osteogenesis imperfecta, homozygous familial hypocholesterolemia,
treatment following meniscectomy, adult periodontitis,
vasculogenesis in patients with severe myocardial ischemia, spinal
cord injury, osteodysplasia, critical limb ischemia, diabetic foot
disease, primary Sjogren's syndrome, osteoarthritis, cartilage
defects, laminitis, multisystem atrophy, amyotropic lateral
sclerosis, cardiac surgery, systemic lupus erythematosis, living
kidney allografts, nonmalignant red blood cell disorders, thermal
burn, radiation burn, Parkinson's disease, microfractures,
epidermolysis bullosa, severe coronary ischemia, idiopathic dilated
cardiomyopathy, osteonecrosis femoral head, lupus nephritis, bone
void defects, ischemic cerebral stroke, after stroke, acute
radiation syndrome, pulmonary disease, arthritis, bone
regeneration, uveitis or combinations thereof.
[0181] In another aspect, the disclosure provides a kit comprising
any of the mesenchymal stromal cells or any preparation of
mesenchymal stromal cells as described above.
[0182] In another aspect, the disclosure provides a kit comprising
the mesenchymal stromal cells or preparation of mesenchymal stromal
cells as described above, wherein said cells or preparation of
cells may be frozen or cryopreserved.
[0183] In another aspect, the disclosure provides a kit comprising
the mesenchymal stromal cells or preparation of mesenchymal stromal
cells as described above, wherein said cells or preparation of
cells may be contained in a cell delivery vehicle.
[0184] In another aspect, the disclosure provides a method for
treating a disease or disorder, comprising administering an
effective amount of mesenchymal stromal cells or a preparation of
mesenchymal stromal cells as described above to a subject in need
thereof.
[0185] The method may further comprise the transplantation of other
cells or tissues. The cells or tissues may comprise retinal, RPE,
corneal, neural, immune, bone marrow, liver or pancreatic cells.
The disease or disorder may be selected from multiple sclerosis,
systemic sclerosis, hematological malignancies, myocardial
infarction, organ transplantation rejection, chronic allograft
nephropathy, cirrhosis, liver failure, heart failure, GvHD, tibial
fracture, left ventricular dysfunction, leukemia, myelodysplastic
syndrome, Crohn's disease, diabetes, chronic obstructive pulmonary
disease, osteogenesis imperfecta, homozygous familial
hypocholesterolemia, treatment following meniscectomy, adult
periodontitis, vasculogenesis in patients with severe myocardial
ischemia, spinal cord injury, osteodysplasia, critical limb
ischemia, diabetic foot disease, primary Sjogren's syndrome,
osteoarthritis, cartilage defects, multisystem atrophy, amyotropic
lateral sclerosis, cardiac surgery, refractory systemic lupus
erythematosis, living kidney allografts, nonmalignant red blood
cell disorders, thermal burn, Parkinson's disease, microfractures,
epidermolysis bullosa, severe coronary ischemia, idiopathic dilated
cardiomyopathy, osteonecrosis femoral head, lupus nephritis, bone
void defects, ischemic cerebral stroke, after stroke, acute
radiation syndrome, pulmonary disease, arthritis, bone
regeneration, or combinations thereof.
[0186] The disease or disorder may be uveitis. Said disease or
disorder may be an autoimmune disorder or an immune reaction
against allogeneic cells. The autoimmune disorder may be multiple
sclerosis.
[0187] In another aspect, the disclosure provides a method of
treating bone loss or cartilage damage comprising administering an
effective amount of mesenchymal stromal cells or preparation of
mesenchymal stromal cells to a subject in need thereof.
[0188] The mesenchymal stromal cells may be administered in
combination with an allogeneic or syngeneic transplanted cell or
tissue. The allogeneic transplanted cell may comprise a retinal
pigment epithelium cell, retinal cell, corneal cell, or muscle
cell.
[0189] In another aspect, the disclosure provides a pharmaceutical
preparation comprising mitotically inactivated mesenchymal stromal
cells. The mesenchymal stromal cells may be differentiated from a
hemangioblast cell.
[0190] The pharmaceutical may comprise at least 10.sup.6
mesenchymal stromal cells and a pharmaceutically acceptable
carrier.
[0191] In another aspect, the disclosure provides a pharmaceutical
preparation comprising mitotically inactivated mesenchymal cell
produced by the method above.
[0192] The preparation may be suitable for administration to a
human patient. The preparation may be suitable for administration
to a non-human veterinarian mammal.
[0193] The pharmaceutical preparation may be devoid of pluripotent
cells.
[0194] The pharmaceutical preparation may comprise an amount of
mesenchymal stromal cells effective to treat an unwanted immune
response in a subject in need thereof.
[0195] The pharmaceutical preparation may comprise an amount of
mesenchymal stromal cells effective to treat a disease or condition
selected from the group consisting of: inflammatory respiratory
conditions, respiratory conditions due to an acute injury, Adult
Respiratory Distress Syndrome, post-traumatic Adult Respiratory
Distress Syndrome, transplant lung disease, Chronic Obstructive
Pulmonary Disease, emphysema, chronic obstructive bronchitis,
bronchitis, an allergic reaction, damage due to bacterial
pneumonia, damage due to viral pneumonia, asthma, exposure to
irritants, tobacco use, atopic dermatitis, allergic rhinitis,
hearing loss, autoimmune hearing loss, noise-induced hearing loss,
psoriasis and any combination thereof.
Preparation of Mesenchymal Stromal Cells
[0196] In an embodiment of the instant invention, a preparation of
the subject mesenchymal stromal cells (e.g., generated by culturing
hemangioblasts) is provided, wherein the desired phenotype of said
mesenchymal stromal cells presents earlier as compared to
mesenchymal stromal cells by ESC culture (See FIG. 5). In a further
embodiment of the instant invention, a preparation of the subject
mesenchymal stromal cells (e.g., generated by culturing
hemangioblasts) is provided, wherein the desired phenotype of said
mesenchymal stromal cells presents earlier as compared to
mesenchymal stromal cells by ESC culture, and wherein said desired
phenotype is defined by the expression of at least two markers
selected from the group comprising CD9, CD13, CD29, CD44, CD73,
CD90, CD105, CD166, and HLA-abc.
[0197] A further embodiment of the instant invention comprises a
preparation of mesenchymal stromal cells, wherein the phenotype of
said mesenchymal stromal cells is defined by the expression of at
least two markers selected from the group comprising CD9, CD13,
CD29, CD44, CD73, CD90, CD105, CD166, and HLA-ABC. A still further
embodiment of the instant invention comprises a preparation of
mesenchymal stromal cells, wherein the phenotype of said
mesenchymal stromal cells is defined by the expression of at least
two markers selected from the group comprising CD9, CD13, CD29,
CD44, CD73, CD90 and CD105, and wherein said mesenchymal stromal
cells do not express CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15,
CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD61, CD62E and
CD133.
[0198] In an embodiment of the instant invention about 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the
subject mesenchymal stromal cells (e.g., generated by culturing
hemangioblasts) present a phenotype defined by the expression of
the markers CD9, CD13, CD29, CD44, CD73, CD90, CD105, CD166, and
HLA-abc after about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, or 30 days in culture. In an embodiment of the instant
invention at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 98%, 99%, or 100% of the subject mesenchymal stromal cells
(e.g., generated by culturing hemangioblasts) present a phenotype
defined by the expression of at least two markers selected from the
group comprising CD9, CD13, CD29, CD44, CD73, CD90, CD105, CD166,
and HLA-abc and a lack of expression of CD2, CD3, CD4, CD5, CD7,
CD8, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD61,
CD62E, CD133 and Stro-1 after about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, or 30 days in culture. The previous
embodiment, wherein said phenotype is further defined by the
markers selected from the group comprising AIRE-1, IL-11, CD10,
CD24, ANG-1, and CXCL1.
[0199] A preferred process of the instant invention is provided,
wherein the number of mesenchymal stromal cells derived from
hemangioblasts is about 8.times.10.sup.7, 8.5.times.10.sup.7,
9.times.10.sup.7, 9.5.times.10.sup.7, 1.times.10.sup.8,
1.25.times.10.sup.8, or 1.5.times.10.sup.8 mesenchymal stromal
cells derived from about 2.times.105 hemangioblasts within about 30
days of culture of mesenchymal stromal cells. In an alternative
embodiment of the instant invention, mesenchymal stromal cells may
be generated from hemangioblasts in a ratio of hemangioblasts to
mesenchymal stromal cells of about 1:200, 1:400, 1:415, 1:425,
1:440; 1:450, 1:465, 1:475, 1:490, and 1:500, within about 30 days
of culture of mesenchymal stromal cells.
[0200] In a preferred embodiment of the instant invention, the
number of mesenchymal stromal cells obtained by hemangioblast
culture is higher than the number of mesenchymal stromal cells
obtained directly from ESCs. In a further preferred embodiment of
the instant invention, the number of mesenchymal stromal cells
obtained by hemangioblast culture is at least 5 times, 10 times, 20
times, 22 times higher than the number of mesenchymal stromal cells
obtained directly from ESCs than the number of mesenchymal stromal
cells obtained directly from ESCs (See FIG. 4).
[0201] In another embodiment of the instant invention, a
preparation of the subject mesenchymal stromal cells does not form
teratomas when introduced into mammalian host.
[0202] An embodiment of the instant invention provides a
preparation of mesenchymal stromal cells generated by culturing
hemangioblasts using any of the process embodiments of the instant
invention. An embodiment of the instant invention comprising a
preparation of mesenchymal stromal cells generated by culturing
hemangioblasts using any of the process embodiments of the instant
invention, wherein the phenotype of said preparation is defined by
the presence of any or all of the markers selected from the group
comprising AIRE-1, IL-11, CD10, CD24, ANG-1, and CXCL1. A further
embodiment of the instant invention comprising a preparation of
mesenchymal stromal cells generated by culturing hemangioblasts
using any of the process embodiments of the instant invention,
wherein the phenotype of said preparation is defined by the
presence of any or all of the markers selected from the group
comprising AIRE-1, IL-11, CD10, CD24, ANG-1, and CXCL1, and wherein
said preparation presents a reduced expression of IL-6, Stro-1 and
VEGF.
[0203] In an embodiment of the instant invention, a preparation of
the subject mesenchymal stromal cells (e.g., generated by culturing
hemangioblasts) is provided, wherein said preparation comprises
substantially similar levels of p53 and p21 protein, or wherein the
levels of p53 as compared to p21 are 1.5, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 times greater. In an embodiment of the instant invention, a
preparation of the subject mesenchymal stromal cells (e.g.,
generated by culturing hemangioblasts) is provided, wherein said
preparation comprises substantially similar levels of p53 and p21
protein, or wherein the levels of p53 as compared to p21 are 1.5,
2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater. In an embodiment of
the instant invention, a pharmaceutical preparation of the subject
mesenchymal stromal cells (e.g., generated by culturing
hemangioblasts) is provided, wherein said pharmaceutical
preparation comprises substantially similar levels of p53 and p21
protein, or wherein the levels of p53 as compared to p21 are 1.5,
2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater.
[0204] In an embodiment of the instant invention, a preparation of
the subject mesenchymal stromal cells (e.g., generated by culturing
hemangioblasts) is provided, wherein said preparation comprises a
substantially similar percentage of cells positive for p53 and p21
protein, or wherein the percentage of cells positive for p53 as
compared to p21 are 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times
greater. In an embodiment of the instant invention, a preparation
of the subject mesenchymal stromal cells (e.g., generated by
culturing hemangioblasts) is provided wherein said preparation
comprises a substantially similar percentage of cells positive for
p53 and p21 protein, or wherein the percentage of cells positive
for p53 as compared to p21 are 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10
times greater. In an embodiment of the instant invention, a
pharmaceutical preparation of the subject mesenchymal stromal cells
(e.g., generated by culturing hemangioblasts) is provided, wherein
said pharmaceutical preparation comprises a substantially similar
percentage of cells positive for p53 and p21 protein, or wherein
the percentage of cells positive for p53 as compared to p21 are
1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater.
[0205] In an embodiment of the instant invention, a preparation of
the subject mesenchymal stromal cells (e.g., generated by culturing
hemangioblasts) is provided, wherein said preparation comprises a
substantially similar percentage of cells having background levels
of aging markers selected from the group comprising S100A1, VIM,
MYADM, PIM1, ANXA2, RAMP, MEG3, IL13R2, S100A4, TREM1,DGKA, TPBG,
MGLL, EML1, MYO1B, LASS6, ROBO1, DKFZP586H2123, LOC854342, DOK5,
UBE2E2, USP53, VEPH1, SLC35E1, ANXA2, HLA-E, CD59, BHLHB2, UCHL1,
SUSP3, CREDBL2, OCRL, OSGIN2, SLEC3B, IDS, TGFBR2, TSPAN6, TM4SF1,
MAP4, CAST, LHFPL2, PLEKHM1, SAMD4A, VAMP1, ADD1, FAM129A, HPDC1,
KLF11, DRAM, TREM140, BHLHB3, MGC17330, TBC1D2, KIAA1191, C5ORF32,
C15ORF17, FAM791, CCDC104, PQLC3, EIF4E3, C7ORF41, DUSP18, SH3PX3,
MYO5A, PRMT2, C8ORF61, SAMD9L, PGM2L1, HOM-TES-103, EPOR, and
TMEM112 or from the group comprising S100A1, VIM, MYADM, PIM1,
ANXA2, RAMP, MEG3, IL13R2, S100A4,TREM1, DGKA, TPBG, MGLL, EMLI,
MYO1B, LASS6, ROBO1, DKFZP586H2123, LOC854342, DOK5, UBE2E2, USP53,
VEPH1, and SLC35E1, or wherein the percentage of cells positive for
aging markers selected from the group comprising S100A1, VIM,
MYADM, PIM1, ANXA2, RAMP, MEG3, IL13R2, S100A4, TREM1,DGKA, TPBG,
MGLL, EML1, MYO1B, LASS6, ROBO1, DKFZP586H2123, LOC854342, DOKS,
UBE2E2, USP53, VEPH1, SLC35E1, ANXA2, HLA-E, CD59, BHLHB2, UCHL1,
SUSP3, CREDBL2, OCRL, OSGIN2, SLEC3B, IDS, TGFBR2, TSPAN6, TM4SF1,
MAP4, CAST, LHFPL2, PLEKHM1, SAMD4A, VAMP1 ADD1, FAM129A, HPDC1,
KLF11, DRAM, TREM140, BHLHB3, MGC17330, TBC1D2, KIAA1191, C5ORF32,
C15ORF17, FAM791, CCDC104, PQLC3, EIF4E3, C7ORF41, DUSP18, SH3PX3,
MYO5A, PRMT2, C8ORF61, SAMD9L, PGM2L1, HOM-TES-103, EPOR, TMEM112
or from the group comprising S100A1, VIM, MYADM, PIM1, ANXA2, RAMP,
MEG3, IL13R2, S100A4, TREM1,DGKA, TPBG, MGLL, EML1, MYO1B, LASS6,
ROBO1, DKFZP586H2123, LOC854342, DOK5, UBE2E2, USP53, VEPH1, and
SLC35E1, are 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than
background. In an embodiment of the instant invention, a
preparation of the subject mesenchymal stromal cells (e.g.,
generated by culturing hemangioblasts) is provided, wherein said
preparation comprises a substantially similar percentage of cells
having background levels of markers selected from the group
comprising HoxB3, HoxB7, MID1, SNAPC5, PPARG, ANXA2, TIPIN, MYLIP,
LAX1, EGR1CRIP1, SULT1A3, STMN1, CCT8, SFRS10, CBX3, CBX1,
FLJ11021, DDX46, ACADM, KIAA0101, TYMS, BCAS2, CEP57, TDG, MAP2K6,
CSRP2, GLMN, HMGN2, HNRPR, EIF3S1, PAPOLA, SFRS10, TCF3, H3F3A,
LOC730740, LYPLA1, UBE3A, SUM02, SHMT2, ACP1, FKBP3, ARL5A,GMNN,
ENY2, FAM82B, RNF138, RPL26L1, CCDC59, PXMP2, POLR3B, TRMT5,
ZNF639, MRPL47, GTPBP8, SUB1, SNHG1, ATPAF1, MRPS24, C16ORF63,
FAM33A, EPSTL1, CTR9, GAS5, ZNF711, MTO1, and CDP2, or wherein the
percentage of cells positive for markers selected from the group
comprising HoxB3, HoxB7, MID1, SNAPC5, PPARG, ANXA2, TIPIN, MYLIP,
LAX1, EGR1, CRIP1, SULT1A3, STMN1, CCT8, SFRS10, CBX3, CBX1,
FLJ11021, DDX46, ACADM, KIAA0101, TYMS, BCAS2, CEP57, TDG, MAP2K6,
CSRP2, GLMN, HMGN2, HNRPR, EIF3S1, PAPOLA, SFRS10, TCF3, H3F3A,
LOC730740, LYPLA1, UBE3A, SUM02, SHMT2, ACP1, FKBP3, ARL5A,GMNN,
ENY2, FAM82B, RNF138, RPL26L1, CCDC59, PXMP2, POLR3B, TRMT5,
ZNF639, MRPL47, GTPBP8, SUB1, SNHG1, ATPAF1, MRPS24, C16ORF63,
FAM33A, EPSTL1, CTR9, GAS5, ZNF711, MTO1, and CDP2 are 1.5, 2, 3,
4, 5, 6, 7, 8, 9, or 10 times less than background.
[0206] In an embodiment of the instant invention, a preparation of
the subject mesenchymal stromal cells (e.g., generated by culturing
hemangioblasts) is provided wherein said preparation comprises a
substantially similar percentage of cells having background levels
of aging markers selected from the group comprising HoxB3, HoxB7,
MID1, SNAPC5, PPARG, ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1,
SULT1A3, STMN1, CCT8, SFRS10, CBX3, CBX1, FLJ11021, DDX46, ACADM,
KIAA0101, TYMS, BCAS2, CEP57, TDG, MAP2K6, CSRP2, GLMN, HMGN2,
HNRPR, EIF3S1, PAPOLA, SFRS10, TCF3, H3F3A, LOC730740, LYPLA1,
UBE3A, SUM02, SHMT2, ACP1, FKBP3, ARL5A,GMNN, ENY2, FAM82B, RNF138,
RPL26L1, CCDC59, PXMP2, POLR3B, TRMT5, ZNF639, MRPL47, GTPBP8,
SUB1, SNHG1, ATPAF1, MRPS24, C16ORF63, FAM33A, EPSTL1, CTR9, GAS5,
ZNF711, MTO1, and CDP2, or from the group comprising HoxB3, HoxB7,
MID1, SNAPC5, PPARG, ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1 and
SULT1A3 or wherein the percentage of cells positive for aging
markers selected from the group comprising HoxB3, HoxB7, MID1,
SNAPC5, PPARG, ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1, SULT1A3,
STMN1, CCT8, SFRS10, CBX3, CBX1, FLJ11021, DDX46, ACADM, KIAA0101,
TYMS, BCAS2, CEP57, TDG, MAP2K6, CSRP2, GLMN, HMGN2, HNRPR, EIF3S1,
PAPOLA, SFRS10, TCF3, H3F3A, LOC730740, LYPLA1, UBE3A, SUM02,
SHMT2, ACP1, FKBP3, ARL5A,GMNN, ENY2, FAM82B, RNF138, RPL26L1,
CCDC59, PXMP2, POLR3B, TRMT5, ZNF639, MRPL47, GTPBP8, SUB1, SNHG1,
ATPAF1, MRPS24, C16ORF63, FAM33A, EPSTL1, CTR9, GAS5, ZNF711, MTO1,
and CDP2 or the group comprising HoxB3, HoxB7, MID1, SNAPC5, PPARG,
ANXA2, TIPIN, MYLIP, LAX1, EGR1, CRIP1, SULT1A3 are 1.5, 2, 3, 4,
5, 6, 7, 8, 9, or 10 times less than background.
[0207] In another embodiment, the hemangioblast-derived MSCs
possess phenotypes of younger cells as compared to adult-derived
MSCs. In one embodiment, the subject MSCs are capable of undergoing
at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or
more population doublings in culture. In contrast, adult-derived
mesenchymal stromal cells typically undergo 2-3 doublings in
culture. In another embodiment, the hemangioblast-derived MSCs have
longer telomere lengths, greater immunosuppressive effects, fewer
vacuoles, divide faster, divide more readily in culture, higher
CD90 expression, are less lineage committed, or combinations
thereof, compared to adult-derived MSCs. In another embodiment, the
hemangioblast-derived MSC have increased expression of transcripts
promoting cell proliferation (i.e., have a higher proliferative
capacity) and reduced expression of transcripts involved in
terminal cell differentiation compared to adult-derived MSCs.
[0208] In an embodiment of the instant invention, a preparation of
mesenchymal stromal cells is generated by any one or more of the
processes of the instant invention, wherein said mesenchymal
stromal cells are capable of undergoing at least or about 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more population
doublings in culture.
[0209] In another embodiment of the instant invention, a
preparation of the subject mesenchymal stromal cells (e.g.,
generated by culturing hemangioblasts) are capable of undergoing at
least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or
more population doublings in culture. In another embodiment of the
instant invention, a preparation of the subject mesenchymal stromal
cells are capable of undergoing at least or about 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, or more population doublings in
culture, wherein after said population doublings less than 50%,
40%, 30%, 20%, 15%, 10%, 5%, or 1% of mesenchymal stromal cells
have undergone replicative senescence. In a further embodiment,
said preparation is a pharmaceutical preparation.
[0210] In another embodiment of the instant invention, a
preparation of mesenchymal stromal cells is provided, wherein said
mesenchymal stromal cells have undergone at least or about 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 doublings in culture,
[0211] In another embodiment of the instant invention, a
preparation of mesenchymal stromal cells is provided, wherein said
mesenchymal stromal cells have undergone at least or about 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 doublings in culture,
wherein less than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% of said
mesenchymal stromal cells have undergone replicative senescence,
wherein said mesenchymal stromal cells retain a youthful phenotype
and potency, and wherein said preparation is a pharmaceutical
preparation. Said preparation may comprise an effective number of
mesenchymal stromal cells for the treatment of disease, such as an
immunological disorder, degenerative disease, or other disease
amenable to treatment using MSCs.
[0212] In another embodiment of the instant invention, a
preparation of the subject mesenchymal stromal cells (e.g.,
generated by culturing hemangioblasts) is provided, wherein said
mesenchymal stromal cells have undergone at least or about 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 doublings in culture,
wherein less than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% of said
mesenchymal stromal cells have undergone replicative senescence
after such doublings, wherein said mesenchymal stromal cells retain
a youthful phenotype and potency, and wherein said preparation is a
pharmaceutical preparation. Said preparation may comprise an
effective number of mesenchymal stromal cells for the treatment of
disease, such as an immunological disorder, degenerative disease,
or other disease amenable to treatment using MSCs.
[0213] In another embodiment of the instant invention, a
preparation of the subject mesenchymal stromal cells (e.g.,
generated by culturing hemangioblasts) is provided, wherein said
mesenchymal stromal cells have undergone about 5, 10, 15, 20, 25,
30, 35, 40, 45, 50, 55, or 60 doublings in culture. The previous
embodiment wherein less than 50%, 40%, 30%, 20%, 15%, 10%, 5% 1% of
said mesenchymal stromal cells have undergone replicative
senescence, wherein said mesenchymal stromal cells retain a
youthful phenotype and potency, wherein said preparation is a
pharmaceutical preparation, wherein said pharmaceutical preparation
comprises an effective number of mesenchymal stromal cells, and
wherein said pharmaceutical preparation is preserved.
[0214] In another embodiment, the instant invention provides a kit
comprising a pharmaceutical preparation of mesenchymal stromal
cells. In another embodiment, the instant invention provides a kit
comprising a pharmaceutical preparation of mesenchymal stromal
cells, wherein said preparation is preserved. In another
embodiment, the instant invention provides a kit comprising a
pharmaceutical preparation of the subject mesenchymal stromal cells
(e.g., generated by culturing hemangioblasts). In another
embodiment, the instant invention provides a kit comprising a
pharmaceutical preparation of the subject mesenchymal stromal cells
(e.g., generated by culturing hemangioblasts), wherein said
preparation is preserved.
[0215] In another embodiment, the instant invention provides for a
method of treating a pathology by administering an effective amount
of mesenchymal stromal cells derived from hemangioblasts to a
subject in need thereof. Said pathology may include, but is not
limited to an autoimmune disorder, uveitis, bone loss or cartilage
damage.
[0216] The mesenchymal stromal cells obtained by culturing
hemangioblasts have improved characteristics as compared to MSCs
derived directly from ESCs. For example, ESC-derived MSCs clump
more, are more difficult to disperse when splitting, do not
generate nearly as many MSCs when starting with equivalent numbers
of ESCs, and take longer to acquire characteristics MSC cell
surface markers compared to hemangioblast-derived MSCs. See Example
2 and FIGS. 3-6.
[0217] In one embodiment, the instant invention provides a
preparation of the subject mesenchymal stromal cells (e.g.,
generated by culturing hemangioblasts), wherein said preparation is
effective at normalizing a pathology. In a further embodiment of
the instant invention a preparation of the subject mesenchymal
stromal cells (e.g., generated by culturing hemangioblasts) is
provided, wherein said preparation is effective at reducing
excessive or unwanted immune responses. In a further embodiment of
the instant invention, a preparation of the subject mesenchymal
stromal cells (e.g., generated by culturing hemangioblasts) is
provided, wherein said preparation is effective at ameliorating an
autoimmune disorder. In a further embodiment of the instant
invention, normalization of a pathology by administering to a host
an effective amount of the subject mesenchymal stromal cells (e.g.,
generated by culturing hemangioblasts) is provided. A further
embodiment of the instant invention provides for normalization of a
pathology, wherein such normalization of a pathology is
characterized by effects selected from the group comprising
cytokine release by said MSCs, stimulating an increase in the
number of regulatory T cells, inhibiting a certain amount of IFN
gamma release from Th1 cells, and stimulating a certain amount of
IL4 secretion from Th2 cells. In a further embodiment,
administration of a preparation of the subject mesenchymal stromal
cells (e.g., generated by culturing hemangioblasts) results in the
release from said mesenchymal stromal cells of cytokines selected
from the group comprising transforming growth factor beta,
indoleamine 2, 3dioxygenase, prostaglandin E2, hepatocyte growth
factor, nitric oxide, interleukin 10, interleukin 6,
macrophage-colony stimulating factor, and soluble human leukocyte
antigen (HLA) G5.
[0218] In a further embodiment of the instant invention,
administration of a preparation of the subject mesenchymal stromal
cells (e.g., generated by culturing hemangioblasts) results in the
release from said mesenchymal stromal cells of cytokines selected
from the group comprising transforming growth factor beta,
indoleamine 2, 3dioxygenase, prostaglandin E2, hepatocyte growth
factor, nitric oxide, interleukin 10, interleukin 6,
macrophage-colony stimulating factor, soluble human leukocyte
antigen (HLA) G5, interleukin 4, 8, 11, granulocyte macrophage
colony stimulating factor, vascular endothelium growth factor,
insulin-like growth factor 1, Phosphatidylinositol-glycan
biosynthesis class F protein, monocyte chemoattractant protein 1,
stromal derived factor 1, tumor necrosis factor 1, transforming
growth factor beta, basic fibroblast growth factor, angiopoietin 1
and 2, monokine induced by interferon gamma, interferon inducible
protein 10, brain derived neurotrophic factor, interleukin 1
receptor alpha, chemokine ligand 1 and 2.
[0219] Pharmaceutical Preparations of MSCs
[0220] MSCs of the instant invention may be formulated with a
pharmaceutically acceptable carrier. For example, MSCs of the
invention may be administered alone or as a component of a
pharmaceutical formulation, wherein said MSCs may be formulated for
administration in any convenient way for use in medicine. One
embodiment provides a pharmaceutical preparation of mesenchymal
stromal cells comprising said mesenchymal stromal cells in
combination with one or more pharmaceutically acceptable sterile
isotonic aqueous or non-aqueous solutions selected from the group
consisting of: dispersions, suspensions, emulsions, sterile powders
optionally reconstituted into sterile injectable solutions or
dispersions just prior to use, antioxidants, buffers,
bacteriostats, solutes or suspending and thickening agents.
[0221] In an embodiment of the instant invention, a pharmaceutical
preparation of mesenchymal stromal cells is provided, wherein said
mesenchymal stromal cells have undergone between about 5 and about
100 population doublings. In a further embodiment of the instant
invention, a pharmaceutical preparation of mesenchymal stromal
cells is provided, wherein said mesenchymal stromal cells have
undergone between about 10 and about 80 population doublings. In a
further embodiment of the instant invention, a pharmaceutical
preparation of mesenchymal stromal cells is provided, wherein said
mesenchymal stromal cells have undergone between about 25 and about
60 population doublings. In a further embodiment of the instant
invention, a pharmaceutical preparation of mesenchymal stromal
cells is provided, wherein said mesenchymal stromal cells have
undergone less than about 10 population doublings. In a still
further embodiment of the instant invention, a pharmaceutical
preparation of mesenchymal stromal cells is provided, wherein said
mesenchymal stromal cells have undergone less than about 20
population doublings. In a further embodiment of the instant
invention, a pharmaceutical preparation of mesenchymal stromal
cells is provided, wherein said mesenchymal stromal cells have
undergone less than about 30 population doublings, wherein said
mesenchymal stromal cells have not undergone replicative
senescence. In a further embodiment of the instant invention, a
pharmaceutical preparation of mesenchymal stromal cells is
provided, wherein said mesenchymal stromal cells have undergone
less than about 30 population doublings, wherein less than about
25% of said mesenchymal stromal cells have undergone replicative
senescence. In a further embodiment of the instant invention, a
pharmaceutical preparation of mesenchymal stromal cells is
provided, wherein said mesenchymal stromal cells have undergone
less than about 30 population doublings, wherein less than about
10% of said mesenchymal stromal cells have undergone replicative
senescence. In a further embodiment of the instant invention, a
pharmaceutical preparation of mesenchymal stromal cells is
provided, wherein said mesenchymal stromal cells have undergone
less than about 30 population doublings, wherein less than about
10% of said mesenchymal stromal cells have undergone replicative
senescence, and wherein said mesenchymal stromal cells express the
markers selected from the group comprising AIRE-1, IL-11, CD10,
CD24, ANG-1, and CXCL1.
[0222] Concentrations for injections of pharmaceutical preparations
of MSCs may be at any amount that is effective and, for example,
substantially free of ESCs. For example, the pharmaceutical
preparations may comprise the numbers and types of MSCs described
herein. In a particular embodiment, the pharmaceutical preparations
of MSCs comprise about 1.times.10.sup.6 of the subject MSCs (e.g.,
generated by culturing hemangioblasts) for systemic administration
to a host in need thereof or about 1.times.10.sup.4 of said MSCs by
culturing hemangioblasts for local administration to a host in need
thereof.
[0223] Exemplary compositions of the present disclosure may be
formulation suitable for use in treating a human patient, such as
pyrogen-free or essentially pyrogen-free, and pathogen-free. When
administered, the pharmaceutical preparations for use in this
disclosure may be in a pyrogen-free, pathogen-free, physiologically
acceptable form.
[0224] The preparation comprising MSCs used in the methods
described herein may be transplanted in a suspension, gel, colloid,
slurry, or mixture. Also, at the time of injection, cryopreserved
MSCs may be resuspended with commercially available balanced salt
solution to achieve the desired osmolality and concentration for
administration by injection (i.e., bolus or intravenous).
[0225] One aspect of the invention relates to a pharmaceutical
preparation suitable for use in a mammalian patient, comprising at
least 10.sup.6, 10.sup.7, 10.sup.8 or even 10.sup.9 mesenchymal
stromal cells and a pharmaceutically acceptable carrier. Another
aspect of the invention relates to a pharmaceutical preparation
comprising at least 10.sup.6, 10.sup.7, 10.sup.8 or even
10.sup.9mesenchymal stromal cells and a pharmaceutically acceptable
carrier, wherein the mesenchymal stromal cells a differentiated
from a hemangioblast cell. Yet another aspect of the invention
provides a cryogenic cell bank comprising at least 10.sup.8,
10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12 or even 10.sup.13
mesenchymal stromal cells. Still another aspect of the invention
provides a purified cellular preparation free of substantially free
of non-human cells and/or non-human animal products, comprising at
least 10.sup.6, 10.sup.7, 10.sup.8 or even 10.sup.9 mesenchymal
stromal cells and less than 1% of any other cell type, more
preferably less than 0.1%, 0.01% or even 0.001% of any other cell
type. Certain preferred embodiments of the above preparations,
compositions and bank include, but are not limited to those listed
in the following paragraphs:
[0226] In certain embodiments, the mesenchymal stromal cells have
replicative capacity to undergo at least 10 population doublings in
cell culture with less than 25, 20, 15, 10 or even 5 percent of the
cells undergoing cell death, senescing or differentiating into
non-MSC cells (such as fibroblasts, adipocytes and/or osteocytes)
by the 10.sup.th doubling.
[0227] In certain embodiments, the mesenchymal stromal cells have
replicative capacity to undergo at least 15 population doublings in
cell culture with less than 25, 20, 15, 10 or even 5 percent of the
cells undergoing cell death, senescing or differentiating into
non-MSC cells (such as fibroblasts, adipocytes and/or osteocytes)
by the 15.sup.th doubling.
[0228] In certain embodiments, the mesenchymal stromal cells have
replicative capacity to undergo at least 20 population doublings in
cell culture with less than 25, 20, 15, 10 or even 5 percent of the
cells undergoing cell death, senescing or differentiating into
non-MSC cells (such as fibroblasts, adipocytes and/or osteocytes)
by the 20.sup.th doubling.
[0229] In certain embodiments, the mesenchymal stromal cells have
replicative capacity to undergo at least 5 passages in cell culture
with less than 25, 20, 15, 10 or even 5 percent of the cells
undergoing cell death, senescing or differentiating into non-MSC
cells (such as fibroblasts, adipocytes and/or osteocytes) by the
5.sup.th passage.
[0230] In certain embodiments, the mesenchymal stromal cells have
replicative capacity to undergo at least 10 passages in cell
culture with less than 25, 20, 15, 10 or even 5 percent of the
cells undergoing cell death, senescing or differentiating into
non-MSC cells (such as fibroblasts, adipocytes and/or osteocytes)
by the 10.sup.th passage.
[0231] In certain embodiments, the mesenchymal stromal cells are
differentiated from a pluripotent stem cell source, such as a
pluripotent stem cell that expresses OCT-4, alkaline phosphatase,
Sox2, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-80 (such as, and
embryonic stem cell line or induced pluripotency stem cell line),
and even more preferably from a common pluripotent stem cell
source.
[0232] In certain embodiments, the mesenchymal stromal cells are
HLA-genotypically identical.
[0233] In certain embodiments, the mesenchymal stromal cells are
genomically identical.
[0234] In certain embodiments, at least 30%, 35%, 40%, 45% or even
50% of the mesenchymal stromal cells are positive for CD10.
[0235] In certain embodiments, at least 60%, 65%, 70%, 75%, 80%,
85% or even 90% of the mesenchymal stromal cells are positive for
markers CD73, CD90, CD105, CD13, CD29, CD44, CD166 and CD274 and
HLA-ABC.
[0236] In certain embodiments, less than 30%, 25%, 20%, 15% or even
10% of the mesenchymal stromal cells are positive for markers CD31,
CD34, CD45, CD133, FGFR2, CD271, Stro-1, CXCR4 and TLR3.
[0237] In certain embodiments, the mesenchymal stromal cells have
replicative rates to undergo at least 10 population doublings in
cell culture in less than 25, 24, 23, 22, 21 or even 20 days.
[0238] In certain embodiments, the mesenchymal stromal cells have a
mean terminal restriction fragment length (TRF) that is longer than
7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 10.5 kb, 11 kb,
11.5 kb or even 12 kb.
[0239] In certain embodiments, the mesenchymal stromal cells do not
undergo more than a 75%, 70%, 65%, 60%, 55%, 50%, or even 45%
percent increase in cells having a forward-scattered light value,
measured by flow cytometry, greater than 5,000,000 over 10, 15 or
even 20 population doublings in culture.
[0240] In certain embodiments, the mesenchymal stromal cells, in a
resting state, express mRNA encoding Interleukin-6 at a level which
is less than 10%, 8%, 6%, 4% or even 2% of the IL-6 mRNA level
expressed by mesenchymal stromal cells preparations, in a resting
state, derived from chord blood, bone marrow or adipost tissue.
[0241] In certain embodiments, the mesenchymal stromal cells are at
least 2, 4, 6, 8, 10, 20, 50 or even 100 times more potent than
MSCs derived from chord blood, bone marrow or adipost tissue.
[0242] In certain embodiments, one million of the mesenchymal
stromal cells, when injected into an MOG35-55 EAE mouse model (such
as C57BL/6 mice immunized with the MOG35-55 peptide) will, on
average, reduce a clinical score of 3.5 to less than 2.5, and even
more preferably will reduce the clinical score to less 2, 1.5 or
even less than 1.
[0243] In certain embodiments, the preparation is suitable for
administration to a human patient, and more preferably pyrogen free
and/or free of non-human animal products.
[0244] In other embodiments, the preparation is suitable for
administration to a non-human veterinarian mammal, such as a dog,
cat or horse.
[0245] Diseases and Conditions Treatable using MSCs Derived from
Culturing Hemangioblasts
[0246] MSCs have been shown to be therapeutic for a variety of
diseases and conditions. In particular, MSCs migrate to injury
sites, exert immunosuppressive effects, and facilitate repair of
damaged tissues. An embodiment of the instant invention is
provided, wherein a pharmaceutical preparation of mesenchymal
stromal cells reduces the manifestations of a pathology. An
embodiment of the instant invention is provided, wherein a
pharmaceutical preparation of mesenchymal stromal cells are
administered to a host suffering from a pathology. In a further
embodiment of the instant invention, a pharmaceutical preparation
of the subject MSCs (e.g., generated by culturing hemangioblasts)
reduces the manifestations of a pathology selected from the group
comprising wound healing, graft-versus-host disease (GvHD),
disease, chronic eye disease, retinal degeneration, glaucoma,
uveitis, acute myocardial infarction, chronic pain, hepatitis, and
nephritis. In a further embodiment of the instant invention, a
pharmaceutical preparation of mesenchymal stromal cells by
culturing hemangioblasts reduces the manifestations of equine
laminitis. As a further example, MSCs may be administered in
combination with an allogeneic transplanted cell or tissue (e.g., a
preparation comprising cells that have been differentiated from ES
cells, such as retinal pigment epithelium (RPE) cells,
oligodendrocyte precursors, retinal, corneal, muscle such as
skeletal, smooth, or cardiac muscle or any combination thereof, or
others) thereby decreasing the likelihood of an immune reaction
against the transplanted cell or tissue and potentially avoiding
the need for other immune suppression. The the subject MSCs (e.g.,
generated by culturing hemangioblasts) described herein may be used
in similar applications. An embodiment of a process of the instant
invention, wherein the administration of a pharmaceutical
preparation of the subject MSCs (e.g., generated by culturing
hemangioblasts) to a host reduces the need for future therapy. An
embodiment of a process of the instant invention is provided,
wherein the administration of a pharmaceutical preparation of the
subject MSCs (e.g., generated by culturing hemangioblasts) to a
host reduces the need for future therapy, wherein said therapy
suppresses immune function.
[0247] In an embodiment of the instant invention, a pharmaceutical
preparation of the subject MSCs (e.g., generated by culturing
hemangioblasts) is administered to a host for the treatment of a
pathology. In an embodiment of the instant invention, a
pharmaceutical preparation of the subject MSCs (e.g., generated by
culturing hemangioblasts) is administered to a host for the
treatment of pathologies selected from the list comprising wound
healing, multiple sclerosis, systemic sclerosis, hematological
malignancies, myocardial infarction, tissue and organ
transplantation, tissue and organ rejection, chronic allograft
nephropathy, cirrhosis, liver failure, heart failure, GvHD, tibial
fracture, left ventricular dysfunction, leukemia, myelodysplastic
syndrome, Crohn's disease, Type I or Type II diabetes mellitus,
chronic obstructive pulmonary disease, pulmonary hypertension,
chronic pain, osteogenesis imperfecta, homozygous familial
hypocholesterolemia, treatment following meniscectomy, adult
periodontitis, vasculogenesis in patients with severe myocardial
ischemia, spinal cord injury, osteodysplasia, critical limb
ischemia associated with diabetes mellitus, diabetic foot disease,
primary Sjogren's syndrome, osteoarthritis, cartilage defects
(e.g., articular cartilage defects), laminitis, multisystem
atrophy, amyotropic lateral sclerosis, cardiac surgery, refractory
systemic lupus erythematosis, living kidney allografts,
nonmalignant red blood cell disorders, thermal burn, radiation
burn, Parkinson's disease, microfractures (e.g., in patients with
knee articular cartilage injury of defects), epidermolysis bullosa,
severe coronary ischemia, idiopathic dilated cardiomyopathy,
osteonecrosis femoral head, lupus nephritis, bone void defects,
ischemic cerebral stroke, after stroke, acute radiation syndrome,
pulmonary disease, arthritis, and bone regeneration.
[0248] In a further embodiment of the instant invention, a
pharmaceutical preparation of the subject MSCs (e.g., generated by
culturing hemangioblasts) is administered to a host for the
treatment of autoimmune pathologies selected from the list
comprising Acute necrotizing hemorrhagic leukoencephalitis,
Addison's disease, Agammaglobulinemia, Alopecia areata,
Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis,
Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune
aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis,
Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune
inner ear disease (AIED), Autoimmune myocarditis, Autoimmune
pancreatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic
purpura (ATP), Autoimmune thyroid disease, Autoimmune urticarial,
Axonal & neuronal neuropathies, Balo disease, Behcet's disease,
Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac
disease, Chagas disease, Chronic fatigue syndrome, Chronic
inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent
multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial
pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans
syndrome, Cold agglutinin disease, Congenital heart block,
Coxsackie myocarditis, CREST disease, Essential mixed
cryoglobulinemia, Demyelinating neuropathies, Dermatitis
herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis
optica), Discoid lupus, Dressler's syndrome, Endometriosis,
Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum,
Experimental allergic encephalomyelitis, Evans syndrome,
Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal
arteritis), Glomerulonephritis, Goodpasture's syndrome,
Granulomatosis with Polyangiitis (GPA) see Wegener's, Graves'
disease, Guillain-Barre syndrome, Hashimoto's encephalitis,
Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein
purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic
thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related
sclerosing disease, Immunoregulatory lipoproteins, Inclusion body
myositis, Insulin-dependent diabetes (type1), Interstitial
cystitis, Juvenile arthritis, Juvenile diabetes, Kawasaki syndrome,
Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus,
Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease
(LAD), Lupus (SLE), Lyme disease, chronic, Meniere's disease,
Microscopic polyangiitis, Mixed connective tissue disease (MCTD),
Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis,
Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica
(Devic's), Neutropenia, Ocular cicatricial pemphigoid, Optic
neuritis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune
Neuropsychiatric Disorders Associated with Streptococcus),
Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal
hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner
syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral
neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS
syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune
polyglandular syndromes, Polymyalgia rheumatic, Polymyositis,
Postmyocardial infarction syndrome, Postpericardiotomy syndrome,
Progesterone dermatitis, Primary biliary cirrhosis, Primary
sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic
pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia,
Raynauds phenomenon, Reflex sympathetic dystrophy, Reiter's
syndrome, Relapsing polychondritis, Restless legs syndrome,
Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis,
Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren's
syndrome, Sperm & testicular autoimmunity, Stiff person
syndrome, Subacute bacterial endocarditis (SBE), Susac's syndrome,
Sympathetic ophthalmia, Takayasu's arteritis, Temporal
arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP),
Tolosa-Hunt syndrome, Transverse myelitis, Ulcerative colitis,
Undifferentiated connective tissue disease (UCTD), Uveitis,
Vasculitis, Vesiculobullous dermatosis, Vitiligo, and Wegener's
granulomatosis (now termed Granulomatosis with Polyangiitis
(GPA).
[0249] Treatment Regimens Using MSCs Derived from Culturing
Hemangioblasts
[0250] The MSCs and pharmaceutical preparations comprising MSCs
described herein may be used for cell-based treatments. In
particular, the instant invention provides methods for treating or
preventing the diseases and conditions described herein comprising
administering an effective amount of a pharmaceutical preparation
comprising MSCs, wherein the MSCs are derived from culturing
hemangioblasts.
[0251] The MSCs of the instant invention may be administered using
modalities known in the art including, but not limited to,
injection via intravenous, intramyocardial, transendocardial,
intravitreal, or intramuscular routes or local
implantationdependent on the particular pathology being
treated.
[0252] The mesenchymal stromal cells of the instant invention may
be administered via local implantation, wherein a delivery device
is utilized. Delivery devices of the instant invention are
biocompatible and biodegradable. A delivery device of the instant
invention can be manufactured using materials selected from the
group comprising biocompatible fibers, biocompatible yarns,
biocompatible foams, aliphatic polyesters, poly(amino acids),
copoly(ether-esters), polyalkylenes oxalates, polyamides, tyrosine
derived polycarbonates, poly(iminocarbonates), polyorthoesters,
polyoxaesters, polyamidoesters, polyoxaesters containing amine
groups, poly(anhydrides), polyphosphazenes, biopolymers;
homopolymers and copolymers of lactide, glycolide,
epsilon-caprolactone, para-dioxanone, trimethylene carbonate;
homopolymers and copolymers of lactide, glycolide,
epsilon-caprolactone, para-dioxanone, trimethylene carbonate,
fibrillar collagen, non-fibrillar collagen, collagens not treated
with pepsin, collagens combined with other polymers, growth
factors, extracellular matrix proteins, biologically relevant
peptide fragments, hepatocyte growth factor, platelet-derived
growth factors, platelet rich plasma, insulin growth factor, growth
differentiation factor, vascular endothelial cell-derived growth
factor, nicotinamide, glucagon like peptides, tenascin-C, laminin,
anti-rejection agents, analgesics, anti-oxidants, anti-apoptotic
agents anti-inflammatory agents and cytostatic agents.
[0253] The particular treatment regimen, route of administration,
and adjuvant therapy may be tailored based on the particular
pathology, the severity of the pathology, and the patient's overall
health. Administration of the pharmaceutical preparations
comprising MSCs may be effective to reduce the severity of the
manifestations of a pathology or and/or to prevent further
degeneration of themanifestation of a pathology.
[0254] A treatment modality of the present invention may comprise
the administration of a single dose of MSCs. Alternatively,
treatment modalities described herein may comprise a course of
therapy where MSCs are administered multiple times over some period
of time. Exemplary courses of treatment may comprise weekly,
biweekly, monthly, quarterly, biannually, or yearly treatments.
Alternatively, treatment may proceed in phases whereby multiple
doses are required initially (e.g., daily doses for the first
week), and subsequently fewer and less frequent doses are
needed.
[0255] In one embodiment, the pharmaceutical preparation of
mesenchymal stromal cells obtained by culturing hemangioblasts is
administered to a patient one or more times periodically throughout
the life of a patient. In a further embodiment of the instant
invention, a pharmaceutical preparation of the subject MSCs (e.g.,
generated by culturing hemangioblasts) is administered once per
year, once every 6-12 months, once every 3-6 months, once every 1-3
months, or once every 1-4 weeks. Alternatively, more frequent
administration may be desirable for certain conditions or
disorders. In an embodiment of the instant invention, a
pharmaceutical preparation of the subject MSCs (e.g., generated by
culturing hemangioblasts) is administered via a device once, more
than once, periodically throughout the lifetime of the patient, or
as necessary for the particular patient and patient's pathology
being treated. Similarly contemplated is a therapeutic regimen that
changes over time. For example, more frequent treatment may be
needed at the outset (e.g., daily or weekly treatment). Over time,
as the patient's condition improves, less frequent treatment or
even no further treatment may be needed.
[0256] In accordance with the present invention, the diseases or
conditions can be treated or prevented by intravenous
administration of the mesenchymal stem cells described herein. In
some embodiments, about 20 million, about 40 million, about 60
million, about 80 million, about 100 million, about 120 million,
about 140 million, about 160 million, about 180 million, about 200
million, about 220 million, about 240 million, about 260 million,
about 280 million, about 300 million, about 320 million, about 340
million, about 360 million, about 380 million, about 400 million,
about 420 million, about 440 million, about 460 million, about 480
million, about 500 million, about 520 million, about 540 million,
about 560 million, about 580 million, about 600 million, about 620
million, about 640 million, about 660 million, about 680 million,
about 700 million, about 720 million, about 740 million, about 760
million, about 780 million, about 800 million, about 820 million,
about 840 million, about 860 million, about 880 million, about 900
million, about 920 million, about 940 million, about 960 million,
or about 980 million cells are injected intravenously. In some
embodiments, about 1 billion, about 2 billion, about 3 billion,
about 4 billion or about 5 billion cells or more are injected
intravenously. In some embodiments, the number of cells ranges from
between about 20 million to about 4 billion cells, between about 40
million to about 1 billion cells, between about 60 million to about
750 million cells, between about 80 million to about 400 million
cells, between about 100 million to about 350 million cells, and
between about 175 million to about 250 million cells.
[0257] The methods described herein may further comprise the step
of monitoring the efficacy of treatment or prevention using methods
known in the art.
[0258] Kits
[0259] The present invention provides for kits comprising any of
the compositions described herein. A preparation of mesenchymal
stromal cells may be contained in a delivery device manufactured
according to methods known by one of ordinary skill in the art, and
include methods in US Patent Application Publication 2002/0103542,
European Patent Application EP 1 454 641, or preserved according to
methods known by one of ordinary skill in the art, and include
methods in U.S. Pat. No. 8,198,085, PCT Application WO2004/098285,
and US Patent Application Publication 2012/0077181. In an
embodiment of the instant invention, a kit comprising a preparation
of about at least 8.times.10.sup.7, 8.5.times.10.sup.7,
9.times.10.sup.7, 9.5.times.10.sup.7, 1.times.10.sup.8,
1.25.times.10.sup.8, or 1.25.times.10.sup.8 MSCs derived from
culturing hemangioblasts. In another embodiment, a kit comprising a
preparation of about 8.times.10.sup.7, 8.5.times.10.sup.7,
9.times.10.sup.7, 9.5.times.10.sup.7, 1.times.10.sup.8,
1.25.times.10.sup.8, or 1.25.times.10.sup.8 the subject MSCs (e.g.,
generated by culturing hemangioblasts) is provided, wherein said
preparation is pharmaceutical preparation. In a still further
embodiment of the instant invention, a kit comprising a
pharmaceutical preparation of about 8.times.10.sup.7,
8.5.times.10.sup.7, 9.times.10.sup.7, 9.5.times.10.sup.7,
1.times.10.sup.8, 1.25.times.10.sup.8, or 1.25.times.10.sup.8 the
subject MSCs (e.g., generated by culturing hemangioblasts) is
provided, wherein said pharmaceutical preparation is preserved. In
a still further embodiment of the instant invention, a kit
comprising a pharmaceutical preparation of about 8.times.10.sup.7,
8.5.times.10.sup.7, 9.times.10.sup.7, 9.5.times.10.sup.7,
1.times.10.sup.8, 1.25.times.10.sup.8, or 1.25.times.10.sup.8 the
subject MSCs (e.g., generated by culturing hemangioblasts) is
provided, wherein said pharmaceutical preparation is contained in a
cell delivery vehicle.
[0260] Additionally, the kits may comprise cryopreserved MSCs or
preparations of cryopreserved MSCs, frozen MSCs or preparations of
frozen MSCs, thawed frozen MSCs or preparations of thawed frozen
MSCs.
[0261] Combinations of Various Embodiments and Concepts
[0262] It will be understood that the embodiments and concepts
described herein may be used in combination. For example, the
instant invention provides for a method of generating MSCs
comprising generating hemangioblasts from ESCs, culturing the
hemangioblasts for at least four days, harvesting the
hemangioblasts, re-plating the hemangioblasts on a Matrigel-coated
plate, and culturing the hemangioblasts as described herein for at
least fourteen days, wherein the method generates at least 85
million MSCs that are substantially free of ESCs.
EXAMPLES
[0263] The following examples are not intended to limit the
invention in any way.
Example 1
Generating MSCs from Hemangioblasts
[0264] Hemangioblasts were generated from the clinical grade,
single-blastomere derived ESC line, MA09 [16], as follows:
[0265] First, early-stage clusters of cells were generated from
MA09 ESC cultured in serum-free medium supplemented with a
combination of morphogens and early hematopoietic cytokines,
specifically bone morphogenetic protein-4 (BMP-4), vascular
endothelial growth factor (VEGF), basic fibroblast growth factor
(bFGF), stem cell factor (SCF), thrombopoietin (Tpo) and
fms-related tyrosine kinase 3 ligand (FL). More specifically, ESCs
from one well of a 6-well tissue-culture treated plate were plated
in one well of a six well ultra low adherence place (Corning) in 3
ml Stemline II medium (Sigma) supplemented with 50 ng/ml of VEGF
and 50 ng/ml of BMP-4 (R & D) and incubated at 37.degree. C.
with 5% CO2. Clusters of cells were formed within the first 24 hr.
After 40-48 hours, half of the medium (1.5 ml) was replaced with
fresh Stemline II medium supplemented with 50 ng/ml of VEGF, 50
ng/ml of BMP-4, and 20-22.5 ng/ml bFGF, and incubation continued
for an additional 40-48 hours (i.e., 3.5-4 days total).
[0266] Clusters of cells were dissociated and plated single cells
in serum-free semisolid blast-colony growth medium (BGM).
Specifically, clusters of cells were dissociated by 0.05%
trypsin-0.53 mM EDTA (Invitrogen) for 2-5 min. The cell suspension
was pipeted up and down and then DMEM+10% FCS was added to
inactivate the trypsin. Cells were then passed through a 40 .mu.m
strainer to obtain a single cell suspension. Cells were then
counted and resuspended in Stemline II medium at
1-1.5.times.10.sup.6 cells/ml.
[0267] The single cell suspension (0.3 ml, 3 to 4.5.times.10.sup.5
cells) was mixed with 2.7 ml of hemangioblast growth medium (H4536
based medium recipe as described above) with a brief vortex, and
let stand for 5 min. The cell mixture was then transferred to one
well of a six-well ultra low adherence plate by using a syringe (3
ml) attached with an 18G needle, and incubated at 37.degree. C.
with 5% CO2.
[0268] Some of the cells developed into grape-like blast colonies
(BCs). Specifically, BCs were visible at 3 days (typically
contained less than 10 cells at the beginning of day 3), and after
4-6 days, grape-like hES-BCs were easily identified under
microscopy (containing greater than 100 cells per BC). The number
of BCs present in the culture gradually increased over the course
of several days. After 6-7 days, BCs could be picked up using a
mouth-glass capillary.
[0269] Hemangioblasts can be harvested between day 7-12 of culture
and replated onto Matrigel-coated tissue culture plates in .alpha.
MEM+20% FCS. Flow cytometry analysis shows that expression levels
of 5 cell surface markers typically found on MSCs are relatively
low in the starting hemangioblast population. (FIG. 2, left panel,
average of 4 experiments +/- standard deviation). However, after
three weeks of culture in MSC growth conditions, a homogenous
adherent cell population arises that stains >90% positive for
these 5 characteristic MSC markers (FIG. 2, right panel-22-23 days,
average of 4 experiments +/- standard deviation). Upon MSC culture
conditions, the amount of time it takes for differentiating cells
to acquire MSC surface markers may vary depending on the specific
ESC line used, the day of hemangioblast harvest, and the number of
hemangioblasts plated onto Matrigel. In some experiments, markers
arise in 90% of the cells by 7-14 days, whereas in other
experiments, it may take 22-24 days for this many cells to acquire
these MSC markers.
[0270] Relating to the above experiments, FIG. 1 shows the
generation of FM-MA09-MSC from pluripotent cells, and a microscopic
view of generating mesenchymal stromal cells from ESCs via
hemangioblasts. In addition, FIG. 2 contains a phenotype of
FM-MA09-MSC obtained from pluripotent cell-derived hemangioblasts
produced as above-described. This figure shows the percentage of
cells positive for MSC surface markers in the initial hemangioblast
population (left side of graph, day 7-11 hemangioblast) and after
culturing hemangioblasts on Matrigel coated plates (right side of
graph) and a microscopic view of the mesenchymal stromal cells
derived from the hemangioblasts (right panel photograph). Also,
relating to the above experiments FIG. 17 depicts the process of
FM-MA09-MSC generation; and the effects of Matrigel, i.e., that
removing cells from Matrigel at an early passage (ie, p2) may
temporarily slow MSC growth as compared to those maintained on
Matrigel until p6.
[0271] FIG. 18 further shows that the obtained BM-MSCs and
FM-MA09-MSCs undergo chondrogenesis.
Example 2
Comparison of Differentiation of ESCs and MSC-Derived
Hemangioblasts.
[0272] This example describes comparison of the differentiation of
ESCs into MSCs by two methods: either direct differentiation (in
which ESCs were directly plated on gelatin or Matrigel) or the
hemangioblast method (in which ESCs were first differentiated into
hemangioblasts and then plated on Matrigel, as described in Example
1). Direct differentiation on gelatin gave rise to MSC-like cells,
but the cells lacked CD105 expression, suggesting incomplete
adoption of MSC fate (FIG. 3, left panel). When ESCs were plated
directly on Matrigel, the resulting cells did express CD105 as
expected for MSCs (FIG. 3, middle panel). However, compared to MSCs
produced by the hemangioblast method, the directly differentiated
MSCs cells grew in clumps, were more difficult to disperse when
splitting, and did not generate nearly as many MSCs when starting
from equivalent numbers of ESCs (FIG. 4).
[0273] MSCs differentiated directly from ESCs also took longer to
acquire characteristic MSC cell surface markers (FIG. 5). Once MSCs
were obtained, extended immunophenotyping shows that MSCs from both
methods are positive for other markers typically found on MSCs,
such as HLA-ABC, while negative for hematopoiesis-associated
markers such as CD34 and CD45 (FIG. 6). These results suggest that
use of a hemangioblast-intermediate stage permits robust production
of homogeneous MSCs from ESCs. Given these findings, additional
studies on MSCs will be conducted with hemangioblast-derived
MSCs.
[0274] In addition, experiments the results of which are contained
in FIGS. 3-6, 13, 15, 16, 19, and 21-27 (described supra) compare
different properties of ESC-MSCs or BM-MSCs versus
hemangioblast-derived MSC's and reveal that these cells exhibit
significant differences which may impact therapeutic efficacy of
these cells and compositions derived therefrom. Particularly, FIG.
3 shows the percentage of cells positive for MSC surface markers
after culturing human embryonic stem cells (ESC) on gelatin coated
plates (left panel), ESC on Matrigel coated plates (middle panel),
and hemangioblasts on Matrigel coated plates (right panel).
Additionally, FIG. 4 shows the MSC yield from pluripotent cells,
FIG. 5 illustrates the acquisition of mesenchymal stromal cell
markers, and FIG. 6 shows phenotypes of mesenchymal stromal cells
derived from different culture methods, including expression of MSC
markers and lack of expression of hematopoiesis and endothelial
markers. Further, FM-MA09-MSCs were assayed to detect notable
differences (relative to BM-MSCs) in potency and inhibitory effects
(FIG. 13), stimulation of Treg expansion (FIG. 15), proliferative
capacity (FIG. 16), PGE2 secretion (FIG. 19), Stro-1 and CD10
expression (FIGS. 21-22), maintenance of size during passaging
(FIG. 23), CD10 and CD24 expression (FIG. 24), Aire-1 and IL-11
expression (FIG. 25), Ang-1 and CXCL1 expression (FIG. 26), and and
IL6 and VEGF expression (FIG. 27).
Example 3
MSCs Derived from Hemangioblasts Differentiate into Other Cell
Types
[0275] MSCs, by definition, should be able to give rise to
adipocytes, osteocytes, and chondrocytes. Using standard methods,
FIG. 7 shows the ability of hemangioblast-derived MSCs to
differentiate into adipocytes and osteocytes, while FIG. 8 shows
their potential to differentiate towards chondrocytes via the
expression of chondrocyte-specific genes and FIG. 18 shows their
potential to differentiate towards chondrocytes via safranin O
staining of pellet mass cultures.
[0276] MSCs derived from hemangioblasts are expected to
differentiate into adipocytes, osteocytes, and chondrocytes. These
differentiation pathways may be examined using methods previously
reported in theart. See Karlsson et al, Stem Cell Research 3: 39-50
(2009) (for differentiation of the hemangioblast-derived and direct
ESC-derived MSCs into adipocytes and osteocytes). Particularly,
FM-MA09-MSC display differentiation capabilities including the
ability to differentiate into adipocytes and osteocytes (FIG. 7).
For chondrocyte differentiation, methods have been adapted from
Gong et al, J. Cell. Physiol. 224: 664-671 (2010) to study this
process and continue to examine the acquisition of chondrocyte
specific genes, (e.g., Aggrecan and Collagen IIa) as well as
glycosaminoglycan deposition through safranin O, alcian blue,
and/or toluene blue staining. Particularly, chondrogenic
differentiation of MA09 ESC hemangioblast-derived mesenchymal
stromal cells was detected by mRNA expression of Aggrecan
(chondroitin proteoglycan sulfate 1) and Collagen IIa (FIG. 8). It
has been reported in the literature that none of these three cell
types, adipocytes, osteocytes, or chondrocytes derived from MSCs
will express the immunostimulatory HLA DR molecule (Le Blanc 2003,
Gotherstrom 2004, Liu 2006). Immunostaining and/or flow cytometry
will be performed on these fully differentiated MSC cell types to
confirm these reported observations. This is important to confirm
so that differentiation of MSCs in an in vivo environment will not
induce an immune response from the host recipient. Of these three
cell types, chondrogenic differentiation may be of particular
interest due to its potential to be used in cartilage replacement
therapies for sports injuries, aging joint pain, osteoarthritis,
etc. For such therapies, MSCs may not need to be fully
differentiated into chondrocytes in order to be used
therapeutically.
Example 4
Confirmation that MSCs Derived from Hemangioblasts are
Substantially Free of ESCs
[0277] MSCs should also be devoid of the ESC propensity to form
teratomas. MSCs were confirmed to contain normal karyotypes (data
not shown) by passage 12 (.about.50 days in culture). To confirm
that the blast-derived MSCs do not contain trace amounts of ESCs,
teratoma formation assays were performed in NOD/SCID mice.
5.times.10.sup.6 MSCs are injected subcutaneously into the left
thigh muscle of 3 mice. CT2 ECs were used as positive controls and
the mice will be monitored over the course of 6 weeks to compare
teratoma formation in MSC versus ESCC-injected mice. No teratomas
formed in the mice injected with MSCs.
Example 5
Reduction of EAE Scores by MSCs Derived from Hemangioblasts
[0278] A pilot study to treat experimental autoimmune
encephalomyelitis (EAE) on 6-8 weeks of C57BL/6 mice with the
hemangioblast-derived ESC-MSCs was conducted. EAE was induced by
s.c. injection into the flanks of the mice on day 0 with 100 pL of
an emulsion of 50 pg of MOG(35-55) peptide and 250 pg of M.
tuberculosis in adjuvant oil (CFA), the mice were also i.p.
injected with 500 ng of pertussis toxin. Six days later the mice
were i.p. injected with either one million ESC-MSCs in PBS (n=3) or
the vehicle as a control (n=4). The clinical scores of the animals
were recorded for 29 days post the immunization. A remarkable
reduction of the disease scores was observed (data not shown).
Example 6
Confirmation of the Efficacy of Hemangioblast-Derived ESC-MSCs in
EAE Treatment and Use of Additional Animal Models of Disease
[0279] A. Test ESC-MSCs on EAE Models in Mice Confirm Their
anti-EAE Effect.
[0280] To confirm the results obtained in Example 5, additional
tests are conducted with increased animal numbers, varying cell
doses, different administration protocols, and more controls.
Clinical score and mortality rate are recorded. The degree of
lymphocyte infiltration in the brain and spinal cord of mice will
also be assessed. MSC anti-EAE effects are generally thought to
involve immunosuppressive activities such as the suppression of
Th17 cells and would be expected to reduce the degree of lymphocyte
infiltration in the CNS.
[0281] B. Compare ESC-MSCs with Mouse Bone Marrow (BM)-MSCs, Human
BM-MSCs and Human UCB-MSCs.
[0282] Mouse BM-MSCs were the first to be used for EAE treatment
and have been thoroughly studied [1]. ESC-MSCs (given their
xenogenic nature) may be directly compared with murine BM-MSCs for
anti-EAE efficacy. Human UCB-MSCs have been shown to also possess
immunosuppressive activity [19]. The anti-EAE activity of human
UCB-MSCs and human BM-MSCs may also be compared with that of
ESC-MSCs in the EAE mouse models. The age or passage number of
these various cell types may influence their anti-EAE behavior,
thus we will also evaluate the consequences of age on the efficacy
of MSCs in the EAE mouse model system.
[0283] C. Optimize the Administration Dose, Route, and Timing of
ESC-MSCs.
[0284] Injection of the ESC-MSCs can reduce the scores of EAE as
recorded within 29 days after immunization. To study long-term
prevention and cure of disease, ESC-MSCs may be administered at
various doses, routes, and times.
[0285] MSCs have been generated from Hlgfp ESCs and confirmed that
they still express GFP in the MSC state. EAE mice can be injected
with these GFP+ ESC-MSCsand their distribution can be tracked in
vivo by using a Xenogen In Vivo Imaging System. Through these
approaches, various administration doses, routes, and timing of
ESC-MSCs will be analyzed and provide information as to the
mechanism of action for MSCs anti-EAE activity (ie, paracrine or
endocrine effects), longevity of the MSCs within the mice and MSC
biodistribution and routes of elimination/clearance.
[0286] Anti-EAE effects may be reflected by one or more of reduced
clinical scores, increased survival, and/or attenuated lymphocyte
infiltration and demyelination of the CNS. Different ESC lines may
have different intrinsic abilities to generate MSCs. Therefore,
multiple ESC lines may be used in this study and acquisition of MSC
markers can be monitored over time and compared for each ESC line.
To further reduce variations between experiments with ESC-MSCs,
large stocks of frozen ESC-MSCs can be made in aliquots and each
stock of aliquots can be used in multiple experiments.
[0287] D. Confirm Efficacy of Hemangioblast-Derived MSCs in Other
Disease Models.
[0288] As mentioned above, MSCs may also have therapeutic activity
against other types of autoimmune disorders such as Crohn's
disease, ulcerative colitis, and the eye-disorder, uveitis. Animal
models for these diseases exist and are well known in the art (see,
e.g., Pizarro et al 2003, Duijvestein et al 2011, Liang et al 2011,
Copland. et al 2008). In vivo studies may be expanded to include an
assessment of MSC therapeutic utility in one or more of these
animal model systems. Such models may allow us to examine the
cytokine secretion profile of human MSCs by isolating and screening
the serum of injected animals for human cytokines. Particularly,
the uveitis model may be useful as a local intraviteal injection
may allow us to study the effects of MSCs in a non-systemic
environment.
[0289] MSCs may also have great therapeutic utility in treating
osteoarthritis conditions, including those that involve loss of
articular cartilage and inflammation of the affected joints (Noth
et al, 2008). Models for examining osteoarthritis, cartilage loss
and joint inflammation are well known in the art (see, e.g.,
Mobasheri et al 2009). In some of these studies, human BM-MSCs are
encapsulated in semi-solid scaffolds or microspheres and
transplanted into an affected joint in human subjects to determine
if the MSCs have a local, non-systemic therapeutic effect in terms
of reduced inflammation and/or restoration of cartilage (Wakitani
et al 2002). Such methods will assist in determining the
therapeutic utility of our ESC heamngioblast-derived MSCs for
treating degenerative joint conditions.
[0290] The life span of injected MSCs is very short [8], which
indicates that long-term survival of the transplanted cells is not
required. Thus, mitotically-inactivated ESC-MSCs (e.g., irradiated
or treated with mitomycin C) may also be tested for an anti-EAE
effect or other anti-disease effect in the animal models mentioned
above. If so, live ESC-MSCs may not be needed, thus further
decreasing the biosafety concern from potential residual ESC
contamination in the transplanted ESC-MSCs.
[0291] E. Results
[0292] MSCs from different donor derive sources (mouse BM-MSCs,
human BM-MSCs and human UCB-MSCs) are expected to harbor anti-EAE
effects. However, their effects may vary between experiments as the
MSCs are from donor-limited sources. In contrast, the ESC-MSCs of
the present disclosure may have more consistent effects. Because
many cell surface markers are used to characterize MSCs and not
every MSC expresses all the markers, a subset of markers, e.g.,
CD73+ and CD45- may be used in order to compare efficacy of MSCs
from different sources.
[0293] ESC-MSCs are expected to have therapeutic utility in animal
models of Crohn's Disease, ulcerative colitis, and uveitis as these
contain autoimmune components and inflammatory reactions.
[0294] Mitotically inactivated MSCs (e.g. irradiated or mitomycin C
inactivated MSCs or ESC-MSCs) may retain, at least partially, the
immunosuppressive function since they still secret cytokines and
express cell surface markers that are related to the function [29].
Their effect may, however, be decreased due to their shortened life
span in vivo. If so, the dose of irradiated or other mitotically
inactived cells and administration frequency may be increased to
enhance the immunosuppressive function. The mitotically inactivated
MSCs and ESC-MSCs may retain, at least partially, the
immunosuppressive function since they still secret cytokines and
express cell surface markers that are related to the function [29].
Their effect may, however, be decreased due to their shortened life
span in vivo. If so, the dose of mitotically inactivated cells and
administration frequency may be increased to enhance the
immunosuppressive function.
[0295] A second pilot study to treat EAE was conducted. Eight to
ten week old C57BL/6 mice were immunized with the MOG35-55 peptide
in complete freund's adjuvant via subQ injection. Thus was done in
conjunction with Intraperitoneal injection of pertussis toxin. Six
days later, 1 million live (or 2 million irradiated)
hemangioblast-derived pluripotent cell-mesenchymal stromal cells
were injected intraperitoneally per mouse. Disease severity was
scored on a scale of 0-5 by monitoring mouse limb/body motion, as
previously published. Results demonstrate a significant reduction
in clinical score as compared to vehicle control with
hemangioblast-derived pluripotent cell-mesenchymal stromal cells at
passage 4 and irradiated hemangioblast-derived pluripotent
cell-mesenchymal stromal cells (data not shown). Scoring for both
pilot studies was performed according to the following protocol: a
score of 1 indicates limp tail, 2 indicates partial hind leg
paralysis, 3 is complete hind leg paralysis, 4 is complete hind and
partial front leg paralysis, 5 is moribund.
[0296] In addition, the efficacy of MSC's according to the
invention and products derivable therefrom for use in different
therapies may be confirmed in other animal models, e.g., other
transplantation or autoimmune models depending on the contemplated
therapeutic indication.
Example 7
Investigation of Functional Components of ESC-MSCs
[0297] MSCs may be defined as plastic adherent cells that express
the following cell surface markers: CD105, CD73, CD29, CD90, CD166,
CD44, CD13, and HLA-class I (ABC) while at the same time being
negative for CD34, CD45, CD14, CD19, CD11b, CD79a and CD31 when
cultured in an uninduced state (eg, culture in regular
.alpha.MEM+20%FCS with no cytokines). Under these conditions, they
must express intracellular HLA-G and be negative for CD40 and HLA
class II (DR). Functionally, such cells must also be able to
differentiate into adipocytes, osteocytes, and chondrocytes as
assessed by standard in vitro culture assays. After 7 days
stimulation with interferon gamma (IFN.gamma.), MSCs should express
HLA-G on their cell surface as well as CD40 and HLA-class II (DR)
on their cell surface. Despite these requirements, MSCs derived
from any source may contain some heterogeneity and due to the
pluripotency of ESCs it is possible that MSC cultures derived from
ESCs may contain cells of any lineage from the three germ layers.
While the culture system described herein indicated that >90% of
cells routinely display the above mentioned immunophenotype and
functional characteristics, small subpopulation(s) of cells within
the MSC culture may exist that lack expression of one or more of
the MSC cell surface markers or express one or more of the markers
that should be absent. The extent of such subpopulations within our
MSC cultures will be examined to determine the degree of
contaminating heterogeneity. Multicolor flow cytometry (8+ colors
simultaneously) can be performed on a BD LSR II flow cytometer in
order to determine the overlap between the above mentioned markers.
This may also help pinpoint the exact cell surface marker profile
that is required for the greatest immunosuppressive activity.
[0298] A. Characterize the Differentiation Stage, Subpopulations,
and Activation Status of ESC-MSCs in Relevance to Their
Immunosuppressive Effects.
[0299] There is a large time window (e.g., at least from day 14 to
28 in the MSC differentiation medium) to harvest ESC-MSCs (see,
e.g., FIG. 1). Several studies have indicated that MSCs tend to
lose their immunosuppressive functions and may senesce as they are
continually passaged and age during long culture periods. As such,
the cells may be harvested at different time points activity in
order to determine is a specific number of days in MSC medium
affords greater immunosuppressive activity. Indeed, MSCs collected
at an early time point (e.g., 14 days in MSC culture conditions)
may contain precursor cells that have not yet fully acquired all of
the characteristic MSC cell surface markers but that harbor highly
potent immunosuppressive effects. To define potentially useful MSC
precursor populations, the expression of a wide range of cell
surface markers are being tracked throughout the MSC
differentiation process, from day 7 through day 28. It has been
observed that at least 50% of the culture will acquire the cell
surface marker CD309 (other names include VEGFR2, KDR) within 14
days of MSC culture conditions. CD309 is largely absent from the
starting hemangioblast population (FIG. 9, first time point, MA09
hemangioblasts harvested at d7 and 8), but rises within the first
two weeks of MSC culture conditions and then declines again back to
less than 5% of the cells by day 28 (FIG. 9, second, third, and
fourth time points). This pattern has been found to occur not only
with MA09 hemangioblast-derived MSCs but also with those from MA01,
H1gfp, and H7 ESCs. In these experiments, hemangioblasts are
routinely negative (less than 5% of cells stain positive) for CD309
regardless of their harvest date (day 6-14). However, the
percentage of developing MSCs that acquire CD309 expression may be
reduced when developing from older hemangioblasts (e.g., d10 or d12
blasts). In a similar fashion, it has been observed that the
expansion properties of hemangioblast-derived MSCs may differ
depending on the harvest date of hemangioblasts. MSCs developing
from younger hemangioblasts (day 6 or 7) do not continue to expand
as robustly as MSCs developing from older (d8-12) hemangioblasts.
The optimal date of hemangioblast harvest may be an intermediate
one (day 8-10) as they may allow adequate acquisition of CD309 as a
surrogate marker of MSC development while still maintaining a
robust ability to expand through day 28 and beyond. Work is ongoing
to optimize these aspects of MSC precursor development.
[0300] Except CD105, CD90 and CD73 that have proved the most
typical markers for MSCs (as noted by the International Society for
Cellular Therapy as the minimum classification of MSCs (Dominici et
al., Cytotherapy 8 (4): 315-317 (2006)), many other cell surface
molecules not mentioned above such as CD49a, CD54, CD80, CD86,
CD271, VCAM, and ICAM have also been proposed or used as MSC
markers [22]. It is therefore possible that ESC-MSCs may contain
subpopulations that express various combinations of other markers
during the differentiation from hemangioblasts, which may possess
varying immunosuppressive activities. Subpopulations may be sorted
(e.g., using FACS) based one or more markers (individually or in
combination) for analysis to compare their immunosuppressive
activity using in vitro or in vivo methods.
[0301] B. Optimize Differentiation and Expansion Conditions to
Obtain Large Quantities of Functional ESC-MSCs.
[0302] While preliminary experiments have indicated that MSCs may
be maintained in IMDM+10% heat-inactivated human serum, we have not
yet tested their derivation in this medium. Different culture
conditions may be tested to determine whether substituting culture
components (eg, base medium, serum source, serum replacement
products, human serum platelet lysate) may enrich the effective
subpopulations described herein. Different basal medium including
animal-free and a defined culture (without FBS) system to culture
ESCs and prepare MSCs will be evaluated. Specifically, StemPro.RTM.
MSC SFM from Invitrogen and the MSCM bullet kit from Lonza will be
used to examine if a serum-free defined culture system would
generate ESC-MSCs with desired quality and quantity. Also, various
growth factors such as FGFs, PDGF, and TGFI3, as well as small
chemicals that regulate signaling pathways or cell structures, may
be used to enhance the quality and quantity of ESC-MSCs.
[0303] C. Results
[0304] The ESC-MSCs express the typical markers CD73
(ecto-5'-nucleotidase [26]), CD90 and CD105. Also, FIG. 20 shows
that FM-MA09-MSCs produced according to the invention maintain
their phenotype over time (based on marker expression detected
during flow cytometry analysis of different MSC populations over
time and successive passaging).
Example 8
Mechanism of Immunosuppression by ESC-MSCs
[0305] A. Study how ESC-MSCs may Suppress Adaptive Immune Responses
Mediated by T Cells.
[0306] A general response of T cells within PBMC is to proliferate
when they are induced with mitotic stimulators such as
phytohemaglutinin (PHA) or phorbol myristate acetate
(PMA)/ionomycin or when they encounter antigen presenting cells
(APCs) such as dendritic cells. This is best exemplified by the
general proliferation of CD4+ and CD8+ T cells in a mixed leukocyte
reaction (MLR) assay. Prior studies indicate that MSCs can suppress
T cell proliferation in an MLR assay.
[0307] The ability of our ESC-hemangioblast derived MSCs to inhibit
T cell proliferation caused by either chemical stimulation
(PMA/ionomycin, FIGS. 10a and 13a) (PHA, FIG. 13b) or exposure to
APCs (dendritic cells, FIGS. 10b and 13c) was examined. It was
observed that MSCs dampened the proliferative response of T cells
due to either chemical stimulation or co-culture with APCs and that
this suppression occurred in a dose dependent manner (FIG. 10b,
graph on right) Moreover, it was found that mitotically inactivated
MSCs (FIG. 10b) were able to suppress T cell proliferation to an
equivalent degree as live MSCs, suggesting that mitotically
inactivated MSCs may indeed be useful in vivo for
immunosuppression.
[0308] Various functional subsets of T cells exist and they carry
out specific roles involved in proinflammatory responses,
anti-inflammatory responses, or induction of T cell anergy.
Regulatory T cells (Tregs) can be thought of as naturally occurring
immunosuppressive T cells and in a normal setting, are responsible
for dampening hypersensitive auto-reactive T cell responses. They
usually represent only a small proportion of the body's T cells but
their prevalence can be influenced by various environmental
factors. MSCs have been shown to induce peripheral tolerance
through the induction of Treg cells [33-35].
[0309] In a short, 5 day co-culture assay, it was found that,
similar to prior studies, the hemangioblast-derived MSCs were able
to increase the percentage of CD4/CD25 double positive Tregs that
are induced in response to IL2 stimulus (FIG. 11a, 14, 15a).
Co-culture of a mixed T cell population from non-adherent
peripheral blood mononuclear cells (PBMCs) with MSCs (at a ratio of
10 PBMCs:1 MSC) shows that Treg induction nearly doubled when MSCs
were included in the IL2 induced culture. This degree of Treg
induction is similar to that observed in the highly cited Aggarwal
et al study published in Blood, 2005. The amount of FoxP3 induced
within the CD4/CD25 double positive population have been examined
to confirm that these are indeed true Tregs (FIG. 15b).
Intracellular flow cytometry,was used to study FoxP3 induction in
the absence and presence of MSCs in the IL2-induced T cell
cultures. Both non-adherent PBMCs and purified CD4+ T cell
populations may be used to study Treg induction in these assays.
Without intent to be limited by theory, it is believed that ES-MSC
are more effective at inducing Tregs because they increase
expresson of CD25 more effectively than BM--MSCs (FIG. 15b)
[0310] Th1 and Th17 cells are thought to play important roles in MS
and in other autoimmune diseases. The differentiation and function
of Th1 and Th17 CD4+ T cells will be analyzed first and foremost
using in vitro assays; they may also be examined in the EAE model
or in other animal models we may employ. The effects of MSCs on Th1
induction in vitro have begun to be examined. Culture conditions
that promote Th1 specification from naive CD4+ T cells are known in
the field (Aggarwal et al). These culture conditions (which include
anti-CD3, anti-CD28, and anti-CD4 antibodies together with human
IL3 and IL12) have been employed to induce Th1 cells from naive,
non-adherent PBMCs in the absence or presence of MSCs (10 PBMCs:1
MSC). After 48 hours of co-culture, non-adherent cells were
isolated, rinsed, and stimulated with PMA/ionomycin for 16 hours in
a new well. After the 16 hour induction, supernants were collected
and analyzed for secretion of the Th1 cytokine, IFN.gamma.. As
anticipated, it was found that the PBMCs cultured with MSCs in the
48 hr Th1 inducing conditions did not produce as much IFN.gamma. as
those cultured without MSCs. This indicates that MSCs can suppress
a major Th1 cell function, i.e., IFN.gamma. secretion. (FIG. 11b)
Similar studies will be performed by differentiating Th17 cells in
vitro and determining the effects of MSCs on pro-inflammatory IL17
secretion using an ELISA assay on culture supernatants.
[0311] Th2 cells are known to secrete cytokines that have
anti-inflammatory effects, such as IL4. MSCs may be able to enhance
Th2 differentiation and secretion of IL4. Similar to the experiment
described above for Th1 cells, Th2 inducing conditions will be used
in a 48 hour culture system to stimulate Th2 differentiation from
naive PBMC containing T cells. The effects of MSC co-culture on IL4
secretion will be examined using an ELISA assay.
[0312] Recently, studies have suggested that CD8 T cells also play
a pivotal role in EAE models and the underlying mechanism of MS
[30]. The inventor will examine if co-culture with ESC-MSCs in
vitro may affect the function of CD8 T cells. To do this,
non-adherent PBMCs or purified CD8+ T cells will be exposed to
EAE-associated MBP110-118 peptide through the use of APCs. This
will cause an antigen-specific CD8+ T cell population to emerge and
such a population can be expanded using CD3/CD28 expander beads
(Invitrogen). Existence of the angiten-specific CD8+ T cells can be
verified using a pentamer reagent specific for the MBP-peptide
(Proimmune) in flow cytometry. Re-stimulation with
MBP110-118-loaded APCs will be performed in order to induce an
antigen specific immune response, which includes both expansion of
the antigen-specific CD8+ T cells and secretion of IFN.gamma.. The
response from T cells cultured in the absence or presence of MSCs
will be compared to determine if the MSCs can suppress the
induction of these cytotoxic EAE-associated antigen specific T
cells. Pentamer specific flow cytometry, BrdU incorporation, and
ELISA assays will be employed for this purpose.
[0313] B. Determine if Inflammatory Factors and Inter-Cellular
Adhesion Molecules (ICAMs) Contribute to the Immunosuppresive
Effect of ESC-MSCs.
[0314] It has been shown that TGFbeta, PGE2, IDO, nitric oxide
(NO), and ICAMs are important for the immunosuppressive function of
MSCs [7]. The secretion of these molecules and expression of ICAMs
by ESC-MSCs will be examined using ELISA assays and flow
cytometry.
[0315] It has been shown that the pro-inflammatory cytokine,
IFN.gamma. is required for the activation of MSCs [23], and various
agonists for Toll-like receptors (TLRs) such as LPS and poly(I:C)
can induce different subsets of MSCs [24]. For example, it has
recently been shown that IFN.gamma.-activated MSCs have greater
therapeutic efficacy in a mouse model of colitis than do untreated
MSCs (Duijvestein et al 2011). The effects of IFN.gamma. on MSC
properties have begun to be examined. ESC-MSCs have been treated in
vitro with IFN.gamma. for up to seven days and striking changes in
cell surface marker expression have resulted. These findings are
consistent with observations made in previous studies (Gotherstrom
et al 2004, Rasmusson et al 2006, Newman et al 2009) and confirm
that the hemangionblast derived ESC-MSCs function similarly to MSCs
isolated from the body. For example, in a resting state, MSCs
typically do not express much (<10%) HLA G on their cell surface
while they do harbor intracellular stores of this special class of
immunotolerant HLA marker. Upon 7 days IFN.gamma. treatment, HLA G
can be readily detected at the cell surface (FIG. 12) and may also
be induced to be secreted (not yet tested). Additionally,
IFN.gamma. treatment causes an upregulation of CD40 expression and
HLA DR expression at the cell surface (FIG. 12). These changes are
proposed to enhance their immunosuppressive effects. For example,
we will determine if pretreatment of MSCs with IFN.gamma. enhances
their ability to induce Treg populations, to suppress Th1 secretion
of IFN.gamma., or to enhance IL4 secretion from Th2 cells by using
in vitro co-culture assays described above. IFN.gamma. may also
influence the ability of MSCs to inhibit general T cell
proliferation in MLR assays. The effects of TNF.alpha., LPS, and/or
poly I:C on these types of MSC immunosuppressive properties may
also be tested.
[0316] C. Results
[0317] It was shown that the CD4/CD25 double positive population of
Tregs induced by MSCs also express the transcription factor, FoxP3
as it has been reported that functional Tregs upregulate its
expression in response to inducing stimuli (FIG. 15b).
[0318] It is expected that MSCs will inhibit, to some degree the
pro-inflammatory secretion of IL17 by Th17 cells and that MSCs can
also significantly enhance IL4 secretion by anti-inflammatory Th2
cells. Such observations have been made in previous studies and
will assist in confirming the true functionality of the
hemangioblast-derived MSCs.
[0319] The ESC-MSCs should inhibit at least partially the
antigen-induced activation of CD8+ T cells. The function of NK
cells, macrophages, and dendritic cells after ESC-MSC co-culture
may also be examined. The effects of ESC-MSCs on maturation,
cytotoxicity, and/or specific cytokine production by these other
types of immune cells will be examined.
[0320] For example, the experiments in FIG. 11A show that
hemangioblast-derived mesenchymal stromal cells increase the
percentage of CD4/CD25 double positive Tregs that are induced in
response to IL2 stimulus. Also, the experiments in FIG. 12 show
that the proinflammatory cytokine IFNg stimulates changes in
FM-MA09-MSC surface marker expression and that interferon gamma
stimulates changes in MSC surface marker expression and may enhance
MSC immunosuppressive effects.
[0321] Moreover, the experiments in FIG. 14 show that FM-MA09-MSCs
enhance Treg induction, and particularly that early passage MSCs
had greater effects than late passage MSCs. Non-adherent PBMCs
(different donors) were cultured with or without IL2 for 4 days in
the absence or presence of FM-MA09-MSCs. The percentage of CD4/CD25
double positive Tregs was assessed by flow cytometry. Young (p6) or
old (p16-18) FM-MA09-MSCs were used. The black bars indicate the
average of 6 experiments. MSCs as a whole had a statistically
significant effect on induction of Tregs. (p=0.02).
Example 9
ESC-MSCs have Increased Potency and Greater Inhibitory Effects than
BM-MSCs
[0322] A mixed lymphocyte reaction (MLR) assay was performed to
determine if different MSC populations have different abilities to
inhibit T cell proliferation. Results suggest that ESC-MSCs are
more potent than BM-MSCs in their ability to inhibit T cell
proliferation in response to either mitogenic stimulus ("one-way
MLR") (see FIGS. 13a and 13b) or to antigen-presenting cells
(dendritic cells, DCs; "two-way" MLR) (see FIG. 13c).
[0323] The "one-way" MLR assay was performed as follows: Human
PBMCs were purchased from AllCells. Upon thawing a frozen vial,
PBMCs were plated for at least 1 hour or overnight in IMDM+10%
heat-inactivated human serum to selectively adhere monocytes. The
non-adherent cells (containing T cells) were used as a crude source
of T cell responders. ESC-derived MSCs or BM-derived MSCs were used
as inhibitors. These MSCs were were either live or
mitotically-arrested with mitomycin C. Non-adherent PBMCs and MSCs
were mixed together at varying ratios and allowed to co-culture for
5 days. On day 3, the mitogens, phorbol-12-myristate 13-acetate
(PMA) and ionomycin or phytohemagglutinin (PHA) were added to the
cultures to induce T cell proliferation. On day 4,
bromodeoxyuridine (BrdU) was added. On day 5, T cell proliferation
was assessed through flow cytometric staining with antibodies
directed against CD4, CD8, and BrdU using the BrdU incorporation
kit (B&D Biosystems). T cell proliferation was assessed as the
% of CD4+ and/or CD8+ cells that had incorporated BrdU into their
DNA (ie, BrdU+) (shown in FIGS. 13a and 13b).
[0324] In the "two-way" MLR, ESC-derived MSCs or BM-derived MSCs
were used as inhibitors, non-adherent peripheral blood mononuclear
cells (PBMCs) were used as a crude source of T cell responders, and
monocyte-derived dendritic cells (DCs) were used as stimulators. To
derive DCs, plastic-adherent monocytes were isolated from PBMCs
PBMCs were plated for at least 1 hour or overnight in IMDM+10%
heat-inactivated human serum (10% HuSer) to selectively adhere
monocytes. Non-adherent cells were removed and the adherent cells
were cultured in IMDM+10% HuSer for 4 days with SCF, FL, GM-CSF,
IL3, and IL4. In this variation of the assay, no mitogen is added
on day 3. BrdU is simply added 16-24 hours before harvesting the
cells for flow cytometry as above. Both MSCs and DCs were
mitotically-inactivated with Mitomycin C in this assay (shown in
FIG. 13c).
Example 10
Improved Induction of Treg Expansion by Young ESC-MSCs Compared to
BM-MSCs and Old ESC-MSCs.
[0325] Co-culture experiments were performed with PBMCs and MSCs to
determine if the presence of MSCs can induce regulatory T cell
(Treg) expansion within the PBMC population. Results suggest that
young ESC-MSCs induced Treg expansion better than both BM-MSCs and
old ESC-MSCs (see FIG. 14 and FIG. 15).
[0326] Co-cultures were established with non-adherent PBMCs and
different types of MSCs ("young" ESC-derived (.about.p5-6), "old"
ESC-derived (--p12 or higher), BM-derived) at a 10:1 ratio
(PBMC:MSC). Co-cultures were incubated in IMDM+10% heat inactivated
Human Serum+300 units/ml recombinant human IL2 for 4 days. The
presence of Tregs was determined by the percentage of PBMCs that
stained positive for CD4, CD25, and FoxP3 using a FoxP3
intracellular flow cytometry staining kit (Biolegend).
Example 11
ESC-MSC have Greater Proliferative Capacity
[0327] The growth rates of different MSC populations were monitored
over time to determine if the source of MSCs affects their
proliferative capacity. Results show that ESC-derived MSCs have
greater proliferative capacity than BM-derived MSCs. Results also
suggest that culturing ESC-MSCs on a substrate (such as Matrigel)
for a longer period of time (up to 6 passages) may help maintain a
higher growth rate than if the cells are moved off of the substrate
at an earlier passage, such as p2 (see FIG. 16 and FIG. 17).
[0328] ESC-derived hemangioblasts were seeded onto Matrigel-coated
tissue-culture plastic at 50,000 cells/cm.sup.2 in .alpha.MEM+20%
Hyclone FBS+1-glutamine+non-essential amino acids (=MSC growth
medium as p0. Bone-marrow mononuclear cells were seeded onto
regular tissue culture plastic at 50,000 cells/cm.sup.2 in MSC
growth medium as p0. Cells were harvested with 0.05% trypsin-edta
(Gibco) when they reached .about.50-60% confluence at p0 or at
70-80% confluence from pl onwards (usually every 3-5 days). Upon
harvest, cells were spun down, counted, and replated at 7000
cells/cm.sup.2. ESC-MSCs were removed from Matrigel and
subsequently grown on regular tissue culture plastic starting at
p3, unless otherwise indicated. Cumulative population doublings
over time are plotted to show the rate of cell growth as the MSCs
are maintained in culture.
Example 12
ESC-MSCs Undergo Chondrogenic Differentiation
[0329] To determine the chondrogenic potential of different MSC
populations, ESC-MSCs or BM-MSCs were seeded as pellet mass
cultures and induced to differentiate into chondrocytes with
differentiation medium (or kept in regular MSC growth media as
negative controls). Results suggest that ESC-MSCs undergo
chondrogenesis in a manner similar to that of BM-MSCs. Both ESC-MSC
and BM-MSC pellets reveal cartilaginous matrix (proteoglycan)
deposition via Safranin O staining (see FIG. 18).
[0330] To form chondrogenic pellet culture, 2.5.times.10.sup.5
cells ESC-MSCs were centrifuged at 500.times.g for 5 min in a 15 mL
conical tube. Culture medium was aspirated and 0.5 mL of
chondrogenic culture medium, consisting of DMEM-HG (Life
Technologies, Gaithersburg, Md.) supplemented with 1 mM Sodium
Pyruvate (Life Technologies), 0.1 mM ascorbic acid 2-phosphate
(Sigma-Aldrich, St. Louis, Mo.), 0.1 .mu.M dexamethasone
(Sigma-Aldrich), 1% ITS (Collaborative Biomedical Products,
Bedford, Mass.), 10 ng/mL TGF-.beta.3 (Peprotech, Rocky Hill,
N.J.), or culture medium (control) was added to the pellet. Pellet
cultures were maintained for 21 days with medium changes every 2-3
days. At the end of the 21 days, pellets were fixed with 4%
paraformaldehyde and sent to MassHistology (Worcester, Mass.) for
paraffin-embedding, sectioning, and Safranin O staining using
standard procedures.
Example 13
Enhanced Sectretion of Prostaglandin E2 (PGE2) under IFN-.gamma. or
TNF-.alpha. Stimulation
[0331] ESC-MSCs exert immunomodulatory effects in part through the
secretion of PGE2. Conditioned medium collected from FM ESC-MSCs
and BM-MSCs show that BM-MSCs secrete higher levels of PGE2 in the
basal state than FM ESC-MSCs. Experiments to determine PGE2
secretion under stimulated conditions (various concentrations of
IFN-.gamma. and/or TNF-.alpha.) show that FM ESC-MSCs greatly
increase their secretion of PGE2 in response to stimulation (see
FIG. 19). In fact, the fold induction for PGE2 secretion from a
basal to stimulated state is much greater for FM ESC-MSCs than for
BM-MSCs. However, the actual raw amounts of PGE2 secretion (in
pg/ml) under stimulated conditions is similar for FM ESC-MSCs and
BM-MSCs.
[0332] ESC-MSCs were plated at 7.5.times.10.sup.6 cells/cm.sub.2 in
6 well plates (BD Falcon, Franklin Lakes, N.J.). Cultures were
maintained in culture medium for 24 hrs, followed by stimulation
with 10, 50, 100, or 200 ng/ml IFN-.gamma. and/or 10, 25, 50 ng/mL
TNF-.alpha. (Peprotech). Supernatant was collected after 3 days of
induction and stored at -20.degree. C. ESC-MSCs were harvested and
counted to normalize PGE2 levels to cell number. PGE.sub.2
concentration was measured with ELISA kits (R&D PGE2 Parameter
or Prostaglandin E2 Express EIA kit, Cayman Chemicals) and used
according to manufacturer's protocol.
Example 14
ESC-MSC Phenotypic Evaluation
[0333] The expression of various cell surface markers was assessed
on different MSC populations to determine their individual
immunophenotypes. ESC-derived MSCs can be differentiated on various
substrates. A panel of cell surface markers were examined to
determine their expression profile on MSCs that had been derived on
three different matrices (Matrigel, fibronectin, or collagen I)
versus their expression on BM-MSCs. Results show similar patterns
of expression for these markers regardless of the substrate used
for their initial differentiation. They were over 95% positive for
CD13, 29, 44, 73, 90, 105, 166, and HLA-ABC while negative for
CD31, 34, 45, HLA-DR, FGFR2, CD271 (see FIG. 20A). Stro-1
expression varied, between approximatey 5% for ESC-MSCs to
approximately 30% for BM-MSCs.
[0334] MSCs slow in growth and population doubling with increasing
passage number. The aim of this experiment was to look at surface
marker expression for a number of different MSC markers from
passage 3 to 17 in FM-ESC-MSCs. Cells in all passages of FM-ESC-MSC
stained positive for CD90, CD73, CD105, HLA-ABC, CD166, CD13, and
CD44. Cells were negative for CD34, CD45, TLR3, HLA-DR, CD106,
CD133, and CD271 (see FIG. 20B).
[0335] For each line/passage number, the same protocol was
followed. Cells were grown in T75 or T175 flasks, in MSC media.
Cells were passaged every 3-4 days. Passaging cells consisted of
washing flasks with PBS, collecting cells using cell dissociation
media TryPLE Express, and washing with MSC media. Cells were
counted for viability with trypan blue and aliquoted at 50-100,000
viable cells per condition. The following antibodies were used:
CD34-Fitc, CD34-PE, CD44-Fitc, CD73-PE, CD106-PE, CD45-APC (BD);
HLA-DR-APC, CD90-Fitc, HLA-ABC-Fitc, CD133-APC, CD29 (ebioscience);
CD166-PE, CD105-APC, CD13-PE, CD13-APC, CD271-Fitc, CD10-Fitc,
Stro-1-AF647, CD10 (Biolegend); TLR3-Fitc (Santa Cruz Biotech).
Propidium Iodide was also added as a viability marker. Cells were
incubated at room temperature for 30 minutes, spun down, passed
through a 40 .mu.m cell strainer, and analyzed with na Accuri C6
Flow Cytometer. For each cell type, cells were gated on the MSC
population (FSC vs. SSC), PI negative. Percent positive was
determined by gating histogram plots and using the unstained cell
population as a negative control. See, Wagner W, et al. Replicative
Senescence of Mesenchymal Stem Cells: A Continuous and Organized
Process. PLoS ONE (2008). 3(5): e2213.
doi:10.1371/journal.pone.0002213; and Musina, R, et al. Comparison
of Mesenchymal Stem Cells Obtained from Different Human Tissues.
Cell Technologies in Biology and Medicine (2005) April. 1(2),
504-509.
[0336] Additionally, FM ESC-MSCs have a greater level of CD10
expression and less Stro-1 expression than FM ESC-MSCs and BM-MSCs
(see FIG. 21). This expression pattern of low Stro-1 (5-10% of
cells) and mid-level CD10 (.about.40% of cells) was confirmed in 10
different lots of FM-MA09-MSCs (see FIG. 22). Flow cytometry was
also used on different populations to evaluate cell size (see FIG.
23). Results show that as the cells are maintained in culture for
longer periods of time, the cell size of BM-MSCs increases while FM
ES-derived MSCs maintain cell size. Cell size was determined by
forward vs. side scatter on flow cytometry dot plots. A quadrant
gate was used to divide the plot into 4 regions. The upper right
quadrant contains the large cells, i.e., cells in that area have
large forward scatter (cell volume) and also high side scatter
(granularity).
[0337] ESC-MSCs were harvested, as previously mentioned, and washed
in 1.times. DPBS (Life Technologies). 75-100.times.10.sup.5 cells
were washed with flow buffer (3% FBS ; Atlas Biologicals, Fort
Collins, Colo.), followed by incubation with 100 .mu.L of flow
buffer containing either primary antibody or isotype control
antibody for 45 min on ice. Cells were washed with 2 mL flow buffer
and incubated in 100 .mu.L flow buffer containing secondary
antibody for 45 min on ice. Cells were washed a final time and
resuspended in flow buffer containing propidium iodide and analyzed
on an Accuri C6 flow cytometer (Accuri Cytometers Inc., Ann Arbor,
Mich.).
Example 15
Gene Expression Analysis in ESC-MSCs
[0338] The purpose of these studies was to determine the
similarities and differences of mRNA expression between FM-ESC-MSC
and BM-MSC. In the first set of experiments (basal experiments),
relative differences of mRNA expression of cells from FM-ESC-MSC
and BM-MSC were compared by Quantitative Polymerase Chain Reaction
(QPCR). Taqman probes (Life Technologies) to the various genes were
used to determine relative expression to the endogenous control,
GAPDH, using the .DELTA..DELTA.Ct method. From a list of 28 genes,
the following genes were upregulated in the basal experiments in
FM-ESC-MSC vs BM-MSC: AIRE, ANGPT1 (ANG-1), CXCL1, CD10, CD24, and
IL11 (see FIGS. 24-26). IL6 and VEGF were downregulated in
FM-ESC-MSC vs BM-MSC (see FIG. 27). There was no significant
difference for the following genes between the sources of MSC:
ALCAM, FGF7, HGF, LGALS1, NT5E, and TNFSF1B (data not shown). The
following genes were not detected in any of the MSC sources:
ANGPT2, CD31, CD34, CD45, HLA-G, IL2RA, IL3, IL12B (data not
shown). As a negative control, all MSCs were tested for expression
of the hematopoietic progenitor markers, CD34, CD41, and CD45. From
these experiments, we have determined that FM-ESC-MSCs do express
some genes at higher or lower levels than the equivalent
BM-MSCs.
[0339] We also challenged the MSCs to an environment that mimics an
immune response by treating the MSC with T cells and then adding
the stimulant, Phytohemagglutinin (PHA). ESC-MSC were grown in the
presence of T cells (unstimulated) or T cells plus PHA (stimulated)
for two days before adding 2.5 .mu.g/ml PHA for an additional 2
days prior to RNA collection. The gene expression of ESC-MSC
unstimulated and stimulated are currently being compared to
unstimulated and stimulated BM-MSC mRNA levels.
[0340] For basal experiments: FM-ESC-MSC and BM-MSC were cultured
for 4 days at a starting density of approximately 500,000 cells in
a 10 cm dish under previously described conditions. Additionally, a
negative control for basal experiments was MA09 ESC derived
hematopoetic progenitors.
[0341] For stimulation experiments: FM-ESC-MSC and BM-MSC were
cultured for 3-4 days at a starting density of approximately
500,000 cells in a 10 cm dish under previously described
conditions. MSCs were then exposed to T cells for 2 days and then
+/- exposure to 2.5 .mu.g/ml PHA. As a control, MSCs were grown in
the presence of T cells without PHA, and separately, T cells plus
PHA (no MSCs) were also grown. Media was aspirated, rinsed 2 times
in PBS, and aspirated dry. RNA was isolated using the RNAeasy kit
(Qiagen) as per manufacturer's directions. The concentration and
purity of RNA was analyzed by using the Nanodrop 2000 (Thermo
Scientific). cDNA synthesis was performed using the SuperScript III
First-Strand Synthesis SuperMix for qRT-PCR (Life Technologies)
using 1 microgram RNA as the starting material. cDNA was diluted
approximately 30 fold for 5 microliters/well. Diluted cDNA, 1
microliter of QPCR Taqman probe (Life Technologies), and 15
microliters of SSO Fast Mastermix (Biorad) were mixed per well.
QPCR was performed on the Biorad CFX 96. Data was analyzed using
CFX manager 2.1 (Biorad). Relative quantities of mRNA expression
were determined using the endogenous control, GAPDH, and the
.DELTA..DELTA.Ct method.
Example 16
Indoleamine 2, 3-Dioxygenase (IDO) Enzyme Activity in ESC-MSCs
[0342] Indoleamine 2, 3-dioxygenase (IDO) is an enzyme involved in
the conversion of tryptophan to kynurenine. IFN.gamma.-activated
MSCs produce IDO and this may be partly responsible for their
ability to suppress T cell proliferation as IDO interferes with T
cell metabolism. In this study, we are testing the IDO activity of
BM-MSCs compared with ESC-MSCs. IDO expression is being measured
before and after stimulation of cells with either IFN.gamma. or by
co-culturing with T cells. Experiments show all MSC populations
greatly increase IDO activity upon stimulation with IFNgamma (see
FIG. 28).
[0343] Cells were stimulated by the addition of either IFN.gamma.
(50 ng/ml) to media, or by co-culture with T cells for 3 days;
measurement of IDO expression is performed using a
spectrophotometric assay. After stimulation, cells were collected,
and 1-2.times.10.sup.6 cells are lysed. Lysates are collected, and
mixed 1:1 with 2.times. IDO buffer (PBS with 40 mM ascorbate, 20
.mu.M methylene blue, 200 .mu.g/ml catalase, and 800 .mu.M
L-tryptophan) and incubated for 30 minutes at 37.degree. C. The
reaction was stopped by addition of 30% trichloroacetic acid, and
incubated for 30 minutes at 52.degree. C. Lysates were spun down,
and supernatants are mixed 1:1 with Ehrlich's reagent (0.8%
p-dimethylaminobenzaldehyde in acetic acid, freshly prepared).
After color development, absorbance was read on a spectrophotometer
at 492 nm. OD values were compared with a standard of kynurenine
from 0-1000 .mu.M for assessing the conversion of tryptophan to
kynurenine.
[0344] See, Meisel R et. al. Human bone marrow stromal cells
inhibit allogeneic T-cell responses by indoleamine
2,3-dioxygenase-mediated tryptophan degradation. Blood. (2004) June
15; 103 (12): 4619-21.
[0345] See, Braun, D et. al. A two-step induction of indoleamine
2,3 dioxygenase (IDO) activity during dendritic-cell maturation.
Blood. (2005) October 1; 106 (7): 2375-81.
Example 17
Expression Levels of Aire-1 and Prion-Protein in ESC-MSCs
[0346] The expression levels of Aire-1 and Prion-Protein (Prp) were
monitored using western blot analysis to determine if there are
differences among different MSC populations (based on cell source,
derivation method, or passage number of the MSCs). Aire-1 helps
induce transcription of rare peripheral tissue-restricted antigens
(PTA) that are subsequently presented on MHC and quell the response
of neighboring T cells. Aire-1 may also suppress expression of
early T cell activation factor-1 (ETA-1) to inhibit T cell
inflammatory response. Prion protein (PrP) has been shown to
enhance the proliferation and self-renewal of various stem cell
populations (hematopoietic, neural, etc) and its expression may
correlate with the growth characteristics of different MSC
populations in culture. Results show age-related decline in both
proteins (after consideration of loading control, actin for each
sample). FM MA09-MSCs appear to maintain expression of both Aire-1
and PrP over time (see FIG. 29).
[0347] MSCs whole cell lysates were run on 12% acrylamide SDS-PAGE
gels according to standard protocols. Proteins were transferred to
nitrocellulose membrane and blocked with 5% milk in PBS+0.05% tween
20. Membranes were probed with antibodies directed against Aire-1
(Santa Cruz Biotechnology) or Prion Protein (Abcam), followed by
HRP-conjugated secondary antibodies. Enhanced chemiluminescent
reagent was used to develop the signal prior to analysis on a
Biorad GelDoc Imaging System.
[0348] See, Parekkadan et al. Molecular Therapy 20 (1): 178-186
(2011).
[0349] See, Mohanty et al. Stem Cells 30: 1134-1143 (2012).
Example 18
ESC-MSC Secretion of Cytokines
[0350] MSCs are known to secrete a variety of cytokines and growth
factors in both the basal state and in response to various stimuli.
More than 20 different secreted factors were analysed using
cytokine arrays. Results show that there are a few key differences
between ESC-MSCs and BM-MSCs with respect to secreted factors in
both the basal and stimulated states. BM-MSCs express higher levels
of VEGF and IL6 than do ESC-MSCs in both the basal and
IFN.gamma.-stimulated state (see FIGS. 30-32).
[0351] Equivalent numbers of MSCs were initially plated and
conditioned medium from MSCs were collected 3-4 days after plating.
CM was spun down briefly to remove cellular debris and then frozen
at -20 C. CM was thawed for analysis on RayBiotech(Norcross, Ga.)
custom membrane arrays or on various R&D Systems (Minneapolis,
Minn.) ready-made cytokine arrays according to manufacturer's
protocols.
Example 19
Human ES Cell Culture for the Differentiation of MSCs
[0352] The purpose of this experiment was to evaluate different
growth media used for hESC culture prior to differentiation into
MSCs.
[0353] Human ES cells were generally cultured on irradiated or
mitomycin-C treated mouse embryonic fibroblasts (MEF) feeder cells
in Human ES Cell Growth Medium (knockout DMEM or DMEM/F12 (1:1)
base medium, 20% serum replacement, 1-glutamine, non-essential
amino acids, and 10 ng/ml bFGF). Passaging is performed using 0.05%
trypsin/EDTA. Alternatively, hESCs were cultured on MEF feeders in
Primate Medium and passaged using Dissociation solution (both are
purchased from ReproCELL). Results showed that Primate Medium
consistently gave "better" looking hESC colonies (rounder, tighter
colonies, less spontaneous differentiation) compared to cells grown
on the Human ES Cell Growth Medium containing knockout DMEM.
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