Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02868733 2014-10-29
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MESENCHYMAL STEM CELLS FOR TREATING INFLAMMATION
This is a divisional application of Canadian patent application
Serial No. 2564679, filed on March 15, 2005.
This invention relates to mesenchymal stem cells. More particularly, this
invention relates to novel uses for mesenchymal stem cells, including
promoting
angiogenesis in various tissues and organs, treating autoimmune diseases,
treating
allergic responses, treating cancer, treating inflammatory diseases and
disorders, and
promoting would healing. Specifically, this divisional application relates to
mesenchymal stem
cells for use in treating inflammation. It should be understood that the
expression "the invention"
or the like encompasses the subject matter of both the parent and this
divisional application.
Mesenchymal stem cells (MSCs) are multipotent stem cells that can
differentiate
readily into lineages including osteoblasts, myocytes, chondrocytes, and
adipocytes
(Pittenger, et al., Science, Vol. 284, pg. 143 (1999); Haynesworth, et al.,
Bone, Vol. 13,
pg. 69 (1992); Prockop, Science, Vol. 276, pg. 71 (1997)). in vitro studies
have
demonstrated the capability of MSCs to differentiate into muscle (Wakitani, et
al.,
Muscle Nerve, Vol. 18õ pg. 1417 (1995)), neuronal-like precursors (Woodbury,
et al., J.
Neurosci. Res., Vol. 69, pg. 908 (2002); Sanchez-Ramos, et al., Exp. Neurol.,
Vol. 171,
pg. 109 (2001)), cardiomyocytes (Toma, et al., Circulation, Vol. 105, pg. 93
(2002);
Fakuda, Artif. Organs, Vol. 25, pg. 187 (2001)) and possibly Other cell types.
In
addition, MSCs have been shown to provide effective feeder layers for
expansion of
hematopoietic and embryonic stem cells (Eaves, et al., Ann. N.Y. Acad. Sci.,
Vol. 938,
pg. 63 (2001); Wagers, et al., Gene Therapy, Vol. 9, pg. 606 (2002)). Recent
studies
with a variety of animal models have shown that MSCs may be useful in the
repair or
regeneration of damaged bone, cartilage, meniscus or myocardial tissues
(DeKok, at
=
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al., Clin. Oral Implants Res., Vol. 14, pg. 481 (2003)); Wu, et al.,
Transplantation, Vol.
75, pg. 679 (2003); Noel, et al., Curr. Opin. Investio. Drugs, Vol. 3, pg.
1000 (2002);
BaIlas, et al., J. Cell. Biochem. Suppl., Vol. 38, pg. 20 (2002); Mackenzie,
et al., Blood
Cells Mol. Dis., Vol. 27 (2002)). Several investigators have used MSCs with
encouraging results for transplantation in animal disease models including
osteogenesis
imperfecta (Pereira, et al., Proc. Nat. Acad. Sc., Vol. 95, pg. 1142 (1998)),
parkinsonism (Schwartz, et al., Hum. Gene Ther., Vol. 10, pg. 2539 (1999)),
spinal cord
injury (Chopp, et al., Neuroreport, Vol. 11, pg. 3001 (2000); Wu, et at., J.
Neurosci.
Res., Vol. 72, pg. 393 (2003)) and cardiac disorders (Tomita, et al.,
Circulation, Vol.
100, pg. 247 (1999). Shake, et al., Ann. Thorac. Surg., Vol. 73, pg. 1919
(2002)).
Importantly, promising results also have been reported in clinical trials for
osteogenesis
imperfecta (Horwitz, et al., Blood, Vol. 97, pg. 1227 (2001); Horowitz, et at.
Proc. Nat.
Acad. Sci., Vol. 99, pg. 8932 (2002)) and enhanced engraftment of heterologous
bone
marrow transplants (Frassoni, et al., Int. Society for Cell Therapy, SA006
(abstract)
(2002); Koc, et at., J. Clin. Oncol., Vol. 18, pg. 307 (2000)).
MSCs express major histocompatibility complex (MHC) class I antigen on their
surface but limited MHC class II (Le Blanc, et al., Exp. Hematol., Vol. 31,
pg. 890
(2003); Potian, et al., J. Immunol., Vol. 171, pg. 3426 (2003)) and no B7 or
CD40 co-
stimulatory molecules (Majumdar, et al., J. Biomed. Sc., Vol. 10, pg. 228
(2003)),
suggesting that these cells have a low-immunogenic phenotype (Tse, et al.,
Transplantation, Vol. 75, pg. 389 (2003)). MSCs also inhibit T-cell
proliferative
responses in an MHC-independent manner (Bartholomew, et al., Exp. Hematol.,
Vol.
30, pg. 42 (2002); Devine, et al., Cancer J., Vol. 7, pg. 576 (2001);
DiNicola, at al.,
Blood, Vol. 99, pg. 3838 (2002)). These immunological properties of MSCs may
enhance their transplant engraftment and limit the ability of the recipient
immune system
to recognize and reject allogeneic cells following transplantation. The
production of
factors by MSCs, that modulate the immune response and support hematopoiesis
together with their ability to differentiate into appropriate cell types under
local stimuli
make them desirable stem cells for cellular transplantation studies (Majumdar,
et at.,
Hematother. Stem Cell Res., Vol. 9, pg. 841 (2000); Haynesworth, et al., J.
Cell.
Physiol., Vol. 166, pg. 585 (1996).
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Applicants presently have examined the interactions of mesenchymal stem cells
with isolated immune cell populations, including dendritic cells (DC1 and
DC2), effector
T-cells (Thl and Th2), and NK cells. Based on such interactions, Applicants
discovered
that mesenchymal stem cells may regulate the production of various factors
that may
regulate several steps in the immune response process. Thus, the mesenchymal
stem
cells may be employed in the treatment of disease conditions and disorders
involving
the immune system, or diseases, conditions, or disorders involving
inflammation or
allergic responses. Such diseases, conditions, and disorders include, but are
not
limited to, autoimmune diseases, allergies, arthritis, inflamed wounds,
alopecia araeta
(baldness), periodontal diseases including gingivitis and periodontitis, and
other
diseases, conditions or disorders involving an immune response.
In addition, it is believed that mesenchymal stem cells stimulate peripheral
blood
mononuclear cells (PBMCs) to produce vascular endothelial growth factor, or
VEGF,
which promotes angiogenesis by stimulating the formation of new blood vessels.
Furthermore, it is believed that mesenchymal stem cells stimulate dendritic
cells
(DCs) to produce Interferon-Beta (IFN-a), which promotes tumor suppression and
immunity against viral infection.
In accordance with an aspect of the present invention, there is provided a
method of treating an autoimmune disease in an animal. The method comprises
administering to the animal mesenchymal stem cells in an amount effective to
treat the
autoimmune disease in the animal.
Although the scope of this aspect of the present invention is not to be
limited to
any theoretical reasoning, it is believed that at least one mechanism by which
the
mesenchymal stem cells suppress autoimmune disease is by causing the release
of
Interleukin-10 (IL-10) from regulatory T-cells (T,g cells) and/or dendritic
cells (DC),
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Autoimmune diseases which may be treated in accordance with the present
invention include, but are not limited to, multiple sclerosis, Type 1
diabetes, rheumatoid
arthritis, uveitis, autoimmune thyroid disease, inflammatory bowel disease,
autoimmune
lymphoproliferative disease (ALPS), demyelinating disease, autoimmune
encephalomyelitis, autoimmune gastritis (AIG), and autoimmune glomerular
diseases.
It is to be understood, however, that the scope of the present invention is
not to be
limited to the treatment of the specific autoimmune diseases mentioned herein.
In one embodiment, the animal to which the mesenchymal stem cells are
administered is a mammal. The mammal may be a primate, including human and non-
human primates.
In general, the mesenchymal stem cell (MSC) therapy is based, for example, on
the following sequence: harvest of MSC-containing tissue, isolation and
expansion of
MSCs, and administration of the MSCs to the animal, with or without
biochemical or
genetic manipulation.
The mesenchymal stem cells that are administered may be a homogeneous
composition or may be a mixed cell population enriched in MSCs. Homogeneous
mesenchymal stem cell compositions may be obtained by culturing adherent
marrow or
periosteal cells, and the mesenchymal stem cell compositions may be obtained
by
culturing adherent marrow or periosteal cells, and the mesenchymal stem cells
may be
identified by specific cell surface markers which are identified with unique
monoclonal
antibodies. A method for obtaining a cell population enriched in mesenchymal
stem
cells is described, for example, in U.S. Patent No. 5,486,359. Alternative
sources for
mesenchymal stem cells include, but are not limited to, blood, skin, cord
blood, muscle,
fat, bone, and perichondriunn.
The mesenchymal stem cells may be administered by a variety of procedures.
The mesenchymal stem cells may be administered systemically, such as by
intravenous, intraarterial, or intraperitoneal administration.
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,
The mesenchymal stem cells may be from a spectrum of sources including
autologous, allogeneic, or xenogeneic.
The mesenchymal stem cells are administered in an amount effective to treat an
autoimmune disease in an animal. The mesenchymal stem cells may be
administered
in an amount of from about 1x105 cells/kg to about 1x107 cells/kg, preferably
from about
1x106 cells/kg to about 5x106 cells/kg. The amount of mesenchymal stem cells
to be
administered is dependent upon a variety of factors, including the age,
weight, and sex
of the patient, the autoimmune disease to be treated, and the extent and
severity
thereof.
The mesenchymal stem cells may be administered in conjunction with an
acceptable pharmaceutical carrier. For example, the mesenchymal stem cells may
be
administered as a cell suspension in a pharmaceutically acceptable liquid
medium for
injection. ,
In accordance with another aspect of the present invention, there is provided
a
method of treating an inflammatory response in an animal. The method comprises
administering to the animal mesenchymal stem cells in an amount effective to
treat the
inflammatory response in the animal.
Although the scope of this aspect of the present invention is not to be
limited to
any theoretical reasoning, it is believed that the mesenchymal stem cells
promote T-cell
maturation to regulatory T-cells (Treg), thereby controlling inflammatory
responses. It is
also believed that the mesenchymal stem cells inhibit T helper 1 cells (Th1
cells),
thereby decreasing the expression of the Interferon1 (IFN-y) in certain
inflammatory
reactions, such as those associated with psoriasis, for example.
In one embodiment, the inflammatory responses which may be treated are those
associated with psoriasis.
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In another embodiment, the mesenchymal stem Cells may be administered to an
animal such that the mesenchymal stem cells contact microglia and/or
astrocytes In the
brain to reduce Inflammation, whereby the mesenchymal stem cells limit
neurodegeneration caused by activated glial cells in diseases or disorders
such as
Alzhelmera Disease, Parkinson's Disease, stroke, or brain cell injuries.
In yet another embodiment, the mesenchymal stem cells may be administered to
an animal such that the mesenchymal stem cells contact keratlnocytes and
Langerhans
cells in the epidermis of the skin to reduce inflammation as may occur in
psoriasis,
chronic dermatitis, and contact dermatitis. Although this embodiment Is not to
be limited =
to any.theoretical reasoning, it is believed that the mesenchymal stem cells
may contact
the keratinocytes and Langerhans cells In the epidermis, and alter the
expression of T-
eell receptors and cytoklne secretion profiles, leading to decreased
expression of tumor
necrosis factor-alpha (TNF-a) and Increased regulatory T-cell (Treg cell)
population.
In a further embodiment, the mesenchymal stem cells may be used to reduce
Inflammation in the bone, as occurs in arthritis and arthritis-like
conditions, including but
not limited to, osteoarthritis and rheumatoid arthritis, and other arthritic
diseases.
Although the scope of this embodiment
Is not intended to be limited to any theoretical reasoning, It Is believed
that the
mesenchymal stem cells may inhibit interleukIn-17 secretion by memory T-cells
In the
synovial fluid.
In another embodiment, the mesenchymal stem cells may be used to limit
inflammation in the gut and liver during Inflammatory bowel disease and
chronic
hepatitis, respectively. Although the scope of this aspect of the present
invention Is not
Intended to be limited to any theoretical reasoning, it is believed that the
mesenchymal
stem cells promote Increased secretion of interieukIn-10 (IL-10) and the
generation of
regulatory T-celis (Treg cells).
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The present invention as claimed relates to:
(1) cultured mesenchymal stem cells for use in treating inflammation
associated with graft vs. host disease (GvHD) in an animal;
(2) the mesenchymal stem cells for use according to (1), wherein the
mesenchymal stem cells inhibit TNF-a secretion and promote IL-10 secretion
from
activated DC1 and DC2 dendritic cells;
(3) the mesenchymal stem cells for use according to (1) or (2), wherein the
mesenchymal stem cells inhibit T helper 1 cells (Th1 cells), thereby
inhibiting
secretion of Interferon-y (IFN-y);
(4) the mesenchymal stem cells for use according to any one of (1)-(3),
wherein the mesenchymal stem cells directly interact with mature T cells and
inhibit
pro-inflammatory Interferon-y (I FN-y) production;
(5) the mesenchymal stem cells for use according to any one of (1)-(4),
wherein the mesenchymal stem cells inhibit excessive neutrophil and macrophage
activation;
(6) the mesenchymal stem cells for use according to (5), wherein the
mesenchymal stem cells inhibit excessive neutrophil and macrophage activation
by
promoting secretion of IL-10;
(7) the mesenchymal stem cells for use according to any one of (1)-(6),
wherein the mesenchymal stem cells promote T-cell maturation to regulatory T-
cells
(Treg);
(8) the mesenchymal stem cells for use according to any one of (1)-(7),
wherein the mesenchymal stem cells promote the regulatory T cell phenotype;
(9) the mesenchymal stem cells for use according to any one of (1) to (8),
wherein the mesenchymal stem cells are for systemic administration;
(10) the mesenchymal stem cells for use according to any one of (1)-(9),
wherein the mesenchymal stem cells are genetically unmanipulated;
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(11) use of cultured mesenchymal stem cells for treating inflammation
associated with graft vs. host disease (GyHD) in an animal;
(12) the use according to (11), wherein the mesenchymal stem cells inhibit
TNF-a secretion and promote IL-10 secretion from activated DC1 and DC2
dendritic
cells;
(13) the use according to (11) or (12), wherein the mesenchymal stem cells
inhibit T helper 1 cells (Th1 cells), thereby inhibiting secretion of
Interferon-y
(IFN-y);
(14) the use according to any one of (1 1 )-(1 3), wherein the mesenchymal
stem cells directly interact with mature T cells and inhibit pro-inflammatory
Interferon-y (IFN-y) production;
(15) the use according to any one of (11)-(14), wherein the mesenchymal
stem cells inhibit excessive neutrophil and macrophage activation;
(16) the use according to (15), wherein the mesenchymal stem cells inhibit
excessive neutrophil and macrophage activation by promoting secretion of IL-
10;
(17) the use according to any one of (1 1)-(1 6), wherein the mesenchymal
stem cells promote T-cell maturation to regulatory T-cells (Treg);
(18) the use according to any one of (11)-(17), wherein the mesenchymal
stem cells promote the regulatory T cell phenotype;
(19) the use according to any one of (11) to (18), wherein the mesenchymal
stem cells are for systemic administration;
(20) the use according to any one of (11)-(19), wherein the mesenchymal
stem cells are genetically unmanipulated;
(21) the mesenchymal stem cells for use according to any one of (1)-(10),
wherein the mesenchymal stem cells are for administration in an amount from
1x105 cell/kg of the animal to 1x107 cells/kg of the animal;
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(22) the mesenchymal stem cells for use according to any one of (1)-(10),
wherein the mesenchymal stem cells are for administration in an amount from
1x106 cell/kg of the animal to 5x106 cells/kg of the animal;
(23) the mesenchymal stem cells for use according to any one of (1)-(10),
(21), and (22), wherein the mesenchymal stem cells are for administration in a
cell
suspension in a pharmaceutically acceptable liquid medium;
(24) the mesenchymal stem cells for use according to any one of (1)-(10)
and
(21)-(23), wherein the mesenchymal stem cells are for intravenous
administration;
(25) the mesenchymal stem cells for use according to any one of (1)-(10)
and
(21)-(24), wherein the mesenchymal stem cells are allogeneic;
(26) the mesenchymal stem cells for use according to any one of (1)-(10)
and
(21)-(25), wherein the animal is human;
(27) the use according to any one of (11)-(20), wherein the mesenchymal
stem cells are for administration in an amount from 1x106 cell/kg of the
animal to
1x107 cells/kg of the animal;
(28) the use according to any one of (11)-(20), wherein the mesenchymal
stem cells are for administration in an amount from 1x106 cell/kg of the
animal to
5x106 cells/kg of the animal;
(29) the use according to any one of (11)-(20), (27) and (28), wherein the
mesenchymal stem cells are for administration in a cell suspension in a
pharmaceutically acceptable liquid medium;
(30) the use according to any one of (11)-(20) and (27)-(29), wherein the
mesenchymal stem cells are for intravenous administration;
(31) the use according to any one of (11)-(20) and (27)-(30), wherein the
mesenchymal stem cells are allogeneic; and
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(32) the use according to any one of (11)-(20) and (27)-(31),
wherein the
animal is human.
In another embodiment, the mesenchymal stem cells may be used to
inhibit excessive neutrophil and macrophage activation in pathological
conditions
such as
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sepsis and trauma, including burn injury, surgery, and transplants. Although
the scope
of this embodiment is not to be limited to any theoretical reasoning, it is
beleived the
mesenchymal stem cells promote secretion of suppressive cytokines such as IL-
10, and
inhibit macrophage migration inhibitory factor.
In another embodiment, the mesenchymal stem cells may be used to control
inflammation in immune privileged sites such as the eye, including the cornea,
lens,
pigment epithelium, and retina, brain, spinal cord, pregnant uterus and
placenta, ovary,
testes, adrenal cortex, liver, and hair follicles. Although the scope of this
embodiment is
not to be limited to any theoretical reasoning, it is believed that the
mesenchymal stem
cells promote the secretion of suppressive cytokines such as IL-10 and the
generation
of Treg cells.
In yet another embodiment, the mesenchymal stem cells may be used to control
end-stage renal disease (ESRD) infections during dialysis and/or
glomerulonephritis.
Although the scope of this embodiment is not to be limited to any theoretical
reasoning,
it is believed that mesenchymal stem cells induce peripheral blood mononuclear
cells to
express vascular endothelial growth factor, or VEGF, which stimulates
glomerular
structuring.
In a further embodiment, the mesenchymal stem cells may be used to control
viral infections such as influenza, hepatitis C, Herpes Simplex Virus,
vaccinia virus
infections, and Epstein-Barr virus. Although the scope of this embodiment is
not to be
limited to any theoretical reasoning, it is believed that the mesenchymal stem
cells
promote the secretion of Interferon-Beta (IFN-I3).
In yet another embodiment, the mesenchymal stem cells may be used to control
parasitic infections such as Leishmania infections and Helicobacter
infections. Although
the scope of this embodiment is not to be limited to any theoretical
reasoning, it is
believed that the mesenchymal stem cells mediate responses by T helper 2 (Th2)
cells,
and thereby promote increased production of lmmunoglobulin E (IgE) by 13-
cells.
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It is to be understood, however, that the scope of this aspect of the present
invention is not to be limited to the treatment of any particular inflammatory
response.
The mesenchymal stem cells may be administered to a mammal, including
human and non-human primates, as hereinabove described.
The mesenchymal stem cells also may be administered systemically, as
hereinabove described. Alternatively, in the case of osteoarthritis or
rheumatoid
arthritis, the mesenchymal stem cells may be administered directly to an
arthritic joint.
The mesenchymal stem cells are administered in an amount effective to treat an
inflammatory response in an animal. The mesenchymal stem cells may be
administered in an amount of from about 1x105 cells/kg to about 1x107
cells/kg,
preferably from about 1x106 cells/kg to about 5x106 cells/kg. The exact dosage
of
mesenchymal stem cells to be administered is dependent upon a variety of
factors,
including the age, weight, and sex of the patient, the inflammatory response
being
treated, and the extent and severity thereof.
The mesenchymal stem cells may be administered in conjunction with an
acceptable pharmaceutical carrier, as hereinabove described.
In accordance with yet another aspect of the present invention, there is
provided
a method of treating cancer in an animal. The method comprises administering
to the
animal mesenchymal stem cells in an amount effective to treat cancer in the
animal.
Although the scope of this aspect of the present invention is not to be
limited to
any theoretical reasoning, it is believed that the mesenchymal stem cells
interact with
dendritic cells, which leads to IFN-j3 secretion, which in turn acts as a
tumor suppressor.
Cancers which may be treated include, but are not limited to, hepatocellular
carcinoma,
cervical cancer, pancreatic cancer, prostate cancer, fibrosarcoma,
medullablastoma,
and astrocytoma. It is to be understood, however, that the scope of the
present
invention is not to be limited to any specific type of cancer.
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The animal may be a mammal, including human and non-human primates, as
hereinabove described.
The mesenchymal stem cells are administered to the animal in an amount
effective to treat cancer in the animal. In general, the mesenchymal stem
cells are
administered in an amount of from about 1x105 cells/kg to about 1x107
cells/kg,
preferably from about 1x106 cells/kg to about 5x105 cells/kg. The exact amount
of
mesenchymal stem cells to be administered is dependent upon a variety of
factors,
including the age, weight, and sex of the patient, the type of cancer being
treated, and
the extent and severity thereof.
The mesenchymal stem cells are administered in conjunction with an acceptable
pharmaceutical carrier, and may be administered sytemically, as hereinabove
described. Alternatively, the mesenchymal stem cells may be administered
directly to
the cancer being treated.
In accordance with still another aspect of the present invention, there is
provided
a method of treating an allergic disease or disorder in an animal. The method
comprises administering to the animal mesenchymal stem cells in an amount
effective
to treat the allergic disease or disorder in the animal.
Although the scope of this aspect of the present invention is not to be
limited to
any theoretical reasoning, it is believed that mesenchymal stem cells, when
administered after an acute allergic response, provide for inhibition of mast
cell
activation and degranulation. Also, it is believed that the mesenchymal. stem
cells
downregulate basophil activation and inhibit cytokines such as TNF-a,
chemokines such
as Interleukin-8 and monocyte chemoattractant protein, or MCP-1, lipid
mediators such
as leukotrienes, and inhibit main mediators such as histamine, heparin,
chondroitin
sulfates, and cathepsin.
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Allergic diseases or disorders which may be treated include, but are not
limited
to, asthma, allergic rhinitis, atopic dermatitis, and contact dermatitis. It
is to be
understood, however, that the scope of the present invention is not to be
limited to any
specific allergic disease or disorder
The mesenchymal stem cells are administered to the animal in an amount
effective to treat the allergic disease or disorder in the animal. The animal
may be a
mammal. The mammal may be a primate, including human and non-human primates.
In general, the mesenchymal stem cells are administered in an amount of from
about
1x105 cells/kg to about 1x107 cells/kg, preferably from about 1x106 cells/kg
to about
5x106 cells/kg. The exact dosage is dependent upon a variety of factors,
including the
age, weight, and sex of the patient, the allergic disease or disorder being
treated, and
the extent and severity thereof.
The mesenchymal stem cells may be administered in conjunction with an
acceptable pharmaceutical carrier, as hereinabove described. The mesenchymal
stem
cells may be administered systemically, such as by intravenous or
intraarterial
administration, for example.
In accordance with a further aspect of the present invention, there is
provided a
method of promoting wound healing in an animal. The method comprises
administering
to the animal mesenchymal stem cells in an amount effective to promote wound
healing
in the animal.
Although the scope of the present invention is not to be limited to any
theoretical
reasoning, it is believed that, as mentioned hereinabove, the mesenchymal stem
cells
cause Treg cells and dendritic cells to release Interleukin-10 (IL-10). The IL-
10 limits or
controls inflammation in a wound, thereby promoting healing of a wound.
Furthermore, the mesenchymal stem cells may promote wound healing and
fracture healing by inducing secretion factors by other cell types. For
example, the
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mesenchymal stem cells may induce prostaglandin E2 (PGE2)-mediated release of
vascular endothelial growth factor (VEGF) by peripheral blood mononuclear
cells
(PBMCs), as well as PGE2-mediated release of growth hormone, insulin, insulin-
like
growth factor 1 (IGF-1) insulin-like growth factor binding protein-3 (IGFBP-
3), and
endothelin-1.
The mesenchymal stem cells are administered to the animal in an amount
effective to promote wound healing in the animal. The animal may be a mammal,
and
the mammal may be a primate, including human and non-human primates. In
general,
the mesenchymal stem cells are administered in an amount of from about 1x106
cells/kg
to about 1x107 cells/kg, preferably from about 1x106 cells/kg to about 5x106
cells/kg.
The exact amount of mesenchymal stem cells to be administered is dependent
upon a
variety of factors, including the age, weight, and sex of the patient, and the
extent and
severity of the wound being treated.
The mesenchymal stem cells may be administered in conjunction with an
acceptable pharmaceutical carrier, as hereinabove described. The mesenchymal
stem
cells may be administered systemically, as hereinabove described.
Alternataively, the
mesenchymal stem cells may be administered directly to a wound, such as in a
fluid on
a dressing or reservoir containing the mesenchymal stem cells.
In accordance with yet another aspect of the present invention, there is
provided
a method of treating or preventing fibrosis in an animal. The method comprises
administering to the animal mesenchymal stem cells in an amount effective to
treat or
prevent fibrosis in an animal.
The mesenchymal stem cells may be administered to the animal in order to treat
or prevent any type of fibrosis in the animal, including, but not limited to,
cirrhosis of the
liver, fibrosis of the kidneys associated with end-stage renal disease, and
fibrosis of the
lungs. It is to be understood that the scope of the present invention is not
to be limited
to any specific type of fibrosis.
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The mesenchymal stem cells are administered to the animal in an amount
effective to treat or prevent fibrosis in the animal. The animal may be a
mammal, and
the mammal may be a primate, including human and non-human primates. In
general,
the mesenchymal stem cells are administered in an amount of from about 1 xl Cr
cells/kg
to about 1x107 cells/kg, preferably from about 1x106 cells/kg to about 5x10r
cells/kg.
The exact amount of mesenchymal stem cells to be administered is dependent
upon a
variety of factors, including the age, weight, and sex of the patient, and the
extent and
severity of the fibrosis being treated or prevented.
The mesenchymal stem cells may be administered in conjunction with an
acceptable pharmaceutical carrier, as hereinabove described. The mesenchymal
stem
cells may be administered systemically, also as hereinabove described.
It is another object of the present invention to promote angiogenesis in a
tissue
or organ of an animal, wherein such tissue or organ is in need of
angiogenesis.
Thus, in accordance with a further aspect of the present invention, there is
provided a method of promoting angiogenesis in an organ or tissue of an
animal. The
method comprises administering to the animal mesenchymal stem cells in an
amount
effective to promote angiogenesis in an organ or tissue of the animal.
Angiogenesis is the formation of new blood vessels from a pre-existing
microvascular bed.
The induction of angiogenesis may be used to treat coronary and peripheral
artery insufficiency, and thus may be a noninvasive and curative approach to
the
treatment of coronary artery disease, ischemic heart disease, and peripheral
artery
disease. Angiogenesis may play a role in the treatment of diseases and
disorders in
tissue and organs other than the heart, as welt as in the development and/or
maintenance of organs other than the heart. Angiogenesis may provide a role in
the
treatment of internal and external wounds, as well as dermal ulcers.
Angiogenesis also
plays a role in embryo implantation, and placental growth, as well as the
development of
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the embryonic vasculature. Angiogenesis also is essential for the coupling of
cartilage
resorption with bone formation, and is essential for correct growth plate
morphogenesis.
Furthermore, angiogenesis is necessary for the successful engineering and
maintenance of highly metabolic organs, such as the liver, where a dense
vascular
network is necessary to provide sufficient nutrient and gas transport.
The mesenchymal stern cells can be administered to the tissue or organ in need
of angiogenesis by a variety of procedures. The mesenchymal stem cells may be
administered systemically, such as by intravenous, intraarterial, or
intraperitoneal
administration, or the mesenchymal stem cells may be administered directly to
the
tissue or organ in need of angiogenesis, such as by direct injection into the
tissue or
organ in need of angiogenesis.
The mesenchymal stem cells may be from a spectrum of sources including
autologous, allogeneic, or xenogeneic.
Although the scope of the present invention is not to be limited to any
theroretical
reasoning, it is believed that the mesenchymal stem cells, when administered
to an
animal, stimulate peripheral blood mononuclear cells (PBMCs) to produce
vascular
endothelial growth factor, or VEGF, which stimulates the formation of new
blood
vessels.
In one embodiment, the animal is a mammal. The mammal may be a primate,
including human and non-human primates.
The mesenchymal stem cells, in accordance with the present invention, may be
employed in the treatment, alleviation, or prevention of any disease or
disorder which
can be alleviated, treated, or prevented through angiogenesis. Thus, for
example, the
mesenchymal stem cells may be administered to an animal to treat blocked
arteries,
including those in the extremities, i.e., arms, legs, hands, and feet, as well
as the neck
or in various organs. For example, the mesenchymal stem cells may be used to
treat
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blocked arteries which supply the brain, thereby treating or preventing
stroke. Also, the
mesenchymal stem cells may be used to treat blood vessels in embryonic and
post-
natal corneas and may be used to provide glomerular structuring. In another
embodiment, the mesenchymal stem cells may be employed in the treatment of
wounds, both internal and external, as well as the treatment of dermal ulcers
found in
the feet, hands, legs or arms, including, but not limited to, dermal ulcers
caused by
diseases such as diabetes and sickle cell anemia.
Furthermore, because angiogenesis is involved in embryo implantation and
placenta formation, the mesenchymal stem sells may be employed to promote
embryo
implantation and prevent miscarriage.
In addition, the mesenchymal stem cells may be administered to an unborn
animal, including humans, to promote the development of the vasculature in the
unborn
animal.
In another embodiment, the mesenchymal stem cells may be administered to an
animal, born or unborn, in order to promote cartilage resorption and bone
formation, as
well as promote correct growth plate morphogenesis.
The mesenchymal stem cells are administered in an amount effective in
promoting angiogenesis in an animal. The mesenchymal stem cells may be
administered in an amount of from about 1x105 cells/kg to about 1x107
cells/kg,
preferably from about 1x106 cells/kg to about 5x106 cells/kg. The amount of
mesenchymal stem cells to be administered is dependent upon a variety of
factors,
including the age, weight, and sex of the patient, the disease or disorder to
be treated,
alleviated, or prevented, and the extent and severity thereof.
The mesenchymal stem cells may be administered in conjunction with an
acceptable pharmaceutical carrier. For example, the mesenchymal stem cells may
be
administered as a cell suspension in a pharmaceutically acceptable liquid
medium for
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injection. Injection can be local, i.e., directly into the tissue or organ in
need of
angiogenesis, or systemic.
The mesenchymal stem cells may be genetically engineered with one or more
polynucleotides encoding a therapeutic agent. The polynucleotides may be
delivered to
the mesenchymal stem cells via an appropriate expression vehicle. Expression
vehicles which may be employed to genetically engineer the mesenchymal stem
cells
include, but are not limited to, retroviral vectors, adenoviral vectors, and
adeno-
associated virus vectors.
The selection of an appropriate polynucleotide encoding a therapeutic agent is
dependent upon various factors, including the disease or disorder being
treated, and the
extent and severity thereof. Polynucleotides encoding therapeutic agents,
and
appropriate expression vehicles are described further in U.S. Patent No.
6,355,239.
It is to be understood that the mesenchymal stem cells, when employed in the
above-mentioned therapies and treatments, may be employed in combination with
other
therapeutic agents known to those skilled in the art, including, but not
limited to, growth
factors, cytokines, drugs such as anti-inflammatory drugs, and cells other
than
mesenchymal stem cells, such as dendritic cells, and may be administered with
soluble
carriers for cells such as hyalurionic acid, or in combination with solid
matrices, such
collagen, gelatin, or other biocompatible polymers, as appropriate.
It is to be understood that the methods described herein may be carried out in
a
number of ways and with various modifications and permutations thereof that
are well
known in the art. It also may be appreciated that any theories set forth as to
modes of
action or interactions between cell types should not be construed as limiting
this
invention in any manner, but are presented such that the methods of the
invention can
be understood more fully.
The invention now will be described with respect to the drawings, wherein:
Fig. 1 MSCs modulate dendritic cell functions. (A) Flow cytometric analysis of
mature monocytic DC1 cells using antibodies against HLA-DR and CD11c and of
plasmacytoid DC2 cells using antibodies against HLA-DR and CD123 (IL-3
receptor). (--
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-): isotype control; ( ): FITC/PE conjugated antibodies. (B) MSCs inhibit
INF-a
secretion (primary y-axis) and increase IL-10 secretion (secondary y-axis)
from
activated DC1 and DC2 respectively. (C) MSCs cultured with mature DC1 cells
inhibit
IFN-y secretion (primary y-axis) by T cells and increase IL-4 levels
(secondary y-axis) as
compared to MSC or DC alone. The decreased production of pro-inflammatory IFN-
y
and increased production of anti-inflammatory IL-4 in the presence of MSCs
indicated a
shift in the T cell population towards an anti-inflammatory phenotype.
Fig. 2 MSCs inhibit pro-inflammatory effector T cell function. (A) Flow
cytometric analysis of TReg cell numbers (in %) by staining PBMCs or non-
adherent
fraction in MSC+PBMC culture (MSC+PBMC) with FITC-conjugated CD4 (x-axis) and
PE conjugated CD25 (y-axis) antibodies. Gates were set based on isotype
control
antibodies as background. Graphs are representative of 5 independent
experiments. (B)
TH1 cells generated in presence of MSCs secreted reduced levels of IFN-y
(primary y-
axis) and TH2 cells generated in presence of MSCs secreted increased amounts
of IL-4
(secondary y-axis) in cell culture supernatants. (C) MSCs inhibit IFN-y
secretion from
purified NK cells cultured for 0, 24, or 48 hours in a 24-well plate. Data
shown are
mean SD cytokine secretion in one experiment and are representative of 3
independent
experiments.
Fig. 3 MSCs lead to increased numbers of Treg cell population and
increased GITR expression. (A) A CD4+ CD25+ Treg cell population from PBMC or
MSC + PBMC (MSC to PBMC ratio 1:10) cultures (cultured without any further
stimulation for 3 days) was isolated using a 2-step magnetic isolation
procedure. These
cells were irradiated (to block any further proliferation) and used as
stimulators in a
mixed lymphocyte reaction (MLR), where responders were allogeneic PBMCs
(stimulator to responder ratio 1:100) in the presence of phytohemagglutinin
(PHA) (2.5
mg/ml). The cells were cultured for 48 hours, following which 3H thymidime was
added,
and incorporated radioactivity was counted after 24 hours. The results showed
that the
Treg population generated in the presence of MSCs (lane 3) was similar
functionally to
the Treg cells generated in the absence of MSCs (lane 2). (B) PBMCs were
cultured for 3
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days in the absence (top plot) or presence (bottom plot) of MSCs (MSC to PBMC
ratio
1:10), following which the non-adherent fraction was harvested and
immunostained with
FITC-labeled GITR and PE-labeled CD4. Results show a greater than twofold
increase
in GITR expression in cells cultured in the presence of MSCs.
Fig. 4 MSCs produce PGE2 and blocking PGE2 reverses MSC-mediated
immuno-modulatory effects . (A) PGE2 secretion (mean SD) in culture
supernatants
obtained from MSCs cultured in the presence or absence of PGE2 blockers NS-398
or
indomethacin (Indometh.) at various concentrations. Inhibitor concentrations
are in 1.1M
and data presented are values obtained after 24 hour culture (B) COX-1 and COX-
2
expression in MSCs and PBMCs using real-time RT-PCR. MSCs expressed
significantly higher levels of COX-2 as compared to PBMCs, and when MSCs were
cultured in presence of PBMCs, there was a >3-fold increase in COX-2
expression in
MSCs. Representative data from 1 of 3 independent experiments is shown. The
MSC+PBMC cultures were setup in a trans-well chamber plate where MSCs were
plated onto the bottom chamber and PBMCs onto the top chamber. (C) Presence of
PGE2 blockers indomethacin (Ind.) or NS-398 increases INF-a secretion from
activated
DCs (D) and IFNI secretion from TH1 cells (n!) as compared to controls. Data
were
calculated as % change from cultures generated in absence of MSCs and PGE2
inhibitors (C) Presence of PGE2 blockers indomethacin (lndo) and NS-398 during
MSC-
PBMC co-culture (1:10) reverses MSC-mediated anti-proliferative effects on PHA-
treated PBMCs. Data shown are from one experiment and are representative of 3
independent experiments.
Fig. 5 Constituitive MSC cytokine secretion is elevated in the presence of
allogeneic PBMCs. Using previously characterized human MSCs, the levels of the
cytokines IL-6 and VEGF, lipid mediator PGE2, and matrix metalloproteinase 1
(pro-
MMP-1) in culture supernatant of MSCs cultured for 24 hours in the presence
(hatched
bars) or absence (open bars) of PBMCs (MSC to PBMC ratio 1:10) were analyzed.
The
MSCs produced IL-6, VEGF, and PGE2 constituitively, and the levels of these
factors
increased upon co-culture with PBMCs, thereby suggesting that MSCs may play a
role
in modulating immune functions in an inflammatory setting.
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Fig. 6 MSCs inhibit mitogen-induced T-cell proliferation in a dose-
dependent manner. Increasing numbers of allogeneic PBMCs were incubated with
constant numbers of MSCs (2,000 cells/well) plated on a 96-well plate in the
presence
or absence of PHA (2.5 mg/ml) for 72 hours, and 3H thymidine incorporation
determined
(in counts per minute, or cpm). There was a dose-dependent inhibition of the
proliferation of PHA-treated PBMCs in the presence of MSCs. Representative
results
from 1 of 3 independent experiments are shown. Similar results were reported
by
LeBlanc, et al., Scand J. Immunol., Vol. 57, pg. 11 (2003).
Fig. 7 Schematic diagram of proposed MSC mechanism of action
MSCs mediate their immuno-modulatory effects by affecting cells from both the
innate (DCs- pathways 2-4; and NK- pathway 6) and adaptive (T- pathways 1 and
5 and
B-pathway 7) immune systems. In response to an invading pathogen, immature DCs
migrate to the site of potential entry, mature and acquire an ability to prime
naive T cells
(by means of antigen specific and co-stimulatory signals) to become protective
effector
T cells (cell-mediated TH1 or humoral TH2 immunity). During MSC-DC
interaction,
MSCs, by means of direct cell-cell contact or via secreted factor, may alter
the outcome
of immune response by limiting the ability of DCs to mount a cell-mediated
response
(pathway 2) or by promoting the ability to mount a humoral response (pathway
4). Also,
when mature effector T cells are present, MSCs may interact with them to skew
the
balance of THI (pathway 1) responses towards TH2 responses (pathway 5), and
probably towards an increased IgE producing B cell activity (pathway 7),
desirable
outcomes for suppression of GvHD and autoimmune disease symptoms. MSCs in
their
ability to result in an increased generation of TReg population (pathway 3)
may result in a
tolerant phenotype and may aid a recipient host by dampening bystander
inflammation
in their local micro-environment. Dashed line (----) represents proposed
mechanism.
The invention now will be described with respect to the following example; it
is to
be understood, however, that the scope of the present invention is not to be
limited
thereby.
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Example 1
Materials and Methods
Culture of human MSCs
= Human MSCs were cultured as described by Pittenger et al., Science, Vol.
284,
pg. 143 (1999). Briefly, marrow samples were collected from the iliac crest of
anonymous donors following informed consent by Poietics Technologies, Div of
Cambrex Biosciences. MSCs were cultured in complete Dulbecco's Modified
Eagle's
Medium-Low Glucose (Life Technologies, Carlsbad, California) containing -I%
antibiotic-
antimyotic solution (Invitrogen, Carlsbad, California) and 10% fetal bovine
serum (FBS,
JRH BioSciences, Lenexa, Kansas). MSCs grew as an adherent monolayer and were
detached with trypsin/EDTA (0.05% trypsin at 37 C for 3 minutes). All MSCs
used were
previously characterized for multilineage potential and retained the capacity
to
differentiate into mesenchymal lineages (chondrocytic, adipogenic, and
osteogenic)
(Pittenger, et al., Science, Vol. 284, pg. 143 (1999)).
Isolation of Dendritic cells
Peripheral blood mononuclear cells (PBMCs) were obtained from Poietics
Technologies, Div of Cambrex Biosciences (Walkersville, MD). Precursors of
dendritic
cells (DCs) of monocytic lineage (CD1c+) were positively selected from PBMCs
using a
2-step magnetic separation method according to Dzionek, et. al., J. Immunol.,
Vol. 165,
pg. 6037 (2000). Briefly, CD1c expressing B cells were magnetically depleted
of CD19+
cells using magnetic beads, followed by labeling the B-cell depleted fraction
with biotin-
labeled CD1c (BDCA1+) and anti-biotin antibodies and separating them from the
unlabeled cell fraction utilizing magnetic columns according to the
manufacturer's
instructions (Miltenyi Biotech, Auburn, California). Precursors of DCs of
plasmacytoid
lineage were isolated from PBMCs by immuno-magnetic sorting of positively
labeled
antibody coated cells (BDCA2+) (Miltenyi Biotech, Auburn, California).
MSC-DC culture
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In most experiments, human MSCs and DCs were cultured in equal numbers for
various time periods and cell culture supernatant collected and stored at ¨80
C until
further evaluation. In selected experiments, MSCs were cultured with mature
DC1 or
DC2 cells (1:1 MSC:DC ratio) for 3 days, and then the combined cultures (MSCs
and
DCs) were irradiated to prevent any proliferation. Next, antibody purified,
naïve,
allogeneic T cells (CD4+,CD45RA+) were added to the irradiated MSCs/DCs and
cultured for an additional 6 days. The non-adherent cell fraction (purified T
cells) was
then collected from the cultures, washed twice and re-stimulated with PHA for
another
24 hours, following which cell culture supematants were harvested and analyzed
for
secreted IFN-y and IL-4 by ELISA.
Isolation of NK cells
Purified populations of NK cells were obtained by depleting non-NK cells that
are
magnetically labeled with a cocktail of biotin-conjugated monoclonal
antibodies (anti -
CD3, -CD14, -CD19, -CD36 and anti-IgE antibodies) as a primary reagent and
anti-
biotin monoclonal antibodies conjugated to Microbeads as secondary labeling
reagent.
The magnetically labeled non-NK cells were retained in MACS (Miltenyi Biotech,
Auburn, California) columns in a magnetic field, while NK cells passed through
and
were collected.
Isolation of TReg cell population
The TReg cell population was isolated using a 2-step isolation procedure.
First
non-CD4+ T cells were indirectly magnetically labeled with a cocktail of
biotin labeled,'
antibodies and anti-biotin microbeads. The labeled cells were then depleted by
separation over a MACS column (Miltenyi Biotech, Auburn, California). Next,
CD41.CD251. cells were directly labeled with CO25 microbeads and isolated by
positive
selection from the pre-enriched CD4+ T cell fraction. The magnetically labeled
CD4+CD25+ T cells were retained on the column and eluted after removal of the
column
from the magnetic field.
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In order to determine whether the increased CD4+CD25+ population generated
in the presence of MSCs were suppressive in nature, CD4+CD25+ Treg cell
populations
were isolated from PBMC or MSC+PBMC (MSC to PBMC ratio 1:10) cultures
(cultured
without any further stimulation for 3 days) using a 2-step magnetic isolation
procedure.
These cells were irradiated to block any further proliferation and used as
stimulators in a
mixed lymphocyte reaction (MLR), where responders were allogeneic PBMCs
(stimulator to responder ratio 1:100) in the presence of PHA (2.5 pg/ml). The
culture
was carried out for 48 hours, following which 3H thymidine was added.
Incorporated
radioactivity was counted after 24 hours.
PBMCs were cultured in the absence or presence of MSCs (MSC to PBMC ratio
1:10), following which the non-adherent fraction was harvested and
immunostained with
FITC-labeled glucocorticoid-induced TNF receptor, or GITR, and PE -labeled
CD4.
Generation of TH1ITH2 cells
Peripheral blood mononuclear cells (PBMCs) were plated at 2x106 cells/mil for
45
min. at 37 C in order to remove monocytes. Non-adherent fraction was incubated
in the
presence of plate-bound anti-CD3 (5 pg/m1) and anti-CD28 (1 1.1g/m1)
antibodies under
TH1 (IL-2 (4 ng/ml) + IL-12 (5 ng/ml) + anti-IL-4 (1fig/m1)) or TH2 (IL-2 (4
ng/ml) + IL-4 (4
ng/ml) + anti-IFN-y (1 gimp) conditions for 3 days in the presence or absence
of MSCs.
The cells were washed and then re-stimulated with PHA (2.5 gimp for another
24 or 48
hours, following which levels of IFN-y and IL-4 were measured in culture
supernatants
by ELISA (R&D Systems, Minneapolis, Minnesota).
Analysis of levels of VEGF, PGE2, and pro-MMP-1 in culture supernatant of
MSCs.
Using previously characterized human MSCs, the levels of Interleukin-6 (IL-6),
VEGF, lipid mediator prostaglandin E2 (PGE2), and matrix metalloproteinase 1
(pro-
MMP-1) were analyzed in culture supernatant of MSCs cultured for 24 hours in
the
presence or absence of PBMCs (MSC to PBMC ratio 1:10).
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Prqliferation of PBMCs
Purified PEINICs were prepared by centrifuging LeukopackTM (Cambrex,
Walkersville, Maryland) on Ficoll-HypaquTMe (Lymphoprep, 'Oslo, Norway).
Separated
cells were cultured (in triplicates) in the presence or absence of MSCs
(plated 3-4 hours
prior to PBMC addition to allow them to settle) for 48 hours in presence of
the mitogen
= PHA (Sigma Chemicals, St. Louis, Missouri). In selected experiments,
PBMCs were
resuspended in medium containing PGE2 inhibitors lndomethacin (Sigma
Chemicals,
St. Louis, Missouri) or NS-938 (Cayman Chemicals, Ann Arbor, Michigan).
(3H)thymidine was added (20 I In a 200 id culture) and the cells harvested
after an
additional 24 hour culture using an automatic harvester. The effects of MSCs
or PGE2
blockers were calculated as the percentage of the control response (100%) in
presence
of PHA.
Quantitative RT-PCR
Total RNA from cell pellets were prepared using a commercially available kit
(Olagen, Valencia, California) and according to the manufacturer's
instructions.
Contaminating genornic DNA was removed using the DNA-free kit (Amblon, Austin,
Texas). Quantitative RT-PCR was performed on a MJ Research Opticon detection
system (South San Francisco, California) using QuantiTect SYBR GreerimRT-PCR
kit
(Qiagen, Valencia, California) with primers at concentration of 0.5 p.M.
Relative changes
in expression levels in cells cultured under different conditions were
calculated by the
difference in Ct values (crossing point) using 13-actin as Internal control.
The sequence
for COX-1 and COX-2 specific primers were: COX-1: 5'-CCG GAT GCC AGT CAG GAT
GAT G-3Tforward), 5'-CTA GAC AGC CAG ATG CTG ACA G-3' (reverse); COX-2: 5'-
ATO TAC OCT OCT CAA GIG CC-3*(forward), 5'-TAG CAG MG GGC AGG ATA
CAG-3' (reverse).
increasing numbers of allogeneic PBMCs were Incubated with constant numbers
of MSCs (2,000 .celis/well) plated on a 96-well plate in the presence of PHA
(2.5 pg/ml) .
for 72 hours, and 3H thymidine Incorporation (counts per minute, cpm) was
determined.
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The PBMCs and MSCs were cultured at ratios of MSC:PBMC of 1:1, 1:3, 1:10,
1:30,
and 1:81.
Results
In the present studies, the interaction of human MSCs with isolated immune
cell
populations, including dendritic cells (DC1 and DC2), effector T cells (TH1
and TH2) and
NK cells was examined. The interaction of MSCs with each immune cell type had
specific consequences, suggesting that MSCs may modulate several steps in the
immune response process. The production of secreted factor(s) that modulate
and may
be responsible for MSC immuno-modulatory effects was evaluated and
prostaglandin
synthesis was implicated.
Myeloid (DC1) and plasmacytoid (DC2) precursor dendritic cells were isolated
by
immuno-magnetic sorting of BDCA1+ and BDCA2+ cells respectively and matured by
incubation with GM-CSF and IL-4 (1x10311U/m1 and 1x103 IU/ml, respectively)
for DC1
cells, or IL-3 (10 ng/ml) for DC2 cells. Using flow cytometry, DC1 cells were
HLA-DR+
and CD11c+, whereas DC2 cells were HLA-DR+ and CD123+ (Fig. 1A). In the
presence
of the inflammatory agent bacterial lipopolysaccharide (LPS, 1 ng/ml), DC1
cells
produced moderate levels of TNF-a but when MSCs were present (ratios examined
1:1
and 1:10), there was >50% reduction in TNF-a, secretion (Fig. 1B). On the
other hand,
DC2 cells produced IL-10 in the presence of LPS and its levels were increased
greater
than 2-fold upon MSC:DC2 co-culture (1:1) (Fig. 1B). Therefore, the MSCs
modified the
cytokine profile of activated DCs in culture towards a more tolerogenic
phenotype.
Additionally, activated DCs, when cultured with MSCs, were able to reduce IFN-
y and
increase IL-4 levels secreted by naïve CD4+ T cells (Fig. 1C) suggesting a MSC-
mediated shift from pro-inflammatory to anti-inflammatory T cell phenotype.
As increased IL-10 secretion plays a role in generation of regulatory cells
(Kingsley, et al., J. Immunol., Vol. 168, pg. 1080 (2002)), T-regulatory cells
(TReg) were
quantified by flow cytometry in co-cultures of PBMCs and MSCs. Upon culture of
PBMCs with MSCs for 3-5 days, there was an increase in TReg cell numbers as
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determined by staining of PBMCs with anti-CD4 and anti-CD25 antibodies (Fig.
2A),
further supporting a MSC-induced tolerogenic response. The CD4+CD25 TReg cell
population, generated in presence of MSCs expressed increased levels of
gluocorticoid-
induced TNF receptor (GITR), a cell surface receptor expressed on TReg cell
populations, and was suppressive in nature as it suppressed allogeneic T cell
proliferation (Fig. 3A,B). Next, MSCs were investigated as to their direct
ability to affect
T cell differentiation. Using antibody selected purified T cells (CD4+ Th
cells), IFN-y
producing TH1 and IL-4 producing TH2 cells were generated in presence or
absence of
MSCs. When MSCs were present during differentiation, there was reduced IFN-y
secretion by 1H1 cells and increased IL-4 secretion by TH2 cells (Fig. 2B). No
significant
change in IFN-y or IL-4 levels were seen when MSCs were added to the culture
after Th
cells had differentiated (at 3 days) into effector TH1 or TH2 types (data not
shown).
These experiments suggest that MSCs can affect effector T cell differentiation
directly
and alter the T cell cytokine secretion towards a humoral phenotype.
Similarly, when MSCs were cultured with purified NK cells (CD3-, CD14-, CD19-,
CD36") at a ratio 1:1 for different time periods (0-48 hrs), there -was
decreased IFN-y
secretion in the culture supernatant (Fig. 2C), thereby suggesting that MSCs
can
modulate NK cell functions also.
Previous work has indicated that MSCs modify T-cell functions by soluble
factor(s) (LeBlanc, et al., Exp. Hematol., Vol. 31, pg. 890 (2003); Tse, et
al.,
Transplantation, Vol. 75, pg. 389 (2003). It was observed that the MSCs
secreted
several factors, including IL-6, prostaglandin E2, VEGF and proMMP-
1constitutively,
and the levels of each increased upon culture with PBMCs (Fig. 5). In order to
investigate MSC-derived factors leading to inhibition of TNF-cx and increase
of IL-10
production by DCs, the potential role of prostaglandin E2 was investigated, as
it has
been shown to inhibit TNF-a. production by activated DCs (Vassiliou, et al.,
Cell.
Immunol., Vol. 223, pg. 120 (2003)). Conditioned media from MSC culture (24
hour
culture of 0.5 x106 cells/mil) contained approx. 1000 pg/ml of PGE2 (Fig. 4A).
There was
no detectable presence of known inducers of PGE2 secretion e.g. TNF-cc, IFNif
or IL-1 13
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WO 28051093044 PCT/1JS2005/008506
(data not shown) in the culture supernatant indicating a constitutive
secretion of PGE2
by MSCs. The PGE2 secretion by hMSCs was inhibited 60-90% in the presence of
known inhibitors of PGE2 production, NS-398 (5 M) and indomethacin (41.1M)
(Fig. 4A).
As the release of PGE2 secretion occurs as a result of enzymatic activity of
constitutively active cycloxygenase enzyme 1 (COX-1) and inducible
cycloxygenase
enzyme 2 (COX-2) (Harris, et al., Trends Immunol., Vol. 23, pg. 144 (2002))
the mRNA
expression for COX-1 and COX-2 in MSCs and PBMCs using trans-well culture
system
was analyzed. MSCs expressed significantly higher levels of COX-2 as compared
to
PBMCs and the expression levels increase >3-fold upon co-culture of MSCs and
PBMCs (MSC to PBMC ratio 1:10) for 24 hours (Fig. 4B). Modest changes in COX-1
levels were seen suggesting that the increase in PGE2 secretion upon MSC-PBMC
co-
culture (Fig. 5) is mediated by COX-2 up-regulation. To investigate whether
the
immunomodulatory effects of MSC on DCs and T-cells were mediated by PGE2, MSCs
were cultured with activated dendritic cells (DC1) or TH1 cells in the
presence of PGE2
inhibitors NS-398 or indomethacin. The presence of NS-398 or indomethacin
increased
TNF-cx secretion by DC1s, and IFNI secretion from TH1 cells (Fig. 40),
respectively,
suggesting that MSC effects on immune cell types may be mediated by secreted
PGE2.
Recent studies have shown that MSCs inhibit T-cell proliferation induced by
various
stimuli (DeNicola, et al., Blood, Vol. 99, pg. 3838 (2002); LeBlanc, et al.,
Scand. J.
Immunol., Vol. 57, pg. 11 (2003)). it was observed that MSCs inhibit mitogen-
induced T
cell proliferation in a dose-dependent manner (Fig. 6) and when PGE2
inhibitors NS-398
(5 u.M) or indomethacin (4 p,M) were present, there was a >70% increase in
(3H)
thymidine incorporation by PHA-treated PBMCs in MSC containing cultures as
compared to controls without inhibitors (Fig. 4D).
In summary, a model of MSC interaction with other immune cell types (Fig. 7)
is
proposed. When mature T cells are present, MSCs may interact with them
directly and
inhibit the pro-inflammatory IFN-y production (pathway 1) and promote
regulatory T cell
phenotype (pathway 3) and anti-inflammatory TH2 cells (pathway 5). Further,
MSCs can
alter the outcome of the T cell immune response through DCs by secreting PGE2,
inhibiting pro-inflammatory DC1 cells (pathway 2) and promoting anti-
inflammatory DC2
cells (pathway 4) or regulatory DCs (pathway 3). A shift towards TH2 immunity
in turn,
CA 02868733 2014-10-29
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'64-150
suggests a change In B cell activity towards increased generation of IgE/IgG1
subtype
antibodies (pathway 7). MSCs, by their ability to inhibit IFNI, secretion from
NK cells
nicely modify NK cell function (pathway 8). This model of MSC:immune cell
interactions
= is consistent with the experimentation performed In several other
laboratories (LeBlanc,
at al., Exp., Hematel., Vol. 31, pg. 890 (2003); Tao. at al.. Transplantation,
Vol. 75, pg.
389 (2003); Mimi% et at., Blood, Val. 99, pg. 3838 (2002)). Further
examination of
the proposed mechanisms is underway and animal studies are now necessary to
examine the In vivo .effects of MSC administration.
It is to be understood, however, that the scope of the present invention Is
not to
be limited to the specific embodiments described above. The invention may be
practiced other than as particularly described and still be within the scope
of the
accompanying claims.
=
26
