U.S. patent application number 12/727971 was filed with the patent office on 2010-07-08 for multipotent adult stem cells and uses of multipotent adult stem cells to treat inflammation.
This patent application is currently assigned to OSIRIS THERAPEUTICS, INC.. Invention is credited to Sudeepta Aggarwal, Mark F. Pittenger.
Application Number | 20100172885 12/727971 |
Document ID | / |
Family ID | 39772836 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100172885 |
Kind Code |
A1 |
Pittenger; Mark F. ; et
al. |
July 8, 2010 |
Multipotent Adult Stem Cells And Uses of Multipotent Adult Stem
Cells To Treat Inflammation
Abstract
Disclosed are cell preparations comprising multipotent adult
stem cells and methods for using multipotent adult stem cells to
treat autoimmune diseases, treat allergic responses, treat cancer,
treat inflammatory diseases, treat fibrotic disorders, reduce
inflammation and/or fibrosis, promote would healing, repair
epithelial damage, and/or promote angiogenesis.
Inventors: |
Pittenger; Mark F.; (Severna
Park, MD) ; Aggarwal; Sudeepta; (Ellicott City,
MD) |
Correspondence
Address: |
MCANDREWS HELD & MALLOY, LTD
500 WEST MADISON STREET, SUITE 3400
CHICAGO
IL
60661
US
|
Assignee: |
OSIRIS THERAPEUTICS, INC.
Columbia
MD
|
Family ID: |
39772836 |
Appl. No.: |
12/727971 |
Filed: |
March 19, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11541853 |
Oct 2, 2006 |
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12727971 |
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11080298 |
Mar 15, 2005 |
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11541853 |
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60555118 |
Mar 22, 2004 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 5/14 20180101; C12N 5/0668 20130101; C12N 5/0665 20130101;
A61P 37/06 20180101; A61P 1/04 20180101; A61P 1/02 20180101; A61K
38/2026 20130101; A61K 38/2066 20130101; A61P 43/00 20180101; A61P
19/08 20180101; A61P 13/08 20180101; A61P 37/02 20180101; A61P 9/00
20180101; A61P 17/14 20180101; A61P 27/02 20180101; A61P 37/00
20180101; C12N 5/0664 20130101; C12N 5/0667 20130101; A61P 17/02
20180101; C12N 5/0666 20130101; A61K 35/28 20130101; A61P 29/00
20180101; A61P 37/08 20180101; A61P 1/00 20180101; A61P 3/10
20180101; A61P 7/04 20180101; A61P 7/06 20180101; C12N 5/0663
20130101; A61P 17/06 20180101; A61P 35/00 20180101; A61P 19/02
20180101; A61P 11/00 20180101; A61P 17/00 20180101; Y02A 50/30
20180101; A61K 2035/124 20130101; C12N 5/0662 20130101; A61P 25/00
20180101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61P 35/00 20060101 A61P035/00; A61P 17/02 20060101
A61P017/02 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The present technology was made with Government support
under Contract No. N66001-02-C-8068 awarded by the Department of
the Navy. The Government has certain rights in this technology
Claims
1. A cell preparation comprising adult bone marrow-derived stem
cells in a dose effective to treat an inflammatory response in a
subject.
2. The cell preparation of claim 1, wherein the stem cells are
capable of differentiating into at least one cell type of each of
the endodermal, ectodermal, and mesodermal embryonic lineages.
3. The cell preparation of claim 1, wherein the stem cells are
capable of differentiating into at least one cell type of at least
one of the endodermal, ectodermal, or mesodermal embryonic
lineages.
4. The cell preparation of claim 1, wherein the dose contains a
sufficient number of stem cells to provide about 1.times.10.sup.5
to about 1.times.10.sup.7 cells per kilogram of the subject.
5. The cell preparation of claim 1, wherein the stem cells are
positive for one or more cell surface markers selected from the
group consisting of integrin al (CD49a); integrin .alpha.2 (CD49b);
integrin .alpha.3 (CD49c); integrin .alpha.5 (CD49e); integrin
.alpha.V (CD51); integrin .beta.1 (CD29); integrin .beta.3 (CD61);
integrin .beta.4 (CD104); IL-1R (CD121a); IL-3R.alpha. (CD123);
IL-4R (CDw124); IL-6R (CD126); IL-7R (CDw127); IFN.gamma.R
(CDw119); TNFIR (CD120a); TNFIIR (CD120b); TGF.beta.1R;
TGF.beta.IIR; bFGFR; PDGFR (CD140a); transferrin (CD71); ICAM-1
(CD54); ICAM-2 (CD102); VCAM-1 (CD106); L-Selectin (CD62L); LFA-3
(CD58); ALCAM (CD166); hyaluronate (CD44); endoglin (CD105); Thy-1
(CD90); and CD9.
6. The cell preparation of claim 1, wherein administration of the
cell preparation elevates interferon-beta levels in the
subject.
7. The cell preparation of claim 1, wherein the subject is a
subject having inflammatory bowel disease.
8. A method of treating inflammatory bowel disease in a subject,
comprising the step of: administering to the subject allogeneic,
multipotent adult bone marrow-derived stem cells in an amount
effective to treat the inflammatory bowel disease.
9. The method of claim 8, wherein the multipotent stem cells are
administered intravenously or intraarterially.
10. The method of claim 8, wherein the multipotent stem cells are
positive for one or more cell surface markers selected from the
group consisting of integrin al (CD49a); integrin .alpha.2 (CD49b);
integrin .alpha.3 (CD49c); integrin .alpha.5 (CD49e); integrin
.alpha.V (CD51); integrin .beta.1 (CD29); integrin .beta.3 (CD61);
integrin .beta.4 (CD104); IL-1R (CD121a); IL-3R.alpha. (CD123);
IL-4R (CDw124); IL-6R (CD126); IL-7R (CDw127); IFN.gamma.R
(CDw119); TNFIR (CD120a); TNFIIR (CD120b); TGF.beta.1R;
TGF.beta.IIR; bFGFR; PDGFR (CD140a); transferrin (CD71); ICAM-1
(CD54); ICAM-2 (CD102); VCAM-1 (CD106); L-Selectin (CD62L); LFA-3
(CD58); ALCAM (CD166); hyaluronate (CD44); endoglin (CD105); Thy-1
(CD90); and CD9.
11. The method of claim 8, wherein the multipotent stem cells are
positive for CD49b, CD49e, and CD140a.
12. The method of claim 8, wherein the multipotent stem cells are
negative for one or more cell surface markers selected from the
group consisting of integrin .alpha.4 (CD49d); integrin .alpha.L
(CD11a); integrin C.beta.2 (CD18); CD4; CD14; CD34; CD45; IL-2R
(CD25); EGFR-3; Fas ligand; ICAM-3 (CD50); E-Selectin (CD62E);
P-Selectin (CD62P); vW Factor; cadherin 5; and Lewis x (CD15).
13. The method of claim 8, wherein the multipotent stem cells are
negative for CD34, CD45, CD62E, and CD62P.
14. A method of treating a gastrointestinal autoimmune disease in a
subject, comprising the step of: administering to the subject
allogeneic multipotent adult stem cells in an amount effective to
repair or regenerate intestinal tissue.
15. The method of claim 14, wherein the autoimmune disease is
selected from the group consisting of Crohn's disease, inflammatory
bowel disease, and autoimmune gastritis.
16. The method of claim 14, wherein the multipotent stem cells are
administered intravenously or intraarterially.
17. The method of claim 14, wherein the multipotent stem cells are
positive for one or more cell surface markers selected from the
group consisting of integrin al (CD49a); integrin .alpha.2 (CD49b);
integrin .alpha.3 (CD49c); integrin .alpha.5 (CD49e); integrin
.alpha.V (CD51); integrin .beta.1 (CD29); integrin .beta.3 (CD61);
integrin .beta.4 (CD104); IL-1R (CD121a); IL-3R.alpha. (CD123);
IL-4R (CDw124); IL-6R (CD126); IL-7R (CDw127); IFN.gamma.R
(CDw119); TNFIR (CD120a); TNFIIR (CD120b); TGF.beta.1R;
TGF.beta.IIR; bFGFR; PDGFR (CD140a); transferrin (CD71); ICAM-1
(CD54); ICAM-2 (CD102); VCAM-1 (CD106); L-Selectin (CD62L); LFA-3
(CD58); ALCAM (CD166); hyaluronate (CD44); endoglin (CD105); Thy-1
(CD90); and CD9.
18. The method of claim 14, wherein the multipotent stem cells are
positive for CD49b, CD49e, and CD140a.
19. The method of claim 14, wherein the multipotent stem cells are
negative for one or more cell surface markers selected from the
group consisting of integrin .alpha.4 (CD49d); integrin .alpha.L
(CD11a); integrin C.beta.2 (CD18); CD4; CD14; CD34; CD45; IL-2R
(CD25); EGFR-3; Fas ligand; ICAM-3 (CD50); E-Selectin (CD62E);
P-Selectin (CD62P); vW Factor; cadherin 5; and Lewis x (CD15).
20. The method of claim 14, wherein the multipotent stem cells are
negative for CD34, CD45, CD62E, and CD62P.
21. A method of repairing or regenerating intestinal tissue in a
human, comprising the step of: treating the human with allogeneic
adult mesenchymal stem cells.
22. The method of claim 21, wherein the mesenchymal stem cells are
administered to the human.
23. The method of claim 22, wherein the administration of the
mesenchymal stem cell comprises intravenous, intraarterial, or
intraperitoneal injection.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
11/541,853, filed Oct. 2, 2006, which is a continuation-in-part of
application Ser. No. 11/080,298, filed Mar. 15, 2005, which claims
priority based on provisional application Ser. No. 60/555,118,
filed Mar. 22, 2004; the contents of each application are hereby
incorporated by reference in their entireties.
BACKGROUND OF THE INVENTION
[0003] Mesenchymal stem cells (MSCs), which are present in adult
bone marrow, are a kind of multipotent stem cell 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. 18,
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 can be useful in the
repair and/or regeneration of damaged bone, cartilage, meniscus or
myocardial tissues (DeKok, et al., Clin. Oral Implants Res., Vol.
14, pg. 481 (2003)); Wu, et al., Transplantation, Vol. 75, pg. 679
(2003); Noel, et al., Curr. Opin. Investig. Drugs, Vol. 3, pg. 1000
(2002); Ballas, 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. Sci., 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 al., 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 al. 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 al., J. Clin. Oncol., Vol. 18, pg. 307 (2000)).
[0004] MSCs express major histocompatibility complex (MHC) class I
antigen on their surface, but do not express 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 do not express B7
or CD40 co-stimulatory molecules (Majumdar, et al., J. Biomed.
Sci., 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, et al., Blood, Vol. 99, pg. 3838 (2002)). These
immunological properties of MSCs can enhance their transplant
engraftment and limit the ability of the recipient's 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 al., Hematother. Stem Cell Res., Vol. 9, pg. 841
(2000); Haynesworth, et al., J. Cell. Physiol., Vol. 166, pg. 585
(1996).
BRIEF SUMMARY OF THE INVENTION
[0005] The present technology generally relates to multipotent
adult stem cells, such as adult bone marrow-derived stem cells.
More particularly, the present technology relates to new and
heretofore unappreciated uses for multipotent adult stem cells,
such as mesenchymal stem cells, including, but not limited to
promoting angiogenesis in various tissues and organs, treating
autoimmune diseases, treating allergic responses, treating cancer,
treating inflammatory diseases and disorders, promoting wound
healing, treating inflammation, and repairing epithelial
damage.
[0006] In accordance with the present technology, multipotent adult
stem cells can be used, for example, to treat an autoimmune
disease, treat an inflammatory response, treat an allergic disease,
treat a pulmonary disease having fibrotic and/or inflammatory
components, repair epithelial damage, and promote wound healing in
subjects, including a human subject. Autoimmune diseases that can
be treated with multipotent adult stem cells include, for example,
Type 1 Diabetes, inflammatory bowel disease, Crohn's disease, and
uveitis. Pulmonary diseases that can be treated with multipotent
adult stem cells include, but are not limited to, Acute Respiratory
Distress Syndrome (ARDS), Chronic Obstructive Pulmonary Disease
(COPD), and asthma. Inflammatory responses, including those
associated with autoimmune diseases or pulmonary diseases, can be
reduced with multipotent adult stem cells. An inflammatory response
can be reduced by, for example, reducing the production or
expression of pro-inflammatory mediators, increasing the production
or expression of anti-inflammatory mediators, or a combination
thereof.
[0007] Multipotent adult stem cells of the present technology have
the capacity to differentiate into at least one cell type of each
of the mesodermal, ectodermal, and endodermal lineages. For
example, the cells can be induced to differentiate into cells of at
least osteoblast, chondrocyte, adipocyte, fibroblast, marrow
stroma, skeletal muscle, smooth muscle, cardiac muscle,
endothelial, epithelial, hematopoietic, glial, neuronal or
oligodendrocyte cell types, among others. Multipotent adult stem
cells of the present technology are capable of differentiating into
at least one cell type of at least one of the endodermal,
ectodermal, or mesodermal embryonic lineages.
[0008] Adult bone marrow is an accessible and renewable source of
adult multipotent stem cells that can be greatly expanded in
culture. For example, bone marrow-derived mesenchymal stem cells
(MSCs) are multipotent cells that have been identified and cultured
from various avian and mammalian species.
[0009] The present technology provides one or more cell
preparations comprising adult bone marrow-derived stem cells in one
or more doses effective to treat an inflammatory response in a
subject. The subject can be a human having, for example,
inflammatory bowel disease. The stem cells can be capable of
differentiating into at least one cell type of each of the
endodermal, ectodermal, and mesodermal embryonic lineages. The
effective dose of the cell preparation can contain a sufficient
number of stem cells to provide about 1.times.10.sup.5 to about
1.times.10.sup.7 cells per kilogram of the subject. In certain
embodiments, the subject can be a mammal. The mammal can be, for
example, a primate, including a human and a non-human primate.
Administration of the cell preparation can, for example, elevate
interferon-beta levels in the subject.
[0010] The present technology also provides a method of treating
inflammatory bowel disease in a subject, comprising administering
to the subject allogeneic multipotent adult stem cells in an amount
effective to treat inflammatory bowel disease. In certain
embodiments, the subject can be an animal. The animal can be, for
example, a primate, including a human and a non-human primate. The
multipotent adult stem cells can be administered systemically, such
as intravenously, intraarterially, or intraperitoneally. The
multipotent adult stem cells can be administered in conjunction
with an acceptable pharmaceutical carrier, such as a
pharmaceutically acceptable liquid medium. The multipotent adult
stem cells can be administered as a suspension of cells. The amount
effective to treat inflammatory bowel disease can be about
1.times.10.sup.5 to about 1.times.10.sup.7 cells per kilogram of
the subject.
[0011] The present technology further provides a method of treating
a gastrointestinal autoimmune disease in a subject, comprising
administering to the subject allogeneic multipotent adult stem
cells in an amount effective to repair or regenerate intestinal
tissue. The autoimmune disease can be selected from the group
consisting of Crohn's disease, inflammatory bowel disease, and
autoimmune gastritis. In certain embodiments, the subject can be an
animal. The animal can be, for example, a primate, including a
human and a non-human primate. The multipotent adult stem cells can
be administered systemically, such as intravenously,
intraarterially, or intraperitoneally. The multipotent adult stem
cells can be administered in conjunction with an acceptable
pharmaceutical carrier, such as a pharmaceutically acceptable
liquid medium. The multipotent adult stem cells can be administered
as a suspension of cells. The amount effective to repair or
regenerate intestinal tissue can be about 1.times.10.sup.5 to about
1.times.10.sup.7 cells per kilogram of the subject.
[0012] The multipotent adult stem cells of the present technology
can be positive for one or more cell surface markers selected from,
for example, integrin al (CD49a); integrin .alpha.2 (CD49b);
integrin .alpha.3 (CD49c); integrin .alpha.5 (CD49e); integrin
.alpha.V (CD51); integrin .beta.1 (CD29); integrin .beta.3 (CD61);
integrin .beta.4 (CD104); IL-1R (CD121a); IL-3R.alpha. (CD123);
IL-4R (CDw124); IL-6R (CD126); IL-7R (CDw127); IFN.gamma.R
(CDw119); TNFIR (CD120a); TNFIIR (CD120b); TGF.beta.1R;
TGF.beta.IIR; bFGFR; PDGFR (CD140a); transferrin (CD71); ICAM-1
(CD54); ICAM-2 (CD102); VCAM-1 (CD106); L-Selectin (CD62L); LFA-3
(CD58); ALCAM (CD166); hyaluronate (CD44); endoglin (CD105); Thy-1
(CD90); or CD9. The stem cells can also be positive for each of
CD49b, CD49e, and CD140a. Further, the stem cells can be negative
for one or more cell surface markers such as integrin .alpha.4
(CD49d); integrin .alpha.L (CD11a); integrin C.beta.2 (CD18); CD4;
CD14; CD34; CD45; IL-2R (CD25); EGFR-3; Fas ligand; ICAM-3 (CD50);
E-Selectin (CD62E); P-Selectin (CD62P); vW Factor; cadherin 5; or
Lewis x (CD15). In addition, the multipotent adult stem cells of
the present technology can be adult bone marrow-derived stem cells
such as, for example, mesenchymal stem cells.
[0013] The present technology also provides a method of repairing
and/or regenerating intestinal tissue in a subject, comprising
treating the subject with allogeneic mesenchymal stem cells. In
certain embodiments, the subject can be an animal. The animal can
be, for example, a primate, including a human and a non-human
primate. The stem cells can be from an adult. The mesenchymal stem
cells can be administered to the subject as part of, for example, a
pharmaceutical formulation having a pharmaceutically acceptable
carrier, such as a liquid injectable carrier, a liquid topical
carrier, a gel injectable carrier, a gel topical carrier, a solid
matrix, and combinations thereof. The mesenchymal stem cell
pharmaceutical formulation can be administered via intravenous,
intraarterial, or intraperitoneal injection. Mesenchymal stem cells
can be administered in an amount effective to repair or regenerate
intestinal tissue in a subject. The amount effective to repair or
regenerate intestinal tissue can be about 1.times.10.sup.5 to about
1.times.10.sup.7 cells per kilogram of the subject.
[0014] Applicants presently have examined the interactions of
multipotent adult stem cells with isolated immune cell populations,
including dendritic cells (DC1 and DC2), effector T-cells (Th1 and
Th2), and NK cells. Based on such interactions, Applicants
discovered that multipotent adult stem cells regulate the
production of various factors that affect several steps in the
inflammatory and/or immune response process. Thus, the multipotent
adult stem cells of the present technology can be employed in the
treatment of disease conditions and disorders involving the immune
system, or diseases, conditions, or disorders involving
inflammation, epithelial damage, 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.
[0015] In addition, it is believed that multipotent adult stem
cells, including mesenchymal stem cells, express and secrete
vascular endothelial growth factor, or VEGF, which promotes
angiogenesis by stimulating the formation of new blood vessels.
Mesenchymal stem cells also stimulate peripheral blood mononuclear
cells (PBMCs) to produce VEGF.
[0016] Furthermore, it is believed that multipotent adult stem
cells stimulate dendritic cells (DCs) to produce Interferon-Beta
(IFN-.beta.), which promotes tumor suppression and immunity against
viral infection.
BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS
[0017] The present technology now will be described with respect to
the drawings.
[0018] 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). (---): isotype
control; (-): FITC/PE conjugated antibodies. (B) MSCs inhibit
TNF-.alpha. 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-.gamma. 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-.gamma. 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.
[0019] FIG. 2. MSCs inhibit pro-inflammatory effector T cell
function. (A) Flow cytometric analysis of T.sub.reg cell numbers
(in %) by staining PBMCs or the 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-.gamma. (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-.gamma. 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.
[0020] FIG. 3. MSCs lead to increased numbers of T.sub.reg cell
population and increased GITR expression. (A) A CD44+CD25+T.sub.reg
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; 3H thymidine was added; and incorporated
radioactivity was counted after 24 hours. The results showed that
the T.sub.reg population, generated in the presence of MSCs (lane
3) was similar functionally to the T.sub.reg cells generated in the
absence of MSCs (lane 2). (B) PBMCs were cultured for 3 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.
[0021] FIG. 4. MSCs produce PGE.sub.2 and blocking PGE.sub.2
reverses MSC-mediated immuno-modulatory effects. (A) PGE.sub.2
secretion (mean.+-.SD) in culture supernatants obtained from MSCs
cultured in the presence or absence of PGE.sub.2 blockers NS-398 or
indomethacin (Indometh.) at various concentrations. Inhibitor
concentrations are in .mu.M 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 are 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 PGE.sub.2
blockers indomethacin (Ind.) or NS-398 increases TNF-.alpha.
secretion from activated DCs (open bars) and IFN-.gamma. secretion
from Th1 cells (hatched bars) as compared to controls. Data were
calculated as % change from cultures generated in absence of MSCs
and PGE.sub.2 inhibitors (D) Presence of PGE.sub.2 blockers
indomethacin (Indo) and NS-398 during MSCPBMC 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.
[0022] 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 PGE.sub.2, 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 PGE.sub.2
constituitively, and the levels of these factors increased upon
co-culture with PBMCs, thereby suggesting that MSCs can play a role
in modulating immune functions in an inflammatory setting.
[0023] 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).
[0024] FIG. 7. Schematic diagram of proposed MSC mechanism of
action. MSCs mediate their immuno-modulatory effects by affecting
cells from both the innate (DC-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, for example,
means of direct cell-cell contact or via secreted factor, can 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 can interact with them to skew
the balance of Th1 (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 T.sub.reg population (pathway 3) can
result in a tolerant phenotype and can aid a recipient host by
dampening bystander inflammation in their local micro-environment.
Dashed line (----) represents proposed mechanism.
DETAILED DESCRIPTION OF THE INVENTION
[0025] In accordance with an aspect of the present technology,
there is provided a method of treating a disease selected from the
group consisting of autoimmune diseases and graft-versus-host
disease in an animal, including, for example, a human. The method
comprises at least the step of administering to the animal
mesenchymal stem cells in an amount effective to treat the disease
in the animal.
[0026] Although the scope of this aspect of the present technology
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 and graft-versus-host disease is by
causing the release of Interleukin-10 (IL-10) from regulatory
T-cells (T.sub.reg cells) and/or dendritic cells (DC).
[0027] Autoimmune diseases which can be treated in accordance with
the present technology include, but are not limited to, multiple
sclerosis, Type 1 diabetes, rheumatoid arthritis, uveitis,
autoimmune thyroid disease, inflammatory bowel disease,
scleroderma, Graves' Disease, lupus, Crohn's disease, autoimmune
lymphoproliferative disease (ALPS), demyelinating disease,
autoimmune encephalomyelitis, autoimmune gastritis (AIG), and
autoimmune glomerular diseases. Also, as noted hereinabove,
graft-versus-host disease can be treated. It is to be understood,
however, that the scope of the present technology is not to be
limited to the treatment of the specific diseases mentioned
herein.
[0028] In certain embodiments, the mesenchymal stem cells are
administered to a mammal. The mammal can be a primate, including
human and non-human primates.
[0029] In general, the multipotent adult stem cell therapy is
based, for example, on the following sequence: harvest of stem
cell-containing tissue such as, for example, bone marrow; isolation
and/or expansion of stem cells; and administration of the stem
cells, with or without biochemical or genetic manipulation, to the
animal.
[0030] The mesenchymal stem cells that are administered can be, for
example, a homogeneous composition or a mixed cell population
enriched in mesenchymal stem cells. The mesenchymal stem cell
compositions can be obtained, for example, by culturing adherent
bone marrow or periosteal cells. Adult bone marrow-derived stem
cells, such as bone marrow-derived mesenchymal stem cells, can be
identified by specific cell surface markers, which are capable of
being bound by unique monoclonal antibodies.
[0031] A method for obtaining a cell population enriched in
mesenchymal stem cells is described in, for example, U.S. Pat. No.
5,486,359, the contents of which are hereby incorporated by
reference in its entirety. Adult bone marrow contains multipotent
stem cells, including mesenchymal stem cells. Alternative sources
for multipotent adult stem cells such as mesenchymal stem cells
include, but are not limited to, blood, skin, cord blood, muscle,
fat, bone, and perichondrium.
[0032] Multipotent adult stem cells can be obtained from a variety
of sources, including bone marrow. For example, multipotent adult
stem cells such as human mesenchymal stem cells can be obtained
from bone marrow from a number of different sources, including
plugs of femoral head cancellous bone pieces, patients with
degenerative joint disease during hip or knee replacement surgery,
and aspirated marrow from normal donors or oncology patients who
have marrow harvested for future bone marrow transplantation.
Harvested marrow can be prepared for cell culture by a number of
different mechanical isolation processes depending upon the source
of the harvested marrow (i.e., the presence of bone chips,
peripheral blood, etc.) that are well known in the art. Exemplary
culture media and culture conditions are identified in, for
example, U.S. Pat. No. 5,486,359 and include media and conditions
that allow for expansion, growth, and isolation of mesenchymal stem
cells, without differentiation.
[0033] Multipotent stem cells isolated from human adult bone marrow
can be surface antigen positive for integrin al (CD49a); integrin
.alpha.2 (CD49b); integrin .alpha.3 (CD49c); integrin .alpha.5
(CD49e); integrin .alpha.V (CD51); integrin .beta.1 (CD29);
integrin .beta.3 (CD61); integrin .beta.4 (CD104); IL-1R (CD121a);
IL-3R.alpha. (CD123); IL-4R (CDw124); IL-6R (CD126); IL-7R
(CDw127); IFN.gamma.R (CDw119); TNFIR (CD120a); TNFIIR (CD120b);
TGF.beta.1R; TGF.beta.IIR; bFGFR; PDGFR (CD140a); transferrin
(CD71); ICAM-1 (CD54); ICAM-2 (CD102); VCAM-1 (CD106); L-Selectin
(CD62L); LFA-3 (CD58); ALCAM (CD166); hyaluronate (CD44); endoglin
(CD105); Thy-1 (CD90); CD9; and combinations thereof. Cell isolated
from human adult bone marrow can be surface antigen negative for
integrin .alpha.4 (CD49d); integrin .alpha.L (CD11a); integrin
C.beta.2 (CD18); CD4; CD14; CD34; CD45; IL-2R (CD25); EGFR-3; Fas
ligand, ICAM-3 (CD50); E-Selectin (CD62E); P-Selectin (CD62P); vW
Factor; cadherin 5; Lewis x (CD15); and combinations thereof.
[0034] Cell preparations having greater than about 95%, usually
greater than about 98%, of multipotent adult human stem cells can
be achieved using techniques for isolation, purification, and
culture expansion of stem cells. For example, isolated, cultured
adult bone marrow-derived stem cells such as mesenchymal stem cells
can comprise a single phenotypic population (about 95% or about 98%
homogeneous) by flow cytometric analysis of expressed surface
antigens. The desired cells in such composition can be identified,
for example, by expression of a cell surface marker (e.g., CD73 or
CD105) specifically bound by an antibody produced from hybridoma
cell line SH2, ATCC accession number HB 10743, an antibody produced
from hybridoma cell line SH3, ATCC accession number HB 10744, or an
antibody produced from hybridoma cell line SH4, ATCC accession
number HB 10745. Such antibodies selectively bind bone
marrow-derived mesenchymal stem cells and, therefore, can be used
to identify, quantify, isolate, or purify mesenchymal stem cells
from bone marrow samples.
[0035] The mesenchymal stem cells can be administered by a variety
of procedures. The mesenchymal stem cells can be administered
systemically, such as by intravenous, intraarterial, or
intraperitoneal administration.
[0036] The mesenchymal stem cells can be from a spectrum of sources
including autologous, allogeneic, or xenogeneic.
[0037] The mesenchymal stem cells are administered in an amount
effective to treat an autoimmune disease or graft-versus-host
disease in an animal. The mesenchymal stem cells can be
administered in an amount of from about 1.times.10.sup.5 cells/kg
to about 1.times.10.sup.7 cells/kg. In other embodiments, the
mesenchymal stem cells are administered in an amount of from about
1.times.10.sup.6 cells/kg to about 5.times.10.sup.6 cells/kg. In
other embodiments, the mesenchymal stem cells are administered in
an amount of from about 2.times.10.sup.6 cells/kg to about
4.times.10.sup.6 cells/kg. In still other embodiments, the
mesenchymal stem cells are administered in an amount of about
3.times.10.sup.6 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 subject and/or patient, the
autoimmune disease to be treated, and the extent and severity
thereof.
[0038] The mesenchymal stem cells can be administered in
conjunction with an acceptable pharmaceutical carrier. For example,
the mesenchymal stem cells can be administered as a cell suspension
in a pharmaceutically acceptable liquid medium or gel for injection
or topical application.
[0039] In accordance with another aspect of the present technology,
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. Without wishing to be bound by
any particular theory, the mesenchymal stem cells prevent or
reverse an inflammatory response by, for example, increasing
expression, production, and/or secretion of pro-inflammatory
cytokines such as, for example, tumor necrosis factor-alpha
(TNF-.alpha.) and Interferon-.gamma. (IFN-.gamma.); decreasing
expression, production, and/or secretion of anti-inflammatory
cytokines such as, for example, IL-10 and IL-4; and/or combinations
thereof.
[0040] Although the scope of this aspect of the present technology
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 (T.sub.reg), 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 IFN-.gamma. in certain inflammatory reactions,
such as those associated with psoriasis, for example.
[0041] In certain embodiments, the inflammatory responses which can
be treated are those associated with psoriasis.
[0042] In other embodiments, the mesenchymal stem cells can be
administered to an animal such that the mesenchymal stem cells
prevent or reduce inflammation in the brain by, for example,
contacting or secreting factors that affect, microglia and/or
astrocytes in the brain. The mesenchymal stem cells limit
neurodegeneration caused by activated glial cells in diseases or
disorders such as Alzheimer's disease, Parkinson's disease, stroke,
or brain cell injuries.
[0043] In yet other embodiments, the mesenchymal stem cells can be
administered to an animal such that the mesenchymal stem cells
reduce skin inflammation as can occur in psoriasis, chronic
dermatitis, and contact dermatitis by, for example, contacting or
secreting factors that affect, keratinocytes and Langerhans cells
in the epidermis of the skin. Although this embodiment is not to be
limited to any theoretical reasoning, it is believed that the
mesenchymal stem cells can contact the keratinocytes and Langerhans
cells in the epidermis, and alter the expression of T-cell
receptors and cytokine secretion profiles, leading to decreased
expression of TNF-.alpha. and increased regulatory T-cell
(T.sub.reg cell) population.
[0044] In further embodiments, the mesenchymal stem cells can 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 such as ankylosing spondylitis, avascular necrosis
(osteonecrosis), fibromyalgia, juvenile dermatomyositis, juvenile
rheumatoid arthritis, juvenile spondyloarthopathy, lyme disease,
marfan syndrome, myositis, osteogenesis imperfecta, osteoporosis,
Paget's disease, Raynaud's Phenomenon, scleroderma, Sjorgren's
Syndrome, and systemic lupus erythematosus. Although the scope of
this embodiment is not intended to be limited to any theoretical
reasoning, it is believed that the mesenchymal stem cells can
inhibit Interleukin-17 secretion by memory T-cells in the synovial
fluid.
[0045] In other embodiments, the mesenchymal stem cells can 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 technology is not intended to
be limited to any theoretical reasoning, it is believed that the
mesenchymal stem cells promote increased secretion of IL-10 and the
generation of T.sub.reg cells.
[0046] In other embodiments, the mesenchymal stem cells can be used
to inhibit excessive neutrophil and macrophage activation in
pathological conditions such as 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
believed the mesenchymal stem cells promote secretion of
suppressive cytokines such as IL-10, and inhibit macrophage
migration inhibitory factor (MIF).
[0047] In other embodiments, the mesenchymal stem cells can 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 T.sub.reg
cells.
[0048] In yet other embodiments, the mesenchymal stem cells can be
used to treat tissue damage associated with 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
can promote renal repair. Mesenchymal stem cells also express and
secrete vascular endothelial growth factor, or VEGF, which
stimulates new blood vessel formation, which should aid in the
repair of damaged kidney tissue.
[0049] In a further embodiment, the mesenchymal stem cells can 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-.beta.).
[0050] In yet other embodiments, the mesenchymal stem cells can 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
Immunoglobulin E (IgE) by B-cells.
[0051] It is to be understood, however, that the scope of this
aspect of the present technology is not to be limited to the
treatment of any particular inflammatory response.
[0052] The mesenchymal stem cells can be administered to a mammal,
including human and non-human primates, as described herein.
[0053] The mesenchymal stem cells also can be administered
systemically, as described herein. Alternatively, in the case of
osteoarthritis or rheumatoid arthritis, the mesenchymal stem cells
can be administered directly to an arthritic joint.
[0054] The mesenchymal stem cells are administered in an amount
effective to treat an inflammatory response in an animal. The
mesenchymal stem cells can be administered in an amount of from
about 1.times.10.sup.5 cells/kg to about 1.times.10.sup.7 cells/kg.
In other embodiments, the mesenchymal stem cells are administered
in an amount of from about 1.times.10.sup.6 cells/kg to about
5.times.10.sup.6 cells/kg. In other embodiments, the mesenchymal
stem cells are administered in an amount of from about
2.times.10.sup.6 cells/kg to about 4.times.10.sup.6 cells/kg. In
still other embodiments, the mesenchymal stem cells are
administered in an amount of about 3.times.10.sup.6 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 subject and/or patient, the inflammatory response being
treated, and the extent and severity thereof.
[0055] The mesenchymal stem cells can be administered in
conjunction with an acceptable pharmaceutical carrier, as described
herein.
[0056] In accordance with another aspect of the present technology,
there is provided a method of treating inflammation and/or
repairing epithelial damage in an animal. The method comprises
administering to the animal mesenchymal stem cells in an amount
effective to treat the inflammation and/or epithelial damage in the
animal.
[0057] Although the scope of this aspect of the present technology
is not to be limited to any theoretical reasoning, it is believed
that the mesenchymal stem cells cause a decrease in the secretion
of the pro-inflammatory cytokines TNF-.alpha. and IFN-.gamma. by
T-cells, and an increase in the secretion of the anti-inflammatory
cytokines IL-10 and Interleukin-4 (IL-4) by T-cells. It is also
believed that the mesenchymal stem cells cause a decrease in
IFN-.gamma. secretion by natural killer (NK) cells.
[0058] The inflammation and/or epithelial damage which can be
treated in accordance with at least this aspect of the present
technology includes, but is not limited to, inflammation and/or
epithelial damage caused by a variety of diseases and disorders,
including, but not limited to, autoimmune disease, rejection of
transplanted organs, burns, cuts, lacerations, and ulcerations,
including skin ulcerations and diabetic ulcerations.
[0059] In certain embodiments, the mesenchymal stem cells are
administered to an animal at least in order to repair epithelial
damage resulting from autoimmune diseases, including, but not
limited to, rheumatoid arthritis, Crohn's Disease, Type 1 diabetes,
multiple sclerosis, scleroderma, Graves' Disease, lupus,
inflammatory bowel disease, autoimmune gastritis (AIG), and
autoimmune glomerular disease. The mesenchymal stem cells also can
repair epithelial damage resulting from graft-versus-host disease
(GvHD).
[0060] This aspect of the present technology is applicable
particularly to the repair of epithelial damage resulting from
graft-versus-host disease, and more particularly, to the repair of
epithelial damage resulting from severe graft-versus-host disease,
including Grades III and IV GvHD affecting, for example, the skin
and/or the gastrointestinal system. Applicants have discovered, in
particular, that mesenchymal stem cells, when administered to a
patient suffering from severe graft-versus-host disease, and in
particular, Grades III and IV GvHD, the administration of the
mesenchymal stem cells resulted in repair of skin and/or ulcerated
intestinal epithelial tissue in the subject and/or patient.
[0061] In other embodiments, the mesenchymal stem cells are
administered to an animal in order to repair epithelial damage to a
transplanted organ or tissue including, but not limited to, kidney,
heart, and lung, caused by rejection of the transplanted organ or
tissue.
[0062] In yet other embodiments, the mesenchymal stem cells are
administered to an animal to repair epithelial damage caused by
burns, cuts, lacerations, and ulcerations, including, but not
limited to, skin ulcerations and diabetic ulcerations.
[0063] The mesenchymal stem cells can be administered to an animal
such as a mammal, including human and non-human primates, as
described herein.
[0064] The mesenchymal stem cells also can be administered
systemically, as described herein.
[0065] The mesenchymal stem cells are administered in an amount
effective to repair epithelial damage in an animal. The mesenchymal
stem cells can be administered in an amount of from about
1.times.10.sup.5 cells/kg to about 1.times.10.sup.7 cells/kg. In
other embodiments, the mesenchymal stem cells are administered in
an amount of from about 1.times.10.sup.6 cells/kg to about
5.times.10.sup.6 cells/kg. In other embodiments, the mesenchymal
stem cells are administered in an amount of from about
2.times.10.sup.6 cells/kg to about 4.times.10.sup.6 cells/kg. In
still other embodiments, the mesenchymal stem cells are
administered in an amount of about 3.times.10.sup.6 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 subject and/or patient, the type of epithelial damage
being repaired, and the extent and severity thereof.
[0066] In accordance with yet another aspect of the present
technology, 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.
[0067] Although the scope of this aspect of the present technology
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-.beta. secretion, which in turn acts as a tumor
suppressor. Cancers which can 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 technology is not to be limited to any specific type of
cancer.
[0068] As noted herein, it shall be appreciated by those of skill
in the art that the term "animal" includes a mammal, such as a
human or a non-human primate.
[0069] 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
1.times.10.sup.5 cells/kg to about 1.times.10.sup.7 cells/kg. In
other embodiments, the mesenchymal stem cells are administered in
an amount of from about 1.times.10.sup.6 cells/kg to about
5.times.10.sup.6 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 subject and/or patient,
the type of cancer being treated, and the extent and severity
thereof.
[0070] The mesenchymal stem cells are administered in conjunction
with an acceptable pharmaceutical carrier, and can be administered
systemically, as described herein. Alternatively, the mesenchymal
stem cells can be administered directly to the cancer being
treated.
[0071] In accordance with still another aspect of the present
technology, 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.
[0072] Although the scope of this aspect of the present technology
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-.alpha., 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.
[0073] Allergic diseases or disorders which can 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 technology is not to be
limited to any specific allergic disease or disorder.
[0074] 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 can be a mammal. The mammal can be a
primate, including human and non-human primates. In general, the
mesenchymal stem cells are administered in an amount of from about
1.times.10.sup.5 cells/kg to about 1.times.10.sup.7 cells/kg. In
other embodiments, the mesenchymal stem cells are administered in
an amount of from about 1.times.10.sup.6 cells/kg to about
5.times.10.sup.6 cells/kg. The exact dosage is dependent upon a
variety of factors, including the age, weight, and sex of the
subject and/or patient, the allergic disease or disorder being
treated, and the extent and severity thereof.
[0075] The mesenchymal stem cells can be administered in
conjunction with an acceptable pharmaceutical carrier, as described
herein. The mesenchymal stem cells can be administered
systemically, such as by intravenous or intraarterial
administration, for example.
[0076] In accordance with a further aspect of the present
technology, 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.
[0077] Although the scope of the present technology is not to be
limited to any theoretical reasoning, it is believed that, as
mentioned hereinabove, the mesenchymal stem cells cause T.sub.reg
cells and dendritic cells to release IL-10, which limits or
controls inflammation in a wound, thereby promoting healing of a
wound.
[0078] Furthermore, the mesenchymal stem cells can promote wound
healing and fracture healing by inducing secretion factors by other
cell types. For example, the mesenchymal stem cells can induce
prostaglandin E.sub.2 (PGE.sub.2)-mediated release of vascular
endothelial growth factor (VEGF) by peripheral blood mononuclear
cells (PBMCs), as well as PGE.sub.2-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.
[0079] Wounds which can be healed include, but are not limited to,
those resulting from cuts, lacerations, burns, and skin
ulcerations.
[0080] The mesenchymal stem cells are administered to the animal in
an amount effective to promote wound healing in the animal. The
animal can be a mammal, and the mammal can be a primate, including
human and non-human primates. In general, the mesenchymal stem
cells are administered in an amount of from about 1.times.10.sup.5
cells/kg to about 1.times.10.sup.7 cells/kg. In other embodiments,
the mesenchymal stem cells are administered in an amount of from
about 1.times.10.sup.6 cells/kg to about 5.times.10.sup.6 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 subject and/or patient, and the extent and severity of
the wound being treated.
[0081] The mesenchymal stem cells can be administered in
conjunction with an acceptable pharmaceutical carrier, as described
herein. The mesenchymal stem cells can be administered
systemically, as described herein. Alternatively, the mesenchymal
stem cells can be administered directly to a wound, such as in a
fluid on a dressing or reservoir containing the mesenchymal stem
cells.
[0082] In accordance with yet another aspect of the present
technology, there is provided a method of treating or preventing
fibrosis or fibrotic disorder in an animal. The method comprises
administering to the animal mesenchymal stem cells in an amount
effective to treat or prevent fibrosis or a fibrotic disorder in an
animal.
[0083] The mesenchymal stem cells can be administered to the animal
in order to treat or prevent any type of fibrosis or fibrotic
disorder and in the animal, including, but not limited to,
cirrhosis of the liver, fibrosis of the kidneys associated with
end-stage renal disease, and lung disorders or diseases having
fibrotic and can include in addition, inflammatory components,
including, but not limited to, Acute Respiratory Distress Syndrome
(ARDS), Chronic Obstructive Pulmonary Disease (COPD). It is to be
understood that the scope of the present technology is not to be
limited to any specific type of fibrosis or fibrotic disorder.
[0084] The mesenchymal stem cells are administered to the animal in
an amount effective to treat or prevent fibrosis or a fibrotic
disorder in the animal. The animal can be a mammal, and the mammal
can be a primate, including human and non-human primates. In
general, the mesenchymal stem cells are administered in an amount
of from about 1.times.10.sup.5 cells/kg to about 1.times.10.sup.7
cells/kg. In other embodiments, the mesenchymal stem cells are
administered in an amount of from about 1.times.10.sup.6 cells/kg
to about 5.times.10.sup.6 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 subject and/or
patient, and the extent and severity of the fibrosis or fibrotic
disorder being treated or prevented.
[0085] The mesenchymal stem cells can be administered in
conjunction with an acceptable pharmaceutical carrier, as described
herein. The mesenchymal stem cells can be administered
systemically, also as described herein.
[0086] It is another object of the present technology to promote
angiogenesis (i.e., the formation of new blood vessels from a
pre-existing microvascular bed) 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 technology, 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.
[0087] The induction of angiogenesis can be used to treat coronary
and peripheral artery insufficiency, and thus can be a noninvasive
and curative approach to the treatment of coronary artery disease,
ischemic heart disease, and peripheral artery disease. Angiogenesis
can play a role in the treatment of diseases and disorders in
tissue and organs other than the heart, as well as in the
development and/or maintenance of organs other than the heart.
Angiogenesis can 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 the embryonic vasculature. Angiogenesis also is
essential for the coupling of cartilage resorption with bone
formation, and is essential for correct growth plate
morphogenesis.
[0088] 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.
[0089] The mesenchymal stem cells can be administered to the tissue
or organ in need of angiogenesis by a variety of procedures. The
mesenchymal stem cells can be administered systemically, such as by
intravenous, intraarterial, or intraperitoneal administration, or
the mesenchymal stem cells can 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.
[0090] The mesenchymal stem cells can be from a spectrum of sources
including autologous, allogeneic, or xenogeneic.
[0091] Although the scope of the present technology is not to be
limited to any theoretical 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.
[0092] In certain embodiments, the animal is a mammal. The mammal
can be a primate, including human and non-human primates.
[0093] The mesenchymal stem cells, in accordance with the present
technology, can 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 can be administered to an animal to treat
blocked arteries, including those in the extremities, such as arms,
legs, hands, and feet, as well as the neck or in various organs.
For example, the mesenchymal stem cells can be used to treat
blocked arteries which supply the brain, thereby treating or
preventing stroke. Also, the mesenchymal stem cells can be used to
treat blood vessels in embryonic and post-natal corneas and can be
used to provide glomerular structuring. In other embodiments, the
mesenchymal stem cells can 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.
[0094] Furthermore, because angiogenesis is involved in embryo
implantation and placenta formation, the mesenchymal stem sells can
be employed to promote embryo implantation and prevent
miscarriage.
[0095] In addition, the mesenchymal stem cells can be administered
to an unborn animal, including humans, to promote the development
of the vasculature in the unborn animal.
[0096] In other embodiments, the mesenchymal stem cells can 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.
[0097] The mesenchymal stem cells are administered in an amount
effective in promoting angiogenesis in an animal. The mesenchymal
stem cells can be administered in an amount of from about
1.times.10.sup.5 cells/kg to about 1.times.10.sup.7 cells/kg. In
other embodiments, the mesenchymal stem cells are administered in
an amount of from about 1.times.10.sup.6 cells/kg to about
5.times.10.sup.6 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 subject and/or patient, the disease
or disorder to be treated, alleviated, or prevented, and the extent
and severity thereof.
[0098] The mesenchymal stem cells can be administered in
conjunction with an acceptable pharmaceutical carrier. For example,
the mesenchymal stem cells can be administered as a cell suspension
in a pharmaceutically acceptable liquid medium for injection.
Injection can be local, such as by administration directly into the
tissue or organ in need of angiogenesis, or systemic, such as
intravenously or intraarterially.
[0099] The mesenchymal stem cells can be genetically engineered
with one or more polynucleotides encoding a therapeutic agent. The
polynucleotides can be delivered to the mesenchymal stem cells via
an appropriate expression vehicle. Expression vehicles which can be
employed to genetically engineer the mesenchymal stem cells
include, but are not limited to, retroviral vectors, adenoviral
vectors, and adeno-associated virus vectors.
[0100] 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. Pat.
No. 6,355,239, the contents of which are hereby incorporated by
reference in its entirety.
[0101] It is to be understood that the mesenchymal stem cells, when
employed in the above-mentioned therapies and treatments, can 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 can be administered with soluble carriers for cells such as
hyaluronic acid, or in combination with solid matrices, such
collagen, gelatin, or other biocompatible polymers, as
appropriate.
[0102] It is to be understood that the methods described herein can
be carried out in a number of ways and with various modifications
and permutations thereof that are well known in the art. It also
can be appreciated that any theories set forth as to modes of
action or interactions between cell types should not be construed
as limiting this technology in any manner, but are presented such
that the methods of the present technology can be understood more
fully.
[0103] It is to be understood that the scope of the present
technology is not to be limited to the specific embodiments
described above. The present technology can be practiced other than
as particularly described and still be within the scope of the
accompanying claims.
[0104] Likewise, the following examples are presented in order to
more fully illustrate the present technology. They should in no way
be construed, however, as limiting the broad scope of the
technology disclosed herein.
EXAMPLES
[0105] The present technology now will be described with respect to
the following examples; it is to be understood, however, that the
scope of the present technology is not to be limited thereby.
Example 1
[0106] 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, Calif.) containing 1% antibiotic-antimyotic
solution (Invitrogen, Carlsbad, Calif.) and 10% fetal bovine serum
(FBS, JRH BioSciences, Lenexa, Kans.). MSCs grew as an adherent
monolayer and were detached with trypsin/EDTA (0.05% trypsin at
37.degree. 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)).
[0107] 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, Calif.). Precursors of DCs
of plasmacytoid lineage were isolated from PBMCs by immuno-magnetic
sorting of positively labeled antibody coated cells (BDCA2+)
(Miltenyi Biotech, Auburn, Calif.).
[0108] MSC-DC culture. 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.degree. 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, naive, 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 supernatants were harvested and analyzed for secreted
IFN-.gamma. and IL-4 by ELISA.
[0109] 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,
Calif.) columns in a magnetic field, while NK cells passed through
and were collected.
[0110] Isolation of T.sub.Reg cell population. The T.sub.Reg cell
population was isolated using a 2-step isolation procedure. First
non-CD4.sup.+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, Calif.). Next,
CD4.sup.+CD25.sup.+ cells were directly labeled with CD25
microbeads and isolated by positive selection from the pre-enriched
CD4.sup.+. T cell fraction. The magnetically labeled
CD4.sup.+CD25.sup.+T cells were retained on the column and eluted
after removal of the column from the magnetic field.
[0111] In order to determine whether the increased CD4+CD25+
population generated in the presence of MSCs were suppressive in
nature, CD4+CD25+T.sub.reg 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 .mu.g/ml).
The culture was carried out for 48 hours, following which .sup.3H
thymidine was added. Incorporated radioactivity was counted after
24 hours.
[0112] 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.
[0113] Generation of Th1/Th2 cells. Peripheral blood mononuclear
cells (PBMCs) were plated at 2.times.10.sup.6 cells/ml for 45 min.
at 37.degree. C. in order to remove monocytes. Non-adherent
fraction was incubated in the presence of plate-bound anti-CD3 (5
.mu.g/ml) and anti-CD28 (1 .mu.g/ml) antibodies under Th1 (IL-2 (4
ng/ml) IL-12 (5 ng/ml)+anti-IL-4 (1 .mu.g/ml)) or Th2 (IL-2 (4
ng/ml)+IL-4 (4 ng/ml)+anti-IFN-.gamma. (1 .mu.g/ml)) conditions for
3 days in the presence or absence of MSCs. The cells were washed
and then re-stimulated with PHA (2.5 .mu.g/ml) for another 24 or 48
hours, following which levels of IFN-.gamma. and IL-4 were measured
in culture supernatants by ELISA (R&D Systems, Minneapolis,
Minn.).
[0114] Analysis of levels of VEGF, PGE.sub.2, and pro-MMP-1 in
culture supernatant of MSCs. Using previously characterized human
MSCs, the levels of Interleukin-6 (IL-6), VEGF, lipid mediator
PGE.sub.2, 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).
[0115] Proliferation of PBMCs. Purified PBMCs were prepared by
centrifuging leukopack (Cambrex, Walkersville, Md.) on
Ficoll-Hypaque (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, Mo.). In selected experiments, PBMCs were resuspended in
medium containing PGE.sub.2 inhibitors Indomethacin (Sigma
Chemicals, St. Louis, Mo.) or NS-938 (Cayman Chemicals, Ann Arbor,
Mich.). (.sup.3H)-thymidine was added (20 .mu.I in a 200 .mu.l
culture) and the cells harvested after an additional 24 hour
culture using an automatic harvester. The effects of MSCs or
PGE.sub.2 blockers were calculated as the percentage of the control
response (100%) in presence of PHA.
[0116] Quantitative RT-PCR. Total RNA from cell pellets were
prepared using a commercially available kit (Qiagen, Valencia,
Calif.) and according to the manufacturer's instructions.
Contaminating genomic DNA was removed using the DNA-free kit
(Ambion, Austin, Tex.). Quantitative RT-PCR was performed on a MJ
Research Opticon detection system (South San Francisco, Calif.)
using QuantiTect SYBR Green RT-PCR kit (Qiagen, Valencia, Calif.)
with primers at concentration of 0.5 .mu.M. Relative changes in
expression levels in cells cultured under different conditions were
calculated by the difference in Ct values (crossing point) using
.beta.-actin as internal control. The sequences for COX-1 and COX-2
specific primers were: COX-1: 5'-CCG GAT GCC AGT CAG GAT GAT G-3'
(forward) (SEQ ID NO:1), 5'-CTA GAC AGC CAG ATG CTG ACA G-3'
(reverse) (SEQ ID NO:2); COX-2: 5'-ATC TAC CCT CCT CAA GTC CC-3'
(forward) (SEQ ID NO:3), 5'-TAC CAG AAG GGC AGG ATA CAG-3'
(reverse) (SEQ ID NO:4).
[0117] 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 of PHA (2.5 .mu.g/ml) for 72 hours, and
.sup.3H thymidine incorporation (counts per minute, cpm) was
determined. The PBMCs and MSCs were cultured at ratios of MSC:PBMC
of 1:1, 1:3, 1:10, 1:30, and 1:81.
[0118] 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 can modulate
several steps in the immune response process. The production of
secreted factor(s) that modulate and can be responsible for MSC
immuno-modulatory effects was evaluated and prostaglandin synthesis
was implicated.
[0119] Myeloid (DC1) and plasmacytoid (DC2) precursor dendritic
cells were isolated by immuno-magnetic sorting of BDCA1.sup.+ and
BDCA2.sup.+ cells respectively and matured by incubation with
GM-CSF and IL-4 (1.times.10.sup.3 IU/ml and 1.times.10.sup.3 IU/ml,
respectively) for DC1 cells, or IL-3 (10 ng/ml) for DC2 cells.
Using flow cytometry, DC1 cells were HLA-DR.sup.+ and CD11c.sup.+,
whereas DC2 cells were HLA-DR.sup.+ and CD123.sup.+ (FIG. 1A). In
the presence of the inflammatory agent bacterial lipopolysaccharide
(LPS, 1 ng/ml), DC1 cells produced moderate levels of TNF-.alpha.
but when MSCs were present (ratios examined 1:1 and 1:10), there
was >50% reduction in TNF-.alpha. 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-.gamma. and increase IL-4 levels secreted by naive
CD4.sup.+T cells (FIG. 1C) suggesting a MSC-mediated shift from
pro-inflammatory to anti-inflammatory T cell phenotype.
[0120] 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 (T.sub.Reg) 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 T.sub.Reg cell
numbers as determined by staining of PBMCs with anti-CD4 and
anti-CD25 antibodies (FIG. 2A), further supporting a MSC-induced
tolerogenic response. The CD4.sup.+CD25.sup.+ T.sub.Reg cell
population, generated in presence of MSCs expressed increased
levels of gluocorticoid-induced TNF receptor (GITR), a cell surface
receptor expressed on T.sub.Reg 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.sup.+Th cells), IFN-.gamma.
producing Th1 and IL-4 producing T.sub.H2 cells were generated in
presence or absence of MSCs. When MSCs were present during
differentiation, there was reduced IFN-.gamma. secretion by Th1
cells and increased IL-4 secretion by Th2 cells (FIG. 2B). No
significant change in IFN-.gamma. 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.
[0121] 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-.gamma. secretion in
the culture supernatant (FIG. 2C), thereby suggesting that MSCs can
modulate NK cell functions also.
[0122] 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 E.sub.2, VEGF and
proMMP-lconstitutively, and the levels of each increased upon
culture with PBMCs (FIG. 5). In order to investigate MSC-derived
factors leading to inhibition of TNF-.alpha. and increase of IL-10
production by DCs, the potential role of prostaglandin E.sub.2 was
investigated, as it has been shown to inhibit TNF-.alpha.
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.times.10.sup.6 cells/ml) contained
approximately 1000 pg/ml of PGE.sub.2 (FIG. 4A). There was no
detectable presence of known inducers of PGE.sub.2 secretion, such
as TNF-.alpha., IFN-.gamma. or IL-10 (data not shown), in the
culture supernatant indicating a constitutive secretion of
PGE.sub.2 by MSCs. The PGE.sub.2 secretion by hMSCs was inhibited
60-90% in the presence of known inhibitors of PGE.sub.2 production,
NS-398 (5 .mu.M) and indomethacin (4 .mu.M) (FIG. 4A). As the
release of PGE.sub.2 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.
[0123] To investigate whether the immunomodulatory effects of MSC
on DCs and T-cells were mediated by PGE.sub.2, MSCs were cultured
with activated dendritic cells (DC1) or Th1 cells in the presence
of PGE.sub.2 inhibitors NS-398 or indomethacin. The presence of
NS-398 or indomethacin increased TNF-.alpha. secretion by DC1s, and
IFN-.gamma. secretion from Th1 cells (FIG. 4C), respectively,
suggesting that MSC effects on immune cell types can be mediated by
secreted PGE.sub.2. 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 PGE.sub.2 inhibitors NS-398 (5 .mu.M) or
indomethacin (4 .mu.M) were present, there was a >70% increase
in (.sup.3H) thymidine incorporation by PHA-treated PBMCs in MSC
containing cultures as compared to controls without inhibitors
(FIG. 4D).
[0124] In summary, a model of MSC interaction with other immune
cell types (FIG. 7) is proposed. When mature T cells are present,
MSCs can interact with them directly and inhibit the
pro-inflammatory IFN-.gamma. production (pathway 1) and promote
regulatory T cell phenotype (pathway 3) and anti-inflammatory
T.sub.H2 cells (pathway 5). Further, MSCs can alter the outcome of
the T cell immune response through DCs by secreting PGE.sub.2,
inhibiting pro-inflammatory DC1 cells (pathway 2) and promoting
anti-inflammatory DC2 cells (pathway 4) or regulatory DCs (pathway
3). A shift towards T.sub.H2 immunity in turn, suggests a change in
B cell activity towards increased generation of IgE/IgG1 subtype
antibodies (pathway 7). MSCs, by their ability to inhibit
IFN-.gamma. secretion from NK cells likely modify NK cell function
(pathway 6). This model of MSC:Immune cell interactions is
consistent with the experimentation performed in several other
laboratories (LeBlanc, et al., Exp. Hematol., Vol. 31, pg. 890
(2003); Tse, et al., Transplantation, Vol. 75, pg. 389 (2003);
DiNicola, et al., Blood, Vol. 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.
Example 2
[0125] Mesenchymal stem cells were given to a 33-year-old female
patient suffering from severe Grade IV gastrointestinal
graft-versus-host disease (GVHD). The patient was refractory to all
other GVHD treatments. Endoscopic views of the patient's colon
showed areas of ulceration and inflammation prior to treatment.
Histology of the patient's colon showed that the graft-versus-host
disease had destroyed the vast majority of the patient's intestinal
crypts, prior to treatment.
[0126] The patient was given an intravenous infusion of allogeneic
mesenchymal stem cells in 50 ml of Plasma Lyte A (Baxter) in an
amount of 3.times.10.sup.6 cells per kilogram of body weight.
[0127] The patient was evaluated at two weeks post-infusion. At two
weeks post-infusion, an endoscopic view of the patient's colon
showed that the areas of inflammation and ulceration visible prior
to treatment were resolved. In addition, a biopsy of the patient's
colon showed significant regeneration of intestinal crypts. Thus,
the administration of the mesenchymal stem cells to the patient
resulted in a significant reduction in the inflammatory component
of gastrointestinal graft-versus-host disease, and resulted in the
regeneration of new functional intestinal tissue.
[0128] The disclosures of all patents, publications, including
published patent applications, depository accession numbers, and
database accession numbers are hereby incorporated by reference in
their entireties to the same extent as if each patent, publication,
depository accession number, and database accession number were
specifically and individually incorporated by reference.
[0129] The presently described technology is now described in such
full, clear, concise and exact terms as to enable any person
skilled in the art to which it pertains, to practice the same. It
is to be understood that the foregoing describes preferred
embodiments of the technology and that modifications can be made
therein without departing from the spirit or scope of the invention
as set forth in the appended claims.
[0130] It is to be understood, however, that the scope of the
present technology is not to be limited to the specific embodiments
described above. The present technology can be practiced other than
as particularly described and still be within the scope of the
accompanying claims.
Sequence CWU 1
1
4122DNAArtificialsynthetic sequences and are primers for PCR
derived from human COX-1 1ccggatgcca gtcaggatga tg
22222DNAArtificialsynthetic sequences and are primers for PCR
derived from human COX-1 2ctagacagcc agatgctgac ag
22320DNAartificialsynthetic sequences and are primers for PCR
derived from human COX-2 3atctaccctc ctcaagtccc
20421DNAArtificialprimer for PCR derived from human COX-2
4taccagaagg gcaggataca g 21
* * * * *