U.S. patent application number 12/188922 was filed with the patent office on 2009-12-31 for method of treating autoimmune disease with mesenchymal stem cells.
This patent application is currently assigned to Genzyme Corporation. Invention is credited to Scott Eisenbeis, Tracey Lodie, Ross Tubo, Michele Youd.
Application Number | 20090324609 12/188922 |
Document ID | / |
Family ID | 39870057 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090324609 |
Kind Code |
A1 |
Lodie; Tracey ; et
al. |
December 31, 2009 |
METHOD OF TREATING AUTOIMMUNE DISEASE WITH MESENCHYMAL STEM
CELLS
Abstract
Methods and compositions for treating an autoimmune disease,
such as new onset type 1 diabetes (T1D) in a subject using
autologous or allogeneic mesenchymal stem cells administered to the
subject prior to autoimmune-induced complete depletion of
insulin-producing pancreatic beta cells, e.g., within six months of
new onset type 1 diabetes (T1D) diagnosis or prior to the onset of
disease in a subject determined to be at high risk for T1D.
Inventors: |
Lodie; Tracey; (Sutton,
MA) ; Youd; Michele; (Lexington, MA) ; Tubo;
Ross; (Quincy, MA) ; Eisenbeis; Scott;
(Westborough, MA) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Genzyme Corporation
Cambridge
MA
|
Family ID: |
39870057 |
Appl. No.: |
12/188922 |
Filed: |
August 8, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60954973 |
Aug 9, 2007 |
|
|
|
Current U.S.
Class: |
424/158.1 ;
424/184.1; 424/93.7 |
Current CPC
Class: |
A61P 3/10 20180101; C12N
2501/21 20130101; C12N 2501/515 20130101; A61K 38/13 20130101; A61K
35/28 20130101; C12N 2510/00 20130101; A61K 35/28 20130101; A61K
2300/00 20130101; A61K 38/13 20130101; C12N 5/0663 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/158.1 ;
424/93.7; 424/184.1 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61K 39/395 20060101 A61K039/395; A61K 39/00 20060101
A61K039/00 |
Claims
1. A method of treating new onset type 1 diabetes (T1D) in a
subject comprising administering autologous or allogeneic
mesenchymal stem cells to the subject prior to autoimmune-induced
complete depletion of insulin-producing pancreatic beta cells.
2. A method of treating new onset type 1 diabetes (T1D) in a human
subject comprising administering autologous or allogeneic
mesenchymal stem cells to the subject within six months of new
onset type 1 diabetes (T1D) diagnosis.
3. A method of treating new onset type 1 diabetes (T1D) in a human
subject determined to be at high risk for the disease comprising
administering autologous or allogeneic mesenchymal stem cells to
the subject.
4. The method of claim 2, wherein the mesenchymal stem cells are
administered within 10 days of T1D diagnosis.
5. The method of claim 2, wherein the mesenchymal stem cells are
administered within 24 hours of T1D diagnosis.
6. The method of claim 2, wherein the mesenchymal stem cells are
administered at the time of or before T1D diagnosis.
7. The method of claim 2, further comprising a second
administration of autologous or allogeneic mesenchymal stem cells
within ten days of the first administration of autologous or
allogeneic mesenchymal stem cells.
8. The method of claim 2, further comprising a second
administration of autologous or allogeneic mesenchymal stem cells
within one month of the first administration of autologous or
allogeneic mesenchymal stem cells.
9. The method of claim 2, further comprising a second
administration of autologous or allogeneic mesenchymal stem cells
within three months of the first administration of autologous or
allogeneic mesenchymal stem cells.
10. The method of claim 2, further comprising a second
administration of autologous or allogeneic mesenchymal stem cells
within six months of the first administration of autologous or
allogeneic mesenchymal stem cells.
11. The method of claim 2, further comprising a second
administration of autologous or allogeneic mesenchymal stem cells
within one year of the first administration of autologous or
allogeneic mesenchymal stem cells.
12. The method of claim 2, further comprising a second
administration of autologous or allogeneic mesenchymal stem cells
within two years of the first administration of autologous or
allogeneic mesenchymal stem cells.
13. The method of claim 2, further comprising a second
administration of autologous or allogeneic mesenchymal stem cells
within five years of the first administration of autologous or
allogeneic mesenchymal stem cells.
14. The method of claim 2, wherein the mesenchymal stem cells are
derived from bone marrow or peripheral blood.
15. The method of claim 14, wherein the bone marrow derived cells
comprise CD271-positive mesenchymal stem cells.
16. The method of claim 2, wherein the mesenchymal stem cells are
derived from umbilical cord blood cells.
17. The method of claim 2, wherein the mesenchymal stem cells are
derived from a population of cells selected from the group
consisting of muscle cells, fat cells, embryonic yolk sac cells,
placenta cells, fetal blood cells, fetal skin cells, and adult skin
cells.
18. The method of claim 2, wherein the mesenchymal stem cells are
administered to a subject having an abnormally low, but measurable,
serum C-peptide level.
19. The method of claim 18, wherein the subject further has an
abnormally high blood glucose level in the absence of exogenous
insulin administration.
20. The method of claim 18, wherein the abnormally high blood
glucose level is a fasting blood glucose level of greater than
about 120 mg/dl in the absence of exogenous insulin
administration.
21. The method of claim 18, wherein the subject has a fasting
C-peptide level of 0.1 nmol/L or greater.
22. The method of claim 18, wherein the subject has a fasting
C-peptide level of about 0.033 nmol/L or greater.
23. The method of claim 18, wherein the subject has a stimulated
C-peptide test integrated C-peptide level of 1.0 nmol/L or
less.
24. The method of claim 23, wherein the subject has a measurable
increase in stimulated C-peptide test integrated C-peptide.
25. The method of claim 23, wherein the subject has a measurable
increase in stimulated C-peptide test integrated C-peptide level of
0.54 nmol/L or less.
26. The method of claim 2, wherein the subject has a detectable
level of pancreatic autoantibody.
27. The method of claim 26, wherein the pancreatic autoantibody is
selected from the group consisting of GADAb, ICA, IA-2Ab, and
IAA.
28. The method of claim 2, wherein the subject has a HbA1c level of
7% or higher.
29. The method of claim 2, wherein the mesenchymal stem cells are
autologous.
30. The method of claim 29, wherein the autologous mesenchymal
stems cells are derived from umbilical cord blood.
31. The method of claim 2, wherein the mesenchymal stem cells are
allogeneic.
32. The method of claim 2, wherein the mesenchymal stem cells are
CD105-positive mesenchymal stem cells.
33. The method of claim 32, wherein the CD105 positive mesenchymal
stem cells are plastic-adherent and spindle-shaped cells.
34. The method of claim 32, wherein the CD105 positive mesenchymal
stem cells are capable of dividing to form a population of CD105
positive mesenchymal stem cells.
35. The method of claim 32, wherein the CD105 positive mesenchymal
stem cells are capable of differentiating into a differentiated
cell type.
36. The method of claim 35, wherein the CD105 positive mesenchymal
stem cells are capable of differentiating into more than one
differentiated cell type including at least one differentiated cell
type selected from the group consisting of osteoblasts,
chondrocytes, myocytes, adipocytes, and neuronal cells.
37. The method of claim 35, wherein the CD105 positive mesenchymal
stem cells are capable of differentiating into a tissue type
selected from the group consisting of bone, cartilage, muscle,
marrow stroma, tendon and connective tissue.
38. The method of claim 2, wherein the mesenchymal stem cells are
positive for one or more mesenchymal stem cell markers selected
from the group consisting of CD105 (endoglin, SH2), and CD73
(ecto-5' nucleotidase, SH3, SH4).
39. The method of claim 38, wherein the mesenchymal stem cells are
negative for the markers CD45, CD34, and/or CD14.
40. The method of claim 2, wherein the mesenchymal stem cells are
positive for the markers CD105, CD73 and CD90.
41. The method of claim 40, wherein the mesenchymal stem cells are
negative for the markers CD45, CD34, and CD14.
42. The method of claim 40, wherein the mesenchymal stem cells are
plastic-adherent when maintained in standard culture conditions and
are capable of differentiating in vitro into osteoblasts,
adipocytes and/or chondrobasts.
43. The method of claim 2, further comprising administering to the
subject an immunosuppressive agent.
44. The method of claim 43, wherein the immunosuppressive agent is
selected from the group consisting of prednisone, azathioprine,
cyclosporine, antibodies against CD3, antibodies against CD20, and
antithymocyte globulin.
45. The method of claim 2, further comprising administering to the
subject a peptide vaccine.
46. The method of claim 45, wherein the vaccine induces tolerance
of insulin-producing cells.
47. The method of claim 46, wherein the vaccine comprises an
autoimmune Type I diabetes (T1D) autoantigen.
48. The method of claim 47, wherein the vaccine comprises an
islet-cell autoantigen selected from the group consisting of
insulin, proinsulin, glutamic acid decarboxylase (GAD65), HSP60,
and IA-2 protein tyrosine phosphatase.
49. The method of claim 2, further comprising administering a
non-mitogenic anti-CD3 active compound selected from the group
consisting of CD3 antibodies and fragments of CD3 antibodies.
50. The method of claim 49, wherein the non-mitogenic anti-CD3
active compound is administered in an injectable form comprising 5
to 20 mg of the non-mitogenic anti-CD3 active compound.
51. The method of claim 43, wherein the immunosuppressive agent is
administered contemporaneously with the autologous or allogeneic
mesenchymal stem cells
52. The method of claim 43, wherein the immunosuppressive agent is
administered within one month of the autologous or allogeneic
mesenchymal stem cells.
53-74. (canceled)
Description
[0001] This application claims the benefit of priority of U.S.
Provisional Patent Application No. 60/954,973, filed Aug. 9, 2007,
the entire contents of which are incorporated by reference.
[0002] Diabetes is characterized by chronic hyperglycemia resulting
from a lack of insulin action, along with various characteristic
metabolic abnormalities. Diabetes can be broadly divided into type
I and type II. Type I diabetes (T1D) is characterized by the loss
of pancreatic .beta.-cells of the Langerhans' islets, while type II
diabetes is characterized by reductions in both insulin secretion
and insulin sensitivity (insulin resistance). In the United States,
the prevalence of diabetes is about 2 to 4 percent of the
population, with type I (insulin-dependent or IDDM) making up about
7 to 10 percent of all cases.
[0003] Type I diabetes mellitus is characterized by the dysfunction
of the pancreas to produce insufficient or no insulin. This
disorder is caused by autoimmune-mediated destruction of the
pancreatic .beta.-cells. Autoimmunity associated with type I
diabetes mellitus involves the participation of both B and T
autoreactive lymphocytes. Indeed, up to 98% of type I diabetes
mellitus patients have antibodies against one or more of their own
.beta.-cell antigens. These include: insulin (Atkinson, et al.,
Diabetes 35:894-98 (1986)); the major of the 2 isoforms of glutamic
acid decarboxylase (GAD) 65 (Atkinson, et al., J. Clin. Invest.
91:350-56 (1993)); two of the protein tyrosine phosphatases,
insulinoma antigen-2 and insulinoma antigen-2b (IA-2 and
IA-2.beta.) (Lu, et al., Proc. Natl. Acad. Sci USA 93:2307-11
(1996); Lan, et al., Proc. Natl. Acad. Sci. USA 93:6367-70 (1996));
and the heterogeneous islet cell cytoplasmic antigens (ICAs)
(Gorus, et al., Diabetologia 40:95-99 (1997); Strebelow, et al.,
Diabetologia 42:661-70 (1999)). A minority of type I diabetes
mellitus patients also have serum antibodies to a glycosylated
islet cell membrane antigen, GLIMA (Aanstoot, et al., J. Clin.
Invest. 97:2772-83 (1996)). More recently, autoantibodies to other
new antigens of protein tyrosine phosphatases, IA-2/ICA512 and
IA-2.beta./phogrin, expressed by pancreatic islet cells, have also
been detected in type I diabetes mellitus patients (Kawasaki, et
al., Diabetes 47:733-42 (1998)).
[0004] The generation of autoantibodies to islet cells can be
observed for as many as 10 years prior to the onset of clinical
diabetes (Luhder, et al., Autoimmunity 19:71-78 (1994)). Despite
this observation, the existence of autoantibodies is not solely
sufficient to cause development of type I diabetes mellitus. This
conclusion is based on the finding that infants born of antibody
positive type I diabetes mellitus mothers can remain free of
disease despite the existence of serum autoantibodies to insulin,
GAD and other islet cell antigens. On the other hand, persons with
severe genetic B cell deficiency can still develop type I diabetes
mellitus (Martin, et al., N. Engl. J. Med. 345:1036-40 (2001)).
Generally, the level of autoantibodies correlates with the state of
.beta.-cell destruction (Irvine, et al., Diabetes 26:138-47 (1997);
Riley, et al., N. Engl. J. Med. 323:1167-72 (1990)). As such,
autoantibodies can serve as indicators of the development of
autoimmune diabetes. A low level of GAD-specific autoantibodies is
associated with a slow breakdown of .beta.-cell function, while a
high level of autoantibodies to IA-2 together with the maturation
of autoantibody responses elicited against ICAs or GAD are signs
for more severe and imminent .beta.-cell failure (Borg, et al., N.
Engl. J. Med. 86:3032-38(2001)).
[0005] The development of type I diabetes mellitus may be mediated
by autoreactive T cells. The most direct indication of this is the
direct examination of biopsy tissues obtained near the time of type
I diabetes mellitus diagnosis, which show that the islets are
infiltrated with activated T cells, primarily of the CD8+
population but also, to a lesser extent, CD4+ cells and macrophages
as well (Bottazzo, et al., N. Engl. J. Med. 313:353-60 (1985);
Hanninen, et al., J. Clin. Invest. 90:1901-10 (1992); Itoh, et al.,
J. Clin. Invest. 92:2313-22 (1993); Imagawa, et al., Diabetes
50:1269-73 (2001)). The association of type I diabetes mellitus
with the major histocompatibility complex (MHC)-associated
susceptibility gene locus, type I diabetes mellitus, has also been
well reported (Froguel, Horm. Res. 48:55-57 (1997)). Recurrence of
organ-specific autoimmunity is responsible for .beta.-cell
destruction in diabetics transplanted with a pancreatic graft
donated by their discordant, non-diabetic monozygotic twins
(Sutherland, et al., Trans. Assoc. Am. Physic. 97:80-87 (1984)).
Furthermore, type I diabetes mellitus is transferable to
non-diabetics given bone marrow transplant donated by diabetic
HLA-identical siblings, or allogeneic donors (Marmont, et al., J.
Rheumatol. 48:13-18 (1997)).
[0006] Autoreactive CD4+ cells of the Th1 subset are potentially
capable of directly and indirectly causing islet damage; directly
via the release of cytotoxic mediators such as nitric oxide or
oxygen radicals (Held, et al., Proc. Natl. Acad. Sci. USA
87:2239-43(1990)), and indirectly through the secretion of IL-2 and
IFN-.gamma. by activating autoreactive CD8+ T cells and macrophages
leading to their infiltration of the islets (Jean-Michel and
Burger, Arthritis. Res. 1:17-20 (1999)). In this regard,
characterization and quantitation of autoreactive T cells in humans
are important for the development of an improved diagnosis of type
I diabetes mellitus, and intervention strategies for arresting
disease progression. However, direct detection of autoreactive T
cells in type I diabetes mellitus is more difficult than the
detection of autoantibodies. The reason is that CD4+ and CD8+
autoreactive T cells generated in the course of type I diabetes
mellitus development are only present at very low frequencies in
the circulation of subjects with recent disease onset (Tisch and
McDevitt, Cell 85:291-97 (1996); Notkins and Lernmar, J. Clin.
Invest. 108:1247-52(2001)).
[0007] Assays dependent on in vitro expansion to allow the
detection of autoreactive CD4+ T cells in the pool of peripheral
blood leucocytes (PBL) of diabetics have been used in some studies.
When employing in vitro proliferation assays, PBL of individuals
with recent onset of type I diabetes mellitus respond to human
insulin (Keller, Autoimmunity 3:321-27(1994)), a spectrum of islet
cell antigens (Roep, et al., Diabetes 44:278-83 (1995);
Brooks-Worrell, et al., J. Immunol. 157:5668-74 (1996); Mayer, et
al., J. Clin. Endocrinol. Metab. 84:2419-24 (1999)), and GAD
(Atkinson, Lancet 339:458-59 (1992)). Regarding detection,
GAD-specific autoreactive T cells can be generated and cloned from
peripheral T cells of recent onset type I diabetes mellitus
patients who are carrying the disease-susceptible HLA-DR alleles
(Endl, et al., J. Clin. Invest. 99:2405-15 (1997)). Furthermore,
endogenous GAD fragments presented by type I diabetes
mellitus-associated HLA class II molecules can be isolated (Nepom,
et al., Proc. Natl. Acad. Sci USA 98:1763-68 (2001)).
[0008] Autoreactive CD8+ T cells have been detected against two
.beta.-cell antigens in diabetic humans, namely GAD 65 and
preproIAPP (precursor human islet amyloid polypeptide protein),
which are co-secreted with insulin in subjects recently diagnosed
with type I diabetes mellitus. GAD 65-specific cytotoxic T cells
(CTLs) carrying the disease-associated allele, HLA-A2, following in
vitro expansion with a HLA-A2 binding peptide, have been generated
from PBL of these individuals (Panina-Bordignon, et al., J. Exp.
Med. 181:1923-27 (1995)). A recent study describes the presence of
an autoreactive CD8+ subset in the circulation of recently
diagnosed patients that recognizes a 9 amino acid long
immunodominant epitope of preproIAPP in the context of HLA-A2 using
an IFN-.gamma.-based ELISPOT assay (Panagiotopoulos, et al.,
Diabetes 52:2647-51 (2003)). The direct detection and quantitation
of circulating autoreactive T cells at early disease onset may
provide a valuable tool for improved diagnosis of type I diabetes
mellitus.
[0009] The discovery that diabetics mount humoral and cellular
immune responses against islet cell antigens (ICAs) has led to the
testing of ICAs and their analogs as candidates for therapeutic
agents for better treatment of type I diabetes mellitus at its
prediabetic and diabetic stages. In addition, various immunological
intervention strategies aimed at direct targeting of the
autoreactive T cells have also been investigated. Nevertheless, new
and alternative methods for treating and/or preventing the onset of
type I diabetes mellitus are needed.
[0010] Thus, the invention provides methods of treating or
preventing the onset of type 1 diabetes (T1D) in a subject by
administering autologous or allogeneic mesenchymal stem cells to
the subject before the complete autoimmune-induced depletion of
insulin-producing pancreatic beta cells. The invention is based, in
part, upon the observation that mesenchymal stem cells, when
administered to a mammalian subject prior to the complete
auto-immune induced depletion of insulin-producing pancreatic beta
cells, can treat, or even prevent the development of, new onset of
type 1 diabetes (T1D).
[0011] In one aspect, the invention provides a method of treating
new onset type 1 diabetes (T1D) in a subject by administering
autologous or allogeneic mesenchymal stem cells to the subject
prior to autoimmune-induced complete depletion of insulin-producing
pancreatic beta cells. In another aspect, the method of treating
new onset type 1 diabetes (T1D) involves administering autologous
or allogeneic mesenchymal stem cells to the subject within six
months of new onset type 1 diabetes (T1D) diagnosis. In still
another aspect, the invention provides a method of treating or
preventing new onset type 1 diabetes (T1D) in a human subject
determined to be at high risk for the disease by preemptively
administering autologous or allogeneic mesenchymal stem cells to
the subject.
[0012] In certain embodiments, the invention provides methods of
treating T1D by administering the mesenchymal stem cells within 10
days of T1D diagnosis. In other embodiments, the mesenchymal stem
cells are administered within 24 hours of T1D diagnosis. In still
other embodiments, the mesenchymal stem cells are administered at
the time of, or even before T1D diagnosis (e.g., following a
determination that the subject is at high risk for developing T1D
such as by the presence of a predisposing genotype or the initial
presence of diabetic auto-antibodies or other pre-diabetic
autoimmune indicators).
[0013] In some embodiments, the method of the invention further
includes a second administration of autologous or allogeneic
mesenchymal stem cells within ten days of the first administration
of autologous or allogeneic mesenchymal stem cells. In further
embodiments, the second administration of autologous or allogeneic
mesenchymal stem cells is made within one month of the first
administration of autologous or allogeneic mesenchymal stem cells.
In still further embodiments, the second administration of
autologous or allogeneic mesenchymal stem cells may be made within
three months, six months, one year, two years, or even five years
of the first administration of autologous or allogeneic mesenchymal
stem cells.
[0014] In certain embodiments, the invention provides methods
wherein the mesenchymal stem cells are derived from bone marrow or
peripheral blood. In particular embodiments, the bone marrow
derived cells comprise CD271-positive mesenchymal stem cells. In
further embodiments, the mesenchymal stem cells may be derived from
umbilical cord blood cells. In other embodiments, the mesenchymal
stem cells may be derived from a population of muscle cells, fat
cells, embryonic yolk sac cells, placenta cells, fetal blood cells,
fetal skin cells, or adult skin cells.
[0015] In general, the invention provides methods of treating or
preventing new onset type 1 diabetes (T1D) by administering
mesenchymal stem cells to a subject in the early stages of
autoimmune-induced loss of pancreatic islet .beta.-cells. The early
stages of autoimmune-induced loss of pancreatic islet .beta.-cells
may be defined by one or more temporal parameters. In certain
embodiments, the mesenchymal stem cells are administered to a
subject having an abnormally low, but measurable, serum C-peptide
level. Serum C-peptide levels decline with the onset of T1D, and a
low, but measurable, level of C-peptide is one indication that the
subject is in the early stages of autoimmune-induced loss of
pancreatic islet .beta.-cells. Other temporal indicators of the
early stages of autoimmune-induced T1D may further be used to
refine the method of the invention.
[0016] In particular embodiments, the therapeutic mesenchymal stem
cells are administered to a subject having both an abnormally low,
but measurable, serum C-peptide level, and an abnormally high blood
glucose level in the absence of exogenous insulin administration.
In certain embodiments, the abnormally high blood glucose level is
a fasting blood glucose level of greater than about 120 mg/dl in
the absence of exogenous insulin administration. In further
embodiments, the subject has a fasting C-peptide level of about
0.033 nmol/L or greater. In particular embodiments, the subject has
a fasting C-peptide level of 0.1 nmol/L or greater. In still
further embodiments, the subject has a fasting C-peptide level of
1.0 nmol/L or less. In particular embodiments, the subject has a
fasting C-peptide level of about 0.033 nmol/L to about 1.0 nmol/L.
In other embodiments, the subject has a fasting C-peptide level of
about 0.1 nmol/L to about 1.0 nmol/L. In still further embodiments,
the subject manifests a measurable increase in post-oral glucose
tolerance test integrated C-peptide level, or, preferably, the
subject manifests a measurable increase in stimulated C-peptide
test integrated C-peptide level. In particular embodiments, the
subject has a measurable increase of 0.54 nmol/L, or less, in
post-oral glucose tolerance test integrated C-peptide levels, or,
more preferably, the subject manifests an increase of 0.54 nmol/L,
or less, in stimulated C-peptide test integrated C-peptide
levels.
[0017] Other parameter(s) may also be used to indicate the
subject's amenability to the method of the invention. For example,
in certain embodiments the subject has a detectable level of
pancreatic autoantibody. In certain embodiments, the pancreatic
autoantibody may be GADAb, ICA, IA-2Ab, or IAA. In further
embodiments, the subject has an HbA1c level of 7% or higher.
[0018] In still other embodiments of the invention, the mesenchymal
stem cells administered to the subject may be autologous
mesenchymal stem cells (i.e., derived from the same subject to
which they are administered). In particular embodiments, the
autologous mesenchymal stem cells are derived from umbilical cord
blood.
[0019] In further embodiments, the mesenchymal stem cells
administered to the subject may be allogeneic mesenchymal stem
cells (i.e., derived from individuals of the same species as the
subject to which they are administered).
[0020] In still further embodiments, the mesenchymal stem cells
administered to the subject are CD105 positive. In particular
embodiments, the CD105 positive mesenchymal stem cells are
plastic-adherent and spindle-shaped cells. In certain embodiments,
the CD105 positive mesenchymal stem cells are capable of dividing
to form a population of CD105 positive mesenchymal stem cells. In
some embodiments, the CD105 positive mesenchymal stem cells are
capable of differentiating into a differentiated cell type. In
particular embodiments, the CD105 positive mesenchymal stem cells
are capable of differentiating into multiple different
differentiated cell types. In certain embodiments, the mesenchymal
stem cells are capable of differentiating into osteoblasts,
chondrocytes, myocytes, adipocytes, and/or neuronal cells. In some
embodiments, the CD105 positive mesenchymal stem cells are capable
of differentiating into a particular tissue type. In certain
embodiments, the mesenchymal stem cells are capable of
differentiating into bone, cartilage, muscle, marrow stroma, tendon
and/or connective tissue.
[0021] In yet other embodiments, the mesenchymal stem cells are
positive for one or more mesenchymal stem cell markers such as
CD105 (endoglin, SH2), and/or CD73 (ecto-5' nucleotidase, SH3,
SH4). In particular embodiments, the mesenchymal stem cells are
negative for the markers CD45, CD34, and/or CD14.
[0022] In certain embodiments, the mesenchymal stem cells are
positive for the markers CD105, CD73 and CD90. In particular
embodiments, the mesenchymal stem cells are negative for the
markers CD45, CD34, and CD14. In some such embodiments, the
mesenchymal stem cells are plastic-adherent when maintained in
standard culture conditions and are capable of differentiating in
vitro into osteoblasts, adipocytes and/or chondrobasts.
[0023] In other useful aspects, the invention provides methods in
which, in addition to the autologous or allogeneic mesenchymal stem
cells, the subject is further administered an immunosuppressive
agent. In particular embodiments, the immunosuppressive agent is
prednisone, azathioprine, cyclosporine, antibodies against CD3,
antibodies against CD20, or antithymocyte globulin. In certain
embodiments, the immunosuppressive agent is administered
contemporaneously with the autologous or allogeneic mesenchymal
stem cells. In other embodiments, the immunosuppressive agent is
administered within one month of the autologous or allogeneic
mesenchymal stem cells.
[0024] In still other useful aspects, the invention provides
methods in which, in addition to the autologous or allogeneic
mesenchymal stem cells, the subject is further administered a
peptide vaccine. In particular embodiments, the vaccine induces
tolerance of insulin-producing cells. In certain embodiments, the
vaccine includes an autoimmune type 1 diabetes (T1D) autoantigen.
In particular embodiments, the autoantigen is insulin, proinsulin,
glutamic acid decarboxylase (GAD65), HSP60, or IA-2 protein
tyrosine phosphatase.
[0025] In further useful aspects, the invention provides methods in
which, in addition to the autologous or allogeneic mesenchymal stem
cells, the subject is further administered a non-mitogenic anti-CD3
active compound, such as a CD3 antibody, or a CD3-binding antibody
fragment. In particular embodiments, the non-mitogenic anti-CD3
active compound is administered in an injectable form having 5 to
20 mg of the non-mitogenic anti-CD3 active compound.
[0026] In another aspect, the invention provides a mesenchymal stem
cell expressing an exogenous PD-L1 and/or PD-L2 gene or activity
(e.g., a mammalian PD-L1 and/or PD-L2 expression vector, such as an
adenovirus vector express PD-L1). In particular embodiments, the
mesenchymal stem cell expresses an exogenous PD-L1 gene or
activity. In other embodiments, the mesenchymal stem cell
overexpresses, relative to a native mesenchymal stem cell, an
endogenous PD-L1 and/or PD-L2 gene or activity (e.g., by insertion
of a strong transcriptional promoter upstream of the PD-L1 and/or
PD-L2 gene, or by selection of epigenetic variants over-expressing
one or more of these genes). In particular embodiments, the
mesenchymal stem cells overexpressing an endogenous PD-L1 and/or
PD-L2 are screened or selected from a group of native mesenchymal
stem cells based upon high PD-L1 and/or PD-L2 expression.
[0027] In still another aspect, the invention provides a method of
treating an autoimmune disease or disorder in a mammal by
administering autologous or allogeneic mesenchymal stem cells
expressing an exogenous PD-L1 and/or PD-L2 gene or activity, or
overexpressing, relative to a native mesenchymal stem cell, an
endogenous PD-L1 and/or PD-L2 gene or activity. In particular
embodiments, the autoimmune disease or disorder is T1D.
[0028] In a further useful aspect, the invention provides a method
of treating an autoimmune disease in a mammal by administering
autologous or allogeneic mesenchymal stem cells in combination with
one or more PD-1-PDL-1/PDL-2 pathway agonists. In certain
embodiments the PD-1-PDL-1/PDL-2 pathway agonist is a small
molecule, an antibody, and/or a fusion protein. In particular
embodiments, the PD-I-PDL-1/PDL-2 pathway agonist is a PD-L1 -Fc
fusion protein. In certain embodiments, the PD-L1 polypeptide of
the PD-L1-Fc fusion protein is a human PD-L1 polypeptide. In
particular embodiments, the PD-I-PDL-1/PDL-2 pathway agonist is a
fusion protein that includes an anti-PD-1 Fab fragment and an Fc
fragment. In further embodiments, the fusion protein includes a
linker, e.g., a flexible polypeptide segment joining the PD-L1
polypeptide portion to the Fc polypeptide portion of the PD-L1-Fc
fusion protein. In certain embodiments, the autoimmune disease or
disorder is T1D.
[0029] In another useful aspect, the invention provides a
mesenchymal stem cell that underexpresses, relative to a native
mesenchymal stem cell, a CXCL10-CXCR3 pathway gene or activity. In
one embodiment, the CXCL10-CXCR3-underexpressing mesenchymal stem
cell may be one to which a CXCL10 siRNA has been administered
(e.g., transfected with). In another embodiment, the mesenchymal
stem cell is engineered to express a CXCL10 siRNA (e.g., from an
siRNA expression vector construct). In still another embodiment,
the CXCL10-CXCR3-underexpressing mesenchymal stem cell may be one
that underexpresses one or more endogenous CXCL10-CXCR3 genes or
activities (e.g., by insertion of a transcriptional silencer
upstream of one or more CXCL10-CXCR3 pathway genes or activities,
or by selection of epigenetic variants underexpressing one or more
of these genes). In particular embodiments, the mesenchymal stem
cells underexpressing an endogenous PD-L1 and/or PD-L2 are screened
or selected from a group of native mesenchymal stem cells based
upon low CXCL10-CXCR3 pathway expression or activity. In still
other embodiments, the CXCL10-CXCR3-underexpressing mesenchymal
stem cell may be one which is treated with a CXCL10-CXCR3 pathway
antagonist. In particular embodiments, the CXCL10-CXCR3 pathway
antagonist is a CXCR3 siRNA and/or a CXCL10 antibody.
[0030] In yet another aspect, the invention provides a method of
treating an autoimmune disease in a mammal by administering
autologous or allogeneic mesenchymal stem cells underexpressing,
relative to native mesenchymal stem cells, a CXCL10-CXCR3 pathway
gene or activity. In certain embodiments, the autoimmune disease or
disorder is T1D. In further embodiments, the method provides for
treating an autoimmune disease in a mammal by administering
autologous or allogeneic mesenchymal stem cells in combination with
one or more antagonists of a CXCL10-CXCR3 pathway gene or activity.
In an exemplary embodiment, the CXCL10-CXCR3 pathway antagonist is
a small molecule, an antibody, and/or a fusion protein.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1 is a graphical representation of experiments
demonstrating that administration of normal mesenchymal stem cells
(MSCs) to prediabetic nonobese diabetic (NOD) mice prevents or
delays the onset of type I diabetes (T1D).
[0032] FIG. 2 is a graphical representation of experiments
demonstrating that green fluorescent protein (GFP) transgenic MSCs
track to pancreatic lymph nodes and spleen when administered to
pre-diabetic (top panels) and diabetic (bottom panels) NOD mice.
Tissues examined (bars from left to right) are spleen, liver,
kidney, mesenteric lymph nodes, pancreatic lymph nodes, and
non-draining peripheral lymph nodes.
[0033] FIG. 3 is a graphical representation of experiments
demonstrating that administration of normal allogeneic MSCs, but
not NOD MSCs, delays the onset of diabetes in pre-diabetic NOD
mice.
[0034] FIG. 4A is a "heat map" expression profile showing the
various up-regulated and down-regulated genes in NOD MSCs after
IL1.beta. treatment.
[0035] FIG. 4B is a summary of the genes differentially expressed
in NOD autoimmune-prone MSCs as compared to normal MSCs following
IL1.beta. treatment.
[0036] FIG. 5A is a graphical representation of experiments
demonstrating that normal (Balb/c or C57BL/6) MSCs, but not NOD
MSCs, up-regulated PD-L1 in response to IL1.beta. treatment.
[0037] FIG. 5B is a flow cytometry analysis of PD-L1 protein on the
surface of normal (Balb/c) and diabetic (NOD) MSCs treated with
IL1.beta..
[0038] FIG. 6 is a graphical representation of experiments
demonstrating that MSCs lacking PD-L1 expression demonstrate a
reduced ability to inhibit T cell proliferation.
[0039] FIG. 7A is a flow cytometry analysis of PD-L1 expression on
the surface of NOD MSCs infected with adenoviral vector encoding
mouse membrane PD-L1 (Ad.mPD-L1).
[0040] FIG. 7B is a graphical representation of experiments
demonstrating that NOD MSCs engineered to over-express PD-L1 delay
onset of diabetes in NOD mice.
[0041] FIG. 8A is a graphical representation of experiments
demonstrating that NOD MSCs, but not normal (Balb/c or C57BL/6)
MSCs, over-express CXCL10-CXCR3 pathway genes.
[0042] FIG. 8B is a graphical representation of experiments
demonstrating that NOD MSCs express CXCL10 in response to
IFN-.gamma. treatment.
[0043] FIG. 9A is a graphical representation of experiments
demonstrating the inhibition by different doses of human MSCs (MSC)
of human T cell (TC) proliferation induced by human allogeneic
dendritic cells (DC), as measured by tritiated thymidine
incorporation after 6 days.
[0044] FIG. 9B is a graphical representation of experiments
demonstrating the inhibition by human MSCs (MSC) from different
donors (M28, M29 and M41).
[0045] FIG. 9C is a graphical representation of experiments
demonstrating the inhibition by different doses of human MSCs (MSC)
or human umbilical vein endothelial cells (HUVECs) of human
peripheral blood mononuclear cell (PBMC) proliferation induced by
anti-CD3/anti-CD28 coated beads, as measured by tritiated thymidine
incorporation after 5 days.
[0046] FIG. 10 is a graphical representation of experiments
demonstrating that MSCs modulate cytokines in vitro as shown by
TNF.alpha. (middle panel) and IL10 (bottom panel) levels following
T cell activation by human peripheral blood mononuclear cells
(PBMC) (proliferation induced by human allogeneic dendritic cells
(DC), as measured by tritiated thymidine incorporation, is shown in
the top panel).
[0047] FIG. 11A is a graphical representation of experiments
demonstrating that mouse MSCs inhibit T cell proliferation in
vitro.
[0048] FIG. 11B is a graphical representation of experiments
demonstrating that mouse MSCs dampen TNF.alpha. in vivo.
[0049] FIG. 12A is a graphical representation of experiments
demonstrating the delay of diabetes onset by administration of
allogeneic MSCs.
[0050] FIG. 12B is a graphical representation of experiments that
PDL-1 knock-out MSCs do not significantly delay the onset of
diabetes in NOD mice.
[0051] FIG. 13A is a graphical representation of experiments
demonstrating the reversal of diabetes with allogeneic MSCs, as
demonstrated by blood glucose levels following administration of
1.times.10.sup.6 allogeneic Balb/c MSCs (top panel), but no
reversal of diabetes with PBS (bottom panel) administered twice
weekly (black arrowheads).
[0052] FIG. 13B is a graphical representation of experiments
demonstrating the reversal of diabetes as indicated by glucose
tolerance tests of MSC-treated mice that did reverse (top graphs,
mice 3830 and 3895) compared to MSC-treated mice that did not
reverse (bottom graphs, mice 4056 and 3926).
[0053] FIG. 13C is a graphical representation of experiments
demonstrating the daily insulin dosage for mice treated with MSCs
that reversed (left panel) versus MSC treated mice that did not
reverse (right panel).
[0054] In general, the invention provides methods and compositions
for treating autoimmune disease, such as new onset T1D, with
mesenchymal stem cells (MSCs). In particular, the invention
provides compositions and beneficial methods of delivery of MSCs to
patients with early onset diabetes. The invention further provides
genes and markers identified by expression profile analysis of
MSCs, including Programmed death 1 (PD-1)--Programmed death ligand
1 (PD-L1) and Programmed death ligand 2 (PD-L2) as well as the
components of the CXCL10-CXCR3 pathway, which provide new
therapeutic targets that may be used in the treatment of patients
with type I diabetes.
Definitions
[0055] The singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise.
[0056] All numbers expressing quantities of ingredients, reaction
conditions, and so forth used in the specification and claims are
to be understood as being modified in all instances by the term
"about," wherein about signifies, e.g., .+-.5%, .+-.10%, .+-.15%,
.+-.20%, or .+-.25%. Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the specification and
attached claims are approximations that may vary depending upon the
desired properties sought to be obtained by the present invention.
At the very least, and not as an attempt to limit the application
of the doctrine of equivalents to the scope of the claims, each
numerical parameter should be construed in light of the number of
significant digits and ordinary rounding approaches.
[0057] The term "agonist" as used herein, is meant to refer to an
agent that mimics or up-regulates (e.g., potentiates or
supplements) the bioactivity of a protein. An agonist can be a
wild-type protein or derivative thereof having at least one
bioactivity of the wild-type protein. An agonist can also be a
compound that up-regulates expression of a gene or increases at
least one bioactivity of a protein. An agonist can also be a
compound that increases the interaction of a polypeptide with
another molecule, e.g., a target peptide or nucleic acid.
[0058] "Antagonist" as used herein is meant to refer to an agent
that down-regulates (e.g., suppresses or inhibits) at least one
bioactivity of a protein. An antagonist can be a compound that
inhibits or decreases the interaction between a protein and another
molecule, e.g., a target peptide or enzyme substrate. An antagonist
can also be a compound that down-regulates expression of a gene or
which reduces the amount of expressed protein present.
[0059] The term "antibody" as used herein refers to both polyclonal
and monoclonal antibody. The term encompasses not only intact
immunoglobulin molecules, but also such fragments and derivatives
of immunoglobulin molecules (such as single chain Fv constructs,
diabodies, and fusion constructs) that retain a desired antibody
binding specificity, as may be prepared by techniques known in the
art.
[0060] The terms "array" or "matrix" is means an arrangement of
addressable locations or "addresses" on a device. The locations can
be arranged in two-dimensional arrays, three-dimensional arrays, or
other matrix formats. The number of locations can range from
several to at least hundreds of thousands. Most importantly, each
location represents a totally independent reaction site. A "nucleic
acid array" refers to an array containing nucleic acid probes, such
as oligonucleotides or larger portions of genes. The nucleic acid
on the array is preferably single stranded. Arrays wherein the
probes are oligonucleotides are referred to as "oligonucleotide
arrays" or "oligonucleotide chips." A "microarray," also referred
to herein as a "biochip" or "biological chip," is an array of
regions having a density of discrete regions of at least about
100/cm.sup.2, and preferably at least about 1000/cm.sup.2. The
regions in a microarray have typical dimensions, e.g., diameters,
in the range of between about 10-250 um, and are separated from
other regions in the array by about the same distance.
[0061] As used herein, the term "autoimmune disease" means a
disease resulting from an immune response against a self tissue or
tissue component, including both self antibody responses and
cell-mediated responses. The term autoimmune disease, as used
herein, encompasses organ-specific autoimmune diseases, in which an
autoimmune response is directed against a single tissue, such as
type I diabetes mellitus (T1D), Crohn's disease, ulcerative
colitis, myasthenia gravis, vitiligo, Graves' disease, Hashimoto's
disease, Addison's disease and autoimmune gastritis and autoimmune
hepatitis. The term autoimmune disease also encompasses non-organ
specific autoimmune diseases, in which an autoimmune response is
directed against a component present in several or many organs
throughout the body. Such autoimmune diseases include, for example,
rheumatoid disease, systemic lupus erythematosus, progressive
systemic sclerosis and variants, polymyositis and dermatomyositis.
Additional autoimmune diseases include pernicious anemia including
some of autoimmune gastritis, primary biliary cirrhosis, autoimmune
thrombocytopenia, Sjogren's syndrome, multiple sclerosis and
psoriasis. One skilled in the art understands that the methods of
the invention can be applied to these or other autoimmune diseases,
as desired.
[0062] The term "biological sample" as used herein, refers to a
sample obtained from a subject, e.g., a human or from components
(e.g., tissues) of a subject. The sample may be of any biological
tissue or fluid. Frequently the sample will be a "clinical sample"
which is a sample derived from a patient. Such samples include, but
are not limited to bodily fluids which may or may not contain
cells, e.g., blood, synovial fluid; tissue or fine needle biopsy
samples, such as from bone, cartilage or tissues containing
mesenchymal cells. Biological samples may also include sections of
tissues such as frozen sections taken for histological
purposes.
[0063] The term "biomarker" of a disease related to bone or
cartilage formation or resorption refers to a gene that is up- or
down-regulated in a diseased cell of a subject having such a
disease, relative to a counterpart normal cell, which gene is
sufficiently specific to the diseased cell that it can be used,
optionally with other genes, to identify or detect the disease.
Generally, a biomarker is a gene that is characteristic of the
disease.
[0064] The terms "cell culture" and "culture" encompass the
maintenance of cells in an artificial, in vitro environment. It is
to be understood, however, that the term "cell culture" is a
generic term and may be used to encompass the cultivation not only
of individual cells, but also of tissues, organs, organ systems or
whole organisms, for which the terms "tissue culture," "organ
culture," "organ system culture," or "organotypic culture" may
occasionally be used interchangeably with the term "cell
culture."
[0065] The term "derivative" refers to the chemical modification of
a compound, e.g., a polypeptide, or a polynucleotide. Chemical
modifications of a polynucleotide can include, for example,
replacement of hydrogen by an alkyl, acyl, or amino group. A
derivative polynucleotide encodes a polypeptide which retains at
least one biological or immunological function of the natural
molecule. A derivative polypeptide can be one modified by
glycosylation, pegylation, or any similar process that retains at
least one biological or immunological function of the polypeptide
from which it was derived.
[0066] The term "expression profile," which is used interchangeably
herein with "gene expression profile," "finger print" and
"expression pattern", refers to a set of values representing the
activity of about 10 or more genes. An expression profile
preferably comprises values representing expression levels of at
least about 20 genes, preferably at least about 30, 50, 100, 200 or
more genes.
[0067] "Genes that are up- or down-regulated" in a particular
process, e.g., in a mesenchymal stem cell, refer to genes which are
up- or down-regulated by, e.g., a factor of at least about 1.1
fold, 1.25 fold, 1.5 fold, 2 fold, 5 fold, 10 fold or more.
Exemplary genes that are up- or down-regulated during bone and
cartilage formation are set forth in Tables 1, 2, 5, 6 and/or 7.
"Genes that are up- or down-regulated in a disease" refer to the
genes which are up- or down-regulated by, e.g., at least about 1.1
fold, 1.25 fold, 1.5 fold, 2 fold, 5 fold, 10 fold or more in at
least about 50%, preferably 60%, 70%, 80%, or 90% of the patients
having the disease.
[0068] The term "isolated," used in reference to a single cell or
clonal cell cluster, e.g., a mesenchymal stem cell or clonal colony
thereof, means that the cell is substantially free of other
nonclonal cells or cell types or other cellular material with which
it naturally occurs in the tissue of origin (e.g., bone or adipose
tissue). A sample of mesenchymal stem cells is "substantially pure"
when it is at least 60%, or at least 75%, or at least 90%, and, in
certain cases, at least 99% free of cells other than cells of
clonal origin. Purity can be measured by any appropriate method,
for example, by fluorescence-activated cell sorting (FACS).
[0069] As used herein, the terms "label" and "detectable label"
refer to a molecule capable of detection, including, but not
limited to, radioactive isotopes, fluorophores, chemiluminescent
moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme
inhibitors, dyes, metal ions, ligands (e.g., biotin or haptens),
and the like. The term "fluoresce" refers to a substance or a
portion thereof, which is capable of exhibiting fluorescence in the
detectable range. Particular examples of labels which may be used
under the invention include fluorescein, rhodamine, dansyl,
umbelliferone, Texas red, luminol, NADPH, alpha-beta-galactosidase,
and horseradish peroxidase.
[0070] A "precursor cell", or "progenitor cell", refers to a cell
that has the capacity to create progeny that are more
differentiated than itself. For example, the term may refer to an
undifferentiated cell or a cell differentiated to an extent short
of final differentiation that is capable of proliferation and
giving rise to more progenitor cells having the ability to generate
a large number of mother cells that can in turn give rise to
differentiated or differentiable daughter cells. In certain
embodiments, the term progenitor cell refers to a generalized
mother cell whose descendants (progeny) specialize, often in
different directions, by differentiation, e.g., by acquiring
completely individual characters, as occurs in progressive
diversification of embryonic cells and tissues. Cellular
differentiation is a complex process typically occurring through
many cell divisions. A differentiated cell may derive from a
multipotent cell which itself is derived from a multipotent cell,
and so on. While each of these multipotent cells may be considered
stem cells, the range of cell types each can give rise to may vary
considerably. Some differentiated cells also have the capacity to
give rise to cells of greater developmental potential. Such
capacity may be natural or may be induced artificially upon
treatment with various factors. By this definition, stem cells may
also be progenitor cells, as well as the more immediate precursors
to terminally differentiated cells. Exemplary precursor cells
include osteoprogenitor cells such as for example, mesenchymal
precursor cells, osteoblasts, and chondroblasts.
[0071] As used herein, a nucleic acid or other molecule attached to
an array is referred to as a "probe" or "capture probe." When an
array contains several probes corresponding to one gene, these
probes are referred to as "gene-probe set." A gene-probe set can
consist of, e.g., 2 to 10 probes, preferably from 2 to 5 probes and
most preferably about 5 probes.
[0072] "Small molecule" as used herein, is meant to refer to a
composition, which has a molecular weight of less than about 5 kD
and most preferably less than about 4 kD. Small molecules can be
nucleic acids, peptides, polypeptides, peptidomimetics,
carbohydrates, lipids or other organic (carbon-containing) or
inorganic molecules. Many pharmaceutical companies have extensive
libraries of chemical and/or biological mixtures, often fungal,
bacterial, or algal extracts, which can be screened with any of the
assays of the invention to identify compounds that modulate a
bioactivity.
[0073] A "subject" can be a mammal, e.g., a human, primate, ovine,
bovine, porcine, equine, feline, canine and a rodent (rat or
mouse).
[0074] The term "treating" a disease in a subject or "treating" a
subject having a disease refers to providing the subject with a
pharmaceutical treatment, e.g., the administration of a drug, such
that at least one symptom of the disease is decreased. Treating a
disease can be preventing the disease, improving the disease or
curing the disease.
[0075] A "variant" of a polypeptide refers to a polypeptide having
the amino acid sequence of the polypeptide, in which one or more
amino acid residues are altered. The variant may have
"conservative" changes, wherein a substituted amino acid has
similar structural or chemical properties (e.g., replacement of
leucine with isoleucine). More rarely, a variant may have
"nonconservative" changes (e.g., replacement of glycine with
tryptophan). Analogous minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
LASERGENE software (DNASTAR). The term "variant," when used in the
context of a polynucleotide sequence, encompasses a polynucleotide
sequence related to that of a gene of interest or the coding
sequence thereof. This definition may also include, for example,
"allelic," "splice," "species," or "polymorphic" variants. A splice
variant may have significant identity to a reference molecule, but
will generally have a greater or lesser number of polynucleotides
due to alternate splicing of exons during mRNA processing. The
corresponding polypeptide may possess additional functional domains
or an absence of domains. Species variants are polynucleotide
sequences that vary from one species to another. The resulting
polypeptides generally will have significant amino acid identity
relative to each other. A polymorphic variant is a variation in the
polynucleotide sequence of a particular gene between individuals of
a given species. Polymorphic variants also may encompass "single
nucleotide polymorphisms" (SNPs) in which the polynucleotide
sequence varies by one base. The presence of SNPs may be indicative
of, for example, a certain population, a disease state, or a
propensity for a disease state.
Mesenchymal Stem Cells
[0076] The invention provides mesenchymal stem cell (MSC)
compositions and methods for the treatment of autoimmune disease,
such as T1D. MSCs are multipotent cells that have the potential to
give rise to cells of various mesenchymal and non-mesenchymal
lineages, including adipose, bone, and cartilage (Pittenger, et
al., Science 284:143-7 (1999)). MSCs are a component of bone marrow
stroma and although bone marrow provides a facile source of MSCs,
MSCs can be isolated from most adult and fetal tissues, including
fat and muscle tissue, umbilical cord blood, and fetal blood using
methods known in the art (see, e.g., daSilvaMeirelles, et al., J.
Cell Sci. 119:2204-13 (2006); Erices, et al., Br. J. Haematol.
109:235-42 (2000); Campagnoli, et al., Blood 98:2396-402 (2001)).
In the bone marrow, MSCs are essential because they provide the
supportive microenvironment for growth, differentiation, and
function of hematopoietic stem cells (HSCs), which give rise to all
components of the immune and blood systems (Dazzi, et al., Blood
Rev. 20:161-71 (2006)). Because MSCs and other multi-potent
progenitor cells have been shown to give rise to multiple cell
types, use of MSCs as an alternative source of cells for cellular
replacement therapies is being investigated.
[0077] MSCs are the formative pluripotential blast cells found
inter alia: in bone marrow, blood, dermis and periosteum that are
capable of differentiating into more than one specific type of
mesenchymal or connective tissue (i.e. the tissues of the body that
support the specialized elements; e.g., adipose, osseous, stroma,
cartilaginous, elastic and fibrous connective tissues) depending
upon various influences from bioactive factors, such as
cytokines.
[0078] Approximately 30% of human marrow aspirate cells adhering to
plastic are considered as MSCs. These cells can be expanded in
vitro and then induced to differentiate. The fact that adult MSCs
can be expanded in vitro and stimulated to form bone, cartilage,
tendon, muscle or fat cells render them attractive for tissue
engineering and gene therapy strategies. In vivo assays have been
developed to assay MSC function. MSCs injected into the circulation
can integrate into a number of tissues described hereinabove.
Specifically, skeletal and cardiac muscle can be induced by
exposure to 5-azacytidine and neuronal differentiation of rat and
human MSCs in culture can be induced by exposure to
p-mercaptoethanol, DMSO or butylated hydroxyanisole (Woodbury, J.
Neurosci. Res. 61:364-370 (2000)). Furthermore, MSC-derived cells
are seen to integrate deep into the brain after peripheral
injection as well as after direct injection of human MSCs into rat
brain; they migrate along pathways used during migration of neural
stem cells developmentally, become distributed widely and start to
lose markers of HSC specialization (Azizi, Proc. Natl. Acad. Sci.
USA 95:3908-3913 )1998)). Methods for promoting mesenchymal stem
and lineage-specific cell proliferation are disclosed in U.S. Pat.
No. 6,248,587.
[0079] Epitopes on the surface of the human mesenchymal stem cells
(hMSCs) such as SH2, SH3 and SH4 described in U.S. Pat. No.
5,486,359 can be used as reagents to screen and capture mesenchymal
stem cell population from a heterogeneous cell population, such as
exists, for example, in bone marrow. Precursor mesenchymal stem
cells that are positive for CD45 are preferably used according to
this aspect of the present invention, since these precursor
mesenchymal stem cells can differentiate into the various
mesenchymal lineages.
[0080] Many different methods have been developed to isolate and
expand MSCs. The criteria for defining multipotent mesenchymal
stromal (stem) cells has been established by the Mesenchymal and
Tissue Stem Cell Committee of the International Society of Cellular
Therapy in its "Position Paper" (Dominici, et al., Cytotherapy
8:315-17 (2006)).
[0081] First, MSCs must be plastic-adherent when maintained in
standard culture conditions. Plastic adherence is a well-described
property of MSC, and even unique subsets of MSC that have been
described maintain this property (Colter, et al., Proc. Natl. Acad.
Sci. USA 97:3213-18 (2000); Jiang, et al., Nature 418:41-49
(2002)). While MSC may be maintained, and possibly expanded,
without adherence (Baksh, et al., Exp. Hematol. 31:723-32 (2003)),
these protocols typically require very specific culture conditions,
and these cells, if maintained under more standard conditions,
would be expected to demonstrate adherence if the cells are to be
considered a population of MSC.
[0082] Second, .gtoreq.95% of the MSC population must express
CD105, CD73 and CD90, as measured, e.g., by flow cytometry.
Additionally, most (.gtoreq.98%) of the MSC population must lack
expression of CD45, CD34, CD14 or CD11b, CD79.alpha. or CD19 and
HLA-DR surface molecules. Surface antigen (Ag) expression, which
allows for a rapid identification of a cell population, has been
used extensively in immunology and hematology. MSCs should express
CD105 (known as endoglin and originally recognized by the MAb SH2),
CD73 (known as ecto 5' nucleotidase and originally recognized by
the mAb SH3 and SH4) and CD90 (also known as Thy-1). To assure that
studies of heterogeneous populations of MSCs are not confounded by
other contaminating cell types, lack of expression of hematopoietic
Ags may be used as additional criteria for identification and
purification of MSCs as they are not known to express these Ag. For
this purpose, a panel of Ags may be used to exclude the
contaminating cells most likely to be found in MSC cultures. CD45
is a pan-leukocyte marker; CD34 marks primitive hematopoietic
progenitors and endothelial cells; CD14 and CD11b are prominently
expressed on monocytes and macrophages, the most likely
hematopoietic cells to be found in an MSC culture; CD79.alpha. and
CD19 are markers of B cells that may also adhere to MSCs in culture
and remain vital through stromal interactions; and HLA-DR molecules
are not expressed on MSCs unless stimulated, e.g. by IFN-.gamma..
Only one of the two macrophage and B-cell markers needs to be
tested. The investigator may select the marker(s) that is (are)
most reliable in their laboratory.
[0083] Third, MSCs must be capable of differentiating into
osteoblasts, adipocytes and chondroblasts in vitro. The biologic
property that most uniquely identifies MSCs is their capacity for
trilineage mesenchymal differentiation. Thus, cells may be shown to
differentiate to osteoblasts, adipocytes and chondroblasts using
standard in vitro tissue culture-differentiating conditions.
Differentiation to osteoblasts can be demonstrated by staining with
Alizarin Red or von Kossa staining. Adipocyte differentiation is
most readily demonstrated by staining with Oil Red O. Chondroblast
differentiation is demonstrated by staining with Alcian blue or
immunohistochemical staining for collagen type II. Most published
protocols for such differentiations are similar, and kits for such
assays are now commercially available. Accordingly, demonstrating
differentiation should be feasible for all investigators.
[0084] Several of the above-listed criteria merit further comment.
First, as many surface markers (both positive and negative) may be
tested as deemed important especially as it relates to the
particular application. The optimum flow cytometric assay would
utilize multicolor analyses (i.e. double staining, triple staining,
etc.) to demonstrate that individual cells co-express MSC markers
and lack hematopoietic Ag. The proposed panel of Ag does not
uniquely identify MSCs compared with some other cell types
(Sabatini, et al., Lab. Invest. 85:962-71 (2005)), however, the
surface phenotype, in conjunction with the other functional
criteria, best identifies MSCs with the current state of
knowledge.
[0085] Second, MSC express HLA-DR surface molecules in the presence
of IFN-.gamma. but not in an unstimulated state. Thus, if HLA-DR
expression is found, and in fact, such expression may be desirable
for some applications, the cells may still be termed MSCs, assuming
the other criteria are met, but should be qualified with
adjectives, such as "stimulated MSC" or other nomenclature to
indicate that the cells are not in the baseline state.
[0086] Third, the level of MSC purity (.gtoreq.95% expression of
CD105, CD73, CD90; .ltoreq.2% expression of hematopoietic Ag) may
be considered as a minimal guideline. Greater levels of
demonstrated purity may be required for certain applications.
[0087] Finally, MSCs have great propensity for ex vivo expansion.
Investigators who utilize extensively passaged cells may be well
served by verifying a normal karyotype to reduce the probability of
chromosomal abnormalities, including potentially transforming
events. Such events could potentially lead to the establishment of
a novel cell line, and the resulting cells should no longer be
considered MSCs. However, karyotype analysis is not being
recommended for routine identification of MSCs.
[0088] As described further below, the human mesenchymal stem cells
can be used as hosts for foreign genes for the expression of gene
products in systemic or localized targets. The human mesenchymal
stem cells of the invention can be engineered (transduced or
transformed or transfected) with genetic material of interest. The
engineered human mesenchymal stem cells can be cultured in
conventional nutrient media modified as appropriate for activating
promoters, selecting transformants or amplifying exogenous genes
therein. The culture conditions, such as temperature, pH and the
like, can be those previously used with engineered human
mesenchymal stem cells. See, for example, Gerson, et al., U.S. Pat.
No. 5,591,625. Mesenchymal stem cells can be treated with
IFN.gamma. to stimulate MHC presentation by the mesenchymal stem
cells.
[0089] Unless otherwise stated, genetic manipulations are performed
as described in Sambrook and Maniatis, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1989.
Treatment Methods and Compositions
[0090] In the prophylaxis or treatment of disease states, the
recipient may be only required to undergo a single administration
after which disease remission is realized on a permanent basis.
Alternatively, depending upon observation of follow-up monitoring,
any subsequent administration may be of greater or lesser doses.
Such procedures and monitoring regimens are well known to those who
are versed in the field of immune therapy, infectious disease,
oncology, epidemiology and the like.
[0091] The dosage of the active ingredient varies within wide
limits and will, of course be fitted to the individual requirements
in each particular case. In general, in the case of parenteral
administration, it is customary to administer from about 0.5 to
about 5 million cells per kilogram of recipient body weight. The
number of cells used will depend on the weight and condition of the
recipient and other variables known to those of skill in the art.
The cells can be administered by a route that is suitable for the
particular disease state to be treated. In the case of non-specific
induction of hyporesponsiveness of the immune response, mesenchymal
stem cells can be administered systemically, i.e., parenterally,
intravenously or by injection. In the case of induction of
genetically engineered or modified MSCs, the antigen-modified
mesenchymal stem cells can be targeted to a particular tissue or
organ.
[0092] The cells can be suspended in an appropriate diluent, at a
concentration of from about 5.times.10.sup.6 to about
50.times.10.sup.6 cells/ml. Suitable excipients for injection
solutions are those that are biologically and physiologically
compatible with the recipient, such as buffered saline solution.
The composition for administration should be sterile, stable, and
the like, as is appropriate for administration into an
individual.
[0093] The methods of the present invention are particularly
applicable to therapy of autoimmune disease, particularly T1D, and
should preferably inactivate or eliminate the response to
autoantigen specifically, without compromising other aspects of the
immune system.
[0094] Although not limited to the treatment of autoimmune disease,
the mesenchymal stem cells and method of the invention can
accordingly be appropriately applied to treatment strategies
requiring immunosuppressive reagents. Also contemplated is the
modification of and expansion of mesenchymal stem cells in vitro
for use in cellular immunotherapy, the in vivo administration of
the immunosuppressive mesenchymal stem cells for treating or
preventing unwanted immune responses. One aspect of the invention
is the development of the mesenchymal stem cells into a vehicle for
delivering inhibitory signals or antigen to target a specific
cellular response, the development of vaccines with the mesenchymal
stem cells modified as described herein for either target specific
or systemic delivery of immunosuppression for prophylaxis and
therapy of disease.
PD-1-PD-L1/PD-L2 Pathway
[0095] The methods and compositions of the invention may optionally
include PD-1-PD-L1/PD-L2 pathway proteins, nucleic acids and
agonists (see Yadav and Sarvetnick, Rev. Diab. Stud. 3:6-10
(2006)). Exemplary nucleic acids and polypeptides of this pathway
are known in the art and include GenBank polypeptide listings as
well as the GenBank nucleic acid listings.
[0096] PD-1 (programmed death-1) is a type I transmembrane protein
and its extracellular region contains a single immunoglobulin V
(IgV) domain. Its cytoplasmic region has two tyrosines, each of
which constitute an immunoreceptor tyrosine-based inhibition motif
(ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM)
(Shlapatska, et al., J. Immunol. 166:5480-87(2001)). It is the ITSM
that is required for the inhibitory activity of PD-1. PD-1 exists
as a monomer on cell surfaces due to the lack of membrane proximal
cysteine (Zhang, et al., Immunity 20:337-47(2004)). Co-localization
of PD-1 with TCR/CD28 on T cells is essential for its inhibitory
function that involves the CD28-mediated activation of
phosphatidylinositol-3-kinase (Pl3K) (Greenwald, et al., Ann. Rev.
Immunol. 23:515-48 (2005)). PD-1 can be induced, not only on CD4
and CD8 T cells, but also on B cells and myeloid cells. NK-T cells
have also been shown to express low levels of PD-1. During thymic
development, PD-1 is predominantly expressed on CD4-CD8-T cells and
also on double negative .gamma..delta. T cells (Nishimura, et al.,
J. Exp. Med. 191:891-898 (2000)). There is also some evidence in
support of the role of PD-1 as a regulator of positive selection
(Blank, et al., J. Immunol. 171:4574-81 (2003)). PD-1-deficient
mice exhibit an overactivation of immune responses and thus support
the development of autoimmune diseases (Nishimura, et al., Int.
Immunol. 10:1563-72 (1998); Nishimura, et al., Immunity 11:141-51
(1999); Nishimura, et al., Science 291:319-22 (2001)). Also, PD-1
knock-out mice display a more vigorous T cell response as compared
to normal controls (Iwai, et al., J. Exp. Med. 198:39-50 (2003)).
These findings suggest that the engagement of PD-1 on T cells
predominantly leads to the generation of negative signals.
[0097] PD-1 has two ligands, namely PD-L1 (B7-H1) and PD-L2
(B7-DC), and their similarity with B7 molecules prompted their
identification using databased search (Freeman, et al., J. Exp.
Med. 192:1027-34 (2000); Latchman, et al., Nat. Immunol. 2:261-68
(2001); Tseng, et al., J. Exp. Med. 193:839-46 (2001)). PD ligands
are type I transmembrane proteins with IgV and IgC domains in their
extracellular region. PD-L2 has been shown to have an affinity for
PD-1 that is two to six times higher than that of PD-L1 (Zhang, et
al., Immunity 20:337-47 (2004)). These PD ligands show a distinct
pattern of expression; PD-L1 is more widely expressed than PD-L2
(Freeman, et al., J. Exp. Med. 192:1027-34 (2000); Latchman, et
al., Nat. Immunol. 2:261-68 (2001); Tseng, et al., J. Exp. Med.
193:839-46 (2001); Dong, et al., Nat. Med. 5:1365-69 (1999)). PD-L1
is expressed on T and B cells, dendritic cells and macrophages and
also becomes upregulated upon activation (Liang, et al., Eur. J.
Immunol. 33:2706-16 (2003); Yamzaki, et al., J. Immunol.
169:5538-45 (2002); Ishida, et al., Immunol. Lett. 84:57-62
(2002)). Interestingly, PD-L1 has also been shown to be expressed
by non-hematopoietic cells including endothelial cells in the
heart, .beta.-cells in the pancreas, and also in non-lymphoid
organs namely lung, muscle and placenta (Liang, et al., Eur. J.
Immunol. 33:2706-16 (2003); Ishida, et al., Immunol. Lett. 84:57-62
(2002); Weidl, et al., Brain 126:1026-35 (2003); Rodig, et al.,
Eur. J. Immunol. 33:3117-26 (2003); Petroff et al., Biol. Reprod.
68:1496-1504 (2003)). The expression of PD-L1 in non-lymphoid
tissues suggests a potential regulatory role of PD-L1 in regulating
autoreactive T and B cells in target organs. On the other hand,
PD-L2 is more restricted and its expression can be observed in
dendritic cells and macrophages. There is also evidence that the
expression of PD-L1 and PD-L2 can be influenced by Th1 and Th2
cytokines, such as IFN-.gamma. and IL-4, which have been shown to
up-regulate PD-L1 and PD-L2, respectively (Loke and Allison, Proc.
Natl. Acad. Sci USA 100:5336-41 (2003)).
CXCL10-CXCR3 Pathway
[0098] The methods and compositions of the invention may optionally
include CXCL10-CXCR3 pathway proteins, nucleic acids and agonists.
Exemplary nucleic acids and polypeptides of this pathway are known
in the art and include the GenBank polypeptide listings as well as
the GenBank nucleic acid listings.
[0099] The foregoing detailed description includes many specific
details. The inclusion of such detail is for the purpose of
illustration only and should be understood not to limit the
invention. In addition, features in one embodiment may be combined
with features in other embodiments of the invention. The patent and
scientific literature referred to in this description establishes
knowledge that is available to those of skill in the art. The
issued U.S. patents, allowed applications, published foreign
applications, and references, including GenBank database sequences,
that are cited herein are hereby incorporated by reference to the
same extent as if each was specifically and individually indicated
to be incorporated by reference. To the extent publications and
patents or patent applications incorporated by reference contradict
the invention contained in the specification; the specification
will supersede any contradictory material.
EXAMPLES
[0100] This invention is further illustrated by the following
examples, which should not be construed as limiting.
Example 1
MSCs to Treat New Onset Type 1 Diabetes
[0101] In these illustrative examples, MSCs were delivered to NOD
mice to prevent and reverse diabetes. Systemic delivery of normal
Balb/c MSCs derived from Balb/c bone marrow delayed the onset of
diabetes and reversed established hyperglycemia if delivered within
one week of onset of autoimmune disease. In contrast, the delivery
of MSCs derived from pre-diabetic female NOD mice bone marrow did
not delay diabetes onset. This data supports the therapeutic
benefit of early delivery of MSCs to patients with developing
autoimmune, early onset diabetes.
[0102] Animals and Injections
[0103] Six to eight week old female Balb/c (B/c) and C57BL/6 (B6)
mice and 4-6 week old female pre-diabetic NOD/Lt (NOD) mice that
had been purchased from the Jackson Laboratory were used to
generate MSCs. For in vivo experiments, 10 week old pre-diabetic
female NOD mice were injected with 500,000 MSCs i.v. each week for
4 weeks. For reversal studies, mice were given one dose of 500,000
B/c MSCs at the various times up to 90 days after 10 weeks of age.
Blood glucose measurements were taken two to three times a week
starting the week before MSC administration. Mice with blood
glucose values greater than 250 mg/dL for three consecutive
readings were considered diabetic.
[0104] MSC Generation and Propagation
[0105] Multiple independent sets of MSCs were generated for use in
these experiments. MSCs were isolated by plastic adherence after
culturing pooled bone marrow cells for 7 days. For each MSC
generation, bone marrow cells were flushed from both femurs and
tibias of 15-40 mice. Cells were flushed with a 27 gauge needle
using high glucose DMEM media (Gibco), then pooled and treated with
Puregene RBC Lysis Solution (Gentra Systems) to lyse red blood
cells. Following RBC lysis, cells were washed with high glucose
DMEM, counted and plated in high glucose DMEM media containing 10%
FBS (Gibco 10099-158, lot 1229021), 1.times.
penicillin/streptomycin (Gibco) and 2 mM L-glutamine (Gibco). Five
days after initial plating, the media was removed and fresh media
added back. On day 7 the cells were harvested by treatment with
trypsin-EDTA (0.05%; Gibco) for 5 minutes at 37.degree. C. followed
by gentle scraping and pooling to form "passage 1" cell pool (p1).
These cells were then washed with Ca.sup.+2/Mg.sup.+2 free PBS
before trypsin-EDTA addition and the reaction was stopped by adding
a 1:1 volume of FBS to trypsin-EDTA.
MSC Tracking
[0106] MSCs were generated from GFP transgenic C57BL/6 mice
purchased from Jackson Laboratories as described above. One million
MSCs were delivered i.p. to diabetic and non-diabetic NOD mice and
4 days later organs were harvested, homogenized on trizol
(Invitrogen) and snap frozen. RNA was isolated using standard
techniques and the expression of GFP was analyzed by quantitative
PCR. The relative GFP copy number for each tissue was extrapolated
using various amounts of plasmid containing a known number of GFP
genes.
[0107] MSCs Derived from Normal Mice Delay Diabetes Onset
[0108] Normal allogeneic MSCs were systemically administered to
pre-diabetic NOD animals to determine whether systemic delivery
could alter the course of disease. MSCs were derived from the bone
marrow of 6-8 week old Balb/c mice. MSCs were isolated by adherence
to plastic in 10% FBS and cultured for several passages. After 2
passages, murine MSCs were positive for CD105 and CD44 and negative
for CD34. MSCs were injected into 10 week old (pre-diabetic) female
mice once a week for weeks as shown in FIG. 1. Groups of NOD
animals treated with Balb/c MSCs were compared to animals treated
with PBS vehicle control. Diabetes development was determined by
blood glucose monitoring of all animals. Onset of diabetes was
determined to be when blood glucose levels were >250 mg/dL.
Onset occurred in vehicle control animals starting at 20 days
post-treatment (top panel), whereas disease onset in Balb/c treated
mice occurred between 43-60 days post-treatment (bottom panel). In
detail, FIG. 1 shows that normal allogeneic MSCs prevent the onset
of diabetes in NOD mice. Pre-diabetic female NOD mice (10 weeks of
age) were injected intravenously once per week for 4 weeks with PBS
(top panel) or 500,000 MSCs (bottom panel) derived from bone marrow
of Balb/c mice. Each line represents a single NOD mouse. This is a
representative Figure depicting data from 3 experiments. For each
experiment, a minimum of 7 mice per group was used.
[0109] Therefore, allogeneic MSC treatment significantly delayed
the onset of diabetes development in this cohort of NOD mice.
[0110] MSCs Track to Pancreatic Lymph Nodes and Spleen
[0111] The effects of MSCs on the course of diabetes development in
NOD animals were further investigated in tracking experiments
designed to detect the presence of MSCs in diabetic target organs,
such as the spleen and pancreatic draining lymph node (PDLN). In
order to track MSCs in vivo, MSCs were generated from the bone
marrow of B6 GFP-transgenic mice. GFP-MSCs were injected into
pre-diabetic and diabetic NOD mice and tissues were harvested and
quantitated by PCR for GFP expression 4 days post injection.
[0112] FIG. 2 shows that transgenic MSCs preferentially tracked to
the PDLN and the spleen in both pre-diabetic (top panels) and
diabetic (bottom panels) animals. FIG. 2 depicts relative GFP copy
number in organs harvested from pre-diabetic (mice 3123, 3124,
3125) and diabetic (mice 1888, 3101, 3103) female NOD mice that had
been administered a one time dose of MSCs generated from the bone
marrow of GFP transgenic C57BL/6 mice. 1.times.10.sup.6 GFP C57BL/6
MSCs were injected i.p., and four days later, organs were harvested
and processed for RNA. The relative GFP copy number detected in
each organ was determined by quantitative PCR and plotted. Each
panel represents data from an individual mouse. Each bar, left to
right, represents a specific organ as indicated: first bar (dark
grey) is spleen, second bar (light grey) is liver, third bar
(darkest grey) is kidney, fourth bar (white) is mesenteric lymph
node, fifth bar (black) is pancreatic lymph node, sixth bar is a
pool of inguinal/brachial/axillary lymph nodes (n.d. indicates "not
detected").
[0113] These results show that MSCs are able to traffic to the PDLN
and the spleen where autoreactive T cells interact with auto
antigens before homing to the beta cells in the islets in the
pancreas. Accordingly, MSCs have an intrinsic ability to home to
areas of inflammation presently in disease and exert their
immunosuppressive functions on T cells that are present in target
organs.
[0114] Normal, but not NOD, Allogeneic MSCs Delay Diabetes
Onset
[0115] The therapeutic potential of MSCs in the treatment of
diabetes was further investigated by treating pre-diabetic NOD mice
with both normal allogeneic Balb/c MSCs and NOD MSCs (derived from
10-week old, pre-diabetic NOD mice) once a week for 4 weeks and
monitoring disease development by blood glucose monitoring. The
diabetic disease status of each animal was monitored beginning 1
week after the first injection of MSCs. Mice were non-diabetic
until the first occurrence of high blood glucose, at which point
they were deemed diabetic.
[0116] The results demonstrated that normal allogeneic MSCs
significantly delay onset of diabetes. FIG. 3 shows that the
administration of normal allogeneic MSCs (Balb/C MSC, triangles)
delays the onset of diabetes, while the administration of NOD MSCs
(NOD MSC, circles) does not. Pre-diabetic female NOD mice (10 weeks
of age) were injected intravenously once per week for 4 weeks with
approximately 500,000 Balb/c or NOD MSCs, or were left untreated.
The results show that at 21 weeks of age, the survival rate for
normal allogeneic Balb/c MSC-treated group was more than twice the
survival rate of the untreated group (PBS, dashed line). In marked
contrast, the NOD MSC-treated group had no survivors at 21 weeks.
Given that NOD MSCs were not protective in delaying disease onset,
there appears to be an intrinsic defect in the stem cell population
derived from autoimmune-prone mice.
[0117] These results show that development of autoimmune diabetes
may be linked to a defect in the MSC pool. Normal allogeneic MSCs
can delay disease onset in NOD mice. Furthermore, MSCs can be used
as early intervention treatment in diabetes as the treatment was
most efficacious in mice that have had disease for only 1-2 weeks.
This data suggests that MSCs would be most useful for treatment of
new onset diabetes. While not wishing to be bound by any single
theory of operability, presumably, these animals undergoing new
onset diabetes have a measurable level of functional endogenous
beta cells that are able to restore blood glucose levels back to
normal once MSCs are administered and control autoimmune T
cells.
Example 2
Gene Expression Profiling of Therapeutic MSCs
[0118] In the following illustrative examples, a gene expression
profile analysis was performed to determine whether normal MSCs and
NOD MSCs are intrinsically different with respect to expression of
genes possibly involved in MSC mediated immune suppression.
Differences in the expression of two genes, Pdcd1Ig1 and CXCL10,
were further characterized. Unlike normal MSCs, NOD MSCs did not
up-regulate the expression of Pdcd1Ig1, a gene encoding the
inhibitory protein PDL1, upon cytokine treatment. MSCs generated
from PDL1 -deficient mice are less suppressive than their normal
counterparts, directly showing that PDL1 expressed by MSCs is
involved in suppressing T cell responses. In addition, NOD MSCs,
but not normal MSCs, over-express CXCL10 upon cytokine treatment.
Further analysis showed that supernatants from NOD MSCs, but not
Balb/c MSCs, were able to attract activated T cells. These results
show that MSCs from NOD mice are intrinsically different from MSCs
from normal mice. NOD MSCs may not protect NOD mice from developing
diabetes because NOD MSCs attract autoreactive T cells via
over-expression of CXCL10 and fail to suppress these T cells since
NOD MSCs do not up-regulate PDL1. The results show that the timely
delivery of MSCs to human subjects with early onset diabetes would
be beneficial and that expression profile analysis of MSCs
identified new potential therapeutic targets for use in the
MSC-based treatment of patients with type I diabetes.
[0119] Microarray Analysis
[0120] Total RNA was isolated from duplicate samples of three
independent sets of B/c and B6 MSCs and 2 independent sets of NOD
MSCs which had been left untreated or treated for 6 hr with 5 ng/ml
recombinant mouse IL1.beta. (R & D Systems). RNA was prepared
using standard techniques. Briefly, media was aspirated from the
flasks, cold trizol was added and the cells were scraped off,
transferred to RNAse free eppendorf tubes and snap frozen. After
initial RNA isolation, the RNA was cleaned up using an RNeasy kit
(Qiagen). Total RNA was then hybridized to the AFFYMETRIX.RTM.
mouse whole genome 430 2.0 array. T-tests were performed on data to
identify differences in gene expression. Fold changes of 2 or more
were considered significantly different.
[0121] Flow Cytometry
[0122] MSCs were harvested by a 1 minute exposure to 0.05%
trypsin-EDTA at 37.degree. C. and then gently scraped. Non-specific
staining was blocked using FcR block (BD Biosciences) for 20
minutes on ice. Cells were stained for 30 minutes on ice followed
by fixation using 2% paraformaldehyde. A minimum of 10,000 events
were acquired using a FACSCanto cytometer and the data was analyzed
with FlowJo. MSCs were stained with anti-mouse CD105 (eBiosciences)
and anti-mouse CD34, anti-mouse PDL1, anti-mouse PDL2 (BD
Biosciences). Appropriate isotype antibodies were used as negative
controls.
[0123] Quantitative ELISA
[0124] CXCL10 was measured in the supernatants of untreated MSCs or
those treated with IL1b as described using the mouse CXCL10 DuoSet
kit (R & D Systems) following the manufacturer's instructions.
For each sample, 200 ul of neat supernatant was added to the top
well with 1:2 dilutions down the plate starting with well 2.
[0125] CFSE Staining
[0126] Splenocytes were washed in PBS then re-suspended in PBS. A
1:1 volume of CFSE (Molecular Probes) at 10 uM in PBS was added and
the cells incubated for 5 minutes in the dark. The reaction was
stopped by adding 1:1 volume of 100% FBS for 1 minute followed by
several washes in RPMI+10% FBS.
[0127] Proliferation Assay
[0128] Two million CFSE labeled Balb/c splenocytes were stimulated
for 4 days with 2 ug/ml soluble anti-mouse CD3.epsilon. or hamster
IgG1 (BD Biosciences) in the absence or presence of 25,000 MSCs. On
the day of culture initiation, splenocytes, MSCs, and stimulating
reagents were added at the same time. On the fourth day, the CFSE
profile of the non-adherent cells was analyzed by flow
cytometry.
[0129] Adenoviral Transduction
[0130] NOD MSCs were infected with an adenoviral vector encoding
mouse membrane PDL1 (Ad.mPDL1) at an MOI of 1000. The cells were
incubated with Ad.mPDL1 for 4 hours in high glucose DMEM without
FBS. The cells were washed twice with DMEM then complete media was
added back. Twenty-four hours later the cells were harvested by 1
minute trypsin-EDTA incubation and injected. The mice were injected
once a week for 4 weeks as described above, and the membrane
expression of PDL1 was assessed by flow cytometry each week.
[0131] Autoimmune-Prone NOD Mouse MSCs Differ from Normal MSCs
[0132] The intrinsic differences in MSCs derived from
autoimmune-prone NOD mice were compared to MSCs derived from normal
mice by gene expression profiling. NOD mice spontaneously develop
an autoimmune disease that resembles type 1 diabetes in humans
(Kikutani and Makino, Adv. Immunol. 51:285-322 (1992)), and
multiple chromosomal abnormalities have been identified which
contribute to disease development. Although defects in multiple
cell types, such as macrophages, dendritic cells, and T cells, have
been described in these mice, defects in the adult stem cell
population have not been described. Accordingly, the contribution
of adult stem cell genotype to disease development was
investigated.
[0133] A gene expression profile analysis using microarray
technology was performed to further investigate the mechanism by
which B/c MSCs afford protection from diabetes while NOD MSCs do
not (and may even accelerate diabetes development). This analysis
was performed on MSCs derived from normal and pre-diabetic NOD
mice. RNA harvested from untreated as well as IL-1.beta. treated
MSCs was analyzed. Multiple differences in gene expression between
normal and autoimmune-prone MSCs were identified. FIG. 4A shows a
"heat map" in which differences in IL-1.beta. treated RNA from NOD
MSCs vs. IL-1.beta. treated normal B6 and B/C MSCs are boxed. The
dark gray box (top) represents genes which are down-regulated
whereas the genes boxed in light gray (bottom) are up-regulated.
FIG. 4B lists the top genes of particular interest that were highly
differentially expressed in IL-1.beta.-treated NOD MSCs as compared
to normal MSCs. A dash means the gene was not up-regulated and an
up arrow means the gene was up-regulated.
[0134] Normal MSCs Up-Regulate PD-L1 Upon Inflammation
Stimulation
[0135] The microarray gene analysis results showed that NOD MSCs
did not up-regulate the negative co-stimulatory molecule PD-L1 upon
IL-1.beta. stimulation (FIG. 5A). While the PD-L1/PD1 pathway has
been implicated in T cell regulation in autoimmune diseases
(Okazaki and Honjo, Trends Immunol. 27:195-201 (2006)) and diabetes
(Ansari, et al., J. Exp. Med. 198:63-9 (2003)), further studies
focused on this molecule and the PD-1 pathway were required to
understand its role in autoimmune disease progression. FACS data
confirmed the microarray results at the protein level and showed
that NOD MSCs did not up-regulate the co-stimulatory molecule PD-L1
upon IL-1.beta. stimulation compared to normal B6 MSCs. (FIG. 5B).
This data indicates that MSCs derived from autoimmune-prone mice
have a dysregulation in the PD-1 negative co-stimulatory pathway
and do not possess the immunosuppressive function necessary to
inhibit T cell proliferation.
[0136] In further detail, FIGS. 5A and 5B show that normal Balb/c
and C57BL/6 MSCs, but not NOD MSCs, up-regulate PD-L1 in response
to IL-1.beta.. FIG. 5A shows the fold change in mRNA expression of
the Pdcd1Ig1 gene, encoding PD-L1, for Balb/c (left bar), C57BL/6
(middle bar), and NOD (right bar). MSCs were determined by dividing
the raw expression data for the gene from IL-1.beta. treated
samples divided by the raw value of the untreated samples for each
strain (n.d. indicates "not detected). FIG. 5B shows the flow
cytometry analysis of PD-L1 protein on the surface of Balb/c and
NOD MSCs cultured in the presence or absence of IL-1.beta. for 6
hr. The black line (arrow) represents cells treated with IL-1.beta.
for 6 hr, the dark gray line represents untreated MSCs and the
light gray line represents untreated cells stained with the
appropriate isotype control antibody.
[0137] Reduced Ability of MSCs Lacking PD-L1 Expression to Inhibit
T Cell Proliferation
[0138] The role of PD-L1 in mediating suppression of T cell
proliferation by MSCs was further investigated. MSCs were derived
from the bone marrow of PD-L1 deficient mice (Latchman, et al.,
Proc. Natl. Acad. Sci USA 101:10691-96 (2004)). Wildtype B/6 MSCs
and B/6-/-PD-L1 MSCs were cultured together with CD3 activated B/C
splenocytes in a mixed lymphocyte reaction (MLR). In FIG. 6, the
left panel depicts 87% of the activated T cells proliferated.
Addition of the wildtype B6 MSCs to the MLR suppressed this T cell
proliferation over five-fold to 16% (middle panel), whereas
addition of B6 PD-L1-/-MSCs resulted in less than 2-fold
suppression of T cell proliferation (right panel). Increasing
numbers of B6 PD-L1-/-MSCs were not able to further suppress T cell
proliferation. This data shows that PD-L1 is an important mediator
in the MSC-mediated suppression of lymphocyte proliferation,
because the absence of PD-L1 ligand on the cell surface of the null
MSCs prevents binding to the PD-1 receptor, and thus prevents the
activation of the negative co-stimulatory pathway in the T cells
allowing T cell proliferation.
[0139] FIG. 6 shows that the ability of MSCs to inhibit T cell
proliferation is reduced when MSCs lack PD-L1 expression. In
further detail, FIG. 6 shows flow cytometry analysis of CFSE
labeled B/c splenocytes cultured with anti-mouse CD3.epsilon.
antibody alone (left panel) or together with 25,000 B6 (middle
panel) or B6.PD-L1-/-(right panel) MSCs. The thick vertical line
demarcates proliferating cells (to the left of the line) from
non-proliferating cells (to the right of the line) and the numbers
represent the percentage of cells in these gates within the
lymphocyte compartment.
[0140] Taken together, this data supports the observation that
autoimmune-prone MSCs, which lack the ability to up-regulate PD-L1
on their cell surface, cannot offer disease protection when
delivered prophylactically to NOD mice.
[0141] NOD MSCs Engineered to Over-Express PD-L1 Delay Diabetes
[0142] The role of PD-L1 in mediating MSC immune suppression was
further analyzed by engineering NOD MSCs to over-express PD-L1
using an adenoviral vector encoding mouse membrane bound PD-L1
(Ad.mPD-L1). FIG. 7A shows FACS analysis of PD-L1 cell surface
expression on Ad.mPD-L1 infected NOD MSCs stained with an isotype
control, vs. uninfected NOD MSCs, vs. Ad.mPD-L1 infected MSCs
stained with an anti-PD-L1 monoclonal antibody, respectively. To
elucidate the role of PD-L1 as the underlying pathway conveying
therapeutic potential of MSCs for the treatment of diabetes,
pre-diabetic NOD mice were again treated with normal allogeneic
Balb/c MSCs (wildtype), NOD MSCs (derived from 10-week old,
pre-diabetic NOD mice), or Ad.mPD-L1 engineered NOD MSCs once a
week for 4 weeks and disease development was monitored by measuring
blood glucose levels. The data confirmed that wildtype NOD MSCs did
not confer protection to disease onset as these cohorts developed
disease starting at 15 weeks of age. In contrast, NOD MSCs
engineered to express PD-L1 on their cells surface conferred
protection by delaying disease onset to 17-19 weeks of age similar
to normal B/C MSCs (FIG. 7B).
[0143] In further detail, FIG. 7A shows flow cytometry analysis of
PD-L1 expression on the surface of NOD MSCs infected with
adenoviral vector encoding mouse membrane PD-L1 (Ad.mPD-L1).
Uninfected (light grey line, center peak) or Ad.mPD-L1 infected
(grey line, right peak) NOD MSCs were stained with an antibody to
PD-L1. The dark line (left peak) represents Ad.mPD-L1 infected NOD
MSCs stained with isotype control antibody. FIG. 7B shows blood
glucose values over time from NOD mice left untreated (circles,
black line) or administered 500,000 uninfected Balb/c MSCs
(triangles), uninfected NOD MSCs (squares, grey line), or Ad.mPD-L1
infected NOD MSCs (X's, light grey line) starting at 10 weeks of
age. Collectively, FIGS. 7A and 7B demonstrate that NOD MSCs
engineered to over-express PD-L1 delay diabetes.
[0144] This data shows that the intrinsic PD-L1 defect resulting in
lack of inducible expression on autoimmune-prone MSCs leading to
early onset disease can be completely reversed by restoring PD-L1
expression to these cells. These results demonstrate that the
expression of the negative co-stimulatory molecule PD-L1 is
critical for the innate immunosuppressive function of MSCs. In
addition, lack of expression of this molecule on the MSC population
may contribute to disease development due to lack of T cell
suppression.
[0145] Construction of PD-L1-Fc Fusion Protein (PD-1-PDL-1/PDL-2
Agonist)
[0146] PD-L1 Fc fusion protein was created by fusing the DNA
sequence encoding the full length mouse PD-L1 protein to the DNA
sequence encoding the Fc portion of human IgG1. The sequence for
the Fc portion encodes the C.sub.H2 and C.sub.H3 C-region domains
of IgG1 and 7 out of 12 amino acids that make up the hinge region
most proximal to the sequence encoding the C.sub.H2 domain. The 7
amino acids include the cysteine residues which make the covalent
disulfide bonds involved in dimer formation. The PD-L1 Fc protein
is a dimer composed of 2 PD-L1-Fc chains. Being a dimer, one PD-L1
Fc protein theoretically should bind 2 receptor molecules.
[0147] NOD MSCs, but not Normal MSCs, Over-Express CXCL10
[0148] Further differences between autoimmune-prone NOD MSCs and
normal MSCs were evident in the gene expression level of
CXCL10/CXCR3 chemokine pathway. FIG. 8A shows that NOD MSCs
over-express the chemokine CXCL10 6-fold over Balb/c and B6 MSCs in
response to IL-1.beta. treatment, respectively. In addition, CXCL9
and CCR17 chemokines are up-regulated 1.5-2.0 fold over normal MSCs
in NOD MSCs. The over-expression of CXCL10 gene expression in
response to inflammatory cytokines by NOD MSCs was confirmed on the
protein level by ELISA (FIG. 8B).
[0149] In further detail, FIG. 8A shows the fold change in CXCL10,
CXCL9, and CCL17 mRNA expression of IL-1.beta. treated to untreated
Balb/c (left bar), C57BL/6 (middle bar), and NOD (right bar) MSC
samples. FIG. 8B shows that supernatants from Balb/c (black bar,
left panel), C57BL/6 (light grey bar, center panel), and NOD (grey
bar, right panel) MSCs incubated for 6 hours .+-. IFN-.gamma. were
analyzed for CXCL10 protein via quantitative ELISA (U=untreated,
T=IFN-.gamma. treated, and n.d.=not detected). Therefore, FIG. 8
demonstrates that NOD MSCs, but not Balb/c and C57BL/6 MSCs,
over-express CXCL10.
[0150] This data confirmed that NOD MSCs secrete higher levels of
the chemokine CXCL10 in response to the pro-inflammatory cytokine
IFN-.gamma.. Given that CXCL10 is an important chemokine for T cell
trafficking, this data further suggests that autoimmune-prone MSCs
may further exacerbate disease by secreting CXCL10, which may
recruit autoreactive T cells.
[0151] Anti-CXCL 10 Antibody Treatment Delays Diabetes Onset
[0152] The results show that NOD MSCs secrete higher levels of the
chemokine CXCL10 in response to an inflammatory stimulus. Based on
this data, activated T cells would be expected to preferentially
migrate towards supernatants collected from stimulated NOD MSCs
cultures compared to supernatants collected from normal MSCs in an
in vitro chemotaxis assay.
[0153] Based on the data showing that NOD MSCs overexpress CXCL10
and other chemokines in this pathway and the observation that
delivery of NOD MSCs to pre-diabetic NOD mice contributes to
disease development, in vivo administration of an anti-CXCL10
antibody would be expected to delay disease development by blocking
additional recruitment of autoreactive T cells that lead to disease
development.
[0154] These results demonstrate that the MSCs from NOD mice are
intrinsically different from normal and may attract alloreactive T
cells by secretion of CXCL10. In addition, NOD MSCs may not
functionally suppress these immune cells due to a decrease in PD-L1
expression, thereby contributing to auto-immunity and explaining
disease acceleration after systemic treatment with NOD MSCs. This
data shows that autoimmune-prone MSCs are defective in PD-L1
expression and link this pathway and a defect in the stem cell pool
to the development of autoimmune diabetes.
Example 3
Further Analysis of MSC Treatment for New Onset Type I Diabetes
[0155] To further demonstrate the effectiveness of MSCs on the
treatment or prevention of diabetes, the MSC-mediated suppression
of T cell responses and inhibition of key inflammatory mediators,
such as TNF.alpha., were further analyzed. Allogeneic murine MSCs
were administered to NOD mice, either prior to (preventive
protocol) or at the time of disease onset (therapeutic protocol).
Prophylactic delivery of allogeneic MSCs to pre-diabetic NOD mice
delayed the onset of disease. Therapeutic treatment at the time of
disease onset was effective in reversing disease, as measured by
restoration of blood glucose levels to the normal range. MSCs were
shown to traffic to the pancreatic draining lymph node and spleen
in pre-diabetic and diabetic mice, implying that MSCs modulated the
autoreactive response at these sites. These findings further
demonstrate that MSCs can effectively alter the autoimmune response
and lead to the amelioration of an ongoing diabetic condition, in
addition to being effective in delaying the onset of a developing
diabetic condition.
[0156] Animals
[0157] MSCs were generated from 6-8 week old female mice (Balb/c,
C57BL/6, C57BL/6-Tg (UBC-GFP) 30 Scha/J) purchased from the Jackson
Laboratory (Bar Harbor, Me.). For the diabetes studies, NOD/LtJ
mice (Jackson Laboratory) were maintained under pathogen-free
conditions and screened for glycosuria using an ACCU-CHEK Compact
Plus Blood Glucose Meter (Roche, Indianapolis, Ind.) by tail vein
puncture three times a week starting at 10 weeks of age. Mice were
deemed diabetic when blood glucose measured above 250 mg/dL for
three consecutive days.
[0158] Cell Therapy
[0159] For prevention studies, 10 week old pre-diabetic female NOD
mice were injected with 500,000 Balb/c MSCs i.v. once a week for 4
weeks. For reversal studies, mice were enrolled the day after the
third blood glucose reading >250 mg/dL and administered Balb/c
MSCs (1.times.10.sup.6 i.v. twice a week for 4 weeks) within 7
days. Once enrolled, hyperglycemic mice (blood glucose >250
mg/dL) received daily insulin glargine (Sonafi Aventis,
Bridgewater, N.J.) injections except mice therapeutically treated
with MSCs who were not given insulin unless blood glucose rose
above 250 mg/dL. MSC treated mice with blood glucose .ltoreq.250
mg/dL for an extended time were considered responders. Mice were
observed for up to 60 days post initial treatment.
[0160] MSC Generation and Propagation
[0161] Human MSCs were generated from BM mononuclear cells obtained
from whole BM aspirates (Lonza, Walkersville, Md.) by density
gradient centrifugation as described previously (Lodie, et al.,
Tissue Eng. 8:739-51 (2002)). Mouse MSCs were generated from BM
cells flushed from both femurs and tibias of 10-30 mice with high
glucose DMEM media (DMEM-H; Invitrogen, Carlsbad, Calif.). Flushed
cells were pooled, treated to lyse red blood cells, and plated at
10-12.times.10.sup.6 cells per 25 cm.sup.2 tissue culture flask in
DMEM-H containing 10% FBS, 1.times. penicillin/streptomycin, and 2
mM L-glutamine. 3-5 days after initial plating, the media
containing non-adherent cells was removed and replaced. On day 7,
the adherent cells were harvested by trypsin-EDTA (Invitrogen)
treatment with gentle scraping and pooled down. Cells were expanded
every 3-4 days once 80-90% confluent for up to 8 passages. MSCs
from multiple harvests were cultured at 37.degree. C. in 5%
CO.sub.2 and used in experiments.
[0162] Cytokine Analysis
[0163] Cytokines were measured in culture supernatants or plasma
using the human Th1/Th2 or mouse inflammation CBA kit (BD
Biosciences, San Jose, Calif.), respectively, following the
manufacturer's instructions.
[0164] MSC Tracking
[0165] One million MSCs generated from GFP transgenic C57BL/6 mice
were delivered i.p. to non-diabetic and diabetic NOD mice and 4
days later organs were harvested, homogenized in trizol, and snap
frozen. RNA was isolated using standard techniques and GFP
expression was analyzed by quantitative PCR using the following GFP
primers: 5'-CTGCTGCCCGACAACCAC-3' (SEQ ID NO: 1) (forward) and
5'-ACCATGTGATCGCGCTTCTC-3' (SEQ ID NO: 2) (reverse) (Integrated DNA
Technologies, Coralville, Iowa). The relative GFP copy number in
each tissue was extrapolated using various amounts of plasmid
containing a known number of GFP genes.
[0166] Dendritic Cell (DC) Preparation
[0167] Normal donor PBMCs (HemaCare Corporation, Van Nuys, Calif.)
were plated at 5.5.times.10.sup.6 cells/150 cm.sup.2 flask in RPMI
1640 (Invitrogen) containing 5% huAB (Sigma, St. Louis, Mo.) for
1-2 hrs. Non-adherent cells were removed and adherent cells were
cultured for 6-7 days in media containing human recombinant IL-4
(20 ng/ml) and GMCSF (100 ng/ml; Peprotech Inc., Rocky Hill, N.J.)
then phenotyped by flow cytometry and cryopreserved for later
use.
[0168] Proliferation Assay
[0169] For human MSC assays, PBMCs (400,000/well) or purified CD3+
cells (100,000/well) were stimulated with anti-CD3/CD28 beads (1
bead:1 PBMC; Invitrogen) or allogeneic DCs (100,000/well).+-.human
MSCs or HUVECs (ATCC, Manassas, Va.), as described in the figures,
respectively. The MSCs were allogeneic to the T cells/PBMCs and to
the DCs.
[0170] For mouse MSC assays, splenocytes (500,000/well) were
stimulated with 2 ug/ml anti-mouse CD3e (BD Biosciences).+-.MSCs as
described in the figures. The splenocytes, MSCs and stimulating
reagents were added at culture initiation.
[0171] Proliferation was measured after incubation with 1 .mu.Ci
.sup.3H thymidine (Perkin Elmer, Boston, Mass.) for the last 18
hours of culture for each condition in triplicate.
[0172] Glucose Tolerance Testing
[0173] The evening before the glucose challenge, non-fasting blood
glucose was monitored and insulin treatment of diabetic animals was
withheld. Mice were fasted for 12 hours before D-glucose (20%;
Sigma) at 2 mg/g body weight was injected i.p. Blood glucose was
measured before and 15, 30, 60, and 120 minutes after the
injection.
[0174] MSCs Suppress T Cell Responses
[0175] The MSCs were subjected to culture conditions under which
they have previously been shown to differentiate into fat,
cartilage, and bone. To confirm that the MSCs were
immunomodulatory, the ability of MSCs to suppress T cell responses
in vitro was further assessed. T cell proliferation in response to
allogeneic DCs was inhibited by MSC addition to the cultures in a
dose-dependent fashion (FIG. 9A). The immunomodulatory activity is
a general characteristic of MSCs because MSCs from multiple donors
suppress T cell proliferation (FIG. 9B), whereas HUVECs, a human
endothelial cell line, do not (FIG. 9C). The MSCs cause an arrest
of T cell proliferation, and not the induction of T cell apoptosis,
because the percentage of cells in MSC treated cultures did not
decrease and no increase in propidium iodine/annexin V staining was
observed. The observation that MSCs alone do not activate T cells
is consistent with the fact that, in contrast to DCs, MCS under
these conditions express little to no HLA class II or
co-stimulatory molecules such as CD80 and CD86 (Jones et al., J.
Immunol. 179:2824-31 (2007)).
[0176] In further detail, FIGS. 9A to 9C show that MSCs inhibit T
cell proliferation. FIG. 9A shows purified human T cells (TC)
cultured with human allogeneic dendritic cells (DC) with or without
the indicated doses of third party human MSCs (MSC) for 6 days.
FIG. 9B shows TCs cultured with allogeneic DC alone or together
with 20,000 MSC from three different donors (donors M28, M29 and
M41) for 5 days. FIG. 9C shows PBMCs cultured with
anti-CD3/anti-CD28 beads with or without the indicated doses of
human MSCs, or the control HUVEC line, for 3 days. Cell
proliferation was measured by tritiated thymidine
incorporation.
[0177] MSCs Modulate TNF.alpha. and IL10 Expression
[0178] To further assess the effect of MSCs on cytokine secretion,
supernatants from MSC-treated cultures were analyzed for the
presence of cytokines. TNF.alpha. and IL10 were elevated in
supernatants from T cell/DC cultures but TNF.alpha. levels
decreased and IL10 levels increased when MSCs were present (FIG.
10). In further detail, FIG. 10 shows the results of experiments
demonstrating that MSCs modulate cytokines in vitro. A
proliferation assay was performed as above (top panel) and
supernatants harvested at the end of the assay were tested for the
presence of TNF.alpha. (middle panel) and IL10 (bottom panel) by
cytometric bead array. This pattern was observed whether the
supernatants were taken early or late in the culture period.
TNF.alpha. is a pro-inflammatory cytokine that is secreted by
activated T cells and IL10 is a T cell derived anti-inflammatory
cytokine. These results show that MSCs shift the cytokine response
from pro-inflammatory to anti-inflammatory. MSC-mediated
suppression of T cell proliferation is reduced when neutralizing
anti-IL10 antibodies are added to the cultures (Rasmusson, et al.
Exp. Cell. Res. 305:33-41 (2005)), suggesting that IL-10
contributes to MSC immunomodulation.
[0179] MSCs Modulate TNF.alpha. Expression In Vivo
[0180] The mechanism by which MSCs down-regulate the
TNF.alpha.-mediated inflammatory response was further assessed in
vivo. First, mouse MSCs were generated to confirm that murine and
human MSCs are phenotypically and functionally similar. Like human
MSCs, BM-derived MSCs from Balb/c and C57BL/6 mice expressed
typical MSC surface markers such as CD44, CD105, and CD73, but
lacked hematopoietic markers like CD34. Mouse MSCs functioned like
human MSCs in that mouse MSCs differentiated into multiple
mesenchymal lineages and suppressed T cell proliferation in vitro
in a dose dependent fashion (FIG. 11A).
[0181] To further show that MSCs modulate TNF.alpha. in vivo, MSCs
were delivered to mice challenged with lipopolysaccharide (LPS).
LPS injection results in a cytokine storm characterized by rapid
TNF.alpha. up-regulation. TNF.alpha. was significantly reduced in
the plasma of mice receiving MSCs regardless of whether the MSCs
were delivered 30 minutes prior to, at the same time as, or 30
minutes post LPS injection (FIG. 11B). TNF.alpha. reduction by MSCs
was similar to the reduction caused by dexamethasone treatment. The
finding that MSCs dampen the TNF.alpha. response shows that MSCs
can be anti-inflammatory. In further detail, FIG. 11A shows the
proliferation of Balb/c spleen cells (S) cultured for 3 days with
soluble anti-CD3 antibody (aCD3) with or without the indicated
doses of C57BL/6 MSCs. FIG. 11B shows the results of an experiment
in which C57BL/6 mice were administered 5 .mu.g LPS i.p. and
treated with PBS i.p. (circle), 40 ug dexamethasone i.p. (inverted
triangle), or 500,000 C57BL/6 MSCs i.v., which were delivered
30.minutes before (squares), at the same time (diamond), or 30
minutes after (triangle) LPS injection. As a control, a group of
mice were given MSCs alone without LPS treatment (cross). The
asterisks represent p values <0.01 when comparing the PBS
treated mice to those treated with dexamethasone or MSCs using a
Dunnett's multiple comparison test.
[0182] MSCs Delay Diabetes Onset in NOD Mice
[0183] Knowing that MSCs suppress T cell responses in vitro and
dampen the TNF.alpha. response in vivo, allogeneic MSCs were
delivered to pre-diabetic NOD animals to determine whether systemic
delivery could alter the course of disease. Type I diabetes results
from the autoimmune destruction of beta cells by T cells and
TNF.alpha. is an early inflammatory mediator of disease and is
thought to be directly toxic to beta cells (La Cava, et al. Curr.
Dir. Autoimmun. 1:56-71 (1999); Bach, J. Autoimmun. 8:439-63
(1995)). To test the effect of MSCs in NOD mice, pre-diabetic NOD
mice were administered Balb/c MSCs or PBS. By the end of the study,
the number of hyperglycemic mice in the MSC treated and control
groups were similar. At 22 weeks of age, 4 of 5 MSC treated mice
and 5 of 6 PBS treated mice had developed diabetes (FIG. 12A);
however, disease onset was delayed by 4 weeks with MSC treatment.
Pre-diabetic NOD mice were administered PBS or 500,000 allogeneic
Balb/c MSCs i.v. once a week for 4 weeks starting at 10 weeks of
age. Blood glucose values for individual mice were monitored and
plotted to assess development of disease. The data is depicted as
percent of non-diabetic mice based on these blood glucose values.
Results are representative of at least 3 independent experiments.
This data shows that MSCs can delay the development of
hyperglycemia in NOD mice. In contrast, MSCs derived from PDL-1
knock-out mice did not significantly delay the onset of diabetes in
NOD mice, demonstrating that PDL-1 is critical to the diabetes
therapeutic potential of the allogeneic MSCs (FIG. 12B).
[0184] MSCs can Reverse Established Disease in NOD Mice.
[0185] To further demonstrate the utility of treating hyperglycemia
with MSCs, allogeneic Balb/c MSCs were delivered therapeutically
after disease onset. Although many agents can prevent disease
development when given during the pre-diabetic phase, few have been
shown to reverse disease effectively in the diabetic setting
(Shoda, et al. Immunity 23:115-26 (2005)). Diabetes is a
progressive and overt disease and is reported not to occur until
the majority of islets have been destroyed (Yoon, et al.
Autoimmunity 27:109-22 (1998)). New onset patients are an important
population because it is believed that some beta cell function is
still present in these patients. To reverse diabetes in new onset
patients, an ideal therapy would dampen the autoimmunity and
inflammatory responses and give support to the surviving beta
cells. Theoretically, MSCs could provide these functions because
MSCs dampen T cell responses and inflammatory responses and the
primary function of MSCs in the BM is to provide support for
developing cells. Accordingly, exogenously administered MSCs may
function similarly by supporting beta cells in the pancreas.
[0186] To further demonstrate that MSCs can reverse hyperglycemia,
MSCs were delivered to newly diabetic NOD mice. Mice with
persistent glucose levels <250 mg/dL were considered to be
responders. Six out of ten mice reversed long term when given MSCs
without any other therapy compared to one out of six mice given PBS
and insulin daily (FIG. 13A). FIG. 13A shows the reversal of
diabetes with allogeneic MSCs in newly diabetic NOD mice treated
with 1.times.10.sup.6 allogeneic Balb/c MSCs (top panel; n=10), but
no reversal of diabetes in newly diabetic NOD mice treated with PBS
(bottom panel; n=6) i.v. twice a week for 4 weeks as indicated by
the black arrowheads (PBS treated mice were also administered
insulin s.c. daily). The blood glucose over time for individual
mice was monitored and plotted to assess reversal of disease. Each
line represents data from an individual mouse. The solid lines in
the top panel represent mice that responded to MSC treatment,
whereas the dotted lines signify mice treated with MSCs but did not
respond. The horizontal line in both panels represents the blood
glucose value at 250 mg/dL. The average blood glucose value of
responder mice dropped from 327.+-.83 mg/dL at the time of
enrollment to 216.+-.58 mg/dL at the end of the study and was
substantially lower than MSC treated mice that did not respond
(465.+-.36.8 mg/dL). The high blood glucose values of mice not
responding to MSC treatment suggests that the likelihood of these
mice having residual beta cell function or the ability to respond
to therapy was low. Furthermore, a blood glucose value >350
mg/dL at enrollment did not correlate with whether the animal
responded to MSC treatment.
[0187] To further assess residual beta cell function in MSC-treated
mice, glucose tolerance tests were performed. The response to
glucose challenge of MSC treated mice that reversed was abnormal at
7 days but much improved at 33 days post the last MSC dose and
similar to the response of non-diabetic NOD mice, showing that
residual beta cell function was intact (FIG. 13B). FIG. 13B shows
glucose tolerance tests (GTT) of mice treated with MSCs that
responded (top row) or did not respond (bottom row) to MSC
treatment. The left panel in the top row depicts the blood glucose
values over time for mice 3830 and 3895 that reversed with MSC
treatment, whereas the left panel in the bottom row shows the blood
glucose values for mice 4056 and 3926 that were treated with MSCs
but did not reverse. The middle and right panels show the response
of mice 3830, 3895, 4056, and 3926 to glucose challenge 7 days
(dashed line) and 33 days (solid line) after the last MSC dose in
comparison to a non-diabetic NOD mouse (dotted line). The glucose
tolerance test accurately reflected beta cell function in these
mice because mouse 4056 never reversed with MSC treatment and
responded abnormally to glucose challenge, whereas mouse 3926
responded better when the mouse was showing signs of reversal (day
7), but worse when the mouse was overtly diabetic (day 33).
[0188] Further supporting these findings is the observation that
responder mice required fewer insulin treatments than MSC treated
mice that did not reverse (FIG. 13C). FIG. 13C shows the daily
insulin dosage for mice treated with MSCs that reversed (left
panel), as compared to MSC treated mice that did not reverse (right
panel). Each line represents data from an individual mouse. This
data shows that mice responding to MSC treatment exhibited improved
glucose tolerance and demonstrate the presence of residual beta
cell function.
[0189] Together, this data shows that MSC treatment alters diabetes
development in NOD mice. MSC treatment delays diabetes onset in
pre-diabetic mice and reverses hyperglycemia in newly diabetic
animals. Those diabetic mice that responded to MSC treatment showed
improved responses to glucose challenge and required few insulin
treatments, indicating that residual beta cell function was intact
in these animals. The observation that MSCs alter diabetes
development when administered early in disease shows that MSCs may
provide an effective alternative strategy for recently diagnosed
type I diabetes patients.
[0190] While MSCs modulate disease in both NOD prevention and
reversal models, they appear to be more efficacious in reversing
disease because half of the MSC treated mice were reversed 30 days
after the last MSC dose whereas all the pre-diabetic MSC treated
mice eventually succumbed to disease within 4 weeks after the last
MSC treatment. While not wishing to be limited to a single theory
of the mechanism of action, this difference could be due to the
fact that MSCs are most effective at suppressing T cell responses
when the response is robust, as in a recently diabetic mouse. The
active disease environment might also favor MSC homing to the right
tissues as shown by the presence of MSCs in the PLN from all the
diabetic mice tested thus far.
[0191] Control of glycemia in MSC treated mice indicates that beta
cells are functioning in the reversed mice even though insulin
staining in the pancreas of these mice was undetected. The lack of
detectable insulin staining might be due to constant degranulation
of the residual beta cells or because conventional methods used to
stain for insulin were inadequate at detecting low insulin amounts
(Sherry, et al., Diabetes 55:3238-45 v). Insulin staining might
have been detected if pancreata were harvested within 3 weeks of
enrollment and treatment initiation and not at the end of the
study, as shown for newly diabetic mice reversed with anti-CD3.
Regardless, MSC therapy alone improved diabetes as indicated by the
control of hyperglycemia in over 50% of the treated mice. This
important observation demonstrates that MSC therapy for diabetes
would be most effective during the beginning phase of disease (new
onset). MSC therapy would control ongoing autoimmunity at a time
when sufficient numbers of functioning beta cells are still present
to restore normal glycemic levels (Keymeulen, et al., N. Engl. J.
Med. 352: 2598-608 (2005)).
[0192] The mechanism(s) by which MSCs lead to reversal are unknown.
The data suggests that MSCs dampen the autoimmunity, blunt
inflammation, and provide support for residual beta cells. While
not wishing to be bound by a single theory of operability, the
observations that MSCs, but not other non-mesenchymal cells such as
HUVECs, suppress T cell proliferation and blunt TNF.alpha., suggest
that MSCs inhibit autoreactive T cell responses and reduce on-going
inflammation. Accordingly, MSCs may sense the inflammatory
environment and elicit an anti-inflammatory response, including
TNF.alpha. down-modulation.
[0193] The data also shows that MSCs preferentially home to the
spleen and PLN where MSCs might inhibit autoreactive T cell
responses before the T cells migrate to the pancreas. In vitro and
in vivo data suggests that MSCs do not effect initial T cell
priming but induce hyporesponsiveness of activated T cells
(Glennie, et al., Blood 105:2821-26 (2005); and Augello, et al.,
Arthritis Rheum. 56:1175-86 (2007)) and exert immune regulatory
effects in clinical therapeutic treatment of GvHD (Ringden, et al.,
Transplantation 81:1390-97 (2006); Dean, et al., Curr. Hematol.
Rep. 2:287-94 (2003)). MSCs might also be inhibiting on-going
immune responses in the pancreas itself since MSCs were detected in
the pancreas but not other major organs. This data suggests that
MSCs have an intrinsic ability to home to areas of inflammation
where they may suppress T cell responses at these sites.
[0194] The durability of reversal with MSC treatment is important
to effective clinical treatment. The data here shows that responder
mice remain reversed for 30 days after the last MSC treatment. MSCs
have not been detected in vivo 2 weeks after injection, presumably
due to normal clearing, suggesting that MSCs are having long
lasting effects on the immune response. This characteristic along
with the observation that MSCs modulate immune responses locally
after homing to sites of injury and inflammation make these cells
ideal for treating diabetes and other autoimmune diseases. The data
indicates that MSC delivery to new onset T1D patients would control
their glycemia and consequently, their daily insulin, making MSC
therapy attractive for type I diabetes patients.
[0195] Cell therapy to treat autoimmune diseases has increased over
the years with the use of BM transplantation and more recently
transplantation of mobilized hematopoietic stem cells. The idea is
to first ablate then re-set the immune system in these patients.
Although data is promising, these procedures are very invasive and
autoimmunity recurs (for review see Tyndall, et al., Arthritis
Rheum 55:521-25 (2006); Burt, et al., JAMA 299:925-36 (2008)). As
shown by the foregoing experiments, autologous or allogeneic MSC
therapy is a more manageable alternative to these other cell
therapies.
Equivalents
[0196] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only and are not
meant to be limiting in any way. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
Sequence CWU 1
1
2118DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1ctgctgcccg acaaccac 18220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2accatgtgat cgcgcttctc 20
* * * * *