U.S. patent application number 12/091391 was filed with the patent office on 2009-07-02 for mesenchymal stem cells expressing tnf-a receptor.
Invention is credited to Simon Bubnic, Diane Carter, Alla Danilkovitch, Michelle Marcelino, Rodney Monroy, Alicia Tyrell.
Application Number | 20090169522 12/091391 |
Document ID | / |
Family ID | 38309740 |
Filed Date | 2009-07-02 |
United States Patent
Application |
20090169522 |
Kind Code |
A1 |
Danilkovitch; Alla ; et
al. |
July 2, 2009 |
MESENCHYMAL STEM CELLS EXPRESSING TNF-A RECEPTOR
Abstract
Mesenchymal stem cells which express TNF-.alpha. receptor Type I
in an amount of at least 13 pg/10.sup.6 cells. Such mesenchymal
stem cells inhibit the proliferation of lymphocytes and may be
employed, in particular, in the treatment of graft-versus-host
disease.
Inventors: |
Danilkovitch; Alla;
(Columbia, MD) ; Carter; Diane; (Huntington,
MD) ; Tyrell; Alicia; (Catonsville, MD) ;
Bubnic; Simon; (Ann Arbor, MI) ; Marcelino;
Michelle; (Ijamville, MD) ; Monroy; Rodney;
(Aberdeen, MD) |
Correspondence
Address: |
MCANDREWS HELD & MALLOY, LTD
500 WEST MADISON STREET, SUITE 3400
CHICAGO
IL
60661
US
|
Family ID: |
38309740 |
Appl. No.: |
12/091391 |
Filed: |
January 5, 2007 |
PCT Filed: |
January 5, 2007 |
PCT NO: |
PCT/US07/00274 |
371 Date: |
September 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60759157 |
Jan 13, 2006 |
|
|
|
Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
C12N 2501/25 20130101;
C12N 5/0662 20130101; A61P 29/00 20180101; G01N 2333/525 20130101;
A61K 2035/122 20130101; A61P 35/00 20180101; C12N 5/0663 20130101;
A61K 35/28 20130101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61P 35/00 20060101 A61P035/00 |
Claims
1-9. (canceled)
10. A composition comprising allogeneic mesenchymal stem cells
wherein said mesenchymal stem cells express TNF-.alpha. receptor
Type I in an amount of at least 27 pg/10.sup.6 cells.
11. The composition of claim 10, wherein said mesenchymal stem
cells are human mesenchymal stem cells.
12. The composition of claim 10, wherein said mesenchymal stem
cells are bone marrow-derived.
13. The composition of claim 10, wherein said mesenchymal stem
cells are exposed to at least one freeze-thaw cycle.
14. The composition of claim 10, wherein said mesenchymal stem
cells are expanded in culture.
15. The composition of claim 14, wherein said mesenchymal stem
cells are expanded for three to eight passages.
16. The composition of claim 15, wherein said mesenchymal stem
cells are expanded for four to six passages.
17. The composition of claim 16, wherein said mesenchymal stem
cells are expanded for five passages.
18. The composition of claim 10, further comprising an acceptable
pharmaceutical carrier.
19. The composition of claim 10, further comprising dimethyl
sulfoxide.
20. A method of treating an immunological response in a patient
comprising: administering to said patient allogeneic mesenchymal
stem cells, wherein said mesenchymal stem cells express TNF-.alpha.
receptor Type I in an amount of at least 13 pg/10.sup.6 cells.
21. The method of claim 20, wherein said mesenchymal stem cells
express TNF-.alpha. receptor Type I in an amount of at least 18
pg/10.sup.6 cells.
22. The method of claim 21, wherein said mesenchymal stem cells
express TNF-.alpha. receptor Type I in an amount of at least 27
pg/10.sup.6 cells.
23. The method of claim 20, wherein said mesenchymal stem cells are
human mesenchymal stem cells.
24. The method of claim 20, wherein said immunological response is
associated with an autoimmune disease.
25. The method of claim 24, wherein said autoimmune disease is from
the group consisting essentially of rheumatoid arthritis, multiple
sclerosis, Type I diabetes, Guillain-Barre syndrome, lupus
erythematosus, myasthenia gravis, optic neuritis, psoriasis,
Graves' disease, Hashimoto's disease, Ord's thyroiditis, aplastic
anemia, Reiter's syndrome, autoimmune hepatitis, primary biliary
cirrhosis, antiphospholipid antibody syndrome, opsoclonus myoclonus
syndrome, temporal arteritis, acute disseminated encephalomyelitis,
Good pasture's syndrome, Wegener's granulomatosis, coeliac disease,
pemphigus, polyarthritis, warm autoimmune hemolytic anemia, and
scleroderma.
26. The method of claim 20, wherein said immunological response is
associated with Crohn's disease.
27. The method of claim 20, wherein said immunological response is
associated with graft versus host disease.
28. A method of determining immunosuppressive potency of a
population of mesenchymal stem cells comprising: obtaining a sample
from the population of mesenchymal stem cells; quantifying
TNF-.alpha. receptor Type I expression in the sample to obtain a
measured value; and comparing the measured value to a threshold
value.
29. The method of claim 28, wherein said threshold value is at
least 13 pg/10.sup.6 cells.
30. The method of claim 29, wherein said threshold value is at
least 18 pg/10.sup.6 cells.
31. The method of claim 30, wherein said threshold value is at
least 27 pg/10.sup.6 cells.
32. The method of claim 28, wherein TNF-.alpha. receptor Type I
expression is quantified using an enzyme-linked immunosorbent
assay.
33. The method of claim 28, further comprising lysing cells in the
sample prior to quantifying TNF-.alpha. receptor Type I expression.
Description
[0001] This application claims priority based on application Ser.
No. 60/759,157, filed Jan. 13, 2006, the contents of which are
incorporated by reference in their entirety.
[0002] This invention relates to mesenchymal stem cells. More
particularly, this invention relates to mesenchymal stem cells
which express tumor necrosis factor-alpha (TNF-.alpha.) receptors,
and in particular, the tumor necrosis factor-alpha (TNF-{acute over
(.alpha.)}) receptor Type I (TNFRI), in an amount of at least 13
pg/10.sup.6 cells. Such mesenchymal stem cells inhibit lymphocyte
proliferation.
[0003] Mesenchymal stem cells (MSCs) are multipotent stem cells
that can differentiate readily into lineages including osteoblasts,
myocytes, chondrocytes, and adipocytes (Pittenger, et al., Science,
Vol. 284, pg. 143 (1999); Haynesworth, et al., Bone, Vol. 13, pg.
69 (1992); Prockop, Science, Vol. 276, pg. 71 (1997)). In vitro
studies have demonstrated the capability of MSCs to differentiate
into muscle (Wakitani, et al., Muscle Nerve, Vol. 18, pg. 1417
(1995)), neuronal-like precursors (Woodbury, et al., J. Neurosci.
Res., Vol. 69, pg. 908 (2002); Sanchez-Ramos, et al., Exp. Neurol.,
Vol. 171, pg. 109 (2001)), cardiomyocytes (Toma, et al.,
Circulation, Vol. 105, pg. 93 (2002); Fakuda, Artif. Organs, Vol.
25, pg. 187 (2001)) and possibly other cell types. In addition,
MSCs have been shown to provide effective feeder layers for
expansion of hematopoietic stem cells (Eaves, et al., Ann. N.Y.
Acad. Sci., Vol. 938, pg. 63 (2001); Wagers, et al., Gene Therapy,
Vol. 9, pg. 606 (2002)). Recent studies with a variety of animal
models have shown that MSCs may be useful in the repair or
regeneration of damaged bone, cartilage, meniscus or myocardial
tissues (DeKok, et al., Clin. Oral Implants Res., Vol. 14, pg. 481
(2003)); Wu, et al., Transplantation, Vol. 75, pg. 679 (2003);
Noel, et al., Curr. Opin. Investig. Drugs, Vol. 3, pg. 1000 (2002);
Ballas, et al., J. Cell. Biochem. Suppl., Vol. 38, pg. 20 (2002);
Mackenzie, et al., Blood Cells Mol. Dis., Vol. 27, pgs. 601-604
(2001)). Several investigators have used MSCs with encouraging
results for transplantation in animal disease models including
osteogenesis imperfecta (Pereira, et al., Proc. Nat. Acad. Sci.,
Vol. 95, pg. 1142 (1998)), parkinsonism (Schwartz, et al., Hum.
Gene Ther., Vol. 10, pg. 2539 (1999)), spinal cord injury (Chopp,
et al., Neuroreport, Vol. 11, pg. 3001 (2000); Wu, et al., J.
Neurosci. Res., Vol. 72, pg. 393 (2003)) and cardiac disorders
(Tomita, et al., Circulation, Vol. 100, pg. 247 (1999). Shake, et
al., Ann. Thorac. Surg., Vol. 73, pg. 1919 (2002)). Importantly,
promising results also have been reported in clinical trials for
osteogenesis imperfecta (Horowitz, et al., Blood, Vol. 97, pg. 1227
(2001); Horowitz, et al. Proc. Nat. Acad. Sci., Vol. 99, pg. 8932
(2002)) and enhanced engraftment of heterologous bone marrow
transplants (Frassoni, et al., Int. Society for Cell Therapy, SA006
(abstract) (2002); Koc, et al., J. Clin. Oncol., Vol. 18, pgs.
307-316 (2000)).
[0004] In addition, in vitro studies from different laboratories
have shown that MSCs can inhibit T-cell proliferation either in
mixed lymphocyte cultures or by other stimuli such as antigens and
mitogens (Di Nicola, et al., Blood, Vol. 99, pgs. 3638-3843 (2002);
Tse, et al., Transplantation, Vol. 75, pgs. 389-397 (2003);
Aggarwal, et al., Blood, Vol. 105, pgs. 1815-1822 (2005)). Recent
in vitro data demonstrate further that MSCs decrease the secretion
of pro-inflammatory cytokines, tumor necrosis factor-.alpha.
(TNF-.alpha.), and Interferon-.gamma. (IFN-.gamma.), and
simultaneously increase production of anti-inflammatory cytokines
Interleukin-10 (IL-10) and Interleukin-4 (IL-4) by immune cells.
(Aggarwal, 2005). These results indicate that due to
immunomodulatory and anti-inflammatory activities, MSCs can be
beneficial for treatment of immunological responses which occur in
graft-versus-host disease (GVHD), solid organ transplantation, and
autoimmune diseases such as multiple sclerosis and rheumatoid
arthritis. A clinical case report demonstrating the therapeutic
effect of MSCs for acute GVHD supports strongly this hypothesis.
(Le Blanc, et al., The Lancet, Vol. 363, pgs. 1439-1441
(2004).)
[0005] The TNF-.alpha. receptors are expressed on the surface of
mesenchymal stem cells. Accumulated data indicate that TNF-.alpha.
is an important regulator of mesenchymal stem cell function.
Incubation of TNF-.alpha. with human mesenchymal stem cells in
culture upregulates prostaglandin E2 (PGE.sub.2) and keratinocyte
growth factor (KGF) secretion, induces indoleamine 2,3 deoxygenase
(IDO) enzyme activity and stimulates cell migration. TNF-.alpha.
has been shown to be present at wound and inflammatory sites,
especially in organs targeted by graft-versus-host disease. (Koide,
et al., Transplantation, Vol. 64, pgs. 518-524 (1997); Kuroiwa, et
al., J. Clin. Invest., Vol. 107, pgs. 1365-1373 (2001); Deans, et
al., Exp. Hematol., Vol. 28, pgs. 875-884 (2002); Ellison, et al.,
J. Clin. Immunol., Vol. 24, pgs. 197-211 (2004)). Thus, such data
indicate that expression of TNF-.alpha. receptors by mesenchymal
stem cells may be critical for immunosuppressive, immunomodulatory,
anti-inflammatory, tissue-repairing, or wound-healing activities,
as well as migration to sites of inflammation.
[0006] There are two types of TNF-.alpha. receptors, or TNFRs: Type
I (TNFRI), also known as p55, and Type II (TNFRII), also known as
p75. (Tartaglia, et al., Proc. Nat. Acad. Sci, Vol. 88, pgs.
9292-9296 (1991).) Both types of TNF-.alpha. receptors are present
on MSCs; however, TNFRI is the predominant type. (Vancheri, et al.,
Am. J. Respir. Cell Mol. Biol., Vol. 22, pgs. 628-634 (2000);
Debets, et al., Cytokine, Vol. 8, pgs. 80-88 (1996).)
[0007] The invention now will be described with respect to the
drawings wherein:
[0008] FIG. 1 is a graph of the correlation between TNFRI
expression and the ability of MSCs to inhibit PBMC proliferation in
vitro;
[0009] FIG. 2 is a graph showing TNFRI expression by human
mesenchymal stem cells stored at -80.degree. C., -70.degree. C.,
-60.degree. C., and -50.degree. C.;
[0010] FIG. 3 is a graph showing TNFRI expression and the ability
to inhibit PBMC proliferation in vitro, of human mesenchymal stem
cells stored at -80.degree. C. and -50.degree. C.; and
[0011] FIG. 4 is a graph showing TNFRI expression by human
mesenchymal stem cells stored at -135.degree. C. or below, and then
thawed and kept at room temperature for 6, 8, 24, or 32 hours.
[0012] In accordance with an aspect of the present invention, there
is provided a composition comprising mesenchymal stem cells. The
mesenchymal stem cells express the TNF-{acute over (.alpha.)}
receptor Type I (TNFRI) in an amount effective to inhibit the
proliferation of lymphocytes. In one embodiment, the mesenchymal
stem cells express TNFRI in an amount of at least 13 pg/10.sup.6
cells. In another embodiment, the mesenchymal stem cells express
TNFRI in an amount of at least 15 pg/10.sup.6 cells. In yet another
embodiment, the mesenchymal stem cells express TNFRI in an amount
of at least 18 pg/10.sup.6 cells.
[0013] Although the scope of the present invention is not to be
limited to any theoretical reasoning, Applicants have found that
mesenchymal stem cells which express the TNF-{acute over (.alpha.)}
receptor Type I in an amount from at least 13 pg/10.sup.6 cells
inhibit the proliferation of lymphocytes. Such mesenchymal stem
cells are particularly useful in inhibiting immune responses, and
more particularly such mesenchymal stem cells are useful in the
treatment of graft-versus-host disease; solid organ transplant
rejection such as, for example, heart transplant rejection, liver
transplant rejection, pancreas transplant rejection, intestine
transplant rejection, and kidney transplant rejection; and
autoimmune diseases such as, for example, rheumatoid arthritis,
multiple sclerosis, Type I diabetes, Crohn's disease,
Guillain-Barre syndrome, lupus erythematosus, myasthenia gravis,
optic neuritis, psoriasis, Graves' disease, Hashimoto's disease,
Ord's thyroiditis, aplastic anemia, Reiter's syndrome, autoimmune
hepatitis, primary biliary cirrhosis, antiphospholipid antibody
syndrome, opsoclonus myoclonus syndrome, temporal arteritis, acute
disseminated encephalomyelitis, Goodpasture's syndrome, Wegener's
granulomatosis, coeliac disease, pemphigus, polyarthritis, warm
autoimmune hemolytic anemia, and scleroderma.
[0014] In one embodiment, the mesenchymal stem cells are obtained
from a mammal. The mammal may be a primate, including human and
non-human primates.
[0015] The mesenchymal stem cells may be a homogeneous composition
or may be a mixed cell population enriched in MSCs. Homogeneous
mesenchymal stem cell compositions may be obtained by culturing
adherent marrow or periosteal cells, and the mesenchymal stem cells
may be identified by specific cell surface markers which are
identified with unique monoclonal antibodies. A method for
obtaining a cell population enriched in mesenchymal stem cells is
described, for example, in U.S. Pat. No. 5,486,359. Alternative
sources for mesenchymal stem cells include, but are not limited to,
blood, skin, cord blood, muscle, fat, bone, and perichondrium.
[0016] The amount of cellular TNF-.alpha. receptor, such as
TNF-.alpha. receptor Type I, that is expressed in a culture of
mesenchymal stem cells may be determined by methods known to those
skilled in the art. Such methods include, but are not limited to,
quantitative assays such as quantitative ELISA assays, for example.
It is to be understood, however, that the scope of the present
invention is not to be limited to any particular method for
determining the amount of TNF-.alpha. receptor.
[0017] In one embodiment, the amount of TNF-.alpha. receptor
expressed by a culture of mesenchymal stem cells is determined by
an ELISA assay. In such an assay, a cell lysate from a culture of
mesenchymal stem cells is added to a well of an ELISA plate. The
well may be coated with an antibody, either a monoclonal or a
polyclonal antibody(ies), against the TNF-.alpha. receptor. The
well then is washed, and then contacted with an antibody, either a
monoclonal or a polyclonal antibody(ies), against the TNF-.alpha.
receptor. The antibody is conjugated to an appropriate enzyme, such
as horseradish peroxidase, for example. The well then may be
incubated, and then is washed after the incubation period. The
wells then are contacted with an appropriate substrate, such as one
or more chromogens. Chromogens which may be employed include, but
are not limited to, hydrogen peroxide and tetramethylbenzidine.
After the substrate(s) is (are) added, the well is incubated for an
appropriate period of time.
[0018] Upon completion of the incubation, a "stop" solution is
added to the well in order to stop the reaction of the enzyme with
the substrate(s). The optical density (OD) of the sample then is
measured. The optical density of the sample is correlated to the
optical densities of samples containing known amounts of
TNF-.alpha. receptor in order to determine the amount of
TNF-.alpha. receptor expressed by the culture of mesenchymal stem
cells being tested.
[0019] Thus, the present invention provides for the selection of a
population of mesenchymal stem cells which express TNF-.alpha.
receptor Type I in an amount of at least 13 pg/10.sup.6 cells. Such
selected mesenchymal stem cells then may be admixed with an
appropriate pharmaceutical carrier for treatment of the diseases
and disorders mentioned hereinabove. For example, the mesenchymal
stem cells may be administered as a cell suspension including a
pharmaceutically acceptable liquid medium for injection.
[0020] The mesenchymal stem cells of the present invention are
administered to an animal in an amount effective to treat one or
more of the above-mentioned diseases or disorders in the animal.
The animal may be a mammal, and the mammal may be a primate,
including human and non-human primates. The mesenchymal stem cells
may be administered systemically, such as, for example, by
intravenous, intraarterial, or intraperitoneal administration. The
exact dosage of mesenchymal stem cells to be administered is
dependent upon a variety of factors, including, but not limited to,
the age, weight, and sex of the patient, the disease(s) or
disorder(s) being treated, and the extent and severity thereof.
[0021] The invention now will be described with respect to the
following examples; however, the scope of the present invention is
not intended to be limited thereby.
EXAMPLE 1
[0022] In order to investigate the role of TNFRI on the
immunosuppressive hMSC activity, hMSCs were transfected transiently
by antisense TNFRI type oligonucleotides with the purpose to
decrease TNFRI expression (Shen et al., J. Biol. Chem., Vol. 272,
pgs. 3550-3553 (1997)). In order to reach different degrees of
TNFRI expression inhibition, three different concentrations of
oligonucleotides were used for transfection experiments.
Non-transfected MSCs and MSCs transfected with a sense
oligonucleotide were used as controls. TNFRI expression on hMSCs
was analyzed in cell lysates by ELISA, and effect of reduction in
TNFRI expression on hMSC capacity to inhibit hPBMC proliferation in
vitro was investigated.
[0023] Human bone marrow-derived MSCs at Passage 5 from 7 different
donors were used for analysis. Cells were obtained from bone marrow
aspirates, and isolated using hespan. The cells then were cultured
through Passage 5, and frozen in a standard cryopreservation
solution containing 5% human serum albumin (HSA) and 10%
dimethylsulfoxide in Plasmalyte A. (Baxter) The cells were stored
at -80.degree. C. prior to analysis. On the day of the experiment,
the hMSCs were thawed, counted, and plated into 6-well tissue
culture plates at 2.5.times.10.sup.5 cells/well. After overnight
incubation, cells were transfected with TNFRI sense or antisense
oligonucleotides at concentrations of 1.25, 2.5 and 5 pg/mL
according to the transfection reagent manufacturer's protocol
(Invitrogen, the Cellfectin transfection reagent product insert).
At 24 hours post-transfection, the cells were collected from the
plates. One group of cells was lysed, and expression of TNFRI in
cell lysates was analyzed by ELISA according to the sTNFRI ELISA
protocol (R&D Systems, product insert). TNFRI expression was
expressed in pg of receptor per 1.times.10.sup.6 cells.
[0024] For the ELISA assay, 2.5.times.10.sup.5 MSCs per well were
lysed directly in wells using 250 .mu.l/well of Cell
Lytic-mammalian cell lysis/extraction reagent (Sigma, Catalog No.
C-2978) containing a complete protein inhibitor cocktail (Roche).
The cell lysates then were centrifuged for 10 minutes at
12,000-14,000 rpm in an Eppendorf centrifuge to remove insoluble
material from the lysis buffer solution. The cell lysates then were
collected in a new tube for use in the ELISA assay.
[0025] An alternative method of cell lysis, i.e., lysis of cell
pellets in tubes, also was carried out for frozen cells and for
cells collected from tissue culture plates or flasks. Both methods,
direct cell lysis in culture plates and lysis of cell pellets in
tubes, gave comparable results.
[0026] A commercially available ELISA kit, Quantikine.RTM., Human
sTNFRI (Catalog No. DRT 100, R&D Systems) was used for the
detection of TNFRI in cell lysates. This assay provides for the
measurement of both soluble as well as cell-associated TNFRI
(Qjwang, et al., Biochemistry, Vol. 36, pg. 6033 (1997).) The assay
employs the quantitative sandwich enzyme immunoassay technique. The
assay employs a microplate that includes wells that have been
pre-coated with a monoclonal antibody specific for TNFRI. TNFRI
present in calibrator samples, quality control samples, or samples
of MSC cell lysates is captured by the immobilized TNFRI antibody.
After washing away any unbound substances, enzyme-linked polyclonal
antibodies specific for TNFRI is added to the wells. Following a
wash step to remove any unbound enzyme-linked antibody, a substrate
solution was added to the wells, and color develops in proportion
to the amount of bound TNFRI. The color development then is
stopped, and the intensity of the color is measured using an ELISA
reader.
[0027] The details of the ELISA are given hereinbelow.
[0028] 50 .mu.l of assay diluent HD1-7, a buffered protein base
with preservative, were added to the wells of an ELISA plate. The
wells were coated with a monoclonal antibody specific for TNFRI.
200 .mu.l of either calibrator samples (containing 500 pg/ml, 250
pg/ml, 125 pg/ml, 62.5 pg/ml, 31.25 pg/ml, 15.625 pg/ml, or 7.813
pg/ml of soluble human TNFRI), quality control samples (containing
45 pg/ml, 100 pg/ml, or 250 pg/ml of human TNFRI), or cell lysates
then were added to the wells. Prior to the addition of the
calibration and quality control sample to the wells, such samples
were treated with the Cell Lytic-mammalian cell lysis extraction
agent (Sigma) and complete protein inhibitor cocktail (Roche) as
hereinabove described. The plate then was covered with an adhesive
strip, and incubated for 2 hours .+-.10 minutes at room
temperature.
[0029] The liquid then was decanted from each well by inverting the
plate over a sink, and then the plate was washed three times. The
plate is washed each time with 400 .mu.l of a wash buffer added to
each well. Residual liquid was removed by inverting the plate and
blotting.
[0030] 200 .mu.l of soluble TNFRI polyclonal antibodies conjugated
to horseradish peroxidase then were added to each well. The plate
then was incubated for 2 hours .+-.10 minutes at room temperature.
The liquid then was decanted from each well, and each well was
washed three times with 400 .mu.l of wash buffer as hereinabove
described.
[0031] 200 .mu.l of a substrate solution of stabilized hydrogen
peroxide and stabilized tetramethylbenzidine chromogen then were
added to each well. The plate then was incubated for 20 minutes
.+-.10 minutes at room temperature in the dark. 50 .mu.l of a
solution of 2N sulfuric acid then were added to each well. The
optical density (OD) of each sample then was measured within 30
minutes with a 450 nm test and a 570 nm reference filter. The
optical density values then were correlated to the amounts of TNFRI
in the cell lysate samples.
[0032] Quantitation was achieved by comparing the signal from
samples of MSC cell lysates to TNFRI standards assayed at the same
time. Each ELISA run provided a calibration curve and included
duplicate quality control samples plated in front and after test
samples. Quality control samples were used for ELISA run validity
assessment. TNFRI expression data were expressed in picograms of
receptor per 1.times.10.sup.6 cells. The raw data (in pg/ml)
reflect TNFRI in picograms per 1.times.10.sup.6 cells
(2.5.times.10.sup.5 cells were lysed in 250 .mu.l of the lysis
reagent, thus corresponding to 1.times.10.sup.6 cells/ml).
[0033] The ELISA values for the calibration samples are given in
Table 1 below.
TABLE-US-00001 TABLE 1 Calculations for ELISA run calibration
standards Theoretical Back Concentration Calculated Calculated Mean
of OD Concentration Concentration Calibrator Calibratiors OD* Mean
Standard for Standards for Standards Sample (pg/mL) Values Value
Deviation (pg/mL) (pg/mL) % DFT* % CV* St01 500 2.431 2.437 0.008
498.003 499.923 -0.015 0.3 2.443 501.842 St02 250 1.487 1.476 0.016
252.746 250.306 0.123 1.1 1.464 247.867 St03 125 0.804 0.815 0.015
122.64 124.447 -0.442 1.8 0.825 126.255 St04 62.5 0.453 0.442 0.016
64.774 63.024 0.839 3.5 0.431 61.274 St05 31.25 0.25 0.239 0.016
32.749 30.939 -0.996 6.8 0.227 29.128 St06 15.625 0.143 0.145 0.002
15.765 16.007 2.446 1.5 0.146 16.249 St07 7.813 0.092 0.093 0.001
7.368 7.537 -3.528 1.5 0.094 7.706 *Note: OD--optical density; %
DFT--% Difference from Theoretical; CV %--% Coefficient of
Variance
[0034] The ELISA values for the quality control samples are given
in Table 2 below.
TABLE-US-00002 TABLE 2 Calculations for ELISA run Quality Control
(QC) samples Back Theoretical Calculated Calculated Mean
Concentrations OD Concentration Concentration QC for QCs OD* Mean
Standard for QCs for QCs Samples: (pg/mL) Values Value Deviation
(pg/mL) (pg/mL) % DFT* % CV* Front QCs QC01 45 0.366 0.372 0.008
50.991 51.938 15.417 2.3 0.378 52.884 QC02 100 0.753 0.733 0.028
113.944 110.572 10.572 3.9 0.713 107.2 QC03 250 1.503 1.509 0.008
256.165 257.454 2.982 0.6 1.515 258.742 Back QCs QC01 45 0.315
0.332 0.024 42.964 45.638 1.418 7.2 0.349 48.312 QC02 100 0.712
0.698 0.021 107.033 104.609 4.609 2.9 0.683 102.185 QC03 250 1.547
1.558 0.015 265.671 267.967 7.187 1 1.568 270.263 *Note:
OD--optical density; % DFT--% Difference from Theoretical; CV %--%
Coefficient of Variance
[0035] Based on the ELISA values for the calibration and quality
control samples shown in Tables 1 and 2 hereinabove, TNFRI
expression in pg per 1.times.10.sup.6 cells for samples of
mesenchymal stem cells from the donors was determined. As described
hereinabove, the mesenchymal stem cells from each donor were
non-transfected, or transfected with a TNFRI sense or antisense
oligonucleotide at a concentration of 1.25, 2.5, or 5 .mu.g/ml. The
ELISA values and the amount of TNFRI expressed by each of the
mesenchymal stem cell samples from each of the donors are given in
Table 3 below.
TABLE-US-00003 TABLE 3 Calculations for ELISA run test samples hMSC
OD Calculated Mean TNFRI in Donor OD* Mean Concentration
Concentration pg per # Sample description: Values Value SD* (pg/mL)
(pg/mL) 1 .times. 10.sup.6 cells % CV* 24 Control 0.385 0.384 0.001
53.989 53.831 53.831 0.4 (non-transfected cells) 0.383 53.674
Control oligo- 0.278 0.266 0.018 37.15 35.186 35.186 6.7
transfected cells 5 0.253 33.221 .mu.g/mL Control oligo- 0.348
0.352 0.006 48.155 48.785 48.785 1.6 transfected cells 2.5 0.356
49.415 .mu.g/mL Control oligo- 0.386 0.378 0.012 54.147 52.806
52.806 3.2 transfected cells 1.25 0.369 51.464 .mu.g/mL TNFRI
anti-sense 0.117 0.113 0.006 11.533 10.79 10.79 5.7
oligo-transfected cells 5 0.108 10.047 .mu.g/mL TNFRI anti-sense
0.254 0.245 0.013 33.378 31.962 31.962 5.2 oligo-transfected cells
0.236 30.546 2.5 .mu.g/mL TNFRI anti-sense 0.321 0.311 0.015 43.907
42.257 42.257 4.8 oligo-transfected cells 0.3 40.607 1.25 .mu.g/mL
007 Control 0.368 0.367 0.002 51.306 51.07 51.07 0.6
(non-transfected cells) 0.365 50.833 Control oligo- 0.226 0.219
0.01 28.97 27.866 27.866 4.5 transfected cells 5 0.212 26.761
.mu.g/mL Control oligo- 0.293 0.272 0.03 39.507 36.128 36.128 11.2
transfected cells 2.5 0.25 32.749 .mu.g/mL Control oligo- 0.308
0.286 0.032 41.864 38.329 38.329 11.1 transfected cells 1.25 0.263
34.793 .mu.g/mL TNFRI anti-sense 0.123 0.114 0.013 12.517 10.949
10.949 11.8 oligo-transfected cells 5 0.104 9.382 .mu.g/mL TNFRI
anti-sense 0.269 0.243 0.037 35.736 31.565 31.565 15.5
oligo-transfected cells 0.216 27.393 2.5 .mu.g/mL TNFRI anti-sense
0.313 0.303 0.014 42.65 41.078 41.078 4.7 oligo-transfected cells
0.293 39.507 1.25 .mu.g/mL 014 Control 0.377 0.38 0.004 52.726 53.2
53.2 1.1 (non-transfected cells) 0.383 53.674 Control oligo- 0.251
0.249 0.003 32.907 32.592 32.592 1.1 transfected cells 5 0.247
32.277 .mu.g/mL Control oligo- 0.338 0.315 0.033 46.581 42.887
42.887 10.6 transfected cells 2.5 0.291 39.193 .mu.g/mL Control
oligo- 0.356 0.347 0.013 49.415 47.919 47.919 3.9 transfected cells
1.25 0.337 46.424 .mu.g/mL TNFRI anti-sense 0.11 0.104 0.008 10.378
9.379 9.379 8.2 oligo-transfected cells 5 0.098 8.379 .mu.g/mL
TNFRI anti-sense 0.211 0.206 0.008 26.603 25.733 25.733 3.8
oligo-transfected cells 0.2 24.864 2.5 .mu.g/mL TNFRI anti-sense
0.3 0.294 0.008 40.607 39.664 39.664 2.9 oligo-transfected cells
0.288 38.722 1.25 .mu.g/mL 015 Control 0.475 0.469 0.009 68.284
67.246 67.246 2 (non-transfected cells) 0.462 66.209 Control oligo-
0.278 0.279 0.001 37.15 37.308 37.308 0.5 transfected cells 5 0.28
37.465 .mu.g/mL Control oligo- 0.34 0.343 0.004 46.896 47.289
47.289 1 transfected cells 2.5 0.345 47.683 .mu.g/mL Control oligo-
0.419 0.413 0.009 59.37 58.34 58.34 2.2 transfected cells 1.25
0.406 57.31 .mu.g/mL TNFRI anti-sense 0.13 0.125 0.007 13.658
12.842 12.842 5.7 oligo-transfected cells 5 0.12 12.025 .mu.g/mL
TNFRI anti-sense 0.253 0.262 0.012 33.221 34.557 34.557 4.6
oligo-transfected cells 0.27 35.893 2.5 .mu.g/mL TNFRI anti-sense
0.377 0.381 0.005 52.726 53.279 53.279 1.3 oligo-transfected cells
0.384 53.831 1.25 .mu.g/mL 23 Control 0.260 0.255 0.008 40.591
39.632 39.632 3.1 (non-transfected cells) 0.249 38.672 Control
oligo- 0.191 0.184 0.010 28.560 27.339 27.339 5.4 transfected cells
5 0.177 26.117 .mu.g/mL Control oligo- 0.216 0.209 0.009 32.919
31.786 31.786 4.4 transfected cells 2.5 0.203 30.653 .mu.g/mL
Control oligo- 0.222 0.222 0.000 33.965 33.965 33.965 0.0
transfected cells 1.25 0.222 33.965 .mu.g/mL TNFRI anti-sense 0.107
0.106 0.001 13.798 13.620 13.620 1.3 oligo-transfected cells 5
0.105 13.441 .mu.g/mL TNFRI anti-sense 0.206 0.187 0.027 31.176
27.860 27.860 14.4 oligo-transfected cells 0.168 24.544 2.5
.mu.g/mL TNFRI anti-sense 0.213 0.212 0.001 32.396 32.222 32.222
0.7 oligo-transfected cells 0.211 32.048 1.25 .mu.g/mL 486 Control
0.249 0.249 0.001 41.244 41.148 41.148 0.3 (non-transfected cells)
0.248 41.053 Control oligo- 0.149 0.136 0.018 22.401 19.981 19.981
13.5 transfected cells 5 0.123 17.560 .mu.g/mL Control oligo- 0.246
0.231 0.022 40.672 37.732 37.732 9.5 transfected cells 2.5 0.215
34.792 .mu.g/mL Control oligo- 0.263 0.253 0.015 43.915 41.913
41.913 5.9 transfected cells 1.25 0.242 39.911 .mu.g/mL TNFRI
anti-sense 0.071 0.068 0.004 7.917 7.361 7.361 6.2
oligo-transfected cells 5 0.065 6.805 .mu.g/mL TNFRI anti-sense
0.142 0.142 0.000 21.096 21.096 21.096 0.0 oligo-transfected cells
0.142 21.096 2.5 .mu.g/mL TNFRI anti-sense 0.193 0.179 0.021 30.644
27.924 27.924 11.5 oligo-transfected cells 0.164 25.204 1.25
.mu.g/mL 13 Control 0.211 0.209 0.003 34.037 33.659 33.659 1.4
(non-transfected cells) 0.207 33.282 Control oligo- 0.134 0.134
0.01 19.606 19.513 19.513 0.5 transfected cells 5 0.133 19.420
.mu.g/mL Control oligo- 0.195 0.188 0.011 31.020 29.611 29.611 5.7
transfected cells 2.5 0.180 28.201 .mu.g/mL Control oligo- 0.207
0.192 0.022 33.282 30.366 38.329 11.4 transfected cells 1.25 0.176
27.451 .mu.g/mL TNFRI anti-sense 0.087 0.080 0.010 10.882 9.585
9.585 12.4 oligo-transfected cells 5 0.073 8.288 .mu.g/mL TNFRI
anti-sense 0.156 0.135 0.030 23.708 19.706 19.706 22.6
oligo-transfected cells 0.113 15.703 2.5 .mu.g/mL TNFRI anti-sense
0.208 0.174 0.048 33.470 27.097 27.097 27.6 oligo-transfected cells
0.140 20.723 1.25 .mu.g/mL *Note: OD--optical density; SD--Standard
Deviation; CV %--% Coefficient of Variance
[0036] From the above data shown in Table 3, the mean TNFRI
expression, in picograms per 1.times.10.sup.6 cells, was determined
for non-transfected (control) mesenchymal stem cells, as well as
mesenchymal stem cells transfected with 1.25, 2.5, or 5 .mu.l/ml of
antisense or sense oligonucleotides. The mean TNFRI expression
values are given in Table 4 below.
TABLE-US-00004 TABLE 4 TNFRI expression by hMSCs transfected with
anti-sense and control (sense) oligonucleotides: summary for 7
tested hMSC donors TNFRI expression in pg per 1 .times. 10.sup.6
cells hMSC donor #: Mean for 486 13 24 007 14 15 23 7 donors SD
Control (non- 41* 34 54 51 53 67 40 48.57 11.09 transfected cells)
TNFRI anti-sense 7 10 11 11 9 13 14 10.71 2.36 oligo-transfected
cells 5 .mu.g/mL TNFRI anti-sense 21 20 32 32 26 35 28 27.71 5.74
oligo-transfected cells 2.5 .mu.g/mL TNFRI anti-sense 28 27 42 41
40 53 32 37.57 9.22 oligo-transfected cells 1.25 .mu.g/mL Control
(sense) 20 20 35 28 33 37 27 28.57 6.85 oligo-transfected cells 5
.mu.g/mL Control (sense) 38 30 49 36 43 47 32 39.29 7.30
oligo-transfected cells 2.5 .mu.g/mL Control (sense) 42 30 53 38 48
58 34 43.29 10.21 oligo-transfected cells 1.25 .mu.g/mL *Note:
These values represent mean TNFRI numbers (from table 3, column 8:
"TNFRI in pg per 1 .times. 10.sup.6 cells") rounded to whole
numbers
[0037] A second group of transfected cells was used for
investigation of the effect of hMSCs on hPBMC proliferation in
vitro. Human PBMCs from two different donors were used for this
assay. PBMCs were isolated from leukopheresed blood using
Ficoll-Paque gradient centrifugation according to the
manufacturer's protocol (Amersham Biosciences, Ficoll-Paque Plus
product insert). Cells were stored frozen at -80.degree. C. in a
medium including 90% FBS and 10% DMSO prior to analysis. On the day
of the experiment hPBMCs were thawed, counted and plated into
96-well tissue culture plates at 1.times.10.sup.5 cells/well
together with hMSCs (1.times.10.sup.4 cells/well). A combination of
anti-CD3 (1 .mu.g/mL) and anti-CD28 (1 pg/mL) antibodies was used
to stimulate lymphocyte proliferation that represents an in vitro
model for immune cell activation characteristics of GVHD and
rejection of allogeneic organs. (Trickett, et al., J. Immunol.
Methods, Vol. 275, pgs. 251-255 (2003); Koulova, et al., J. Exp.
Med., Vol. 173, No. 3, pgs. 759-762 (1991); Foster, et al.,
Transplantation, Vol. 76, No. 6; Czitrom, Clin. Ortho. Relat. Res.,
Vol. 326, pgs. 11-24 (1996)). The plates then were incubated in a
humidified atmosphere containing 5% CO.sub.2. The proliferation of
PBMCs alone and in the presence of MSCs was measured at day 5 from
culture initiation by the addition of [Methyl-.sup.3H]-thymidine at
1 .mu.Ci/well for the final 18-20 hrs of culture. After labeling,
the cells were transferred onto a glass filter using a 96-well
plate harvester, and radioactivity incorporated into DNA was
measured by a liquid scintillation beta-counter. The uptake of
[Methyl-.sup.3H]-thymidine into DNA in counts per minute (cpm)
represents hPBMC proliferation. Final results were expressed as %
inhibition of PBMC proliferation in the presence of MSCs calculated
as:
100%-[Proliferation (PBMC+MSC, cpm).times.100/Proliferation (PBMC,
cpm)]
[0038] The results for the mesenchymal stem cells from each of the
donors are given in Table 5 below.
TABLE-US-00005 TABLE 5 Inhibition of CD3/CD28-induced hPBMC
proliferation by hMSCs transfected with anti-sense and control
(sense) oligonucleotides: summary for 7 tested hMSC donors %
inhibition of hPBMC proliferation by hMSCs hMSC donor #: Mean % 486
13 24 007 14 15 23 for 7 hPBMC donor #: 2 3 2 3 3 3 3 2 2 3 donors
SD Control (non- 65 73 82 94 70 66 82 62 68 91 75.30 11.26
transfected cells) TNFRI anti-sense 40 45 46 68 32 10 39 19 38 52
38.90 16.29 oligo-transfected cells 5 .mu.g/mL TNFRI anti-sense 83
90 59 86 ND 73 ND 63 47 58 69.88 15.48 oligo-transfected cells 2.5
.mu.g/mL TNFRI anti-sense 62 74 86 ND 72 64 57 ND 72 80 70.88 9.58
oligo-transfected cells 1.25 .mu.g/mL Control (sense) 38 87 60 77
58 77 62 44 52 53 60.80 15.50 oligo-transfected cells 5 .mu.g/mL
Control (sense) 60 91 67 ND ND 62 66 57 70 95 71.00 14.22
oligo-transfected cells 2.5 .mu.g/mL Control (sense) 87 ND 68 71 66
68 36 ND 49 85 70.57 12.77 oligo-transfected cells 1.25 .mu.g/mL
Note: ND--no data
[0039] The above data with respect to inhibition of CD3/CD28
induced PBMC proliferation were correlated to the mean TNFRI
expression data shown in Table 4 hereinabove. The correlated data
with respect to mean TNFRI expression and inhibition of CD3/CD28
induced PBMC proliferation are given in Table 6 below.
TABLE-US-00006 TABLE 6 TNFRI expression and effect on hPBMC
proliferation in vitro by hMSCs transfected with TNFRI
oligonucleotides % Inhibition of TNFRI Oligonucleotide hPBMC
expression in concentration proliferation pg/1 .times. 10.sup.6
MSCs Human MSCs condition (.mu.g/mL) (Mean .+-. SD) (Mean .+-. SD)
Untransfected (Control Not applicable 75.30 .+-. 11.26 48.57 .+-.
11.09 MSCs) Antisense oligonucleotide 1.25 70.88 .+-. 9.58 37.57
.+-. 9.22 2.5 69.88 .+-. 15.48 27.71 .+-. 5.74 5 38.90 .+-. 16.29
10.71 .+-. 2.36 Sense oligonucleotide 1.25 70.57 .+-. 12.77 43.29
.+-. 10.21 (control oligonucleotide) 2.5 71.00 .+-. 14.22 39.29
.+-. 7.30 5 60.80 .+-. 15.50 28.57 .+-. 6.85
[0040] The results from these experiments show that hMSCs with
decreased expression of TNFR type I (TNFRI) lose their ability to
suppress hPBMC proliferation in vitro. The data support the premise
that the expression of TNFRI is an essential link to the
suppression of PBMC proliferation by MSCs. Thus, TNFRI can be used
as a potency marker for MSC immunomodulative activity. Based on the
obtained data, a potency threshold of 13.07 pg of TNFRI
(mean.+-.SD) per 1.times.10.sup.6 cells correlates with less than
50% inhibition of hPBMC proliferation (Table 6, FIG. 1). Thus,
non-potent MSCs are cells expressing less than 13 pg TNFRI per
1.times.10.sup.6 cells.
EXAMPLE 2
TNFRI is a Temperature-Sensitive Marker of hMSC Functionality
[0041] Ex vivo handling of mammalian cells is restricted by a
number of factors including temperature. For example, low
temperatures such as -80.+-.5.degree. C., or lower, even as low as
-135.degree. C. or below (liquid nitrogen) are required for cell
storage whereas ex vivo cell expansion requires a temperature of
37.+-.0.5.degree. C. Cell exposure to temperatures outside of the
optimal ranges may lead to a decrease in cell functionality or cell
death. Mammalian cells are able to withstand short-term minor
temperature fluctuations; however, each type of cells has its own
temperature tolerance range for cell culture maintenance, shipping,
and storage.
[0042] The expression level of TNFRI on hMSCs correlates with hMSC
immunosuppressive activity. The level of TNFRI expression by hMSCs
of less than 13 pg/10.sup.6 cells has been determined as a
threshold, below which hMSCs begin to lose their ability to
suppress an immune response (See FIG. 1). Thus, TNFRI expression is
a marker of hMSC immunosuppression, an activity that is believed
essential for MSCs to be efficacious for treatment of immunological
reactions taking place in GVHD, organ rejection, autoimmune
diseases, and other diseases. Here, effects of temperature
fluctuations during storage of frozen hMSCs as well as the effect
of time of exposure of cells to room temperature on expression of
TNFRI on hMSCs was investigated.
Effect of Store Temperature Fluctuations on TNFRI Expression and
hMSC immunosuppressive potential.
[0043] The objective of these experiments was to investigate the
ability of hMSCs to retain their functional characteristics after
an exposure to temperatures above -80.degree. C., which are not
optimal temperatures for storage of frozen cells. Human MSCs were
frozen at passage 5 and placed for storage in a freezer at
-80.+-.5.degree. C. After several weeks, bags of frozen cells were
removed from the -80.+-.5.degree. C. freezer and placed at either
-70.+-.5.degree. C., -60.+-.5.degree. C., or -50.+-.5.degree. C.
for 72.+-.2 hours. After 72.+-.2 hours, the bags were returned to
storage at -80.+-.5.degree. C. for at least 24 hours before thaw
and analysis. A set of bags moved from one -80.+-.5.degree. C.
freezer to another, following the same schedule as the other bags,
served as a control. On the day of the experiment the bags
containing the cells were thawed, cells were counted, and cell
lysates for the TNFRI ELISA were prepared as described in Example
1. The TNFRI ELISA was performed as described in Example 1. Results
are summarized in FIG. 2 (bars show mean TNFRI values .+-.SD for 3
hMSC bags). The data showed that exposure of hMSCs to temperatures
of -60.+-.5.degree. C. or -50.+-.5.degree. C. decreases the TNFRI
expression level: the level of TNFRI detected by ELISA was below
the determined hMSC potency threshold of 13 pg/10.sup.6 cells
(represented by the solid line on the graph).
[0044] Parallel with TNFRI measurement, two bags with hMSCs stored
at -80.+-.5.degree. C. (optimal storage temperature served as a
control) and at -50.+-.5.degree. C. (corresponding to a
.+-.30.degree. C. greater than the -80.+-.5.degree. C. optimal
storage temperature) were used for investigation of hMSC
immunosuppressive activity. The ability of the MSCs to suppress
anti-CD3/CD28-induced proliferation of hPBMCs in vitro was
evaluated as described in Example 1. The results showed that hMSCs
stored at -50.+-.5.degree. C. lost their ability to suppress hPBMC
proliferation, whereas cells stored at -80.+-.5.degree. C.
inhibited hPBMC proliferation by 92% (FIG. 3, dark bars represent
mean.+-.SD % inhibition of hPBMC proliferation. Numbers inside the
dark bars show numerical values). The immunosuppressive activity of
MSCs is dependent on the level of TNFRI expression: cells
expressing more than 13 pg/10.sup.6 cells of TNFRI, which was
determined as an MSC immunosuppressive potential threshold, are
biologically active, and cells with the TNFRI level below 13
pg/10.sup.6 cells are not (FIG. 3, light bars represent mean.+-.SD
of the TNFRI expression level. Numbers inside the light bars show
numerical values). Thus, non-optimal storage temperatures decrease
TNFRI expression on hMSCs, and which correlates with decrease in
hMSC functionality.
Effect of Cell Exposure Time to Room Temperature on TNFRI
Expression on hMSC.
[0045] The results of this experiment serve as additional evidence
that TNFRI expression on hMSCs is decreasing under cell exposure to
non-optimal temperatures. In this experiment the effect of cell
suspension storage at room temperature on TNFRI expression was
studied. Two hMSC lots were used in the experiment. Bags containing
hMSCs were stored at .ltoreq.-135.degree. C. prior to the
experiment. On the day of the experiment the cells were thawed and
diluted with Plasmalyte A physiological solution (Baxter) in a
manner that mimics the current cell processing for intravenous hMSC
administration at clinical sites. The thawed and diluted hMSCs were
kept at room temperature (22.degree. C.-24.degree. C.), and samples
were taken and tested for the amount of TNFRI at 0 (immediately
post-thaw-baseline), 6, 8, 10, 24, and 32 hours post-thawing. The
results showed that exposure of hMSCs to room temperature decreased
the TNFRI expression level on the hMSCs (FIG. 4, bars represent
mean.+-.SD of the TNFRI expression level for 2 hMSC lots. The solid
line represents the TNFRI expression level of 13 pg/10.sup.6 cells,
which is the hMSC potency threshold). The significant decrease in
TNFRI expression was observed at 24 hours and 32 hours, and it
correlated with a significant decrease in cell viability (below
20%, data not shown).
[0046] Thus, the experiments described above show that TNFRI
expression by hMSCs is sensitive to temperature, and TNFRI can be
used as a marker of functionality of hMSC that were exposed to
non-optimal temperatures during storage, shipping or cell
processing.
[0047] The disclosures of all patents, publications, including
published patent applications, depository accession numbers, and
database accession numbers are hereby incorporated by reference to
the same extent as if each patent, publication, depository
accession number, and database accession number were specifically
and individually incorporated by reference.
[0048] It is to be understood, however, that the scope of the
present invention is not to be limited to the specific embodiments
described above. The invention may be practiced other than as
particularly described and still be within the scope of the
accompanying claims.
* * * * *