Mesenchymal stem cells expressing TNF-α receptors

Information

  • Patent Grant
  • 11821004
  • Patent Number
    11,821,004
  • Date Filed
    Monday, November 2, 2020
    3 years ago
  • Date Issued
    Tuesday, November 21, 2023
    7 months ago
Abstract
Mesenchymal stem cells which express TNF-α receptor Type I in an amount of at least 13 pg/106 cells. Such mesenchymal stem cells inhibit the proliferation of lymphocytes and may be employed, in particular, in the treatment of graft-versus-host disease.
Description
BRIEF SUMMARY OF THE INVENTION

This invention relates to mesenchymal stem cells. More particularly, this invention relates to mesenchymal stem cells which express tumor necrosis factor-alpha (TNF-α) receptors, and in particular, the tumor necrosis factor-alpha (TNF-α) receptor Type I (TNFRI), in an amount of at least 13 pg/106 cells. Such mesenchymal stem cells inhibit lymphocyte proliferation.


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)).


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-α (TNF-α), and Interferon-γ (IFN-γ), 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).)


The TNF-α receptors are expressed on the surface of mesenchymal stem cells. Accumulated data indicate that TNF-α is an important regulator of mesenchymal stem cell function. Incubation of TNF-α with human mesenchymal stem cells in culture upregulates prostaglandin E2 (PGE2) and keratinocyte growth factor (KGF) secretion, induces indoleamine 2,3 deoxygenase (IDO) enzyme activity and stimulates cell migration. TNF-α 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-α 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.


There are two types of TNF-α 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-α 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).)





BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

The invention now will be described with respect to the drawings wherein:



FIG. 1 is a graph of the correlation between TNFRI expression and the ability of MSCs to inhibit PBMC proliferation in vitro;



FIG. 2 is a graph showing varying TNFRI expression by human mesenchymal stem cells stored at −80° C., −70° C., −60° C., and −50° C.;



FIG. 3 is a graph showing TNFRI expression at varying levels and the correlating ability to inhibit PBMC proliferation in vitro. of human mesenchymal stem cells stored at −80° C. and −50° C.; and



FIG. 4 is a graph showing TNFRI expression by human mesenchymal stem cells stored at −135° C. or below, and then thawed and kept at room temperature for 6, 8, 24, or 32 hours.





DETAILED DESCRIPTION OF THE INVENTION

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-α receptor Type I (TNFR1) in a varying 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/106 cells. In another embodiment, the mesenchymal stem cells express TNFRI in an amount, for example, of at least 15 pg/106 cells. In yet another embodiment, the mesenchymal stem cells express TNFRI in an amount of at least 18 pg/106 cells.


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-α receptor Type I in an amount from, for example, at least 13 pg/106 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-Barré 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.


In one embodiment, the mesenchymal stem cells are obtained from a mammal. The mammal may be a primate, including human and non-human primates.


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.


The varying amount, but at least 13 pg/106 cells, of cellular TNF-α receptor, such as TNF-α 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-α receptor and it should be appreciated that the amount of expression can vary.


In one embodiment, the amount of TNF-α 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-α receptor. The well then is washed, and then contacted with an antibody, either a monoclonal or a polyclonal antibody(ies), against the TNF-α 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.


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-α receptor in order to determine the amount of TNF-α receptor expressed by the culture of mesenchymal stem cells being tested.


Thus, the present invention provides for the selection of a population of mesenchymal stem cells which express TNF-α receptor Type 1 in an amount, for example, of at least 13 pg/106 cells. However, it should be appreciated by those skilled in the art that the selected population of mesenchymal stem cells of the present invention express the desired amount of TNF-α receptor Type 1 while other populations may not express the desired amount of receptor due to the biological nature of the present invention. It is believed, for example, that approximately 98% of the selected mesenchymal stem cell population of the present invention express the desired TNF-α receptor Type 1 level while the remaining portion of that population may not. 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.


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.


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

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.


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° C. prior to analysis. On the day of the experiment, the hMSCs were thawed, counted, and plated into 6-well tissue culture plates at 2.5×105 cells/well. After overnight incubation, cells were transfected with TNFRI sense or antisense oligonucleotides at concentrations of 1.25, 2.5 and 5 μg/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 1×106 cells.


For the ELISA assay. 2.5×105 MSCs per well were lysed directly in wells using 250 μl/well of Cell Lytic-mammalian cell lysis/extraction reagent (Sigma, Catalog No. 0-2978) 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.


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.


A commercially available ELISA kit, Quantikine®, 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.


The details of the ELISA are given hereinbelow.


500 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 μ1 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.


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 μl of a wash buffer added to each well. Residual liquid was removed by inverting the plate and blotting.


200 μl 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 μl of wash buffer as hereinabove described.


200 μl 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 μl 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.


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 1×106 cells. The raw data (in pg/ml) reflect TNFRI in picograms per 1×106 cells (2.5×105 cells were lysed in 250 μl of the lysis reagent, thus corresponding to 1×106 cells/ml).


The ELISA values for the calibration samples are given in Table 1 below.









TABLE 1







Calculations for ELISA run calibration standards




















Back
Calculated





Theoretical



Calculated
Mean





Concentration

OD

Concentration
Concentration




Calibrator
of Calibrations
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






The ELISA values for the quality control samples are given in Table 2 below.









TABLE 2







Calculations for ELISA run Quality Control (QC) samples




















Back
Calculated





Theoretical



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





QCO2
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






Based on the ELISA values for the calibration and quality control samples shown in Tables 1 and 2 hereinabove, TNFRI expression in pg per 1×106 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 μg/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 3







Calculations for ELISA run test samples






















TNFRI in



hMSC


OD

Calculated
Mean
pg per



Donor
Sample
OD*
Mean

Concentration
Concentration
1 × 106
%


#
description:
Values
Value
SD*
(pg/mL)
(pg/mL)
cells
CV*


















24
Control (non-
0.385
0.384
0.001
53.989
53.831
53.831
0.4



transfected
0.383


53.674






cells)










Control oligo-
0.278
0.266
0.018
37.15
35.186
35.186
6.7



transfected
0.253


33.221






cells 5 ug/mL










Control oligo-
0.348
0.352
0.006
48.155
48.785
48.785
1.6



transfected
0.356


49.415






cells 2.5










μg/mL










Control oligo-
0.386
0.378
0.012
54.147
52.806
52.806
3.2



transfected
0.369


51.464






cells 1.25










μg/mL










TNFRI anti-
0.117
0.113
0.006
11.533
10.79
10.79
5.7



sense oligo-
0.108


10.047






transfected










cells 5 μg/mL










TNFRI anti-
0.254
0.245
0.013
33.378
31.962
31.962
5.2



sense oligo-
0.236


30.546






transfected










cells 2.5










μg/mL










TNFRI anti-
0.321
0.311
0.015
43.907
42.257
42.257
4.8



sense oligo-
0.3


40.607






transfected










cells 1.25









007
Control (non-
0.368
0.367
0.002
51.306
51.07
51.07
0.6



transfected
0.365


50.833






cells)










Control oligo-
0.226
0.219
0.01
28.97
27.866
27.866
4.5



transfected
0.212


26.761






cells 5 μg/mL










Control oligo-
0.293
0.272
0.03
39.507
36.128
36.128
11.2



transfected
0.25


32.749






cells 2.5










μg/mL










Control oligo-
0.308
0.286
0.032
41.864
38.329
38.329
11.1



transfected
0.263


34.793






cells 1.25










μg/mL










TNFRI anti-
0.123
0.114
0.013
12.517
10.949
10.949
11.8



sense oligo-
0.104


9.382






transfected










cells 5 μg/mL










TNFRI anti-
0.269
0.243
0.037
35.736
31.565
31.565
15.5



sense oligo-
0.216


27.393






transfected










cells 2.5










μg/mL










TNFRI anti-
0.313
0.303
0.014
42.65
41.078
41.078
4.7



sense oligo-
0.293


39.507






transfected










cells 1.25










μg/mL









014
Control (non-
0.377
0.38
0.004
52.726
53.2
53.2
1.1



transfected
0.383


53.674






cells)










Control oligo-
0.251
0.249
0.003
32.907
32.592
32.592
1.1



transfected
0.247


32.277






cells 5 μg/mL










Control oligo-
0.338
0.315
0.033
46.581
42.887
42.887
10.6



transfected
0.291


39.193






cells 2.5










μg/mL










Control oligo-
0.356
0.347
0.013
49.415
47.919
47.919
3.9



transfected
0.337


46.424






cells 1.25










μg/mL










TNFRI anti-
0.11
0.104
0.008
10.378
9.379
9.379
8.2



sense oligo-
0.098


8.379






transfected










cells 5 μg/mL










TNFRI anti-
0.211
0.206
0.008
26.603
25.733
25.733
3.8



sense oligo-
0.2


24.864






transfected










cells 2.5










μg/mL










TNFRI anti-
0.3
0.294
0.008
40.607
39.664
39.664
2.9



sense oligo-
0.288


38.722






transfected










cells 1.25










μg/mL









015
Control (non-
0.475
0.469
0.009
68.284
67.246
67.246
2



transfected
0.462


66.209






cells)










Control oligo-
0.278
0.279
0.001
37.15
37.308
37.308
0.5



transfected
0.28


37.465






cells 5 μg/mL










Control oligo-
0.34
0.343
0.004
46.896
47.289
47.289
1



transfected
0.345


47.683






cells 2.5










μg/mL










Control oligo-
0.419
0.413
0.009
59.37
58.34
58.34
2.2



transfected
0.406


57.31






cells 1.25










μg/mL










TNFRI anti-
0.13
0.125
0.007
13.658
12.842
12.842
5.7



sense oligo-
0.12


12.025






transfected










cells 5 μg/mL










TNFRI anti-
0.253
0.262
0.012
33.221
34.557
34.557
4.6



sense oligo-
0.27


35.893






transfected










cells 2.5










μg/mL










TNFRI anti-
0.377
0.381
0.005
52.726
53.279
53.279
1.3



sense oligo-
0.384


53.831






transfected










cells 1.25










μg/mL









23
Control (non-
0.260
0.255
0.008
40.591
39.632
39.632
3.1



transfected
0.249


38.672






cells)










Control oligo-
0.191
0.184
0.010
28.560
27.339
27.339
5.4



transfected
0.177


26.117






cells 5 μg/mL










Control oligo-
0.216
0.209
0.009
32.919
31.786
31.786
4.4



transfected
0.203


30.653






cells 2.5










μg/mL










Control oligo-
0.222
0.222
0.000
33.965
33.965
33.965
0.0



transfected
0.222


33.965






cells 1.25










μg/mL










TNFRI anti-
0.107
0.106
0.001
13.798
13.620
13.620
1.3



sense oligo-
0.105


13.441






transfected










cells 5 μg/mL










TNFRI anti-
0.206
0.187
0.027
31.176
27.860
27.860
14.4



sense oligo-
0.168


24.544






transfected










cells 2.5










μg/mL










TNFRI anti-
0.213
0.212
0.001
32.396
32.222
32.222
0.7



sense oligo-
0.211


32.048






transfected










cells 1.25










μg/mL









486
Control (non-
0.249
0.249
0.001
41.244
41.148
41.148
0.3



transfected
0.248


41.053






cells)










Control oligo-
0.149
0.136
0.018
22.401
19.981
19.981
13.5



transfected
0.123


17.560






cells 5 μg/mL










Control oligo-
0.246
0.231
0.022
40.672
37.732
37.732
9.5



transfected
0.215


34.792






cells 2.5










μg/mL










Control oligo-
0.263
0.253
0.015
43.915
41.913
41.913
5.9



transfected
0.242


39.911






cells 1.25










μg/mL










TNFRI anti-
0.071
0.068
0.004
7.917
7.361
7.361
6.2



sense oligo-
0.065


6.805






transfected










cells 5 μg/mL










TNFRI anti-
0.142
0.142
0.000
21.096
21.096
21.096
0.0



sense oligo-
0.142


21.096






transfected










cells 2.5










μg/mL










TNFRI anti-
0.193
0.179
0.021
30.644
27.924
27.924
11.5



sense oligo-
0.164


25.204






transfected










cells 1.25










μg/mL









13
Control (non-
0.211
0.209
0.003
34.037
33.659
33.658
1.4



transfected
0.207


33.282






cells)










Control oligo-
0.134
0.134
0.01
19.606
19.513
19.513
0.5



transfected
0.133


19.420






cells 5 μg/mL










Control oligo-
0.195
0.188
0.011
31.020
29.611
29.611
5.7



transfected
0.180


28.201






cells 2.5










μg/mL










Control oligo-
0.207
0.192
0.022
33.282
30.366
38.329
11.4



transfected
0.176


27.451






cells 1.25










μg/mL










TNFRI anti-
0.087
0.080
0.010
10.882
9.585
9.585
12.4



sense oligo-
0.073


8.288






transfected










cells 5 μg/mL










TNFRI anti-
0.156
0.135
0.030
23.708
19.706
19.706
22.6



sense oligo-
0.113


15.703






transfected










cells 2.5










μg/mL










TNFRI anti-
0.208
0.174
0.048
33.470
27.097
27.097
27.6



sense oligo-
0.140


20.723






transfected










cells 1.25










μg/mL





*Note:


OD—optical density;


SD—Standard Deviation;


CV %—% Coefficient of Variance






From the above data shown in Table 3, the mean TNFRI expression, in picograms per 1×106 cells, was determined for non-transfected (control) mesenchymal stem cells, as well as mesenchymal stem cells transfected with 1.25, 2.5, or 5 μl/ml of antisense or sense oligonucleotides. The mean TNFRI expression values are given in Table 4 below.









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 × 106 cells
Mean for

















hMSC donor #:
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-
 7
10
11
11
9
13
14
10.71
2.36


sense oligo-











transfected cells











5 μg/mL











TNFRI anti-
21
20
32
32
26
35
28
27.71
5.74


sense oligo-











transfected cells











2.5 μg/mL











TNFRI anti-
28
27
42
41
40
53
32
37.57
9.22


sense oligo-











transfected











cells 1.25 μg/mL











Control (sense)
20
20
35
28
33
37
27
28.57
6.85


oligo-transfected











cells 5 μg/mL











Control (sense)
38
30
49
36
43
47
32
39.29
7.30


oligo-transfected











cells 2.5 μg/mL











Control (sense)
42
30
53
38
48
58
34
43.29
10.21


oligo-transfected











cells 1.25 μg/mL





*Note:


These values represent mean TNFRI numbers (from table 3, column 8: “TNFRI in pg per 1 × 106 cells”) rounded to whole numbers






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° 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 1×105 cells/well together with hMSCs (1×104 cells/well). A combination of anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/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% CO2. The proliferation of PBMCs alone and in the presence of MSCs was measured at day 5 from culture initiation by the addition of [Methyl-3H]-thymidine at 1 μCi/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-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)×100/Profiteration(PBMC,cpm)]


The results for the mesenchymal stem cells from each of the donors are given in Table 5 below.









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 #:



















486
13
24
007
14
15
23
Mean %




















hPBMC donor #:
2
3
2
3
3
3
3
2
2
3
for 7 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 μg/mL














TNFRI anti-sense
83
90
59
86
ND
73
ND
63
47
58
69.88
15.48


oligo-transfected














cells 2.5 μg/mL














TNFRI anti-sense
62
74
86
ND
72
64
57
ND
72
80
70.88
9.58


oligo-transfected














cells 1.25 μg/mL














Control (sense)
38
87
60
77
58
77
62
44
52
53
60.80
15.50


oligo-transfected














cells 5 μg/mL














Control (sense)
60
91
67
ND
ND
62
66
57
70
95
71.00
14.22


oligo-transfected














cells 2.5 μg/mL














Control (sense)
87
ND
68
71
66
68
36
ND
49
85
70.57
12.77


oligo-transfected














cells 1.25 μg/mL





Note:


ND—no data






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 6







TNFRI expression and effect on hPBMC proliferation in


vitro by hMSCs transfected with TNFRI oligonucleotides













TNFRI




% inhibition
Expression



Oligonucleotide
of hPBMC
in pg/1 ×


Human MSCs
concentration
proliferation
106 MSCs


condition
(pg/mL)
(Mean ± SD)
(Mean ± SD)





Untransfected
Not applicable
75.30 ± 11.26
 48.57 ± 11.09


(Control MSCs)


Antisense
1.25
70.88 ± 9.58 
37.57 ± 9.22


oligonucleotide
2.5
69.88 ± 15.48
27.71 ± 5.74



5
38.90 ± 16.29
10.71 ± 2.36


Sense
1.25
70.57 ± 12.77
 43.29 ± 10.21


oligonucleotide
2.5
71.00 ± 14.22
39.29 ± 7.30


(control
5
60.80 ± 15.50
28.57 ± 6.85


oligonucleotide)









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, for example, TNFRI (mean±SD) per 1×106 cells correlates with less than 50% inhibition of hPBMC proliferation (Table 6, FIG. 1). Thus, the present invention illustrates that those non-potent MSCs of a selected MSC population of at least one embodiment of the present invention are those cells expressing less than 13 pg TNFRI per 1×106 cells.


EXAMPLE 2
TNFRI is a Temperature-Sensitive Marker of hMSC Functionality

Ex vivo handling of mammalian cells is restricted by a number of factors including temperature. For example, low temperatures such as −80±5° C., or lower, even as low as −135° C. or below (liquid nitrogen) are required for cell storage whereas ex vivo cell expansion requires a temperature of 37±0.5° 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.


As discussed herein, it is believed that the expression level of TNFRI on some hMSCs of the selected population of the present invention correlates with hMSC immunosuppressive activity. For example, the level of TNFRI expression by some of those hMSCs of less than 13 pg/106 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

The objective of these experiments was to investigate the ability of the selected hMSCs to retain their functional characteristics after an exposure to temperatures above −80° 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° C. After several weeks, bags of frozen cells were removed from the −80±5° C. freezer and placed at either −70±5° C., −60±5° C., or −50±5° C. for 72±2 hours. After 72±2 hours, the bags were returned to storage at −80±5° C. for at least 24 hours before thaw and analysis. A set of bags moved from one −80±5° 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° C. or −50±5° C. decreases the TNFRI expression level: the level of TNFRI detected by ELISA was below the determined hMSC potency threshold of 13 pg/106 cells (represented by the solid line on the graph).


Parallel with TNFRI measurement, two bags with hMSCs stored at −80±5° C. (optimal storage temperature served as a control) and at −50±5° C. (corresponding to a ±30° C. greater than the −80±5° 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° C. lost their ability to suppress hPBMC proliferation, whereas cells stored at −80±5° C. inhibited hPBMC proliferation by 92%, but not up to 100% (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: for example, some cells expressing more than 13 pg/106 cells of TNFRI, which was determined as an MSC immunosuppressive potential threshold, are biologically active, while others cells with the TNFRI level below 13 pg/106 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

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 ≤−135° 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° C.-24° 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 those 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/106 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).


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.


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.


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.

Claims
  • 1. A method of treating a disease or disorder involving an activated immunological response in a human subject comprising, (i) determining the amount of cellular membrane-bound TNF-α receptor Type I expressed by at least one population of human mesenchymal stem cells;(ii) selecting a population of human mesenchymal stem cells that express cellular membrane-bound TNF-α receptor Type I in an amount of at least 27.7 pg/106 cells; and(iii) administering to the subject the selected population of mesenchymal stem cells.
  • 2. The method of claim 1, wherein the population of mesenchymal stem cells express cellular membrane-bound TNF-α receptor Type I in an amount of at least 37.5 pg/106 cells.
  • 3. The method of claim 1, wherein the population of mesenchymal stem cells express cellular membrane-bound TNF-α receptor Type I in an amount of at least 48.5 pg/106 cells.
  • 4. The method of claim 1, wherein the activated immunological response is associated with an autoimmune disease.
  • 5. The method of claim 4, where the autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, Type I diabetes, Crohn's disease, Guillain-Barré 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.
  • 6. The method of claim 1, wherein the activated immunological response is associated with graft versus host disease.
  • 7. The method of claim 1, wherein the cellular membrane-bound TNF-α receptor Type I expression is determined using an enzyme-linked immunosorbent assay.
  • 8. The method of claim 1, wherein the population of human mesenchymal stem cells was obtained from a human donor.
  • 9. The method of claim 1, wherein the selected population of human mesenchymal stem cells is administered by intravenous, intraarterial, or intraperitoneal administration.
  • 10. The method of claim 1, wherein the selected population of human mesenchymal stem cells has been frozen at −80° C. or below before administering to the subject.
  • 11. The method of claim 1, wherein the population of human mesenchymal stem cells was obtained from a sample selected from the group consisting of adherent marrow, periosteal cells, blood, skin, cord blood, muscle, fat, bone, and perichondrium.
  • 12. The method of claim 1, wherein the administering of the selected population of human mesenchymal stem cells to the human subject suppresses peripheral blood mononuclear cell (PBMC) proliferation.
  • 13. The method of claim 12, wherein the PBMCs are CD3/CD28-induced PBMCs.
RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/693,754, filed on Sep. 1, 2017, which is a continuation of U.S. patent application Ser. No. 14/228,327, filed on Mar. 28, 2014, which is a continuation of U.S. patent application Ser. No. 13/906,592, filed on May 31, 2013, which is a divisional of U.S. patent application Ser. No. 13/402,498, filed on Feb. 22, 2012, now U.S. Pat. No. 8,486,695, which is a continuation of U.S. patent application Ser. No. 12/091,391, filed on Sep. 15, 2008, which is a National Stage Entry of international patent application number PCT/US07/00274, filed on Jan. 5, 2007, which claims priority to U.S. Provisional Patent Application Ser. No. 60/759,157, filed on Jan. 13, 2006; the contents of all of which are hereby incorporated by reference in their entireties.

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Related Publications (1)
Number Date Country
20210171913 A1 Jun 2021 US
Provisional Applications (1)
Number Date Country
60759157 Jan 2006 US
Divisions (1)
Number Date Country
Parent 13402498 Feb 2012 US
Child 13906592 US
Continuations (4)
Number Date Country
Parent 15693754 Sep 2017 US
Child 17087124 US
Parent 14228327 Mar 2014 US
Child 15693754 US
Parent 13906592 May 2013 US
Child 14228327 US
Parent 12091391 US
Child 13402498 US