Patent Publication Number: US-2021171913-A1

Title: Mesenchymal Stem Cells Expressing TNF-alpha Receptors

Description:
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, now expired; the contents of all of which are hereby incorporated by reference in their entireties. 
    
    
     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/10 6  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. Neural.,  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.,    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 (PGE 2 ) 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/10 6  cells. In another embodiment, the mesenchymal stem cells express TNFRI in an amount, for example, of at least 15 pg/10 6  cells. In yet another embodiment, the mesenchymal stem cells express TNFRI in an amount of at least 18 pg/10 6  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/10 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&#39;s disease, Guillain-Barré syndrome, lupus erythematosus, myasthenia gravis, optic neuritis, psoriasis, Graves&#39; disease, Hashimoto&#39;s disease, Ord&#39;s thyroiditis, aplastic anemia, Reiter&#39;s syndrome, autoimmune hepatitis, primary biliary cirrhosis, antiphospholipid antibody syndrome, opsoclonus myoclonus syndrome, temporal arteritis, acute disseminated encephalomyelitis, Goodpasture&#39;s syndrome, Wegener&#39;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/10 6  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/10 6  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 2.5×10 5  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&#39;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&amp;D Systems, product insert). TNFRI expression was expressed in pg of receptor per 1×10 6  cells. 
     For the ELISA assay. 2.5×10 5  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&amp;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 1×10 6  cells. The raw data (in pg/ml) reflect TNFRI in picograms per 1×10 6  cells (2.5×10 5  cells were lysed in 250 μl of the lysis reagent, thus corresponding to 1×10 6  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 1×10 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 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 × 10 6   
                 % 
               
               
                 # 
                 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 1×10 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 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 × 10 6  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 × 10 6  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&#39;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 1×10 5  cells/well together with hMSCs (1×10 4  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% CO 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- 3 H]-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- 3 H]-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 
                 10 6  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 1×10 6  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 1×10 6  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/10 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 
     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/10 6  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/10 6  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/10 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 
     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/10 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). 
     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.