Potency assay

Abstract

The present disclosure relates to potency assays for cell therapy products. To provide a potency assay of a cell population comprising mesenchymal progenitor or stem cells.SOLUTION: The present invention relates to a method of measuring the biological activity or therapeutic efficacy of cultured mesenchymal lineage precursor or stem cells based on the levels of TGF-9 they release during culture. The invention also relates to an isolated population of mesenchymal lineage precursor or stem cells selected on the basis of the level of TGF-9 levels that the cells release in culture. The invention further relates to treating a subject suffering from degenerative disc disease by administering a cell population so selected.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to assays for cell therapy products. Assays for cell populations containing mesenchymal progenitor or stem cells are provided. [Background technology]

[0002] Several cell therapy products for regenerative or immunotherapy have progressed to the stage of clinical evaluation and marketing approval, but their commercialization is hindered by their complexity and heterogeneity, making it difficult to identify relevant biological activities and, therefore, to define consistent qualities of cell therapy products.

[0003] Physicochemical parameters (e.g., size characterization, morphology, light scattering properties, tensile strength, cell number, confluence, identification of phenotypic markers, secreted substances, genotype, gene expression profile) are routinely used to identify and quantify active substances, intermediates, impurities, and contaminants. However, physicochemical parameters cannot confirm whether a product is biologically active and efficacious (i.e., induces the desired effect). In contrast, biological characterization considers the effect of a product on a biological system, whether in vitro models or in vivo models in animals and ultimately in the clinic.

[0004] US and European pharmaceutical regulations require that active substances whose molecular structure cannot be fully defined be evaluated for potency before they are marketed. Potency evaluation of each batch of licensed cell therapy products is a legal requirement.

[0005] Potency testing should demonstrate one or more relevant biological activities of a product. Potency testing is not required to reflect all biological functions of the product, but should demonstrate one or more relevant biological functions. It is desirable to establish the precision, sensitivity, specificity, and reproducibility of analytical methods used in potency testing, and that they are suitably robust.

[0006] There is a need to identify parameters critical to the efficacy of cell therapy products and control them (e.g., via potency testing) to ensure consistent quality of the product. Summary of the Invention

[0007] The present inventors have developed an assay to measure the biological activity or therapeutic efficacy of cell therapy products containing mesenchymal progenitor cells or mesenchymal stem cells (hereinafter "mesenchymal progenitor or stem cells").

[0008] Thus, the present disclosure: (i) obtaining a population containing mesenchymal progenitor or stem cells; (ii) culturing the cells in a medium; and (iii) measuring the amount of TGFβ1 released by the cells into the medium, wherein the amount is at least about 2800 pg / 10 6 The amount of TGFβ1 in the cells provides a method for measuring the potency of mesenchymal progenitor or stem cells, including indicative of biological activity or therapeutic efficacy. For example, a TGFβ1 level of at least about 2810 pg / 10 6 Cellular TGFβ1, at least approximately 2820 pg / 10 6 Cellular TGFβ1, at least about 2830 pg / 10 6 Cellular TGFβ1, at least about 2840 pg / 10 6 Cellular TGFβ1, at least about 2850 pg / 10 6 Cellular TGFβ1, at least about 2860 pg / 10 6 Cellular TGFβ1, at least about 2870 pg / 10 6 Cellular TGFβ1, at least about 2880 pg / 10 6 Cellular TGFβ1, at least about 2890 pg / 10 6 Cellular TGFβ1, at least about 2900 pg / 10 6 Cellular TGFβ1, at least about 2910 pg / 10 6 Cellular TGFβ1, at least about 2920 pg / 10 6 Cellular TGFβ1, at least about 2930 pg / 10 6 Cellular TGFβ1, at least about 2940 pg / 106 Cellular TGFβ1, at least about 2950 pg / 10 6 Cellular TGFβ1, at least about 2960 pg / 10 6 Cellular TGFβ1, at least about 2970 pg / 10 6 Cellular TGFβ1, at least about 2980 pg / 10 6 Cellular TGFβ1, at least about 2990 pg / 10 6 cellular TGFβ1, or at least about 3000 pg / 10 6 The amount of TGFβ1 in the cells is indicative of biological activity or therapeutic efficacy.

[0009] The present disclosure provides: (i) obtaining a population containing mesenchymal progenitor or stem cells; (ii) culturing the cells in a medium; and (iii) measuring the amount of TGFβ1 released by the cells into the medium, wherein an amount of at least about 400 pg TGFβ1 per ml of medium indicates biological activity or therapeutic efficacy. For example, at least about 405 pg TGFβ1 per ml of medium, at least about 410 pg TGFβ1 per ml of medium, at least about 415 pg TGFβ1 per ml of medium, at least about 420 pg TGFβ1 per ml of medium, at least about 425 pg TGFβ1 per ml of medium, at least about 430 pg TGFβ1 per ml of medium, at least about 435 pg TGFβ1 per ml of medium, at least about 440 pg TGFβ1 per ml of medium, at least about 445 pg TGFβ1 per ml of medium, at least about 450 pg TGFβ1 per ml of medium, at least about 460 pg TGFβ1 per ml of medium, at least about 470 pg TGFβ1 per ml of medium, at least about 480 pg TGFβ1 per ml of medium, at least about 490 pg TGFβ1 per ml of medium, at least about 500 pg TGFβ1 per ml of medium, at least about 510 pg TGFβ1 per ml of medium, at least about 520 pg TGFβ1 per ml of medium, at least about 530 pg TGFβ1 per ml of medium, at least about 540 pg TGFβ1 per ml of medium, at least about 550 pg TGFβ1 per ml of medium, at least about 560 pg TGFβ1 per ml of medium, at least about 570 pg TGFβ1 per ml of medium, at least about 580 pg TGFβ1 per ml of medium, at least about 590 pg TGFβ1 per ml of medium, at least about 600 pg TGFβ1 per ml of medium, at least about 610 pg TGF Amounts of about 455 pg TGFβ1 per ml, at least about 460 pg TGFβ1 per ml of medium, at least about 465 pg TGFβ1 per ml of medium, at least about 470 pg TGFβ1 per ml of medium, at least about 475 pg TGFβ1 per ml of medium, at least about 480 pg TGFβ1 per ml of medium, at least about 485 pg TGFβ1 per ml of medium, at least about 490 pg TGFβ1 per ml of medium, at least about 495 pg TGFβ1 per ml of medium, or at least about 500 pg TGFβ1 per ml of medium demonstrate biological activity or therapeutic efficacy.

[0010] In one embodiment, the biological activity of the cells comprises the ability to stimulate collagen production in human annulus fibrosus cells in vitro.

[0011] In one embodiment, the therapeutic benefit includes therapeutic benefit in treating degenerative disc disease.

[0012] In one embodiment, the method is used to measure the potency of mesenchymal progenitor or stem cells that have been previously expanded in culture. In an alternative embodiment, the method is used to measure the potency of freshly isolated mesenchymal progenitor or stem cells.

[0013] In one embodiment, the population is enriched for mesenchymal progenitor or stem cells.

[0014] In one embodiment, the method further comprises enriching the mesenchymal progenitor or stem cells to obtain an enriched population, for example, the mesenchymal progenitor or stem cells are enriched by selecting for STRO-1+ cells and / or tissue-nonspecific alkaline phosphatase (TNAP)+ cells.

[0015] In one embodiment, the mesenchymal precursor or stem cells are human mesenchymal precursor or stem cells.

[0016] In one embodiment, the method comprises culturing a culture vessel at a density of about 50,000 viable cells / cm. 2 This involves seeding the cells with

[0017] In one embodiment, the method comprises culturing the cells in chondrogenic basal medium supplemented with 0.5% bovine serum albumin.

[0018] In one embodiment, the method comprises culturing the adherent cells for at least 68-76 hours. In one embodiment, the adherent cells are obtained by first culturing a population of cells overnight in chondrogenic basal medium supplemented with 0.5% bovine serum albumin to allow them to adhere to the culture vessel.

[0019] In one embodiment, the method comprises taking a sample of the medium in which the cells were cultured, hi one embodiment, the sample taken comprises all of the medium in which the cells were cultured.

[0020] In one embodiment, the method comprises first activating latent TGFβ1 in the culture medium and then measuring the amount of TGFβ1 in the culture medium.

[0021] In one embodiment, activating latent TGFβ1 comprises adding an acid, e.g., 1N HCl, to the medium to lower the pH of the medium. In one embodiment, the method comprises concentrating the medium sample before lowering the pH. In one embodiment, the method comprises neutralizing the pH of the medium to 7.2-7.6 after adding the acid, e.g., by adding 1.2N NaOH / 0.5M HEPES or 1N NaOH.

[0022] In one embodiment, the method comprises measuring the amount of TGFβ1 in the culture medium by enzyme-linked immunosorbent assay (ELISA).

[0023] In one example, ELISA is (i) diluting the medium 1:5 with sample diluent; (ii) adding the diluted medium to wells of a microplate precoated with a monoclonal antibody specific for TGFβ1; (iii) adding sample dilutions to each well of the microplate; (iv) incubating the microplate at room temperature for 2 hours; (v) washing the microplate; (vi) adding TGFβ1 complex to the well; (vii) incubating the microplate at room temperature for 2 hours; (viii) washing the microplate; (ix) adding a substrate solution to the wells; (x) incubating the microplate at room temperature for 30 minutes; (xi) adding a stop solution to the wells; (xii) reading the optical density on a microplate reader set at 450 nm with wavelength correction at 570 nm; (xiii) Measuring the concentration of TGFβ1 corrected for dilution.

[0024] In one embodiment, the sample diluent is chondrogenic basal medium supplemented with 0.5% bovine serum albumin.

[0025] In one embodiment, the method further comprises: Prepare serial dilutions of TGFβ1 standard solution with sample diluent to final concentrations ranging from 31.2 to 2000 pg / ml; adding a standard solution to the microplate before step (iii); Constructing a standard curve using four-parameter logistic curve fitting, and This involves measuring the concentration of TGFβ1 in the medium against a standard curve.

[0026] The present disclosure also provides a method for measuring the potency of mesenchymal precursor cells, comprising: (i) obtaining a population of mesenchymal progenitor cells; (ii) 50,000 viable cells / cm in a culture vessel. 2 seeding the cells with (iii) culturing the cells in chondrogenic basal medium supplemented with 0.5% bovine serum albumin; (iv) harvesting the medium; (v) activating latent TGFβ1 released by the cells into the medium by adding 1N HCl to lower the pH of the medium; (vi) neutralizing the pH of the medium to 7.2-7.6 by adding 1.2 N NaOH / 0.5 M HEPES or 1 N NaOH; (vii) diluting the medium 1:5 with chondrogenic basal medium supplemented with 0.5% bovine serum albumin; (viii) adding the diluted medium to wells of a microplate precoated with a monoclonal antibody specific for TGFβ1; (ix) adding sample dilutions to each well of the microplate; (x) incubating the microplate at room temperature for 2 hours; (xi) washing the microplate; (xii) adding a TGFβ1 complex to the well; (xiii) incubating the microplate at room temperature for 2 hours; (xiv) washing the microplate; (xv) adding a substrate solution to the wells; (xvi) incubating the microplate at room temperature for 30 minutes; (xvii) adding a stop solution to the wells; (xviii) reading the optical density on a microplate reader set at 450 nm with wavelength correction at 570 nm; (xix) Measuring the concentration of TGFβ1 corrected for dilution.

[0027] In one embodiment, the method further comprises: Serial dilutions of TGFβ1 standard solution with final concentrations ranging from 31.2 to 2000 pg / ml were prepared in chondrogenic basal medium supplemented with 0.5% bovine serum albumin; adding a standard solution to the microplate before step (ix); Constructing a standard curve using four-parameter logistic curve fitting, and This involves measuring the concentration of TGFβ1 in the medium against a standard curve.

[0028] The present disclosure also provides a population of cells comprising mesenchymal progenitor or stem cells selected for use in therapy, wherein such cell population has a cell count of 2800 pg / 10 when assessed by the disclosed method. 6 Releases TGFβ1 from cells.

[0029] The present disclosure also provides an isolated cell population comprising mesenchymal progenitor or stem cells selected for use in therapy, wherein such cell population releases 400 pg of TGFβ1 per ml of medium when assessed by the disclosed method.

[0030] The present disclosure also provides isolated cell populations comprising mesenchymal progenitor or stem cells, wherein such cell populations have been selected for therapeutic use by measuring TGFβ1 release under culture conditions.

[0031] In one embodiment, the isolated cell population comprises mesenchymal progenitor or stem cells that have been expanded in culture. In an alternative embodiment, the isolated cell population comprises freshly isolated mesenchymal progenitor or stem cells. In one embodiment, the isolated cell population comprises mesenchymal progenitor or stem cells that have been assayed to measure TGFβ1 release under culture conditions. In another embodiment, the isolated cell population comprises mesenchymal progenitor or stem cells derived from a population that has been sampled to measure TGFβ1 release under culture conditions (i.e., the cells of the isolated population have not themselves been assayed to measure TGFβ1 release under culture conditions).

[0032] In one embodiment, the mesenchymal progenitor or stem cells comprise at least 5% of the isolated cell population.

[0033] In one embodiment, a composition is provided comprising one of the above-described isolated cell populations and a cryoprotectant. In one embodiment, the cryoprotectant in the composition is DMSO or Profreeze™. In one embodiment, the composition comprises the isolated cell population dissolved in 42.5% (v / v) Profreeze™ / 50% αMEM (v / v) / 7.5% (v / v) DMSO.

[0034] In one embodiment, the present disclosure provides a composition comprising one of the above-described isolated cell populations and hyaluronan, e.g., at least about 0.5% HA or an HA salt, at least about 0.6% HA or an HA salt, at least about 0.7% HA or an HA salt, at least about 0.8% HA or an HA salt, at least about 0.9% HA or an HA salt, at least about 1% HA or an HA salt, at least about 1.5% HA or an HA salt, at least about 2% HA or an HA salt, at least about 2.5% HA or an HA salt, at least about 3% HA or an HA salt, at least about 3.5% HA or an HA salt, at least about 4% HA or an HA salt, at least about 4.5% HA or an HA salt, at least about 5% HA or an HA salt, at least about 6% HA or an HA salt, at least about 7% HA or an HA salt, at least about 8% HA or an HA salt, at least about 9% HA or an HA salt, or at least about 10% HA or an HA salt.

[0035] The present disclosure also provides a method of treating a subject suffering from degenerative disc disease, the method comprising administering to the subject a disclosed composition.

[0036] In one embodiment, the cryopreserved disclosed compositions are thawed and mixed with hyaluronan (HA) or an HA salt, such as sodium HA, prior to administration.

[0037] The present disclosure also provides a method of treating a subject suffering from degenerative disc disease, the method comprising administering to the subject a medium comprising at least about 400 pg of TGFβ1 per ml of medium. [Brief explanation of the drawings]

[0038] [Figure 1] Figure 1A: Representative diagram showing the location of the intervertebral disc (IVD) between two vertebral bodies. Figure 1B: A healthy disc showing the location and structure of the IVD, with the nucleus pulposus (NP) in the center surrounded by the annulus fibrosus (AF) and the vertebral endplates (Figure credit (Raj, 2008)). [Figure 2]Figure 2 shows the effect of MPC CM on human NPC proliferation and matrix composition in micromass cultures. Figure 2A: Data showing EdU incorporation during nucleus pulposus cell (NPC) proliferation in response to mesenchymal progenitor cell (MPC)-conditioned medium (CM). Data are presented as mean ± SD for percent positive EdU incorporation. n = 3 replicates per condition. Figure 2B: Representative images of Alcian blue staining for sulfated GAG proteoglycans in human NPC micromass cultures. Figure 2C: Semiquantification of proteoglycans extracted from NP micromass cultures. Data are presented as mean ± SD. n = 3 replicates per condition. Significance level (p ≤ 0.05) compared to control basal medium. [Figure 3] In vitro effects of MPC CM on proliferation and matrix production of human AFCs in micromass cultures. Figure 3A: Data showing proliferation of annulus fibrosus cells (AFCs) in response to MPC CM. Data are presented as the mean ± SD of the percent positive for EDU incorporation. n = 3 replicates per condition. Figure 3B: Quantification of total collagen produced in AF micromass cultures in response to MPC CM. Representative data from one donor are presented as the mean ± SD. n = 3 replicates per condition. Significance level (p ≤ 0.05) compared to control basal medium. [Figure 4] Levels of TGFβ1 detected in MPC CM. Detection of TGFβ1 in MPC CM measured by ELISA. Data are presented as the mean of duplicate samples. MPC lots derived from 5 different donors, n=15. [Figure 5]Effect of TGFβ1 and MPC CM on hydroxyproline content in human AFC micromass cultures. Figure 5A: Dose response for recombinant human TGFβ1 (rhTGFβ1)-induced collagen production in AF micromass cultures from three AFC donors. Data show significant rhTGFβ1 inhibition in response to anti-TGFβ1 neutralizing antibody. Figure 5B: Quantification of total collagen produced by AF micromass cultures in response to MPC CM after receiving anti-TGFβ1 neutralization or IgG control. Data are from three AFC donors and four to seven lots of MPC CM. Data are presented as mean ± SD. n = 3 replicates per condition. Significance level (p ≤ 0.05) with *comparison with control basal medium; ‡IgG control. [Figure 6] Effect of rhTGFβ1 on hydroxyproline content in fetal and adult human AFC micromass cultures. Collagen production in response to rhTGFβ1 by fetal and adult AFC micromass cultures is shown. Each data point represents three fetal or adult AFC donors. Data are presented as mean ± SD. n = 6–9 replicates per condition. [Figure 7] Effect of TGFβ and MPC CM on hydroxyproline content in adult human AFC micromass cultures. Figure 7A: Collagen production by adult AFC micromass cultures obtained from three AFC donors in response to rhTGFβ1 or MPC CM. Data are presented as mean ± SD. n = 3-8 replicates per condition. Figure 7B: Collagen production by adult AFC micromass cultures obtained from three AFC donors in response to rhTGFβ1 or MPC CM. Data are presented as mean ± SD. n = 3-8 replicates per condition. Figure 7C: Collagen production by adult AFC micromass cultures obtained from three AFC donors in response to rhTGFβ1 or MPC CM. Data are presented as mean ± SD. n = 3-8 replicates per condition. Figure 7D: Mean hydroxyproline content of three adult AFC lots. Significance level (p≦0.05) vs. control basal medium (0 ng / ml rhTGFβ1). [Figure 8]TGFβ1 levels in CM from MPCs grown in different basal media. Detection of TGFβ1 in MPC CM after expansion by ELISA, comparing optimal and suboptimal media compositions for CM generation. Dark bars = chondrogenic basal medium + 0.5% bovine serum albumin (CBM + 0.5% BSA). Light bars = EBM-2 + 0.5% BSA. Data are presented as the average of duplicate samples. All MPC lots were derived from a single donor. [Figure 9] Levels of TGFβ1 detected in MPC CM. Detection of TGFβ1 required acid treatment of the samples, so measurements reflect total TGFβ1 in the CM. Data are presented as mean ± SD. MPC lots derived from four different donors, n=18. [Figure 10] Effect of TGFβ1 and MPC CM on matrix composition of fetal human AFC (lot 4729) in micromass culture. Figure 10A: MPC products from four lots were transfected with siRNA targeting TGFβ1 or a scrambled negative control. TGFβ1 levels in CM generated from transfected cells are shown. Data indicate that the biological activity of CM containing TGFβ1 siRNA is significantly inhibited. Data are presented as mean ± SD. n = 18 normal CM (3-8 replicates / CM) and four scrambled MPC or TGFβ1 siRNA-transfected MPC (triplicates / condition). Significance level p < 0.05 vs. unstimulated basal control (*) or scrambled control (+). Figure 10B: Mean collagen production by fetal AFC (lot 4729) in response to CM derived from untreated MPC or siRNA-transfected MPC. Data indicate that the biological activity of CM containing TGFβ1 siRNA is significantly inhibited. Data are presented as mean ± SD. n = 18 normal CMs (3–8 replicates / CM) and 4 scrambled MPCs or TGFβ1 siRNA-transfected MPCs (triplicates / condition). Significance level: p ≤ 0.05 vs. unstimulated basal control (*) or scrambled control (+). [Figure 11]Regression analysis of TGFβ1 levels on collagen production comparing MPC CM and AFC. [Figure 12] TGF-β1 secretion by two MPC lots as a function of initial cell seeding density, time, and operator. Figure 12A: Time course analysis of cells seeded at 25,000 cells / cm2. Figure 12B: Time course analysis of cells seeded at 50,000 cells / cm2. [Figure 13A] Linearity of the TGFβ1 standard curve using standard curves from three lots of ELISA kits. Standard curve generated with calibrator dilutions. [Figure 13B] Linearity of TGFβ1 standard curve using standard curves from three lots of ELISA kits. Standard curve generated in CBM + 0.5% BSA. [Figure 14] Standard curves for the TGFβ1 ELISA comparing calibrator diluents with chondrogenic basal medium (CBM) + 0.5% BSA. Matrix effects were assessed by comparing standard curves generated with calibrator diluents and CBM + 0.5% BSA (analyzed in parallel on the same plate). Each standard curve was represented in duplicate, and two independent experiments were performed. The OD for the standard curve generated with CBM was slightly higher than that for the standard curve generated with calibrator diluents, indicating a matrix effect. DETAILED DESCRIPTION OF THE INVENTION

[0039] General Techniques and Definitions Throughout this specification, unless specifically stated otherwise or the context dictates otherwise, references to a single step, composition, group of steps or compositions should be construed to encompass both one and more than one (i.e., one or more) such step, composition, group of steps or compositions.

[0040] Those skilled in the art will understand that the disclosure described herein is susceptible to changes and modifications other than those specifically described. The present disclosure is to be understood to include all such changes and modifications. The present disclosure also includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0041] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended as merely illustrative. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0042] Any example in this specification should be construed as applying mutatis mutandis to any other example, unless specifically stated otherwise.

[0043] Unless otherwise defined, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the fields of cell culture, molecular genetics, stem cell differentiation, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0044] Unless otherwise specified, the stem cell, cell culture, and surgical techniques utilized in this disclosure are standard procedures well known to those skilled in the art. Such techniques are fully described and explained in the original literature, such as (Perbal, 1984), (Sambrook & Green, 2012), (Brown, 1991), (Glover & Hames, 1995, 1996), (Ausubel FM, 1987, including all updates to date), (Harlow & Lane, 1988), and (Coligan, Kruisbeek, Margulies, Shevach, & Strober, 1991, including all updates to date).

[0045] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be understood to clearly support both meanings or either meaning.

[0046] As used herein, the term about, unless otherwise specified, refers to + / - 10%, more preferably + / - 5% of the specified value.

[0047] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0048] Mesenchymal progenitor cells As used herein, the term "mesenchymal progenitor or stem cell" refers to an undifferentiated multipotent cell that has the ability to self-renew while maintaining pluripotency and to differentiate into multiple cell types, such as cell types derived from the mesenchyme, e.g., osteoclasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons, or cell types not derived from mesodermal tissue, e.g., hepatocytes, neurons, and epithelial cells.

[0049] The term "mesenchymal precursor or stem cell" includes both the parent cell and its undifferentiated progeny. This term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells, perivascular mesenchymal precursor cells, and their undifferentiated progeny.

[0050] Mesenchymal progenitor or stem cells can be autologous, xenogeneic, syngeneic, or isogenic. Autologous cells are isolated from the same individual into whom the cells are to be transplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0051] Mesenchymal progenitor or stem cells reside primarily in bone marrow, but have also been found to reside in a variety of host tissues, including umbilical cord blood and cord, adult peripheral blood, adipose tissue, cancellous bone, and dental pulp.

[0052] Mesenchymal progenitor or stem cells can be isolated from host tissue and enriched by selecting STRO-1+ cells.For example, bone marrow aspirate obtained from a subject can be further treated with antibodies against STRO-1 or TNAP to allow for the selection of mesenchymal progenitor or stem cells.In one example, mesenchymal progenitor or stem cells can be enriched using STRO-1 antibodies as described in (Simmons & Torok-Storb, 1991).

[0053] STRO-1+ cells are found in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph nodes, thymus, bone, ligaments, tendons, skeletal muscle, dermis, and periosteum, and can differentiate into germ cells such as mesoderm and / or endoderm and / or ectoderm. Thus, STRO-1+ cells can differentiate into numerous cell types, including, but not limited to, adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue, and fibrous connective tissue. The specific lineage commitment and differentiation pathways these cells enter depend on a variety of influences, including mechanical influences and / or endogenous bioactive factors, such as growth factors and cytokines, and / or local microenvironmental conditions established by the host tissue. As used herein, the term "enriched" refers to a cell population in which the proportion of a particular cell type or the proportion of multiple particular cell types is enhanced compared to an untreated cell population (e.g., its native environment). In one example, a population enriched for STRO-1+ cells comprises at least about 0.1%, or 0.5%, or 1%, or 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 50%, or 75% STRO-1+ cells. In this regard, the term "cell population enriched for STRO-1+ cells" will be construed to expressly support the term "cell population comprising X% STRO-1+ cells," where X% is a percentage recited herein. In some examples, STRO-1+ cells are capable of forming clonogenic colonies, for example, CFU-F (fibroblasts) or a subset thereof (e.g., 50%, 60%, 70%, 70%, 90%, or 95%) may have this activity.

[0054] In one example, the cell population is enriched with a cell preparation comprising STRO-1+ cells in a selectable form. In this regard, the term "selectable form" will be understood to mean that the cells express a marker (e.g., a cell surface marker) that allows for the selection of STRO-1+ cells. The marker can be, but need not be, STRO-1. For example, as described and / or exemplified herein, cells (e.g., MPCs) expressing STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 are enriched for STRO-1 (also referred to as STRO-1 bright (which may be TNAP+). Thus, when cells are designated as STRO-1+, it does not mean that the cells were selected by STRO-1 expression. In one example, the cells are selected based on at least STRO-3 expression, e.g., the cells are STRO-3+ (TNAP+).

[0055] The selection criteria for the cells or populations thereof do not necessarily require selection from a particular tissue source. The STRO-1+ cells described herein can be selected or isolated or enriched from a wide variety of sources. However, in some instances, these terms support selection from any tissue containing STRO-1+ cells, or vascularized tissue, or tissue containing pericytes (e.g., STRO-1+ pericytes), or any one or more of the tissues recited herein.

[0056] In one example, the disclosed mesenchymal progenitor or stem cells express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G5+.

[0057] By the term "individually and separately," it is meant that the disclosure encompasses the markers or groups of markers described separately, and that even if each marker or group of markers is not separately listed herein, the appended claims may define such markers or groups of markers as separate and separable from one another.

[0058] By the term "collectively," it is meant that the disclosure encompasses any number or combination of the described markers or peptides, and that even if such number or combination of markers or markers is not specifically recited herein, such combination or subcombination may be defined separately and separably from any other combination of markers or markers according to the appended claims.

[0059] In one example, STRO-1+ cells are STRO-1 bright (syn.STRO-1 bri ) In one example, STRO-1 bri STRO-1 cells dim or STRO-1 intermediate Selectively enriched for cells.

[0060] In one example, STRO-1 bright The cells may further be one or more of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), and / or CD146+. For example, the cells may be selected for and / or exhibit expression of one or more of the above-mentioned markers. In this regard, cells that exhibit expression of a marker do not necessarily need to be tested in detail; rather, they may be used after testing previously enriched or isolated cells, and the isolated or enriched cells may naturally also be considered to express the same markers.

[0061] In one example, STRO-1 bright The cells are perivascular mesenchymal progenitor cells as defined in WO2004 / 85630 and are characterized by the presence of the perivascular marker 3G5.

[0062] Cells that are "positive" for a given marker can express that marker at low (lo or dim) or high (bright, bri) levels, depending on how present the marker is on the cell surface; each term refers to the fluorescence intensity or other marker used in the cell sorting process. The difference between lo (or dim or dull) and bri will be understood in the context of the marker used in the particular cell population being sorted. Cells that are "negative" for a given marker are not necessarily completely absent from the cell. This term means that the cell expresses the marker at a relatively low level and that, when potentially labeled, the marker gives off a very low signal or is undetectable above background levels, e.g., levels detected using an isotype control antibody.

[0063] As used herein, the term "bright" or "bri" refers to a cell surface marker that, when utilizable, gives a relatively high signal. Without wishing to be bound by theory, it is proposed that "bright" cells express more of the target marker protein (e.g., the antigen recognized by the STRO-1 antibody) than other cells in the sample. For example, STRO-1 bri Cells were labeled with FITC-conjugated STRO-1 antibody, resulting in non-bright cells (STRO-1) as measured by fluorescence activated cell sorting (FACS) analysis. dull / dim) produce a stronger fluorescent signal than STRO-1. In one example, mesenchymal progenitor or stem cells are isolated from bone marrow and enriched by selection for STRO-1+ cells. In this example, the "bright" cells comprise at least about 0.1% of the most brightly labeled bone marrow mononuclear cells contained in the starting sample. In other examples, the "bright" cells comprise at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the most brightly labeled bone marrow mononuclear cells contained in the starting sample. In one example, the STRO-1 bright Cells have a 2-log increase in STRO-1 surface expression relative to "background" i.e., STRO-1- cells. dim and / or STRO-1 intermediate Cells have STRO-1 surface expression less than 2 logs above "background," typically about 1 log or less.

[0064] The term "TNAP" as used herein is intended to encompass all isoforms of tissue-nonspecific alkaline phosphatase. For example, this term encompasses the liver isoform (LAP), the bone isoform (BAP), and the kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP refers to a molecule capable of binding to the STRO-3 antibody produced by the hybridoma cell line deposited with the American Type Culture Collection (ATCC) on December 19, 2005 under the provisions of the Budapest Treaty, with deposit accession number PTA-7282.

[0065] Furthermore, in one example, STRO-1+ cells are capable of generating clonogenic CFU-F.

[0066] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two different germ cell types. Non-limiting examples of lineages into which cells can be committed include bone progenitor cells; hepatocyte progenitor cells, which are multipotent cells for bile duct epithelial cells and hepatocytes; neural-committed cells that can give rise to glial progenitor cells that develop into oligodendrocytes and astrocytes; neural progenitor cells that develop into neurons; cardiac muscle and cardiac muscle cell progenitor cells; and glucose-responsive insulin-secreting pancreatic beta cell lines. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as the following progenitor cells: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal duct epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendon, ligament, cartilage, adipocytes, fibroblasts, marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, vascular, epithelial, glial, neuronal, astrocyte, and oligodendrocyte cells.

[0067] In one example, the mesenchymal progenitor or stem cells are MSCs. The MSCs may be a homogenous composition or a mixed cell population enriched for MSCs. Homogeneous MSC compositions may be obtained by culturing adherent bone marrow or periosteal cells, and MSCs may be identified by specific cell surface markers identified with specific monoclonal antibodies. Methods for obtaining MSC-enriched cell populations are described, for example, in U.S. Patent No. 5,486,359. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium.

[0068] The isolated or enriched mesenchymal precursor or stem cells can be expanded in vitro by culture. As will be understood by those skilled in the art, the isolated or enriched mesenchymal precursor or stem cells can be cryopreserved, thawed, and then expanded in vitro by culture.

[0069] In one example, isolated, enriched, or cultured mesenchymal progenitor or stem cells are cultured in serum-supplemented medium, such as alpha minimum essential medium (αMEM) supplemented with 10% fetal bovine serum (FBS) and glutamine, at a density of 50,000 viable cells / cm. 2 Cells were seeded in a 100% O2 culture vessel and allowed to adhere overnight at 37°C and 20% O2. The medium was then replaced with chondrogenic basal medium (CBM; Lonza, Walkersville, MD) supplemented with 0.5% bovine serum albumin (BSA). The cells were then cultured for an additional 68–72 hours at 37°C and 5% O2 before measuring the amount of TGFβ1 released into the medium by the cells.

[0070] The cultured mesenchymal progenitor or stem cells are phenotypically distinct from the cells in vivo, for example, in one embodiment, the cells express one or more of the following markers: CD44, NG2, DC146, and CD140b.

[0071] Cultured mesenchymal progenitor or stem cells are biologically distinct from cells in vivo, and have a rapid rate of proliferation compared to mostly non-cycling (quiescent) cells in vivo.

[0072] The mesenchymal precursor or stem cells may be cryopreserved prior to administration to a subject.

[0073] Measurement of TGFβ1 levels The present disclosure contemplates all types of assays, including Western blot, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), competitive assay, radioimmunoassay, lateral flow immunoassay, flow-through immunoassay, electrochemiluminescence, nephelometric assay, turbidimetric assay, fluorescence activated cell sorting (FACS) assay for detecting TGFβ1 in medium used to culture mesenchymal or progenitor cells, and surface plasmon resonance (SPR by Biacore).

[0074] One suitable form of assay is, for example, an ELISA or a FLISA.

[0075] In one form, such an assay involves immobilizing a TGFβ1-binding protein on a solid matrix, such as a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g., a glass slide). The test sample is then directly contacted with the TGFβ1-binding protein, resulting in binding, or capture, of TGFβ1 in the sample. After washing to remove any unbound proteins in the sample, a protein that binds to TGFβ1 at a different epitope is directly contacted with the captured TGFβ1. This detector protein is typically labeled with a detectable reporter molecule, such as an enzyme (e.g., horseradish peroxidase (HRP)), alkaline phosphatase (AP), or β-galactosidase) in the case of ELISA, or a fluorophore in the case of FLISA. Alternatively, a second, labeled protein can be used that binds to the detector protein. After washing to remove any unbound protein, the detectable reporter molecule is detected by adding a substrate such as hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indole-beta-D-galactopyranoside (x-gal) in the case of ELISA. Of course, the immobilized (capture) protein and the detector protein may be reversed.

[0076] The level of antigen in the sample is then determined using a standard curve generated using known amounts of marker or by comparison with a control sample.

[0077] The various assays described above are easily modified to use chemiluminescence or electrochemiluminescence as the basis for detection.

[0078] Those skilled in the art will appreciate that other immunosorbent-based detection methods are also useful in the practice of the present disclosure, such as immunosorbent methods based thereon using detectable radioactive labels, or detectable gold labels (e.g., colloidal gold), or liposomes, e.g., encapsulating detectable NAD+, or acridinium-linked immunosorbent methods.

[0079] In some disclosed examples, TGFβ1 levels are measured using a surface plasmon resonance detector (e.g., BIAcore™, GE Healthcare, Piscataway, NJ), a flow-through device (e.g., as described in U.S. Pat. No. 7,205,159), a micro- or nano-immunoassay device (e.g., as described in U.S. Pat. No. 7,271,007), a lateral flow device (e.g., as described in U.S. Pat. No. 20040228761 or U.S. Pat. No. 20040265926), a fluorescence polarization immunoassay (FPIA, e.g., as described in U.S. Pat. No. 4,593,089 or U.S. Pat. No. 4,751,190), or an immunoturbidimetric assay (e.g., as described in U.S. Pat. No. 5,571,728 or U.S. Pat. No. 6,248,597).

[0080] Compositions and Administration Compositions comprising mesenchymal precursor or stem cells may be formulated in a pharmacologically acceptable carrier. As used herein, the term "pharmacologically acceptable carrier" refers to a composition that facilitates the storage, administration, and / or maintenance of the biological activity of mesenchymal precursor or stem cells.

[0081] In one example, the carrier does not cause significant local or systemic adverse effects in the recipient. A pharmacologically acceptable carrier may be solid or liquid. Useful examples of pharmacologically acceptable carriers include, but are not limited to, diluents, solvents, surfactants, additives, suspending agents, buffers, lubricants, adjuvants, vehicles, emulsifiers, absorbing agents, dispersion media, coating agents, stabilizers, protective colloids, adhesives, thickeners, thixotropic agents, penetrating agents, sequestering agents, scaffolds, and isotonic and absorption retarding agents that do not affect the viability and activity of mesenchymal progenitor cells or stem cells. The selection of a suitable carrier is within the skill of one of ordinary skill in the art.

[0082] Suitable pharmaceutical carriers include, but are not limited to, hyaluronan, chemically modified hyaluronan, saline, phosphate buffered saline, chondroitin sulfate, glucosamine, mannosamine, proteoglycans, proteoglycan fragments, chitin, chitosan, or other polysaccharide or polymeric substances.

[0083] Mesenchymal progenitor cells or stem cells can be incorporated or embedded within the scaffold. Suitable scaffolds include, but are not limited to, biologically degradable scaffolds. Natural biodegradable scaffolds include, but are not limited to, collagen, fibronectin, and laminin scaffolds. Biodegradable synthetic scaffolds include, but are not limited to, polyglycolic acid scaffolds (e.g., as described in (Vacanti, Morse, & Saltzman, 1988) (Cima, Ingber, Vacanti, & Langer, 1991) (Vacanti, Langer, Schloo, & Vacanti, 1991)), synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid, and resorbable gelatin sponges such as Gelform™ (Pfizer).

[0084] The disclosed compositions may be conveniently presented in unit dosage form and may be prepared by any method known in the art. As used herein, the term "dosage unit form" refers to a physically discrete unit suitable as a unitary dose for a subject to be treated, containing a predetermined amount of active compound calculated to achieve the desired therapeutic or prophylactic effect together with a pharmaceutical carrier. The dose of mesenchymal progenitor cells or stem cells may vary depending on factors such as the disease state, age, sex, and weight of the subject to be treated.

[0085] An exemplary dose is at least about 1 x 10 6 For example, the dose may be about 1.0 x 10 6 ~approx. 1x10 10 cells, e.g., about 1.1 x 10 6 ~approx. 1x10 9 Cells, e.g., about 1.2 x 10 6 ~Approx. 1×10 8 cells, e.g., about 1.3 x 10 6 ~Approx. 1×10 7 cells, e.g., about 1.4 x 10 6 ~Approx. 9×10 6 cells, e.g., about 1.5 x 10 6 ~Approx. 8×10 6 cells, e.g., about 1.6 x 10 6 ~Approx. 7×10 6 cells, e.g., about 1.7 x 10 6 ~about 6×10 6 Cells, e.g., about 1.8 x 10 6 ~Approx. 5×10 6 cells, e.g., about 1.9 x 10 6 ~Approx. 4×10 6 Cells, e.g., about 2 x 10 6 ~Approx. 3×10 6 It may contain cells.

[0086] In one example, the dose is about 5 x 10 5 ~2x10 7 Cells, e.g., about 6 x 10 6 Cells ~ approx. 1.8×10 7 The dose may be, for example, about 6 x 10 cells. 6 cells or approximately 1.8 x 10 7It may be a cell.

[0087] Mesenchymal progenitor or stem cells comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% of the cell population of the composition.

[0088] The disclosed compositions may be cryopreserved. Cryopreservation of mesenchymal progenitor or stem cells can be performed using slow-cooling or "rapid" freezing protocols known in the art. Preferably, the cryopreservation method maintains the phenotype, cell surface markers, and proliferation rate of the cryopreserved cells similarly compared to unfrozen cells.

[0089] The cryopreserved composition may comprise a cryopreservation solution, the pH of which is typically between 6.5 and 8, preferably 7.4.

[0090] Cryopreservation media may include a pyrogen-free, sterile, isotonic solution such as PlasmaLyte A™. 100 mL of PlasmaLyte A™ contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H 11 Contains 368 mg of sodium acetate trihydrate, USP (C2H3NaO2·3H2O); 37 mg of potassium chloride, USP (KCl); and 30 mg of magnesium chloride, USP (MgCl2·6H2O). Contains no antimicrobial agents. pH adjusted with sodium hydroxide. pH is 7.4 (6.5-8.0).

[0091] The cryopreservation medium may include Profreeze™. The cryopreservation medium may additionally or alternatively include culture medium, such as αMEM.

[0092] To facilitate freezing, cryoprotectants, such as dimethyl sulfoxide (DMSO), are typically added to cryopreservation solutions. Ideally, cryoprotectants should be non-toxic to cells and patients, non-antigenic, chemically inert, provide high post-thaw survival rates, and allow transplantation without washing. However, DMSO, the most commonly used cryoprotectant, exhibits some degree of cytotoxicity. To reduce the cytotoxicity of cryopreservation solutions, hydroxylethyl starch (HES) may be used as a substitute or in combination with DMSO.

[0093] The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein bulking agents. In one example, the cryopreservation solution includes about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further include one or more of methycellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0094] In one embodiment, cells are suspended in 42.5% Profreeze™ / 50% αMEM / 7.5% DMSO and cooled in a controlled-rate freezer.

[0095] The cryopreserved composition may be thawed and administered directly to a subject or added to another solution, such as a solution containing HA. Alternatively, the cryopreserved composition may be thawed and the mesenchymal progenitor or stem cells resuspended in an alternative carrier prior to administration.

[0096] The disclosed compositions can be administered by a route suitable for the particular disease to be treated. For example, the disclosed compositions can be administered systemically, i.e., parenterally, intravenously, or by injection. The disclosed compositions can be targeted to a specific tissue or organ.

[0097] The dosage regimen may be adjusted to obtain the optimal therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally increased or decreased according to the indications required by the therapeutic situation. For ease of administration and uniformity of dosage, it may be advantageous to formulate parenteral compositions in unit dosage form.

[0098] In some embodiments, it may not be necessary or desirable to immunosuppress a patient prior to initiating treatment with a cell composition. Indeed, transplantation of allogeneic STRO-1+ cells in sheep has been well tolerated without immunosuppression. However, in other cases, it may be desirable or appropriate to administer pharmacological immunosuppression to a patient prior to initiating cell therapy. Pharmacological immunosuppression may be achieved through the use of systemic or local immunosuppressants, or by cell delivery within an encapsulation device. The cells may be encapsulated within a capsule that is permeable to the nutrients and oxygen required by the cells and therapeutic factors, but is impermeable to immune humoral factors and cells. Preferably, the encapsulation medium is hypoallergenic, readily and stably located in the target tissue, and provides additional protection to the implanted structure. These and other means of reducing or eliminating immune responses to transplanted cells are known in the art. Alternatively, the cells may be genetically modified to reduce their immunogenicity.

[0099] It will be appreciated that the mesenchymal progenitor or stem cells may be administered with other beneficial drugs or biological molecules (growth factors, trophic factors). When administered with other agents, they may be administered together in a single pharmaceutical composition, or in separate pharmaceutical compositions administered simultaneously with the other agent or sequentially (before or after administration of the other agent). Bioactive factors that may be co-administered include anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors); anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST™, TRANILAST™, REMICADE™, SIROLIMUS™, and nonsteroidal anti-inflammatory drugs (NSAIDs), such as TEPOXALIN™, TOLMETIN™, and SUPROFEN™; immunosuppressants / immunomodulators (e.g., calcineurin inhibitors such as cyclosporine and tacrolimus); mTOR inhibitors (e.g., SIROLIMUS™, EVEROLIMUS™); antiproliferative agents (e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g., prednisolone, hydrocortisone); antibodies, such as anti-IL-2R alpha receptor monoclonal antibodies (e.g., antithrombotic agents (e.g., heparin, heparin derivatives, urokinase, PPack (dextrophenylalanine-proline-arginine-chloromethylketone), antithrombin compounds, platelet receptor antagonists, antithrombin antibodies, antiplatelet receptor antibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandin inhibitors, and platelet inhibitors); and antioxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine), and local anesthetics.

[0100] Treatment of degenerative disc disease The intervertebral disc (IVD) is a functional unit connecting the vertebral bodies of the spine and is responsible for shock absorption and mobility of the spinal unit (Raj, 2008). The disc is composed of a central nucleus pulposus (NP) and a surrounding annulus fibrosus (AF), separated from the vertebral body by two cartilaginous endplates (EP) (Figure 1). The NP forms the gel-like inner core of the IVD. It contains irregular meshworks of type II collagen fibers and a large amount of aggrecan, a proteoglycan that provides viscoelasticity, hardness, and compression resistance through its high content of anionic glycosaminoglycans (GAGs) and water binding (Watanabe, Yamada, & Kimata, 1998). The AF is further subdivided into a lateral and medial portion. The lateral AF is formed by distinct lamellae, composed of type I collagen fibers oriented obliquely to each other (Marchand & Ahmed, 1990). The medial AF is characterized by a lack of fibers, poor organization, a transition to type II collagen, and a high proteoglycan content (Humzah & Soames, 1988). This structure allows the AF to limit hydrostatic pressure within the NP when compressed and facilitate mobility between spinal segments (Guerin & Elliott, 2007) (Schmidt, Kettler, Heuer, Simon, Claes, & Wilke, 2007). Except for the outermost layer of the AF, the IVD is aneural (Roberts, Eisenstein, Menage, Evans, & Ashton, 1995) and is virtually devoid of blood vessels (Crock & Goldwasser, 1984), so it relies on diffusion through the EP for nutrient and oxygen delivery (Urban, Smith, & Fairbank, 2004). Homeostasis of the IVD as a unit requires optimal functioning of all three structures, and impairment of one or more of these structures can result in IVD degeneration.

[0101] The integrity of the IVD is maintained by an exquisite balance of cytokines, growth factors, enzymes, and enzyme inhibitor activities, which collectively regulate the balance between extracellular matrix (ECM) synthesis / apposition and degradation in a paracrine and / or autocrine manner.

[0102] In IVD degeneration, this delicate balance is disrupted by multiple etiologic factors, including aging, infection, smoking, genetic predisposition, abnormal biomechanical loading, or nutritional status of the IVD (Roberts, Evans, Trivedi, & Menage, 2006) (Cheung, et al., 2009). Histopathological changes are not necessarily observed at the primary site of the defect, but are first observed in the NP, accompanied by evidence of increased ECM breakdown, altered matrix synthesis (a switch from predominantly type II collagen to type I collagen production and a decrease in aggrecan synthesis), apoptosis, and cell loss due to in situ replication and cluster formation of surviving cells (Adams & Roughley, 2006) (Johnson & Roberts, 2007) (Le Maitre, Pockett, Buttle, Freemont, & Hoyland, 2007). The resulting loss of turgor pressure within the NP disrupts the normal balance of forces between the NP and AF, extending the degenerative process to the AF, resulting in microtrauma ("tears") that provide access for blood vessels and nerves to the IVD (Hilton & Ball, 1984), generating the pain associated with degenerative disc disease.

[0103] Regardless of the specific initial events, IVD degeneration is thought to be triggered by the abnormal synthesis and secretion of pro-inflammatory molecules by both resident NP cells (NPC) and AF cells (AFC), as well as by non-resident immune system cells such as macrophages and T cells (reviewed by (Freemont, 2009) (Risbud & Shapiro, 2014)). Proinflammatory mediators secreted during disc degeneration include tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-6, IL-17, and IL-17, as well as various chemokines (Risbud & Shapiro, 2014) (Seguin, Pilliar, Roughley, & Kandel, 2005) (Le Maitre, Hoyland, & Freemont, 2007) (Shamji et al., 2010) (Purmessur, Walter, Roughley, Laudier, Hecht, & Iatridis, 2013), of which the roles of TNFα and IL-1β have been most extensively studied. Both cytokines induce the upregulation of genes involved in ECM degradation (Le Maitre, Hoyland, & Freemont, 2007) (Le Maitre, Freemont, & Hoyland, 2005) (Le Maitre, Hoyland, & Freemont, 2007). IL-1β and its receptor are both upregulated in degenerating IVD tissue (Le Maitre, Hoyland, & Freemont, 2007) (Le Maitre, Hoyland, & Freemont, 2007), and TNFα expression is also involved in neurite ingrowth and inflammation (Murata, Onda, Rydevik, Takahashi, & Olmarker, 2006) (Wang, Markova, Anderson, Zheng, Shapiro, & Risbud, 2011).

[0104] In one embodiment, mesenchymal progenitor or stem cells are injected into the NP to restore normal mechanical and / or physiological properties to the damaged disc.

[0105] Numerous biological and synthetic substances are contemplated for co-injection into the NP with mesenchymal progenitor cells or stem cells. For example, one or more natural or synthetic glycosaminoglycans (GAGs) or mucopolysaccharides, such as hyaluronan (hyaluronic acid; HA), chondroitan sulfate, dermatan sulfate, keratan sulfate, heparin, heparin sulfate, galactosaminoglycuronglycan sulfate (GGGS), and their physiological salts, may be injected directly into the NP. HA stimulates endogenous HA synthesis by synovial cells and proteoglycan synthesis by chondrocytes, inhibits the release of chondrodegradative enzymes, and acts as a scavenger of oxygen free radicals known to be involved in cartilage degradation. Chondroitin sulfate and glucosamine injections have also been shown to block the progression of articular cartilage degeneration. Arguably, other GAGs may offer similar protective or restorative properties with therapeutic value, making them ideal candidates for injection into DDD-affected discs. Another beneficial property of GAGs is their strong hydrophilicity and water-retaining capacity. Therefore, it may be appropriate to mix GAGs with water or other water-soluble substances to form a viscous gel that can be injected into the space created by aspirating NPs or, alternatively, added as a supplement to existing NPs. This may form a natural "hydrogel" that can fill the space three-dimensionally, act like a non-collapsible filler, and allow the disc to properly absorb shock associated with movement.

[0106] Synthetic hyaluronic acid gels, such as Euflexxa® (Ferring Pharmaceuticals) or Restylane™ (Q-Med Aktiebolag Co., Sweden), are also suitable for use.

[0107] Other examples of injectable synthetic materials that may be used for co-administration include medical-grade silicone, Bioplastique™ (solid silicone particles suspended in a polyvinylpyrrolidone carrier; Uroplasty BV, The Netherlands), Arteplast™ (polymethyl methacrylate (PMMA) microparticles suspended in a gelatin carrier; Artcs Medical, USA), and Artecoll™ (smooth PMMA spheres suspended in a bovine cartilage carrier; Artepharma Pharmazeu Tische, GMBH Co., Germany). Additionally, synthetic hydrogel compositions may be used as fillers to restore normal shape to the intervertebral disc, thereby restoring normal biomechanical function.

[0108] Antioxidants with known chondroprotective properties are also candidates for NP injection. Examples include tocophereol (vitamin E), superoxide dismutase (SOD), ascorbate (vitamin C), catalase, etc. Additionally, amphiphilic derivatives of sodium alginate, etc., are also contemplated for injection herein. Furthermore, recombinant bone morphogenetic protein-1 (OP-1) is a good candidate for injection due to its ability to promote proteoglycan-rich matrix formation by NPCs and AFCs.

[0109] The use of synthetic injectable materials is also contemplated. These may be particularly applicable in situations where the primary goal is to restore biomechanical function to the disc.

[0110] HA alone or in combination with other GAGs may be used as a carrier for delivering mesenchymal progenitor cells or stem cells. The concentration and viscosity of the HA / GAG composition can be routinely determined. In one embodiment, the composition contains at least about 0.5% HA or HA salt. For example, a population of cells containing mesenchymal progenitor cells or stem cells can be suspended in Euflexxa™ (1% sodium hyaluronate) at a 1:1 ratio.

[0111] In another example, mesenchymal progenitor cells or stem cells may be delivered in a mixture with fibrin glue. As used herein, the term "fibrin glue" refers to an insoluble matrix formed by crosslinking fibrin polymers in the presence of calcium ions. Fibrin glue may be formed from fibrinogen, or its derivatives or metabolites, fibrin (soluble monomer or polymer), and / or complexes thereof, derived from biological tissues or fluids that form the fibrin matrix. Alternatively, fibrin glue may be formed from fibrinogen, or its derivatives or metabolites, or fibrin produced by recombinant DNA technology.

[0112] Fibrin glue may be formed by the interaction of fibrinogen with a fibrin glue-forming catalyst (such as thrombin and / or factor XIII). As will be understood by those skilled in the art, fibrinogen is proteolytically cleaved and converted to fibrin monomers in the presence of a catalyst (such as thrombin). The fibrin monomers can then form polymers that can crosslink to form a fibrin glue matrix. The crosslinking of fibrin polymers can be enhanced by the presence of a catalyst such as factor XIII. The catalyst for fibrin glue formation may be derived from plasma, cryoprecipitate, or other plasma fractions containing fibrinogen or thrombin. Alternatively, the catalyst may be produced by recombinant DNA technology.

[0113] Combining fibrinogen and thrombin results in the formation of a clot. The rate at which the clot forms depends on the concentration of thrombin mixed with the fibrinogen. Because it is an enzyme-dependent reaction, the higher the temperature (up to 37°C), the faster the clot will form. The tensile strength of the clot depends on the concentration of fibrinogen used.

[0114] Once a fibrin clot forms in the presence of hyaluronan, it interacts and interdigitates into a cross-linked matrix, which is known to play a key role in tissue regeneration and to exert a cytoregulatory function in tissue repair (Weigel, Fuller, & Le Boeuf, 1986). The hyaluronan-fibrin matrix also prolongs the rate of dissolution of hyaluronan, which may be beneficial in prolonging the therapeutic effect of this GAG (Wadstrom & Tengblad, 1993).

[0115] Several publications describe the use of fibrin glue to deliver therapeutic agents. For example, U.S. Patent No. 4,983,393 discloses a composition for use as an intravaginal insert, comprising agarose, agar, saline, glycosaminoglycan, collagen, fibrin, and an enzyme. Furthermore, U.S. Patent No. 3,089,815 discloses an injectable pharmaceutical preparation composed of fibrinogen and thrombin, and U.S. Patent No. 6,468,527 discloses a fibrin glue that facilitates the delivery of various biological and non-biological agents to specific sites in the body.

[0116] The disclosed compositions can be "surgically added" to the disc space. That is, the compositions can be added by the intervention of a medical professional, as opposed to being "added" by the body's natural growth or regeneration processes. Preferably, the surgical procedure involves injection via a hypodermic needle, although other surgical methods of introducing the composition into the disc may also be used. For example, the compositions may be introduced into the disc by extrusion through an expanded annular opening, infusion via a catheter, insertion through an opening created by trauma or a surgical incision, or other invasive or minimally invasive means of depositing the composition within the disc space.

[0117] genetically modified cells In one embodiment, the mesenchymal progenitor or stem cells are genetically engineered to express and / or secrete, for example, a protein of interest, e.g., a protein that provides a therapeutic and / or prophylactic benefit, e.g., insulin, glucagon, somatostatin, trypsinogen, chymotrypsinogen, elastase, carboxypeptidase, pancreatic lipase or amylase, or a polypeptide associated with or responsible for enhanced angiogenesis or a polypeptide associated with cell differentiation into pancreatic or vascular cells.

[0118] Methods for genetically modifying cells will be clear to those skilled in the art. For example, the nucleic acid to be expressed in cells is functionally linked to a promoter to induce expression in cells. For example, the nucleic acid is linked to a promoter that is functional in various target cells, such as a viral promoter, for example, a CMV promoter (e.g., a CMV-IE promoter) or an SV-40 promoter. Other suitable promoters are known in the art.

[0119] Preferably, the nucleic acid is provided in the form of an expression construct. As used herein, the term "expression construct" refers to another nucleic acid capable of conferring expression of one operably linked nucleic acid (e.g., a reporter gene and / or a counterselectable reporter gene) in a cell. In the context of the present disclosure, an expression construct should be understood to be or include a plasmid, bacteriophage, phagemid, cosmid, viral subgenomic or genomic fragment, or other nucleic acid capable of maintaining and / or replicating heterologous DNA in an expressible format.

[0120] Methods for constructing suitable expression constructs for practicing the present invention will be clear to those skilled in the art and are described, for example, in (Ausubel FM, 1987 and all updates to date) or (Sambrook & Green, 2012). For example, each component of the expression construct may be amplified from a suitable template nucleic acid, for example using PCR, and then cloned into a suitable expression construct, such as a plasmid or phagemid.

[0121] Suitable vectors for such expression constructs are known in the art and / or described herein. For example, expression vectors suitable for the methods of the invention in mammalian cells are, for example, the pcDNA vector package vectors (Invitrogen), the pCI vector package vectors (Promega), the pCMV vector package vectors (Clontech), the pM vectors (Clontech), the pSI vectors (Promega), the VP16 vectors (Clontech), or the pcDNA vector package vectors (Invitrogen).

[0122] Those of skill in the art will be aware of additional vectors and sources of such vectors, such as, for example, Invitrogen Corporation, Clontech, or Promega.

[0123] Means for introducing isolated nucleic acid molecules or genetic constructs containing them into cells for expression are known to those of skill in the art. The technique used for a given organism will depend on techniques known to be successful. Means for introducing recombinant DNA into cells include, among others, microinjection, DEAE-dextran-induced transfection, liposome-induced transfection such as using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-induced DNA uptake, electroporation, and microparticle bombardment such as using DNA-coated tungsten or gold particles (Agracetus Inc., Wisconsin, USA).

[0124] Alternatively, the expression construct of the present invention is a viral vector. Suitable viral vectors are known in the art and commercially available. Conventional systems using viruses to deliver nucleic acids and integrate them into the host cell genome include, for example, retroviral vectors, lentiviral vectors, or adeno-associated viral vectors. Alternatively, adenoviral vectors are useful for introducing nucleic acids that remain episomal into host cells. Viral vectors are an efficient and versatile method for introducing genes into target cells and tissues. Moreover, high transduction efficiencies have been observed in many different cell types and target tissues.

[0125] For example, retroviral vectors generally contain cis-acting long terminal repeats and have the capacity to package up to 6-10 kb of foreign sequence. Minimal cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate expression constructs into target cells for long-term expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SrV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., International Publication WO 1994 / 026877, (Buchschacher & Panganiban, 1992), (Johann, Gibbons, & O'Hara, 1992), (Sommerfelt & Weiss, 1990), (Wilson, Reitz, Okayama, & Eiden, 1989), (Miller, Garcia, von See Suhr, Lynch, Wilson, & Eiden, 1991) (Miller & Rosman, 1989) (Miller, 1990) (Scarpa, Cournoyer, Munzy, Moore, Belmont, & Caskey, 1991) (Burns, Friedmann, Driever, Burrascano, & Yee, 1993) j).

[0126] Various adeno-associated virus (AAV) vector systems have also been developed for nucleic acid delivery. AAV vectors can be easily constructed using techniques known in the art. (See, for example, U.S. Patent Nos. 5,173,414 and 5,139,941, International Publication Nos. WO92 / 01070 and WO93 / 03769, (Lebkowski, McNally, Okarma, & Lerch, 1988), (Vincent, Moore, & Haigwood, 1990), (Carter, 1992), (Muzyczka, 1992); (Kotin, 1994), (Shelling & Smith, 1994), (Zhou, et al., 1994).

[0127] Additional viral vectors useful for delivery of the expression constructs of the invention include vectors derived from poxviruses, such as vaccinia virus and avian poxviruses, or alphavirus or conjugative viral vectors (e.g., those described in (Fisher-Hoch, et al., 1989)). [Example]

[0128] Example 1 Materials and Methods Immunoselection of MPCs by selection of STRO-3+ cells Bone marrow (BM) is collected from normal, healthy adult volunteers (20-35 years old). Briefly, 40 ml of BM is aspirated from the posterior border of the iliac crest into a tube containing lithium-heparin anticoagulant.

[0129] BM mononuclear cells (BMMNCs) are prepared by density gradient separation using Lymphoprep™ (Nycomed Pharma, Oslo, Norway) as previously described (Zannettino, Buhring, Niutta, Watt, Benton, & Simmons, 1998). After centrifugation at 400 × g and 4°C for 30 min, the buffy layer was removed with a transfer pipette and washed three times with "HHF," which consisted of Hank's Balanced Salt Solution (HBSS; Life Technologies, Gaithersburg, MD) containing 5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).

[0130] Subsequently, STRO-3+ (or TNAP+) cells were isolated by magnetic activated cell sorting as previously described (Gronthos & Simmons, 1995) (Gronthos, 2003). Briefly, approximately 1–3 × 10 8BMMNCs were incubated on ice for 20 minutes in blocking buffer consisting of HHF containing 10% (v / v) normal rabbit serum. The cells were then incubated on ice for 1 hour with 200 μl of a 10 μg / ml solution of STRO-3 mAb dissolved in blocking buffer. The cells were then washed twice in HHF by centrifugation at 400 × g. A 1 / 50 dilution of goat anti-mouse γ-biotin (Southern Biotechnology Associates, Birmingham, UK) in HHF buffer was added, and the cells were incubated on ice for 1 hour. The cells were then resuspended in MACS buffer (1% BSA, 5 mM EDTA, and 0.01% sodium azide) supplemented with CaCl2 as described above. 2+ and Mg 2+ The cells are washed twice in PBS (without HCl) and resuspended in a final volume of 0.9 ml MACS buffer.

[0131] 100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) were added to the cell suspension and incubated on ice for 15 minutes. The cell suspension was washed twice, resuspended in 0.5 ml of MACS buffer, and then loaded onto a mini MACS column (MS Columns, Miltenyi Biotec). The column was washed three times with 0.5 ml of MACS buffer to recover cells that did not bind to STRO-3 mAb (deposited with the American Type Culture Collection (ATCC) on December 19, 2005, under accession number PTA-7282; see International Publication WO 2006 / 108229). After adding an additional 1 ml of MACS buffer, the column was removed from the magnet and positive pressure was applied to isolate TNAP+ cells. An aliquot of cells from each fraction was stained with streptavidin-FITC, and their purity could be assessed by flow cytometry.

[0132] Mesenchymal progenitor cells (MPCs) isolated by this method are STRO-1 bright It is MPC.

[0133] Preparation of MPC CM Conditioned medium (CM) was prepared from representative lots of available MPC product lots (263873, 22-12-001US, 22-12-02US, 345938, 2011CC053, 2011CC011, 2012CC010, 322509, 376232, 376233, 380505, 380507, 385470, 385471, and 1857469), each with different donors and manufacturing processes for clinical and product development. Cryopreserved MPC products were thawed and cultured in serum-supplemented growth medium at a density of 50,000 cells / cm. 2 MPCs were seeded at 100°C and allowed to adhere overnight at 37°C and 20% O. To generate CMs compatible with functional analysis using intervertebral disc cells, MPC growth medium was mixed with chondrogenic basal medium (CBM; Lonza, Walkersville, MD) supplemented with 0.5% bovine serum albumin (BSA) alone at 209 μl medium / cm. 2 The volume was replaced with 0.01 ml of PBS, and the cells were cultured at 37°C and 5% O for 3 days. At the end of this culture period, the medium was harvested and centrifuged to remove floating cells, and the resulting supernatant was collected and stored at -80°C until use.

[0134] NPC proliferation bioassay Nucleus pulposus cell (NPC) proliferation in response to MPC CM was assessed by quantifying 5-ethynyl-2'-deoxyuridine (EdU) incorporation into DNA in actively dividing cells. Human NPCs were cultured at 2,500 cells / cm in serum-containing growth medium on poly-L-lysine-coated culture dishes. 2 Cells were plated at 1000 x g for 1 hour. Cells were incubated at 37°C with 5% O2 to allow overnight attachment and then serum-starved for 48 hours. After serum deprivation, cells were stimulated with MPC CM for 48 hours. EdU was added to cells according to the manufacturer's instructions (Click-iT™ Kit, Invitrogen, Carlsbad, CA) for the final 18 hours of culture. Cells were then detached with trypsin and stained for viability. Cells were then fixed, stained for EdU incorporation, and analyzed by flow cytometry. The percentage of EdU in the viable population relative to control cells not stained with EdU was 0.01. + The cells were identified.

[0135] Bioassay of NPC proteoglycan synthesis The effect of MPC CM on NPC matrix production in vitro was investigated by semiquantitatively measuring Alcian blue dye extracted from micromass cultures after staining of proteoglycans deposited within the extracellular matrix (ECM). To establish human NPC micromass cultures, NPCs were cultured in a 5 μg / cm solution with human fibronectin. 2 High-density two-dimensional (2D) cultures were seeded in 10 μl of growth medium containing 100,000 cells per well of a 48-well plate coated with PEG. After allowing cells to adhere for 2 hours, complete growth medium was added to fill the wells and the cells were incubated overnight. The following day, cells were washed once with warm PBS, serum-starved for 48 hours, and then stimulated with MPC CM for 7 days. At the end of the culture period, cells were fixed in situ in 10% zinc formalin and stained with Alcian blue to detect proteoglycans. Digital images of representative wells were captured, and the plates were air-dried for 1–2 hours. The Alcian blue stain was extracted from each well with 6 N guanidine HCl containing 0.25% Triton X-100, and the optical density (OD) at 600 nm was measured for each sample using a microplate reader (Bjornsson, 1993).

[0136] Bioassay of AFC proliferation Annulus fibrosus cell (AFC) proliferation in response to MPC CM was measured by EdU incorporation, similar to the method used to measure NPC proliferation, but the culture period was modified to a period found to be appropriate for AFCs. Briefly, AFCs were cultured at 2,500 cells / cm in serum-supplemented AFC growth medium. 2 After overnight attachment, cells were serum-starved for 48 hours and then treated with MPC CM for 3 days. Cells were pulsed with EdU for 18 hours before the end of culture. Cells were then harvested, stained, and analyzed by flow cytometry.

[0137] AFC collagen synthesis bioassay To establish micromass cultures of annulus fibrosus (AF), 5 μg / cm of human fibronectin was used. 2 Annulus fibrosus cells (AFCs) were seeded in high-density two-dimensional (2D) culture by adding 10 μl of growth medium containing 100,000 cells to each well of a 48-well plate coated with PEG. After allowing the cells to adhere for 2 hours, complete growth medium was added to fill the wells and the cells were incubated overnight. The following day, the cells were washed once with warm PBS, serum-starved for 48 hours, and then stimulated with MPC CM for 7 days. At the end of this culture period, collagen production stimulated by MPC CM was measured by a hydroxyproline assay using a commercially available kit (Sigma, St. Louis, MO). The medium was aspirated, and the cells were washed with water. The deposited collagen was hydrolyzed in hydrochloric acid, and the resulting supernatant was collected and evaporated. Each sample was briefly incubated in the presence of chloramine T to oxidize hydroxyproline. Finally, 4-(dimethylamino)benzaldehyde was added to each sample, and the colorimetric product was read at 560 nm. In each experiment, a standard curve was generated using known amounts of hydroxyproline (0.2-1 μg) to allow quantitative measurement of collagen synthesis in response to MPC CM treatment.

[0138] Isolation and culture of adult human AFCs Adult cadaveric intervertebral disc tissue was obtained from donors screened for the following exclusion criteria: [Table 1]

[0139] The tissues were immersed in a storage medium consisting of DMEM-Ham's F12 (1:1)-10% fetal bovine serum (FBS) supplemented with antibiotics (e.g., penicillin (200 U / mL), streptomycin (200 mg / mL), and fungizone (1.25 mg / mL) or gentamicin (50 mg / mL)) to a final concentration of 0.1% (v / v) for transport to the laboratory and digested. To prepare the tissues for digestion, the AF tissue was carefully excised from the NP tissue, which was subjected to digestion and isolation separately. Each intervertebral disc was excised and cultured separately. All digestions were performed in 45 ml of sterile DMEM-Ham's F12-10% FBS as previously described (Melrose, Ghosh, Taylor, Latham, & Moore, 1997) (Melrose, Smith, Ghosh, & Taylor, 2001) (Shen, Melrose, Ghosh, & Taylor, 2003). Briefly, under sterile conditions, tissue was finely diced with a scalpel. Approximately 2.5 g of the diced tissue was transferred to a 50 ml conical tube containing an enzyme solution of 0.2% w / v pronase and 0.01% w / v DNAase. The tissue was digested at 37°C for 90 min. The remaining tissue was washed with 10 ml PBS, and the supernatant was discarded. The remaining tissue was then digested for several hours using 0.05% Clostridium histolyticum bacterial collagenase type 1A and 0.01% DNase (45 ml / tube) in antibiotic-containing DMEM-Ham's F12-10% FBS for 10 minutes until the tissue was completely disaggregated. Cells were harvested by centrifugation (800 g x 10 min) and washed once with DMEM-Ham's F12-10% FBS. The resulting cell suspension was passed through a 70 μm strainer and resuspended for cell count and viability determination. Both NPCs and AFCs were seeded in tissue culture-treated flasks in DMEM-Ham's F12-5% FBS and 2 mM L-glutamine. Primary NPCs and AFCs were cultured until 80–90% confluent and shipped to Mesoblast Laboratories, Houston. Primary cultures were harvested with 0.05% trypsin / 0.1% EDTA and cryopreserved. Primary cells from adult AFCs were thawed and cultured at passage 1 at 10,000 cells / cm. 2 and then harvested and prepared for bioassays.

[0140] Fetal AFCs were obtained from a commercial supplier (ScienCell, Carlsbad, CA).

[0141] Measurement of TGFβ1 levels in MPC CM by ELISA TGFβ1 levels in MPC CM were measured by ELISA according to the manufacturer's instructions (R&D Systems).

[0142] Prior to use, MPC CM was concentrated approximately 40-fold to facilitate the acid activation step required for measuring total TGFβ1 levels, according to the manufacturer's instructions. After acid treatment, samples were reconstituted in CBM to their original volume for use in the bioassay.

[0143] Perform the ELISA according to the manufacturer's instructions, modifying the diluent used to reconstitute and prepare the standard and sample dilutions. Reconstitute the TGFβ1 standard provided in the kit in CBM supplemented with 0.5% BSA (CBM + 0.5% BSA). Prepare serial dilutions in CBM + 0.5% BSA to achieve final concentrations ranging from 31.2 to 2000 pg / ml. Acid-activate the samples and dilute them 1:5 in CBM + 0.5% BSA. Add the standard, sample, and control to a microplate pre-coated with a monoclonal antibody specific for TGFβ1. After 2 hours of incubation at room temperature (RT), wash the plate. Add the TGFβ1 complex to each well and incubate the plate at room temperature for 2 hours. Then, wash the plate again, add substrate solution to each well, and incubate for 30 minutes at room temperature. Add stop solution to each well, and read the optical density (OD) of each sample using a microplate reader set at 450 nm with wavelength correction at 570 nm. A standard curve is constructed using a four-parameter logistic curve fit. The TGFβ1 concentration in each sample is derived from the standard curve and corrected for dilution to obtain the final result.

[0144] Generation of TGFβ1 knockdown MPCs Freshly thawed MPC preparations (n=4) were transfected with TGFβ1 siRNA or a scrambled oligonucleotide control. Cells were suspended in serum-free αMEM and combined with a transfection mixture containing TGFβ1 siRNA or scrambled siRNA (500 pmol, Life Technologies, Carlsbad, CA) and Lipofectamine (Life Technologies). Cells were then plated at high density on fibronectin-coated plates and allowed to adhere overnight. The following day, cells were washed, and the medium was replaced with CBM + 0.5% BSA. Cells were returned to the incubator at 37°C, 5% O2 / 95% CO2 for 72 hours. At the end of this incubation period, culture supernatants were harvested, and suspended cells or debris were pelleted by centrifugation, aliquoted, and stored at -80°C until assayed.

[0145] Example 2: Proof-of-Concept Experiment With a view to establishing an appropriate efficacy assay for MPC in intervertebral disc repair, a series of in vitro studies were performed to model and evaluate the potential mechanisms by which MPC may induce therapeutic benefit in degenerative disc disease (DDD).

[0146] Under laboratory conditions, MPCs possess the potential for multilineage differentiation, including the ability to differentiate into chondrocytes in vitro in response to appropriate inductive cues. The classic in vitro chondrogenic assay involves culturing cells in high-density pellets in the presence of TGFβ1 for three weeks (Johnstone, Hering, Caplan, Goldberg, & Yoo, 1998). The pellets are then fixed, sectioned, and stained to detect the presence of proteoglycans, a hallmark of chondrogenic activity. Given the time required to perform this assay and the nonquantitative evaluation method, the assay was deemed inapplicable for qualification and validation as a release assay. Therefore, further development of an in vitro chondrogenic release assay was not pursued. Instead, efforts focused on identifying the paracrine mechanism of action of MPCs on intervertebral disc (IVD) cells as a basis for assay development.

[0147] MPCs secrete a wide range of bioactive humoral factors, including those known to attenuate inflammation, promote cell proliferation, and stimulate matrix production (see, e.g., [unclear text], [unclear text], [unclear text], and [unclear text]). Cytokine profiles of multiple lots of MPC products confirmed the secretion of a wide range of bioactive molecules. Based on these data, we hypothesized that MPCs, once introduced into the IVD, may stimulate endogenous repair processes via a paracrine mechanism through the release of soluble molecules that act on resident disc cells. We focused on identifying secreted factors that contribute to the survival, proliferation, and differentiation of NPCs and / or AFCs, potentially resulting in sustained enhancement of disc function. An extensive literature search was conducted to identify secreted factors with anabolic effects on disc cells. Factors identified in this study were then screened for MPC CMs using immunoassays and these data, identifying TGF-β1 as the leading candidate.

[0148] To determine whether a TGFβ1-mediated mechanism of action could provide a basis for the development of an assay to evaluate MPC products, an in vitro proof-of-concept experiment was performed with the following objectives: 1. To determine whether MPC CM stimulates NPC proliferation and matrix production; and 2. To determine whether MPC CM stimulates AFC proliferation and matrix production, and 3. To determine whether TGFβ1 in MPC CM induces the biological activity of MPC CM on intervertebral disc cells in vitro.

[0149] MPC-derived humoral factors stimulate NPC proliferation and proteoglycan production The NP is primarily composed of proteoglycans, aggrecans, and water-binding molecules, which are substrates that provide turgor pressure within the IVD. NPCs are the primary source of proteoglycans for the IVD, thereby playing an important role in maintaining tissue structure and function. Furthermore, in vitro studies have shown that an intact proteoglycan matrix repels neurite ingrowth (Johnson, Caterson, Eisenstein, Hynds, Shaw, & Roberts, 2002), suggesting that proteoglycan deposition by NPCs may be important in maintaining the aneurysmal tissue environment of healthy, pain-free discs. Conversely, DDD is associated with NPC death, matrix breakdown and loss, and neurite ingrowth (Loreto, Musumeci, Castorina, Loreto, & Martinez, 2011) (Melrose, Roberts, Smith, Menage, & Ghosh, 2002). Therefore, repair of damaged NP tissue may require both maintenance of a resident NPC population and stimulation of matrix synthesis. Meanwhile, NPC support may improve disc structure and function and attenuate pain sensation. To investigate whether MPC CM influences NPC function, we established a bioassay to measure the effects of MPC CM on human NPC proliferation and proteoglycan production in vitro.

[0150] CM samples from various lots of MPC were tested for activity in the NPC proliferation assay, as determined by EdU incorporation. Figure 2A shows that 9 of 10 lots tested stimulated a significant increase in the proportion of actively dividing NPCs in culture (mean % EdU + Cells = 44.9 ± 15.7%, range = 38.9–66.9%, n = 10 lots from 4 donors). The remaining lots showed no significant effect on NPC proliferation compared to control basal medium.

[0151] In 2D high-density cultures, NPCs constitutively produced low levels of proteoglycan, as confirmed by Alcian blue staining (Figure 2B). CM obtained from three different lots of MPC significantly enhanced proteoglycan synthesis beyond these baseline levels, as indicated by even stronger Alcian blue staining than unstimulated control cells. To quantify proteoglycan synthesis, Alcian blue staining was extracted for absorbance measurement. Treatment of NPCs with MPC CM increased proteoglycan content approximately twofold over control cells grown in basal medium (mean OD = 0.40 ± 0.02, range = 0.38–0.42, n = 3 lots from one donor) (Figure 2C).

[0152] Together, these data provide evidence that MPC CM contains factor(s) with anabolic activity on NPCs, as measured by their effects on proteoglycan proliferation and synthesis. Thus, MPC treatment may stimulate repair mechanisms in injured discs through paracrine effects on NPCs.

[0153] Humoral factors derived from MPC stimulate proliferation and collagen synthesis of AFC The AF of the IVD is primarily composed of fibrillar type I and type II collagen, forming multiple lamellae surrounding the NP. Type I collagen levels are high in the outer layer and decrease near the interface with the NP, whereas type II collagen content increases away from the outer layer and becomes highly abundant toward the tissue center. This collagen gradient provides both tensile strength and elasticity to the disc, thereby supporting the structure and function of the IVD. An intact AF plays a critical role in creating a barrier that prevents nerves and blood vessels from extending into the disc tissue. In contrast, disruption of the AF, as occurs in DDD, leads to impaired IVD structure and function and pain associated with vascular and nerve invasion. The collagen matrix of the AF is maintained by resident AFCs. AFCs obtained from degenerated discs have been shown to exhibit phenotypic abnormalities, characterized by downregulation of genes related to ECM components and cell proliferation (Gruber, Hoelscher, & Hanley, 2010). The AF population in DDD tissues has been shown to contain a high proportion of senescent cells, concomitantly with a low proportion of proliferating cells (Gruber, Ingram, Davis, & Hanley, 2009). Therapeutic strategies that help maintain the AFC pool and activity may provide long-term benefits by restoring AF structure and function. To determine whether MPC-derived factors affect AFC function, we established a bioassay to measure the effects of MPC CM on human AFC proliferation and collagen synthesis in vitro.

[0154] Figure 3A shows that CM samples generated from separate lots of MPCs increased AFC proliferation, as measured by EdU incorporation, above the levels observed in cells grown in basal medium alone. In this experiment, CM from all seven lots tested stimulated a significant increase in the percentage of actively dividing cells in culture (mean %EdU 0.01) compared to control medium (3.6%). + cells = 27.1 ± 15.1%, range = 7.8–53.2%, n = 10 lots from 3 donors).

[0155] MPC CM stimulated AFC proliferation and increased collagen synthesis in AFCs. AFCs treated with MPC CM contained significantly higher levels of hydroxyproline (0.01 μg / ml) than cells grown in basal medium alone (mean hydroxyproline content = 0.3 ± 0.1 μg / ml, range = 0.2–0.5 μg / ml, n = 6 lots of MPCs from 3 donors) (Figure 3B). Despite the inherent variability observed in these types of bioassays, taken together, these data clearly demonstrate that MPC CM contains humoral factors that stimulate AFC activity.

[0156] The role of TGF-β1 in collagen synthesis in AFC To investigate the possible contribution of TGFβ1, we examined TGFβ1 levels in CM derived from multiple MPC production lots encompassing five different donors, and then investigated the putative causal role of TGFβ1 on in vitro collagen synthesis in AFCs in the presence and absence of anti-TGFβ1 neutralizing antibodies.

[0157] TGFβ1 levels in MPC CM were measured by ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). TGFβ1 levels in MPC CM ranged from 1083.1 to 4202.8 pg / ml (mean = 2981.6 ± 1054.3 pg / ml, n = 15 lots, prepared from five different donors) (Figure 4).

[0158] The data confirmed that MPCs reproducibly secreted robust levels of TGFβ1 into their CM. As shown in Figure 5A, three different lots of AFCs demonstrated a clear TGFβ1 dose-dependent collagen synthesis, reaching a plateau at 1–3 ng / ml TGFβ1. In the presence of anti-TGFβ1 neutralizing antibodies, collagen synthesis by AFCs in response to 1 ng / ml TGFβ1 was reduced to a level slightly above that obtained in the absence of TGFβ1. These data validate the linear dose-responsiveness of the target AFC population to TGFβ1 and the efficacy of the anti-TGFβ1 neutralizing antibodies.

[0159] To investigate whether MPC-derived TGFβ1 plays a causative role in AFC collagen production, MPC CM samples were pretreated with a neutralizing antibody against TGFβ1 before being added to AF cultures. As shown in Figure 5B (left panel), CM from each of seven MPC lots stimulated a statistically significant increase in AFC hydroxyproline content compared to control basal medium. Neutralization of TGFβ1 activity resulted in a significant decrease in hydroxyproline content in five of the seven lots. The remaining two lots showed a trend toward decreased collagen synthesis, but these also contained the lowest levels of activity in the assay. When CM samples were tested between different lots of AFCs obtained from different donors (Figure 5B, center and right panels), a similar pattern of results was observed in each experiment. Several lots of CM demonstrated complete inhibition of collagen synthesis in the presence of anti-TGFβ1 neutralizing antibody (compared to equivalent levels of control antibody), demonstrating that TGFβ1 was the sole causative factor in promoting collagen synthesis in these specific cases.

[0160] Proof-of-concept conclusions The data demonstrate that MPC CM contains humoral factors that stimulate proliferation and matrix production of NPCs and AFCs. TGFβ1, known to have anabolic effects on disc cells, was detected in CM from multiple MPC lots. Furthermore, TGFβ1 was shown to be a major effector of collagen synthesis in cells treated with MPC CM. The data suggest that MPC-derived TGFβ1 stimulates AFC collagen synthesis, thereby potentially contributing to the repair and long-term therapeutic benefits of AF associated with DDD. Thus, detection of TGFβ1 levels in MPC CM represents a powerful surrogate measure of MPC efficacy for disc repair.

[0161] The data also demonstrate the establishment of quantitative assays to measure TGFβ1-induced biological activity: the EdU incorporation assay as a measure of NPC and AFC proliferation, and the hydroxyproline assay as a measure of AFC collagen synthesis. Hydroxyproline assays were performed using different lots of AFC, and the findings support the reproducibility of the collagen synthesis-promoting effect of MPC CM on AFC, regardless of target cell donor, and the contribution of TGFβ1 to this effect. A similar approach to comparing the performance of different lots of NP and AFC was performed in the evaluation of the EdU incorporation assay. Some variability in TGFβ1 levels was observed between MPC product lots (range of screened lots = 1083.1–4202.8 pg / ml). Importantly, TGFβ1 levels within this range were shown to have measurable activity, statistically significantly above baseline controls, in the AFC collagen synthesis assay using recombinant human TGFβ1 (rhTGFβ1). Taken together, these data support the use of this bioassay to measure the biological activity of TGFβ1 in MPC CMs.

[0162] Example 3 Comparability of fetal and adult AFC We previously developed a bioassay to measure the effect of MPC CM on AFC collagen synthesis. The assay was developed using fetal AFC due to their commercial availability. This factor represents an important advantage of availability when approaching the development of a potency assay. In contrast, adult AFC is not currently commercially available. We sought to determine whether fetal AFC would be a suitable substitute for adult AFC in a collagen synthesis bioassay. Therefore, we compared the effects of rhTGFβ1 and MPC CM on fetal and adult AFC micromass cultures.

[0163] Similar to fetal cells, adult AFCs responded to rhTGFβ1 in a dose-dependent manner (0.1–3 ng / ml) (Fig. 6 and Table 2). Compared with fetal cells, adult AFC cultures had higher baseline collagen levels and responded more strongly to low levels of TGFβ1 (100 pg / ml) (Fig. 6 and Table 2). Production in response to 500 pg / ml was similar in fetal and adult AFC. At 1–3 pg / ml of TGFβ1, the dose-response curve began to plateau in adult AFC cultures but remained linear in fetal AFC.

[0164] Treatment of adult AFC with MPC CM stimulated a robust and significant increase in collagen production in adult AFC (Figure 7 and Table 3). [Table 2] [Table 3]

[0165] Taken together, these data demonstrate that stimulation of fetal and adult AFC micromass cultures with TGFβ1 dose-dependently increases collagen production. Treatment of adult AFC with MPC CM resulted in a robust increase in hydroxyproline content, similar to the effect previously observed in fetal AFC. Taken together, these data indicate that fetal AFC are a suitable surrogate for adult AFC when assessing the effect of MPC CM on collagen production in this cell type.

[0166] Example 4 Development of a TGFβ1 potency assay for MPC products for DDD Using an in vitro model, we investigated a number of potential paracrine mechanisms by which MPC may have a beneficial effect in DDD (Example 2). Among these, stimulation of collagen production by AFC may be an important step toward long-term therapeutic benefit. Increased collagen production by AFC may restore the structural integrity of the disc and inhibit ingrowth of blood vessels and nerves, thereby improving the biomechanical function of the disc and reducing pain.

[0167] To examine the effects of humoral factors derived from MPCs on AFC collagen production, we developed a quantitative assay to measure the levels of hydroxyproline (a major component of collagen) in micromass cultures of AFCs (Example 2). The assay was established using three different lots of fetal AFCs and rhTGFβ1. We showed that rhTGFβ1 (throughout the concentration range of 100–3000 pg / ml) stimulated collagen production in AFCs in a dose-dependent manner (Figure 5A). We also demonstrated that MPC CM stimulated collagen production in this bioassay. The collagen-stimulating effect of MPC CM was at least in part due to TGFβ1 activity, as an anti-TGFβ1 neutralizing antibody inhibited these effects (Figure 5B). Importantly, the collagen-stimulating effects of both rhTGFβ1 and MPC CM were reproducible across multiple lots of AFCs representing different donors, indicating that these effects are independent of the AFC donor. We also confirmed that commercially available fetal AFC is a suitable surrogate for adult AFC in this bioassay (Example 3). Taken together, these data provide two key elements for assay development: first, they provide proof-of-concept that MPC can stimulate collagen production by AFC via a paracrine mechanism and that TGFβ1 plays an important role in this setting. Second, they support the utility of the AFC collagen synthesis assay to measure MPC CM bioactivity. Thus, given the robust levels of TGFβ1 observed in MPC CM and the causal role TGFβ1 plays in the effects of MPC CM on AFC collagen production, the data also suggest that TGFβ1 is a reasonable candidate as a surrogate marker for MPC bioactivity in the context of DDD.

[0168] As a potency assay of MPC for DDD, a method using enzyme-linked immunosorbent assay (ELISA) was adopted to detect TGFβ1 in MPC CM.

[0169] A commercially available ELISA was used to measure TGFβ1 levels in CM obtained from MPCs grown in two different basal media. The data show significant differences in TGFβ1 levels in CM as a function of media composition (Figure 8). These samples were generated during experiments to determine the optimal basal media for generating CM for evaluation of MPC TGFβ1 production and in vitro bioactivity. CBM supplemented with only 0.5% BSA (Figure 8, dark bars) was selected for use in these experiments, reflecting a balance between supporting MPC function in functional bioassays using IVDs and compatibility with downstream applications. By extension, these data demonstrate that the TGFβ1 ELISA can be used to detect changes in manufacturing processes that adversely affect TGFβ1 synthesis by MPCs.

[0170] MPC CM stimulates collagen production by human AFCs and contains robust levels of TGF-β1 Figure 7 and Table 4 show the effect of MPC CM on collagen production in three independent lots of fetal AFC. The data show that each AFC lot responded dose-dependently to rhTGFβ1 (100–3000 pg / ml), which was included in the experiment to confirm the suitability of the system. CM from GMP lots 1–18 was evaluated with AFC lot 4729 (Figure 7A). When aliquots were available, CM samples from AFC lots 5945 and 4755 were also evaluated (Figures 7B and 7C). Each MPC lot stimulated a statistically significant increase in collagen production relative to the unstimulated control group of AFC lot 4729 (Figure 7A). This effect was replicated with AFC lots 5945 and 4755 (Figures 7B and 7C). Figure 7D shows the average response of all AFC lots to each MPC CM sample.

[0171] MPC CM collagen-stimulating activity was associated with the presence of distinct levels of TGFβ1 in each sample (Figure 9 and Table 4). Under standardized culture conditions, total TGFβ1 levels in MPC supernatants ranged from 1979.12 to 4202.82 pg / ml, with a mean of 3303.16 ± 569.56 pg / ml.

[0172] Establishing a minimum threshold effect of MPC CMs in the AFC collagen synthesis bioassay To establish a preliminary release specification for MPC based on TGFβ1 secretion, we first sought to identify a minimum threshold effect of MPC CM in the AFC collagen synthesis bioassay. We speculated that this threshold would be related both to the characteristics of the sample type (CM) and the bioassay itself. As shown in Figures 7 and 9, MPC CM samples from the test lots contained a range of levels of TGFβ1 (1979.12 to 4202.82 pg / ml), all of which significantly increased collagen synthesis above baseline unstimulated controls. Furthermore, the inventors noted that although the mean level of TGFβ1 samples in MPC CM was 3303.16±569.56 pg / ml, the effect of MPC CM on collagen production was lower than that exhibited by 3000 pg / ml of rhTGFβ1 alone, consistent with the assay hypothesis that MPC CM may contain factors that inhibit the action of TGFβ1. Therefore, to establish a level of bioactivity in MPC CM that would reflect the lower than usual activity in the bioassay, the inventors generated CM from MPC lots in which TGFβ1 was reduced using siRNA technology.

[0173] Characterization of CMs obtained from TGFβ1 knockdown MPCs We verified TGFβ1 knockdown by measuring TGFβ1 levels in MPC CM by ELISA. Figure 10A and Table 5 show TGFβ1 levels in CM from each MPC lot transfected with control scrambled oligonucleotide or TGFβ1 siRNA. Transfection of MPCs with 500 pmol of siRNA reduced TGFβ1 levels by approximately 90% compared with the scrambled control group, without any direct effect on viability (data not shown).

[0174] Having confirmed TGFβ1 knockdown, we tested MPC CM samples in the AFC hydroxyproline assay. Figure 10B and Table 5 show that CM from scrambled siRNA-transfected MPCs stimulated collagen production in AFCs at levels comparable to the average level stimulated by all tested MPC lots. In contrast, AFC collagen synthesis in response to TGFβ1 knockdown MPC CM was significantly reduced compared to AFCs stimulated with MPC CM from the scrambled siRNA control group. However, the effect of CM from TGFβ1 knockdown MPCs remained significantly greater than that of the baseline control group, suggesting the presence of residual TGFβ1 activity and / or other contributing factors.

[0175] In summary, the data show that knockdown MPCs secreted an average of 204.81 ± 52.07 pg / ml of TGFβ1, resulting in the production of 0.17 ± 0.06 μg of hydroxyproline in AFCs (Figure 10 and Table 5). This level of hydroxyproline represents the minimum effect of MPC CMs observed in the AFC bioassay. We consider this minimum effect level to define the threshold between less potent and more potent cells based on the currently available data set. To increase stringency for more potent cells, we set the minimum effect level at 0.23 μg of hydroxyproline, 1 SD above the threshold level of 0.17 μg. [Table 4] [Table 5]

[0176] Establishment of provisional specifications for MPC release based on TGF-β1 secretion Using data from our experiments with untreated and TGFβ1 knockdown MPCs, we performed statistical analyses to examine the relationship between TGFβ1 levels and AFC collagen production in vitro and to identify the threshold level of TGFβ1 required for the release of a clinical MPC product for DDD.

[0177] Relationship between TGF.BETA.1 levels and activity of MPC CMs in the AFC collagen synthesis bioassay We first determined whether there was a relationship between the levels of TGFβ1 present in MPC CM and the effect of MPC CM on collagen production by AFCs in vitro. Data from untreated and knockdown MPCs totaled 26 samples. In this data set, TGFβ1 levels ranged from 143.8 to 4202.8 pg / ml, and collagen levels ranged from 0.10 to 0.53 μg (Tables 4 and 5). Pearson's correlation revealed a statistically significant relationship between TGFβ1 levels and collagen production (r = 0.65, p < 0.001). Regression analysis was performed to determine the best-fit line between TGFβ1 levels and collagen production (p < 0.001, see Figure 11).

[0178] Using the minimally effective level of MPC CM in the AFC collagen synthesis bioassay established above, this linear regression model predicts that 405 pg / ml of TGFβ1 is required to stimulate 0.23 μg of collagen production in the AFC bioassay.

[0179] Assessment of model sensitivity and specificity The sensitivity and specificity of the model were examined. A threshold of 405 pg / ml was set and a contingency analysis was performed. The sensitivity was 100%, i.e., 22 of the 22 samples predicted to be positive (stimulating more than 0.23 μg of hydroxyproline in the AFC bioassay) were found to be in fact truly positive. The inventors found the specificity to be 100%, i.e., 4 of the 4 samples predicted to be below the threshold (stimulating less than 0.23 μg of hydroxyproline) were in fact truly negative. There were no false positive samples and no false negative samples.

[0180] The data presented here support the use of TGFβ1 as a surrogate marker for the efficacy of MPC in stimulating collagen production by AFCs in vitro. Using the AFC collagen synthesis bioassay, CM samples generated from 18 lots of MPC products were shown to stimulate AFC-induced collagen production in vitro and contain significant levels of TGFβ1, as measured by ELISA. This effect was attenuated in CM obtained from TGFβ1-knockdown MPCs, demonstrating a causal role for TGFβ1 in this setting and consistent with previously provided data from neutralizing antibody studies. Data from untreated and TGFβ1-knockdown MPCs were combined to define a threshold level of TGFβ1 in MPC CM required for bioactivity in the AFC collagen synthesis bioassay. Statistical analysis of the experimental data confirmed that the minimum TGFβ1 level required to stimulate a significant increase in AFC-induced collagen production in vitro was 405 pg / ml. Therefore, 405 pg / ml of TGFβ1 represents a tentative release specification for MPCs in DDD. Taken together, these data indicate that detection of TGFβ1 in MPC CM using ELISA provides a reasonable measure of the potential of MPCs to stimulate endogenous repair processes in human intervertebral discs.

[0181] Example 5 Optimization of the TGFβ1 potency assay The TGFβ1 potency assay measures the levels of TGFβ1 released by cultured and harvested MPC products. The assay consists of two parts: (1) generation of MPC CM from the cultured and harvested MPC products, and (2) detection of TGFβ1 levels in the CM using a commercially available enzyme-linked immunosorbent assay (ELISA, R&D Systems Human TGFβ1 Quantikine ELISA). Evaluation of the effects of cell seeding density, culture time, and inter-operator variability on TGFβ1 levels in MPC CM.

[0182] MPC lots 345938 and 2011cc063 were used in this study. MPC products were thawed, washed, counted, and plated in αMEM supplemented with 10% fetal bovine serum (FBS) at 25,000 or 50,000 viable cells / cm in 6-well plates. 2 Cells were seeded at 100°C for 1 hour. The next day, the cells were washed with PBS, and the medium was replaced with CBM + 0.5% BSA. CM was collected at 24, 48, 68, 70, 72, 74, 76, and 120 hours after the CBM medium change. Triplicates were collected for each time point, and each CM sample was analyzed in duplicate for TGFβ1 content by ELISA. To measure inter-operator variability, a single vial of each cell lot was thawed and divided into two aliquots. Experiments from cell counting to CM collection were performed in parallel by two operators.

[0183] Figure 12 shows TGFβ1 secretion by two MPC lots as a function of initial cell seeding density, time, and operator. Figure 12A shows TGFβ1 secretion by two MPC lots as a function of initial cell seeding density, time, and operator. Figure 12B shows TGFβ1 secretion by two MPC lots at 25,000 viable cells / cm 2 Data are shown for cells seeded at 50,000 viable cells / cm. For both lots 345938 and 2011cc063, TGFβ1 levels increased over time (24-120 hours), but remained stable between 68 and 76 hours. There was significant inter-analyst variability in TGFβ1 levels measured at each of the test time points in CM samples from lot 345938. For lot 2011cc063, inter-analyst variability in results was less pronounced. 2Data from cells seeded at 50,000 viable cells / cm are shown in Figure 12B. 2 TGFβ1 levels obtained from cells seeded at 25,000 cells / cm 2 However, similar to the CM samples obtained from cells seeded at a lower density, the levels were higher than those observed when cells were seeded at 50,000 viable cells / cm. 2 TGFβ1 levels in CM from cells seeded at 50,000 viable cells / cm increased over time and remained constant within the 68-76 hour time frame. Results from each analyst for each lot were obtained by seeding cells at 50,000 viable cells / cm. 2 It was possible to compare the results when sowing in the same manner.

[0184] These data are based on 25,000 viable cells / cm 2 Compared to cell seeding at 50,000 viable cells / cm 2 These results suggest that seeding cells at 62°C yields more consistent data between analysts. The data also show that TGFβ1 levels remain constant between 68 and 76 hours, indicating that this time frame (72±4 hours after CBM medium change) represents an acceptable time frame for harvesting CM for TGFβ1 potency assays.

[0185] Neutralization of acid-activated samples using 1N NaOH versus 1.2N NaOH / 0.5M HEPES Prior to the assay, CM samples must be treated with acid to activate latent TGF-β1 and make it an immunoreactive protein detectable by the TGFβ1 ELISA. This is accomplished by adding 1N HCl to the sample and then neutralizing it to a pH of 7.2-7.6. The neutralization step can be performed using 1.2N NaOH / 0.5M HEPES (according to the TGFβ1 ELISA manufacturer's operating instructions) or 1N NaOH. The equivalence of unbuffered and buffered NaOH for neutralization of acidified samples for the TGFβ1 ELISA was determined. CM samples from three MPC lots (345938, 2011cc063, and 2011cc048) were treated with acid. Duplicate samples were then neutralized to pH 7.2–7.4 using 1 N NaOH or 1.2 N NaOH / 0.5 M HEPES. TGF-β1 levels were then measured in duplicate samples by ELISA.

[0186] The data are shown in Table 6. TGFβ1 levels were similar in replicate samples of acid-activated CM neutralized with NaOH and HEPES-buffered NaOH (p>0.05, Student's t-test). Therefore, HEPES-buffered NaOH can be substituted for unbuffered NaOH in CM sample preparation for TGFβ1 potency assays. [Table 6]

[0187] conclusion Two separate operators performed two different initial cell densities (25,000 viable cells / cm 2 and 50,000 viable cells / cm 2 ) was used to test the secretion of TGFβ1 over time. The results showed that the cells were cultured at 50,000 viable cells / cm. 2 These results indicate that when cells were seeded at a high density, values ​​obtained from two operators were more consistent than when cells were seeded at a lower density. Importantly, there was little variability in TGFβ1 levels between samples taken between 68 and 76 hours. Based on these data, MPC CMs can be grown at a density of 50,000 viable cells / cm. 2 It is recommended that MPC CM be prepared by seeding at 72±4 hours after the addition of CBM+0.5% BSA, and that MPC CM for TGFβ1 potency assay be harvested 72±4 hours after the addition of CBM+0.5% BSA.

[0188] The data show that TGFβ1 levels in acid-activated CM samples neutralized with 1N NaOH are comparable to those neutralized with 1.2N NaOH / 0.5M HEPES. Therefore, 1N NaOH is an acceptable alternative to 1.2N NaOH / 0.5M HEPES for neutralizing samples for TGFβ1 ELISA.

[0189] Example 6: Performance of the TGFβ1 assay The performance of the TGFβ1 ELISA was assessed by evaluation of the following parameters: 1. Assay linearity: Standard curves were generated with calibrator diluents or CBM + 0.5% BSA; 2. Matrix interference: rhTGFβ1 was diluted in calibrator diluent or CBM + 0.5% BSA; 3 Assay accuracy and sample linearity: Experiments were performed to determine spike recovery, and TGFβ1 was serially diluted into CM samples.

[0190] Assay Linearity The fit of the standard curve was evaluated. TGFβ1 standard solutions from three separate kits were reconstituted and diluted in the calibrator diluent provided with the ELISA kit or in CBM + 0.5% BSA. Serial dilutions were prepared starting from 2000 pg / ml. The standard curve prepared by the kit manufacturer consisted of seven measurement time points and a zero time point. Therefore, the established standard curve range was 31.2–2000 pg / ml. Each standard concentration was analyzed in duplicate. A four-parameter logistic nonlinear regression curve fit was used to calculate the correlation coefficient (R). 2 ) was measured. Typically, an acceptable standard curve R 2 The correlation coefficients for the three standard curves prepared using the calibrator dilutions ranged from 0.991 to 1.000, and the R for the three standard curves prepared using CBM + 0.5% BSA was ≥ 0.95. 2 = 1.000. The standard curve is shown in Figure 13. To ensure the accuracy of the standard curve, the TGFβ1 concentrations were back-calculated and the % recovery was determined. Overall, the % recovery was found to be within 80%-120% (Table 7). [Table 7]

[0191] Matrix Interference To test for matrix interference, rhTGF-β1 (R&D Systems) was prepared at 0 pg / ml, 50 pg / ml, 250 pg / ml, and 1,500 pg / ml in calibrator diluent or CBM + 0.5% BSA. Each concentration was prepared in duplicate. The TGF-β1 concentration in each sample was measured by ELISA. The mean concentration and percent recovery for each sample are presented in Table 8. For calibrator diluent, percent recovery ranged from 70.4 to 74.7%, and for CBM + 0.5% BSA, recovery ranged from 94.5 to 106.1%. [Table 8]

[0192] Additionally, matrix effects were assessed by comparing standard curves generated with calibrator dilutions to those generated with CBM + 0.5% BSA (parallel analysis on the same plate). Each standard curve was represented in duplicate, and two independent experiments were performed. Figure 14 shows that the mean OD was slightly higher for the standard curve generated with CBM than for the standard curve generated with calibrator dilutions, indicating a matrix effect.

[0193] Assay accuracy A spike recovery experiment was performed to assess the accuracy of the assay. Three different concentrations of rhTGFβ1 (50 pg / ml, 250 pg / ml, and 500 pg / ml) were spiked into CM derived from three MPC lots (345938, 2011cc063, and 2011cc048). Each condition was evaluated in duplicate. The percent recovery of TGFβ1 at each concentration was calculated using the following formula: [average measured concentration / predicted concentration] x 100

[0194] The mean measured concentrations correspond to the TGFβ1 concentrations in the spiked samples, as determined from the standard curve. Results from three sets of data are shown in Table 9. Acceptable spike recoveries are typically in the range of 80-120%. Percent recovery values ​​for all samples ranged from 96.17% to 126.87%. The mean TGFβ1 recoveries at each test concentration were calculated to be 112.1% at 50 pg / ml, 103.8% at 250 pg / ml, and 97.8% at 500 pg / ml. [Table 9]

[0195] Sample Linearity Sample linearity was assessed by testing CM samples from three different MPC lots (22-12-002US, 1857469, 345938) undiluted and at 2-, 5-, and 10-fold dilutions. Each sample was evaluated in duplicate. Precision (% drift) at each dilution of a given sample was calculated as follows: % Drift = (Result - Mean of all dilution results) / Mean of all dilution results x 100

[0196] The results are summarized in Table 10. The % drift ranged from -17.8 to 10.5%. The allowable % drift is typically ±20%. [Table 10] conclusion The data support the suitability of the TGFβ1 ELISA for measuring TGFβ1 in CM harvested from MPC cultures. 2 The values ​​ranged from 0.991 to 1.000, and the TGFβ1 recoveries at each standard concentration were found to be within 80-120%. Experiments performed to investigate matrix effects showed that TGFβ1 recoveries were higher with CBM + 0.5% BSA compared to the calibrator diluent, and the standard curve constructed with CBM + 0.5% BSA was slightly shifted upward and to the right compared to the calibrator diluent. Therefore, it is recommended that the standard curve be constructed with CBM + 0.5% BSA.

[0197] [References] Adams, M. A., & Roughley, P. J. (2006). Spine, 31, 2151 - 2161. Ausubel, F. M. (Ed.). (1987 including all updates untill present). Current Protocols in Molecular Biology. New York: John Wiley & Sons. Bae, W. C., & Masuda, K. (2011). The Orthopedic Clinics of North America, 2011, 585 - 601, ix. Bjornsson, S. (1993). Analytical Biochemistry, 210, 282 - 291. Brown, T. A. (Ed.). (1991). Essential Molecular Biology: A Practical Approach (Vol.1 and 2). Oxford: IRL Press at Oxford University Press. Buchschacher, G. L., & Panganiban, A. T. (1992). Journal of Virology, 2731 - 2739. Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., & Yee, J. K. (1993). Proceedings of the National Academy of Sciences USA, 8033 - 8037. Carter, B. J. (1992). Current Opinion in Biotechnology, 533 - 539. Cheung, K. M., Karppinen, J., Chan, D., Ho, D. W., Song, Y. Q., Sham, P., et al. (2009). Spine, 34, 934 - 940. Cima,L.G.,Ingber,D.E.,Vacanti,J.P.,&Langer,R.(1991).Biotechnology Bioengineering,38,145-158. Coligan,J.E.,Kruisbeek,A.M.,Margulies,D.H.,Shevach,E.M.,&Strober,W.(Eds.).(1991 including all updates until present).Current Protocols in Immunology.New York: John Wiley &Sons. Crock,H.V.,&Goldwasser,M.(1984).Spine,9,702-706. Fisher-Hoch,S.P.,McCormick,J.B.,Auperin,D.,Brown,B.G.,Castor,M.,Perez,G.,et al.(1989).Proceedings of the National Academy of Sciences USA,56,317-321. Freemont,A.J.(2009).Rheumatology,48,5-10. Glover,M.,&Hames,B.D.(Eds.).(1995 and 1996).DNA Cloning: A Practical Approach (Vols.1-4). Gronthos,S.(2003).Journal of Cell Science,116(Pt 9),1827-1835. Gronthos,S.,&Simmons,P.J.(1995).Blood,85(4),929-940. Gruber ,H.E.,Ingram,J.A.,Davis,D.E.,&Hanley,E.N.(2009).The Spine Journal : Official journal of the American Spine Society,9,210-215. Gruber,H.E.,Hoelscher,G.L.,&Hanley,E.N.(2010).The Spine Journal : Official journal of the North American Spine Society,10,721-727. Guerin,H.L.,&Elliott,D.M.(2007).Journal of Orthopaedic Research - Official publication of the Orthopaedic Research Society,25,508-516. Harlow,E.,&Lane,D.(1988).Antibodies: A Laboratory Manual.New York: Cold Spring Harbor Laboratory Press. Hilton,R.C.,&Ball,J.(1984).Annals of the Rheumatic Diseases,43,302-307. Humzah,M.D.,&Soames,R.W.(1988).The Anatomical Record,220,337-356. Johann,S.V.,Gibbons,J.J.,&O’Hara,B.(1992).Journal of Virology,65,1635-1640. Johnson,W.E.,&Roberts,S.(2007).Biochemical Society Transactions,35,680-682. Johnson,W.E.,Caterson,B.,Eisenstein,S.M.,Hynds,D.L.,Show,D.M.,&Roberts,S.(2002).Arthritis and Rheumatism,46,2658-2664. Johnstone,B.,Hering,T.M.,Caplan,A.I.,Goldberg,V.M.,&Yoo,J.U.(1998).Experimental Cell Research,238,265-272. Kotin,R.M.(1994).Human Gene Therapy,793-801. Le Maitre,C.L.,Freemont ,A.J.,&Hoyland,J.A.(2005).Arthritis Research and Therapy,7,R732-745. Le Maitre,C.L.,Hoyland,J.A.,&Freemont,A.J.(2007).Arthritis Research and Therapy,9,R77. Le Maitre,C.L.,Hoyland,J.A.,&Freemont,A.J.(2007).Arthristis Research and Therapy,9,R83. Le Maitre,C.L.,Pockert,A.,Buttle,D.J.,Freemont,A.J.,&Hoyland,J.A.(2007).Biochemical Society Transactions,2007,652-655. Lebkowski,J.S.,McNally,M.M.,Okarma,T.B.,&Lerch,L.B.(1988).Molecular and Cellular Biology,3988-3996. Loreto,C.,Musumeci,G.,Castorina,A.,Loreto,C.,&Martinez,G.(2011).Annals of Anatomy = Anatomischer Anzeiger : Official organ of the Anatomische Gesellschaft,193,156-162. Marchand,F.,&Ahmed,A.M.(1990).Spine,15,402-410. Masuda,K.(2008).European Spinel Journal : Official publication of the European Spine Society,the European Spinal Deformity Society,and the European Section of the Cervical Spine Research Society,17 Suppl 4,441-451. Melrose,J.,Ghosh,P.,Taylor,T.,Latham,J.,&Moore,R.(1997).Journal of Spinal Disorders,10(1),56-67. Melrose,J.,Roberts,S.,Smith,S.,Menage,J.,&Ghosh,P.(2002).Spine,27,1278-1285. Melrose,J.,Smith,S.,Ghosh,P.,&Taylor,T.(2001).Cells Tissues Organs,168,137-146. Miller,A.D.(1990).Human Gene Therapy,7,5-14. Miller,A.D.,&Rosman,G.J.(1989).Biotechniques,7,980-990. Miller,A.D.,Garcia,J.V.,von Suhr,N.,Lynch,C.M.,Wilson,C.,&Eiden,M.V.(1991).Journal of Virology,65,2220-2224. Murata,Y.,Onda,A.,Rydevik,B.,Takahashi,I.,&Olmarker,K.(2006).Spine,31,530-535. Muzyczka,N.(1992).Current Topics in Microbiology and Immunology,158,97-129. O’Halloran,D.M.,&Pandit,A.S.(2007).Tissue Engineering,13,1927-1954. Perbal,B.V.(1984).A Practical Guide to Molecular Cloning.New York: Wiley. Purmessur,D.,Walter,B.A.,Roughley,P.J.,Laudier,D.M.,Hecht,A.C.,&Iatridis,J.A.(2013).Biochemical and Biophysical Research Communications,433,151-156. Raj,P.P.(2008).Pain Practice,8,18-44. Risbud,M.V.,&Shapiro,I.M.(2014).Nature Reviews.Rheumatology,10,44-56. Roberts,S.,Eisenstein,S.M.,Menage,J.,Evans,E.H.,&Ashton,I.K.(1995).Spine,20,2645-2651. Roberts,S.,Evans,H.,Trivedi,J.,&Menage,J.(2006).The Journal of Bone and Joint Surgery.American Volume,88 Suppl 2,10-14. Sambrook,J.,&Green,M.R.(2012).Molecular Cloning: A Laboratory Manual (Fourth Edition).New York: Cold Spring Harbour Laboratory Press. Scarpa,M.,Cournoyer,D.,Munzy,D.M.,Moore,K.A.,Belmont,J.W.,&Caskey,C.T.(1991).Virology,75,849-852. Schmidt,H.,Kettler,A.,Heuer,F.,Simon,U.,Claes,L.,&Wilke,H.J.(2007).Spine,32,748-755. See,F.,Seki,T.,Psaltis,P.J.,Sondermeijer,H.P.,Gronthos,S.,Zannettino,A.C.,et al.(2011).Journal of Cellular and Molecular Medicine,15,2117-2129. Seguin,C.A.,Pilliar,R.M.,Roughley,P.J.,&Kandel,R.A.(2005).Spine,30,1940-1948. Shamji,M.F.,Setton,L.A.,Jarvis,W.,So,S.,Chen,J.,Jing,L.,et al.(2010).Arthritis and Rheumatism,62,1974-1982. Shelling,A.N.,&Smith,M.G.(1994).Gene Therapy,7,165-169. Shen,B.,Melrose,J.,Ghosh,P.,&Taylor,T.(2003).European Spine Journal,12,66-75. Sommerfelt,M.A.,&Weiss,R.A.(1990).Virology,76,58-59. Urban,J.P.,Smith,S.,&Fairbank,J.C.(2004).Spine,29,2700-2709. Vacanti,C.A.,Langer ,R.,Schloo,B.,&Vacanti,J.P.(1991).Plastic Reconstructive Surgery,88,753-749. Vacanti,J.P.,Morse,M.A.,&Saltzman,W.M.(1988).Journal of Pediatric Surgery,23,3-9. Vincent,K.A.,Moore,G.K.,&Haigwood,N.L.(1990).Vaccine,353-359. Wadstrom,J.,&Tengblad,A.(1993).Journal of Medical Science,98,159-167. Wang,J.,Markova,D.,Anderson,D.G.,Zheng,Z.,Shapiro,I.M.,&Risbud,M.V.(2011).The Journal of Biologoical Chemistry,286,39738-39749. Watanabe,H.,Yamada,Y.,&Kimata,K.(1998).Journal of Biochemistry,124,687-693. Weigel,P.H.,Fuller,G.M.,&Le Boeuf,R.D.(1986).Journal of Theoretical Biology,119,219-234. Wilson,C.,Reitz,M.S.,Okayama,H.,&Eiden,M.V.(1989).Journal of Virology,63,2374-2378. Zannettino,A.C.,Buhring,H.J.,Niutta,S.,Watt,S.M.,Benton,M.A.,&Simmons,P.J.(1998).Blood,92(8),2613-2628. Zhou,S.Z.,Cooper,S.,Kang,L.Y.,Ruggieri,L.,Heimfeld,S.,Srivastava,A.,et al.(1994).The Journal of Experimental Medicine,179,1867-1875.

Claims

1. (i) obtaining a population comprising mesenchymal progenitor or stem cells; (ii) culturing the cells in a medium; and (iii) measuring the amount of TGFβ1 released into the medium by the cells, wherein the amount of TGFβ1 released into the medium is at least about 2800 pg / 10 ６ A method for measuring the potency of mesenchymal progenitor or stem cells, comprising determining the amount of TGFβ1 in the cells to indicate biological activity or therapeutic efficacy.

2. The method of claim 1 , wherein the population is enriched in mesenchymal progenitor or stem cells.

3. The method of claim 1 or 2, wherein the mesenchymal progenitor or stem cells are human mesenchymal progenitor or stem cells.

4. The method of any one of claims 1 to 3, wherein the biological activity comprises the ability of the cells to stimulate collagen production in human annulus fibrosus cells in vitro.

5. The method of any one of claims 1 to 4, wherein the therapeutic benefit comprises therapeutic benefit in treating degenerative disc disease.

6. The method comprises culturing a culture vessel at about 50,000 viable cells / cm. ２ 6. The method of claim 1, comprising seeding the cells at a density of

7. The method of any one of claims 1 to 6, wherein the method comprises culturing the cells in chondrogenic basal medium supplemented with 0.5% bovine serum albumin.

8. The method of any one of claims 1 to 7, wherein the cells are cultured for at least 68 to 76 hours.

9. The method of any one of claims 1 to 8, wherein the method comprises taking a sample of the medium in which the cells have been cultured.

10. The method of claim 9, wherein the method comprises first activating latent TGFβ1 in the medium and then measuring the amount of TGFβ1 in the medium.

11. 11. The method of claim 10, wherein activating latent TGFβ1 comprises adding an acid to the medium sample to lower the pH of the medium.

12. 12. The method of claim 11, wherein the method comprises first concentrating the medium sample and then lowering the pH.

13. The method according to claim 11 or 12, wherein after the addition of the acid, the medium is neutralized to a pH of 7.2 to 7.

6.

14. The method according to any one of claims 1 to 13, wherein the method comprises measuring the amount of TGFβ1 in the culture medium by enzyme-linked immunosorbent assay (ELISA).

15. 15. An isolated cell population comprising mesenchymal progenitor or stem cells selected for use in therapy, wherein the cell population has a concentration of 2800 pg / 10 when assessed by the method of any one of claims 1 to 14. ６ The cell population releases cellular TGFβ1.

16. 1. An isolated cell population comprising mesenchymal progenitor or stem cells, the cell population being selected for therapeutic use by measuring TGFβ1 release under culture conditions.

17. 17. The isolated population of claim 15 or claim 16, wherein the mesenchymal progenitor or stem cells comprise at least 5% of the cell population.

18. A composition comprising the isolated population of any one of claims 15 to 17 and a cryoprotectant.

19. 18. A composition comprising the isolated population of any one of claims 15 to 17, wherein the composition comprises hyaluronan.

20. 18. A method of treating a subject suffering from degenerative disc disease, comprising administering to the subject a composition comprising the isolated cell population of claim 16 or 17.

Images