Method for treating lower back pain

Abstract

To solve the problem in which: although a variety of therapeutic agents have been used for treating pain and / or inflammation, including chronic pain and / or inflammation, the treatment is often still ineffective; in particular, back pain is often poorly managed or controlled even by chronic administration of such agents; this may be due to the loss of potency of the agent and / or the development of side effects associated with chronic treatment with the agent.SOLUTION: A method of treating lower back pain in a subject in need thereof comprises administering to the subject a composition comprising mesenchymal lineage precursor or stem cells (MLPSCs), where the lower back pain is associated with an intervertebral disc that has an intervertebral disc height that is not substantially reduced compared to that of an adjacent healthy intervertebral disc in the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to compositions comprising mesenchymal precursor or stem cells for treating low back pain. [Background technology]

[0002] Low back pain is a chronic condition associated with inflammation that affects approximately two-thirds of the adult population in the United States, leads to a significant increase in hospital visits, and has a significant effect on disability.

[0003] Intervertebral disc degeneration, which primarily manifests as low back pain, represents a considerable social and economic burden to society. The condition is associated with increased long-term disability and reduced quality of life. An estimated 80% of the population will experience at least one significant episode of low back pain during their lifetime, and approximately 2.5% of the working population will take several sick days per year due to low back pain. The direct costs of low back pain in Western countries today are estimated to be in the billions of dollars, most of which is spent on consultations with general practitioners, physiotherapists, and other traditional medical practitioners. 2、3 Total indirect expenditures, including surgical procedures, can be more than 10 times higher. 4

[0004] Low back pain symptoms often resolve spontaneously as patients adapt their lifestyles to accommodate limited mobility. However, surgical intervention is often required, with the "gold standard" spinal fusion providing immobilization at one or more painful levels. Long-term studies suggest that fusion may actually promote degeneration at adjacent levels. 5 Pseudarthrosis may also occur, and subjects undergoing repeated surgical fixation may still experience fusion failure.

[0005] Oxycodone, morphine, and oxymorphone have been used in clinical studies of patients with chronic back pain. Oxycodone sustained-release and immediate-release compositions have been used in clinical studies of patients with stable, chronic, moderate-to-severe low back pain, as described by Hale et al., Clin. J. Pain, 15, 179-183 (1999). A morphine sulfate sustained-release product identified as AVINZA® (Ligand Pharmaceuticals Incorporated, San Diego, Calif., USA) is approved for once-daily administration and is indicated for the relief of moderate-to-severe pain requiring long-term, 24-hour opioid therapy. Oxymorphone sustained-release compositions have been used in clinical studies of ambulatory patients with moderate-to-severe chronic low back pain, as described by Hale et al., Clin. J. Pain, 6(1), 21-28 (2005). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 7,205,159 [Patent Document 2] U.S. Patent No. 7,271,007 [Patent Document 3] U.S. Patent Application Publication No. 20040228761 [Patent Document 4] U.S. Patent Application Publication No. 20040265926 [Patent Document 5] U.S. Patent No. 4,593,089 [Patent Document 6] U.S. Patent No. 4,751,190 [Patent Document 7] U.S. Patent No. 5,571,728 [Patent Document 8] U.S. Patent No. 6,248,597 [Patent Document 9] International Publication No. 1994 / 026877 [Patent Document 10] U.S. Patent No. 5173414

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Patent document 14

Non-licensed literature

[0007]

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Non-licensed Document 5

[0008] Although various therapeutic agents are used to treat pain and / or inflammation, including chronic pain and / or inflammation, treatments are often ineffective. In particular, back pain is often inadequately managed or controlled, even with chronic administration of such agents. This may be due to a loss of efficacy of the agents and / or the occurrence of side effects associated with chronic treatment with the agents. [Means for solving the problem]

[0009] The present disclosure relates to an improved, commercially available, ex vivo expanded allogeneic mesenchymal progenitor or stem cell (MLPSC) product that has been shown to substantially relieve low back pain associated with conditions other than degenerative disc disease. One potential advantage of the product is that administration may provide long-term treatment of pain after only a single dose.

[0010] Accordingly, the present disclosure provides a method of treating low back pain in a subject in need thereof, comprising administering to the subject a composition comprising mesenchymal progenitor or stem cells (MLPSCs), wherein the low back pain is associated with a condition other than a substantially degenerated intervertebral disc.

[0011] In one embodiment, the low back pain is associated with an intervertebral disc having a disc height that is not substantially reduced compared to the height of adjacent healthy discs in the subject.

[0012] In one embodiment, the low back pain is associated with a disc that has a loss of disc height of <30% compared to the height of adjacent healthy discs in the subject.

[0013] It will be appreciated that disc height can be measured by any suitable method. For example, disc height can be assessed by radiographs, such as an anteroposterior (AP) or lateral radiograph, a flexicon and extension radiograph, or an MRI scan.

[0014] In one embodiment, the back pain is non-radicular in origin or does not result from irritation by compressive forces or from spinal nerve ganglia or dorsal root ganglia.

[0015] In one embodiment, the lower back pain is associated with a herniated disc with a protrusion of up to 3 mm. In one example, there is no radiographic evidence of nerve compression.

[0016] In one embodiment, the subject has a visual analog scale (VAS) back pain score of > 40 prior to treatment. In one embodiment, the subject has an Oswestry Disability Index (ODI) of > 30 prior to treatment.

[0017] In one embodiment, the back pain is associated with nerve penetration into the intervertebral disc.

[0018] In one embodiment, the back pain is associated with inflammation in the intervertebral disc. In one example, the inflammation is associated with pro-inflammatory monocytes, T cells, or other immune cells that secrete pro-inflammatory cytokines.

[0019] The nerve invasion or inflammation is in the disc space, or in the nucleus pulposus, or in the annulus fibrosus of the disc.

[0020] In one embodiment, the subject has chronic back pain. For example, the subject may have chronic back pain for at least six months.

[0021] In one embodiment, a subject is evaluated or selected for treatment based on the subject exhibiting one or more of the conditions described above.

[0022] In one embodiment, the MLPSCs are at least about 2800 pg / 10 6 cells, or at least about 2810 pg / 10 6 cells, or at least about 2820 pg / 10 6 cells, or at least about 2830 pg / 10 6 cells, or at least about 2840 pg / 10 6 cells, or at least about 2850 pg / 10 6 cells, or at least about 2860 pg / 10 6 cells, or at least about 2870 pg / 10 6 cells, or at least about 2880 pg / 106 cells, or at least about 2890 pg / 10 6 cells, or at least about 2900 pg / 10 6 cells, or at least about 2910 pg / 10 6 cells, or at least about 2920 pg / 10 6 cells, or at least about 2930 pg / 10 6 cells, or at least about 2940 pg / 10 6 cells, or at least about 2950 pg / 10 6 cells, or at least about 2960 pg / 10 6 cells, or at least about 2970 pg / 10 6 cells, or at least about 2980 pg / 10 6 cells, or at least about 2990 pg / 10 6 cells, or at least about 3000 pg / 10 6 When cultured in large amounts, they release TGFβ1.

[0023] In one embodiment, the MLPSCs have a saturation level of at least about 400 pg / ml, or at least about 405 pg / ml, or at least about 410 pg / ml, or at least about 415 pg / ml, or at least about 420 pg / ml, or at least about 425 pg / ml, or at least about 430 pg / ml, or at least about 435 pg / ml, or at least about 440 pg / ml, or at least about 445 pg / ml, or at least about 450 pg / ml. pg / ml, or at least about 455 pg / ml, or at least about 460 pg / ml, or at least about 465 pg / ml, or at least about 470 pg / ml, or at least about 475 pg / ml, or at least about 480 pg / ml, or at least about 485 pg / ml, or at least about 490 pg / ml, or at least about 495 pg / ml, or at least about 500 pg / ml.

[0024] In one embodiment, the composition comprises MLPSCs that have been assayed to determine their release of TGFβ1 under culture conditions. In another embodiment, the composition comprises MLPSCs from a population that has been sampled to determine their release of TGFβ1 under culture conditions (i.e., the cells in the composition have not themselves been assayed to determine their release of TGFβ1 under culture conditions).

[0025] In one embodiment, the MLPSCs release TGFβ1 sufficient to stimulate collagen production in human annulus fibrosus cells in vitro. In one embodiment, the MLPSCs release TGFβ1 in an amount sufficient to enhance Sema3A expression in annulus fibrosus cells in vitro.

[0026] In one embodiment, the isolated population of cells comprises culture-expanded mesenchymal progenitor or stem cells. In an alternative embodiment, the isolated population of cells comprises freshly isolated mesenchymal progenitor or stem cells.

[0027] In one embodiment, MLPSCs are isolated by immunoselection. In one embodiment, the cells are immunoselected for expression of TNAP. In one embodiment, the immunoselected cells co-express TNAP and STRO-1. In one embodiment, the immunoselected cells co-express TNAP and STRO-1. bright In one embodiment, the immunoselected cells are culture-expanded prior to administration.

[0028] In one embodiment, the MLPSCs are mesenchymal stem cells. In one embodiment, the mesenchymal stem cells are culture-expanded prior to administration.

[0029] In one embodiment, the MLPSCs comprise at least about 5%, or at least about 10%, or at least about 20%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or 100% of the total cell population of the composition.

[0030] In one embodiment, the composition comprises MLPSCs and a cryopreservation agent.

[0031] In one embodiment, the cryopreservative in the composition is DMSO or Profreeze®.

[0032] In one embodiment, the composition comprises MLPSCs in 42.5% (v / v) Profreeze® / 50% αMEM (v / v) / 7.5% (v / v) DMSO.

[0033] In one embodiment, the composition further comprises 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.

[0034] In one embodiment, the composition is cryopreserved in 42.5% Profreeze™ / 50% αMEM / 7.5% DMSO. In one embodiment, the composition is cryopreserved in Plasmalyte-A, 25% HSA, and DMSO.

[0035] In one embodiment, the composition contains about 1×10 6 Cells ~ approx. 20×10 6 In one embodiment, the composition is administered to a subject at a dose of about 6×10 cells. 6 In one embodiment, the composition is administered to a subject at a dose of about 18×10 cells.6 The cells are administered to the subject in doses.

[0036] In one embodiment, the composition is administered as a single dose.

[0037] In one embodiment, the composition is administered into the nucleus pulposus or annulus fibrosus of the intervertebral disc.

[0038] In one embodiment, administration of MLSPC results in at least a 50% reduction in pain as determined by visual analog scale (VAS) for at least 1 month, or at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months after administration.

[0039] In one embodiment, a 50% pain reduction as determined by (VAS) is achieved without further intervention following administration of the MLPSCs.

[0040] In one embodiment, administration of MLSPC results in at least a 15 point reduction as determined by the Oswestry Disability Index (ODI) for at least 1 month, or at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months after administration.

[0041] In one embodiment, a reduction of at least 15 points as determined by the ODI is achieved without further intervention following administration of the MLPSCs. [Brief explanation of the drawings]

[0042] [Figure 1] Exposure of human annulus fibrosus cells to recombinant TGFβ1 enhances Sema3A expression as measured by intracellular flow cytometry. [Figure 2]Least-squares (LS) mean VAS low back pain (LBP) change from baseline score. Prespecified LS mean VAS LBP change from baseline analysis was adjusted to remove the confounding effect of post-treatment intervention / rescue. Subjects who failed treatment due to post-treatment intervention were imputed with baseline observation carried forward (BOCF) for all post-intervention visits. Subjects with missing data at the time point were excluded from the analysis. Ostelo et al. (Spine 33, 1, 90-94) established minimally important changes (MICs) for the most frequently used questionnaires to assess pain and function in patients with chronic low back pain. [Figure 3] VAS category distribution at 24 months. Subjects who failed treatment due to the intervention were imputed with BOCF for all post-intervention visits. BOCF imputation for patients with missing data. [Figure 4] 12-month results. Ostelo et al. (Spine 33, 1, 90-94) established MICs for the most frequently used questionnaires to assess pain and function in patients with chronic low back pain. Market and payer forecasts are based on a study conducted by LEK and Navigant for Mesoblast. [Figure 5] Patients demonstrating a 50% reduction in VAS back pain without intervention up to 12 months and up to 24 months. To be considered a responder, a subject had to have a 50% reduction in pain as measured by VAS at each specified time point and without intervention up to the most recent specified time point. Missing subjects were considered non-responders. [Figure 6]Mean VAS improvement compared to current treatment. Abdel Shaheed Christina, Maher Chris G, Williams Kylie A, Day Richard, McLachlan Andrew, J. Efficacy, Tolerability, and Dose-Dependent Effects of Opioid Analgesics for Low Back Pain: A Systema'c Review and Meta-analysis. JAMA Internal Medicine. American Medical Association; 2016 July 1; 176(7):958-68. **Subjects who failed treatment due to post-treatment intervention had their BOCF imputed for all post-intervention visits. Subjects with missing data were not included in the analysis. ***Chou Roger, Deyo Richard, Friedly Janna, Skelly Andrea, Weimer Melissa, Fu Rochelle, Dana Tracy, Kraegel Paul, Griffin Jessica, Grusing Sara, Systemic Pharmacologic Therapies for Low Back Pain: A Systematic Review for an American College of Physicians Clinical Practice Guideline, Annals of internal medicine. American College of Physicians;April 4, 2017;166(7):480~492. [Figure 7]LS mean ODI change from baseline score. Prespecified LS mean ODI change from baseline analysis was adjusted to remove the confounding effect of post-treatment intervention / rescue. Subjects who failed treatment due to post-treatment intervention were imputed with BOCF for all post-intervention visits. Subjects with missing data at the missing time points were excluded from the analysis. Ostelo et al. (Spine 33, 1, 90-94) established MICs for the most frequently used questionnaires to assess pain and function in patients with chronic low back pain. [Figure 8] Percentage of responders by points of ODI improvement. Ostelo et al. (Spine 33(1):90-94) established MICs for the most frequently used questionnaires to assess pain and function in patients with chronic low back pain. Historically, the FDA required a 15-point ODI improvement to demonstrate sufficient improvement to support marketing of a spinal implant device. [Figure 9] Percentage of responders showing a 15-point ODI reduction without intervention up to 12 months and up to 24 months. To be considered a responder, a subject had to have a 15-point ODI score reduction at each specified time point and be without intervention up to the most recent specified time point. Missing subjects were considered non-responders. [Figure 10]Comparison of mean ODI with standard of care. *Abdel Shaheed Christina, Maher Chris G, Williams Kylie A, Day Richard, McLachlan Andrew, J. Efficacy, Tolerability, and Dose-Dependent Effects of Opioid Analgesics for Low Back Pain: A Systematic Review and Meta-analysis. JAMA Internal Medicine. American Medical Association; 2016 July 1; 176(7):958-68. **Subjects who failed treatment due to post-treatment interventions had their BOCF imputed for all post-intervention visits. Subjects with missing data were not included in the analysis. ***Chou Roger, Deyo Richard, Friedly Janna, Skelly Andrea, Weimer Melissa, Fu Rochelle, Dana Tracy, Kraegel Paul, Griffin Jessica, Grusing Sara, Systemic Pharmacologic Therapies for Low Back Pain: A Systematic Review for an American College of Physicians Clinical Practice Guideline. Annals of internal medicine. American College of Physicians;April 4, 2017;166(7):480~492. [Figure 11] Composite treatment success by time point. Subjects with missing data will be classified as non-responders. A composite treatment successful responder had a 50% reduction in LBP as measured by VAS at the specified time point and a 15-point improvement in function as measured by ODI at the specified time point and was intervention-free up to the specified time point. [Figure 12]Successful combined treatment over 24 months. Subjects with missing data will be classified as non-responders. Successful combined treatment responders had a 50% reduction in LBP as measured by VAS at the specified time point, a 15-point improvement in function as measured by ODI at the specified time point, and remained intervention-free up to the most recent specified time point. DETAILED DESCRIPTION OF THE INVENTION

[0043] General implementation methods and definitions Throughout this specification, unless specifically stated otherwise or the context dictates otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter is intended to encompass one and more (i.e., one or more) of those steps, compositions of matter, group of steps or group of compositions of matter.

[0044] Those skilled in the art will understand that the disclosure described herein is susceptible to variations and modifications other than those specifically described. The present disclosure should be understood to include all such variations and modifications. The present disclosure also includes all of the steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of said steps or features, or any two or more thereof.

[0045] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0046] Any example disclosed herein shall be construed to apply mutatis mutandis to any other example unless expressly stated otherwise.

[0047] Unless expressly defined otherwise, all technical and scientific terms used herein shall be construed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, stem cell differentiation, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0048] 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 described and explained throughout the literature in sources such as Perbal, 1984; Sambrook & Green, 2012; Brown, 1991; Glover & Hames, 1995 and 1996; Ausubel, 1987 (including all current revisions), Harlow & Lane, 1988; and Coligan et al., 1991 (including all current revisions).

[0049] As used in this specification and the appended claims, the singular and singular terms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise, for example, as the case may be.

[0050] The term "subject," as used herein, refers to a mammal, including humans and non-human animals. In one embodiment, the mammal is a human. Terms such as "subject," "patient," or "individual" are terms that can be used interchangeably in this disclosure, where context permits. In certain examples, the subject may be an adult or a child (pediatric) subject.

[0051] An "effective amount" refers to at least an amount effective, at the dosage and for the time necessary, to achieve the desired therapeutic or preventive result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" is used to refer to the amount necessary to perform treatment of a disease or condition as described above. The effective amount may vary depending on the disease or condition being treated, as well as on the body weight, age, ethnic background, sex, health and / or physical condition, and other factors associated with the mammal being treated. Typically, the effective amount falls within a relatively broad range (e.g., a "dosage" range) that can be determined by routine trial and experimentation by a practitioner. The effective amount can be administered once or several times over the treatment period, in a single dose or repeated doses.

[0052] The term "and / or", e.g., "X and / or Y", shall be understood to mean either "X and Y" or "X or Y" and shall be interpreted as providing explicit support for both meanings or either meaning.

[0053] As used herein, the term "about" refers to + / - 10%, more preferably + / - 5% of the specified value, unless otherwise specified.

[0054] Throughout this specification, the term "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.

[0055] Mesenchymal progenitor or stem cells As used herein, the term "mesenchymal progenitor or stem cells" refers to undifferentiated multipotent cells that have the ability to self-renew while maintaining pluripotency and to differentiate into multiple cell types, either of mesenchymal origin, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts and tendons, or of non-mesodermal origin, such as hepatocytes, neurons and epithelial cells.

[0056] The term "mesenchymal precursor or stem cell" includes both the parent cell and its undifferentiated progeny. The term also includes mesenchymal precursor cells (MPCs), multipotent stromal cells, mesenchymal stem cells, perivascular mesenchymal precursor cells, and their undifferentiated progeny.

[0057] Mesenchymal progenitor or stem cells can be autologous, allogeneic, xenogeneic, syngeneic, or isogeneic. Autologous cells are isolated from the same individual into whom they will be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or isogeneic cells are isolated from genetically identical organisms such as twins, clones, or highly inbred research animal models.

[0058] Mesenchymal progenitor or stem cells reside primarily in bone marrow, but have also been shown to reside in a variety of host tissues, including, for example, umbilical cord blood and cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp.

[0059] Mesenchymal progenitor or stem cells can be isolated from host tissue and enriched by immunoselection. For example, bone marrow aspirate from a subject can be further processed 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 by using STRO-1 antibodies as described in Simmons & Torok-Storb, 1991.

[0060] STRO-1+ cells are cells 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 lineages such as mesoderm and / or endoderm and / or ectoderm. STRO-1+ cells can therefore differentiate into numerous cell types, including, but not limited to, adipose, bone, cartilage, elastic, muscle, and fibrous connective tissue. The specific lineage-commitment and differentiation pathways these cells enter depend on a variety of influences from mechanical and / or endogenous bioactive factors, such as growth factors, cytokines, and / or local microenvironmental conditions established by the host tissue.

[0061] The term "enriched," as used herein, describes a population of cells in which the proportion of one particular cell type or the proportion of multiple particular cell types is increased when compared to an untreated population of cells (e.g., cells in their natural environment). In one example, a population enriched for STRO-1+ cells comprises at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% STRO-1+ cells. In this regard, the term "population of cells enriched for STRO-1+ cells" is used to expressly support the term "a population of cells comprising X% STRO-1+ cells," where X% is a percentage as described herein. STRO-1+ cells, in some examples, can form and possess the activity of clonogenic colonies, such as CFU-F (fibroblasts) or a subset thereof (e.g., 50%, 60%, 70%, 70%, 90%, or 95%). In one example, a population enriched for TNAP+ cells comprises at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% TNAP+ cells. In this regard, the term "population of cells enriched for TNAP+ cells" is used to expressly support the term "population of cells comprising X% TNAP+ cells," where X% is a percentage as described herein. In one example, a population enriched for STRO-1+ and TNAP+ cells comprises at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% STRO-1+ and TNAP+ cells. In this regard, the term "a population of cells enriched for STRO-1+ and TNAP+ cells" is used to expressly support the term "a population of cells comprising X% STRO-1+ and TNAP+ cells," where X% is a percentage as described herein.

[0062] In one example, a cell population is enriched from a cell preparation that contains STRO-1+ cells in a selectable form. In this regard, the term "selectable form" is 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 may be, but need not be, STRO-1. For example, as described and / or exemplified herein, cells (e.g., MPCs) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (and STRO-1 bright (It may be that the cells are STRO-1+. Thus, the indication that cells are STRO-1+ 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., they are STRO-3+ (TNAP+).

[0063] Reference to the selection of cells or populations thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells may be selected from, isolated from, or enriched from a wide variety of sources. However, in some instances, these terms support selection from any one or more tissues, including STRO-1+ cells, or vascularized tissue, or tissue containing pericytes (e.g., STRO-1+ pericytes), or tissues described herein.

[0064] In one example, the mesenchymal progenitor or stem cells of the present disclosure 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+.

[0065] "Individually" means that the present disclosure encompasses the described markers or groups of markers separately, and that notwithstanding that individual markers or groups of markers may not be separately recited herein, the appended claims may define such markers or groups of markers separately and separately from one another.

[0066] "Collectively" means that the present disclosure encompasses any number or combination of the described markers or groups of markers, and that notwithstanding that such number or combination of markers or groups of markers may not be specifically recited herein, the appended claims may define such combinations or subcombinations separately and apart from any other combinations of markers or groups of markers.

[0067] Cells referred to as "positive" for a given marker can express either low (lo or dim or dull), intermediate (median), or high (bright, bri) levels of that marker, depending on the extent to which the marker is present on the cell surface; these terms refer to the intensity of fluorescence or other markers used in the cell sorting process or flow cytometry analysis of cells. The distinctions of low (lo or dim or dull), intermediate (median), and high (bright, bri) are understood in the context of the marker used on the particular cell population being sorted or analyzed. Cells referred to as "negative" for a given marker do not necessarily mean that the cell is completely absent. This term means that the marker is expressed at a relatively very low level by the cell, producing a very low signal when detectably labeled, or is undetectable above background levels, e.g., levels detected using an isotype control antibody.

[0068] The term "bright" or "bri," as used herein, refers to a marker on the cell surface that generates a relatively high signal when detectably labeled. Without wishing to be limited 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 labeled with FITC-conjugated STRO-1 antibody were found to be non-bright cells (STRO-1) as determined by fluorescence-activated cell sorting (FACS) analysis. lo / dim / dull / intermediate / median ) produce a fluorescent signal greater 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 The cells have a 2 log increase in STRO-1 surface expression compared to "background," i.e., STRO-1- cells. lo / dim / dull and / or STRO-1 intermediate / median The cells exhibit less than 2 log orders of magnitude higher expression of STRO-1 surface expression, typically about 1 log or below "background."

[0069] In one example, STRO-1+ cells are STRO-1 bright In one example, STRO-1 bright STRO-1 cells lo / dim / dull or STRO-1 intermediate / median are preferentially enriched relative to cells.

[0070] In one example, STRO-1 brightThe 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 shown to express one or more of the aforementioned markers. In this regard, cells shown to express a marker need not be specifically tested; rather, previously enriched or isolated cells can be tested, and then it can be reasonably assumed that they also express the same marker.

[0071] In one example, STRO-1 bright The cells are perivascular mesenchymal progenitor cells as defined in WO 2004 / 85630, characterized by the presence of the perivascular marker 3G5.

[0072] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue-nonspecific alkaline phosphatase. For example, this term encompasses liver isoform (LAP), bone isoform (BAP) and kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP refers to a molecule capable of binding to STRO-3 antibody produced by the hybridoma cell line deposited with ATCC on December 19, 2005 under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0073] Furthermore, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0074] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two different germ lineages. Non-limiting examples of lineages that cells can commit include bone progenitors; hepatocyte precursors that are multipotent for bile duct epithelial cells and hepatocytes; neural-restricted cells that can generate glial cell precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; cardiac muscle and cardiomyocyte precursors; glucose-responsive insulin-secreting pancreatic beta cell lineages. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as the following: 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, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, neurons, astrocytes, and oligodendrocyte precursors.

[0075] In one example, the mesenchymal progenitor or stem cells are mesenchymal stem cells (MSCs). MSCs can be a homogenous composition or a mixed cell population enriched for MSCs. A homogenous MSC composition can be obtained by culturing adherent bone marrow or periosteal cells, and MSCs can be identified by specific cell surface markers identified with specific monoclonal antibodies. A method for obtaining a cell population enriched for MSCs using plastic adhesion technology is described, for example, in U.S. Patent No. 5,486,359. MSCs prepared by conventional plastic adhesion isolation rely on the nonspecific plastic adhesion properties of CFU-Fs. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium.

[0076] The mesenchymal precursor or stem cells can be cryopreserved prior to administration to a subject.

[0077] In a preferred embodiment of the invention, the mesenchymal progenitor or stem cells are obtained from a master cell bank derived from mesenchymal progenitor or stem cells enriched from the bone marrow of healthy volunteers. The use of mesenchymal progenitor or stem cells from such a source is particularly advantageous for subjects who do not have suitable family members available who can serve as mesenchymal progenitor or stem cell donors or who require immediate treatment and who are at high risk of relapse, disease-related decline, or death during the time required to generate mesenchymal progenitor or stem cells.

[0078] The present inventors demonstrate that the mesenchymal progenitor cells of the present disclosure have unexpectedly high potency in their ability to inhibit T cell proliferation after cryopreservation and thawing. In contrast, previous disclosures teach that cryopreserved mesenchymal stem cells exhibit impaired immunosuppressive properties after thawing (Francois et al., 2012; Chinnadurai et al., 2016).

[0079] Isolated or enriched mesenchymal progenitor or stem cells can be expanded ex vivo or in vitro by culture. As will be appreciated by those skilled in the art, isolated or enriched mesenchymal progenitor or stem cells can be cryopreserved, thawed, and then further expanded ex vivo or in vitro by culture.

[0080] Cultured mesenchymal progenitor or stem cells are phenotypically distinct from cells in vivo, for example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b.

[0081] Cultured mesenchymal progenitor or stem cells are biologically distinct from cells in vivo, having a higher proliferation rate compared to mostly non-cycling (quiescent) cells in vivo.

[0082] In one example, a cell population enriched for mesenchymal progenitor or stem cells can be cultured at approximately 6,000-7,000 viable cells / cm in serum-supplemented culture medium, e.g., Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine. 2 and allowed to adhere to the culture vessel overnight at 37° C. and 20% O. In one embodiment, the cells are at about 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6810, 6820, 6830, 6840, 6850, 6860, 6870, 6880, 6890, 6890, 6900, 6910, 6920, 6930, 6940, 6970, 6980, 6990, or 7000 viable cells / cm. 2 , preferably about 6850-6860 viable cells / cm 2 The culture medium is then replaced and the cells are cultured at 37°C and 5% O for a total of 68-72 hours before co-culture with T cells and determination of the amount of IL-2Rα expressed by the T cells.

[0083] Determination of TGFβ1 levels The present disclosure contemplates any form of assay for determining TGFβ1 levels, including Western blot, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), competitive assay, radioimmunoassay, lateral flow immunoassay, flow-through immunoassay, electrochemiluminescence assay, nephelometry-based assay, turbidity-based assay, fluorescence-activated cell sorting (FACS)-based assay for detection of TGFβ1 in culture medium used to culture mesenchymal or progenitor cells, and surface plasmon resonance (SPR or Biacore).

[0084] One form of suitable assay is, for example, ELISA or FLISA.

[0085] 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, and TGFβ1 in the sample is bound or captured. After washing to remove any unbound protein in the sample, a protein that binds to TGFβ1 at a different epitope is directly contacted with the captured TGFβ1. This detection protein is generally 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 detection protein. After washing to remove any unbound protein, the detectable reporter molecule is detected by addition of a substrate such as, for example, hydrogen peroxide, TMB, or toluene, or 5-bromo-4-chloro-3-indole-beta-D-galactopyranoside (x-gal) in the case of an ELISA. Of course, the immobilized (capture) protein and detection protein can be used in the reverse manner.

[0086] The level of antigen in the sample is then determined using a standard curve generated using known amounts of marker or by comparison to a control sample.

[0087] The assay described above is easily modified to use chemiluminescence or electrochemiluminescence as the detection principle.

[0088] As will be apparent to one of skill in the art, other detection methods based on immunosorbent assays are useful in the practice of the present disclosure, such as immunosorbent methods based on the above description using radioactive labels for detection, or gold labels (e.g., colloidal gold) for detection, or liposomes, e.g., encapsulated NAD+ for detection, or acridinium-linked immunosorbent assays.

[0089] In some examples of the present disclosure, the level of TGFβ1 is determined 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. Patent Application Publication No. 20040228761 or U.S. Patent Application Publication 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).

[0090] In one embodiment, the method comprises seeding MLPSCs at about 50,000 viable cells / cm 2 in a culture vessel.

[0091] In one embodiment, the method comprises culturing MLPSCs in chondrogenic basal medium supplemented with 0.5% bovine serum albumin.

[0092] In one embodiment, the method includes 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.

[0093] In one embodiment, the method comprises collecting a sample of the culture medium in which the MLPSCs were cultured, hi one embodiment, the collected sample comprises the entire culture medium in which the cells were cultured.

[0094] In one embodiment, the method comprises the step of activating latent TGFβ1 in the culture medium before determining the amount of TGFβ1 in the culture medium.

[0095] In one embodiment, activating latent TGF-β1 comprises adding an acid, e.g., 1N HCl, to the culture medium to lower the pH of the culture medium. In one embodiment, the method comprises concentrating the culture medium sample prior to lowering the pH. In one embodiment, the method comprises neutralizing the pH of the culture medium to 7.2-7.6 after adding the acid, e.g., by adding 1.2N NaOH / 0.5M HEPES or 1N NaOH.

[0096] In one embodiment, the method comprises determining the amount of TGFβ1 in the culture medium by enzyme-linked immunosorbent assay (ELISA).

[0097] In one example, ELISA is (i) diluting the culture medium 1:5 with sample diluent; (ii) adding the diluted culture medium to wells of a microplate pre-coated 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 a TGFβ1 conjugate 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 in a microplate reader set at 450 nm with wavelength correction at 570 nm; (xiii) determining the concentration of TGFβ1 corrected for dilution Includes.

[0098] In one embodiment, the sample diluent is chondrogenic basal medium supplemented with 0.5% bovine serum albumin.

[0099] In one embodiment, the method comprises: preparing serial dilutions of TGFβ1 standard in sample diluent with final concentrations ranging from 31.2 to 2000 pg / ml; adding standards to the microplate prior to step (iii); constructing a standard curve using a four parameter logistic curve fit; and determining the concentration of TGFβ1 in the culture medium by reference to a standard curve Further includes:

[0100] In one embodiment, the method for determining the potency of MLPSCs comprises: (i) obtaining a population of MLPSCs; (ii) seeding the cells at 50,000 viable cells / cm2 in a culture vessel; (iii) culturing the cells in chondrogenic basal medium supplemented with 0.5% bovine serum albumin; (iv) recovering the culture medium; (v) activating the latent TGFβ1 released by the cells in the culture medium by adding 1N HCl to lower the pH of the culture medium; (vi) neutralizing the pH of the culture medium to 7.2-7.6 by adding 1.2 N NaOH / 0.5 M HEPES or 1 N NaOH; (vii) diluting the culture medium 1:5 with chondrogenic basal medium supplemented with 0.5% bovine serum albumin; (viii) adding the diluted culture medium to wells of a microplate pre-coated 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 conjugate 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 in a microplate reader set at 450 nm with wavelength correction at 570 nm; (xix) determining the concentration of TGFβ1 corrected for dilution Includes.

[0101] In one embodiment, the method comprises: preparing serial dilutions of TGFβ1 standard in chondrogenic basal medium supplemented with 0.5% bovine serum albumin at final concentrations ranging from 31.2 to 2000 pg / ml; adding standards to the microplate prior to step (ix); constructing a standard curve using a four parameter logistic curve fit; and determining the concentration of TGFβ1 in the culture medium by reference to a standard curve Further includes:

[0102] Compositions and Administration Compositions comprising mesenchymal progenitor or stem cells can be prepared in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a composition of matter that facilitates storage, administration, and / or maintains the biological activity of mesenchymal progenitor or stem cells.

[0103] In one example, the carrier does not cause significant local or systemic adverse effects in the recipient. Pharmaceutically acceptable carriers can be solid or liquid. Useful examples of pharmaceutically acceptable carriers include, but are not limited to, diluents, solvents, surfactants, excipients, 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, isotonic and absorption delaying agents that do not affect the viability and activity of mesenchymal progenitor cells or stem cells. The selection of an appropriate carrier is within the skill of one of ordinary skill in the art.

[0104] 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 materials.

[0105] Mesenchymal progenitor cells or stem cells can also be incorporated or impacted 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. Synthetic biodegradable scaffolds include, but are not limited to, polyglycolic acid scaffolds (e.g., as described in (Vacanti, Morse, and Saltzman, 1988) (Cima, Ingber, Vacanti, and Langer, 1991) (Vacanti, Langer, Schloo, and Vacanti, 1991)), synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid, and gelatin resorbable sponges such as Gelform™ (Pfizer).

[0106] The composition of the present disclosure can be conveniently provided in unit dosage form and can be prepared by any method known in the art.The term "dosage unit form" as used herein refers to a physically discrete unit suitable as a single dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic or preventive effect in association with a pharmaceutical carrier.The dosage of mesenchymal precursor cells or stem cells can vary depending on factors such as the disease state, age, sex, and weight of the subject to be treated.

[0107] An exemplary dose is at least about 1 x 10 6 For example, the dose may be about 1.0 x 10 cells. 6 ~Approx. 1.1×10 10 pieces, for example, about 1.1 x 10 6 ~Approx. 1×10 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.

[0108] In one embodiment, the dose is about 5×10 5 ~about 2×10 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. 6cells or approximately 1.8 x 10 7 It may be a cell.

[0109] The 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.

[0110] The compositions of the present disclosure can be cryopreserved. Cryopreservation of mesenchymal progenitor or stem cells can be performed using slow cooling methods or "rapid" freezing protocols known in the art. Preferably, the cryopreservation method maintains a similar phenotype, cell surface markers, and proliferation rate of the cryopreserved cells compared to non-frozen cells.

[0111] The cryopreservation composition may comprise a cryopreservation solution, the pH of which is typically 6.5 to 8, preferably 7.4.

[0112] The cryopreservation solution may contain a sterile, non-pyrogenic, isotonic solution, such as PlasmaLyteA™. 100 mL of PlasmaLyteA™ contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H11NaO7), 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). It does not contain antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5-8.0).

[0113] The cryopreservation solution may include Profreeze™. The cryopreservation solution may additionally or alternatively include culture medium, for example, αMEM.

[0114] 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, and chemically inert, resulting in high post-thaw survival rates and allowing transplantation without washing. However, DMSO, the most commonly used cryoprotectant, exhibits some cytotoxicity. Hydroxyethyl starch (HES) can be used as a substitute or in combination with DMSO to reduce the cytotoxicity of cryopreservation solutions.

[0115] 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 methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0116] In one embodiment, cells are suspended in 42.5% Profreeze™ / 50% αMEM / 7.5% DMSO and cooled in a controlled rate freezer.

[0117] The cryopreserved composition can be thawed and administered directly to a subject or added to another solution containing, for example, HA. Alternatively, the cryopreserved composition can be thawed and the mesenchymal progenitor or stem cells can be resuspended in an alternative carrier prior to administration.

[0118] The compositions of the present disclosure can be administered by a route appropriate for the particular disease state to be treated. For example, the compositions of the present disclosure can be administered systemically, i.e., parenterally, intravenously, or by injection. The compositions of the present disclosure can be targeted to a specific tissue or organ.

[0119] Dosage regimens can be adjusted to provide optimal therapeutic responses. For example, a single bolus can be administered, multiple divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, parenteral compositions can be advantageously formulated in dosage unit form.

[0120] In some embodiments, it may not be necessary or desirable to immunosuppress a patient before initiating treatment with a cell composition. However, in other instances, it may be desirable or appropriate to pharmacologically immunosuppress a patient before initiating cell therapy. This can be achieved through the use of systemic or local immunosuppressants, or by delivering the cells in an encapsulated device. The cells may be encapsulated within a capsule that allows the cells to receive the nutrients and oxygen they need, as well as immune humoral factors and therapeutic factors that are still impermeable to the cells. Preferably, the encapsulating agent is hypoallergenic, easily and stably located within the target tissue, and provides additional protection to the transplanted structure. These and other means for reducing or eliminating the immune response to transplanted cells are known in the art. Alternatively, the cells may be genetically modified to reduce their immunogenicity.

[0121] It is understood 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, simultaneously with the other agent or sequentially (either 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, nonsteroidal anti-inflammatory drugs (NSAIDs) such as PEMIROLAST™, TRANILAST™, REMICADE™, SIROLIMUS™, and TEPOXALIN™, TOLMETIN™, 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); monoclonal anti-IL-2R alpha receptor antibodies (e.g., basal Antibodies such as antithrombotic agents (e.g., heparin, heparin derivatives, urokinase, PPack (dextrophenylalanine proline arginine chloromethyl ketone), 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.

[0122] genetically modified cells In one embodiment, the mesenchymal precursor or stem cells are not genetically modified. In one embodiment, the mesenchymal precursor or stem cells are genetically modified to express and / or secrete a protein of interest, e.g., a protein that provides a therapeutic and / or prophylactic benefit.

[0123] Methods for genetically modifying cells are apparent to those skilled in the art. For example, the nucleic acid to be expressed in the cell is operably linked to a promoter for inducing expression in the cell. For example, the nucleic acid is linked to a promoter operable in various cells of interest, such as a viral promoter, for example, a CMV promoter (e.g., a CMV-IE promoter) or an SV-40 promoter. Additional suitable promoters are known in the art.

[0124] Preferably, the nucleic acid is provided in the form of an expression construct. The term "expression construct," as used herein, refers to a nucleic acid capable of conferring expression in a cell of an operably linked nucleic acid (e.g., a reporter gene and / or a counter-selectable reporter gene). Within the context of the present disclosure, it should be understood that an expression construct may comprise or be a plasmid, bacteriophage, phagemid, cosmid, viral subgenomic or genomic fragment, or other nucleic acid capable of carrying and / or replicating heterologous DNA in an expressible form.

[0125] Methods for constructing suitable expression constructs for carrying out the present invention will be clear to those skilled in the art and are described, for example, in Ausubel FM, 1987 (including all revisions to date); or Sambrook and 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.

[0126] Suitable vectors for such expression constructs are known in the art and / or described herein. For example, suitable expression vectors for the methods of the invention in mammalian cells are, for example, the pcDNA suite of vectors (Invitrogen), the pCI suite of vectors (Promega), the pCMV suite of vectors (Clontech), the pM vectors (Clontech), the pSI vectors (Promega), the VP16 vectors (Clontech), or the pcDNA suite of vectors (Invitrogen).

[0127] 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.

[0128] Means for introducing isolated nucleic acid molecules or gene 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 known success. Means for introducing recombinant DNA into cells include, inter alia, microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection such as using Lipofectamine (Gibco, MD, USA) and / or Cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and microparticle bombardment such as using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0129] Alternatively, the expression construct of the present invention is a viral vector. Suitable viral vectors are known in the art and commercially available. Conventional viral systems for delivering nucleic acids and integrating 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 episomal nucleic acids into host cells. Viral vectors are a highly efficient and versatile method for gene transfer into target cells and tissues. Furthermore, high transduction efficiency has been observed in many different cell types and target tissues.

[0130] For example, retroviral vectors generally contain cis-acting long terminal repeats (LTRs) with packaging capacity for up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which is then used to integrate an expression construct into target cells to provide 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., WO 1994 / 026877; Buchschacher and Panganiban, 1992; Johann et al., 1992; Sommerfelt and Weiss, 1990; Wilson et al., 1989; Miller et al., 1991; Lynch et al., 1991; Miller and Rosman, 1989; Miller, 1990; Scarpa et al., 1991; Burns et al., 1993).

[0131] 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; WO 92 / 01070 and WO 93 / 03769; Lebkowski et al., 1988; Vincent et al., 1990; Carter, 1992; Muzyczka, 1992; Kotin, 1994; Shelling and Smith, 1994; Zhou et al., 1994).

[0132] Additional viral vectors useful for delivering expression constructs of the invention include those derived from the poxvirus family, such as vaccinia virus and avian poxviruses, or alphaviruses, or conjugated viral vectors (e.g., those described in Fisher-Hoch et al., 1989).

[0133] Treatment results Various methods can be used to assess the effectiveness of treating low back pain by administering MLPSCs.

[0134] For example, the reduction of lower back pain can be evaluated by using a visual analogue scale (VAS). A visual analogue scale (VAS) is a psychometric response scale that can be used in questionnaires. It is a measurement device for subjective characteristics or attitudes that cannot be measured directly. When answering a VAS item, respondents indicate their level of agreement with a statement by indicating their position along the solid line between the two endpoints. See, for example, Reips, U.-D.; Funke, F (2008). "Interval level measurement with visual analogue scales in Internet-based research: VAS Generator," Behavior Research Methods 40:699-704.

[0135] In another example, reduction in low back pain can be assessed by using the Oswestry Disability Index (ODI), an index derived from the Oswestry Low Back Pain Questionnaire used by clinicians and researchers to quantify disability due to low back pain. This validated questionnaire was first published by Jeremy Fairbank in Physiotherapy 1980;66:271-273, and subsequently in Fairbank JC, Pynsent PB, The Oswestry Disability Index. Spine, 2000 Nov. 15;25(22):2940-52.

[0136] The self-administered questionnaire includes 10 topics related to the intensity of pain, suspension, ability to care for oneself, walking ability, sitting ability, sexual function, standing ability, social life, sleep quality, and ability to travel. Each topic category is followed by six references that describe different possible scenarios in the patient's life related to the topic. The patient then checks the reference that most closely resembles their situation. Each question is scored on a scale of 0 to 5, with the first reference being scored as zero, indicating the least amount of disability, and the last reference being scored as 5, indicating the most severe disability. The scores for all answered questions are summed and then multiplied by two to obtain an index (ranging from 0 to 100). Zero is equivalent to no disability, and 100 is the greatest possible disability.

[0137] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. [Example]

[0138] Example 1 Materials and Methods Mesenchymal progenitor or stem cells (MLPSCs) prepared using plastic adhesion techniques MLPSCs were generated de novo from bone marrow as described in U.S. Patent No. 5,837,539. Approximately 80–100 ml of bone marrow was aspirated into a sterile heparin-containing syringe and sent to MDACC Cell Therapy Laboratory for MSC generation. Bone marrow mononuclear cells were isolated using ficoll-hypaque and placed into two T175 flasks with 50 ml per flask of MLPSC expansion medium containing alpha-modified MEM (αMEM) containing gentamicin, glutamine (2 mM), and 20% (v / v) fetal bovine serum (FBS) (Hyclone).

[0139] The cells were cultured at 37°C and 5% CO2 for 2-3 days, at which time non-adherent cells were removed and the remaining adherent cells were continuously cultured until the cells reached 70% or greater confluence (7-10 days), at which point the cells were trypsinized and transferred to six T175 flasks with expansion medium (50 ml of medium per flask).

[0140] Immunoselection of mesenchymal progenitor or stem cells (MLPSC) Bone marrow (BM) was collected from healthy adult volunteers (20-35 years old). Briefly, 40 ml of BM was aspirated from the posterior iliac crest into a tube containing lithium-heparin anticoagulant.

[0141] Bone marrow mononuclear cells (BMMNC) were prepared by density gradient separation using Lymphoprep™ (Nycomed Pharma, Oslo, Norway) as previously described by Zannettino et al., 1998. After centrifugation at 400 × g for 30 minutes at 4°C, the buffy layer was removed with a transfer pipette and washed three times in "HHF" consisting of Hank's balanced salt solution (HBSS; Life Technologies, Gaithersburg, MD) containing 5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).

[0142] STRO-3+ (or TNAP+) cells were isolated by magnetic-activated cell sorting as previously described by Gronthos & Simmons, 1995; and Gronthos, 2003. Briefly, approximately 1-3 × 10 8BMMMNCs are incubated on ice for 20 minutes in blocking buffer consisting of 10% (v / v) normal rabbit serum in HHF. Cells are incubated on ice for 1 hour with 200 μl of a 10 μg / ml solution of STRO-3 mAb in blocking buffer. Cells are 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 is added, and cells are incubated on ice for 1 hour. Cells are then resuspended in MACS buffer (CaCl2+) supplemented with 1% BSA, 5 mM EDTA, and 0.01% sodium azide 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.

[0143] 100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) are added to the cell suspension and incubated on ice for 15 minutes. The cell suspension is washed twice, resuspended in 0.5 ml of MACS buffer, and then loaded onto a miniMACS column (MS column, Miltenyi Biotec) and washed three times with 0.5 ml of MACS buffer to recover cells that do not bind to STRO-3 mAb (deposited with the American Type Culture Collection (ATCC) on December 19, 2005 under accession number PTA-7282 - see WO 2006 / 108229). After adding an additional 1 ml of MACS buffer, the column is removed from the magnet, and TNAP+ cells are isolated by positive pressure. An aliquot of cells from each fraction is stained with streptavidin-FITC, and purity can be assessed by flow cytometry.

[0144] Example 2 Effect of TGF-β1 on neuropathic pain Immunoselected TNAP+ MPCs are selected for incorporation into a product for the treatment of low back pain based on their threshold level of TGFβ1 secretion. Previous studies (e.g., Chen et al., J Clin. Invest 2015;125(8):3226-3240) have shown the following: Neuroinflammation, marked by the presence of II-1β, IL-6 and TNF, is involved in the development of neuropathic pain; ·CCI induced upregulation of Iba1, Il6 and TNF transcripts in the L4-L5 spinal dorsal horn; ·TGFβ1 and IL-10 secreted from BMSCs can regulate neuropathic pain through their anti-inflammatory properties.

[0145] These studies also showed: ·BMSCs secrete large amounts of TGFβ1 but not IL-10 in the presence of TNF and LPS. TGFβ1 was increased in the CSF of CCI mice treated with BMSCs, but not in controls. The effects of BMSCs in CCI mice were counteracted by neutralization of TGF-β1, but not IL-10. · Knockdown of TGFβ1 expression in BMSCs resulted in a 67% reduction in TGFβ1 release and a 55% reduction in TGFβ1 expression in BMSC cultures. The BMSC induction effect was impaired for several days after administration of TGFβ1 knockdown BMSCs. Exogenous TGF-β1 inhibited acute neuropathic pain. Exogenous TGF-β1 inhibited chronic neuropathic pain. TGF-β1 receptor 1 eliminated the effects of TGF-β1.

[0146] Previous studies (Tolofari et al., Arthritis Res Ther 12 (2010)) have also shown that Sema3A functions as a chemopreferent for neurons and blood vessels. Current evidence suggests that sema3A in AF functions as a repellent for neurons in healthy IVDs and that expression is progressively lost with increasing levels of IVD degeneration.

[0147] The results herein show that exposure of human annulus fibrosus cells to recombinant TGF-β1 enhances Sema3A expression as measured by intracellular flow cytometry (Figure 1). This data indicates that MPCs selected for the treatment of low back pain based on a threshold level of TGF-β1 secretion can significantly reduce neurite ingression via a TGF-β1-mediated pathway.

[0148] Example 3 Phase 2 clinical trial Clinical study design This is a randomized, multicenter study with four study arms, two receiving different doses of MPC and two receiving different control injections (saline or hyaluronic acid). The saline control was chosen because it was expected that saline would have no pharmacological effect. Hyaluronic acid was used to serve as a vehicle control and to evaluate any therapeutic effect of hyaluronic acid alone.

[0149] Treatment assignment occurred in chronological order according to a master randomization list. Randomization was performed centrally and allocations were distributed to the 13 participating centers. All subjects and radiographic assessors at the core laboratory were blinded to the randomized treatment assignment. The study center investigators, associated study staff, and sponsor were not blinded to the randomization assignment.

[0150] The study had some preliminary efficacy results based on data collected at day 30 and 3, 6, 12, 24, and 36 months after injection. Comparisons of efficacy results were made between the two MPC dose groups, between each MPC dose group and each control group, and among the four study treatment groups.

[0151] Clinical study patient population

[0152] [Table 1]

[0153] treatment Subjects received one of four treatments: Approximately 6 million homogeneous MPCs (1.0 mL of 30 million per 5 mL of MPC product) mixed with 1.0 mL of 1% sodium hyaluronate (Euflexxa®) Approximately 18 million allogeneic MPCs mixed with 1.0 mL of 1% sodium hyaluronate (90 million at 1.0 mL per 5 mL of MPC product) 1% sodium hyaluronate control, 2 mL Sterile saline control, 2 mL

[0154] The investigational or control treatment was injected directly into the nucleus pulposus of the target disc using a fluoroscopically guided transhipetral approach with a sterile pressure manometer syringe and a 23-gauge Luer-Lok™ needle.

[0155] Investigational Products and Administration The investigational product was STRO-3 selected allogeneic MPCs, which were derived from culture-expanded and then cryopreserved adult bone marrow mononuclear cells. Allogeneic MPCs were formulated at concentrations of 30 million and 90 million nucleated cells in a volume of 5 mL and cryopreserved in 7.5% dimethyl sulfoxide / 50% alpha modified Eagle's medium and 42.5% ProFreeze®.

[0156] Investigational products were stored in the vapor phase of liquid nitrogen at -140°C to 196°C until ready for use. Investigational products must be properly identified and segregated from other products.

[0157] Subjects randomized to receive a total dose of 6 million MPCs received a mixture of 2.0 mL of 30 million / 5 mL cell product mixed 1:1 by volume with hyaluronic acid, which was effectively 1.0 mL of 30 million / 5 mL cell product mixed with 1.0 mL of hyaluronic acid. Subjects randomized to receive a total dose of 18 million MPCs received a mixture of 2.0 mL of 90 million / 5 mL cell product mixed 1:1 by volume with hyaluronic acid, which was effectively 1.0 mL of 90 million / 5 mL cell product mixed with 1.0 mL of hyaluronic acid.

[0158] The control medication administered was either 2.0 mL of hyaluronic acid alone or 2.0 mL of saline. Institutions used sterile saline from their institution's regular supply. Hyaluronic acid was supplied by Mesoblast.

[0159] The volume of injection for each subject randomized to either the investigational or control treatment was to be 2.0 mL to avoid possible differences in injection volume affecting the results.

[0160] All subjects in this study received a single target injection of the investigational product or control into the nucleus pulposus of the intervertebral disc. Each eligible subject was randomized to one of two doses of allogeneic MPC with an equal volume of hyaluronic acid, or one of two control treatment groups: hyaluronic acid or saline injection.

[0161] Clinical study results Safety - Serious Adverse Events The procedure and treatment were well tolerated: No clinical signs of allergic or immune reaction to allogeneic MPC - The proportion of subjects with Class I PRA ≥ 5% and Class II PRA ≥ 20% remained relatively stable from screening to subsequent visits across all four study treatment arms - The number of subjects with DSA responses was similar across all treatment groups: 3 / 16 (18.8%); 2 / 14 (14.3%); 5 / 23 (21.7%) and 4 / 21 (19.0%) subjects in the saline, HA, 6M and 18M groups, respectively. Serious adverse events - Saline caused SAEs in 10% of subjects -Occurrence rate of fatigue - Single occurrence rate of back pain -Rate of occurrence of lower limb pain - 5.0% of subjects experienced SAEs due to HA - Single occurrence rate of back pain - 6M MPC resulted in 13.3% of subjects experiencing SAEs - Rate of occurrence of dermoid cysts - Single incidence of discitis (associated with the procedure) -Two occurrences of back pain - 18M MPC resulted in 6.7% of subjects experiencing SAEs -Two incidences of back pain

[0162] 24-month clinical trial As shown in Table 1 below, the MPC treatment group had significantly less intervention than the control over 12 months:

[0163] [Table 2]

[0164] LS mean change in VAS low back pain The MPC treatment group had significantly greater LS mean back pain improvement from baseline compared with saline at 12 and 24 months (Figure 2).

[0165] VAS category distribution at 24 months Patients treated with 6M MPC showed a significant shift to a lower VAS compared to saline controls: the median VAS score for 6M MPC was substantially reduced, while saline was virtually unchanged at 24 months (Figure 3).

[0166] 12-month outcome of VAS responder rate by response threshold Both MPC groups had a higher proportion of patients than either control group who achieved a pain reduction of greater than 50% at 12 months (Figure 4).

[0167] The 6 million MPC group had a higher proportion of patients with at least a 50% reduction in pain and no post-treatment intervention out to 12 months and out to 24 months compared to controls (Figure 5).

[0168] VAS comparison with standard treatment MPC treatment resulted in significant improvements in mean LBP improvement compared to NSAIDs and opioids, and exceeded the threshold for substantial improvement at all times (Figure 6).

[0169] Mean change in ODI function MPC had a mean ODI improvement of at least the FDA threshold at all time points after 1 month and was superior to saline at both 24 and 36 months (Figure 7).

[0170] 24-month results The MPC treatment group had greater functional improvement than controls at 12 months (Figure 8).

[0171] The MPC treatment group had a higher proportion of patients with an ODI reduction of at least 15 points out to 24 months compared to controls (Figure 9).

[0172] ODI comparison with standard treatment MPC treatment significantly improved mean functional improvement compared to NSAIDs and opioids, and exceeded the FDA improvement threshold at all times (Figure 10).

[0173] Success of the combined treatment with respect to time points The MPC group demonstrates treatment benefit within 3 months, a benefit that persists for at least 24 months, consistent with a potential regenerative process (Figure 11).

[0174] Successful 24-month combined treatment The MPC group shows significant differences in successful responders compared to saline controls up to 12 months, and 6M MPC shows significant differences compared to saline up to 24 months (Figure 12).

[0175] [References] Ausubel, FM (Ed.). (1987 including all updates untill present). Current Protocols in Molecular Biology. New York: John Wiley & Sons. Brown, TA (Ed.). (1991). Essential Molecular Biology: A Practical Approach (Vol. 1 and 2). Oxford: IRL Press at Oxford University Press. Buchschacher & Panganiban (1992). Journal of Virology, 2731-2739. Burns et al., (1993). Proceedings of the National Academy of Sciences USA, 8033-8037. Carter (1992). Current Opinion in Biotechnology, 533-539. Chinnadurai et al., (2016). Translational and Clinical Research, 34(9), 2429-2442. 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. Fisher-Hoch et al., (1989). Proceedings of the National Academy of Sciences USA, 56, 317-321. Francois et al., (2012). Cytotherapy, 14(2), 147-152. Glover, M., & Hames, B. D. (Eds.). (1995 and 1996). DNA Cloning: A Practical Approach (Vols. 1-4). Gronthos (2003). Journal of Cell Science, 116(Pt 9), 1827-1835. Gronthos & Simmons (1995). Blood, 85(4), 929-940. Harlow, E., & Lane, D. (1988). Antibodies: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press. Johann et al., (1992). Journal of Virology, 65, 1635-1640. Kotin (1994). Human Gene Therapy, 793-801. Lebkowski et al., (1988). Molecular and Cellular Biology, 3988-3996. Miller (1990). Human Gene Therapy, 7, 5-14. Miller & Rosman (1989). Biotechniques, 7, 980-990. Miller et al., (1991). Journal of Virology, 65, 2220-2224. Muzyczka (1992). Current Topics in Microbiology and Immunology, 158, 97-129. Perbal, B. V. (1984). A Practical Guide to Molecular Cloning. New York: Wiley. Sambrook, J., & Green, M. R. (2012). Molecular Cloning: A Laboratory Manual (Fourth Edition). New York: Cold Spring Harbour Laboratory Press. Scarpa et al., (1991). Virology, 75, 849-852. Sommerfelt & Weiss (1990). Virology, 76, 58-59. Vincent et al., (1990). Vaccine, 353-359. Wilson et al., (1989). Journal of Virology, 63, 2374-2378. Zannettino et al., (1998). Blood, 92(8), 2613-2628.

Claims

1. 1. A method of treating low back pain in a subject in need thereof, comprising administering to the subject a composition comprising mesenchymal progenitor or stem cells (MLPSCs), wherein the low back pain is associated with an intervertebral disc having a disc height that is not substantially reduced compared to the height of healthy adjacent discs in the subject.

2. 10. The method of claim 1, wherein the low back pain is associated with an intervertebral disc having a loss of disc height of <30% compared to the height of adjacent healthy discs in the subject.

3. 3. The method of claim 1 or 2, wherein the lower back pain is non-radicular in origin.

4. 4. The method of any one of claims 1 to 3, wherein the lower back pain is associated with a herniated disc with a protrusion of up to 3 mm.

5. 5. The method of any one of claims 1 to 4, wherein the lower back pain is associated with nerve entry into the intervertebral disc.

6. 6. The method of any one of claims 1 to 5, wherein the lower back pain is associated with inflammation in the intervertebral disc.

7. 7. The method of claim 5 or 6, wherein the nerve invasion or inflammation is in the disc space, or the nucleus pulposus, or the annulus fibrosus of the disc.

8. MLPSC is at least approximately 2800 pg / 10 6 cells, or at least about 2810 pg / 10 6 cells, or at least about 2820 pg / 10 6 cells, or at least about 2830 pg / 10 6 cells, or at least about 2840 pg / 10 6 cells, or at least about 2850 pg / 10 6 cells, or at least about 2860 pg / 10 6 cells, or at least about 2870 pg / 10 6 cells, or at least about 2880 pg / 10 6 cells, or at least about 2890 pg / 10 6 cells, or at least about 2900 pg / 10 6 cells, or at least about 2910 pg / 10 6 cells, or at least about 2920 pg / 10 6 cells, or at least about 2930 pg / 10 6 cells, or at least about 2940 pg / 10 6 cells, or at least about 2950 pg / 10 6 cells, or at least about 2960 pg / 10 6 cells, or at least about 2970 pg / 10 6 cells, or at least about 2980 pg / 10 6 cells, or at least about 2990 pg / 10 6 cells, or at least about 3000 pg / 10 6 8. The method of claim 1, wherein the cells release TGFβ1 when cultured in a quantity of cells.

9. or at least about 400 pg / ml, or at least about 405 pg / ml, or at least about 410 pg / ml, or at least about 415 pg / ml, or at least about 420 pg / ml, or at least about 425 pg / ml, or at least about 430 pg / ml, or at least about 435 pg / ml, or at least about 440 pg / ml, or at least about 445 pg / ml, or at least about 450 pg / ml, or at least about 45 8. The method of claim 1, wherein the cell culture medium releases TGFβ1 when cultured in an amount of at least about 5 pg / ml, or at least about 460 pg / ml, or at least about 465 pg / ml, or at least about 470 pg / ml, or at least about 475 pg / ml, or at least about 480 pg / ml, or at least about 485 pg / ml, or at least about 490 pg / ml, or at least about 495 pg / ml, or at least about 500 pg / ml.

10. The method of claim 8 or 9, wherein the MLPSCs release TGFβ1 in an amount sufficient to enhance Sema3A expression in the annulus fibrosus of the intervertebral disc space.

11. 11. The method of any one of claims 1 to 10, wherein the mesenchymal progenitor or stem cells are isolated by immunoselection.

12. 11. The method of any one of claims 1 to 10, wherein the immunoselected cells are culture-expanded prior to administration.

13. 11. The method of any one of claims 1 to 10, wherein the mesenchymal progenitor or stem cells are culture-expanded mesenchymal stem cells.

14. The composition is about 1 × 10 6 Cells ~ approx. 20×10 6 14. The method of any one of claims 1 to 13, wherein the cell is administered to the subject in a dose of 500 mg / kg of the cell.

15. The composition is about 6 × 10 6 15. The method of any one of claims 1 to 14, wherein the cell is administered to the subject in a dose of 500 mg / kg of the cell.

16. The composition is approximately 18 x 10 6 15. The method of any one of claims 1 to 14, wherein the cell is administered to the subject in a dose of 500 mg / kg of the cell.

17. 17. The method of any one of claims 1 to 16, wherein the composition is administered as a single dose.

18. 18. The method of any one of claims 1 to 17, wherein the composition is administered into the nucleus pulposus or annulus fibrosus of the intervertebral disc.

19. 19. The method of any one of claims 1 to 18, wherein administration of the MLSPC results in at least a 50% reduction in pain as determined by a visual analog scale (VAS) for at least 1 month, or at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months after administration.

20. 20. The method of any one of claims 1 to 19, wherein administration of the MLSPC results in a reduction in pain of at least 15 points as determined by the Oswestry Disability Index (ODI) for at least 1 month, or at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months after administration.

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