Compositions and methods for treating pain or related conditions

Abstract

Disclosed herein are compositions and methods for the treatment of pain and / or related conditions. In particular, the disclosure provides methods and compositions for the treatment of pain, for example in complex regional pain syndrome, including administration of mesenchymal stem cells with high immunomodulatory potency.

Description

2023-144-02 CCF-42973.601COMPOSITIONS AND METHODS FOR TREATING PAIN OR RELATED CONDITIONS FIELD

[0001] The present disclosure provides compositions and methods for the treatment of pain and / or related conditions. In particular, the disclosure provides methods and compositions for the treatment of pain, for example in complex regional pain syndrome, including administration of mesenchymal stem cells with high immunomodulatory potency.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 735,140, filed December 17, 2024, the content of which is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under NS 127258 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] Chronic pain afflicts more than 50 million American adults and costs our nation $560 to $635 billion annually. Complex regional pain syndrome (CRPS) represents one of the most disabling and difficult-to-treat chronic pain conditions in clinical practice. Managing CRPS is a leading challenge in pain medicine. Patients with CRPS frequently fail to respond to the continuum of treatments, ranging from physical, cognitive-behavioral, pharmacological, interventional, to surgical approaches, which only achieve a remission rate of 5.4%. Thus, novel, and more efficacious treatment strategies are needed to relieve the burden of pain, suffering, and disability caused by CRPS.SUMMARY

[0005] Disclosed herein are compositions and methods for treating pain or a pain-related condition in a subject in need thereof. In some embodiments, the methods comprise administering to the subject an effective amount of mesenchymal stem cells with high immunomodulatory potency. In some embodiments, the methods comprise at least one additional administration separated from a first administration by a period of time greater than 6 months.

[0006] In some embodiments, mesenchymal stem cells with high immunomodulatory potency show a 50% or greater reduction in one or more pro-inflammatory cytokines in in vitro co-cultured LPS- stimulated microglia cells as compared to LPS-stimulated microglia cells not co-cultured with mesenchymal stem cells.2023-144-02 CCF-42973.601

[0007] In some embodiments, the mesenchymal stem cells with high immunomodulatory potency have high analgesic efficacy.

[0008] In some embodiments, the mesenchymal stem cells with high immunomodulatory potency are selected for therapeutic applications with or without cultured expansion from cryopreserved mesenchymal stem cells. In select embodiments, the mesenchymal stem cells with high immunomodulatory potency are cultured-expanded from cryopreserved mesenchymal stem cells.

[0009] In some embodiments, the mesenchymal stem cells with high immunomodulatory potency are derived from umbilical cord, bone marrow, or adipose tissue.

[0010] In some embodiments, the pain or pain-related condition comprises chronic pain. In some embodiments, the pain or pain-related condition comprises acute pain.

[0011] In some embodiments, the pain or pain-related condition is complex regional pain syndrome. In some embodiments, the complex regional pain syndrome is complex regional pain syndrome type I. In some embodiments, the complex regional pain syndrome is complex regional pain syndrome type II.

[0012] In some embodiments, the mesenchymal stem cells with high immunomodulatory potency are administered systemically. In some embodiments, the mesenchymal stem cells with high immunomodulatory potency are administered by intravenous administration.

[0013] In some embodiments, the mesenchymal stem cells with high immunomodulatory potency are administrated at a dosage of about lxl04cells / kg to about lxl07cells / kg. In some embodiments, the mesenchymal stem cells with high immunomodulatory potency are administrated at a dosage of about lxlO6cells / kg to about lxl07cells / kg.

[0014] In some embodiments, the methods further comprise measuring pain experience by the subject before and after treatment with the high immunomodulatory potency mesenchymal stem cells.

[0015] In some embodiments, the methods decrease mechanical allodynia and thermal hyperalgesia, normalize the expression of proinflammatory cytokines and chemokines, promote tissue repair, mitigates demyelination, increase neural function, or a combination thereof.

[0016] Also disclosed herein are compositions comprising mesenchymal stem cells with high immunomodulatory potency. In some embodiments, the mesenchymal stem cells with high immunomodulatory potency are derived from umbilical cord, bone marrow, or adipose tissue.

[0017] In some embodiments, the compositions further comprise a cryopreservative. In some embodiments, the compositions further comprise a carrier.

[0018] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.2023-144-02 CCF-42973.601BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is a flow scheme for an exemplary method for the production of clinical grade human mesenchymal stem cells from umbilical cord (hUC-MSCs). hi this example, isolation is from umbilical cord (multiple runs per cord are possible).

[0020] FIG. 2. In vitro assays of neuroimmune modulatory potency of hMSCs from 3 tissue sources. Experimental setup and workflows (left) and qPCR data of gene expression of IL-, TNFa, and IL- 1 [3 by BV2 cells treated with LPS in the presence (right) or absence (middle) of hMSCs. Upon LPS exposure, BV2 cells increased gene expression of TNF-a, IL- 10, and IL-6 in a dose- and timedependent manner. Gene expressions were higher at 6 hours compared with those measured at 24 hours. Inflammatory factors expressed by BV2 cells treated with LPS in the presence of hMSCs: BV2 cells were treated with LPS (lOOOng / ml) for 6 hours, washed out, and then co-cultured with hMSCs for 24 hours. These data represent four individual experiments. ***p<0.001.

[0021] FIGS. 3A-3D. Validation of in vitro Assays. FIGS. 3A-3B show that pain behavioral (PWT) efficacy (FIG. 3A) predicts in vitro assay potencies of hMSCs (FIG. 3B). FIG. 3A. Chronic constriction injury (CCI) model, mean+SD, * p<0.01, Sham, sham surgery control; PBS, placebo control; UCMSC, hUC-MSCs from 7 different donors (R, S, L, P, R, M, N), n=9-10 / group. FIG. 3B. In vitro assays of IL-6, TNFa, and IL-10 mRNAs, mean+SD n=4 independent experiments. Controls, “No” group indicate BV2 cell culture without LPS challenge, “LPS” group denotes BV2 cell with LPS stimulation. “MSC only” group indicates MSC culture without LPS challenge. MSC 2330 and MSC 2338 indicate hBM-MSCs of passage #4 from two different donors. MSC#R and MSC#S indicate hUC-MSCs of passage #3 from donors R and S respectively. mean+SD, 4 independent experiments. FIGS. 3C-3D show in vitro assay potency (FIG. 3C) predicts analgesic efficacies (FIG. 3D). FIG. 3C. In vitro assay of IL6 mRNA. Labels as in FIG. 3B. FIG. 3D. CCI model, Mean+SD. *** p<0.01, n=5 / group. Sham, sham control, PBS, placebo control.

[0022] FIG. 4. Neuroimmune modulatory potency of hMSCs from three tissue sources. Mean+SD, 4 independent experiments; raw data (lower panel) and normalized data (upper panel). “No” group indicate BV2 cell culture without LPS challenge, “LPS” group denotes BV2 cell with LPS stimulation. “MSC only” group indicates MSC culture with no LPS challenge. AD, human adipose tissue derived MSCs. BM, human bone marrow derived MSCs. UC, human umbilical cord tissue derived MSCs. All cells are at passage #2.

[0023] FIGS. 5A-5F. IgG transfer of human CRPS to mice. FIG. 5 A. Patients with CRPS donated blood for IgG purification. FIG. 5B. Protocols to generated IgG transfer model of CRPS in mice.2023-144-02 CCF-42973.601FIGS. 5C-5D. Animals developed CRPS-like symptoms including inflammation of the injured paw FIG. 5C. In vivo images of L-012-derived bioluminescence obtained during general anesthesia, with red color indicating strong bioluminescence, ROS, and inflammation (Helyes, Z. et al. Proc Natl Acad Sci U SA 116, 13067-13076 (2019)) and mechanical hyperalgesia (FIG. 5D) in the patient IgG injected group but not so much in the control groups (Saline or healthy volunteer IgG). FIGS. 5E-5F. Both male and female mice developed long-lasting and robust pain behavior evaluated with paw withdrawal threshold (E, PWT, mean+SD) to mechanical stimulation or paw withdrawal latency (F, PWL, mean+SD) to thermal stimulation in our experiments. AUC, Areas under curve; CRPS IgG, treated with CRPS patient’s IgG: HC, treated with healthy control IgG; FT, treated flow through proteins from CRPS patients; PI, plantar incision only control. n=4 / group.

[0024] FIGS. 6A-6D. Analgesic efficacy of hUC-MSCs and dose-response, 0.2, 0.5, 1, or 2 million cell / mouse as indicated. FIGS. 6A-6B. Analgesic efficacy evaluated with von Frey tests: Paw withdrawal threshold (PWT) to mechanical stimulation mean+SD (FIG. 6A) and area under the curve (AUC) (FIG. 6B). FIGS. 6C-6D. Analgesic efficacy evaluated with Hargreaves tests: Paw withdrawal latency to thermal stimulation mean+SD (FIG. 6C) and area under the curve (AUC) (FIG. 6D). FT, treated flow through proteins from CRPS patients; n=12 / group. Statistical analysis (Two-way ANOVA, followed by Tukey’s paired comparisons) reveal statistically significant differences between treatment groups and the PBS control group. No significant differences were found between treatment groups with different doses.

[0025] FIGS. 7A-7C. Locomotor function, anxiety-like response, and body weight change index. FIG. 7A. Example results of video tracking and heatmapping of locomotion activities. FIG. 7B. Summary data of locomotor function (distance, velocity, acceleration maximum) and anxiety-like responses (including in zone Arena frequency). FIG. 7C. Body weight change index in male and female animals. (ANOVA followed by Tukey’s paired comparisons, n=12 / group, 6 males and 6 females).

[0026] FIG. 8. Organ / tissue integrity and functions of the liver, kidneys, and pancreas. n=6 / group, *p<0.05, ** p<0.01), ANOVA, followed by Tukey’s paired comparisons.

[0027] FIGS. 9A-9C. Flow cytometry of immune cells in blood. FIG. 9A. Gating strategy. FIGS. 9B-9C. Number (FIG. 9B) and percent (FIG. 9C) of immune cells. Mean+SD, ANOVA, followed by Tukey’s paired comparisons.

[0028] FIGS. 10A-10E. hUC-MSC treatment mitigated pain and modulated immune function. FIG. 10 A. Behavioral measurement of mechanical allodynia and thermal hyperalgesia with or without hUC-2023-144-02 CCF-42973.601MSC treatment in CCI mice (n=12 / group). FIG. 10B. Expression of proinflammatory cytokines and chemokines (TNF-a, IL-ip, CCL2) at the injured sciatic nerve per qRT-PCR in response to hUC-MSC treatment or placebo (PBS), (n=6) / group). FIGS. 10C-10D. Infiltration of immune cells in the injured sciatic nerve, quantified by flow cytometry. Data show examples of flow cytometry (FIG. IOC) and summary data (FIG. 10D) of two individual experiments. *p<0.05; **p<0.01; ****p<0.001. FIG. 10E. RNA-seq analysis of differentially expressed genes (DEGs) (FDR<0.05 and a threshold of logFC > 111) at the sciatic nerve at day 28 post-CCI between sham control, CCI+PBS, and CCI+hUC-MSC groups n=3 / group).

[0029] FIGS. 11A-1 ID. hUC-MSC treatment normalized gene expressions. FIG. 11A. Principal Component Analysis of RNA libraries sequenced for RNA-seq shows three distinct clusters with excellent separation between three groups of animals: Sham control, PBS control, and hUC-MSCs. FIG. 11B. Venn diagram shows overlap and differences of differentially expressed genes (DEGs) associated with CCI and with MSC treatment. Specimen of the sciatic nerve were compared at post- CCI day 28 between sham control. CCI+PBS, and CCI+hUC-MSC groups n=3 / group) (FDR<0.05 and a threshold of logFC > 111). FIG. 11C. Heat maps show hierarchical clustering of DEGs between the Sham control, PBS control, and MSC treatment groups respectively. In clustering analysis, up- regulated and down-regulated genes are colored in red and blue, respectively. FIG. 11D. RNAseq data confirmation of expression of proinflammatory factors at CCI site per qRT-PCR in response to hUC- MSC treatment or placebo (PBS), n=6) / group.DETAILED DESCRIPTION

[0030] Disclosed herein are methods and compositions for treating chronic pain conditions, for example complex regional pain syndrome (CRPS), comprising administration of MSCs with high immunomodulatory characteristics. One challenge in hMSC therapy is optimizing treatment outcomes due to variability in the source of the stem cells (e.g., tissue from which the cells are derived from and the donor’s age, health, and genetics will impact stem cell function), as well as the cells’ quality, purity, and delivery method, all which impact the potency and efficacy of hMSC’s.

[0031] As shown herein in the IgG transfer model of CRPS, intravenous administration of four different doses hUC-MSCs with high immunomodulatory potency completely reversed mechanical allodynia and thermal hyperalgesia, far exceeding 50% reversal of pain behavior measurement. There were no differences between male and female animals in the development of CRPS following IgG injection, nor in the therapeutic efficacy of high immunomodulatory potency hUC-MSC treatment. The safety of treatment was demonstrated in various ways, including normal locomotor function, absence2023-144-02 CCF-42973.601 of anxiety-like behavior, and normal body weight gain. High immunomodulatory potency hUC-MSC treatment effectively modulated differentially expressed genes (DEGs) caused by chronic constriction injury (CCI) and substantially normalized DEGs associated with disease modifying processes including immune regulation, neutrophil degranulation, collagen degradation, and demyelination thereby providing a conducive environment for tissue repair, nerve regeneration, and pain resolution.

[0032] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.1. Definitions

[0033] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. However, two or more copies are also contemplated. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0034] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, e.g.. “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0035] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of’ or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (e.g.,2023-144-02 CCF-42973.601“one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of’ “only one of’ or “exactly one of.”

[0036] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0037] As used herein, the term “mesenchymal stem cells” or “MSCs” refers to multipotent stromal stem cells that have the ability to differentiate into a variety of cell types, including osteocytes, adipocytes, myocytes, chondrocytes, skeletal muscle cells and endothelial cells. MSCs are present in the bone marrow, adipose tissue, peripheral blood, chorionic placenta, amniotic placenta, umbilical cord blood, and dental pulp, among other tissues. In some embodiments, the MSCs are derived from a subject. The MSCs may be derived from any tissue in which they are present. In some embodiments, the MSCs are derived from umbilical cord or chorionic placenta. In some embodiments, the MSCs are derived from bone marrow, hi some embodiments, the MSCs are derived from adipose tissue.

[0038] As used herein, the terms “administering,” “providing”, and “introducing,” are used interchangeably herein and refer to the placement of the mesenchymal stem cells or compositions of the disclosure into a subject by a method or route which results in at least partial localization to a desired site. The compounds or compositions can be administered by any appropriate route which results in delivery to a desired location in the subject.

[0039] As used herein, “treat.” “treating” and the like means a slowing, stopping, or reversing of progression of a disease, disorder, condition, or status when provided the mesenchymal stem cells or compositions described herein to an appropriate subject. The term also means a reversing of the progression of such a disease, disorder, or condition. As such, “treating” means an application or administration of the methods described herein to a subject, where the subject has a disease or a symptom of a disease, disorder, condition, or status where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease, disorder, condition, or status.

[0040] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, a patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the methods herein. Examples of mammals include, but are not limited to, any member of the2023-144-02 CCF-42973.601Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents such as rats, mice, and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0041] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0042] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.2. Methods

[0043] Embodiments of the present disclosure include methods for treating pain or a pain-related condition in a subject. The methods comprise administering to the subject an effective amount of mesenchymal stem cells with high immunomodulatory potency.

[0044] In some embodiments, the methods further comprise administering one or more additional doses of mesenchymal stem cells with high immunomodulatory potency separated from a previous dose by a period of time (e.g., greater than 1 month, 6 months, or 12 months). For example, the method may comprise administering only one dose to treat the pain or pain-related condition, or administering a first dose followed by administering one or more second doses.

[0045] If one or more second doses are administered, the second dose may be administered before the maximum time of the pain-relieving effect of the first or previous dose is achieved, or before the pain-relieving effect of the first or previous dose is expired. The methods may achieve a reduction in pain that lasts at least about one month, about two months, about three months, about four months, about six months, or about twelve months. Thus, the separation between the first dose and a second dose, or sequential additional doses may be greater than about one month, greater than about two2023-144-02 CCF-42973.601 months, greater than about three months, greater than about four months, greater than about six months, or greater than about twelve months.

[0046] The disclosed methods may be used to treat, or provide relief from, any type of pain, including but not limited to, inflammatory pain, complex regional pain syndrome, lumbosacral pain, musculoskeletal pain, neuropathic pain, chronic pain, cancer-related pain, acute pain, postoperative pain, etc. The pain relief may be palliative, providing pain relief independent of improvement of the disease or condition or the underlying cause of the pain. For example, although the underlying disease may not improve, or may continue to progress, an individual suffering from the disease may experience pain relief.

[0047] Pain evaluation can include capturing features such as quality (e.g., burning, cramping, aching, deep, superficial, boring, shooting, etc.), severity, location, radiation pattern, duration, timing (e.g.. including pattern and degree of fluctuation and frequency of remissions), and exacerbating and relieving factors. Additionally, pain evaluation can also assess aspects of pain relating to a patient's level of functioning, for example, by focusing on activities of daily living (e.g., dressing, bathing), employment, and personal relationships (e.g., sexual activity / ability). Use of pain measurement scales can assist in the evaluation process by measuring a patient's pain intensity and / or functionality, for example. Pain scales, as well as other data that can be collected in a pain evaluation of a patient, are based on self-report, observational (behavioral), or physiological data. Self-report is considered the primary form and common for children, adolescents, adults and seniors who are able to communicate; however, additional pain scales are also available for neonates, infants, and other persons whose communication is impaired.

[0048] The methods may reduce a level of pain as reported on a pain scale (e.g., Visual Analog Scale, a graphic faces pain scale, a Wrong-Baker FACES Pain Rating Scale, a colored analogue scale, a Face Legs Anns Cry Consolability Scale, a word descriptor scale, a verbal scale and a functional pain scale). The methods may further comprise measuring the pain, for example using one or more pain scales, before and after treatment with the high immunomodulatory potency mesenchymal stem cells. For example, levels of pain may be captured using a numerical rating scale (NRS) pain score measured using a 0-10 scale. In some embodiments, the disclosed methods decreased the NRS pain score by at least about 0.1, at least about 0.5, at least about 0.8, at least about 1, at least about 1.5, up to about 5, or up to about 10.

[0049] In some embodiments, the pain or pain related condition is complex regional pain syndrome. Complex regional pain syndrome is a debilitating pain syndrome characterized by severe pain in a limb2023-144-02 CCF-42973.601 that can be accompanied by edema, and autonomic, motor and sensory changes. The disclosed methods may be suitable for use to relieve any form of complex regional pain syndrome, such as complex regional pain syndrome type I (CRPS-I), complex regional pain syndrome type II (CRPS-II), CRPS-NOS, or another type of CRPS. In select embodiments, the complex regional pain syndrome is complex region pain syndrome type I. In select embodiments, the complex regional pain syndrome is complex region pain syndrome type II.

[0050] The disclosed methods may not only relieve pain but may also modify the disease processes through anti-inflammatory and neurotrophic / neuroprotective effects. Thus, in some embodiments, the underlying disease may improve the underlying condition in addition to providing pain relief.

[0051] In some embodiments, the disclosed methods may decrease mechanical allodynia and thermal hyperalgesia in the acute and chronic phases of CRPS. In some embodiments, the disclosed methods normalize the expression of proinflammatory cytokines and chemokines. For example, the disclosed methods may decrease the expression of immune factors, such as TNF-a, IL-ip, and or CCL2, as compared to CRPS subjects not treated with high immunomodulatory potency mesenchymal stem cells. Additionally, high immunomodulatory potency mesenchymal stem cells treatment showed restorative effects for CRPS, including mitigating inflammation, demyelination, and collagen degradation. The disclosed methods may promote tissue repair, remyelination, and neural function in CRPS. Neutrophil degranulation, which releases proteolytic enzymes and reactive oxygen species, can exacerbate inflammation and tissue damage following nerve injury. In some embodiments, the disclosed methods inhibit neutrophil degranulation. Overall treatment with high immunomodulatory potency mesenchymal stem cells may reduce inflammation and promote a more favorable healing environment. Thus, in some embodiments, treatment with high immunomodulatory potency mesenchymal stem cells not only relieve pain, but also modify the CRPS and other disease processes through anti-inflammatory and neurotrophic / neuroprotective effects. a. High immunomodulatory potency mesenchymal stem cells

[0052] As described herein, mesenchymal therapies are inconsistent due to variability within mesenchymal stem cell preparations. The disclosed methods utilize mesenchymal stem cells with high immunomodulatory potency. As used herein, “high immunomodulatory potency” means causing, or having the capacity to cause, a detectable change in an immune response, and the ability to cause a detectable change in an immune response.

[0053] Mesenchymal stem cells with high immunomodulatory potency may be selected based on potency assay. In some embodiments, the potency assay comprises an in vitro co-culture assay with2023-144-02 CCF-42973.601 immune-challenged (e.g., lipopolysaccharide (LPS)-stimulated) microglia cells. The in vitro co-culture assay assesses the neuroimmune modulatory properties of MSCs, to serve as a measure of quality control, and to predict clinical outcomes of MSC therapy. In some embodiments the in vitro co-culture assay includes co-culturing immune-challenged (e.g., lipopolysaccharide (LPS)-stimulated) microglia cells with mesenchymal stem cells and quantifying gene expression of one or more one or more pro- inflammatory markers, for example, one or more pro-inflammatory cytokines or chemokines (e.g., IL- 6, TNF-a, IL-1 p, and combinations thereof) in the immune-challenged (e.g., lipopolysaccharide (LPS)- stimulated) microglia cells following co-culture as described below in Example 2.

[0054] In some embodiments, mesenchymal stem cells with high immunomodulatory potency show a 50% or greater reduction in one or more pro-inflammatory cytokines or chemokines in in vitro cocultured LPS- stimulated microglia cells as compared to LPS- stimulated microglia cells not co-cultured with mesenchymal stem cells. In some embodiments, high immunomodulatory potency mesenchymal stem cells have high analgesic efficacy.

[0055] The mesenchymal stem cells may be isolated or derived from a subject, e.g., a mammal such as a human, monkey, pig, horse, cow, sheep, dog, cat, mouse, and rabbit. In some embodiments, the mesenchymal stem cells may be derived from a human. The mesenchymal stem cells may be from any tissue source. In some embodiments, the mesenchymal stem cells are derived from umbilical cord, bone marrow, or adipose tissue. The mesenchymal stem cells may be autologous, allogeneic, xenogeneic, syngeneic, or isogenic.

[0056] In some embodiments, the mesenchymal stem cells are genetically unmodified. “Genetically unmodified” refers to cells that have not been modified by transfection with a nucleic acid.

[0057] In some embodiments, the mesenchymal stem cells with high immunomodulatory potency are selected for therapeutic applications with or without cultured expansion from cryopreserved mesenchymal stem cells. Mesenchymal stem cells may be expanded ex vivo, cryopreserved, and thawed, yet maintain their high immunomodulatory potency. Expanded mesenchymal stem cells are cultured ex vivo in a cell culture medium and passaged (e.g., sub-cultured). The expanded mesenchymal stem cells may be expanded for any number of passages, e.g., about 4 to about 10 passages. The mesenchymal stem cells can be expanded ex vivo to produce multiple therapeutic doses. The mesenchymal stem cells may be culture expanded from a cryopreserved intermediate(s). In some embodiments, the mesenchymal stem cells can be expanded before being cryopreserved and be expanded again following cryopreservation. In some embodiments, the mesenchymal stem cells have the same or similar ex vivo age, e.g., same generation or passage number.2023-144-02 CCF-42973.601

[0058] Methods for generating the mesenchymal stem cells may include that as shown and described in FIG. 1. In some embodiments, the mesenchymal stem cells are expanded ex vivo from a single donor to generate multiple therapeutic doses. In some embodiments, the mesenchymal stem cells are expanded ex vivo from multiple donors to generate multiple therapeutic doses.

[0059] Also provided herein are preparations or compositions of high immunomodulatory potency mesenchymal stem cells. In some embodiments, the preparations further comprise a cryopreservative, e.g., DMSO. b. Administration

[0060] The high immunomodulatory potency mesenchymal stem cells may be administered systemically, such as by intravenous, intraarterial, or intraperitoneal administration. Alternatively, the high immunomodulatory potency mesenchymal stem cells may be administered directly to the site of pain, e.g.. locally, at site(s) of injury or disease.

[0061] The high immunomodulatory potency mesenchymal stem cells may be delivered in a suspension, gel, or other formulation to enhance localization and efficacy at the target site. In some embodiments, the high immunomodulatory potency mesenchymal stem cells are delivered in a liquid suspension.

[0062] The pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactically effective amount” of the high immunomodulatory potency mesenchymal stem cells. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease or condition, the prophylactically effective amount will be less than the therapeutically effective amount.

[0063] The high immunomodulatory potency mesenchymal stem cells are administered in an amount effective to provide a reduction in pain or analgesic effect to the subject systemically or at the site of specific pain. The high immunomodulatory potency mesenchymal stem cells may be administered in an amount of from about IxlO4cells / kg to about IxlO7cells / kg. In another2023-144-02 CCF-42973.601 embodiment, the mesenchymal stem cells are administered in an amount of from about lxl06cells / kg to about IxlO7cells / kg. The exact dosage of high immunomodulatory potency mesenchymal stem cells to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient, the type of pain or pain-related condition.

[0064] The high immunomodulatory potency mesenchymal stem cells may be administered as a pharmaceutical composition comprising a earner. Accordingly, provided herein are compositions comprising high immunomodulatory potency mesenchymal stem cells and a carrier. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Also provided herein are compositions comprising high immunomodulatory potency mesenchymal stem cells, and optionally a pharmaceutically acceptable carrier. A carrier may include a single ingredient or a combination of two or more ingredients. The administration route and the form of the composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral).

[0065] The pharmaceutical composition may contain, for example, sterile water, physiological saline, conventional buffers (phosphoric acid, citric acid, other organic acids, and the like), stabilizers, salts, antioxidants (ascorbic acid and the like), surfactants, suspending agents, tonicity agents, or preservatives. The pharmaceutical composition may contain organic substances such as biopolymers and inorganic substances such as hydroxyapatite, and specifically, a collagen matrix, a polylactic acid polymer or copolymer, a polyethylene glycol polymer or copolymer, and a combination of chemical derivatives thereof. In a case where the pharmaceutical composition according to an embodiment is prepared into a dosage form suitable for injection, mesenchymal stem cells may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution state. For example, mesenchymal stem cells can be administered as a cell suspension in a pharmaceutically acceptable liquid medium or gel for injection.

[0066] The pharmaceutical composition according to the present invention may appropriately contain a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a pain reliever, a buffer, a reducing agent, an antioxidant, and the like, if necessary, depending on the administration method or dosage form. Pharmaceutically acceptable earners and agents suitable for the present invention, including those exemplified above, are described in detail and may generally be found in2023-144-02 CCF-42973.601“Remington’s Pharmaceutical Sciences.” (Meade Publishing Co., Easton, Pa.). The pharmaceutical composition according to the present invention may be prepared in a unit dose form or by being packaged in a multi-dose container through formulation using pharmaceutically acceptable carriers and / or excipients according to methods that can be easily performed by those skilled in the art to which the invention belongs. At this time, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium.

[0067] In some embodiments, the high immunomodulatory potency mesenchymal stem cells is delivered in a saline solution, a phosphate buffered saline solution, Plasmalyte A, a 5% dextrose solution, a saline solution comprising heparin, or a sodium lactate solution, also known as Ringer's lactate solution, lactated Ringer's solution, or Hartmann' s solution.3. ExamplesExample 1 Clinical-grade mesenchymal stem cells (MSCs) from human bone marrow (hBM-MSCs), adipose tissue (hAD-MSCs), and umbilical cord (hUC-MSCs)

[0068] Umbilical cord tissues were harvested during the C-section delivery of male babies from healthy mothers who had consented prior to delivery for appropriate screening using the standard battery of tests for bone marrow donors. The tissues were transferred and cells were expanded in a cGMP facility. Cells at Passage 1 were frozen for future use in animal studies and human clinical trials. Aliquots of the hMSCs will be thawed and expanded in culture for 14 days to achieve the required numbers for clinical trial infusion at Passage 2. For each lot, hMSC immunophenotyping is performed using flow cytometry (FIG. 1). Lot release criteria: viability >70% (by cellometer), percentage of positive markers (CD105, CD90, and CD73) >95%, and negative markers (CD45, CD31, HLAII) <5%; negative gram staining of infusion product, and negative bacterial, fungal, endotoxin, and mycoplasma test at first re-plating between days 7-10. Demonstrated safety and efficacy in animal models are the best release criteria of hMSCs for clinical trials.Example 2Determination of immunomodulatory profiles of hMSCs from different tissue sources

[0069] In vitro assays for neuroimmune modulatory potency of hMSCs

[0070] Using a BV2-MSC co-culture system, in vitro assays were developed to evaluate the neuroimmune- modulatory potencies of hMSCs. Since microglia in the spinal cord play a key role in the development of neuropathic pain and MSCs mitigate neuropathic pain through modulating microglia functions, protocols for co-cultures of hMSCs were developed and refined with BV2 cells2023-144-02 CCF-42973.601 derived from primary mouse microglia, which keep many of the functions and features that microglia express in vivo. Real-time (RT)-PCR quantified BV2 cell gene expression of critical pro-inflammatory cytokines upon exposure to lipopolysaccharide (LPS). In response to LPS, BV2 cells dose-dependently increased gene expression of TNF-a, IL-10, and IL-6 by as much as 18,000-fold (IL-6). Co-cultures with hMSCs dramatically inhibited LPS-induced BV2 cell gene expression of TNF-a, IL-6, and IL- 10. A 50% suppression of relative gene expressions of IL-6, TNFa, and IL- 10 indicated hMSCs with strong neuroimmune modulatory potencies.Example 3Validation of in vitro assays for neuroimmune modulatory potency of hMSCs

[0071] Analgesic efficacy of hMSCs predicts in vitro neuroimmune modulatory potencies ofMSCs

[0072] To validate the in vitro assays, the anti-hyperalgesia efficacy of hUC-MSCs (passage #3) from 7 different donors was evaluated in the chronic constriction injury (CCI) model of neuropathic pain in mice, a model of CRPS type II. Anti-hyperalgesia efficacies were significantly different between cells from different donors, confirming that all cells are not created equal and that cells from certain donors provided better therapeutic efficacy than others. High (UCMSC-R) and low (UCMSC- S) performing donor cells were selected and their respective neuroimmune modulatory efficacy was compared using the in vitro assay described above by experimenters who were blind to the behavioral outcomes. The hUC-MSCs with the higher analgesic efficacy (UCMSC-R) also showed superior neuroimmune modulatory effectiveness in suppression gene expression of IL-6, TNFa, and IL-10, compared to the low performer (UCMSC-S). The analgesic efficacy of MSCs can predict the neuroimmune modulatory potencies of MSCs in in vitro assays.

[0073] In vitro neuroimmune modulatory potency of hMSCs predicts in vivo analgesic efficacies

[0074] The order of the validation was reversed by evaluating hBM-MSCs from two donors (MSC230 and MSC2338) in addition to UCMSC-R and UCMSC-S in the in vitro assay described above. MSC2330 achieved >90% suppression of IL-6 gene expression, while MSC2338 achieved <50% suppression. These cells were then evaluated for analgesic efficacy in CCI mice by experimenters blinded to the in vitro assay results. MSC2330 significantly outperformed MSC2338. The neuroimmune modulatory potency of MSCs can predict their analgesic efficacies.

[0075] The neuroimmune modulatory potency of hMSCs from 3 different tissue sources (hUC- MSCs, hAD-MSCs, and hBM-MSCs) was compared by measuring the suppression of relative gene expression of IL-6, TNFa, and IL- 10. With LPS stimulation normalized to 100% as control, all three tissue sources of hMSCs met the criteria of greater than 50% suppression of inflammatory cytokines2023-144-02 CCF-42973.601(IL-6, TNFa, and IL-ip). Suppression of IL-6 is the most sensitive measure among all, which is consistent will all previous in vitro assay experiments. In addition, the cells were tested by T cell suppression: > 70% suppression of T cell proliferation in a tritiated thymidine incorporation assay.Example 4Treatment Protocol Determinations

[0076] Development of IgG transfer model of CRPS in mice.

[0077] Patients with CRPS (n=16) were enrolled to donate their blood, from which, IgG and flow- through (FT) proteins were purified following published protocols (Helyes, Z. et al. Proc Natl Acad Sci U SA 116. 13067-13076 (2019). Cuhadar, U. et al. Pain 160, 2855-2865 (2019), and Tekus, V. et al. Pain 155, 299-308 (2014)). A robust model of CRPS in mice (trauma IgG transfer model) was produced that demonstrated mechanical allodynia and thermal hyperalgesia that lasted for 2-3 months. Longitudinal observation of robust pain behavior extended the 2-3 weeks observation time. These data for the first time demonstrated the entire course of pain behavioral changes in this model. Multiple controls were designed and used including plantar incision-only control (PI), healthy volunteer IgG control (HC), and flow-through protein control (FT). Both male and female animals were utilized and demonstrated that there were no differences between sexes, animals of both sexes developed robust mechanical allodynia and thermal hyperalgesia.

[0078] Analgesic efficacy ofhUC-MSCs and dose-response relationship.

[0079] The analgesic efficacy of clinical- grade hUC-MSCs was investigated. Although the in vitro neuroimmune modulatory potency assays demonstrated that all three sources of hMSC were equally potent in suppressing proinflammatory cytokine gene expressions, predicting an equal neuroimmune modulatory potency, hUC-MSCs are the cells of choice due to their noninvasive harvest, ample supply, and lower cost to produce.

[0080] hUC-MSCs or vehicle (PBS) were injected intravenously at Day 21 post IgG CRPS transfer. Four dose groups were treated with 0.2, 0.5, 1, or 2 million cell / mouse (n=12 / group, 6 male, 6 female per group). hUC-MSC treatment provided remarkable analgesic efficacy. One single-dose treatment produced a total reversal of the pain behavior. The analgesic effects lasted for more than 30 days without any sign of waning. All four dose groups achieved significant improvement in both mechanical allodynia and thermal hyperalgesia. There were no statistical differences between treatment groups. AUC data suggests that the 1 million / mouse group achieved the best numeric analgesic efficacy evaluated by PWT. All four dose groups also achieved significant reduction in2023-144-02 CCF-42973.601 thermal hyperalgesia, compared to the placebo group. The analgesic efficacy of nearly 100% reversal of pain behavioral measures far exceeded a goal of greater than 50% reversal of pain.

[0081] Safety ofhUC-MSC treatment.

[0082] The safety of hMSC treatment was evaluated by measuring the locomotor function, anxietylike responses, and body weight change index, as well as liver / renal / pancreatic functions, tissue / organ damage, and immune function per flow cytometry quantification of peripheral blood immune cells.

[0083] Normal locomotor function and absence of anxiety -like response. Animals treated with hMSCs showed normal locomotion function and absence of anxiety-like responses in open-field evaluation through video tracking and heatmapping. Key kinematic parameters, such as distance traveled, velocity, and acceleration maximum, were analyzed and no differences between any of the treatment groups and the control groups were observed regardless of the sex of the animals in between treatment and control groups. Thigmotaxis, which is when the animals spend a large amount time close to the walls of the arena, was studied and there was no evidence for anxiety-like responses. There were also no differences in measurements of in zone thigl frequency, mean distance to center, in zone center frequency, total distance to center, and in zone Arena frequency between the treatment groups and the control groups (ANOVA followed by Tukey’s paired comparisons).

[0084] Body weight change index, calculated by the equation (D54-D-l) / D-l*100%), is an important measure of animal health and well-being. No significant differences in body weight change index were observed between any of the hMSC treatment groups and the control groups (n=12 / group).

[0085] Organ / tissue integrity and normal functions of the liver, kidneys, and pan creas. Injection of CRPS patient IgG to mice led to a significant increase of lactate dehydrogenase (LDH) (p<0.01 ), which is a highly sensitive biomarker of organ / tissue damage. LDH has been recommended as a general marker of cell / tissue injury as it is released from cells in response to cell damage. Consistent with CRPS symptoms and signs, animals treated with IgG from CRPS patients had significantly elevated LDH. In contrast, hMSC treatments showed a reduction in LDH levels. In fact, the 0.2 million / animal group reached a statistically significant decrease in LDH compared to the PBS control group (Tukey’s paired test, p<0.05). Liver function measures (albumin, alkaline phosphatase, ALT, and AST) were all within normal range and were not different from control groups. Similarly, the pancreatic (Amylase and Lipase) and kidney functions (creatine kinase) were not significantly elevated (ANOVA followed by Tukey’s paired comparisons, n=6 / group).

[0086] Flow cytometry of immune cells in the blood. The number and percentage of immune cells in the peripheral blood were measured by flow cytometry to determine if hMSC treatment leads to2023-144-02 CCF-42973.601 immune compromise. The data did not reveal any significant differences between the hMSC treatment groups and control groups (n=6 / group) in both male and female animals. Thus, the hMSC treatment did not induce significant immunity compromise. In parallel studies in a CCI model, the hUC-MSC treatment significantly reduced the infiltration of inflammatory immune cells in the injured nerve and substantially increased the anti-inflammatory macrophages (M2) in the injured nerve both in the early and late phase of CCI.

[0087] Clinical evidence of safety. In two ongoing clinical trials in pediatric and adult patient populations, intravenous administration of hUC-MSC s is well tolerated without any significant side effects or adverse events observed. These hUC-MSCs are the same cells that were used in the preclinical studies above.Example 5Biomarkers of immunomodulatory properties of hMSCs to predict therapeutic efficacy

[0088] Using a novel methodology developed for flow cytometric analysis of inflammatory cells in the single sciatic nerve in mice, it was found that infiltration of immune cells in the injured nerve was increased in the early phase and decreased in the late phase of the injury, suggesting that hMSCs promoted inflammation resolution. Interestingly, the anti-inflammatory phenotype macrophages (M2) were increased both in the early and late phases of the injury, consistent with a function of proresolution of neuroinflammation.

[0089] hUC-MSCs effectively modulated the differentially expressed genes (DEGs). RNA-seq and enrichment analyses found that CCI induced 5.882 DEGs (FDR<0.05 and a threshold of logFC > 111) at the injured sciatic nerve at day 28 post-CCI compared to controls. The upregulated DEGs were primarily enriched in immune processes, including immune cell activation and expression of cytokines and chemokines, while the downregulated DEGs were mainly enriched in neuronal functions including synaptic transmission, synaptic organization, and axonal outgrowth. The dominant upstream molecules in the upregulated immune responses include IL-6. IL- 1 , TNFa, IFN-y, and IL-17A. The upregulation of immune factors induced by CCI was significantly normalized in animals treated with hUC-MSCs. The findings were further confirmed by qRT-PCR which demonstrated profound restorative effects of hUC-MSC treatment on mitigating inflammation, demyelination, and collagen degradation.

[0090] hUC-MSCs normalized DEGs associated with neutrophil de granulation. Neutrophil degranulation, which releases proteolytic enzymes and reactive oxygen species, can exacerbate inflammation and tissue damage following nerve injury. hUC-MSC treatment inhibited neutrophil degranulation induced by CCI, compared to PBS control and sham control groups, revealing a2023-144-02 CCF-42973.601 promising therapeutic mechanism in pain management and tissue repair. hMSCs, with their immunomodulatory properties, mitigated this response, reducing inflammation and promoting a more favorable healing environment. These findings underscore the potential of hUC-MSC therapy as an innovative approach to managing chronic pain conditions.

[0091] hUC-MSCs normalized DEGs associated with collagen degradation. Collagen degradation by specific enzymes, such as matrix metalloproteinases (MMPs) and cathepsins, which break down collagen fibers, is a critical process in tissue remodeling, wound healing, and various pathological conditions. hUC-MSC treatment normalized the DEGs associated with collagen degradation, indicating that hMSCs provided a more permissive environment for tissue repair. Regulatory factors such as cytokines, growth factors, and tissue inhibitors of metalloproteinases (TIMPs) influence the expression of these collagen-degrading enzymes. Shifting the gene expression profiles related to collagen degradation by hMSCs may represent the underlying molecular mechanisms and potential therapeutic targets for CRPS.

[0092] hUC-MSCs normalized DEGs associated with demyelination. Demyelination, the loss or damage of the myelin sheath surrounding nerve fibers, is a hallmark of peripheral neuropathies. hMSC treatment normalized DEGs associated with demyelination, indicating that hMSCs may promote remyelination, restore neural function, and reduce pain. This normalization process involves the regulation of key molecular pathways and factors involved in myelin repair, including cytokines, growth factors, and neurotrophic factors. The ability of hUC-MSCs to target and adjust the expression of genes associated with demyelination not only aids in understanding the pathophysiology of nerve injury but also opens new avenues for developing effective treatments, enhancing neural repair, and improving patient outcomes.

[0093] hMSCs not only relieve pain, but also modify the disease processes through antiinflammatory and neuro trophic / neuroprotective effects.

[0094] Similar analysis of the efficacy of hBM-MSCs and hAD-MSCs will allow comparison of the therapeutic efficacy between three commonly used sources of hMSCs. Additionally, additional models for CRPS (e.g., rat O Ring model or chronic post-ischemia pain model, CPIP) will determine whether hMSC treatment is also efficacious in another species and in the CPIP model of CRPS.

[0095] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.2023-144-02 CCF-42973.601

[0096] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope thereof.

Claims

2023-144-02 CCF-42973.601CLAIMSWhat is claimed is:

1. A method for treating pain or a pain-related condition in a subject in need thereof comprising administering to the subject an effective amount of mesenchymal stem cells with high immunomodulatory potency.

2. The method of claim 1, wherein mesenchymal stem cells with high immunomodulatory potency show a 50% or greater reduction in one or more pro-inflammatory cytokines in in vitro co-cultured LPS- stimulated microglia cells as compared to LPS- stimulated microglia cells not co-cultured with mesenchymal stem cells.

3. The method of claim 1 or 2, wherein the mesenchymal stem cells with high immunomodulatory potency have high analgesic efficacy.

4. The method of any of claims 1-3, wherein the mesenchymal stem cells with high immunomodulatory potency are selected for therapeutic applications with or without cultured expansion from cryopreserved mesenchymal stem cells.

5. The method of any of claims 1-4, wherein the mesenchymal stem cells with high immunomodulatory potency are derived from umbilical cord, bone marrow, or adipose tissue.

6. The method of any of claims 1-5, wherein the method comprises at least one additional administration separated from a first administration by a period of time greater than 6 months.

7. The method of any of claims 1-6, wherein the pain or pain-related condition comprises chronic pain.

8. The method of any of claims 1-7, wherein the pain or pain-related condition comprises acute pain.

9. The method of any of claims 1-8, wherein the pain or pain-related condition is complex regional pain syndrome.2023-144-02 CCF-42973.60110. The method of claim 9, wherein the complex regional pain syndrome is complex regional pain syndrome type I.

11. The method of claim 10, wherein the complex regional pain syndrome is complex regional pain syndrome type II.

12. The method of any of claims 1-11, wherein the mesenchymal stem cells with high immunomodulatory potency are administered systemically.

13. The method of any of claims 1-12, wherein the mesenchymal stem cells with high immunomodulatory potency are administered by intravenous administration.

14. The method of any of claims 1-13, wherein the mesenchymal stem cells with high immunomodulatory potency are administered locally, at site(s) of injury or disease.

15. The method of any of claims 1-14, wherein the mesenchymal stem cells with high immunomodulatory potency are administrated at a dosage of about lxl04cells / kg to about IxlO7cells / kg.

16. The method of any of claims 1-15, wherein the mesenchymal stem cells with high immunomodulatory potency are administrated at a dosage of about lx 106cells / kg to about IxlO7cells / kg.

17. The method of any of claims 1-16, further comprising measuring pain experience by the subject before and after treatment with the high immunomodulatory potency mesenchymal stem cells.

18. The method of any of claims 1-17, wherein the method decreases mechanical allodynia and thermal hyperalgesia, normalizes the expression of proinflammatory cytokines and chemokines, promotes tissue repair, mitigates demyelination, increases neural function, or a combination thereof.

Images