Systems and methods for screening and selecting mesenchymal stem cells for therapeutic use

Abstract

Disclosed herein are systems and methods for selecting mesenchymal stem cells for clinical use. Particularly, in vitro co-culture methods and systems which characterize the response of immune cells (e.g., expression of IL-6, TNF-α, IL-1β) to immune challenges (e.g., LPS injection) as an assessment of the analgesic effect of mesenchymal stem cells.

Description

2023-143-02 CCF-42972.601SYSTEMS AND METHODS FOR SCREENING AND SELECTING MESENCHYMAL STEM CELLS FOR THERAPEUTIC USEFIELD

[0001] The present disclosure provides systems and methods for selecting mesenchymal stem cells for clinical use. In particular, the disclosure provides in vitro co-culture methods and systems which characterize the response of immune cells (e.g., expression of IL-6, TNF-a, IL- 1P) to immune challenges (e.g., LPS injection) as an assessment of the analgesic effect of mesenchymal stem cells.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 735,146, 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] Mesenchymal stem cells (MSCs) are multipotent cells that retain their ability to expand for long period of time without losing their desired characteristics and can differentiate into a variety of cell types, including osteoblasts, chondrocytes, myocytes, and adipocytes. MSCs have immunomodulatory properties, secreting certain cytokines and chemokines useful to modify the surrounding immune environment. Thus, unlike other stem cells, MSCs have properties that are useful for cell-based therapies. However, use of MSCs on large clinical scale is challenging. MSCs come from different tissue sources and different individuals with wide heterogeneity and diverse secretomes. Further, the immunomodulatory nature and overall potency and efficacy of MSCs is unpredictable, hindering development of standardized MSC- based therapies with consistent outcomes.SUMMARY

[0005] Disclosed herein are methods for screening mesenchymal stem cells for clinical uses.

[0006] In some embodiments, the methods comprise: co-culturing lipopolysaccharide (LPS)- stimulated microglia cells with mesenchymal stem cells; quantifying gene expression of one or2023-143-02 CCF-42972.601 more pro-inflammatory cytokines and / or chemokines in the LPS- stimulated microglia cells following co-culture; and classifying the immunomodulatory potency of the mesenchymal stem cells based on level of reduction of one or more pro-inflammatory cytokines and / or chemokines in the LPS-stimulated microglia cells following co-culture as compared to LPS-stimulated microglia cells not co-cultured with the mesenchymal stem cells.

[0007] In some embodiments, mesenchymal stem cells are classified as having high immunomodulatory potency when there is about 50% or greater reduction in one or more pro- inflammatory cytokines and / or chemokines in the LPS-stimulated microglia cells following coculture as compared to LPS-stimulated microglia cells not co-cultured with mesenchymal stem cells.

[0008] In some embodiments, the one or more pro-inflammatory cytokines and / or chemokines are selected from IL-6, TNF-a, IL-ip, and combinations thereof. In some embodiments, the one or more pro-inflammatory cytokines is IL-6.

[0009] In some embodiments, the methods further comprise treating microglia cells with about 100 ng / mL to about 1000 ng / mL LPS prior to co-culturing. In some embodiments, the treating is carried out for at least 20 minutes. In some embodiments, the treating is carried out for about 6 hours.

[0010] In some embodiments, the LPS-stimulated microglia cells are LPS-stimulated BV2 microglia cells.

[0011] In some embodiments, the methods further comprise quantifying gene expression of one or more pro-inflammatory cytokines and / or chemokines in the microglia cells prior to LPS stimulation.

[0012] In some embodiments, the mesenchymal stem cells are derived from umbilical cord, bone marrow, or adipose tissue.

[0013] In some embodiments, the co-culturing is an indirect or compartmentalized co-culture. In some embodiments, the indirect or compartmentalized co-culture uses a transwell system. In some embodiments, the mesenchymal stem cells are positioned in a transwell upper chamber and the LPS-stimulated microglia cells are positioned in a transwell lower chamber.

[0014] In some embodiments, the co-culturing is carried out for at least about 6 hours. In some embodiments, the co-culturing is carried out for about 12 to about 36 hours. In some embodiments, the co-culturing is carried out for about 24 hours.2023-143-02 CCF-42972.601

[0015] In some embodiments, the methods further comprise quantifying gene expression of one or more pro-inflammatory cytokines and / or chemokines in the LPS- stimulated microglia prior to co-culturing.

[0016] In some embodiments, mesenchymal stem cells have high immunomodulatory potency when there is about 50% or greater reduction in one or more pro-inflammatory cytokines in the LPS-stimulated microglia cells following co-culture as compared to LPS-stimulated microglia cells prior to co-culture.

[0017] In some embodiments, the methods further comprise quantifying gene expression of one or more pro-inflammatory cytokines and / or chemokines in the mesenchymal stem cells prior to and / or following co-culturing.

[0018] In some embodiments, the quantifying of gene expression comprises isolating RNA from the microglia cells and quantifying mRNA of the one or more pro-inflammatory cytokines and / or chemokines. In some embodiments, the quantifying mRNA comprises real-time reverse transcription polymerase chain reaction (RT-qPCR).

[0019] In some embodiments, the methods further comprise designating the mesenchymal stem cells suitable for assays and therapies based on the immunomodulatory potency.

[0020] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1. In vitro assays of neuroimmune modulatory potency of hMSCs from 3 tissue sources. Experimental co-culture setup and workflows (left) and qPCR data of gene expression of IL-6, TNFa, and IL-ip 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-ip, and IL-6 in a dose- and time-dependent 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.

[0022] PIGS. 2A-2D. Validation of in vitro assays. PIGS. 2A-2B show that pain behavioral (PWT) efficacy (PIG. 2A) predicts in vitro assay potencies of hMSCs (PIG. 2B). PIG. 2A. 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=9-2023-143-02 CCF-42972.60110 / group. FIG. 2B. In vitro assays of IL-6, TNFa, and IL-ip 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. 2C-2D show in vitro assay potency (FIG. 2C) predicts analgesic efficacies (FIG. 2D). FIG. 2C. In vitro assay of IL6 mRNA. Labels as in FIG. 2B. FIG. 2D. CCI model, Mean+SD. *** p<0.01, n=5 / group. Sham, sham control, PBS, placebo control.

[0023] FIG. 3. 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.DETAILED DESCRIPTION

[0024] Disclosed herein are methods for screening mesenchymal stem cells (MSCs). One challenge in hMSC therapy is optimizing treatment outcomes considering there are several sources of variability that impact the potency and efficacy of hMSCs. These variables include the source of the stem cells (e.g., umbilical cord), the donor (e.g., a patient’s age, health, and genetics will impact stem cell function), cell quality, cell purity, and delivery methods, among others. Several screening techniques and assays are used today, however, none are specifically designed to assess the analgesic effect of MSCs on the basis of their immunomodulatory profile or potency.

[0025] As described herein, a MSC-microglia co-culture assay provides the reliability, precision, dynamic range, and sensitivity for selecting an MSC source and donor based on efficacy in modulating the response of immune cells (e.g., IL-6, TNF-a, IL-ip) to immune challenges (e.g., LPS injection). The assay is suitable for use on MSCs isolated from a variety of tissues, including umbilical cord, adipose tissue, and bone marrow, for example, from human and animal sources. The assay assesses the neuroimmune modulatory properties of MSCs, to serve as a measure of quality control, and to predict clinical outcomes of MSC therapy. The assays were validated by a bidirectional validation method in a double-blind manner. The analgesic efficacy2023-143-02 CCF-42972.601 of hMSCs predicted the neuroimmune modulatory potencies of MSCs in vitro, and inversely the neuroimmune modulatory potency of hMSCs in vitro predicted the analgesic efficacy of hMSCs in vivo. Three sources of hMCSs from different tissues (hUC-MSCs, hAD-MSCs, and hBM- MSCs) demonstrated greater than 50% suppression of IL-6 gene expression by BV2 microglial cells derived from primary mouse microglia, which keep many of the functions and features that microglia express in vivo. By taking into consideration MSC variability, the disclosed methods and systems overcome a major challenge and limitation in MSC-based therapies, and enable optimal donor cell selection, dosages, and other therapeutic parameters to achieve beneficial treatment, for example for patients with chronic pain conditions.

[0026] 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

[0027] 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.

[0028] 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.2023-143-02 CCF-42972.601

[0029] 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., “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.”

[0030] 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.

[0031] The term “stem cell” refers to a cell that retains the ability to renew itself through mitotic cell division and that can differentiate into a diverse range of specialized cell types. Mammalian stem cells can be divided into three broad categories: embryonic stem cells, which are derived from blastocysts, adult stem cells, which are found in adult tissues, and cord blood stem cells, which are found in the umbilical cord. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body by replenishing specialized cells. Totipotent stem cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Multipotent stem cells can produce only cells of a closely related family of cells (e.g., hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can produce only one cell type, but have the property of self-renewal, which distinguishes them from non-stem cells.

[0032] 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 some2023-143-02 CCF-42972.601 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. In some embodiments, the MSCs are derived from adipose tissue.

[0033] As used herein, the term “co-culturing” refers to culturing two or more different types of cells together in a single culture, either in the same compartment (direct co-culturing) or separate compartments (indirect co-culturing). In some embodiments, the compartments are cultured under the same culturing conditions (e.g., same culture medium, oxygen concentration, temperature, etc.).

[0034] The terms “cell culture medium,” “culture medium,” and “medium” refer to any media for culturing cells containing nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc. Cell culture media used for particular cell types are known to those skilled in the art.

[0035] 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). Examples of mammals include, but are not limited to, any member of the Mammalian 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.

[0036] 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.2023-143-02 CCF-42972.601

[0037] 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. Mesenchymal stem cell immunomodulatory potency

[0038] Embodiments of the present disclosure include methods for determining the immunomodulatory potency of mesenchymal stem cells. The methods utilize a co-culture of immune-challenged (e.g., lipopolysaccharide (LPS)-stimulated) microglia cells with the mesenchymal stem cells to determine the changes in the expression levels of one or more pro- inflammatory markers (e.g., pro-inflammatory cytokines and / or chemokines) in the immune- challenged (e.g., LPS -stimulated) microglia cells indicative of the immunomodulatory nature of the mesenchymal stem cells.

[0039] The methods comprise co-culturing immune-challenged (e.g., LPS-stimulated) microglia cells with mesenchymal stem cells. In some embodiments, the immune-challenged (e.g., LPS-stimulated) microglia cells are immune-challenged (e.g., LPS-stimulated) BV2 microglia cells. In some embodiments, the immune-challenged (e.g., LPS-stimulated) microglia cells are immune-challenged (e.g., LPS-stimulated) primary microglia cells.

[0040] The methods may further comprise generating immune-challenged (e.g., LPS- stimulated) microglia cells. In some embodiments, the methods further comprise treating microglia cells (e.g.. BV2 cells) with LPS prior to co-culturing. Treating the microglia cells cell may comprise culturing microglia cells in the presence of LPS. The method of generating LPS- stimulated microglia cells is not limited by length of treatment or the amount of LPS.

[0041] The microglia cells may be treated with about 50 ng / mL to about 5000 ng / mL LPS. In some embodiments, the microglia cells are treated with about 100 ng / mL to about 1000 ng / mL LPS. For example, the microglia cells may be treated with about 100 ng / mL, about 200 ng / mL, about 300 ng / mL, about 400 ng / mL, about 500 ng / mL, about 600 ng / mL, about 700 ng / mL, about 800 ng / mL, about 600 ng / mL, or about 1000 ng / mL LPS.

[0042] The microglia cells may be treated with LPS for at least about 10 minutes (e.g.. at least about 20 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, or more). In some embodiments, the2023-143-02 CCF-42972.601 microglia cells are treated with LPS for about 2 to about 48 hours, e.g., about 2 to about 36 hours, about 4 to about 28 hours, about 6 to about 24 hours, about 12 to about 20 hours. In select embodiments, the microglia cells are treated with LPS for about 6 hours.

[0043] The mesenchymal stem cells may be isolated or derived from a subject, e.g., a mammal such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits. In some embodiments, the mesenchymal stem cells may be derived from humans. 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.

[0044] The microglia cells are cultured in cell numbers which allow for sensitive and consistent downstream analysis. For example, the number of microglia cells may be at least about 1 x 104cells, at least about 5 x 104cells, at least about 1 x 105cells, or at least about 1 x 106cells (e.g., at least about 0.1 million cells, at least about 0.2 million cells, at least about 0.5 million cells).

[0045] The MSCs may be at any confluency in the co-culture. For example, the MSC confluency may be from about 20% to about 80% (e.g., about 30%, about 40%, about 50%, about 60%, about 70%). In some embodiments, the MSCs are at high confluency (e.g., greater than 60% confluency).

[0046] Methods for preparing and culturing cells can be found in standard textbooks and reviews in cell biology, tissue culture, and embryology. Culture vessels suitable for use for culturing the cell(s) include, but are not limited to: flask, flask for tissue culture, spinner flask, dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CellSTACK Chambers, culture bag. and roller bottle, as long as it is capable of culturing the cells therein. The cells may be cultured at any desired volume. The culture vessel surface can be prepared with cellular adhesive to improve the adhesiveness of the vessel surface to the cells.

[0047] The co-culturing may be compartmentalized or indirect co-cultures. For example, the mesenchymal stem cells are spatially segregated from LPS-stimulated microglia cells separated by a permeable barrier, such as a porous filter, membrane, or hydrogel. Thus, a first culture compartment comprises the LPS-stimulated microglia cells and a second culture compartment comprises the mesenchymal stem cells, and the two compartments are separated by permeable barrier that allows fluid, cell media materials, bioactive molecules secreted by the cells, and the like to pass through into the bulk culture solution in both compartments.2023-143-02 CCF-42972.601

[0048] In some embodiments, the indirect or compartmentalized co-culture uses a transwell system. In specific embodiments, the mesenchymal stem cells are positioned in the transwell upper chamber and the LPS-stimulated microglia cells are positioned in the transwell lower chamber, and the mesenchymal stem cells and the LPS-stimulated microglia cells may be cocultured in a medium, for example, while sharing the same medium or in the respective culture media while being spatially separated from each other.

[0049] The co-culture may be performed for about 6 hours or more, for example about 8 hours or more, about 12 hours or more, about 16 hours or more, about 20 hours or more, about 24 hours or more, about 30 hours or more, about 36 hours or more, or about 40 hours or more. In select embodiments, the co-culturing is carried out for at least about 6 hours. In select embodiments, the co-culturing is carried out for about 12 to about 36 hours. In select embodiments, the co-culturing is carried out for about 24 hours.

[0050] MSCs and microglia cells can be cultured using established conditions (e.g., media, temperatures, oxygen concentration, etc.). For example, the co-culturing may be performed at a temperature of 35° C to 40° C, e.g., 36° C to 38° C and 4% to 6% CO2, but is not limited thereto.

[0051] The methods comprise quantifying gene expression of one or more pro-inflammatory cytokines and / or chemokines in the immune-challenged (e.g., LPS-stimulated) microglia cells following co-culture. Proinflammatory cytokines are secreted from immune cells like helper T cells, CD4+cells, macrophages, dendritic cells, and certain other cell types that promote inflammation. Proinflammatory cytokines include, for example, interleukin- 1 (IL- 1), IL-6, IL-12, IL-17, and IL-18, tumor necrosis factor alpha (TNF-a), interferon gamma (IFNy), and granulocyte-macrophage colony stimulating factor (GM-CSF). Chemokines are cytokines with chemotactic activities. They are classified into four main subfamilies including CXC, CC, CX3C, and XC chemokines with both structural and functional differences. Pro- inflammatory chemokines, include for example, CCL2, CCL3, CCL4, CCL5, CCL11, CCL20, CXCL8. In some embodiments, the pro-inflammatory cytokines and / or chemokines are selected from IL-6, TNF-a, IL-1 , and combinations thereof. In select embodiments, the one or more pro- inflammatory cytokines and / or chemokines comprise IL-6.

[0052] The term “expression” as used herein refers to the biosynthesis of the one or more pro- inflammatory cytokines and / or chemokines, including the transcription and / or translation. Thus, expression of a nucleic acid molecule may refer to transcription of the nucleic acid fragment(e.g., transcription resulting in mRNA or other functional RNA) and / or translation of RNA into a2023-143-02 CCF-42972.601 precursor or mature protein (polypeptide). In some embodiments, expression of the one or more pro-inflammatory cytokines and / or chemokines refers to RNA expression. In some embodiments, expression of the one or more pro-inflammatory cytokines and / or chemokines refers to protein expression. Each of the one or more pro-inflammatory cytokines and / or chemokines may be assayed for expression using RNA expression, protein expression, or both.

[0053] Quantifying the gene expression of the one or more pro-inflammatory cytokines and / or chemokines in may comprise isolating RNA from the microglia cells and quantifying mRNA of the one or more pro-inflammatory cytokines and / or chemokines, e.g., by real-time reverse transcription polymerase chain reaction (RT-qPCR).

[0054] In some embodiments, the methods further comprise quantifying gene expression of one or more pro-inflammatory cytokines and / or chemokines in the immune-challenged (e.g., LPS-stimulated) microglia cells prior to co-culturing. For example, a population of immune- challenged (e.g., LPS-stimulated) microglia cells may be isolated and the rate of induction of the pro-inflammatory cytokines and / or chemokines following the immune challenge (e.g., LPS treatment) but prior to culture may determined in a similar manner as to that used following coculture.

[0055] The methods comprise classifying the immunomodulatory potency of the mesenchymal stem cells based on level of reduction of one or more pro-inflammatory cytokines and / or chemokines in the immune-challenged (e.g., LPS-stimulated) microglia cells following co-culture. The reduction may be compared to immune-challenged (e.g., LPS-stimulated) microglia cells not co-cultured with the mesenchymal stem cells. Alternatively, the reduction may be compared to immune-challenged (e.g., LPS-stimulated) microglia cells prior to coculturing with the mesenchymal stem cells.

[0056] In some embodiments, mesenchymal stem cells are considered to have high or strong immunomodulatory potency when there is about 50% or greater (e.g., about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more) reduction in one or more pro-inflammatory cytokines and / or chemokines in the immune- challenged (e.g., LPS-stimulated) microglia cells. Conversely, those mesenchymal stem cells that confer a less than 50% or greater reduction in one or more pro-inflammatory cytokines and / or chemokines in the immune-challenged (e.g.. LPS-stimulated) microglia cells are considered to have low or weak immunomodulatory potency.2023-143-02 CCF-42972.601

[0057] Also provided herein are preparations or compositions having or enriched for high immunomodulatory potency mesenchymal stem cells. In some embodiments, the preparations or compositions comprise a earner, such as a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers and agents suitable for the present invention, including those exemplified above, are described in detail and may generally be found in “Remington’s Pharmaceutical Sciences,” (Meade Publishing Co., Easton, Pa.). Accordingly, provided herein are compositions comprising high immunomodulatory potency mesenchymal stem cells and a carrier.

[0058] Following determination of the immunomodulatory potency, the mesenchymal stem cells can be designated as suitable for a variety of assays and therapies.3. ExamplesExample 1Determination of immunomodulatory profiles of hMSCs from different tissue sources

[0059] In vitro assays for neuroimmune modulatory potency of hMSCs

[0060] 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 cells 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-1 , 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 2 Validation of in vitro assays for neuroimmune modulatory potency of hMSCs

[0061] Analgesic efficacy of hMSCs predicts in vitro neuroimmune modulatory potencies of MSCs

[0062] 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. Anti-hyperalgesia efficacies were significantly different between cells2023-143-02 CCF-42972.601 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.

[0063] In vitro neuroimmune modulatory potency of hMSCs predicts in vivo analgesic efficacies

[0064] 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 CO mice by experimenters blind to the in vitro assay results. MSC2330 significantly outperformed MSC2338. The neuroimmune modulatory potency of MSCs can predict their analgesic efficacies.

[0065] The neuroimmune modulatory potency of hMSCs from 3 different tissue sources (hUC-MSCs. hAD-MSCs, and hBM-MSCs) that are produced at Duke 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 from Duke met the criteria of >50% suppression of inflammatory cytokines (IL-6. TNFa. and IL- 10). Suppression of IL-6 is the most sensitive measure among all, which is consistent with 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.

[0066] 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.

[0067] 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

2023-143-02 CCF-42972.601CLAIMSWhat is claimed is:

1. A method for determining the immunomodulatory potency of mesenchymal stem cells, comprising: co-culturing lipopolysaccharide (LPS)-stimulated microglia cells with mesenchymal stem cells; quantifying gene expression of one or more pro-inflammatory cytokines and / or chemokines in the LPS-stimulated microglia cells following co-culture; and classifying the immunomodulatory potency of the mesenchymal stem cells based on level of reduction of one or more pro-inflammatory cytokines and / or chemokines in the LPS- stimulated microglia cells following co-culture as compared to LPS-stimulated microglia cells not co-cultured with the mesenchymal stem cells.

2. The method of claim 1, wherein the mesenchymal stem cells are classified as having high immunomodulatory potency when there is about 50% or greater reduction in one or more pro- inflammatory cytokines and / or chemokines in the LPS-stimulated microglia cells following coculture as compared to LPS-stimulated microglia cells not co-cultured with mesenchymal stem cells.

3. The method of claim 1 or 2, wherein the one or more pro-inflammatory cytokines and / or chemokines are selected from IL-6, TNF-a, IL-ip, and combinations thereof.

4. The method of any of claims 1-3, wherein the one or more pro-inflammatory cytokines is IL- 6.

5. The method of any of claims 1-4, wherein the method further comprises treating microglia cells with about 100 ng / mL to about 1000 ng / mL LPS prior to co-culturing.

6. The method of claim 5, wherein the treating is earned out for at least 20 minutes.

7. The method of claim 5 or 6, wherein the treating is carried out for about 6 hours.2023-143-02 CCF-42972.6018. The method of any of claims 1-7, wherein the LPS-stimulated microglia cells are LPS- stimulated BV2 microglia cells.

9. The method of any of claims 1-8, wherein the method further comprises quantifying gene expression of one or more pro-inflammatory cytokines and / or chemokines in the microglia cells prior to LPS stimulation.

10. The method of any of claims 1-9, wherein the mesenchymal stem cells are derived from umbilical cord, bone marrow, or adipose tissue.

11. The method of any of claims 1-10, wherein the co-culturing is an indirect or compartmentalized co-culture.

12. The method of claim 11, wherein the indirect or compartmentalized co-culture uses a transwell system.

13. The method of claim 12, wherein the mesenchymal stem cells are positioned in a transwell upper chamber and the LPS-stimulated microglia cells are positioned in a transwell lower chamber.

14. The method of any of claims 1-13, wherein the co-culturing is earned out for at least about 6 hours.

15. The method of any of claims 1-14, wherein the co-culturing is earned out for 12 to 36 hours.

16. The method of any of claims 1-15, wherein the co-culturing is carried out for about 24 hours.

17. The method of any of claims 1-16, wherein the method further comprises quantifying gene expression of one or more pro-inflammatory cytokines and / or chemokines in the LPS-stimulated microglia prior to co-culturing.2023-143-02 CCF-42972.60118. The method of claim 17, wherein mesenchymal stem cells have high immunomodulatory potency when there is about 50% or greater reduction in one or more pro-inflammatory cytokines in the LPS-stimulated microglia cells following co-culture as compared to LPS-stimulated microglia cells prior to co-culture.

19. The method of any of claims 1-18, wherein the method further comprises quantifying gene expression of one or more pro-inflammatory cytokines and / or chemokines in the mesenchymal stem cells prior to and / or following co-culturing.

20. The method of any of claims 1-19, wherein the quantifying of gene expression comprises isolating RNA from the microglia cells and quantifying mRNA of the one or more pro- inflammatory cytokines and / or chemokines.

21. The method of claim 20, wherein the quantifying mRNA comprises real-time reverse transcription polymerase chain reaction (RT-qPCR).

22. The method of any of claims 1-21, further comprising designating the mesenchymal stem cells suitable for assays and therapies based on the immunomodulatory potency.

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