Methods, compositions and their implementation for culturing mesenchymal stem cells

Abstract

Provided herein is a method of generating a population of exosomes derived from primed mesenchymal stem cells. The method according to the present disclosure may include expanding a population of mesenchymal stem cells (MSCs) in culture, administering one or more priming agents during culture to prime the population of MSCs to obtain a population of primed MSCs, expanding the population of primed MSCs in culture to produce conditioned medium from the primed MSCs, recovering the primed MSC conditioned medium, and purifying a population of exosomes from the primed MSC conditioned medium. The one or more priming agents may include a conditioned medium derived from a population of stem cells different from the population of MSCs, an Nrf2 activator, or a combination thereof. Also provided herein is a method of treating tissues such as the cornea and liver with a population of exosomes from primed MSC conditioned medium.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Indian Application No. 202141036331 filed on August 11, 2021. All applications are incorporated herein by reference in their entirety. [Background technology]

[0002] There are several compelling preclinical and clinical studies demonstrating the efficacy of cell-free therapy using mesenchymal stem cells (MSCs) and cell-derived exosomes in treating fibrosis, inflammation, wound healing and promoting tissue regeneration. The therapeutic effects of MSCs are largely attributed to paracrine factors secreted by the cells, including exosomes.

[0003] MSCs secreting exosomes function as mediators of intercellular communication, such as, but not limited to, tumors, and play several roles in tumorigenesis, angiogenesis, metastasis, and intracellular communication. Exosomes are nanoscale extracellular vesicles (EVs) that act as mediators of crosstalk between cells. MSC-derived exosomes (MSC-Exo) contain as cargo proteins, for example, growth factors, cytokines, lipid moieties, as well as nucleic acids, such as miRNA, mRNA, and transfer RNA (tRNA), and other non-coding RNAs (ncRNAs), which may provide the anti-fibrotic, anti-inflammatory, and pro-regenerative therapeutic effects of exosomes in humans. MSC-Exo has also been found to activate several signaling pathways (Akt, ERK, and STAT3) important in tissue regeneration and inflammation, as well as induce the expression of numerous growth factors. While MSCs themselves can be used as therapeutic agents, the advantages of using MSC-Exo over MSCs include less immunogenicity, lower risk of rejection, and less tumorigenicity. MSC-Exo are also less likely to suffer from pulmonary first-pass effects, which is important for both safety and systemic delivery. Therefore, exosomes derived from MSCs have been attempted in the treatment of certain diseases and disorders.

[0004] However, the complement of exosomal cargo is influenced by priming agents in culture that prime MSCs and affect their activity and transcriptional profile. As a result, priming can dramatically affect the therapeutic efficacy of exosomes produced by a given MSC population in unanticipated ways, for better or worse.

[0005] Moreover, MSC-Exo is obtained from MSC that secretes MSC-Exo.Mass expansion of MSC is one of the characteristics of MSC-Exo-based therapy.However, conventional methods available in the art cannot adequately scale up the production of MSC and its secreted products for commercial therapeutic application. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need to identify priming agents and related MSC culture methods to produce primed MSCs and the MSC-Exos produced thereby, thereby providing a sufficient supply of exosomes with high and improved therapeutic efficacy. [Means for solving the problem]

[0007] Provided herein are embodiments of methods for generating a population of exosomes derived from primed mesenchymal stem cells, the methods including (a) expanding a population of cultured mesenchymal stem cells (MSCs), (b) priming the population of MSCs with conditioned medium from cells derived from a cell population different from the population of MSCs and at least one defined priming agent to obtain a population of primed MSCs, (c) expanding the population of primed MSCs in culture to produce primed MSC-derived conditioned medium, and (d) recovering the primed MSC conditioned medium. In some variations, the methods include purifying exosomes from the primed MSC conditioned medium.

[0008] In some variations, priming the population of MSCs comprises (1) contacting the population of MSCs with conditioned medium from the cells and (2) contacting the population of MSCs with at least one defined priming agent. Optionally, the population of MSCs is contacted with conditioned medium from the cells from seeding to about 60% to about 90% confluency, and the population of MSCs is contacted with at least one defined priming agent starting from about 60% to about 90% confluency. Optionally, the population of MSCs is contacted with conditioned medium from the cells from seeding to about 60% to about 90% confluency, and then contacted with at least one defined priming agent. Optionally, the population of MSCs is contacted with at least one defined priming agent for about 12 hours to about 72 hours.

[0009] In some variations, the conditioned medium from cells from a distinct cell population is conditioned medium from corneal stromal stem cells.

[0010] In some variations, the at least one defined priming agent is a nuclear factor erythroid 2-related factor 2 (Nrf2) activator, a silencing information regulator 1 (SIRT1) activator, or all-trans retinoic acid (ATRA). Optionally, the at least one priming agent is an Nrf2 activator. Optionally, the Nrf2 activator is dimethyl fumarate (DMF) or 4-octyl itaconate (4-OI).

[0011] In some variations, the population of MSCs is a population of bone marrow-derived MSCs (BM-MSCs), a population of umbilical cord-derived MSCs (UM-MSCs), a population of induced pluripotent stem cell (iPSC)-derived MSCs (iPSC-MSCs), or a population of Wharton's jelly-derived MSCs (WJ-MSCs). Optionally, the population of BM-MSCs is a population of human BM-MSCs.

[0012] In some variations, the cell-derived conditioned medium is corneal stem cell-derived conditioned medium and the defined priming agent is DMF or 4-octyl itaconate (4-OI). Optionally, the corneal stem cell-derived conditioned medium is present at a concentration of about 10% to about 30%. Optionally, the DMF is present at a concentration of about 50 μM to about 100 μM.

[0013] Also provided herein are embodiments of populations of primed MSC-derived exosomes produced by the above method embodiments.

[0014] Also provided herein is an embodiment of a population of exosomes derived from primed MSCs, characterized by having one or more of (a) a low expression level of vascular endothelial growth factor (VEGF); and (b) a high expression level of nerve growth factor (NGF) compared to exosomes derived from unprimed MSCs. Optionally, exosomes derived from primed MSCs are characterized by one or more of (c) a high expression level of hepatic growth factor (HGF); and (d) a high expression level of sFLT1 compared to exosomes derived from unprimed mesenchymal stem cells. Optionally, exosomes derived from primed MSCs are characterized by one or a combination of two or more, a combination of three or more, or all of (a) at least two-fold higher expression level of sFLT1; (b) an expression level of VEGF that is one-quarter or less than that in exosomes derived from unprimed MSCs; (c) at least two-fold higher expression level of HGF; and (d) at least three-fold higher expression of NGF compared to exosomes derived from unprimed mesenchymal stem cells. In some variations, the primed MSCs are prepared by (1) contacting a population of MSCs with corneal stem cell-derived conditioned medium; and (2) contacting a population of MSCs with an Nrf2 activator. Optionally, the Nrf2 activator is DMF or 4-octyl itaconate (4-OI). Optionally, the population of MSCs is contacted with corneal stem cell-derived conditioned medium from seeding to about 60% to about 90% confluency, and the population of MSCs is contacted with the Nrf2 activator starting from about 60% to about 90% confluency. Optionally, the population of MSCs is contacted with the Nrf2 activator for about 12 hours to about 72 hours.

[0015] Also provided herein are embodiments of methods for treating corneal defects, comprising administering a therapeutic dose of a population of exosomes disclosed herein to a corneal surface having a corneal defect. Optionally, the corneal defect is selected from the group consisting of corneal scarring, keratitis, corneal ulcer, corneal abrasion, corneal epithelial damage, corneal stromal damage, infection-based corneal damage, trachoma, keratoconus, corneal perforation, corneal limbal injury, corneal dystrophy, angiogenesis, vernal keratoconjunctivitis, and dry eye. In some variations, the population of exosomes is included in an ophthalmic composition formulated for application to the corneal surface. Optionally, the composition is an eye drop. Optionally, the eye drop comprises a biocompatible polymer. Optionally, the biocompatible polymer is crosslinkable, and the method comprises administering the eye drop to the corneal surface and crosslinking a sufficient portion of the crosslinkable polymer to convert the eye drop into a hydrogel.

[0016] Also provided herein are embodiments of ophthalmic compositions formulated for application onto the corneal surface, comprising a population of exosomes as disclosed herein. Optionally, the ophthalmic composition is in an ophthalmic solution. Optionally, the ophthalmic solution comprises a biocompatible polymer. Optionally, the ophthalmic composition is a hydrogel, and at least a portion of the biocompatible polymer is crosslinked.

[0017] Also provided herein are embodiments of a method for generating a population of exosomes derived from primed mesenchymal stem cells, the method comprising: (a) culturing a population of mesenchymal stem cells (MSCs) in a medium; (b) priming the population of MSCs with an Nrf2 activator to obtain a population of primed MSCs; (c) growing the population of primed MSCs in a collection medium, such that the collection medium becomes enriched with exosomes produced by the primed MSCs, thereby producing a conditioned medium from the primed MSCs; and (d) collecting the primed MSC conditioned medium. Optionally, the method further comprises (e) purifying exosomes from the primed MSC conditioned medium. Optionally, the population of MSCs is grown in a first medium from seeding to about 60% to about 90% confluency, and then contacted with an Nrf2 activator. Optionally, the population of MSCs is contacted with the Nrf2 activator for about 12 hours to 72 hours. Optionally, the Nrf2 activator is dimethyl fumarate (DMF) or 4-octyl itaconate (4-OI). Optionally, the DMF is present at a concentration of about 50 μM to about 100 μM. Optionally, the population of MSCs is a population of bone marrow MSCs (BM-MSCs), a population of umbilical cord-derived MSCs (UM-MSCs), a population of induced pluripotent stem cell (iPSC)-derived MSCs (iPSC-MSCs), or a population of Wharton's jelly-derived MSCs (WJ-MSCs).

[0018] Also provided herein are embodiments of a population of primed MSC-derived exosomes produced by the above methods.

[0019] Also provided is a population of exosomes derived from primed MSCs, characterized by having one or more of (a) high expression levels of hepatic growth factor (HGF); and (b) high expression levels of nerve growth factor (NGF) compared to exosomes derived from unprimed MSCs. Optionally, exosomes derived from primed MSCs are characterized by (a) high expression levels of HGF; and (b) high expression levels of NGF compared to exosomes derived from unprimed MSCs. Optionally, exosomes derived from primed MSCs are characterized by one or both of (a) at least 1.2-fold higher expression levels of HGF; and (b) at least 2-fold higher expression levels of NGF compared to exosomes derived from unprimed MSCs.

[0020] In some variations, the primed MSCs are prepared by (1) growing a population of MSCs in a first medium; and (2) contacting the population of MSCs with an Nrf2 activator. Optionally, the Nrf2 activator is DMF or 4-octyl itaconate (4-OI). Optionally, the DMF is present at a concentration of about 50 μM to about 100 μM. Optionally, the MSCs are grown in the first medium from seeding to about 60% to about 90% confluency, and then contacted with the Nrf2 activator. Optionally, the population of MSCs is contacted with the Nrf2 activator for about 12 hours to about 72 hours, and then replaced with a harvest medium.

[0021] Also provided herein is an embodiment of a method for treating a liver condition, the method comprising administering to a subject having a liver condition a therapeutic amount of a population of exosomes provided herein. Optionally, the liver condition is non-alcoholic fatty liver disease (NAFLD). Optionally, the NAFLD is non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH). Optionally, the population of exosomes is administered to the liver via an intravenous route. Optionally, the intravenous route is via the hepatic portal vein.

[0022] Also provided herein is an embodiment of a composition comprising a population of exosomes disclosed herein above.Optionally, the composition is for use in treating a liver condition.Optionally, the liver condition is non-alcoholic fatty liver disease (NAFLD).Optionally, the NAFLD is non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH).

[0023] Provided herein are embodiments of a method for increasing secretion of exosomes by a population of mesenchymal stem cells (MSCs), the method comprising: (a) culturing a population of MSCs in a medium; (b) priming the population of MSCs with an Nrf2 activator to obtain a population of primed MSCs; and (c) expanding the primed population of MSCs in a collection medium, such that the collection medium is enriched with exosomes produced by the primed MSCs. Optionally, the population of MSCs is expanded in a first medium from seeding to about 60% to about 90% confluency and then contacted with an Nrf2 activator. Optionally, the population of MSCs is contacted with the Nrf2 activator for about 12 hours to 72 hours. Optionally, the Nrf2 activator is dimethyl fumarate (DMF) or 4-octyl itaconate (4-OI). Optionally, the DMF is present at a concentration of about 50 μM to about 100 μM. Optionally, the population of MSCs is a population of bone marrow MSCs (BM-MSCs), a population of umbilical cord-derived MSCs (UM-MSCs), a population of induced pluripotent stem cell (iPSC)-derived MSCs (iPSC-MSCs), or a population of Wharton's jelly-derived MSCs (WJ-MSCs).

[0024] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This Summary is provided to introduce a selection of concepts in a simplified form. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0025] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The present disclosure may be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0026] [Figure 1] 1A-1E show bar graphs of quantification of the secretome of human bone marrow-derived MSCs (hBM-MSCs) primed with corneal stromal stem cell-derived conditioned medium (CSSC-CM). Quantification was based on enzyme-linked immunoassay (ELISA) of the medium in which hBM-MSCs were grown or fractions thereof. hBM-MSC cells were primed with CSSC-CM (10% of the 20% of the medium was replaced with CSSC-CM) and the secretome profile of primed hBM-MSC cells was characterized according to an embodiment of the present disclosure, in particular the secretion levels of HGF (FIG. 1A), VEGF (FIG. 1B), sFLT1 (FIG. 1C), IL-6 (FIG. 1D), and NGF (FIG. 1E). [Diagram 2] 2A-2E show bar graphs of quantification of the anti-inflammatory effect of primed hBM-MSC-derived exosomes on RAW264.7 macrophage cells stimulated with lipopolysaccharide (LPS) binding protein in the presence of the indicated primed exosomes. Cytokine expression was measured by ELISA for IL-6 (FIG. 2A), IL-1β (FIG. 2B), IL-10 (FIG. 2C), TNF-α (FIG. 2D), and IFNγ (FIG. 2E) according to embodiments of the present disclosure. [Diagram 3] Figures 3A-3E show bar graphs of quantification of cytokine expression from RAW264.7 macrophage cells stimulated with LPS in the presence of the indicated primed exosomes (500 million exosomes). Cytokine expression was measured by quantitative PCR (qPCR) for IL-6 (Figure 3A), IL-1β (Figure 3B), TNF-α (Figure 3C), IL-10 (Figure 3D) and IFNγ (Figure 3E), respectively. [Figure 4]4A-4F show representative immunofluorescence images of antifibrotic characterization of different exosome variants on human dermal fibroblasts treated with TGF-β. Human dermal fibroblasts (HDFs) were treated simultaneously with TGF-β (FIG. 4B) and the indicated exosomes (FIG. 4C-4F) for 24 hours. Cells were fixed and α-SMA expression was assessed by immunostaining. TGF-β induced α-SMA expression in cells treated with TGF-β alone, which was blocked in the presence of CSSC-primed BM-MSC-exosomes (FIG. 4D-4E) and CSSC-exosomes (FIG. 4F) and in small amounts by naive hBM-MSC-exosomes (FIG. 4C) according to an embodiment of the present disclosure. [Diagram 5] FIG. 5 shows a bar graph illustrating the effect of priming of BM-MSCs mediated by Nrf2 activators (DMF or 4-OI) and characterizes the secretome and exosome profiles under different priming conditions. Data represent three technical replicates ± SEM. The NRF2 activator DMF shows a higher yield of exosomes according to an embodiment of the present disclosure. [Figure 6] 6A-6F show bar graphs of quantification of secretome marker profiling of primed cells under different priming conditions. Secretion of HGF (FIG. 6A), IL-6 (FIG. 6B), VEGF (FIG. 6C), sFLT1 (FIG. 6D), NGF (FIG. 6E) and SDF-1 (FIG. 6F) was quantified at protein level by ELISA. Expression of HGF (FIG. 6A) was high in all primed variants, while VEGF and sFLT1 levels were unchanged (FIG. 6C-6D). Curcumin and Nrf2 activators attenuated expression of IL-6 (FIG. 6B), while NGF secretion levels were enhanced by Nrf2 activator 4-OI and DMF (FIG. 6E). The combination of conditioned medium + DMF with curcumin did not seem to have a significant effect on any of the readouts according to embodiments of the present disclosure. [Figure 7]Figures 7A-7E show bar graphs of quantification profiling the cargo (e.g., exosomal proteins) of exosomes secreted by MSCs under priming with different priming agents such as curcumin (CUR) and Nrf2 activators DM and 4-OI. Quantified cargoes include exosomal HGF (Figure 7A), exosomal VEGF (Figure 7B), exosomal sFLT1 (Figure 7C), exosomal NGF (Figure 7D), exosomal TGF-β (Figure 7E), and exosomal SDF-1 (Figure 7F). Exosomal HGF was higher in all primed variants compared to naïve MSCs. Expression of exosomal NGF was induced by Nrf2 activator priming (especially DMF). sFLT1, TGF-β and SDF-1 levels were unchanged. [Figure 8] 8A-8E show bar graphs of quantification of the anti-inflammatory effect of primed hBM-MSC-derived exosomes (hBM-MSC-Exo) in RAW264.7 macrophage cells. RAW264.7 macrophage cells were stimulated with LPS in the presence of the indicated primed exosomes (400 million exosomes). Cytokine expression was measured by ELISA for IL-6 (FIG. 8A), IL-1β (FIG. 8B), IL-10 (FIG. 8C), TNF-α (FIG. 8D), and IFNγ (FIG. 8E) according to an embodiment of the present disclosure. [Figure 9] 9A-9D show bar graphs of quantification of exosomal cargo proteins of exosomes derived from hBM-MSCs primed in combination with CSSC-CM and Nrf2 activators. Quantification was based on ELISA applied to purified exosomes produced according to embodiments of the present disclosure. Exosomal HGF (FIG. 9A), exosomal VEGF (FIG. 9B), exosomal sFLT1 (FIG. 9C), and exosomal NGF (FIG. 9D) were quantified at the protein level by ELISA. [Figure 10]10A-10E show bar graphs of quantification of the characterization of anti-inflammatory activity of different exosome variants primed with CSSC-CM with Nrf2 activator (DMF). RAW264.7 macrophage cells were stimulated with LPS in the presence of the indicated primed exosomes (approximately 500 million exosomes). Cytokine expression was measured by ELISA for IL-6 (FIG. 10A), IL-1β (FIG. 10B), TNF-α (FIG. 10C), IL-10 (FIG. 10D) and IFNγ (FIG. 10E), respectively, according to an embodiment of the present disclosure. [Figure 11] 11A-11F show representative immunofluorescence images of the characterization of the antifibrotic activity of different exosome variants derived from BM-MSCs primed with CSSC-CM and / or Nrf2 activator (DMF) on human dermal fibroblasts treated with TGF-β. Human dermal fibroblasts were treated with TGF-β and the indicated exosome variants for 24 hours and probed for α-SMA expression. TGF-β induced α-SMA in cells treated with TGF-β alone (FIG. 11B), whereas exosomes inhibited the induction of a-SMA to different extents, as shown above. [Figure 12] 12 shows representative images of wound healing activity characterization of different exosome variants, hBM-MSCs primed with CSSC-CM and / or Nrf2 activator (DMF) observed over multiple time points. Representative images showing time course of wound closure (2D scratch assay) on epithelial cell monolayer observed over multiple time points of 0 hours, 24 hours, 48 ​​hours, and 72 hours. According to an embodiment of the present disclosure, BM-MSCs; CSSC-CM (20%); Nrf2 activator: DMF. [Figure 13] FIG. 13 shows graphs of cell migration / cell proliferation assays tracking the confluence of cells treated with different exosome variants containing naive exosomes, exosomes derived from hBM-MSCs primed with CSSC-CM, Nrf2 activators, or combinations thereof. [Figure 14]FIG. 14 shows representative images of a wound healing assay using rabbit corneas with open epithelial wounds containing an untreated control, liquid corneal biopolymer, and a combination of liquid corneal biopolymer with CSSC-CM and Nrf2 activator, exosomes derived from hBM-MSCs primed with DMF. [Figure 15] Figure 15A shows representative immunofluorescence images stained for CYP34A in healthy liver spheroids, NASH-induced liver spheroids, or NASH-induced liver spheroids treated with exosomes. Figure 15B shows bar graphs of quantification of secreted albumin levels in human liver spheroids, including NASH-induced liver spheroids and NASH-induced liver spheroids treated with exosomes, after 24 hours, 48 ​​hours, and 72 hours. Figure 15C shows representative immunofluorescence images stained for collagen, a fibrosis marker, in NASH-induced liver spheroids and liver spheroids treated with exosomes. Figure 15D shows bar graphs of quantification of collagen deposition coverage in human liver spheroids, including NASH-induced liver spheroids and NASH-induced liver spheroids treated with exosomes. Figure 15E shows representative immunofluorescence images stained for fibrosis marker α-SMA in healthy liver spheroids, NASH-induced liver spheroids, and exosome-treated NASH-induced liver spheroids. Figure 15F shows a bar graph of quantification of α-SMA intensity in human liver spheroids, including NASH-induced liver spheroids or exosome-treated NASH-induced liver spheroids. [Figure 16] Figure 16A shows a gene expression heat map of the global gene expression profile of NASH-induced liver spheroids treated with primed exosomes derived from BM-MSCs. Figure 16B shows a principal component analysis plot comparing the differentially expressed genes of NASH-induced liver spheroids treated with primed exosomes. [Figure 17]FIG. 17A shows a gene expression heat map of liver-specific genes of the profile of NASH-induced liver spheroids treated with naive or primed exosomes. FIG. 17B shows a gene expression heat map of non-alcoholic steatohepatitis (NASH) / fibrosis-related genes of the profile of NASH-induced liver spheroids treated with naive or primed exosomes. FIG. 17C shows a gene expression heat map of stellate cell-specific genes of the profile of NASH-induced liver spheroids treated with naive or primed exosomes. FIG. 17D shows a gene expression heat map of genes related to xenobiotic metabolic processes of the profile of NASH-induced liver spheroids treated with naive or primed exosomes. FIG. 17E shows a gene expression heat map of fatty acid metabolism genes of the profile of NASH-induced liver spheroids treated with naive or primed exosomes. Figure 17F shows a gene expression heat map of genes related to the epoxygenase p450 pathway in the profile of NASH-induced liver spheroids treated with naive or primed exosomes. Figure 17G shows a gene expression heat map of fatty fiber-related genes in the profile of NASH-induced liver spheroids treated with naive or primed exosomes. [Figure 18] Figure 18A shows the plot based on the Jaccard similarity index for NASH / fibrosis-related genes, global genes, and liver-specific genes in liver spheroids. Figure 18B shows the plot based on the Jaccard similarity index for genes related to neurogenesis, angiogenesis, and inflammatory response genes in liver spheroids. Figure 18C shows the plot based on the Jaccard similarity index for genes related to extracellular matrix, wound healing, and tissue remodeling in liver spheroids. [Figure 19]FIG. 19 shows gene expression heatmap of genes associated with enriched biological processes in untreated, NASH-induced, or primed exosome-treated NASH-induced liver spheroids. [Figure 20] FIG. 20 shows a flowchart of a method for generating primed exosomes according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Those skilled in the art will recognize that the present disclosure is subject to variations and modifications other than those specifically described.The present disclosure should be understood to include all such variations and modifications.The present disclosure also includes all such steps, features, compositions, and compounds mentioned or indicated herein, individually or collectively, and any or all combinations of any or more of such steps or features.

[0028] definition For convenience, before further description of the present disclosure, certain specific terms and examples employed herein are outlined here.These definitions should be read in light of the remaining parts of the present disclosure and understood by those skilled in the art.Terms used herein have meanings that are recognized and known to those skilled in the art, but for convenience and completeness, certain terms and their meanings are described below.

[0029] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.

[0030] The terms "comprise" and "comprising" are used in an inclusive, open sense, meaning that additional elements may be included. It is not intended to be interpreted as "consisting only of."

[0031] Throughout this specification, unless the context otherwise requires, the term "comprise", and variations such as "comprises" and "comprising", are meant to include the stated elements or steps, or groups of elements or steps, but not to exclude any other elements or steps, or groups of elements or steps.

[0032] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0033] For purposes of this document, the term "expanded primed mesenchymal stem cell population" refers to a population of mesenchymal stem cells that has an increased cell number compared to the population of mesenchymal stem cells originally obtained for culture. The culture process does not differentiate the cells, but merely increases the number of cell manifolds. Additionally, priming refers to the use of a small molecule priming agent.

[0034] The term "three-dimensional culture" or "3D culture" refers to a system for culturing cells in vitro that allows biological cells to grow and interact with their surroundings in all three dimensions.

[0035] The term "two-dimensional culture" or "2D culture" refers to a method of culturing cells as a monolayer on a surface where biological cells can interact with their surroundings in two dimensions.

[0036] The term "spheroid-based system" refers to the process of culturing mesenchymal stem cells (MSCs) in three dimensions by the formation of spheroids according to the methods described in this disclosure.

[0037] The term "microcarrier-based system" refers to the process of culturing mesenchymal stem cells (MSCs) in three dimensions by the formation of alginate-gelatin (Alg / Gel) microcarriers or microbeads according to the methods described in this disclosure.

[0038] The terms "microcarriers" and "microbeads" are used interchangeably and refer to the alginate-gelatin (Alg / Gel) microcarriers or microbeads described in this disclosure.

[0039] The term "conditioned medium from mesenchymal stem cells" or "MSC-CM" refers to the medium obtained after the expansion of MSCs. The conditioned medium thus obtained contains secreted cell modulators and multiple factors important for tissue regeneration. The conditioned medium thus obtained also contains secretome and exosomes that need to be purified from the conditioned medium before it can be applied for therapeutic purposes. As described herein, the process for obtaining expanded MSCs also results in the formation of MSC-CM, and therefore, it can be said that a single process results in the procurement of a population of expanded primed MSCs as well as MSC-CM.

[0040] The term "exosome" refers to extracellular vesicles secreted by cells in the nanoscale range (e.g., 20-200 nm range), which contain as cargo biomolecules such as proteins, DNA, and RNA (including various types such as mRNA and miRNA) from the biological cells that secrete them. Some of the biomolecules can have anti-inflammatory, anti-fibrotic and regenerative properties and may be of clinical interest.

[0041] The term "micromolecule" or "small molecule" is defined as a synthetic or naturally occurring chemical modifier of cell behavior and induces therapeutic properties. Small molecules have a molecular weight of less than 800 Da.

[0042] The term "macromolecule" refers to a biological agent having a molecular weight greater than 800 Da. In this disclosure, macromolecules include proteins, lipids, nucleic acids, growth factors, cytokines, and components of conditioned media.

[0043] For purposes of this document, the term "limbal stem cells" refers to a population of stem cells that reside in the limbal stem cell niche. Limbal stem cells refer to a population of stem cells that are primarily represented by corneal stromal stem cells (CSSCs) and limbal epithelial stem cells (LESCs).

[0044] The term "conditioned medium from corneal stromal stem cells" or "CSSC-CM" refers to a medium for growing corneal stromal stem cells (CSSCs). The CSSC-CM described herein is obtained by culturing CSSCs according to methods known in the art or by culturing CSSCs according to the methods disclosed herein.

[0045] The term "xeno-free" as described in this disclosure refers to a medium that does not contain any products derived from animals other than humans. The method being xeno-free is an important advantage for the relevance of its clinical application.

[0046] The term "subject" refers to an animal subject that may be administered with a therapeutic agent, such as a composition comprising an exosome. The animal subject may be a mammalian subject. The mammalian subject may be suffering from or diagnosed with a condition referred to in this disclosure. The mammalian subject may be a human subject.

[0047] The term "therapeutically effective amount" refers to the amount of a composition needed to treat a subject's condition.

[0048] The term "naive cells" as used herein refers to unprimed mesenchymal stem cells that have not been primed with any conditioned medium. Thus, the terms unprimed and naive are used interchangeably in this disclosure.

[0049] Despite the great variability of MSCs through different in vitro cell culture methods, there are various limitations associated with conventional methods that have limited the success of MSC therapy in clinical trials. The high susceptibility of MSCs to the harsh microenvironment of immune-mediated, inflammatory, and degenerative diseases remains a major obstacle to the success of MSC-based therapy. The hostile tissue environment may limit the function and survival of transplanted MSCs. Furthermore, the use of homogenous populations of MSCs has limited their use in therapeutic applications. Also, many other limitations compromise MSC-based therapy, such as cellular senescence due to overexpansion in vitro, loss of function after cryopreservation, and discrepancies in in vivo therapeutic efficacy between preclinical and clinical trials.

[0050] To address the challenges faced in the art, one aspect of the present disclosure provides a method for the generation of mesenchymal stem cells (MSCs) and the production of exosomes purified from MSCs. In some variations, the methods provided in the present disclosure include isolating subpopulations of mesenchymal stem cells from various sources that express a signature set of markers. In some variations, the subpopulations of mesenchymal stem cells can be further modified with hTERT (human telomerase reverse transcriptase), which can extend the doubling potential of engineered MSCs (eMSCs), to facilitate scalable and uniform production of cells and therapeutic exosomes derived from said MSCs.

[0051] Another aspect of the method of the present disclosure is to prime MSCs with one or more priming agents, such as small and macromolecules and / or conditioned medium from other stem cell populations. The cells and exosomes derived from the method of the present disclosure can be used as such or in combination with each other for clinical applications. In some variations, conditioned medium from a population of naive MSCs can be used to prime a different population of naive MSCs from different tissue sources. In some variations, the use of two or more priming agents, i.e., combinatorial priming strategies, can enhance one or more of the regenerative, anti-inflammatory, and anti-fibrotic properties of MSCs and / or exosomes secreted by MSCs. For ease of presentation, exosomes secreted by naive MSCs can be referred to herein as "naive exosomes", and exosomes secreted by MSCs primed with one or more priming agents and / or conditioned medium from other cells can be referred to herein as "primed exosomes" or "primed exosome variants". Naive or primed exosomes in addition to naive / primed MSCs can be used in different cell-based therapies as such or in combination for therapeutic applications and to address multiple unmet clinical needs.

[0052] The present disclosure refers to the in vitro culture of umbilical cord blood-derived mesenchymal stem cells (UC-MSCs) / Wharton's jelly-derived MSCs (WJ-MSCs) / bone marrow-derived MSCs (BM-MSCs) and the subsequent selection of unique subpopulations with enriched factors associated with one or more of anti-fibrotic, anti-inflammatory / immunomodulatory and pro-angiogenic activities. To carry out said method, exosomes can be isolated from defined MSC populations and comprehensively characterized. The present disclosure describes protocols and / methodologies to prime various MSC populations such as UC-MSCs, WJ-MSCs, and BM-MSCs with different priming agents (in some examples, clinically approved priming agents), including but not limited to Nrf2 activators, SRT1 activators, ATRA, conditioned media, alone or in combination, to enhance cell regeneration, stemness and anti-inflammatory properties. Different exosome variants can be generated with specific / enriched cargo-loading factors using single or combinatorial priming. In some variations, exosome variants can be characterized at both physical and molecular levels for their functional effectiveness. In some variations, exosome variants can be classified based on their function on different inflammatory and fibrosis-related diseases such as pulmonary dysfunction, acute respiratory distress, inflammation-related disorders, including but not limited to rheumatoid arthritis, systemic juvenile idiopathic arthritis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome (ARDS), pneumonia, bronchitis, chronic obstructive pulmonary disease (COPD), COVID-19, coronavirus class infections, cystic fibrosis, hantavirus, influenza, tuberculosis, systemic lupus, osteoarthritis, NASH, hepatic fibrosis, Mooren's ulcer, neurotrophic ulcer, myocardial infarction, etc.

[0053] In some variations, the priming agent may be one of the following: (a) Priming with an Nrf2 activator: Without being bound by theory, in some variations, priming with an Nrf2 activator enhances the anti-inflammatory properties of primed MSCs and exosomes secreted by MSCs. Exosomes secreted by primed MSCs ("primed exosomes") may be enriched with cargo advantageous for the treatment of inflammation-related disorders. Examples of inflammation-related disorders include rheumatoid arthritis, systemic juvenile idiopathic arthritis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome (ARDS), pneumonia, bronchitis, chronic obstructive pulmonary disease (COPD), COVID-19, coronavirus class infections, cystic fibrosis, hantavirus, influenza, tuberculosis, systemic lupus, osteoarthritis, nonalcoholic fatty liver disease (NAFLD) (which may be nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH)), hepatic fibrosis, Mooren's ulcer, neurotrophic ulcer, and myocardial infarction; (b) priming with SIRT1 activator; (c) combinatorial priming with Nrf2 activator + SIRT1 activator: Without being bound by theory, in some variants, the regenerative therapeutic effect is enhanced by priming MSCs with SRT1 activator as well as Nrf2 activator. The concentrated therapeutic grade exosomes can be applied to vascular tissue regeneration. In some variations, combinatorial priming with SRT1 activators and Nrf2 activators may induce MSCs to produce exosomes with enhanced therapeutic cargo-loaded factors having one or more of anti-inflammatory, anti-fibrotic, and pro-angiogenic effects; (d) combinatorial priming with Nrf2 activators + CSSC-derived conditioned medium (CCSC-CM): In some variations, without being bound by theory, combinatorial priming with CSSC-CM with an Nrf2 activator such as DMF induces MSCs to produce exosomes enriched in anti-inflammatory factors but reduced in angiogenic factors.Thus, primed exosomes derived from MSCs combinatorially primed with Nrf2 activators and CSSC-CM can be used for regeneration of avascular tissues such as the cornea; (e) NRF2 activators + SIRT1 activators + all-trans retinoic acid + CSSC-CM. Furthermore, induction of hypoxia via physical (creating a hypoxic microenvironment) or chemical (HIF-1α) inducers in the priming process increases the viability and stemness of primed MSCs. The present disclosure also discloses a protocol for combinatorial priming with SRT1 activators and Nrf2 activators in specific BM-MSCs / UC-MSCs / WJ-MSCs in the presence and absence of hypoxia to generate more significant therapeutically enriched exosomes for regenerative therapy treatments in bioengineering and 3D bioprinting of lung, liver, and vascular tissues. The objective of the present disclosure is to significantly improve cell yields and address larger patient cohorts while maintaining the same number of product generation runs and downstream processing.

[0054] In some variations, MSC populations can be modified with hTERT (human telomerase reverse transcriptase), which extends the doubling potential of engineered MSCs (eMSCs), to facilitate scalable and uniform production of cells and therapeutic exosomes. Modulating specific pathways in MSCs, eMSCs, or induced pluripotent stem cells (iPSCs) and / or applying various combinatorial priming protocols can be used to generate exosomes that are customized for downstream applications.

[0055] The present disclosure provides as feedstocks MSCs derived from a variety of sources including human bone marrow, adipose tissue, umbilical cord, unrestricted somatic stem cells, Wharton's jelly, MSCs derived from dental pulp, iPSCs, engineered cells, limbal stem cells. MSCs may be expanded and primed to generate unique subpopulations or variants that express a signature set of markers and produce exosomes that are therapeutically effective for a given condition or set of conditions.

[0056] One aspect of the present disclosure is to prime MSCs derived from various tissue sources such as bone marrow, adipose, umbilical cord, etc. with specific combinations of inducers to activate certain pathways for the production of therapeutic exosomes having enriched factors as cargo, including one or more combinations of anti-inflammatory, anti-fibrotic, wound pre-healing, angiogenic (pro / anti), and re-innervation factors for the regeneration of avascular or vascular tissues.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art who belong to this disclosure.Any method and material similar or equivalent to those described herein can be used in the implementation or testing of this disclosure, but preferred method and material are described here.All publications mentioned herein are incorporated herein by reference.

[0058] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure, as described herein.

[0059] Methods for generating exosomes derived from primed mesenchymal stem cells In some embodiments of the present disclosure, a method 100 is provided for generating a population of exosomes derived from primed mesenchymal stem cells. Figure 20 shows a flow chart of an embodiment of the method 100. The method may include the following steps: step 101 - expanding a population of mesenchymal stem cells (MSCs) in culture; step 103 - administering one or more priming agents during culture to prime the population of MSCs to obtain a population of primed MSCs; step 105 - expanding the population of primed MSCs in culture to produce primed MSC-derived conditioned medium; and step 107 - recovering the primed MSC-conditioned medium. In some variations, the method further includes step 109 - purifying the population of exosomes from the primed MSC-conditioned medium.

[0060] Types of MSC populations In some variations, the population of MSCs may be selected from the group consisting of bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, Wharton's jelly-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, induced pluripotent stem cell-derived mesenchymal stem cells, limbal stem cells, and corneal stromal stem cells. In some variations, the MSCs may be primary cells or engineered cells. In some variations, the MSCs may be freshly derived from a primary source or cryopreserved and then thawed.

[0061] In some variations, the MSCs are CD34 - , α-SMA - , CD46 + , CD47 + , CD73 + , CD90 + , CD105 + / - , CD54 + , CD58 + , CD106 + , CD142 + / - , CD146 + , CD166 + , CD200 + , CD273 + , CD274 + , CD276+ In some variations, the subpopulation of stem cells can be a subpopulation of stem cells expressing a stem cell marker selected from the group consisting of CD34, CD40, CD50, CD60, CD80, CD90, CD100, CD120, CD25, CD31, CD42, CD125, CD140, CD260, CD180, CD270, CD31, CD42, CD160, CD180, CD270, CD180, CD180, CD280, CD180, CD290, CD31, CD42, CD180, CD180, CD29 ... - , α-SMA - , CD73 + , CD90 + , and CD166 + and (b) selecting a second subpopulation of mesenchymal stem cells from the stem cell subpopulation, the second subpopulation of mesenchymal stem cells expressing a positive marker selected from the group consisting of CD146. + , CD54 + , CD58 + , and CD142 + / - The vector expresses a marker selected from the group consisting of:

[0062] In some variations, the MSCs may comprise non-viral human telomerase enzyme reverse transcriptase (hTERT).

[0063] Culture medium In some variations, MSCs can be expanded in xeno-free medium. In some variations, MSCs can be grown under hypoxic conditions, with oxygen in the medium ranging from 0.2-10%. In some variations, MSCs can be cultured with minimal essential medium and at least one type of collagenase enzyme ranging from 5-20 IU / μl, where the at least one type of collagenase enzyme is a combination of collagenase-I and collagenase-II, to obtain expanded stem cells.

[0064] Priming Agent In some embodiments, priming agent can be applied to MSC to change the activity or gene expression pattern of cells. The change induced by priming agent to MSC can lead to or induce the change of one or more characteristics of exosomes produced and secreted by affected MSC, such as the presence of a certain biomolecule or the relative expression of a certain biomolecule in the cargo. Priming agent can be a defined substance, such as a large molecule or a small molecule. A large molecule is a biomolecule, such as a protein, DNA, or RNA (such as mRNA, miRNA, or siRNA). A protein can be a small molecule. In some variations, the priming agent may be a small molecule selected from the group consisting of Nrf2 activators, SIRT1 (silencing information regulator 1) activators, all-trans retinoic acid (ATRA), ML228, MDL800, isoquercetin, fucoidan, luteolin, quercetin, 5-aminoimidazole-4-carboxamide riboside (AICAR), 5-phenylalkoxypsoralen (Psora-4), thienopyridone (A-769662), metformin, rapamycin, 5-azacytidine (5-Aza), UM171, SB203580, fisetin, atorvastatin, valproic acid, sphingosine-1-phosphate (S1P), astaxanthin (ATX), succinic acid, and combinations thereof.

[0065] In some variations, the Nrf2 activator may be selected from the group consisting of dimethyl fumarate (DMF), optionally having a concentration in the range of 10 to 250 μM, 4-octyl itaconate (4-OI), optionally having a concentration in the range of 10 to 500 μM, and the imidazole derivative of 2-cyano-3,12-dioxoleana-1,9(11)-dien-28-oic acid (CDDO-Im), optionally having a concentration in the range of 0.1 to 10 μM, curcumin, optionally having a concentration in the range of 1 to 20 μM, and berberine, optionally having a concentration in the range of 0.1 to 100 μM.

[0066] In some variations, the SIRT1 activator may be selected from the group consisting of SRT-2104, optionally having a concentration in the range of 0.01 nM to 10 nM, trans-resveratrol, optionally having a concentration in the range of 0.1 μM to 10 μM, trans-resveratrol, optionally having a concentration in the range of 10 to 200 μM, SRT-1720, optionally having a concentration in the range of 0.1 to 10 μM, nicotinamide adenine dinucleotide (NAD), optionally having a concentration in the range of 50 to 200 μM, nicotinamide mononucleotide (NMN), optionally having a concentration in the range of 0.08 to 2.25 μM, or nicotinamide riboside (NR), optionally having a concentration in the range of 1 to 10,000 μM, 1 to 100 μM, 100 to 10,000 μM, 10 to 100 μM, or 100 to 1000 μM.

[0067] In some variations, the at least one defined priming agent is selected from the group consisting of all-trans retinoic acid (ATRA), optionally having a concentration in the range of 0.1-500 μM; ML228, optionally having a concentration in the range of 1-10 μM; MDL800, optionally having a concentration in the range of 5-500 μM; isoquercetin, optionally having a concentration in the range of 0.01-5000 μM; fucoidan, optionally having a concentration in the range of 0.00001-0.001 μM; luteolin, optionally having a concentration in the range of 10-100 μM; quercetin, optionally having a concentration in the range of 0.1-10 μM; 5-aminoimidazole-4-carboxamide riboside (AICAR), optionally having a concentration in the range of 1000-10000 μM; 5-phenylalkoxypsoralen (Psora-4), optionally having a concentration in the range of 0.01-200 μM; nopyridone (A-769662), metformin having a concentration in the range of 1 to 10000 μM as appropriate, rapamycin having a concentration in the range of 0.001 to 0.1 μM as appropriate, 5-azacytidine (5-Aza) having a concentration in the range of 0.1 to 1 μM as appropriate, UM171 having a concentration in the range of 0.01 to 0.1 μM as appropriate, SB203580 having a concentration in the range of 1 to 10 μM as appropriate, fumarate having a concentration in the range of 1 to 50 μM as appropriate, The composition may include isetin, atorvastatin having a concentration in the range of 0.1 to 20 μM, valproic acid having a concentration in the range of 500 to 5000 μM, sphingosine-1-phosphate (S1P) having a concentration in the range of 0.01 to 0.1 μM, astaxanthin (ATX) having a concentration in the range of 0.01 to 100 μM, succinate having a concentration in the range of 10 to 500 μM, or a combination thereof.

[0068] In some variations, the at least one priming agent may comprise a conditioned medium derived from a population of cells, optionally a population of stem cells, different from the population of MSCs primed with the at least one priming agent. In some variations, the conditioned medium is selected from the group consisting of conditioned medium derived from corneal stromal stem cells and conditioned medium derived from limbal epithelial stem cells. In some variations, the volume percentage (concentration) of conditioned medium added to the medium to serve as a priming agent ranges from 5-50%, 10-50%, 15-40%, about 10%, about 15%, about 20%, about 25%, or about 30% with respect to the medium in which the MSCs are growing.

[0069] In some variations, the time for exposure of the MSCs to the at least one priming agent can range from 12 to 72 hours, 24 to 72 hours, or 24 to 48 hours, or about 24 hours or about 48 hours. In some variations, the time for exposure of the MSCs to the at least one priming agent can be from seeding to about 60% to about 90% confluency, about 70% to 90% confluency, about 70% to 80% confluency, about 60% confluency, about 70% confluency, or about 80% confluency.

[0070] Combinatorial priming In some variations, MSCs may be exposed to two or more priming agents simultaneously or sequentially with partial or no overlap. In some variations, the time for priming for each or both priming agents may range from 12 to 72 hours. In some variations, MSCs may be exposed to a first priming agent contained in a first medium from seeding to about 60% to about 90% confluency, about 70% to 90% confluency, about 60% confluency, about 70% confluency, or about 80% confluency, and then replaced with a second medium containing a second priming agent and exposed to the second priming agent for a period ranging from 12 to 72 hours, 24 to 72 hours, or 24 to 48 hours, as appropriate.

[0071] In some variations, the population of MSCs can be primed with at least one Nrf2 activator and at least one SIRT1 activator. In some variations, the MSCs can be primed with at least one Nrf2 activator and CSSC-CM. In some variations, the MSCs can be primed with at least one Nrf2 activator, at least one SIRT1 activator, and ATRA.

[0072] In some variations, the population of MSCs may be primed with a combination of at least one Nrf2 activator and at least one SIRT1 activator. The Nrf2 activator may be selected from the group consisting of dimethyl fumarate (DMF) optionally having a concentration in the range of 10-250 μM, 4-octyl itaconate (4-OI) optionally having a concentration in the range of 10-500 μM, or imidazole derivative of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Im) optionally having a concentration in the range of 0.1-10 μM. The at least one SIRT1 activator may be selected from the group consisting of SRT-2104 optionally having a concentration in the range of 0.01-10 nM, trans-resveratrol optionally having a concentration in the range of 0.1-10 μM, or SRT-1720 optionally having a concentration in the range of 0.1-10 μM. In some variations, the Nrf2 activator and the SIRT1 activator may be administered to the MSCs simultaneously or sequentially with partial or no overlap, with the time for priming for each or both defined priming agents being within the range of 12 to 72 hours.

[0073] In some variations, the MSC population can be primed with at least one Nrf2 activator and CSSC-CM combination. Without being bound by theory, the exosomes secreted by the MSC primed with Nrf2 activator and CSSC-CM can advantageously have relatively reduced angiogenic factors, while relatively enriched in anti-inflammatory factors, compared with exosomes from a comparable naive MSC population. Thus, the exosomes from the MSC primed with Nrf2 activator and CSSC-CM combination can be useful for treating or inducing regeneration in non-vascular tissues, such as the cornea. The Nrf2 activator may be selected from the group consisting of dimethyl fumarate (DMF) having a concentration ranging from 10 to 250 μM, 4-octyl itaconate (4-OI) having a concentration ranging from 10 to 500 μM, or imidazole derivative of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Im) having a concentration ranging from 0.1 to 10 μM. In some variations, the volume percentage of CSCC-CM added to the medium to serve as a priming agent may be in the range of 5 to 50%, 10 to 50%, 15 to 40%, about 10%, about 15%, about 20%, about 25%, or about 30% with respect to the medium in which the MSCs are growing. In some variations, the priming of MSCs may be performed under hypoxic conditions with oxygen ranging from 0.2 to 10% as appropriate. In some variations, a population of MSCs can be grown in a first medium containing CSSC-CM at a concentration of about 20% from seeding to about 60% to about 90% confluency, then replaced with a second medium containing DMF at concentrations of about 50 μM and about 100 μM, and grown in the second medium for a time ranging from 24 to 28 hours.

[0074] Culture method In some variations, the MSCs may be cultured in a 3D bioreactor system in a method selected from the group consisting of hollow fiber-based methods, microcarrier-based methods, and spheroid-based methods.

[0075] In some variations, the hollow fiber based method may include the steps of: (i) providing or obtaining a hollow fiber bioreactor system; (ii) culturing the stem cells obtained in step (a) in a xeno-free medium to obtain a suspension of cells; (iii) injecting the suspension of cells into a cartridge of the hollow fiber bioreactor system; (iv) incubating the suspension of stem cells for a period ranging from 21 to 35 days to obtain an expanded population of stem cells; (v) adding a protease, optionally trypsin EDTA, to the excess capillary space of the hollow fiber bioreactor system containing the expanded population of stem cells to obtain expanded stem cells; and (vi) treating the expanded stem cells with a buffer to obtain expanded stem cells.

[0076] In some variations, the microcarrier-based method may include the steps of: (i) suspending microcarriers in a medium to obtain a suspension; (ii) seeding the suspension, as obtained in step (a), with stem cells; (iii) culturing the stem cells of step (ii) in a medium to obtain an expanded stem cell population attached to the microcarriers; and (iv) contacting the microcarriers of step (iii) with a lysis buffer comprising sodium chloride and trisodium citrate to lyse the microcarriers to obtain the expanded stem cells.

[0077] In some variations, the spheroid-based method may include: (i) pelleting the stem cells obtained in step (a) to obtain a stem cell pellet; (ii) resuspending the stem cell pellet in a medium comprising a basal medium to obtain a stem cell suspension; (iii) providing or obtaining stem cell spheroids from the stem cell suspension obtained in step (ii), wherein the stem cell spheroids have a stem cell density in the range of 600-10,000 cells per spheroid; and (iv) culturing the stem cell spheroids of step (iii) in a medium comprising an MSC basal medium to obtain expanded stem cells.

[0078] Exosome purification methods Exosomes contained in the conditioned medium from primed MSCs can be purified by an exosome purification process.

[0079] In some variations, the exosome purification process may include the steps of: (i) centrifuging the primed conditioned medium at a speed ranging from 90,000 to 120,000×g for a time ranging from 70 to 110 minutes at a temperature ranging from 2 to 6° C. to obtain a pellet; (ii) dissolving the pellet in a low serum xeno-free medium to obtain crude exosomes; and (iii) performing density gradient ultracentrifugation on the crude exosomes to obtain an exosome fraction; and (c) purifying the exosome fraction by size exclusion chromatography to obtain enriched exosomes.

[0080] In some variations, the exosome purification process may include the steps of: (i) subjecting the primed conditioned medium or conditioned medium to a first centrifugation at a speed ranging from 200 to 400×g for a time ranging from 5 to 20 minutes, followed by a second centrifugation at a speed ranging from 2000 to 4000×g for a time ranging from 10 to 40 minutes to obtain a supernatant; (ii) centrifuging the supernatant at a speed ranging from 200 to 400×g for a time ranging from 5 to 20 minutes, followed by filtering the supernatant to obtain a secretome; (c) centrifuging the secretome to obtain a pellet; (d) dissolving the pellet in a low serum xeno-free medium to obtain a crude solution; (e) performing density gradient ultracentrifugation on the crude solution to obtain an exosome-containing fraction; and (f) purifying the exosome-containing fraction by size exclusion chromatography to obtain an enriched population of exosomes.

[0081] Molecular characterization of primed exosomes Priming of MSC induces characteristic changes in the expression levels of certain exosomal proteins, for example, as measured by ELISA of enriched exosomes. In some variations, combinatorial priming of BM-MSC with CSSC-CM and Nrf2 activator can result in the production of exosomes characterized by having a low exosomal expression level of vascular endothelial growth factor (VEGF) and a combination of one or more of a high expression level of nerve growth factor (NGF), a high expression level of hepatic growth factor (HGF), and a high expression level of sFLT1, compared to exosomes from non-primed MSC. In some variations, the higher expression level of HGF can be at least 1.5-fold, at least 1.7-fold, at least 2-fold, or about 2.2-fold higher expression. In some variations, the higher expression level of NGF can be at least 2-fold, at least 2.2-fold, at least 2.5-fold, at least 3-fold, or about 3.2-fold higher expression. In some variations, the higher expression level of sFLT1 can be at least 1.5-fold, at least 1.7-fold, at least 2-fold, or about 2.2-fold higher expression. In some variations, the lower expression level of VEGF can be half or less, one-third or less, or one-quarter or less expression.

[0082] In some variations, priming of BM-MSCs with an Nrf2 activator (such as DMF or 4-OI) can result in the production of exosomes characterized by having a higher expression level of HGF and / or a higher expression level of NGF compared to exosomes derived from non-primed MSCs. In some variations, the higher expression level of NGF can be at least 1.5-fold, at least 1.7-fold, at least 2-fold, or about 2.2-fold higher expression. In some variations, the higher expression level of HGF can be at least 1.1-fold, at least 1.2-fold, at least about 1.3-fold, or about 1.4-fold higher expression.

[0083] Compositions based on the above method In an aspect of the present disclosure, there is provided a population of primed MSCs obtained by the methods described herein.

[0084] In an aspect of the disclosure, there is provided a primed conditioned medium obtained by the methods described herein.

[0085] In an embodiment of the present disclosure, a population of primed exosomes purified from conditioned medium obtained by the method described herein is provided. In an embodiment of the present disclosure, a composition comprising the purified primed exosomes described herein is provided. In some variations, the composition can be in a form for parenteral administration. In some variations, the composition can be in an ophthalmic composition or eye drop formulated for application to the corneal surface. In some variations, the eye drop or ophthalmic composition can include a biocompatible polymer. In some variations, the ophthalmic composition can be a hydrogel in which at least a portion of the biocompatible polymer is crosslinked. In some variations, the biocompatible polymer can include one polymer or a combination of two or more polymers selected from collagen, hyaluronic acid, cellulose, polyethylene glycol, polyvinyl alcohol, poly(N-isopropylacrylamide), silk, gelatin, and alginic acid. In some variations, one or more of the biocompatible polymers can be modified to be crosslinkable, for example, via thiolation or methacrylation.

[0086] In an aspect of the present disclosure, a composition is provided comprising the primed MSCs described herein.

[0087] In an aspect of the present disclosure, there is provided a composition comprising at least two components selected from the group consisting of: (a) primed stem cells as described herein; (b) primed conditioned medium as described herein; and (c) enriched exosomes as described herein.

[0088] Therapeutic Methods or Compositions for Use in Treating a Condition In certain embodiments of the present disclosure, a method for treating a condition in a subject is provided, the method comprising: (a) providing or obtaining enriched exosomes as described herein; and (b) administering the exosomes to a subject to treat the condition. In some variations, administration may be onto the corneal surface. In some variations, corneal surface administration may be with eye drops that may include a biocompatible polymer. The biocompatible polymer may be a crosslinkable polymer, such that the liquid becomes a hydrogel upon crosslinking. In some variations, administration may be parenteral or intravenous. Intravenous administration may be via the hepatic portal vein.

[0089] In certain embodiments of the present disclosure, a method is provided for treating a condition in a subject, the method comprising: (a) providing or obtaining a primed conditioned medium as described herein; and (b) administering a therapeutically effective amount of the conditioned medium to the subject to treat the condition.

[0090] In certain embodiments of the present disclosure, a method is provided for treating a condition in a subject, the method comprising: (a) providing or obtaining a primed stem cell as described herein; and (b) administering a therapeutically effective amount of an expanded primed mesenchymal stem cell population to the subject to treat the condition.

[0091] In certain embodiments of the present disclosure, a method is provided for treating a condition in a subject, the method comprising: (a) providing or obtaining a composition described herein, e.g., enriched exosomes, conditioned cell medium, primed mesenchymal stem cell population; and (b) administering a therapeutically effective amount of the composition to the subject to treat the condition.

[0092] In certain variations, the condition may be selected from the group consisting of rheumatoid arthritis, systemic juvenile idiopathic arthritis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), pneumonia, bronchitis, chronic obstructive pulmonary disease (COPD), COVID-19, coronavirus class infections, cystic fibrosis, hantavirus, influenza, tuberculosis, systemic lupus, myocardial infarction, osteoarthritis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), hepatic fibrosis, Mooren's ulcer, neurotrophic ulcer, corneal keratitis (CK), dry eye disease ulcer, herpetic simple keratitis, post-LASIK ectasia, postoperative corneal melt, post-keratoprosthesis melt, corneal perforation, neurotrophic keratitis (NK), keratoconus Sjogren's syndrome, mucous membrane pemphigoid, Stevens-Johnson syndrome, chemical burns, and thermal burns.

[0093] In certain embodiments of the present disclosure, the present invention is directed to treating rheumatoid arthritis, systemic juvenile idiopathic arthritis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), pneumonia, bronchitis, chronic obstructive pulmonary disease (COPD), COVID-19, coronavirus class infections, cystic fibrosis, hantavirus, influenza, tuberculosis, systemic lupus, myocardial infarction, osteoarthritis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic fibrosis, Mooren's ulcer, neurotrophic pulmonary fibrosis, and the like. In one embodiment, a composition comprising the enriched exosomes, conditioned cell media, or primed mesenchymal stem cell population as described herein for use in treating a condition selected from the group consisting of chronic keratitis, corneal ulcer, corneal keratitis (CK), dry eye disease ulcer, herpetic simple keratitis, post-LASIK ectasia, post-operative corneal melt, post-keratoprosthesis melt, corneal perforation, neurotrophic keratitis (NK), keratoconus, Sjogren's syndrome, mucous membrane pemphigoid, Stevens-Johnson syndrome, chemical burn, and thermal injury is provided.

[0094] Certain embodiments of the present disclosure include, but are not limited to, rheumatoid arthritis, systemic juvenile idiopathic arthritis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), pneumonia, bronchitis, chronic obstructive pulmonary disease (COPD), COVID-19, coronavirus class infections, cystic fibrosis, hantavirus, influenza, tuberculosis, systemic lupus, myocardial infarction, osteoarthritis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic fibrosis, Mooren's ulcer, neurotrophic ulcer, corneal keratitis, and keratitis of the cornea. In one embodiment, the present invention provides a method for treating a condition selected from the group consisting of chronic kidney disease (CK), dry eye disease ulcer, herpetic simple keratitis, post-LASIK ectasia, post-operative corneal melt, post-keratoprosthesis melt, corneal perforation, neurotrophic keratitis (NK), keratoconus Sjogren's syndrome, mucous membrane pemphigoid, Stevens-Johnson syndrome, chemical burn, and thermal burn.

[0095] Although the subject matter has been described in some detail with reference to certain embodiments and implementations thereof, other implementations are possible. EXAMPLES

[0096] Here, the present disclosure is illustrated using working examples, which are intended to illustrate the operation of the present disclosure and are not intended to be taken as limiting to mean any limitation in the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs. In the implementation of the disclosed methods and compositions, similar or equivalent methods and materials to those described herein can be used, but exemplary methods, devices and materials are described herein. The present disclosure is not limited to the specific methods and experimental conditions described, and it should be understood that such methods and conditions can be applied.

[0097] material and method Stem Cell Sources For the purposes of this disclosure, mesenchymal stem cells (MSCs) derived from sources such as human bone marrow (BM), limbal stem cells, umbilical cord (UC), unrestricted somatic stem cells, Wharton's jelly (WJ), dental pulp (DP) and adipose tissue (AD), induced pluripotent stem cells (iPSCs), engineered cells, MSCs primed with conditioned medium from corneal stromal stem cells (CSSCs) can be used in the methods and cell-derived products described herein. The engineered cells referred to in this context are cells immortalized with hTERT. The selection of stem cell type is target-directed and tissue-specific.

[0098] Sources of immortalized adult stem cell lines (non-virally immortalized MSC cell lines): (1) hTERT-immortalized human bone marrow mesenchymal stem cells (hBM-MSCs): clinically approved CD105 + , CD90 + , CD73 + Primary BM-MSCs bearing markers (naive) were used for naive exosome production, and naive BM-MSCs primed with CSSC-conditioned medium were used for primed mutant exosome production for avascular tissue regeneration. Clinically approved non-viral human telomerase enzyme reverse transcriptase (hTERT)-induced immortalized BM-MSCs were used for steady-state production of exosomes.

[0099] (2) hTERT-immortalized human Wharton's jelly-derived MSC (WJMSC) / umbilical cord-derived MSC (UC-MSC) cell lines: clinically approved CD166 + , CD90 + , CD73 + -Markers and CD34 - , α-SMA - UC-MSCs bearing the markers were selected and expanded in xeno-free medium, and naïve exosomes were produced from the above-mentioned cell populations expressing the aforementioned markers. Clinically approved non-viral hTERT-induced immortalized UCMSCs were used for routine production of exosomes.

[0100] [Example 1] Obtaining and culturing corneal stromal stem cells (CSSCs) and naïve cells (hBM-MSCs, UC-MSCs and WJ-MSCs) This example describes the process of obtaining or culturing stem cells, such as corneal stromal stem cells (CSSCs), hBM-MSCs, umbilical cord UC-MSCs, and WJ-MSCs, as well as the process of enriching stem cells to obtain expanded stem cell populations under xeno-free culture conditions. This example also describes the process of obtaining conditioned medium from stem cells as described above.

[0101] 1.1 Culture CSSCs under xeno-free conditions and collect conditioned medium from CSSCs. 1.1.1. Isolation and culture of CSSCs under xeno-free conditions Corneal tissues stored in corneal storage medium were obtained with a validity of 4-5 days. Tissues with the following details in the "Tissue Specifications" were used for cell extraction and culture: (1) tissue with an expiration date for transplantation / cell harvest; (2) absence of major cause of death, HIV (human immunodeficiency virus), HCV (hepatitis C virus), HbsAg (hepatitis B surface antigen) and syphilis; (3) cell count per square mm; (4) absence of sepsis and scarring, any systemic infection, any ocular medical history that would make the tissue unsuitable for cell harvest.

[0102] After thorough screening, corneas from human donors were used to induce CSSCs using a protocol under xeno-free conditions. The process for culturing CSSCs under xeno-free conditions is described below.

[0103] Corneas from human donors were washed with antibiotic-enhanced buffered saline (PBS) and then the limbus containing CSSCs was extracted. In sterile conditions, a 360° annular ring was excised using surgical instruments, washed with buffered saline, and minced into smaller fragments. The minced tissue fragments were collected in incomplete medium (MEM medium) and subjected to Liberase digestion by adding 20 μL of reconstituted Liberase (Roche) at a concentration of 0.5 U to the tissue suspension.

[0104] After 16 h of incubation, the enzymatic digestion was stopped by adding 2 mL of complete medium enriched with 2% human platelet lysate (HPL).

[0105] The digested tissue was centrifuged at 200×g for 3 min at room temperature in saline supplemented with penicillin and streptomycin, followed by various levels of passage.

[0106] Passage 0 (P0): During passage 0, digested explants were resuspended in 5 mL of xeno-free complete medium (MEM + 2% HPL, 1x ITS, 10 ng / mL EGF) and cultured in T25 Corning CellBIND flasks for 14 days. Medium was changed every 3 days.

[0107] Passage 1 (P1): During passage 1, cells isolated from explants were trypsinized with Tryple (1X, Gibco) and resuspended in fresh complete medium at the end of day 14. Cells were cultured at passage 1 at 8000 cells / cm in T75 CellBIND flasks. 2 The seeds were seeded at 0.7 × 10 6 Cells were seeded into three T-75 flasks at P1, and for the next passage (passage 2; P2), the cells were split into two T-75 flasks, and then for the next passage (passage 3; P3), they were further split into four T-75 flasks according to cell doubling.

[0108] Complete medium changes at P1, P2 and P3 were performed at the following times: Day P1-3: 50% medium was replenished, i.e. 2.5 mL medium. Day 5: 5 mL medium was replaced with 5 mL fresh medium. Day P2-3: Replenished with 50% medium, i.e., 5 mL. Day 5: 10 mL medium was replaced with 10 mL fresh medium. Day 7: 10 mL medium was replaced with 10 mL fresh medium. P3-Day 3: Replenished with 50% medium, i.e. 5 mL. Day 5: 10 mL medium was replaced with 10 mL fresh medium. Day 7: 10 mL medium was replaced with 10 mL fresh medium.

[0109] For P2 and P3, approximately 1.2 × 10 6 Recover CSSSCs at approximately 8,000 cells / cm 2 Cell seeding was performed at density.

[0110] Table 1 shows the volumes of CSSC spent medium harvested at passage 1 (P1), passage 2 (P2), and passage 3 (P3).

[0111] [Table 1]

[0112] For quality control, cells in P1, P2 and P3 were characterized using markers such as stem cell markers like CD90, CD73 and CD105, corneal cell specific markers like PAX6, and negative markers like SMA, CD34 (immunofluorescence images).

[0113] 1.1.2. Harvesting of conditioned medium from CSSC cultures (CSSC-CM) As mentioned above, from passage 1-2-3, all medium changes were accompanied by collection of spent / conditioned medium from flasks. The spent medium was further pretreated by centrifuging the medium at 300×g for 10 minutes to collect the supernatant. The supernatant was further centrifuged at 3000×g for 20 minutes at 4° C., followed by recentrifuging the supernatant at 13000×g for 30 minutes at 4° C. to collect the further treated supernatant. The medium was double filtered through a 0.45 micron filter, and the supernatant was further collected using a 0.22 micron filter.

[0114] The harvested supernatant (conditioned medium) was stored at 4°C for short-term (1–2 days) or at −80°C for long-term storage.

[0115] 1.2. Cultivation of naive hBM-MSCs and collection of conditioned medium from naive hBM-MSCs 1.2.1. Culture of naive hBM-MSCs under xeno-free conditions To culture naïve hBM-MSCs, 10 mL of human BM-MSC (hBM-MSC) cells (passage 2) were obtained from the manufacturer with their recommended medium (hBM-MSC High Performance Medium Kit XF) and the hBM-MSC cells were cultured according to the manufacturer's protocol. Briefly, 10 mL of Booster-MSC-Xenofree and Basal-MSCs were thawed at room temperature, transferred aseptically in a biosafety cabinet, and reconstituted in 500 mL medium.

[0116] For culturing purposes, a vial hBM-1M-XF was removed from the liquid nitrogen (LN) box and thawed in a 37 °C water bath for 2-3 min. The cell vial was aseptically transferred to a 50 / 15 mL centrifuge tube and 4 mL medium was added dropwise to the cells. The cell pellet obtained after centrifugation at 200 × g for 10 min was dissolved in 5 mL of complete medium and, as a quality control, the cell count was recorded. The volume of medium in the tube was brought up to 30 mL (recommended by the manufacturer's protocol) and the cells were cultured in CELLBIND T225cm 2 Flasks with 2000-3000 cells / cm 2 The cells were seeded at a density of 100–150 μg / ml and the medium volume was increased to 40–45 mL in each flask and incubated at 37°C and 5% CO 2 and incubated at 4°C for 1 hour.

[0117] To determine the percentage of confluent cells, cells were observed daily from day 3 onwards. The medium was adjusted to the concentration of cells when the cells were 80% confluent, i.e. (43,000–50,000) cells / cm. 2 The medium was not allowed to be exchanged until it reached 0.05% and then the cells were ready to be harvested. During harvesting, the cells were transferred to a biosafety cabinet, the spent medium was removed, and 10 mL of spent medium was placed in a sterile tube (15-50 mL) to quench the trypsin enzyme. The medium was removed and the cells were washed with 1x PBS, followed by the addition of 10 mL of TrypLE and incubation in a 37 °C incubator. The cells were checked every 5 min for cell detachment from the surface. An equal volume of quench (fresh medium) or spent medium was added to the cells to stop TrypLE activity.

[0118] The cell suspension was transferred to a sterile 50 mL centrifuge tube and centrifuged at 200 g for 10 min. The supernatant was discarded, the cells were resuspended in 4 to 5 mL of fresh medium, and the total volume of the cell suspension was measured. After obtaining the suspension, 0.1 mL of the cell suspension was transferred to a microcentrifuge tube for cell counting and diluted with Dulbecco's phosphate-buffered saline (DPBS) to obtain a counting range of (0.1 to 1) x 10. 6 Cells / mL were obtained and cells were stored frozen until needed.

[0119] 1.2.2 Collection of conditioned medium from naive hBM-MSCs 1M hBM-MSC cell vials were allowed to recover and observed from day 3 onwards, as described above. Images were taken with a phase contrast microscope on days 3, 4 / 5 and 6 / 7, and the cell densities are shown in Table 2. Cells were cultured at a cell density (43,000–50,000 cells / cm) of 1000 µg / ml. 2 On day 4 / 5, depending on the incubation time, the cells were washed either 1-2x with 20mL PBS and the medium was replaced with Rooster EV collection medium. After 48 hours of incubation, the conditioned medium was collected and cells were harvested according to the protocol described in Example 1.2.1 above. Cells were then counted using a cell counter. The collected conditioned medium was immediately processed with the pretreatment step as described in Example 1.1.2 above, and then the pretreated conditioned medium was stored at 4°C (short-term storage up to 24 hours) or -80°C (long-term storage up to 1 month).

[0120] Table 2 shows the cell densities (cells / cm) on days 3, 4, and 6 / 7. 2 ) is shown.

[0121] [Table 2]

[0122] 1.3. Cultivation of naïve UCMSCs and collection of conditioned medium from naïve UCMSCs 1.3.1. Culture of naïve UCMSCs UC-MSC cells were procured from the manufacturer and cultured in the recommended xeno-free medium according to the manufacturer's protocol. Briefly, MSC-XF and basal-MSC were thawed in the dark at room temperature and reconstituted in one bottle of 500 mL of medium. Additionally, a vial-human umbilical cord-1X-XF (xeno-free) (hUC-1M-XF) obtained after passaging was thawed in a 37 °C water bath and the cells were transferred aseptically in a biosafety cabinet. The cells were transferred to 15 / 50 mL centrifuge tubes in 10 mL medium volume and centrifuged at 280 × g for 6 min. The supernatant was discarded and the cell pellet was dissolved in 20 mL of medium. The cell suspension was further divided into four T75 flasks and two T225 flasks, with the seeding density of the T75 flasks being 2000–3000 cells / cm. 2 The seeding density of the T225 was kept within the range of 2000-3000 cells / cm. 2 was within the range.

[0123] For T225 and T75 flasks, a medium volume of 45 mL and 15 mL, respectively, was used and the medium was maintained at 37° C. To determine the percentage of confluence, cells were observed under a microscope from day 3 onwards and images were acquired, followed by cell counting using Image J software.

[0124] Additionally, cells were observed on days 4 and 5 when the cultures were 80% confluent (e.g., cells were 60k-100k cells / cm for a T225 flask). 2The cell density was 100-fold higher at 280 × g for 6 min). The cells were harvested by transferring the flasks to a biosafety cabinet and collecting the spent medium in a sterile container (approximately 10 mL) to quench the trypsin enzyme. After removing the medium, 10 mL or 3 mL of TrypLE was added to the respective T225 or T75 flasks and the flasks were incubated in a 37 °C incubator. The cell culture was checked every 5 min until the cells were detached from the surface or the cells were dislodged by gentle tapping. To stop TrypLE activity, an equal volume of quench or spent medium was added to the cell suspension and transferred to a 15 / 50 mL centrifuge tube for centrifugation at 280 × g for 6 min. After discarding the supernatant, the cells were resuspended in 4–5 mL of fresh medium and the total volume of the cell suspension was measured. It was diluted in DPBS / medium to obtain (0.1–1) × 10 6 After obtaining the counting range of cells / mL, cell counting was performed using 0.1 mL of cell suspension and cells were stored frozen until further use.

[0125] 1.3.2. Collection of conditioned medium from naïve UCMSCs To generate extracellular vesicles (EVs) from naïve UC-MSCs, a 1M cell vial was recovered as described above and cells were observed from day 3 onwards to acquire images using a phase contrast microscope on days 3, 4 / 5 and 6 / 7 using cell densities as provided in Table 3. On days 4 / 5, cell densities (60-100k) cells / cm were used. 2 Depending on the number of cells (T225 flasks), i.e., at approximately 80% confluence, cells were washed twice with 20 mL of PBS and the medium was replaced with extracellular vesicle (EV) collection medium. After 48 hours of incubation, the conditioned medium was collected and cells were harvested and counted in a cell counter. The collected conditioned medium was immediately processed with the pretreatment step as described in Example 1.1.2 above.

[0126] [Table 3]

[0127] 1.4. Cell sorting for selection of stem cell populations and their subtypes One of the important aspects of the present disclosure is the isolation of a unique subpopulation of mesenchymal stem cells (MSCs) from stem cells such as UC-MSCs / WJ-MSCs that express a signature set of markers to produce exosomes with desired therapeutic effects such as anti-inflammation, anti-fibrosis, wound pre-healing, (pro / anti)angiogenesis, and re-innervation. This feature is important and differs from conventional methods known in the literature because conventional methods use a heterogeneous population of stem cells such as UC-MSCs. Meanwhile, the method of the present disclosure deploys a unique subpopulation of MSCs that provides superior therapeutic activity (anti-inflammation, anti-fibrosis, wound pre-healing, (pro / anti)angiogenesis, re-innervation).

[0128] A common signature set of markers expressed by stem cells such as UCMSCs and WJMSCs is listed in Table 4.

[0129] [Table 4-1] [Table 4-2]

[0130] Furthermore, aside from the markers listed in the table above, other cell markers that are expressed at higher levels in MSCs include, but are not limited to, CD44, CD73, and CD90.

[0131] 1.4.1 Isolation of UCMSC subpopulations At the end of the first passage (P4) or after expansion of a vial of hUC-1M-XF cells as described in Example 1.3.1, cells were sorted based on the clinically approved MSC surface markers CD90, CD73, and CD166 using a flow cytometry-based sorting protocol to obtain two subpopulations of UC-MSCs. The two subpopulations of UC-MSCs were as follows:

[0132] First subpopulation: Clinically approved MSC surface markers, e.g., CD90 + , CD73 + and CD166 + A first subpopulation of stem cells expressing

[0133] Second subpopulation: CD166 + , CD90 + , CD73 + MSCs with positive populations were re-sorted to provide the first subpopulation of two subtypes (enriched therapeutically specific UC-MSC populations): (i) CD146+, CD54+, CD58+ and CD142+ positive populations; (ii) CD146+, CD54+, CD58+ and CD142- (low / negative) population.

[0134] The above UC-MSC subtypes were maintained in xeno-free medium with two further passages (P5 and P6), followed by harvesting of conditioned medium as described in Example 1.1.2 of the present disclosure.

[0135] 1.5. Cultivation of hTERT-immortalized WJ-MSCs 1.5.1 Cell recovery and expansion of WJ-MSCs Culture flasks were pre-coated with animal component-free cell attachment substrate and then allowed to recover. Briefly, substrate (1:300, diluted in 1× PBS) was added to the culture flask and incubated for at least 2 hours at room temperature. Excess substrate solution was removed and the flask was rinsed twice with PBS (1×). After rinsing, 6 ml of growth medium was added to 25 cm 2 Culture flasks were added and placed in an incubator for at least 30 minutes to allow the medium to reach normal pH. Frozen cell vials were removed from liquid nitrogen, rinsed on the outside with 70% ethanol, and pre-warmed by hand until the last frozen cell debris was visible. The thawed vials were transferred to 15 mL centrifuge tubes pre-filled with 9 mL of medium pre-chilled to 4°C.

[0136] The cells were centrifuged for an additional 5 minutes at room temperature at 400×g, the supernatant was discarded, and the cells were then resuspended in 1 mL of pre-warmed medium. The cells were then transferred to a prepared culture flask (T25cm 2 ) and incubated at 37°C. As a quality control (QC), cells were counted and recorded, followed by a medium change after 24 hours and passage of cells at approximately 70-80% confluency.

[0137] 1.5.2 Subculture of WJMSC / hTERT-immortalized WJMSC Subcultures were performed at a cell density of 28,000 cells / cm 2 This was done using pre-coated culture flasks with 70-80% confluency. To detach the cells, TrypLE selection enzyme solution (20 µL / cm 2 ) was added and the cells were soaked in PBS (160 μL / cm 2 The flasks were incubated at 37°C for approximately 2-3 minutes and observed under a microscope for cell detachment. Growth medium was added to the cells and centrifuged at 400 × g for 5 minutes, followed by resuspension in 1 mL of medium and coating with trypan blue. The cells were then plated at 240 μL / cm 2 Cells were seeded (7000 cells / cm) onto coated culture vessels supplemented with growth medium. 2 ), 80% confluence (e.g., approximately 28,000 cells / cm 2 ), maintaining a split ratio of 1:4 twice a week, and then the cells were trypsinized using TrypLE selection enzyme.

[0138] The cells were then resuspended in growth medium and centrifuged at 400 × g for 5 min. The cells were then diluted to 5 × 10 5 Cells were suspended in 1 mL of cryopreservation medium, CryoStor (CS10), corresponding to cells / mL. 1 mL of cell suspension was transferred into pre-chilled cryovials and transferred to -80°C overnight or liquid nitrogen for long-term storage. For further use, cells were expanded in MesenCult-ACF Plus medium supplemented with MesenCult-ACF Plus 500X Supplement, 200 pg / mL G418.

[0139] [Example 2] How to expand stem cells in 3D culture MSCs (naive / hTERT immortalized) from different sources were cultured in 3D culture-based systems to obtain expanded stem cells, as described in Example 1. The different 3D culture-based methods are as follows: (i) Culture on 3D microcarriers: The culture of MSCs on 3D microcarriers is described in detail in pending application PCT / IN2020 / 050622, which is incorporated in its entirety into this disclosure. (ii) Culture as 3D spheroids: The culture of MSCs on 3D microcarriers is described in detail in pending application PCT / IN2020 / 050622, which is incorporated in its entirety into this disclosure. (iii) Cultivation in hollow fiber bioreactors.

[0140] 2.1 2D culture of hMSCs in CellSTACK flasks Human mesenchymal stem cells (hMSCs) were purchased at passages 1-2 and expanded according to the manufacturer's protocol to generate a working cell bank. For large-scale expansion of hMSCs, i.e., expansion in CellSTACK culture chambers (10 stacks), 20 million hBM-MSCs from the working cell bank were cultured at 3145 cells / cm. 2 Cells were seeded into the chamber at a seeding density of 100 μg / ml (Corning, cat. #3271). Complete medium was prepared as recommended by the manufacturer's protocol, and cells were grown for 4 days until cell confluency reached approximately 80-90%.

[0141] To harvest cells from the CellSTACK flask, the medium was removed and 0.25% trypsin-EDTA was added, after which the cells were incubated at 37 °C for 6-8 min. Furthermore, to quench the trypsin activity, 200 µL of 2% MSC-screened FBS prepared in DPBS (without Ca++, Mg++) was added to the cells, and subsequently, the suspension was collected in a 50 mL centrifuge tube and centrifuged at 200 x g for 10 min. The suspension was further resuspended in a final volume of 20 mL of complete medium and injected into the hollow fiber bioreactor system.

[0142] 2.2 3D Culture of hMSCs in FiberCell Hollow Fiber Bioreactors The cells are 90–220 × 10 6 Cells / cartridge (20kD MWCO, 4000cm 2 Cells were seeded in a hollow fiber bioreactor (polysulfone fiber cartridge) and maintained in xeno-free complete medium, and the hollow fiber bioreactor system was prepared and used according to the manufacturer's instructions. All pre-inoculation steps were performed using sterile D-PBS- / -. Prior to injection of the cell suspension, 1 mL of medium was taken from the medium reservoir and the total glucose content was confirmed using a glucose meter, and L(+)-lactate using a lactate assay kit (50 µL of 1000-fold diluted medium). To inoculate the bioreactor system with cells, the prepared cell suspension (20 mL) was injected into the cartridge according to the manufacturer's procedures. The flow rate of the pulsatile perfusion pump was set to 22 times / min for the first 2-3 days of the 28-day cell inoculation period.

[0143] The medium volume in the extracellular capillary lumen was maintained at 250 mL and circulated at a system flow rate of 25 times / min through the bioreactor from days 3–17 of the 28-day cell seeding period. After 17 days, the medium volume was doubled to 500 mL at the same flow rate. A 1 mL aliquot of medium from the medium reservoir was withdrawn every 2–3 days to monitor the glucose content and pH. At the end of the 25-day culture period, hMSCs were harvested using 40 mL of trypsin-EDTA0, followed by collection of conditioned medium. 25% cells were injected into the extracellular capillary lumen and incubated at 37 °C for 10 min. Trypsinized cells were extruded using PBS until 60 mL of cell suspension was obtained. The harvested cell suspension was further quenched with an equivalent volume of 2% MSC-screened FBS prepared in DPBS (without Ca++, Mg++), centrifuged at 200 × g for 10 min, and used for cell viability counting using a trypan blue exclusion kit before being processed for downstream analysis.

[0144] [Example 3] Stem cell priming This example shows one of the important aspects of the present disclosure, which is the priming of stem cells from various sources, as described in Example 1. Stem cell priming is performed in the presence of various priming agents, such as small molecules with molecular weights less than 800 Da, and macromolecules with molecular weights greater than 800 Da. Stem cells are primed with one or more priming agents (each alone or combinatorial priming) to enhance the regenerative, stemness, anti-inflammatory, and anti-fibrotic properties of MSCs. Naive MSCs or primed MSCs can be used as or in addition to naive exosomes or primed exosomes (obtained by the method described in the following example) for therapeutic applications.

[0145] 3.1 Priming Agents Used in the Present Disclosure 3.1.1. Priming stem cells with small molecules Enhanced regeneration by priming (approved to enhance stemness, viability and engraftment capacity in vitro and in vivo) was performed with various small molecules (hydrophobic substances), including but not limited to SIRT1 activators, Nrf2 activators, in the absence or presence of physical inducers (hypoxia). Since most of these compounds (small molecules) are hydrophobic in nature and therefore not water-soluble, they are first dissolved at high concentrations in non-toxic organic solvents such as dimethyl sulfoxide (DMSO), ethanol, acetone, etc., and then diluted in aqueous media (PBS, saline or cell culture media) to generate working concentrations for the treatment of MSCs or to obtain lipid-based carriers such as liposomes.

[0146] Table 5 lists the priming agents (small molecules) with working concentrations and treatment duration.

[0147] [Table 5-1] [Table 5-2]

[0148] Silencing information regulator 1 (SIRT1) activators: In one example, the small molecule is a silencing information regulator 1 (SIRT1) activator. SIRT1 is an NAD-dependent histone deacetylase that plays an important role in cell metabolism, cell survival and senescence, DNA repair, inflammation, cell proliferation, and neurodegenerative diseases (Zhu, Yg, et al., Human mesenchymal stem cell microvesicles for treatment of Escherichia coli endotoxin-induced acute lung injury in mice. Stem cells, 2014. 32(1): p. 116-125). SIRT1 activators include, but are not limited to, SRT-2104, SRT-1720, and trans-resveratrol.

[0149] Nuclear factor erythroid 2-related factor 2 (Nrf2) activators: Nuclear factor erythroid 2-related factor 2 (Nrf2) is ubiquitously expressed in most eukaryotic cells and functions to induce a broad range of cellular defenses against extrinsic and endogenous stresses, including oxidants, xenobiotics, and excess nutrient / metabolite supply. Nrf2 activators act as key regulators of stem cell quiescence, survival, self-renewal, proliferation, senescence, and differentiation.

[0150] Nrf2 activators include, but are not limited to, dimethyl fumarate (DMF), the imidazole derivative of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Im), and 4-octyl itaconate (4-OI).Other activators include the families: arylcyclohexylpyrazoles, sulfonylcoumarins, 1,4-diaminonaphthalene core-containing, benzenesulfonyl-pyrimidone, and 1,2,3,4-tetrahydroisoquinoline core-containing compounds.

[0151] Nrf2-inducing peptides (blockers of Nrf2 / Keap interaction) that can be used as priming agents in the present disclosure: LDEETGEFL-NH2, (NH2-RKKRRQRRR-PLFAERLDEETGEFLPNH2), Ac-DPETGEL-OH, Ac-DEETGEF-OH, LQLDEETGEFLPIQGK(MR121)-OH, Ac-LDEETGEFL-NH, AcDPETGEL-NH2, Ac-NPETGEL-OH.

[0152] 3.1.2. Hypoxia-mediated priming Hypoxia priming mimics the in vivo MSC niche microenvironment and may improve the regeneration, survival and angiogenic potential of MSCs. Hypoxia regulates cell metabolism during MSC expansion, provides resistance to oxidative stress, and improves engraftment and survival in ischemic microenvironments. HIF-1α induction was detected in hypoxia priming, and overexpression of HIF-1α showed induction of miR-15, miR-16, miR-17, miR-31, miR-126, miR-145, miR-221, miR-222, miR-320 and miR-424, which are associated with the angiogenic potential of MSCs.

[0153] In the present disclosure, hypoxia-mediated priming was performed in the presence of oxygen ranging from 0.2 to 10%.

[0154] 3.1.3 Light-mediated priming Light-mediated priming or photobiomodulation is another inducer platform that involves the use of non-ionizing forms of light sources in the visible and near-infrared spectrum. This non-thermal process results in both photophysical and photochemical processes at the biological scale under the influence of endogenous chromophores. The wavelengths of the light sources involved are in the following ranges (300-650 nm and 800-1400 nm), while the light energy is between 0.5 and 4 J / cm. 2 The proliferation of MSCs at low and high densities was also investigated using irradiation (5-20 mW / cm), which can be either single or multiple doses of irradiation. 2 Studies have shown that the brain is influenced by

[0155] 3.1.4. Priming stem cells with macromolecules In this disclosure, macromolecules are used to prime stem cells, and macromolecules are referred to as biological factors such as proteins, lipids, nucleic acids, growth factors, cytokines, components of conditioned medium, etc. For the purposes of this disclosure, conditioned medium from naive MSCs described in Example 1 was used to prime naive MSCs from different sources.

[0156] In this disclosure, naive MSCs are referred to as A, and exosomes derived from naive MSCs (A) are referred to as B. Priming of A with small and macromolecules such as nuclear factor erythroid-related factor 2 (Nrf2) activator, silencing information regulator (SIRT1) activator, etc. is referred to as A', and exosomes derived from primed MSCs (A') are referred to as B'. The process of single priming with single inducer molecules and combinatorial priming with small or macromolecules are described below in the next examples.

[0157] 3.2. Single priming protocol for hBM-MSCs with CSSC-conditioned medium hBM-MSCs, passage 4 cells, were maintained according to the process described in Example 1.2. Priming of hBM-MSCs was performed in medium supplemented with conditioned medium (macromolecules) from corneal stromal stem cells (CSSCs) with a volume percentage ranging from 10-20%. Xeno-free culture of CSSCs and collection of conditioned medium for priming with a final concentration of 20% were performed during CSSC maintenance. hBM-MSCs were cultured at 80-85% confluence (i.e., 43000-50000 cells / cm). 2 ) and used for exosome production. Conditioned medium was harvested and processed as described above in Examples 1.2 and 1.1.2.

[0158] Table 6: Single priming protocol of hBM-MSCs with CSSC-conditioned medium.

[0159] [Table 6]

[0160] 3.3. A single priming protocol for hBM-MSCs with Nrf2 activators hBM-MSC, passage 4 cells were maintained according to the process described in Example 1.2 above, and cells were treated with Nrf2 activators or inducers (DMF, 4-OI). Once cells reached 70-80% confluence, they were washed with PBS and replenished with fresh media with 100 μM DMF or 100 μM 4-OI for 24 hours before switching to EV collection, followed by the conditioned media collection and processing procedures as described in Examples 1.2 and 1.1.2 above.

[0161] Table 7: Single priming protocol of hBM-MSCs with Nrf2 activators.

[0162] [Table 7]

[0163] 3.4 Single priming protocol of UCMSCs with SIRT1 activators (SRT2104 or trans-resveratrol) (EXO variant B') The two UC-MSC cell types were separately cultured in the presence of SIRT1 activators (SRT-2104 or trans-resveratrol) at concentrations less than the IC50 value of SRT-2104. IC50 values ​​were determined for both subpopulations using a concentration range of 0.04-3.78 nM or 24-1962 ng / mL for SRT-2104 in UC-MSC cells. The working concentration range of RSV was 0.1-2.5 μM or 22.85-571.25 μg / mL for priming in UC-MSC. UC-MSC were cultured to 80% confluence in the presence of SRT-2104 or RSV. Then, EV recovery procedures, conditioned media collection were performed for primed exosome production. After priming, the UC-MSC primed conditioned medium / secretome was screened using ELISA assays to detect the expression of TNF-α, IFN-γ, IL-10 and HGF for both cases.

[0164] The primed exosome variants were characterized by ELISA to detect the expression levels of exosomal cargo molecules such as SIRT1, HGF, IDO, IL-10, NRF2, VEGF, and NGF, and based on these results, the concentrations of SRT2104 or RSV treatment were finalized for UC-MSC priming.

[0165] 3.5 Single priming protocol of UC-MSCs with Nrf2 activators (DMF or 4-OI or CDDO-IM) - (EXO variant B') UC-MSCs were cultured according to the process as described in Example 1.3. Cells passaged to the 5-6 passage stage were used for primed exosome production. (B'). UC-MSCs were treated with Nrf2 activators (DMF, 4-OI or CDDO-IM) with working concentrations ranging from DMF ((1.44-36mg / mL) or (10-250μM)), 4OI ((0.0024-0.060)mg / mL or (10-250))μM, CDDO-Im ((2.56-128μg / mL) or (0.2-1))μM, etc.

[0166] After priming, the UC-MSC primed conditioned medium / secretome was screened using ELISA assay kits to detect the expression levels of TNF-α, IFN-γ, IL-10, and HGF. The primed exosome variants were characterized by ELISA assays to detect the expression levels of exosomal cargo molecules such as Nrf2, IL-10, HGF, and VEGF, and the concentrations of DMF or 4-OI or CDDO-Im were finalized for UC-MSC priming.

[0167] 3.6 Combinatorial priming of hBM-MSCs with CSSC-derived conditioned medium and Nrf2 activators - (EXO variant B') Priming of hBM-MSCs was performed in medium supplemented with conditioned medium from CSSCs (volume percentage ranging from 10 to 20%). Collection of conditioned medium for xeno-free culture of corneal stromal stem cells (CSSCs) and priming to a final concentration of 20% was performed during CSSC maintenance. hBM-MSC cells were thawed and cultured in T225cm 2 2000-3000 cells / cm in flask 2 hBM-MSCs were seeded at 1000 x g for 24 h and the medium was supplemented with 10–20% conditioned medium from CSSCs. Once the hBM-MSCs reached approximately 70–80% confluency, the cells were washed in PBS and then supplemented with Nrf2 activators (e.g., 100 μM DMF or 100 μM 4-OI) for 24–72 h before switching to extracellular vesicle (EV) collection medium.

[0168] Table 8 shows the protocol for combinatorial priming of hBM-MSCs with CSSC-conditioned medium and Nrf2 activators - (Exo variant B').

[0169] [Table 8]

[0170] 3.7 Combinatorial priming of UC-MSCs with SIRT1 and Nrf2 activators in the absence of hypoxia - (EXO variant B') UC-MSCs and selected subpopulations of UC-MSCs were cultured according to the protocol described in Example 1.3, followed by priming the expanded population of UC-MSC stem cells, which are subpopulations, in the presence of SIRT1 activators such as SRT-2104 (0.00001-0.01M) or trans-resveratrol (RSV) (0.1-10M) until 80% confluence (e.g., 60-100K cells / cm). 2The cells were subsequently treated with DMF ranging from 10 to 250 μM, or Nrf2 activators such as 4-OI ranging from 10 to 250 μM, for 24 to 72 h. The conditioned media were further screened to detect anti-inflammatory molecule expression using ELISA. Furthermore, UC-MSC primed exosome variants (B') were isolated from the combinatorial priming set (SIRT1 activators and Nrf2 activators in the absence of hypoxia). The exosome primed variants were then characterized by ELISA detecting cargo molecules.

[0171] 3.8 Combinatorial priming of US-MSCs with SIRT1 and NRF2 activators in the presence of hypoxia - (EXO variant B') UC-MSCs were cultured according to the protocol described in Example 1.3, followed by priming with SRT-2104 in the range of 0.00001-0.01 μM or trans-resveratrol (RSV) in the range of 0.1-10 μM. Hypoxia was produced by hypoxia / normoxia cycles (8-20 cycles at 30-90 min intervals). In hypoxia, oxygen concentrations were 0.5-10%, and in normoxia, oxygen concentrations were 14-22%. Next, cells were treated with Nrf2 activators, DMF in the range of 10-250 μM, or 4-OI in the range of 10-250 μM, or 24-72 h after they reached 80% confluence. EV collection exposure, conditioned media collection were performed as described above in Examples 1.2 and 1.1.2. Conditioned media were further screened to detect anti-inflammatory molecule expression using ELISA. UC-MSC-primed exosome variants were isolated from a combinatorial priming set (SRT1 activator and Nrf2 activator in the presence of hypoxia), and the exosome primed variants were characterized by ELISA to detect various cargo molecules.

[0172] 3.9. Combinatorial priming protocol of UC-MSCs with SIRT1 activators (SRT-2104 or trans-resveratrol) in the presence of hypoxia (EXO variant B') UC-MSCs were cultured up to passage 5-6 according to the protocol described in Example 1.3 and hypoxia was produced by hypoxia / normoxia cycles (8-20 cycles at 30-90 min intervals). Hypoxic concentrations were 0.5-10% and in normoxia, oxygen concentrations were 14-22% using a triggered gas chamber incubator setup. Once UC-MSCs reached 80% confluence in hypoxic treatment, cell viability, HIF-1α, HGF, VEGF and TNF-α expression were checked in secretome and inducible exosome mutants. Secretome and exosome profiles were compared to exosomes derived from UC-MSCs that maintained cells in normoxic conditions. Additionally, UC-MSC priming was induced in the presence of alternate hypoxia / normoxia cycles with SRT-2104 in the range of 0.00001-0.01 µM, or trans-resveratrol (RSV) in the range of 0.1-10 µM. EV collection exposure, conditioned media collection were performed as described above in Examples 1.2 and 1.1.2.

[0173] 3.10 Combinatorial priming protocol of UC-MSCs with Nrf2 activators (DMF or 4-OI under hypoxic conditions) (EXO variant B') UC-MSCs were cultured until passage and hypoxia was produced as described in Example 3.8 above. Prior to the shift to EV collection, UC-MSCs were treated with Nrf2 activators, DMF ranging from 10 to 250 μm, or 4-OI ranging from 10 to 250 μm, for 24 to 72 hours. EV collection exposure, conditioned media collection were performed according to the protocols described in Examples 1.2 and 1.1.2 above. Secretomes were characterized by detecting levels of Nrf2, HIF-1α, HGF, VEGF, sFLT1 by ELISA, whereas primed exosome variants (B') were characterized by ELISA detecting levels of exosomal cargo molecule expression such as Nrf2, HIF-1α, VEGF, sFLT1, IL-10, SIRT1, etc.

[0174] 3.11 A single stem cell priming protocol with all-trans retinoic acid (ATRA) UC-MSC / hBM-MSC were cultured according to the protocol described above. Passages 5–6 were used for primed exosome production, and UC-MSC / hBM-MSC were treated with ATRA inducers with a range of working concentrations (0.1–500) μM for 24–72 h before shifting to EV harvesting. Rooster EV harvesting incubation, conditioned medium harvesting were performed as described above. After priming, UC-MSC / hBM-MSC primed conditioned medium / secretome were screened with ELISA assay kits to detect the expression levels of COX-2, HIF-1, CXCR4, CCR2, VEGF, Ang-2 and Ang-4. The primed exosome variants were then characterized by ELISA assays to detect the expression levels of exosomal cargo molecules such as COX-2, HIF-1, CXCR4, CCR2, VEGF, Ang-2 and Ang-4. Based on the results obtained, the concentration of ATRA was finalized for UC-MSC / hBM-MSC priming.

[0175] ATRA has been found to increase the viability of MSCs. This was confirmed when MSCs were treated with various concentrations of ATRA (0.1 μM-500 μM) for 24 and 48 h and their viability was examined by MTT assay. MSC viability was significantly higher in all treated MSCs except for 0.1 μmol / L ATRA. ATRA increased PGE2 levels. Pretreatment of MSCs with various concentrations of ATRA (1, 10, 100 μmol / L) significantly increased PGE2 levels in MSCs in a dose-dependent manner. ATRA increased the expression of genes involved in MSC survival, migration and angiogenesis. The mRNA levels of COX-2, HIF-1, CXCR4, CCR2, VEGF, Ang-2 and Ang-4 were estimated by quantitative real-time PCR and were elevated in a dose-dependent manner when MSCs were treated with ATRA (1, 10, 100 μmol / L).

[0176] 3.12. Combinatorial Priming Protocol of Stem Cells with Nrf2 Activator, SIRT1 Activator, and ATRA in the Presence of Hypoxia (EXO Mutant B') UC-MSC / hBM-MSC were cultured as described above until passage (5-6) and hypoxia was produced as described herein. Prior to the shift to EV collection, UC-MSC / hBM-MSC were treated with Nrf2 activator (DMF, 4-OI) for 24-72 h. EV collection exposure, conditioned media collection were performed as described above.

[0177] The secretome was characterized by detecting the levels of Nrf2, HIF-1α, HGF, VEGF, and sFLT1 by ELISA.

[0178] The primed exosome variants were characterized by ELISA detecting the levels of exosomal cargo molecule expression, including Nrf2, HIF-1α, VEGF, sFLT1, IL-10, and SIRT1.

[0179] 3.13. Combinatorial priming protocol of UC-MSC / hBM-MSC with ATRA inducers in the presence of hypoxia (Exo variant B') UC-MSC / hBM-MSC were cultured as above until passage (5-6) and hypoxia was produced as described. Prior to shifting to EV harvesting, UC-MSC / hBM-MSC were treated with ATRA inducer (0.1-500) μM for 24-72 h. Rooster EV harvesting exposure, conditioned media collection were performed as above. Secretome was further characterized by detecting the levels of COX-2, HIF-1, CXCR4, CCR2, VEGF, Ang-2 and Ang-4 by ELISA.

[0180] The primed exosome variants were characterized by ELISA detecting the levels of exosomal cargo molecule expression, such as COX-2, HIF-1, CXCR4, CCR2, VEGF, Ang-2 and Ang-4.

[0181] 3.14. Combinatorial priming of UC-MSC / hBM-MSC with SIRT1 inducers and ATRA in the presence of hypoxia (Exo variant B') UC-MSCs / hBM-MSCs were cultured as described above after SRT2104 activator or RSV-induced priming. Hypoxia was produced by hypoxia / normoxia cycles (8–20 cycles at 30–90 min intervals). Oxygen concentrations were maintained at 0.5–10% during hypoxia and 14–22% during normoxia. After cells reached 80% confluence, they were treated with ATRA inducers (0.1–500) μM for 24–72 h. Rooster EV exposure and conditioned media collection were performed as described above.

[0182] Conditioned media was screened to detect COX-2, HIF-1, CXCR4, CCR2, VEGF, Ang-2 and Ang-4 expression using ELISA.

[0183] Following the optimized exosome isolation protocol, UC-MSC / hBM-MSC primed exosome variants were isolated from the combinatorial priming set (SRT1 activator and ATRA inducer in the presence of hypoxia). The exosome primed variants were characterized by ELISA to detect various cargo molecules.

[0184] Different UC-MSC / hBM-MSC primed variants of exosomes were thoroughly characterized by mass spectrometry, protein profiling and miRNA profiling via techniques such as Nanostring analysis. The functional efficacy of each exosome variant was tested using in vitro assays such as 2D scratch assay, anti-inflammatory assay, anti-fibrosis, pro / anti-angiogenesis and re-innervation assay. Based on the functional efficacy of different variants, the exosome variant with the highest score is selected and continues for in vivo preclinical testing in anti-inflammatory disease models. LPS-induced ARDS-induced and bleomycin-treated lung injury models are used for in vivo efficacy testing of the exosome with the highest score.

[0185] [Example 4] Exosome isolation and purification Conditioned medium was harvested from hBM-MSCs and UC-MSCs according to the processes described in Examples 1.2.2 and 1.3.2, respectively.

[0186] The resulting conditioned medium was either used directly as a secretome or subjected to ultracentrifugation to isolate exosomes. Isolation of exosomes from conditioned medium / secretomes was performed using three methods: (i) single-step ultracentrifugation; (ii) sucrose-based cushion density ultracentrifugation, and (iii) iodixanol density gradient ultracentrifugation.

[0187] Isolation of exosomes from conditioned media / secretomes was performed by three methods:

[0188] 4.1 Sucrose-based cushion density ultracentrifugation Exosomes were purified using sucrose-based cushion density centrifugation according to the following steps.

[0189] (i) After the cells reached 80% confluency, the medium was removed and the cells were washed in 1× PBS (20 mL), followed by adding 260 mL of EV recovery medium to the flask, which was then incubated at 37° C. and 5% CO for 72 h. 2 and incubated at 4°C for 1 hour.

[0190] (ii) The supernatant was collected and subjected to pretreatment steps as follows: a. The medium was centrifuged at 300 xg for 10 minutes at 4°C, and the supernatant was collected. b. The supernatant was centrifuged at 3000 xg for 20 minutes at 4°C and the supernatant was collected. c. The supernatant was centrifuged at 13,000 xg for 30 minutes at 4°C, and the supernatant was collected. d. The medium was filtered through a 0.45 micron filter. e. The medium was then filtered through a 0.22 micron filter.

[0191] (iii) Conditioned medium was stored at 4° C. for short term (24 hours) or at −80° C. for long term (1 month). However, if the medium was processed immediately or frozen, the conditioned medium was brought to 4° C. and the protocol described below was followed: a. Conditioned medium was centrifuged at 100,000 x g for 90 minutes at 4°C. b. The supernatant was carefully removed. A clear pellet was observed at the bottom of the tube. c. Enriched exosomes were transferred onto ultracentrifuge tubes containing 30% sucrose (1M) as described. d. The speed was set at 100000g for 2 hours at 4°C and acceleration / deceleration was set to zero. e. The supernatant was carefully removed and the exosomes were resuspended in sterile 1x PBS. f. Exosomes were aliquoted and stored at -80°C.

[0192] 4.2 Single-step ultracentrifugation: Exosomes were purified using a single step of centrifugation according to the following steps. (i) After the cells were allowed to reach 80% confluence, the medium was removed and the cells were washed in 1x PBS (20 mL). The PBS was discarded and 40-45 mL / flask of EV recovery medium was added to the flasks, followed by incubation at 37 °C and 5% CO for 72 h. 2 The supernatant was collected and the pretreatment process was carried out as follows: a. The medium was centrifuged at 300 xg for 10 minutes at 4°C, and the supernatant was collected. b. The supernatant was centrifuged at 3000 xg for 20 minutes at 4°C and the supernatant was collected. c. The supernatant was centrifuged at 13,000 xg for 30 minutes at 4°C, and the supernatant was collected. d. The medium was filtered through a 0.45 micron filter. e. The medium was then filtered through a 0.22 micron filter.

[0193] Conditioned media was stored at 4°C for short term (24 hours) or at -80°C for long term (1 month), however, when freshly processed or thawed samples were used, the following protocol was used: a. Conditioned medium was centrifuged at 100,000 x g for 90 minutes at 4°C. b. The supernatant was carefully removed. A clear pellet was observed at the bottom of the tube. c. The pellet was dissolved in PBS / saline. 0.5 mL of crude exosomes was stored at -80°C for QC.

[0194] 4.3. Iodixanol density gradient ultracentrifugation When cells are 80% confluent (e.g., 43,000–50,000 cells / cm), 2 After reaching 100% CO, the medium was removed and the cells were washed in 1x PBS (20 mL), followed by adding 40-45 mL / flask of EV collection medium to the flasks and incubating for 72 h at 37 °C and 5% CO. 2 The supernatant was collected and immediately proceeded to the pretreatment step as follows: a. The medium was centrifuged at 300 xg for 10 minutes at 4°C, and the supernatant was collected. b. The supernatant was centrifuged at 3000 xg for 20 minutes at 4°C and the supernatant was collected. c. The supernatant was centrifuged at 13,000 xg for 30 minutes at 4°C, and the supernatant was collected. d. The medium was filtered through a 0.45 micron filter. e. The medium was then filtered through a 0.22 micron filter.

[0195] Conditioned medium was stored for short term storage (24 hours) at 4° C. or for long term storage (1 month) at −80° C. For processing immediately or with frozen samples, the following protocol was followed: 1. Conditioned medium was centrifuged at 100,000 x g for 90 minutes at 4°C. 2. The supernatant was carefully removed. A clear pellet was observed at the bottom of the tube. 3. The pellet was dissolved in 36 mL of EV recovery medium (36 mL per 300 mL of starting conditioned medium). 0.5 mL of crude exosomes was stored at -80°C for QC.

[0196] Density Gradient Ultracentrifugation (DGUC): An iodixanol (IDX) gradient was prepared by floating 3 mL of 10% w / v IDX solution (Sigma #D1556) containing NaCl (150 mM) and 25 mM Tris:HCl (pH 7.4) in 3 mL of 55% w / v IDX solution. Concentrated conditioned medium (6 mL) was floated on top of the IDX cushion and ultracentrifuged at 100,000 × g (4 °C) for 4.5 h using a Beckman Coulter SW 40 Ti rotor. Twelve fractions (1 mL each) were collected from the top of the gradient on ice and each fraction was collected in a pre-chilled 1.5 mL tube. Fraction-9 was transferred to a fresh ultracentrifuge tube and 11 mL of PBS was added to the 1 mL fraction. Ultracentrifugation was repeated at 100,000 × g for 4 h at 4 °C in an Optima XPN-100 ultracentrifuge using a Beckman Coulter SW 40 Ti rotor. The supernatant was discarded and the exosomes were resuspended in 1 mL PBS. Different aliquots of 50-100 μL were prepared and stored at 4 °C for short-term (2-3 days) and at -80 °C for long-term storage.

[0197] All three methods above were followed by a second round of purification using size exclusion chromatography (with a Captocore 700 column). The process of exosome purification is described in detail in pending applications PCT / IN2020 / 050622, PCT / IN2020 / 050623, PCT / IN2020 / 050653, which are incorporated in their entirety into this disclosure. This example shows the isolation and purification of exosomes from conditioned medium collected from hBM-MSCs and UC-MSCs, however, those skilled in the art may consider that exosomes can be obtained from conditioned medium collected from stem cells, including but not limited to CSSCs, WJMSCs. The stem cells described herein can be naive stem cells or stem cells primed with different priming agents as described in Example 3, where naive stem cells (A) / primed stem cells (A') are used to further harvest conditioning that can be used to obtain naive exosomes (B) / primed exosomes (B'), respectively, by following the protocol described in this Example.

[0198] [Example 5] Characterization of exosomes Harvested or purified exosomes were characterized by methods such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Western blotting, mass spectrometry, and analysis of RNA content by real-time PCR and RNAseq, as described in Example 4. The exosome variants characterized were naive exosomes (B) or primed exosomes (B').

[0199] 5.1 NTA analysis Purified exosomes were dissolved in sterile PBS and a separate aliquot (20-50 μl) of the exosome fraction was stored at -80°C. Autoclaved Milli-Q was filtered through a 0.22 μm syringe filter / nuclease-free water and used for sample dilution. A 1:500 dilution of the exosome sample was used for NTA data acquisition. After mixing by pipetting, 2 μl of the exosome sample was taken from the aliquot. This was added to 998 μl of Milli-Q in a 1.5 ml microcentrifuge tube and mixed multiple times with a 1 ml pipette. Instrument information and data acquisition settings were performed by using a Nanosight LM10 from Malvern for data acquisition with the following settings: camera level 16, gain 3 and 3 runs, each run and threshold of 30 seconds.

[0200] 5.2 Transmission electron microscope images of exosomes The exosome pellet was fixed with 1 mL of 2.5% glutaraldehyde in 0.1 M sodium cacodylate solution (pH 7.0) for 1 hour at 4°C. The fixative was removed and the pellet was rinsed with 1 mL of 0.1 M sodium cacodylate buffer at room temperature. This was repeated three times, with each cycle lasting 10 minutes. The sample was fixed with 1 mL of 2% osmium tetroxide for 1 hour at 4°C. The fixative was removed and rinsed three times with 0.1 M sodium cacodylate buffer for 10 minutes each. The sample was incubated with a graded acetone series (50%, 60%, 70%, 80%, 90%, 95%, and 100%, respectively) for 10 minutes on a shaker. The acetone was removed and a solution of 3:1 acetone:low viscosity embedding mixture was incubated for 30 minutes to obtain the exosome pellet. Furthermore, 1:1 acetone:low viscosity embedding mixture medium was added again and incubated for 30 minutes. The medium was removed and 1:3 acetone:low viscosity embedding mix was added followed by a 30 minute incubation, after which the medium was removed and 100% low viscosity embedding mix was added followed by overnight incubation at room temperature.

[0201] The samples were embedded in pure low-viscosity embedding mixture using embedding molds and baked for 24 h at 65° C. Sections 60 nm thick were obtained using an ultramicrotome, double stained with 2% uranyl acetate for 20 min and lead citrate for 10 min, and grids were viewed under a transmission electron microscope at 80 kV.

[0202] 5.3: Western blotting Western blotting was performed according to the two-step process described below.

[0203] 5.3.1: Protocol 1 20 μl of exosome lysate, corresponding to 200 million particles, was mixed with 20 μl of 2× Laemmli sample buffer (without β-mercaptoethanol for CD63, CD9 and CD81). After heating at 95° C. for 10 min and vortexing, the gel was loaded (12% SDS-PAGE). For Alix and TSG101, 2× Laemmli sample buffer with β-mercaptoethanol (e.g., under reducing conditions) was used according to the antibody data sheet sample preparation.

[0204] 5.3.2 Protocol 2 Approximately 0.4 2n exosomes were lyophilized, and then 20 μl of nuclease-free water was added to the lyophilized exosomes. This was followed by the addition of 20 μl of 2× Laemmli sample buffer, which was heated at 95° C. for 10 minutes. After vortexing, the gel was loaded (12% SDS-PAGE).

[0205] The PVDF membrane was cut to the appropriate size along with the two layers of paper in which it was embedded. The white membrane was separated with the help of forceps and immersed in 50 mL of methanol for activation of the membrane. It was kept for 1 min and rinsed with distilled water. The activated membrane was then transferred to 50 mL of 1× transfer buffer. The transfer apparatus cassette was washed and the absorbent paper was wetted in about 30 ml of 1× transfer buffer. One blot of absorbent filter paper was placed in the cassette followed by the membrane, gel and filter paper. A blot roller was used to ensure that air bubbles were removed between the blot and gel. The transfer was set at 25 V and 2.5 A for 60 min.

[0206] After the transfer was completed, the PVDF membrane was carefully removed using forceps and incubated in blocking solution (50 mL of 5% non-fat milk solution in 1×TBST) for 1 hour at room temperature on a shaker. The PVDF membrane was then removed from the blocking solution using forceps and given a 1×TBST wash for 10 minutes at room temperature on a shaker. The membrane was carefully cut into stripes according to the size of the protein using clean forceps.

[0207] The PVDF stripes were incubated in the respective primary antibodies (diluted in 0.1% BSA in 1x TBS) for the proteins of interest overnight on a shaker at 40°C. The PVDF membranes were removed from the primary antibodies the next day and rinsed six times in 1x TBST buffer (each rinse for 5 min). The primary antibodies were reused by storing at -20°C. The membranes were incubated with HRP-conjugated secondary antibodies (diluted in 0.1% BSA in 1x TBS) for 2 hours at room temperature on a shaker. The membranes were washed six times with 1x TBST buffer (each rinse for 5 min) and after the last wash the membranes were kept in transfer buffer prior to development. The blots were developed in the dark with ECL chemiluminescence reagent (active reagent was prepared according to the manufacturer's guidelines).

[0208] 5.4 Mass spectrometry 5.4.1 Sample preparation: Samples were thawed at 2-8 °C and 25 μL of sample was mixed with 25 μl of lysis buffer (0.1% Triton-X100, 100 mM DTT, 150 mM Tris-HCl pH 8.0), followed by incubation at room temperature for 1 h and brief sonication. The resulting samples were loaded into three different lanes on a pre-cast SDS-PAGE gel (Invitrogen NuPage 4-12% Bis-Tris gradient gel). A short period (less than 10 min) of electrophoresis was performed at constant voltage to remove detergent from the protein samples. Protein bands were visualized using Gelcode Blue staining. Proteins were digested using an in-gel tryptic protein digestion method that involves reduction and alkylation of cysteine ​​residues. The resulting tryptic peptides were pooled and clarified using a C18 zip-tip. The Zip-tip eluate was concentrated to near dryness and dissolved in 8 μL of 0.1% FA. For protein identification, three replicate injections of 2 μL each were performed.

[0209] 5.4.2 Mass spectrometry-based protein identification Tryptic peptides were separated on a reversed-phase liquid chromatography column through a linear gradient of 0.1% formic acid (FA) using a nanoflow setup and developed over 110 min (total run time 140 min). Data were collected under well-optimized conditions in data-dependent mode. Under standard optimized conditions, He-La cell tryptic digestion with 2 μg load provided 6000 protein identifications. The acquired MS and MS / MS data of the test samples were searched against the human proteome database using Maxquant Software. Protein identification was performed with the following criteria: (a) tryptic digest peptides that did not tolerate 4 cleavages, (b) peptide tolerance <10 ppm, (c) ≥ 1 unique peptide, (d) FDR < 1%, (e) fixed modification - carbamidomethylation of cysteine ​​and variable modification - oxidation of methionine.

[0210] Table 9 shows a list of protein biomarkers that can be present as cargo in primed exosome variants and can be detected using one or more standard methods of protein detection such as Western blot, ELISA, or mass spectrometry.

[0211] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7]

[0212] 5.5 Exosomal RNA isolation 5.5.1 Exosomal RNA isolation protocol RNA extraction was performed from naive BM-MSC-derived exosomes using the RNeasy Mini kit from Qiagen according to the manufacturer's protocol. 5 billion exosomes were considered to isolate exosomal RNA. RNA quantification was performed with Nanodoprop and Qubit and the presence of miRNA / small RNA molecules was checked using Qubit microRNA assay.

[0213] 5.5.2 Isolation of exosomal RNA from 10 billion lyophilized exosomes RNA extraction was performed using miRVana miRNA isolation kit (Cat#:AM1560) and four elutions were performed in volumes of 25 μl, 25 μl, 50 μl, and 50 μl, respectively. RNA quantification was performed with Nanodoprop and Qubit and the presence of miRNA / small RNA molecules was checked using Qubit microRNA assay.

[0214] 5.5.3 Exosomal miRNA profiling The different eluates of extracted exosomal RNA were run on a bioanalyzer to check for the presence of small RNAs and miRNAs. The eluates were pooled together and prepared for miRNA profiling using the NanoString platform. All processes were performed according to the manufacturer's instructions.

[0215] Table 10 shows the list of miRNAs analyzed in the primed exosome variants.

[0216] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4]

[0217] Table 11 shows a list of mRNAs analyzed in the primed exosome variants.

[0218] [Table 11-1] [Table 11-2]

[0219] 5.6 Real-time PCR Total RNA isolated using RNAeasy and mirVanaTM (n=3) was DNase treated using Turbo DNase-free kit (Ambion). First strand cDNA synthesis and real-time PCR were performed using miRCURYTM LNATM micro-PCR system according to the manufacturer's protocol. Briefly, each cDNA synthesis was performed in duplicate using a fixed volume of total RNA, miR-451 specific reverse primer (gene ID: 574411), and first strand cDNA synthesis kit reagents, incubated at 50°C for 30 minutes, followed by incubation at 85°C for 10 minutes. Each cDNA sample was then diluted 1:10 and used in duplicate using miRCURYTM LNATM SYBR® Green master mix, universal primers, and LNATM PCR miR-451 specific primers. PCR was performed at 95°C for 10 minutes; 40 cycles of 95°C for 10 seconds + 60°C for 5 seconds, and finalized for each 0.5°C by melting curve for 5 seconds. Control samples were run in parallel. The CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) was utilized for both the cDNA and real-time PCR reactions.

[0220] [Example 6] Functional characterization of exosomes Exosomes were functionally characterized by assessing the following assays: a) scratch assay (wound healing ability); b) anti-inflammatory assay; c) anti-fibrosis assay; d) re-innervation assay; e) angiogenesis (anti- / pro-) assay.

[0221] 6.1 Scratch assay Immortalized human corneal epithelial cells (hTCEPI) were used for the 2D scratch assay. hTCEPI cells were grown at 5000 cells / cm in serum-free medium. 2 Cells were seeded into tissue culture-treated culture dishes at a density of 10 x 10 and allowed to grow until confluent before creating a scratch across the center of the well. The medium was removed and cells were washed with 1x PBS to remove floating cells, followed by (1–20) x 108 Media containing exosomes / mL was added to each well. Cells were incubated at 37°C, 5% CO 2 Cells were incubated at 4°C for 1 h and scratch closure was assessed every 6 h until complete closure. Images of cells were taken at different time points (every 6 h) and wound width was quantified using ImageJ. Controls employed were either medium only (no exosomes) or exosome-depleted control / medium.

[0222] 6.2 Anti-inflammatory assay RAW264.7 macrophage cells were cultured at 5000 cells / cm in complete medium (RPMI + 10% FBS). 2 The cells were seeded in tissue culture dishes at a density of 100 × 10 and grown to 80% confluence. The cells were starved in serum-free medium for 16 h and replenished with (1–20) × 10 8 Cells were stimulated with LPS (10 ng / mL) in the presence or absence of exosomes / mL for 4 hours. Media was collected after treatment and secreted cytokine levels were measured by ELISA. Additionally, cells were lysed and cytokine transcript levels were measured by qPCR to complement secreted protein levels. Controls employed were either media only (no exosomes) or exosome-depleted control / media.

[0223] 6.3 Antifibrotic Assays Human corneal epithelial cells were cultured at 5000 cells / cm in serum-free medium. 2 The cells were seeded at a density of 10 × 10 in tissue culture-treated culture dishes and grown to 80% confluence. 8 Cells were treated with TGF-β (10 ng / mL) for 24 h in the presence or absence of exosomes / mL. The degree of induction of fibrosis was assessed by characterizing the expression of collagen type I, alpha-smooth muscle actin, and fibronectin by immunofluorescence. Controls employed were either medium only (no exosomes) or exosome-depleted control / medium.

[0224] 6.4 Reinnervation assay PC12 cells at 5000 cells / cm 2 The cells were seeded on collagen-coated plates at a seeding density of (1–20) × 10, and the medium was changed to (1–20) × 10 at 24 hours after cell seeding. 8 Serum-free medium was replaced with exosomes / mL treatment. Images were acquired at 24-h intervals from days 3 to 5. Controls employed were either medium only (no exosomes) or exosome-depleted control / medium as a negative control, while NGF (20 ng / mL) served as a positive control.

[0225] 6.5 Angiogenesis (anti- / pro-) assays Human vascular endothelial cells (HUVEC) or coronary artery endothelial cells (CAEC) were used for the assay. HUVEC were grown for 24 h in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U / mL penicillin, and 100 μg / mL streptomycin. One day before the assay, HUVEC cells were serum starved as follows: the medium from the cells was aspirated and serum-reduced medium of DMEM supplemented with 0.2% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U / mL penicillin, and 100 μg / mL streptomycin was added, and the cells were grown for another 24 h. After this step, 300 μL of Matrigel (reduced growth factors) was added to the 24-well plate and allowed to solidify for 30 min at 37 °C. HUVECs (2 × 10 4 / well) to exosomes (1-20) × 10 8 The cells were suspended in medium supplemented with VEGF (concentrated) in the presence or absence of 100 μg / mL for 24 hours. Cells were stained with Cell Tracker™ Green CMFDA according to the manufacturer's instructions, and tube formation was detected using immunofluorescence staining.

[0226] 6.6. Cell transformation assay Mouse 3T3 cells in DMEM / HAM's F-12 containing 3 g / l D-glucose, 5% fetal bovine serum, and 1% penicillin / streptomycin were used for the cell transformation assay. 3T3 cells (5000 cells / well) were seeded into Corning® Primaria™ 6-well plates and incubated under standard conditions (37° C., 5% CO 2 The cells were cultured at 4°C (37°F, 95% humidity) for 42 days. 24 hours after seeding, the cells were treated with samples, and the medium was changed 3 days after treatment. In addition, the tumor promoter TPA (12-O-tetradecanoyl-phorbol-13-acetate, 0.3 μg / ml, Sigma#79346) was added on days 8, 11, 15, 18, and up to day 21. After 42 days, the cells were washed twice with PBS, fixed with PBS / methanol (50:50) for 3 min, washed with 100% ice-cold methanol for 10 min, and finally washed twice with methanol.

[0227] For the purposes of this disclosure, controls employed were: (i) medium only (no exosomes); (ii) exosome-depleted control / medium as a negative control; (iii) VEGF as a positive control.

[0228] [Example 7] Investigating the efficacy of exosomes in preclinical trials Based on the functional efficacy of different variants, the exosome variants with the highest scores were selected and continued for in vivo preclinical testing on anti-inflammatory disease models, as described in Example 8, and the application of the selected exosome variants was used for the regeneration of specific tissues, i.e., avascular and vascular tissues.

[0229] 7.1 In vivo efficacy studies Preclinical efficacy studies were conducted in multiple in vivo acute respiratory distress syndrome (ARDS) models, as described below. MSC-derived exosomes were used in one or more in vivo mouse models of COVID-19-associated ARDS: LPS-induced lung injury model, or Bleomycin-induced pulmonary fibrosis model

[0230] 7.2 Animal models A mouse model (8-10 weeks old) was used for the purpose of in vivo studies. The mouse strain used was C57BL / 6 strain.

[0231] 7.3 Mode of Administration Exosomes were administered via intravenous mode (iv). Groups: Group 1: saline control; Group 2: UC-MSC-Exo (naive / primed).

[0232] 7.4 Dosage Calculations The dose of exosomes was determined based on an evaluation of available clinical and preclinical data regarding the use of UC-MSCs and exosomes for therapeutic applications.

[0233] 7.5 Preclinical Study Dosage (Mouse Model) Dose equivalence formulas for humans and animals were calculated based on differences in body weight and surface area. (i) Mouse dose (per kg body weight) = Human dose (per kg body weight) × 12.3 (ii) Human dose: Exosomes were administered at doses ranging from 80 to 160 billion cells (1.3–2.6 billion cells / kg body weight for an average body weight of 70 kg).

[0234] Exosomes were administered at high doses of 16–32 billion exosomes / kg body weight.

[0235] 7.6 LPS dose (LPS-induced lung injury model) Given the body of work available, doses between 10 mg and 100-125 mg were considered appropriate to ensure that the study covered sublethal and lethal dose concentrations. A dose of 100 mg was expected to induce death 48-72 hours after LPS administration.

[0236] 7.7 Bleomycin Dose (Bleomycin-Induced Pulmonary Fibrosis Model) A single intratracheal administration of bleomycin (50 μL, 3 U / kg (2 mg / kg)) was determined.

[0237] 7.8 In vivo readout: Terminal Read: ·Survival rate Histology: HandE, Masson's Trichrome or Sirius Red Evaluation of total and differential blood cell counts: Automated analyzer Characterization of fibrosis and inflammatory markers -Inflammatory cytokine profiling in serum and BAL samples Inflammatory cell type and subpopulation analysis

[0238] Temporal Readout: Evaluation of total and differential blood cell counts: Automated analyzer - Inflammatory cytokine profiling in serum samples.

[0239] [Example 8] Generation of therapeutically enriched exosomes derived from primed BM-MSCs for avascular tissue (cornea) regeneration Based on protein expression and higher regenerative potential vs. cargo protein or correlation with biomarkers, the highest scoring exosomes were used for avascular tissue regeneration. This example shows avascular tissue, i.e., corneal regeneration with enriched exosomes using macromolecule (CSSC-derived conditioned medium (CSSC-CM))-mediated priming of bone marrow-derived mesenchymal stem cells (hBM-MSCs).

[0240] 8.1 Exosomes derived from hBM-MSCs primed with CSSC-CM 8.1.1 Induction of hBM-MSC priming with CSSC-conditioned medium hBM-MSCs were cultured in xeno-free medium with 10% and 20% replacement of CSSC-derived conditioned medium (CSSC-CM) to reach 80-90% confluence. Then, the medium was subsequently shifted to EV collection for 24-72 h, the primed conditioned medium was collected (as described in Examples 1.1 and 1.2), and exosome isolation was performed by iodixanol density gradient method (as described in Example 4.4) to obtain enriched exosomes. The homogenous fraction F9 was considered for further functional analysis. Secretome analysis was performed using ELISA to detect the levels of HGF, VEGF, sFLT1, IL-6, and NGF.

[0241] 1A-1E show secretome profiles of enriched exosomes derived from hBM-MSCs primed with CSSC-CM-conditioned medium (see Example 3.2). FIG. 1A shows a bar graph showing increased levels of secreted hepatocyte growth factor (HGF) from exosomes derived from hBM-MSCs primed with CSSC-CM compared to control exosomes. Similarly, FIG. 1C-1E show increased levels of secreted sFLT1, IL-6, and nerve growth factor (NGF) from exosomes derived from hBM-MSCs primed with CSSC-CM compared to control exosomes, respectively. FIG. 1B shows significantly lower levels of vascular endothelial growth factor (VEGF) from exosomes derived from hBM-MSCs primed with CSSC-CM compared to control exosomes. These results indicate that hBM-MSCs can be primed with CSSC-conditioned medium to generate therapeutically enriched exosomes.

[0242] 8.1.2 Characterization of the anti-inflammatory activity of exosome variants primed with different CSSC-conditioned media Different exosome variants derived from hBM-MSCs primed with CSSC-conditioned medium (CSSC-CM) were tested to detect their anti-inflammatory activity using RAW264.7 cells. RAW264.7 cells were treated with lipopolysaccharide (LPS)-binding protein to induce inflammation in the presence of exosomes to check the pro-inflammatory / preventive effect of exosomes on inflammation. As shown in Figures 2A-2B and 2D-2E, exosome variants derived from hBM-MSCs primed with CSSC-CM reduced inflammatory cytokine protein expression of key inflammatory cytokines (e.g., IL-6, IL-1β, TNF-α and IFN-γ) in RAW264.7 cells treated with LPS. Moreover, the overall inflammatory cytokine gene expression was also reduced in RAW264.7 cells stimulated with LPS and treated with exosome variants derived from hBM-MSCs primed with CSSC-CM. Figures 2A-3E show that exosome variants derived from hBM-MSCs and conditioned with CSSC medium have anti-inflammatory activity by reducing inflammatory cytokine expression and inflammatory cytokine gene expression of key inflammatory cytokines (e.g., IL-6, IL-10, IL-1β, TNF-α and IFN-γ). Furthermore, from Figures 3A-3E, it can be inferred that exosomes derived from hBM-MSCs primed with both CSSC-CM and Nrf2 activator DMF (see Example 3.6) show increased expression levels of anti-inflammatory cytokine IL-10 and decreased levels of IL-6, TNFα, IL-1β in RAW264.7 cells treated with LPS.

[0243] 8.1.3 Characterization of the antifibrotic properties of different exosome variants primed with CSSC-conditioned medium Different exosome variants derived from hBM-MSC primed with CSSC-conditioned medium (CSSC-CM) (see Example 3.2) were tested to detect the anti-fibrotic activity of primed exosomes. To test the anti-fibrotic activity of exosome variants, human dermal fibroblasts were treated with TGF-β (Figure 4B) for 24 hours or co-treated with TGF-β and the indicated exosomes (Figures 4C-4F) to induce fibrosis (similar to Example 6.3). α-SMA expression was monitored as a fibrosis marker to check the effectiveness of exosome variants. With reference to Figures 4D-4E, it can be observed that exosomes derived from hBM-MSC primed with 20% and 10% CSSC-CM can inhibit TGF-β-induced α-SMA expression in fibroblasts. Cells treated with naive exosomes derived from hBM-MSCs expressed less α-SMA (Figure 4C) compared to primed exosomes (Figures 4D-4E). Exosomes derived from hBM-MSCs primed with 20% and 10% CSSC-CM were able to efficiently inhibit fibrosis in fibroblasts compared to control and naive exosome treatments.

[0244] 8.2 Exosomes derived from hBM-MSCs primed with Nrf2 activators (DMF or 4-OI) 8.2.1 NRF2 activators (DMF or 4-OI) mediated priming of hBM-MSC cells and characterization of secretome and exosome profiles hBM-MSCs were cultured in xeno-free medium as recommended by the manufacturer. hBM-MSC cells were grown to reach (80-90)% confluency and treated with Nrf2 activator (DMF-100 μM) for 24 h, after which the cells were shifted and maintained in EV collection medium for 24-72 h (see Example 3.3). After 72 h, conditioned medium was collected and exosome isolation was performed using an iodixanol density gradient protocol (described in Example 4.4).

[0245] FIG. 5 shows a bar graph quantifying the yield of naive exosomes, exosomes derived from hBM-MSCs primed with various Nrf2 activators (e.g., DMF or 4-OI), and exosomes derived from hBM-MSCs primed with other priming agents such as curcumin. From FIG. 5, it can be observed that exosomes derived from hBM-MScCs and primed with Nrf2 activators DMF or 4-OI produced higher yields of exosomes compared to untreated (naive) exosomes and exosomes primed with other priming agents (e.g., curcumin or a combination of curcumin and DMF), respectively. Alternatively, FIG. 5 also suggests that Nrf2 activators do not have an inhibitory effect on cellular exosome secretion, making them good priming agents.

[0246] 8.2.2 Secretome marker profiling of primed cells with priming agents hBM-MSCs were primed with various priming agents (small molecules) and secretomes were harvested (see Examples 3.2-3.6). Secretome analysis was performed using ELISA to detect levels of HGF, VEGF, NGF, IL-6, sFLT1, and SDF1. Figures 6A-6F show bar graphs of quantification of secreted protein levels of HGF, VEGF, NGF, IL-6, sFLT1, and SDF-1 from hBM-MSCs primed with curcumin, 4-OI, DMF, or a combination of curcumin-conditioned medium and DMF.

[0247] The Nrf2 activators, 4-OI and DMF, each produced a significant increase in HGF secretion compared to the other primed mutants and the untreated control (Figure 6A). VEGF secretion levels were unchanged regardless of the priming agent used (Figure 6C). Priming with DMF resulted in increased secretion of sFLT1 (Figure 6D), NGF (Figure 6E), and SDF (Figure 6F). Furthermore, with reference to Figure 6B, it can be observed that curcumin and Nrf2 activators (4-OI and DMF) each attenuated the secretion of IL-6. It is pertinent to note that NGF secretion levels were enhanced by each of the Nrf2 activators (4-OI and DMF) (Figure 6E). Also, from Figures 6A-6F, it was observed that the combination of curcumin conditioned medium + DMF did not seem to have a significant effect on the levels of HGF, VEGF, NGF, IL-6, sFLT1, SDF1.

[0248] 8.2.3. Profiling of exosomal cargo from exosomes derived from cells primed with different priming agents See Figures 7A-7F. hBM-MSCs were primed with the indicated priming agents, secretomes were harvested, and exosome isolation was performed using Pandorum's optimized iodixanol density gradient method (see Example 4.3), and levels of HGF (Figure 7A), VEGF (Figure 7B), sFLT1 (Figure 7C), NGF (Figure 7D), TGF-β (Figure 7E), and SDF1 (Figure 7F) were detected in purified fraction 9 using ELISA. As shown in Figure 7D, exosomes derived from hBM-MSCs primed with DMF contain significantly higher levels of exosomal NGF compared to naive exosomes or exosomes derived from hBM-MSCs primed with other priming agents such as 4-OI, curcumin (CUR) or combined curcumin and DMF (CUR / DMF). Similarly, priming of hBM-MSCs with DMF resulted in increased exosomal TGF-β compared to naive exosomes, curcumin-primed, or 4-OI-primed exosomes.

[0249] 8.2.4. Anti-inflammatory activity of exosomes derived from primed BM-MSCs Exosome variants derived from hBM-MSCs primed with Nrf2 activator DMF (see Example 6.2) were tested to detect their anti-inflammatory activity using RAW264.7 cells. RAW264.7 cells were treated with LPS to induce inflammation in the presence and absence of exosomes derived from primed hBM-MSCs, and major inflammatory cytokines were measured by ELISA to determine the preventive effect of exosomes on inflammation. As shown in Figures 8A-8E, exosomes derived from hBM-MSCs primed with DMF or curcumin showed a decrease in IL-6, IL-1β, TNF-α and IFN-γ expression at protein level. Furthermore, the anti-inflammatory cytokine IL-10 was significantly increased in exosomes derived from hBM-MSCs primed with DMF or curcumin, indicating that exosomes primed with DMF and curcumin not only reduced general inflammatory cytokines but also had anti-inflammatory effects.

[0250] 8.3 Exosomes derived from hBM-MSCs primed with CSSC-CM and Nrf2 activators 8.3.1 Profiling of exosomal cargo from exosomes derived from hBM-MSCs primed with CSSC-conditioned medium, Nrf2 activators, or both hBM-MSCs were cultured in xeno-free medium as recommended by the manufacturer. hBM-MSCs were grown in the presence of CSSC-CM at a concentration of 20% of the total medium volume until the cells reached 80-90% confluency. Then, the medium was replaced and hBM-MSCs were treated with Nrf2 activator (DMF-100 μM) for 24 h, after which the cells were shifted in EV collection medium and maintained in EV collection medium for 72 h. After 72 h, the conditioned medium was collected and exosome isolation (purification) was performed using an iodixanol density gradient protocol (described in Example 4.4). The levels of HGF, VEGF, sFLT1 and NGF were detected in the different combinatorially primed exosome variants using ELISA (Figures 9A-9D). While singly primed (Nrf2 activators or CSSC-conditioned medium) exosomes showed increased exosomal HGF, sFLT1 and NGF, combinatorially primed (CSSC-CM and DMF) exosomes showed the highest levels of exosomal HGF, sFLT1 and NGF.

[0251] Both CSSC-CM-primed and DMF-primed exosomes showed an increase in exosomal HGF. The expression level of exosomal HGF in DMF-primed exosomes was about 1.2-fold (1.2×) that of naive exosomes, and the expression level of exosomal HGF in CSSC-CM-primed exosomes was about 2-fold (2×) that of naive exosomes. That being said, combinatorially primed (CSSC-CM+DMF) showed the highest increase in exosomal HGF, with more than 2-fold (2×) or about 3-fold (3×) expression levels of exosomal HGF compared to naive exosomes, and about 2-fold expression levels of exosomal HGF compared to DMF-only priming (Figure 9A). Furthermore, CSSC-CM-primed exosomes and combined primed (CSSC-CM+DMF) exosomes both showed substantially reduced exosomal VEGF expression, less than one-quarter (1 / 4 or 25%) of VEGF expression, compared to naive or DMF-primed exosomes (Figure 9B).

[0252] Single-primed (CSSC-CM or DMF) exosomes showed increased exosomal sFLT1 and NGF (Figure 9C-9D). The expression level of exosomal NGF in DMF-primed exosomes was more than two-fold (2×) or about three-fold (3×) that of naive exosomes, and the expression level of exosomal NGF in CSSC-CM-primed exosomes was about two-fold (2×) that of naive exosomes. However, combinatorially primed (CSSC-CM and DMF) exosomes had the highest exosomal sFLT1 and NGF, indicating that combinatorial priming with CSSC-CM and DMF may result in more desirable exosomal cargo compared to single priming of exosomes or naive exosomes. Exosomal sFLT expression levels from combinatorially primed (CSSC-CM and DMF) exosomes were more than twice as high as naive exosomes and approximately twice as high as DMF-primed exosomes. Exosomal NGF expression levels from combinatorially primed (CSSC-CM and DMF) exosomes were more than three times (3×) as high as naive exosomes and approximately 1.5× as high as DMF-primed exosomes.

[0253] 8.3.2 Characterization of the anti-inflammatory activity of different exosome variants primed with CSSC-conditioned medium and NRF2 activator (DMF) Exosomes derived from hBM-MSCs primed with either CSSC-conditioned medium, Nrf2 activator DMF, or their combination were tested with LPS-treated RAW264.7 cells to detect the anti-inflammatory activity of the primed exosomes. Pro- and anti-inflammatory cytokines were measured by ELISA. As shown in Figures 10A-10E, exosomes derived from hBM-MSCs primed in combination with CSSC-CM and Nrf2 activator (DMF) could reduce inflammation by decreasing IL-6 (Figure 10A), IL-1β (Figure 10B), TNF-α (Figure 10C), and IFN-γ (Figure 10E) expression at the protein level. Furthermore, the expression of anti-inflammatory cytokine IL-10 was increased by treatment with single-primed exosomes (CSSC-CM or DMF). Moreover, the greatest increase in IL-10 expression was achieved with exosomes derived from combinatorial priming of hBM-MSCs with CSSC-CM and DMF.

[0254] These data indicate that exosomes derived from hBM-MSCs primed with both CSSC-CM and DMF effectively reduced the expression of pro-inflammatory cytokines while increasing the expression of anti-inflammatory cytokines.

[0255] 8.3.3 Characterization of the antifibrotic activity of exosomes derived from cells primed with CSSC-conditioned medium + NRF2 activator (DMF) Exosomes derived from hBM-MSC primed with CSSC-CM and DMF were tested to detect their anti-fibrotic activity. Human dermal fibroblasts were treated with TGF-β to induce fibrosis (see Example 6.3). Fibroblasts were treated with primed exosomes to test the anti-fibrotic activity of exosomes. α-SMA expression, a fibrosis marker, was monitored using immunofluorescence to check the effectiveness of the anti-fibrotic activity of exosomes after treatment. Representative immunofluorescence images shown in Figures 11A-11F show that even single primed exosomes (CSSC-CM or DMF) reduced fibrosis (Figures 11D and 11E). However, exosomes derived from hBM-MSC primed with a combination of CSSC-CM and DMF showed the greatest reduction in fibrosis (Figure 11F).

[0256] 8.3.4 Characterization of the wound healing activity of exosomes derived from hBM-MSCs primed with CSSC-conditioned medium, Nrf2 activator (DMF), or their combination See Figure 12. Exosomes derived from hBM-MSC primed with CSSC-CM, Nrf2 activator (DMF) or their combination were tested to detect their effects in 2D scratch assay (see Example 6.2). Immortalized human corneal epithelial cells (hTCEPi) were labeled with green fluorescent dye (CMFDA) and scratches were generated through hTCEPi cells. hTCEPi cells were treated with naive exosomes (naive BM-MSC), single primed exosomes (CSSC-CM or DMF) or their combination, and wound closure was observed at the following time points 0, 24 hours, 48 ​​hours, and 72 hours using a fluorescent microscope. Single primed exosome treatment and its combination led to wound closure by 72 hours, indicating that primed exosome treatment led to significant directional cell migration. Similarly, quantitative assays of corneal cell migration and proliferation after exosome treatment were performed, as shown in Figure 13. Corneal cells treated with exosomes derived from CSSC-CM and DMF-primed hBM-MSCs showed the highest cell proliferation at almost all time points, while corneal cells treated with DMF-primed exosomes showed increased cell proliferation at later time points compared to corneal cells treated with CSSC-CM-primed exosomes or naive exosomes alone.

[0257] 8.3.5 Characterization of the wound-healing activity of exosomes derived from CSSC-CM and DMF-primed hBM-MSC in rabbit cornea FIG. 14 shows representative microscopic images of rabbit corneas with open epithelial wounds after 1, 7, and 14 days post-surgery. To test the ability of primed exosomes derived from hBM-MSCs to effectively induce wound healing in a 3D model, injured rabbit corneas were treated with liquid corneal biopolymer combined with exosomes derived from hBM-MSCs primed with CSSC-CM and Nrf2 activator DMF, liquid corneal biopolymer alone, or untreated control. On day 7 post-surgery, the combination of liquid corneal biopolymer and exosomes derived from hBM-MSCs primed with CSSC-CM and Nrf2 activator DMF showed significant wound healing compared to liquid corneal biopolymer alone or untreated control. On day 14, the combination of liquid corneal biopolymer and exosomes derived from hBM-MSCs primed with CSSC-CM and Nrf2 activator DMF showed complete and stable epithelialization of the rabbit cornea. Treatment with liquid corneal biopolymer alone showed nearly complete epithelialization with some disrupted patches on day 14, whereas untreated controls showed defective epithelialization. Taken together, Figures 12, 13, and 14, exosomes derived from hBM-MSCs primed with a combination of both CSSC-CM and DMF demonstrated superior wound healing activity based on 2D scratch assays and corneal cell migration and proliferation analyses, and demonstrated robust wound healing in rabbit corneas in vivo.

[0258] [Example 9] Generation of therapeutically enriched exosomes derived from primed UC-MSC / WJ-MSC for vascularized multi-tissue regeneration (liver, lung) This example shows the in vitro culture of umbilical cord blood-derived mesenchymal stem cells (UC-MSC) / Wharton's jelly-derived MSC (WJ-MSC). The protocol for culturing UC-MSC and WJ-MSC is described in Example 1.5. Furthermore, unique subpopulations of stem cells (UC-MSC and WJ-MSC) were selected based on the method described in Example 1.4. Then, an expanded population of stem cells was obtained and primed with different priming agents: (a) Nrf2 activators such as DMF or 4-OI, or CDDO-Im; and (b) SRT1 activators: SRT-2104, or resveratrol, alone or in combination. Priming of stem cells is possible in the absence or presence of hypoxic conditions. Priming of stem cells with the priming agents mentioned herein can provide or obtain primed stem cells and primed conditioned medium with enhanced regenerative, stemness and anti-inflammatory properties. MSCs primed with a single priming agent, such as Nrf2 activator or SIRT1 activator, or a combination of priming agents, such as Nrf2 activator + SIRT1 activator, were used as a source for the production of different exosome variants with specific / enriched cargo loading factors. The exosome variants were then characterized at both physical and molecular levels for their functional efficacy. The characterized exosome variants were classified based on their function against different inflammatory and fibrosis-related diseases, such as pulmonary dysfunction, acute respiratory distress, inflammation-related disorders, including but not limited to rheumatoid arthritis, systemic juvenile idiopathic arthritis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome (ARDS), pneumonia, bronchitis, chronic obstructive pulmonary disease (COPD), COVID-19, coronavirus class infections, cystic fibrosis, hantavirus, influenza, tuberculosis, systemic lupus, osteoarthritis, NASH, hepatic fibrosis, Mooren's ulcer, neurotrophic ulcer, and myocardial infarction.

[0259] Overall, the present disclosure provides a method for providing or obtaining primed stem cells and primed conditioned medium. The method involves isolating a population of mesenchymal stem cells, such as UC-MSCs, and WJ-MSCs that express a signature set of markers. The selected population of MSCs is then modified with hTERT (human telomerase reverse transcriptase), which expands the doubling potential of MSCs, which helps promote a scalable and homogenous population of cells. The stem cells are further cultured in a 3D culture system (microcarrier-based system, or spheroid-based system, or hollow fiber bioreactor) to obtain an expanded population of stem cells. The expanded population of stem cells is further primed with different priming agents (small and macromolecules). The presence of the priming agents (small and macromolecules), along with the disclosed concentration ranges, and the duration of treatment of the priming agents on the cells can be important to provide or obtain primed stem cells and primed conditioned medium. Priming of naive stem cells with different priming agents used alone or in combination serves to enhance the regenerative, stemness and anti-inflammatory properties of the cells. The primed stem cells are further used as a source of different exosome variants enriched with anti-inflammatory, anti-fibrotic and pro-angiogenic factors. The enriched therapeutic grade exosomes are then further applied for vascular tissue regeneration (lung or liver) or avascular tissue regeneration (cornea).The disclosed methods, high yields of enriched primed stem cells, or primed exosomes of the present disclosure can be used to treat, but are not limited to, rheumatoid arthritis, systemic juvenile idiopathic arthritis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), pneumonia, bronchitis, chronic obstructive pulmonary disease (COPD), COVID-19, coronavirus class infections, cystic fibrosis, hantavirus, influenza, tuberculosis, systemic lupus, The majority of patients suffering from diseases including myocardial infarction, osteoarthritis, nonalcoholic fatty liver disease (NASH), hepatic fibrosis, Mooren's ulcer, neurotrophic ulcer, corneal keratitis (CK), dry eye disease ulcer, herpetic simple keratitis, post-LASIK ectasia, post-operative corneal melt, post-keratoprosthesis melt, corneal perforation, neurotrophic keratitis (NK), keratoconus, Sjogren's syndrome, mucous membrane pemphigoid, Stevens-Johnson syndrome, chemical burns, and thermal burns can be treated.

[0260] [Example 10] Treatment of nonalcoholic steatohepatitis (NASH)-induced liver spheroids with primed exosomes This example shows the in vitro treatment of induced non-alcoholic steatohepatitis (NASH) phenotype in human liver spheroids with exosomes derived from naive hBM-MSCs (naive exosomes) grown under standard conditions or from hBM-MSCs primed with DMF (primed exosomes). Protocols for culturing and priming hBM-MSCs and obtaining exosomes from primed hBM-MSCs are described in Examples 3 and 4, as well as Example 8. In particular, the protocol for producing the DMF-primed MSC-derived exosomes used in the treatment of NASH-induced liver spheroids in this example is described in Section 8.2.1 of Example 8.

[0261] Nonalcoholic fatty liver disease (NAFLD) is a condition in which fat accumulates in the liver of a subject, regardless of the subject's alcohol consumption. The disease is divided into several stages. When the liver has excess fat but does not cause inflammation or fibrosis, it can be called fatty liver or nonalcoholic fatty liver (NAFL). Once the disease progresses further and liver tissue develops inflammation and fibrosis, the disease is called nonalcoholic steatohepatitis (NASH).

[0262] In this study, the protocol for generating human hepatic spheroids and inducing the NASH phenotype in human hepatic spheroids was as follows: a mixture of primary hepatocytes and hepatic stellate cells from human donors was co-cultured at a 70:30 ratio (70 hepatocytes to 30 hepatic stellate cells). The cell mixture was seeded on ultra-low attachment plates and cultured to form viable hepatic spheroids. Once viable hepatic spheroids were obtained, the NASH phenotype was induced in the hepatic spheroids through sequential treatments of free fatty acids to induce steatosis followed by TGF-β to induce fibrosis, so that the hepatic spheroids exhibited the aspects of a steatotic fibrotic liver. The NASH induction protocol consisted of treating the spheroids with a 600 μM free fatty acid (FFA) mixture consisting of oleic acid and palmitic acid at a weight ratio of 2:1 for 6 days on day 7 after seeding, with medium changes every other day. Six days after FFA treatment (13 days after seeding), 20ng / mL of TGFβ1 was added to the FFA-containing medium, and FFA and TGFβ1 treatment were combined for another 2 days. 15 days after seeding (8 days after FFA treatment and 2 days after combined FFA and TGFβ1 treatment), the medium was changed to TGFβ1 treatment alone, containing 20ng / mL of TGFβ1 and no FFA mix. Two days later, on the 17th day after seeding, spheroids were designated as NASH-inducible (i.e., exhibiting aspects of lipofibrosis) and subjected to therapeutic treatment without FFA or TGFβ1 (e.g., with naive or primed exosomes, HGF, or vehicle control). Two days later, on the 19th day after seeding, spheroids and conditioned medium were harvested and characterized. The NASH-inducible protocol described above was as follows: Day 0: Seeding of primary hepatocytes Day 7: Treatment with 600 μM FFA mixture (2:1 mixture of oleic acid and palmitic acid) Day 13: Addition of TGFβ1 to combined FFA and TGFβ1 treatment Day 15: TGFβ1 treatment only Day 17: Therapeutic treatment (naive or primed exosomes, HGF, or vehicle control) Day 19: Collect for analysis This can be summarized as follows.

[0263] After induction of NASH with a free fatty acid mixture followed by sequential treatment with TGF-β, liver spheroids exhibited a NASH-like phenotype, including decreased spheroid size, increased collagen deposition, and reduced albumin secretion. NASH-induced liver spheroids were then subjected to administration of exosomes from naive or primed hBM-MSCs and used for further assays described herein below.

[0264] FIG. 15A shows representative immunofluorescence images stained for CYP3A4 and DAPI in liver spheroids under four conditions: (1) healthy (no NASH induction); (2) NASH induction (Disease / reversed control); (3) NASH induction followed by treatment with naive exosomes ("Naive-Exo"); and (4) NASH induction followed by treatment with primed exosomes ("Primed-Exo"). DAPI is a nuclear marker and CYP3A4 is a marker used to indicate healthy liver tissue, so a decrease in CYP3A4 staining indicates a decrease in liver tissue health and an increase in CYP3A4 staining indicates an improvement in liver tissue health. Liver spheroids were imaged as described in Example 6.3.

[0265] It was found that NASH-induced liver spheroids showed reduced CYP3A4 staining compared to healthy liver spheroids that were not subjected to NASH induction, and treatment of NASH-induced liver spheroids treated with primed exosomes ("Primed-Exo") from hBM-MSCs primed with DMF showed partial restoration of CYP3A4 staining, indicating that exosome treatment is effective in at least partially restoring the health of NASH-induced spheroids. In contrast, treatment with naive exosomes ("naive exo") did not show restored CYp3A4 staining compared to naive exosome treatment.

[0266] Secreted albumin levels are another marker of liver health. Figure 15B shows a bar graph showing the albumin levels secreted into the medium by hepatic spheroids under different conditions: (1) healthy (no NASH induction) at D19; (2) NASH induction at D19 ("Disease / reverse control"); (3) NASH induction at D17-D19 followed by treatment with 40ng / ml hepatic growth hormone ("HGF"); (4) NASH induction at D17-D19 followed by treatment with naive exosomes ("naive-Exo"); and (4) NASH induction at D17-D19 followed by treatment with exosomes primed with DMF ("Primed-Exo"). Albumin secreted into the medium was quantified based on ELISA measurement. Measurements were taken from each medium sample 24 hours, 48 ​​hours, and 72 hours after treatment (or equivalent time frames for healthy and NASH-induced conditions). It was found that NASH-induced liver spheroids treated with DMF-primed exosomes showed upregulated albumin secretion, indicating improved spheroid health. In contrast, treatment of NASH-induced liver spheroids with HGF or naive exosomes did not increase albumin secretion by the spheroids.

[0267] Collagen can serve as a marker of fibrosis in liver tissue, including liver spheroids. Figure 15C shows representative immunofluorescence images stained for collagen 1 in liver spheroids under three conditions: (1) NASH induction at D17-D19 and treatment with vehicle control ("Vehicle Control"); (2) NASH induction at D17-D19 followed by treatment with naive exosomes; and (3) NASH induction at D17-D19 followed by treatment with exosomes primed with DMF ("Primed Exo"). Liver spheroids were imaged as described in Example 6.3. Treatment of NASH-induced liver spheroids with primed exosomes reduced cell surface collagen protein expression compared to treatment with vehicle control or naive exosomes.

[0268] FIG. 15D shows a bar graph of quantification of coverage of collagen deposition in hepatic spheroids under the following conditions:

[0269] Healthy D19 serves as healthy control for the reversal group, vehicle-treated (Disease-D19), while healthy controls on the right correspond to the D17 disease-induced group and serve as controls for FFA and FFA+TGFb1 treatments.

[0270] (1) before NASH induction at D19 ("healthy"); (2) NASH was induced and treated with vehicle control from D17-D19 and harvested on D19 ("vehicle treatment"). (3) HGF treatment on D17-D19, harvested on D19 “reversal by T1”; (4) simultaneous treatment with naive exosomes during fibrosis induction by TGFβ1 at D13-D17 ("simultaneous naive exosome treatment"); (5) co-treatment with primed exosomes during fibrosis induction by TGFβ1 at D13-D17 ("co-primed exosome treatment"); (6) treatment with naive exosomes at D17-D19 after NASH induction ("naive exosome treatment"); (7) treatment with primed exosomes at D17-D19 after NASH induction ("primed exosome treatment"); (8) Not subjected to NASH induction and harvested at D17 (“healthy control”). (9) steatosis induction - treated only with FFA mix without TGFβ1 and harvested at D15; (10) Lipofibrosis induction with sequential treatment with FFA and TGFβ1, harvested at D19 (Figure 15G; see "Lipofibrosis"). Comparing "healthy control" and "steatosis", we show that induction of NASH by FFA and TGFβ1 induced fibrosis in liver spheroids, but not by FFA treatment alone. Based on collagen 1 staining alone, the addition of either naive or primed exosomes appeared to prevent TGFβ1-induced fibrosis. Also based on collagen 1 staining, cessation of TGFβ1 treatment and subsequent treatment with vehicle for 2 days appeared to be sufficient to reverse TGFβ1-induced fibrosis. However, treatment of NASH-induced liver spheroids with naive and primed exosomes was found to be both more effective than vehicle in reducing fibrosis compared to vehicle treatment alone.

[0271] FIG. 15E shows representative immunofluorescence images of NASH-induced liver spheroids, including exosome-treated NASH-induced liver spheroids stained for another fibrosis marker, α-SMA, using the same set of conditions for liver spheroids as shown for collagen staining in FIG. 15C. FIG. 15F shows a bar graph of the relative intensity of α-SMA positive cells compared to healthy cells using the same set of conditions for liver spheroids as shown for collagen staining in FIG. 15D. Based on α-SMA staining alone, NASH induction by FFA mixture and TGFβ1, as well as steatosis induction by FFA mixture alone, both appear to induce fibrosis. Also, based on α-SMA staining alone, both naive and primed exosomes appear to be more effective than vehicle in reducing NASH-induced fibrosis. Furthermore, based on α-SMA staining alone, co-treatment with primed exosomes, but not naive exosomes, appeared to prevent the NASH-induced increase in α-SMA.

[0272] As described herein above and shown in Figures 15A-15F, the effect of treatment with disease state inducers such as FFA mixtures or TGFβ1, and / or therapeutic agents such as exosomes, on liver spheroids can be assayed one marker (or a new marker) at a time using techniques such as immunostaining and ELISA. However, a much larger count of markers (tens, hundreds, thousands) can be assayed simultaneously using high-throughput techniques such as microarrays or next-generation sequencing (NGS). Figure 16A shows a heat map of gene expression changes in liver spheroids based on microarray hybridization data. Microarray hybridization data was acquired as follows: after growth of liver spheroid samples and treatment with NASH inducers and / or therapeutic agents as described above, liver spheroids were isolated, lysed, and mRNA was isolated and then converted to sequences of complementary DNA (cDNA). The cDNA samples were then applied to Agilent® microarrays and read on a microarray reader. Microarray analysis was performed using standard methods to obtain normalized expression values ​​for a given transcriptome set of 22,473 genes (included in the microarray). Microarray analysis was performed with liver spheroid samples under the following conditions ("liver spheroid conditions"). (1) treatment with 40 ng / ml HGF on D17-D19 after NASH induction ("HGF"); (2) treatment with naive exosomes at D17-D19 after NASH induction ("naive exosome treatment"); (3) NASH induction at D19 ("disease"); (4) were not subjected to NASH induction at D19 ("healthy"); (5) treatment with primed exosomes at D17-D19 after NASH induction (“primed exosome treatment”); (6) Simultaneous treatment with naive exosomes during fibrosis induction by TGFβ1 at D13-D17 ("naive exosome simultaneous"); and (7) Co-treatment with primed exosomes during fibrosis induction by TGFβ1 at D13-D17 ("primed exosome co-treatment"). Each condition was based on a pool of 30 spheroids, and heat maps were generated based on expression profile data from the pooled samples.

[0273] In the heatmap, each row represents a gene and each column represents a liver spheroid condition. The columns are arranged based on a hierarchical cluster analysis of the gene expression patterns in each of the liver spheroid conditions. Pairs of liver spheroid conditions that cluster close to each other with respect to their respective gene expression patterns are arranged as adjacent columns, and brackets connecting the columns reflect the degree of similarity between the respective gene expression patterns of the different liver spheroid conditions. As shown in Figure 16A, the cluster analysis of the gene expression profiles of the liver spheroid conditions indicated that among liver spheroid conditions 1, 2, and 5-7, in which NASH-induced liver spheroids were subjected to potential therapeutic treatments, the gene expression profile of the primed exosome-treated liver spheroid condition most closely resembled the gene expression profile of the healthy liver spheroid condition. In contrast, the gene expression profile of the naive exosome-treated liver spheroid state most closely resembled that of the diseased liver spheroid state, indicating that primed exosomes were effective in at least partially reversing NASH induction and improving the health of NASH-induced liver spheroids, whereas naive exosomes were ineffective in achieving such results.

[0274] The gene expression levels of liver spheroids in each of the different liver spheroid states were also represented and stored as feature vectors ("spheroid state feature vectors"), respectively, with each element of a given spheroid state feature vector being the expression level of one of the genes assayed in the microarray. The similarity (or lack of similarity) of gene expression profiles between different states was determined based on a measure of distance between pairs of feature vectors in n-dimensional space, where n is the number of genes represented in each of the feature vectors. Also based on this distance analysis, it was found that the spheroid state feature vector representing the primed exosome treatment condition (NASH-induced liver spheroids were treated after induction with DMF-primed exosomes) had the shortest Euclidean distance to the spheroid feature vector representing healthy liver spheroids (not subjected to NASH induction) compared to the other treatment conditions (i.e., naive exosome co-condition, primed exosome co-condition, and naive exosome treatment condition). In other words, treatment with exosomes from DMF-primed hBM-MSCs was shown to be the most effective treatment tested in reverting the gene expression profile of NASH-induced liver spheroids to that of healthy liver spheroids.

[0275] Figure 16B shows a plot based on principal component analysis of differentially expressed genes in healthy control liver spheroids, NASH-induced liver spheroids, and NASH-induced liver spheroids treated with exosomes.The figure shows a two-dimensional projection of the n-dimensional space that includes various spheroid state feature vectors representing the same seven liver spheroid conditions described with reference to Figure 16A: HGF, naive exosome treatment, diseased, healthy, primed exosome treatment, naive exosome simultaneous, and primed exosome simultaneous.The two-dimensional projection is based on principal component analysis.Induction of the NASH phenotype in liver spheroids produced differentially expressed genes that shifted gene profile downward and leftward compared to healthy control gene profile. Based on the microarray-based transcriptome analysis visualized in two-dimensional projections, it is readily apparent that treatment with naive exosomes or 40ng / ml HGF is not effective in treating NASH-induced livers, and as shown in the figure, the gene expression profile of NASH-induced liver spheroids treated with naive exosomes or HGF did not change substantially from untreated NASH-induced liver spheroids. In contrast, treatment of NASH-induced liver spheroids with exosomes derived from hBM-MSCs primed with the Nrf2 activator DMF resulted in treated NASH-induced liver spheroids with a gene expression profile that shifted back upward and to the right, closer to that of healthy control liver spheroids, indicating that primed exosome treatment was substantially more effective than the other evaluated treatments (naive exosomes and HGF) in reversing NASH-induced genetic changes in liver spheroids and altering the gene expression profile of NASH-induced liver spheroids to closely resemble that of healthy liver spheroids.

[0276] In examining the preventive effect of exosome treatment in NASH-induced spheroids, simultaneous exosomes (primed and naive) were used to treat NASH-induced liver spheroids. Treatment with simultaneous exosomes, whether naive or primed, significantly shifted the gene profile of differentially expressed genes to the right compared to diseased controls, indicating some preventive effects related to the suppression of fibrosis progression in NASH-induced liver spheroids with simultaneous exosomes. There were some notable differences in the differentially expressed genes between naive simultaneous exosome treatment in NASH-induced liver spheroids and primed simultaneous exosome treatment in NASH-induced liver spheroids. That being said, simultaneous exosome treatment with either naive or primed exosomes was substantially less effective than a series of non-simultaneous treatments with primed exosomes in changing the gene expression profile of NASH-induced liver spheroids to one that closely resembles that of healthy liver spheroids.

[0277] Figure 17A shows a heatmap of 287 liver-specific genes, which is a subset of the genes assayed in the microarray study described above with reference to Figure 16A.As shown in Figure 17A, cluster analysis of gene expression profiles of the same set of liver spheroid states shown in Figure 16A showed that among the liver spheroid states in which NASH-induced liver spheroids were subjected to potentially therapeutic treatment, the gene expression profile of primed exosome-treated liver spheroid state was most similar to that of healthy liver spheroid state.In contrast, the gene expression profile of HGF-treated liver spheroids was most similar to that of diseased liver spheroid state, followed by naive exosome-treated liver spheroid state.

[0278] Referring to Figure 17B, by repeating the analysis with different gene subsets, DMF-primed exosome treatment was consistently shown to be the most effective in reversing NASH pathology. Figures 17B-17G show heat maps with cluster analysis for the following gene subsets: Figure 17B: 184 NASH / fibrosis-related genes (selected based on Hoang et al., Gene Expression Predicts Histological Severity and Reveals Distinct Molecular Profiles of Nonalcoholic Fatty Liver Disease, Scientific Reports 9 (12541) 2019, Govaere et al. Transcriptomic profiling across the nonalcoholic fatty liver disease spectrum reveals gene signatures for steatohepatitis and fibrosis, Science Translational Medicine 12(572), Dec 2020 and Gu C et al., Identification of Common Genes and Pathways in Eight Fibrosis Diseases, Frontiers in Genetics, January 2021). Figure 17C: 294 hepatic stellate cell-specific genes (selected based on Payen et al., Single-cell RNA sequencing of human liver reveals hepatic stellate cell heterogeneity, JHEP Reports 3(3) June 2021). FIG. 17D: 75 genes related to xenobiotic metabolic processes (selection based on gene ontology reference GO:0006805). FIG. 17E: 50 genes related to fatty acid metabolism (selection based on gene ontology reference GO:0006631). FIG. 17F: 15 genes related to the epoxygenase P450 pathway (selection based on gene ontology reference GO:0019373). Figure 17G: 24 genes associated with lipofibrosis and expression in diseased tissues associated with progression of histologically defined NAFLD severity to lipofibrosis and fibrosis in two independent cohorts of patients (gene selection based on Govaere et al. Transcriptomic profiling across the nonalcoholic fatty liver disease spectrum reveals gene signatures for steatohepatitis and fibrosis, Science Translational Medicine 12(572), Dec 2020).

[0279] Figures 18A-C show graphs of liver spheroid status before and after exosome treatment, including global genes, liver-specific genes, and NASH / fibrosis-related genes on the X, Y, and Z axes, respectively. Each of Figures 18A-18C is a three-dimensional projection showing a respective n-dimensional space including spheroid status feature vectors representing the following liver spheroid statuses: Healthy (Heathy), Diseased (NASH-induced), HGF-treated, naive exosome-treated, and primed exosome-treated. Each of Figures 18A-18C shows the three-dimensional space shown in each of Figures 18A-18C, which is a combination of three two-dimensional projections, with each of the X, Y, and Z axes being based on a principal component analysis (PCA) of a matrix of Jaccard similarity index scores of a subset of gene assays in the microarray study described herein above (a portion of which is shown in Figures 17A-17G). In Figure 18A, the X-axis is based on a global gene set with 18,936 genes, the Y-axis is based on a set of 287 liver-specific genes (shown as a heatmap in Figure 17A), and the Z-axis is based on a set of 184 NASH / fibrosis genes (shown as a heatmap in Figure 17B). In Figure 18B, the X-axis is based on a set of 476 inflammatory response genes based on GO:0006954, the Y-axis is based on a set of 386 angiogenesis genes based on GO:0001525, and the Z-axis is based on a set of 937 neurogenesis genes based on GO:0022008. In Figure 18C, the X-axis is based on a set of 315 wound healing genes based on GO:0042060, the Y-axis is based on a set of 127 tissue remodeling genes (based on GO:0048771), and the Z-axis is based on a set of 405 extracellular matrix genes based on GO:0031012. Each two-dimensional projection, which is one side of the three-dimensional space shown in Figures 18A-18C, uses two of the three axes provided in the respective three-dimensional space.

[0280] In each projection, the healthy liver spheroid state is assigned a coordinate of [1,1], and the coordinates of the remaining states are determined based on the Jaccard similarity index. As shown in each of Figures 18A-18C, DMF-primed exosome treatment of NASH-induced liver spheroids induces partial recovery of the NASH-induced liver spheroids back to a healthy state based on gene expression patterns: the coordinate values ​​of the DMF-primed exosome-treated state are shifted forward on the Z axis, to the right on the X axis, and upward on the Y axis, away from the coordinate values ​​of the diseased spheroids and toward the coordinate values ​​of the healthy state. In contrast, the NASH-induced liver spheroids treated with naive exosomes, as well as the NASH-induced liver spheroids treated with HGF, had coordinate values ​​in all three axes that were substantially more similar to the diseased state than the DMF-primed exosome-treated state or the healthy state.

[0281] Based on the above results, administration of therapeutically effective doses of DMF-primed exosomes to human subjects with NASH is expected to treat NASH in subjects.In addition, DMF-primed exosomes are expected to be effective in treating NAFLD and NAFL.In addition, DMF-primed exosomes are expected to be effective in treating liver fibrosis.

[0282] Figure 19 shows a heat map of the shortlisted selection of 87 genes whose expression levels were most robustly reversed from diseased to healthy expression levels after treatment with DMF-primed exosomes. These genes can be used as markers to determine healthy or NASH induction in liver tissue in vivo as well as in liver spheroids. These 87 genes include genes representative of various signaling pathways related to liver function as well as NAFLD and NASH disease progression, such as secretion (GL:0046903), cell homeostasis (GO:0019725), wound healing (GO:0042050), and lipid biosynthesis (GO:0008610). Thus, it is shown that the gene expression patterns of liver spheroids during NASH induction and its treatment with DMF-primed exosomes as described herein are mechanistically linked to the clinical manifestations of NAFLD and NASH in vivo, as well as the recovery from these conditions.

[0283] Of the 87 genes, the following genes were determined to be particularly robust and useful as markers for liver health in hepatic spheroids and for restoration of said health by treatment with DMF-primed exosomes: FOXA1, FOXA3, MMP10, FGFR2, FGFR3, ANGPT2, ANG, ATP1B, and ICAM2.

[0284] Table 12 shows the normalized values ​​of gene expression in Log2 for these 9 genes.

[0285] [Table 12]

[0286] Advantages of the present disclosure The present disclosure discloses methods of priming MSCs derived from various tissue sources such as bone marrow, adipose, umbilical cord with specific combinations of inducers to activate certain pathways for the production of therapeutic exosomes with enriched factors including anti-inflammatory, anti-fibrotic, wound pre-healing, angiogenic (pro / anti), and reinnervation factors for tissue avascularity and revascularization. Priming of MSCs is performed with various priming agents (small and macromolecules). The advantages of the present disclosure are as follows:

[0287] 1. The present disclosure provides for the selection of unique populations of stem cells, such as UC-MSC / WJ-MSC, based on a signature set expression of markers to produce exosomes with desired therapeutic effects, e.g., anti-inflammatory, anti-fibrotic, wound pre-healing, angiogenesis (pro / anti), and re-innervation.

[0288] 2. The present disclosure also provides for the immortalization / engineering of human MSCs using hTERT (human telomerase reverse transcriptase) to extend the doubling potential of MSCs (eMSCs) to facilitate scalable and uniform production of cells and therapeutic exosomes.

[0289] 3. The present disclosure provides methods involving priming agents such as Nrf2 activators, SIRT1 activators, all-trans retinoic acid (ATRA), conditioned medium from CSSCs, which can be used alone or in combination. The methods deploying priming agents are useful for obtaining concentrated therapeutic grade exosomes with substantially enhanced regenerative therapeutic efficacy.

[0290] 4. The present disclosure also provides inducible and activated exosomes for treating inflammation-related disorders including rheumatoid arthritis, systemic juvenile idiopathic arthritis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), pneumonia, bronchitis, chronic obstructive pulmonary disease (COPD), COVID-19, coronavirus class infections, cystic fibrosis, hantavirus, influenza, tuberculosis, systemic lupus, myocardial infarction, osteoarthritis, nonalcoholic fatty liver disease (NASH), hepatic fibrosis, Mooren's ulcer, neurotrophic ulcer, keratitis of the cornea (CK), dry eye disease ulcer, herpetic simple keratitis, post-LASIK ectasia, post-operative corneal melt, post-keratoprosthesis melt, corneal perforation, neurotrophic keratitis (NK), keratoconus Sjogren's syndrome, mucous membrane pemphigoid, Stevens-Johnson syndrome, chemical burns, and thermal injuries.

[0291] 5. The present disclosure also provides a cost-effective method since the amount of cell-derived products of MSCs required to have a therapeutic effect in animal models is about 50 μg of protein or close to 10 billion particles. To perform physical, molecular and transcriptomic analyses, a scale-up strategy is then the way forward that significantly reduces the costs involved.

[0292] 6. Overall, the present disclosure discloses a process for culturing, expanding, and priming MSCs with different priming agents to obtain primed MSCs and primed conditioned medium. Thus, the scalability of the process described herein, along with the fact that the process is a xeno-free process, gives a viable option of scalability to meet commercial requirements and provides clinical grade end products in terms of primed MSCs, primed conditioned medium. The conditioned medium (CSSC-CM) or primed conditioned medium can be further processed to treat rheumatoid arthritis, systemic juvenile idiopathic arthritis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), pneumonia, bronchitis, chronic obstructive pulmonary disease (COPD), COVID-19, coronavirus class infections, cystic fibrosis, hantavirus, influenza, tuberculosis, systemic lupus, myocardial infarction, osteoarthritis, non-alcoholic fatty liver disease (NASH), liver fibrosis, Obtain clinical grade exosomes, secretomes, and other sero-derived products that can be used to treat conditions selected from the group consisting of Mooren's ulcer, neurotrophic ulcer, corneal keratitis (CK), dry eye disease ulcer, herpetic simple keratitis, post-LASIK ectasia, post-operative corneal melt, post-keratoprosthesis melt, corneal perforation, neurotrophic keratitis (NK), keratoconus, Sjogren's syndrome, mucous membrane pemphigoid, Stevens-Johnson syndrome, chemical burns, and thermal burns. Exosome yields according to the present disclosure are scalable without impacting production costs.

Claims

1. 1. A method for generating a population of exosomes derived from primed mesenchymal stem cells, comprising: (a) culturing a population of mesenchymal stem cells (MSCs) in a medium; (b) contacting the population of MSCs with an Nrf2 activator to obtain a population of primed MSCs; (c) expanding the population of primed MSCs in collection medium such that the collection medium becomes enriched with exosomes produced by the primed MSCs, thereby producing primed MSC-derived conditioned medium; (d) collecting the primed MSC-conditioned medium; A method comprising:

2. (e) Purifying exosomes from primed MSC-conditioned medium The method of claim 1 further comprising:

3. 10. The method of claim 1, wherein the population of MSCs is grown in a first culture medium from seeding to about 60% to about 90% confluency and then contacted with an Nrf2 activator.

4. 4. The method of claim 3, wherein the population of MSCs is contacted with the Nrf2 activator for about 12 to 72 hours.

5. 2. The method of claim 1, wherein the Nrf2 activator is dimethyl fumarate (DMF) or 4-octyl itaconate (4-OI).

6. 6. The method of claim 5, wherein DMF is present at a concentration of about 50 μM to about 100 μM.

7. 2. The method of claim 1, wherein the population of MSCs is a population of bone marrow MSCs (BM-MSCs), a population of umbilical cord-derived MSCs (UM-MSCs), a population of induced pluripotent stem cell (iPSC)-derived MSCs (iPSC-MSCs), or a population of Wharton's jelly-derived MSCs (WJ-MSCs).

8. The method of claim 7, wherein the population of MSCs is a population of BM-MSCs.

9. A population of primed MSC-derived exosomes produced by the method of any one of claims 1 to 8.

10. Compared to exosomes derived from unprimed MSCs, (a) high expression levels of hepatic growth factor (HGF); and (b) High expression levels of nerve growth factor (NGF) A population of exosomes derived from primed MSCs, characterized by having one or more of:

11. Exosomes derived from primed MSCs compared to exosomes derived from non-primed MSCs (a) a high expression level of HGF; and (b) High expression levels of NGF 11. The population of primed MSC-derived exosomes of claim 10, characterized by:

12. Exosomes derived from primed MSCs compared to exosomes derived from non-primed MSCs (a) an expression level of HGF that is at least 1.2-fold higher than HGF; and (b) an expression level of NGF that is at least two-fold higher than that of NGF; 11. The population of primed MSC-derived exosomes of claim 10, characterized by one or both of:

13. Primed MSCs (1) expanding a population of MSCs in a first culture medium; and (2) contacting a population of MSCs with an Nrf2 activator; 11. The population of primed MSC-derived exosomes of claim 10, prepared by:

14. The population of primed MSC-derived exosomes of claim 13, wherein the Nrf2 activator is DMF or 4-octyl itaconate (4-OI).

15. 15. The population of primed MSC-derived exosomes of claim 14, wherein DMF is present at a concentration of about 50 μM to about 100 μM.

16. 14. The population of primed MSC-derived exosomes of claim 13, wherein the MSCs are grown in a first culture medium from seeding to about 60% to about 90% confluency and then contacted with an Nrf2 activator.

17. 17. The population of primed MSC-derived exosomes of claim 16, wherein the population of MSCs is contacted with an Nrf2 activator for about 12 hours to about 72 hours, and then replaced with a collection medium.

18. 11. A pharmaceutical composition comprising a therapeutic amount of the population of exosomes of claim 10 for treating a liver condition.

19. 19. The pharmaceutical composition of claim 18, wherein the liver condition is non-alcoholic fatty liver disease (NAFLD).

20. 20. The pharmaceutical composition of claim 19, wherein the NAFLD is nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH). 。

21. 21. The pharmaceutical composition of any one of claims 18 to 20, wherein the population of exosomes is administered to the liver via an intravenous route.

22. 22. The pharmaceutical composition of claim 21, wherein the intravenous route is via the hepatic portal vein.

23. 10. A composition comprising the population of exosomes of claim 9.

24. 24. The composition of claim 23 for use in treating a liver condition.

25. 25. The composition of claim 24, wherein the liver condition is non-alcoholic fatty liver disease (NAFLD).

26. 26. The composition of claim 25, wherein the NAFLD is nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH).

27. 27. The composition of claim 26, wherein the NAFLD is NASH.

28. 1. A method for increasing exosome secretion by a population of mesenchymal stem cells (MSCs), comprising: (a) culturing a population of MSCs in a medium; (b) contacting the population of MSCs with an Nrf2 activator to obtain a population of primed MSCs; (c) expanding the population of primed MSCs in collection medium, such that the collection medium becomes enriched with exosomes produced by the primed MSCs; A method comprising:

29. 29. The method of claim 28, wherein the population of MSCs is grown in a first culture medium from seeding to about 60% to about 90% confluency and then contacted with an Nrf2 activator.

30. 29. The method of claim 28, wherein the population of MSCs is contacted with the Nrf2 activator for about 12 to 72 hours.

31. The method of claim 28, wherein the Nrf2 activator is dimethyl fumarate (DMF) or 4-octyl itaconate (4-OI).

32. 32. The method of claim 31 , wherein DMF is present at a concentration of about 50 μM to about 100 μM.

33. 33. The method of any one of claims 28 to 32, wherein the population of MSCs is a population of bone marrow MSCs (BM-MSCs), a population of umbilical cord-derived MSCs (UM-MSCs), a population of induced pluripotent stem cell (iPSC)-derived MSCs (iPSC-MSCs), or a population of Wharton's jelly-derived MSCs (WJ-MSCs).

34. 1. A method for generating a population of exosomes derived from primed mesenchymal stem cells, comprising: (a) expanding a population of mesenchymal stem cells (MSCs) in culture; (b) contacting the population of MSCs with conditioned medium from cells derived from a cell population different from the population of MSCs and at least one defined priming agent to obtain a population of primed MSCs; (c) expanding the population of primed MSCs in culture to produce primed MSC-derived conditioned medium; (d) collecting the primed MSC-conditioned medium; A method comprising:

35. moreover, (e) Purifying exosomes from primed MSC-conditioned medium 35. The method of claim 34, comprising:

36. 35. The method of claim 34, wherein the population of MSCs is contacted with conditioned medium from the cells from seeding to about 60% to about 90% confluency, and the population of MSCs is contacted with at least one defined priming agent starting from about 60% to about 90% confluency.

37. 37. The method of claim 36, wherein the population of MSCs is contacted with conditioned medium from the cells from seeding to about 60% to about 90% confluency, and then contacted with at least one defined priming agent.

38. 37. The method of claim 36, wherein the population of MSCs is contacted with the at least one defined priming agent for about 12 hours to about 72 hours.

39. The method of claim 34, wherein the conditioned medium derived from cells from a different cell population is conditioned medium derived from corneal stromal stem cells.

40. 35. The method of claim 34, wherein the at least one defined priming agent is a nuclear factor erythroid 2-related factor 2 (Nrf2) activator, a silencing information regulator 1 (SIRT1) activator, or all-trans retinoic acid (ATRA).

41. 41. The method of claim 40, wherein at least one priming agent is an Nrf2 activator.

42. 42. The method of claim 41, wherein the Nrf2 activator is dimethyl fumarate (DMF) or 4-octyl itaconate (4-OI).

43. 35. The method of claim 34, wherein the population of MSCs is a population of bone marrow-derived MSCs (BM-MSCs), a population of umbilical cord-derived MSCs (UM-MSCs), a population of induced pluripotent stem cell (iPSC)-derived MSCs (iPSC-MSCs), or a population of Wharton's jelly-derived MSCs (WJ-MSCs).

44. 44. The method of claim 43, wherein the population of BM-MSCs is a population of human BM-MSCs.

45. The method of claim 34, wherein the cell-derived conditioned medium is a corneal stem cell-derived conditioned medium, and the defined priming agent is DMF or 4-octyl itaconate (4-OI).

46. 46. The method of claim 45, wherein the corneal stem cell-derived conditioned medium is present at a concentration of about 10% to about 30%.

47. 47. The method of claim 46, wherein DMF is present at a concentration of about 50 μM to about 100 μM.

48. 48. A population of primed MSC-derived exosomes produced by the method of any one of claims 34 to 47.

49. Compared to exosomes derived from unprimed MSCs, (a) low expression levels of vascular endothelial growth factor (VEGF); and (b) High expression levels of nerve growth factor (NGF) A population of exosomes derived from primed MSCs, characterized by having one or more of:

50. Exosomes derived from primed MSCs compared to exosomes derived from unprimed mesenchymal stem cells (c) high expression levels of hepatic growth factor (HGF); and (d) High expression levels of sFLT1 50. The population of primed MSC-derived exosomes of claim 49, characterized by one or more of the following:

51. Exosomes derived from primed MSCs compared to exosomes derived from unprimed mesenchymal stem cells (a) at least two-fold higher expression levels of sFLT1; (b) VEGF expression levels that are four-fold or less than those in exosomes derived from unprimed MSCs; (c) at least two-fold higher expression levels of HGF; and (d) at least three-fold higher expression of NGF 50. The population of primed MSC-derived exosomes of claim 49, characterized by one or more of the following:

52. Primed MSCs (1) contacting a population of MSCs with corneal stem cell-derived conditioned medium; and (2) contacting a population of MSCs with an Nrf2 activator; 50. The population of primed MSC-derived exosomes of claim 49, prepared by:

53. 53. The population of primed MSC-derived exosomes of claim 52, wherein the Nrf2 activator is DMF or 4-octyl itaconate (4-OI).

54. 53. The population of primed MSC-derived exosomes of claim 52, wherein the population of MSCs is contacted with corneal stem cell-derived conditioned medium from seeding until about 60% to about 90% confluency, and the population of MSCs is contacted with an Nrf2 activator starting from about 60% to about 90% confluency.

55. 55. The population of primed MSC-derived exosomes of claim 54, wherein the population of MSCs is contacted with the Nrf2 activator for about 12 hours to about 72 hours.

56. 50. A pharmaceutical composition comprising a therapeutic dose of the population of exosomes of claim 10 or 49 for treating a corneal defect.

57. 57. The pharmaceutical composition of claim 56, wherein the corneal defect is selected from the group consisting of corneal scarring, keratitis, corneal ulcer, corneal abrasion, corneal epithelial damage, corneal stromal damage, infection-based corneal damage, trachoma, keratoconus, corneal perforation, limbal injury, corneal dystrophy, angiogenesis, vernal keratoconjunctivitis, and dry eye.

58. 58. The pharmaceutical composition of claim 57, wherein the corneal defect is keratitis.

59. The pharmaceutical composition of claim 56, which is an ophthalmic composition formulated for application to the corneal surface.

60. The pharmaceutical composition described in claim 59, which is an eye drop.

61. 61. The pharmaceutical composition of claim 60, wherein the ophthalmic solution comprises a biocompatible polymer.

62. 62. The pharmaceutical composition of claim 61, wherein the biocompatible polymer is crosslinkable, and when the ophthalmic solution is administered to the corneal surface, a sufficient portion of the crosslinkable polymer is crosslinked to convert the ophthalmic solution into a hydrogel.

63. A pharmaceutical composition comprising a therapeutic dose of a population of exosomes according to claim 48 for treating a corneal defect.

64. The pharmaceutical composition described in claim 63, wherein the corneal defect is selected from the group consisting of corneal scarring, keratitis, corneal ulcer, corneal abrasion, corneal epithelial damage, corneal stromal damage, corneal damage due to infection, trachoma, keratoconus, corneal perforation, corneal limbal injury, corneal dystrophy, angiogenesis, vernal keratoconjunctivitis, and dry eye.

65. The pharmaceutical composition described in claim 64, wherein the corneal defect is keratitis.

66. The pharmaceutical composition described in claim 63, which is an ophthalmic composition formulated for application to the corneal surface.

67. The pharmaceutical composition described in claim 66, wherein the composition is an eye drop.

68. The pharmaceutical composition described in claim 67, wherein the eye drops contain a biocompatible polymer.

69. 67. The pharmaceutical composition of claim 66, wherein the ophthalmic composition is a hydrogel and at least a portion of the biocompatible polymer is crosslinked.

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