KR20220139816A - Use for treating intractable immune diseases using single process culture that simultaneously enhances stem cell antioxidant activity, engraftment rate and stemness - Google Patents

Abstract

The present invention relates to therapeutic use of intractable immune diseases using a single process culture method, and more specifically, to a single process culture technology which simultaneously enhances stem cell antioxidant activity, engraftment rates and stemness through primed fresh OCT-4 enrichment (PFO) process. As a result of evaluating a stemness cultured by the PFO process of the present invention through the glutathione (GSH)-recovering capacity (GRC) analysis, it is confirmed that the stemness of PFO MSCs is enhanced, treating effects of graft-versus-host disease (GVHD), asthma, and bladder disease are improved.

Description

Use for treating intractable immune diseases using single process culture that simultaneously enhances stem cell antioxidant activity, engraftment rate and stemness

The present invention relates to a use for treating intractable immune diseases using a single process culture method that simultaneously enhances stem cell antioxidant capacity, engraftment rate, and stem cell properties.

Mesenchymal stem cells (MSCs) can repair tissue damage through their ability to engraft and directly regenerate tissue-specific cells after in vivo administration. In addition, these progenitor cells supply growth factors or matrix proteins and mediate cell-cell interactions, thereby providing an advantageous microenvironment that includes immunomodulatory, anti-inflammatory or angiogenesis-promoting activities. These pluripotent cells are isolated from several adult or fetal tissues, such as bone-marrow (BM), umbilical cord blood, and amniotic fluid. Recently, direct induction of MSCs from pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) has been shown as an effective alternative. This is to obtain a sufficient number of cells while overcoming limitations compared to adult tissues such as unpredictable loss of function and heterogeneity due to ex vivo expansion.

Because of the practical benefits of multiple causes and the effects of these multiple aspects, MSC-based therapies have been proposed to treat a variety of intractable cardiovascular, musculoskeletal, neurological, immunological and urological disorders, but their performance is controversial. impeding its application. More importantly, the lack of information about the basic characteristics that drive the in vivo behavior of these cells after transplantation has made it difficult to advance promising preclinical studies of MSC therapy into clinical trials. Therefore, it is emphasized that the dynamic monitoring of the biological and molecular properties of engrafted MSCs in a pathological environment not only facilitates processes such as delivery but also enables early prediction of therapeutic effects. However, in-depth information about the MSC engraftment process, particularly the interactions between microenvironment and molecular profiles at the genome-wide level, is still lacking.

Korean Patent No. 10-2097191 (registered on March 30, 2020)

An object of the present invention is ascorbic acid 2-glucoside (AA2G), sphingosine-1-phosphate (S1P) and valproic acid; VPA) to provide a method for promoting mesenchymal stem cell activity comprising the step of processing.

In addition, another object of the present invention is to provide a composition for promoting mesenchymal stem cell activity comprising AA2G, S1P and VPA as active ingredients.

In addition, another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of intractable immune diseases comprising mesenchymal stem cells whose activity is promoted by treatment with AA2G, S1P and VPA or a culture medium thereof as an active ingredient.

In order to achieve the above object, the present invention comprises the steps of: 1) treating the isolated mesenchymal stem cells with ascorbic acid 2-glucoside (AA2G) and subculture; And 2) provides a method for promoting mesenchymal stem cell activity comprising the step of adding sphingosine-1-phosphate (S1P) and valproic acid (VPA) to the subculture.

In addition, the present invention provides a composition for promoting mesenchymal stem cell activity comprising 0.1 to 0.75 mM of AA2G, 10 to 50 nM of S1P, and 0.1 to 1.0 mM of VPA as active ingredients.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of intractable immune diseases comprising mesenchymal stem cells whose activity is promoted by treatment with AA2G, S1P and VPA or a culture medium thereof as an active ingredient.

The present invention relates to a use for the treatment of intractable immune diseases using a single process culture method that simultaneously enhances stem cell antioxidant capacity, engraftment rate, and stem cell properties. Specifically, stem cell antioxidant through the PFO process (Primed Fresh OCT-4 enrichment). It is about a single process culture technology that simultaneously enhances potency, engraftment rate, and stem cellularity. As a result of evaluating the stem cell capacity cultured by the PFO process of the present invention through glutathione (GSH) recovery capacity (GRC) analysis, the stem cell capacity of PFO MSCs was improved, and graft-versus-host disease ( Graft-versus-host disease (GVHD), asthma and bladder diseases were also confirmed to be improved.

1 shows ascorbic acid 2-glucoside (AA2G), low concentrations of sphingosine-1-phosphate (S1P, 50 nM) and valproic acid (VPA, 0.5). mM) to provide an optimal environment for preserving primitive MSCs characterized by small size and high glutathione (GSH) kinetics, a schematic diagram of the Primed Fresh OCT-4 enrichment (PFO) process is shown.
Figure 2 shows the results that the PFO process activates the NRF2 pathway.
Figure 3 shows the results that the PFO process maintains the primitive small size UC-MSCs.
4 is a result showing the improved immunomodulatory function of small-sized UC-MSCs with high GSH kinetics.
Figure 5 shows the effect of the PFO process on the properties of MSCs.
Figure 6 shows the effect of the PFO process in human AD-MSCs.
7 shows the results that the PFO process protects the replication-induced senescence of human UC-MSCs.
8 shows the expression results of OCT4 -A and SOX2 in PFO UC-MSCs.
Figure 9 shows the effect of the PFO process on the core function of human UC-MSCs.
Figure 10 shows the beneficial effects of the PFO process in human AD-MSCs.
11 shows the gene expression profile results of PFO UC-MSCs.
12 shows the results that high GRC MSCs enhance the therapeutic effect of GVHD.
13 shows the results for improving the in vivo treatment effect and engraftment effect of PFO UC-MSCs in the treatment of GVHD.
Figure 14 shows the cytokine profile results in the serum of GVHD mice.
15 shows the results of analysis of the transcriptome of PFO MSCs.
16 to 20 show the results of PFO MSCs transcriptome gene ontology and gene network analysis.
21 to 23 show methylome analysis results of PFO MSCs.
24 to 26 show the results of analysis of the protein body (Proteome) and the protein secretion body (Secretome) of PFO MSCs.
27 to 29 show the results of single-cell transcriptome analysis of PFO MSCs.
30 to 32 show the evaluation results of PFO-MSCs treatment efficacy in the Th2 immune response-induced asthma disease model.
33 to 39 show the results of the in vivo treatment effect of diabetic underactive bladder (streptozotocin induced detrusor underactivity, STZ-DUA) by PFO-MSC.
40 shows the evaluation results of PFO-MSCs treatment efficacy in a chronic bladder ischemia (CBI) injury-induced hypoactive bladder (CBI induced DUA) disease model.
41 shows the evaluation results of PFO-MSCs treatment efficacy in chronic bladder ischemia-induced overactive bladder (CBI induced OAB) disease model.
42 to 44 show in vitro efficacy evaluation results for PFO-MSC characteristic titer protein.
45 shows the in vivo efficacy evaluation results for the PFO-MSC characteristic titer protein.

The present invention comprises the steps of 1) subculturing the separated mesenchymal stem cells by treating ascorbic acid 2-glucoside (AA2G); And 2) provides a method for promoting mesenchymal stem cell activity comprising the step of adding sphingosine-1-phosphate (S1P) and valproic acid (VPA) to the subculture.

Preferably, the mesenchymal stem cells may be umbilical cord derived MSCs (UC-MSCs) or adipose derived MSCs (AD-MSCs), but are limited thereto. not.

Preferably, step 1) may be subcultured for 4 to 11 days by treating 0.1 to 0.75 mM of AA2G, and in step 2), 10 to 50 nM of S1P and 0.1 to 1.0 mM of VPA are added. can, but is not limited thereto.

Preferably, the mesenchymal stem cells whose activity is promoted by the method have improved intracellular glutathione levels and glutathione-recovering capacity (GRC), stem cell characteristics, colony forming units - fibroblasts (colony forming) unit-fibroblast; CFU-F) activity, chemotaxis to platelet-derived growth factor (PDGF), self-renewal, cell migration, engraftment ability, anti-inflammatory and any selected from the group consisting of T-cell inhibitory ability One or more stem cell activity may be promoted, but is not limited thereto.

In addition, the present invention provides a composition for promoting mesenchymal stem cell activity comprising 0.1 to 0.75 mM of AA2G, 10 to 50 nM of S1P, and 0.1 to 1.0 mM of VPA as active ingredients.

Preferably, the mesenchymal stem cells may be umbilical cord derived MSCs (UC-MSCs) or adipose derived MSCs (AD-MSCs), but are limited thereto. not.

Preferably, the mesenchymal stem cells have improved intracellular glutathione levels and glutathione-recovering capacity (GRC), and stem cell, colony forming unit-fibroblasts (colony forming unit-fibroblast; CFU-F). ) activity, platelet-derived growth factor (PDGF) for chemotaxis, self-renewal, cell migration, engraftment, anti-inflammatory and T-cell inhibitory ability to promote any one or more stem cell activity selected from the group consisting of may be, but is not limited thereto.

As used herein, the term “priming” refers to a phenomenon in which reactivity (activity) is improved in order to enhance the therapeutic efficacy of stem cells.

In addition, the composition for promoting the activity can not only enhance the in vivo effect of the cell therapy agent by mixing it with the cell therapy agent for treatment and injecting it in vivo, but also a cell therapy agent whose function is increased after treating the stem cells themselves with the composition. It can also be used as a method for in vivo transplantation.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of intractable immune diseases comprising mesenchymal stem cells whose activity is promoted by treatment with AA2G, S1P and VPA or a culture medium thereof as an active ingredient.

Preferably, the AA2G may be treated at a concentration of 0.1 to 0.75 mM, the S1P may be treated at a concentration of 10 to 50 nM, and the VPA may be treated at a concentration of 0.1 to 1.0 mM, but is not limited thereto.

Preferably, the mesenchymal stem cells may be umbilical cord derived MSCs (UC-MSCs) or adipose derived MSCs (AD-MSCs), but are limited thereto. not.

Preferably, the mesenchymal stem cells have improved intracellular glutathione levels and glutathione-recovering capacity (GRC), and stem cell, colony forming unit-fibroblasts (colony forming unit-fibroblast; CFU-F). ) activity, platelet-derived growth factor (PDGF) for chemotaxis, self-renewal, cell migration, engraftment, anti-inflammatory and T-cell inhibitory ability to promote any one or more stem cell activity selected from the group consisting of may be, but is not limited thereto.

Preferably, the intractable immune disease may be graft-versus-host disease (GVHD), asthma, underactive bladder or overactive bladder, but is not limited thereto.

Preferably, the mesenchymal stem cells may specifically express any one or more selected from the group consisting of SV2A, CD2AP and ALDH6A1, but is not limited thereto.

In the present invention, the "culture medium" includes a medium capable of supporting the growth and survival of stem cells in vitro, the cultured stem cell secretions contained in the medium, and the like. The medium used for culture includes all of the conventional medium used in the art suitable for culturing stem cells. Depending on the type of cell, the medium and culture conditions can be selected. The medium used for the culture is preferably a cell culture minimum medium (CCMM), and generally contains a carbon source, a nitrogen source, and a trace element component. Such cell culture minimal medium includes, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (α Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), Iscove's Modified Dulbecco's Medium, etc., but is not limited thereto.

In addition, the medium may contain antibiotics such as penicillin, streptomycin, and gentamicin.

On the other hand, the present invention provides a form including all of the stem cells, their secretions, and medium components, a form containing only secretions and medium components, a form that separates only secretions and uses them alone or together with stem cells, or administers only stem cells. It is also possible to use it in a form that produces secretions in the body.

The stem cells can be obtained using any method known in the art.

The mesenchymal stem cells whose activity is promoted by treatment with AA2G, S1P and VPA of the present invention can be used as a cell therapy agent for the treatment of specific diseases, and the treatment can be direct treatment or pre-treatment of the molecules.

The "cell therapy agent" refers to a method that proliferates, selects, or otherwise changes the biological properties of living autologous, allogenic, and xenogenic cells in vitro in order to restore the functions of cells and tissues. It refers to medicines used for treatment, diagnosis, and prevention purposes through a series of actions such as

The cell therapeutic agent may be administered to the human body through any general route as long as it can reach the target tissue.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, and a lubricant. Alternatively, a solubilizing agent such as a flavoring agent may be used. The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

The pharmaceutical formulation of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained-release formulations of the active compound. can The pharmaceutical composition of the present invention can be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. can be administered as The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for prevention or treatment of a disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, weight, general health status, sex and diet of the patient, administration time, administration route and composition It can be adjusted according to various factors including secretion rate, duration of treatment, and drugs used at the same time.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

< Experimental example >

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Study Approval

All animal experiments were approved by the Animal Experimental Ethics Committee of Ulsan University College of Medicine (IACUC-2020-12-141). Human umbilical cord (UC) and Fat samples were obtained. Prior to UC collection, informed consent was obtained from all pregnant women.

2. Cell Culture and MSCs In vitro characterization

10% heat inactivated fetal bovine serum (FBS), 5 ng/ml human epidermal growth factor (Sigma-Aldrich), 10 ng/ml basic FGF and 50 ng/ml long-R3 insulin-like growth factor- In low-glucose DMEM containing 1 (ProSpec, Rehovot, Israel), human UC MSCs (UC-MSCs) were cultured. Human adipose tissue derived MSCs (AD-MSCs) were established as previously reported and maintained by the same procedure as UC-MSCs. All MSCs used in the present invention were extended to 7 passages or less to ensure functionality, and were maintained at 37° C. in a humidified atmosphere of 5% CO ₂ . Trans-well migration in response to colony forming unit (CFU)-fibroblasts, platelet-derived growth factors for cell proliferation, self-renewal (10 ng/mL PDGF-AA, R&D Systems, Minneapolis, MN, USA), Mart Angiogenesis by Liesel tube-forming assay, in vitro anti-inflammatory, immunomodulatory, and inhibitory properties in response to allogeneic stimuli via allogeneic mixed lymphocyte reaction (MLR) assay. In vitro ) MSCs cell activity assay was performed as previously reported. The core functions of MSCs were quantified through digital image analysis using Image Pro 5.0 software (Media-Cybernetics, Rockville, MD, USA). FACS data analysis was performed using FlowJo software 7.6.5 (FlowJo, LLC, Ashland, OR, USA).

3. Humanized GVHD animal model

For GVHD animal model production, 9-week-old male NOD/ShiLtJ -Prkdc ^em1AMC Il2rg ^em1AMC (NSG) mice (26-29 g) were purchased from JOONGAH BIO (JA BIO, Suwon-si, Gyeonggi-do, Republic of Korea). , was irradiated at 2.0 Gy using an X-RAD 320 X-ray irradiator (Precision X-ray, North Branford, CT, USA). To induce GVHD, within 24 hours after irradiation, 1.0 × 10 ⁶ human PBMCs (#70025; STEMCELL Technologies, Vancouver, Canada) or an equal volume of phosphate-buffered saline (PBS; Sham group) were administered. Injected via tail vein. As a treatment option, human UC-MSCs expanded under normal culture (naive) or PFO process were injected via tail vein at a density of 1 × 10 ⁵ cells in 100 μL PBS 18 days after injection of human PBMCs. For the Sham and GVHD groups, PBS alone was injected as a control. Mice were randomly assigned to treatment groups, and the sequence of irradiation, cell transplantation, or vehicle infusion and daily testing was randomized. The treatment group was hidden from investigators involved in the GVHD induction procedure.

4. GVHD animal evaluation

Clinical symptoms of GVHD were assessed daily by measuring weight loss, survival rate, waist curvature and fur texture, and for 60 days after GVHD induction, 5 mice per group were recorded every other day. Six weeks after injection of human PBMCs, histological evaluation of GVHD target organs (lung, liver, kidney and small intestine), immunological analysis of donor T cell populations and multiple human astrocytes using an independent experimental set of 5 mice per group. Cain analysis was performed. All GVHD symptoms, histological analyzes, cytokine and splenic lymphocyte evaluations were performed by blinded investigators.

For donor T cell proliferation, cells isolated from GVHD mouse spleen were resuspended in 500 μl PBS and reacted with FITC- or PE-conjugated primary antibodies at room temperature for 30 minutes. Primary antibodies against the human antigens CD3 (#300412), CD4 (#300512) and CD45 (#555483) and the mouse antigen CD45 (#553079) were purchased from BD Biosciences. Cell fluorescence intensity was analyzed using a BD FACS Canto II flow cytometer (BD Biosciences). Data were analyzed using FlowJo software 7.6.5 (FlowJo).

For multiple cytokine analysis, humanized GVHD mouse serum was analyzed using the Magnetic Luminex Screening Assay Human Premixed Multi-Analyte Kit (#LXSAHM-28, R&D Systems). Measurement and quantification of 28-plex human cytokines is KOMA BIOTECH INC. (Seoul, Korea), and was performed according to the manufacturer's instructions using a Varioskan Flash Reader (Invitrogen/Thermo Fisher Scientific).

5. Alive in MSCs Real-time observation of GRC

In order to track the GSH changes of all living single cells in real time under various culture conditions, we used the Operetta High-Content Imaging System (HH12000000; PerkinElmer, Waltham, MA, USA) with ×200 or ×400 magnification, GRC Analysis was performed. The GRC analysis was performed as a non-destructive, integrated and image-based high-throughput assay for qualitative and quantitative analysis of GSH kinetics in live MSCs. GRC analysis was performed based on the unique properties of FreSHtracer (Fluorescent real-time thiol tracer ; Cell2in , Inc., Seoul, Korea), a reversible chemical probe for GSH. In the reaction with GSH, FreSHtracer exhibited a λ _max spectral shift of UV-Vis absorbance from 520 nm to 430 nm, which was shown to decrease the fluorescence emission intensity at 580 nm (F ₅₈₀ , λ _ex 520 nm). , the fluorescence intensity at 510 nm (F ₅₁₀ , λ _ex 430 nm) was shown to increase. To obtain the fluorescence ratio (FR) values of FreSHtrace, fluorescence emission excited at 430 and 520 nm was detected at 510 and 580 nm, respectively. The fluorescence signal of FreSHtrace was analyzed using Harmony High-Content Imaging and Analysis Software 3.1 (PerkinElmer) with confocal mode.

6. Gene Expression and western blotting analysis

Quantitative measurements of the transcripts of the indicated genes were performed using 50 ng of total RNA as previously reported. Relative expression levels of these genes were measured using the 2 ^{- ΔΔCt} method, and GAPDH was used as an endogenous control gene.

For Western blot analysis, a cell extract (30 μg) was prepared with RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and separated on an SDS-PAGE gel. Expression levels of the indicated proteins were measured using specific antibodies as probes. Signal densities for the indicated proteins were measured and quantified using NIH Image J software.

7. chromatin -immunoprecipitation (Chromatin- immunoprecipitation ; ChIP ) analysis

ChIP analysis was performed using the Magna ChIP G Kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. The detailed procedure used 3 μg of ChIP-grade antibody against acetylated histone-3 (H3Ac), tri-methylation at lysine-4 of histone-3 (H3K4me3) and tri-methylation at lysine-27 of histone-3 (H3K27me3). Thus, it was performed as previously reported.

8. Immunostaining

To detect OCT4-A protein, human MSCs were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min, stained with an antibody specific for OCT4-A (#Ab62352; Abcam), and then Alexa 488- Labeled with conjugated anti-rabbit antibody (#A11008, Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were counterstained with DAPI (Sigma-Aldrich). Images were acquired using a ZEISS LSM800 confocal microscope system (Carl Zeiss, Munich, Germany).

9. Statistical Analysis

Data were statistically analyzed using the non-parametric Mann-Whitney test or ANOVA (one- or two-way) with the Bonferroni post-hoc test. All analyzes were performed using GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA). p < 0.05 was judged to be statistically significant.

10. transcript analysis

For transcript analysis, RNA was isolated from Naive and PFO MSCs as described above. For analysis using the Affymetrix GeneChip Human (for hESC-derived M-MSCs) 20 ST Array (Affymetrix, Santa Clara, CA, USA), 1 microgram of total RNA was applied. Microarray image data were processed with GeneChip GCS3000 Scanner and Command Console software (Affymetrix). After importing CEL files of 6 samples (3 independent samples from each group), data were summarized and normalized using a robust multi-average (RMA) method implemented in Affymetrix Expression Console Software.

For functional analysis of transcriptome databases for gene networks, biological functions, and canonical pathways, either MetaCore microarray software (Clarivate Analytics, Philadelphia, PA, USA) or geneset enrichment analysis (GSEA; Broad Institute, Cambridge, MA, USA) with default settings ) was used. In the MetaCore analysis, genes with p < 0.05 and more than 15-fold up- or down-regulated genes were defined as having significant changes. For GSEA, gene sets were obtained from published literature or filtered from a selected functional gene set (C2) database. Significant differences were determined based on false discovery rate (FDR) <0.25.

11. Single Cell transcript analysis

After RNA extraction from single-cell isolated PFO MSCs for single-cell transcriptome analysis, single-cell cDNA library synthesis was amplified with a T7-primed single-cell cDNA library as previously reported. This experiment was commissioned by eBiogen, and the experimental method was used in the initial screening of the cDNA library of PFO MSC, using the T7-primed PCR amplification product diluted 20 times, by examining the expression level of human GAPDH, and it is possible to analyze the transcriptome. Only high-level single-cell cDNA libraries were selected for genome-wide analysis, and gel-eluted T7-primed libraries were biotin-labeled using the GeneChip 3' in vitro transcription kit (Affymetrix, Santa Clara, CA, USA).

Biotin-labeled aRNA derived from a single cell library was fragmented and hybridized with Affymetrix GeneChip™ Human Genome U133 Plus 2.0 Array. Microarray image data were processed with GeneChip GCS3000 Scanner and Command Console software (Affymetrix). Raw data was automatically extracted from the Affymetrix data extraction protocol using software provided by the Affymetrix GeneChip™ Command Console™ Software (AGCC).

Functional analysis of transcriptome and core analysis of gene networks, biofunctionality and regulatory pathways were performed using MetaCore (Clarivate Analytics, Philadelphia, PA, USA) or GSEA (Broad Institute, Cambridge, MA, USA) microarray software default settings. did. In MetaCore analysis, 1.5-fold up- or down-regulation of p<0.05 was defined as the cutoff value for a significant change. In the GSEA, gene sets were obtained from published literature or filtered from a selected functional gene set (C2) database.

12. T helper ( Th ) type 2 response-induced asthma animal model and PFO - MSc's administration

All animal experiments were approved by the Animal Experiment Ethics Committee (IACUC-2015-12-061) of Ulsan University College of Medicine, and to produce an asthma mouse model, 6-week-old BALB/c mice (Orient Bio, Gapyeong, Gyeonggi-do) , Korea) was used. On days 0 and 7, it was sensitized by intraperitoneal administration with 2 mg Alum (Thermo, #77161, USA), and on days 14, 15, 16, 21, 22, and 23 with 50 μg OVA (Sigma, #A5503, USA). After anesthesia, the challenge was caused by intranasal administration. The mice were injected with 1.0×10 ⁵ Navie MSCs and PFO MSCs via intravenous injection on the 17th day. BALF, lymph nodes, and lung tissues were obtained from mice 24 hours after the last immunization. The number of monocytes, basophils, neutrophils, and lymphocytes and changes in cytokine expression in BALF and lung tissues were measured as previously described. For histopathological evaluation, the lungs were perfused with 5 ml PBS through the right ventricle and inflated with 1 ml PBS through the bronchus. The inflated lungs were fixed by immersion in 10% neutral buffered formalin solution for 24 hours. The fixed lung tissue was embedded in paraffin and sectioned to a thickness of 4 μm, and the size of inflammation in the bronchial and perivascular regions was tested by hematocillin and eosin staining. Engraftment of the injected MSCs was determined by immunofluorescence analysis of human β2-microglobulin (ab15976; Abcam, USA) and visualized using a FITC-labeled secondary antibody. Nuclei were relatively stained using 4'-6-diamino-2-phenylindole (DAPI; Sigam, USA).

13. Diabetes hypoactivity bladder( streptozotocin induced detrusor underactivity, STZ-induced DUA ) animal models and PFO - MSc's administration

All animal experiments were carried out with the approval of the Institutional Animal Care and Use Committee of Ulsan University College of Medicine (IACUC-2019-12-129). STZ-induced DUA (streptozotocin induced detrusor underactivity) STZ injection into the rat model was performed as previously described. To create the STZ-DUA animal model, diabetes was induced by intraperitoneal injection of Streptozotocin (STZ, Sigma) in 8-week-old female Sprague-Dawley rats (OrientBio, Gapyong, Gyeonggi-do, Korea). After titrating 0.1M citrate buffer to pH 4.5, it was intraperitoneally injected once at a dose of 50mg/kg, and the normal group was intraperitoneally injected with the same amount of 0.1M citrate buffer. After 72 hours, blood glucose was measured and mice with a blood sugar level of 200 mg/dL or more were used for the experiment, thinking that diabetes was induced. An abdominal incision was made 3 weeks after STZ injection, and PBS vehicle or PFO-MSC was injected into the anterior wall of the bladder and the submucosal layer of the dome using a 500 μm syringe and a 26-gauge needle as previously reported. was injected directly. After one week of PFO-MSC administration, bladder voiding function and tissue damage were measured through non-anesthetic and unrestricted cystometry (arousal cystometry) and histological staining, respectively.

14. CBI (chronic bladder ichemia ) Establishment of induced overactive bladder (OAB) and detrusor underactivity (DUA) models

16-week-old Sprague Dawley rat (Orient) experimental animals were anesthetized by intraperitoneal administration of 30 mg/kg zoletyl and 5 mg/kg xylazine. After anesthesia, to induce damage to the lumen of the iliac artery supplying blood to the bladder, the iliac artery was dissected by longitudinal incision in the lower abdomen on both sides in the supine position, and a 2 French Fogarty catheter (Edwards Lifesciences LLC, Irvine, CA) was placed in the lumen of the dissected iliac artery. ) using the iliac artery lumen (10; overactive bladder, OAB), 30; Underactive bladder, Dtrusor underactivity, DUA) was applied, and then a 1.25% high-fat diet (D12336, Research Diets, Brunswick, NJ, USA) was applied for 8 weeks. For the control group, sham surgery was performed on the same site as the experimental group and a high-fat diet was applied for 8 weeks. After 8 weeks of iliac artery lumen injury, bladder pressure was measured to confirm that OAB and DUA models were created.

15. awake In awake cystometry by bladder manometry diagram (cystometrogram)

Cystometry was performed in non-anesthetized, unsuppressed rats in metabolic cages. For the recording of intravesical pressure (IVP) and intraabdominal pressure (IAP), as previously reported, simultaneous catheterization was performed for 3 days before intravesical pressure measurement. The bladder was connected to a pressure transducer (Research Grade Blood Pressure Transducer; Harvard Apparatus, Holliston, MA, USA) and a microinjection pump (PHD22/2000 pump; Harvard Apparatus) with an inflated PE-50 catheter (Clay Adams, Parsippany, NJ) was used. The micturition volume (MV) was continuously recorded by a bodily fluid collection tube connected to a Research Grade Isometric Transducer (Harvard Apparatus), and sterile saline was injected into the bladder at about 0.4 ml/min. IVP, IAP and MV were continuously recorded using Acq Knowledge 3.8.1 software and MP150 data acquisition system (Biopac Systems, Goleta, CA, USA) at a sampling rate of 50 Hz. Mean values were calculated from 3 repeated urinations using 5 animals in each group (n=5). Non-voiding contraction (NVC) was calculated from when the increase in IVP exceeded 2 cmH ₂ O from baseline without urination. BP (bladder basal pressure) is the lowest bladder pressure during bladder filling, MP (micturition pressure) is the maximum bladder pressure during the voiding cycle, MV (micturition volume) is the volume of urine voided, and RV (residual volume) ) is the volume of urine remaining after urination. BC (bladder capacity) is the sum of MV and RV, and MI (micturition interval) is the interval between voiding contractions.

16. Evaluation of Bladder Function by Measurement of Bladder Smooth Muscle Tension

To measure the bladder flat muscle tension, the entire layer of the bladder was excised to a size of 2 × 1 cm, and the excised tissue was pre-saturated with a gas mixed with 5% O2 and 95% CO2 at 25°C in a bicarbonate buffer solution ( bicarbonate buffered solution) and quickly transported to the laboratory. The bladder mucosa layer was carefully removed using a microsurgical tool under a 7.5x stereo-microscopy field of view, and a pre-designed tissue fixation frame was made by making a bladder muscle longitudinal section of a certain size in the microscope field of view. Both ends of the section were ligated and fixed with 5-0 silk (tissue holder). It was hung in a 5 ml vertical bath and the isometric tension was recorded. After equilibrating for about 50 minutes before starting the experiment, the optimal length is obtained, and the experiment is performed based on the optimal length to maximize the contraction response (maximum contraction is shown at 200% of the resting period in the preliminary experiment). This length was maintained throughout the experiment. Tissue reactions to all drugs were terminated with only one experiment and replaced with another tissue section. For the recording of tension, the isotonic contraction signal from the bladder muscle section was computerized and recorded using a personal computer and computer software using four force transducers connected to four baths to evaluate bladder function.

< Example 1> PFO (Primed Fresh OCT-4 enrichment) Stem cell antioxidant capacity, engraftment rate and stem cell enhancement effect of MSCs through a single process culture

The present inventors have reported that ascorbic acid 2-glucoside (AA2G), a vitamin-C derivative (vitamin-C; VitC), that exhibits beneficial effects in preserving the native state and epigenetic integrity of MSCs through a CREB1-dependent mechanism. ) was applied. Human umbilical cord derived MSCs (UC-MSCs) cultured under 0.74 mM AA2G conditions promoted NRF2 protein translocation into the nucleus and upregulates key CREB1-NRF2 target genes that maintain redox homeostasis in MSCs. (eg, GCLM , GCLC , PRDX1 , and GSR ), indicating activation of the NRF2 pathway ( FIG. 2 ). In addition to AA2G priming, to improve the in vivo engraftment rate of MSCs, low concentrations of sphingosine-1-phosphate (S1P, 50 nM) and valproic acid (VPA, 0.5 mM) ), which may be a key part of the treatment of GVHD. The novel ex vivo expansion process was named PFO ( primed fresh O CT-4 enrichment) (FIG. 1).

First, we tested whether various AA2G treatment times affect the maintenance of small-sized primitive MSCs (Fig. 3a). Through fluorescence-activated cell sorting and microscopic analysis, it was confirmed that the UC-MSCs cell population with enlarged cell size gradually increased under normal culture conditions (naive). However, it was confirmed that replication-induced UC-MSCs cell size expansion was protected in the PFO process (PFO UC-MSCs) ( FIGS. 4a and 4b ). In two independent donor-derived UC-MSCs, the protective effect started from day 4 of AA2G treatment and was maintained until at least 8 passages (day 11) expansion (Fig. 3b-d), and their pluripotency and cell surface phenotype There was no change at all ( FIG. 5 ). In addition, the PFO process was effective in protecting small-sized cells even from adipose derived MSCs (AD-MSCs) ( FIG. 6 ).

Small-sized UC-MSCs enriched by the PFO process were resistant to replicative senescence and exhibited enhanced proliferative activity (Fig. 4c and Fig. 7a), and senescence-associated β-galactosidase (β-galactosidase; SA- β-gal) staining was not expressed at all ( FIGS. 4D and 7B ). Compared with naive cultured cells, PFO UC-MSCs showed low expression of cyclin-dependent kinase inhibitors such as p21 ^CIP1 and p53, but high expression of stem cell marker BMI-1 (Fig. 4e). and Figure 7c). Consistently, PFO UC-MSCs expressed pluripotent-specific transcripts OCT-4A and SOX2 (Fig. 4f), which was further verified through intranuclear staining of OCT-4 and SOX2 proteins (Fig. 4g and Fig. 8a), The open chromatin structure in the OCT4 promoter with low DNA methylation and the enrichment of transcriptionally preferred histone modifications were also confirmed ( FIGS. 8b and 8c ).

We tested whether the PFO process affects the GSH kinetics of MSCs. To this end, the present inventors used FreSHtracer, which is a reversible chemical probe for GSH, capable of non-destructive, integrated, and image-based high-throughput analysis, in real time to measure qualitative and quantitative aspects of GSH-recovering capacity (GRC). observed (Fig. 1). Compared with naive cells, PFO UC-MSCs showed higher basal GSH levels and GRC activity after brief exposure to 100 μM diamide, a thiol-specific oxidizing agent (Fig. 4h). PFO UC-MSCs have colony forming unit-fibroblast (CFU-F) activity, chemotaxis against platelet-derived growth factor (PDGF) and anti-inflammatory activity. was found to be high (FIG. 9). In addition, the beneficial effect of the PFO process was also observed in AD-MSCs from two independent donors (Fig. 10). And, genes related to stem cell characteristics, cell migration, growth factors, chemokines, inflammation and immune regulation were up-regulated in PFO UC-MSCs (FIG. 11).

In terms of immuno-modulatory capacity, PFO UC-MSCs are CD3 ⁺ T- in human peripheral blood mononuclear cells (PBMC) through phytohaemagglutinin (PHA) stimulation in vitro . Cell proliferation was inhibited (Fig. 4i). As a result of allogeneic mixed lymphocyte reaction (MLR) analysis, proliferation of PBMCs following allogeneic stimulation was strongly inhibited in PFO UC-MSCs (FIG. 4j), which supports improved immunomodulatory activity. Taken together, these results indicate that the PFO process can enrich small-sized primitive MSCs with high GSH kinetics, which can be resistant to aging and enhance self-renewal, anti-inflammatory and immunomodulatory abilities.

< Example 2> PFO Through single process culture MSCs GVHD treatment improvement effect

To confirm the in vivo relevance of the above results, the present inventors investigated the therapeutic effect of primitive UC-MSCs enriched by the PFO process in a GVHD mouse model humanized by transplantation of human PBMCs. All mice transplanted with human PBMCs alone (GVHD) died within 60 days, whereas the survival rate of GVHD mice increased upon injection of UC-MSCs in naive culture (60%) and PFO process (90%) (Fig. 12a). Consistently, the weight loss of GVHD mice was protected by transplantation of PFO UC-MSCs, and the beneficial effect was higher than that of naive cultured cells ( FIGS. 12b and 13a ). In the GVHD target organs, the small intestine, lung, liver and kidney, injection of PFO UC-MSCs significantly improved clinical scores and histological damage in GVHD mice ( FIGS. 12C and 12D ). Consistent with the results for the above GVHD symptoms, PFO UC-MSCs were shown to have excellent in vivo homing and engraftment ability into GVHD target organs ( FIG. 13b ).

In addition, 40 days after transplantation of human PBMCs, multiplex cytokine profiling and flow cytometry were performed using serum and splenic lymphocytes from GVHD mice, respectively. Consistent with in vitro results, PFO UC-MSCs are Th17 (eg, IL17, IL6 and GM-CSF), Th1 (eg, INF-γ and IL18) helper T cells and pro-inflammatory (eg, CXCL13) , TNF-a, IL8 and IL23) effectively reduced the levels of human cytokines and chemokines associated with the response ( FIGS. 12E and 14 ). In addition, the cell populations of human CD45 ⁺ , CD3 ⁺ and CD3 ⁺ CD4 ⁺ cells in GVHD mice were further reduced in the spleen of GVHD mice injected with PFO UC-MSCs ( FIG. 12h ), indicating that in vivo anti-inflammatory and It supports the improved immunomodulatory activity.

< Example 3> PFO MSCs transcript ( Transcrtiptome ) analysis

In order to demonstrate the difference in characteristics between naive MSC (cells treated with nothing; control group) and PFO MSC cell groups at a genome-wide level, transcriptome analysis was performed using RNA extracted from both cells. At this time, by further analyzing transcriptome datasets of MSCs that have undergone mixed priming with AA2G and S1P+VPA, two priming techniques used for PFO-MSC production, PFO-MSC and AA2G alone or S1P+VPA mixed priming MSC transcripts and A comparison of the properties was carried out.

As a result of PCA analysis of these stem cell transcripts, naive MSCs showed a diverse distribution in PCA, confirming that they were cells with high heterogeneity. Cells with distinct differences in genome-wide gene expression characteristics from AA2G alone or S1P+VPA priming MSCs was confirmed (FIG. 15A).

The results of the PFO-MSC transcript showing a distinct difference from the naive MSC and AA2G alone or S1P+VPA mixed priming MSC transcripts were verified through heatmap clustering analysis (FIG. 15B).

As a result of individual scatter plotting analysis of PFO-MSC transcripts with naive MSC and AA2G alone or S1P+VPA mixed priming MSC transcripts, it was confirmed that PFO-MSC was a stem cell group with distinct gene expression differences from these cells (Fig. 15C).

Therefore, through the transcriptome analysis of these stem cell groups, it was verified that PFO-MSC is a stem cell group with different molecular characteristics from AA2G alone or S1P+VPA mixed priming MSC as well as naive MSC.

< Example 4> PFO MSCs transcript ( Transcrtiptome ) Gene Ontology, Gene network analysis

As a result of in-depth analysis to study the specificity of gene ontology, pathway map, gene network, etc. of transcriptome characteristics from naive MSC and PFO MSC, genes involved in inflammatory response, angiogenesis, differentiation, etc. in PFO MSC compared to naive MSC A significant difference in expression between the groups was confirmed ( FIG. 16 ).

Next, the results were analyzed using 'MetaCore (Clarivate Anlaytics)', a program that can compare the differences between groups and predict interactions and expected targets based on previously reported information. When analyzing the signaling pathway, it is involved in the inflammatory response induced by Interleukins (IL) that occurs in bronchial asthma, IL-1 signaling in melanoma, and the IL-17 signaling system in immune response. Genes known to do so showed mainly changes. When analyzed based on the gene ontology (GO) classification criteria, it was analyzed that the cytokines involved in cell signal transduction and the response signaling system of the cells caused by this showed the greatest difference. In addition, in terms of disease, it was analyzed that there was a difference in the genes involved in the pathogenesis of joint diseases and diseases (FIG. 17).

In addition, the expression of proteins that play a key role in the inflammatory response, such as NFkB, p38 MAPK, and IL-6, is increased, and it is predicted that they will be largely involved in the inflammatory response of the cells. Since the proteins identified in the above results overlap with the results predicted to interact in the pathway analysis, high reliability results were obtained that the corresponding results would be involved in the inflammatory response (FIG. 18).

GSEA (Gene Set Enrichment Analysis), an analysis method that secures a previously announced specific gene group and compares it with the analysis result, is widely used to predict a new target among other analysis methods that can predict effective targets after transcriptome analysis. By substituting and analyzing the transcript analysis results of PFO MSCs, a list of 89 predicted target genes that are highly expressed and overlapped in multiple genes were obtained (Fig. 19), and verified through real-time gene expression analysis technique (qRT-PCR). Results It was confirmed that there was a large difference (increase) in gene expression levels such as FAM113A, NFkB2, NFkBID, IL-6, ICAM, QN1, YIPF5, ZC2H12A, LOH11CR2A, MPV, ZNF175, and ZFP90 in PFO MSC (FIG. 20).

< Example 5> PFO MSCs methylome ( Methylome ) analysis

The characteristics of naive MSC and PFO MSC were compared and analyzed by analyzing the degree of methylation of the genome, not the level of individual genes, for methylation, a very important step in determining the expression or non-expression (suppression) of a gene. In order to increase the accuracy, a group of three replicates was prepared (FIG. 21). The meaning of each indicator in FIG. 21 is as follows. Correlation Matrix means the similarity between each group, and AVG_Beta means the degree of methylation. In addition, BMIQ is an index comparing homogeneity within a sample group, and M value and log2 value indicate the degree of methylation.

To look more specifically, 99.27% of cells in which methylation occurred at the CpG site in the promoter of the PFO MSC gene were less than that of the naive MSC, and a significantly reduced gene group was discovered (FIG. 22).

When naive and PFO MSC were prepared by repeating 3 times each, a total of 6 samples were divided into groups showing similar patterns, and it was confirmed that methylation of Naive and PFO MSC showed completely different characteristics, and increased methylation of 170 genes or By confirming the opposite results of the decreasing pattern, it was confirmed that the PFO MSC had a distinct change compared to the naive MSC ( FIG. 23 ).

Based on the transcriptome and DNA methylome multiple omics analysis results, the experimental evidence was presented that PFO-MSCs are stem cells with distinct molecular characteristics from those of naive MSCs cultured in general.

< Example 6> PFO MSCs protein body ( Proteome ) and protein secretory ( Secretome ) analysis

In order to more clearly demonstrate the difference in cell characteristics between normally cultured naive MSCs and PFO-MSCs, proteome or secretome analysis that directly indicates cell activity was conducted through high-efficiency mass spectrometry ( Fig. 24).

Among the proteins extracted from naive and PFO MSC, it was confirmed that 3556 proteins were expressed in common, 165 were expressed only in naive, and 71 were expressed only in PFO MSC. In addition, when the characteristics of 6 samples (Naive and PFO each were 3 repeated samples) were analyzed and grouped, it was found that Naive and PFO MSC had different characteristics ( FIG. 25 ).

In addition, as a result of mass spectrometry analysis, proteins such as WASHC3, COMMD6, NUDT4, INTS4, FYTTD1, DHRS3, GOLIM4, SNAPIN, and PARVB were highly expressed in Naive, but not detected in PFO MSC. Conversely, proteins such as SAAL1, POLR2D, TGOLN2, ALDH6A1, FIBP, CC2B1B, PIH1D1, AASS, CD2AP, and SV2A were highly expressed only in PFO and hardly detected in Naive ( FIG. 26 ).

Through Transcriptome, DNA methylome, Proteosome, and Secretome multi-omics analysis, PFO-MSC is a stem cell group that is molecularly different from that of naive MSC cultured in general. This was verified.

< Example 7> PFO MSCs single cell transcript analysis

As a result of analyzing the single-cell transcriptome dataset of PFO-MSC cells, it was confirmed that PFO MSCs are largely divided into six cell clusters with different characteristics, and it was confirmed that the expression patterns of the gene groups in each cluster are different. (Fig. 27).

In order to find genes that can be used as markers for each cluster, genes showing different aspects for each cluster were selected and confirmed, and as a result, it was confirmed that the difference between the clusters was clearly shown ( FIG. 28 ).

Genes that can be used as markers for each cluster (Cluster 1: SLBP et al., Cluster 2: CDCA8 et al., Cluster 3: HIST1H4C et al., Cluster 4: MXD1 et al., Cluster 5: CCNB1, Cluster 6: FAM111B et al.) were discovered ( Fig. 29).

< Example 8> Th2 In an immune response-induced asthma disease model PFO - MSCs Evaluation of treatment efficacy

In order to expand the indication of the excellent therapeutic efficacy of PFO-MSC for intractable immune diseases, the therapeutic efficacy of PFO-MSC was evaluated in an asthma disease model, an intractable immune disorder induced by a Type 2 T helper immune response. As a result of evaluating the anti-asthma efficacy after intravenous injection of 1 × 10 ⁵ Naive MSCs and PFO-MSCs using the OVA-induced asthma mouse model, the BALF of the PFO MSC-administered mouse group was more immune to the Navie MSC-administered mouse group. It was confirmed that the infiltration of immune cells was significantly inhibited (FIG. 30).

As a result of histological evaluation of lung tissue, it was confirmed that inflammation of the bronchus was reduced in the mouse group administered with PFO MSC than the mouse group administered with Navie MSC, confirming the enhanced therapeutic efficacy of PFO-MSC (FIG. 31) .

In order to evaluate the engraftment rate of PFO-MSC stem cells in the lung tissue, as a result of immunofluorescence analysis of human β2 microglobulin (B2MG) protein, the mice administered with PFO MSC compared to the group of mice administered with Navie MSC. It was confirmed that human-derived cells expressing human B2MG were significantly increased in the lung tissue of the group, confirming the improved in vivo engraftment ability of PFO-MSC after transplantation (FIG. 32).

< Example 9> PFO - on MSc Diabetes mellitus hypoactivity bladder( streptozotocin induced detrusor underactivity, STZ-DUA) in vivo ( in vivo ) therapeutic effect

For a study to expand the indications for the excellent therapeutic efficacy of PFO-MSC, the therapeutic efficacy for hypoactivity, a typical complication induced by diabetes, was evaluated, and for this, the STZ-DUA animal model was used. Analysis of bladder function using awake cystometry showed that STZ intraperitoneal injection (STZ-DUA group) induced in DUA rats was compared with the control group, micturition interval (MI), residual volume (residual volume, RV), bladder capacity (BC) was significantly increased, and micturition press (MP) was decreased. Transplantation of 5×10 ⁵ PFO-MSCs significantly alleviated defective urination parameters ( FIGS. 33 and 34 ).

In combination with awake cystometry data, injection of 5×10 ⁵ PFO-MSC restored histologic changes such as severe muscle tissue changes, mast cell infiltration, and fibrosis, which are characteristic of the bladder of DUA patients. Therefore, it was confirmed that PFO-MSC has a significant effect in preventing or treating underactive bladder induced by STZ intraperitoneal injection (FIG. 35).

As a result of observing the long-term treatment efficacy, it was confirmed that the single administration of PFO-MSC stem cells had a therapeutic effect lasting up to 2 or 4 weeks after stem cell transplantation (FIG. 36).

To analyze the differentiation characteristics of the transplanted cells, co-staining of beta 2 microglobulin and muscle marker alpha-smooth muscle antibody in bladder tissue confirmed that the PFO-MSC group was clearly integrated into the muscle layer (FIG. 37). ).

Therefore, it was confirmed that the paracrine effect, which was a limitation of the existing stem cell therapeutics, now shows therapeutic efficacy through direct differentiation.

The differentiation of these transplanted PFO-MSCs into muscle cells was also observed in bladder tissue 2 and 4 weeks after transplantation, demonstrating the long-term therapeutic effect of direct muscle tissue damage caused by the high engraftment rate of PFO-MSC stem cells ( 38 and 39).

Accordingly, the present inventors optimized a hypoactive bladder disease model representing the clinical type, demonstrated the efficacy of PFO-MSC stem cells, and optimized the preclinical protocol for the number of stem cell administration cells, administration frequency and administration route, etc.

< Example 10> Chronic bladder ichemia ; CBI ) damage induction I Evaluation of PFO-MSCs Therapeutic Efficacy in Active Bladder (CBI-induced DUA) Disease Model

In order to evaluate the excellent therapeutic efficacy of PFO-MSC stem cells for underactive bladder caused by various pathological factors, the CBI induced DUA rat model, a disease model representing aging-induced underactive bladder, was used. To evaluate the therapeutic efficacy of PFO-MSCs, 7 weeks after iliac artery lumen injury, 1 × 10 ⁶ PFO-MSCs were directly injected into the bladder of DUA rats. In order to accurately confirm the results of bladder function one week after injection, the present inventors performed non-sleep intravesical manometry. Compared to DUA rats, in the DUA + PFO-MSCs group, maximum intravesical pressure (56.24 ± 7.57 vs. 88.65 ± 17.76 cmH ₂ O) and voiding pressure (MP; 17.37 ± 1.36 vs. 50.64 ± 8.67 cmH ₂ O; p; <0.05) increased, voiding volume (MV; 2.77 ± 0.11 vs. 2.16 ± 0.15 mL; p<0.01), voiding interval (MI; 273.7 ± 14.3 vs. 244 ± 11.46 sec), residual volume (RV; 0.25 ± 0.01) vs. 0.15 ± 0.01 mL; p<0.005) and bladder capacity (BC; 3.03 ± 0.11 vs. 2.32 ± 0.16 mL; p<0.001) decreased ( FIG. 40 ).

< Example 11> Chronic bladder ischemia-induced overactive bladder ( CBI induced OAB ) in the disease model PFO - MSCs Evaluation of treatment efficacy

In the case of chronic bladder ischemia, depending on the degree of damage, underactive bladder and overactive bladder are induced. To evaluate the therapeutic efficacy of PFO-MSCs using the CBI-induced overactive bladder rat model, 7 weeks after iliac artery lumen injury, 1 × 10 ⁶ PFO-MSCs were directly injected into the bladder of OAB rats. In order to accurately confirm the results of bladder function one week after injection, the present inventors performed non-sleep intravesical manometry. Compared to OAB rats, voiding interval (MI; 75.44 ± 3.79 vs. 204.2 ± 4.3 sec; p<0.001), voiding volume (MV; 0.57 ± 0.03 vs. 1.23 ± 0.09 mL; p<0.001) in the OAB + PFO-MSCs group. , maximum intravesical pressure (25.09 ± 1.25 vs. 58.72 ± 5.28 cmH ₂ O; p<0.001), voiding pressure (MP; 17.29 ± 1.56 vs. 51.04 ± 4.78 cmH ₂ O; p<0.001), residual volume ( RV; 0.06 ± 0.01 vs. 0.08 ± 0.01 mL) and bladder capacity (BC; 0.50 ± 0.02 vs. 1.36 ± 0.02 mL; p<0.001) were characteristically increased ( FIG. 41 ).

< Example 12> PFO - MSC characteristic titer In vitro efficacy evaluation for proteins

Through proteomic analysis, among proteins such as SAAL1, POLR2D, TGOLN2, ALDH6A1, FIBP, CC2B1B, PIH1D1, AASS, CD2AP, and SV2A that are specifically expressed in PFO-MSC, SV2A, CD2AP, shRNA for ALDH6A1 genes (shSV2A, UC-MSCs stably expressing lentiviruses expressing shCD2AP, shADLH6A1) were obtained. As a control, empty shRNA control (shControl) lentivirus was used.

It was confirmed that the expression of each gene was reduced in UC-MSC stably expressing the shRNA (FIG. 42A), and after inducing PFO-MSC using these cells, the expression of the PFO-MSC-specific titer protein was As a result of measuring the self-renewal ability according to the CFU-F assay, it was confirmed that PFO-MSC expressing shSV2A, shCD2AP, and shADLH6A1 significantly decreased CFU-F activity compared to the shControl control cells (FIG. 42B). and Figure 42C).

In order to confirm the difference in cell mobility, chemotaxis assay for PDGF-AA was performed using Transwell. As a result, when PFO-MSC was induced in MSC knocked down (KD) of SV2A, CD2AP, and ALDH6A1, shControl MSC Compared with cells, it was confirmed that PDGF-responsive chemotaxis activity was significantly reduced ( FIGS. 43A and 43B ).

As a result of measuring the ability to secrete TNF-a pro-inflammation protein secreted from LPS-stimulated MH-S cells, conditioned media (CM) obtained from PFO-MSC expressing shSV2A, shCD2AP, and shADLH6A1 was compared with control cells, -It was confirmed that the ability to inhibit TNF-a protein secretion by inflammation activity was significantly reduced (FIG. 44).

Therefore, the main functions of MSCs, such as self-renewal ability (CFU-F), cell migration ability (Transwell chemotatic response to PDGF), and anti-inflammatory response, as performed above, were observed in PFO-MSC induced by SV2A, CD2AP, and ALDH6A1 KD MSC. By confirming the inhibition, the significance of the PFO-MSC-specific titer proteins was verified in vitro.

< Example 13> PFO - MSC characteristic titer protein in vivo (in in vivo ) effectiveness evaluation

For in vivo efficacy evaluation of the importance of the PFO-MSC titer protein identified above, 1 × 10 ⁵ PFO-MSC (shControl , shSV2A, shCD2AP, shADLH6A1) were administered intravenously and the anti-asthma efficacy was evaluated. In mice administered with PFO-MSC induced by Control MSC, the bronchial inflammatory response in lung tissue induced by asthma was effectively inhibited, whereas PFO-MSC induced by SV2A, CD2AP, ALDH6A1 KD MSC had a reduced inhibitory effect on bronchial inflammatory response. was confirmed (FIG. 45A).

As with the histological examination results, when evaluating the degree of infiltration of immune cells in pulmonary bronchial lavage (BALF), SV2A, CD2AP, ALDH6A1 KD MSC-derived PFO-MSC was compared with control cells By comparison, it was confirmed that the anti-asthma ability to inhibit immune cell infiltration into lung tissue was significantly reduced ( FIGS. 45B and 45C ).

Therefore, the in vivo significance of SV2A, CD2AP, and ALDH6A1 titer proteins in anti-asthma efficacy by PFO-MSC was verified in the same manner as in the in vitro efficacy evaluation experiment.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

1) subculture by treating the isolated mesenchymal stem cells with ascorbic acid 2-glucoside (AA2G); and
2) A method for promoting mesenchymal stem cell activity comprising the step of adding sphingosine-1-phoshate (S1P) and valproic acid (VPA) to the subculture. The intermediate according to claim 1, wherein the mesenchymal stem cells are umbilical cord derived MSCs (UC-MSCs) or adipose derived MSCs (AD-MSCs). A method for promoting mesenchymal stem cell activity. [Claim 2] The method of claim 1, wherein in step 1), 0.1 to 0.75 mM of AA2G is treated and subcultured for 4 to 11 days. The method of claim 1, wherein in step 2), 10 to 50 nM of S1P and 0.1 to 1.0 mM of VPA are added. The method according to any one of claims 1 to 4, wherein the mesenchymal stem cells whose activity is promoted by the method have improved intracellular glutathione levels and glutathione-recovering capacity (GRC), and stem cell characteristics. , colony forming unit-fibroblast (CFU-F) activity, chemotaxis to platelet-derived growth factor (PDGF), self-renewal, cell migration, engraftment ability, anti-inflammatory and A method for promoting mesenchymal stem cell activity, characterized in that the activity of any one or more stem cells selected from the group consisting of T-cell inhibitory ability is promoted. A composition for promoting mesenchymal stem cell activity comprising 0.1 to 0.75 mM of AA2G, 10 to 50 nM of S1P, and 0.1 to 1.0 mM of VPA as active ingredients. The intermediate according to claim 6, wherein the mesenchymal stem cells are umbilical cord derived MSCs (UC-MSCs) or adipose derived MSCs (AD-MSCs). A composition for promoting mesenchymal stem cell activity. The method according to claim 6, wherein the mesenchymal stem cells have improved intracellular glutathione levels and glutathione-recovering capacity (GRC), and stem cell, colony forming unit-fibroblasts (colony forming unit-fibroblast; CFU). -F) activity, any one or more stem cell activity selected from the group consisting of chemotaxis, self-renewal, cell migration, engraftment ability, anti-inflammatory and T-cell inhibitory activity for platelet-derived growth factor (PDGF) A composition for promoting mesenchymal stem cell activity, characterized in that this is promoted. A pharmaceutical composition for the prevention or treatment of intractable immune diseases comprising mesenchymal stem cells whose activity is promoted by treatment with AA2G, S1P and VPA or a culture medium thereof as an active ingredient. The pharmaceutical composition for preventing or treating intractable immune diseases according to claim 9, wherein the AA2G is treated at a concentration of 0.1 to 0.75 mM, the S1P is at a concentration of 10 to 50 nM, and the VPA is treated at a concentration of 0.1 to 1.0 mM. . The intractable according to claim 9, wherein the mesenchymal stem cells are umbilical cord derived MSCs (UC-MSCs) or adipose derived MSCs (AD-MSCs). A pharmaceutical composition for preventing or treating immune diseases. 10. The method of claim 9, wherein the mesenchymal stem cells have improved intracellular glutathione levels and glutathione-recovering capacity (GRC), stem cell, colony forming unit-fibroblasts (colony forming unit-fibroblast; CFU) -F) activity, any one or more stem cell activity selected from the group consisting of chemotaxis, self-renewal, cell migration, engraftment ability, anti-inflammatory and T-cell inhibitory activity for platelet-derived growth factor (PDGF) A pharmaceutical composition for preventing or treating intractable immune diseases, characterized in that this is promoted. The pharmaceutical composition for preventing or treating intractable immune diseases according to claim 9, wherein the intractable immune disease is graft-versus-host disease (GVHD), asthma, underactive bladder or overactive bladder. The pharmaceutical composition for preventing or treating intractable immune diseases according to claim 9, wherein the mesenchymal stem cells specifically express any one or more selected from the group consisting of SV2A, CD2AP and ALDH6A1.

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