KR102500377B1 - Use for treating dysuric diseases using functionally improved stem cells by activating FOS or CDK1 gene - Google Patents

Abstract

The present invention relates to the use of stem cells with improved functions through FOS or CDK1 gene activation to treat dysuria disorders. In the model, the in vivo behavior of engrafted multipotent-MSCs (M-MSCs) derived from human embryonic-SCs (hESCs) was analyzed. Longitudinal two-photon image analysis visualized engrafted MSC-bladder vasculature interactions over time in the same living organism, revealing progressive perivascular localization. As a result of single-cell transcriptome analysis of the engrafted M-MSCs, expression changes in several pathways related to pericytes, cell-adhesion, and cell stress were revealed. In particular, FOS and CDK1 play an important role in regulating migration/engraftment and anti-inflammatory functions of M-MSCs, which determine the in vivo therapeutic ability of M-MSCs. Taken together, the present invention provides accurate characterization of transplanted MSCs, which may improve understanding of the therapeutic mechanism of MSC therapy and improve its efficacy and safety.

Description

{Use for treating dysuric diseases using functionally improved stem cells by activating FOS or CDK1 gene}

The present invention relates to the use of stem cells with improved functions through FOS or CDK1 gene activation to treat dysuria disorders.

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 a favorable microenvironment including immune modulatory, anti-inflammatory or angiogenic activity. These pluripotent cells have been isolated from several adult or fetal tissues such as bone-marrow (BM), umbilical cord blood and amniotic fluid. Recently, direct derivation of MSCs from pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), has emerged 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 in vitro expansion.

Because of the substantial benefits due to the multiple causes and effectiveness of these multiple aspects, MSC-based therapies have been proposed to treat a variety of refractory cardiovascular, musculoskeletal, neurological, immunological and urological disorders, but controversy over their performance is prevalent in clinical trials. Interfering with enemy application. More importantly, the lack of information on the fundamental features that drive the in vivo behavior of these cells after transplantation makes it difficult to advance promising preclinical studies of MSC therapy into clinical trials. Accordingly, it is emphasized that 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.

Recent advances in single cell analysis or bio-imaging methods can provide methods for cellular and molecular mapping or direct visualization of stem cells at high resolution at the single cell level, in living cells or animals, respectively. For example, as a result of microfluidic single cell analysis of transplanted human iPSC-derived cardiomyocytes, significant levels of angiogenesis-promoting and survival-promoting factors were released in an acute myocardial infarction animal model. Several bio-image analysis platforms, including fluorescence microscopy, photoacoustic image analysis, and multimodal image analysis of positron emission tomography, computed tomography, and bioluminescence, have been proposed to investigate stem cell trafficking and engraftment, intracellular interactions, and vascular changes. It is used to study the kinetics of various intracellular processes such as in vivo . By providing both spatial and temporal information at the single cell level about the dynamic interactions between engrafted cells and their microenvironment over time in the same animal, intravital microscopy (IVM) techniques can be used for mechanistic analysis, It is commonly practiced to overcome the limitations of commonly used methods, such as immunohistochemistry, which can only provide static analysis at discrete time intervals in different animals.

In our previous study, which applied a combination of confocal fluorescence image analysis and microcystoscopy in vivo in living animals, the present inventors demonstrated that human ESCs (hESCs)-derived transplanted pluripotent MSCs (M-MSCs) in vivo ( In vivo ) Observation of distribution and phenotypic characteristics longitudinally, engraftment after transplantation into two animal models for acute or chronic interstitial cystitis/bladder pain syndrome (IC/BPS) Characteristics during the process were visualized. Importantly, hESC-derived M-MSCs were shown to improve engraftment and viability in vivo , which showed superior therapeutic effects to their BM-derived counterparts without any side effects in several IC/BPS animal models. However, in-depth information on the MSC engraftment process, especially on the interaction between the microenvironment and molecular profile at the genome-wide level, is still lacking.

US Patent Publication US 2016-0017433 A1 (published on 2016.01.21)

An object of the present invention is to treat mesenchymal stem cells (MSCs) containing one or more proteins selected from the group consisting of Fos Proto-Oncogene (FOS) protein and cyclin dependent kinase-1 (CDK1) or genes encoding them It is to provide a biomarker composition for predicting engraftment after transplantation.

Another object of the present invention is a composition for predicting engraftment ability after transplantation of mesenchymal stem cells comprising, as an active ingredient, an agent capable of measuring the expression level of any one or more selected from the group consisting of FOS and CDK1, and an intermediate comprising the same It is to provide a kit for predicting engraftment ability after transplantation of mesenchymal stem cells.

In addition, another object of the present invention is to provide information necessary for predicting engraftment ability after transplantation of mesenchymal stem cells, which includes measuring the expression level of any one or more selected from the group consisting of FOS and CDK1 in isolated mesenchymal stem cells. It is to provide a way to provide.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating dysuria comprising mesenchymal stem cells overexpressing at least one selected from the group consisting of FOS and CDK1 or a culture medium thereof as an active ingredient.

In addition, another object of the present invention is to provide a composition for promoting mesenchymal stem cell activity comprising at least one selected from the group consisting of FOS and CDK1 as an active ingredient.

In addition, another object of the present invention is to provide a screening method for treating dysuria disease, comprising the step of selecting a test substance having an increased expression level of one or more selected from the group consisting of FOS and CDK1 in mesenchymal stem cells.

In order to achieve the above object, the present invention provides mesenchymal stem cells containing at least one protein selected from the group consisting of Fos Proto-Oncogene (FOS) protein and cyclin dependent kinase-1 (CDK1) or genes encoding them. A biomarker composition for predicting engraftment after transplantation of stem cells (MSCs) is provided.

In addition, the present invention provides a composition for predicting engraftment ability after transplantation of mesenchymal stem cells comprising, as an active ingredient, an agent capable of measuring the expression level of at least one selected from the group consisting of FOS and CDK1.

In addition, the present invention provides a kit for predicting engraftment ability after transplantation of mesenchymal stem cells containing the above composition.

In addition, the present invention (1) measuring the expression level of any one or more selected from the group consisting of FOS and CDK1 in the isolated mesenchymal stem cells; and (2) comparing the measured expression level with a control sample.

In addition, the present invention provides a pharmaceutical composition for preventing or treating dysuria comprising mesenchymal stem cells overexpressing at least one selected from the group consisting of FOS and CDK1 or a culture medium thereof as an active ingredient.

In addition, the present invention provides a composition for promoting mesenchymal stem cell activity comprising at least one selected from the group consisting of FOS and CDK1 as an active ingredient.

In addition, the present invention (1) contacting the test substance to the separated mesenchymal stem cells;

(2) measuring the expression level of one or more selected from the group consisting of FOS and CDK1 in mesenchymal stem cells contacted with the test substance; and (3) selecting a test substance having an increased expression level of at least one selected from the group consisting of FOS and CDK1 compared to a control sample.

The present invention relates to the use of stem cells with improved functions through FOS or CDK1 gene activation to treat dysuria disorders. In the model, the in vivo behavior of engrafted multipotent-MSCs (M-MSCs) derived from human embryonic-SCs (hESCs) was analyzed. Longitudinal two-photon image analysis visualized engrafted MSC-bladder vasculature interactions over time in the same living organism, revealing progressive perivascular localization. As a result of single-cell transcriptome analysis of the engrafted M-MSCs, expression changes in several pathways related to pericytes, cell-adhesion, and cell stress were revealed. In particular, FOS and CDK1 play an important role in regulating migration/engraftment and anti-inflammatory functions of M-MSCs, which determine the in vivo therapeutic ability of M-MSCs. Taken together, the present invention provides accurate characterization of transplanted MSCs, which may improve understanding of the therapeutic mechanism of MSC therapy and improve its efficacy and safety.

Figure 1 shows the results of two-photon in vivo microscopic analysis of M-MSCs transplanted in living animals.
Figure 2 shows the results of two-photon in vivo microscopic analysis.
Figure 3 shows the 3D result of a two-photon IVM z-stack image of the bladder.
Figure 4 shows the results of quantitative analysis of two-photon IVM in the bladder of live animals.
5 shows the results of isolation and single cell transcriptome analysis of engrafted M-MSCs.
Figure 6 shows the isolation results of engrafted M-MSCs without host cell contamination.
7 shows the results of single-cell transcriptome profiling by analyzing the characteristics of the engrafted M-MSCs.
Figure 8 shows the results of a single cell transcriptome profile characteristic of engrafted cells.
Figure 9 shows the gene set results characteristic of cells engrafted from the IC/BPS bladder.
10 shows the results of enrichment of pericyte-related gene sets in engrafted cells.
11 shows the results of upregulation of FOS and CDK1 in engrafted M-MSCs.
12 shows the gene expression analysis results of cells engrafted in HCl-IC bladder.
13 shows the results of the critical roles of FOS and CDK1 in the key functions of M-MSCs.
14 shows the results of the role of FOS and CDK1 in the characteristics of M-MSCs.
Figure 15 shows the results for FOS and CDK1 determining the therapeutic ability of M-MSCs in HCl-IC rats.
Figure 16 shows the results of difficult histological recovery by FOS or CDK1 silenced M-MSCs in HCl-IC bladder.
Figure 17 shows the results of engraftment damage in vivo of FOS or CDK1 silenced M-MSCs.
18 shows the results of immunofluorescence analysis of engrafted M-MSCs.
19 shows the bioluminescence image analysis results of transplanted M-MSCs.
20 and 21 show results of measurement of bladder pressure after administration of M-MSC stem cells in an animal model of streptozotocin induced detrusor underactivity (STZ-induced DUA).
22 shows histological staining results after administration of M-MSC stem cells in an STZ-induced DUA animal model.

The present invention provides direct evidence of the dynamic association between engrafted M-MSCs and the bladder vasculature through two-photon IVM image analysis in living animals, which suggests that transplanted M-MSCs are progressively incorporated into perivascular-like structures. supports Moreover, the present inventors analyzed the characteristics of the single-cell transcriptome profile of M-MSCs engrafted from affected IC/BPS bladders, including Fos Proto-Oncogene (FOS; AP-1 transcription factor subunit) and cyclin dependent kinase-1 ( CDK1) was found to be important in regulating migration, homing, function, and in vivo engraftment of transplanted M-MSCs, which determine the therapeutic ability of MSCs, and completed the present invention.

Transplantation of mesenchymal stem cells (MSCs) containing one or more proteins or genes encoding them selected from the group consisting of Fos Proto-Oncogene (FOS) protein and cyclin dependent kinase-1 (CDK1) Provided is a biomarker composition for predicting post engraftment.

Preferably, the mesenchymal stem cells may be human embryonic stem cells (hESCs)-derived pluripotent mesenchymal stem cells (multipotent-MSCs; M-MSCs), but are not limited thereto.

In addition, the present invention provides a composition for predicting engraftment ability after transplantation of mesenchymal stem cells comprising, as an active ingredient, an agent capable of measuring the expression level of at least one selected from the group consisting of FOS and CDK1.

Preferably, the agent is a primer or probe that specifically binds to any one or more genes selected from the group consisting of FOS and CDK1, an antibody that specifically binds to any one or more proteins selected from the group consisting of FOS and CDK1 , It may be a peptide, aptamer or compound, but is not limited thereto.

Preferably, the transplantation of mesenchymal stem cells may be transplantation of mesenchymal stem cells in the bladder, but is not limited thereto.

In addition, the present invention provides a kit for predicting engraftment ability after transplantation of mesenchymal stem cells containing the above composition.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3' hydroxyl group, capable of base pairing with a complementary template, and serving as a starting point for template strand copying. refers to the sequence of nucleic acids. Primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to several hundred bases in length that can specifically bind to mRNA and is labeled, so that the presence or absence of a specific mRNA, expression quantity can be checked. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. Selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art.

As used herein, the term "antibody" is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to SPP1 of the present invention, and the antibody can be prepared according to a conventional method in the art. The type of antibody includes polyclonal antibodies or monoclonal antibodies, and all immunoglobulin antibodies are included. The antibody is meant in its complete form with two full-length light chains and two full-length heavy chains. In addition, the antibody also includes special antibodies such as humanized antibodies.

As used herein, the term "peptide" has the advantage of having high binding ability to a target substance, and does not undergo denaturation even during heat/chemical treatment. In addition, because of its small molecular size, it can be attached to other proteins and used as a fusion protein. Specifically, since it can be used by attaching it to a polymer protein chain, it can be used as a diagnostic kit and a drug delivery material.

As used herein, the term "aptamer" refers to a special kind of single-stranded nucleic acid (DNA, RNA or modified nucleic acid) having a stable tertiary structure and being able to bind to a target molecule with high affinity and specificity. ) means a type of polynucleotide composed of As described above, aptamers can specifically bind to antigenic substances in the same way as antibodies, but are more stable than proteins, have a simple structure, and are composed of polynucleotides that are easy to synthesize, so they can be used instead of antibodies. can

In addition, the present invention (1) measuring the expression level of any one or more selected from the group consisting of FOS and CDK1 in the isolated mesenchymal stem cells; and (2) comparing the measured expression level with a control sample.

Preferably, the transplant of mesenchymal stem cells may be transplantation of mesenchymal stem cells in the bladder, but is not limited thereto.

In detail, the method for measuring the expression level is 1) a method for measuring the mRNA expression level, RT-PCR, competitive RT-PCR (Competitive RT-PCR), real-time RT-PCR (Real-time RT-PCR) , RNase protection assay (RPA; Northern blotting) and DNA chip can be used, and 2) methods for measuring protein expression levels include Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunization Radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay Fixation Assay), FACS and protein chips may be used, but are not limited thereto.

The present invention provides a pharmaceutical composition for preventing or treating dysuria comprising mesenchymal stem cells overexpressing at least one selected from the group consisting of FOS and CDK1 or a culture medium thereof as an active ingredient.

Preferably, the mesenchymal stem cells may promote one or more stem cell activities selected from the group consisting of cell migration, engraftment after transplantation, and anti-inflammation, but are not limited thereto.

Preferably, the dysuria disorder may be interstitial cystitis/bladder pain syndrome (IC/BPS) or hypoactive bladder, but is not limited thereto.

In the present invention, "culture medium" includes a medium capable of supporting the growth and survival of stem cells in vitro, secretions of cultured stem cells contained in the medium, and the like. The medium used for the culture includes all conventional mediums used in the art suitable for culturing stem cells. Media and culture conditions can be selected according to the type of cell. The medium used for culture is preferably a cell culture minimum medium (CCMM), and generally contains a carbon source, a nitrogen source, and trace elements. Such cell culture minimal media include, 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 are not limited thereto.

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

On the other hand, the present invention is a form containing all of the stem cells, their secretions, and medium components, a form containing only secretions and medium components, a form in which only secretions are separated and used alone or together with stem cells, or by administering only stem cells It is also possible to use it in a form that produces secretions in the body.

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

Mesenchymal stem cells overexpressing at least one selected from the group consisting of FOS and CDK1 of the present invention can be used as a cell therapy agent for the treatment of a specific disease, and the treatment can be direct treatment or pre-treatment of the molecules. there is.

The "cell therapy product" means a product that changes the biological properties of cells by proliferating or selecting living autologous, allogenic, or xenogenic cells in vitro in order to restore the function of cells and tissues or by other methods. Drugs used for treatment, diagnosis, and prevention through a series of actions such as

The cell therapy 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 can 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, an expanding agent, a lubricant, and a glidant. Alternatively, a solubilizer such as a flavoring agent may be used. The pharmaceutical composition of the present invention may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, 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 bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

The pharmaceutical formulation form 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 preparations of active compounds. can The pharmaceutical composition of the present invention can be administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered with An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for preventing or treating a disease. Therefore, the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health condition, sex and diet, administration time, administration route and composition It can be controlled by various factors including secretion rate, duration of treatment, and drugs used concurrently.

In addition, the present invention provides a composition for promoting mesenchymal stem cell activity comprising at least one selected from the group consisting of FOS and CDK1 as an active ingredient.

Preferably, the mesenchymal stem cell activity may be any one or more selected from the group consisting of cell migration, engraftment after transplantation, and anti-inflammation, but is not limited thereto.

In addition, the composition for promoting activity can enhance the in vivo effect of the cell therapy agent by mixing it with a cell therapy agent for treatment and injecting it into the body, as well as a cell therapy agent whose function is increased after treating the stem cells themselves with the present composition. It can also be used as a method of transplanting in vivo.

The present invention (1) contacting the test substance to the separated mesenchymal stem cells; (2) measuring the expression level of one or more selected from the group consisting of FOS and CDK1 in mesenchymal stem cells contacted with the test substance; and (3) selecting a test substance having an increased expression level of at least one selected from the group consisting of FOS and CDK1 compared to a control sample.

Specifically, the mesenchymal stem cells may be hESCs-derived M-MSCs, but are not limited thereto.

Specifically, the dysuria disorder may be interstitial cystitis/bladder pain syndrome (IC/BPS) or hypoactive bladder, but is not limited thereto.

The term "test substance" used while referring to the screening method of the present invention is used to examine whether or not it affects the expression level of a gene, affects the expression or activity of a protein, or affects the binding between proteins. It means an unknown candidate substance used in screening. The samples include, but are not limited to, chemical substances, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural product extracts.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining 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. Research Approval

All animal experiments were approved by the Animal Experiment Ethics Committee of Ulsan University School of Medicine (IACUC-2019-12-128 and IACUC-2017-12-054).

2. hESCs -Origin M- MSCs culture

M-MSCs differentiated from H9 hESCs were prepared in EGM2-MV medium (Lonza, San Diego, CA, USA) on plates coated with rat tail collagen type I (Sigma-Aldrich, St. Louis, MO, USA) at 37°C. was maintained in a humidified atmosphere of 5% CO ₂ . M-MSCs of 10 passages or less were used for in vitro cell culture and in vivo animal experiments. For ectopic expression of shRNA specific for GFP, FOS or CDK1 , lentiviruses with the indicated constructs were injected into M-MSCs. The therapeutic effect in cells in vitro and in vivo was confirmed 4 days after infection with lentivirus.

3. Animal Models and M- MSCs Injection

An acute IC/BPS rat model induced by HCl administration was constructed according to a previous report. Briefly, 10-week-old female Sprague-Dawley rats (Orient Bio, Gapyong, Gyeonggi-do, Korea) were induced by administering 0.2 M HCl, and 1 week after HCl injury, the anterior wall and dome of the bladder ) was directly injected with 1 × 10 ⁶ M-MSCs or PBS vehicle into the outer layer. One week after M-MSC administration, bladder voiding function and tissue damage were measured by non-anesthetic and unrestricted cystometry (awakening cystometry) and histological staining, respectively. In this treatment evaluation assay, evaluations were performed in two independent sets of 5 independent animals per group. For histological analysis, 2 representative regions per slide were randomly selected from 10 independent animals ( n =20). Quantitative digital image analysis was performed using Image Pro 5.0 software (Media-Cybernetics, Rockville, MD, USA).

For bioimage analysis, after HCl-IC/BPS induction in 10-week-old female BALB/c-nu (CAnN.Cg -Foxn1 ^nu /CrlCrlj) immune-compromised mice (Charles River Laboratories, Yokohama, Japan), two-photon microscopy analysis or For IVIS bioluminescence image analysis, 1×10 ⁵ M-MSCs or Nano-lantern constructs stably expressing GFP were administered, respectively. In the rat animal model, M-MSCs were directly injected into the serosa of the bladder using the same procedure. Bioluminescence resonance energy transfer image analysis was performed using the IVIS Spectrum Pre-clinical In vivo Imaging System and Living Imaging 4.4 software (PerkimElmer, Waltham, MA) according to the manufacturer's instructions and previously published protocols.

4. Two-photon IVM image analysis

Five independent mice were used for two-photon IVM analysis on the same living animal from 3 to 28 days post implantation. Mice were anesthetized by intraperitoneal administration of 30 mg/kg zoletyl and 5 mg/kg xylazine, and fixed in a supine position on an externally heated image analysis plate. After anesthesia, 1 mg of Texas-Red conjugated Dextran (70,000 MW; Sigma-Aldrich) in 100 μl per mouse was injected into the orbit to label blood vessels. After a 3 mm incision was made in the abdomen to expose the bladder, it was mounted and fixed between organ plates prepared internally by 3D printing with a glass coverslip and polylactic acid. Using a two-photon IVM (IVM-MS, IVIM technology, Republic of Korea), GFP ⁺ engrafted cells and luminal walls of blood vessels were labeled with Texas red and visualized.

An ultrafast femtosecond pulse laser at 920 nm (Alcor 920, Spark Lasers, France) was used as a two-photon excitation source. Fluorescence signals of GFP and Texas red were detected at 502.5 - 537.5 nm and 569.5 - 604.5 nm. In vivo images were obtained using a high numerical aperture water-immersion objective lens (CFI75 Apochromat 25XW, NA1.1, Nikon, Japan) with a field of view of 454 × 454 μm ² . First, the microscope was aligned near the M-MSCs injection site, and video-speed image analysis was recorded. Once GFP ⁺ engrafted cell location and blood flow were confirmed, z-image stacks were acquired at 1-5 μm z-step intervals with an average total depth of 223.4 μm (114 - 334 μm) from the outer layer of the bladder. In the negative control group, almost no GFP signal was observed in the bladder before transplantation of GFP-nonexpressing M-MSCs and until 28 days after transplantation.

5. Image Processing and Analysis

Image processing and analysis were performed using commercial software IMARIS 9.5 (Oxford Instruments, USA). For volume analysis, Z-stack images were reconstructed three-dimensionally along with surface features. Using the statistical function, the volumetric area of GFP ⁺ cells and the luminal wall of the vessel were calculated with the same threshold of fluorescence intensity. The sphericity of the cells was calculated by the following formula (where V _p is the volume of the cell and A _p is the surface of the cell):

Using the distance conversion function, the distance between each GFP ⁺ cell and blood vessel was determined by measuring the shortest distance between the outer boundary of the volume reconstructed surface of the GFP ⁺ cell and the inner lumen wall of the vessel. Considering the endothelial cell surface layer with a thickness of about 1 μm, each cell whose measured distance was less than 1 μm was considered to be in contact with the blood vessel.

6. M- MSCs magnetic nanoparticles on Resovist sign

M-MSCs stably expressing GFP were inoculated at a cell density of 2 × 10 ⁶ 2 hours before Resovist labeling. Labeled by reacting for 10 minutes in a culture medium containing 500 μg/ml Perucarbotran (Resovist; Bayer schering pharma, Berlin, Germany) and 5 μg/ml protamine sulfate (P3369, Sigma-Aldrich) has been completed. After washing off the labeling medium, before in vivo administration, the labeled cells were further cultured for 24 hours.

7. engrafted M- to MSCs 2-step continuous MACS- FACS separation

Bladder was prepared from 3 IC/BPS rats of the indicated DAT, and was reacted with 2 ml of Accutase (A1110501, Gibco, Waltham, MA, USA) for 10 min at room temperature to small pieces before preparing bladder tissue cells. Chopped. Erythrocytes were removed with RBC Lysis Solution (158902, QIAGEN, Hilden, Germany), and the single cell suspension of bladder tissue was centrifuged at 6,000 × g for 20 minutes at 4°C, followed by 1 ml of MACS buffer (2.5% FBS and and resuspended in low glucose DMEM containing 1% HEPES). MACS separation was performed by placing the resuspended cells in a magnetic device (12321D, Invitrogen, Waltham, MA, USA) at room temperature for 3 minutes, and then washing the unbound cells with MACS buffer twice. Resovist ⁺ cells by MACS positivity were additionally isolated based on GFP expression through live FACS cell separation using an ARIAIII Flow Cytometer System (BD Biosciences, San Jose, CA). To verify contamination with cells of host origin, the expression levels of rat B2mg and human GAPDH housekeeping genes were quantified in the isolated cells at each isolation step.

8. Single cells transcriptome analyze

Single cell cDNA library synthesis was performed as previously reported. Briefly, Resovist ⁺ /GFP ⁺ cells or trypsinized and cultured M-MSC cells isolated through bladder-derived MACS - FACS were plated in a 96-well plate containing 4.5 μl of lysis buffer using an ARIAIII Flow Cytometer System. Thermo Scientific, Waltham, MA, USA) were distributed as single cells into each well. RNA from each well was amplified with a T7-primed single cell cDNA library. In the initial screening of the Resovist ⁺ /GFP ⁺ single cell cDNA library, using a 20-fold diluted T7-primed PCR amplification product, host-derived rat B2mg Alternatively, expression of indicated genes related to stem cell function was measured. Only single-cell cDNA libraries with little host cell contamination were selected for genome-wide analysis. A gel-eluted T7-primed library was generated using the GeneChip® 3' in vitro transcription kit (Affymetrix, Santa Clara, CA, USA). was biotin-labeled.

Biotin-labeled aRNA from the single cell library was fragmented and hybridized to the Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array. Microarray image data was processed with GeneChip GCS3000 Scanner and Command Console software (Affymetrix). Raw data was automatically extracted in the Affymetrix data extraction protocol using software provided by Affymetrix GeneChip® Command Console® Software (AGCC). The microarray data set used in the present invention has been registered with NCBI's Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo) and is accessible through the GEO Series accession number GSE162982.

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

9. M- MSCs In vitro ( in vitro ) molecular and cellular characterization

RNA and protein levels for the indicated genes were measured and quantified by RQ-PCR and Western blot analysis, respectively. Levels of protein indicated are monoclonal antibodies specific for FOS (#4384, Cell Signaling Technology), CDK1 (sc-54, Santa Cruz Biotechnology, Dallas, TX, USA) and standardized β-ACTIN (A5441, Sigma-Aldrich). It was measured by probing with .

Including cell viability, proliferation, self-renewal (CFU-F), pluripotency (in vitro differentiation into chondrocyte, osteocyte or adipocyte lineages), transwell transfer, in vitro anti-inflammatory and neovascular assays In vitro cell function analysis of M-MSCs to be performed was performed as previously reported. Each cell activity was quantified through digital image analysis using Image Pro 5.0 software (Media-Cybernetics, Rockville, MD, USA).

10. Data and Software Availability

Transcriptome data mentioned in the present invention has been registered in NCBI Gene Expression Omnibus and is accessible through GEO Series accession number GSE162982. To check the data set, access https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE162982 and enter "axohkkqutpibjit".

11. Statistical Analysis

Data are presented as mean ± standard error of the mean (SEM). Statistical significance was analyzed by one-way or two-way ANOVA followed by Bonferroni post-hoc test using GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA). If the p value was less than 0.05, it was judged to be statistically significant.

< Example 1> Implanted M- MSCs and two-photon to bladder vasculature IVM image analysis

To directly visualize the dynamic interaction between transplanted M-MSCs and the bladder vasculature, we employed minimal scattering, reduced non-specific excitation, low phototoxicity, and effective detection with image analysis depth increased by photobleaching in the surrounding tissue. Therefore, two-photon IVM has been applied for repeated and long-term image analysis of living tissue. After inducing acute IC/BPS through administration of hydrochloride (HCl) to BALB/c-nu immune-compromised mice, 1×10 ⁵ M-MSCs stably containing green fluorescent protein (GFP) were inoculated. It was implanted directly into the outer layer of the anterior wall and dome of the bladder. On the first 3 days after transplantation (DAT), the animals were anesthetized, the bladder was surgically exposed by making a small incision in the abdomen, and the bladder was irradiated via two-photon IVM of the outer layer of the bladder near the site of M-MSC injection. (FIG. 1A). To delineate blood vessels, Texas Red conjugated dextran was injected into the orbit at the time of image analysis.

To minimize movement during image analysis, images were acquired by clamping the bladder between a glass cover stage and a custom organ plate (FIG. 2A). Although the minimal incision and bladder maintenance process for image analysis can change the observable M-MSC engraftment phenotype or in vivo behavior, the present inventors conducted a two-photon IVM process through histological experiments with minimal pain. Almost no inflammatory response and overall tissue damage were observed (Fig. 2B). Once GFP ⁺ signal localization was confirmed in the initial scanning of the entire bladder, video-velocity recordings were performed, after which high-resolution z-stack images were acquired at an average of 223.4 μm (114 - 334 μm) from the outer layer of the bladder. After image acquisition, the XYZ stack was input into IMARIS software to generate a 3-dimension (3D) image (FIGS. 1B and 3).

Our initial goal was to longitudinally visualize the biodistribution of engrafted M-MSCs and their dynamic interactions with bladder vasculature in the same living animals several weeks after transplantation. As a result of two-photon IVM image analysis, the number of GFP ⁺ engrafted cells in all 5 independent mice tested in the present invention decreased rapidly from 7 DAT, was gradually damaged until 21 DAT, and disappeared at 28 DAT (Fig. 1B). The biological kinetic results of the high apoptosis rate of engrafted M-MSCs in the early engraftment stage are consistent with previous studies. In the early stages of the present invention (3 and 5 DAT), several areas with GFP ⁺ cells were identified throughout the bladder, showing a heterogeneous distribution in the bladder vasculature over a wide range of distances to the bladder vessels (Fig. 1B and Fig. 3A). The amount of engrafted cells decreased from 7 DAT, and in all 5 independent animals, GFP ⁺ cells were mainly observed near the effective blood flow supplying oxygen and nutrients. At 14 DAT, perivascular localization of GFP ⁺ cells was clearly confirmed, and several GFP ⁺ cells bound along the vessels in a pericyte-like morphology were observed (FIGS. 1A, 1B and 3B). Although not all GFP ⁺ cells engrafted to the vessel surface at 21 DAT, they were preferentially localized near the bladder vasculature and disappeared at 28 DAT.

< Example 2> M- to bladder MSCs Quantitative analysis of in vivo images for engraftment

To further analyze the quantitative information on the two-photon IVM images, we characterized the number, volume and sphericity of engrafted GFP ⁺ cells. In addition, the present inventors quantified the shortest distance to three adjacent blood vessels for GFP ⁺ cells and the contact area when the engrafted cells were localized around blood vessels (FIGS. 4A and 4B). As shown in Figures 4C and 4D, GFP ⁺ engrafted cells at 3 and 5 DAT had a more heterogeneous distribution in the bladder vessels (101.4 ± 53.29 and 100.1 ± 62.29 μm) and the highest total cell counts (43.8 ± 10.03 and 52.2 ± 52.29 μm). 35.63 within approximate dimensions of 454 × 454 × average 50 μm ³ ), which is consistent with the video-speed two-photon IVM results. Compared to the initial engraftment period, the distance between engrafted cells and bladder vasculature became shorter (52.87 ± 49.58 μm) from 7 DAT and decreased to an average of 20.72 μm by 14 DAT, supporting perivascular localization (Fig. 4D). . As the distance to the vasculature decreased, the sphericity defining perfectly round cells as 1 decreased from 7 DAT (Fig. 4E), indicating their spindle-like morphology (Fig. 4B). During the whole course of the experiment in the present invention, the GFP ⁺ cells localized around the vessels selected at 7 DAT and 14 DAT, respectively, were similar in the area in contact with blood vessels (Fig. 4F) and the volume of engrafted cells (Fig. 4G).

To further analyze the perivascular binding of transplanted M-MSCs, bladder tissues were immunostained with antibodies specific for human β2 microglobin (hB2MG) and pericyte-specific marker NG2. Consistent with the two-photon IVM results, at 14 DAT most hB2MG ⁺ cells were perivascularly localized with NG2 expression, supporting a contribution to perivascular cells (Fig. 4H). Taken together, two-photon IVM image analysis allows us to observe the full spectrum of M-MSC behavior in vivo in the bladder of living IC/BPS animals, indicating that transplanted M-MSCs progressively develop pericytes. support that it contributes to

< Example 3> IC/BPS in acute animal models engrafted M- MSCs single cell transcriptome analyze

Next, the present inventors attempted to understand the engraftment process of M-MSCs through genome-wide molecular profiling of engrafted cells in a pathological microenvironment, but the number of cells was limited due to the low engraftment efficiency of MSCs in vivo . has not progressed In addition, direct fluorescence activated cell sorting (FACS) of cells engrafted at a low frequency takes a long time, and yield and purity are reduced. In order to overcome these technical limitations, the present inventors performed a two-step continuous sorting process by performing FACS to increase purity after magnetic activated cell sorting (MACS) of a positive enrichment method, and high purity Molecular characteristics of engrafted cells isolated from were analyzed through single cell transcriptome analysis (FIG. 5A).

For positive MACS classification, we labeled GFP ⁺ M-MSCs with Resovist, a clinically approved superparamagnetic iron oxide, and injected 1×10 ⁶ Resovist-labeled into the outer layer of IC/BPS-induced rat bladder via HCl injection. GFP ⁺ M-MSCs were transplanted. During the 7 days of in vitro cell culture, the Resovist label had little effect on cell viability, and the magnetic beads remained inside the cells (FIGS. 6A and 6B). When the engrafted cells were separated through two consecutive MACS - FACS sorting, the frequency of Resovist ⁺ /GFP ⁺ double positive cells was very low, about 0.475% of the MACS sorted cells (Fig. 5B). Using an ARIAIII cell sorter, the isolated cells were distributed as single cells in each well of a 96-well plate (FIG. 5B), and then a single cell transcript library was synthesized through a T7-primed amplification process.

For single cell libraries established from normal cultured M-MSCs (Cultured_#1 - 6) and IC/BPS bladder-derived 3 DAT Resovist ⁺ /GFP ⁺ engrafted cells (Eng_#1 - 8), no host cell contamination was detected. It was initially screened for absence. In the above libraries, expression of the rat-specific β2 microglobin (r B2mg ) transcript was hardly confirmed (FIGS. 5C and 5D). Conversely, unlike the first-step FACS sorting, cells engrafted by 1-step MACS showed a high frequency of host animal-derived cell contamination (FIGS. 6C, 6D, and 6E).

Next, the synthesized single-cell transcript libraries were tested for i) cytokines, ii) chemokines, iii) matrix metalloproteases, iv) growth factors, v) cell survival and vi) stem cell activity. It was characterized by measuring the expression of genes related to stem cell engraftment. As shown in Figure 5E, they were shown to up-regulate the expression of these genes, including interleukin-6 ( IL6 ), platelet derived growth factor subunit-C ( PDGFC ), High expression of PDGFD , vascular endothelial growth factor-A ( VEGFA ) , VEGFR1 , and lysyl oxidase ( LOX ) was characteristic. Therefore, the present inventors optimized a method for rapidly isolating cells that rarely engraft after transplantation without contamination of host-derived cells with high purity and a method for characterizing genome-wide molecular characteristics at the single cell level.

< Example 4> IC/BPS in acute animal models engrafted cellular transcriptome characterization

To identify the molecular mechanisms regulating the engraftment process, we compared single cell transcriptome profiles of engrafted M-MSCs and cultured M-MSCs. As a result of principal component analysis (PCA) and scatter plot, the cells engrafted to the IC/BPS bladder showed different molecular characteristics from the cells cultured before transplantation (FIGS. 7A and 7B). According to the PCA results, the engrafted cells showed a higher degree of heterogeneity than the cultured cells (FIG. 7A).

As a result of MetaCore pathway analysis, engrafted cells showed expression changes in YAP/TAZ and EGFR functions, as well as pathways and biological processes involved in extracellular matrix (ECM), cell adhesion and cytoskeleton (Fig. 7C). and Figure 8A). In addition, when the engrafted cells are divided into two sub-clusters (sub1: Eng_#3, #5-7; sub2: Eng_#1, #2, #4), cell adhesion, ECM, and cytoskeleton regulatory pathways are enriched. was observed (Fig. 8B and Fig. 8C), supporting the importance of this biological process in M-MSC engraftment to the IC/BPS bladder. Consistent with the above results, as a result of gene set enrichment analysis (GSEA), the single-cell transcript dataset of engrafted cells was higher than that of cultured cells, neural cell adhesion molecule-1. ;NCAM-1) interaction (NES = 1.720), NCAM signaling (NES = 1.690), WNT pathway (NES = 1.710) and lysine-specific histone demethylase-1a (LSD1) A set of genes associated with the target (NES = 1.730) was found to be enriched (FIGS. 7D, 9A and 9B).

< Example 5> FOS and CDK1 high in protein expressed engrafted M- MSCs

To identify the effector genes to orchestrate the M-MSC engraftment process, we performed gene network (MetaCore) and advanced (GSEA) analyzes. In MetaCore analysis, engrafted cell-derived single cell transcripts were characterized by CREB1- and NFκB-related gene networks, as well as WNT- and cyclin G-related gene networks (FIGS. 7E and 8D). In particular, GSEA advanced analysis revealed that genes encoding FOS and collagen Type IV α chain ( COL4A1 , COL4A2 and COL4A5 ) were significantly up-regulated in engrafted cells (FIG. 9C and COL4A5). Figure 9D). Consistent with the results of perivascular localization observed in two-photon IVM, GSEA analysis focusing on pericyte-related gene sets showed that engrafted cells significantly enriched PDGF- and PECAM1-related pathways (Fig. 7F, Fig. 10A and Figure 10B). Importantly, in the PDGF-related pathway enriched in M-MSCs engrafted with IC/BPS bladders, FOS and AP-1 constituting a heterodimer with FOS were found to be central genes (FIGS. 7F and 10C).

To verify the results, the present inventors performed real-time quantitative PCR (RQ-PCR) analysis on the single cell cDNA library. As a result, genes representing cell cycle, proliferation, migration, mitochondria, oxygen-containing, endoplasmic reticulum stress, and pericytes as well as WNT-, CREB1- and NFκB-signaling were highly expressed in the engrafted cells (FIG. 11A to 11E and 12). Among these, we paid attention to FOS and CDK1 , identified as major biomarkers in both GSEA and MetaCore analyses, which represent pericyte- and cell cycle-related processes, respectively. Both genes were significantly upregulated in the bladders of IC/BPS rats transplanted with M-MSCs (FIG. 11F). Consistent with the above results, immunofluorescence staining analysis showed that M-MSCs engrafted from the IC/BPS bladder expressed high levels of FOS and CDK1 proteins (FIG. 11G).

< Example 6>M- MSCs in migratory and anti-inflammatory functions FOS and of CDK1 role

To study the biological significance of these results, we expressed FOS or CDK1- specific shRNA (sh FOS or sh CDK1 ) in M-MSCs (FIGS. 13A and 14A), confirming the key functions of these cells. did Silencing of FOS or CDK1 induced a significant deficit in chemotaxis to PDGF, indicating impaired migration and engraftment (FIG. 13B). Compared to M-MSCs with control blank shRNA (shEmpty), knock-down (KD) of FOS or CDK1 reduced colony forming unit-fibroblast (CFU-F) activity. (FIGS. 13C and 14B), but had little effect on the pluripotent differentiation ability of M-MSCs (FIG. 14C). MSCs expressing sh CDK1 showed a decrease in cell proliferation rate, which was only observed on day 5 with FOS silencing (FIG. 13D). Importantly, conditioned medium (CM) derived from M-MSCs with sh FOS or sh CDK1 had reduced anti-inflammatory activity, suggesting that lipopolysaccharide (LPS)-promoted macrophage-induced tumor necrosis factor ; Regarding the anti-inflammatory ability reduced by the silencing of FOS or CDK1 , it was further verified based on the increased expression levels of cytokines including Tnf -a , Il -6 , Il -18 , Ccl2 and Ccl7 in LPS-promoted macrophages. (FIG. 13G). In the matrigel tube formation assay, the angiogenic ability by CM derived from FOS or CDK1 silenced MSCs was hardly affected (Fig. 14E). Taken together, the in vitro functional assay results support that FOS and CDK1 play an important role in the migration and anti-inflammatory ability of MSCs, in that they have therapeutic ability to treat IC/BPS. It is important.

< Example 7> M- of IC-BPS MSc in treatment FOS and of CDK1 in vivo ( in vivo ) importance

In order to confirm whether the lack of migration and anti-inflammatory function by FOS or CDK1 silencing affects the therapeutic ability of M-MSCs, the present inventors treated 1 × 10 ⁶ M-MSCs with sh FOS or sh CDK1 in HCl-induced It was injected directly into the bladder of IC/BPS rats, and its treatment results were compared to cells with control blank shRNA. In order to accurately confirm the functional results, we performed wake filling cystometry, which makes it possible to evaluate bladder voiding function long-term in freely moving animals. As previously reported, compared to sham-treated rats, an irregular voiding pattern was observed in the HCl-IC group, with voiding interval (MI; 31.34 ± 1.15 vs. 109.1 ± 3.42 sec; p<0.001), voiding Volume (MV; 0.16 ± 0.01 vs. 0.66 ± 0.02 mL; p<0.001), voiding pressure (MP; 36.29 ± 2.19 vs. 63.32 ± 3.12 cmH ₂ O; p<0.001), bladder capacity (BC; 0.20 ± 0.01 vs. 0.73 ± 0.02 mL; p<0.001) and residual volume (RV; 0.04 ± 0.00 vs. 0.07 ± 0.01 mL; p<0.05) were characteristically reduced (FIGS. 15A and 15B).

M-MSCs single transplanted with blank shRNA into IC/BPS rats significantly improved the voiding parameters (FIGS. 15A and 15B). M-MSC treatment significantly improved contraction frequency during the non-voiding period (non-voiding contraction (NVC)), the most common factor for evaluating bladder voiding function in clinical practice. Importantly, these beneficial results were hardly observed in IC/BPS rats transplanted with M-MSCs expressing sh FOS or sh CDK1 (FIGS. 15A and 15B). Consistent with the arousal cystometry results, administration of FOS or CDK1 -silenced M-MSCs was not effective in reversing the histological damage, including severe urothelial detachment, mast cell invasion and cell death, typically observed in the bladders of IC/BPS patients. Failed (FIGS. 15C and 16). Thus, the above results support that FOS and CDK1 play important roles in the therapeutic ability of M-MSCs to treat IC/BPS.

< Example 8> In IC/BPS animals FOS and CDK1 silence M- MSCs lack of engraftment

Next, we confirmed whether FOS and CDK1 have a critical effect on the engraftment of M-MSCs in the IC/BPS bladder. As a result of immunofluorescence staining, hB2MG ⁺ cells were found to be lower in the bladders administered with FOS or CDK1 -silenced M-MSCs compared to shEmpty control cells (FIGS. 17A, 17B and 18).

In order to compare the in vivo engraftment ability of the cells in a living animal, the present inventors developed an improved Renilla luciferase and Venus fluorescent protein with high-efficiency bioluminescence resonance energy transfer ability. M-MSCs stably expressing the chimeric Nano-lantern were applied, and the bioluminescence intensity of transplanted cells was compared through optical image analysis. Consistent with the results of hB2MG immunofluorescence staining, IC/BPS immunocompromised mice transplanted with M-MSCs with sh FOS and sh CDK1 showed significantly reduced bioluminescent activity than mice with shEmpty during the entire experimental period (Fig. 17C, FIG. 17D and FIG. 19). Taken together, these results support that FOS and CDK1 are key regulators of engraftment of M-MSCs into the IC/BPS bladder, which may determine the therapeutic capacity of these cells.

< Example 9> Diabetic low activity bladder( streptozotocin induced detrusor underactivity, STZ -induced DUA ) with animal models MSc stem cell administration

STZ injection into the STZ-induced streptozotocin induced detrusor underactivity (DUA) rat model to reproduce diabetic hypoactive bladder was performed as previously described (Shin, J., et al. J. Clin. Med. 2020, 9). (9), 2853). To create the STZ-DUA animal model, diabetes was induced by intraperitoneal injection of streptozotocin (STZ, Sigma) in 8-week female Sprague-Dawley rats (OrientBio, Gapyong, Gyeonggi-do, Korea). After titrating 0.1M citrate buffer to pH 4.5, 50mg/kg was intraperitoneally injected once, and the normal group was intraperitoneally injected with the same amount of 0.1M citrate buffer. After 72 hours, blood glucose was measured and rats with a blood sugar level of more than 200 mg/dL were considered to have diabetes and were used in the experiment. Abdominal incision was made 3 weeks after STZ injection, and M-MSCs treated with PBS vehicle or shFOS , shCDK1 , or blank shRNA (shEmpty) lentivirus were treated with a 500 μm syringe and 26-gauge as previously reported. It was injected directly into the anterior wall of the bladder and the submucosa of the dome using a needle (Song, M., et al. Stem Cells and Development 24, 1648-1657 (2015); Kim, A., et al. Scientific Reports 6, 30881 (2016); and Song, M., et al. Stem Cells and Development 23, 654-663 (2013)). One week after administration of 1 × 10 ⁶ M-MSC stem cells, bladder voiding function and tissue damage were measured by non-anesthetic and unrestricted cystometry (awakening cystometry) and histological staining, respectively.

The in vivo importance of FOS and CDK1 in STZ-DUA's M-MSC treatment was confirmed. In order to confirm whether the lack of migration and anti-inflammatory function by FOS or CDK1 silencing affects the therapeutic ability of M-MSCs, the present inventors treated 1 × 10 ⁶ M-MSCs with sh FOS or sh CDK1 as STZ-induced DUA was injected directly into the bladder of rats, and its treatment results were compared to cells with control blank shRNA. In order to accurately confirm the functional results, we performed wake filling cystometry, which makes it possible to evaluate bladder voiding function long-term in freely moving animals. As previously reported, significant increases in micturition interval (MI), residual volume (RV), and bladder capacity (BC) in the STZ-DUA group compared to sham-treated rats and urination pressure (micturition press, MP) showed a reduced urination pattern (FIGS. 20 and 21). M-MSCs single transplanted with empty shRNA into STZ-DUA rats significantly improved the voiding parameters (FIG. 21). Importantly, these beneficial results were hardly observed in STZ-DUA rats transplanted with M-MSCs expressing sh FOS or sh CDK1 (FIGS. 20 and 21).

Consistent with the arousal cystometry results, administration of FOS or CDK1 -silenced M-MSCs failed to restore histological damage such as changes in muscle tissue, mast cell infiltration, and fibrosis typically observed in the bladders of patients with DUA (Fig. 22). Thus, these results support that FOS and CDK1 play important roles in the therapeutic ability of M-MSCs to treat DUA.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and 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 equivalents thereof.

Claims (16)

Transplantation of mesenchymal stem cells (MSCs) containing one or more proteins selected from the group consisting of Fos Proto-Oncogene (FOS) protein and cyclin dependent kinase-1 (CDK1) protein or genes encoding them into the bladder Biomarker composition for confirming engraftment after. The mesenchymal stem cells according to claim 1, wherein the mesenchymal stem cells are multipotent-MSCs (M-MSCs) derived from human embryonic stem cells (hESCs). A biomarker composition for confirming engraftment after intravesical transplantation. A composition for confirming engraftment after intravesical transplantation of mesenchymal stem cells comprising, as an active ingredient, an agent capable of measuring the expression level of any one or more proteins selected from the group consisting of FOS protein and CDK1 protein or genes encoding them. The method of claim 3, wherein the agent is a primer or probe that specifically binds to any one or more genes selected from the group consisting of the FOS gene and the CDK1 gene, to any one or more proteins selected from the group consisting of the FOS protein and the CDK1 protein A composition for confirming engraftment after intravesical transplantation of mesenchymal stem cells, characterized in that they are specifically binding antibodies, peptides, aptamers or compounds. delete A kit for confirming engraftment after intravesical transplantation of mesenchymal stem cells comprising the composition of claim 3 or 4. (1) measuring the expression level of any one or more proteins selected from the group consisting of FOS protein and CDK1 protein or genes encoding them in mesenchymal stem cells isolated after intravesical transplantation; and
(2) A method for providing information necessary for confirming engraftment after transplantation of mesenchymal stem cells into the bladder, comprising comparing the measured expression level with a control sample. delete Interstitial cystitis/bladder pain syndrome (IC/BPS) comprising mesenchymal stem cells overexpressing any one or more proteins selected from the group consisting of FOS protein and CDK1 protein or a culture solution thereof as an active ingredient, or A pharmaceutical composition for preventing or treating hypoactive bladder disease. The method of claim 9, wherein the mesenchymal stem cells are interstitial cystitis/bladder pain syndrome characterized in that the activity of one or more stem cells selected from the group consisting of cell migration, engraftment after transplantation, and anti-inflammatory is promoted. / Bladder pain syndrome; IC / BPS) or a pharmaceutical composition for preventing or treating hypoactive bladder disease. delete A composition for promoting engraftment after intravesical transplantation of mesenchymal stem cells comprising at least one protein selected from the group consisting of FOS protein and CDK1 protein as an active ingredient. delete (1) contacting the test substance to the separated mesenchymal stem cells;
(2) measuring the expression level of any one or more proteins selected from the group consisting of FOS protein and CDK1 protein or genes encoding them in mesenchymal stem cells contacted with the test substance; and
(3) Interstitial cystitis/bladder pain syndrome comprising the step of selecting a test substance having an increased expression level of any one or more proteins selected from the group consisting of the FOS protein and CDK1 protein or genes encoding them compared to the control sample ( A screening method for drugs for interstitial cystitis/bladder pain syndrome (IC/BPS) or hypoactive bladder disease. 15. The method according to claim 14, wherein the mesenchymal stem cells are hESCs-derived M-MSCs. delete

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