KR20200047330A - Stem cell-derived microvesicles with enhanced efficacy, use thereof and method for enhancing efficacy - Google Patents

Abstract

The present invention relates to a stem cell-derived microvesicle with enhanced efficacy, a use thereof, and a method for strengthening efficacy thereof, and more particularly to a use of a stem cell-derived microvesicle with an enhanced expression level of microRNAs for preventing or treating stroke and a method for promoting a production and reinforcing an efficacy of the microRNAs in the stem cell-derived microvesicle, wherein the method is performed by subjecting stem cells to three-dimensional culture or ischemic stimulus to promote the production of stem cell-derived microvesicles and microRNAs in the microvesicles and also reinforce the efficacy of the stem cells and the microvesicles thereof. According to the present invention, the method has an excellent effect of not only promoting the production of the stem cell-derived microvesicle and microRNAs in the microvesicle, but also reinforcing the efficacy of the stem cells or microvesicles isolated therefrom. Accordingly, it is possible to obtain the stem cell-derived microvesicles containing a high level of substances including therapeutic microRNAs in large quantities and thus it is expected that the microvesicle will be useful in related fields of study and future clinical studies.

Description

Stem cell-derived microvesicles with enhanced efficacy, use thereof and method for enhancing efficacy}

The present invention relates to a stem cell-derived microvesicle having enhanced efficacy, a use thereof, and a method for enhancing the efficacy, more specifically, a stroke prevention or treatment use of a stem cell-derived microvesicle having enhanced expression level of microRNA, stem cell It relates to a method for promoting the production of microRNAs and enhancing the efficacy of the derived microvesicles, and thereby promoting the production of microRNAs in the stem cells derived microvesicles and the microvesicles by stimulating the cells in three dimensions or by ischemia stimulation. It relates to a method of enhancing the efficacy of vesicles.

Recently, several clinical trials using mesenchymal stem cells (MSC) have been conducted for intractable diseases such as stroke, spinal cord injury, multiple sclerosis, Alzheimer's disease, liver sclerosis, myocardial infarction, kidney disease and graft versus host disease. have. Although positive clinical results have been reported so far, the treatment using stem cells still has some problems in applying to the clinic. First, in the case of cell therapy, stem cells are engrafted to the tissue and then there is a risk of tumor formation. Second, in the case of stem cells, there is a possibility of causing arterial occlusion and brain infarction due to the large size. Third, in the case of stem cells, acute phase As shown, the brain-vascular barrier is easily moved into the brain in the open state, but in the chronic phase, movement is limited due to its large size. Lastly, in the case of cell therapy products, there is a limit to inducing the propensity to cells specialized to a desired propensity.

Recently, microvesicles (MV) secreted from stem cells in regenerative medicine have increased their short-term secretion due to increasing reports that the clinical effectiveness of mesenchymal stem cells is mainly due to the paracrine effect. It is attracting attention in that it mediates various effects. Microvesicles are small vesicles with a diameter of 0.1 to 1 μm or less, and mean that a part of the cell membrane, such as endothelial cells and platelets, is released into the blood by stimulation. Microvesicles derived from stem cells contain nuclear components as well as receptors and proteins. It is known to mediate intercellular communication. In addition, stem cell-derived microvesicles have the following important characteristics as an alternative to current stem cell transplantation therapy (Nephrol. Dial. Transplant. 27, 3037-3042 (2012)). Specifically, the vesicle structure composed of nano-sized and lipid is more safe and advantageous for long-term blood circulation and long-term therapeutic activity than MSC, and the stem cell membrane protein present on the microvesicle surface has the ability to target diseases such as injected stem cells. It has the advantage of being able to confer, and the risk of symptoms (zoonosis) caused by animal serum infection is also excluded because it contains relatively little animal serum compared to stem cells.

However, there has not been established a method for mass separation and obtaining microvesicles derived from MSC for research and clinical purposes, and this is regarded as a major limiting factor in the development of stem cell-derived microvesicles as pharmaceuticals, and its efficacy will be further enhanced. Research on how to do this is also lacking, and research on this is needed.

Accordingly, the present inventors have researched to develop a method capable of solving the above problems, and produce PEG hydrogel microwell arrays to dynamically cultivate stem cells in 3D or to apply ischemic stimulation to stem cells. It has been experimentally confirmed that the production of microvesicles derived from stem cells, which contains a large amount of various therapeutic substances and exhibits neurogenic and angiogenic effects, has been completed based on this.

Accordingly, the present invention is a microRNA-137 (miR-137), microRNA-184 (miR-184) and microRNA-210 (miR-210) selected from the group consisting of one or more expression level is enhanced, stem cells It provides a pharmaceutical composition for preventing or treating stroke, comprising the derived microvesicles.

In addition, another object of the present invention is to provide a method for promoting the production of microRNAs (microRNAs or miRNAs) in microvesicles derived from stem cells, including the step of culturing stem cells in three dimensions.

In addition, another object of the present invention is to provide a method for promoting the production of microRNAs (microRNAs) in stem cells-derived microvesicles, which includes ischemia-stimulating stem cells.

In addition, another object of the present invention is to provide a method for enhancing the efficacy of stem cells or microvesicles separated therefrom, including the step of culturing the stem cells in three dimensions.

In addition, another object of the present invention is to provide a method for enhancing the efficacy of stem cells or microvesicles separated therefrom, comprising the step of ischemia-stimulating the stem cells.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention is selected from the group consisting of microRNA-137 (miR-137), microRNA-184 (miR-184) and microRNA-210 (miR-210) 1 Provided is a pharmaceutical composition for preventing or treating stroke, including stem cell-derived microvesicles with enhanced expression level of species or higher.

In addition, it provides a method for promoting the production of microRNAs (microRNAs) in microvesicles derived from stem cells, comprising the step of culturing the stem cells in three dimensions.

In addition, the present invention provides a method for promoting the production of microRNAs (microRNAs) in microvesicles derived from stem cells, comprising the step of stimulating ischemia of the stem cells.

In one embodiment of the present invention, the microRNA may be one or more selected from the group consisting of miR-137, miR-184, and miR-210.

In another embodiment of the present invention, the stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSC) or adult stem cells.

In another embodiment of the present invention, the adult stem cells are at least one adult selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and multipotent stem cells. It may be a stem cell.

In another embodiment of the present invention, the three-dimensional culture may be to divide the cells and incubate for 5 to 9 days while rotating and stirring at 20 to 40 rpm after 6 to 18 hours in the incubator.

In another embodiment of the present invention, the ischemic stimulation may be by treatment of an ischemic brain tissue extract.

In another embodiment of the present invention, the ischemia stimulation may be made for 12 hours to 48 hours.

In addition, the present invention provides a method for enhancing the efficacy of stem cells or microvesicles separated therefrom, including the step of culturing stem cells in three dimensions.

In addition, the present invention provides a method for enhancing the efficacy of stem cells or microvesicles separated therefrom, including the step of ischemia-stimulating the stem cells.

In one embodiment of the present invention, the potentiation may be that the expression of growth factor, cytokine, or microRNA (microRNA) in stem cells is enhanced.

In another embodiment of the present invention, the growth factor is fibroblast growth factor (FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), trait It may be one or more selected from the group consisting of transforming growth factor (TGFβ) and bone morphogenetic protein (BMP2).

In another embodiment of the invention, the cytokine is in the group consisting of CH13L1, CD105, CD147, ICAM-1, IP-10, MIP-1β, IL-6, IL-8, GRO, TIMP-1 and SerpineE1 It may be one or more selected.

In another embodiment of the present invention, the microRNA may be one or more selected from the group consisting of miR-137, miR-184, and miR-210.

The method according to the present invention not only promotes the production of microvesicles derived from stem cells and microRNAs in the microvesicles, but also has an excellent effect of enhancing the efficacy of stem cells or microvesicles isolated therefrom. It is expected that the microvesicles derived from stem cells containing substances including therapeutic microRNAs at a high level can be efficiently and mass-produced, and thus the microvesicles can be useful in related research fields and clinical trials in the future.

1 is a diagram showing the overall contents of the three-dimensional stem cell culture method and effect according to the present invention.
Figure 2 shows the results of forming and culturing mesenchymal stem cells (MSC) -spheroids through three-dimensional (3D) culture, Figure 2a is a process of manufacturing a PEG microwell array used for 3D culture of MSC in the present invention Figure 2b is a result showing that a spherical cell aggregate was formed within 12 hours after dispensing MSC at a constant density in the microwell, Figure 2c is a cell for MSC-spheroids on the 5th day of dynamic 3D culture As a result of confirming survival, FIGS. 2D and 2E are results of hematoxylin and eosin (H & E) and Mason Trichrome (M & T) staining, respectively. FIG. 2F is a static 2D culture of MSC, respectively, and dynamic 2D for continuously stirring. Proliferation capacity of MSC by measuring the number of cells on culture 3 (D3), 5 (D5), and 7 days (D7) while culturing (2D w / shaking), static 3D culture, and dynamic 3D culture (3D w / shaking) It is the result of comparing.
Figure 3 is a result of analyzing the gene expression profile of the dynamic 3D cultured MSC-spheroids, Figure 3a is a 2D culture or dynamic 3D culture method (Day 1 and 7) MSC culture through PCR for the characteristics of MSC It is a result of measuring the expression level of 84 major genes related to and presented as a cluster gram, and FIG. 3B compares the expression levels of genes related to stem cell function and stem cell marker of MSC in each experimental group. 3c shows the expression of genes showing the characteristics of hMSCs having an average Ct value of less than 30 in a scatter plot, and shows comparison between experimental groups.
4 is a result of confirming an increase in microvesicle production by dynamic 3D-MSC culture, and FIG. 4A shows static 2D culture, continuous dynamic 2D culture (2D w / shaking), static 3D culture, and dynamic 3D culture, respectively. The results of the flow cytometry analysis by separating the microvesicles on 3D (D3), 5 (D5), and 7 (D7) cultures in 3D w / shaking MSC, and FIG. 4B shows the culture time for each culture method. The result of comparing the amount of microvesicles generated with a certain number of cells is compared, and FIG. 4C shows the result of correcting and comparing the total protein concentration in the microvesicles generated according to each culture method to a certain number of cells on the 7th day of culture.
5 is a result of analyzing the levels of therapeutic substances in hMSC-derived microvesicles (IBE-MVs) treated with ischemia-brain tissue extract and dynamic 3D cultured hMSC-derived microvesicles (3D-MVs), FIGS. 5A and 5B Is a result of measuring the content of various cytokines in the above two types of microvesicles, and FIG. 5C shows rMSC-derived microvesicles (MSC-MV) and fibroblast-derived microvesicles (Fibroblast-MV) treated with ischemia-brain tissue extracts. ) This is a result of comparing and measuring the level of growth factors related to my stroke treatment by Western blot.
6 is important for angiogenesis and / or neurogenic signaling in hMSC-derived microvesicles (IBE-MVs) treated with ischemia-brain tissue extracts and hMSC-derived microvesicles (3D-MVs) cultured by a dynamic 3D culture method. As a result of analyzing the level of microRNAs known to be, FIG. 6A is a result of measuring and comparing the expression level of the microRNAs in the two types of microvesicles, and FIG. 6B is a 2D culture method, a dynamic 3D culture method using exosome-free FBS , And hMSC-derived microvesicles (2D-MVs, Exo-free 3D-MVs, 3D-MVs, respectively) cultured through a dynamic 3D culture method are measured and compared, and FIG. 6C shows 2D culture method ( 2D-MVs) or a result of comparing expression levels of microRNAs in hMSC-derived microvesicles (3D-MVs) cultured by a dynamic 3D culture method, and FIG. 6D shows 3D-MVs as human umbilical vein endothelial cells (HUVECs) or neural lines The number of microRNAs expressed after treatment with gastric cells (NSCs) Results of the analysis of the quasi, Figure 6e is a result of measuring and comparing the expression level of the microRNAs in the microvesicles (MSC-MV) and fibroblast-derived microvesicles (MSC-MV) treated with ischemia-brain tissue extract to be.
7 is a result of confirming the stimulation effect of angiogenesis of MSC-derived microvesicles, and FIG. 7A shows ischemic-brain tissue extract-treated MSC-derived microvesicles (IBE-MVs) and dynamic 3D culture in human umbilical vein endothelial cells (HUVECs) The result of treatment of hMSC-derived microvesicles (3D-MVs) cultured by the method and evaluation of angiogenesis level, FIG. 7B is an evaluation result of angiogenesis of 3D-MVs and miR-210 transfected cells, and FIG. 7c is a result of evaluating the degree of angiogenesis after treatment with MSC-derived microvesicles (2D-MVs and Exo-free 3D-MVs, respectively) cultured by a 2D culture method and a dynamic 3D culture method using exosome-free FBS.
8 is a result of confirming the neural stimulation ability of MSC-derived microvesicles, and FIGS. 8A and 8B are MSC-derived microvesicles (IBE-MVs) treated with rat-derived neural stem cells treated with ischemic-brain tissue extracts and dynamic 3D culture method As a result of processing the microvesicles (3D-MVs) derived from hMSCs cultured with the cells, and evaluating the ability to stimulate nerve development through microscopic observation (FIG. 8A) and immunocytochemical staining (FIG. 8B) on the 4th day of culture, FIGS. 8C and 8 8d is a 2D culture method, MSC-derived microvesicles (2D-MVs, Exo-free 3D-MVs, respectively) cultured by a dynamic 3D culture method using exosome-free FBS, followed by microscopic observation on the 4th day of culture (FIG. 8C) and immune cells It is a result of evaluating the ability to stimulate nerve development through a chemical staining method (FIG. 8D), and FIG. 8E is a result of evaluating the ability to proliferate neural stem cells of cells transfected with 3D-MVs and miR-184.
Figure 9a is a result of evaluating the degree of angiogenesis ability and neural stem cell proliferation, respectively, compared to the case of transfection of microRNA-210 or microRNA-184 with rMSC-derived microvesicles (rMSC-MV) treated with ischemia-brain tissue extract , Figure 9b is a result of confirming the inhibition of the expression of the target protein of Ephrin A3 and Numbl of the microRNAs in cells treated with rMSC-MV or miR-210 and miR-184, respectively, through Western blot, Figure 9c Is a result of confirming the inhibition of the expression of the target proteins of Ephrin A3 and Numbl of the microRNAs in cells treated with 3D-MVs or miR-210 and miR-184, respectively, through Western blot.

The present inventors produce a PEG hydrogel microwell array to dynamically generate 3D culture of stem cells or to apply ischemic stimulation to stem cells, and generate various therapeutic substances including microvesicles derived from stem cells and therapeutic microRNAs in the microvesicles. This is promoted, and the effect of stimulation of angiogenesis and neurogenesis by the microvesicles derived from the stem cells was experimentally confirmed, and based on this, the present invention was completed.

Accordingly, the present invention is a microRNA-137 (miR-137), microRNA-184 (miR-184) and microRNA-210 (miR-210) selected from the group consisting of one or more expression level is enhanced, stem cells It provides a pharmaceutical composition for preventing or treating stroke, comprising the derived microvesicles.

In addition, the present invention provides a method for promoting the production of microRNAs (microRNAs) in microvesicles derived from stem cells, comprising the step of culturing the stem cells in three dimensions.

In addition, the present invention provides a method for promoting the production of microRNAs (microRNAs) in microvesicles derived from stem cells, comprising the step of stimulating ischemia of the stem cells.

The microRNA may be one or more selected from the group consisting of miR-137, miR-184, and miR-210.

The present inventors can promote the production of microvesicles derived from stem cells and microRNAs in the microvesicles through the three-dimensional culture method and the ischemic stimulation of stem cells according to the present invention, and accordingly the stem cells and their fines It has been found that the efficacy of vesicles can be enhanced.

In one embodiment of the present invention, a PEG hydrogel microwell array was prepared for a three-dimensional culture according to the present invention, and spontaneous spheroid formation was performed by dispensing mesenchymal stem cells (MSC) into each of the microwells at a constant density. After induction, 3D culture was performed while rotating and stirring at a constant rate in the incubator for 7 days. As a result, it was confirmed that the stem cells were densely packed to form a spheroid, secrete the extracellular matrix, and that most of the cells constituting the spheroid survived. In addition, as a result of observing cell proliferation according to the cultivation time while conducting 2D or 3D cultivation with or without agitation, it was confirmed that in the case of 3D culturing according to the present invention, the number of cells did not increase until the 7th day of cultivation and the initial cell number was maintained (See Example 1).

In another embodiment of the present invention, as a result of analyzing a change in the expression profile of 84 genes related to the characteristics of stem cells after static 2D culture of MSC or dynamic 3D culture of the method according to the present invention, the expression of various genes is upward Or it was observed to be down-regulated, and through this analysis, it was found that the differentiation potential for cartilage formation and bone formation in the 3D cultured MSC according to the present invention was significantly improved (see Example 2).

In another embodiment of the present invention, after performing the dynamic 3D culture according to the present invention and culturing the MSC through other conventional culture methods as described above, the microvesicles are separated from the MSC cultured by each culture method to produce and fine As a result of measuring the total protein amount of vesicles, it was confirmed that a significantly higher amount of microvesicles was generated when cultured by the method according to the present invention compared to other methods (see Example 3).

In another embodiment of the present invention, the microvesicles derived from the MSC-derived microvesicles cultured by the dynamic 3D culture method according to the present invention and the ischemic-brain tissue extract on stem cells are obtained, respectively, to obtain the microvesicles derived from the ischemia-stimulated MSC The therapeutic substances contained in the vesicles were analyzed. As a result, it was confirmed that although there is a difference in the expression level in both types of microvesicles, cytokines related to immune regulation and angiogenesis are generally contained. In addition, it was confirmed that a large amount of microRNAs known to be related to neurogenesis and angiogenesis were present (see Example 4).

In another embodiment of the present invention, the vascular stimulation and neurogenic stimulation effects of each of the microvesicles treated with the dynamic 3D cultured MSC derived microvesicles and the ischemic-brain tissue extract treated MSC derived microvesicles were examined. As a result, it was confirmed that both types of microvesicles stimulated the formation of ducts of human umbilical vein endothelial cells, the proliferation of neural stem cells, and the differentiation into neurons at a significant level. Compared, it was confirmed that there is a significant effect. Furthermore, the microvesicles derived from the dynamic 3D cultured MSC and the microvesicles treated with the ischemic-brain tissue extract were similar or higher compared to those transfected with miR-210 and miR-184, which are known to be related to angiogenesis and neurogenesis. It exhibited angiogenic and neurogenic stimulation effects at the level, and it was confirmed that the miRNAs inhibit the expression of target proteins Ephrin A3 and Numbl, respectively (see Example 5).

These results promote the production of microvesicles and microRNAs in the microvesicles by stimulating ischemia or 3D culturing the stem cells by the method according to the present invention, thereby enhancing the efficacy of stem cells or microvesicles derived from the stem cells. Is to prove that it can.

In the present invention, the stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSC) or adult stem cells, preferably, adult stem cells are mesenchymal stem cells, human tissue-derived mesenchymal matrix Cells, human tissue-derived mesenchymal stem cells, and may be one or more selected from the group consisting of multipotent stem cells, but is not limited thereto.

The overall concept and effect of the dynamic 3D mesenchymal stem cell culture method according to the present invention is illustrated in FIG. 1 by the method, the stem cell-derived microvesicles are simple and There is an advantage that can be obtained in large quantities in an efficient manner. Specifically, the dynamic three-dimensional culture is performed in the PEG hydrogel microwell prepared in the present invention, and more specifically, a predetermined amount of stem cells are dispensed into the microwell and for 6 to 18 hours, more preferably for about 12 hours. After inducing spontaneous spheroid formation, it may be incubated for 5 to 9 days with 20 to 40 rpm, more preferably 30 rpm in the incubator, and more preferably for about 6 to 7 days Process.

Ischemic stimulation of stem cells according to the present invention is achieved through a process of processing brain tissue extract of an individual inducing brain ischemia, and processing ischemic brain tissue extract on mesenchymal stem cells for 12 to 48 hours, more preferably Was cultured for 24 hours to induce ischemia stimulation.

In the present invention, the potentiation may include an increase in the expression of growth factors, cytokines, or microRNAs (microRNAs) in stem cells, the growth factors being fibroblast growth factor (FGF). , Hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFβ) and bone morphogenetic protein (Bone Morphogenetic Protein 2; BMP2) It may be one or more selected from the group consisting of, the cytokine is the cytokine is CH13L1, CD105, CD147, ICAM-1, IP-10, MIP-1β, IL-6, IL-8, GRO, TIMP- 1 and SerpineE1 may be one or more selected from the group consisting of, and the microRNA may be one or more selected from the group consisting of miR-137, miR-184, and miR-210, but is not limited thereto.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example  One. Mesenchyme  3D culture of stem cells

1-1. Limited in size hMSC - Spheroid  Formation and culture

For 3-dimensional cell culture of mesenchymal stem cells, polyethylene is produced through a soft-lithography process using a poly (dimethylsiloxane) (PDMS) mold as shown in FIG. 2A. A glycol (polyethyleneglycol; PEG) microwell array was prepared, and then sterilized with 70% ethanol and PBS was added to perform UV sterilization for 20 minutes. The custom microwell array manufactured by the present inventors is composed of a cylindrical microwell with an inverted pyramid opening, so that there is no cell loss when culturing hMSC-spheroids in large quantities, and the microscopy is performed through an optimized PEG hydrogel soft-lithography technology. Cell adhesion to the well substrate was prevented. In addition, in order to control the size and cell number of the hMSC-spheroids in a constant manner, the size of a microwell array containing 1,225 microwells with a diameter of 200 μm each fits each well of a commercial 6-well plate 20 ×. It was made in 20 mm size. Thereafter, in order to dispense the cells in the microwell, human-derived medium cultured in DMEM medium containing 10% fetal bovine serum (FBS) or exosome-free FBS (FBS) and 1% antibiotic Cells were collected and counted by treating trypsin on mesenchymal stem cells (hMSCs, PT2501, Lonza, Basel, Switzerland), followed by dispensing hMSCs at a density of 5 × 10 ⁵ cells / array (~ 400 cells / microwell) and then CO ₂ It was cultured in an incubator (37 ° C., 5% CO ₂ ), and it was confirmed that spherical cell aggregates spontaneously formed within 12 hours after dispensing as shown in FIG. 2B. The h-MSC-spheroid was uniformly formed to a diameter of about 150 μm smaller than the size of the microwell, and then further incubated for 7 days at 30 rpm in an orbital shaker in a CO ₂ incubator (3D w / shaking) ).

1-2. Through dynamic 3D culture (3D w / shaking) Spheroid  Observation and cell Proliferative capacity  analysis

For hMSCs cultured by the method of Example 1-1, first, to verify the viability of cells constituting the spheroid, LIVE / DEAD Viability / Cytotoxicity Kit (Invitrogen, Carlsbad, CA, USA) was used on the 5th day of culture. Was used to conduct the experiment. As a result, it was confirmed that most of the cells survived (green fluorescence) as shown in FIG. 2C.

In addition, hematoxylin and eosin (H & E) and Masson trichrome (M & T) staining were performed on hMSC-spheroids on the 5th day of culture, as shown in FIGS. 2D and 2E. Likewise, it was found that hMSCs are densely aggregated to form a spheroid and secrete the extracellular matrix (ECM).

Furthermore, in order to check the proliferation ability of hMSCs, 3 days (D3), 5 days while incubating hMSCs with 2D culture method (2D or 2D w / shaking) without stirring or with static 3D culture method without stirring, respectively with the dynamic 3D culture method Cell numbers were measured using DNA quantifcation assay kit (CyQUANT NF Cell Proliferation Assay Kit, Invitrogen) on (D5) and 7th (D7). As a result, as shown in FIG. 2f, when cultured in a static 2D culture method (2D) without agitation, the number of hMSCs increased according to the culture time, and continuously applied when cultured in a dynamic 2D culture method (2D w / shaking) The number of cells decreased after about 5 days due to the susceptibility of adherent cells to shear stress. On the other hand, in the case of the static 3D culture method, hMSCs were formed in a microwell to form a spheroid, then transferred to a petri dish and cultured without agitation. Interestingly, the hMSC-spheroid was attached to the bottom of the dish after 1 day of culture and propagated to the surrounding area. Was observed. This phenomenon was presumed that the ECM secreted from hMSCs initially diffused on the petri dish, and then hMSCs constituting the spheroid were attached to them. Therefore, in the case of static 3D culture (3D), hMSCs were found to be almost double the number of cells at the 5th day of culture (D5) and maintained until the 7th day of culture. In contrast, in the case of dynamic 3D culture (3D w / shaking) according to the present invention, it was found that the number of cells did not increase during the culture period. These results were consistent with the results of previous studies that MSC-spheroids cultured using fetal bovine serum (FBS) medium maintained initial cell numbers while maintaining biological characteristics such as stem cells.

Example  2. Dynamic 3D cultured MSC - Spheroidal  Gene expression profile analysis

Next, the present inventors performed expression profiling of 84 major genes related to the general characteristics of hMSCs through PCR in order to verify differences between stem cell characteristics of 2D or dynamic 3D cultured hMSCs, and PCR array in FIG. 3A. Gene expression was relatively different between MSCs cultured in 2D or dynamic 3D using a cluster gram of. In the case of 3D culture, the hMSC (3D-MSC D1) on the first day of culture seemed to temporarily change the gene expression profile because these cells were in the process of forming 3D-spheroids for 7 days in culture. In addition, as shown in Figure 3b, FGF2, LIF and POU5F1 related to stem cell genes (stemness) were found to be highly expressed in all culture methods (2D, 3D-D1, 3D-D7). Heavy FGF2 and LIF generally decreased somewhat during hMSC-spheroid formation (3D-D1) and then increased back to 3D culture (3D-D7). Moreover, various hMSCs marker genes were found to be highly expressed in dynamic 3D-MSC compared to 2D-MSC.

Furthermore, the expression of genes showing the characteristics of hMSCs having an average Ct value of less than 30 was expressed by a scatter plot and comparison between groups was performed. At this time, in order to avoid overestimating, the case of up-regulation or down-regulation of 30 times or less in the relative comparison was not regarded as a significant difference. As a result of the analysis, as shown in FIG. 3C, on the first day of hMSC-spheroid formation (3D-MSC D1), GDF15 and TGFB3 were found to be up-regulated about 40 times compared to the 2D cultured control (2D-MSC). On the other hand, BMP4 was down regulated about 60 times. When comparing between dynamic 3D cultured hMSCs, IL1B, BDNF and BMP2 were up-regulated more than 30 times on day 7 of culture (3D-MSC D7), and COL1A1 was compared to the initial stage of culture day 1 (3D-MSC D1) It was found to be adjusted down about 50 times. A significant decrease in COL1A1 as it progresses from D1 to D7 means that an increase in ECM secretion in the early stage of culture is only necessary for the structural composition of 3D hMSCs aggregates. Next, when 3D-MSCs D7 were compared to 2D-MSCs, IL1B and GDF15 were up-regulated by about 40-fold and 90-fold, respectively, and in particular, BMP2 was found to be very up-regulated up to about 230-fold. Through these results, it was found that the differentiation potential for cartilage formation (upregulation of TGFB3) and bone formation (upregulation of BMP2) in 3D cultured MSC was significantly improved, which is consistent with the results of previous studies.

Example  3. Dynamic 3D- MSC  Analysis of microvesicle production by culture

3-1. From MSC  Microvesicle separation

In order to separate microvesicles from MSCs, the culture solution of the MSCs cultured by each method is collected, and then centrifuged at low speed (2,500 xg, 10 ° C, 10 minutes) to remove impurities from the culture supernatant, and then again, high-speed centrifugation (14,000 xg) , 10 ° C., 45 minutes) to obtain microvesicles derived from stem cells.

3-2. MSC  Confirmation of the increase of the origin microvesicle

The present inventors isolated from MSCs cultured by static 2D culture (2D), dynamic 2D culture (2D w / shaking), static 3D culture (3D) and dynamic 3D culture (3D w / shaking) methods using flow cytometry. The aim was to measure the phenotype and amount of microvesicles. To this end, as shown in FIG. 4A, particles having a size of 1.0 μm or less (red solid square) and anti-measurement measured using standard sized beads on 3 days (D3), 5 days (D5), and 7 days (D7) of culture and anti Double positive staining (blue dotted square) for -CD105 (hMSC surface marker) and anti-annexin V (lipid surface marker) were counted as microvesicles derived from MSC. The counted dots (purple solid squares) were used to calculate the absolute number of microvesicles, and the final microvesicle count was corrected by the number of cells in the culture group. As a result, in the case of dynamic 3D culture (3D w / shaking) as shown in FIG. 4B, the number of microvesicles derived from hMSC was measured to be the highest, which is about 100 times more than a static 2D culture control in which a very small number of microvesicles were measured. It was found to be high. In the case of dynamic 2D culture (2D w / shaking), the number of microvesicles collected was not significant, but was higher than that of static 2D culture. In addition, in static culture, the formation of hMSC-spheroids was 3D culture (3D). As observed in one case, it has been shown to increase microvesicle production, although not in significant amounts. In addition, as can be seen in Figure 4c, the protein analysis results corrected by the number of cells in each culture group on the 7th day of culture supported the above results, and the total protein concentration of the microvesicles obtained in the case of dynamic 3D culture compared to other groups It was confirmed that it was significantly higher than the other groups. Moreover, through the results obtained using exosome-free FBS (Exo-free 3D-MVs), it was confirmed that the increase in the production of MSC-derived microvesicles confirmed in this experiment was not affected by the particles contained in the FBS.

Example  4. MSC  Analysis of expression of therapeutic substances in derived microvesicles

The present inventors tried to verify the therapeutic properties of microvesicles isolated from hMSCs cultured in a large scale by a dynamic 3D culture method. Along with this, the present inventors confirmed that the efficacy of MSCs can be improved when the ischemic-brain tissue extract is pre-treated to MSCs through a previous study, by isolating microvesicles derived from MSC treated with ischemic-brain tissue extract. We also tried to analyze its therapeutic properties.

4-1. Ischemia-brain tissue extract preparation and treatment

Transient middle cerebral artery occlusion (tMCAo) was induced in rats for 90 minutes, and after 3 days, damaged hemisphere tissue was crushed with DMEM medium at a concentration of 150 mg / ml. Next, the pulverized tissue solution was centrifuged at 10,000 xg for 10 minutes, and then the supernatant was taken to obtain an ischemic brain extract (IBE), and the obtained ischemic brain tissue extract was dispensed in equal amounts until use- Stored at 70 ° C. Then, to apply ischemia stimulation to the MSC, the stored ischemic brain tissue extract was centrifuged at 2,500 xg for 10 minutes to remove impurities, then diluted 5 times in DMEM, then centrifuged again at 14,000 xg for 45 minutes and 0.2 um filter. Filtered with. The ischemic-brain tissue extract prepared by the above method was treated with marrow-derived adult stem cells (rMSCs) or hMSCs from 220-250 g of Sprague-Dawley (SD) male rat femur and tibia for 24 hours.

4-2. IBE - MVs  And 3D- MVs  Check my therapeutic substance level

The purpose of this study was to determine whether therapeutic substances are contained in MSC-derived microvesicles (IBE-MVs) treated with hMSC-derived microvesicles (3D-MVs) and ischemia-brain tissue extracts cultured by a dynamic 3D culture method. To this end, first, representative cytokines included in the microvesicles were analyzed using various cytokine array kits. As a result, it was confirmed that IBE-MVs contain various cytokines related to immune regulation and angiogenesis as shown in FIGS. 5A and 5B. In addition, in general, it was found that cytokines detected in IBE-MV are also included in 3D-MVs. In particular, IP-10, MIP-1, βIL-8, GRO, and TIMP-1 were found to be at a high level with some differences in both IBE-MVs and 3D-MVs. In addition, some cytokines appeared differently in the two groups, while 3D-MVs contained a large amount of ICAM-1, bFGF, CHI3L1, CD147, and CD105, whereas IBE-MVs contained a large amount of IL-6 and SerpineE1. Turned out to be.

In addition, according to the method of Example 4-1, ischemia-brain tissue extract was treated to isolate microvesicles from mouse bone marrow-derived mesenchymal stem cells and expression of growth factors affecting stroke with fibroblast-derived microvesicles Were compared via Western blot. As a result, as shown in Figure 5c, it was confirmed that the microvesicle markers Flotillin-1 and HSP70 proteins are expressed in both microvesicles derived from the two cells, and fibroblast-derived microvesicles treated with ischemia-brain tissue extract (Fibroblast- Compared to MV), it was confirmed that the expression of proteins related to stroke treatment was increased in microvesicles derived from mesenchymal stem cells (MSC-MV).

4-3. IBE - MVs  And 3D- MVs  For my treatment microRNA  Level check

In addition to the results of Example 4-2, the present inventors performed neuronal and / or angiogenesis signaling in hMSC-derived microvesicles treated with ischemia-brain tissue extracts and dynamic 3D cultured hMSC by performing qPCR. The expression level of microRNAs known to be important was analyzed. As a result, as shown in Figure 6a, it was confirmed that miR-137, and miR-184, which are reported to be related to neurogenesis, are present in a large amount of microvesicles (IBE-MVs) derived from ischemia-brain tissue extract treated hMSC. In the case of miR-210, which is known to be related to both neurogenesis and angiogenesis, it was found to be highly expressed in both IBE-MVs and 3D-MVs, and was relatively more contained in 3D-MVs than IBE-MVs. Was confirmed.

In addition to the above results, in order to verify whether the dynamic 3D culture method is effective in increasing the expression of therapeutic microRNAs in microvesicles derived from MSC, cultured through 2D culture method, dynamic 3D culture method using exosome-free FBS, and dynamic 3D culture method, respectively. The microRNA content in microvesicles derived from hMSC (2D-MVs, Exo-free 3D-MVs, and 3D-MVs, respectively) was compared. As a result, as shown in Figure 6b, in the case of 2D culture method, all of the therapeutic microRNAs were weakly expressed, whereas in two cases of dynamic 3D culture, miR-134 and miR-137 were found to be expressed at high levels, and miR- In the case of 210, it was confirmed that it is highly expressed in 3D-MVs.

In addition, miR137, miR-184 and miR, which are known to be important for angiogenesis and / or neurogenic signaling in microvesicles (3D-MVs) derived from hMSCs cultured by 2D culture (2D-MVs) or dynamic 3D culture methods The expression level of -210 was analyzed. As a result, it was confirmed that miR137, miR-184 and miR-210 are higher in 3D-MVs than in 2D-MVs, as shown in FIG. It was found that the expression of microRNAs increased.

In addition, 3D-MVs were treated with human umbilical vein endothelial cells (HUVECs) or neural stem cells (NSCs), and then expression levels of microRNAs were analyzed. As a result, as shown in Fig. 6d, when 3D-MVs were treated with human umbilical vein endothelial cells (HUVECs), it was confirmed that miR-210 was expressed higher than the control (CTRL), and 3D-MVs were expressed as neural stem cells ( NSCs), it was confirmed that miR-137 and miR-184 were expressed higher than the control (CTRL).

Furthermore, as a result of comparing the microRNA content of rMSC-derived microvesicles treated with ischemia-brain tissue extract and fibroblast-derived microvesicles, miR-210, miR in rMSC-derived microvesicles (MSC-derived MVs) as shown in FIG. It was confirmed that the expression of -184, and miR-137 was significantly increased. Through this, it was found that the expression of the therapeutic microRNAs specifically increased in MSC by ischemia-brain tissue extract treatment.

From the above results, ischemia-brain tissue extract-treated MSC-derived microvesicles and dynamic 3D cultured MSC-derived microvesicles are rich in various therapeutic cytokines and micro RNAs related to immunoregulation, angiogenesis, and neurogenesis. Was confirmed.

Example  5. MSC  The effect of the resulting microvesicles to produce new blood vessels and Neurogenesis  Stimulation effect verification

5-1. IBE -MV and 3D-MV's ability to produce blood vessels

The present inventors investigated the therapeutic effect of the collected microvesicles using an in vitro model for angiogenesis and neurogenesis. First, human umbilical vein endothelial cells inoculated on matrigel in order to evaluate the angiogenic ability of hMSC-derived microvesicles (IBE-MV) and hMSC-derived microvesicles cultured in dynamic 3D, each treated with ischemia-brain tissue extract, HUVECs) were treated with 3 μg / mL of IBE-MV and 3D-MV, respectively, and then generated with the control or vascular endothelial growth factor (VEGF) treated group with only basic medium added. The degree of angiogenesis was compared through loop numbers, branch numbers, and branch length.

As a result, as shown in FIG. 7A, when the VEGF treatment was performed, the degree of tube formation was significantly increased compared to the control group, and when the IBE-MV treatment was performed, it was found that the level was similar to that of the VEGF treatment group. In addition, it was confirmed that when 3D-MV treatment was performed, tube formation was induced at a higher level.

In addition, the therapeutic effect of 3D-MVs and miR-210 collected using an in vitro model for angiogenesis was investigated. First, in order to evaluate the ability to generate 3D-MVs and miR-210 angiogenesis, non-specific miRNA and / or miR-210 were transfected into human umbilical vein endothelial cells (HUVECs) inoculated on Matrigel, and then 3D-MV was respectively injected. After 3 μg / mL treatment, loop numbers and branch numbers generated with the control group (CTRL) or vascular endothelial growth factor (VEGF) treated group with only basic medium added , And the degree of angiogenesis was compared through branch length.

As a result, as shown in FIG. 7B, when the VEGF treatment was performed, the degree of tube formation was significantly increased compared to the control group, and when the 3D-MVs treatment was performed, it was found that the level was similar to that of the VEGF treatment group. In addition, it was confirmed that the tube formation was induced at a high level even when miR-210 was transfected.

Furthermore, in order to verify once again the improvement of the ability of MSC-derived microvesicles to generate blood vessels according to the dynamic 3D culture method, hMSC-derived microvesicles (2D-MVs, respectively) cultured through 2D culture method and dynamic 3D culture method using exosome-free FBS, respectively. Exo-free 3D-MVs) were used to treat HUVECs cells in the same manner as above, and the results were compared. As a result, as shown in FIG. 7c, it was confirmed that when the Exo-free 3D-MVs were treated, vascular production was induced at a significantly higher level than when the VEGF or 2D-MVs was treated.

5-2. IBE -Verifying neurogenic stimulation ability of MV and 3D-MV

Next, to investigate the ability to stimulate the neurogenesis of hMSC-derived microvesicles (IBE-MV) and hMSC-derived microvesicles cultured in dynamic 3D, each treated with ischemia-brain tissue extract, SD rats at 14.5 days Ischemic-brain tissue extract-treated hMSC-derived microvesicles (IBE-MV) or dynamic 3D-cultured hMSC-derived microvesicles (3D-MV) on primary cultured neural stem cells (NSCs) from the cerebral cortex isolated from embryos After treatment with 3 μg / mL, the ability to generate neurons was compared with the control or nerve growth factor (NGF) -treated group with only basic medium added.

First, as shown in FIG. 8A, microvesicles were treated by the above method and observed under a microscope on the 4th day of culture. As a result, differentiation into neurons was induced in the NGF, IBE-MV and 3D-MV treated groups compared to the control group. Found. In addition, as shown in FIG. 8B, the degree of differentiation into neurons was evaluated by quantifying Tuj1 expression in NSC on the 4th day of culture through immunocytochemical staining, and Ki67 expression was quantified to evaluate the proliferation rate of neural stem cells. , In the case of IBE-MV, it was confirmed that the neural stem cell's neurogenic stimulation ability was the highest, and in the case of 3D-MV, it was confirmed that induction of neuronal differentiation and proliferation of NSC was similar to that of the NGF-treated group.

In addition to the above results, hMSC-derived microvesicles (2D each) cultured through dynamic 3D culture using 2D culture and exosome-free FBS, respectively, in order to verify once again the ability to stimulate MSC-derived microvesicles by dynamic 3D culture -MVs, Exo-free 3D-MVs) were treated with 3 μg / mL of the primary cultured neural stem cells (NSCs), and then compared with a control or NGF treatment group. As a result, the microvesicles were treated with the above method and observed under a microscope on the 4th day of culture. As shown in FIG. 8C, NGF, 2D-MVs and Exo-free 3D-MV treated groups compared to the control group to neurons. It was found that differentiation was induced. In addition, as shown in FIG. 8D, as a result of performing immunocytochemical staining, there was no significant difference in the proliferation of neural stem cells through Ki67 expression, while quantifying Tuj1 expression resulted in NGF and 2D-MVs treatment groups to neurons. It was found that the differentiation increased to a significant level, and it was confirmed that differentiation into neurons was induced at a higher level in the Exo-free 3D-MVs treatment group.

Furthermore, in order to evaluate the ability of 3D-MVs and miR-184 to proliferate neural stem cells, non-specific miRNA and / or miR-184 were transfected into neural stem cells (NSCs), and then 3D-MV was treated at 3 μg / mL, respectively. After that, Ki67 / DAPI expression level was quantified together with the control (CTRL) to which only basic medium was added to compare the proliferation rate of neural stem cells.

As a result, as shown in Figure 8e, compared to the control group, it was confirmed that the proliferation of neural stem cells was significantly increased in the group transfected with miR-184 or treated with 3D-MVs.

From the above results, it was found that the ability to stimulate the angiogenesis and neurogenic stimulation of stem cells-derived microvesicles is improved by the dynamic 3D culture method according to the present invention compared to the 2D culture method.

5-3. IBE -For treatment in MV microRNAs  Effect verification

From the results of the above examples, the present inventors Ischemic-brain tissue extract-treated hMSC-derived microvesicles and dynamic 3D-cultured hMSC-derived microvesicles contain high levels of therapeutic microRNAs, and confirm the ability to stimulate angiogenesis and neurovascular formation by each microvesicle treatment. In order to verify whether the therapeutic microRNAs contained in the microvesicles affect the ability of the microvesicles as described above.

To this end, first, miR-210 was transfected into HUVEC cells or treated with rMSC-derived microvesicles (rMSC-MV) treated with ischemia-brain tissue extracts, and the degree of angiogenesis was compared, and also in the neural stem cell line (ReN cell). After transfection of miR-184 related to neurogenesis or treatment of rMSC-derived microvesicles (rMSC-MV) treated with ischemia-brain tissue extracts and incubation for 48 hours, neurogenic ability was compared. As a result, as shown in FIG. 9A, when miR-210 was transfected, angiogenesis was significantly increased compared to the negative control group, and when the rMSC-MV treatment was performed, angiogenesis was more generated than when miR-210 was transfected. It was found to be induced at a high level, and also when miR-184 was introduced compared to the negative control (CTRL) transfected with non-specific miRNA instead of miR-184, proliferation of neural stem cells was significantly increased, similarly to the micro Even when the vesicles were treated, it was confirmed that the proliferation of neural stem cells increased.

In addition, Western blot was performed to analyze whether the target protein expression was inhibited by the microRNAs. More specifically, the control and transfected cells were washed with PBS buffer, lysed with Lysis buffer, and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a certain amount of the lysate to separate proteins by size. Next, it was transferred to a nitrocellulose membrane and the expression levels of Ephrin A3 and Numbl, target proteins of miR-210 and miR-184, respectively, were observed. As a result, when miR-210 and miR-184 were transfected as shown in FIGS. 9B and 9C, the expression levels of Ephrin A3 and Numbl were decreased, respectively, and cells treated with rMSC-MV or 3D-MV were It was confirmed that the expression of Ephrin A3 and Numbl was significantly suppressed in the treated cells, respectively.

From the above results, the miR-210 and miR-184 contained in the microvesicles in each cell treated with rMSC-MV or each cell treated with 3D-MV inhibited the expression of Ephrin A3 and Numbl, respectively, thereby generating blood vessels and generating neurons. It was found that it mediates stimulation.

The above-described description of the present invention is for illustration only, and a person having ordinary knowledge in the technical field to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (17)

MicroRNA-137 (miR-137), microRNA-184 (miR-184) and microRNA-210 (miR-210) selected from the group consisting of one or more expression levels enhanced, including stem cell-derived microvesicles A pharmaceutical composition for preventing or treating stroke.
A method of promoting the production of microRNAs (microRNAs) in microvesicles derived from stem cells, comprising culturing the stem cells in three dimensions.
A method for promoting the production of microRNAs (microRNAs) in stem cells-derived microvesicles, comprising stimulating ischemia to the stem cells.
The method of claim 2 or 3,
The microRNA is characterized in that at least one member selected from the group consisting of miR-137, miR-184, and miR-210, production promoting method.
The method of claim 2 or 3,
The stem cells are embryonic stem cells, induced pluripotent stem cells (induced pluripotent stem cells; iPSC) or adult stem cells, characterized in that the production promoting method.
The method of claim 5,
The adult stem cells are mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and characterized in that one or more adult stem cells selected from the group consisting of multipotent stem cells, produced Promotion method.
According to claim 2,
The three-dimensional culture is characterized in that cells are cultured and cultured for 5 to 9 days while rotating and stirring at 20 to 40 rpm after 6 to 18 hours in the incubator.
According to claim 3,
The ischemia stimulation, characterized in that by the treatment of the brain tissue extract of ischemic individuals, production promotion method.
According to claim 3,
The ischemia stimulation, characterized in that made for 12 hours to 48 hours, production promoting method.
A method of enhancing the efficacy of stem cells or microvesicles separated therefrom, comprising culturing the stem cells in three dimensions.
A method of enhancing the efficacy of stem cells or microvesicles separated therefrom, comprising the step of stimulating ischemia of the stem cells.
The method of claim 10 or 11,
The efficacy enhancement is characterized in that the expression of growth factors, cytokines (cytokine), or microRNAs (microRNAs) in stem cells is enhanced, the efficacy enhancement method.
The method of claim 12,
The growth factors are fibroblast growth factor (FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), transforming growth factor beta TGFβ) and bone-forming protein (Bone Morphogenetic Protein 2; BMP2), characterized in that at least one member selected from the group consisting of, efficacy enhancement method.
The method of claim 12,
The cytokine is characterized in that at least one member selected from the group consisting of CH13L1, CD105, CD147, ICAM-1, IP-10, MIP-1β, IL-6, IL-8, GRO, TIMP-1 and SerpineE1, Efficacy enhancement method.
The method of claim 12,
The microRNA is characterized in that at least one member selected from the group consisting of miR-137, miR-184, and miR-210, potency enhancing method.
The method of claim 10 or 11,
The stem cells are embryonic stem cells, induced pluripotent stem cells (induced pluripotent stem cells; iPSC) or adult stem cells, characterized in that the efficacy enhancement method.
The method of claim 16,
The adult stem cells are mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and one or more adult stem cells selected from the group consisting of multipotent stem cells, efficacy How to strengthen.

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