A method for reprogramming cells using environmental influx induced by physical stimuli

Abstract

To provide a method for reprogramming cells using environmental influx by physical stimulation.SOLUTION: A method of reprogramming cells comprises: providing a mixture of differentiated or undifferentiated cells and a culture medium with a physical stimulus of any one of ultrasound, laser, light-emitting diode, and heat treatment that can promote environmental influx; culturing the physically stimulated mixture for 1 to 6 days; and mixing exosome-containing extracellular vesicles isolated from the culture medium with differentiated or undifferentiated cells, followed by culturing the mixture for a certain period of time to obtain reprogrammed cells.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for reprogramming cells using environmental influx with physical stimuli such as ultrasound, laser or heat treatment.

[0002] The Korea National Research and Development Project which supported this invention is an advanced medical technology development project implemented by the Ministry of Health and Welfare and the Korea Health Industry Development Institute as the lead implementing agency, the project ID number is "HI14C3297", the research title is "Development of a method for monitoring in vivo stem cell distribution and neural differentiation using a microRNA tracking system in an ischemic brain injury model", and was supported by the lead implementing agency, Catholic Gwangdong University Industry Technology Holdings Company Limited.

[0003] In addition, the Korea National Research and Development Project that supported this invention is a mid-career researcher support project implemented by the Ministry of Science, ICT and Future Planning and the Korea Research Foundation as the lead implementing agency, the project unique number is "2013R1A2A2A01068140", the research title is "Development of radiation biomolecular imaging method for tracking stem cell differentiation based on microRNA", and the project was supported by the lead implementing agency, Catholic Gwangdong University Industry Technology Holdings Company Limited. [Background technology]

[0004] Methods for reprogramming somatic cells into other types of cells, such as progenitor cells and stem cells, are clinically important techniques in cell therapy, disease models and transplantation. In recent years, these techniques have been attempted by molecular and chemical methods targeting some pluripotency genes and various differentiation-specific expression genes. However, conventional methods have been criticized for their stability and efficiency, and have disadvantages such as complicated processes.

[0005] Cells are exposed to various environments, which affect the expression of genes in cells over short or long periods of time as cells undergo generations, and the stress caused by environmental changes regulates the gene expression program of cells. The medium components in the cell culture environment contain various substances and ions, and the inflow of such environments into cells can be a groundbreaking method to promote cellular changes. However, due to the diversity of polarity and size of cells, the cell membrane, which is composed of phospholipids, does not allow smooth cell transmission and inflow into the various components of the cell culture medium. In recent years, it has been reported that the external environment can flow into cells due to the microbubbles and cavitation effect generated by ultrasound, and that ultrasound stimulation has a positive effect on cell development. It has also been reported that ultrasound induces ATP, which reacts with receptors on the cell membrane to induce the transport of substances.

[0006] From this perspective, the inventors have devised a method of temporarily damaging somatic cell membranes using physical stimuli such as ultrasound, and of transmitting various substances into cells using the cavitation effect of the culture medium caused by ultrasound. They have developed a method of reprogramming cells using environmental influx caused by physical stimuli, known as "Physical stimulation-mediated permeation of Environmental transition guided cellular reprogramming; ENTER cells," thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0007] It is an object of the present invention to provide a method for reprogramming differentiated cells by physical stimuli that can promote environmental influx. [Means for solving the problem]

[0008] In order to achieve the above objective, The present invention provides a method for producing a culture medium comprising the steps of: providing a mixture of differentiated or undifferentiated cells and a culture medium with a physical stimulus capable of promoting environmental influx; The present invention provides a method for reprogramming cells, the method comprising culturing the mixture to which the physical stimulus has been applied for a certain period of time to obtain reprogrammed cells.

[0009] The present invention further comprises providing a physical stimulus capable of promoting environmental influx to a mixture of differentiated or undifferentiated cells and a culture medium, The mixture to which the physical stimulus has been applied is cultured for 1 to 6 days, The present invention provides a method for reprogramming cells, comprising mixing exosome-containing extracellular vesicles separated from the culture with differentiated or undifferentiated cells and culturing the mixture for a certain period of time to obtain reprogrammed cells. Effect of the Invention

[0010] The present invention provides differentiated cells with physical stimuli such as ultrasound, laser or heat treatment that can promote the influx of the environment, without the introduction of reverse differentiation inducers or chemicals into the differentiated cells, and enables cells to be reprogrammed into pluripotent cells or any differentiated cell with a different phenotype from the differentiated cells simply by the influx of the external environment. This type of induction has the effect of increasing the possibility of autologous cell therapy, since the production process is simple and efficient. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing a method for reprogramming into various ENTER (Environmental transition guided cellular reprogramming) cells according to the present invention. [Diagram 2] The results show the damage and repair of cell membranes by ultrasound stimulation. Figure 2a is a cell surface image taken by SEM, and Figure 2b is a live / dead kit stained image. [Diagram 3] The results are shown in (a) Ca influx into cells, (b) ATP response analysis, and (c) RT-PCR analysis of ATP receptor gene expression. [Figure 4] The results show the possibility of influx of external substances into cells by ultrasound stimulation using QD605. Figure 4a shows the analysis results of QD605 influx in usMC-S (ultrasound-exposed medium and cells) spheroids and single cells, and Figures 4b and 4c show the analysis of QD605 influx and transcription factor expression results in usMC-S spheroids (b) and single cells (c) under each environmental condition. [Diagram 5] This shows the results of RT-PCR analysis of exosome RNA in sonicated culture medium. [Figure 6] The results show the transfer of substances via exosomes. Figure 6a is an image capturing the process of QD605 being transferred to other cells via exosomes, Figure 6b shows the expression of protein markers within exosomes, and Figure 6c shows the experimental results of the transfer of poly(A)27-Cy5.5 via exosomes. [Figure 7] Figure 7 shows the cellular changes associated with exosomes in culture medium of sonicated cells and cavity culture of non-sonicated cells. Figure 7a shows the change in cell morphology with culture time, and Figure 7b shows the expression of Oct4 in cells cultured with exosomes for 6 days. [Figure 8] 1 shows a method for direct differentiation of human dermal fibroblasts according to the present invention. [Figure 9] FIG. 9 shows the results of differentiation of human dermal fibroblasts treated with ultrasound in an adipocyte differentiation induction medium into adipocytes. FIG. 9a shows the changes in cells and the results of Oil Red O staining of adipocytes on the 20th day after differentiation induction, and FIG. 9b shows the results of RT-PCR analysis of the expression of adipocyte marker genes in the differentiated cells. [Figure 10]FIG. 10 shows the results of differentiation into neural progenitor cells using neural stem cell differentiation induction medium and ultrasonically treated human dermal fibroblasts. FIG. 10a shows the changes in cells three days after differentiation induction, FIG. 10b shows the expression of neural progenitor cell markers after differentiation induction, and FIG. 10c shows the results of analyzing the expression of neural progenitor cell markers in attached cells. [Figure 11] This shows the results of flow cytometry analysis of the expression of Pax6 and Nestin in cells induced to differentiate into neural progenitor cells (n / ENTER cells). [Figure 12] This shows the expression of Ki67, a proliferation marker, in n / ENTER cells. [Figure 13] FIG. 13a shows the process of cell expansion in a single sphere of n / ENTER cells, and FIG. 13b shows the results of maintaining the attributes of neural progenitor cells in the expanded cells. [Figure 14] The results show the differentiation of n / ENTER cells after transplantation into a mouse brain. Figure 14a shows the expression of an astrocyte marker (Gfap) in transplanted cells (HNA stained cells), and Figure 14b shows the secretion of synapsin (Synl) in cells differentiated after transplantation. [Figure 15] The results show the differentiation of MEFs into neural progenitor cells using neural stem cell differentiation induction medium and ultrasonic stimulation. Figure 15a shows the change in cell morphology, Figure 15b shows the expression of neural progenitor cell markers in the spheres, and Figure 15c shows the results of an analysis of the expression of neural progenitor cell markers using flow cytometry. [Figure 16] This shows the results of an analysis of the expression of differentiation markers for neural precursor cells (mouse n / ENTER cells) induced to differentiate in MEFs. [Figure 17] Schematic representation of directed differentiation into hepatocytes. [Figure 18] FIG. 18 shows the results of differentiation into hepatocytes from HDFs treated with a hepatocyte differentiation-inducing medium and ultrasonic treatment. FIG. 18a shows the change in cell morphology, and FIG. 18b shows the expression of hepatocyte markers. [Figure 19]FIG. 19 shows the results of differentiation of HeLa cells treated with hepatocyte differentiation induction medium and ultrasound into hepatocytes (HeLa h / ENTER), where FIG. 19a shows the change in cell morphology, FIG. 19b shows the results of hepatocyte marker expression using qPCR, and FIG. 19c shows the results of hepatocyte marker expression using immunocytochemistry. [Figure 20] FIG. 20 shows the results of differentiation of Hep3B cells into hepatocytes (Hep3B h / ENTER cells) treated with a hepatocyte differentiation induction medium and ultrasonic treatment. FIG. 20a shows the change in cell morphology, and FIG. 20b shows the expression of hepatocyte markers. [Figure 21] FIG. 21a shows the results of differentiation into es / ENTER cells from human ES culture medium and sonicated HDFs, and FIG. 21b shows the changes in cell morphology, and FIG. 21b shows the changes in Oct4 expression over the culture time. [Figure 22] 1 shows the expression of pluripotency marker genes (a) and proteins (b) in es / ENTER cells. [Figure 23] These are the results of an analysis of pluripotency attributes in es / ENTER cells. Figure 23a shows the results of an analysis of pluripotency markers using flow cytometry, Figure 23b shows the results of an analysis of pluripotency gene expression patterns using microarrays, and Figure 23c shows the results of an analysis of the presence or absence of methylation of the Oct4 and Nanog promoters using bisulfite sequencing. [Figure 24] This shows the expression of three germ layer marker genes (a) and proteins (b) in es / ENTER cells. [Diagram 25] This shows the changes in expression of Oct4 and three germ layer markers in es / ENTER cells according to the culture time. [Figure 26] The figure shows the expression of three germ layer marker proteins in adherently cultured es / ENTER cells (a) and the results of methylation analysis of the three germ layer marker DNA of es / ENTER cells using bisulfite sequencing (b). [Figure 27] The figures show the results of in vitro differentiation of es / ENTER cells into neurons (a), cardiomyocytes (b) and hepatocytes (c). [Figure 28]The figures show the results of analyzing the presence or absence of expression of neuronal (a), cardiomyocyte (b), and hepatocyte (c) markers in HDF. [Figure 29] 1 shows the results of RT-PCR analysis of the expression of differentiation marker genes for neurons (a), cardiomyocytes (b), and hepatocytes (c) in differentiation-induced es / ENTER cells. [Diagram 30] 1 shows the results of karyotype analysis by chromosomal G-banding of es / ENTER cells. [Diagram 31] 31 shows the results of muscle transplantation and in vivo differentiation of es / ENTER cells. FIG. 31a shows the results of differentiation of the transplanted cells into skeletal muscle, and FIG. 31b shows the expression of Oct4 and the presence or absence of proliferation (K167) of the transplanted cells. [Diagram 32] The results are shown in Fig. 32A, which shows the coordinates where the cells were transplanted, Fig. 32B, which shows the secretion of astrocyte differentiation markers (Gfap) and neuronal functional markers (synapsin, Synl, and vesicular glutamate transporter (vGlutl)) of the transplanted cells, and Fig. 32C, which shows the expression of Oct4 and the presence or absence of proliferation (Ki67) of the transplanted cells. [Diagram 33] The results show the differentiation of MEFs into mouse ES / ENTER cells using human ES culture medium and ultrasonic stimulation. FIG. 33a shows the changes in cell morphology and Oct4-GFP expression over the culture time, FIG. 33b shows the spheroid formation rate over the culture time, and FIG. 33c shows the changes in Oct4 expression. [Diagram 34] The results of pluripotency attribute analysis in mouse es / ENTER cells are shown in Figure 34a, where Figure 34a shows the expression of pluripotency marker proteins by immunocytochemistry, Figure 34b shows the expression of pluripotency marker genes by RT-PCR, Figure 34c shows the analysis results of the expression ratio of pluripotency marker genes by flow cytometry, and Figure 34d shows the results of alkaline phosphatase (AP) staining of mouse es / ENTER cells. [Diagram 35]These are the results of an analysis of the three germ layer characteristics in mouse es / ENTER cells. Figure 35a shows the expression of three germ layer markers by immunocytochemistry, and Figures 35b and 35c show the results of an analysis of the expression patterns of three germ layer marker genes (b) and proteins (c) according to the culture time. [Diagram 36] The figures show the results of in vitro differentiation of mouse es / ENTER cells into neurons (a) and cardiomyocytes (b), and the results of karyotype analysis of mouse es / ENTER cells by chromosome G-band analysis (c). [Figure 37] The results show the differentiation of L132 cells into L132 es / ENTER cells using human ES culture medium and ultrasonic stimulation. Figure 37a shows the change in cell morphology over time in culture, and Figures 37b and 37c show the pluripotency (b) and three germ layer (c) attributes of L132 es / ENTER cells. [Figure 38] The results show the differentiation of MSCs into MSC es / ENTER cells using human ES culture medium and ultrasonic stimulation. Figure 38a shows the change in cell morphology over culture time, and Figures 38b and 38c show the pluripotency (b) and three germ layer (c) attributes of MSC es / ENTER cells. [Figure 39] The results show the differentiation of human dermal fibroblasts into SF es / ENTER cells using human ES culture medium and ultrasonic stimulation. Figure 39a shows the change in cell morphology over time in culture, and Figures 39b and 39c show the pluripotency (b) and three germ layer (c) attributes of SF es / ENTER cells. [Diagram 40] The results show the differentiation of HDFs into es / ENTER cells using heat treatment and hES medium. Figure 40a shows the differentiation-induced HDF spheroids, and Figures 40b and 40c show the pluripotency (b) and three germ layer (c) attributes of es / ENTER cells. [Diagram 41] The results show the differentiation of HDFs into es / ENTER cells using laser stimulation and hES medium. Figure 41a shows the differentiation-induced HDF spheroids, and Figures 41b and 41c show the pluripotent (b) and three germ layer (c) attributes of es / ENTER cells. [Diagram 42]These are the results of confirming whether or not extracellular vesicles (EVs) are transmitted to normal somatic cells. [Diagram 43] These are the results of confirming whether EVs induce reprogramming of human fibroblasts. [Diagram 44] This shows changes in cell morphology over time in EVs-treated human fibroblasts harvested upon induction of es / ENTER. [Diagram 45] This is the result of flow cytometry analysis confirming the presence or absence of Oct4 expression in HDFs cultured for 6 days depending on the amount of EVs added. [Figure 46] The results show the presence or absence of expression of pluripotency marker proteins (a: ICC image) and genes (b: qPCR analysis) in EVs-treated HDFs cultured for 3 days, which were collected upon induction of es / ENTER. [Figure 47] The results show the presence or absence of expression of neural stem cell marker proteins (a: ICC image) and genes (b: qPCR analysis) in EVs-treated HDFs cultured for 3 days, which were collected upon induction of n / ENTER. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The configuration of the present invention will be specifically described below.

[0013] The present invention relates to a method for reprogramming cells, which comprises applying a physical stimulus capable of promoting environmental influx to a mixture of differentiated or undifferentiated cells and a culture medium, and culturing the mixture to which the physical stimulus has been applied for a certain period of time to obtain reprogrammed cells.

[0014] The present invention is characterized in that it is possible to induce cellular reprogramming in pluripotency cells, or in any differentiated cell having a phenotype different from that of the differentiated or undifferentiated cells, such as hepatocytes, osteoblasts, adipocytes, muscle cells, nerve cells, astrocytes, keratinocytes, hair root cells, pancreatic β cells, or cardiac muscle cells, by culturing differentiated or undifferentiated cells in any medium capable of inducing the desired reprogrammed cells while applying a physical stimulus capable of promoting environmental influx to the differentiated or undifferentiated cells, such as ultrasound, laser, or heat treatment.

[0015] According to one example, if the goal is to make the reprogrammed cells pluripotent cells, the differentiated cells can be reprogrammed into pluripotent cells by mixing the differentiated cells with stem cell culture medium, applying physical stimuli, and culturing for a certain period of time.

[0016] In another example, when aiming to turn a reprogrammed cell into any differentiated cell having a different phenotype from a differentiated cell, the differentiated cell can be reprogrammed into any differentiated cell having a different phenotype by mixing the differentiated cell with a differentiation-inducing medium of the desired differentiated cell, applying physical stimulation and culturing for a certain period of time.

[0017] In yet another example, undifferentiated cells, such as induced pluripotent stem cells or embryonic stem cells, can be mixed with a differentiation-inducing medium for the desired differentiated cells, and then the cells can be cultured for a certain period of time while being given physical stimulation, resulting in reprogramming into the desired differentiated cells with an improved differentiation rate compared to conventional techniques.

[0018] It is recognized that the cell reprogramming method of the present invention induces reprogramming of differentiated or undifferentiated cells by influx of extracellular environment caused by physical stimulation of differentiated or undifferentiated cells. Such environmental influx refers to the influx of genetic material, chemicals, small molecules, exosomes, or exosome-containing extracellular vesicles; or culture medium components, etc., discharged from a differentiated cell to which a physical stimulation has been applied, into adjacent differentiated or undifferentiated cells.

[0019] According to the cell reprogramming method of the present invention, it is recognized that the influx of the environment into a differentiated or undifferentiated cell can determine the direction of reprogramming into a pluripotent cell that stably expresses pluripotency markers and three germ layer markers, or a differentiated cell that has a phenotype different from the differentiated or undifferentiated cell.

[0020] In addition, it is recognized that the directionality of reprogramming can be determined by the type of culture medium.

[0021] That is, as described above, reprogramming of differentiated or undifferentiated cells into pluripotent cells can be induced when a physical stimulus is applied to a mixture of differentiated cells and stem cell culture medium, reprogramming of differentiated cells into any differentiated cell with a different phenotype can be induced when a physical stimulus is applied to a mixture of differentiated cells and differentiation induction medium for any differentiated cell, and when a physical stimulus is applied to a mixture of undifferentiated cells and differentiation induction medium for any differentiated cell, they can be reprogrammed into any differentiated cell.

[0022] Regarding the influx of the environment into differentiated or undifferentiated cells, the present inventors have considered, in particular, cell membrane damage caused by physical stimuli and cell secreted substances (exosomes or exosome-containing extracellular vesicles). That is, ultrasound, laser, or heat treatment increases the temperature by energy, vibrates microbubbles generated by ultrasound, and induces the generation of liquid flow, i.e., induces the generation of microstreams along the cell membrane. These effects cause minute damage to the cell membrane and induce the generation of holes, thereby increasing the absorption of external substances. This is due to the increase in intracellular Ca 2+ Changes in intracellular Ca 2+ Concentration change analysis shows that when the cell membrane is damaged or its fluidity increases, the intracellular (cytosolic) Ca 2+ According to one embodiment of the present invention, the Ca concentration in the cell membrane is increased immediately after sonication, which reflects an increase in Ca concentration in the cell membrane. 2+The concentration increased quickly, then decreased gradually, and finally decreased to the level of the control group without ultrasound treatment, indicating that the cell membrane was damaged and then restored. It is also known that the generation and increase of ATP by ultrasound is a response to various cellular stresses and induces endocytosis by reacting with ATP receptors in the cell membrane. In other words, there is a relationship between ATP concentration and cell damage and the influx of substances into cells. To confirm this, the ATP concentration in cells was analyzed after ultrasound treatment, and the result was that the ATP concentration was higher than that of the untreated control group. In addition, the expression of ionotropic P2X receptors and metabolic P2Y receptors in the cell membrane, which are affected by ATP, was also activated in the ultrasound-treated cells compared to the control group. These results suggest that ultrasound may not only cause intracellular damage, but also the influx of the external environment into cells.

[0023] Meanwhile, exosomes or exosome-containing extracellular vesicles are known to contain genetic information materials (DNA, mRNA, microRNA, protein) inside, and the genetic information materials present in the exosomes or exosome-containing extracellular vesicles can be transferred through the process in which the exosomes or exosome-containing extracellular vesicles discharged outside the cell membrane due to damage to the cell membrane re-enter other surrounding cells. Thus, the expression of pluripotent markers, three germ layer markers, or differentiated cell markers that are lowly expressed or maintained in a suppressed state within the cell by stimulation with ultrasonic treatment is induced or promoted, and at the same time, the exosomes or exosome-containing extracellular vesicles present in the cells containing the pluripotent markers, three germ layer markers, or differentiated cell markers whose expression is induced or promoted due to damage to the cell membrane are discharged to the outside and transferred to the surrounding cells. Here, since the surrounding cells also have partially damaged cell membranes, it is presumed that the fluidity of the cell membrane increases, and the efficiency of exosomes or exosome-containing extracellular vesicles entering the cells is higher than in the normal state, and the expression-induced or promoted pluripotency, development, and differentiation-related genetic information present in such exosomes or exosome-containing extracellular vesicles is transmitted to produce pluripotent cells or any differentiated cells. In one embodiment of the present invention, the culture medium was collected during the pluripotent cell induction process, and exosomes or exosome-containing extracellular vesicles in the medium were extracted to confirm whether pluripotent cell-related pluripotency markers or differentiation markers were present therein. As a result, it was confirmed that known pluripotency markers and differentiation markers were expressed at high levels, which was considered to support the present inventors' hypothesis. In addition, even after such ultrasound, laser, or heat treatment, the karyotype was not malformed and was normal.

[0024] Such a hypothesis makes it possible that pluripotent or differentiated cells can be produced by inducing the release of exosomes or exosome-containing extracellular vesicles by damaging the cell membrane.

[0025] The differentiated cells can be somatic cells including mammalian dermal fibroblasts, skin fibroblasts, etc.; cancer cells including uterine cancer cells (HeLa), liver cancer cells (Hep3B), etc.; or organ tissue cells including lung epithelial cells (L132 cells).

[0026] As used herein, the term "somatic cells" refers to cells that constitute an adult and have limited differentiation and self-production capabilities. According to one embodiment, the somatic cells may be somatic cells that constitute mammalian skin, hair, and fat, and preferably may be mammalian-derived fibroblasts, but are not limited thereto.

[0027] As used herein, the term "undifferentiated cells" refers to cells that have differentiation potential and self-production potential, such as induced pluripotent stem cells, embryonic stem cells, and progenitor cells.

[0028] As used herein, the term "pluripotent cells" refers to cells that have acquired pluripotency after physical stimulation, in the strict sense, ultrasound, laser, magnetic field, plasma, light-emitting diode, electrical stimulation, chemical exposure, heat treatment, or acid treatment. As used herein, pluripotency generally refers to a state in which pluripotency markers expressed in stem cells are stably expressed. It also refers to a state in which three types of germ layer markers, i.e., endoderm, ectoderm, and mesoderm, are expressed. The pluripotent cells are " E mbryonic stem cell meaia-based En vironmental t ransition-guided c e llular r eprogramming;es / ENTER" cells.

[0029] The pluripotent cells according to the present invention have a feature that is differentiated from known induced pluripotent stem cells in that they are well induced to differentiate by the external environment and have stronger attributes of progenitor cells, which have stronger differentiation attributes than stem cell attributes. That is, when embryonic stem cells such as induced pluripotent stem cells are used as cell therapy agents, a certain degree of preparation process is required, they contain a risk of turning into cancer, and safety issues are raised by using a viral vector for introducing a reverse differentiation inducer. However, the pluripotent cells of the present invention are induced without separately introducing a reverse differentiation inducer for gene mutation or a reverse differentiation inducer such as a chemical substance, so there is no need to culture through co-culture with other types of cells, so there is no problem of cell contamination (problem of mixing with other cells), and they do not form teratomas similar to cancer cells in in vivo experiments, so there is no problem of cancer development, and safety is ensured. In other words, the pluripotent cells of the present invention have the advantage that the induction process is simple and short, so the time required to process autologous cells and transplant can be dramatically shortened.

[0030] The pluripotent cells are characterized by stably expressing any one of pluripotency markers OCT3 / 4, S0X2, NANOG, c-MYC, KLF4, TDGF1, SSEA4, TRA-1-60, PAX6, Nestin, Brachyury, SMA, GATA4, or AFP, or a triple germ layer marker gene consisting of mesoderm and endoderm.

[0031] In this specification, the term "reprogramming" refers to a process in which differentiated cells that exist in different ways, such as cells without differentiation potential or cells with some differentiation potential, can be restored or switched to a state having a new type of cell or a new type of differentiation potential. It also includes a process in which a cell with differentiation potential can be switched to a new type of cell. According to the present invention, a differentiated cell may be reprogrammed into any desired differentiated cell that has a different phenotype from a pluripotent cell or a differentiated cell when it is subjected to a physical stimulus that can promote environmental influx. In addition, an undifferentiated cell may be reprogrammed into any differentiated cell while showing a significantly excellent differentiation rate when it is subjected to a physical stimulus that can promote environmental influx.

[0032] The differentiated cells may include, but are not limited to, for example, neural cells expressing any one of PAX6, SOX1, SOX2, Nestin, MAP2, TuJ1, GFAP or 04 (referred to as "neuronal stem cell media-based ENTER; n / ENTER"); muscle cells expressing any one of Desmin, Pax3, Actinin, SMA, GATA4 or NKX2-5 (referred to as "muscle differentiation media-based ENTER; m / ENTER"); hepatocytes expressing any one of AFP, HNFla, HNF4a, CK18 or ALB (referred to as "hepatocyte differentiation media-based ENTER; h / ENTER"); or adipocytes expressing any one of Pparc2, C / ebpa, aP2 or Fabp4 (referred to as "adipocyte differentiation media-based ENTER; a / ENTER").

[0033] As used herein, the term "culture medium" refers to a medium used in in vitro cell culture in a comprehensive sense, and in the present invention, refers to a stem cell culture medium or a differentiation-inducing medium, and more specifically, the stem cell culture medium refers to an embryonic stem cell culture medium. In addition, the term "differentiation-inducing medium" refers to a medium used to induce normal stem cells into differentiated cells, and examples that can be used include, but are not limited to, a multipotent cell differentiation-inducing medium, a hepatocyte differentiation-inducing medium, an osteogenic differentiation-inducing medium, an adipocyte differentiation-inducing medium, a muscle cell differentiation-inducing medium, an astrocyte differentiation-inducing medium, a nervous system cell differentiation-inducing medium, a vascular endothelial cell differentiation-inducing medium, a keratinocyte differentiation-inducing medium, a pancreatic β cell differentiation-inducing medium, and a cardiomyocyte differentiation-inducing medium.

[0034] The cell reprogramming method of the present invention will be described in detail with reference to FIG.

[0035] First, a culture medium and differentiated or undifferentiated cells are mixed, and a physical stimulus is applied to the mixture.

[0036] Prior to applying the physical stimulus to the mixture containing differentiated or undifferentiated cells, the culture medium can be subjected to a physical stimulus to enhance the efficiency of reprogramming the cells.

[0037] The physical stimulus may be any one of an ultrasound, a laser, a plasma, a light-emitting diode, an electrical stimulus, a chemical exposure, a heat treatment, or an acid treatment.

[0038] The culture medium was sonicated at an output intensity of 1 W / cm 2 ~20W / cm 2 Ultrasound for 1-20 minutes, specifically, output intensity 2W / cm 2 ~10W / cm 2 of ultrasound for 5-15 minutes, more specifically, with an output intensity of 3W / cm 2 ~7W / cm 2 Ultrasound can also be performed for 7 to 13 minutes.

[0039] The laser treatment of the culture medium can be performed by irradiating a pulsed laser beam in a wavelength range of 300 to 900 nm for 1 to 20 minutes, more specifically, for 3 to 10 minutes, and even more specifically, for 4 to 6 minutes. The wavelength range can be, for example, 400 nm, 808 nm, or 880 nm.

[0040] The heat treatment of the culture medium can be carried out at a temperature of 40 to 50° C. for 5 to 20 minutes.

[0041] When applying a physical stimulus to differentiated or undifferentiated cells, it is preferable to expose them to a certain intensity, and if the intensity is outside this range, the cell survival rate may decrease.

[0042] Therefore, sonication of a mixture of culture medium and differentiated or undifferentiated cells was performed at a power intensity of 0.5 W / cm 2 ~3W / cm 2 for 1-5 seconds, more specifically, with a power intensity of 0.7 W / cm 2 ~2W / cm 2 for 1 to 5 seconds, more specifically, with a power intensity of 0.8 W / cm 2 ~1.5W / cm 2 This can be done for 1 to 5 seconds.

[0043] The mixture of culture medium and differentiated or undifferentiated cells may be laser-treated with a pulsed laser beam in a wavelength range of 300 to 900 nm for 1 to 20 seconds, more specifically, for 3 to 10 seconds, and even more specifically, for 4 to 6 seconds. The wavelength range may be, for example, 400 nm, 808 nm, or 880 nm.

[0044] Heat treatment of a mixture of culture medium and differentiated or undifferentiated cells may be performed by exposing it to a temperature condition of 40 to 50°C for 1 to 10 minutes, and then exposing it to a temperature condition of 0 to 4°C for 5 to 10 seconds.

[0045] The mixture to which the physical stimuli have been applied is then cultured for a certain period of time to obtain reprogrammed cells.

[0046] The mixture to which the physical stimuli have been applied may be cultured for a period during which spheroids stably expressing pluripotency markers or differentiation markers are formed through suspended culture or monolayer culture, i.e., for 2 to 10 days, but is not particularly limited thereto.

[0047] According to one embodiment of the present invention, suspension culture exhibits higher spheroid formation efficiency than adherent culture, and the number and size of spheroids in suspension culture are larger and have a more consistent size distribution than adherent culture.

[0048] According to one embodiment of the present invention, when ultrasonically or laser-treated human dermal fibroblasts are cultured in suspension, the expression of pluripotency markers or differentiation markers increases or stabilizes from about day 3, and reprogramming is considered to begin from this time. Also, when heat-treated human dermal fibroblasts are cultured in suspension, the expression of pluripotency markers increases or stabilizes from about day 8, and reprogramming is considered to begin from this time.

[0049] The pluripotency of the spheroids can be confirmed by the expression of the pluripotency markers, such as OCT3 / 4, SOX2, NANOG, c-MYC, KLF4, TDGF1, SSEA4, TRA-1-60, etc. The pluripotency markers can be confirmed by RT-PCR or immunocytochemistry, but are not limited thereto.

[0050] Furthermore, the pluripotent cells of the present invention are characterized by high levels of expression of three germ layer markers, namely ectoderm (PAX6, Nestin), mesoderm (Brachyury, SMA), and endoderm (GATA4, AFP) markers.

[0051] In another specific example, when a physical stimulus is applied to skin fibroblasts in a differentiation-inducing medium, spheroids can be formed within about 1 to 20 days after culture.

[0052] When reprogramming to neural cells, the differentiation marker may be any one of PAX6, SOX1, SOX2, Nestin, MAP2, TuJl, GFAP or 04.

[0053] When reprogramming into muscle cells, it may be any one of Desmin, Actinin, Pax3, SMA, GATA4 or NKX2-5.

[0054] When reprogramming into hepatocytes, it may be any one of AFP, HNFla, HNF4a, CK18 or ALB.

[0055] When reprogrammed into adipocytes, they are stained with Oil Red O and may be any one of Pparc2, C / ebpa, aP2 or Fabp4.

[0056] Furthermore, the pluripotent cells of the present invention are characterized by expressing the proliferation marker protein Ki-67 and having proliferation ability.

[0057] Furthermore, when the reprogrammed pluripotent cells are co-cultured with nutrient cells, the proliferation of the pluripotent cells may be increased.

[0058] Furthermore, the cell reprogramming method of the present invention may further comprise a step of culturing the pluripotent cells in a differentiation-inducing medium. Depending on the type of the differentiation-inducing medium, the pluripotent cells can be differentiated into a target differentiated cell.

[0059] The differentiation-inducing medium may be, but is not limited to, a multipotent cell differentiation-inducing medium, a hepatocyte differentiation-inducing medium, an osteogenic differentiation-inducing medium, an adipocyte differentiation-inducing medium, a muscle cell differentiation-inducing medium, an astrocyte differentiation-inducing medium, a nervous system cell differentiation-inducing medium, a vascular endothelial cell differentiation-inducing medium, a keratinocyte differentiation-inducing medium, a pancreatic β cell differentiation-inducing medium, or a cardiomyocyte differentiation-inducing medium.

[0060] The present invention also provides a method for producing a culture medium comprising the steps of: providing a physical stimulus capable of promoting environmental influx to a mixture of differentiated or undifferentiated cells and a culture medium; The mixture to which the physical stimulus has been applied is cultured for 1 to 6 days, The present invention relates to a method for reprogramming cells, comprising mixing exosome-containing extracellular vesicles separated from the culture with differentiated or undifferentiated cells and culturing the mixture for a certain period of time to obtain reprogrammed cells.

[0061] The cell reprogramming method of the present invention is characterized in that exosome-containing extracellular vesicles separated from a differentiated or undifferentiated cell that has been subjected to a physical stimulus can be cultured with a differentiated or undifferentiated cell for a certain period of time to reprogram the cell into any differentiated cell.

[0062] The exosome-containing extracellular vesicles can be recovered by applying a physical stimulus capable of promoting inflow into the environment to a mixture of differentiated or undifferentiated cells and a culture medium, culturing the mixture to which the physical stimulus has been applied for 1 to 6 days, and then centrifugally recovering the exosome-containing extracellular vesicles.

[0063] The physical stimulation of the differentiated or undifferentiated cells has been described above, and therefore, a description thereof will be omitted to avoid repetition.

[0064] The exosome-containing extracellular vesicles may express any one of a pluripotency marker or a three germ layer marker, among OCT3 / 4, SOX2, NANOG, c-MYC, KLF4, TDGF1, SSEA4, TRA-1-60, PAX6, Nestin, Brachyury, SMA, GATA4, or AFP; any one of a neuronal marker, among PAX6, Nestin, Sox1, Sox2, MAP2, TuJl, GFAP, or O4; any one of a muscle cell marker, among Desmin, Pax3, Actinin, SMA, GATA4, or NKX2-5; any one of a hepatocyte marker, among AFP, HNFla, HNF4a, CK18, or ALB; or any one of an adipocyte marker, among Pparc2, C / ebpa, aP2, or Fabp4, stained with Oil Red O.

[0065] For example, when the exosome-containing extracellular vesicles contain a pluripotency marker, the differentiated cells can be reprogrammed into pluripotent cells by culturing them with differentiated cells. When the exosome-containing extracellular vesicles contain a differentiation marker, the differentiated cells can be reprogrammed into any differentiated cells with different phenotypes by culturing them with differentiated cells. When the exosome-containing extracellular vesicles contain a differentiation marker, the differentiated cells can be reprogrammed into any differentiated cells by culturing them with undifferentiated cells.

[0066] According to one embodiment of the present invention, the expression of various pluripotency markers was confirmed in extracellular vesicles (EVs) stained for CD63, an exosome marker, collected during induction of es / ENTER, and after culturing the EVs in treated normal human somatic cells for three days, the expression of pluripotency markers Oct4, Sox2, and Nanog was confirmed, confirming cell reprogramming.

[0067] In addition, expression of neural stem cell markers such as Pax6 was confirmed in extracellular vesicles (EVs) stained for CD63, an exosome marker, collected during induction of n / ENTER, and after culturing the EVs in treated normal human somatic cells for three days, expression of neural stem cell markers Sox1, Sox2, Pax6, and Nestin was confirmed.

[0068] Furthermore, the expression of muscle cell markers such as Pax3 was confirmed in extracellular vesicles (EVs) stained for CD63, an exosome marker, collected during induction of m / ENTER, and the expression of hepatic cell markers such as HNFla was confirmed in extracellular vesicles (EVs) stained for CD63, an exosome marker, collected during induction of h / ENTER.

[0069] Therefore, it has been found that differentiated or undifferentiated cells that receive physical stimuli secrete extracellular vesicles containing reprogramming factors. By treating differentiated or undifferentiated cells with these and culturing them for 1 to 20 days using suspension or adherent culture methods, they can be reprogrammed into any pluripotent or differentiated cell.

[0070] The cells that can be reprogrammed through the cell reprogramming method of the present invention may be pluripotent cells or differentiated cells of the types mentioned above, and the description will be omitted to avoid duplication.

[0071] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples. EXAMPLES

[0072] <Example 1> Experiment to prove the influx of the environment into cells by physical stimuli This example is a verification experiment for the influx of the environment into cells by physical stimuli. For this purpose, primary HDF cells purchased from Invitrogen were cultured in DMEM supplemented with 10% FBS (Gibco) and 1% penicillin / streptomycin (Gibco). The culture medium was sonicated at 5 W / cm. 2 The cells were treated with 1x10 6 1W / cm to the cell 2 After treatment with 500 mM NaCl for 5 seconds, 2x10 cells were plated in a 35 mm culture dish together with the treated culture medium. 5 The cells were cultured.

[0073] For SEM image analysis, untreated HDF cells, cells immediately after treatment as described above, and cells cultured for 2 hours in a 37°C, 5% CO2 incubator were fixed with 4% paraformaldehyde for 12 hours at 4°C, then treated with 0.1% tannic acid solution for 1 hour and 1% osmium tetroxide solution for 2 hours. After dehydration with acetone at each concentration level, the cells were dried with liquid CO2, fixed on a gold-palladium coated surface, and observed under an electron microscope (1555VP-FESEM, Carl Zeiss).

[0074] For live / dead image analysis, cells were stained using a Live / dead viability / cytotoxicity assay kit (Molecular Probes, Eugene, OR, USA) for untreated HDFs, immediately after sonication, and for cells cultured for 2 h in a 5% CO2 incubator at 37°C. The staining process consisted of adding 2 μM calcein (live cell staining dye) and 4 μM ethidium homodimer-l (EthD-l, dead cell staining dye) to the cell culture medium for live cells, followed by incubation for 30 min in a 5% CO2 incubator at 37°C, and then analyzing the red (EthD-1 staining, dead or damaged cells, excitation / emission, 528 / 617 nm) and green fluorescence (calcein staining, live cells, excitation / emission, 494 / 517 nm) under a fluorescence microscope (IX3-ZDC, Olympus).

[0075] As shown in FIG. 2a, ultrasound damaged the cell membrane, forming holes through which the external environment could enter, and this damage was repaired after 2 hours.

[0076] In addition, after ultrasonic stimulation, cells were stained with a Live / dead kit used to analyze cell death in order to confirm damaged cells and cell repair.

[0077] As can be seen from Figure 2b, cells were treated with ultrasound, stained immediately after treatment, and then stained two hours later. As a result, cells in which both green and red fluorescence coexisted were observed immediately after treatment, and after two hours, the number of cells showing red fluorescence was significantly reduced, indicating that the red fluorescence was reduced due to the repair of the cell membrane, as shown in Figure 2a.

[0078] This suggests that although ultrasonic stimulation causes cell damage, it is possible for it to be repaired, and that such damage to the cell membrane caused by stimulation allows the inflow of the culture medium environment. Based on this, we analyzed the phenomenon that occurs due to the inflow of intracellular substances.

[0079] On the other hand, in FIG. 2, usMC (ultrasound-exposed medium and cells) refers to the case where the cells and culture medium were each treated with ultrasound, and usMC-S refers to usMC cultured in suspension.

[0080] <Example 2> Experiment to prove the influx of external substances into cells by physical stimulation To measure changes in intracellular calcium concentration, which is sensitive to the influx of external substances into cells, and to confirm the possibility of substance influx accompanying the generation of ATP by ultrasound, we used RT-PCR to measure intracellular ATP and to analyze the expression of ATP receptors, which are known to open the influx pathway for external substances in the cell membrane through ATP reactions.

[0081] Calcium concentration analysis was performed using the Fluo-4 NW Calcium Assay Kit (Molecular Probes). HDF cells without any treatment and those treated with ultrasound (1W / cm 2 , 5 s) followed by ultrasonication in medium (5 W / cm 2 Cells (usMC-S) exposed to 1000 ng / mL of PBS were mixed with the assay buffer from the kit and added to a 96-well plate at 3 x 10 mL / well. 4 After dispensing the cells, they were mixed with 50 μl of Fluo-4NW reagent per well, and the fluorescence was measured in the range of excitation wavelength 494 nm / emission wavelength 516 nm every 10 s for 15 min using a Varioskan Flash Fluorescent Microplate Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).

[0082] ATP was measured using an adenosine 5'-triphosphate (ATP) bioluminescent assay kit. Cells were either untreated or treated with ultrasound (1 W / cm 2 , 5 s) followed by ultrasonication in medium (5 W / cm 2, 10 min) were placed in 3x10 cells / well of a 96-well plate. 4 After dispensing the cells, 100 μl of ATP assay mix and ATP standard were dispensed per well and incubated at room temperature for 3 minutes, after which the luminescence intensity was measured using a Varioskan Flash Fluorescent Microplate Fluorometer (Thermo Fisher Scientific).

[0083] For RT-PCR to analyze ATP receptor expression, RNA was extracted from treated cells using RNeasy plus mini kit (Qiagen, Hilden, Germany), and cDNA was synthesized using SuperScripII kit (Invitrogen, Carlsbad CA, USA). PCR was performed using a Thermal cycler dice PCR machine (TP600, TAKARA, Otsu, Japan) under the following conditions: denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, gradient (50-65°C) for 30 s, and 72°C for 1 min, followed by 72°C for 15 min.

[0084] [Table 1]

[0085] As shown in Figure 3, after ultrasonic stimulation, the influx of calcium into the cells increased for up to 60 seconds, the intracellular ATP concentration increased to its maximum at 60 minutes, and the expression of ATP receptors in the cell membrane also increased at 1 hour and 4 hours. This shows that the initial influx of external substances into the cells due to ultrasonic stimulation was confirmed by the increase in calcium concentration, and that ultrasonic waves generated ATP, which caused the ATP receptor to react and open the cell membrane passage, allowing the influx of external substances.

[0086] <Example 3> Experiment to prove the influx of external substances into cells by physical stimulation using QD605 Using QD605 as a foreign substance, we confirmed whether QD605 would flow into cells by ultrasound. QD605 is a fluorescent nano-substance known to have poor permeability in living cells, so we confirmed the flow of foreign substances into cells by ultrasound using QD605.

[0087] For this purpose, HDFs were subjected to ultrasonic stimulation in the same manner as in Example 1, and then treated with 100 pmol of QD605. After 24 hours, the presence of QD605 in single cells and spheroids was confirmed.

[0088] As shown in Figure 4, no fluorescence was observed from the HDFs that were not subjected to ultrasound stimulation, but fluorescence was observed from the HDFs that were subjected to ultrasound stimulation.

[0089] In addition, to confirm the possibility of cellular changes due to the influx of external substances, ultrasonic treatment was performed in each culture medium environment (ES, Neuroprogenitor, Hepatocyte, muscle), and then 100 pmol of QD605 was added. After 24 hours, the expression of transcription factors involved in each differentiation process (ES: Oct4, Neuroprogenitor: Pax6, Hepatocyte: HNFla, Muscle: Pax3) was confirmed by ICC.

[0090] As shown in Figure 4b and Figure 4c, transcription factors were observed in the cells and spheroids to which an external substance (QD605) had been injected, respectively, suggesting the possibility of cell reprogramming following the injection of an external substance.

[0091] During the experiment, of the four types of environmental inflow samples, only es / ENTER and n / ENTER formed spheroids. However, m / ENTER and h / ENTER did not form spheroids. This is due to the characteristics of the cells and the medium preparation. ES and neural progenitor cells are cells that form spheroids and spheres during the suspension culture process, but muscle cells and liver cells do not form spheroids. This is because they were cultured by attaching them to a coated culture dish during the differentiation induction process. In particular, the medium for muscle cells contains FBS, but FBS increases cell adhesion, which is thought to prevent spheroids from forming.

[0092] Example 4: Analysis of exosomes in cell culture medium subjected to physical stimulation Since cell stimulation by ultrasound does not stimulate all cells uniformly, cell reprogramming may occur in some cells, and we considered the possibility of cell exchange between cells that have been changed in this way and cells that have not. In recent years, based on the possibility of material exchange between cells by exosomes, it is possible that exosomes in the culture medium discharged from sonicated cells contain genetic material, which means that the material secreted from reprogrammed cells may contain genetic material that plays an important role in reprogramming. Therefore, exosomes in the culture medium cultured after sonication were collected when changing the medium at different culture times, and RNA from exosomes in the culture medium was extracted using Amicon Ultra-0.5 kit (Millipore), and cDNA synthesis was performed using Super ScripII kit (Invitrogen, Carlsbad CA, USA). For PCR, cDNA and primers were mixed with PCR premix (Bioneer, Daejeon, Korea), and RT-PCR analysis was performed using a Thermal cycler dice PCR machine (TP600, TAKARA, Otsu, Japan) under the following conditions: denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, gradient 30 s, and 72°C for 1 min, and 72°C for 15 min (Table 2).

[0093] As shown in FIG. 5, expression of pluripotent RNA was confirmed among exosomal RNAs.

[0094] On the other hand, in FIG. 5, usMC refers to the case where the cells and the culture medium were each treated with ultrasound, and usMC-A refers to adherent cultured usMC.

[0095] [Table 2] TIFF0007648124000003.tif51166

[0096] <Example 5> Experiment to prove substance transfer by exosomes Since the expression of pluripotency markers was confirmed in exosomes in the culture medium of sonicated cells in Example 4, it was examined whether genetic material and proteins could be transferred by such exosomes.

[0097] After ultrasonic treatment, QD605 was added and images of living cells were taken. As a result, as shown in Figure 6a, it was possible to confirm that at around 7 hours and 45 minutes, QD605 from the cells into which QD605 had been introduced was shed along with part of the cytoplasm and moved to other cells.

[0098] Assuming that some of the shed cytoplasm was exosomes, usMC-treated cells were exposed to various medium environments, fixed in 4% paraformaldehyde for 10 min, and soaked in PBS containing 0.1% Triton X-100 for 40 min. Blocked with PBS containing 5% (v / v) goat serum for 1 h, and stained with primary antibodies for exosome marker CD63 (1:100, Santa Cruz Biotechnology) and early expression markers of cells to be induced by each differentiation-inducing medium, such as embryonic stem cells (Oct41:200; Nanog1:200; abeam), neural stem cells (Pax6, 1:200; abeam), muscle cells (Pax3, 1:200; abeam), and hepatocytes (HNFla, 1:200; Cell Signaling Technology), overnight at 4°C. The sections were then washed with PBS buffer containing 0.03% Triton X-100, and stained with secondary antibodies, anti-rabbit and anti-mouse antibodies conjugated with Alexa-488 or -594 (1:1000, Thermo, excitation / emission, 495 / 519 nm, excitation / emission, 590 / 617 nm) at room temperature for about 1.5 hours. After that, the sections were washed with PBS buffer containing 0.03% Triton X-100, and mounted with DAPI-containing mounting sol (Vector Laboratories, Inc., Burlingame, CA, excitation / emission, 420 / 480 nm), and the images were analyzed using a confocal laser scanning fluorescence microscope (LSM700; Carl Zeiss).

[0099] As shown in Figure 6b, what is presumed to be a portion of the cytoplasm that was protruding from around the cells was stained with CD63, and upon induction of es / ENTER, the expression of pluripotent cell markers such as Oct4 and Nanog was confirmed in extracellular vesicles (EVs) stained with the exosome marker CD63. Upon induction of n / ENTER, the expression of neural stem cell markers such as Pax6 was confirmed in extracellular vesicles (EVs) stained with the exosome marker CD63. Upon induction of m / ENTER, the expression of muscle cell markers such as Pax3 was confirmed in extracellular vesicles (EVs) stained with the exosome marker CD63. Upon induction of h / ENTER, the expression of liver cell markers such as Hnfla was confirmed in extracellular vesicles (EVs) stained with the exosome marker CD63.

[0100] Based on the above results, we hypothesized that exosomes are released from the cytoplasm and contain genetic material and proteins, which can be transmitted to surrounding cells and induce changes in the surrounding cells. To prove this, we extracted exosomes, stained them with CD63 (exosomes are also present in cultured cells, so we stained them to distinguish them from newly injected exosomes), and then detected poly(A) in the exosomes. 27 -When genetic material modified with Cy5.5 was introduced into exosomes and then cultured with untreated HDFs, the exosomes produced poly(A) 27 - We thought that Cy5.5 might be transmitted.

[0101] As shown in Figure 6c, CD63-stained exosomes were found in the cells, and it was confirmed that cy5.5 was expressed together with CD63, which indicates that the injected gene (poly-A) was delivered by the exosomes.

[0102] Example 7: Co-culture of physically stimulated cells and normal cells Because exosomes can transmit genetic material, we hypothesized that exosomes secreted from cells cultured in human ES medium could change the attributes of surrounding cells or even untreated cells. To prove this, we extracted exosomes from the medium of sonicated cells cultured in human ES medium for two days, and mixed the exosome extract with untreated cells in human ES medium and DMEM, a fibroblast culture medium, and cultured them for six days.

[0103] As a result, spheroids were generated in the exosome-added group (Fig. 7a), and the expression of Oct4, a pluripotency marker, was confirmed in these cells (Fig. 7b), indicating that exosome-mediated genetic material transfer can induce cell reprogramming.

[0104] Example 8: Direct reprogramming of fibroblasts In Examples 1 to 7, it was demonstrated that cell reprogramming is possible by changing the culture medium environment and that even surrounding cells can be reprogrammed. Based on this, cell reprogramming was confirmed by applying various culture medium environments.

[0105] To achieve this, human fibroblasts were cultured in 1 mL of differentiation-inducing medium at 1 x 10 6 After collecting the cells at a density, the ultrasound was adjusted to 1W / cm 2 After treatment with 100% CO for 5 seconds, 2x10 5 After dispensing in / well, ultrasonic waves were applied at 10W / cm 2 The cells were cultured in 2 mL of differentiation induction medium and treated with an intensity of 100 μg / ml for 10 minutes.

[0106] Although there were differences depending on the type of differentiation-inducing medium, spheroids were formed approximately 2 to 6 days after culture.

[0107] In addition, after treating usMC with adipocyte differentiation-inducing medium and culturing for 20 days, intracellular bubbles were observed to be formed, and analysis of these bubbles revealed that they were stained with Oil Red O, a lipid staining reagent used to distinguish adipocytes, which is an indicator that the cells were producing fat (Figure 9a).

[0108] In addition, the expression of adipocyte marker genes, Pparc2, C / ebpa, aP2, and Fabp4, was confirmed by extracting RNA from the cells and using RT-PCR, and it was shown that expression increased after differentiation induction (Figure 9b).

[0109] Furthermore, to confirm the differentiation of HDFs into neural progenitor cells using neural stem cell (neural progenitor cell) differentiation induction medium and ultrasound, the expression of neural progenitor cell markers Oct4, Sox2, Pax6, and Nestin was confirmed by immunocytochemical staining in the spheroids generated on the third day after differentiation induction and the attached cells.

[0110] Figure 10a shows the appearance of differentiation-induced cells, and Figure 10b shows neural progenitor cell markers in spheroids. It was confirmed that upon differentiation, Oct4 expression decreased, while Sox2, Pax6, and Nestin expression increased. Figure 10c shows the results of examining expression in attached cells, which also showed the same expression pattern as above. In this case, the markers for neural progenitor cells and neural stem cells were Sox2, Pax6, and Nestin, and Oct4 was a pluripotent marker, and the expression ability of Oct4 decreased in adult stem cells and progenitor cells.

[0111] [Table 3]

[0112] Figure 11 shows the results of flow cytometry analysis of the expression pattern of Pax6 / Nestin in cells that had been induced to differentiate for 7 days. Pax6 and Nestin were relatively expressed by more than 50% on the first day after treatment, and the expression of Pax6 and Nestin was highest on the third day.

[0113] FIG. 12 shows the results of confirming the expression of ki67 on the third day after differentiation induction to confirm the presence or absence of proliferation in cells expressing neural progenitor cell markers (Pax6 / nestin). Looking at the cells indicated by the white arrows, it was possible to confirm the expression of ki67 in cells expressing Nestin. Such results indicate that the differentiation-induced cells have the ability to proliferate. In FIG. 12, the arrows are markers indicating that Nestin-stained cells are proliferating.

[0114] Figure 13 shows the experimental results confirming the self-renewal of differentiation-induced cells (n / ENTER cells). Figure 13a shows the video in which cells proliferated from a single spheroid express Pax6 and nestin, indicating that the neural progenitor cell attributes are transmitted directly to the cells proliferated thereafter.

[0115] Next, the differentiated cells (n / ENTER cells) were injected into the brains of 5-week-old mice, and the brains were harvested 4 weeks later. After that, the differentiation of the injected cells into surrounding cells was confirmed, and the function of the differentiated cells was confirmed.

[0116] As shown in Figure 14a, the cells injected into the brain (n / ENTER cells) were stained with Human Nuclear antigen (HNA) and then stained with Gfap antibody, confirming that Gfap was expressed in the injected cells.

[0117] To confirm whether Gfap-expressing cells function normally, we stained them with a synapsin 1 antibody (1:500, R&D system) to see whether they secreted synapsin. As a result, we observed the expression of synapsin 1 in Gfap-expressing cells among HNA-expressing cells (Figure 14b).

[0118] Next, the expression of neural progenitor cell markers (Oct4, Sox2, Pax6, Nestin) in the spheroids generated on day 3 after differentiation induction was confirmed by immunocytochemistry staining.

[0119] For this, spheroids and attached cells were fixed in 4% paraformaldehyde for 10 min, soaked in PBS containing 0.1% Triton X-100 for 40 min, blocked with PBS containing 5% (v / v) goat serum for 1 h, and stained with primary antibodies, including Oct4 (1:200), Sox2 (1:200), Pax6 (1;200), and Nestin (1:200, Cell Signaling Technology), overnight at 4°C. The sections were then washed with PBS buffer containing 0.03% Triton X-100, and stained with secondary antibodies, anti-rabbit and anti-mouse antibodies conjugated with Alexa-488 or -594 (1:1000, Thermo, excitation / emission, 495 / 519 nm, excitation / emission, 590 / 617 nm) at room temperature for about 1.5 hours. After that, the sections were washed with PBS buffer containing 0.03% Triton X-100, mounted with mounting sol containing DAPI (Vector Laboratories, Inc., excitation / emission, 420 / 480 nm), and images were analyzed using a confocal laser scanning fluorescence microscope (LSM700; Carl Zeiss).

[0120] The formation of spheroids was confirmed in FIG. 15a, and the expression of neural progenitor cell markers Pax6 and Nestin was highly expressed in the spheroids (FIG. 15b).

[0121] Figure 15c shows the results of flow cytometry analysis of the expression pattern of Pax6 / Nestin in cells induced to differentiate for three days after induction of differentiation. Pax6 and Nestin were expressed by more than 70% on the first day after treatment, and the expression of Pax6 and Nestin was highest on the third day.

[0122] Figure 16 shows the results of immunocytochemical staining to confirm the expression of neural progenitor cell markers (Sox2, Pax6, Nestin) in cells 20 days after differentiation induction. The upper and middle photographs show high expression of Sox2, Pax6, and Nestin, and the lower photograph shows the result of examining the expression of oligodendrocyte markers, which also showed the same expression pattern as above.

[0123] This result means that the differentiation-induced cells have a differentiation ability similar to that of neural precursor cells.

[0124] Example 9: Direct hepatocyte differentiation As shown diagrammatically in FIG. 17, HDFs, HeLa cells, and Hep3B cells were cultured in 1 mL of differentiation-inducing medium at 1×10 6 After collecting the cells at a density, the ultrasound was adjusted to 1W / cm 2 After 5 seconds at 100 Hz, the cells were transferred to a 35 mm laminin-coated culture dish with 2x10 5 After dispensing at 10W / cm, ultrasonic waves were applied at 10W / cm 2 The cells were cultured in 2 mL of hepatocyte differentiation-inducing medium treated with an intensity of 100 μg / mL for 10 minutes. The cells induced to differentiate using the hepatocyte differentiation-inducing medium were named h / ENTER.

[0125] Figure 18 shows the change in cell appearance 20 days after inducing differentiation of HDFs treated with hepatocyte differentiation induction medium into hepatocytes (h / ENTER). Twenty days after induction of HDF differentiation, hepatocyte markers (AFP, HNF4a, CK18, ALB) were confirmed by immunocytochemistry, and the expression of hepatocyte markers was confirmed in h / ENTER cells.

[0126] Next, the differentiation of HeLa cells into hepatocytes (HeLa h / ENTER) was induced using hepatocyte differentiation induction medium and sonication. The figure shows the change in the external appearance of the cells (HeLa h / ENTER) 19 days after induction of HeLa cell differentiation, and the expression of hepatocyte markers (ALB, HNF4a, CYP3A4F, CYP3A7F, AIAT, SOX7, GATA6) was confirmed by qPCR 20 days after induction of HeLa cell differentiation (HeLa h / ENTER).

[0127] As shown in FIG. 19a, it was confirmed that most of the hepatocyte markers were increased in the differentiation-induced HeLa cells compared to the HeLa cells.

[0128] In addition, three weeks after induction of HeLa cell differentiation, we confirmed the expression of hepatocyte markers (HNF4a, CK18, ALB) by immunocytochemistry and found that the expression of hepatocyte markers was increased in differentiated HeLa cells (HeLa h / ENTER) compared to HeLa cells (Figure 19b).

[0129] Three weeks after induction of HeLa cell differentiation, we confirmed the expression of hepatocyte markers (HNF4a, CK18, ALB) by immunocytochemistry, and found that the expression of hepatocyte markers was increased in differentiated HeLa cells (HeLa h / ENTER) compared to HeLa cells (Figure 19c).

[0130] Next, the differentiation of Hep3B cells into hepatocytes (Hep3B h / ENTER cells) was induced using hepatocyte differentiation induction medium and sonication. The change in cell morphology 19 days after the induction of Hep3B cell differentiation, and the expression of hepatocyte markers (HNF4a, CK18, ALB) by immunocytochemistry 3 weeks after the induction of Hep3B cell differentiation were confirmed.

[0131] As shown in FIG. 20b, it was confirmed that the expression of hepatocyte markers was increased in differentiated Hep3B cells (Hep3B h / ENTER) compared to Hep3B cells.

[0132] Next, human ES culture medium and sonicated HDFs were induced to differentiate into es / ENTER cells. Figure 21a shows the change in cell morphology over culture time, and Figure 21b shows the difference in Oct4 expression over culture time. Spheroids were formed on the first day after differentiation induction in human ES medium, and the expression of Oct4, a pluripotency marker, increased over culture time.

[0133] Spheroids formed after 6 days of culture were harvested and the expression of pluripotency markers was confirmed by RT-PCR and ICC. As a result, the expression of pluripotency marker genes (a) and proteins (b) was confirmed in es / ENTER cells.

[0134] The pluripotency attributes of the es / ENTER cells were analyzed by flow cytometry to confirm the expression of SSEA4 and TRA-1-60 in the es / ENTER cells (Figure 23a). The expression pattern of pluripotency genes was analyzed by microarray (affymetrix chip), and the pattern was similar to that of ES cells compared to HDF (Figure 23b). In addition, the methylation of the promoter region where the synthesis of the expressed DNA gene begins was confirmed by bisulfite sequencing, and it was confirmed that the important pluripotency genes Oct4 and Nanog were released. These results show that es / ENTER differentiated in human ES medium has pluripotency attributes (Figure 23c).

[0135] [Table 4]

[0136] Next, differentiation markers were confirmed in es / ENTER cells. Figure 24 shows the expression of three germ layer marker genes (a) and protein expression (b) in es / ENTER cells, Figure 25 shows the change in Oct4 and three germ layer marker gene expression in es / ENTER cells according to the culture time, and Figure 26 shows the expression of three germ layer marker proteins in adherent cultured es / ENTER cells (a) and the analysis of the DNA methylation of three germ layer markers in es / ENTER cells using bisulfite sequencing (b).

[0137] [Table 5] TIFF0007648124000007.tif229166TIFF0007648124000008.tif204166

[0138] The expression of three germ layer markers in es / ENTER spheroids was confirmed by RT-PCR and ICC, which indicates that es / ENTER cells have multilineage differentiation attributes.

[0139] Therefore, we investigated the expression pattern of Oct4, a pluripotency marker, over time in culture, and found that after 6 days, the expression of Oct4, which is a pluripotency attribute, decreased, while the expression of the three germ layer markers increased. These results indicate that the differentiation of es / ENTER cells proceeds with pluripotency attributes. Therefore, it was confirmed that the cells emerging from the spheroids that were cultured for 2 days after attachment of es / ENTER spheroids expressed the three germ layer markers, and the DNA methylation of representative three germ layer marker genes was analyzed, and it was confirmed that the three germ layer marker genes were open.

[0140] Figure 27 shows the results of an in vitro differentiation experiment of es / ENTER cells into neurons (a), cardiomyocytes (b), and hepatocytes (c). es / ENTER cells were induced to differentiate for four weeks using a medium that induces differentiation into cells differentiated from each of the three types of germ layers. As a result, the cells were induced to differentiate into neurons (ectoderm), cardiomyocytes (mesoderm), and hepatocytes (endoderm), and the respective differentiation markers were expressed.

[0141] FIG. 28 shows the results of confirming the expression of differentiation markers for neurons (a), cardiomyocytes (b) and hepatocytes (c) in HDF, and differentiation markers were not expressed in HDF.

[0142] Figure 29 shows the results of RT-PCR analysis showing the expression of differentiation marker genes in neurons (a), cardiomyocytes (b) and hepatocytes (c) of differentiation-induced es / ENTER cells, and the expression of differentiation marker genes in differentiation-induced es / ENTER increased. These results prove the pluripotency of es / ENTER.

[0143] FIG. 30 shows the karyotype of es / ENTER cells by chromosome G-band analysis. When es / ENTER was generated, the presence or absence of mutations in the cellular chromosomes due to ultrasonic stimulation was analyzed, and the results showed that they were normal.

[0144] Next, the es / ENTER cells were transplanted into the leg muscles of 5-week-old SCID mice, and the transplanted cells were confirmed using HNA 4 weeks later.

[0145] As shown in Figure 31, it was confirmed that the es / ENTER cells were differentiated into skeletal muscle. It was also confirmed that Oct4 was not expressed or proliferated in the transplanted cells.

[0146] The es / ENTER cells were then transplanted into mouse brains to confirm in vivo differentiation.

[0147] To this end, es / ENTER cells were transplanted into the brains of 5-week-old SCID mice, and the transplanted cells were identified using HNA 4 weeks later.

[0148] As a result, it was confirmed that es / ENTER cells were differentiated into astrocytes (Gfap), and that synapsin and vesicular glutamate transporter were secreted. This means that the transplanted cells were normally differentiated and functioning. It was also confirmed that Oct4 was not expressed in the transplanted cells, and they were not proliferating (Figure 32).

[0149] Next, MEF (mouse embryonic fibroblasts) were also induced to differentiate into mouse ES / ENTER cells in the same manner as HDF, using hES medium. The MEF used in this experiment was OG2-MEF, a mouse embryonic fibroblast transformed with a vector carrying the Oct4 promoter. In this case, when Oct4 is expressed, GFP fluorescence is expressed, and this was used to observe the expression of Oct4.

[0150] As shown in FIG. 33, the cells induced by sonication increased the number and size of spheroids as well as GFP expression over time.

[0151] The pluripotent attributes in the mouse es / ENTER cells were then analyzed.

[0152] As shown in FIG. 34, ICC, RT-PCR, flow cytometry, and AP staining of mouse es / ENTER showed similar trends to those of mouse ES.

[0153] Next, the characteristics of the three germ layers in the mouse es / ENTER cells were analyzed.

[0154] As a result of the experiment, the three germ layer attributes that appeared in human es / ENTER were also present in mouse es / ENTER cells, and the expression differences over time were revealed by RT-PCR and ICC analysis according to the culture time. These results were similar to those of human es / ENTER (Figure 35).

[0155] Figure 36 shows in vitro differentiation of mouse es / ENTER into neurons (a) and cardiomyocytes (b), and Figure 36c shows the results of karyotype analysis of mouse es / ENTER by chromosome G-banding analysis. Karyotype analysis was performed using GTG banding chromosome analysis (GenDix, Inc.; Seoul, Korea).

[0156] As a result of the experiment, differentiation of mouse es / ENTER cells was confirmed by checking for differentiation markers into neurons and cardiomyocytes, and karyotype analysis using ultrasound confirmed that no mutations had occurred.

[0157] Example 10: Differentiation induction experiment of es / ENTER using other cells Based on these results, we concluded that this method could be applied not only to HDFs but also to cells from other individuals, and tried applying it to a variety of cells (L132, MSC, and patient skin fibroblasts).

[0158] Using L132 (lung epithelial cells), MSCs (mesenchymal stem cells), and skin fibroblasts (patient-derived skin fibroblasts), differentiation was induced using the same method as es / ENTER, and cell spheroids were formed, and it was found that pluripotency markers and three germ layer markers were expressed in a manner similar to es / ENTER.

[0159] Figure 37 shows the results showing the differentiation of L132 cells into L132 es / ENTER cells using human ES culture medium and ultrasonic stimulation, where Figure 37a shows the change in cell morphology over culture time, and Figures 37b and 37c show the pluripotency (b) and three germ layer (c) attributes of L132 es / ENTER cells.

[0160] Figure 38 shows the results showing the differentiation of MSCs into MSC es / ENTER cells using human ES culture medium and ultrasonic stimulation, where Figure 38a shows the change in cell morphology over culture time, and Figures 38b and 38c show the pluripotency (b) and three germ layer (c) attributes of MSC es / ENTER cells.

[0161] Figure 39 shows the results showing the differentiation of human dermal fibroblasts into SF es / ENTER cells using human ES culture medium and ultrasonic stimulation, where Figure 39a shows the change in cell morphology over culture time, and Figures 39b and 39c show the pluripotency (b) and three germ layer (c) attributes of SF es / ENTER cells.

[0162] <Example 11> Differentiation induction into es / ENTER cells using other physical stimuli This time, we used heat shock and laser as physical stimuli to induce differentiation in the same culture medium environment of human ES cells.

[0163] First, the differentiation of es / ENTER cells from HDFs was induced using heat shock and hES medium. For heat treatment, HDFs were exposed to 42°C for 2 minutes and then placed on ice for about 5 seconds. Figure 40a shows the differentiation-induced HDF spheroids, and Figures 40b and 40c show the pluripotency (b) and three germ layer (c) attributes of es / ENTER cells.

[0164] Next, the differentiation of es / ENTER cells from HDFs was induced using laser stimulation and hES medium. The laser treatment conditions were Ocla therapeutic laser (Ndlux), and the cells were cultured after irradiation with an 808 nm laser for 5 seconds. Figure 41a shows the differentiation-induced HDF spheroids, and Figures 41b and 41c show the pluripotency (b) and three germ layer (c) attributes of es / ENTER cells.

[0165] As shown in Figures 40 and 41, both types of stimulation confirmed that cell spheroids were formed and pluripotency and three germ layer markers were expressed in a manner similar to the effect of ultrasound. These results indicate that cell reprogramming by influx of culture medium environment can be applied to various cells, and that various methods can be adopted for physical stimulation for influx of environment in addition to culture medium environment, and as in the previous results, the attributes of cells can be changed depending on the environment.

[0166] Example 12: Cell reprogramming using extracellular vesicles (EVs) Cells were cultured in DMEM supplemented with 10% FBS (Gibco) and 1% penicillin / streptomycin (Gibco) from primary HDF purchased from Invitrogen. The culture medium was sonicated at 5 W / cm. 2 , 10 min, and cell treatment was performed with lx10 6 1W / cm for HDF 2 After 5 seconds of treatment, 2x10 5 The cells were cultured for 1 day at 37°C and 5% CO2. The culture medium was collected and placed in an Amicon Ultra centrifugal filter (Millipore), and centrifuged at 14,000 rpm for 20 minutes to filter and collect EVs in the culture medium.

[0167] Next, the HDFs were cultured in a culture dish so that the dish was filled with approximately 70-80% of the original volume. The culture medium was then collected and washed twice with D-PBS. After that, 10 μl / mL (v / v) of concentrated EVs collected from the culture medium on day 1 of es / ENTER and n / ENTER were added to embryonic stem cell medium or neural stem cell differentiation medium (Gibco), respectively, and then mixed with the washed HDFs and cultured for 3 days.

[0168] <Example 13> EVs transduction experiment in normal somatic cells (HDF) To confirm the reprogramming of somatic cells using EVs, EVs obtained after one day of culture of physically stimulated cells were concentrated as in Example 12, and then the EVs were labeled with Did dye. The EVs were then transferred to normal somatic cells, and the expression of the pluripotency marker Oct4 and the neural stem cell marker Pax6 was confirmed in the transferred cells.

[0169] To this end, 50 μl of EVs obtained in Example 12 was mixed and diluted with 450 μl of D-PBS, and 2.5 μl of Vybrant DiD cell-labelling solution (molecular probe, excitation / emission, 644 / 667 nm) was added thereto to stain exosomes at 37 ° C for 30 minutes. After staining, the Did-stained EVs were concentrated by centrifugation at 14000 rpm for 20 minutes using an Amicon Ultra Centrifugal Filter (Millipore), and then diluted with D-PBS twice. The EVs were then added to 3 mL of HDF culture medium (DMEM (Gibco) culture medium containing 5% FBS) and cultured at 37 ° C and 5% CO2 for 24 hours. The HDFs cultured for 24 hours were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.2% Triton X100 in PBS buffer for 10 minutes. Next, after blocking with 3% BSA in PBS buffer for 1 hour, the primary antibodies rabbit anti-Oct4 (1:250, Abeam) and Pax6 (1:200, Abeam) were stained overnight at 4°C, and then the secondary antibody anti-rabbit conjugated Alexa-488 (1:1000, Thermo, excitation / emission, 495 / 519 nm) was stained for 1 hour. The secondary antibody-stained samples were analyzed using a mounting solution containing DAPI (4',6-diamidino-2-phenylindole dihydrochloride) (Vector Laboratories, excitation / emission, 420 / 480 nm) and images were analyzed using a confocal laser scanning microscope (LSM700; Carl Zeiss). The results are shown in Figure 42. In this figure, green indicates Oct4 and red indicates EVs stained with Did dye.

[0170] As shown in Figure 42, it was confirmed that EVs secreted from cells after physical stimulation contained various pluripotency marker genes and proteins depending on the cell culture environment, and these factors could be transmitted to neighboring cells by EVs. The results suggest that EVs secreted from cells subjected to physical stimulation in various culture environments may induce reprogramming of normal somatic cells.

[0171] Example 14: Reprogramming effect of human fibroblasts by EVs In Example 13, EVs secreted from cells subjected to physical stimulation in various medium environments may induce reprogramming of normal somatic cells, and to verify this, experiments were performed using DMEM medium, which is a culture medium for human fibroblasts, and hESC medium, which is a culture medium for human embryonic stem cells or iPS cells. The control group was cultured for 3 days in each medium without EVs, and the treatment group was cultured for 3 days in each medium with 10 μl / mL (v / v) of EVs added.

[0172] The cultured cells were stained with rabbit anti-Oct4 (1:250, abeam) as the primary antibody and anti-rabbit conjugated Alexa-488 (1:1000, Thermo, excitation / emission, 495 / 519 nm) as the secondary antibody, as in Example 13, and mounted with a mounting solution containing DAPI. Images were analyzed using a confocal laser microscope and shown in Figure 43. In the figure, hESC represents human ESC medium, DMEM represents fibroblast culture medium, and EVs represents EVs collected during induction of es / ENTER.

[0173] As shown in Figure 43, Oct4 expression was not observed in the control group, but Oct4 expression and cell spheroid formation were observed in the treatment group. The results indicate that cell reprogramming was not influenced by the culture medium, but was simply induced by EVs.

[0174] Example 15: Changes in cell morphology of EVs-treated human fibroblasts depending on the culture time Human fibroblasts were treated with 10 μl / mL (v / v) of EVs collected during es / ENTER induction, and then cultured for 6 days to observe changes in cell morphology.

[0175] As shown in FIG. 44, the morphology of the cells changed with the passage of culture time, and it was observed that spheroids were formed on the third day.

[0176] <Example 16> Confirmation experiment on the expression of pluripotency markers in HDFs cultured for 6 days according to the amount of EVs added collected during es / ENTER induction To determine the optimal concentration of EVs for cell reprogramming, HDFs were treated with different amounts of EVs and cultured for 6 days. In addition, cells expressing Oct4, a pluripotency marker, were analyzed by flow cytometry to use EVs collected during es / ENTER induction.

[0177] To this end, EVs collected during the induction of es / ENTER were added to the culture of fibroblasts at concentrations of 0, 5, 12.5, 25, 50 and 100 μl / mL (v / v), respectively, and cultured for 6 days under conditions of 37° C. and 5% CO 2. The cultured cells were stained with rabbit anti-Oct4 (1:250, abeam) as the primary antibody and anti-rabbit conjugated Alexa-488 (1:1000, Thermo, excitation / emission, 495 / 519 nm) as the secondary antibody, as in Example 13, and analyzed using a BDA ccuri™ C6 flow cytometer (BD biosciences).

[0178] As shown in Figure 45, when 12.5 μl / mL (v / v) of EVs was treated, Oct4 expression was confirmed in the highest proportion of cells, 84.6%.

[0179] <Example 17> Confirmation experiment on expression of pluripotency markers in EVs-treated 3-day cultured HDFs collected upon induction of es / ENTER Human fibroblasts were treated with 10 μl / mL (v / v) of EVs collected during the induction of ES / ENTER, and then cultured for 3 days to confirm the cell reprogramming effect. For ICC analysis, the cultured cells were mounted with a mounting solution containing DAPI using rabbit anti-Oct4 (1:250, abeam), Sox2 (1:250, abeam) and Nanog (1:250, abeam) as primary antibodies, and anti-rabbit conjugated Alexa-488 (1:1000, Thermo, excitation / emission, 495 / 519 nm) as secondary antibodies, as in Example 13, and images were analyzed using a confocal laser microscope. qPCR analysis was performed by recovering all RNA from the cells cultured for 3 days using Trizol (Takara), and synthesizing cDNA using Superscrip 2 kit (Invitrogen). PCR analysis was performed using a real-time PCR instrument (ab step one plus, AB) for pluripotency markers Oct4, Sox2 and Nanog.

[0180] As shown in FIG. 46, ICC analysis revealed that expression of pluripotency markers Oct4, Sox2, and Nanog was observed in the nuclei of human fibroblasts, and real-time qPCR analysis of gene expression revealed that Oct4, Sox2, and Nanog genes were overexpressed by approximately 50-fold compared to normal fibroblasts not treated with EVs.

[0181] <Example 18> Confirmation experiment on the expression of neural stem cell markers in EVs-treated 3-day cultured HDFs collected upon induction of n / ENTER Human fibroblasts were treated with 10 μl / mL (v / v) of EVs collected during n / ENTER induction and then cultured for 3 days to confirm the cell reprogramming effect. For ICC analysis, the cultured cells were mounted in a mounting solution containing DAPI using rabbit anti-Sox1 (1:200, abeam), Sox2 (1:250, abeam), Pax6 (1:200, abeam) and mouse anti-Nestin (1:250, Thermo Scientific) as primary antibodies and anti-rabbit conjugated Alexa-488 (1:1000, Thermo excitation / emission, 495 / 519 nm) and anti-mouse conjugated Alexa-594 (1:1000, Thermo, alexa488 excitation / emission, 495 / 519 nm; alexa594 excitation / emission, 590 / 617 nm) as secondary antibodies, as in Example 13, and images were analyzed using a confocal laser microscope. qPCR analysis was performed by recovering total RNA from cells cultured for 3 days using Trizol (Takara), synthesizing cDNA with the Superscript 2 kit (Invitrogen), and analyzing the neural stem cell markers Sox1, Sox2, and Nestin using a real-time PCR instrument (ab step one plus, AB).

[0182] As shown in Figure 47, ICC analysis revealed the expression of neural stem cell markers Sox1, Sox2, and Pax6 in the nuclei of human fibroblasts, and Nestin in the cytoplasm. Real-time PCR qPCR analysis of gene expression revealed that Sox1, Sox2, Pax6, and Nestin genes were overexpressed by approximately 200-fold compared to normal fibroblasts not treated with EVs.

[0183] [Table 6] [Industrial Applicability]

[0184] The present invention can be used in the field of cell therapy agents.

Claims

1. The mixture of the first differentiation cells and the culture medium is Applying physical stimulation by ultrasonic treatment that can promote environmental influx; Incubating the sonicated mixture for 1 to 6 days; and mixing the exosome-containing extracellular vesicles separated from the culture with second differentiated cells and culturing the mixture for a certain period of time to obtain reprogrammed cells from the second differentiated cells. The ultrasonic treatment was performed at an output intensity of 0.5 W / cm 2 ~3 W / cm 2 for 1 to 5 seconds, The environmental influx includes the influx of genetic material, chemicals, small molecules, exosomes or exosome-containing extracellular vesicles in the mixture to which the physical stimulus is applied; or the influx of culture medium components into the cells. The culture medium is any one of a multipotent cell differentiation induction medium, a hepatocyte differentiation induction medium, an osteogenic differentiation induction medium, an adipocyte differentiation induction medium, a muscle cell differentiation induction medium, an astrocyte differentiation induction medium, a nervous system cell differentiation induction medium, a vascular endothelial cell differentiation induction medium, a keratinocyte differentiation induction medium, a pancreatic β cell differentiation induction medium, and a cardiomyocyte differentiation induction medium; The cells to be reprogrammed are determined by selecting the type of culture medium. Methods for cell reprogramming.

2. The method for reprogramming cells according to claim 1 , wherein the first differentiated cell and the second differentiated cell are any one of somatic cells, cancer cells, and tissue cells in an organ.

3. Before mixing the culture medium with the first differentiated cells, the culture medium was exposed to a power intensity of 1 W / cm 2 ~20W / cm 2 The method for reprogramming cells according to claim 1, further comprising treating the cells with ultrasound at a dose of 1 to 20 nm for 1 to 20 minutes.

4. The method for reprogramming cells according to claim 1, further comprising irradiating the culture medium with a pulsed laser beam having a wavelength band of 300 to 900 nm for 1 to 20 minutes before mixing the culture medium with the first differentiated cells.

5. The method for reprogramming cells according to claim 1, further comprising heat-treating the culture medium at a temperature of 40 to 50° C. for 5 to 20 minutes before mixing the culture medium with the first differentiated cells.

6. The method for reprogramming cells according to claim 1 , wherein the mixture to which the physical stimulus is applied is cultured through a suspension culture or an adherent culture method.

7. The method for reprogramming cells according to claim 1 , wherein the exosome-containing extracellular vesicles are collected by centrifugation of the culture.

8. Exosome-containing extracellular vesicles are any one of the following pluripotency markers or three germ layer markers: OCT3 / 4, SOX2, NANOG, c-MYC, KLF4, TDGF1, SSEA4, TRA-1-60, PAX6, Nestin, Brachyury, SMA, GATA4, or AFP; any one of the neuronal markers PAX6, Nestin, Sox1, Sox2, MAP2, TuJl, GFAP, or O4; any one of muscle cell markers: Desmin, Actinin, Pax3, SMA, GATA4, or NKX2-5; any one of the following hepatocyte markers: AFP, HNFla, HNF4a, CK18, or ALB; or The method for reprogramming cells according to claim 1 , wherein the cells are stained with Oil Red O and express any one of the adipocyte markers Pparc2, C / ebpa, aP2, or Fabp4.

9. The method for reprogramming cells according to claim 1, wherein the exosome-containing extracellular vesicles and the second differentiated cells are cultured for 1 to 20 days in a suspension or adherent culture mode.

10. The method for reprogramming a cell according to claim 1, wherein the reprogrammed cell is any one of a pluripotency cell; or a differentiated cell including a hepatocyte, an osteoblast, an adipocyte, a muscle cell, a neuron, an astrocyte, a keratinocyte, a hair root cell, a pancreatic beta cell, or a cardiomyocyte, and the first differentiated cell, the second differentiated cell, and the reprogrammed cell have different cell phenotypes.

11. The method for reprogramming cells according to claim 10, wherein the pluripotent cells express any one of a pluripotency marker gene, Oct3 / 4, SOX2, NANOG, c-MYC, KLF4, TDGF1, SSEA4, TRA-1-60, PAX6, Nestin, Brachyury, SMA, GATA4, and AFP, or a three germ layer marker gene.

12. The method of claim 10, wherein the differentiated cell is any one of a neuronal cell expressing any one of PAX6, Sox1, Sox2, Nestin, MAP2, TuJ1, GFAP, or O4; a muscle cell expressing any one of Desmin, Pax3, Actinin, SMA, GATA4, or NKX2-5; a hepatic cell expressing any one of AFP, HNF1a, HNF4a, CK18, or ALB; or an adipocyte stained with Oil Red O and expressing any one of Pparc2, C / ebpa, aP2, or Fabp4.

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