KR102416171B1 - Reprosomes, the exosomes capable of inducing reprogramming of cells and the preparation method thereof - Google Patents

Abstract

It is characterized in that it contains RNA of a gene involved in chromatin remodeling, wherein the gene includes a kinase gene and a gene having histone modification activity on MAPK (mitogen-activated protein kinase) signaling system. A liposome, an exosome that induces reprogramming of cells, is disclosed. Such liposomes can be obtained from difficult-to-handle stem cells as well as from somatic cells that can be easily obtained and maintained through a simple process of sonication, and through a simple treatment of co-culturing the liposomes with cells, one type of cell can be reprogrammed with high efficiency and in a short time into other cells with the desired function, and the obtained cells have the desired function without the introduction of chemicals or foreign transcription factors into the genome, so they have a more suitable safety for cell replacement therapy. . Also disclosed are a composition comprising a liposome and its use for tissue regeneration by treating a body part with the same to promote reprogramming of cells present at the treated site into target cells having a desired function.

Description

Liposome inducing reprogramming of cells and method for manufacturing the same

The present invention relates to a liposome, which is an exosome capable of inducing reprogramming of cells, and a method for manufacturing the same, and more particularly, a liposome containing a large amount of chromatin remodeling factor, and a liposome It relates to a method capable of producing in a high yield, and to the use of the liposome prepared by the method.

Exosomes are naturally secreted nanovesicles with a diameter of 30 to 200 nm, and it is known that they can act as important nanomediators that transport genetic material from derived cells. Since the discovery that exosomes can change the phenotype of target cells through mRNA delivery, several studies have revealed that exosomes are involved in cell differentiation and the like. In these previous studies, stem cells and progenitor cells were used as targets for inducing differentiation or phenotypic changes using cells or exosomes to obtain exosomes. There is a problem with the side. Therefore, it induces secretion of exosomes containing factors that can change cells in a desired direction from somatic cells of the patient, which can be easily obtained, and also converts somatic cells, which can be obtained easily by using these exosomes, into other cells with desired functions. If it can be changed, it will be a huge leap forward for the clinical application of cell replacement therapy.

Additionally, the possibility of using the exosome itself directly on the human body for therapeutic purposes is being explored in various ways. In the field of biologics, exosomes are in an intermediate position between small molecules, peptides, growth factors, antibodies, and biopharmaceuticals such as nucleic acids and large pharmaceuticals such as various cells and platelets. That is, compared to biopharmaceuticals, exosomes contain a variety of proteins and nucleic acids, and thus can produce more complex and long-lasting effects. In addition, it has other advantages compared to cells. That is, when cells are injected into a living body, there is a concern about safety, for example, the injected cells may metastasize to cancer cells. In addition, cells injected into blood vessels for cell therapy are generally filtered and trapped in organs with a filtering function, such as lung, liver, spleen, or kidney. is known to be Therefore, when an exosome having a small size while containing an active ingredient that can be secreted from the cells is administered, it is possible to more safely produce a spatiotemporal therapeutic effect different from that of an intact cell.

However, as described above, exosomes with high potential as a tool for cell therapy or as a therapeutic agent themselves are currently difficult to mass-produce. For example, exosomes that can be obtained from 60 million mesenchymal stem cells cultured in 1 liter of medium are at a level that can obtain 1-2 mg based on the included protein content. This is an amount that can be used for a treatment experiment in several mice, and in humans, for example, exosomes used for the treatment of Graft-versus-host disease (GVHD) are used for one patient based on protein content. It is known that 0.05 to 0.6 mg per kg body weight is required. Therefore, in order to use the exosomes clinically, a technology capable of increasing the yield is required.

The present invention responds to various needs, including the aforementioned needs, that exist in the current cell therapy and exosome therapy. That is, stem cells generally used for cell therapy are difficult to isolate, amplify, and maintain, and it is difficult to predict the direction of their differentiation, and there are problems that they may develop into cancer. In addition, the process of obtaining cells having a desired function also takes a long time through several stages of differentiation, has low conversion efficiency, and is difficult to secure safety due to chemical treatment. Therefore, there is a need for a technology that allows easily obtainable cells to be used for cell therapy and a technology that can safely and efficiently obtain cells with a desired function in a short time. In the case of exosome therapeutics, there is a lack of technology to obtain a sufficient amount for treatment.

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International Publication WO2013/163296 (2013.10.31.)

The present invention has been devised to solve the above-mentioned problem, and an embodiment of the present invention is characterized in that it includes RNA of a gene involved in chromatin remodeling, and the gene is MAPK (mitogen-activated protein kinase) ) It provides a liposome capable of inducing reprogramming of cells in a desired direction, including a kinase gene on a signal transduction system and a gene having histone modification activity.

In addition, an embodiment of the present invention provides ultrasonic stimulation to cells, ultrasonic stimulation to a cell-free culture medium, mixes the cells with the culture medium, and incubates the above-mentioned liposomes in a short time. A manufacturing method capable of mass production is provided.

In addition, an embodiment of the present invention provides a method for efficiently reprogramming the cell in a desired direction by treating the liposome to the cell.

In addition, there is provided a composition comprising the liposome inducing reprogramming of the cell of the present invention.

The technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. There will be.

As a technical means for achieving the above-described technical problem, the liposome capable of inducing reprogramming of a cell according to an aspect of the present invention includes RNA of a gene involved in chromatin remodeling, The gene is characterized in that it includes a kinase gene on a mitogen-activated protein kinase (MAPK) signal transduction system and a gene having histone modification activity.

Here, the kinase gene on the MAPK signaling system may be at least one selected from the group consisting of BRAF, MAP2K3, MAP3K10, MAP3K4, MAP3K5, MAP3K7, MAPK12, RPS6KA4 (MSK2), TAOK1 and TAOK2.

The gene having the histone modification activity may be selected from the group consisting of ASH1L, CREBBP, DOT1L, EP300, GTF3C1, KAT2A, KAT6B, KDM1A, KDM3B, KDM6A, KMT2A, KMT2E, NCOA3, NSD1, SETD1A and SETD2. .

It may be characterized in that the ratio of small RNA (small RNA) of the total RNA in the liposome is 40% or more, and the ratio of microRNA (miRNA) of the small RNA is 40% or more.

Another aspect of the present invention is to provide ultrasonic stimulation to cells, and to provide ultrasonic stimulation to a cell-free culture medium; Mixing the cells and the culture medium and culturing for a predetermined time; and separating the liposomes from the mixture; provides a method for producing liposomes inducing reprogramming of cells.

Here, the cells may be any one of mammalian-derived fibroblasts or tissue cells in organs.

The culture medium includes embryonic stem cell medium, neural stem cell medium, cardiac stem cell medium, dermal papilla cell medium, mesenchymal stem cell medium, osteogenic medium, myogenic medium, hematopoietic stem cell medium, neuron medium, astrocyte medium , oligodendrocyte medium, hepatocyte medium, adipocyte medium, muscle cell medium, vascular endothelial cell medium, pancreatic beta cell medium, or cardiomyocyte medium may be any one.

The culture medium may be any one of neural stem cell medium, dermal papilla cell medium, hepatocyte medium, and adipocyte medium.

The ultrasonic stimulation provided to the cells may be performed for 1 to 10 seconds at 10 to 30 KHz, 0.5 to 3 W/cm ² .

Ultrasonic stimulation provided to the culture medium may be performed at 10 to 30 KHz, 1 to 20 W/cm ² for 1 to 20 minutes.

The culture of the mixture may be characterized in that it proceeds for 1 to 10 days.

Separating the liposome may include centrifuging the cultured mixture to obtain a supernatant; filtering the supernatant with a filter to obtain a filtrate; and concentrating the filtrate. Here, the step of storing the supernatant at 4° C. or less for 7 days to 1 month before filtering with a filter may be further included. The isolated liposome may have a diameter of 50 to 200 nm.

Another aspect of the present invention comprises the steps of incorporating a liposome into the first culture medium; culturing the first cells in the mixture; and obtaining a second cell after the culturing; provides a method for reprogramming cells, including.

Here, the first cell may be any one of mammalian-derived fibroblasts or tissue cells in organs.

The second cell may be characterized as a cell having a differentiation capacity of less than pluripotency (pluripotency).

The second cell may be any one of embryonic stem cells, neural stem cells, cardiac stem cells, dermal papilla cells, mesenchymal stem cells, and hematopoietic stem cells.

The second cell is any one of neural stem cells, neurons, astrocytes, oligodendrocytes, hepatocytes, adipocytes, hair follicle cells, muscle cells, vascular endothelial cells, keratinocytes, pancreatic beta cells, or cardiomyocytes can be

The second cell may be characterized in that it is different from the first cell.

The liposome may be characterized in that it is incorporated into the first culture medium at a concentration of 10 ⁷ to 10 ¹⁵ pieces/ml.

The first culture medium may be characterized as the same as the culture medium used to prepare the liposome.

The second cell may be any one of a stem cell, a progenitor cell, or a precursor cell, and the culture of the first cell may be performed for 1 to 6 days.

The second cell is any one of neurons, astrocytes, oligodendrocytes, hepatocytes, adipocytes, hair follicles, muscle cells, vascular endothelial cells, keratinocytes, pancreatic beta cells, or cardiomyocytes; The culture of one cell may be characterized in that it proceeds for 10 to 60 days.

Another aspect of the present invention provides a composition comprising a liposome inducing reprogramming of the above-described cells.

The liposome according to an embodiment of the present invention contains various factors capable of inducing reprogramming of cells, particularly chromatin remodeling factors, and has a phospholipid-based membrane structure to facilitate cell membrane penetration, resulting in high material delivery efficiency. Therefore, it is possible to induce reprogramming into a cell having a desired function with high efficiency. For example, the direct differentiation efficiency of neurons using conventional human somatic cells is less than 0.1%, but the method using liposomes reaches about 70%.

The method for producing liposomes according to an embodiment of the present invention induces a large amount of liposome secretion through a simple process called sonication from easily obtainable somatic cells as well as stem cells and progenitor cells, which are difficult to separate and amplify. can do. The yield of the exosome induced in this way is higher than that of the conventional method, and the amount and number of various factors contained in the exosome is also high.

The cell reprogramming method according to an embodiment of the present invention can safely induce reprogramming into a cell having a desired function without introducing a chemical or foreign transcription factor into the genome. In addition, the reprogramming method can efficiently reprogram one type of cell into another cell within a relatively short time without having to go through several development steps through a simple process of culturing cells by adding liposomes to a culture medium. .

The composition including the liposome according to an embodiment of the present invention may promote tissue regeneration of the treated site by treating the body part to promote reprogramming of cells present in the treated part.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 is liposome generation data having neural progenitor cell (NPC) inducing ability induced according to Example 1 of the present invention, exosomes secreted from sonicated cells (UHDF) and untreated cells (NHDF) Electron microscope image (a) confirming the morphology of (iExo (liposome) and nExo, respectively), immunofluorescence staining image showing CD63 induction (b), size distribution and number of exosomes (cd), time after incubation Changes in the amount of exosome secretion according to (e).
2 is component data of a liposome having neural progenitor cell (NPC) inducibility induced according to Example 1 of the present invention, the RNA concentration in the exosome (a), the number of genes expressed by the RNA (b) ), the amount of small RNA and miRNA in the RNA (c), the protein concentration in the exosome (d), the NPC marker mRNA (Sox1, Sox2, Pax6 and Nestin) and miRNA (miR9, miR124a, miR125b and miR128-) in the exosome 1) Relative expression level (ef), neuronal-related mRNA (left), and heat map showing miRNA expression (g), and intracellular co-distribution of NPC markers (Sox1, Sox2, Pax6 and Nestin) and exosome marker (CD63) Immunofluorescence staining image showing (h).
Figure 3 is the NPC inducing ability analysis data of the liposome having neural progenitor cell (NPC) inducing ability isolated according to Example 1 of the present invention. iExo) Confocal microscopy image showing intracellular influx of liposomes according to time after treatment (b), morphological change of HDF (c), and qRT-PCR data for NPC markers (Sox1, Sox2, Pax6 and Nestin) RNA (d), immunofluorescence staining image and flow cytometry data for the marker protein (ef), cluster analysis data for NSC-related genes (g), and immunofluorescence staining image for cell division marker (Ki-67) (h) , The number of spheroids formed according to the concentration of exosome treatment and the expression levels of NPC markers (Sox1, Sox2, Pax6 and Nestin) (ij), and the growth curve of subcultured cells (k).
4 is an analysis data of the induction mechanism of neural progenitor cells (rNPC) according to Example 1 of the present invention, chromatin remodeling-related gene expression heat map (a), liposomes shown in nExo and liposome (iExo)-treated cell group. Western blot results showing the phosphorylation level of MAPK signaling system proteins (Erk1/2, p38 and Msk1) according to time after treatment, intracellular immunofluorescence staining images (bc), and NPC markers (Sox1, Sox2, Pax6 and Nestin) RNA qRT-PCR data for (d), immunofluorescence staining data for chromatin remodeling indicators (HP1α, H3K4me3, H3K27me3) and the distribution of the markers relative to the counterstained DAPI (e) and neuronal-related gene expression hits in each cell group map (f).
Figure 5 is the in vitro and in vivo differentiation capacity analysis data of rNPC according to Example 1 of the present invention, the neural markers of rNPC differentiated by culturing in a neural differentiation medium for 4 weeks (neuron markers Map2 and Tuj1, and astrocyte marker Gfap) , Immunofluorescence staining data for oligodendrocyte marker O4) (a), voltage clamp data and action potential data (bc) for the differentiated rNPC, in vitro differentiation potential data, and GFP 4 weeks after transplantation into rat brain -rNPC distribution (d), immunofluorescence staining results using human mitochondrial antibody and GFP (e) and immunofluorescence staining results for the neuronal markers (f).
Figure 6 is the generation data of a liposome having adipocyte inducing ability according to Example 2 of the present invention, Nanosight and electron microscope images confirming the form of iExo (liposome) (a), exosome marker Immunofluorescence staining image (bc) showing the induction of phosphorus CD63 and the coexistence of CD63 and UCP1, a brown adipocyte marker, and analysis of brown adipocyte-related mRNA/miRNA and lipid synthesis-related mRNA characteristics in isolated liposomes RNA-seq data ( d).
7 is immunofluorescence staining data of brown adipocytes (rBA) according to Example 2 of the present invention. Immunofluorescence using AdipoRed, a lipid staining reagent, human mitochondrial antibody (HuMito), and an antibody against UCP1, a brown adipocyte marker. dyed image.
Figure 8 is data of the generation of liposomes having hepatocyte inducibility according to Example 3 of the present invention, Nanosight and electron microscope images confirming the form of iExo (liposomes) (a), exosome markers Immunofluorescence staining images showing the induction of CD63 and coexistence of CD63 with the hepatocyte marker HNF1α (bc), and RNA-seq data for characterization of hepatocyte-associated mRNA/miRNA in isolated liposomes (d).
9 is immunofluorescence staining data of hepatocytes (rH) according to Example 3 of the present invention, and immunofluorescence staining images for hepatocyte markers AFP, HNF4α, CK18 and ALB.
Figure 10 is the generation data of the liposome having the ability to induce hair tissue differentiation according to Example 4 of the present invention, Nanosight and electron microscope images confirming the form of iExo (liposome) (a), the exosome marker CD63 Immunofluorescence staining image (bc) showing the induction of CD63 and the coexistence of Shh, a protein important for hair regeneration, and RNA-seq data (d) for hair regeneration-related mRNA characterization in isolated liposomes.
11 is in vivo gene expression change data in C57 and nude mouse skin by liposomes having hair regeneration ability according to Example 4 of the present invention, and skin application of liposomes labeled with the lipophilic marker Did Fluorescent staining image showing post-distribution (ab), protein (β-Catenin, Shh, Ki-67) immunofluorescent staining image (cd) and mRNA (Shh, β-Catenin, KRT-25, VCAN, Gli1, Lef1, Pct1, Tyrp1, Tyr, Mitf, Dct, Sfrrp4, DKK) qRT-PCR data (ef).
12 is tissue change data in C57 and nude mouse skin by liposomes having hair regeneration ability according to Example 4 of the present invention, H&E staining image of skin tissue (ab), and hair follicles in subcutaneous and whole skin Numeric data (cd).
13 is hair regeneration data in C57 and nude mouse skin by liposomes having hair regeneration ability according to Example 4 of the present invention, and an image of hair generation according to the treatment concentration of the exosomes and time after treatment (ab) ).
14 is an immunofluorescence staining image showing the induction of CD63, an exosome marker, as data for generation of liposomes having wound healing ability according to Example 5 of the present invention (a), and wound healing in the induced liposomes. qRT-PCR data (b) and RNA-seq data (c) analyzing the RNA of a gene.
15 is an in vitro effect data of the liposome having wound healing ability according to Example 5 of the present invention. Immunofluorescence staining showing the expression of the cell proliferation marker Ki67 in HDF treated with the liposome at various concentrations. Image (a), time-dependent growth rate (b) for each liposome treatment concentration of HDF measured by MTT assay, mobility capacity for each liposome treatment concentration of HDF measured by scratch assay (cd) over time (cd), liposome treatment An optical microscope image showing the tube formation of HUVECs by concentration, the length of the formed tube and number of intersections (eg), and qRT-PCR data analyzing RNA of wound healing-related genes in cells treated with liposomes (h).

Hereinafter, the present invention will be described in more detail. However, the present invention may be embodied in various different forms, and the present invention is not limited by the embodiments described herein, and the present invention is only defined by the claims to be described later.

In addition, the terms used in the present invention are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the entire specification of the present invention, 'including' any component means that other components may be further included, rather than excluding other components, unless otherwise stated.

According to one aspect of the present invention, the liposome capable of inducing reprogramming of cells includes RNA of a gene involved in chromatin remodeling, and the gene is a mitogen-activated protein kinase (MAPK) signal. It is characterized in that it contains a kinase gene and a gene having histone modification activity on the delivery system.

Here, the kinase gene on the MAPK signaling system is BRAF (B-Raf proto-oncogene), MAP2K3 (Mitogen-activated protein kinase kinase 3), MAP3K10 (Mitogen-activated protein kinase kinase kinase 10), MAP3K4 (Mitogen-activated protein kinase kinase kinase 4), MAP3K5 (Mitogen-activated protein kinase kinase kinase kinase 5), MAP3K7 (Mitogen-activated protein kinase kinase kinase 7), MAPK12 (Mitogen-activated protein kinase 12), RPS6KA4 (Ribosomal protein S6 kinase A4, also known as Msk2), TAOK1 (TAO kinase 1), and TAOK2 (TAO kinase 2) may be selected from the group consisting of at least one or more. Preferably, the kinase gene may include at least MAP3K10, RPS6KA4, and TAOK1 among the 10 genes, and more preferably, at least MAP2K3, MAP3K10, MAP3K7, MAPK12, RPS6KA4, TAOK1 and TAOK2.

The gene having the histone modification activity is ASH1L (ASH1 like histone lysine methyltransferase), CREBBP (CREB binding protein), DOT1L (DOT1 like histone lysine methyltransferase), EP300 (E1A binding protein P300), GTF3C1 (General transcription factor IIIC subunit 1) , KAT2A(Lysine acetyltransferase 2A), KAT6B(Lysine acetyltransferase 6B), KDM1A(Lysine demethylase 1A), KDM3B(Lysine demethylase 3B), KDM6A(Lysine demethylase 6A), KMT2E(Lysine methyltransferase 2A), KMT2E(Lysine methyltransferase 2A), KMT2E(Lysine methyltransferase 2A) At least one may be selected from the group consisting of Nuclear receptor coactivator 3 (NCOA3), Nuclear receptor binding SET domain protein 1 (NSD1), SET domain containing 1A (SETD1A), and SET domain containing 2 (SETD2). Preferably, the gene having the histone modification activity may include at least ASH1L, CREBBP, DOT1L, EP300, GTF3C1, KAT6B, KDM1A, KDM3B, KMT2A, KMT2E and NSD1 among the 16 genes, more preferably at least ASH1L, CREBBP , DOT1L, EP300, GTF3C1, KAT6B, KDM1A, KDM3B, KMT2A, KMT2E, NCOA3, NSD1 and SETD2.

Small RNA is RNA having a size of less than 200 nt (nucleotides) and is mainly non-coding RNA. Small RNA includes microRNA, short interfering RNA, short nucleolar RNA, piwi-interacting RNA (piRNA), and the like. Among them, miRNA has a length of about 22 nt and is known to be involved in RNA silencing and post-transcriptional gene regulation. The ratio of small RNA among the total RNA in the liposome is 40% or more, and it may be characterized in that the ratio of miRNA in the small RNA is 40% or more. More preferably, the ratio of small RNA in the total RNA in the liposome is 50% or more, the ratio of miRNA in the small RNA is 50% or more, Most preferably, the ratio of small RNA in the total RNA in the liposome is 60% or more, and it may be characterized in that the miRNA ratio among the small RNAs is 60% or more.

The liposome capable of inducing reprogramming of the cells described above may be characterized in that it is prepared by a manufacturing method to be described later.

Another aspect of the present invention is to provide ultrasonic stimulation to cells, and to provide ultrasonic stimulation to a cell-free culture medium; Mixing the cells and the culture medium and culturing for a predetermined time; and isolating liposomes from the mixture; It provides a method for producing a liposome that induces reprogramming of the cell, comprising a.

Here, the cells may be preferably selected from cells other than germ cells, and more preferably may be any one of mammalian-derived fibroblasts or organ tissue cells. According to the method for producing a liposome inducing reprogramming of a cell according to an aspect of the present invention, the liposome can be obtained using any cell, and thus it is difficult to obtain and amplify stem cells or progenitor cells. This is because cells that are easier to obtain, maintain, and amplify, such as fibroblasts or intra-organ tissue cells, are easier and more efficient. The cell may be any one of autologous, allogeneic, or heterologous, depending on the future use of the liposome for other cells or living organisms to be described later. may have been obtained. Since there is a possibility of immune rejection, it is preferably allogeneic, and most preferably autologous.

The step of applying ultrasonic stimulation to the cells may be performed in a state in which the cells are directly sonicated, or the cells contain only a minimal amount so as to barely cover the initial culture medium. The initial culture medium at this time is a general medium used to maintain the cells in a healthy state, and may be a medium suitable for conventional culture of the cells, for example, DMEM medium in which the cells are fibroblasts and contain antibiotics and serum. .

The culture medium (the medium to be sonicated) includes embryonic stem cell medium, neural stem cell medium, cardiac stem cell medium, dermal papilla cell medium, mesenchymal stem cell medium, osteogenic medium, myogenic medium, hematopoietic stem cell medium, neurons ( neuron) medium, astrocyte medium, oligodendrocyte medium, hepatocyte medium, adipocyte medium, myocyte medium, vascular endothelial cell medium, pancreatic beta cell medium, or cardiomyocyte medium. The culture medium may be any one of a medium for differentiation induction or maintenance and amplification purposes. According to another aspect of the present invention, which will be described later, when it is desired to reprogram the first cell to the second cell by treating the liposome, preferably, a medium in which the second cell to be obtained can be maintained in good health and amplified. can be selected. According to another aspect of the present invention, which will be described later, when it is desired to promote reprogramming of cells in the site to target cells by treating a body part with a composition containing liposomes, preferably, the target cells in When cultured in vitro , a medium that can be maintained and amplified in good health can be selected.

The culture medium may be any one of neural stem cell medium, dermal papilla cell medium, hepatocyte medium, and adipocyte medium.

Ultrasonic stimulation provided to the cells may be performed for 1 to 10 seconds at 10 to 30 KHz, 0.5 to 3 W/cm ² , preferably 15 to 25 KHz, 0.5 to 1.5 W/cm ² for 3 to 7 seconds It may be carried out during

Ultrasonic stimulation provided to the culture medium may be performed for 1 to 20 minutes at 10 to 30 KHz, 1 to 20 W/cm ² , and preferably 7 to 13 at 15 to 25 KHz, 0.5 to 1.5 W/cm ² may be performed in minutes

The culture of the mixture may be characterized in that it proceeds for 1 to 10 days, preferably for 1 to 6 days, and most preferably for 1 to 2 days. This is because liposomes are secreted the most on the first day after sonication, and the amount of secretion decreases over time.

Separating the liposome may include centrifuging the cultured mixture to obtain a supernatant; filtering the supernatant with a filter to obtain a filtrate; and concentrating the filtrate. The centrifugation is performed to remove cell debris and dead cells, and may be preferably performed at 1000 to 5000 g for 10 to 60 minutes. The step of filtering the supernatant with a filter is performed to further remove cell debris and to leave only particles of a specific size or less, and the filter used here may preferably be a syringe filter. Concentrating the filtrate may preferably be performed using a centrifugal filter. If the centrifugal filter is used, it is possible to remove particles having a specific size or smaller while concentrating the filtrate. Separating the liposome may further include storing the supernatant at 4° C. or lower for 7 days to 3 months before filtering with a filter. The storage may be preferably within 7 days at 4° C. or less, more preferably within 1 month at -20° C. or less, and most preferably within 3 months at -80° C. or less. The active ingredients of liposomes are mRNA and protein, and the higher the temperature or the closer to the temperature at which the enzyme activity is high, the more easily these components can be denatured or degraded. Here, the isolated liposome may be characterized in that it has a diameter of 50 to 200 nm, preferably it may be characterized in that it has a diameter of 100 to 150 nm.

Another aspect of the present invention comprises the steps of incorporating a liposome into the first culture medium; culturing the first cells in the mixture; and obtaining a second cell after the culturing; provides a method for reprogramming cells, including.

Here, the first cell may be any one of mammalian-derived fibroblasts or tissue cells in organs. This is because the method of reprogramming cells according to an aspect of the present invention can obtain a second cell no matter which somatic cell is used, fibroblasts or organ tissue cells, etc. , because cells that are easy to maintain and amplify are easier and more efficient. When the second cell is later transplanted into the human body, the first cell may preferably be a human-derived cell, and most preferably an autologous cell derived from a subject to be transplanted. When a cell derived from a living body that is genetically close to the transplant target is used as the first cell, the possibility of side effects such as rejection during cell transplantation can be reduced.

The second cell may be characterized as a cell having a differentiation capacity of less than pluripotency (pluripotency).

The second cell may be any one of embryonic stem cells, neural stem cells, cardiac stem cells, dermal papilla cells, mesenchymal stem cells, and hematopoietic stem cells.

The second cell is any one of neural stem cells, neurons, astrocytes, oligodendrocytes, hepatocytes, adipocytes, hair follicle cells, muscle cells, vascular endothelial cells, keratinocytes, pancreatic beta cells, or cardiomyocytes can be The differentiation capacity of non-terminal differentiated cells is divided into totipotency, pluripotency, multipotency, oligopotency, and unipotency from high to low levels. divide and call Pluripotency refers to the ability to differentiate into all cells of an organism and to form an organism from one cell. It means that it can differentiate into all germ cells. Multipotency refers to the ability to differentiate into several cells of a lineage or a minority lineage, oligopotency refers to the ability to differentiate into several cells in a smaller range, and unipotency refers to the ability to differentiate into several cells in a smaller range. It means that each can be differentiated into a cell type. Accordingly, the cells having a differentiation capacity less than or equal to the pluripotency include pluripotency, multipotency, oligopotency and unipotency cells and terminally differentiated cells. do. When the second cell is later transplanted into the human body, the second cell is preferably a stem cell, progenitor cell or progenitor cell having multipotency, oligopotency or unipotency ( precursor cells). In the case of pluripotent cells, the question of the possibility of mutation into cancer cells has been continuously raised, and in the case of terminally differentiated cells, there is a possibility that the lifespan is short or the effect of cell therapy may decrease.

The second cell may be characterized in that it is different from the first cell. For example, the first cell may be a cell obtained from fibroblasts or other tissues that are easy to obtain and maintain, and according to the cell reprogramming method of the present invention, a desired second cell can be easily obtained therefrom.

The liposome may be characterized in that it is incorporated into the first culture medium at a concentration of 10 ⁷ to 10 ¹⁵ cells/ml, preferably, it is incorporated into the first culture medium at a concentration of 10 ¹⁰ to 10 ¹² pieces/ml can be done with The inventors of the present invention were able to confirm that if the concentration of the incorporated liposomes was too low or high, the reprogramming efficiency decreased (Fig. 3i).

The first culture medium may be characterized as the same as the culture medium used to prepare the liposome, but is not limited thereto. This has the effect that, when the first culture medium is the same as the culture medium used to prepare the liposome, reprogramming into the desired second cell occurs more rapidly, but even when a different culture medium is used, the liposome is This is because, upon incorporation, reprogramming into a second cell may occur.

The second cell may be any one of a stem cell, a progenitor cell, or a precursor cell, and the culture of the first cell may be performed for 1 to 6 days. The second cell is any one of neurons, astrocytes, oligodendrocytes, hepatocytes, adipocytes, hair follicles, muscle cells, vascular endothelial cells, keratinocytes, pancreatic beta cells, or cardiomyocytes; The culture of one cell may be characterized in that it proceeds for 10 to 60 days. This is because there is a difference in the time taken for reprogramming into cells with high differentiation potential and reprogramming into cells with lower differentiation potential, and the latter tends to take a little longer, the latter preferably being cultured for 15 to 25 days. can

The composition according to another aspect of the present invention includes the above-described liposome inducing reprogramming of cells. Here, the composition may be used for, for example, treating a body part to promote regeneration of tissues present in the treated part. The body part may include the epidermis, dermis, and scalp, and in this case, the composition may be applied or injected to the part to have tissue regeneration effects such as wound healing or hair regeneration.

Since it is self-evident that various additives may be added to the composition in addition to the liposome, such as a pharmaceutically acceptable carrier and/or other additives that can further enhance the tissue regeneration effect by increasing the penetration rate into the treatment site. A detailed description thereof will be omitted.

As described above, the liposome according to an embodiment of the present invention contains various reprogramming factors, particularly chromatin remodeling factors, and has a phospholipid-based membrane structure to induce reprogramming into cells having desired functions with high efficiency. can do.

The method for producing a liposome according to an embodiment of the present invention is a method of preparing reprogramming factors of various cells from stem cells and progenitor cells, which are difficult to separate and amplify, as well as easily obtainable somatic cells, through a simple process of sonication. It can induce liposome secretion.

The cell reprogramming method according to an embodiment of the present invention can safely induce a cell having a desired function without introducing a chemical substance or foreign transcription factor into the genome. In addition, since the reprogramming method does not need to go through several development steps, it is possible to efficiently reprogram one type of cell into another cell within a relatively short time through a simple process of culturing by adding a liposome to a culture medium.

The composition comprising a liposome according to an embodiment of the present invention may be treated on a body part to promote tissue regeneration present in the treated part.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

All cell cultures in all Examples and Experimental Examples of the present invention were performed at 37° C., 5% CO ₂ conditions.

Example 1. Preparation of liposomes having neural progenitor cell induction ability and induction of reprogramming of fibroblasts into neural progenitor cells using the same.

In order to obtain liposomes having neural progenitor cell inducibility, ultrasonic stimulation of 20 KHz, 1.0 W/cm ² was applied to 1 Х10 ⁶ HDFs using UltraRepro 1001 (STEMON Inc., Seoul, Republic of Korea) for 5 seconds. Directly applied (hereinafter the stimulated HDF is referred to as UHDF). 2 Х10 ^Five UHDFs were incubated in 35-mm Petri dishes with sonicated (20 KHz, 5.0 W/cm ² , 10 min) hNSC medium for one day. Liposomes were separated from the culture medium in which the UHDF was cultured as follows: The culture medium was centrifuged at 3,000 X g for 20 minutes to remove cell debris and dead cells, and the supernatant was filtered with a 0.22-mm filter (Minisart®). Syringe Filter, Sartorius, Goettingen, Germany). The medium passed through was put into an Amicon® Ultra-15 100,000 kDa device (Millipore, Billerica, MA, USA) and centrifuged at 14,000 X g for 20 minutes to concentrate the liposomes (iExo).

To prepare rNPC (neural progenitor cells obtained by reprogramming with liposomes), 1 Х10 ⁵ HDFs were seeded in a 35-mm Petri dish and cultured for one day. The medium was changed and cultured for 5 days. The culture medium was changed every 2 days.

Example 2. Preparation of liposomes having adipocyte inducing ability and induction of reprogramming of fibroblasts into brown adipocytes using the same.

To obtain liposomes having adipocyte inducibility, 20 KHz, 1.0 W/cm ² ultrasonic stimulation was directly applied to 1 Х10 ⁶ HDFs for 5 seconds using UltraRepro 1001 (STEMON Inc., Seoul, Republic of Korea). added. 2 Х10 ⁵ UHDFs in a 35-mm Petri dish sonicated (20 KHz, 5.0 W/cm ² , 10 min) for one day with MesenCult ^TM adipogenetic Differentiation Medium, Stemcell technologes cultured. Liposomes were separated from the culture medium in which UHDF was cultured in this way through the same liposome separation process as in Example 1.

To prepare rBA (brown adipocytes obtained by reprogramming with liposomes), 1 Х10 ⁵ HDFs were seeded in a 35-mm Petri dish and cultured for one day, and then adipocytes including exosomes from which the medium was separated. Changed to differentiation induction medium and cultured for 20 days. The culture medium was changed every 2 days.

Example 3. Preparation of liposomes having hepatocyte inducing ability and induction of reprogramming of fibroblasts into hepatocytes using the same.

To obtain liposomes with hepatocyte inducibility, 20 KHz, 1.0 W/cm ² ultrasonic stimulation was directly applied to 1 Х10 ⁶ HDFs using UltraRepro 1001 (STEMON Inc., Seoul, Republic of Korea) for 5 seconds. did 2 Х10 ⁵ UHDFs were incubated in a 35-mm Petri dish with sonicated (20 KHz, 5.0 W/cm ² , 10 min) hepatocyte culture medium (HCM ^TM hepatocyte culture medium, Lonza) for one day. Liposomes were separated from the culture medium in which UHDF was cultured in this way through the same liposome separation process as in Example 1.

To prepare rH (hepatocytes obtained by reprogramming with liposomes), 1 Х10 ⁵ HDFs were seeded in a 35-mm Petri dish and cultured for one day, and then the medium was separated into hepatocyte culture medium containing exosomes. and cultured for 24 days. The culture medium was changed every 2 days.

Example 4. Preparation of liposomes having hair regeneration ability and induction of hair regeneration using the same.

To obtain liposomes with hair regeneration ability, 20 KHz, 1.0 W/cm ² ultrasonic stimulation was directly applied to 1 Х10 ⁶ HDFs using UltraRepro 1001 (STEMON Inc., Seoul, Republic of Korea) for 5 seconds. did 2 Х10 ^Five UHDFs were incubated in a 35-mm Petri dish with sonicated (20 KHz, 5.0 W/cm ² , 10 minutes) dermal papilla cell medium (Dermal Papilla cell medium, PromoCell) for one day. Liposomes were separated from the culture medium in which UHDF was cultured in this way through the same liposome separation process as in Example 1.

Example 5. Preparation of liposomes having wound healing properties.

To obtain liposomes with wound healing properties, 20 KHz, 1.0 W/cm ² ultrasonic stimulation was directly applied to 1 Х10 ⁶ HDFs using UltraRepro 1001 (STEMON Inc., Seoul, Republic of Korea) for 5 seconds. did ² Х10 embryonic stem cell culture medium (DMEM/F12, 15% FBS, ^2mM GlutaMAX, 0.1% NEAA, 0.1 % penicillin/streptomycin, 0.1 mM β-mercaptoethanol, 1000 unit/ml leukemia inhibitory factor (LIF)) and cultured for one day. Liposomes were separated from the culture medium in which UHDF was cultured in this way through the same liposome separation process as in Example 1.

Experimental Example 1. Experiments on the generation of liposomes with neural progenitor cell induction ability

In order to induce the secretion of liposomes having neural progenitor cell inducing ability, HDF was exposed to ultrasonic stimulation according to Example 1 (UNDF) and then cultured in sonicated human neural stem cell medium (hNSC medium) for one day. After culturing for one day, it was confirmed that a large amount of endoplasmic reticulum was induced in UHDF compared to NHDF using CD63, an exosome-specific marker (FIG. 1b). As a result of isolating the exosomes (iExo) induced in the UHDF culture medium and the exosomes secreted from NHDF (nExo) by the liposome separation method of Experimental Example 1, transmission electron microscopy images showed that all common exosome vesicles It was observed to have a sexual structure (Fig. 1a). As a result of the nanoparticle tracking analysis, the particle size of iExo was distributed in the range of about 50 to 200 nm, and the average was 155.6 +- 4.2 nm (Fig. 1c). iExo with a size of 50 to 200 nm was 9 Х 10 ⁸ , which was 3.2 times higher than that of nExo (Fig. 1d). These results show that exosomes can be efficiently induced by applying ultrasound to HDF. The secretion amount of iExo was highest on day 1 based on the time of incubation after sonication, and showed a tendency to decrease over time. could be confirmed (Fig. 1e).

To analyze the components of iExo, the total RNA and microRNA (miRNA) concentration and quality were measured from the exosomes isolated from the primary culture medium using the Agilent 2100 Bioanalyzer, and the protein through exosome RNA sequencing (RNA-Seq). The number of coding genes was determined. The total RNA concentration in iExo was 5.3 times higher than that of nExo (FIG. 2a), and the number of genes expressed with the RNA was 8400, which was 4.7 times higher than that of nExo, which recorded 1762 (FIG. 2b). Through open database search and gene ontology analysis, it was revealed that some mRNAs in iExo are related to neurogenesis (Fig. 2g). It was confirmed that the expression level of NPC-specific marker genes such as Sox1, Sox2, Pax6, and Nestin in iExo was increased through qRT-PCR (quantitative reverse transcription polymerase chain reaction; FIG. 2e). Among the non-coding small RNAs in the exosome, the proportion of miRNA of iExo (60.57%) was much higher than that of nExo (8.52%) ( FIG. 2c ). In addition, 53 out of a total of 72 in iExo were identified by Parsons et al. It was found to belong to the nervous system-specific miRNA according to (Fig. 2g). The total protein concentration of iExo was also increased by 20 times compared to that of nExo (Fig. 1d). Through immunofluorescence staining and flow cytometry, it was confirmed that iExo contained NPC-specific marker proteins Sox1, Sox2, Pax6 and Nestin, and it was confirmed that the protein expression of the genes increased as well as mRNA expression (Fig. 1h). The above results demonstrate that UHDF could be induced to secrete liposomes rich in mRNA, miRNA and protein involved in neurodevelopment. UHDF-induced a dramatic increase in the number of liposome transcripts and proteins, many of which play a role in maintaining differentiation capacity in stem cells. means there is

Experimental Example 2. Neural progenitor cell inducibility analysis experiment of liposomes according to Example 1

In order to confirm whether cellular reprogramming in the direction of a neuronal phenotype can be induced by liposomes, the present inventors checked whether iExo is delivered into HDF. iExo was labeled with DiD (DiD-labeled iExo), a lipophilic tracer, and then treated with HDF and cultured for one day. DiD-labeled iExo was confirmed in the cytoplasm of iExo-treated HDF (Fig. 3c, left panel). Additionally, to check whether the reprogramming factor inside iExo was delivered, intact HDF was labeled with Cy5.5-labeled poly(A)27, and then went through the process of preparing UHDF to include poly(A)27-Cy5.5 The exosomes (Cy5.5-exo) were induced. After HDF was treated with iExo containing Cy5.5-exo and cultured for one day, Cy5.5-exo was observed in the cytoplasm of HDF (Fig. 3c right panel). Interestingly, an increase in the expression of Pax6 was induced from HDF treated with iExo 24 hours after treatment, indicating that liposomes were delivered into HDF with high efficiency.

Next, we examined whether iExo could induce cellular reprogramming. HDF (1X10 ⁵ pieces, 7th subculture) was exposed to exosomes (hereinafter, hNSC-Exo, nExo, iExo, respectively) isolated from the culture medium in which hNSC, NHDF and UHDF were cultured for one day. Interestingly, when the three exosomes and HDF were co-cultured for 5 days, only iExo-treated cells (iExo-HDF) formed dense colonies (> 100 μm in diameter) ( FIG. 3b ), and NPC-specific An increase in the specific marker was observed from the first day of culture (Figure 3d-f). When cells were treated with 20 Х 10 ¹¹ iExos/ml, iExo-HDF showed maximum levels of spheroid formation and NPC-specific marker expression ( FIG. 3i ), and the effect of iExo was confirmed using this concentration below. . After 5 days of exosome treatment. The number of iExo-HDF colonies reached 1,500, and the expression of NPC-specific genes and proteins such as Sox1, Sox2, Pax6, and Nestin increased to a level comparable to that of hNSC, indicating NPC-like properties (Fig. 3d). -e). Finally, as a result of double positive flow cytometry, 74.7%, 68.5%, and 75.8% of Pax6/Nestin, Sox1/Nestin, and Sox2/Nestin cells in iExo-HDF on day 5, respectively (Fig. 3f). As a result of RNA-seq analysis, the neuronal-specific gene expression profile of iExo-HDF was similar to that of hNSC and showed a different pattern from HDF (Fig. 3g). Through gene ontology analysis, a number of genes overexpressed in iExo-HDF were neurogenesis was confirmed to be related to These results show that iExo can rapidly induce cell reprogramming to generate NPC-like cells in high yield from HDF in 5 days. On day 5, iExo-HDF, which had similar characteristics to NPC, was designated as rNPC, and dense colonies of iExo-HDF were designated as rNPC primary subculture group (p1).

The spheroids of rNPC p1 were collected and a homogeneous cell population was obtained through several passages. As a result of analyzing NPC-specific marker genes and proteins, rNPCs of p2, p4, p6 and p10 all expressed Sox1, Sox2, Pax6 and Nestin at high levels. As a result of Ki-67 immunofluorescence staining, it was found that rNPC was actively proliferating (Fig. 3h). The proliferative ability of P6 rNPC was maintained for several weeks ( FIG. 3k ). Taken together, the above results prove that the cells are a homogeneous and amplifiable group of cells that maintain the expression of Ki67 and NPC markers while going through several passages, indicating that rNPC possesses the amplification and self-renewal ability of NPCs. it means.

Experimental Example 3. rNPC induction mechanism assay

In order to elucidate the mechanism by which rapid cell reprogramming by liposomes appears, the inventors of the present invention focused on epigenetic regulation of gene expression by cellular stress. Interestingly, iExo contained mRNA and protein related to histone degeneration and mitogen-activated protein kinase (MAPK) pathway-related genes (FIG. 4a). Next, we examined whether iExo could induce chromatin remodeling by stimulating the MAPK signaling pathway in target cells. As a result of Western blot and flow cytometry, the expression of p38, Erk, and Msk1 in HDF increased sharply from one day after iExo treatment (Fig. 4b,c). In addition, when SB203580 and/or U0126, which are inhibitors of p38 and Erk of the MAPK signaling pathway, were treated with iExo-HDF, each target protein and the downstream proteins of the signaling pathway, Msk1 and Sox1, Sox2, Pax6 and It was confirmed that the expression of NPC-specific genes and proteins such as Nestin was significantly reduced (FIG. 4d). These results indicate that the MAPK signaling system acts as an important mechanism for liposomes to induce rNPC through rapid chromatin remodeling. Additionally, the changes in local chromatin density and histone degeneration in the nucleus of HDF on day 3 after iExo treatment were tracked. The local chromatin density was confirmed by transfection of H2B protein (H2B-GFP) labeled with green fluorescent protein (GFP). As time passed after iExo treatment, the H2B-GFP distribution appeared to spread (Fig. 4e). In addition, nuclear expression of HP1α (heterochromatin protein 1α) and inhibitory histone degeneration H3K27me3 decreased, and activated histone degeneration H3K4me324 increased in HDF with time after iExo treatment (Fig. 4f). In particular, as a result of confirming the DNA methylation profile of the neurogenesis-related gene, it was found that methylation was reduced (FIG. 4g). These results indicate that liposomes induce rapid NPC-like reprogramming through several epigenetic regulators as well as activation and chromatin remodeling of the MAPK signaling system.

Experimental Example 4. rNPC's in vitro and in vivo pluripotency assay

In order to evaluate the ability of rNPCs to differentiate into neurons, we checked whether rNPCs can differentiate into neural lineages such as neurons, astrocytes and oligodendrocytes. Day 5 rNPC spheroids were cultured in gelatin-coated culture plates according to a recently published neural differentiation protocol. After 4 weeks of neuronal differentiation, it was confirmed that Map2, Tuj1 (neuron marker), Gfap (astrocytic marker), and O4 (oligodendrocyte marker) were expressed in the differentiated cells (Figure 5a). qRT-PCR analysis of Map2, Tuj1, Gfap, S100b (astrocyte marker), Mbp (oligodendrocyte marker), and Oligo1 (oligodendrocyte marker) also indicated that rNPCs successfully differentiated into neurons. Next, the functional properties of rNPC-derived neurons were verified through whole-cell patch-clamp recordings. First, expression of genes associated with potassium ion channels (EAG1, Kv4.3, and Kv7.2) and sodium ion channels (Nav 1.3, Nav1.6, and Nav1.7) was significantly increased after differentiation from rNPCs into neurons. confirmed that. Inward sodium and outward potassium currents were confirmed in neurons derived from rNPC during current clamping (FIG. 5b). In addition, it was observed that sodium current was inhibited in neurons derived from rNPC by TTX (tetrodotoxin), a sodium ion channel blocker. Neurons derived from rNPCs evoked action potentials in response to current clamps (Fig. 5c). The above results indicate that rNPC has multipotency and effectively differentiated into neurons, astrocytes and oligodendrocytes in vitro .

To confirm that rNPC can differentiate pluripotently in vivo , using HDF transfected with a GFP reporter expressed by CMV (cytomegalovirus) promoter, cells stably expressing the reporter with G418 antibiotic (GFP-HDF) has been established. Established day 5 GFP-rNPC spheroids (about 1,500 clusters) were transplanted into the brain (striata, striatum) of normal Sprague Dawley rats (n = 5), and the differentiation status of these cells was confirmed 4 weeks later. As a result, GFP-positive cells could be identified in various parts of the brain, and many of them formed long neurites (Fig. 5d), indicating that these cells survived, migrated and successfully integrated into the host. . GFP-positive cells were confirmed to be stained with human mitochondrial antibody, confirming that these cells were GFP-rNPC (FIG. 5e). Some of the Gfap-positive astrocytes, Map2 and Tuj1-positive neurons, and O4-positive oligodendrocytes were GFP-positive ( FIG. 5f ). The above results indicate that the rNPC derived from the transplanted HDF could have the pluripotency to differentiate into three different cell types of the nervous system in vivo in 5 days.

Experimental Example 5. Liposome assay with adipocyte inducibility

As a result of analyzing UHDF cultured according to the liposome induction method having adipocyte inducing ability of Example 2 through immunofluorescence staining for the CD63 exosome marker (in this case, the counterstaining was performed with the nuclear staining agent DAPI), a large amount exosomes were being generated (FIG. 6a), and as a result of immunofluorescence staining of the marker and the brown adipocyte marker UCP1, it was confirmed that UCP1 was expressed in the secreted exosomes (FIG. 6b). The morphology of liposomes obtained according to the method for preparing liposomes having adipocyte inducibility of Example 2 was analyzed using Nanosight and TEM (transmission electron microscopy), and as a result, a normal morphology of exosomes was observed ( FIG. 6c), as a result of RNA-Seq analysis of the liposome, it was found that brown adipocyte-related mRNA and microRNA and lipid synthesis-related mRNA were significantly increased compared to the control group ( FIG. 6d ). In summary, it can be confirmed that liposomes having adipocyte inducing ability were successfully induced and obtained from HDF by the liposome preparation method of Example 2.

Experimental Example 6. rBA assay

As a result of staining the rBA prepared according to the rBA preparation method of Example 2 with AdipoRed, which can confirm a large amount of fat droplets found in adipocytes (in this case, the counterstaining was performed with a nuclear staining agent, DAPI), AdipoRed negative control showed a distinct positivity compared to that (Fig. 7 upper panel). As a result of immunofluorescence staining of the rBA with UCP1, a brown adipocyte marker (in this case, the counterstaining was performed using the nuclear staining agent DAPI and the human mitochondrial antibody HuMito), it was also clearly positive compared to the negative control group (Fig. 7 bottom). panel). The above results show that HDF can be reprogrammed into rBA through the liposome prepared according to the preparation method of Example 2 and the rBA preparation method using the same.

Experimental Example 7. Liposome assay with hepatocyte inducibility

As a result of analyzing UHDF cultured according to the liposome induction method having hepatocyte induction ability of Example 3 through immunofluorescence staining for the CD63 exosome marker (in this case, the counterstaining was performed using the nuclear staining agent DAPI), a large amount of Exosomes were being generated (FIG. 8a), and as a result of immunofluorescence staining of the marker and the hepatocyte marker HNF1a, it was confirmed that HNF1a was expressed in the secreted exosomes (FIG. 8b). The morphology of liposomes obtained according to the method for preparing liposomes having hepatocyte inducibility of Example 3 was analyzed using Nanosight and TEM (transmission electron microscopy), and as a result, a normal morphology of exosomes was observed (Fig. 8c), as a result of RNA-Seq analysis of the liposomes, it was found that hepatocyte-related mRNA and microRNA significantly increased compared to the control group (Fig. 8d). In summary, it can be confirmed that liposomes having adipocyte inducing ability were generated and secreted from HDF by the liposome preparation method of Example 2.

Experimental Example 8. rH assay

As a result of immunofluorescence staining of rH prepared according to the rH production method of Example 3 with hepatocyte markers AFP, HNF4a, CK18, and ALB (in this case, the counterstaining was performed using the nuclear staining agent DAPI), it was clearly positive (Fig. 9). The above results show that HDF can be reprogrammed into rH through the liposome prepared according to the preparation method of Example 3 and the rH preparation method using the same.

Experimental Example 9. Liposome analysis experiment with hair regeneration ability

As a result of analyzing UHDF cultured according to the method of inducing liposomes having hair regeneration ability of Example 4 through immunofluorescence staining for the CD63 exosome marker (in this case, the counterstaining was performed with the nuclear staining agent DAPI), a large amount of Exosomes were being generated (FIG. 10b), and as a result of immunofluorescence staining of the above marker and Sonic hedgehog (Shh), an important marker for hair regeneration, it was confirmed that Shh was expressed in the secreted exosomes (FIG. 10c). As a result of analyzing the morphology of liposomes obtained according to the method for preparing liposomes having hair regeneration ability of Example 4 using Nanosight and TEM (transmission electron microscopy), a normal morphology of exosomes was observed (Fig. 10a), as a result of RNA-Seq analysis on the liposome, it was found that mRNA and microRNA of hair regeneration-related hair regeneration, hair growth promotion, Jak-Stat signaling system and Wnt signaling system increased significantly compared to the control group ( Fig. 10d). Taken together, it can be confirmed that liposomes having hair regeneration ability were successfully induced from HDF by the liposome preparation method of Example 4.

Experimental Example 10. Hair regeneration experiment by liposome with hair regeneration ability

After staining the liposomes obtained according to the method for preparing liposomes having hair regeneration ability of Example 4 with DiD, a lipid marker, the liposomes were applied to the epidermis of Nude mice and control C57 mice. It was confirmed that it penetrated (FIGS. 11a-b, counterstaining was performed with DAPI, a nuclear staining agent). 1X10 ⁹ , 1X10 ¹⁰ , 1X10 ¹¹ pieces/ml concentration of liposomes diluted in D-PBS culture medium were applied to the skin of C57 mice and nude mice from which the hair on the dorsal side was removed, respectively, after 1 and 2 weeks As a result of confirming each effect, in the case of nude mice, hairs were not observed with the naked eye in the group not treated with liposomes, whereas in the group treated with liposomes, it was confirmed that a large number of hairs grew from the first week. There was, and it was confirmed that a large number of hairs grew longer in C57 mice than in the control group (FIGS. 13a-b). In the group treated with liposomes for 2 weeks, hair grew more than 3 times longer than that of the control group, and as a result of H&E staining, a number of areas stained with dark purple indicating hair follicles were observed in the skin treated with liposomes (Fig. 12a-b). ), a much larger number of hair follicles than the control group was confirmed in the entire skin layer, especially in the subcutis (Fig. 12c-d), and it was confirmed at the tissue level that hair production was promoted. In addition, compared to the control group, the liposome-treated group showed increased expression of β-Catenin, Shh and Ki67, which are proteins related to hair follicle cell regeneration, through tissue immunofluorescence staining (FIG. 10c-d), and the hair growth promoting factor qRT-PCR showed that the mRNA expression of Shh, β-Catenin, KRT-25, VCAN, Gli1, Lef1, Pct1, Tyrp1, Tyr, Mitf, and DCT was increased and the mRNA expression of Sfrp4 and DKK, the hair growth inhibitory factors, decreased. through (Fig. 11e-f). In summary, the liposome having hair regeneration ability was predicted in the in vitro experiment according to Experimental Example 9, meaning that it exhibited an excellent effect on hair regeneration in vivo as well.

Experimental Example 11. Liposome analysis experiment with tissue regeneration ability

As a result of analyzing UHDF cultured according to the method for producing liposomes having tissue regeneration ability of Example 5 through immunofluorescence staining for the CD63 exosome marker (in this case, the counterstaining was performed with the nuclear staining agent DAPI), a large amount It could be confirmed that the exosomes were being generated (Fig. 14a). As a result of analyzing the exosomes (liposomes) isolated from the cell culture solution through qRT-PCR, Collagen 1α (Col1α), Collagen 3α (Col3α), which are genes related to the extracellular matrix associated with wound healing and Elastin (ELN), and proliferating cell nuclear antigen (PCNA), which are genes related to cell proliferation, it was found that the expression of N-Cadherin and Cyclin-D1 was increased compared to the exosomes isolated from the control group (FIG. 14b). In addition, as a result of RNA-seq, it was confirmed that many wound healing-related genes were expressed in liposomes compared to nExo (FIG. 14c).

Experimental Example 12. Tissue regeneration effect analysis experiment by liposome with tissue regeneration ability

The liposomes prepared according to the method for preparing liposomes having tissue regeneration ability of Example 5 were treated with HDF, and the effect according to the concentration of the liposomes was checked. First, as a result of analyzing the cell proliferation effect through immunofluorescence staining for the related marker Ki67, the expression of the marker was higher in the experimental group than in the control group, especially liposomes at concentrations of 5X10 ¹¹ /ml and 10X10 ¹¹ /ml. It was found that the effect was more excellent when treated with , and it was confirmed that the number of cells increased more (Figs. 15a-b). Next, after culturing the HDF to fill the plate, a line was drawn with a 20 μl yellow tip to decrease the distance between cells, and a wound healing assay was performed in which liposomes were treated and cultured. As a result, in the liposome-treated group It appeared that the empty space was filled relatively quickly (FIGS. 15c-d), and it was confirmed that the cell migration rate of the control group was high.

Next, in order to analyze the blood vessel formation efficiency during the tissue regeneration process according to the concentration of the liposomes prepared according to Example 5, endothelial cells, HUVECs, were treated with liposomes at different concentrations, and after 10 hours, a tube (tube) ) formation was confirmed. As a result, it was confirmed with the naked eye that the liposome-treated group formed a tube better than the control group (FIG. 15e), and the length of the tube and the number of agglutination points were also higher (FIG. 15f-g), especially 5X10 In the group treated with ¹¹ /ml and 10X10 ¹¹ /ml concentrations, the values were high.

Finally, the expression of wound healing-related genes in liposome-treated cells was analyzed by qRT-PCR. As a result, the expression of wound healing-related genes was higher in the experimental group than in the control group (FIG. 15h).

Taken together, the liposome prepared according to Example 5 can improve tissue wound healing and regeneration ability by promoting cell proliferation, migration, and blood vessel development.

Comparative Example 1. Control for liposomes

Unsonicated HDF (NHDF) was treated with hNSC medium (StemPro®NSC) containing 2 mM GlutaMAX ^TM -I Supplement (Gibco), 6 U/mL heparin (Sigma-Aldrich), and 200 μM ascorbic acid (Sigma-Aldrich). SFM, Gibco) or fibroblast medium (DMEM (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco)). When separating the exosomes (nExo) from the culture medium in which the NHDF was cultured, the same procedure as the liposome separation method of Example 1 was performed.

Comparative Example 2. Control of cells induced by liposomes

After seeding 1 Х10 ⁵ HDFs in a 35-mm Petri dish and culturing for one day, the medium was changed to a medium containing exosomes obtained according to Comparative Example 1 and cultured for 5 days. At this time, the medium including exosomes was consistent with each experimental group. For the control groups for rNPC, rBA, and rH, hNSC medium, adipocyte differentiation induction medium for stem cells, and hepatocyte culture medium were used, respectively, and the culture medium was 2 days. was replaced every

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

Claims (25)

forming a mixture by incorporating a composition comprising a liposome inducing reprogramming of mammalian-derived cells into a first culture medium;
culturing the first cells in the mixture; and
obtaining a second cell after the culturing;
As a method of reprogramming a mammal-derived cell comprising a,
The liposome is
providing ultrasound stimulation to mammalian-derived cells and providing ultrasound stimulation to a cell-free culture medium;
culturing a mixture of the ultrasonically stimulated mammalian-derived cells and the ultrasonically stimulated culture medium for a predetermined time;
centrifuging the cultured mixture to obtain a supernatant;
filtering the supernatant with a filter to obtain a filtrate; and
Concentrating the filtrate
It is manufactured by a manufacturing method comprising
The liposome has a diameter of 50 to 200 nm, and is included in the composition at a concentration of 1 × 10 ¹¹ to 50 × 10 ¹¹ pieces/ml,
A small RNA (small RNA) ratio among the total RNA in the liposome is 40% or more, and a microRNA (miRNA) ratio among the small RNAs is 40% or more,
The incorporated medium is a culture medium in which the second cells can be maintained and expanded,
The liposome includes RNA of a gene involved in chromatin remodeling, and the gene includes a kinase gene and a gene having histone modification activity on a mitogen-activated protein kinase (MAPK) signaling system. How to characterize. According to claim 1,
The kinase gene on the MAPK signaling system is at least one selected from the group consisting of BRAF, MAP2K3, MAP3K10, MAP3K4, MAP3K5, MAP3K7, MAPK12, RPS6KA4 (MSK2), TAOK1 and TAOK2. According to claim 1,
The gene having the histone modification activity is characterized in that at least one selected from the group consisting of ASH1L, CREBBP, DOT1L, EP300, GTF3C1, KAT2A, KAT6B, KDM1A, KDM3B, KDM6A, KMT2A, KMT2E, NCOA3, NSD1, SETD1A and SETD2 how to do it with delete delete According to claim 1,
The method wherein the mammalian-derived cells are mammalian-derived fibroblasts or tissue cells in mammalian-derived organs. According to claim 1,
The cell-free culture medium includes embryonic stem cell medium, neural stem cell medium, cardiac stem cell medium, dermal papilla cell medium, mesenchymal stem cell medium, osteogenic medium, myogenic medium, hematopoietic stem cell medium, neuron medium, A method of any one of astrocyte medium, oligodendrocyte medium, hepatocyte medium, adipocyte medium, myocyte medium, vascular endothelial cell medium, pancreatic beta cell medium, and cardiomyocyte medium. According to claim 1,
The cell-free culture medium is any one of a neural stem cell medium, a dermal papilla cell medium, a hepatocyte medium, and an adipocyte medium. According to claim 1,
Ultrasonic stimulation provided to the mammalian-derived cells is 10 to 30 KHz, 0.5 to 3 W/cm ² The method is performed for 1 to 10 seconds. According to claim 1,
Ultrasonic stimulation provided to the cell-free culture medium is 10 to 30 KHz, 1 to 20 W / cm ² The method is performed for 1 to 20 minutes. According to claim 1,
The method of claim 1, wherein the step of culturing a mixture of the ultrasonically stimulated mammalian-derived cells and the ultrasonically stimulated culture medium for a predetermined time is performed for 1 to 10 days. delete According to claim 1,
The liposome is
Filtering the supernatant with a filter to obtain a filtrate, the method prepared by a manufacturing method further comprising the step of storing the supernatant at 4 ° C. or less for 7 days to 1 month. delete delete According to claim 1,
The first cell is a mammal-derived fibroblast or a tissue cell in a mammal-derived organ. According to claim 1,
The second cell is a cell having a differentiation capacity of less than pluripotency (pluripotency) method. According to claim 1,
The second cell is any one of embryonic stem cells, neural stem cells, cardiac stem cells, dermal papilla cells, mesenchymal stem cells, and hematopoietic stem cells. According to claim 1,
The second cell is any one of neural stem cells, neurons, astrocytes, oligodendrocytes, hepatocytes, adipocytes, hair follicle cells, muscle cells, vascular endothelial cells, keratinocytes, pancreatic beta cells, and cardiomyocytes. A method characterized in that According to claim 1,
The method, characterized in that the second cell is a different type from the first cell. delete According to claim 1,
The method of claim 1, wherein the first culture medium is the same as the cell-free culture medium. According to claim 1,
The second cell is any one of stem cells, hepatocytes (progenitor cells) and precursor cells (precursor cells), and the step of culturing the first cell is characterized in that it is carried out for 1 to 6 days. According to claim 1,
The second cell is any one of neurons, astrocytes, oligodendrocytes, hepatocytes, adipocytes, hair follicles, muscle cells, vascular endothelial cells, keratinocytes, pancreatic beta cells, and cardiomyocytes; The method of culturing one cell is characterized in that it proceeds for 10 to 60 days. delete

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