KR20200090657A - Reprosomes, the exosomes capable of inducing reprogramming of cells and the preparation method thereof - Google Patents

Abstract

Disclosed is a reprosome, which is an exosome to induce reprogramming of cells, including RNA of a gene participating in chromatin remodeling, wherein the gene includes a kinase gene in a mitogen-activated protein kinase (MAPK) signal transfer system and a gene having histone transform activity. According to the present invention, such a reprosome can be acquired from somatic cells easy to acquire and maintain as well as stem cells difficult to handle through a simple process such as ultrasonic treatment. Through a simple process of co-cultivating the reprosome and the cells, one kind of cell can be reprogrammed into other cells having a desired function in a short time, and the cells acquired through such a way have the desired function without introducing chemical materials or foreign transcription factors, thereby providing stability suitable for a cell replacement therapy. Moreover, a portion of the body is treated by the reprosome and a composition including the same, such that cells existing in the treated portion are promoted to be reprogrammed into target cells having the desired function, thereby being used for tissue regeneration.

Description

REPROSOMES, THE EXOSOMES CAPABLE OF INDUCING REPROGRAMMING OF CELLS AND THE PREPARATION METHOD THEREOF}

The present invention relates to a liposome which is an exosome capable of inducing reprogramming of a cell and a method for manufacturing the same, and more specifically, a liposome containing a large amount of chromatin remodeling factor, and a liposome. It relates to a method that can be produced in a high yield and the use of the liposomes produced by the above method.

It is known that exosomes are naturally secreted nano-vesicles with a diameter of 30 to 200 nm, and can act as an important nano-media that carries genetic material from a derived cell. Since the discovery that exosomes can change the phenotype in target cells through mRNA delivery, several studies have revealed that exosomes are involved in cell differentiation and the like. In these existing studies, stem cells and progenitor cells were used as targets for inducing differentiation or phenotypic changes by using cells or exosomes to obtain exosomes, and these cells require a careful process for separation and amplification, resulting in efficiency and economic efficiency. There is a side problem. Therefore, inducing secretion of exosomes containing factors that can change cells in a desired direction from somatic cells of easily obtainable patients, and also using such exosomes, somatic cells that can be easily obtained by using these exosomes to other cells having desired functions If changeable, this would be a great leap forward in the clinical application of cell replacement therapy.

In addition, in recent years, various possibilities for directly using the exosomes themselves for treatment purposes have been explored. Exosomes are in the middle position of biopharmaceuticals, such as small molecules, peptides, growth factors, antibodies, nucleic acids, and large pharmaceutical products such as various cells or platelets in the field of biologics. In other words, compared to biopharmaceuticals, exosomes contain a variety of proteins and nucleic acids, which can have a more complex and long-lasting effect. In addition, it has other advantages compared to cells. That is, when the 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 usually filtered and trapped in filtration organs such as the lung, liver, spleen, or kidney, and the filtered cells are eventually broken down into small vesicles to affect the human body. It is said to be. Therefore, when administering an exosome of small size while containing an active ingredient that can be secreted from the cells, it is possible to more safely produce a space-time treatment effect different from that of an intact cell.

However, as described above, exosomes having high potential as a tool for cell therapy or the therapeutic agent itself are currently difficult to produce in large quantities. For example, exosomes obtained from 60 million mesenchymal stem cells cultured in 1 liter of medium are levels capable of obtaining 1-2 mg based on the contained protein content. This is an amount that can be used in the treatment experiment of several mice, and in humans, for example, exosomes used for treatment of graft-versus-host disease (GVHD) are 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 exosome clinically, a technique capable of increasing yield is required.

The present invention addresses a variety of needs, including the aforementioned needs present in cell therapy and exosome therapy. In other words, stem cells generally used for cell therapy are difficult to separate, amplify, and maintain, and it is difficult to predict the direction of differentiation, and there is a problem that they may develop into cancer. In addition, the process of obtaining a cell having a desired function also takes a long time after different stages of differentiation, conversion efficiency is low, and it is difficult to secure safety due to chemical treatment. Therefore, there is a need for a technology that allows cells that can be easily obtained to be used for cell therapy and a technology that can safely obtain cells with desired functions in a short time with high efficiency. In the case of an exosome treatment, there is a lack of a technique for obtaining a sufficient amount for treatment.

The present invention has been devised to solve the above-described problem, and one embodiment of the present invention is characterized in that it comprises RNA of a gene involved in chromatin remodeling, and the gene is a MAPK (mitogen-activated protein kinase). ) Provides a liposome capable of inducing reprogramming of cells in a desired direction, including a gene having a phosphatase gene and a histone modification activity on a signaling system.

In addition, an embodiment of the present invention provides ultrasonic stimulation to cells, provides ultrasonic stimulation to a cell-free culture medium, and mixes the cells and the culture medium for a certain period of time to cultivate the aforementioned reprosomes in a short time. It provides a manufacturing method capable of producing in large quantities.

In addition, an embodiment of the present invention provides a method for efficiently reprogramming cells in a desired direction by treating the cells with the liposomes.

Also provided is a composition comprising the reprosome which induces reprogramming of the cells 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 skilled in the art from the following description. There will be.

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

Here, the phosphatase 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 histone modification activity may be selected from at least one 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 among the total RNA in the reprosome is 40% or more, and the ratio of microRNA (miRNA) among the small RNA is 40% or more.

Another aspect of the present invention provides an ultrasound stimulation to the cells, and providing the ultrasound stimulation to the cell-free culture medium; Mixing the cells and the culture medium and culturing for a predetermined time; And separating the prolosome from the mixture. It provides a method for producing a prolosome that induces reprogramming of cells.

Here, the cell may be either a fibroblast derived from a mammal or a tissue cell in an organ.

The culture medium is an embryonic stem cell medium, a neural stem cell medium, a heart stem cell medium, a papillary cell medium, a mesenchymal stem cell medium, an osteogenic medium, a myogenic medium, a hematopoietic stem cell medium, a neuron medium, astrocyte medium , Oligodendrocyte medium, hepatocyte medium, adipocyte medium, muscle cell medium, vascular endothelial cell medium, pancreatic beta cell medium, or cardiomyocyte medium.

The culture medium may be any one of a neural stem cell medium, a papillary cell medium, a hepatocyte medium, and an adipocyte medium.

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

The 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.

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

The step of separating the liposomes may include centrifuging the mixture after the culture 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 for 7 days to 1 month at 4 ℃ or less before filtering with a filter may be further included. The isolated reprosome may be characterized in that the diameter is 50 to 200 nm.

Another aspect of the present invention comprises the steps of incorporating reprosome into the first culture medium; Culturing the first cell in the mixture; And obtaining a second cell after the culture. It provides a method for reprogramming a cell.

Here, the first cell may be either a fibroblast derived from a mammal or a tissue cell in an organ.

The second cell may be characterized as being a cell having pluripotency or less differentiation ability.

The second cell may be any one of embryonic stem cells, neural stem cells, heart 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 reprosome may be characterized in that it is incorporated into the first culture medium at a concentration of 10 ⁷ to 10 ¹⁵ cells/ml.

The first culture medium may be characterized by the same culture medium used to prepare the liposomes.

The second cell is any one of a stem cell, a progenitor cell, or a procursor 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 follicle cells, muscle cells, vascular endothelial cells, keratinocytes, pancreatic beta cells, or cardiomyocytes. The cultivation of 1 cell may be characterized by proceeding for 10 to 60 days.

Another aspect of the present invention provides a composition comprising reprosomes that induce reprogramming of the aforementioned cells.

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

The method for producing a liposome according to an embodiment of the present invention induces a large amount of reprosome secretion through a simple process of ultrasonic treatment, from stem cells and progenitor cells, which are difficult to separate and amplify, as well as somatic cells that are easily obtained. can do. The yield of exosomes derived in this way is higher than that of conventional methods, and the amount and number of various factors included in the exosomes are also high.

The cell reprogramming method according to an embodiment of the present invention can safely induce reprogramming to 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 to another cell in a relatively short time without having to go through several developmental steps through a simple process of culturing cells by adding reprosome to the culture medium. .

A composition comprising a reprosome according to an embodiment of the present invention may promote tissue regeneration of the treatment site by treating a body part to promote reprogramming of cells present at the treatment site.

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 deduced from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 is a reprosome generation data having a neuronal progenitor cell (NPC) inducing ability induced in accordance with Example 1 of the present invention, exosomes secreted from cells treated with ultrasound (UHDF) and untreated cells (NHDF) (Emicroscopic image (a) confirming the form of iExo (reprosome) and nExo respectively (a), immunofluorescence staining image showing induction of CD63 (b), size distribution and number of exosomes (cd), time after initiation of culture Changes in the amount of exosome secretion according to (e).
Figure 2 is a component data of a reprosome having a neuronal progenitor cell (NPC) inducing ability induced according to Example 1 of the present invention, 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), protein concentration in exosomes (d), NPC marker mRNA in exosomes (Sox1, Sox2, Pax6 and Nestin) and miRNA (miR9, miR124a, miR125b and miR128- 1) Relative expression level (ef), heat map (g) showing neuronal mRNA (left) and miRNA expression, and intracellular co-distribution of NPC markers (Sox1, Sox2, Pax6 and Nestin) and exosome markers (CD63) Immunofluorescence staining image showing (h).
Figure 3 is an NPC inducing ability analysis data of a liposome having a neural progenitor cell (NPC) inducing ability isolated according to Example 1 of the present invention, a schematic diagram of the protocol for inducing the cell (a), reprosome ( iExo) Confocal microscopy image showing intracellular influx of reprosomes over time after treatment (b), morphological changes in HDF (c), 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), 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 level of NPC markers (Sox1, Sox2, Pax6 and Nestin) (ij), and the growth curve of passage 6th cell (k).
Figure 4 is an induction mechanism analysis data of neuronal progenitor cells (rNPC) according to Example 1 of the present invention, the chromatin remodeling related gene expression heat map (a), reprosomes shown in nExo and reprosome (iExo) treated cell group Western blot results showing the degree of phosphorylation of MAPK signaling system proteins (Erk1/2, p38 and Msk1) over time after treatment, intracellular immunofluorescence staining images (bc), NPC markers (Sox1, Sox2, Pax6 and Nestin) RNA QRT-PCR data for (d), immunofluorescence staining data for chromatin remodeling indicators (HP1α, H3K4me3, H3K27me3) and distribution of these indicators relative to control staining DAPI (e) and neurological gene expression hits in each cell group Map (f).
5 is an in vitro and in vivo differentiation analysis data of rNPC according to Example 1 of the present invention, neuronal markers of rNPC differentiated by culturing for 4 weeks in neuronal differentiation medium (Map2 and Tuj1 neuronal markers, Gfap astrocyte markers) , In vitro differentiation data represented by immunofluorescence staining data for the oligodendrocyte marker O4) (a), voltage clamp data for the differentiated rNPC and action potential data (bc), 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 marker (f).
Figure 6 is the production data of the liposomes having adipocyte (adipocyte) inducing ability according to Example 2 of the present invention, Nanosight and electron microscope image (a) confirming the form of iExo (reprosome), exosome marker Immunofluorescence staining image (bc) showing the induction of phosphorus CD63 and the coexistence of CD63 with the brown adipocyte marker UBC1 and brown adipocyte-associated mRNA/miRNA and lipid synthesis-related mRNA characterization RNA-seq data in isolated liposomes ( d).
7 is immunofluorescence staining data of brown adipocytes (rBA) according to Example 2 of the present invention, immunofluorescence using an antibody against lipid-staining reagent AdipoRed, human mitochondrial antibody (HuMito) and brown adipocyte marker UCP1 Dyed image.
FIG. 8 is a generation data of a reprosome having hepatocyte inducing ability according to Example 3 of the present invention, a nanosight and electron microscope image (a) confirming the form of iExo (reprosome), an exosome marker Immunofluorescence staining image (bc) showing induction of CD63 and the coexistence of CD63 with the hepatocyte marker HNF1α and hepatocyte-related mRNA/miRNA characterization RNA-seq data (d) in isolated reprosomes.
9 is immunofluorescence staining data of hepatocytes (rH) according to Example 3 of the present invention, immunofluorescence staining images for hepatocyte markers AFP, HNF4α, CK18 and ALB.
FIG. 10 is a generation data of reprosome having hair tissue differentiation inducing ability according to Example 4 of the present invention, Nanosight and electron microscope image (a) confirming the form of iExo (reprosome), CD63 as an exosome marker Immunofluorescence staining image (bc) showing the coexistence of Shh, a protein important for induction and CD63 and hair regeneration, and RNA-seq data (d) for mRNA regeneration-related mRNA characterization in isolated reprosomes.
FIG. 11 shows in vivo gene expression change data in C57 and nude mouse skin by reprosomes having hair regeneration ability according to Example 4 of the present invention, and application of the liposomes labeled with lipophilic marker Did Fluorescence staining image (ab) showing post-distribution, protein (β-Catenin, Shh, Ki-67) immunofluorescence 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 a tissue change data in C57 and nude mouse skin by reprosome having hair regeneration ability according to Example 4 of the present invention, H&E stained image (ab) of skin tissue and hair follicles in subcutaneous and whole skin Number data (cd).
Figure 13 is hair regeneration data in the skin of C57 and nude mice by reprosomes having hair regeneration ability according to Example 4 of the present invention, the concentration of the exosomes and the hair generation image with time after treatment (ab ).
Figure 14 is the production data of the liposomes having wound healing ability according to Example 5 of the present invention, immunofluorescence staining image showing the induction of the exosome marker CD63 (a), wound healing in the induced liposomes QRT-PCR data (b) and RNA-seq data (c) of RNA analysis of genes.
15 is an in vitro effect data by the liposomes 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 various concentrations of the liposomes Image (a), Proliferation rate by hour for each reprosome treatment concentration of HDF measured by MTT assay (b), Mobility per hour for each reprosome treatment concentration of HDF measured by scratch assay (cd), Reprosome treatment Optical microscope image showing tube formation of HUVEC by concentration, length and number of intersections of formed tube (eg), and qRT-PCR data analyzing RNA of wound healing-related genes in cells treated with reprosomes (h).

Hereinafter, the present invention will be described in more detail. However, the present invention can be implemented in many different forms, and the present invention is not limited by the examples described herein, and the present invention is only defined by the claims below.

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. Singular expressions include plural expressions unless the context clearly indicates otherwise. 'Inclusion' of a component in the specification of the present invention means that other components may be further included instead of excluding other components, unless otherwise specified.

According to an aspect of the present invention, a reprosome capable of inducing reprogramming of a cell includes RNA of a gene involved in chromatin remodeling, and the gene is a MAPK (mitogen-activated protein kinase) signal It is characterized by including a gene having a phosphatase gene and a histone modification activity on a delivery system.

Here, the phosphatase 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 5), MAP3K7 (Mitogen-activated protein kinase kinase 7), MAPK12 (Mitogen-activated protein kinase 12), RPS6KA4 (Ribosomal protein S6 kinase) At least one may be selected from the group consisting of known as Msk2), TAOK1 (TAO kinase 1), and TAOK2 (TAO kinase 2). Preferably, the phosphatase gene may include at least MAP3K10, RPS6KA4, and TAOK1 among the 10 genes, more preferably at least MAP2K3, MAP3K10, MAP3K7, MAPK12, RPS6KA4, TAOK1 and TAOK2.

Genes having the histone modification activity include 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) , Lysine acetyltransferase 2A (KAT2A), Lysine acetyltransferase 6B (KAT6B), Lysine demethylase 1A (KDM1A), Lysine demethylase 3B (KDM3B), Lysine demethylase 6B (KDM6A), KMT2A (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 a RNA having a size of less than 200 nt (nucleotide), and is mainly non-coding RNA. Small RNAs include microRNAs, short interfering RNAs, short nucleolar RNAs, and piwi-interacting RNAs (piRNAs). Among them, miRNA has a length of 22 nt and is known to be involved in RNA silencing and post-transcriptional gene regulation. Among the total RNA in the reprosome, the small RNA ratio may be 40% or more, and the small RNA may have a miRNA ratio of 40% or more. More preferably, the ratio of small RNA among total RNA in the reprosome is 50% or more, and the ratio of miRNA among the small RNA is 50% or more, and most preferably, the proportion of small RNA among the total RNA in the reprosome is 60%. That is, the ratio of miRNA among the small RNAs may be 60% or more.

Reprosomes capable of inducing the reprogramming of the above-described cells may be characterized by being prepared by a manufacturing method to be described later.

Another aspect of the present invention provides an ultrasound stimulation to the cells, and providing the ultrasound stimulation to the cell-free culture medium; Mixing the cells and the culture medium and culturing for a predetermined time; And separating the reprosomes from the mixture. It provides a method for producing a reprosome that induces the reprogramming of cells, including.

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

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

The culture medium (the medium subjected to ultrasonic treatment) includes embryonic stem cell medium, neural stem cell medium, heart 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, muscle cell medium, vascular endothelial cell medium, pancreatic beta cell medium, or cardiomyocyte medium. The culture medium may be any one of differentiation induction or medium for maintenance and amplification purposes. According to another aspect of the present invention to be described later, when reprogramming a first cell into a second cell by processing a liposome, preferably, a medium in which the second cell to be obtained can be maintained and amplified in a healthy manner You can choose According to another aspect of the present invention to be described later, when a composition comprising a liposome is treated on a body part to promote the reprogramming of cells at the site to target cells, preferably, the target cells are in When cultured in vitro , a medium that can be maintained and amplified in a healthy manner can be selected.

The culture medium may be any one of a neural stem cell medium, a papillary cell medium, a hepatocyte medium, and an adipocyte medium.

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

The ultrasonic stimulation provided in the culture medium may be performed at 10 to 30 KHz, 1 to 20 W/cm ² for 1 to 20 minutes, preferably 15 to 25 KHz, 0.5 to 1.5 W/cm ² at 7 to 13 Can be done for a minute

Incubation of the mixture may be characterized by proceeding for 1 to 10 days, preferably for 1 to 6 days, most preferably for 1 to 2 days. This is because the liposomes are most often secreted on the first day after sonication, and the secretion amount decreases with time, because there is a possibility that there may be a change in the components due to the decrease in the secretion amount.

The step of separating the liposomes may include centrifuging the mixture after the culture to obtain a supernatant; Filtering the supernatant with a filter to obtain a filtrate; And concentrating the filtrate. The centrifugation proceeds to remove cell debris and dead cells, and preferably may be 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 the cell debris and leave only particles of a specific size or less, and the filter used here may preferably be a syringe filter. The step of concentrating the filtrate may preferably be performed using a centrifugal filter. By using a centrifugal filter, the filtrate can be concentrated while removing particles below a certain size. Separating the reprosome may further include storing the supernatant at 4° C. or lower for 7 days to 3 months before filtering the supernatant. The storage may be preferably within 4 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 the liposomes are mRNA and protein, etc., and the higher the temperature of these components or the higher the enzyme activity, the more easily they can be denatured or degraded. Here, the isolated reprosome may be characterized by having a diameter of 50 to 200 nm, and preferably having a diameter of 100 to 150 nm.

Another aspect of the present invention comprises the steps of incorporating reprosome into the first culture medium; Culturing the first cell in the mixture; And obtaining a second cell after the culture. It provides a method for reprogramming a cell.

Here, the first cell may be either a fibroblast derived from a mammal or a tissue cell in an organ. This is because the method for reprogramming cells according to an aspect of the present invention can obtain second cells regardless of any somatic cells, thus obtaining fibroblasts or tissue cells in organs rather than stem cells or progenitor cells that are difficult to obtain and difficult to amplify. This is because cells that are easy to maintain and amplify are easier and more efficient. When the second cell is subsequently transplanted into the human body, the first cell may preferably use a cell derived from a human, and most preferably, an autologous cell derived from a target to be transplanted. When a cell derived from a living organism genetically close to the transplant target is used as the first cell, the possibility of side effects such as a rejection reaction during cell transplantation may be reduced.

The second cell may be characterized as being a cell having pluripotency or less differentiation ability.

The second cell may be any one of embryonic stem cells, neural stem cells, heart 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 differentiation cells is as follows: from high to low levels, totipotency, pluripotency, multipotency, oligopotency and unipotency. It is called divided. Pluripotency refers to the ability to differentiate into all cells of an organism and form one organism from one cell, and pluripotency is three types of endoderm, mesoderm and ectoderm. It means that it can differentiate into all germ cells. Multipotency refers to the ability to differentiate into several cells of one lineage or a small number of lines, and oligopotency is a few cells with a smaller range, and unipotency is one. It means that each can differentiate into cell types. Therefore, cells having differentiation capacity below the pluripotency include pluripotency, multipotency, oligopotency and unipotency cells, and terminally differentiated cells. do. When the second cells are subsequently transplanted into the human body, the second cells are preferably multipotency, oligopotency or unipotency stem cells, hepatocytes (progenitor cells) or progenitor cells ( precursor cell). In the case of pluripotent cells, 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 is poor.

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 a method for reprogramming the cell of the present invention, a desired second cell can be easily obtained therefrom.

The reprosome may be characterized in that it is incorporated into the first culture medium at a concentration of 10 ⁷ to 10 ¹⁵ pieces/ml, and is preferably 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 the reprogramming efficiency decreases when the concentration of the mixed prolosome is too low or too high (FIG. 3i).

The first culture medium may be characterized by the same culture medium used to prepare the reprosome, but is not limited thereto. This has the effect that reprogramming to the desired second cell occurs more rapidly when the first culture medium is the same as the culture medium used to prepare the liposomes. This is because reprogramming to the second cell may occur when incorporated.

The second cell is any one of a stem cell, a progenitor cell, or a procursor 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 follicle cells, muscle cells, vascular endothelial cells, keratinocytes, pancreatic beta cells, or cardiomyocytes. The cultivation of 1 cell may be characterized by proceeding for 10 to 60 days. This is because there is a difference in the time required for reprogramming to cells with high differentiation ability and reprogramming to cells with less differentiation potential, and the latter tends to take a little longer, and the latter is preferably cultured for 15 to 25 days. Can.

The composition according to another aspect of the present invention includes the above-described reprosome which induces reprogramming of cells. Here, the composition may be used, for example, to promote regeneration of tissues present in the treatment site by treating the body part. The body part may include the epidermis, dermis, and scalp, and in this case, the composition may have a tissue regeneration effect such as wound healing or hair regeneration by being applied or injected into the site.

Since it is obvious that various additives, such as a pharmaceutically acceptable carrier and/or a penetration rate to the treatment site, and other additives that can further enhance the tissue regeneration effect, may be added to the composition in addition to the reprosome. A detailed description of this will be omitted.

As described above, the reprosome 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 to cells having a desired function with high efficiency. can do.

The method of manufacturing a reprosome according to an embodiment of the present invention provides reprogramming factors of various cells through a simple process called sonication, from stem cells and progenitor cells that are difficult to separate and amplify, as well as somatic cells that can be easily obtained. The phlegm may induce secretion of reprosomes.

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

The composition comprising reprosomes according to an embodiment of the present invention may be treated on a body part to promote tissue regeneration existing at the treatment site.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art to which the present invention pertains can easily practice. However, the present invention can be implemented in many 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 and 5% CO ₂ conditions.

Example 1. Preparation of reprosomes having neuroprogenitor cell inducing ability and induction of reprogramming of fibroblasts into neuroprogenitor cells using the same.

In order to obtain reprosomes having neuroprogenitor cell inducing ability, ultrasonic stimulation of 20 KHz, 1.0 W/cm ² was performed for 5 seconds using UltraRepro 1001 (STEMON Inc., Seoul, Republic of Korea) in 1 Х10 ⁶ HDFs. Directly applied (hereinafter referred to as stimulated HDF UHDF). 2 Х10 ⁵ UHDF were incubated for 1 day with hNSC medium sonicated in a 35-mm Petri dish (20 KHz, 5.0 W/cm ² , 10 min). The 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 0.22-mm filter (Minisart® Syringe Filter, Sartorius, Goettingen, Germany). The medium passed through was placed in an Amicon® Ultra-15 100,000 kDa device (Millipore, Billerica, MA, USA), and centrifuged at 14,000 X g for 20 minutes to concentrate reprosome (iExo).

To prepare rNPC (neuroprogenitor cells obtained by reprogramming with reprosomes), 1 Х10 ⁵ HDFs were seeded in a 35-mm Petri dish and cultured for one day, followed by hNSC including the reprosomes from which the medium was separated. The medium was changed and cultured for 5 days. 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.

In order to obtain a liposome with adipocyte inducing ability, ultrasonic stimulation of 20 KHz, 1.0 W/cm ² was directly performed for 5 seconds using UltraRepro 1001 (STEMON Inc., Seoul, Republic of Korea) in 1 Х10 ⁶ HDFs. Added. 2 Х10 ⁵ UHDFs in a 35-mm Petri dish (20 KHz, 5.0 W/cm ² , 10 min) with adipocyte differentiation medium for stem cells (MesenCult ^TM adipogenetic Differentiation Medium, Stemcell technologes) for 1 day Cultured. As described above, the liposomes were separated from the culture medium in which the UHDF was cultured through the separation process of the liposomes as in Example 1.

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

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

To obtain a reprosome having hepatocyte inducing ability, ultrasonic stimulation of 20 KHz, 1.0 W/cm ² was directly applied for 5 seconds using UltraRepro 1001 (STEMON Inc., Seoul, Republic of Korea) in 1 Х10 ⁶ HDFs. Did. 2 Х10 ⁵ UHDF were incubated with a hepatocyte culture medium (HCM ^TM hepatocyte culture medium, Lonza) sonicated in a 35-mm petri dish (20 KHz, 5.0 W/cm ² , 10 min). As described above, the liposomes were separated from the culture medium in which the UHDF was cultured through the separation process of the liposomes as in Example 1.

In order to prepare rH (hepatocytes obtained by reprogramming with reprosomes), 1 Х10 ⁵ HDFs were seeded in a 35-mm Petri dish and cultured for one day, followed by a hepatocyte culture medium containing exosomes from which the medium was separated. The culture was reversed for 24 days. Culture medium was changed every 2 days.

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

In order to obtain reprosomes with hair regenerating ability, ultrasonic stimulation of 20 KHz, 1.0 W/cm ² was directly applied for 5 seconds using UltraRepro 1001 (STEMON Inc., Seoul, Republic of Korea) on 1 Х10 ⁶ HDFs. Did. 2 Х10 ⁵ UHDF were incubated for 1 day with dermal papilla cell medium (PromoCell) sonicated in a 35-mm Petri dish (20 KHz, 5.0 W/cm ² , 10 min). As described above, the liposomes were separated from the culture medium in which the UHDF was cultured through the separation process of the liposomes as in Example 1.

Example 5. Preparation of liposomes having wound healing ability.

In order to obtain a reprosome having wound healing ability, 20 kHz, 1.0 W/cm ² of 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 UHDF sonicated in a 35-mm Petri dish (20 KHz, 5.0 W/cm ² , 10 min) embryonic stem cell culture medium (DMEM/F12, 15% FBS, 2 mM GlutaMAX, 0.1% NEAA, 0.1 % Penicillin/Streptomycin, 0.1 mM β-mercaptoethanol, 1000 unit/ml Leukemia Inhibitor (LIF)). As described above, the liposomes were separated from the culture medium in which the UHDF was cultured through the separation process of the liposomes as in Example 1.

Experimental Example 1. Reprosome production experiment with neuroprogenitor cell inducing ability

In order to induce the secretion of reprosomes having neuroprogenitor cell-inducing ability, HDF was exposed to ultrasound stimulation according to Example 1 (UNDF) and cultured for 1 day in ultrasound-treated human neural stem cell medium (hNSC medium). After one day of incubation, it was confirmed that a large amount of endoplasmic reticulum was induced in UHDF compared to NHDF using the exosome-specific marker CD63 (FIG. 1B ). As a result of separating the exosomes (iExo) and exosomes (nExo) secreted from NHDF in the medium in which the UHDF was cultured by the method of separating the liposomes of Experimental Example 1, vesicles of common exosomes through transmission electron microscope images It was observed to have a sex structure (Fig. 1a). As a result of nanoparticle tracking analysis, the particle size of iExo was distributed in the range of about 50 to 200 nm, and averaged 155.6 +- 4.2 nm (FIG. 1C). The iExo with a size of 50 to 200 nm was 9 x 10 ⁸ , which was 3.2 times higher than nExo (FIG. 1D). These results show that exosomes can be efficiently induced by applying ultrasound to HDF. The secretion amount of iExo was the highest on day 1 based on the time of incubation after sonication and showed a tendency to decrease with time, but nonetheless, the analyzed secretion amounts on days 1, 3, and 5 were higher than nExo. It was confirmed (Fig. 1E).

To analyze the components of iExo, total RNA and microRNA (miRNA) concentrations and qualitative portions were analyzed from exosomes separated from primary culture medium using Agilent 2100 Bioanalyzer, and proteins were analyzed through exosome RNA sequencing (RNA-Seq). The number of coding genes was measured. The total RNA concentration in iExo was 5.3 times higher than nExo (FIG. 2A), and the number of genes expressed by the RNA was 8400, which was 4.7 times higher than nExo recording 1762 (FIG. 2B). Through an open database search and gene ontology analysis, it was revealed that a part of the mRNA in iExo is related to neurogenicity (FIG. 2G ). It was confirmed that the expression level of NPC specific marker genes such as Sox1, Sox2, Pax6, Nestin in iExo increased through qRT-PCR (quantitative reverse transcription polymerase chain reaction; FIG. 2e). Among the non-coding small RNAs in exosomes, the proportion of miRNA of iExo (60.57%) was much higher than that of nExo (8.52%) (FIG. 2c ). In addition, 53 of the total 72 in iExo were Parsons et al. According to the neural system specific miRNA according to the results (Fig. 2g). The total protein concentration of iExo was also increased 20-fold compared to nExo (Fig. 1d). Through the results of 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 with mRNA expression (FIG. 1h). These results demonstrate that UHDF could be induced to secrete liposomes rich in mRNA, miRNA and protein associated with neurodevelopment. The number of transcripts and proteins in UHDF-derived reprosomes has been dramatically increased. Many of these genes play a role in maintaining the differentiation ability in stem cells. This increase may serve as a significant factor for cell reprogramming. It means there is.

Experimental Example 2. Neuroprogenitor cell inducibility assay of reprosome according to Example 1

In order to confirm whether cell reprogramming in the direction of neurophenotype can be induced by the reprosome, the inventors of the present invention have confirmed that iExo is delivered into HDF. iExo was labeled with DiD-labeled iExo (DiD), a lipophilic tracer, and then treated with HDF to incubate for one day. DiD-labeled iExo was confirmed in the cytoplasm of iF-treated HDF (FIG. 3C, left panel). Additionally, in order to confirm that the reprogramming factor inside iExo was delivered, intact HDF was labeled with Cy5.5 labeled poly(A)27, and then, through the process of manufacturing UHDF, poly(A)27-Cy5.5 was included. The exosomes (Cy5.5-exo) were induced. After incubating the HDF with iExo containing Cy5.5-exo for one day, Cy5.5-exo was observed in the cytoplasm of HDF (Fig. 3c right panel). Interestingly, increased expression of Pax6 was induced 24 hours after treatment from HDF treated with iExo, which means that the liposomes are transferred into HDF with high efficiency.

Next, we confirmed whether iExo can induce cell reprogramming. HDF (1×10 ⁵ , 7th passage) was exposed to exosomes (hNSC-Exo, nExo, iExo, respectively) separated from the culture medium in which hNSC, NHDF and UHDF were cultured for one day. Interestingly, when the above three types of exosomes and HDF were co-cultured for 5 days, only iExo treated cells (iExo-HDF) formed dense colonies (>100 μm in diameter) (Figure 3b), and NPC specificity An increase in markers was seen from the first day of culture (Figure 3d-f). When the cells were treated with 20 Х 10 ¹¹ iExos/ml, iExo-HDF showed spheroid formation and NPC specific marker expression at the maximum level (FIG. 3i), and the effect of iExo was confirmed below using this concentration. . 5 days after exosome treatment. iExo-HDF colonies reached 1,500, and NPC specific genes and proteins such as Sox1, Sox2, Pax6, and Nestin increased to a level comparable to that of hNSC, showing properties such as NPC (Fig. 3d) -e). Finally, as a result of double positive flow cytometry, cells of Pax6/Nestin, Sox1/Nestin and Sox2/Nestin in day 5 iExo-HDF reached 74.7%, 68.5% and 75.8%, respectively (FIG. 3F). As a result of RNA-seq analysis, iExo-HDF's neuro-specific gene expression profile was similar to hNSC and showed a distant pattern from HDF (FIG. 3G ), and multiple genes overexpressed in iExo-HDF through gene ontology analysis were neurogenic. It was confirmed that it is related to. These results show that iExo can rapidly induce cell reprogramming to produce NPC-like cells in 5 days with high yield from HDF. On day 5, iExo-HDF, which had similar properties to NPC, was designated as rNPC, and dense colonies of iExo-HDF were designated as rNPC primary passage (p1).

The spheroids of rNPC p1 were collected and subjected to several passages to obtain a uniform cell population. As a result of analyzing NPC-specific marker genes and proteins, rNPCs of p2, p4, p6, and p10 expressed Sox1, Sox2, Pax6, and Nestin at high levels. As a result of Ki-67 immunofluorescence staining, rNPC was found to be actively proliferating (FIG. 3H). It has been shown that the proliferative capacity of P6 rNPC is maintained for several weeks (FIG. 3K). Synthesizing the above results, it has been proved that the cells are cells of a uniform and amplifiable group that maintains Ki67 and NPC marker expression while undergoing several passage cultures, which indicates that rNPC retains the amplification and self-renewal capability of NPC. it means.

Experimental Example 3. rNPC induction mechanism assay

To elucidate the mechanism by which rapid cell reprogramming by liposomes occurs, the inventors of the present invention have focused on epigenetic regulation of gene expression by cell stress. Interestingly, iExo contained mRNAs and proteins associated with genes related to histone degeneration and mitogen-activated protein kinase (MAPK) pathways (FIG. 4A ). Next, it was confirmed that iExo can induce chromatin remodeling by stimulating the MAPK signaling pathway of the target cell. As a result of Western blot and flow cytometry analysis, expression of p38, Erk, and Msk1 increased rapidly in HDF 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 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 decreased (FIG. 4D ). These results indicate that the MAPK signaling system acts as an important mechanism for reprosome to rapidly induce rNPC through chromatin remodeling. Additionally, changes in local chromatin density and histone degeneration were tracked in the nucleus of HDF on day 3 after iExo treatment. Local chromatin density was confirmed by transfecting H2B protein (H2B-GFP) labeled with green fluorescent protein (GFP). After iExo treatment, the pattern of H2B-GFP distribution spread over time (FIG. 4e). In addition, the expression of HP1α (heterochromatin protein 1 α) in the nucleus and the inhibitory histone degeneration H3K27me3 decreased, and the 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 gene related to neurogenesis, it was found that methylation is reduced (FIG. 4g ). These results indicate that reprosomes induce activation of the MAPK signaling system and chromatin remodeling, as well as rapid reprogramming to NPC-like cells through several epigenetic regulators.

Experimental Example 4. rNPC in vitro And in vivo Differentiation analysis

To evaluate the ability of rNPC to differentiate into neurons, it was confirmed whether rNPC can differentiate into neural systems such as neurons, astrocytes, and oligodendrocytes. Day 5 rNPC spheroids were cultured in gelatin coated culture plates according to a recently published neuronal differentiation protocol. After 4 weeks of neuronal differentiation, it was confirmed that Map2 and Tuj1 (neuron markers), Gfap (astrocyte markers), and O4 (diploid cell markers) were expressed in differentiated cells (Figure 5a). The results of qRT-PCR analysis of Map2, Tuj1, Gfap, S100b (astrocyte markers), Mbp (dipotential cell markers), and Oligo1 (dipotential cell markers) also showed that rNPC successfully differentiated into neurons. Next, the functional properties of neurons derived from rNPC were confirmed by whole cell patch clamp recording. First, gene expression associated with potassium ion channels (EAG1, Kv4.3, and Kv7.2) and sodium ion channels (Nav 1.3, Nav1.6, and Nav1.7) increased significantly after differentiation from rNPC to neurons. Was confirmed. In the current clamp, neurons derived from rNPC were able to confirm inward sodium and outward potassium currents (FIG. 5B ). In addition, it was observed that sodium current is inhibited in neurons derived from rNPC by the sodium ion channel blocker TTX (tetrodotoxin). Neurons derived from rNPC induced an action potential in response to a current clamp (FIG. 5C ). These results indicate that rNPC has multipotency and effectively differentiated into neurons, astrocytes and oligodendrocytes in vitro .

Cells stably expressing the reporter with G418 antibiotic using HDF transfected with the GFP reporter expressed by the CMV (cytomegalovirus) promoter to confirm that rNPC is capable of multipotent differentiation in vivo (GFP-HDF) Established. The established Day 5 GFP-rNPC spheroid (about 1,500 clusters) was implanted into the brain (striata, striatum) of normal Sprague Dawley rats (n = 5) and the differentiation status of these cells was confirmed after 4 weeks. As a result, cells that were GFP-positive were identified in various parts of the brain, many of which formed long neurites (FIG. 5D), indicating that these cells were efficiently survived, migrated, and host integrated. . GFP-positive cells were confirmed to be stained with human mitochondrial antibody, confirming that these cells are GFP-rNPC (FIG. 5E ). Gfap-positive astrocytes, Map2 and Tuj1-positive neurons, and O4-positive oligodendrocytes showed GFP-positivity (FIG. 5F ). The above results indicate that rNPC derived from the transplanted HDF was able to retain 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 inducing ability

As a result of analyzing UHDF cultured according to the method for inducing liposomes having adipocyte inducing ability of Example 2 through immunofluorescence staining for a CD63 exosome marker (at this time, the control staining is referred to as the nuclear staining agent DAPI), large amount Exosomes were 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). As a result of analyzing the form of the liposomes obtained according to the method for preparing a liposome having the adipocyte inducing ability of Example 2 using Nanosight and transmission electron microscopy (TEM), a normal form of the exosome was observed ( 6c), as a result of RNA-Seq analysis for the reprosome, it was found that brown adipocyte related mRNA and microRNA and lipid synthesis related mRNA increased significantly compared to the control group (FIG. 6d ). Taken together, it can be confirmed that the liposomes having adipocyte inducing ability from HDF were successfully induced and obtained by the method for preparing the liposomes 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 capable of identifying a large amount of fat droplets found in adipocytes (in this case, the control staining is referred to as DAPI, a nuclear staining agent), to the control group shown as AdipoRed negative It showed a clear positive compared to (Fig. 7 top panel). As a result of immunofluorescence staining of the rBA with the brown adipocyte marker UCP1 (at this time, the control staining is referred to as the nuclear staining agent DAPI and the human mitochondrial antibody HuMito), similarly, it showed a clear positive compared to the negative control (Fig. panel). The above results show that HDF can be reprogrammed to rBA through the liposomes prepared according to the production method of Example 2 and the rBA production method using the same.

Experimental Example 7. Liposome assay with hepatocyte inducing ability

As a result of analyzing UHDF cultured according to the method for inducing reprosome having hepatocyte inducing ability of Example 3 through immunofluorescence staining for a CD63 exosome marker (in this case, the control staining is referred to as the nuclear staining agent DAPI), a large amount Exosome was 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 exosome (Fig. 8b). As a result of analyzing the form of the liposomes obtained according to the method for preparing a liposome having hepatocyte inducing ability of Example 3 using Nanosight and transmission electron microscopy (TEM), a normal form of exosome was observed (Fig. 8c), RNA-Seq analysis of the reprosome showed that the hepatocyte-related mRNA and microRNA were significantly increased compared to the control group (FIG. 8D). Taken together, it can be confirmed that the liposomes having adipocyte-inducing ability were produced and secreted from HDF by the method for preparing the liposomes of Example 2.

Experimental Example 8. rH assay

The result of immunofluorescence staining of the rH prepared according to the rH preparation method of Example 3 with the hepatocyte markers AFP, HNF4a, CK18, and ALB (at this time, the control staining is referred to as the nuclear staining agent DAPI), showed distinct positive (Fig. 9). The above results show that HDF can be reprogrammed to rH through the reprosome prepared according to the preparation method of Example 3 and the rH manufacturing method using the same.

Experimental Example 9. Reprosome assay with hair regeneration

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

Experimental Example 10. Hair regeneration experiment by reprosome having hair regeneration ability

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

Experimental Example 11. Reprosome assay with tissue regeneration

As a result of analyzing the UHDF cultured according to the method for preparing the reprosome having the tissue regeneration ability of Example 5 through immunofluorescence staining for the CD63 exosome marker (in this case, the control staining is referred to as the nuclear staining agent DAPI), a large amount It was confirmed that the exosomes of the are generated (Fig. 14a). As a result of analyzing the exosomes (reprosomes) isolated from the culture of the cells through qRT-PCR, collagen 1α (Col1α), collagen 3α (Col3α), which are genes related to wound healing and extracellular matrix And Elastin (ELN), and the proliferating cell nuclear antigen genes related to cell proliferation (PCNA), N-Cadherin and Cyclin-D1 expression of the control was found to be higher than the exosomes isolated from the control (Fig. 14b). In addition, as a result of RNA-seq, it was confirmed that more wound healing-related genes are expressed in reprosomes compared to nExo (FIG. 14C).

Experimental Example 12. Tissue regeneration effect analysis experiment by reprosome having tissue regeneration ability

The liposomes prepared according to the production method of the liposomes having tissue regeneration ability of Example 5 were treated with HDF, and the effect according to the concentration of the liposomes was confirmed. First, as a result of analyzing the cell proliferation effect through immunofluorescence staining for the related marker Ki67, the experimental group had a higher expression of the marker than the control group, particularly 5X10 ¹¹ /ml and 10X10 ¹¹ /ml concentration of reprosomes When it was treated, it was found that the effect was more excellent, and it was confirmed that the number of cells increased more (FIGS. 15A-B ). Next, after incubating the HDF to fill the plate, a line was drawn with a 20 μl yellow tip to reduce the distance between cells, and then a wound healing assay was performed to incubate by processing the liposomes. It appears that the empty space is filled relatively quickly (Fig. 15c-d), it was confirmed that the cell migration rate of the control is high.

Next, in order to analyze the efficiency of blood vessel formation during the tissue regeneration process according to the concentration of the liposomes prepared according to Example 5, endothelial cells were treated with different concentrations of liposomes, and after 10 hours, ) Confirmed formation. As a result, it could be confirmed with the naked eye that the reprosome-treated group formed the 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. The levels were high in the groups treated with ¹¹ /ml and 10X10 ¹¹ /ml concentrations.

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

Taken together, the liposomes prepared according to Example 5 can improve cell wound healing and regeneration ability by promoting cell proliferation, migration, and vascular development.

Comparative Example 1. Control for liposomes

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 media (DMEM (Gibco)) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco)). When separating the exosome (nExo) from the culture medium in which the NHDF was cultured, the same procedure as in the method for separating the reprosome from Example 1 was performed.

Comparative Example 2. Control of cells induced by liposomes

After 1 Х10 ⁵ HDFs were seeded in a 35-mm Petri dish and cultured 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 containing the exosomes was matched with each experimental group, hNSC medium, adipocyte differentiation inducing medium for stem cells, and hepatocyte culture medium were used for the controls for rNPC, rBA, and rH, respectively. It was replaced every time.

The above description of the present invention is for illustration only, 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 distributed manner, and similarly, 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 modifications or variations derived from the meaning and scope of the claims and their equivalent concepts should be interpreted to be included in the scope of the present invention.

Claims (25)

Characterized in that it contains the RNA of the gene involved in chromatin remodeling (chromatin remodeling), the gene is characterized in that it contains a gene having a phosphatase gene and histone modification activity on the mitogen-activated protein kinase (MAPK) signaling system Reprosome that induces reprogramming of the body. According to claim 1,
Phosphorylase gene on the MAPK signaling system, characterized in that at least one selected from the group consisting of BRAF, MAP2K3, MAP3K10, MAP3K4, MAP3K5, MAP3K7, MAPK12, RPS6KA4 (MSK2), TAOK1 and TAOK2, cell reprogramming Reprosome that induces. According to claim 1,
The gene having histone modification activity is selected from 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 A reprosome that induces reprogramming of cells. According to claim 1,
The ratio of small RNA among total RNAs in the reprosome is 40% or more,
Of the small RNA, characterized in that the microRNA (miRNA) ratio is 40% or more, a reprosome inducing reprogramming of cells. Providing ultrasonic stimulation to the cells and providing ultrasonic stimulation to the cell-free culture medium;
Mixing the cells and the culture medium and culturing for a predetermined time; And
Separating reprosomes from the mixture;
A method of manufacturing a reprosome comprising induction of reprogramming of a cell. The method of claim 5,
The cell is any one of a mammalian-derived fibroblast or a tissue cell in an organ, a method for producing a reprosome that induces reprogramming of a cell. The method of claim 5,
The culture medium is an embryonic stem cell medium, a neural stem cell medium, a heart stem cell medium, a papillary cell medium, a mesenchymal stem cell medium, an osteogenic medium, a myogenic medium, a hematopoietic stem cell medium, a neuron medium, astrocyte medium , Oligodendrocyte medium, hepatocyte medium, adipocyte medium, muscle cell medium, vascular endothelial cell medium, pancreatic beta cell medium, or myocardial cell medium. Manufacturing method. The method of claim 5,
The culture medium is any one of a neural stem cell medium, a papillary cell medium, a hepatocyte medium, and an adipocyte medium, a method for producing reprosomes inducing reprogramming of cells. The method of claim 5,
The ultrasound stimulation provided to the cells is performed for 1 to 10 seconds at 10 to 30 KHz, 0.5 to 3 W/cm ² , and a method for producing reprosomes inducing reprogramming of cells. The method of claim 5,
The ultrasonic stimulation provided in the culture medium is performed for 1 to 20 minutes at 10 to 30 KHz, 1 to 20 W/cm ² , and a method for producing reprosomes inducing reprogramming of cells. The method of claim 5,
The culture of the mixture is characterized in that proceeds for 1 to 10 days, a method for producing a reprosome inducing reprogramming of cells. The method of claim 5,
The step of isolating the liposomes
Centrifuging the mixture after the culture to obtain a supernatant;
Filtering the supernatant with a filter to obtain a filtrate; And
Concentrating the filtrate;
Method for producing a reprosome that induces reprogramming of a cell comprising a. The method of claim 12,
The step of isolating the liposomes
Further comprising the step of storing for 7 days to 1 month at 4 ℃ or less before filtering the supernatant with a filter, a method for producing a reprosome to induce reprogramming of cells. The method of claim 12,
The isolated reprosome is characterized in that the diameter is 50 to 200 nm, a method for producing a reprosome inducing reprogramming of cells. Incorporating the reprosome according to claim 1 into a first culture medium;
Culturing the first cell in the mixture; And
Obtaining a second cell after the culture;
A method of reprogramming a cell comprising a. The method of claim 15,
The first cell is any one of a mammalian-derived fibroblast or tissue cell in an organ, a method for reprogramming a cell. The method of claim 15,
The second cell is a cell having a differentiation ability of pluripotency or less, characterized in that, the method for reprogramming the cell. The method of claim 15,
The second cell is any one of embryonic stem cells, neural stem cells, heart stem cells, dermal papilla cells, mesenchymal stem cells, and hematopoietic stem cells, a method for reprogramming cells. The method of claim 15,
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. A method of reprogramming cells, characterized in that. The method of claim 15,
The second cell is characterized in that different from the first cell, the method of reprogramming the cell. The method of claim 15,
The reprosome is characterized in that it is incorporated into the first culture medium at a concentration of 10 ⁷ to 10 ¹⁵ pcs/ml, the method for reprogramming cells. The method of claim 15,
The first culture medium is characterized in that the same as the culture medium used to prepare the liposomes, a method for reprogramming cells. The method of claim 15,
The second cell is any one of a stem cell, a progenitor cell or a procursor cell, and the culture of the first cell is performed for 1 to 6 days, and the method for reprogramming the cell . The method of claim 15,
The second cell is any one of neurons, astrocytes, oligodendrocytes, hepatocytes, adipocytes, hair follicle cells, muscle cells, vascular endothelial cells, keratinocytes, pancreatic beta cells, or cardiomyocytes. Method for reprogramming cells, characterized in that culturing of 1 cell is performed for 10 to 60 days. A composition according to claim 1 comprising a reprosome which induces reprogramming of a cell.

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