KR102137883B1 - High-efficiency cell culture medium additive including sodium phenylbutyrate - Google Patents

Abstract

The present invention relates to a cell conversion method capable of high-efficiency and high-function cell conversion by applying sodium phenylbutyrate, a new regulator capable of controlling the stress and controlling the homeostasis of organelles generated during dedifferentiation. More specifically, according to the present invention, by adding sodium phenylbutyrate to the culture process for cell conversion, it is possible to not only improve the efficiency of cell conversion, but also significantly reduce the time required for cell conversion. At this time, sodium phenylbutyrate effectively improves the cell conversion-inducing culture conditions by suppressing aging induction and oxidative stress occurring during the process of cell conversion, and inducing cell proliferation and mitochondrial activity-enhancing effects.

Description

High-efficiency cell culture medium additive including sodium phenylbutyrate}

The present invention relates to a cell conversion method for converting cells by culturing cells in a medium containing sodium phenylbutyrate, and more specifically, searching for homeostasis of cell organelles occurring during cell conversion such as dedifferentiation and controlling stress. It relates to a cell conversion method capable of high-efficiency and high-function cell conversion by applying a new modulator that can be used, sodium phenylbutyrate, in cell culture.

In the conventional cell conversion technology research, many studies and interests have been focused on changes occurring in the cell nucleus. Nevertheless, there are still very few cell conversion efficiencies and many questions regarding the basis of cell fate determination. The reason for this is that the mechanism of cell fate conversion centering on the cell nucleus and the change of numerous organelles in the cytoplasm are not sufficiently accompanied, and in particular, there is little understanding and research on mitochondrial metabolism/change and endoplasmic reticulum stress.

These existing cell conversion technologies face the following problems:

Traditional natural differentiation techniques using embryonic stem cells to differentiate into specific target cells have low differentiation efficiency, high tumorigenicity by undifferentiated stem cells, and various ethical issues, including immune rejection problems or chromosomal abnormalities in clinical applications. They exist. In the process of differentiation into target cells, the time required is relatively long and the efficiency of each step is very low, such as ~3 weeks for cardiomyocytes, 1-2 weeks for neural and neural stem cells, and ~4 weeks for hepatocytes. In addition, it is very difficult to obtain a purified cell without the risk of tumor formation, and has a limitation of low efficiency and high cost due to the costly nature of culturing embryonic stem cells into culture and target cells.

Since the first dedifferentiated pluripotent stem cells have been reported, various researchers have developed various low-molecular compounds, vectors, and signaling mechanisms to increase the efficiency of dedifferentiation, and recently, non-coding RNA microRNA ( miR-302 cluster) or a method of introducing a protein to mediate a combination protein or synthesized mRNA, etc. was devised, but still has a problem that the efficiency of inducing pluripotent stem cell production is very low.

Introducing 4 factors of dedifferentiation into somatic cells, it is possible to observe a population of cells that form a cluster after about 14 days. More than 90-95% of the cell clusters that first appeared are incomplete dedifferentiated cells. Differentiated cells are known to not only spontaneously dedifferentiate over time, but also have a significantly low expression of a starch-potential specific gene. Therefore, it is expected that we will be able to secure better and more complete stem cell treatment resources based on in-depth research and understanding of numerous incomplete dedifferentiated stem cells that emerge during this dedifferentiation process. Unfortunately, to date, there is a lack of understanding and research on the incomplete dedifferentiated cell population in the middle stage of dedifferentiation, and domestic research on the organelles of the cell is very poor.

Cell conversion based on cross-differentiation technology in various cases has the advantage of short time and relatively low cost when producing target cells because it induces cross-differentiation into specific somatic cells without going through an omnipotent phase due to the nature of the technology. In general, the time required for cross-differentiation is usually about 1 to 3 weeks, and the purity of the produced cells is relatively high, so it is possible to produce high-efficiency target cells in a short period of time compared to a reverse-differentiation technique that requires at least two steps. However, according to a recent study (Cell Stem Cell 2012), cells established by the cross-differentiation method are known to be similar to primary cells in terms of the expression of cell-specific markers and in vitro and functional functions in the body. The results of the genome-wide analysis, including aspects, still show great differences from cells derived from the body. These differences must be improved in order for the cross-differentiated cells to be used in clinical interim studies in the future, and since the efficiency of cross-differentiation in most cells is still low, the source of maximizing the efficiency of cross-differentiation through cross-differentiation mechanism research Development of technology is necessary.

Although much research and attention has been focused on changes occurring in the cell nucleus to date (transcriptome and epigenome), there are still many cell conversion efficiencies and many questions regarding the mechanism of cell fate determination. The reason for this is that the mechanism of cell fate conversion centered on the cell nucleus and the change of numerous organelles in the cytoplasm have not been sufficiently accompanied.

Currently, studies on the presence or absence of endoplasmic reticulum stress and its effects are mainly focused on what appears in the process of individual development or cell differentiation. Is unprecedented.

In the development of cancer or the development of metabolic diseases, it is known that in many cases, it is caused not only by overall gene expression or epigenetic changes, but also by problems of organelles. That is, in order for cells to function properly, it is known that maintenance of the organelle's homeostasis is very important, but little is known about the mode of change of the organelle and the mechanism of homeostasis control that changes during cell conversion.

According to the results of a series of studies, several types of specific membrane proteins and secreted proteins are made in the endoplasmic reticulum during normal development of the individual, and accordingly, ER stress caused by abnormal synthesis of proteins in the endoplasmic reticulum occurred.

In the process of differentiation of keratinocytes, unfolded protein response (UPR) expression is very low in undifferentiated cells, but ER stress markers such as BiP, ATF6, HRD1, PDI, and CHOP during differentiation into keratinocytes. It has been reported that the expression of genes is markedly increased.

In the process of cell differentiation, if the ER stress is not properly relieved, the stem cell differentiation (stemness) is significantly reduced. For example, when ER stress occurs, the expression of various stem cell markers of visceral stem cells is significantly reduced, and eventually, these cells' signatures as stem cells disappear.

Based on these results, it can be seen that endoplasmic reticulum plays an important role in cell differentiation and cell transformation. However, such studies have been mainly studied in the developmental process and disease development model, and there are no studies on the stresses of the organelles that may occur during the somatic cell dedifferentiation process or the cross-differentiation process.

As described above, after the development of reverse differentiation/cross-differentiation, various technologies have been attempted to develop new cell conversion technologies, but despite many studies and efforts (Korean Patent Application No. 10-2013-0132270), there are still problems of low efficiency and low functionality. Did not overcome.

The present invention has been devised to solve the above problems, and while studying the organelle changes and stress-inducing mechanisms and the control factors, the activity of inhibiting the organelle stress of sodium phenylbutyrate was confirmed. The present invention was completed.

An object of the present invention is to provide a medium additive for suppressing ER stress, comprising sodium phenylbutylate.

Another object of the present invention is to provide a medium composition for inducing dedifferentiation into induced pluripotent stem cells, comprising the medium additive.

Another object of the present invention is to provide a method for inducing dedifferentiation into induced pluripotent stem cells, comprising the step of treating sodium phenylbutyrate with somatic cells or incomplete dedifferentiated cells.

Another object of the present invention is to provide cells differentiated by the above method.

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

In order to achieve the above object, the present invention

It provides a medium additive for suppressing ER stress, comprising sodium phenylbutylate.

In one embodiment of the present invention, the medium additive may be added to the medium at a concentration of 1 to 100 μM.

In addition, the present invention provides a medium composition for inducing reverse differentiation into induced pluripotent stem cells, comprising the medium additive.

In addition, the present invention provides a method for inducing dedifferentiation into induced pluripotent stem cells, comprising the step of treating sodium phenylbutyrate to somatic cells or incomplete dedifferentiated cells.

In one embodiment of the present invention, the somatic cell may be introduced with a factor for inducing dedifferentiation.

In another embodiment of the present invention, the dedifferentiation inducer may be selected from the group consisting of Oct4, Sox2, Klf4 and c-Myc.

In addition, the present invention provides cells differentiated by the above method.

According to the present invention, by adding sodium phenylbutyrate to the culture process for cell conversion, it is possible to not only improve the efficiency of cell conversion, but also significantly reduce the time required for cell conversion. Sodium phenylbutylate effectively improves cell conversion-inducing culture conditions by inhibiting aging induction and oxidative stress occurring during the process of cell conversion, and inducing cell proliferation and mitochondrial activity-enhancing effects.

The present invention is a process for producing induced pluripotent stem cells from a small amount of patient-specific somatic cells obtained from various sources, a process for producing patient-specific somatic cells having different functions from patient-specific somatic cells, stem cells to somatic cells It can be expected to contribute to optimizing the manufacturing process, and thus, it is expected to significantly improve the development of a new drug development process and personalized stem cell therapeutics that can be clinically applied, and accelerate the time to commercialization.

In addition, it is expected that the present invention can be usefully used to develop a system capable of culturing desired cells in large quantities during cell conversion.

Figure 1 shows the results of confirming the reverse differentiation efficiency according to the sodium phenylbutyrate treatment according to the present invention, Figure 1a is a result of confirming the reverse differentiation efficiency using SSEA4 and TRA1-60, the differentiation markers in a partially reprogrammed cell , FIG. 1b is a result of confirming the expression of a dedifferentiation specific marker in a partially reprogrammed cell, and FIG. 1c shows the dedifferentiation efficiency using SSEA4 and TRA1-60, which are dedifferentiation markers in iPSC induced by human fibroblast cells. It is the result of confirmation.
2 is a result confirming the efficiency of dedifferentiation through a positive reaction in Alkaline Phosphatase (AP) staining for iPSC derived from human fibroblast cells.
3 is a result of confirming ER-stress related gene expression according to treatment with sodium phenylbutyrate by concentration using qRT-PCR when inducing dedifferentiation using human fibroblast cells.

Hereinafter, the present invention will be described in detail.

As a result of research on efficient cell conversion, the present inventors searched for cell organelle changes and stress-induced mechanisms and search for control factors based on the fact that organelle stress plays an important role in cell differentiation and cell conversion processes. The present invention was completed by confirming the activity of sodium phenyl butyrate to inhibit organelle stress.

Accordingly, the present invention provides a medium composition for inducing reverse differentiation into induced pluripotent stem cells comprising a medium additive for inhibiting ER stress and containing the medium additive.

In the present invention, sodium phenylbutylate is a compound represented by the following Chemical Formula 1, and is a salt of 4-phenylbutyrate (4-PBA), which is an aromatic fatty acid, or a salt of 4-phenylbutyric acid.

[Formula 1]

As used in the present invention, the term "cell conversion" refers to natural differentiation of embryonic stem cells (spontaneous differentiation), reverse differentiation induced pluripotent stem cell reprogramming and direct conversion, and somatic cell nuclear transfer stem cells (SCNT) ) It means the cell conversion technology that covers the establishment process.

The natural differentiation is to differentiate stem cells into somatic cells or progenitor cells, the reverse differentiation is to differentiate somatic cells or progenitor cells into stem cells, and the cross-differentiation cross-differentiates somatic cells into somatic or progenitor cells of other functions. Can be

The stem cells may be appropriately selected without limitation depending on the purpose, and may be derived from adult cells such as mammals including humans, preferably all known tissues or cells derived from humans, for example, bone marrow, Cord blood, placenta (or placental tissue cells), fat (or adipose tissue cells), and the like.

For example, the stem cells are restricted from bone marrow, adipose tissue, muscle tissue, ex vivo cultured autologous mesenchymal stem cells, allogeneic mesenchymal stem cells, umbilical cord blood, embryonic yolk sac, placenta, umbilical cord, periosteum, fetal and adolescent skin, and blood It may be stem cells obtained without, or may be stem cells derived from a fetus or immediately after birth or from an adult.

The stem cells are, for example, embryonic stem cells, induced pluripotent stem cells, placental stem cells, umbilical cord stem cells, peripheral blood stem cells and bone marrow stem cells, neural stem cells, hematopoietic stem cells, induced blastodermal epithelial stem cells, fat Stem cells, vascular endothelial progenitor cells and mesenchymal stem cells may be any one cell selected from the group consisting of, but is not limited thereto.

The somatic cells include fibroblst, epithelial cells, muscle cells, nerve cells, gastric mucosal cells, goblet cells, G cells, B cells, epidermal cells, astrocytes, blood cells, cord blood-derived hematopoietic stem cells, and It may be any one cell selected from the group consisting of vascular endothelial cells. The somatic cells may mean all cells except germ cells, for example, fibroblasts, muscle cells, nerve cells, gastric mucosal cells, goblet cells, G cells, pericyte, astrocytes ), B cells, blood cells, epithelial cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, cord blood-derived hematopoietic stem cells, vascular endothelial cells, or cord blood stem cells may also be used. However, cross-differentiation is not limited to the above, since it can be applied irrespective of whether or not a specific tissue cell is a starting cell.

The term "differentiation" as used in the present invention refers to a phenomenon in which cells divide and proliferate and the structure or function of cells is specialized during the growth of an entire individual. In other words, it refers to a process in which cells, tissues, etc. of an organism are transformed into a suitable form and function to perform a role given to each. For example, the process of transforming an omnipotent stem cell such as an embryonic stem cell into ectodermal, mesodermal, and endoderm cells is also differentiated, and narrowly, the process by which hematopoietic stem cells turn into red blood cells, white blood cells, platelets, etc. also corresponds to differentiation. It is also referred to as "natural differentiation."

The term "differentiated cell" used in the present invention refers to a cell that has a certain shape and function through the differentiation process. The differentiated cells of the present invention are not particularly limited, but are preferably germ cells, somatic cells or progenitor cells. In addition, it is preferably a human-derived cell.

As used herein, the term "de-differentiation" refers to a process by which differentiated cells can be restored to a state with the potential for a new type of differentiation. In addition, the dedifferentiation is used in the same sense as cell reprogramming in the present invention. The mechanism of dedifferentiation of these cells means establishing a different set of epigenetic marks after the epigenetic marks in the nucleus (the DNA state associated with causing genetic changes in function without changes in the nucleotide sequence) are deleted. However, while multicellular organisms differentiate and grow, different cells and tissues acquire different gene expression programs.

The term "high-efficiency cell conversion" used in the present invention means that the process of cell conversion occurs rapidly in the process of cell conversion or the efficiency of cell conversion is increased. In other words, it includes the meaning of improving the efficiency of cell conversion in terms of speed or ratio.

The term "de-differentiation inducer" used in the present invention is a substance that induces finally differentiated cells to de-differentiate into induced pluripotent stem cells having a new type of differentiation potential. The dedifferentiation inducer may include, without limitation, any substance that induces dedifferentiation of the finally differentiated cells, and may be selected according to the type of cells to be differentiated. Although not limited thereto, preferably, a protein selected from the group consisting of Oct4, Sox2, KlF4, c-Myc, Nanog, Lin-28, and Rex1 as a dedifferentiation inducer or a nucleic acid molecule encoding these proteins is additionally included. It may, and more preferably, may include Oct4 protein or nucleic acid molecules encoding these proteins, may include Oct4, Sox2, KlF4 and c-Myc proteins or nucleic acid molecules encoding these proteins.

The term “cross-differentiation” (or hereinafter also referred to as “direct reprogramming/direct conversion/transdifferentiation”), as used in the present invention, is between mature (differentiated) cells with completely different cell types in higher organisms. As a process of inducing conversion, unlike the process of reprogramming into induced pluripotent stem cells (iPSCs) and re-differentiating it into a desired cell, it does not go through an induced pluripotent stem cell stage, but instead becomes a desired cell immediately. It has a difference in that it induces the conversion of the present.It is currently recognized that it can be used for disease modeling and discovery of new drugs, and is expected to be applied to gene therapy and regenerative medicine in the future.

The term "somatic cell" used in the present invention refers to all cells that have completed differentiation constituting animals and plants except germ cells, and has a characteristic that the chromosome maintains 2n.

The term "progenitor cell" used in the present invention refers to a parent cell that does not express a differentiation trait but has a differentiation fate when the cell corresponding to a progeny expresses a specific differentiation trait. For example, neurons (neurons) correspond to progenitor cells for neurons (neurons), and myoblasts to progenitor cells for myotubes.

The term "pluripotent stem cell" used in the present invention refers to a pluripotent or totipotent self-renewal capable of differentiating into cells of all tissues of an individual. Refers to a stem cell having, and includes, but is not limited to, embryonic stem cells and induced pluripotent stem cells. The pluripotent stem cells include pluripotent stem cells derived from humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, etc., but are preferably pluripotent stem cells derived from humans. .

As used in the present invention, the term "embryonic stem cell" refers to an inner cell mass extracted from the blastocyst embryo just before the fertilized egg implants into the mother's uterus and cultured in vitro, and differentiates into cells of all tissues of the individual. Refers to a cell having a pluripotent or omnipotent self-renewal capable, and broadly includes embryoid bodies derived from embryonic stem cells. The embryonic stem cells of the present invention include embryonic stem cells derived from humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, etc., but are preferably human-derived embryonic stem cells.

The term "derived pluripotent stem cell" used in the present invention refers to cells induced to have pluripotent differentiation ability through artificial dedifferentiation process from differentiated cells, also called induced pluripotent stem cells (iPSCs) do. The artificial dedifferentiation process is performed by retroviral and lentiviral virus-mediated or non-viral vector use, introduction of non-viral-mediated dedifferentiation factors using protein and cell extracts, or by stem cell extracts, compounds, etc. Includes a dedifferentiation process. Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically showing similar cell shapes, similar to gene and protein expression patterns, and capable of differentiating in vitro and in vivo . It forms a teratoma, and when inserted into the blastocyst of a mouse, forms a chimera mouse, and germline transmission of the gene is possible.

The term "vector" used in the present invention is an expression vector capable of expressing a target protein in a host cell, and may mean a gene construct comprising essential regulatory elements operably linked to express a gene insert. The vector includes a signal sequence or a leader sequence for membrane targeting or secretion in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, and may be variously prepared according to the purpose. The promoter of the vector can be constitutive or inducible. In addition, the expression vector includes a selectable marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, the origin of replication. Vectors can be self-replicating or integrated into host DNA.

The term "culture media" used in the present invention refers to a medium capable of supporting stem cell growth and survival in vitro (in-vitro), and is conventionally used in the art suitable for culturing cells. All media are included. Media and culture conditions can be selected according to the cell type. The medium used for culture is preferably a cell culture minimum medium (CCMM), and generally contains a carbon source, a nitrogen source, and a trace element component. These cell culture minimal media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI1640, F-10, F-12, aMEM (a Minimal Essential Medium), GMEM (Glasgow's Minimal essential Medium), Iscove's Modified Dulbecco's Medium, and the like. In addition, the medium may include antibiotics such as penicillin, streptomycin, and gentamicin.

The term "nucleic acid molecule encoding a protein" used in the present invention may be in a form operably linked to a promoter or the like so as to express the protein itself when delivered in a cell, and is also inserted into a chromosome in a cell and the corresponding protein Nucleic acid molecules capable of expressing a wide range. For example, a nucleic acid molecule encoding any one or more proteins selected from the group consisting of Oct4, Sox2, KlF4, c-Myc, Nanog, Lin-28 and Rex1 as dedifferentiation inducers is operably linked in an expression vector. It may be delivered into the cell, or may be delivered into the cell in a form inserted into the host cell's chromosome.

As used in the present invention, the term "cell therapy agent" is a pharmaceutical product (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention with cells and tissues prepared through separation, culture, and special authoring from humans, and functions of cells or tissues Refers to a medicine used for the purpose of treatment, diagnosis and prevention through a series of actions such as proliferation, selection of living autologous, allogeneic, or xenogeneic cells in vitro, or altering the biological properties of cells by other methods to restore them. .

In one embodiment of the present invention, when inducing dedifferentiation using a dedifferentiation inducing factor in human blood cells (human mononuclear cells), a majority of partially reprogrammed cells appear, and sodium phenylbutyrate according to the present invention After treatment, after quantitative analysis of the dedifferentiation efficiency through FACS analysis, it was confirmed that ER-stress was suppressed according to sodium phenylbutylate treatment, and that dedifferentiation was significantly increased, and also human fibroblasts. After the treatment of sodium phenylbutyrate according to the present invention from the initial stage of dedifferentiation induction using a dedifferentiation inducing factor, the quantitative analysis of the dedifferentiation efficiency through FACS analysis showed that, according to the treatment with sodium phenylbutylate, ER -It was specifically confirmed that the stress was suppressed to significantly increase the reverse differentiation, and as a result of performing Alkaline Phosphatase (AP) staining, it was confirmed that AP positive colonies were formed at a level similar to that of the control group (Example 1 and 2).

In addition, in another embodiment of the present invention, as a result of confirming the expression of ER-stress related genes (ATF3,ATF4, ATF6, GADD34, CHOP, BIP, tXBP1, SxBP1) according to sodium phenylbutate treatment, sodium phenylbutylate It was confirmed that the expression of the ER-stress-related gene was reduced by the treatment (see Example 3).

According to the results, it was confirmed that the effect of effectively suppressing the organelle stress by sodium phenylbutyrate and the effect of improving the dedifferentiation efficiency accordingly, can be usefully used as a cell conversion technology for culturing desired cells in large quantities during cell conversion. Suggests

In another aspect of the present invention, the present invention provides a method for inducing dedifferentiation into induced pluripotent stem cells, comprising the step of treating sodium phenylbutyrate to somatic cells or incomplete dedifferentiated cells.

The term “partially reprogrammed cell” used in the present invention is introduced into somatic cells by introducing four inducers (Yamanaka factor; OCT4, SOX2, KLF4, c-MYC) to induce first dedifferentiation. After 14 days, a population of cells forming a colony can be observed. At this time, about 90-95% or more of the appeared cell populations are incomplete dedifferentiated cells (TRA1-60 marker factor negative), and such incomplete dedifferentiated cells spontaneously dedifferentiate over time (TRA1-60 positive). Refers to cells that are known to not only occur but also have a significantly low expression of starch-specific genes. Thus, incomplete dedifferentiated cells are intermediate cells of dedifferentiation that can migrate to fully dedifferentiated cells when there is signal stimulation within the new cells.

In this case, when sodium phenylbutyrate is treated on somatic cells, the somatic cells may be introduced with dedifferentiation inducers, and the method for introducing the dedifferentiation inducers into somatic cells is a nucleic acid to cells commonly used in the art. A method for providing a molecule or protein may be used without limitation, but preferably, a method for inducing a dedifferentiation inducer to a culture medium of differentiated cells, a method for injecting a dedifferentiation inducer directly to differentiated cells, or a dedifferentiation inducer It is possible to use a method that infects cells differentiated with a virus obtained from packaging cells transfected with a viral vector into which the gene of the gene has been inserted.

The method for directly injecting the dedifferentiation inducer into differentiated cells may be used by selecting any method known in the art, but is not limited thereto, microijection, electroporation, and particles It can be appropriately selected and applied from a method using a particle bombardment, a direct muscle injection method, an insulator, and a transposon.

In the present invention, the dedifferentiation inducer may be selected according to the type of cells to be dedifferentiated, but is not limited thereto, preferably Oct4, Sox2, KlF4, c-Myc, Nanog, Lin-28, Rex1 and It may further include any one or more proteins selected from the group consisting of microRNA-302 clusters or any one or more nucleic acid molecules encoding these proteins, more preferably Oct4 proteins or nucleic acid molecules encoding these proteins. And may include Oct4, Sox2, KlF4 and c-Myc proteins or nucleic acid molecules encoding these proteins.

In the present invention, the packaging cells can be used by selecting various cells known in the art according to the viral vector used, but is not limited thereto, preferably, GP2-293 packaging cells can be used.

In addition, the viral vectors are retroviruses, for example, human immunodeficiency virus (HIV), Murine leukemia virus (MLV), Avian sarcoma/leukosis (ASLV), Spleen necrosis virus (SNV), Rous sarcoma virus (RSV) , mMTV (Mouse mammary tumor virus), Lentiviruses, Adenovirus, Adeno-associated virus, Herpes simplex virus, Sendai virus and It may include vectors derived from epizomal vectors and the like, but is not limited thereto, preferably retroviral vectors may be used, and more preferably retroviral vector pMXs may be used.

As another aspect of the present invention, the present invention provides cells differentiated by the above method.

The dedifferentiated pluripotent stem cells prepared through the cell conversion method may include all the cell cultures in vitro obtained by treating the differentiated cells with sodium phenylbutylate and a dedifferentiation inducer. The cell culture may include various cells in the process of reverse differentiation, various proteins and enzymes obtained from the process of culturing them, and even transcripts containing the same.

In a preferred embodiment of the present invention, cell growth and proliferation are enhanced as compared to cells differentiated with a differentiated pluripotent stem cell cultured in a culture medium containing sodium phenylbutyrate by transduction of a dedifferentiation inducer. , Apoptosis is inhibited, mitochondrial activity is enhanced, aging is suppressed, oxidative stress is reduced, p53 signaling is suppressed, dedifferentiation induction time is shortened, and dedifferentiation induction efficiency is enhanced. Confirmed.

The delivery step and the culture step may be performed simultaneously, sequentially or in reverse order.

When the cell conversion is cross-differentiation, the cell conversion method of the present invention may include the following steps:

(A) introducing a differentiation factor into the somatic cells; And

(b) Incubating the incomplete dedifferentiated cells obtained in step (a) in a medium containing sodium phenylbutylate.

In the step (a), a vector may be used for the introduction of the cross-differentiation factor.

The cross-differentiation factors include OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2, Hnf4a, Foxa1, Foxa3, Gata4, Hnf1a, FLI1 (Friend leukemia virus integration 1), ETV2 (Ets variant gene 2), It may be any one or more selected from the group consisting of GATA1, TAL1, LMO2, KLF1 and RUNX1

The OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, and NKX6.2 genes may be provided in the form of a protein or a nucleic acid encoding a protein thereof, the proteins being human and mouse, horse, sheep, pig, goat, It can contain any protein from animals such as camels, antelopes, and dogs. In addition, the OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 proteins used in the present invention include proteins having amino acid sequences of their wild type, as well as protein variants of each gene.

Variant of the protein means a protein in which the natural amino acid sequence of the protein and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. The variant may be a functional equivalent that exhibits the same biological activity as a natural protein, or a variant in which the physical and chemical properties of the protein are modified as necessary, or may be a variant in which structural stability to the physical and chemical environments is increased or a physiological activity is increased. have.

In addition, the nucleic acid encoding the protein is a wild-type or a nucleotide sequence encoding a protein in a variant form as described above, and one or more bases may be mutated by substitution, deletion, insertion, or a combination thereof. It can be prepared using a chemical synthesis method. The nucleic acid having the nucleotide sequence encoding the protein may be a single chain or a double chain, and may be a DNA molecule (genomic DNA, cDNA) or an RNA molecule.

In one embodiment of the present invention, in the present invention, the nucleic acid molecules encoding OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, and NKX6.2 proteins are vectors expressing proteins containing nucleic acids encoding each protein. Can.

Vectors usable in the present invention include plasmid vectors, cosmid vectors, viral vectors, epiisomal vectors, and the like. Preferably, it can be a viral vector. Virus vectors include lentiviral vectors, retroviruses, such as Human immunodeficiency virus (HIV), Murineleukemia virus (MLV), Avian sarcoma/leukosis (ASLV), Spleen necrosis virus (SNV), Rous sarcoma virus (RSV) , MMTV (Mouse mammary tumor virus), adenovirus (Adenovirus), adeno-associated virus (Adeno-associated virus), herpes simplex virus (Herpes simplex virus), Sendai virus (Sendai virus) and epizomal (episomal) vector It includes, but is not limited to, one vector. Such a vector system is used for the purpose of inducing cross-differentiation by overexpressing a gene related to a specific cell in somatic cells, and can exhibit the effect of the present invention regardless of using any vector system. In one embodiment of the present invention, the vector may be a pMX-based retroviral vector expressing OCT4.

In addition, the nucleic acid encoding the protein can be introduced into cells using known methods in the art, for example, naked DNA in the form of vectors, or introduced into cells using liposomes, cationic polymers, and the like. Can.

The liposome is a phospholipid membrane prepared by mixing cationic phospholipids such as DOTMA or DOTAP for gene transfer. When cationic liposomes and anionic nucleic acids are mixed at a certain ratio, nucleic acid-liposomal complexes can be formed and introduced into cells. .

Specifically, in the present invention, the nucleic acid molecule encoding the protein is included in a vector and a virus designed to express each gene by transforming and infecting a viral vector containing the nucleic acid encoding the protein with packaging cells. Can be introduced into somatic cells. The virus includes, but is not limited to, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and the like.

The medium is not particularly limited, and a conventionally known medium can be used, and specific examples thereof include dexamethasone, ascorbic acid, insulin, transferrin, L-alanyl-L-glutamine, glycerol 2-phosphate, fibroblast growth factor ( bFGF) and DMEM (Dulbeco's Modified Eagle's Media) medium containing a compound selected from the group consisting of selenium salt, the insulin, transferrin, and selenium salt may be used commercially available ITS (Gibco, USA) . In addition, the medium may further include fetal bovine serum (FBS) and antibiotics, if necessary, for example, about 10% FBS, and about 1% penicillin-streptomycin.

The cells include cells derived from humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, etc., but are preferably human-derived cells.

The culture may be performed on a conventional culture plate in the form of pellet culture, for example, cultured at 37°C, 5% CO ₂ . The culture period is not particularly limited, but may be performed, for example, for about 3 weeks. After incubation, the cells are obtained in pellet form and can be isolated by removing the medium.

In addition, the culture may be a plurality of continuous passage culture.

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

[Example]

Example 1. Experiment preparation and experiment method

1-1. Culture of induced pluripotent stem cells (iPSC)

In order to confirm the efficiency of sodium phenylbutyrate in the process of reverse differentiation, human blood cell-derived partially reprogrammed cells and fibroblast cells were cultured.

First, in order to cultivate partially reprogrammed cells, 10 ml of patient blood was obtained from the hospital to obtain mononucleocytes using Ficoll-paque, and in RPMI1640 medium containing SCF, TPO, Flt3, IL-6, and IL-3. Incubation was carried out for about 6 days. Subsequently, a Sendai virus (cytotune-iPS reprogramming kit, Invitrogen) was used to introduce a de-differentiation factor (Yamanaka factor; Oct4, Sox2, Klf4, c-Myc), and thereafter, culture was performed for 4 days with the culture medium. Then, when the culture was completed, it was transferred to a culture dish coated with Vitronectin, and then cultured in mTesR-E8 medium.

After incubation for a period of time, fully reprogrammed iPS cells and partially reprogrammed cells were obtained.

Next, in order to culture induced pluripotent stem cells using human fibroblast cells, the human fibroblast line (BJ1) was cultured in DMEM medium containing 10% FBS, and then Sendai virus (cytotune-iPS reprogramming) kit, Invitrogen) was used to introduce de-differentiation factors (Oct4, Sox2, Klf4, c-Myc).After this, the culture was performed for 4 days, and when the culture was completed, mouse embryonic fibroblst (MEF) was generated. After transferring to a culture dish or a culture dish coated with Vitronectin, the culture was performed in hESC only or mTesR-E8 medium.

1-2. FACS (Fluorescence-activated cell sorting) analysis

Partially reprogrammed cells obtained from Example 1-1 were treated with 50 uM of sodium phenylbutylate for 6 days in 5x10 ⁴ /24 wells, and expressing the initial dedifferentiation marker SSEA4 and the final dedifferentiation marker TRA1-60 Cells were analyzed using FACS (Fluorescence-activated cell sorting).

Similarly, human fibroblasts (BJ1) treated with a reverse differentiation factor (Yamanaka factor; Oct4, Sox2, Klf4, c-Myc) 5x10 ⁴ /24 wells containing 10, 50 and 100 uM of sodium phenylbutyrate in E8 (Vitronectin) After inducing dedifferentiation for 14 days in the coated culture dish), cells expressing the initial dedifferentiation marker SSEA4 and the final dedifferentiation marker TRA1-60 were analyzed using FACS (Fluorescence-activated cell sorting).

1-3. Confirmation of colony formation rate (Alkaline Phosphatase staining)

MMC treated-MEF 5x10 ⁴ /24 wells were prepared to prepare human fibroblasts (BJ1) treated with dedifferentiation factors (Yamanaka factor; Oct4, Sox2, Klf4, c-Myc) 2.5x10 ⁴ /24 wells at 10 and 100 uM. After inducing reverse differentiation for 14 days in hESC culture containing sodium phenylbutylate, Alkaline Phosphatase (AP) staining (Staemgent) was performed according to the manufacturer's protocol.

1-4. Confirmation of genetic expression related to ER-stress (qRT-PCR)

In order to confirm the expression change of the ER-stress related gene in the process of dedifferentiation of cells by treatment with sodium phenylbutyrate according to the present invention.

More specifically, when inducing dedifferentiation using human fibroblast cells, ER-stress related genes (ATF3,ATF4, ATF6, GADD34) according to sodium phenylbutylate treatment concentrations (10, 50 and 100 uM) , CHOP, BIP, tXBP1, SxBP1) was confirmed by performing qRT-PCR.

Example 2. Confirmation of cell conversion efficiency according to sodium phenylbutylate treatment

2-1. Check the efficiency of reverse differentiation of partially reprogrammed cells

In order to confirm the efficiency of dedifferentiation of partially reprogrammed cells according to the treatment with sodium phenylbutyrate according to the present invention, the dedifferentiation efficiency is quantified using the embryonic stem cell specific markers SSEA4 and TRA1-60 according to Example 1-2 above. Did.

As a result, as shown in Figure 1a, the ER-stress inducer TG was significantly reduced by the dedifferentiation, but specifically the de-differentiation by the sodium phenylbutyrate treatment according to the invention specifically increased I could confirm.

In addition, as shown in FIG. 1B, SSEA4 and TRA1-60 expression could be specifically confirmed by treatment with sodium phenylbutylate.

2-2. Determination of iPSC-induced differentiation efficiency from human fibroblast cells

In order to confirm the efficiency of dedifferentiation of iPSC derived from human fibroblast cells according to the treatment with sodium phenylbutyrate according to the present invention, the embryonic stem cell specific markers SSEA4 and TRA1-60 according to Example 1-2 above The reverse differentiation efficiency was quantified using.

As a result, as shown in Figure 1c, when using a human fibroblast (Fibroblast cell), there was little difference in the expression of the final marker of dedifferentiation (TRA1-60), but the initial marker of dedifferentiation according to the concentration of sodium phenylbutyrate treatment. It can be seen that (SSEA4) increases significantly.

2-3. Confirmation of a positive response in Alkaline Phosphatase (AP) staining of iPSC derived from human fibroblast cells

Next, according to Examples 1-3, the efficiency of reverse differentiation according to each concentration shows a positive reaction in the staining of hESC-specific factor Alkaline Phosphatase (AP), and the number of dyed colonies is counted to calculate the phenylbutyl according to the present invention. Quantification of reverse differentiation efficiency according to sodium acid treatment.

As a result, as shown in FIG. 2, it was confirmed that AP positive colonies were formed at the same level as control in PBA 10 uM.

Example 3. Confirmation of ER-stress reduction effect according to sodium phenylbutylate treatment

In order to confirm the effect of reducing ER-stress according to the treatment of sodium phenylbutyrate of the present invention, the ER-stress related genes (ATF3,ATF4, ATF6, GADD34, CHOP according to the treatment with sodium phenylbutylate according to Examples 1-4 above) , BIP, tXBP1, SxBP1) was confirmed.

As a result, as shown in FIG. 3, it was confirmed that the expression of the ER-stress related gene was reduced by treatment with sodium phenylbutylate. From the above results, it was confirmed that sodium phenylbutylate can increase the efficiency of reverse differentiation by directly controlling ER-stress.

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

Claims (7)

A medium composition for promoting the induction of dedifferentiation into induced pluripotent stem cells, comprising sodium phenylbutylate. The medium composition of claim 1, wherein the sodium phenylbutylate inhibits vesicle stress. The medium composition of claim 1, wherein the sodium phenylbutylate is added at a concentration of 1 to 100 μM. A method of promoting induction of dedifferentiation into induced pluripotent stem cells, comprising the step of treating sodium phenylbutate with somatic cells or incomplete dedifferentiated cells. The method of claim 4, wherein the somatic cell is characterized in that a factor for inducing dedifferentiation is introduced. The method of claim 5, wherein the dedifferentiation inducer is selected from the group consisting of Oct4, Sox2, Klf4 and c-Myc. delete

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