WO2019117454A1 - Medium additive for highly efficient cell transformation using cell organelle stress regulation factor - Google Patents

Abstract

The present invention relates to a cell transformation method that enables highly efficient and functional cell transformation by applying sodium phenylbutyrate, tauroursodeoxycholic acid, or butylated hydroxyanisole, which are new regulation factors which can be used to explore cell organelle homeostasis during dedifferentiation and to control stress, during culturing. More particularly, according to the present invention, the addition of at least one selected from the group consisting of sodium phenylbutyrate, tauroursodeoxycholic acid, and butylated hydroxyanisole in the culturing process for cell transformation can not only promote cell transformation induction efficiency, but also dramatically reduces the time required to induce cell transformation. At such time, the sodium phenylbutyrate, tauroursodeoxycholic acid, or butylated hydroxyanisole inhibit ageing induction and oxidization stress that occur during the cell transformation induction process, and induces a cell proliferation and mitochondrial activity promotion effect, thereby effectively improving cell transformation inducing culture conditions.

Description

High-efficiency cell culture medium additives using cell organelle stress modulator

The present invention relates to a cell transformation method for transforming cells by culturing the cells in a medium containing at least one selected from the group consisting of sodium phenylbutyanoate, taurourosoldioxycholinic acid and butylated hydroxyanisole, More specifically, the present invention relates to a novel regulator that can detect the homeostasis of cell organelles during cell transformation such as degeneration and control stress, and is composed of sodium phenylbutanoate, taururous dioxycholic acid, and butylated hydroxyanisole The present invention relates to a cell switching method capable of high-throughput / high-performance cell switching by applying at least one selected from the group of cells to cell culturing.

Conventional cell switching technology research has been focused on the changes occurring in the nucleus. Nevertheless, there is still a very low cell conversion efficiency and there are many questions about the basis of cell fate determination. The reason for this is that the mechanism of cell fate, which is centered on the nucleus of the nucleus, and the change of numerous cytoplasmic organelles in the cytoplasm are not fully accompanied by the understanding and study of mitochondrial metabolism / change and endoplasmic reticulum stress.

These existing cell switching techniques face the following problems:

Traditional natural differentiation techniques using embryonic stem cells to differentiate into specific target cells have low efficiency of differentiation, high possibility of tumor formation by undifferentiated stem cells, and various clinical and ethical issues including immune rejection problem and chromosomal abnormality Lt; / RTI > In the process of differentiation into target cells, the time required for ~ 3 weeks for myocardial cells, 1 ~ 2 weeks for neurons and neural stem cells, ~ 4 weeks for hepatocytes is relatively long and the efficiency of each step is very low. In addition, it is very difficult to obtain purified cells without the risk of tumor formation, and there are limitations on low efficiency and high cost due to the characteristic that embryonic stem cells are costly to cultivate and differentiate into target cells.

Since the first degenerate pluripotent stem cells have been reported, many researchers have developed various low-molecular compounds, vectors, and signaling mechanisms to improve the differentiation efficiency. Recently, miR-302 clusters), or a method of introducing a recombinant protein or synthesized mRNA into the medium. However, the production efficiency of the pluripotent pluripotent pluripotent stem cells is still very low.

When four factors are introduced into somatic cells, it is possible to observe a population of cells constituting a cluster after about 14 days. About 90 to 95% of the initial cell clusters are incompletely degenerated cells, It is known that the differentiated cells do not undergo spontaneous regeneration according to the passage of time, but also have remarkably low pre-differentiation-specific gene expression. Therefore, it is anticipated that it will be possible to obtain more excellent and complete stem cell treatment resources based on deep research and understanding of the many imperfect degenerated stem cell emerging in this degeneration process. Until now, however, understanding and study of the incompletely degenerate cell group at the intermediate stage of de-differentiation has been lacking, and domestic studies on cell organelles have been insufficient.

Cell transduction based on cross-differentiation technology in various cases is advantageous in that the time required for producing target cells is short and the cost is relatively low, because it induces cross-differentiation into specific somatic cells without going through an osmotic step due to the nature of the technology. Generally, the time required for cross-differentiation is usually 1 to 3 weeks, and the purity of the produced cells is relatively high, so that it is possible to produce high-efficiency target cells in a short period of time as compared with the degeneration technique which requires at least two steps. However, according to a recent study (Cell Stem Cell 2012), it is known that the cells established by the cross-differentiation method are similar to the primary cells in the expression of cell-specific markers and in vitro and in the functional part of the body, The results of the genome-wide analysis, including aspects, still differ significantly from those of cells derived from the body. These differences are due to the fact that crossed differentiated cells must be improved in order to be used in clinical intervention studies in the future and the cross-differentiation efficiency is still low in most cells. Therefore, a cross-differentiation mechanism study is needed to maximize the cross- Technology development is required.

Although much research and attention has been focused on transcriptome and epigenome, there is still a very low cell conversion efficiency and there are many questions about the mechanism of cell fate determination. The reason for this is that the mechanism of cell fate, which is centered on the nucleus of the cell, and the change of numerous cytoplasmic organelles in the cytoplasm are not sufficiently accompanied, and understanding and research on mitochondrial metabolism, change and endoplasmic reticulum are rarely conducted.

Currently, studies on the presence or effect of the presence of stress on the endoplasmic reticulum are mainly focused on the development of the somatic or cell differentiation process, and the study on the stress of the cell organelle, especially the endoplasmic reticulum, .

It is known that in the onset of cancer or metabolic diseases, not only the whole gene expression and the morphological change, but also the problem of the cell organelle become more frequent. In other words, it is known that the homeostasis of cell organelles is very important for the proper function of cells. However, there is little information about the change of cell organelles and the mechanism of homeostasis.

According to a series of studies, various kinds of specific membrane proteins and secretory proteins are produced in the endoplasmic reticulum during the normal development of the subject, resulting in ER stress due to abnormal synthesis of proteins in the endoplasmic reticulum.

In the differentiation process of keratinocytes, UPR (Unfolded Protein Response) expression is very low in the undifferentiated cells. However, ER stress markers such as BiP, ATF6, HRD1, PDI, and CHOP in keratinocyte differentiation process, Significant increases in the expression of genes have been reported.

If the ER stress is not resolved properly in the cell differentiation process, the stemness of the stem cell is remarkably reduced. For example, when ER stress occurs, the expression of markers of stem cells of a stem cell is significantly reduced, and the stem cell signature of these cells is lost.

Based on these results, it can be seen that endoplasmic reticulum stress plays an important role in cell differentiation and cell conversion process. However, such studies have been studied mainly in the developmental process of the somatic and disease models, and there is no study on the stress of the cell organelles that may occur during somatic cell degeneration or cross-differentiation process.

Although various techniques have been attempted to develop a new cell switching technology after the development of de-differentiation / cross-differentiation as described above, despite many researches and efforts (Korean Patent Application No. 10-2013-0132270), still low efficiency and low functionality .

DISCLOSURE Technical Problem The present invention has been conceived in order to solve the above-mentioned problems, and it is an object of the present invention to provide a method for detecting a cell organelle change and a stress inducing mechanism, The present inventors have completed the present invention based on this finding.

An object of the present invention is to provide a medium additive for suppressing ER stress, which comprises at least one selected from the group consisting of sodium phenylbutanoate, taurourosoldioxycholinic acid and butylated hydroxyanisole .

Another object of the present invention is to provide a medium composition for inducing the differentiation into induced pluripotent stem cells, which comprises the above-mentioned medium additive.

It is still another object of the present invention to provide a method for inducing an induction, comprising the step of treating somatic cells or incompletely degenerated cells with at least one selected from the group consisting of sodium phenylbutanoate, taurourosoldioxycholinic acid and butylated hydroxyanisole Thereby providing a method of inducing the differentiation into pluripotent stem cells.

It is yet another object of the present invention to provide cells differentiated by the above method.

It is still another object of the present invention to provide a method of treating a somatic cell or an incompletely degenerated cell, which comprises treating at least one selected from the group consisting of sodium phenylbutanoate, taurourosoldioxycholinic acid and butylated hydroxyanisole, And to provide a method for inhibiting stress (ER stress).

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

In order to achieve the above object,

(EN) Disclosed is a media additive for suppressing ER stress, which comprises at least one selected from the group consisting of sodium benzoate, sodium phenylbutanoate, taururous dioxycholic acid, and butylated hydroxyanisole.

In one embodiment of the present invention, the culture medium additive may be added to the medium at a concentration of 1 to 100 [mu] M.

In addition, the present invention provides a culture medium composition for inducing the differentiation into an inducible pluripotent stem cell comprising the culture medium additive.

The present invention also relates to a method for producing an inducible pluripotent stem, comprising the step of treating somatic cells or incompletely degenerated cells with at least one selected from the group consisting of sodium phenylbutanoate, taurourosoldioxycholinic acid and butylated hydroxyanisole, Lt; RTI ID = 0.0 > cell. ≪ / RTI >

In one embodiment of the present invention, the somatic cell may be one in which a dedifferentiation inducing factor is introduced.

In another embodiment of the present invention, the de-differentiation inducing factor may be selected from the group consisting of Oct4, Sox2, Klf4 and c-Myc.

The present invention also provides cells differentiated by the above method.

The present invention also relates to a method of treating a subject suffering from endothelial dysfunction, comprising the step of treating somatic cells or incompletely degenerated cells with at least one selected from the group consisting of sodium phenylbutyanoate, taururous dioxycholinic acid and butylated hydroxyanisole, ER stress. ≪ / RTI >

According to the present invention, by adding at least one selected from the group consisting of sodium phenylbutanoate, taurousdioxycholinic acid and butylated hydroxyanisole to the culturing process for cell conversion, And the time required to induce cell conversion can be drastically reduced. Sodium phenylbutanoate, taururous dioxycholinic acid or butylated hydroxyanisole inhibits aging induction and oxidative stress that occur during induction of cell transformation, induces cell proliferation and promotes mitochondrial activity, Effectively improving the condition.

The present invention relates to a process for producing pluripotent stem cells derived 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, This will contribute to optimizing the manufacturing process, thereby greatly improving the development process of a custom-made stem cell cell therapeutic agent and clinical drug development, and accelerating the practical use time.

In addition, the present invention is expected to be useful for developing a system capable of culturing a large number of desired cells at the time of cell transformation.

FIG. 1A shows the results of confirming the de-differentiation efficiency by using the dedifferentiation markers SSEA4 and TRA1-60 in partially reprogrammed cells treated with sodium phenylbutyanoate.

FIG. 1B shows the results of confirming the expression of the de-differentiation-specific marker in a partially reprogrammed cell treated with sodium phenylbutyanoate.

FIG. 1C shows the results of confirming the de-differentiation efficiency using SSEA4 and TRA1-60, which are dedifferentiation markers, in partially reprogrammed cells treated with taururous dioxycholinic acid.

FIG. 1D shows the results of confirming the dedifferentiation efficiency using the degeneration markers SSEA4 and TRA1-60 in partially reprogrammed cells treated with butylated hydroxyanisole.

FIG. 1E shows the results of confirming the dedifferentiation efficiency by using SSEA4 and TRA1-60, which are degeneration markers, in iPSC derived from human fibroblast cells treated with sodium phenylbutyanoate.

Fig. 1F shows the results of confirming the de-differentiation efficiency using iPSC derived from human fibroblast cells by treatment with tauroloxodioxycholate, using SSEA4 and TRA1-60, which are degeneration markers.

Fig. 1G shows the results of confirming the dedifferentiation efficiency using iPSC derived from human fibroblast cells by treatment with butylated hydroxyanisole using SSEA4 and TRA1-60, which are degeneration markers.

FIG. 2A shows the result of confirming the de-differentiation efficiency through positive reaction in the Alkaline Phosphatase (AP) staining of iPSC derived from human fibroblast cells according to sodium phenylbutyanoate treatment.

FIG. 2B shows the result of confirming the de-differentiation efficiency through positive reaction in the iPSC-induced Alkaline Phosphatase (AP) staining of human fibroblast cells following treatment with tauroloxodoxycholic acid.

FIG. 2C shows the result of confirming the reverse-differentiation efficiency through positive reaction in the iPSC-induced Alkaline Phosphatase (AP) staining from human fibroblast cells according to the treatment with butylated hydroxyanisole.

FIG. 3A shows the results of qRT-PCR for ER-stress-related gene expression by sodium phenylbutyrate treatment at different concentrations when inducing differentiation using human fibroblast cells.

FIG. 3B shows the results of qRT-PCR for ER-stress-related gene expression following treatment with tauroloxo-dioxycholic acid at different concentrations when inducing differentiation using human fibroblast cells.

FIG. 3c shows the results of qRT-PCR for expression of ER-stress-related genes following treatment with butylated hydroxyanisole at different concentrations when inducing differentiation using human fibroblast cells.

Hereinafter, the present invention will be described in detail.

The present inventors have studied efficient cell transformation and found that cell organelle stress plays an important role in the cell differentiation and cell transformation process. In searching for control factors and exploring cell organelle changes and stress inducing mechanisms, The present inventors have completed the present invention by confirming the activity of inhibiting cell organelle stress of sodium phenylbutanoate, taururous deoxycholic acid, and butylated hydroxyanisole.

Accordingly, the present invention relates to a medium additive for suppressing ER stress, which comprises at least one selected from the group consisting of sodium phenylbutanoate, taurourosoldioxycholinic acid and butylated hydroxyanisole, The present invention provides a medium composition for inducing the differentiation into induced pluripotent stem cells.

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

[Chemical Formula 1]

In the present invention, tauroursodeoxycholic acid is a compound represented by the following formula (2), and the compound is named 2 - [[(4R) -4 - [(3R, 5S, 7S, 8R, 9S, 13R, 14S, 17R) -3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetra Decahydro-1H-cyclopenta [a] phenanthren-17-yl] pentanoyl] amino] ethanesulfonic acid or 3?, 7? -Dihydroxy-5? -Colanoyl taurine. It is a kind of bile acid. It is produced from liver by using cholesterol as a precursor. It may exist in various salt forms, pharmaceutically acceptable salt forms, and may exist in the form of alkali metal salts such as sodium and potassium.

(2)

In the present invention, butylated hydroxyanisole is a compound represented by the following general formula (3), wherein 2-tertiary-butyl-4-hydroxyanisole represented by the following general formula (4) - tertiary-butyl-4-hydroxyanisole.

(3)

[Chemical Formula 4]

[Chemical Formula 5]

As used herein, the term "cell transformation" is intended to include spontaneous differentiation of embryonic stem cells, reprogramming and direct conversion of dedifferentiated pluripotent stem cells, and somatic cell nuclear transfer (SCNT) ) Establishment process.

The natural differentiation is to differentiate stem cells into somatic cells or progenitor cells, and the de-differentiation is to dedifferentiate somatic or progenitor cells into stem cells, and the cross-differentiation is to cross-differentiate somatic cells into different somatic cells or progenitor cells Can be

The stem cell may be selected from any and all without limitation, and may be derived from adult cells such as all known tissues and cells derived from mammals, including humans, preferably from humans. For example, 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 sac, placenta, cord, periosteum, fetal and pubic skin, And may be embryonic stem cells, stem cells immediately after birth or from an adult.

The stem cells may be, for example, embryonic stem cells, inducible pluripotent stem cells, placental stem cells, cord blood stem cells, peripheral blood stem cells and bone marrow stem cells, neural stem cells, hematopoietic stem cells, A stem cell, a vascular endothelial progenitor cell, and an mesenchymal stem cell. However, the present invention is not limited thereto.

The somatic cell may be a fibroblast, an epithelial cell, a muscle cell, a neural cell, a gastric mucosa cell, a germ cell, a G cell, a B cell, a hippocampus, an astrocyte, a blood cell, Vascular endothelial cells, and vascular endothelial cells. The somatic cell may be any cell except for germ cells, and examples thereof include fibroblasts, muscle cells, nerve cells, gastric mucosal cells, goblet cells, G cells, pericyte, astrocyte ), B cells, blood cells, epithelial cell neural stem cells, hematopoietic stem cells, mesenchymal stem cells, umbilical cord blood-derived hematopoietic stem cells, vascular endothelial cells or cord blood stem cells. However, cross-differentiation is not limited to the above, as the starting cells can be applied to somatic cells regardless of whether they are specific tissue cells or not.

As used herein, the term "differentiation" refers to a phenomenon in which the structure or function of a cell is specialized while cells are divided and proliferated and the entire individual grows. In other words, it refers to a process in which cells, tissues, etc. of a living organism are changed into appropriate forms and functions to perform their respective roles. For example, pluripotent stem cells such as embryonic stem cells are differentiated into ectoderm, mesoderm, and endoderm cells, and narrowly, the processes in which hematopoietic stem cells change into red blood cells, white blood cells, platelets, etc. are also differentiated. It may also be referred to as "natural differentiation ".

The term "differentiated cell" used in the present invention refers to a cell that has undergone the differentiation process and has a certain form and function. The differentiated cells of the present invention are not particularly limited, but are preferably germ cells, somatic cells, or progenitor cells. It is also 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 having the potential of a new type of differentiation. In addition, the de-differentiation is used in the same sense as the cell reprogramming in the present invention. The de-differentiation mechanism of these cells is to establish a different set of welfare genetic markers after deletion of the marking in the nucleus (the DNA state associated with the genetic change in function without changes in the nucleotide sequence) in the nucleus . While differentiation and growth of multicellular organisms, different cells and tissues acquire different gene expression programs.

As used herein, the term " high-efficiency cell conversion "means that the cell conversion process occurs rapidly or the efficiency of cell conversion is increased in the process of cell conversion. That is, it means that the efficiency of cell conversion is improved in terms of rate or ratio.

The term " dedifferentiation inducing factor "as used herein is a substance that induces finally differentiated cells to reverse differentiate into induced pluripotent stem cells having a potential for a new type of differentiation. The dedifferentiation factor may be any substance that induces the differentiation of the finally differentiated cells, and may be selected depending on the type of cells to be differentiated. But are not limited thereto, preferably further comprise a protein selected from the group consisting of Oct4, Sox2, KlF4, c-Myc, Nanog, Lin-28 and Rex1 or a nucleic acid molecule encoding these proteins More preferably an Oct4 protein or a nucleic acid molecule encoding these proteins, and may include Oct4, Sox2, KlF4 and c-Myc proteins or nucleic acid molecules encoding these proteins.

The term "direct reprogramming / direct conversion / transdifferentiation ", as used herein, is intended to encompass mature (differentiated) cells with totally different cell types in higher organisms Unlike the process of reprogramming into Induced Pluripotent Stem Cells (iPSCs) and regenerating them into desired cells, it is a process of inducing the conversion. It is expected that cross-differentiation will be used for disease modeling and drug discovery, and it will be applied to gene therapy and regenerative medicine in the future.

As used herein, the term "somatic cell" refers to all cells that have undergone differentiation constituting an animal or plant, excluding germ cells, and 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 it is found that a cell corresponding to a progeny expresses a specific differentiation trait. For example, neurons (neuron hepatocytes) correspond to progenitor cells for neurons (neurons), and myocytes correspond to progenitor cells for canaliculus cells

As used herein, the term "pluripotent stem cell" refers to a pluripotent or totipotent self-renewal capable of differentiating into cells of all tissues of the individual 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 all of human, monkey, pig, horse, cattle, sheep, dog, cat, mouse, rabbit and the like, but preferably human pluripotent stem cells .

The term "embryonic stem cell" used in the present invention refers to a cell obtained by extracting an inner cell mass from a blastocyst embryo immediately before fertilization of the embryo into the uterus of a mother and culturing the same in vitro, Pluripotent, or omnipotent, self-renewal cells, and broadly includes embryoid bodies derived from embryonic stem cells. The embryonic stem cells of the present invention include all embryonic stem cells derived from human, monkey, pig, horse, cattle, sheep, dog, cat, mouse, rabbit and the like, but are preferably human-derived embryonic stem cells.

As used herein, the term "induced pluripotent stem cells" refers to cells induced to have pluripotential differentiation ability through artificial reprogramming from differentiated cells, which is also referred to as induced pluripotent stem cells (iPSCs) do. An artificial reprogramming process may be performed by introduction of a non-viral-mediated reprogramming factor using virus-mediated or non-viral vector utilization, retroviruses and lentiviruses, proteins and cell extracts, or by stem cell extracts, And includes a de-differentiation process. Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically showing similar cell shapes, similar in gene and protein expression pattern, and in vitro and in vivo, , Which forms a teratoma and is inserted into a blastocyst of a mouse to form a chimera mouse and enable germline transmission of the gene.

As used herein, the term "vector" refers to an expression vector capable of expressing a target protein in a host cell, which gene construct comprises an essential regulatory element operably linked to the expression of the gene insert. The vector may include a signal sequence or a leader sequence for membrane targeting or secretion in addition to an expression regulatory element such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, an enhancer, and the like. The promoter of the vector may 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, a replication origin. The vector may be self-replicating or integrated into the host DNA.

The term "culture media " as used in the present invention means a medium capable of supporting stem cell growth and survival in vitro, and includes a culture medium, . Depending on the type of cells, medium and culture conditions can be selected. The medium used for the culture is preferably a cell culture minimum medium (CCMM), which generally contains a carbon source, a nitrogen source and a trace element component. For example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, aMEM Glasgow's Minimal Essential Medium, and Iscove's Modified Dulbecco's Medium. In addition, the medium may include antibiotics such as penicillin, streptomycin, and gentamicin.

As used herein, the term "nucleic acid molecule encoding a protein" refers to a nucleic acid molecule that is operatively linked to a promoter or the like so that the protein can be expressed therein, Lt; RTI ID = 0.0 > nucleic acid < / RTI > molecules. 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 is operably linked to an expression vector May be delivered into the cell, or may be delivered into the cell in a form that is inserted into the chromosome of the host cell.

The term "cell therapeutic agent" used in the present invention is a medicament (US FDA regulation) used for the purpose of treatment, diagnosis and prevention with cells and tissues prepared by isolation, Refers to a drug used for therapeutic, diagnostic, and prophylactic purposes through a series of actions, such as alive, homologous, or xenogeneic cell propagation, screening, or otherwise altering the biological characteristics of a cell .

In one embodiment of the present invention, most of the cells are partially reprogrammed cells by inducing the differentiation of human mononuclear cells using a differentiation inducing factor. In the present invention, sodium phenylbutanoate , Taururous dioxycholinic acid, or butylated hydroxyanisole, respectively, and then subjected to FACS analysis to quantitatively analyze the de-differentiation efficiency. As a result, it was found that sodium phenylbutanoate, taururous dioxycholinic acid, or It was confirmed that ER-stress was inhibited by treatment with butylated hydroxyanisole, and that the de-differentiation was significantly increased. Also, human fibroblasts were observed from the beginning After treatment of sodium phenylbutanoate, taururous dioxycholinic acid, or butylated hydroxyanisole according to the invention, respectively, Were quantitatively analyzed. As a result, it was confirmed that ER-stress was inhibited by treatment with sodium phenylbutanoate, taururous dioxycholinic acid, or butylated hydroxyanisole, respectively, , And Alkaline Phosphatase (AP) staining revealed that AP-positive colonies were formed at a level similar to that of the control group (see Examples 1 and 2).

In another embodiment of the present invention, ER-stress related genes (ATF3, ATF4, ATF6, GADD34, CHOP (SEQ ID NO: , BIP, tXBP1, and SxBP1), the expression of ER-stress related genes was reduced by treatment with sodium phenylbutanoate, taururous dioxycholate, or butylated hydroxyanisole (See Example 3).

Based on the above results, it was confirmed that the effects of sodium phenylbutyanoate, taururodioxycholinic acid, and butylated hydroxyanisole on the cell organelle stress were effectively inhibited and the effect of improving the reprogramming efficiency was improved. As a result, Suggesting that it can be usefully used as a cell transformation technology to cultivate a large amount of cells.

In another aspect of the present invention, the present invention includes a step of treating somatic cells or incompletely degenerated cells with at least one selected from the group consisting of sodium phenylbutanoate, taurourus deoxycholic acid, and butylated hydroxyanisole Induced pluripotent stem cells. ≪ / RTI >

The term " partially reprogrammed cell "used in the present invention means that when four inducers (Yamanaka factor: OCT4, SOX2, KLF4, c-MYC) are introduced into somatic cells to induce the initial de- differentiation, After 14 days, a population of cells forming a colony can be observed. At this time, about 90 to 95% of the emerging cell clusters are incompletely degenerated cells (negative for TRA1-60 marker), and the incompletely degenerated cells are spontaneously degenerated (TRA1-60 positive) Refers to a cell that is known to exhibit a significantly lower pre-differentiation-specific gene expression as well as a lesser degree of expression. Thus, incomplete degenerated cells are de-differentiated mesenchymal cells that can be transferred into fully degenerated cells when new intracellular signal stimuli are present.

At this time, when somatic cells are treated with somatic cells selected from the group consisting of sodium phenylbutanoate, taururous dioxycholinic acid and butylated hydroxyanisole, the somatic cell may be one in which a differentiation inducing factor is introduced The method of introducing the dedifferentiation inducing factor into a somatic cell may be any method of providing a nucleic acid molecule or a protein to a cell commonly used in the art without limitation. Preferably, the regeneration inducing factor is added to a culture medium of the differentiated cells A method of directly injecting a differentiation inducing factor into a differentiated cell, or a method of infecting a cell differentiated with a virus obtained from a packaging cell transfected with a virus vector into which a gene of a differentiation inducing factor is inserted Method can be used.

The method of directly injecting the dedifferentiation inducing factor into the differentiated cells may be selected from any method known in the art and may be performed by a variety of methods including, but not limited to, microinjection, electroporation, Particle bombardment, direct muscle injection, insulator, and transposon.

In the present invention, the reprogramming inducing factor may be selected according to the type of cell to be reprogrammed, and is preferably selected from the group consisting of Oct4, Sox2, KlF4, c-Myc, Nanog, Lin-28, microRNA-302 clusters, or any one or more nucleic acid molecules that encode these proteins. More preferably, the nucleic acid molecule may encode an Oct4 protein or a nucleic acid molecule encoding the protein. , Oct4, Sox2, KlF4 and c-Myc proteins or nucleic acid molecules encoding these proteins.

In the present invention, the packaging cells may be selected from various cells known in the art depending on the viral vector used, and preferably, GP2-293 packaging cells can be used.

In addition, the viral vector may be a retroviruses such as human immunodeficiency virus (HIV), murine leukemia virus (MLV), Avian sarcoma / leukosis (ASLV), Spleen necrosis virus (SNV), Rous sarcoma virus , mouse mammary tumor virus (mMTV), lentiviruses, adenovirus, adeno-associated virus, herpes simplex virus, Sendai virus, An episomal vector, and the like. However, it is preferable to use a retroviral vector, and more preferably, a retroviral vector pMXs can be used.

In another aspect of the present invention, the present invention provides a cell differentiated by the above method.

The degenerated pluripotent stem cells prepared by the cell transformation method may include all cell cultures in vitro obtained by treating the differentiated cells with sodium phenylbutanoate and a dedifferentiation inducer. The cell culture may include various cells in the differentiation process, various proteins and enzymes obtained in the culturing thereof, transcripts and a culture solution containing the same.

In a preferred embodiment of the present invention, the cells are differentiated from the differentiated pluripotent stem cells cultured in the culture medium containing the sodium phenylbutyanoate, and the cell growth and proliferation are promoted , The cell death is suppressed, the mitochondrial activity is enhanced, the aging is suppressed, the oxidative stress is reduced, the p53 signaling is inhibited, the dedifferentiation induction time is shortened, and the dedifferentiation inducing efficiency is enhanced Respectively.

The delivery and incubation steps may be performed simultaneously, sequentially or in reverse order.

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

(a) introducing a cross-differentiation factor into somatic cells; And

(b) culturing the incomplete degenerated cells obtained in the step (a) in a medium containing sodium phenylbutanoate.

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

The cross-differentiation factor may be selected from the group consisting of OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2, Hnf4a, Foxa1, Foxa3, Gata4, Hnf1a, FLT1 (Friend leukemia virus integration 1), ETV variant gene 2 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 nucleic acid encoding a protein or a protein thereof, wherein the protein is human, mouse, horse, sheep, pig, Camel, nutrition, dog, and the like. In addition, the OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, and NKX6.2 proteins used in the present invention include protein variants of respective genes as well as proteins having a wild type amino acid sequence thereof.

A variant of the protein means a protein having a sequence which differs from the native amino acid sequence of the protein by one or more amino acid residues by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. The mutant may be a functional equivalent exhibiting the same biological activity as the natural protein or may be a mutant in which the physicochemical properties of the protein are modified as needed and may be a mutant having increased structural stability against physiological or chemical environment or increased physiological activity have.

In addition, the nucleic acid encoding the protein may be a wild-type or a nucleotide sequence encoding a protein in the mutant form as described above, wherein one or more bases may be mutated by substitution, deletion, insertion, or a combination thereof, Can be prepared by chemical synthesis. The nucleic acid having the nucleotide sequence encoding the above-mentioned protein may be short or double-stranded, and may be a DNA molecule (genomic DNA, cDNA) or an RNA molecule.

In one embodiment of the present invention, the nucleic acid molecule encoding the OCT4, SOX1, SOX2, SOX10, OLIG2, NKX2.2, NKX6.2 protein in the present invention is a vector expressing a protein comprising a nucleic acid encoding each protein .

Vectors usable in the present invention include a plasmid vector, a cosmid vector, a viral vector, and an episomal vector. Preferably, it may be a viral vector. The viral vectors may be selected from the group consisting of lentivirus vectors, retroviruses, human immunodeficiency virus (HIV), murine leukemia virus (MLV), Avian sarcoma / leukosis (ASLV), Spleen necrosis virus (SNV), Rous sarcoma virus , Mouse mammary tumor virus (MMTV), etc., adenovirus, adeno-associated virus, herpes simplex virus, Sendai virus and episomal vector. But are 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 a somatic cell, and any vector system can be used to exhibit the effect of the present invention. In one embodiment of the invention, the vector may be a pMX-based retroviral vector expressing OCT4

In addition, the nucleic acid encoding the protein may be introduced into cells by known methods in the art, for example, naked DNA in vector form, or introduced into cells using liposome, cationic polymer, or the like .

The liposome is a phospholipid membrane prepared by mixing a cationic phospholipid such as DOTMA or DOTAP for gene transfer. When a cationic liposome and an anionic nucleic acid are mixed at a certain ratio, a nucleic acid-liposome complex is formed and introduced into cells .

Specifically, in the present invention, the nucleic acid molecule encoding the protein is contained in a vector produced to express each gene by transforming and infecting a viral vector containing a nucleic acid encoding the protein with a packaging cell, and a virus And introduced into somatic cells. Such viruses include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses and the like.

The medium is not particularly limited and conventionally known media can be used. Examples of the medium include dexamethasone, ascorbic acid, insulin, transferrin, L-alanyl-L-glutamine, glycerol 2-phosphate, fibroblast growth factor bFGF) and selenium salts, and the insulin, transferrin, and selenium salts may be commercially available ITS (Gibco, USA). . In addition, the medium may further comprise, if desired, fetal bovine serum (FBS) and antibiotics, for example, about 10% FBS, about 1% penicillin-streptomycin.

The cells include all the cells derived from human, monkey, pig, horse, cattle, sheep, dog, cat, mouse, rabbit and the like, but are preferably human-derived cells.

The culture may be carried out on conventional culture plates in pellet culture form, for example, it is incubated at 37 ℃, conditions of 5% CO _2. The incubation period is not particularly limited, but can be carried out for about three weeks, for example. After completion of the culture, the cells are obtained in the form of pellets and can be separated by removing the medium.

The culture may also be a number of continuous passages.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental Preparation and Experimental Method

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

In order to confirm the efficiency of sodium phenylbutanoate, taururodioxycholinic acid, or butylated hydroxyanisole in the differentiation process, human blood cell-derived partially reprogrammed cells and fibroblast cells ) Were cultured.

First, to cultivate the partially reprogrammed cells, 10 ml of patient blood was obtained from the hospital, mononucleocytes were obtained using Ficoll-paque, and RPMI1640 medium containing SCF, TPO, Flt3, IL-6 and IL-3 The culture was continued for about 6 days. Subsequently, the Yamanaka factor (Oct4, Sox2, Klf4, c-Myc) was introduced using Sendai virus (cytotune-iPS reprogramming kit, Invitrogen). After that, the cells were cultured for 4 days When the culture was completed, the cells were transferred to a Vitronectin-coated culture dish and cultured in mTesR-E8 medium.

After a period of incubation, 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 cell line (BJ1) was cultured in a DMEM medium containing 10% FBS, and then transfected with Sendai virus (cytotune-iPS reprogramming (Oct4, Sox2, Klf4, c-Myc) was introduced into the culture medium using the above-mentioned culture medium for 4 days. After completion of the culture, MEF (mouse embryonic fibroblast) And cultured in hESC-exclusive or mTesR-E8 medium after transferring to a culture dish or Vitronectin-coated culture dish.

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

After the above-described embodiment 1-1 Partially reprogrammed cell 5x10 ^4/24 wells phenyl-butyric acid, sodium tauro-low Russo deoxy choline acid, or butylated hydroxy anisole in 50 uM each in treatment 6 days obtained from the initial Cells expressing the dedifferentiation marker SSEA4 and the final degeneration marker TRA1-60 were analyzed by fluorescence-activated cell sorting (FACS).

Similarly, de-differentiation factor (Yamanaka factor; Oct4, Sox2, Klf4, c-Myc) a process of human fibroblasts (BJ1) 5x10 ^4/24 wells 10, 50 and 100 uM of the phenyl-butyric acid, sodium tauro-low Russo deoxy After 14 days of regeneration was induced in E8 medium (Vitronectin-coated culture dish) containing cholinic acid or butylated hydroxyanisole, cells expressing the early degeneration marker SSEA4 and the final degeneration marker TRA1-60 Were analyzed by fluorescence-activated cell sorting (FACS).

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

MMC treatment -MEF 5x10 to prepare the ^4/24 wells de-differentiation factor (Yamanaka factor; Oct4, Sox2, Klf4, c-Myc) of the the treatment of human fibroblasts ^{(BJ1) 2.5x10 4/24 wells} 10 and 100 uM After induction of the differentiation in hESC culture medium containing sodium phenylbutylate, taururous dioxycholinic acid, or butylated hydroxyanisole for 14 days, Alkaline Phosphatase (AP) staining (Staemgent) was performed according to the manufacturer's protocol Respectively.

1-4. Identification of ER-stress related gene expression (qRT-PCR)

The expression of ER-stress related gene in the cell degeneration process by sodium phenylbutyanoate, taururodioxycholinic acid, or butylated hydroxyanisole according to the present invention was examined.

More specifically, when dedifferentiation is induced by using human fibroblast cells, the treatment concentration (10, 50) is determined for each of sodium phenylbutylate, taururous dioxycholinic acid, or butylated hydroxyanisole, Expression of ER-stress related genes (ATF3, ATF4, ATF6, GADD34, CHOP, BIP, tXBP1, SxBP1) was confirmed by qRT-PCR.

Example 2. Confirmation of cell conversion efficiency by treatment with sodium phenylbutyrate

2-1. Identify the reprogramming efficiency of partially reprogrammed cells

In order to confirm the reprogramming efficiency of the partially reprogrammed cells according to the treatment with sodium phenylbutyanoate, taururodioxycholinic acid, or butylated hydroxyanisole of the present invention, embryonic stem cell specific Markers, SSEA4 and TRA1-60, were used to quantify the dedifferentiation efficiency. The results were as follows.

Degradation Efficiency of Partially Reprogrammed Cells by Sodium Phenylbutyrate Treatment:

As shown in FIG. 1A, the de-differentiation was significantly reduced by the ER-stress inducer, TG, but it was confirmed that the de-differentiation was significantly increased by the treatment with sodium phenylbutyrate according to the present invention .

In addition, as shown in Fig. 1B, the expression of SSEA4 and TRA1-60 could be specifically confirmed by treatment with sodium phenylbutyrate.

Degradation Efficiency of Partially Reprogrammed Cells by Treatment with Taururous Dioxycholinic Acid:

As shown in FIG. 1C, the de-differentiation was significantly reduced by the ER-stress inducer, TG, but the depletion was significantly increased by the treatment with tauroloxodoxycholic acid according to the present invention. Specifically, I could confirm. More specifically, the expression of SSA4 was significantly increased by taurosuronic deoxycholic acid, but the expression of TRA1-60 induced a simple increase.

Degradation efficiency of partially reprogrammed cells by treatment with butylated hydroxyanisole

As shown in FIG. 1D, the de-differentiation was significantly decreased by TG which is an ER-stress inducer, but it was confirmed that the degeneration was significantly increased by the butylated hydroxyanisole treatment according to the present invention I could. More specifically, butylated hydroxyanisole induced a simple increase in SSEA4 and TRA1-60.

2-2. Identification of iPSC dedifferentiation efficiency from human fibroblast cells

In order to confirm the dedifferential efficiency of iPSC derived from human fibroblast cells according to the treatment with sodium phenylbutylate, taururodioxycholinic acid, or butylated hydroxyanisole of the present invention, -2, the dedifferentiation efficiency was quantified using SSEA4 and TRA1-60, which are embryonic stem cell-specific markers, and the results are as follows.

Degradation efficiency of iPSC derived from human fibroblast cells by treatment with sodium phenylbutanoate:

As shown in FIG. 1E, there was almost no difference in the expression of the dedifferentiation end marker (TRA1-60), but it can be confirmed that the initial differentiation early marker (SSEA4) was significantly increased depending on the concentration of sodium phenylbutanoate treatment.

Degradation Efficiency of iPSC Derived from Human Fibroblast Cells Following Treatment with Taururous Dioxycholinic Acid:

As shown in FIG. 1F, there was almost no difference in the expression of the dedifferentiation end marker (TRA1-60), but it was confirmed that the initial stage of dedifferentiation marker (SSEA4) was significantly increased according to the concentration of tauroloxodioxycholine acid treatment .

Degradation Efficiency of iPSC Derived from Human Fibroblast Cells Following Treatment with Butylated Hydroxy Anisole:

As shown in FIG. 1G, there was almost no difference in the expression of the dedifferentiation end marker (TRA1-60), but it was confirmed that the initial differentiation early marker (SSEA4) was significantly increased according to the concentration of butylated hydroxyanisole treatment have.

2-3. Positive response to iPSC-induced Alkaline Phosphatase (AP) staining from human fibroblast cells

Next, according to the above Example 1-3, the reprogramming efficiency according to each concentration was positive in the hESC-specific factor Alkaline Phosphatase (AP) staining. The number of stained colonies was counted, and the phenylbutyl The dedifferentiation efficiency by treatment with sodium acid, taururous dioxycholinic acid, or butylated hydroxyanisol was quantified.

As a result, as shown in Fig. 2A, it was confirmed that AP-positive colony was formed at 10 mu M of sodium phenylbutanoate at the same level as control, and as shown in Fig. 2B, 10 mu M of taurosuronic deoxycholic acid As shown in FIG. 2C, AP-positive colony was formed at 10 uM of butylated hydroxyanisole as compared with the control, and AP-positive colony was formed at 10 uM of butylated hydroxyanisole .

Example 3. Confirmation of ER-stress relieving effect by treatment with sodium phenylbutyrate

In order to confirm the ER-stress relieving effect of the sodium phenylbutylate, taururous dioxycholinic acid, or butylated hydroxyanisole treatment of the present invention, sodium phenylbutylate, tau Expression of ER-stress related genes (ATF3, ATF4, ATF6, GADD34, CHOP, BIP, tXBP1, SxBP1) following treatment with rou- rodeoxycholic acid or butylated hydroxyanisole was confirmed, same.

Identification of ER-stress relief effect by treatment with sodium phenylbutyrate:

As shown in FIG. 3A, it was confirmed that the expression of the ER-stress related gene was alleviated by treatment with sodium phenylbutyrate. From the above results, it was confirmed that sodium phenylbutyrate can increase the de-differentiation efficiency by directly controlling ER-stress.

Confirmation of ER-stress relief effect by treatment with taurolurso-dioxycholinic acid:

As shown in Fig. 3B, it was confirmed that the expression of the ER-stress related gene was alleviated by treatment with taurosulfite deoxycholic acid. From the above results, it was confirmed that taurosuronic deoxycholic acid can directly increase ERP-induced regeneration efficiency by directly controlling ER-stress.

Identification of ER-stress relief effect by butylated hydroxyanisole treatment:

As shown in Fig. 3C, it was confirmed that the expression of the ER-stress related gene was alleviated by the treatment with butylated hydroxyanisole. From the above results, it was confirmed that the butylated hydroxyanisole can increase the reprogramming efficiency by directly controlling ER-stress.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

According to the present invention, by adding at least one selected from the group consisting of sodium phenylbutanoate, taurousdioxycholinic acid and butylated hydroxyanisole to the culturing process for cell conversion, And the time required to induce cell conversion can be drastically reduced. Sodium phenylbutanoate, taururous dioxycholinic acid or butylated hydroxyanisole inhibits aging induction and oxidative stress that occur during induction of cell transformation, induces cell proliferation and promotes mitochondrial activity, Effectively improving the condition.

The present invention relates to a process for producing pluripotent stem cells derived 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, This will contribute to optimizing the manufacturing process, thereby greatly improving the development process of a custom-made stem cell cell therapeutic agent and clinical drug development, and accelerating the practical use time.

In addition, the present invention is expected to be useful for developing a system capable of culturing a large number of desired cells at the time of cell transformation.

Claims (8)

1. Wherein the medium contains at least one selected from the group consisting of sodium phenylbutanoate, taururous dioxycholic acid, and butylated hydroxyanisole.
2. The media additive of claim 1, wherein the medium additive is added to the medium at a concentration of 1 to 100 μM.
3. A medium composition for inducing the differentiation into induced pluripotent stem cells, comprising the culture medium additive of claim 1.
4. Comprising treating the somatic cells or the incompletely degenerated cells with at least one member selected from the group consisting of sodium carboxymethylcellulose, sodium carboxymethylcellulose, sodium carboxymethylcellulose, sodium phenylbutylate, taururous dioxycholy acid and butylated hydroxyanisole, Induction method.
5. 5. The method of claim 4, wherein the somatic cell is introduced with a dedifferentiation inducing factor.
6. 6. The method of claim 5, wherein the de-differentiation inducing factor is selected from the group consisting of Oct4, Sox2, Klf4, and c-Myc.
7. A cell differentiated by the method of claim 4.
8. A method for inhibiting ER stress, comprising the step of treating somatic cells or incompletely degenerated cells with at least one selected from the group consisting of sodium phenylbutanoate, taururous dioxycholinic acid and butylated hydroxyanisole .

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