KR20190128961A - A method for producing directly reprogrammed induced neural stem cells from non-neuronal cells using Sox2 and c-Myc - Google Patents

Abstract

The present invention relates to a method for generating neural stem cells induced from non-neural cells through direct reprogramming and a use thereof. As the method for generating induced neural stem cells according to the present invention allows the generation of induced neural stem cells from non-neural cells even when only two inducing factors such as Sox2 and c-Myc are used, the method can generate induced neural stem cells more effectively than conventional generation methods using four or five inducing factors. The induced neural stem cells generated by the method exhibit excellent differentiation potency and proliferation ability, and thus can be applied to therapeutic purposes in the future.

Description

A method for producing directly reprogrammed induced neural stem cells from non-neuronal cells using Sox2 and c-Myc}

The present invention relates to a method for producing neural stem cells derived from non-neuronal cells via direct reprogramming and use thereof.

Stem cells are generally classified as omnipotent or pluripotent. Pluripotent stem cells are wholly differentiated cells, which can produce all of the different cell types in the body. Fertilized egg cells are an example of pluripotent stem cells. Pluripotent stem cells produce any cell type in the body derived from the three main germ layers or the embryo itself.

Pluripotent stem cells such as embryonic stem cells (ESC) can be useful for cell transplantation treatments because they proliferate rapidly while maintaining pluripotency, ie the ability to differentiate into various cell types. To date, pluripotent stem cells have been produced mainly by nuclear transplantation and cell fusion (Philos Trans R Soc Lond B Biol Sci. 363 (1500): 2079-2087). However, because both methods use embryonic stem cells, an ethical dilemma arises for both research and treatment.

Recently, the discovery of induced pluripotent stem cells (iPSC) has been able to overcome these problems by using embryonic stem cells. "Induced pluripotent stem cells (iPSCs)" are cells that exhibit properties similar to embryonic stem cells (ESCs). Induced pluripotent stem cells expressed expression of defined factors from mouse fibroblasts (Cell 126: 663-676 (2006) in 2006) and human fibroblasts (Science 318: 1917-1920 (2007), in 2007). Was first created by augmenting These studies included Oct-3 / 4, Sox2, Klf4 and c-Myc to initiate reprogramming of mature somatic cells into iPSCs; Oct4, Sox2, Nanog and Lin28 were used. However, iPSC has a limitation in that it does not control the differentiation of cells into teratoma as well as differentiation when transplanted into a living body, as in embryonic stem cells.

Therefore, in order to overcome these limitations, recently, a technique for differentiating cells of a specific lineage through direct induction or direct reprogramming has been attracting attention. This technology introduces specific lineage-specific genes into fully differentiated cells, ie, fibroblasts, and directly induces specific cells without undergoing a pluripotent state, thereby reducing the risk of teratoma in pluripotent cells. It is a technology that can be excluded.

Especially in the case of neurons that can be permanently damaged once damaged, researchers around the world have actively tried to reprogram them. However, induced neurons without self-renewal are difficult to cultivate in vitro for a certain period of time and thus cannot obtain a sufficient amount of cells, and thus sufficient amounts for cell therapy, including molecular cytological mechanisms involved in direct reprogramming. It is practically impossible to obtain cells.

Under these circumstances, the present inventors have made diligent efforts to develop a method for producing induced neuronal stem cells that are genetically safe and have excellent differentiation ability. As a result, the present inventors have precisely regulated the MOI of viruses including Sox2 and c-Myc, and thus, Sox2 and c-Myc. It was found that the induced neural stem cells can be produced only by the introduction of the present invention. The present invention was completed by confirming the stability and differentiation capacity of the prepared induced neural stem cells.

One object of the invention is (a) introducing Sox2 and c-Myc into isolated cells; And (b) culturing the cells of step (a) to induce direct reprogramming from the isolated cells to the cells into which the strain is converted.

Another object of the present invention to provide a neural stem cell prepared according to the above method.

Still another object of the present invention is to provide a cell therapy agent comprising the neural stem cells prepared according to the above method as an active ingredient.

Still another object of the present invention is to provide a pharmaceutical composition for treating or preventing neurological diseases, including neural stem cells prepared according to the above method as an active ingredient.

Another object of the present invention comprises the step of identifying a therapeutic agent for neurological diseases tailored to the individual from which the neural stem cells are derived, by treating a candidate material with neural stem cells or neural cells differentiated therefrom prepared according to the above method. Methods of screening for personalized neurological disease therapies are provided.

Each description and embodiment disclosed in the present application may also apply to each other description and embodiment. That is, all combinations of the various elements disclosed in this application are within the scope of the present application. In addition, the scope of the present application is not to be limited by the specific description described below.

One aspect for achieving the object of the present invention comprises the steps of (a) introducing Sox2 and c-Myc into isolated cells; And (b) culturing the cells of step (a) to induce direct reprogramming from the isolated cells to the cells into which the strain is converted.

In the present invention, step (a) is a step of introducing Sox2 and c-Myc into the isolated cells.

The term “isolated cell” of the present invention is not particularly limited, and specifically, may be a cell in which lineage is already specified, such as a somatic cell, a germ cell or a progenitor cell, and an adult stem. Cells, bone marrow cells, mesoderm stem cells, etc. may be a stem cell with limited differentiation capacity.

Isolated cells of the present invention may include both cells in vivo or ex vivo, and specifically, may be cells isolated from the body. In the present invention, "somatic cells" refers to all cells that have completed differentiation constituting plants and plants except germ cells, somatic cells of the present invention may be somatic cells except neurons.

In addition, the "progenitor cell" 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 the progeny expresses a specific differentiation trait.

"Adult stem cells" of the present invention refers to stem cells appearing in the stage of development or adult formation of each organ of the embryo as the process of development, which is generally limited to cells constituting specific tissues. Specifically, adult stem cells may be derived from the group consisting of breast, bone marrow, umbilical cord blood, blood, liver, skin, gastrointestinal tract, placenta, and uterus, and adult stem cells of the present invention may be adult stem cells other than neural stem cells. Can be.

In addition, the adult stem cells may be mesenchymal stem cells, but is not limited thereto. "Mesenchymal stem cells" of the present invention is a cell that helps to create cartilage, bone, fat, myeloid epilepsy, muscle, nerves, skin, etc., in adults generally stay in the bone marrow, but umbilical cord, cord blood, peripheral blood, other tissues It means cells that can also be obtained from, etc. Specifically, the cell may be derived from a cell selected from the group consisting of umbilical cord, cord blood, bone marrow, fat, muscle, skin, amniotic membrane, and placenta, but is not limited thereto. Mesenchymal stem cells of the umbilical cord blood may be mesenchymal stem cells, but is not limited thereto.

In addition, the isolated cells may be non-neuronal cells. In the present invention, "non-neuronal cell" includes all differentiated or undifferentiated cells which are not neurons, and serve as target cells of the present invention. The non-neuronal cells of the present invention may be cells derived from various animals such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits, and specifically, may be cells derived from humans, This does not limit the cells that can be reprogrammed to induced neural stem cells.

In one embodiment of the present invention, Sox2 and c- in non-neuronal fibroblasts (Example 1), Niemann-Pick disease-derived fibroblasts and cord blood-derived mesenchymal stem cells (Example 2) Neural stem cells were prepared by introducing Myc.

As the non-neuronal cells used in the present invention, fibroblasts are representative examples of somatic cells, cord blood-derived mesenchymal stem cells are representative examples of adult stem cells, and these results indicate that Sox2 and c-Myc are reprogramming inducers. It is shown that adult stem cells as well as somatic cells can be effectively reprogrammed into induced neural stem cells.

The “Sox2” gene of the present invention is also known as SRY (Sex determining region Y) -box2 gene and is known as a transcription factor essential for maintaining pluripotency in undifferentiated embryonic stem cells, and is a gene involved in the neural cloning of neural stem cells. Known.

The “c-Myc” gene of the present invention is one of the prototype cancer genes that encode DNA binding proteins in the nucleus and is involved in cell proliferation.

Sox2 gene and c-Myc gene or a protein expressed therefrom of the present invention includes all SOX2 and c-Myc derived from an animal such as human or rat, specifically may be Sox2 and c-Myc derived from human. In addition, the protein expressed from the Sox2 and c-Myc genes of the present invention may include not only proteins having amino acid sequences of their wild type but also variants thereof. Variants of Sox2 and c-Myc proteins include proteins whose sequences differ from the natural amino acid sequence of SOX2 and c-Myc and one or more amino acid residues 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, and may be a variant in which the structural stability to the physical and chemical environment is increased or the physiological activity is increased. .

In addition, in the present invention, "Sox2 or c-Myc gene" is a nucleotide sequence encoding a Sox2 or c-Myc protein in the form of a wild type of Sox2 or c-Myc protein or a variant as described above, one or more bases are substituted, deleted Can be mutated by means of insertion, insertion, or a combination thereof, and can be isolated from nature or prepared using chemical synthesis.

The Sox2 and c-Myc genes are transcription factors that are very important in the production of induced pluripotent stem cells (iPSC), but the combination of Sox2 and c-Myc has not yet been used in the study of induced neural stem cell production through direct reprogramming. There is no successful case for the production of neural stem cells.

The nucleic acid molecule encoding the Sox2 protein and the nucleic acid molecule encoding the c-Myc protein of the present invention may each independently have a form contained in the expression vector.

Specifically, the Sox2 protein or c-Myc protein may be a protein of a form expressed from a cell line in vitro using an expression vector comprising a nucleic acid molecule encoding them. The expression vector may include a viral vector, a non-viral vector, a plasmid vector, a cosmid vector, and the expression vector may include a Baculovirus expression vector, a Mammalian expression vector, or a Bacterial expression vector. Mammalian cell lines or bacterial cell lines may be used, but this does not limit the expression vectors and cell lines available in the present invention.

The term “expression vector” of the present invention refers to a gene construct that is a vector capable of expressing a protein of interest in a suitable host cell and includes an essential regulatory element operably linked to express a gene insert.

The expression vector of the present invention includes a signal sequence or 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, an enhancer, and may be variously prepared according to the purpose. . The promoter of the expression 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. Expression vectors can be self-replicating or integrated into host DNA.

Specifically, the viral vector may be a retrovirus, a lentivirus, for example, HIV (Human immunodeficiency virus), MLV (Murineleukemia virus), ASLV (Avian sarcoma / Leukosis), SNV (Spleen necrosis virus), Derived from RSV (Rous sarcoma virus), MMTV (Mouse mammary tumor virus), Adenovirus, Adeno-associated virus, Herpes simplex virus, Sendai virus, etc. It can contain one vector. In addition, more specifically, it may be an RNA-based viral vector, but is not limited thereto.

The term "operably linked" of the present invention refers to the functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. Operative linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art and the like.

The nucleic acid molecule encoding the Sox2 or c-Myc protein may be messenger RNA (mRNA).

In the present invention, the "step of introducing Sox2 and c-Myc" refers to an expression vector, mRNA encoding Sox2 or c-Myc protein, genetic modification, foreign expression gene introduction, treatment of a substance having an expression inducing effect, etc. It may be a method of increasing the expression level of the Sox2 and c-Myc genes and proteins expressed therefrom, but is not limited so long as it increases the expression level of the genes and proteins. In particular, introducing the gene may be a method of inducing expression of the gene under a desired time and condition.

Specifically, the method of introducing the gene of step (a) into the cell can be used without limitation to provide nucleic acid molecules (DNA or RNA) to cells commonly used in the art. For example, a nucleic acid molecule encoding Sox2 and / or c-Myc protein can be operably linked in an expression vector and transferred into the cell, and can be delivered into the cell in the form of being inserted into the chromosome of the host cell. In addition, the method of administering the Sox2 and / or c-Myc to the culture of the isolated cells or the method of direct injection into the isolated cells, the method of directly injecting mRNA encoding the Sox2 or c-Myc protein into the isolated cells, etc. May be used, but is not limited thereto.

The method of directly injecting the Sox2 and / or c-Myc into the isolated cells can be used by selecting any method known in the art, but is not limited thereto, microinjection (microinjection), electroporation (electroporation) ), Particle bombardment, direct muscle injection, insulator, and transposon method may be appropriately selected and applied.

In the present invention, a method of introducing Sox2 and / or c-Myc into an isolated cell may be by using a virus, and specifically, a virus in which a nucleic acid molecule encoding Sox2 protein and a nucleic acid molecule encoding c-Myc protein are respectively inserted. The virus obtained from the packaging cells transformed with the vector may be introduced into Sox2 and / or c-Myc through a process of treating the culture medium of the isolated cells, but is not limited thereto.

The viral vector may be a vector derived from a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes simplex virus, Sendai virus, and the like, but is not limited thereto, and specifically, a retroviral vector may be used. . In addition, the packaging cells can be used to select a variety of cells known in the art depending on the viral vector used.

In the present invention, the method of introducing the virus may be a method of treating with a certain level of MOI.

The term “MOI” in the present invention is an abbreviation of “Multiplicity of infection” which means “multiplicity of infection”. For example, if 1,000,000 vectors were used to transduce 100,000 host cells, the MOI is 10. When using a viral vector, MOI represents the ratio of the inoculated virus amount to the number of cells inoculated with the virus, ie, the average number of virus particles infecting a single cell. The use of this term is not limited to cases involving transduction, but instead includes the introduction of the vector into the host by means such as lipofection, microinjection, calcium phosphate precipitation, and electroporation.

In the present invention, the MOI value used to introduce Sox2 and c-Myc is greater than 0 to less than 20, greater than 0 to less than 15, greater than 0 to less than 10, greater than 0 to less than 5, greater than 0.5 to less than 10, greater than 0.5 and less than 5 , More than 0.5, less than 4, more than 0.5, less than 3, or more than 0.5, less than 2, MOI, specifically 1 MOI, but is not limited so long as it is within the range causing direct cross-differentiation into neural stem cells.

In one embodiment of the present invention, it was confirmed that neural stem cells were prepared by treating the fibroblasts with c-Myc and Sox-2-encoding retroviral cells with MOI 1 (Example 1).

In addition, in the present invention, the method of introducing Sox2 and / or c-Myc into an isolated cell may be by using a non-viral vector, and specifically, a non-viral epi containing a nucleic acid molecule of Sox2 and / or c-Myc. The som vector may be introduced into isolated cells.

The non-viral episomal vector of the present invention is a non-viral non-insertable vector and is known to have a characteristic of expressing a gene included in the vector without being inserted into a chromosome. For the purposes of the present invention, cells comprising episomal vectors include both cases where the episomal vector is inserted into the genome or is present in the cell without being inserted into the genome.

In addition, in the present invention, the method of introducing Sox2 and / or c-Myc into isolated cells may be to directly introduce mRNA of Sox2 or c-Myc into isolated cells. The method of directly introducing the mRNA into an isolated cell can be used by selecting any method known in the art, but is not limited thereto.

Next, step (b) of the present invention is a method of producing neural stem cells by inducing direct reprogramming from cells isolated from cells separated from cells by culturing the cells of step (a).

As used herein, the term “lineage-converted cell” refers to a cell in which a unique lineage characteristic of the cell has been changed into a cell type having a different lineage characteristic by changing genetically or artificially. To convert to a cell having a characteristic of a cell type that is completely different from the characteristic. Specifically, the present invention may be neural stem cells converted from non-neuronal cells through reprogramming.

In the present invention, the term "reprogramming" refers to a method of converting a cell into a desired cell by controlling a global gene expression pattern of a specific cell. In other words, in the present invention, reprogramming refers to a method of artificially manipulating the fate of a cell and converting it into a cell having completely different characteristics. For the purpose of the present invention, the reprogramming is a vector containing a foreign gene or RNA or DNA. May be performed by introducing into a cell. In one example, reprogramming may include, but is not limited to, dedifferentiation of cells, direct reprogramming or direct conversion, or direct cross-differentiation.

As used herein, the term "reprogramming factor" refers to a gene (or polynucleotide encoding it), or a protein that can be finally or partially introduced into differentiated cells to induce reprogramming. The reprogramming factor may vary depending on the cell of interest to which reprogramming is intended, and on the type of isolated cell from which reprogramming is induced. For example, in one embodiment of the present invention, by introducing only Sox2 and c-Myc, the initial cells were reprogrammed into induced neuronal stem cells, which are target cells, but the scope of the present invention is not limited to the reprogramming factor, and thus, neural stem cells are prepared. Reprogramming factors introduced when Sox2, c-Myc. It may include one or more factors selected from the group consisting of Lin28, Asc11, Pitx3, Nurr1, Lmx1a, Nanog, Oct3, Oct4 and Klf4. In addition, it may include factors that induce overexpression of Sox21 and induce overexpression of the neuroectodermal lineage genes Tapa1, Atbf1, NeuroD1, Mash1, Hes1, Hes6, Id2, and can produce neural stem cells. All known factors may be included. In addition, the reprogramming factor can be used to induce direct reprogramming into neural stem cells. Reprogramming using reprogramming genes regulates the entire gene expression pattern of the cells and induces the conversion to the cells of interest. Therefore, the reprogramming genes are introduced into cells and cultured for a certain period of time, The initial cell can be reprogrammed with the target cell having the gene expression pattern of the cell.

The "direct reprogramming" of the present invention is differentiated from the technology of producing induced pluripotent stem cells having pluripotency through the reprogramming process, and is a technology of directly inducing direct conversion to a desired target cell through reprogramming culture. . Existing somatic cell nuclear transfer has the disadvantage of using an egg, which is less useful than other cell reprogramming techniques, and when induced pluripotent stem cell reprogramming technology is inherently passed through pluripotent stem cells, There is a disadvantage that it needs to be verified whether or not it remains and ensures safety. However, the present invention is expected to provide an alternative that can overcome the problems of the above technology, such as production time, cost, efficiency and safety by directly producing neural stem cells, which are target cells, through direct reprogramming. . For the purposes of the present invention, direct reprogramming may be mixed with direct dedifferentiation, direct differentiation, direct conversion, direct cross-differentiation, cross-differentiation and the like. In the present invention, direct reprogramming may refer to direct reverse differentiation or cross differentiation into neural stem cells.

The "induced neural stem cell (iNSC)" of the present invention is similar to neural stem cells using reprogramming from differentiated cells existing in different aspects, such as non-differentiating cells or cells with partial differentiation ability. It includes cells made in such a way as to establish undifferentiated stem cells with the same multipotency. Induced neural stem cells have the same or similar characteristics as neural stem cells, specifically, show similar cell morphology, gene and protein expression patterns are similar, and may have multipotent ability in and out of the body. Therefore, the induced neural stem cells of the present invention may be capable of differentiating into various neurons such as neurons (nerve cells), astroglia (astrocytes), oligodendrocytes, GABA-like neurons or dopaminergic neurons. The term "nerve stem cell" of the present invention has the same meaning as "derived neural stem cell" unless otherwise indicated, and is used interchangeably.

In the embodiment of the present invention, it was confirmed that neural stem cells prepared according to the method of the present invention can differentiate into neurons, astrocytes and oligodendrocytes (Example 1). In addition, safety was confirmed through the mutation of the p53 gene did not occur in the prepared neural stem cells (Example 3).

Another aspect of the present invention provides a neural stem cell prepared according to the above method. The neural stem cells are as described above. Neural stem cells prepared in the present invention may be differentiated into one or more neurons selected from the group consisting of neurons, astrocytes and oligodendrocytes, but is not limited thereto.

Another aspect of the present invention provides a cell therapeutic agent comprising the neural stem cells prepared according to the above method as an active ingredient. The neural stem cells are as described above.

As used herein, the term "cell therapeutic agent" refers to a medicine (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared through isolation, culture, and special manipulation from an individual. Means a medicine used for the purpose of treatment, diagnosis and prevention through a series of actions, such as proliferating and screening the living autologous, allogeneic, or heterologous cells in vitro or by altering the biological characteristics of the cells in order to restore.

The cell therapy agent of the present invention may include 1.0 × 10 to 1.0 × 10 ⁹ cells per ml, but is not limited thereto. The cell therapy of the invention can be used unfrozen or frozen for future use. If frozen, standard cryopreservatives (eg DMSO, glycerol, Epilife cell freezing medium (Cascade Biologics)) can be added to the cell population before freezing. In addition, the cell therapeutic agent may be administered in a unit dosage form suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field, and the agent may be administered in an effective dosage by one or several administrations. Include. Suitable formulations for this purpose can be used as parenteral formulations, injections such as ampoules for injection, injections such as infusion bags, sprays such as aerosol formulations and the like. The injection ampoule may be mixed with the injection solution immediately before use, and physiological saline, glucose, mannitol, and Ringer's solution may be used as the injection solution. The infusion bag may also be made of polyvinyl chloride or polyethylene and may be Baxter, Becton ickinson, Medcep, National Hospital Products or Terumo. The injection bag of the yarn can be illustrated.

The cell therapy product includes one or more pharmaceutically acceptable conventional inert carriers such as preservatives, analgesics, solubilizers or stabilizers in the case of injections, and in the case of formulations for topical administration , Excipients, lubricants or preservatives.

The cell therapeutic agent of the present invention thus prepared may be administered with other stem cells used for transplantation and other uses or in the form of a mixture with such stem cells using administration methods commonly used in the art. It is possible, but not limited to, to engraft or implant directly at the disease site of the patient in need or to implant or inject directly into the abdominal cavity. In addition, the administration can be both non-surgical administration using a catheter and surgical administration methods such as injection or transplantation after dissection of the disease site. In addition to parenteral administration, for example, in addition to direct lesions, transplantation by intravascular injection, which is a general method of hematopoietic stem cell transplantation, is also possible according to a conventional method.

The cell therapy agent may be administered once or in divided doses. However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender, and therefore, the dosage may be It does not limit the scope of the present invention in terms of aspects.

In the present invention, "prophylaxis" includes all actions of inhibiting or delaying the onset of neuronal damage disease by administration of the composition.

In the present invention, "treatment" includes all actions that improve or benefit from the symptoms of neuronal cell damage by administration of the composition.

Another aspect of the invention provides a pharmaceutical composition for preventing or treating neuronal cell damage disease, comprising the neural stem cells prepared according to the above method as an active ingredient. The neural stem cells are as described above.

In the present invention, "neuronal cell damage disease" is a disease caused by neuronal transformation, loss, and the like, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease (Huntington's disease), muscular dystrophy ( amyotriophiclateral sclerosis, ischemic brain disease, stroke, demyelinating disease, multiple sclerosis, epilepsy, neurodegenerative disease, spinal cord injury, etc., but are not limited to the above examples.

The composition may comprise a pharmaceutically acceptable carrier.

The "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not interfere with the biological activity and properties of the compound to be injected without stimulating the organism. The kind of the carrier usable in the present invention is not particularly limited, and any carrier can be used as long as it is a conventionally used and pharmaceutically acceptable carrier in the art. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more thereof.

The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.

Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose in the compound. , Gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups.In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In another aspect, the present invention provides a method for preventing or treating a neuronal cell damage disease, comprising administering the pharmaceutical composition for preventing or treating the neuronal cell disease. The neural stem cells are as described above.

The composition may be administered in a pharmaceutically effective amount.

The "pharmaceutically effective amount" means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of subject and its severity, age, sex, type of virus infected, drug Activity, sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.

The administration means introducing the composition of the present invention to the patient in any suitable way, and the route of administration of the composition can be administered via any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, but is not limited thereto.

The composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into two or three times. The frequency of administration in the case where the two active ingredients are single drugs may be the same or different times. In addition, the compositions of the present invention can be used alone or in combination with other drug treatments for the prevention or treatment of neuronal cell damage diseases. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

The subject includes all humans, including monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs, who may or may have developed neuronal cell disease. Means. If the pharmaceutical composition of the present invention can be effectively prevented or treated by administering to the subject, any kind of subject is included without limitation.

In a specific embodiment of the present invention, it was confirmed that the neural stem cells produced by direct reprogramming can be differentiated into various kinds of neurons, the neural stem cells of the present invention can be used as a therapeutic agent for various neuronal cell damage diseases have.

In another aspect of the present invention, by treating the neural stem cells or neural cells differentiated therefrom prepared according to the method of the candidate material, identifying a therapeutic agent for neurological diseases tailored to the individual from which the neural stem cells are derived. To provide a method for screening a therapeutic agent for personalized neurological diseases.

Specifically, in order to select a personalized neurological disease treatment agent according to the constitution or environment of an individual patient, by treating a candidate substance to a neural stem cell or a differentiated neuronal cell prepared by the method of the present invention, By confirming the change in the mechanism and the expression change of the protein and the like, it is possible to confirm and verify whether the isolated cells used to generate the neural stem cells can be a customized neurological disease treatment agent for the individual from which the stem cells are derived. .

In the method of producing induced neuronal stem cells of the present invention, since induced neural stem cells can be produced from non-neuronal cells using only two inducers of Sox2 and c-Myc as inducers, there are four factors and five inducers. Induced neural stem cells can be produced more efficiently than the production method using the induced neural stem cells prepared by the method is excellent in the differentiation and proliferative capacity, it can be used for future therapeutic purposes.

Figure 1 shows a retroviral vector for the overexpression of hc-Myc and hSox-2 and its manufacturing process. FIG. 1A is a schematic diagram of a structure in which hc-Myc and hSox2 are introduced into a pMX vector. 1B to 1D are diagrams illustrating retroviral vector production and titer adjustment process for introducing hSox2 and hc-Myc into human fibroblasts (hDF).
Figure 2 shows the results of treatment of hSox2 and hc-Myc in human fibroblasts (hDF). FIG. 2A shows the number of transduced cells over time after treatment with hSox2 retrovirus with MOI 1 and hc-Myc retrovirus with MOI 1, 5, and 10, respectively. As compared with the case where the MOI is 5 or 10, it can be seen that the number of cells increased significantly when the MOI was 1. FIG. 2B shows the morphology of hDF when hDF treated with 1 of MOS of hSox2 and hc-Myc retroviral vectors. As a result, it was confirmed that reprogramming occurred in a form similar to induced nerve stem cells (iNSC). Figure 2C is the result of culturing iNSC (hDF-iNSC) prepared by treating hSox2 and hc-Myc with MOI 1 in hDF. It can be seen that the culture is possible in both the attached culture (attached culture), or the suspension culture (suspension culture). 2D shows the result of thawing again after freezing hDF-iNSC. It is confirmed that iNSC maintains its specific morphology and proliferative capacity even after freezing and thawing. Figure 2E shows the results of genetic fingerprint analysis of hDF-iNSC. As a result of genetic fingerprint analysis, it can be confirmed that the manufactured iNSC is derived from the hDF used for the production. Figure 2F is the result of analyzing the proportion of cells positive for CD133, a marker of neural stem cells. As a result of comparative analysis of direct cross-differentiation efficiency, it can be seen that there is a difference of 0.2-0.5% for each batch of hDF.
3 is a measure of the expression level of p53 according to the change in the MOI value of hSox2 and hc-Myc. 3A is a graph showing the transcription level of p53. In the case of MOI 1, the expression level of p53 was not significantly increased compared to the control group, whereas in the case of MOI 5 and MOI 10, the expression levels were significantly increased. 3B is an experimental result of measuring the expression level of p53 protein, indicating that p53 protein expression is increased in proportion to MOI.
4 confirms that the manufactured iNSC does not have pluripotency. Figure 4A is a graph confirming the amount of Oct4 expression of hDF-iNSC. It can be seen that the transcription and expression levels of Oct4 are lower than that of human induced pluripotent stem cells (hiPSC). (P <0.01) By confirming the low expression level of Oct4, a representative pluripotency marker, it can be seen that direct cross-differentiation occurred without going through a step having pluripotency. 4B is a result of culturing cells in which genes were introduced in the presence of Oct4 (O), Sox-2 (S), c-Myc (M), and Klf-4 (K), which are factors used in iPSC preparation. When one of the OSMKs was excluded from the experimental conditions of the team, alkaline phosphatase positive colony was not formed at all even if the cells into which the gene was introduced were cultured under favorable conditions for iPSC reprogramming. When the OMK gene, except for hSox-2, was introduced into the somatic cells, many colonies were formed, but all were alkaline phosphatase negative colony.
Figure 5 confirms the neural stem cell characteristics, proliferative capacity and differentiation capacity of the prepared hDF-iNSC. 5A and 5B confirm that the neuronal stem cell specific markers Sox2, Nestin and Pax6 are overexpressed at transcription level (5A) and protein level (5B). This confirms that hDF-iNSC is reprogrammed into neural stem cells. 5C shows the doubling time of the prepared hDF-iNSC. The prepared hDF-iNSC shows a doubling time of about 21.3 h, it can be seen that the self-proliferation. Figure 5D is a measure of the amount of MAP2, GFAP and Olig1 expression of the prepared hDF-iNSC. MAP2 is a neurite, GFAP is a central nervous system (CNS) cell including astrocytes and Ependymal cells, and Olig1 is a gene involved in the formation of oligodendrocytes. )can confirm.
Figure 6 shows the results of treatment of Sox2 and c-Myc with MOI 1 in human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC). Figure 6A confirms the marker pattern of hUCB-MSC used in the experiment. It can be confirmed that hUCB-MSC has the properties of mesoderm stem cells. FIG. 6B shows the morphology of hUCB-MSC when hSox2 and hc-Myc were treated with MOI 1 in hUCB-MSC. hUCB-MSC is similar to iNSC and reprogramming can be seen. Figure 6C confirms the direct cross-differentiation efficiency of the produced hUCBMSC-iNSC. Direct cross-differentiation can be seen with an efficiency of 1.0-2.4%. FIG. 6D confirms the expression of the neural stem cell markers Sox2, Nestin, and Pax6 in the manufactured hUCBMSC-iNSC. As shown in FIG. 6E shows that hUCBMSC-iNSC was differentiated into neuronal forms. This can confirm the differentiation capacity of hUCBMSC-iNSC.
Figure 7 shows the results of treatment of Sox2 and c-Myc with MOI 1 in human Niemann-Pick Type C disease derived dermal fibroblasts (hNPCDF). Figure 7A shows the location of the genetic variation of the hNPCDF used in the experiment compared to normal hDF. FIG. 7B shows that hSox2 and hc-Myc were treated with MOI 1 in hNPCDF to show a similar form to iNSC. This confirms that reprogramming to neural stem cells occurs. Figure 7C shows the colony formation rate of hNPCDF-iNSC, it can be seen that the direct cross-differentiation efficiency is about 1.3%. 7D and 7E confirm the expression levels of the neuron-specific markers Nestin and Pax6 (FIG. 7D) and the fibroblast markers COL1A2 and S100A4 (FIG. 7E) in hNPCDF-iNSC. hNPCDF-iNSC, like H9NSC, which is a neural stem cell, had a high expression level of a neuronal marker and a low expression level of a fibroblast marker. Figure 7F confirms the differentiation capacity of the prepared hNPCDF-iNSC. Tuj 1 (Alexa 594) is an antibody that specifically binds to nerves, indicating that the prepared iNPCDF-iNSC differentiated into neurons. In addition, it is possible to confirm the differentiation ability of the prepared iNPCDF-iNSC through the expression of Olig2 involved in neuronal differentiation.
Figure 8 shows the experimental results verifying the genetic stability of the prepared hDF-iNSC. 8A shows the result of analyzing karyotype of the prepared hDF-iNSC. In the same form as the general karyotype, it can be seen that the prepared hDF-iNSC is genetically stable from a macroscopic point of view. Figure 8B is the result of PCR amplification of p53 genomic DNA in the prepared hDF-iNSC. It shows the same degree of amplification as normal hDF, and it can be confirmed that the number of p53 alleles is normal. 8C and 8D show the results of sequencing sites where mutations of the p53 gene occur frequently and comparing them with normal cells (hDF). This confirms that p53 mutation did not occur in hDF-iNSC.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes only and the scope of the present invention is not limited to these Examples and Experimental Examples.

Example 1 Preparation of iNSC from Sox2, c-Myc Overexpression Control from Human Fibroblasts (hDF)

Example 1-1 Preparation of Sox2, c-Myc Retrovirus Vector

Retroviruses for overexpressing hc-Myc and hSox-2 in somatic cells were prepared.

First, pMXs-hc-MYC and pMXs-hSOX2 were prepared by introducing hcMyc and hSOX-2 into a pMX retrovirus vector, a retroviral expression vector, respectively (FIG. 1A).

Then, hcMyc or hSOX-2 retroviruses for the introduction of hcMyc or hSOX-2 were prepared using the pMXs-hc-MYC and pMXs-hSOX2 vectors prepared above. Specifically, 24 ul Convoy, pMXs-hc-Myc or pMXs-hSox-2 4 ug, VSV-G 2 ug and Gag-Pol 2 ug were mixed in phosphate buffered saline (PBS) and allowed to stand at room temperature for 10 minutes. . The mixture was then added to 2 × 10 ⁶ 293FT cells attached to a 100 mm cell culture dish and transfected for one day at 37 ° C. 5% CO ₂ conditions. In addition, the pMXs-GFP vector was transfected under the same conditions for titration. Virus soup generated after transfection was collected at 24, 48 and 72 hours after transfection, respectively, and stored at 4 ° C.

After filtering the virus soup to 0.45 um for the concentration of the virus, Retro-X concentrator (Clontech) was added to 1/3 of the virus soup and incubated at 4 ℃ for one day. After 24 hours the mixture was centrifuged at 4,000 rpm at 4 ° C. for 60 minutes. After centrifugation, the soup was discarded, the retroviral pellet was suspended in PBS, aliquoted, and stored at -80 ° C until use.

For titration of the virus, GFP retroviruses enriched in 5 × 10 ⁵ 293FT cells seeded in 6-well plates were transduced (overnight transduction) at 50, 5, 0.5 and 0 ul, respectively. Two days later, the percentage of GFP expressing cells in the cells was analyzed using a flow cytometer. Titers were calculated by introducing them into the formula shown in FIG. 1D using results within 1-20% (FIGS. 1B-D).

Example 1-2: Confirmation of the production of induced neural stem cells according to the difference in the MOI value of Sox2 and c-Myc retrovirus

By Sox2 and c-Myc retroviruses prepared in Example 1-1 it was confirmed whether the induced neural stem cells can be prepared using human fibroblasts.

First, the hSOX-2 introduction retrovirus prepared in Example 1-1 was treated with MOI 1, and the hcMyc introduction retroviruses were prepared with MOI 1, 5 and 10, respectively, in order to prepare induced nerve stem cells (iNSC). Treatment with blasts induced direct reprogramming.

Specifically, 1.25 × 10 ⁴ human fibroblasts were plated on 24-well tissue culture plates the day before retroviral transduction. The next day, Sox2 and c-Myc retroviruses prepared in Example 1-1 were added to the cells with a defined MOI and spinfected at 800xg for 60 minutes at 20 ° C. After spinfection, the cells were cultured in DMEM / F12 containing 20% (v / v) FBS (fetal bovine serum) and 1X primocin.

As a result of comparing the number of cells on the second day, the number of cells when the retrovirus for introducing hcMyc was treated with MOI 1 was significantly increased compared to MOI 5 or 10 (P <0.01) (FIG. 2A).

In addition, 5 × 10 ³ cells were seeded in poly-L-ornithine / Fibronectin-coated 6-well plates on day 3 after Sox2 and c-Myc retroviruses were transformed as described above. After seeding, the cells were attached to the plate on day 5 and then replaced with iNSC medium (1x supplement, 1X primocin, 20 ng / ml bFGF, Stempro NSC medium with 20 ng / ml EGF). Medium exchange was performed every 2 days. On 14-21 days after starting the culture in the iNSC medium, the iNSC colonies were mechanically picked to continue the culture on the poly L-ornithine / Fibronectin coated plate. INSC in culture in the attached state for neurosphere culture was detached using accutase and suspended in Petri dish to form neurosphere.

As a result of the experiment, when the retrovirus for the introduction of hcMyc retrovirus treatment with MOI 1 showed similar morphology to induced neuronal stem cells and directly cross-divided (Fig. 2B), attached culture (attached culture), culture medium In suspension culture (suspension culture) it was confirmed that the culture is possible in all (Fig. 2C), even after slowly freezing using iNSC medium added with 5% DMSO and thawed slowly in a 37 ℃ water bath induced nerve cord It was confirmed that the stromal cells maintained their specific morphology and proliferative capacity (FIG. 2D). However, when the MOI was treated with 5 or more, similar morphology to induced neural stem cells was not observed (FIG. 2B).

Genetic fingerprinting was performed to confirm whether the prepared hDF-iNSC was derived from the used hDF. As a result, it was confirmed that the parental hDF origin was used (FIG. 2E).

In addition, direct cross-differentiation efficiency was analyzed by the proportion of cells positive for CD133, an NSC marker. Specifically, 1 × 10 ⁵ to 1 × 10 ⁶ cells were suspended in 100 ul of PBS. Then, after mixing 3 ul of antibody with Fluorescein isothiocyanate (FITC) bound to the cells, binding was induced for 30 minutes at room temperature in a light-blocked space. The antibody used is anti-CD133 / 1-VioBright / FITC (Miltenyi Biotec). After washing with 3 ml of PBS, the cells were subjected to flow cytometry using MACSQuant VYB (Miltenyi Biotec). As a result, the difference was 0.2-0.5% for each batch of hDF (FIG. 2F).

In addition, as the MOI of Sox2 and c-Myc retroviruses treated with hDF increases, the following experiments were performed to confirm the expression of p53 transcription and p53 protein, which are major factors that interfere with reprogramming. .

Specifically, qRT-PCT was performed to confirm the transcriptional expression level of p53. To perform the qRT-PCT, mRNA was isolated from cell pellets using PureLink RNA Mini Kit (Invitrogen), and cDNA was synthesized from mRNA isolated using AccuPower RT Premix (Bioneer). The cDNA, PowerUp SYBR Green Master Mix (Appliedbiosystems), p53 forward and reverse primers (SEQ ID NOs: 19 and 20) and distilled water were mixed to make 20 ul, and the PCR reaction was performed using Quant Studio3 (Appliedbiosystems), p53 gene. The relative fold value of was calculated.

The primers used in the following examples as well as p53 are listed in Table 1 below.

In addition, Western blotting was performed to confirm the amount of p53 protein expression. In order to perform the experiment, Sox2 and c-Myc retroviruses were introduced into the cells, and on day 6, the cells were lysed with lysis buffer to separate proteins from the cells, and the process of denaturing the proteins by heating was performed. Sampled through. Thereafter, the prepared sample was subjected to electrophoresis on an SDS-PAGE gel (Bio-Rad, Hercules, CA, USA), and the protein of the gel was transferred to a polyvinylidene fluoride (Bio-Rad) membrane. The membrane was blocked using skim milk powder. The membrane was then treated with anti-p53 antibody (1: 1,000; Santacruz) at 4 ° C. for one day. After washing the next day, HRP conjugated secondary antibody (1: 1,000; Santacruz) was treated for 1 hour at room temperature, and after washing, the membrane was developed with ECL solution (iNTRON) to check the expression level of p53 protein.

As a result of the qRT-PCR and Western blot, it was confirmed that the transcription of p53 and the expression of p53 protein were significantly increased in proportion to MOI (P <0.05 and P <0.01) (FIGS. 3A and B).

Based on the above results, the higher MOI values of Sox2 and c-Myc retroviruses treated with human fibroblasts, the more difficult it is to induce reprogramming to induced neuronal stem cells, and when treated with less than MOI 5, hDF cells to induced neuronal stem cells We confirmed that we could reprogram it.

Example 1-3: Direct Cross-Differentiation Confirmation Without Steps Having Starch Capacity

As a result of confirming Oct4 expression level of hDF-iNSC through qRT-PCR experiment described in Example 1-2, it was confirmed that the transcription and expression level of Oct4 was lower than that of human induced pluripotent stem cells (P <0.01) (FIG. 4A) . By confirming the low expression level of Oct4, a representative pluripotency marker, it was confirmed that direct cross-differentiation occurred without the step having pluripotency.

In addition, the experimental conditions of the team to determine the omnipotence of any one of the reprogramming 4 factors (hOct-4, hSox-2, hc-Myc, and hKlf-4; OSMK) of the iPSC technology is The same experiment was performed.

Specifically, as in the retroviral transduction procedure described in Example 1-1, the reprogramming factor was introduced in four combinations except one of hOct-4, hSox-2, hc-Myc and hKlf-4. Thereafter, the cells were incubated in DMEM / F12 containing 20% (v / v) FBS for 3 days. On day 3, STO feeder cells were passaged to seeded tissue culture plates. Thereafter, the medium was changed daily with iPSC medium (DMEM / F12, 20% serum replacement, 1 × primocin, 4 ng / ml bFGF) until 5-21 days. Alkaline phosphatase staining was performed on day 21 and the reprogramming efficiency was quantified by counting the number of alkaline phosphatase positive colonies.

As a result, alkaline phosphatase-positive colonies were cultured under iPSC reprogramming conditions in cells into which a combination containing at least one of the reprogramming 4 factors (hOct-4, hSox-2, hc-Myc, and hKlf-4; OSMK) was introduced. Was not formed at all. Interestingly, when the OMK gene except for hSox-2 in OSMK was introduced into somatic cells, colony was formed in large numbers, but all were alkaline phosphatase negative colonies (FIG. 4B).

Example 1-4: Confirmation of multipotency of the prepared induced stem cells

It was confirmed whether hDF-iNSC prepared by the method of the present invention has neural stem cell characteristics, and the proliferative and differentiating ability of the cells was confirmed.

First, in order to confirm whether the hDF-iNSC has neural stem cell characteristics, expression patterns of neural stem cell specific markers were confirmed.

Specifically, through the qRT-PCR experiment described in Example 1-2, the hDF-iNSC prepared in Example 1-2 overexpressed the neural stem cell specific markers endogenous Sox2, Nestin, Pax6 at the transcription level It was confirmed (FIG. 5A).

In addition, the immunostaining method was confirmed that neural stem cell specific markers are overexpressed at the protein level. The immunostaining method was performed by the following procedure. First, anti-Nestin (Abcam) and anti-PAX6 (BioLegent) primary antibodies (1: 100, respectively) for the purpose of experiments on the hDF-iNSC, which had been fixed, permeabilized, and blocked. Was treated at 4 ° C. for one day. The next day, after washing with PBS, Alex Fluor 488 goat anti-mouse (Invitrogen), Alex Fluor 594 goat anti-mouse (Invitrogen), Alex Fluor 594 goat anti-rabbit (Invitrogen) secondary antibody (1 each) : 1,000) was treated for 2 hours at room temperature. Subsequently, the extra secondary antibody was washed with PBS, removed, and the cell nuclei were stained with DAPI (4 ′, 6′-Diamidino-2-Phenylindole, Dihydrochloride). Observation of the stained cells using a Nikon ECLIPSE Ti-U microscope (Nikon). Excitation / emission wavelength was 358/461 nm for DAPI, 488/525 nm for Alex Fluor 488 and 594/617 nm for Alex Fluor 594. As a result of immunostaining, it was confirmed that Nestin and Pax6 were overexpressed at the protein level (FIG. 5B).

Through this, it was confirmed that the cells prepared in the present invention are reprogrammed into neural stem cells.

In addition, the proliferative and differentiating ability of the prepared hDF-iNSC was measured. Specifically, spontaneous differentiation of iNSC was induced by culturing for 3 weeks in bFGF / EGF-free iNSC medium. Medium exchange was performed every 2-3 days, the culture was performed at 37 ℃ 5% CO ₂ conditions. The differentiated cells were confirmed the transcription level of MAP2, GFAP and Olig1 by the qRT-PCR method described in Example 1-1. As a result, the prepared hDF-iNSC showed self-renewal with a doubling time of about 21.3 h, and neurons and glial cells (astroglia and oligodendrocytes) by spontaneous differentiation. It was confirmed that it has a multipotent ability to differentiate into (Fig. 5C and D).

Example 2: Preparation of iNSCs from Various Somatic Cells

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) to confirm whether the somatic-derived induced neural stem cell manufacturing method identified in Example 1 can be generally applied to various somatic cells in addition to human fibroblasts. ) And human Niemann-Pick Type C disease derived dermal fibroblasts (hNPCDF).

Example 2-1 Preparation of iNSC Using hUCB-MSC

First, to identify typical MSC specific marker expression patterns, anti-HLA-ABC (BD), anti-HLA-DR (BD), anti-CD34 (BD), anti-CD45 (BD), anti-CD73 ( BD), flow cytometry performed in Examples 1-2 using anti-CD105 (BD) antibodies. As a result, hUCB-MSC showed a typical MSC specific marker expression pattern with HLA-ABC +, HLA-DR-, CD34-, CD45-, CD73 +, CD105 + (FIG. 6A), through which the hUCB-MSC used in the experiment It was confirmed that the mesenchymal stem cells have the characteristics.

When the retrovirus was treated with MOI 1 as in Example 1 to introduce hc-Myc and hSox-2 into the cells, it was confirmed that iNSC-specific attached colony morphology was shown (FIG. 6B). Flow cytometry described in Examples 1-2 using anti-CD133 / 1-VioBright / FITC (Miltenyi Biotec) antibody showed direct crossover at an efficiency of about 1.0-2.4% in each of the two types of hUCB-MSCs. It was confirmed that differentiation occurs (FIG. 6C).

In addition, by performing the qRT-PCR experiment described in Example 1-1, it was confirmed that the hUCB-MSC-iNSC prepared above expressed NSC-specific markers endogenous Sox2, Nestin, Pax6, and differentiated into neuron-like forms. Bars (FIGS. 6D and E), the cells were reprogrammed directly into induced nerve stem cells, it can be seen that there is a differentiation ability to neurons.

Through this, it can be seen that the induced neural stem cell manufacturing method of Example 1 is also applied to hUCB-MSC.

Example 2-2: iNSC Preparation Using hNPCDF

HNPCDF, which was the subject of experiment, had a point mutation in the NPC1 gene, and isoleucine of the NPC1 protein was replaced with threonine (FIG. 7A). That is, somatic cells in which mutations exist in a gene of human fibroblasts.

As a result of treating retroviruses with MOI 1 as in Example 1 for introducing hc-Myc and hSox-2 into the cells, the hNPCDFs were directly cross-differentiated with an efficiency of about 1.3% to give a morphology similar to that of neural stem cells ( 7B and C). In addition, the qRT-PCR experiments described in Examples 1-2 revealed that hNPCDF-iNSC expressed NSC-specific markers Nestin and Pax6, whereas the fibroblast markers COL1A2 and S100A4 did not express (Fig. 7D and E), which confirmed that hNPCDF can also be reprogrammed to neural stem cells and its efficiency.

In addition, the prepared hNPCDF-iNSC was differentiated through an immunostaining method (described in Examples 1-3) using anti-Olig2 (Millipore) and anti-Tuj 1 (Abcam) primary antibodies (1: 100, respectively). As a result, it was confirmed that the neuron and glia (oligodendrocyte) differentiation (Fig. 7F), the hNPCDF-iNSC also has a differentiation capacity.

Example 3: verification of the stability of the prepared induced stem cells

In order to verify the stability of the induced nerve stem cells prepared in the present invention, the following experiment was performed.

Specifically, as a result of confirming the karyotype of hDF-iNSC prepared in the present invention, it was confirmed that the general karyotype (46, XX) was shown, through which the induced neuronal stem cells produced in the present invention is genetically stable from a macroscopic perspective Confirmation (FIG. 8A).

In addition, the following experiments were performed to verify whether mutations occurred in p53, which is known to be the most important to prevent cancer cellization.

Specifically, gDNA was isolated from hDF-iNSC using AccuPrep Genomic DNA Extraction Kit (Bioneer). PCR amplification was carried out using the isolated gDNA as a template using AccuPower PCR Premix (Bioneer), p53 forward and reverse primers (SEQ ID NOs: 19 and 20) shown in Table 1, and Genetouch Thermal Cycler (Hanzhou bioer technology). After the PCR was confirmed whether the gene amplification by gel electrophoresis (Mupid) (Fig. 8B), the PCR product was purified using a MEGAquick-spin plus fragment DNA purification kit (iNtRON). The purified PCR product was sequencing in macrogen (Seoul, Korea) with the forward primer (SEQ ID NO: 19) used for PCR (Fig. 8C).

As a result, no mutation was observed in all six mutation hotspots where the mutation of the p53 gene occurred (FIG. 8C).

 In addition, in order to completely exclude the possibility that the p53 gene was mutated even in a few cells, the following experiment was performed.

Specifically, for in-depth genotyping, PCR products were cloned using TOPcloner TA Kit (Enzynomics), and transformed into DH5a chemically competent E. coli (Enzynomics) using a heat shock method. The transformed E. coli was plated on LB Agar LOP plate (Narae biotech) and incubated at 37 ° C. for one day. The next day 10 colonies were incubated for one day at 37 ° C. in LB broth (Gibco). Thereafter, plasmids were isolated from E. coli grown using AccuPrep Plasmid Mini Extraction Kit (Bioneer), and sequencing was performed in macrogen (Seoul, Korea). As a result, no mutation of the p53 gene was observed in all 10 individually sequenced sequences (FIG. 8D).

Through the above results, it was confirmed that the induced neural stem cells produced by the method of the present invention is genetically stable.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

<110> Kangstem Biotech CO., LTD. <120> A method for producing directly reprogrammed induced neural stem          cells from non-neuronal cells using Sox2 and c-Myc <130> KPA180273-KR <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COL1A2_F <400> 1 acaaggcatt cgtggcgata 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COL1A2_R <400> 2 accatggtga ccagcgatac 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Endogeneous Sox2_F <400> 3 gagagtgttt gcaaaagggg g 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Endogeneous Sox2_R <400> 4 cgccgccgat gattgttatt a 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GADPH_F <400> 5 gtcagtggtg gacctgacct 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GADPH_R <400> 6 tgctgtagcc aaattcgttg 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GFAP_F <400> 7 gctcaatgac cgctttgcca g 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GFAP_R <400> 8 cctgtgccag attgtccctc t 21 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MAP2_F <400> 9 ggcagatgaa cggaaagatg aagc 24 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> MAP2_R <400> 10 gtatttgaat aggtgccctg tgacc 25 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nestin_F <400> 11 gcatgtgaat gggggagtga 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nestin_R <400> 12 tctccctcag agactagcgg 20 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NPC1_F <400> 13 atgagattcc tgcccatgtt cc 22 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NPC1_R <400> 14 gacacaccga ggttgaagat agtg 24 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oct-4_F <400> 15 gaaggatgtg gtccgagtgt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oct-4_R <400> 16 gtgaagtgag ggctcccata 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Olig1_F <400> 17 tcctccacct cctccacttc 20 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Olig1_R <400> 18 ctatcttgga gagcttgcgg c 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> p53_F <400> 19 tctgtgactt gcacgtactc c 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> p53_R <400> 20 gagaggagct ggtgttgttg g 21 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pax6_F <400> 21 cagaghagac aggccagcaa 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pax6_R <400> 22 tggttggtag acactggtgc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S100A4_F <400> 23 gggcaaagag ggtgacaagt 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S100A4_R <400> 24 cctgttgctg tccaagttgc 20

Claims (12)

(a) introducing Sox2 and c-Myc into the isolated cells; And
(b) culturing the cells of step (a) to induce direct reprogramming from the isolated cells to the cells whose lines are switched
Containing, neural stem cell manufacturing method. The method of claim 1, wherein the method of introducing Sox2 and c-Myc of step (a) is to use a virus. The method of claim 2, wherein in step (a), the virus is treated with a MOI greater than 0 to less than 5, 4. The method of claim 1, wherein the method of introducing Sox2 and c-Myc of step (a) uses a non-viral vector. The method of claim 1, wherein the method of introducing Sox2 and c-Myc of step (a) is to introduce mRNA of Sox2 or c-Myc directly into the isolated cells, neural stem cell manufacturing method. The method of claim 1, wherein the isolated cells are somatic cells other than neurons. The method of claim 1, wherein the isolated cells are adult stem cells other than neural stem cells. Neural stem cells prepared according to the method of any one of claims 1 to 7. The neural stem cell of claim 8, wherein the neural stem cell can be differentiated into one or more neurons selected from the group consisting of neurons, astrocytes and oligodendrocytes. Cell therapy comprising a neural stem cell prepared according to any one of claims 1 to 7 as an active ingredient. Claims 1 to 7, comprising a neural stem cell prepared according to any one of the methods as an active ingredient, neuronal cell damage disease treatment or prevention pharmaceutical composition. A method of identifying a therapeutic agent for neurological diseases tailored to an individual derived from the neural stem cells, by treating a candidate substance with the neural stem cells or the differentiated neural cells prepared according to the method of claim 1. A method for screening a personalized neurological disease therapeutic agent, comprising.

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