WO2019216667A9 - Procédé de génération de cellules souches neurales induites reprogrammées directement à partir de cellules non neurales en utilisant la sox2 et le c-myc - Google Patents

Procédé de génération de cellules souches neurales induites reprogrammées directement à partir de cellules non neurales en utilisant la sox2 et le c-myc Download PDF

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WO2019216667A9
WO2019216667A9 PCT/KR2019/005566 KR2019005566W WO2019216667A9 WO 2019216667 A9 WO2019216667 A9 WO 2019216667A9 KR 2019005566 W KR2019005566 W KR 2019005566W WO 2019216667 A9 WO2019216667 A9 WO 2019216667A9
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cells
neural stem
myc
stem cells
sox2
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WO2019216667A1 (fr
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강경선
서광원
권대기
지민준
한미정
안희진
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주식회사 강스템바이오텍
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a method for producing a neural stem cell derived from a non-nerve cell through direct reprogramming and a use thereof.
  • a fertilized egg cell is an example of an omnipotent stem cell.
  • Pluripotent stem cells produce any cell type in the body that derives from the three major germ layers or embryos themselves.
  • pluripotent stem cells such as embryonic stem cells (ESCs) proliferate rapidly while maintaining pluripotency, that is, the ability to differentiate into various cell types
  • the embryonic stem cells can be usefully used for cell transplantation treatment.
  • pluripotent stem cells have been mainly produced by nuclear transfer and cell fusion (Philos Trans R Soc Lond B Biol Sci. 363(1500): 2079-2087).
  • nuclear transfer and cell fusion Philos Trans R Soc Lond B Biol Sci. 363(1500): 2079-2087.
  • both of these methods use embryonic stem cells, an ethical dilemma is raised in both research and treatment purposes.
  • iPSCs induced pluripotent stem cells
  • ESCs embryonic stem cells
  • Induced pluripotent stem cells express defined factors from mouse fibroblasts (Cell 126: 663-676 (2006)) in 2006 and human fibroblasts (Science 318: 1917-1920 (2007), in 2007).
  • ESCs embryonic stem cells
  • Induced pluripotent stem cells express defined factors from mouse fibroblasts (Cell 126: 663-676 (2006)) in 2006 and human fibroblasts (Science 318: 1917-1920 (2007), in 2007).
  • Oct-3/4, Sox2, Klf4 and c-Myc to initiate reprogramming of mature somatic cells into iPSCs; Oct4, Sox2, Nanog and Lin28 were used.
  • iPSCs have a limitation in that they cannot be converted into desired cells in vivo because teratoma cancer occurs and at the same time, when transplanted into a living body, iPSC is not well controlled for differentiation.
  • This technology is a technology that directly induces specific cells without going through a pluripotent state by introducing a specific lineage-specific gene into completely differentiated cells, i.e., fibroblasts, thereby reducing the risk of teratoma of pluripotent cells. It is a technology that can be excluded.
  • the MOI of the virus containing Sox2 and c-Myc is precisely controlled to induce only the introduction of Sox2 and c-Myc. It was found that neural stem cells can be produced, and the present invention was completed by confirming the stability and differentiation ability of the prepared induced neural stem cells.
  • One object of the present invention is (a) introducing Sox2 and c-Myc into the isolated cells; And (b) culturing the cells of the step (a) to induce reprogramming directly from the isolated cells to cells whose lineage has been converted to provide a method for producing neural stem cells.
  • Another object of the present invention is to provide a neural stem cell prepared according to the above method.
  • Another object of the present invention is to provide a cell therapy product comprising, as an active ingredient, neural stem cells prepared according to the above method.
  • Another object of the present invention is to provide a pharmaceutical composition for treating or preventing nervous system diseases, comprising as an active ingredient neural stem cells prepared according to the above method.
  • Another object of the present invention comprises the step of identifying a neurological disease therapeutic agent tailored to the individual from which the neural stem cells are derived by treating a candidate substance on the neural stem cells prepared according to the above method or the neurons differentiated therefrom, It provides a screening method for personalized neurological disease treatment.
  • induced neural stem cells of the present invention since it is possible to produce induced neural stem cells from non-nerve cells by using only two inducers of Sox2 and c-Myc as inducing factors, the existing four factors, five inducers The induced neural stem cells can be produced more efficiently than the production method using, and the induced neural stem cells produced by the above method have excellent differentiation and proliferation ability, and thus can be used for future therapeutic purposes.
  • 1 shows a retroviral vector for overexpressing hc-Myc and hSox-2 and a process of its construction.
  • 1A is a schematic diagram of a structure in which hc-Myc and hSox2 are introduced into a pMX vector.
  • Figures 1b, 1c, and 1d are schematic diagrams of a retroviral vector construction and titer adjustment process for introducing hSox2 and hc-Myc into human fibroblasts (hDF).
  • Figure 2 shows the results of treatment with hSox2 and hc-Myc in human fibroblasts (hDF).
  • Figure 2a is a measure of the number of cells transduced over time after treating hDF with hSox2 retrovirus with MOI 1 and hc-Myc retrovirus with MOIs of 1, 5, and 10, respectively. Compared to the case where the MOI is 5 or 10, it can be seen that the number of cells is greatly increased when the MOI is 1.
  • Figure 2b shows the morphology of hDF when the MOIs of hSox2 and hc-Myc retroviral vectors were treated with 1 in hDF.
  • 2C is a result of culturing iNSC (hDF-iNSC) produced by treating hSox2 and hc-Myc with MOI 1 in hDF. It can be seen that cultivation is possible in either attached culture or suspension culture.
  • Figure 2d is a result of freezing hDF-iNSC and then thawing again. It can be seen that even after freezing and thawing, the iNSC's specific shape and proliferative capacity are maintained.
  • Figure 2e shows the results of the genetic fingerprint analysis of hDF-iNSC.
  • Figure 3 is a measure of the expression level of p53 according to the change of the MOI value of hSox2 and hc-Myc.
  • 3A is a graph showing the level of transcription of p53. In the case of MOI 1, the expression level of p53 did not increase significantly compared to the control group, whereas in the case of MOI 5 and MOI 10, the expression level significantly increased.
  • 3B is an experimental result of measuring the expression level of p53 protein, indicating that the amount of p53 protein expression increases in proportion to the MOI.
  • Figure 4 confirms that the prepared iNSC does not have pluripotency.
  • Figure 4a is a graph confirming the Oct4 expression level of hDF-iNSC. It can be seen that the amount of transcription and expression of Oct4 is lower than that of human induced pluripotent stem cells (hiPSC). (P ⁇ 0.01) By confirming the low expression level of Oct4, which is 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 the cells into which the gene was introduced in the presence of Oct4(O), Sox-2(S), c-Myc(M), and Klf-4(K), which are factors used in iPSC production.
  • Figure 5 shows the neural stem cell characteristics, proliferation and differentiation ability of the prepared hDF-iNSC.
  • 5A and 5B confirm that the neural stem cell-specific markers Sox2, Nestin, and Pax6 are overexpressed at the transcription level (5a) and protein level (5b). Through this, it can be confirmed that hDF-iNSC has been reprogrammed into neural stem cells.
  • Figure 5c is a measurement of the doubling time (doubling time) of the prepared hDF-iNSC. It can be seen that the prepared hDF-iNSC exhibits a doubling time of about 21.3 h and self-proliferates.
  • 5D is a measurement of the expression levels of MAP2, GFAP and Olig1 of the prepared hDF-iNSC.
  • MAP2 is a neurite
  • GFAP is a central nervous system (CNS) cell including astrocytes and ependymal cells
  • Olig1 is a gene involved in the formation of oligodendrocytes.
  • 6 shows the results of treatment with MOI 1 of Sox2 and c-Myc in human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC).
  • 6A shows the marker pattern of hUCB-MSC used in the experiment. It can be confirmed that hUCB-MSC has the characteristics of mesoderm stem cells.
  • 6B shows the morphology of hUCB-MSC when hUCB-MSC was treated with hSox2 and hc-Myc with MOI 1. It can be seen that hUCB-MSC has a similar form to iNSC and reprogramming takes place.
  • Figure 6c confirms the direct cross-differentiation efficiency of the produced hUCBMSC-iNSC.
  • 6D shows the expression of the neural stem cell markers Sox2, Nestin, and Pax6 in the hUCBMSC-iNSC, and it can be seen that reprogramming has occurred with neural stem cells.
  • 6E is a result of observing that hUCBMSC-iNSC is differentiated into a neuron form. Through this, the differentiation ability of hUCBMSC-iNSC can be confirmed.
  • FIG. 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 mutations in 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 confirm that they showed a similar form to iNSC. Through this, it can be confirmed that reprogramming has occurred with neural stem cells.
  • 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 show the expression levels of Nestin and Pax6 (Fig.
  • hNPCDF-iNSC Like H9NSC, which is a neural stem cell, hNPCDF-iNSC showed a high relative expression level of a neuron-specific marker and a low relative expression level of a fibroblast marker.
  • Figure 7f confirms the differentiation ability of the prepared hNPCDF-iNSC.
  • Tuj 1 Alexa 594 is an antibody that specifically binds to nerves, indicating that the prepared iNPCDF-iNSC has differentiated into nerve cells.
  • the differentiation ability of the prepared iNPCDF-iNSC can be confirmed through the expression of Olig2 involved in neuronal differentiation.
  • FIG. 8 shows the experimental results verifying the genetic stability of the prepared hDF-iNSC.
  • Figure 8a is the result of analyzing the karyotype of the prepared hDF-iNSC. It can be seen that the hDF-iNSC produced through the same shape as a general karyotype is genetically stable from a macroscopic point of view.
  • 8B is a result of PCR amplification of p53 genomic DNA in the prepared hDF-iNSC. It shows the same degree of amplification as that of normal hDF, and through this, it can be confirmed that the number of p53 alleles is normal.
  • 8C and 8D are the results of sequencing a site where mutations of the p53 gene occur frequently and comparing it with a normal cell (hDF). Through this, it can be confirmed that the p53 mutation did not occur in hDF-iNSC.
  • One aspect for achieving the object of the present invention is (a) introducing Sox2 and c-Myc into the isolated cells; And (b) culturing the cells of the step (a) to provide a method for producing neural stem cells comprising the step of inducing reprogramming directly from the isolated cells to cells whose lineage has been converted.
  • step (a) is a step of introducing Sox2 and c-Myc into the isolated cells.
  • isolated cell of the present invention is not particularly limited, and specifically, may be a cell whose lineage is already specified, such as a somatic cell, a germ cell, or a progenitor cell, and an adult stem Cells, bone marrow cells, mesenchymal stem cells, etc. may be stem cells with limited differentiation ability.
  • the isolated cells of the present invention may include both in vivo or ex vivo cells, and specifically, may be cells isolated from the living body.
  • "somatic cell” refers to all cells in which differentiation of animals and plants is completed except for germ cells, and the somatic cells of the present invention may be somatic cells excluding nerve cells.
  • progenitor cell refers to a parent cell that does not express the differentiation trait, but has the differentiation fate, when it is found that the cell corresponding to the progeny expresses a specific differentiation trait.
  • the “adult stem cell” of the present invention refers to a stem cell that appears in the stage in which each organ of the embryo is formed or the adult stage is formed by the development process, and the differentiation ability is generally limited to cells constituting a specific tissue.
  • adult stem cells may be derived from a group consisting of breast, bone marrow, cord blood, blood, liver, skin, gastrointestinal tract, placenta, and uterus, and the adult stem cells of the present invention are adult stem cells excluding neural stem cells. I can.
  • the adult stem cells may be mesenchymal stem cells, but are not limited thereto.
  • mesenchymal stem cells are cells that aid in making cartilage, bone, fat, bone marrow stroma, muscle, nerve, skin, etc., and generally stay in the bone marrow in adults, but umbilical cord, umbilical cord blood, peripheral blood, and other tissues. It means cells that can also be obtained from the back.
  • the mesenchymal stem cell of the present invention is a cord blood mesenchymal stem cell. However, it is not limited thereto.
  • the isolated cells may be non-neuronal cells.
  • non-neuronal cells include all differentiated or undifferentiated cells other than 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, cells derived from humans, Accordingly, the cells that can be reprogrammed into induced neural stem cells are not limited.
  • non-neuronal fibroblasts (Example 1), Niemann-Pick disease-derived fibroblasts, and umbilical cord blood-derived mesenchymal stem cells (Example 2) were treated with Sox2 and c- Neural stem cells were prepared by introducing Myc.
  • fibroblasts are representative examples of somatic cells
  • umbilical cord blood-derived mesenchymal stem cells are representative examples of adult stem cells.
  • Sox2 gene of the present invention is also referred to 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 self-replication of neural stem cells. Is known.
  • c-Myc gene of the present invention is one of the prototypical cancer genes encoding DNA-binding proteins in the nucleus, and is involved in cell proliferation.
  • the Sox2 gene and c-Myc gene of the present invention or proteins expressed therefrom include all SOX2 and c-Myc derived from animals such as humans or mice, and specifically, may be Sox2 and c-Myc derived from humans.
  • the protein expressed from the Sox2 and c-Myc genes of the present invention may include not only a protein having its wild-type amino acid sequence, but also a variant thereof.
  • Variants of Sox2 and c-Myc proteins include proteins in which the natural amino acid sequence of SOX2 and c-Myc and one or more amino acid residues have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.
  • the variant may be a functional equivalent exhibiting the same biological activity as a natural protein, or a variant in which the physical and chemical properties of the protein have been modified as needed, and may be a variant with increased structural stability or increased physiological activity to a physical or chemical environment. .
  • Sox2 or c-Myc gene is a nucleotide sequence encoding a wild type of Sox2 or c-Myc protein or a variant of Sox2 or c-Myc protein as described above, wherein one or more bases are substituted or deleted. , Insertion, or a combination thereof, and may be isolated from nature or prepared using chemical synthesis.
  • Sox2 and c-Myc genes are transcription factors that are very importantly used in the production of induced pluripotent stem cells (iPSCs), but the combination of Sox2 and c-Myc is still used in research on the production of induced neural stem cells through direct reprogramming. Therefore, there is no case of successful 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 included in the expression vector.
  • the Sox2 protein or c-Myc protein may be a protein expressed from a cell line in vitro using an expression vector containing a nucleic acid molecule encoding them.
  • the expression vector may include a viral vector, a non-viral vector, a plasmid vector, or a cosmid vector, and the expression vector may be a Baculovirus expression vector, a Mammalian expression vector, or a bacterial expression vector, and the cell line is an insect cell line, A mammalian cell line or a bacterial cell line may be used, but expression vectors and cell lines usable in the present invention are not limited thereto.
  • expression vector of the present invention is a vector capable of expressing a protein of interest in a suitable host cell, and refers to a genetic construct comprising essential regulatory elements 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, and can be variously prepared according to the purpose.
  • the promoter of the expression vector may be constitutive or inducible.
  • the expression vector includes a selectable marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, the origin of replication is included. Expression vectors can be self-replicating or integrated into host DNA.
  • the viral vector is retrovirus, 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), etc., Adenovirus, Adeno-associated virus, Herpes simplex virus, Sendai virus, etc. It can contain one vector. Further, more specifically, it may be an RNA-based viral vector, but is not limited thereto.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function.
  • the operative linkage with a recombinant vector can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage use enzymes generally known in the art.
  • the nucleic acid molecule encoding the Sox2 or c-Myc protein may be messenger RNA (mRNA).
  • mRNA messenger RNA
  • the "step of introducing Sox2 and c-Myc” refers to an expression vector, an mRNA encoding a Sox2 or c-Myc protein, a genetic modification, introduction of a foreign-expressed gene, and the treatment of a substance having an expression-inducing effect. It may be a method of increasing the expression level of the existing Sox2 and c-Myc genes and proteins expressed therefrom, but is not limited as long as the expression levels of the genes and proteins are increased. In particular, the step of introducing the gene may be a method of inducing expression of the gene under a desired time and condition.
  • the method of introducing the gene of step (a) into a cell may be used without limitation in providing a nucleic acid molecule (DNA or RNA) to cells commonly used in the art.
  • a nucleic acid molecule encoding a Sox2 and/or c-Myc protein may be operably linked into an expression vector and delivered into a cell, and may be delivered into a cell in a form that is inserted into a chromosome of a host cell.
  • the method of administering the Sox2 and/or c-Myc to the culture medium of the isolated cells or the method of injecting directly into the isolated cells, the method of directly injecting the mRNA encoding Sox2 or c-Myc protein into the isolated cells, etc. Can be used, but is not limited thereto.
  • the method of directly injecting Sox2 and/or c-Myc into the isolated cells may be used by selecting any method known in the art, but is not limited thereto, but microinjection, electroporation ), particle bombardment, direct muscle injection, an insulator, and a method using a transposon can be appropriately selected and applied.
  • the method of introducing Sox2 and/or c-Myc into isolated cells 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 inserted respectively Sox2 and/or c-Myc may be introduced through the process of treating the virus obtained from the packaging cells transformed with the vector into the culture medium of the isolated cells, but is not limited thereto.
  • the viral vector may be a vector derived from a retrovirus, a lentivirus, an adenovirus, an adeno-related virus, a herpes simplex virus, a Sendai virus, etc., but is not limited thereto, but specifically, a retroviral vector may be used.
  • the packaging cells may be used by selecting various cells known in the art according to the viral vector used.
  • the method of introduction using the virus may be a method of treating with a certain level of MOI.
  • MOI of 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. In the case of using a viral vector, the MOI represents the ratio of the amount of inoculated virus to the number of cells inoculated with the virus, that is, the average number of virus particles infecting a single cell. The use of this term is not limited to cases relating to transduction, but instead includes introduction of the vector into the host by means such as lipofection, microinjection, calcium phosphate precipitation, and electroporation.
  • the MOI value used for Sox2 and c-Myc introduction 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 and less than 4, more than 0.5 and less than 3, or more than 0.5 may be less than 2 MOI, specifically, may be 1 MOI, as long as it is within the range causing direct cross-differentiation into neural stem cells, it is not limited.
  • neural stem cells were prepared by treating fibroblasts with a retroviral vector encoding c-Myc and Sox-2 with MOI 1 (Example 1).
  • the method of introducing Sox2 and/or c-Myc into isolated cells in the present invention may be using a non-viral vector, and specifically, a non-viral epitaxial containing a nucleic acid molecule of Sox2 and/or c-Myc. It may be to introduce a cotton vector into the isolated cell.
  • the non-viral episomal vector of the present invention is a non-viral non-insertable vector, and is known to have a characteristic capable of expressing a gene included in the vector without being inserted into a chromosome.
  • cells containing an episomal vector include all cases in which the episomal vector is inserted into the genome or is present in the cell without being inserted into the genome.
  • the method of introducing Sox2 and/or c-Myc into the isolated cells may be direct introduction of the mRNA of Sox2 or c-Myc into the isolated cells.
  • the method of directly introducing the mRNA into the isolated cells may be used by selecting any method known in the art, but is not limited thereto.
  • step (b) of the present invention is a method for producing neural stem cells by culturing the cells of step (a) to induce reprogramming directly from the isolated cells to cells whose lineage has been converted.
  • the term "lineage-converted cell” refers to a cell that has been converted into a cell type having different lineage characteristics by altering the inherent lineage characteristics of the cell by embryological or artificial methods. It refers to the conversion into cells with characteristics of a cell type that are completely different from those of characteristics. Specifically, in the present invention, it may be a neural stem cell converted from a non-neural cell through reprogramming.
  • reprogramming refers to a method of converting a cell into a desired cell by controlling the global gene expression pattern of a specific cell.
  • reprogramming refers to a method of artificially manipulating the fate of a cell and converting it into a cell having completely different characteristics, and for the purposes of the present invention, the reprogramming is a foreign gene or a vector containing RNA or DNA. It may be performed by introducing into the cell.
  • reprogramming may include dedifferentiation of cells, direct reprogramming or direct conversion, or trans-differentiation, but is not limited thereto.
  • the term "reprogramming factor” refers to a gene (or a polynucleotide encoding the same), or a protein that is finally or introduced into a partially differentiated cell to induce reprogramming.
  • the reprogramming factor may vary depending on the target cell to induce reprogramming and the type of isolated cells from which reprogramming is induced.
  • initial cells were reprogrammed into induced nerve stem cells as target cells by introducing only Sox2 and c-Myc, but the scope of the present invention is not limited to the reprogramming factor, and neural stem cells are prepared.
  • the reprogramming factors introduced are Sox2, c-Myc.
  • it may contain factors that induce overexpression of Sox21 and induce overexpression of neuroectodermal lineage genes Tapa1, Atbf1, NeuroD1, Mash1, Hes1, Hes6, and Id2, and are capable of producing neural stem cells. It may include all known factors.
  • direct reprogramming into neural stem cells can be induced by using the reprogramming factor.
  • Reprogramming using a reprogramming genetic factor is to induce conversion to a target cell by regulating the entire gene expression pattern of the cell. Therefore, the reprogramming genetic factor is introduced into the cell and cultured for a certain period of time. Initial cells can be reprogrammed with target cells having the cell's gene expression pattern.
  • the "direct reprogramming" of the present invention is differentiated from the technology of producing pluripotent induced pluripotent stem cells through a reprogramming process, and is a technology that induces direct conversion to a desired target cell directly through reprogramming culture.
  • Existing somatic cell nuclear transplantation has the disadvantage of using an egg, so it is less likely to be utilized compared to other cell reprogramming techniques, and when using the induced pluripotent stem cell reprogramming technology, it is inherently via pluripotent stem cells. There is a drawback that it is necessary to verify whether it remains or not to secure safety.
  • 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, from initial cells through direct reprogramming.
  • direct reprogramming can be mixed with direct dedifferentiation, direct differentiation, direct conversion, direct cross-differentiation, and cross-differentiation.
  • direct reprogramming may mean direct dedifferentiation or cross-differentiation into neural stem cells.
  • the "induced neural stem cell (iNSC)" of the present invention is similar to a neural stem cell using a reprogramming technique from differentiated cells that exist in different aspects, such as cells without differentiation ability or cells with a certain ability to differentiate or 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, they show similar cell morphology, have similar gene and protein expression patterns, and may have pluripotency in vitro and in vivo.
  • the induced neural stem cells of the present invention may be differentiated into various neurons such as neurons (nerve cells), astrocytes, oligodendrocytes, GABA neurons or dopaminergic neurons.
  • the term "neural stem cell” of the present invention has the same meaning as "induced neural stem cell” unless otherwise indicated, and is used interchangeably.
  • neural stem cells prepared according to the method of the present invention can differentiate into neurons, astrocytes, and oligodendrocytes (Example 1).
  • the safety was confirmed through that the mutation of the p53 gene did not occur in the prepared neural stem cells (Example 3).
  • the neural stem cells are as described above.
  • the 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 therapy comprising the neural stem cells prepared according to the above method as an active ingredient.
  • the neural stem cells are as described above.
  • cell therapy of the present invention is a drug (US FDA regulation) used for treatment, diagnosis, and prevention of cells and tissues manufactured through isolation, culture, and special manipulation from an individual, and functions of cells or tissues It refers to a drug used for treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting live autologous, allogeneic, or xenogeneic cells in vitro to restore or altering the biological properties of cells in other ways.
  • the cell therapy agent of the present invention may contain 1.0 ⁇ 10 to 1.0 ⁇ 10 9 cells per 1 ml, but is not limited thereto.
  • the cell therapy agent of the present invention can be used unfrozen or frozen for later use. If frozen, standard cryopreservatives (eg DMSO, glycerol, Epilife Cell Freezing Medium (Cascade Biologics)) can be added to the cell population prior to freezing.
  • standard cryopreservatives eg DMSO, glycerol, Epilife Cell Freezing Medium (Cascade Biologics)
  • the cell therapy agent may be formulated and administered in a unit dosage form suitable for intra-body administration of a patient according to a conventional method in the pharmaceutical field, and the formulation may be administered once or several times to obtain an effective dose Include.
  • injections such as ampoules for injection, infusions such as infusion bags, and sprays such as aerosol formulations may be used as parenteral formulations.
  • the ampoule for injection may be mixed with an injection solution immediately before use, and physiological saline, glucose, mannitol, Ringer's solution, and the like may be used as the injection solution.
  • the infusion bag may be made of polyvinyl chloride or polyethylene, and Baxter, Becton ickinson, Medcep, National Hospital Products or Terumo The company's infusion bag can be illustrated.
  • one or more pharmaceutically acceptable conventional inert carriers for example, preservatives, painless agents, solubilizers or stabilizers in the case of injections, and a base in the case of topical administration preparations , Excipients, lubricants or preservatives, etc. may additionally be included.
  • the cell therapeutic agent of the present invention thus prepared can be administered with other stem cells used for transplantation and other uses or in the form of a mixture with such stem cells using an administration method commonly used in the art. It is possible to engraft or transplant directly into the diseased site of a necessary patient, or directly into the abdominal cavity, but is not limited thereto.
  • the administration may be non-surgical administration using a catheter and surgical administration methods such as injection or transplantation after incision at the disease site.
  • transplantation by intravascular injection which is a general method of hematopoietic stem cell transplantation, is also possible.
  • the cell therapy agent may be administered once or divided into several times. However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the weight, age, and sex of the patient. It is not intended to limit the scope of the present invention to any aspect.
  • prevention includes all actions of inhibiting or delaying the onset of neuronal damage disease by administration of the composition.
  • treatment includes all actions in which symptoms of neuronal damage disease are improved or benefited by administration of the composition.
  • Another aspect of the present invention provides a pharmaceutical composition for preventing or treating neuronal damage disease, comprising as an active ingredient neural stem cells prepared according to the above method.
  • the neural stem cells are as described above.
  • neural cell damage disease is a disease that occurs due to deformation or loss of nerve cells, such as Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, and amyotrophic lateral sclerosis ( amyotriophiclateral sclerosis), ischemic brain disease (stroke), demyelinating disease (demyelinating disease), multiple sclerosis, epilepsy, neurodegenerative disease, spinal cord injury, and the like, but is not limited to the above examples.
  • nerve cells such as Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, and amyotrophic lateral sclerosis ( amyotriophiclateral sclerosis), ischemic brain disease (stroke), demyelinating disease (demyelinating disease), multiple sclerosis, epilepsy, neurodegenerative disease, spinal cord injury, and the like, but is not limited to the above examples.
  • composition may contain a pharmaceutically acceptable carrier.
  • the "pharmaceutically acceptable carrier” may mean a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the compound to be injected.
  • the kind of the carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used.
  • 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.
  • composition including a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms.
  • a pharmaceutically acceptable carrier is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.
  • solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid formulations include at least one excipient, such as starch, calcium carbonate, sucrose, and lactose, in the compound. , Gelatin, etc. can be mixed to prepare.
  • excipients such as starch, calcium carbonate, sucrose, and lactose
  • lubricants such as magnesium stearate and talc may also be used.
  • Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, and other excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents. have.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.
  • injectable ester such as ethyl oleate
  • suppositories Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.
  • the present invention provides a method for preventing or treating neuronal damage disease comprising administering a pharmaceutical composition for preventing or treating neuronal damage disease.
  • the neural stem cells are as described above.
  • composition can be administered in a pharmaceutically effective amount.
  • the "pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the subject, age, sex, type of infected virus, and Activity, sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.
  • the administration means introducing the composition of the present invention to a patient by any suitable method, and the administration route of the composition may be administered through 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, or intranasal administration are not limited thereto.
  • 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 2-3 times.
  • the number of administrations may be the same or different.
  • the composition of the present invention may be used alone or in combination with other drug treatments for the prevention or treatment of neuronal damage diseases. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.
  • the individual means all animals including humans who have or may develop neuronal damage disease, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, mice, rabbits, or guinea pigs. Means.
  • the type of individual is included without limitation as long as the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to an individual.
  • neural stem cells produced by direct reprogramming can differentiate into various types of nerve cells, and the neural stem cells of the present invention can be used as therapeutic agents for various neuronal damage diseases. have.
  • a neuronal stem cell prepared according to the method or a neuronal cell differentiated therefrom with a candidate substance to identify a therapeutic agent for neurological diseases tailored to the individual from which the neural stem cell is derived.
  • a candidate substance is treated on the neural stem cells prepared by the method of the present invention or the neurons differentiated therefrom, and related to the treatment of neurological diseases.
  • the isolated cells used to generate the neural stem cells can be tailored therapeutic agents for neurological diseases for the derived individual. .
  • Example 1 Preparation of iNSC from human fibroblasts (hDF) by controlling Sox2, c-Myc overexpression
  • Example 1-1 Sox2, c-Myc retroviral vector preparation
  • a retrovirus was constructed to overexpress hc-Myc and hSox-2 in somatic cells.
  • pMXs-hc-MYC and pMXs-hSOX2 were produced by introducing hcMyc and hSOX-2 into a retroviral expression vector pMX retroviral vector (Fig. 1A).
  • hcMyc or hSOX-2 retrovirus for introduction of hcMyc or hSOX-2 was prepared using the pMXs-hc-MYC and pMXs-hSOX2 vectors produced above. Specifically, 24 ul of Convoy, 4 ug of pMXs-hc-Myc or pMXs-hSox-2, 2 ug of VSV-G and 2 ug of Gag-Pol were mixed with PBS (phosphate buffered saline), and then allowed to stand at room temperature for 10 minutes. . Thereafter, the mixture was 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 . In addition, pMXs-GFP vector was also transfected under the same conditions for titration. Virus soup generated after transfection was collected 24, 48, and 72 hours after transfection, respectively, and stored at 4°C.
  • the virus soup was filtered with 0.45 um, and Retro-X concentrator (Clontech) was added in 1/3 volume of the virus soup and incubated at 4° C. 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, and the retrovirus pellet was suspended in PBS and then dispensed, and stored at -80°C until use.
  • the GFP retrovirus concentrated in 5x10 5 293FT cells seeded in a 6-well plate was transduced overnight at 50, 5, 0.5 and 0 ul, respectively. After 2 days, the proportion of GFP-expressing cells among the cells was analyzed using a flow cytometer. The titer was calculated by introducing the result value within 1-20% into the calculation formula shown in Fig. 1D (Figs. 1B to 1D).
  • Example 1-2 Confirmation of production of induced neural stem cells according to the difference in MOI values of Sox2 and c-Myc retrovirus
  • induced nerve stem cells could be prepared using human fibroblasts by the Sox2 and c-Myc retroviruses prepared in Example 1-1.
  • the retrovirus for introducing hSOX-2 prepared in Example 1-1 to prepare induced nerve stem cells (iNSC) was treated with MOI 1, and the retrovirus for introducing hcMyc was MOI 1, 5 and 10 respectively. Reprogramming was directly induced by treatment on blast cells.
  • Example 1-1 the day before retrovirus transduction, 1.25 ⁇ 10 4 human fibroblasts were plated in a 24-well tissue culture plate. The next day, Sox2 and c-Myc retroviruses prepared in Example 1-1 were added to the cells at a predetermined MOI, followed by spinfection at 800xg for 60 minutes at 20°C. After spinfection, it was cultured in DMEM/F12 containing 20% (v/v) FBS (fetal bovine serum) and 1X primocin.
  • DMEM/F12 20% (v/v) FBS (fetal bovine serum) and 1X primocin.
  • the transformed cells were seeded in a 6-well plate coated with poly L-ornithine/Fibronectin by 5 ⁇ 10 3 each. After seeding, on the 5th day, after confirming that the cells were attached to the plate, it was replaced with iNSC medium (1X supplement, 1X primocin, 20 ng/ml bFGF, 20 ng/ml EGF-added Stempro NSC medium). The medium was exchanged once every 2 days.
  • Neurospheres Neurospheres The iNSC in culture attached to the culture was removed using accutase and then suspended in a petri dish to form a neurosphere.
  • the direct cross-differentiation efficiency was compared and analyzed by the proportion of cells in which CD133, an NSC marker, was positive.
  • 1x10 5 to 1x10 6 cells were suspended in 100 ul of PBS. Thereafter, 3 ul of an antibody conjugated with Fluorescein isothiocyanate (FITC) was mixed with the cells, and binding was induced at room temperature for 30 minutes in a light-blocked space.
  • the antibody used was anti-CD133/1-VioBright/FITC (Miltenyi Biotec).
  • the cells were analyzed by flow cytometry using MACSQuant VYB (Miltenyi Biotec). As a result, it showed a difference of 0.2-0.5% by hDF batch (batch) (Fig. 2f).
  • qRT-PCT was performed to confirm the amount of transcriptional expression of p53.
  • mRNA was isolated from the cell pellet using PureLink RNA Mini Kit (Invitrogen), and cDNA was synthesized from the isolated mRNA 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 PCR reaction was performed using Quant Studio3 (Appliedbiosystems), and p53 gene The relative multiple values of are calculated.
  • the membrane was treated with an anti-p53 antibody (1:1,000; Santacruz) at 4° C. for one day. After washing the next day, the HRP-conjugated secondary antibody (1:1,000; Santacruz) was treated at room temperature for 1 hour, and the membrane was developed with ECL solution (iNTRON) after washing, and the expression level of the p53 protein was confirmed.
  • Example 1-3 Confirmation of direct cross-differentiation without going through a step having pluripotency
  • reprogramming factors were introduced in four combinations of hOct-4, hSox-2, hc-Myc, and hKlf-4 each excluded. Then, it was cultured in DMEM/F12 containing 20% (v/v) FBS for 3 days. On the third day, STO feeder cells were passaged to the seeded tissue culture plate. Thereafter, the medium was changed daily with iPSC medium (DMEM/F12, 20% serum replacement, 1x primocin, 4ng/ml bFGF) until day 5-21. Alkaline phosphatase staining was performed on the 21st day, and reprogramming efficiency was quantified by counting the number of alkaline phosphatase-positive colonies.
  • DMEM/F12 20% serum replacement, 1x primocin, 4ng/ml bFGF
  • Example 1-4 Confirmation of multipotency of the prepared induced nerve stem cells
  • hDF-iNSC prepared by the method of the present invention has neural stem cell characteristics, and the proliferation and differentiation capacity of the cells were confirmed.
  • the expression pattern of a neural stem cell specific marker was confirmed.
  • Example 1-2 hDF-iNSC prepared in Example 1-2 overexpressed endogenous Sox2, Nestin, Pax6, which are neural stem cell-specific markers, at the transcription level. Confirmed (Fig. 5a).
  • the immunostaining method was performed as follows. First, anti-Nestin (Abcam), anti-PAX6 (BioLegent) primary antibodies (each 1:100) to the hDF-iNSC, which has been fixed, permeabilization, and blocking, according to the experimental purpose. Were treated at 4° C. for one day. After washing with PBS the next day, according to the origin of the primary antibody, 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 at room temperature for 2 hours.
  • the proliferation and differentiation capacity of the prepared hDF-iNSC were measured. Specifically, spontaneous differentiation of iNSC was induced by culturing with iNSC medium without bFGF/EGF for 3 weeks. Medium exchange was performed once every 2-3 days, and the cultivation was performed at 37° C. 5% CO 2 . In the cells in which differentiation was completed, the transcription levels of MAP2, GFAP and Olig1 were confirmed by the qRT-PCR method described in Example 1-1. As a result, the prepared hDF-iNSC showed a doubling time of about 21.3 h and self-renewal, and neurons and glial cells (astroglia and oligodendrocytes) due to spontaneous differentiation. It was confirmed that it has a pluripotency that can be differentiated into (Figs. 5c and 5d).
  • Example 1 In order to confirm whether the method for preparing somatic cell-derived induced nerve stem cells identified in Example 1 can be generally applied to various somatic cells other than human fibroblasts, human umbilical cord blood-derived mesenchymal stem cells: hUCB-MSC ) And human Niemann-Pick Type C disease derived dermal fibroblasts (hNPCDF) were subjected to the following experiments.
  • human umbilical cord blood-derived mesenchymal stem cells hUCB-MSC
  • hNPCDF human Niemann-Pick Type C disease derived dermal fibroblasts
  • anti-HLA-ABC anti-HLA-DR
  • anti-CD34 anti-CD34
  • anti-CD45 anti-CD73
  • an anti-CD105 (BD) antibody was used to perform flow cytometric analysis performed in Example 1-2.
  • hUCB-MSC showed typical MSC-specific marker expression patterns as HLA-ABC+, HLA-DR-, CD34-, CD45-, CD73+, and CD105+ (Fig. 6a), through which hUCB-MSC used in the experiment was It was confirmed that it has the characteristics of mesodermal stem cells.
  • Example 6B As a result of treating the retrovirus with MOI 1 as in Example 1 for the introduction of hc-Myc and hSox-2 into the cells, it was confirmed that iNSC-specific attached colony morphology was shown (FIG. 6B), As a result of performing the flow cytometric analysis described in Example 1-2 using an anti-CD133/1-VioBright/FITC (Miltenyi Biotec) antibody, direct crossing with 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).
  • Example 1-1 by performing the qRT-PCR experiment described in Example 1-1, it was confirmed that the hUCB-MSC-iNSC prepared above expressed endogenous Sox2, Nestin, Pax6, which are NSC-specific markers, and differentiated into a neuron-like form. Bars (Figs. 6D and 6E), it can be seen that the cells were directly reprogrammed into induced nerve stem cells, and have the ability to differentiate into nerve cells.
  • Example 1 the method for preparing induced nerve stem cells of Example 1 is also applied to hUCB-MSC.
  • the hNPCDF used for the experiment has a point mutation in the NPC1 gene, so isoleucine of the NPC1 protein is replaced with threonine (FIG. 7A). In other words, it is a somatic cell in which a mutation exists in one gene of human fibroblasts.
  • hNPCDF-iNSC expressed NSC-specific markers Nestin and Pax6, and on the contrary, fibroblast markers COL1A2 and S100A4 were not expressed (Fig. 7d and 7e), through this, it was confirmed that hNPCDF can also be reprogrammed into neural stem cells and its efficiency.
  • the prepared hNPCDF-iNSC was differentiated through an immunostaining method (described in Example 1-3) using anti-Olig2 (Millipore) and anti-Tuj 1 (Abcam) primary antibodies (each 1:100). As a result, it was confirmed that the differentiation into neuron and glia (oligodendrocyte) was confirmed (Fig. 7f), and it can be seen that the hNPCDF-iNSC also has differentiation ability.
  • Example 3 Verification of the stability of the prepared induced nerve stem cells
  • gDNA was isolated from hDF-iNSC using AccuPrep Genomic DNA Extraction Kit (Bioneer). PCR amplification was performed using AccuPower PCR Premix (Bioneer), p53 forward and reverse primers shown in Table 1 (SEQ ID NOs: 19 and 20), and gDNA isolated using Genetouch Thermal Cycler (Hanzhou bioer technology) as a template. After PCR was finished, it was confirmed whether or not gene amplification was performed through gel electrophoresis (Mupid) (FIG. 8B), and 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) together with the forward primer (SEQ ID NO: 19) used for PCR (Fig. 8c).
  • iNtRON MEGAquick-spin plus fragment DNA purification kit
  • the PCR product was cloned using TOPcloner TA Kit (Enzynomics), and this was transformed into DH5a chemically competent E.coli (Enzynomics) using a heat shock method.
  • the transformed E. coli was plated on an LB Agar LOP plate (Narae biotech) and incubated at 37°C for one day. The next day, 10 colonies were incubated in LB broth (Gibco) for one day at 37°C. Thereafter, the plasmid was isolated from E. coli grown using the AccuPrep Plasmid Mini Extraction Kit (Bioneer), and sequencing was performed in Macrogen (Seoul, Korea) using this. As a result, the mutation of the p53 gene was not observed in all of the results of sequencing 10 individually (Fig. 8D).

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Abstract

La présente invention concerne un procédé de génération de cellules souches neurales induites à partir de cellules non neurales par reprogrammation directe et leur utilisation. Du fait que le procédé de génération de cellules souches neurales induites selon la présente invention permet de générer des cellules souches neurales induites à partir de cellules non neurales même lorsque seuls les deux facteurs d'induction Sox2 et c-Myc sont utilisés, le procédé peut générer des cellules souches neurales induites plus efficacement que les procédés de génération classiques qui emploient quatre ou cinq facteurs d'induction. Les cellules souches neurales induites générées par le procédé présentent une excellente puissance de différenciation et une excellente activité de prolifération et peuvent ainsi être appliquées à des fins thérapeutiques à l'avenir.
PCT/KR2019/005566 2018-05-09 2019-05-09 Procédé de génération de cellules souches neurales induites reprogrammées directement à partir de cellules non neurales en utilisant la sox2 et le c-myc WO2019216667A1 (fr)

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