WO2022163466A1 - オリゴデンドロサイトの作製方法 - Google Patents

オリゴデンドロサイトの作製方法 Download PDF

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WO2022163466A1
WO2022163466A1 PCT/JP2022/001814 JP2022001814W WO2022163466A1 WO 2022163466 A1 WO2022163466 A1 WO 2022163466A1 JP 2022001814 W JP2022001814 W JP 2022001814W WO 2022163466 A1 WO2022163466 A1 WO 2022163466A1
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cells
oligodendrocytes
producing
inducer
progenitor cells
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French (fr)
Japanese (ja)
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剛士 田邊
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I Peace Inc
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I Peace Inc
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Priority to US18/258,778 priority patent/US20240093146A1/en
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Definitions

  • the present invention relates to cell technology, and to methods for producing oligodendrocytes and their progenitor cells.
  • Oligodendrocytes which are myelin-forming cells in the central nervous system, may be useful for cell transplantation of hereditary and acquired leukodystrophies (see, for example, Patent Documents 1 and 2).
  • conventional methods for inducing oligodendrocytes from stem cells are problematic in that they are time consuming and inefficient.
  • One of the objects of the present invention is to provide an efficient method for producing oligodendrocytes and their progenitor cells.
  • a method for producing oligodendrocytes and their progenitor cells comprises introducing inducers including OLIG and SOX into iPS cells or ES cells.
  • methods for producing oligodendrocytes and their progenitor cells comprise introducing inducers including OLIG and NKX into iPS cells or ES cells.
  • a method for producing oligodendrocytes and their progenitor cells which comprises introducing inducers including OLIG, NKX, and SOX into iPS cells or ES cells.
  • the inducer may further contain a factor that promotes cell proliferation.
  • the factor that promotes cell proliferation is at least one selected from the group consisting of p53 gene-repressing factors, Rb gene-repressing factors, and MYC.
  • MYC may be c-MYC.
  • the inducer may further contain ASCL.
  • a method for producing oligodendrocytes and their progenitor cells which comprises introducing inducers including OLIG and factors that promote cell proliferation into iPS cells or ES cells.
  • the factor that promotes cell proliferation is at least one selected from the group consisting of p53 gene-repressing factors, Rb gene-repressing factors, and MYC.
  • MYC may be c-MYC.
  • the inducer may contain at least one selected from the group consisting of SOX, ASCL, and NKX.
  • an oligodendro oligodendro which includes producing iPS cells without cloning cells into which a reprogramming factor has been introduced, and introducing an inducer including OLIG into the iPS cells.
  • the steps include seeding cells into which a reprogramming factor has been introduced to produce iPS cells without cloning, and introducing an inducer including OLIG into the iPS cells.
  • an inducer including OLIG into the iPS cells.
  • the cells into which the reprogramming factor has been introduced are detached from the culture vessel, and at least a portion of the detached cells are mixed and seeded to produce iPS cells; Introducing an inducer comprising OLIG is provided.
  • the cells into which the reprogramming factor has been introduced are recovered from the culture vessel, and at least a portion of the recovered cells are mixed and seeded to produce iPS cells; Introducing an inducer comprising OLIG is provided.
  • iPS cells are produced without picking up each of a plurality of colonies formed by cells into which a reprogramming factor has been introduced, and an inducer containing OLIG is introduced into the iPS cells.
  • a method for producing oligodendrocytes and their progenitor cells comprising:
  • iPS cells are prepared by seeding a mixture of cells into which a reprogramming factor has been introduced, which are derived from different single cells, and iPS cells containing OLIG. Introducing a factor is provided.
  • the cells do not have to be cloned in seeding.
  • cells into which reprogramming factors have been introduced may be mixed during seeding.
  • clones of cells into which reprogramming factors have been introduced may be mixed during seeding.
  • oligodendrocytes and their progenitor cells In the method for producing oligodendrocytes and their progenitor cells described above, different clones of reprogramming factor-introduced cells may be mixed in seeding.
  • the above-described method for producing oligodendrocytes and their progenitor cells may not include cloning a single colony formed by cells into which a reprogramming factor has been introduced.
  • the above method for producing oligodendrocytes and their progenitor cells may not include picking up colonies formed by cells into which reprogramming factors have been introduced.
  • the cells into which the reprogramming factor has been introduced and which are attached to the culture vessel are collected, and at least part of the collected cells are seeded in a medium.
  • the cells into which the reprogramming factor has been introduced may be seeded without discrimination by gene expression state.
  • the cells into which the reprogramming factor has been introduced may be seeded without distinguishing the degree of reprogramming.
  • the inducer may contain at least one selected from the group consisting of SOX, ASCL, and NKX.
  • a method for producing oligodendrocytes and their progenitor cells which comprises introducing inducers including OLIG, SOX, ASCL, and NKX into somatic cells.
  • the inducer may further contain a factor that promotes cell proliferation.
  • a method for producing oligodendrocytes and their progenitor cells which comprises introducing inducers including OLIG and factors that promote cell proliferation into somatic cells.
  • the inducer may contain at least one selected from the group consisting of SOX, ASCL, and NKX.
  • the factor that promotes cell proliferation is at least one selected from the group consisting of p53 gene-repressing factors, Rb gene-repressing factors, and MYC.
  • MYC may be c-MYC.
  • the somatic cells need not be iPS cells or ES cells.
  • the somatic cells may be blood cells.
  • the somatic cells may be mononuclear cells.
  • the somatic cells may be fibroblasts.
  • FIG. 4 is a graph showing measurement results by a flow cytometer according to Example 1.
  • FIG. 4 is a graph showing the results of PCR according to Example 1.
  • FIG. 1 is a photograph showing TRA1-60 positive cells according to Example 1.
  • FIG. 4 is a graph showing clonal efficiencies according to Examples 1 and 2.
  • FIG. 4 is a fluorescence micrograph of cells according to Example 3.
  • FIG. 4 is a fluorescence micrograph of cells according to Example 3.
  • FIG. 4 is a fluorescence micrograph of cells according to Example 4.
  • FIG. 10 is a fluorescence micrograph of cells according to Examples 5 to 9.
  • FIG. 10 is a fluorescence micrograph of cells according to Example 10.
  • FIG. 11 is a fluorescence micrograph of cells according to Example 11.
  • FIG. 12 is a fluorescence micrograph of cells according to Example 12.
  • FIG. 2 is a graph showing the results of Examples 13-18.
  • a method for producing oligodendrocytes and their progenitor cells includes introducing inducers including OLIG, SOX, ASCL, and NKX into somatic cells.
  • the method for producing oligodendrocytes and their progenitor cells includes introducing inducers including OLIG and SOX into iPS cells (induced pluripotent stem cells) or ES cells (embryonic stem cells).
  • inducers including OLIG and SOX into iPS cells (induced pluripotent stem cells) or ES cells (embryonic stem cells).
  • the method for producing oligodendrocytes and their progenitor cells includes introducing inducers including OLIG and NKX into iPS cells or ES cells.
  • the method for producing oligodendrocytes and their progenitor cells is to produce iPS cells without cloning cells into which a reprogramming factor has been introduced, and to introduce an inducer containing OLIG into the iPS cells.
  • the method for producing oligodendrocytes and their progenitor cells includes producing iPS cells by seeding cells into which a reprogramming factor has been introduced without cloning, and iPS cells containing an inducer containing OLIG. and introducing
  • the cells into which the reprogramming factor has been introduced are detached from the culture vessel, and at least part of the detached cells are mixed and seeded to produce iPS cells. and introducing an inducer comprising an OLIG into iPS cells.
  • cells into which a reprogramming factor has been introduced are recovered from a culture vessel, and at least a portion of the recovered cells are mixed and seeded to produce iPS cells. and introducing an inducer comprising an OLIG into iPS cells.
  • the method for producing oligodendrocytes and their progenitor cells comprises producing iPS cells without picking up each of a plurality of colonies formed by cells into which a reprogramming factor has been introduced; introducing an inducer comprising
  • iPS cells are produced by seeding a mixture of cells into which a reprogramming factor has been introduced, which are derived from different single cells. and introducing an inducer comprising an OLIG into the iPS cells.
  • Inducers may include OLIG and ASCL. Inducers may include OLIG, ASCL, and SOX. Inducers may include OLIG, SOX, ASCL, and NKX.
  • OLIG is, for example, OLIG2.
  • SOX is, for example, SOX10.
  • ASCL is, for example, ASCL1.
  • NKX is, for example, NKX2.2 or NKX6.1.
  • the inducer may further contain a factor that promotes cell proliferation.
  • factors that promote cell proliferation include factors that repress the p53 gene, factors that repress the Rb gene, and MYC.
  • a factor that promotes cell proliferation can be a factor that promotes canceration, a factor that suppresses apoptosis, or a factor that induces chromatin to euchromatin.
  • p53 is a cancer suppressor protein.
  • Factors that repress the p53 gene are, for example, dominant-negative mutants of p53.
  • the dominant-negative mutant of p53 is not particularly limited as long as it can act competitively with the wild-type p53 protein endogenously present in somatic cells to inhibit the function of the wild-type p53 protein.
  • Examples of dominant-negative mutants of p53 include p53P275S, in which proline at position 275 (position 278 in humans) located in the DNA-binding region of mouse p53 is mutated to serine; p53DD in which amino acids at positions 11 to 304 in p53) are deleted, p53S58A in which serine at position 58 (position 61 in human) of mouse p53 is mutated to alanine, position 135 in human p53 (position 135 in mouse) p53C135Y with a point mutation of cysteine at position 132 in the case of mouse p53 to tyrosine, p53A135V with a point mutation of alanine at position 135 in mouse p53 (position 138 in the case of humans) to valine, and position 172 of mouse p53 (position 132 in the case of humans).
  • the agent that suppresses the p53 gene can be an RNA such as short hairpin RNA (shRNA) and siRNA that interferes with the p53 gene.
  • Rb is a cancer suppressor protein.
  • Agents that suppress the Rb gene can be RNAs such as, for example, short hairpin RNAs (shRNAs) and siRNAs that interfere with the Rb gene.
  • shRNAs short hairpin RNAs
  • siRNAs that interfere with the Rb gene.
  • MYC loses the function of cell cycle control and induces canceration.
  • MYC may be c-MYC.
  • the inducer may be DNA or RNA.
  • RNA may be mRNA.
  • human gene symbols are used here, capital letters and small letters are not intended to limit species. For example, all capital letters do not exclude the inclusion of mouse or rat genes. However, in the examples, gene symbols are indicated according to the species actually used.
  • Somatic cells are, for example, cells that are not stem cells. Somatic cells are, for example, cells other than iPS cells and ES cells. Examples of somatic cells include fibroblasts, blood cells, dental pulp stem cells, keratinocytes, dermal papilla cells, oral epithelial cells, and somatic stem progenitor cells. Examples of blood cells include T cells and blood cells other than T cells (non-T cells) such as macrophages, monocytes, monocytes, B cells, and non-rosette forming cells. The somatic cells may be cells contained in urine. Examples of cells contained in urine include bladder epithelial cells.
  • Somatic cells may be human-derived or non-human animal-derived.
  • a somatic cell may be derived from one human, or may be derived from multiple humans.
  • a somatic cell may be derived from one non-human animal or may be derived from multiple non-human animals.
  • Somatic cells may be of fetal origin.
  • iPS cells and ES cells may be human-derived or non-human animal-derived.
  • the iPS cells and ES cells may be derived from one human, or may be derived from multiple humans.
  • iPS cells and ES cells may be derived from one non-human animal, or may be derived from multiple non-human animals.
  • iPS cells may be of fetal origin.
  • culturing cells introduced with a reprogramming factor, collecting the cells introduced with the reprogramming factor, seeding at least a part of the collected cells in a medium and subculturing may be induced by a culture method, including
  • Cells into which reprogramming factors are introduced are not particularly limited, but examples include fibroblasts, blood cells, dental pulp stem cells, keratinocytes, dermal papilla cells, oral epithelial cells, and somatic stem progenitor cells.
  • Cells into which reprogramming factors are introduced may be cells contained in urine. Examples of cells contained in urine include bladder epithelial cells.
  • Cells into which reprogramming factors are introduced may be derived from humans or non-human animals.
  • a cell into which a reprogramming factor is introduced may be derived from one human, or may be derived from multiple humans.
  • the cells into which the reprogramming factor is introduced may be derived from one non-human animal or multiple non-human animals.
  • a reprogramming factor introduced into a cell is, for example, RNA.
  • RNA is, for example, mRNA.
  • Reprogramming factors introduced into cells include, for example, OCT RNA such as OCT3/4, SOX RNA such as SOX2, KLF RNA such as KLF4, and MYC RNA such as c-MYC.
  • OCT3/4-modified M 3 O may be used as a reprogramming factor RNA.
  • reprogramming factor RNA is LIN28A, FOXH1, LIN28B, GLIS1, p53-dominant negative, p53-P275S, L-MYC, NANOG, DPPA2, DPPA4, DPPA5, ZIC3, BCL-2, E-RAS, TPT1, SALL2 , NAC1, DAX1, TERT, ZNF206, FOXD3, REX1, UTF1, KLF2, KLF5, ESRRB, miR-291-3p, miR-294, miR-295, NR5A1, NR5A2, TBX3, MBD3sh, TH2A, TH2B, and P53DD It may further contain RNA of at least one factor selected from the group consisting of. These RNAs are available from TriLink.
  • p53 is a cancer suppressor protein.
  • the dominant-negative mutant of p53 is not particularly limited as long as it can act competitively with the wild-type p53 protein endogenously present in somatic cells to inhibit the function of the wild-type p53 protein.
  • Examples of dominant-negative mutants of p53 include p53P275S, in which proline at position 275 (position 278 in humans) located in the DNA-binding region of mouse p53 is mutated to serine; p53DD in which amino acids at positions 11 to 304 in p53) are deleted, p53S58A in which serine at position 58 (position 61 in human) of mouse p53 is mutated to alanine, position 135 in human p53 (position 135 in mouse) p53C135Y with a point mutation of cysteine at position 132 in the case of mouse p53 to tyrosine, p53A135V with a point mutation of alanine at position 135 in mouse
  • p53R172H with a point mutation of arginine to histidine at position 175) of mouse p53
  • p53R270H with a point mutation of arginine to histidine at position 270 (position 273 in humans) of mouse p53
  • position 278 position 281 in humans of mouse p53.
  • p53D278N in which the aspartic acid at position ) is mutated to asparagine.
  • RNA may be modified with pseudouridine ( ⁇ ) or 5-methyluridine (5meU). RNA may be polyadenylated.
  • the RNA introduced into the cell is, for example, single-stranded RNA, and double-stranded RNA may be substantially removed. Moreover, it is preferable that impurities such as short-chain RNA and contaminants are substantially removed from the RNA to be introduced into the cells.
  • Single-stranded RNA introduced into cells may be purified and/or concentrated to substantially remove double-stranded RNA. Methods for purifying single-stranded RNA to be introduced into cells include a purification method using high performance liquid chromatography (HPLC). For example, HPLC removes 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of double-stranded RNA.
  • HPLC high performance liquid chromatography
  • the RNA introduced into the cells may be treated with a ribonuclease that degrades double-stranded RNA to substantially remove double-stranded RNA.
  • the RNA to be introduced into the cell may further comprise MYOD transcriptional activation domain (TAD) RNA directly connected to the full-length OCT3/4 RNA.
  • TAD MYOD transcriptional activation domain
  • the lipofection method is a method in which a complex of a nucleic acid, which is a negatively charged substance, and a positively charged lipid is formed through electrical interaction, and the complex is taken up into cells by endocytosis or membrane fusion. .
  • the lipofection method has advantages such as little damage to cells, excellent transfection efficiency, simple operation, and short time.
  • a reprogramming factor is introduced into cultured cells using, for example, an RNA transfection reagent.
  • an RNA transfection reagent for example, if the cells are mononuclear cells, the RNA may be introduced into the mononuclear cells immediately after their isolation from the blood.
  • a viral vector may be an RNA viral vector.
  • the RNA virus vector may be a Sendai virus vector.
  • the Sendai virus vector may be a temperature-sensitive Sendai virus vector in which the stability of the viral nucleic acid is reduced above a predetermined temperature.
  • the viral nucleic acid of the temperature-sensitive Sendai virus vector is stable below a certain temperature.
  • the viral nucleic acid may be viral DNA or viral RNA.
  • a viral nucleic acid may be a viral genome. The reduction in viral nucleic acid stability may be at least one of degradation of the viral nucleic acid and inhibition of replication or proliferation of the viral nucleic acid.
  • the predetermined temperature is, for example, 36.5° C. or higher and 37.5° C. or lower.
  • the stability of the viral nucleic acid of the temperature-sensitive Sendai virus vector ie at least one of the growth, replication rate and gene expression level, is high below a predetermined temperature and low above a predetermined temperature.
  • the temperature-sensitive Sendai virus vector has a growth rate or gene expression level in cells cultured at 37°C that is half or less than the growth rate or gene expression level in cells cultured at 32°C. is.
  • the Sendai virus encodes the N gene, P gene, M gene, F/HN gene, and L gene.
  • the HN protein recognizes sialic acid on the cell surface and tethers the virus particles to the cell when the Sendai virus attaches to the cell.
  • the F protein is cleaved and activated by an extracellular protease to catalyze the fusion of the tethered Sendai virus envelope and the plasma membrane of the target cell, thereby establishing infection.
  • the L protein, together with its modification protein, the P protein catalyzes replication of viral nucleic acids and transcription from the replicated multi-copy nucleic acids in the cytoplasm after infection.
  • the Sendai virus vector By deleting the F gene in the Sendai virus vector, it is possible to suppress the production of infectious virus particles from transfected cells.
  • the Sendai virus vector can be made temperature-sensitive by introducing a mutation into at least one of the L gene and the P gene.
  • TS7 Y942H/L1361C/L1558I mutation of L protein
  • TS12 D433A/R434A/K437A mutation of P protein
  • TS13 D433A/R434A/K437A of P protein mutation and L1558I mutation of L protein
  • TS14 D433A/R434A/K437A mutation of P protein and L1361C mutation of L protein
  • TS15 D433A/R434A/K437A mutation of P protein and L1361C/L1558I mutation of L protein
  • the Sendai virus vector has, for example, G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, L511F mutation in the P protein, and N1197S in the L protein. and an F gene-deleted ( ⁇ F) Sendai virus vector having the K1795E mutation, which is a Sendai virus vector into which the above-mentioned TS7, TS12, TS13, TS14, or TS15 mutation has been introduced.
  • temperature-sensitive mutations of Sendai virus vectors are not limited to these.
  • the Sendai virus vector is, for example, SeV(PM)/TS ⁇ F, SeV18+/TS ⁇ F, or SeV(HNL)/TS ⁇ F, and is a Sendai virus vector introduced with the above TS7, TS12, TS13, TS14, or TS15 mutation. be.
  • temperature-sensitive mutations of Sendai virus vectors are not limited to these.
  • the Sendai virus vector to be introduced into cells may be a combination of a temperature-sensitive Sendai virus vector and a temperature-insensitive Sendai virus vector.
  • the Sendai virus vector to be introduced into the cell may be only the temperature-sensitive Sendai virus vector and may not contain the temperature-insensitive Sendai virus vector.
  • the Sendai virus vector to be introduced into cells may be only a temperature-sensitive Sendai virus vector introduced with the TS7, TS12, TS13, TS14, or TS15 mutation, and may not contain a temperature-insensitive Sendai virus vector.
  • the Sendai virus vector to be introduced into cells is only a Sendai virus vector that has the same or higher temperature sensitivity than the temperature-sensitive Sendai virus vector into which the TS7, TS12, TS13, TS14, or TS15 mutation is introduced. It does not have to contain a viral vector.
  • the Sendai virus vector to be introduced into cells is only a Sendai virus vector having temperature sensitivity equal to or higher than that of the temperature-sensitive Sendai virus vector introduced with the TS7, TS12, TS13, TS14, or TS15 mutation.
  • the Sendai virus vector which is less temperature sensitive than the temperature-sensitive Sendai virus vector into which the TS13, TS14, or TS15 mutation is introduced, may not be included.
  • the Sendai virus vector introduced into cells carries any reprogramming factor.
  • the Sendai virus vector to be introduced into the cell contains, for example, KLF RNA, OCT RNA, and SOX RNA in this order, a temperature-sensitive Sendai virus vector that does not contain MYC RNA, and a temperature-sensitive Sendai virus vector that contains MYC RNA and KLF.
  • RNA, OCT RNA, and a temperature-sensitive Sendai virus vector that does not contain SOX RNA are arbitrary and are not particularly limited.
  • the Sendai virus vector to be introduced into the cell may contain a Sendai virus vector that contains KLF RNA and does not contain OCT RNA and SOX RNA.
  • the Sendai virus vector containing KLF RNA and not containing OCT RNA and SOX RNA may be a temperature-sensitive Sendai virus vector or a temperature-insensitive Sendai virus vector.
  • a temperature-sensitive Sendai virus vector containing KLF RNA, OCT RNA, and SOX RNA has, for example, G69E, T116A, and A183S mutations in the M protein and A262T, G264R, and K461G mutations in the HN protein.
  • Temperature-sensitive mutations are, for example, TS7 or TS12, or TS12.
  • a temperature-sensitive Sendai virus vector comprising KLF RNA, OCT RNA, and SOX RNA is, for example, SeV(PM)KOS/TS7 ⁇ F or SeV(PM)KOS/TS12 ⁇ F, or SeV(PM)KOS/TS12 ⁇ F. be.
  • a temperature-sensitive Sendai virus vector containing MYC RNA has, for example, G69E, T116A, and A183S mutations in the M protein, A262T, G264R, and K461G mutations in the HN protein, and L511F mutation in the P protein. and the F gene-deleted Sendai virus vector having the N1197S and K1795E mutations in the L protein, which is a Sendai virus vector having the above mutations of TS7, TS12, TS13, TS14, or TS15.
  • a temperature sensitive mutation is for example TS15.
  • a temperature-sensitive Sendai virus vector containing MYC RNA is, for example, SeV(HNL)MYC/TS12 ⁇ F, SeV(HNL)MYC/TS13 ⁇ F, or SeV(HNL)MYC/TS15 ⁇ F, or SeV(HNL)MYC/TS15 ⁇ F. be.
  • a Sendai virus vector containing KLF RNA and not containing OCT RNA and SOX RNA has, for example, G69E, T116A, and A183S mutations in the M protein and A262T, G264R, and K461G mutations in the HN protein. It is an F gene-deleted Sendai virus vector having an L511F mutation in the P protein and N1197S and K1795E mutations in the L protein.
  • a Sendai virus vector containing KLF RNA and not containing OCT RNA and SOX RNA is, for example, weaker in temperature sensitivity than the Sendai virus vector introduced with the above-mentioned TS7, TS12, TS13, TS14, or TS15 mutation,
  • the KLF gene can be expressed even at temperatures above .
  • a Sendai virus vector containing KLF RNA and not containing OCT RNA and SOX RNA is, for example, SeV18+KLF4/TS ⁇ F.
  • Sendai virus vectors When introducing multiple types of Sendai virus vectors into cells, for example, multiple types of Sendai virus vectors are simultaneously introduced into cells. Alternatively, it is preferable to introduce all types of Sendai virus vectors into cells within 48 hours after the introduction of one type of Sendai virus vector into cells.
  • the multiplicity of infection (MOI) of the Sendai virus vector when infecting cells is, for example, 0.1 or more. Moreover, MOI is 100 or less, for example.
  • the temperature at which the cells are infected with the Sendai virus vector should be below a predetermined temperature at which the stability of the viral nucleic acid of the temperature-sensitive Sendai virus vector decreases, that is, the temperature at which the viral nucleic acid of the temperature-sensitive Sendai virus vector is stable. or above a predetermined temperature.
  • the temperature at which cells are infected with the Sendai virus vector reduces the stability of the viral nucleic acid of the temperature-sensitive Sendai virus vector.
  • the temperature is preferably lower than the predetermined temperature, that is, the temperature at which the viral nucleic acid of the temperature-sensitive Sendai virus vector is stable.
  • Cells into which reprogramming factors are introduced may be adherent or suspension cultured.
  • Somatic cells introduced with reprogramming factors are feeder-free using basement membrane matrices such as Matrigel (Corning), CELLstart®, Laminin 511 (iMatrix-511, nippi), fibronectin, and vitrotin. It may be cultured.
  • basement membrane matrices such as Matrigel (Corning), CELLstart®, Laminin 511 (iMatrix-511, nippi), fibronectin, and vitrotin. It may be cultured.
  • Examples of media in which cells into which reprogramming factors are introduced are cultured include human ES/iPS media such as Primate ES Cell Medium (ReproCELL), Stemfit AK02N, Stemfit AK03 (Ajinomoto), and TeSR-E8 (STEMCELL Technologies).
  • Stem cell culture medium such as can be used.
  • Stem cell culture medium is placed in an incubator such as, for example, a dish, well, or tube.
  • At least 2 days, or 2 to 10 days after infecting the cells with the Sendai virus vector, at a temperature lower than a predetermined temperature at which the stability of the viral nucleic acid of the temperature-sensitive Sendai virus vector decreases, that is, the temperature-sensitive Sendai virus Cells may be cultured at a temperature at which the viral nucleic acid of the vector is stable. After that, the cells may be cultured at a predetermined temperature or higher. While culturing the cells at a predetermined temperature or higher, the medium may be changed, for example, once every two days.
  • the cells After infecting the cells with the Sendai virus vector, the cells may be cultured at a temperature of at least 2 days, or 2 to 10 days, for example, at a temperature of 4.0°C or higher and lower than 37.0°C. Thereafter, the temperature is raised, and the cells may be cultured at a temperature of 36.5°C or higher and 40.0°C or lower. The temperature may be raised once or may be raised stepwise. After raising the temperature, the medium may be changed while culturing the cells, eg every two days.
  • the viral nucleic acid of the temperature-sensitive Sendai virus vector is below a predetermined temperature at which the stability of the viral nucleic acid of the temperature-sensitive Sendai virus vector decreases until stem cell-like colonies begin to appear.
  • Cells may be cultured at a temperature that is stable. After stem cell-like colonies begin to appear, the cells may be cultured at a predetermined temperature or higher. While culturing the cells at a predetermined temperature or higher, the medium may be changed, for example, once every two days.
  • the cells may be cultured at a temperature of, for example, 4.0°C or higher and lower than 37.0°C until stem cell-like colonies begin to appear. After stem cell-like colonies begin to appear, the temperature is increased and the cells may be cultured at temperatures above 36.5°C and below 40.0°C. The temperature may be raised once or may be raised stepwise. After raising the temperature, the medium may be changed while culturing the cells, eg every two days.
  • the cells introduced with the reprogramming factor are recovered, and at least a part of the recovered and mixed cells is seeded in a medium for at least one passage.
  • the reprogramming factor-introduced cells may be collected, and at least a portion of the collected mixed cells may be seeded in culture medium and passaged until stem cells are established. In addition, all of the collected mixed cells may be seeded in the medium.
  • collecting the reprogramming factor-introduced cells and seeding at least a part of the collected and mixed cells in a culture medium for passage means, for example, reprogramming factor-introduced cells, gene It refers to passaging without distinguishing between expression states.
  • cells introduced with reprogramming factors may be seeded in the same culture vessel without being differentiated by gene expression state.
  • recovering the cells introduced with the reprogramming factor, seeding at least a part of the recovered and mixed cells in a medium for passage means, for example, reprogramming the cells introduced with the reprogramming factor. It means to pass without distinguishing at the degree of.
  • cells into which reprogramming factors have been introduced may be seeded in the same culture vessel without being differentiated by the degree of reprogramming.
  • recovering the reprogramming factor-introduced cells and seeding at least a portion of the recovered and mixed cells in a medium for passaging means, for example, that the reprogramming factor-introduced cells are morphologically Passage without discrimination.
  • cells into which reprogramming factors have been introduced may be seeded in the same culture vessel without being differentiated by morphology.
  • recovering the reprogramming factor-introduced cells and seeding at least a portion of the recovered and mixed cells in a medium for passaging means, for example, reprogramming factor-introduced cells to a size It means to pass without distinction.
  • cells into which reprogramming factors have been introduced may be seeded in the same culture vessel without size discrimination.
  • recovering the reprogramming factor-introduced cells and seeding at least a portion of the recovered and mixed cells in a medium for passage means without cloning the reprogramming factor-introduced cells. It means to pass. For example, when passage without cloning, it is not necessary to pick up colonies formed by cells into which a reprogramming factor has been introduced. For example, when passaging without cloning, multiple colonies formed by cells into which a reprogramming factor has been introduced may not be separated from each other. For example, during passaging, cells forming different colonies may be mixed and seeded in the same culture vessel.
  • a single colony formed by cells into which a reprogramming factor has been introduced may not be cloned.
  • colonies may be mixed together and seeded in the same culture vessel during passaging.
  • the adherently cultured cells may be collected, and at least a portion of the collected and mixed cells may be seeded in a medium for passage. good.
  • cells may be detached from a culture vessel and at least a portion of the detached and mixed cells may be seeded into the same culture vessel.
  • the cells may be detached from the incubator with a detachment solution, and the detached and mixed whole cells may be passaged.
  • non-colony forming cells may be passaged.
  • the whole cells in suspension culture may be subcultured.
  • the cells When subculturing cells into which a reprogramming factor has been introduced, the cells may be seeded in a medium or incubator at a low concentration.
  • the low concentration is, for example, 1 cell/cm 2 or more, 0.25 ⁇ 10 4 cells/cm 2 or less, 1.25 ⁇ 10 3 cells/cm 2 or less, or 0.25 ⁇ 10 3 cells/cm 2 or less. cm 2 or less, 0.25 ⁇ 10 2 cells/cm 2 or less, or 0.25 ⁇ 10 1 cells/cm 2 or less.
  • low concentration means that 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less cells can contact each other.
  • the concentration at which 11 or more cells do not come into contact with each other is a concentration at which 11 or more cells do not come into contact with each other.
  • the state where the entire bottom surface of the cell container is covered with cells is 100% confluent, and low concentration means 5% or less confluent, 4% or less confluent, 3% or less confluent, 2% or less confluent, 1% or less confluent, 0 0.5% confluent, 0.1% confluent, 0.05% confluent, or 0.01% confluent.
  • the low concentration is, for example, a concentration at which single cells do not contact each other in seeded cells.
  • wells of a well plate may be seeded with single cells.
  • a well plate may be a 12-well plate or a 96-well plate.
  • the cells may be cultured at a predetermined temperature or higher at which the stability of the viral nucleic acid of the temperature-sensitive Sendai virus vector decreases after passage. After passage, the cells are cultured at a temperature of, for example, 36.5°C or higher and lower than 38.0°C. After passage, for example, the cells are cultured at a temperature of 36.5 ° C. or higher and less than 38.0 ° C. until intercellular adhesion starts, and after intercellular adhesion starts, a higher temperature until intercellular adhesion starts, For example, cells may be cultured at a temperature of 37.5°C or higher and 42.0°C or lower. After subculturing, the cells may be cultured at a temperature of 37.5° C. or higher and 42.0° C. or lower before cell-cell adhesion starts.
  • Cells introduced with reprogramming factors may be cultured and passaged in a closed incubator.
  • a closed incubator does not exchange, for example, gases, viruses, microorganisms and impurities with the outside.
  • cells into which a reprogramming factor has been introduced may be expanded by two-dimensional culture, or expanded by three-dimensional culture.
  • the whole adherent cultured cells may be cryopreserved as pluripotent stem cells.
  • whole cells detached from the incubator with a detachment solution may be cryopreserved as pluripotent stem cells.
  • the whole cells in suspension culture may be cryopreserved as pluripotent stem cells.
  • Induced pluripotent stem cells can form flat-shouldered colonies similar to ES cells and express alkaline phosphatase. Induced pluripotent stem cells can express undifferentiated cell markers such as Nanog, OCT4, and SOX2. Induced pluripotent stem cells can express TERT. Induced pluripotent stem cells may exhibit temoutherase activity.
  • a cytoflow meter which are cell surface markers indicating undifferentiated TRA-1-60, TRA-1-81, SSEA-1, and It may be done by analyzing whether at least one surface marker selected from SSEA5 is positive.
  • TRA-1-60 is an antigen specific to iPS/ES cells. Since iPS cells can be generated only from the TRA-1-60 positive fraction, TRA-1-60 positive cells are considered to be iPS cell seeds.
  • the method of introducing an inducer to oligodendrocytes into cells is not particularly limited.
  • Inducers are introduced into cells, for example, by electroporation.
  • inducers are introduced into cells by, for example, the lipofection method.
  • the inducer is introduced into the cell using, for example, a viral vector.
  • the inducer may be introduced into the cell integration-free.
  • the cells into which the oligodendrocyte inducer is introduced may be adherent or suspension cultured.
  • Somatic cells introduced with an inducer to oligodendrocytes are treated with basement membrane matrices such as Matrigel (Corning), CELLstart (registered trademark, ThermoFisher), Laminin511 (iMatrix-511, nippi), fibronectin, and vitrotin. , may be cultured feeder-free.
  • basement membrane matrices such as Matrigel (Corning), CELLstart (registered trademark, ThermoFisher), Laminin511 (iMatrix-511, nippi), fibronectin, and vitrotin.
  • stem cell medium such as Stemfit (Ajinomoto) and mTeSR (Stem Cell Technologies) can be used.
  • a gel medium for example, is used when cells into which an inducer has been introduced are cultured in suspension or three-dimensionally.
  • a gel medium is prepared, for example, by adding gellan gum to a stem cell medium.
  • the gel medium contains gellan gum, hyaluronic acid, rhamsan gum, diutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, deltaman sulfate, rhamnan sulfate, and It may contain at least one polymer compound selected from the group consisting of salts thereof. Moreover, the gel medium may contain methylcellulose. Containing methylcellulose further suppresses aggregation between cells.
  • the gel medium contains poly(glycerol monomethacrylate) (PGMA), poly(2-hydroxypropyl methacrylate) (PHPMA), Poly (N-isopropylacrylamide) (PNIPAM), amine terminated, carboxylic acid terminated, maleimide terminated, N-hydroxysuccinimide ( NHS) ester terminated, triethoxysilane terminated, Poly (N-isopropylacrylamide-co-acrylamide), Poly (N-isopropylacrylamide-co-acrylic acid), Poly (N-isopropylacrylamide-co-butylacrylate), Poly (N-isopropylacrylamide-co- methacrylic acid), Poly (N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-isopropylacrylamide.
  • PGMA poly(glycerol monomethacrylate)
  • PPMA poly(2-hydroxypropyl methacrylate)
  • the gel medium may or may not contain growth factors such as basic fibroblast growth factor (bFGF).
  • the gel medium may contain growth factors such as bFGF at low concentrations of 400 ⁇ g/L or less, 40 ⁇ g/L or less, or 10 ⁇ g/L or less.
  • the gel medium may contain TGF- ⁇ , may not contain TGF- ⁇ , or may contain TGF- ⁇ at a low concentration of 600 ⁇ g/L or less, 300 ⁇ g/L or less, or 100 ⁇ g/L or less.
  • the gel medium does not have to be stirred. Also, the gel medium may not contain feeder cells.
  • the gel medium may contain at least one substance selected from the group consisting of cadherin, laminin, fibronectin, and vitronectin.
  • the cells are produced within, for example, 30 days, 20 days, 10 days, or 7 days after the introduction of the inducer into the cells. It can be directed to oligodendrocytes.
  • Immature oligodendrocytes such as oligodendrocyte progenitor cells and mature oligodendrocytes express at least one selected from O4 and PLP (Myelin Proteolipid Protein) 1 .
  • Mature oligodendrocytes express MOG (Myelin Oligodendrocyte Glycoprotein) and PDGFR ⁇ (platelet-derived growth factor receptor ⁇ ).
  • Example 1 A dish coated with laminin 511 was used as a dish for inducing pluripotent stem cells.
  • human peripheral blood mononuclear cells were suspended in a blood medium, and the number of mononuclear cells was measured using a hemocytometer to adjust the number of mononuclear cells in the blood medium. After that, the mononuclear cells were two-dimensionally cultured on a dish for pluripotent stem cell induction at 37° C. for 1 to 7 days.
  • SeV(PM) hKOS/TS12 ⁇ F and SeV(HNL) hC-Myc/TS15 ⁇ F were added to two-dimensionally cultured mononuclear cells at an MOI of 5, and a dish for inducing pluripotent stem cells was prepared. was placed in an incubator at 34° C. to culture the cells. Two days after infection, the blood medium was replaced with iPS cell medium. After that, the medium was changed once every two days using the iPS cell medium. On the way, the temperature was raised stepwise to 37°C and 38°C.
  • stem cell-like cell clusters were generated. Almost all the cells became TRA1-60-positive cells on the 14th day after the infection, and exhibited an iPS cell-like morphology. 14 days after the infection, a cell detachment agent, Triple Select, was added to the dish, allowed to stand at room temperature for 1 minute, then the cell-containing solution was aspirated, and the cell-containing solution was incubated at 37°C for 5 to 10 minutes. . Thereafter, an iPS cell medium was added, and the iPS cell medium containing the cells was collected in a 15 mL tube.
  • a cell detachment agent Triple Select
  • Example 2 The number of cells was measured using a hemocytometer, the concentration of the cell-containing solution was adjusted, and the cells were seeded in a well plate so that the concentration was 0.25 ⁇ 10 4 cells/cm 2 or less. took over.
  • Example 1 during the first passage, all cells were detached from the well plate and the detached and mixed cells were indiscriminately seeded in the next well plate.
  • Example 2 colonies were picked and cloned during the first passage. In both Examples 1 and 2, the cells were seeded so that 11 or more cells did not come into contact with each other during passage.
  • the well dish was placed in a 37° C. incubator to two-dimensionally culture the cells. After the cells began to divide, the culture temperature was raised to 38°C. After that, in both Examples 1 and 2, all the cells were collected every time the cells became 60% to 80% confluent, and at least a part of the collected and mixed cells was seeded in the medium and subcultured. . Cells were seeded in well plates at a concentration of 0.25 ⁇ 10 4 cells/cm 2 or less for the second and subsequent passages. Also in this case, 11 or more cells did not contact each other.
  • the number of colonies formed 5 days after the Sendai virus disappeared in the cells was counted.
  • clonal efficiency was calculated by dividing the number of colonies by the number of cells plated. The results of three tests are shown in FIG. When all cells are harvested at the first passage using mTeSR Plus as the medium, and some of the harvested and mixed cells are plated in the medium and passaged, the clonal efficiency ranges from about 5%. It was about 8%, and the variation was small. When using mTeSR Plus as the medium and cloning by colony picking at the first passage, the clonal efficiency is sometimes less than 1% and sometimes about 6%. There was variability.
  • Clonal efficiency is about 10% to about 10% when all cells are harvested at the first passage using StemFit in the medium and some of the harvested mixed cells are plated in the medium and passaged. 15%, and the variation was small.
  • the clonal efficiency is sometimes less than 1% and sometimes about 16%, and the clonal efficiency varies. was there.
  • Example 3 A well dish coated with a solubilized basement membrane preparation (Matrigel, Corning) was prepared, and ROCK (Rho-associated coiled-coil forming kinase) inhibitor (Selleck) was added to each well at a concentration of 10 nmol/mL.
  • ROCK Rho-associated coiled-coil forming kinase inhibitor
  • Human iPS cells were dispersed with a tissue/culture cell detachment/separation/dispersion solution (Accutase, Innovative Cell Technologies) and seeded in a well dish. After that, the iPS cells were cultured for 24 hours in a feeder-free medium under gaseous conditions of 5% carbon dioxide concentration and 20% oxygen concentration.
  • a single-cell suspension was prepared by detaching the iPS cells from the wells using a detachment agent.
  • OLIG2, SOX10, ASCL1, and NKX2.2 as inducers were introduced into iPS cells by electroporation using a Human Stem Cell Nucleofector Kit (registered trademark, LONZA) and an episomal plasmid. After that, the inducer-introduced cells were seeded in a well dish and cultured in mTeSR.
  • Oligodendrocyte medium contains DMEM/F12, N2 (1X), B27 (1X), penicillin-streptomycin (1X), NEAA (1X), insulin (25 ⁇ g/mL, Sigma), PDGF-AA (10 ng/ml, PeproTech), IGF (10 ng/mL, PeproTech), NT3 (1 ng/mL, PeproTech), biotin (100 ng/mL, Sigma), and cAMP (1 ⁇ mol/L, Sigma).
  • the wells were also supplemented with oligodendrocyte medium.
  • the inducer-introduced cells were seeded on human fibroblasts or human glial cells, and the inducer-introduced cells were cultured until 21 days.
  • Cells on day 7 after introduction of the inducer were stained with an anti-O4 antibody.
  • the cells 21 days after introduction of the inducer were stained with anti-O4 antibody, anti-PLP1 antibody, and anti-MOG antibody.
  • the cells 7 days after the introduction of the inducer expressed O4, a marker for oligodendrocytes and their progenitor cells.
  • O4, PLP1, and MOG were expressed in the cells 21 days after introduction of the inducer. Whether the iPS cells to be introduced with the inducer are the iPS cells prepared in Example 1 or the iPS cells prepared in Example 2, the expression of markers for oligodendrocytes and their progenitor cells is confirmed.
  • Example 4 Inducing factors were introduced into iPS cells in the same manner as in Example 3, except that OLIG2, SOX10, ASCL1 and NKX6.1 were used as inducers. As shown in FIG. 7, the inducer-introduced cells expressed O4.
  • Example 5 Inducing factors were introduced into iPS cells in the same manner as in Example 3, except that OLIG2, SOX10, and ASCL1 were used as inducers. As shown in FIG. 8(a), the inducer-introduced cells expressed O4.
  • Example 6 Inducing factors were introduced into iPS cells in the same manner as in Example 3, except that OLIG2 and SOX10 were used as inducers. As shown in FIG. 8(b), the inducer-introduced cells expressed O4.
  • Example 7 Inducing factors were introduced into iPS cells in the same manner as in Example 3, except that OLIG2 and ASCL1 were used as inducers. As shown in FIG. 8(c), the inducer-introduced cells expressed O4.
  • Example 8 An inducer was introduced into iPS cells in the same manner as in Example 3, except that OLIG2 was used as the inducer. As shown in FIG. 8(d), the inducer-introduced cells expressed O4.
  • Example 9 Inducing factors were introduced into iPS cells in the same manner as in Example 3, except that OLIG2 and NKX2.2 were used as inducers. As shown in FIG. 8(e), the inducer-introduced cells expressed O4, a marker for oligodendrocytes and their progenitor cells.
  • Example 10 A well dish coated with a solubilized basement membrane preparation (Matrigel, Corning) was prepared and media was added to each well. In addition, Ficoll (GE)-separated human adult peripheral blood mononuclear cells were seeded in well dishes. Mononuclear cells were then cultured in blood medium (StemSpan®, SFEM II, STEMCELL TECHNOLOGIES) at 37° C. for 1 to 7 days. Medium contained 20 ng/mL FLT3, 10 ng/mL TPO, 50 ng/mL IL6, 10 ng/mL GCSF, 50 ng/mL SCF, 20 ng/mL IL3, 10 ng/mL GM-CSF. . It was cultured on a dish under gaseous conditions of 5% carbon dioxide concentration and 20% oxygen concentration.
  • the mononuclear cells were collected from the wells and a single cell suspension was prepared. Next, OLIG2, SOX10, ASCL1 and NKX2.2 as inducers were introduced into mononuclear cells by electroporation using a Nucleofector kit for CD34+ cells (registered trademark, LONZA) and an episomal plasmid. After that, the inducer-introduced cells were seeded in a well dish and cultured in blood medium.
  • OLIG2, SOX10, ASCL1 and NKX2.2 as inducers were introduced into mononuclear cells by electroporation using a Nucleofector kit for CD34+ cells (registered trademark, LONZA) and an episomal plasmid. After that, the inducer-introduced cells were seeded in a well dish and cultured in blood medium.
  • oligodendrocyte medium was added to the wells.
  • the wells were also supplemented with oligodendrocyte medium.
  • all medium in the wells was replaced with fresh oligodendrocyte medium.
  • the inducer-introduced cells were seeded on human fibroblasts, and the inducer-introduced cells were cultured until 28 days.
  • Cells 28 days after introduction of the inducer were stained with anti-O4 antibody, anti-PLP1 antibody and anti-MOG antibody, respectively. As shown in FIG. 9, cells 28 days after introduction of the inducer expressed O4, PLP1 and MOG.
  • Example 11 A well dish coated with a solubilized basement membrane preparation (Matrigel, Corning) was prepared and medium (DMEM containing 10% FBS) was added to each well. In addition, human neonatal fibroblasts were seeded in well dishes. The fibroblasts were then cultured on the dish at 37° C. for 1 to 7 days under gaseous conditions of 5% carbon dioxide concentration and 20% oxygen concentration.
  • Fibroblasts were detached from the wells using a detachment agent to prepare a single-cell suspension.
  • OLIG2, SOX10, ASCL1, and NKX2.2 as inducers were introduced into fibroblasts by electroporation using a Human Dermal Fibroblast Nucleofector Kit (registered trademark, LONZA) and an episomal plasmid.
  • LONZA Human Dermal Fibroblast Nucleofector Kit
  • the inducer-introduced cells were seeded in a well dish and cultured in a 10% FBS DMEM medium.
  • oligodendrocyte medium was added to the wells.
  • the wells were also supplemented with oligodendrocyte medium.
  • all medium in the wells was replaced with fresh oligodendrocyte medium.
  • the inducer-introduced cells were seeded on human fibroblasts, and the inducer-introduced cells were cultured until 28 days.
  • Cells 28 days after introduction of the inducer were stained with anti-O4 antibody, anti-PLP1 antibody and anti-MOG antibody, respectively. As shown in FIG. 10, cells 28 days after introduction of the inducer expressed O4, PLP1 and MOG.
  • Example 12 A well dish coated with a solubilized basement membrane preparation (Matrigel, Corning) was prepared and media was added to each well. In addition, human adult peripheral blood-derived T cells were seeded in well dishes. After that, the T cells were expanded on a dish at 37° C. for 1 to 7 days under gaseous conditions of 5% carbon dioxide concentration and 20% oxygen concentration. Note that the expansion culture does not necessarily have to be performed.
  • a serum-free medium (X-VIVO10, Lonza) supplemented with 30 U/mL interleukin-2 and beads for human T cell proliferation stimulation (Dynabeas CD3/CD28, BD) was used.
  • T cells were collected from the wells and a single cell suspension was prepared.
  • OLIG2, SOX10, ASCL1 and NKX2.2 as inducers were introduced into mononuclear cells by electroporation using a Tcell electroporation kit (registered trademark, LONZA) and an episomal plasmid. After that, the inducer-introduced cells were seeded in a well dish, and serum-free medium (X-VIVO10 , Lonza).
  • oligodendrocyte medium was added to the wells.
  • the wells were also supplemented with oligodendrocyte medium.
  • all medium in the wells was replaced with fresh oligodendrocyte medium.
  • the inducer-introduced cells were seeded on human fibroblasts, and the inducer-introduced cells were cultured until 28 days.
  • Cells 28 days after introduction of the inducer were stained with anti-O4 antibody, anti-PLP1 antibody and anti-MOG antibody, respectively. As shown in FIG. 11, cells 28 days after introduction of the inducer expressed O4, PLP1 and MOG.
  • Example 13 Oligodendrocytes were induced from human iPS cells in the same manner as in Example 3, except that p53DD was added as an inducer. The results are shown in FIG. Addition of p53DD increased the percentage of cells showing O4 positivity.
  • Example 14 Oligodendrocytes were induced from human iPS cells in the same manner as in Example 3, except that RB gene shRNA was added to the inducer. The results are shown in FIG. Addition of Rb Sh increased the percentage of cells showing O4 positivity.
  • Example 15 Oligodendrocytes were induced from human iPS cells in the same manner as in Example 3, except that c-MYC was added as an inducer. The results are shown in FIG. Addition of c-MYC increased the percentage of cells showing O4 positivity.
  • Example 16 Oligodendrocytes were induced from human adult peripheral blood-derived mononuclear cells in the same manner as in Example 10, except that p53DD was added as an inducer. The results are shown in FIG. Addition of p53DD increased the percentage of cells showing O4 positivity.
  • Example 17 Oligodendrocytes were induced from human adult peripheral blood-derived mononuclear cells in the same manner as in Example 10, except that RB Sh was added as an inducer. The results are shown in FIG. Addition of Rb Sh increased the percentage of cells showing O4 positivity.
  • Example 18 Oligodendrocytes were induced from human adult peripheral blood-derived mononuclear cells in the same manner as in Example 10, except that c-MYC was added as an inducer. The results are shown in FIG. Addition of c-MYC increased the percentage of cells showing O4 positivity.

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