The present invention relates to a nerve cell degeneration inhibitor. The present invention provides an agent that is highly effective in inhibiting the formation of RNA foci and DPRs for neurodegenerative diseases associated with abnormal expansion of the G4C2 repeat of the C9orf72 gene, and can be used as a prophylactic or therapeutic agent for said diseases.

The present invention relates to agents (medicines, pharmaceutical compositions) containing compounds that has an inhibitory activity on motor neuron degeneration (e.g., amyotrophic lateral sclerosis, progressive bulbar paralysis, progressive muscular atrophy, primary lateral sclerosis, progressive pseudobulbar paralysis, spinal muscular atrophy, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy) and is useful for treating motor neuron disorder and the like.

The present invention provides a drug kit for rebuilding a damaged neural pathway, wherein the drug kit comprises a first formulation comprising axonal elongation-inducing protein, nucleic acid encoding said protein, or a vector that expressibly contains DNA encoding said protein that induces axonal elongation of tranplanted neuronal cells. The present invention also provides a method for rebuilding a damaged neural pathway in a patient, the method comprising the administration of the first formulation of this drug kit.

The present invention provides a method for producing skeletal muscle stem cells with improved purity, comprising a step for inducing differentiation of pluripotent stem cells into skeletal muscle cells, and a step for recovering, from a cell population in the process of inducing differentiation, cells positive for the cell surface marker FGFR4, CDH13-positive cells, or FGFR4-positive and CDH13-positive cells.

The present invention provides a method for producing skeletal muscle stem cells with improved purity, comprising a step for inducing differentiation of pluripotent stem cells into skeletal muscle cells, and a step for recovering, from a cell population in the process of inducing differentiation, cells positive for the cell surface marker FGFR4, CDH13-positive cells, or FGFR4-positive and CDH13-positive cells.

The present invention provides a method for producing a skeletal muscle cell of which the contraction activity can be induced by an optical stimulation, the method comprising (1) a step for producing a pluripotent stem cell capable of stably expressing channelrhodopsin, (2) a step for differentiating/inducing the cell produced in step (1) into a skeletal muscle cell, and (3) a step for irradiating the cell of which the differentiation/induction is initiated with light.

Provided is an efficient method for producing genome-edited cells, in which targeted DNA double strand breaks (DSBs) in the genome are repaired by homology-directed repair (HDR) or comparable pathway (alternative pathway), the method improving the efficiency of HDR or comparable pathway and thus performing accurate genome editing. In one embodiment, the method comprises the steps of: (1) synchronizing the cell cycle of the cells; (2) treating the cells with an agent that modifies a DNA repair pathway; (3) introducing a donor DNA into the cells; (4) inducing a sequence-specific DSB in the genomic DNA of the cells in the presence of the donor DNA; and (5) repairing DSB according to all or part of the donor DNA sequence by HDR or comparable pathway, thereby obtaining genome-edited cells.

Provided is an efficient method for producing genome-edited cells, in which targeted DNA double strand breaks (DSBs) in the genome are repaired by homology-directed repair (HDR) or comparable pathway (alternative pathway), the method improving the efficiency of HDR or comparable pathway and thus performing accurate genome editing. In one embodiment, the method comprises the steps of: (1) synchronizing the cell cycle of the cells; (2) treating the cells with an agent that modifies a DNA repair pathway; (3) introducing a donor DNA into the cells; (4) inducing a sequence-specific DSB in the genomic DNA of the cells in the presence of the donor DNA; and (5) repairing DSB according to all or part of the donor DNA sequence by HDR or comparable pathway, thereby obtaining genome-edited cells.

Provided is a method for producing mature myotube cells from skeletal muscle stem cells, comprising the steps of: (1) seeding skeletal muscle stem cells in a cell culture container and culturing the cells using a growth medium; (2) exchanging the medium with a differentiation medium and continuing the culture until some of the cells show a spindle shape; and (3) overlaying a gel, which contains an extracellular matrix component, on the cells and performing the culture using the differentiation medium. The mature myotube cells produced according to the invention from a patient with a genetic muscle disease can suitably be used for the pathological study of the disease and for drug screening.

Provided is a lysosomal disease preventive and/or therapeutic agent comprising an HMG-CoA reductase inhibitor. The inventors found that HMG-CoA reductase inhibitors decreased autophagosome accumulation in a skeletal muscle cell differentiated from an iPS cell derived from patients of Pompe Disease and/or GNE Myopathy, and thereby completed this invention.

Provided is a method for producing a cell having a scarless genome sequence wherein an exogenous nucleic acid sequence inserted into a targeted region in the genome is completely excised, wherein the exogenous nucleic acid sequence comprises a nucleic acid sequence homologous to a genome sequence in the targeted region at each end and one or more sequence-specific nuclease-recognizing site(s) between the two homologous nucleic acid sequences, and wherein the method comprises: (1) introducing the sequence-specific nuclease or a nucleic acid encoding the same into a host cell having a genome sequence into which the exogenous nucleic acid sequence is inserted; and (2) culturing the cell obtained in step (1), thereby causing double-strand break at the sequence-specific nuclease-recognizing site(s) and the subsequent microhomology-mediated end joining or single-strand annealing between the resulting broken ends that contain the homologous nucleic acid sequences to generate a cell having a scarlessly reverted genome sequence.

The invention provides a method for selecting neural progenitor cells, comprising steps of introducing into the cells a mRNA (miRNA switch) in which a sequence recognized by a miRNA expressed in the neural progenitor cells and a sequence encoding a marker protein is functionally linked (or a DNA that encodes said mRNA) and selecting the cells in which the expression of said marker protein is suppressed.

Provided is a therapeutic or prophylactic agent for ectopic ossification, which contains (i) a Plasminogen Activator Inhibitor-1 (PAI1) inhibitor and/or (ii) a Matrix Metalloproteinase-1 (MMP1) inhibitor as active ingredient(s). Also provided is a method for screening such agent, comprising steps of (a) differentiating neural crest cells having an ACVR1 mutation (incl. those differentiated from pluripotent stem cells) into mesenchymal stromal cells in the presence or absence of a test compound, (b) differentiating the mesenchymal stromal cells obtained in (a) into chondrocytes, (c) measuring the cartilage tissue mass in the culture of (b), and (d) selecting the test compound as a therapeutic or prophylactic agent for ectopic ossification when the presence of the test compound in (a) has decreased the cartilage tissue mass.

Provided is a method for producing differentiation of cerebral cortex neurons from pluripotent stem cells, comprising steps of (i) suspension culture of pluripotent stem cells in a medium containing a TGFβ inhibitor, bFGF, a Wnt inhibitor, and a BMP inhibitor, (ii) suspension culture of the cells obtained in (i) in a medium containing a Wnt inhibitor and a BMP inhibitor, and (iii) further culturing the cells obtained in (ii). Subsequent to step (iii), the method may further comprise a step (4) selecting the cell which expresses at least one of the marker proteins selected from the group consisting of CD231, PCDH17 and CDH8.

Provided is a method for producing, from a donor cell, a low-antigenic cell that causes a reduced immune rejection when allogenically transplanted into a recipient, the method comprising a selective deletion of HLA alleles that are present in the donor cell but not in the recipient (donor-specific HLA alleles) through a targeted disruption or modification of the donor-specific HLA alleles by means of genome-editing technology. Being distinct from B2M knock-out cells (HLA-null cells), the low-antigenic cells of the invention are capable of evading NK cell attack (missing-self response) while having antigen-presenting ability. One aspect of the invention pertains to those cells in which both HLA-A alleles and HLA-B alleles are disrupted but at least one allele of HLA-C is retained (HLA-C retained cells). The HLA-C retained cells are especially useful in the field of cell therapy, since 12 kinds of HLA-C retained cells cover more than 93% of the world's population.（The low-antigenic cells of the invention are capable of evading NK cell attack (missing-self response) while having antigen-presenting ability. Especially the cells in which both HLA-A and HLA-B alleles are disrupted but HLA-C is retained (HLA-C retained cells) are useful in the field of cell therapy, since 12 kinds of HLA-C retained cells cover more than 93% of the world's population.）

Provided is a method for producing, from a donor cell, a low-antigenic cell that causes a reduced immune rejection when allogenically transplanted into a recipient, the method comprising a selective deletion of HLA alleles that are present in the donor cell but not in the recipient (donor-specific HLA alleles) through a targeted disruption or modification of the donor-specific HLA alleles by means of genome-editing technology. Being distinct from B2M knock-out cells (HLA-null cells), the low-antigenic cells of the invention are capable of evading NK cell attack (missing-self response) while having antigen-presenting ability. One aspect of the invention pertains to those cells in which both HLA-A alleles and HLA-B alleles are disrupted but at least one allele of HLA-C is retained (HLA-C retained cells). The HLA-C retained cells are especially useful in the field of cell therapy, since 12 kinds of HLA-C retained cells cover more than 93% of the world's population.（The low-antigenic cells of the invention are capable of evading NK cell attack (missing-self response) while having antigen-presenting ability. Especially the cells in which both HLA-A and HLA-B alleles are disrupted but HLA-C is retained (HLA-C retained cells) are useful in the field of cell therapy, since 12 kinds of HLA-C retained cells cover more than 93% of the world's population.）

Provided is a method for evaluating the neurotoxicity of a compound by use of neurons differentiated from an iPS cell derived from a Charcot-Marie-Tooth disease (CMT) patient. This invention is based on a finding that the neurons from a CMT patient was more susceptible than neurons from a control subject, in terms of mitochondrial aggregation in a neurite after exposure to anti-cancer drugs which are known to cause neurotoxicity in humans.

Provided is a method for inducing differentiation into skeletal muscle progenitor cells from pluripotent stem cells comprising the steps (1) and (2A) or (2B): (1) a step of culturing the pluripotent stem cells in a medium containing a TGF-β inhibitor and a GSK3β inhibitor; (2A) a step of culturing the cells obtained in (1) in a medium containing HGF optionally with IGF1; or (2B) a step of culturing the cells obtained in (1) successively (i) in a medium containing a TGF-β inhibitor, a GSK3β inhibitor, IGF1, HGF and bFGF, (ii) in a medium containing a TGF-β inhibitor, a GSK3β inhibitor and IGF1, and (iii) in a medium containing a TGF-β inhibitor, IGF1 and HGF.

Provided is a method for controlling the activity of a nuclease in response to a cell-specific miRNA, said method comprising linking a nucleic acid sequence specifically recognized by the miRNA to a nucleic acid sequence encoding the nuclease.

Provided is a method for skipping a target exon of a desired gene in a genome using CRISPR-Cas and a guide RNA, characterized in that said guide RNA has such a spacer sequence that the CRISPR-Cas cleavage site is positioned within 80 bases apart either from a splice donor site immediately before the target exon or from a splice acceptor site immediately after the target exon.

The present invention provides a method for selecting a skeletal muscle progenitor cell from a population of cells including skeletal muscle progenitor cells induced from pluripotent stem cells, comprising a step of introducing into the population of cells a mRNA which contains (i) a nucleic acid containing one or more sequence specifically recognized by miRNA selected from the group consisting of miR-1, MiR-133 and miR-206 that is specifically expressed in skeletal muscle progenitor cells, and (ii) a nucleic acid containing a sequence that codes a marker protein functionally linked to the nucleic acid (i).

Provided is a method for screening preventive or therapeutic anti-tauopathy agents, comprising steps of bringing a test substance into contact with neurons containing a mutant MAPT (microtubule-associated protein tau) and measuring for the neurons any one indicator selected from the group consisting of (a) quantity of tau oligomers in culture supernatant, (b) quantity of intracellular tau oligomers, (c) frequency of depolarization, (d) concentration of intracellular calcium ions, and (e) viable cell count.

Provided is a method for efficiently producing synchronized motor neurons from pluripotent stem cells comprising performing the following steps (1) and (2) in this order: (1) a step for introducing into the pluripotent stem cells a nucleic acid that encodes Lhx3, Ngn2, and Isl1; and (2) a step for maintaining the expression of Lhx3, Ngn2, and Isl1 for three days or more.

This invention relates to a method for producing astrocytes from neural progenitor cells, the method comprising; (1) culturing neural progenitor cells in a culture medium compring a neurotrophic factor; (2) dissociating the cells obtained in the step 1; and (3) subjecting the cells obtained in the step (2) to adherent culture in a culture medium comprising a neurotrophic factor using an uncoated culture vessel.

Provided is a method for screening therapeutic or prophylactic agents for Alzheimer's disease comprising steps of: (1) contacting neurons or astrocytes differentiated from iPS cells of the disease patients with or without APP E693 mutation with a compound, and (2) measuring the amount of Aβoligomer, ER stress (BiP), cleaved caspase 4 activities, Transgelin, or oxidative stress (PRDX4 or ROS) in the neurons or astrocytes.

Highly efficient method for generating an iPS cell comprising introduction into a somatic cell of: (1) one or more episomal vectors carrying nucleic acids encoding nuclear reprogramming factors and (2) an episomal vector which is different from the episomal vector (1) and carries a nucleic acid encoding EBNA-1.

Highly efficient method for generating an iPS cell comprising introduction into a somatic cell of: (1) one or more episomal vectors carrying nucleic acids encoding nuclear reprogramming factors and (2) an episomal vector which is different from the episomal vector (1) and carries a nucleic acid encoding EBNA-1.

Provided is a method for efficiently producing a matured skeletal muscle cell from a pluripotent stem cell comprising a step of expressing one or more exogenous factors selected from MyoD, Myf5 and nucleic acids encoding them in the pluripotent stem cell.

This invention, in one major embodiment, relates to a method for producing an iPS cell, which comprises bringing a somatic cell into contact with a combination of (a) Oct3/4 or a nucleic acid encoding the same, (b) Klf4 or a nucleic acid encoding the same, and (c) Sox2 or a nucleic acid encoding the same, as well as (d1) L-Myc or a nucleic acid encoding the same and/or (d2) a functional inhibitor of p53. Preferably, the reprogramming factors are introduced into a somatic cell by means of an episomal vector, especially of the early self-removal type, containing loxP sequences.

This invention relates to a method for inducing differentiation of pluripotent stem cells into neural precursor cells, which comprises culturing pluripotent stem cells in the presence of a small molecule BMP inhibitor.

Invention relating to the method of improving the efficiency of establishment of induced pluripotent stem cells by inhibiting the p53 function in the step of somatic cell nuclear reprogramming. In this invention, the following substances are exemplified as method of inhibiting p53 function; (1)chemical inhibitors of p53, (2) dominant-negative mutants of p53, (3)siRNA and shRNA against p53, (4）inhibitors of p53 pathway.

Invention relating to a method of producing iPS cells, comprising the step of introducing a non-viral expression vector (preferably a plasmid vector) incorporating at least one gene that encodes a reprogramming factor into a somatic cell. iPS cells wherein nuclear reprogramming factors are not incorporated into the chromosome can be produced by the method of this invention.

・Invention related to the method for producing an iPS cell efficiently, comprising the step of introducing 3 genes (Oct family gene, Klf famil gene and Sox family gene) into the somatic cell.
・Invention relating to the method for producing an iPS cell efficiently, comprising the step of introducing 2 genes (either a combination of Oct family genes and Sox family genes or that of Oct family genes and Klf family genes) and at least one gene selected from 3 genes (L-Myc, Sall1 and Sall4) into the somatic cell.