WO2015131788A1 - Traitement de troubles neurologiques - Google Patents

Traitement de troubles neurologiques Download PDF

Info

Publication number
WO2015131788A1
WO2015131788A1 PCT/CN2015/073472 CN2015073472W WO2015131788A1 WO 2015131788 A1 WO2015131788 A1 WO 2015131788A1 CN 2015073472 W CN2015073472 W CN 2015073472W WO 2015131788 A1 WO2015131788 A1 WO 2015131788A1
Authority
WO
WIPO (PCT)
Prior art keywords
neuronal
cell
cells
inhibitor
subject
Prior art date
Application number
PCT/CN2015/073472
Other languages
English (en)
Inventor
Gang Pei
Jian Zhao
Lin Cheng
Wenxiang Hu
Binlong QIU
Original Assignee
Shanghai Institutes For Biological Sciences,Chinese Academy Of Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Institutes For Biological Sciences,Chinese Academy Of Sciences filed Critical Shanghai Institutes For Biological Sciences,Chinese Academy Of Sciences
Publication of WO2015131788A1 publication Critical patent/WO2015131788A1/fr

Links

Images

Classifications

    • 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
    • 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
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • 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
    • G01N33/502—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 for testing non-proliferative effects
    • 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
    • G01N33/502—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 for testing non-proliferative effects
    • G01N33/5026—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 for testing non-proliferative effects on cell morphology
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/065—Modulators of histone acetylation
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts

Definitions

  • the present disclosure relates to systems and methods of use. In particular, it relates to methods of producing a phenotype in a non ⁇ neuronal cell and methods of treating a subject having a neurological condition.
  • Neuron related diseases are a great threat to public health. Because neurons have little or no regenerative capacity, conventional therapies neither effectively nor efficiently alleviate symptoms of these diseases.
  • cell ⁇ based methods such as introducing exogenous neural stem cells or neurons have been adopted to improve treatment of the diseases.
  • these cell ⁇ based methods face various challenges such as limited cell resources, unclear delivery strategies, and complexity of cell integration as well as cellular maturation during the treatment. For example, while adult fibroblasts may be reprogrammed and become induced pluripotent stem cells, reprogramming ⁇ based therapies for neuron related diseases still have concerns related to, for example, safety and efficiency.
  • Embodiments of the present disclosure relate to a method of producing a neuronal phenotype in a glial cell, the method comprising: contacting a glial cell with a composition comprising an inhibitor of a histone deacetylase (HDAC) .
  • HDAC histone deacetylase
  • Embodiments of the present disclosure further relate to another method of treating a subject having a neurological condition, the method comprising administering a therapeutically effective does of a composition comprising an inhibitor of a HDAC, thereby producing a neuronal phenotype in a glial cell of the subject to ameliorate the neurological condition of the subject.
  • Embodiments of the present disclosure further relate to yet another method of treating a subject having a neurological condition, the method comprising providing a therapeutically effective does of a composition comprising an inhibitor of a HDAC, and delivering the therapeutically effective does of the composition to a nervous system of the subject, the composition producing a neuronal phenotype in a glial cell of the nervous system of the subject to ameliorate the neurological condition of the subject.
  • the neuronal phenotype may comprise at least one of: a neuronal morphological characteristic, a neuronal immunological characteristic, or a neuronal physiological characteristic.
  • the neuronal phenotype may comprise at least one of: presence of a marker of a neuron, an electrophysiological characteristic of a neuron, synapse formation, or release of a neurotransmitter.
  • the neuronal phenotype may comprise presence of at least one of doublecortin (DCX) , polysialylated neural cell adhesion molecule (PSA ⁇ NCAM) , hexaribonucleotide binding protein ⁇ 3 (NeuN) , or Neuron ⁇ specific class III beta ⁇ tubulin (Tuj1) in the glial cell.
  • DCX doublecortin
  • PSA ⁇ NCAM polysialylated neural cell adhesion molecule
  • NeN hexaribonucleotide binding protein ⁇ 3
  • Tuj1 Neuron ⁇ specific class III beta ⁇ tubulin
  • the neuronal phenotype may comprise presence of NeuN and/or Tuj1 in the glial cell.
  • NeuN or Tuj1 is expressed in the glial cell after at least one of DCX, SRY (sex determining region Y) ⁇ box 2 (Sox2) , PSA ⁇ NCAM is expressed in the glial cell.
  • the neuronal phenotype may comprise a neuronal phenotype associated with a neuroblast and/or a mature neuron.
  • the composition further comprise at least one of an inhibitor of a GSK ⁇ 3 kinase, an inhibitor of a TGF ⁇ pathway, or an antiallergic compound.
  • the inhibitor of the histone deacetylase may comprise valproic acid (VPA) .
  • the composition may comprise VPA and at least one of CHIR99021, RepSox, or Tranilast.
  • the embodiments may further comprise expressing in the glial cell an exogenous fibroblast growth factor 2 (FGF2) and/or an exogenous epidermal growth factor (EGF) .
  • FGF2 exogenous fibroblast growth factor 2
  • EGF epidermal growth factor
  • the glial cell may be characterized by absence of glial fibrillary acidic protein (GFAP) .
  • GFAP glial fibrillary acidic protein
  • the producing the neuronal phenotype in the glial cell may comprise producing a neuronal phenotype of a neuroblast before producing a neuronal phenotype of a mature neuron.
  • the neuronal phenotype of the neuroblast may comprise presence of DCX and/or PSA ⁇ NCAM in the glial cell, and the neuronal phenotype of the mature neuron may comprise presence of at least one of PSA ⁇ NCAM, NeuN, or Tuj1 in the glial cell.
  • the glial cell may comprise an astrocyte and/or a NG2 cell.
  • the glial cell is in vivo or in vitro.
  • the glial cell is a human glial cell.
  • the neurological condition may comprise at least one of Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis (ALS) , stroke, brain injuries, or spinal cord injuries.
  • Alzheimer disease Parkinson disease
  • amyotrophic lateral sclerosis ALS
  • stroke stroke
  • brain injuries or spinal cord injuries.
  • the neurological condition comprises an injury to a central nervous system and/or peripheral nervous system.
  • the neurological condition is characterized by presence of reactive astrocytes.
  • Embodiments of the present disclosure further relate to a method of producing a neuronal phenotype in a somatic non ⁇ neuronal cell, the method comprising: contacting the a somatic non ⁇ neuronal cell with a composition comprising at least one of an activator of a cyclic AMP pathway, an inhibitor of a GSK ⁇ 3 kinase, an inhibitor of protein kinase C (PKC) , or an inhibitor of Rho ⁇ associated kinase (ROCK) .
  • a composition comprising at least one of an activator of a cyclic AMP pathway, an inhibitor of a GSK ⁇ 3 kinase, an inhibitor of protein kinase C (PKC) , or an inhibitor of Rho ⁇ associated kinase (ROCK) .
  • Embodiments of the present disclosure further relate to a method of treating a subject having a neurological condition, the method comprising: culturing a somatic non ⁇ neuronal cell in vitro with an effective amount of a composition comprising at least one of an activator of a cyclic AMP pathway, an inhibitor of a GSK ⁇ 3 kinase, an inhibitor of protein kinase C (PKC) , or an inhibitor of Rho ⁇ associated kinase (ROCK) for a predetermined time period such that the somatic non ⁇ neuronal cell are induced to produce a neuronal phenotype; and transplanting the induced somatic non ⁇ neuronal cell into a location of a central nerve system of the subject, thereby ameliorating the neurological condition of the subject.
  • a composition comprising at least one of an activator of a cyclic AMP pathway, an inhibitor of a GSK ⁇ 3 kinase, an inhibitor of protein kinase C (PKC) , or an inhibitor of Rh
  • Embodiments of the present disclosure further relate to a method for screening for a therapeutic agent for a neurological condition of a subject, the method comprising: obtaining a somatic non ⁇ neuronal cell from the subject; culturing the somatic non ⁇ neuronal cell in vitro with an effective amount of a composition comprising at least one of an activator of a cyclic AMP pathway, an inhibitor of a GSK ⁇ 3 kinase, an inhibitor of protein kinase C (PKC) , or an inhibitor of Rho ⁇ associated kinase (ROCK) for a predetermined time period such that the somatic non ⁇ neuronal cell are induced to produce a neuronal phenotype; obtaining the induced somatic non ⁇ neuronal cell; contacting the induced somatic non ⁇ neuronal cell with a candidate therapeutic agent; and detecting a physiological or a morphological changes in response to the contacting with the candidate therapeutic agent.
  • a composition comprising at least one of an activator of a cycl
  • the somatic non ⁇ neuronal cell cell comprises at least one of a fibroblast, a blood cells, or a glial cell.
  • the composition comprises the activator of the cyclic AMP pathway, the inhibitor of the GSK ⁇ 3 kinase, the inhibitor of the PKC, and the inhibitor of the ROCK.
  • composition comprises at least one of Forskolin, CHIR99021, GO6983, and Y ⁇ 27632.
  • somatic non ⁇ neuronal cell is obtained from the subject.
  • the neuronal phenotype comprises at least one of: a neuronal morphological characteristic, a neuronal immunological characteristic, or a neuronal physiological characteristic.
  • the neuronal phenotype comprises at least one of: presence of a marker of a neuron, an electrophysiological characteristic of a neuron, synapse formation, or release of a neurotransmitter.
  • the neuronal phenotype comprises presence at least one of hexaribonucleotide binding protein ⁇ 3 (NeuN) , Neuron ⁇ specific class III beta ⁇ tubulin (Tuj1) , or synapsin (SYN) in the somatic non ⁇ neuronal cell.
  • the neuronal phenotype comprises presence of NeuN or Tuj1 in the somatic non ⁇ neuronal cell, a combination thereof.
  • the composition further comprise at least one of an inhibitor of the HDAC, an inhibitor of a TGF ⁇ pathway, or an inhibitor of c ⁇ Jun N ⁇ terminal kinase (JNK) .
  • the composition further comprises at least one of VPA, RepSox, or SP600125.
  • the neurological condition comprises at least one of the neurological condition is Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis (ALS) , Huntington's disease, amyotrophic lateral sclerosis, stroke, progressive supranuclear palsy, Creutzfeldt–Jakob disease, epilepsy, or dementia.
  • Alzheimer disease Parkinson disease
  • amyotrophic lateral sclerosis ALS
  • Huntington's disease amyotrophic lateral sclerosis
  • stroke progressive supranuclear palsy
  • Creutzfeldt–Jakob disease epilepsy
  • dementia dementia
  • the neurological condition comprises an injury to a central nervous system or peripheral nervous system, or a combination thereof.
  • the subject is a mammal.
  • the location of a central nerve system comprises a ventricle of the central nerve system. In certain embodiments, the location of a central nerve system comprises at least one of cerebral cortex, hippocampus, thalamus, or striatum.
  • the transplanting the induced somatic non ⁇ neuronal cell comprises transplanting the induced fibroblasts with a pharmaceutically acceptable carrier.
  • FIGs. 1a and 1b show induced neuroblasts from resident astrocytes by small molecules after brain injury. Data is presented as mean ⁇ s.e.m. DAPI, 4’ , 6 ⁇ diamidino ⁇ 2 ⁇ phenylindole. Scale bars: 50 ⁇ m in (Aand B) , 10 ⁇ m in (C, D and E) . Arrows highlight individual GFP + cells coexpressing neuroblast markers.
  • FIGs. 2a and 2b show that small molecules treatment induces neurogenic potential of astrocytes specifically labelled by retrovirus expressing GFAP: : GFP in mouse cortex and striatum after brain injury.
  • FIGs. 3a and 3b show that induced neuroblasts mature into functional neurons in vivo. Data is presented as mean ⁇ s.e.m.; scale bars: 10 ⁇ m. Arrows highlight individual GFP + cells co ⁇ expressing mature neuronal cell markers.
  • FIG. 4 shows that astrocytes are converted into neuronal cells in situ two weeks after small molecules treatment. Arrows highlight individual cells expressing GFP and neuronal cell markers; Scale bars: 10 ⁇ m.
  • FIGs. 5a and 5b show that cultured astrocytes are induced into functional neurons by small molecules. Data is presented as mean ⁇ s.e.m.; scale bars: 10 ⁇ m.
  • FIGs. 6a, 6b, and 6c show that cultured astrocytes are converted into neurons by small molecules.
  • FIGs. 7a and 7b show that small molecules converts astrocytes into neuronal cells and alleviates brain dysfunction of Parkinson’s disease mouse model. Data is presented as mean ⁇ s.e.m.; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001; scale bars: 10 ⁇ m. Arrows highlight individual GFP + cells co ⁇ expressing neuronal cell markers. Arrowheads indicate GFP + cells without expressing neuronal markers.
  • FIGs. 8a and 8b show small molecules activating NeuroD1 and NeuroG2 in vitro induces astrocytes into neuroblasts in striatum after brain injury and alleviates brain dysfunction of mouse model with Parkinson’s disease.
  • FIGs. 9a, 9b, and 9c show induction of human neuronal cells by small molecules.
  • A Scheme of induction of hciNs from human adult fibroblasts.
  • FIGs. 10a, 10b, 10c, and 10d show screening chemicals for neuronal cells induction and characterization of hciN cells during induction process.
  • FIGs. 11a, 11b, and 11c show that hciN cells expression profiling during chemical reprogramming.
  • FIGs. 12a, 12b, 12c, and 12d show whole ⁇ genome profile of HAFs, hciN cells at day 3, 7 and 14 and control neurons analyzed by cDNA microarray.
  • FIGs. 13a, 13b, and 13c show hciN cells integration in vivo.
  • A Diagram showing the transplantation of GFP ⁇ hciN cells into the cerebral ventricle of E13.5 embryo.
  • FIGs. 14a and 14b show that hciN cells develop into different mature neurons in vivo but control fibroblasts and VCRFSGY ⁇ treated HAFs at day 2 could’t.
  • FIGs. 15a and 15b show generation of hciN cells from FAD fibroblasts.
  • FIG. 16 is a schematic diagram illustrating the possible mechanism of neuronal conversion from HAFs.
  • an element means one element or more than one element.
  • polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, rRNA, cDNA or DNA.
  • the term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA and RNA.
  • polynucleotide variant and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
  • polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51%to at least 99%and all integer percentages in between, e.g., 90%, 95%, or 98%) sequence identity with a reference polynucleotide sequence described herein.
  • polynucleotide variant and variant also include naturally ⁇ occurring allelic variants and orthologs that encode these enzymes.
  • exogenous refers to a polynucleotide sequence that does not naturally ⁇ occur in a wild ⁇ type cell or organism, but is typically introduced into the cell by molecular biological techniques.
  • exogenous polynucleotides include vectors, plasmids, and/or man ⁇ made nucleic acid constructs encoding a desired protein.
  • endogenous or “native” refers to naturally ⁇ occurring polynucleotide sequences that may be found in a given wild ⁇ type cell or organism.
  • polynucleotide sequences that is isolated from a first organism and transferred to second organism by molecular biological techniques is typically considered an “exogenous” polynucleotide with respect to the second organism.
  • polynucleotide sequences can be “introduced” by molecular biological techniques into a microorganism that already contains such a polynucleotide sequence, for instance, to create one or more additional copies of an otherwise naturally ⁇ occurring polynucleotide sequence, and thereby facilitate overexpression of the encoded polypeptide.
  • mutants or “deletion, ” in relation to the genes of a “blood group antigen biosynthesis or storage pathway” refer generally to those changes or alterations in a stem cell that render the product of that gene non ⁇ functional or having reduced function with respect to the synthesis and/or storage of the blood group antigen.
  • Polypeptide, ” “polypeptide fragment, ” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non ⁇ naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally ⁇ occurring amino acid polymers.
  • polypeptides may include enzymatic polypeptides, or “enzymes” , which typically catalyze (i.e., increase the rate of) various chemical reactions.
  • polypeptide variant refers to polypeptides that are distinguished from a reference polypeptide sequence by the addition, deletion or substitution of at least one amino acid residue.
  • a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non ⁇ conservative.
  • the polypeptide variant may include conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide.
  • Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acid residues.
  • “Substantially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99%or greater of some given quantity.
  • “Pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • a pharmaceutically acceptable carrier may include one or more inactive pharmaceutical ingredients.
  • the inactive pharmaceutical ingredients in the pharmaceutically acceptable carrier system may include stabilizers, preservatives, additives, adjuvants, aerosols, compressed air or other suitable gases, or other suitable inactive pharmaceutical ingredients formulated with the therapeutic compound (i.e., an active ingredient (API) ) .
  • an active ingredient i.e., an active ingredient (API)
  • the pharmaceutically acceptable carrier may include the pharmaceutically suitable inactive ingredients known in the art for use in various inhalation dosage forms, such as aerosol propellants (e.g., hydrofluoroalkane propellants) , surfactants, additives, suspension agents, solvents, stabilizers and the like. Alcohol is a good example of an ingredient that may be considered either active or inactive depending on the product formulation.
  • aerosol propellants e.g., hydrofluoroalkane propellants
  • surfactants e.g., hydrofluoroalkane propellants
  • additives e.g., suspension agents, solvents, stabilizers and the like.
  • Alcohol is a good example of an ingredient that may be considered either active or inactive depending on the product formulation.
  • Embodiments of the present disclosure relate to a discovery that somatic non ⁇ neuron cells (e.g., glial cells and fibroblasts) may be converted into neuronal cells using small molecules.
  • somatic non ⁇ neuron cells e.g., glial cells and fibroblasts
  • astrocytes in the mouse brain may be induced into neuroblasts by delivering a composition including small molecules. These induced neuroblasts may further mature into functional neurons, for example expressing mature neuron markers and resembling functional neuron phenotypes.
  • administration of the composition may convert astrocytes into neuronal cells in the brain of a mouse model with Parkinson’s disease to attenuate brain dysfunction and to cause improvement in the mouse model’s behavior.
  • Embodiments of the present disclosure further relates to systems and methods of producing a neuronal phenotype in a glial cell.
  • the embodiments include contacting a glial cell with a composition.
  • Embodiments of the present disclosure further relates to systems and methods of treating a subject having a neurological condition.
  • the embodiments include administering a therapeutically effective does of a composition.
  • the embodiments may include providing a therapeutically effective does of a composition, and delivering the therapeutically effective does of the composition to a nervous system of the subject.
  • the composition may include an inhibitor of a HDAC.
  • the glial cell may produce a neuronal phenotype in a glial cell of the subject to ameliorate the neurological condition of the subject.
  • Embodiments of the present disclosure further relates to systems and methods of producing a neuronal phenotype in a contractile cell (e.g., pericyte) .
  • the embodiments include contacting a pericyte with a composition.
  • the composition may include one or more agents.
  • agent as used herein means any compound or substance such as a small molecule, nucleic acid, polypeptide, peptide, drug, ion, vectors, etc.
  • An “agent” can be any chemical, entity or moiety, including synthetic and naturally ⁇ occurring proteinaceous and non ⁇ proteinaceous entities.
  • the agent is a compound that can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
  • the composition may include one or more small molecules.
  • a small molecule may be a low molecular weight compound that may help regulate a biological process (e.g., a signaling pathway) .
  • a small molecule may include an inhibitor of HDAC, an inhibitor of GSK ⁇ 3 kinases, an inhibitor of TGF ⁇ pathways, an antiallergic compound, an activator of cyclic AMP pathways (e.g., Forskolin) , an inhibitor of Notch signaling pathways, and/or an inhibitor of P53 signaling pathways, etc.
  • Example of small molecules may include a cAMP activator (e.g., Forskolin) , an ALK5 inhibitor (e.g., SB431542) , a BMP inhibitor (e.g., LDN193189, Dorsomorphin, DMH1) , a GSK3 inhibitor (e.g., CHIR99021) , a COX ⁇ 2 inhibitor (e.g., Isoxazole) , a Retinoic acid receptor agonist (e.g., Retinoid acid) , 2' ⁇ O ⁇ Dibutyryl ⁇ cAMP (e.g., db ⁇ cAMP) , a notch inhibitor (e.g., DAPT) , a p38 MAPK inhibitor (e.g., SB202190) , an inhibitor of PI3K/AKT (e.g., LY294002) , a TGF ⁇ inhibitor (e.g., RepSox) , an Erk inhibitor (e.g.,
  • the inhibitor of HDAC may include VPA.
  • the inhibitor of HDAC may further include sodium butyrate (NaB) and Trichostatin A (TSA) .
  • the composition may include 0.2 ⁇ 1 mM of VPA, such as 0.3 ⁇ 0.8 mM of VPA or 0.4 ⁇ 0.6 mM of of VPA, 0.2 ⁇ 1 mM of NaB, such as 0.3 ⁇ 0.8 mM or 0.4 ⁇ 0.6 mM of NaB, and/or 5 ⁇ 20 nM of TSA, such as 8 ⁇ 15 nM of TSA or 10–12 nM of TSA.
  • the composition may include at least one of an inhibitor of the HDAC, an inhibitor of a GSK ⁇ 3 kinase, an inhibitor of a TGF ⁇ pathway, or an antiallergic compound.
  • the composition may include an inhibitor of a HDAC or an antiallergic compound (e.g., Tranilast) , or a combination thereof.
  • phenotype refers to one or more well ⁇ known detectable characteristics of cells, such as glial cells and neurons.
  • a neuronal phenotype may include at least one of: neuronal morphology, neuronal immunology, neuronal physiology such as synapse formation and/or release of neurotransmitter, the presence of one or more neuronal markers, electrophysiological characteristics of neurons.
  • the one or more neuronal markers may include a neuroblast marker: DCX, a neural progenitor cell marker: Sox2, a neuroblast marker: PSA ⁇ NCAM, mature neuron markers: Tuj1 and/or NeuN.
  • the presence of a marker refers to a condition that an amount of a marker protein and/or gene product (e.g. mRNA) is significantly increased in a glial cell that has been contacted with the composition for a predetermined time period as compared to the amount of the same marker present in a glial cell that has not been contacted with the composition.
  • the absence of a marker refers to another condition that an amount of a marker protein and/or gene product (e.g. mRNA) is significantly decreased in the glial cell that has been contacted with the composition for a predetermined time period as compared to the amount of the same marker present in a glial cell that has not been contacted with the composition.
  • a "marker” as used herein is used to describe a characteristic and/or phenotype of a cell. Markers may be used for selection of cells including characteristics of interests. Markers vary with specific cells. Markers may include characteristics including morphological, functional or biochemical (enzymatic) characteristics of the cell of a particular cell type, or molecules expressed by the cell type. In some embodiments, the markers may include proteins. In certain embodiments, the markers are an epitope for an antibody and/or other binding molecules available in the art.
  • a marker may include molecules found in a cell including proteins (peptides and polypeptides) , lipids, polysaccharides, nucleic acids and steroids.
  • morphological characteristics may include shape, size, and synapse formation.
  • functional characteristics may include the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, ability to release transmitters, and the ability to differentiate along particular lineages. Markers may be detected by any methods available in the art. Markers may also be the absence of a morphological characteristic or absence of proteins, lipids etc. Markers may be a combination of a panel of unique characteristics of the presence and absence of polypeptides as well as other morphological characteristics.
  • a glial phenotype such as astrocyte phenotype and/or reactive astrocyte phenotypes may include: morphological aspects of astrocytes and/or reactive astrocytes, such as a generally “star ⁇ shaped” morphology.
  • a glial phenotype may further include present of a glial cell marker, such as GFAP.
  • a glial cell may be converted into a cell having neuronal phenotype by using a composition.
  • composition converted neuron and “converted neuron” are used herein to refer to a cell that has contacted a composition and has a consequent neuronal phenotype.
  • the neuronal phenotype may include at least one of a neuronal morphological characteristic, a neuronal immunological characteristic, or a neuronal physiological characteristic. In certain embodiments, the neuronal phenotype may include at least one of presence of a marker of a neuron, an electrophysiological characteristic of a neuron, synapse formation, or release of a neurotransmitter.
  • the neuronal phenotype may include characteristic morphological aspects of a neuron such as presence of dendrites, an axon and dendritic spines; characteristic neuronal protein expression and distribution, such as presence of synaptic proteins in synaptic puncta, presence of Microtubule ⁇ associated protein 2 (MAP2) in dendrites; and characteristic electrophysiological signs such as spontaneous and evoked synaptic events of viable neurons.
  • characteristic morphological aspects of a neuron such as presence of dendrites, an axon and dendritic spines
  • characteristic neuronal protein expression and distribution such as presence of synaptic proteins in synaptic puncta, presence of Microtubule ⁇ associated protein 2 (MAP2) in dendrites
  • MAP2 Microtubule ⁇ associated protein 2
  • a neuronal phenotype may include a neuronal phenotype of a neuroblast and/or a mature neuron.
  • the neuronal phenotype may include presence of one or more neuronal markers including at least one of DCX, Sox2, PSA ⁇ NCAM, NeuN, or Tuj1 in a glial cell.
  • these neuronal markers may be detected using immunophysiological assays, molecular biology assays and/or other methods available in the art.
  • a neuronal phenotype may include presence of a marker of a mature neuron, such as NeuN and Tuj1, in glial cell, a marker of a neural progenitor cell, such as Sox2, or a marker of a neuroblast, such as DCX and PSA ⁇ NCAM.
  • NeuN or Tuj1 may be expressed in the glial cell after at least one of DCX, Sox2, PSA ⁇ NCAM is expressed in the glial cell.
  • the glial cell the glial cell may be characterized by absence of GFAP.
  • the glial cell may stop expressing GFAP or decrease expression of GFAP such that GFAP is not detectable in the glial cell.
  • a neuronal phenotype of a neuroblast in the glial cell may be produced before a neuronal phenotype of a mature neuron is produced.
  • the neuronal phenotype of the neuroblast may include presence of DCX and/or PSA ⁇ NCAM in the glial cell
  • the neuronal phenotype of the mature neuron may include presence of at least one of PSA ⁇ NCAM, NeuN, or Tuj1 in the glial cell.
  • the glial cell may include at least one of an astrocyte, a reactive astrocyte, microglial cell, Schwann cell, or an oligodendrocyte (e.g., NG2) .
  • the glial cell may be in vivo or in vitro.
  • the glial cell may be a mammalian glial cell, such as a human glial cell.
  • treating and “treatment” are used to refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health ⁇ related condition, and includes at least one of: (1) preventing the disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it; (2) inhibiting the disease or condition, i.e., arresting its development; (3) relieving the disease or condition, i.e., causing regression of the disease or condition; or (4) relieving the symptoms resulting from the disease or condition, i.e., relieving pain without addressing the underlying disease or condition.
  • treatment of a subject may include ameliorating abnormal behaviors and/or relieving the symptoms associated with neurological conditions (e.g., Parkinson’s diseases) by delivering a composition to the subject.
  • neurological conditions e.g., Parkinson’s diseases
  • the terms “disease” and “condition” may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians.
  • the condition may include any conditions of the central and/or peripheral nervous system of a subject which is alleviated, ameliorated or prevented by additional neurons.
  • a disease and/or condition in the present disclosure may include injuries or diseases which result in loss or inhibition of neurons and/or loss or inhibition of neuronal function.
  • injuries or diseases which result in loss or inhibition of neurons and/or loss or inhibition of neuronal functions including Alzheimer's disease, Parkinson disease, Amyotrophic lateral sclerosis (ALS) , stroke, physical injury such as brain or spinal cord injury, and tumor, are neurological conditions for treatment by methods herein.
  • injuries or diseases may result in loss or inhibition of at least one of glutamatergic, glutaminergic, GABAergic, cholinergic, dopaminergic, norepinephrinergic, or seratonergic neurons.
  • the neurological condition may include an injury to a central nervous system and/or peripheral nervous system.
  • the neurological condition may include at least one of the neurological condition is Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis (ALS) , brain injuries, spinal cord injuries, or stroke.
  • the neurological condition may be characterized by presence of reactive astrocytes.
  • administered and “delivered” are used to describe the process by which a composition of the present disclosure is administered or delivered to a subject, a target (e.g., a cell, a tissue, an organ, a portion of a system, etc. ) or are placed in direct juxtaposition with the target.
  • a target e.g., a cell, a tissue, an organ, a portion of a system, etc.
  • “administrated” and “delivered” may include a process that inhalation of a subject such that a composition of the present disclosure is delivered to the subject, a target (e.g., a cell, a tissue, an organ, a portion of a system, etc. ) or are placed in direct juxtaposition with the target.
  • a composition of the present disclosure may be administered orally, intravenously, intraperitoneally, subcutaneously, intramuscularly, intrathecally, intradermally, nasally, enterically, pessaries, suppositories.
  • a target may be a region of a nervous system, such as cortex and striatum of the subject.
  • the region of a nervous system may have loss or inhibition of neurons and/or loss or inhibition of neuronal functions induced by injuries or diseases.
  • administered and “delivered” are used interchangeably.
  • contacted and “exposed” when applied to a target are used to describe the process by which a compound of the present disclosure is administered or delivered to a target or are placed in direct juxtaposition with the target.
  • a target e.g., a cell, a tissue, an organ, etc.
  • administered and “delivered” are used interchangeably with “contacted” and “exposed” .
  • the terms “patient” , “subject” and “individual” are used interchangeably herein, and mean a mammalian subject to be treated and/or to obtain a biological sample from.
  • the mammalian includes humans and domestic animals, such as cats, dogs, swine, cattle, sheep, goats, horses, rabbits, and the like.
  • the composition may reprogram the glial cell into neurons in vitro and in vivo and alleviate brain dysfunction of a mouse model with Parkinson’s disease.
  • the regeneration of neuronal cells in a never system (e.g., a brain) by resident astrocytes after acute or chronic injuries may be used to treat a neurological condition.
  • the composition may include an inhibitor and/or an antagonist.
  • An inhibitor/antagonist is a molecule that binds to another molecule and therefore decreases the activity of the other molecule.
  • an enzyme inhibitor may bind to an enzyme and decrease the function of the enzyme.
  • the composition may include one or more inhibitors or antagonists, such as an inhibitor of HDAC, an inhibitor of TGF ⁇ pathways, an inhibitor of GSK ⁇ 3 kinases or an antiallergic compound.
  • An activator is a molecule that bind to another molecule and therefore enhances the activity of the other molecule.
  • the composition may include at least one of an inhibitor of the HDAC, an inhibitor of GSK ⁇ 3 kinases, an inhibitor of TGF ⁇ pathways, or an antiallergic compound.
  • the inhibitor of the HDAC may include VPA.
  • the inhibitor of GSK ⁇ 3 kinases may include CHIR99021, LiCl, or Li 2 CO 3 .
  • the composition may include 1–5 ⁇ M of CHIR99021, such as 2–4 ⁇ M of CHIR99021, 0.5 ⁇ 3 ⁇ M of LiCl, such as 1–2 ⁇ M, and/or 0.05 ⁇ 1 mM of Li 2 CO 3 , such as 0.1 ⁇ 0.8 mM or 0.2 ⁇ 0.5 mM of Li 2 CO 3 .
  • the composition may include 0.2–3 ⁇ M of RepSox, such as 0.5–2 ⁇ M of RepSox, 0.2–3 ⁇ M of SB431542, such as 0.5–2 ⁇ M, and/or 10–50 ⁇ M of Tranilast, such as 20–40 ⁇ M.
  • the composition may include VPA and least one of CHIR99021, RepSox, or Tranilast.
  • the methods may further include expressing in the glial cell an exogenous fibroblast growth factor 2 (FGF2) and/or an exogenous epidermal growth factor (EGF) .
  • FGF2 exogenous fibroblast growth factor 2
  • EGF epidermal growth factor
  • the delivery vehicle carrying a polynucleotides encoding FGF2 and/or EGF may be a vector, a liposome, a polymer, a pharmaceutically acceptable composition, or a device which facilitates delivery of such delivery vehicle.
  • the vector may be selected from the group consisting of adenovirus vectors, retrovirus vectors, adeno ⁇ associated virus vectors, herpes simplex virus vectors, SV40 vectors, polyoma virus vectors, papilloma virus vectors, picarnovirus vectors, vaccinia virus vectors, lentiviral vectors, alphaviral vectors, a helper ⁇ dependent adenovirus, and a plasmid.
  • adenovirus vectors retrovirus vectors, adeno ⁇ associated virus vectors, herpes simplex virus vectors, SV40 vectors, polyoma virus vectors, papilloma virus vectors, picarnovirus vectors, vaccinia virus vectors, lentiviral vectors, alphaviral vectors, a helper ⁇ dependent adenovirus, and a plasmid.
  • an “effective” means adequate to accomplish a desired, expected, or intended result.
  • an “effective amount” may be an amount of a compound sufficient to produce a therapeutic benefit.
  • the terms “therapeutically effective” or “therapeutically beneficial” refers to anything that promotes or enhances the well ⁇ being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the onset, frequency, duration, or severity of the signs or symptoms of a disease.
  • the term “therapeutically effective amount” is meant an amount of a composition as described herein effective to yield the desired therapeutic response.
  • the amount of a compound of the disclosure which constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and its severity, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to her own knowledge and to this disclosure.
  • diagnosis means identifying the presence or nature of a pathologic condition.
  • safe and effective amount refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used as described herein.
  • the specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any) , and the specific formulations employed and the structure of the compounds or its derivatives.
  • Embodiments of the present disclosure further relate to a method of producing a neuronal phenotype in a somatic non ⁇ neuronal cell, the method comprising: contacting the a somatic non ⁇ neuronal cell with a composition comprising at least one of an activator of a cyclic AMP pathway, an inhibitor of a GSK ⁇ 3 kinase, an inhibitor of protein kinase C (PKC) , or an inhibitor of Rho ⁇ associated kinase (ROCK) .
  • a composition comprising at least one of an activator of a cyclic AMP pathway, an inhibitor of a GSK ⁇ 3 kinase, an inhibitor of protein kinase C (PKC) , or an inhibitor of Rho ⁇ associated kinase (ROCK) .
  • Embodiments of the present disclosure further relate to a method of treating a subject having a neurological condition, the method comprising: culturing a somatic non ⁇ neuronal cell in vitro with an effective amount of a composition comprising at least one of an activator of a cyclic AMP pathway, an inhibitor of a GSK ⁇ 3 kinase, an inhibitor of protein kinase C (PKC) , or an inhibitor of Rho ⁇ associated kinase (ROCK) for a predetermined time period such that the somatic non ⁇ neuronal cell are induced to produce a neuronal phenotype; and transplanting the induced somatic non ⁇ neuronal cell into a location of a central nerve system of the subject, thereby ameliorating the neurological condition of the subject.
  • a composition comprising at least one of an activator of a cyclic AMP pathway, an inhibitor of a GSK ⁇ 3 kinase, an inhibitor of protein kinase C (PKC) , or an inhibitor of Rh
  • Embodiments of the present disclosure further relate to a method for screening for a therapeutic agent for a neurological condition of a subject, the method comprising: obtaining a somatic non ⁇ neuronal cell from the subject; culturing the somatic non ⁇ neuronal cell in vitro with an effective amount of a composition comprising at least one of an activator of a cyclic AMP pathway, an inhibitor of a GSK ⁇ 3 kinase, an inhibitor of protein kinase C (PKC) , or an inhibitor of Rho ⁇ associated kinase (ROCK) for a predetermined time period such that the somatic non ⁇ neuronal cell are induced to produce a neuronal phenotype; obtaining the induced somatic non ⁇ neuronal cell; contacting the induced somatic non ⁇ neuronal cell with a candidate therapeutic agent; and detecting a physiological or a morphological changes in response to the contacting with the candidate therapeutic agent.
  • a composition comprising at least one of an activator of a cycl
  • germline cells also known as “gametes”
  • gametes are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body—apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells—is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells.
  • the somatic cell is an “adult somatic cell” , by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro.
  • a somatic cell e.g., fibroblast
  • the methods for direct conversion of a somatic cell, e.g., fibroblast to a neuron can be performed both in vivo and in vitro (where in vivo is practiced when somatic a somatic cell, e.g., fibroblast are present within a subject, and where in vitro is practiced using isolated somatic a somatic cell, e.g., fibroblast maintained in culture) .
  • the somatic cell is a “somatic non ⁇ neuronal cell” , by which is meant a somatic cell that is not a neuron (e.g., a motor neuron and a Purkinje cell) or a neuron in the brain) , and/or a cell having a neuronal phenotype.
  • the somatic non ⁇ neuronal cell may include at least one of a fibroblast, a blood cells, or a glial cell.
  • the composition may include the activator of the cyclic AMP pathway, the inhibitor of the GSK ⁇ 3 kinase, the inhibitor of the PKC, and the inhibitor of the ROCK.
  • the composition may include at least one of Forskolin, CHIR99021, GO6983, and Y ⁇ 27632.
  • the somatic non ⁇ neuronal cell is obtained from the subject.
  • the neuronal phenotype may include at least one of: a neuronal morphological characteristic, a neuronal immunological characteristic, or a neuronal physiological characteristic. In some embodiments, the neuronal phenotype may include at least one of: presence of a marker of a neuron, an electrophysiological characteristic of a neuron, synapse formation, or release of a neurotransmitter.
  • the neuronal phenotype may include presence at least one of NeuN, Tuj1, or SYN in the somatic non ⁇ neuronal cell. In certain embodiments, the neuronal phenotype may include presence of NeuN or Tuj1 in the somatic non ⁇ neuronal cell, a combination thereof.
  • the composition further may include at least one of an inhibitor of the HDAC, an inhibitor of a TGF ⁇ pathway, or an inhibitor of JNK. In certain embodiments, the composition further may include at least one of VPA, RepSox, or SP600125.
  • the neurological condition may include at least one of the neurological condition is Alzheimer disease, Parkinson disease, ALS, Huntington's disease, amyotrophic lateral sclerosis, stroke, progressive supranuclear palsy, Creutzfeldt–Jakob disease, epilepsy, or dementia.
  • the neurological condition may include neurological degeneration and/or an injury to a central nervous system or peripheral nervous system, or a combination thereof.
  • the location of a central nerve system may include a ventricle of the central nerve system. In certain embodiments, the location of a central nerve system may include at least one of cerebral cortex, hippocampus, thalamus, or striatum.
  • the transplanting the induced somatic non ⁇ neuronal cell may include transplanting the induced fibroblasts with a pharmaceutically acceptable carrier.
  • suitable pharmaceutically acceptable carriers include water, salt solutions (such as Ringer's solution) , alcohols, oils, gelatins and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, and polyvinyl pyrrolidine.
  • Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the cells of the present disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the cells of the present disclosure.
  • Molecules Induce Astrocytes into Neuroblasts after Brain Injury
  • Retroviruses were generated to examine whether local astrocytes can be reprogrammed into neuronal cells by small molecules for brain repair.
  • the retrovirus expresses green fluorescent protein (GFP) under the control of human glial fibrillary acidic protein (GFAP) promoter, which selectively label proliferative astrocytes, but not neuronal cells.
  • GFP green fluorescent protein
  • GFAP human glial fibrillary acidic protein
  • GFAP GFP retrovirus into the mouse cortex and striatum, it was found that reactive astrocytes were induced by stab injury ( Figure 2A and 2B) and GFP ⁇ infected cells were immunopositive for astrocyte marker GFAP, no GFP ⁇ positive (GFP + ) cell expressed neural progenitor cell marker Sox2 or neuroblast marker doublecortin (DCX) , mature neuron marker Tuj1 and NeuN ( Figure 2C and 2D) .
  • VCR VPA
  • CHIR99021 C
  • RepSox R
  • GFP + cells in both cortex and striatum showed extensive signals of DCX and polysialylated neural cell adhesion molecule (PSA ⁇ NCAM) , another marker for neuroblast one week after VCR treatment.
  • the number of double positive cells (GFP + DCX + and GFP + PSA ⁇ NCAM + ) was relatively low.
  • No GFP + DCX + cell was detected in cortex and only rare GFP + DCX + cells (less than 0.3%among total GFP + cells) in striatum injected with virus without chemical treatment ( Figure 1C and 1D) .
  • FIG. 1 shows induced neuroblasts from resident astrocytes by small molecules after brain injury.
  • FIGs. 1A and 1B are schematic diagrams illustrating the location of virus injection sites in cortex and striatum.
  • Retrovirus expressing GFPA GFP was injected into cortex (position: AP ⁇ 1.25 mm, ML 1.4 mm, DV ⁇ 1.5 mm) or striatum (AP ⁇ 1 mm, ML 2 mm, DV ⁇ 3 mm) of 4 ⁇ week ⁇ old mouse.
  • Cx cortex
  • CC corpus callosum
  • LV lateral ventricle
  • Str striatum
  • D3V dorsal 3rd ventricle.
  • FIG. 1C and 1D show that no astrocyte ⁇ derived neuroblast was observed in cortical and striatal sites by brain injury itself, but VCR treatment induced subset of astrocytes into DCX + or PSA ⁇ NCAM + neuroblasts in both sites after brain injury.
  • FIG. 1E illustrates that VCR and GFs (FGF2 and EGF) together promoted neuronal reprogramming from astrocytes in vivo.
  • FIG. 1F shows the percentages of GFP + cells expressing DCX or PSA ⁇ NCAM in the cortex and striatum under various conditions after the brain injuries.
  • FIG. 2 shows that VCR treatment induces neurogenic potential of astrocytes specifically labelled by retrovirus expressing GFAP: : GFP in mouse cortex and striatum after brain injury.
  • FIGs. 2 A and 2B show that stab injury itself significantly activated astrocyte proliferation around the injection sites, which highlighted by dashed line. The needle injection of virus itself is considered as a model of stab injury in brain.
  • mice were anesthetized for cardiac perfusion with PBS and fixation in 4%PFA.
  • Mouse brains were harvested for immunofluorescent staining of astrocyte marker and neuronal cell markers (Scale bars: 50 ⁇ m) .
  • FIG. 2C and 2D show that GFP+ cells were immunostained negative for neuroblast marker DCX, neuron markers Tuj1 and NeuN at both injection sites three days after viral injection (Scale bars: 10 ⁇ m) .
  • FIG. 2E shows that no Sox2+ cells appeared around the injection sites in both cortex and striatum three and seven days after VCR treatment (SVZ, subventricular zone; LV, lateral ventricle, Scale bars: 20 ⁇ m) .
  • FIG. 2F shows that VCR treatment caused subsets of GFP + cells losing astrocyte maker GFAP, which is much more obviously in striatum comparing with that in cortex. Arrows indicate GFP + GFAP + cells.
  • FIG. 2G shows astrocytes in striatum labelled by GFP gradually lost astrocytic shape and showed bipolar or branching morphology resembling neuroblast, three or seven days after brain injury with VCR treatment (Scale bars: 10 ⁇ m) .
  • GFP + NeuN + cells were detected from week two then slightly increased between week two and four post chemical treatment (approximately from 2%to 4%in cortex and from 4%to 7%in striatum) .
  • GFP + DCX + cells emerged from week one but dramatically decreased later (roughly from 11%to 5%in cortex and from 25%to 9%in striatum) and became hardly detectable after four weeks (FIGs. 3D, 4A , and 4B) .
  • FIG. 3 shows that induced neuroblasts mature into functional neurons in vivo.
  • FIGs. 3A and 3B shows GFP + cells expressed the mature neuronal markers Tuj1 (A) and NeuN (B) in both injection sites two and four weeks (w) respectively after VCR treatment.
  • FIG. 3C illustrates quantification of cell numbers in FIG. 3A and FIG. 3B.
  • FIG. 3D shows comparison of the numbers of GFP + cells co ⁇ expressing DCX or NeuN at different time points after VCR treatment.
  • FIG. 4 shows that astrocytes are converted into neuronal cells in situ two weeks after VCR treatment.
  • FIGs. 4A and 4B shows that at two weeks after brain injury with VCR treatment, GFP + DCX + cells were still detected at both injection sites, while with lower cell numbers in comparison with that at one week. GFP + NeuN + cells were also detected at this time point and cell number of NeuN+ cells among GFP + cells is higher in striatum than that in cortex.
  • Molecules Convert Astrocytes into Neuronal Cells In Vitro
  • astrocyte were treated with VCR in vitro to verify the cell transition from astrocytes into neurons by small molecules in vivo.
  • VCR treatment changed the cell morphology from astrocytes into neuronal cells in vitro, as shown by the phase contrast images (FIG. 5A) .
  • the isolation protocol were followed and the isolated astrocytes were passaged for at least three passages and labelled the cells by retrovirus expressing GFAP: : GFP (FIGs. 6A ⁇ 6C) to exclude contamination of neuronal cells and neural progenitor cells in primary astrocytes.
  • Cultured mouse astrocytes may be induced directly and efficiently into DCX + neuroblasts (Up to ⁇ 35%) (FIG.
  • the expression of transcription factors were checked to analyze the potential underling mechanisms.
  • the transcription factors include NeuroG2, NeuroD1, Ascl1, Pax6, Dlx2 and Sox2, which alone was reported to sufficiently reprogram astrocytes into neuronal cells in vitro or in vivo.
  • no chemical or chemical mixture was able to activate Dlx2, Sox2 and Pax6 expression.
  • Ascl1 can also be significantly activated by VR, but not by other chemical (s) (FIG. 5H) .
  • Notch signaling regulating bHLH factors is an conserved module and also regulates neurogenic program of astrocytes after brain stroke.
  • the expression pattern of genes involved in this pathways was investigated. It was not found that the chemical (s) caused any significant change in the expression of Notch receptors 1/2/3/4 and Notch ligands Dll1/3/4, and Jagged1/2 and Hes1/5 (FIG. 6H) .
  • the small molecules inducing astrocytic ⁇ neuronal transition act by activating NeuroG2 and NeuroD1 expression independently of Notch signaling.
  • FIG. 5 shows that cultured astrocytes are induced into neurons by small molecules.
  • FIG. 5A shows cultured astrocytes had neuronal morphology 12 days after VCR treatment.
  • GFP + cells stands for astrocytes labelled by retrovirus expressing GFAP: : GFP.
  • FIG. 5B shows that cultured astrocytes were converted into DCX + neuoroblasts 12 days after VCR treatment.
  • FIG. 5C show astrocyte ⁇ converted neurons expressed mature neuronal cell markers Tuj1 and NeuN 18 days after VCR treatment in vitro.
  • FIG. 5D shows that patch clamp recording was conducted on VCR ⁇ induced neurons from cultured astrocytes identified by differential interference contrast microscopy and fluorescence.
  • FIG. 5A shows cultured astrocytes had neuronal morphology 12 days after VCR treatment.
  • GFP + cells stands for astrocytes labelled by retrovirus expressing GFAP: : GFP.
  • FIG. 5B shows that cultured astrocytes were converted
  • FIG. 5E shows current ⁇ clamp recordings of neurons derived from cultured astrocytes with VCR treatment showed a representative train of action potentials with stepwise current injection.
  • FIG. 5F shows representative traces of spontaneous postsynaptic currents in VCR ⁇ induced neurons were recorded.
  • FIG. 5G illustrates GFP + cells co ⁇ expressing DCX and NeuN induced by removing chemical (s) from VCR.
  • FIG. 5H shows heat map depicting the relative fold change of gene expression in astrocytes one week after chemical treatment in vitro. The value in the color indicates log2 changes (relative to HPRT and normalized to ctrl) .
  • FIG. 6 shows that cultured astrocytes are converted into functional neurons by small molecules.
  • FIGs. 6A, 6B, and 6C show characterization of primary astrocytes isolated from mouse cortex. Cortical astrocytes were isolated from P1 mouse pups. In order to exclude the contamination of neuronal cells, mixed cells were passaged for three generations. No exist of Tuj1 + neurons or Sox2 + neural progenitor cells in cultured astrocytes at passage 3. Pure astrocytes showed typical astrocytic shape demonstrated by cell morphology and immunofluorescent staining of well ⁇ established astrocyte marker GFAP, which were highly different from primary neurons (B) and primary neural progenitor cells (NPCs) .
  • B primary neurons
  • NPCs primary neural progenitor cells
  • FIGs. 6D, 6E, and 6F show screening of essential small molecule (s) in VCR inducing astrocytes into neuroblasts and neurons.
  • FIG. 6D shows changes in cell morphology of cultured astrocytes treated with diverse small molecules, removing one or two component (s) from VCR, under conditional medium for 11 days.
  • GFAP retrovirus expression GFAP
  • FIG. 6E shows these chemical ⁇ treated astrocytes were further fixed by 4%PFA for immunofluorescent staining of neuroblast marker DCX.
  • FIG. 6F shows that cultured astrocytes treated with diverse small molecules under conditional medium for 18 days were fixed and immunostained of mature neuron marker NeuN.
  • Treatment of VCR, VR, VC or V alone resulted in generation of DCX + or NeuN + neurons from astrocytes in vitro, but not the treatment of CR, C or R.
  • Chemical ⁇ induced neurons also showed typical neuronal cell morphology (Arrowheads highlight individual cells co ⁇ expressing GFP and NeuN.V, VPA; C, CHIR99021; and R, RepSox. Scale bars: 20 ⁇ m) .
  • FIG. 6G and 6H show that astrocytic ⁇ neuronal transition in vitro is unrelated with neural progenitor cell state and Notch signaling.
  • FIG. 6G shows no significant expression of Sox2 during the entire cell transition from astrocytes to neuronal cells at day 1, 3, 5 and 7 post VCR treatment. Sample data is normalized to that of control (Ctrl) , which is considered as 1. Data is presented as mean ⁇ s.e.m.
  • FIG. 6H shows a heat map depicting the relative fold change of expression of genes involved in Notch signaling pathways, including Notch receptors 1, 2, 3 and 4, and Notch ligands Jagged 1 and 2, Dll 1 and 2, and Hes 1 and 5, in astrocytes one week after chemical treatment in vitro.
  • FIG. 6I shows small molecules converting astrocytes into neuronal cells activate NeuroD1 expression.
  • VT ⁇ induced neuroblasts from resident astrocytes can also develop into mature neurons expressing Tuj1 and NeuN in both wild ⁇ type mice and MPTP ⁇ treated mice (FIGs. 7C and 7D) .
  • FIG. 7 shows that drug mixture converts astrocytes into neuronal cells and alleviates brain dysfunction of Parkinson’s disease mouse model.
  • FIGs. 7A and 7B show that DCX + , Tuj1 + and NeuN + cells were generated from cultured astrocytes under the treatment of drug mixture VT.
  • FIGs. 7C and 7D shows systematic administration of VT for four weeks caused astrocytes ⁇ derived mature neurons to appear in mouse striatum after brain injury with or without MPTP intoxication.
  • FIGs. 7E and 7F show brain function of mice subjected to MPTP intoxication followed by i.p. injection of chemical compounds was evaluated by pole test and wire hanging maneuver. T ⁇ LA, locomotor activity time.
  • 7 G, 7H, and 7I shows mean densities of striatal dopaminergic termini (G) , striatal astrocytes (H) and numbers of dopaminergic neurons in substantia nigra (SN) (I) were determined and quantified by digital image analysis.
  • FIG. 8 show drug mixture activating NeuroD1 and NeuroG2 in vitro induces astrocytes into neuroblasts in striatum after brain injury and alleviates brain dysfunction of mouse model with Parkinson’s disease.
  • FIG. 8A shows application of Q ⁇ PCR for detecting expression level of transcription factors reported to convert astrocytes into neuronal cells.
  • FIG. 8B shows systematic administration of drug mixture, VT converts local astrocytes in striatum of adult mice into DCX + neuroblasts.
  • FIG. 8C shows schematic representation of the experiment design. Two days before MPTP intoxication, 8 ⁇ week ⁇ old C57/BL6 mice were trained for pole test and wire hanging maneuver.
  • mice displaying symptoms of Parkinson’s disease were kept and systematic administrated with drugs including L ⁇ 3, 4 ⁇ dihydroxyphenylalanine (L ⁇ DOPA, 20 mg/kg) increasing dopamine levels in the striatum, VPA (V, 150 mg/kg) and/or Tranilast (T, 100 mg/kg) twice daily for one month.
  • drugs including L ⁇ 3, 4 ⁇ dihydroxyphenylalanine (L ⁇ DOPA, 20 mg/kg) increasing dopamine levels in the striatum, VPA (V, 150 mg/kg) and/or Tranilast (T, 100 mg/kg) twice daily for one month.
  • L ⁇ 3, 4 ⁇ dihydroxyphenylalanine L ⁇ DOPA, 20 mg/kg
  • VPA V, 150 mg/kg
  • Tranilast Tranilast
  • FIG. 8D and 8E show IHC staining of tyrosine hydroxylase (TH) , GFAP in striatum and substantia nigra (SN) .
  • TH tyrosine hydroxylase
  • SN substantia nigra
  • Retrovirus was produced by transfection of plat ⁇ E cells with retroviral vectors, using FugeneHD transfection reagent (Roche) as previously described. Retroviral solution was collected and concentrated by Retro ⁇ Concentin virus precipitation solution (System Biosciences, catalogue #EMB100A ⁇ 1) following protocol provided by the manufacturer.
  • mice Surgeries of viral injection before local administration of small molecules were performed on 4 ⁇ week ⁇ old mice and 8 ⁇ week ⁇ old mice were used for Parkinson’s disease model before systematic delivery of drug mixture. Mice anesthetized with pentobarbital sodium were attached to stereotaxic injection device. Each mouse received lateral injection with virus solution.
  • One injection into the cortex with the coordinate as follows: anterior/posterior (AP) ⁇ 1.25 mm, medial/lateral (ML) 1.4 mm, dorsal/ventral (DV) ⁇ 1.5 mm; the other injecting position in the striatum was AP ⁇ 1 mm, ML 2 mm, DV ⁇ 3 mm.
  • the injection volume and rate were set at 3 ⁇ l with 0.2 ⁇ l/min by a 5 ⁇ l syringe with 34 gauge needle. After the injection, the cannula was left for additional 5 min then slowly withdrawn at a speed of 0.5 mm/min.
  • the virus solution was supplemented with growth factors, including 0.9 ⁇ g/ml FGF2 and 0.9 ⁇ g/ml EGF, in some experiments.
  • mice were also subjected to intracerebroventricular administration of either VCR (3 mM VPA, 1 ⁇ M CHIR99021, and 1 ⁇ M RepSox) or vehicle (1 ⁇ PBS) using osmotic pump (ALZET) , which allowed supplying chemical solution for 30 days.
  • VCR 3 mM VPA, 1 ⁇ M CHIR99021, and 1 ⁇ M RepSox
  • vehicle 1 ⁇ PBS
  • AZAT osmotic pump
  • pump was installed in a subcutaneous pocket in the midscapular area of mouse back and the cannula connecting catheter with the pump was inserted into the lateral ventricle.
  • the injection coordinate was AP 0.1 mm, ML 1 mm, DV ⁇ 2.5 mm.
  • DMEM/F12 medium including B27, N2, 20 ng/ml BDNF and 20 ng/ml GDNF, with indicated chemical (s) .
  • Medium containing chemical compounds was changed every four days. After twelve days, cells were maintained in DMEM/F12 medium without chemicals (400 ng/ml shh, 100 ng/ml FGF8, 10 ng/ml bFGF, 20 ⁇ M L ⁇ Ascorbic acid, 20 ng/ml BDNF, 20 ng/ml GDNF, B27 and N2) .
  • Mouse brain sections were prepared as described. In brief, cardiac PBS ⁇ perfused mouse brains were perfused with 4%PFA. After being cryopreserved in 30%sucrose, the mouse brains were cryosectioned at 20 ⁇ m for further immunofluorescent staining analysis. Cells cultured on glass coverslips were fixed in 4%PFA solution for 10 min then incubated in blocking buffer (1%bovine serum albumin in PBS) with or without 0.5%Triton X ⁇ 100 for 30 min at room temperature (RT) . Afterwards, samples were incubated with primary antibodies at 4 °C overnight and then with appropriate fluorescent probe ⁇ conjugated secondary antibodies for one hour at RT. Nuclei were counterstained with DAPI.
  • mice brain tissues were performed with an ABC kit using DAB detection as described previously and the primary antibodies included GFAP (1: 1000, Millipore, catalogue #AB5804) and Tyrosine Hydroxylase (TH, 1: 1000, Sigma ⁇ Aldrich, catalogue #T2928) .
  • GFAP 1: 1000, Millipore, catalogue #AB5804
  • TH Tyrosine Hydroxylase
  • TH+ neuron numbers in the substantia nigra were automated by stereological analysis and the densities of dopaminergic neuron axonal termini were determined by quantitative densitometric analysis of TH ⁇ stained termini.
  • the astrocytic density in striatum was quantified by measuring the optical density of GFAP ⁇ stained striatal cells.
  • astrocytes were isolated from P1 (one day post birth) mouse brain as previously described. Briefly, mouse pup was sprayed with 70%ethanol then sacrificed by decapitation. After taking out the brain into dissecting dish filled with HBSS on ice, following dissection procedures were performed under a stereomicroscope. Olfactory bulbs, cerebellum and meninges were all carefully removed using the fine dissecting forceps. The remaining cortex pieces were transferred into Falcon tube and digested with 1.25%trypsin in water bath at 37 °Cfor 30 min. Mixed by occasional shaking every 10 min. Pelleted cortex tissue pieces by centrifuge at 300 ⁇ g for 5 min and decanted supernatant.
  • astrocyte culturing medium Added 10 ml astrocyte culturing medium and vigorously pipetted to dissociate tissue pieces into single cells proofing under a hematocytometer.
  • Dissociated single cell suspension was plated on poly ⁇ D ⁇ lysine (Sigma ⁇ Aldrich, catalogue #P0899) coated dish and incubated at 37 °C in the CO2 incubator for one week. Dishes were rigorously shaken daily to remove neurons and non ⁇ astorcytic cells and obtain an enriched astrocyte culture. After reaching confluence, astrocytes were further passaged three times for further experiments. Astrocyte culture purity was characterized by microscopy morphological studies and cell marker expression.
  • Astrocyte culture medium was DMEM/F12 (Gibco, catalogue #11330 ⁇ 032) supplemented with 10%fetal bovine serum, B27 (Gibco, catalogue #17504) , 10 ng/ml EGF (Gibco, catalogue #PHG0311) , 10 ng/ml FGF2 (Gibco, catalogue #PHG0021) and penicillin/streptomycin.
  • mouse neural progenitor cells were derived from E12.5 mouse embryos and expanded in neural expansion medium (Millipore, catalogue #SCM003) supplemented with 30 ng/ml heparin, 20 ng/ml EGF and 20 ng/ml bFGF as described.
  • Electrophysiological analysis was performed just as previous report. Whole ⁇ cell patch clamp recordings were carried out on ciNPC ⁇ derived neurons. Recordings were made using Multiclamp 700B amplifier (Molecular Devices) . The bath was constantly perfused with fresh artificial cerebrospinal fluid (ACSF) at RT. The ACSF contained (in mM) 126 NaCl, 3 KCl, 1.25 KH2PO4, 1.3 MgSO4, 3.2 CaCl2, 26 NaHCO3, and 10 glucose, bubbled with 95%O2 /5%CO2. Signals were sampled at 10 kHz with a 2 kHz low ⁇ pass filter. The whole ⁇ cell capacitance was fully compensated. Recordings with Ra > 50M or fluctuation > 20 %were excluded.
  • the intracellular solution contained (in mM) : 93 K ⁇ gluconate, 16 KCl, 2 MgCl2, 10 HEPES, 4 ATP ⁇ Mg, 0.3 GTP ⁇ Na2, 10 creatine phosphate, 0.5%Alexa Fluor 568 hydrazide (Invitrogen) (pH 7.25, 290/300 mOsm) , and 0.4%neurobiotin (Invitrogen) .
  • Membrane potentials were hold around ⁇ 70 mV, and step currents with an increment of 3 pA were injected to elicit action potentials. Data were analyzed using pClamp10 (Clampfit) .
  • RNA samples were extracted from cells using Trizol reagent according to the manufacturer's instructions (Sigma ⁇ Aldrich, catalogue #T9424) .
  • RNA was reverse ⁇ transcribed to cDNA using random hexamers and M ⁇ MLV Reverse Transcriptase (Promega, catalogue #M5301) .
  • cDNA samples were then mixed with 2 ⁇ PCR Mix (Qiagen) and Eva Green (Biotium) and subjected to real ⁇ time quantitative PCR (Q ⁇ PCR) analysis with an MX3000P Stratagene PCR machine. The relative expression values were normalized against the internal controls (HPRT) . Primers used were listed in table 1.
  • the pole test has been used to detect bradykinesia and motor coordination in PD mice.
  • the mice were placed facing upwards near the top of a pole (50 cm long and 1 cm in diameter) with rough surface that led into their home cage.
  • Two days before MPTP injections mice were trained to turn to orient downward and traverse the pole into their home cage.
  • the mice were tested for the amount of time to turn to orient downward and the total time to descend the pole from the time that the mouse is placed on the pole until it reaches the base of the pole in the home cage. Five trials were performed for each mouse. Wire hanging maneuver tests neuromuscular and locomotors development.
  • mice suspended by their forepaws from a horizontal rod tend to support themselves with their hind limbs, preventing them from falling and aiding in progression along the rod.
  • a bedding ⁇ filled box at the base served as protection for the falling. Suspension latencies were recorded and the cut ⁇ off time was 60 sec.
  • HAFs human adult fibroblasts
  • FS090609 derived from a 28 ⁇ year old male foreskin
  • induced neuronal cells Most of the induced neuronal cells survived until day 10 ⁇ 12 but died before developing into more mature neurons. Additional chemicals were screened to promote neuron survival and maturation. It has found that CHIR99021, Forskolin and Dorsomorphin are beneficial for neuron survival and maturation. It was found that adding these chemicals CFD (CHIR99021, Forskolin and Dorsomorphin) into neuron maturation medium with neurotropic factors significantly promote neuronal cells survival and maturation. 2 or 3 weeks after chemicals treatment, the induced neuronal cells (hciN cells) were positive for mature neuronal markers Tau, NeuN and synapsin (SYN) (FIG. 9C ⁇ 9E) .
  • Tuj1 ⁇ or Map2 ⁇ positive hciN cells The percentage of Tuj1 ⁇ or Map2 ⁇ positive hciN cells relative to initial HAFs were about 13% (FIG. 10C) . It was further found that more than 80%of Tuj1 ⁇ positve cells also expressed vGluT1, however, GABAergic, Cholinergic and Dopaminergic neurons were rarely detected (FIG. 9F and 10D) . Another HAFs (SF002, derived from 25 ⁇ year old male skin) were also treated with these chemicals using the same induction protocol (FIG. 9A) . Consistently, after 14 days induction, cells expressing neuronal markers Tuj1 and Map2 with neuronal morphology were also observed (FIG. 10E) .
  • RNA expression profiling by qRT ⁇ PCR and immunostaining analysis showed that neural progenitor genes Sox2, Pax6 or FoxG1 never expressed during the hciN cells induction procedure (FIG. 10G) .
  • the proliferation marker Ki67 were not detectable since day 3 (FIG. 10H) , suggesting that the induction protocol might trigger a fast cell cycle exit of human fibroblasts, and the conversion of human fibroblasts into hciN cells using chemical strategy is probably direct.
  • FIG. 9 shows induction of human neuronal cells by small molecules.
  • A Scheme of induction of hciNs from human adult fibroblasts. Initial fibroblasts were plated in DMEM and this day is termed as “Day–2” . 2 days later, cells were transferred into induction medium with chemical compounds for 8 days, and then further cultured in maturation medium with small molecules and neurotrophic factors for 2 ⁇ 3 weeks. Bottom panels show phase contrast images of human foreskin fibroblasts (left) or hciN cells at day7 (middle) and day 14 (right) .
  • B hciN cells display bipolar neuronal morphologies and express DCX (green) , Tuj1 (red) and Map2 (green) at day 7. HAFs were treated with indicated chemicals for 1 week, and stained positive for DCX, Tuj1 and Map2. Scale bars: 20 ⁇ m.
  • C ⁇ F hciN cells express mature neuronal markers and show complex neuronal morphologies at day 14 or day 21.
  • HAFs were treated with indicated chemicals for 2 weeks, and stained positive for Tau (C) , NeuN (D) , SYN (E) and vGluT1 (F) . Scale bars: 20 ⁇ m.
  • G Current ⁇ clamp recordings of hciN cells generated from HAFs showing a representative train of action potentials (top panel) . Step currents were injected from ⁇ 10pA to 56pA in 6pA increments (bottom panel) .
  • H Representative traces of tetrodotoxin ⁇ sensitive fast inward currents recorded in voltage ⁇ clamp mode from hciN cells at day 14. The inset shows sodium currents (left panel) .
  • Cells were depolarized from ⁇ 20 mV to 60 mV in 10 mV increments.
  • FIG. 10 shows screening chemicals for neuronal cells induction and characterization of hciN cells during induction process.
  • B Screening chemicals for neuronal cells induction. Morphology change (left panel) and immunostaining images (right panel) of HAFs treated with indicated chemicals for day 7.
  • Scale bars 100 ⁇ m.
  • D Quantification of the percentage of different subtype neurons in Tuj1 ⁇ positive hciN cells.
  • hciNs from HAFs possess basic electrophysiological properties of neurons, such as firing action potential and induction of membrane current
  • whole ⁇ cell patch ⁇ clamp recording of hciN cells were performed with complex neuron morphology at day 14 post chemicals treatment.
  • TTX tetrodotoxin
  • HAFs were infected with lentivirus encoding green fluorescent protein (GFP) and seeded GFP ⁇ positive hciN cells (6 days after VCRFSGY ⁇ treatment) to a monolayer astrocyte culture.
  • GFP green fluorescent protein
  • FIG. 9J whole ⁇ cell patch ⁇ clamp recording on GFP ⁇ positive hciN cells with complex neuron morphology was carried out.
  • hciN cells possess neuronal genes expression signature
  • FIG. 11A A qRT ⁇ PCR analysis was performed to monitor the gene profiles during neuronal conversion of HAFs. It was found that, during the induction process, the expression of neuronal genes were elevated (FIG. 11A) , whereas typical fibroblast specific genes were dramatically down ⁇ regulated (FIG. 11B) .
  • the voltage ⁇ gated sodium channel subunit Scn3b and potassium channel subunit Kcnj6 were also obviously increased as early as day 6 after chemicals treatment (FIG. 12A) , which may be responsible for electrophysiological properties of hciN cells.
  • FIG. 12B glial genes were almost unaltered in hciN cells (FIG. 11D, middle panel) .
  • upregulation of some neuronal genes were also observed in VCRFSGY ⁇ treated HAFs as early as day 3 and 7 (FIG. 12C) .
  • fibroblast specific genes including Col12A1, Tgfb1i1, Thy1 and Ctgf were dramatically downregulated during neuronal conversion (FIG. 11D, right panel and FIG. 12D) .
  • Genes that show a more than 2 folds alteration were subjected to further gene ontology (GO) function enrichment analysis.
  • FIG. 11 shows that hciN cells expression profiling during chemical reprogramming.
  • a and B Induced expression of defined neuronal transcriptional factors (A) and downregulation of certain fibroblast specific genes (B) during neuronal conversion. All sample data are normalized to that of d0, which is considered as 1. Data of three independent experiments are shown as means ⁇ SEM.
  • E Gene ontology (GO) analysis of the overlapping genes whose expression changes are >2 ⁇ fold indentified in hciN cells (day 14 ⁇ 1 and day 14 ⁇ 2) and control neurons compared to that of HAFs. See also Figure S2.
  • FIG. 12 shows whole ⁇ genome profile of HAFs, hciN cells at day 3, 7 and 14 and control neurons analyzed by cDNA microarray.
  • A Channels for potassium (Kcnj6) , calcium (Cacna1h) and sodium (Scn3b) were induced during neuronal conversion. HAFs were collected and analyzed at day 3, 6 post VCRFSGY treatment. All sample data are normalized to that of d0, which is considered as 1. Data of three independent experiments are shown as means ⁇ SEM.
  • B Heatmap for subsets of selected neuronal, glial and fibroblast ⁇ specific genes.
  • C and D Pairwise gene expression comparisons show that some neural markers are increased (C) , whereas some fibroblast specific genes are silenced (D) in hciN cells at day 3 and 7.
  • E Gene ontology (GO) analysis of the downregulated genes in hciN cells, whose expression changes are >2 ⁇ fold indentified in hciN cells (day 14 ⁇ 1 and day 14 ⁇ 2) and control neurons compared to that of HAFs.
  • GFP ⁇ labeled pre ⁇ hciN cells (VCRFSGY ⁇ treated HAFs at day 6) were transplanted to ventricles of E13.5 pups mouse brain (FIG. 13A) .
  • Two additional groups of transplantation were also performed with GFP ⁇ labeled VCRFSGY ⁇ treated HAFs at day 2 and GFP ⁇ labeled HAFs, respectively.
  • the pups were perfused and analyzed 1 week after transplantation.
  • Figure 3B obvious appearances of GFP ⁇ labeled grafts were found in mouse brain sections.
  • GFP ⁇ positive cells develop to be Dcx ⁇ and Tuj1 ⁇ positive (FIG.
  • Glutamatergic neurons, GABAergic neurons and Dopaminergic neurons derived from GFP ⁇ labeled pre ⁇ hciN cells can be observed (FIG. 14B) , which demonstrated that engrafted pre ⁇ hciN cells could develop into several subtype neurons.
  • GFP ⁇ grafts in the brain sections from groups transplanted with VCRFSGY ⁇ treated HAFs for 2 days or GFP ⁇ labeled HAFs were rarely detected, suggesting the loss and death of these engrafted cells possibly due to the failure of integration and immunoreactivity to host mouse brains.
  • Tuj1 + and NeuN + cells co ⁇ labeled with GFP were still detectable (FIG.
  • FIG. 13 shows hciN cells integration in vivo.
  • A Diagram showing the transplantation of GFP ⁇ hciN cells into the cerebral ventricle of E13.5 embryo.
  • B Distribution of GFP ⁇ labeled grafts in mouse brains 1 week after transplantation. GFP ⁇ pre ⁇ hciN cells (VCRFSGY ⁇ treated HAFs at day 6) were transplanted to brains of E13.5 pups. 1 week after transplantation, the brain sections were collected and analyzed. Scale bars: 100 ⁇ m.
  • C In vivo generation of Dcx + (left panel) and Tuj1 + (right panel) immature neurons from transplanted GFP ⁇ pre ⁇ hciN cells (VCRFSGY ⁇ treated HAFs at day 6) .
  • GFP ⁇ pre ⁇ hciN cells were transplanted to brains of E13.5 pups. 1 week after transplantation, the brain sections at the implantation site were collected and subjected to immunostaining. Scale bars: 10 ⁇ m.
  • FIG. 14 shows that hciN cells develop into different mature neurons in vivo but control fibroblasts and VCRFSGY ⁇ treated HAFs at day 2 could’t.
  • A No DCX + (left panel) , Tuj1 + (middle panel) or NeuN + (right panel) neurons were generated from control GFP ⁇ HAFs and VCRFSGY ⁇ treated HAFs at day 2 1 week after transplantation. Scale bars: 20 ⁇ m. 7 pups were transplanted with control fibroblasts, 13 pups were transplanted with VCRFSGY ⁇ treated HAFs at day 2, and representative images were shown.
  • B Different subtype neuron markers were detected from GFP ⁇ pre ⁇ hciN cells were detected 1 month after transplantation.
  • GFP ⁇ pre ⁇ hciN cells were transplanted to brains of E13.5 pups. 1 month after transplantation, the brain sections at the implantation site were collected and subjected to immunostaining with SYN, vGluT1, GAD67 and TH. Scale bars: 10 ⁇ m. 21 pups were transplanted with GFP ⁇ hciN cells, representative images were shown.
  • hciN cells neuronal conversion of human skin fibroblasts derived from patients with familial Alzheimer’s disease (FAD) carrying mutation in APP (V717I) or presenilin1 (I167del) were induced.
  • FAD familial Alzheimer’s disease
  • APP V717I
  • I167del presenilin1
  • the hciN cells derived from FAD fibroblasts display similar neuronal characteristics comparing to hciN cells derived from normal fibroblasts (FIG. 15A) .
  • the induction efficiency of hciN cells from FAD fibroblasts was comparable to that of unaffected fibroblasts (FIG. 15B) .
  • FIG. 15 shows generation of hciN cells from FAD fibroblasts.
  • A Characterization of hciN cells generated from FAD fibroblasts. FAD fibroblasts (AD p109 and AD p131) were treated with chemicals for 2 weeks, and stained positive for Tuj1, Map2 and NeuN.
  • B Quantification of Tuj1 + hciN cells derived from a panel of human fibroblast lines. The conversion efficiency was calculated by dividing the number of Tuj1 ⁇ or Map2 ⁇ positive hciN cells by the number of initial seeding cells in each visual field at day 14.
  • C ⁇ E Higher A ⁇ 42/40 ratio in FAD hciN cell cultures comparing to that of WT hciN cell cultures.
  • FIG. 16 is a schematic diagram illustrating the possible mechanism of neuronal conversion from HAFs.
  • neuronal induction medium DMEM: F12 (Life Technologies, 11330 ⁇ 032) : Neurobasal (Life Technologies, 21103 ⁇ 049) (1: 1) , 0.5%N ⁇ 2 (Invitrogen) , 1%B ⁇ 27 (Invitrogen) , cAMP (100 ⁇ M, Sigma ⁇ Aldrich) and bFGF (10 ng/ml, Invitrogen) ) with indicated chemicals.
  • DMEM neuronal induction medium
  • F12 Life Technologies, 11330 ⁇ 032
  • Neurobasal Neurobasal (Life Technologies, 21103 ⁇ 049)
  • DMEM neuronal induction medium
  • 0.5%N ⁇ 2 Invitrogen
  • 1%B ⁇ 27 Invitrogen
  • cAMP 100 ⁇ M, Sigma ⁇ Aldrich
  • bFGF 10 ng/ml, Invitrogen
  • neuronal maturation medium F12: Neurobasal (1: 1) , 0.5%N ⁇ 2 (Invitrogen) , 1%B ⁇ 27 (Invitrogen) , cAMP (100 ⁇ M, Sigma ⁇ Aldrich) , 10 ng/ml bFGF (Invitrogen) , 20 ng/ml BDNF (PeproTech) , 20 ng/ml GDNF (PeproTech) and 20 ng/ml NT3 (PeproTech)) with indicated chemicals.
  • DMEM F12: Neurobasal (1: 1) , 0.5%N ⁇ 2 (Invitrogen) , 1%B ⁇ 27 (Invitrogen) , cAMP (100 ⁇ M, Sigma ⁇ Aldrich) , 10 ng/ml bFGF (Invitrogen) , 20 ng/ml BDNF (PeproTech) , 20 ng/ml GDNF (PeproTech) and 20 ng/ml NT3 (P
  • VPA concentration of chemicals used is listed as below: VPA, 0.5 mM; CHIR99021, 3 ⁇ M; Repsox, 1 ⁇ M; Forskolin, 10 ⁇ M; SP600125, 10 ⁇ M; GO6983, 5 ⁇ M; Y ⁇ 27632, 5 ⁇ M; Dorsomorphin, 1 ⁇ M.
  • Human adult fibroblasts were derived from human foreskin or skin biopsies of healthy and patient individuals. Institutional ethical committees approved collection and use of human samples.
  • mice were anesthetized on ice or with chloral hydrate and brains section were prepared as described above for further analysis.
  • Primary human adult fibroblasts (FS090609) were established from dissociated foreskin tissue of a healthy donor collected by Shanghai Renji Hospital.
  • Primary human adult fibroblasts (SF002) were obtained from skin biopsy of a healthy donor collected by Guangzhou Institutes of Biomedicine and Health.
  • FAD patient fibroblasts (FAD p109 and p131) were derived from skin biopsies of familial Alzheimer disease patients collected by Hunan Xiangya Hospital. The Ethical Committees of the Shanghai Renji Hospital, Guangzhou Institutes of Biomedicine and Health and Hunan Xiangya Hospital approved collection and use of human samples. Informed consents were obtained from all subjects.
  • Primary fibroblasts cultures were established from biopsies using established methods.
  • Human fibroblasts were maintained in DMEM (Life Technologies, C11965) supplemented with 10%fetal bovine serum (PAA Laboratories, A15101) , 1mM GlutaMAX (Life Technologies, 35050 ⁇ 061) , 0.1 mM non ⁇ essential amino acid (NEAA, Millipore, TMS ⁇ 001 ⁇ C) and penicillin/streptomycin at 37°C with 5%CO 2 .
  • H9 human ES cells (as gift from Dr. Xiaoqing Zhang) were expanded in mTeSR1 (Stem Cell Technologies) and passaged as clumps with dispase or EDTA. Induction of neural stem cells from hES cells were completed by dual inhibition of SMAD signaling as previously described.
  • Derivation of neurons from hES ⁇ derived NPCs were performed in neuronal differentiation medium with BDNF, GDNF, IGF (all at 10 ng/ml, PeproTech) , cAMP (1 ⁇ M, Sigma ⁇ Aldrich) and ascorbic acid (200 ng/ml, Sigma ⁇ Aldrich) were added to promote neural differentiation.
  • the calculation of conversion efficiency was carried out as previously described. Briefly, 10–20 visual fields were randomly selected for each sample on an Olympus IX ⁇ 51 microscope at indicated time points, and counted the total cell number of Tuj1 ⁇ or Map2 ⁇ positive cells with neuron morphology. The conversion efficiency was calculated by dividing the number of Tuj1 ⁇ or Map2 positive hciN cells by the number of initial seeding cells in each visual field. Quantitative data are means ⁇ SEM from at least three independent experiments.
  • Immunostaining of cells were done as previously described. In brief, cells plated on glass coverslips were fixed by 4%PFA solution for 10 min at room temperature. After two times washing, permeabilization and blocking was then carried out in blocking buffer (1%BSA in PBS) with or without 0.5%Triton ⁇ X for 30 min at room temperature. Afterwards, primary antibody incubation was performed at 4°C overnight and appropriate fluorescent probe ⁇ conjugated secondary antibody was subsequently incubated at room temperature for 1 h. Nuclei counterstaining was performed with DAPI. Images were captured with fluorescence microscope (Olympus IX ⁇ 51) or Leica SP ⁇ 8 confocal microscope.
  • the primary antibodies and dilutions were used as follows: DCX (1: 200, Abcam) , Tuj1 (1: 500, Covance) , MAP2 (1: 500, Millipore) , NeuN (1: 500, Millipore) , SYN1 (1: 500, Millipore) , vGluT1 (1: 500, Synaptic system) , GAD67 (1: 500, Millipore) , Chat (1: 200, Millipore) , TH (1: 1000, Sigma ⁇ Aldrich) , Nestin (1:1000, Millipore) , Sox2 (1: 500, R&D) .
  • cardiac ⁇ perfused transplantation mouse brains were fixed with 4%PFA overnight, cryoprotected in 30%sucrose and then cryosectioned into 20 ⁇ m coronal sections for further immunofluorescence analysis.
  • RNA samples were extracted from human fibroblasts, hciN cells after chemicals treatment for 3, 7 and 14 days and control neurons derived from hES cell line. The total RNAs were reverse ⁇ transcribed and hybridized to PrimeView TM Human Gene Expression Array (Affymetrix) under the manufacturer’s instruction. Microarray hybridization and whole genome expression analysis were performed by Shanghai OE Biotech Co., Ltd. Affymetrix GeneChip Command Console (version 4.0, Affymetrix) was used to analyze array images to get raw data. Next, Genespring software (version 12.5, Agilent Technologies) was employed to finish the basic analysis with the raw data.
  • Hierarchical clustering analysis was applied using a Euclidean distance matrix and the complete ⁇ linkage clustering method in selected genes, which show 10 ⁇ fold change in hciN cells (day 14) versus HAFs. Afterwards, differentially expressed genes with expression levels change ⁇ 2 ⁇ fold were applied for GO analysis by DAVID Bioinformatics Resources.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Neurology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Public Health (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Neurosurgery (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Wood Science & Technology (AREA)
  • Food Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)

Abstract

L'invention concerne des procédés de production d'un phénotype neuronal dans une cellule gliale ou dans une cellule non neuronale somatique. Ces procédés consistent à mettre en contact la cellule gliale ou la cellule non neuronale somatique avec une composition comprenant de petites molécules. L'invention concerne également des procédés de criblage d'un agent thérapeutique chez un patient présentant un trouble neurologique.
PCT/CN2015/073472 2014-03-03 2015-03-02 Traitement de troubles neurologiques WO2015131788A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410075246.5 2014-03-03
CN201410075246.5A CN104894060B (zh) 2014-03-03 2014-03-03 诱导体细胞转分化为神经干细胞的方法及其应用

Publications (1)

Publication Number Publication Date
WO2015131788A1 true WO2015131788A1 (fr) 2015-09-11

Family

ID=54027020

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2015/073472 WO2015131788A1 (fr) 2014-03-03 2015-03-02 Traitement de troubles neurologiques
PCT/CN2015/073549 WO2015131797A1 (fr) 2014-03-03 2015-03-03 Procédé pour induire la trans-différenciation de cellules somatiques en cellules souches neurales, et son application

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/073549 WO2015131797A1 (fr) 2014-03-03 2015-03-03 Procédé pour induire la trans-différenciation de cellules somatiques en cellules souches neurales, et son application

Country Status (2)

Country Link
CN (1) CN104894060B (fr)
WO (2) WO2015131788A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3055410A4 (fr) * 2014-07-29 2017-04-26 Shenzhen Cell Inspire Biotechnology Co., Ltd. Milieu pour la préparation d'une cellule neuronale et son utilisation
WO2017143207A1 (fr) 2016-02-18 2017-08-24 The Penn State Research Foundation Génération de neurones gabaergiques dans des cerveaux
WO2018232258A1 (fr) * 2017-06-15 2018-12-20 University Of North Texas Health Science Center Reprogrammation de fibroblastes en cellules rétiniennes
US11453661B2 (en) 2019-09-27 2022-09-27 Takeda Pharmaceutical Company Limited Heterocyclic compound
WO2023034440A1 (fr) * 2021-09-01 2023-03-09 Case Western Reserve University Traitement de maladies neurodégénératives avec des inhibiteurs de hdac

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3423069A4 (fr) * 2016-03-02 2020-04-08 Frequency Therapeutics, Inc. Procédés de prolifération contrôlée de cellules souches vestibulaires/de production de cellules ciliées de l'oreille interne à l'aide de l'inhibition de wnt et de tgf-bêta
CN106399248B (zh) * 2016-09-30 2020-01-14 浙江大学 一种诱导成纤维细胞转分化为神经细胞的方法
CN106381285A (zh) * 2016-09-30 2017-02-08 浙江大学 一种诱导成纤维细胞转分化为神经细胞的诱导培养基及其应用
CN108070549A (zh) * 2016-11-07 2018-05-25 昆明学院 小分子化合物组合及利用该小分子化合物组合诱导分化的细胞制备血管内皮细胞的方法
CN108060126B (zh) 2016-11-07 2020-09-08 云南济慈再生医学研究院有限公司 一种诱导分化的细胞制备间充质干细胞的方法及调控靶点的组合
KR102097191B1 (ko) * 2016-11-10 2020-04-06 울산대학교 산학협력단 히스톤 탈아세틸화 저해제 및 프라이밍 인자를 유효성분으로 포함하는 줄기세포 활성 촉진용 조성물
CN108148808B (zh) * 2016-12-05 2020-12-11 同济大学 有助于诱导生成神经前体细胞的诱导培养基
CN106635991A (zh) * 2016-12-30 2017-05-10 东莞惠恩生物工程有限公司 人皮肤成纤维细胞诱导为神经细胞的方法与应用
CN108300686B (zh) * 2017-01-13 2022-08-26 云南美颜美龄生物科技有限公司 用于预防、延缓或逆转细胞、组织、器官、机体衰老的小分子化合物/组合，产品及其用途
CN107674859B (zh) * 2017-08-28 2021-01-05 浙江大学 一种利用小分子组合物诱导小鼠成纤维细胞成软骨的方法
WO2019073055A1 (fr) 2017-10-13 2019-04-18 Imba - Institut Für Molekulare Biotechnologie Gmbh Reprogrammation améliorée de cellules somatiques
US20210246422A1 (en) * 2018-06-14 2021-08-12 Academia Sinica Method to generate induced oligodendrocyte-lineage cells and treatment using such cells
CN110714024A (zh) * 2018-07-13 2020-01-21 中国科学院上海生命科学研究院 建立自外周血单核细胞的神经前体细胞及其构建方法
WO2020037326A1 (fr) 2018-08-17 2020-02-20 Frequency Therapeutics, Inc. Compositions et méthodes pour produire des cellules ciliées par la régulation à la baisse de foxo
JP2020115792A (ja) * 2019-01-24 2020-08-06 成夫 齋藤 幹細胞の製造方法、及び癌細胞化のリスク低減方法
CN111718890B (zh) * 2019-03-19 2022-08-09 中国科学院动物研究所 一种将成纤维细胞转分化为腺上皮细胞的方法及其培养体系和应用
CN110760476B (zh) * 2019-09-03 2020-11-03 广州瑞臻再生医学科技有限公司 一种大脑皮层神经干细胞及谷氨酸能神经元的制备方法
CN110551688B (zh) * 2019-09-26 2023-03-21 上海交通大学医学院附属瑞金医院 一种诱导体细胞重编程为造血干/祖细胞且促进造血干/祖细胞体外扩增的组合物及其应用
CN111909901B (zh) * 2020-08-05 2023-12-29 上海市第十人民医院 一种个性化诱导性神经干细胞外泌体的制备方法及应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011159726A2 (fr) * 2010-06-14 2011-12-22 The Scripps Research Institute Reprogrammation de cellules pour leur conférer un nouveau destin
CN102604894A (zh) * 2012-02-29 2012-07-25 中国科学院广州生物医药与健康研究院 用于制备神经干细胞的培养基及其用途
CN103068974A (zh) * 2010-08-19 2013-04-24 弗·哈夫曼-拉罗切有限公司 体细胞至诱导的重编程神经干细胞(irNSC)的转化
WO2013086570A1 (fr) * 2011-12-13 2013-06-20 Flinders University Of South Australia Procédé de production de cellules souches multipotentes
CN104195108A (zh) * 2014-07-29 2014-12-10 深圳市三启生物技术有限公司 蛋白激酶抑制剂在从非神经细胞制备神经细胞中的用途

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2009225665B9 (en) * 2008-03-17 2015-01-15 The Scripps Research Institute Combined chemical and genetic approaches for generation of induced pluripotent stem cells
KR101685209B1 (ko) * 2008-07-30 2016-12-09 고쿠리츠 다이가쿠 호진 교토 다이가쿠 유도된 다능성 줄기 세포의 효율적인 확립 방법
CN102260646B (zh) * 2010-05-26 2015-09-16 北京瑞普晨创科技有限公司 一种制备诱导多潜能干细胞的方法,试剂盒及用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011159726A2 (fr) * 2010-06-14 2011-12-22 The Scripps Research Institute Reprogrammation de cellules pour leur conférer un nouveau destin
CN103068974A (zh) * 2010-08-19 2013-04-24 弗·哈夫曼-拉罗切有限公司 体细胞至诱导的重编程神经干细胞(irNSC)的转化
WO2013086570A1 (fr) * 2011-12-13 2013-06-20 Flinders University Of South Australia Procédé de production de cellules souches multipotentes
CN102604894A (zh) * 2012-02-29 2012-07-25 中国科学院广州生物医药与健康研究院 用于制备神经干细胞的培养基及其用途
CN104195108A (zh) * 2014-07-29 2014-12-10 深圳市三启生物技术有限公司 蛋白激酶抑制剂在从非神经细胞制备神经细胞中的用途

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GIUSEPPE FARACO ET AL.: "Histone deacetylase (HDAC) inhibitors reduce the glial inflammatory response in vitro and in vivo", NEUROBIOLOGY OF DISEASE, 25 July 2009 (2009-07-25), pages 269 - 279, XP026676555, ISSN: 0969-9961 *
JIN XING.: "nNOS from NSCs Regulates Neurogenesis Via HDAC2", CHINESE MASTER'S THESES FULL-TEXT DATABASE, 15 February 2013 (2013-02-15), ISSN: 1674-0246 *
LIN CHENG ET AL.: "Generation of neural progenitor cells by chemical cocktails and hypoxia", CELL RESEARCH, 18 March 2014 (2014-03-18), pages 665 - 679, XP055222906, ISSN: 1001-0602 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3055410A4 (fr) * 2014-07-29 2017-04-26 Shenzhen Cell Inspire Biotechnology Co., Ltd. Milieu pour la préparation d'une cellule neuronale et son utilisation
US10053666B2 (en) 2014-07-29 2018-08-21 Shenzhen Cell Inspire Biotechnology Co., Ltd. Medium for preparing a neural cell and usage thereof
WO2017143207A1 (fr) 2016-02-18 2017-08-24 The Penn State Research Foundation Génération de neurones gabaergiques dans des cerveaux
CN109069544A (zh) * 2016-02-18 2018-12-21 宾州研究基金会 脑内生成gaba能神经元
US10973930B2 (en) 2016-02-18 2021-04-13 The Penn State Research Foundation Generating GABAergic neurons in brains
EP3881857A1 (fr) * 2016-02-18 2021-09-22 The Penn State Research Foundation Génération de neurones gabaergiques dans des cerveaux
WO2018232258A1 (fr) * 2017-06-15 2018-12-20 University Of North Texas Health Science Center Reprogrammation de fibroblastes en cellules rétiniennes
JP2020524501A (ja) * 2017-06-15 2020-08-20 ユニバーシティ オブ ノース テキサス ヘルス サイエンス センター 網膜細胞への線維芽細胞の再プログラミング方法
JP7376092B2 (ja) 2017-06-15 2023-11-08 ザ ユニバーシティ オブ ノース テキサス ヘルス サイエンス センター アット フォート ワース 網膜細胞への線維芽細胞の再プログラミング方法
US11453661B2 (en) 2019-09-27 2022-09-27 Takeda Pharmaceutical Company Limited Heterocyclic compound
US11958845B2 (en) 2019-09-27 2024-04-16 Takeda Pharmaceutical Company Limited Heterocyclic compound
WO2023034440A1 (fr) * 2021-09-01 2023-03-09 Case Western Reserve University Traitement de maladies neurodégénératives avec des inhibiteurs de hdac

Also Published As

Publication number Publication date
CN104894060A (zh) 2015-09-09
CN104894060B (zh) 2018-11-06
WO2015131797A1 (fr) 2015-09-11

Similar Documents

Publication Publication Date Title
WO2015131788A1 (fr) Traitement de troubles neurologiques
JP6756786B2 (ja) 移植用中脳ドーパミン（ｄａ）ニューロン
US20240052306A1 (en) METHODS FOR EFFICIENT GENERATION OF GABAergic INTERNEURONS FROM PLURIPOTENT STEM CELLS
US20190099452A1 (en) Methods for promoting oligodendrocyte regeneration and remyelination
JP2019507802A (ja) ＧＳＫ３α阻害剤を用いる、幹細胞の制御された増殖／内耳有毛細胞の発生のための方法
JP2002515071A (ja) 系列限定ニューロン前駆体
JP7471084B2 (ja) 幹細胞由来星状細胞、作製方法および使用方法
JP2023525297A (ja) 幹細胞をドーパミン作動性前駆細胞に分化させるための方法
Terrigno et al. Neurons generated by mouse ESCs with hippocampal or cortical identity display distinct projection patterns when co-transplanted in the adult brain
Luo et al. Inhibition of CSPG receptor PTPσ promotes migration of newly born neuroblasts, axonal sprouting, and recovery from stroke
US20030082802A1 (en) Method for neural stem cell differentiation using 5ht1a agonists
Xu et al. Neurotrophic factor expression in expandable cell populations from brain samples in living patients with Parkinson's disease
JP2023116525A (ja) 網膜細胞への線維芽細胞の再プログラミング方法
US20210246422A1 (en) Method to generate induced oligodendrocyte-lineage cells and treatment using such cells
Pacioni et al. Fast, potent pharmacological expansion of endogenous hes3+/sox2+ cells in the adult mouse and rat hippocampus
Tsonis Regeneration of the lens in amphibians
US20220241345A1 (en) Induced pluripotent stem cell derived glial enriched progenitor cells for the treatment of white matter stroke
KR102657342B1 (ko) 희소돌기아교세포로 분화가 특화된 신경줄기세포로부터 희소돌기아교세포를 포함하는 오가노이드의 제조방법, 상기 방법으로 제조된 오가노이드 및 이의 용도
CN109620844A (zh) 一种过表达Nurr1的细胞混合液及其制备方法和应用
US20240173356A1 (en) Extracellular Vesicles From Human Induced Pluroptent Stem Cells
US20240060040A1 (en) Methods for generating inner ear hair cells
Tiberi A glial side to the neurotrophin field: studying the effects of neurotrophins on glial cells in the CNS
Todd The Signaling Pathways that Regulate the Proliferative and Neurogenic Capacity of Muller glia
Sharma Role of MECP2 in Transcriptional Regulation of Central Nervous System Myelination
Stanke Beyond Neuronal Replacement: Embryonic Retinal Cells Protect Mature Retinal Neurons

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15758809

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15758809

Country of ref document: EP

Kind code of ref document: A1