WO2009146098A2 - Cellules souches et leurs utilisations - Google Patents

Cellules souches et leurs utilisations Download PDF

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Publication number
WO2009146098A2
WO2009146098A2 PCT/US2009/039274 US2009039274W WO2009146098A2 WO 2009146098 A2 WO2009146098 A2 WO 2009146098A2 US 2009039274 W US2009039274 W US 2009039274W WO 2009146098 A2 WO2009146098 A2 WO 2009146098A2
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cell
subject
ips
cells
kit
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PCT/US2009/039274
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WO2009146098A3 (fr
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Kevin Eggan
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President And Fellows Of Harvard College
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • 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
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
    • 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
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
    • 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
    • C12N2501/40—Regulators of development
    • C12N2501/41—Hedgehog proteins; Cyclopamine (inhibitor)
    • 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
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention relates to the production of cell types, e.g., neurons (e.g., motor neurons) or glial cells, from somatic cells of a subject, e.g., a subject having a disorder which affects those cell types.
  • cell types e.g., neurons (e.g., motor neurons) or glial cells
  • somatic cells e.g., a cell type having a disorder which affects those cell types.
  • ALS is a neurodegenerative disorder in which motor neuron loss in the spinal cord and motor cortex leads to progressive paralysis and death of the patient (i).
  • Two aspects of ALS have contributed to difficulties encountered in generating efficient treatments for this condition: first, it is not possible to culture motor neurons from patients, and secondly, the genetic basis of 90% of cases remains unknown, rendering them refractory to modeling in rodents.
  • Studies aimed at understanding the root causes of motor neuron death in ALS and efforts to develop new therapeutics would be greatly advanced if a robust supply of human motor neurons carrying the genes responsible for this condition could be generated.
  • mice (2-5) and human (6) skin fibroblasts can be reprogrammed to a pluripotent state, similar to that of an embryonic stem (ES) cell, following retroviral transduction with four genes.
  • ES embryonic stem
  • iPS cells can be generated directly from elderly patients with chronic disease using material that has been exposed to disease-causing agents for a lifetime, and whether such patient-specific iPS cells could be differentiated into the particular cell types that would be needed to treat or study the patient's condition.
  • the invention features, a method of providing a differentiated cell of a selected cell type from a subject.
  • the subject is (1) a subject having or at risk for having a disorder which causes cells of said cell type to be absent, diseased, inactive, or in general to possess an unwanted phenotype.
  • the subject has the disorder; or (2) a subject who has been selected on the basis of having a preselected disorder, e.g., a disorder which causes cells of said cell type to be absent, diseased, inactive, or in general to possess an unwanted phenotype, and on another preselected parameter related to the patient's medical history.
  • the method includes: (1) converting the somatic cell of the subject to an iPS cell; and
  • the differentiated cell is from a subject having or at risk for having a disorder which causes cells of said cell type to be absent, diseased, inactive, or in general to possess an unwanted phenotype.
  • the subject has the disorder.
  • the subject is one which is selected on the basis of having or at risk of having a preselected disorder, e.g., a disorder which causes cells of said cell type to be absent, diseased, inactive, or in general to possess an unwanted phenotype, and on a preselected parameter related to the patient's medical history.
  • a preselected disorder e.g., a disorder which causes cells of said cell type to be absent, diseased, inactive, or in general to possess an unwanted phenotype
  • a preselected parameter related to the patient's medical history e.g., a disorder which causes cells of said cell type to be absent, diseased, inactive, or in general to possess an unwanted phenotype
  • a preselected parameter related to the patient's medical history e.g., a disorder which causes cells of said cell type to be absent, diseased, inactive, or in general to possess an unwanted phenotype
  • preselected parameters can include, the age of the patient, how long the patient has displayed symptoms, the
  • the somatic cell is taken from the subject after said subject has exhibited at least one symptom of said disorder, e.g., some or all of the cells of the selected cell type has been affected by the disorder and, e.g., are absent, diseased, inactive, or in general possess an unwanted phenotype.
  • the subject has exhibited symptoms of the disorder for at least 2, 4, 5, or ten years.
  • the subject has a neurodegenerative disorder, e.g., ALS, and the selected cell type is a neuron.
  • a neurodegenerative disorder e.g., ALS
  • the selected cell type is a neuron.
  • the method further comprises isolating a population of the iPS cells (e.g., wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 50%, 75% or greater are of the subject cell type).
  • a plurality of iPS cells are converted to a plurality of neurons.
  • the method further comprises isolating a population of the neurons (e.g., wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 50%, 75% or greater are of the subject cell type).
  • a plurality of iPS cells are converted to a plurality of glial cells.
  • the method further comprises isolating a population of the glials (e.g., wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 50%, 75% or greater are of the subject cell type).
  • the somatic cell has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the iPS cell has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the subject is a human.
  • the subject disorder is a neurological disorder (e.g., ALS).
  • a neurological disorder e.g., ALS
  • the subject is suffering from a familiar form of neurological disorder (e.g., a familiar form of ALS).
  • a familiar form of neurological disorder e.g., a familiar form of ALS.
  • the subject has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the subject is suffering from a sporadic form of neurological disorder (e.g., a sporadic form of ALS).
  • a sporadic form of neurological disorder e.g., a sporadic form of ALS.
  • the subject is a human with advanced age (e.g., at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100).
  • advanced age e.g., at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100.
  • the somatic cell is obtained from a sample, e.g. a hair follicle, a blood sample, a biopsy (e.g., a skin biopsy or an adipose biopsy) or a swab sample (e.g., an oral swab sample).
  • a sample e.g. a hair follicle, a blood sample, a biopsy (e.g., a skin biopsy or an adipose biopsy) or a swab sample (e.g., an oral swab sample).
  • the somatic cell is converted to an iPS cell by overexpressing one or more transcription factor(s).
  • the transcription factor is selected from a group comprising KLF4, SOX2, OCT4 and C-MYC.
  • the somatic cell is converted to an iPS cell by overexpressing four transcription factors (e.g., KLF4, SOX2, OCT4, and C-MYC). In one embodiment, the somatic cell is converted to an iPS cell by overexpressing at least three transcription factors (e.g., KLF4, SOX2 and OCT4).
  • four transcription factors e.g., KLF4, SOX2, OCT4, and C-MYC.
  • the somatic cell is converted to an iPS cell by overexpressing at least three transcription factors (e.g., KLF4, SOX2 and OCT4).
  • the somatic cell is converted to an iPS cell by overexpressing at least two transcription factors (e.g., SOX2 and OCT4).
  • at least two transcription factors e.g., SOX2 and OCT4
  • the iPS cell maintains a normal karyotype.
  • the expression of a marker selected from the group consisting of: AP, SSEA-3, SSEA-4, TRA1-60, TRA1-81, NANOG, REX1/ZFP42, F0XD3, TERT and CRIPTO/TDFGl is upregulated to by a statistically significant amount in the iPS cell relative to a somatic cell.
  • the iPS cell is converted to a neuron by culturing the cell in a medium comprising at least one compound selected from a group comprising retinoic acid (RA) and sonic hedgehog (SHH) agonist.
  • a medium comprising at least one compound selected from a group comprising retinoic acid (RA) and sonic hedgehog (SHH) agonist.
  • RA retinoic acid
  • SHH sonic hedgehog
  • the expression of a marker selected from the group consisting of: TuJl, HB9, ISL1/2 and ChAT is upregulated to by a statistically significant amount in the neuron relative to an iPS cell.
  • the iPS cell is converted to a glial cell by culturing the cell in a medium comprising at least one compound selected from a group comprising retinoic acid (RA) and sonic hedgehog (SHH) agonist.
  • a medium comprising at least one compound selected from a group comprising retinoic acid (RA) and sonic hedgehog (SHH) agonist.
  • RA retinoic acid
  • SHH sonic hedgehog
  • the expression of a marker selected from the group consisting of GFAP and SlOO is upregulated to by a statistically significant amount in the glial cell relative to an iPS cell.
  • the method further comprises implanting the differentiated cell, e.g., neuron or glial cells into the subject (e.g., wherein the subject has a neurological disorder (e.g., ALS, e.g., a familiar or sporadic ALS)).
  • a neurological disorder e.g., ALS, e.g., a familiar or sporadic ALS
  • the method further comprises implanting the differentiated cell, e.g., neurons or glial cells into a recipient other than the subject, e.g., into a person who is a relative, e.g., sibling, child, parent, or cousin of the subject.
  • the differentiated cell e.g., neurons or glial cells
  • a recipient other than the subject e.g., into a person who is a relative, e.g., sibling, child, parent, or cousin of the subject.
  • the neurons or glial cells are from a donor different than the subject (e.g., a relative of the subject) are implanted into the subject.
  • the neurons or glial cells are surgically implanted.
  • the disclosure features a method of converting a human iPS cell to a neuron or glial cell, the method comprising providing a human iPS cell, and culturing the cell in a medium comprising at least one compound selected from a group comprising retinoic acid (RA) and sonic hedgehog (SHH) agonist.
  • RA retinoic acid
  • SHH sonic hedgehog
  • the iPS cell is converted from a somatic cell.
  • the iPS cell has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the somatic cell has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the somatic cell is from a subject.
  • the subject is suffering from a neurological disorder (e.g., ALS).
  • a neurological disorder e.g., ALS
  • the subject is suffering from a familiar form of neurological disorder (e.g., a familiar form of ALS).
  • a familiar form of neurological disorder e.g., a familiar form of ALS.
  • the subject has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the subject is suffering from a sporadic form of neurological disorder (e.g., a sporadic form of ALS).
  • a sporadic form of neurological disorder e.g., a sporadic form of ALS.
  • the human iPS cell can be from a subject described herein.
  • a plurality of iPS cells are converted to a plurality of neurons.
  • the method further comprises isolating a population of the neurons (e.g., wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 50%, 75%, or greater are of the subject cell type).
  • a plurality of iPS cells are converted to a plurality of glial cells.
  • the method further comprises isolating a population of the glials (e.g., wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 50%, 75%, or greater are of the subject cell type).
  • a marker selected from the group consisting of: TuJl, HB9, ISL1/2 and ChAT is upregulated to by a statistically significant amount in the neuron relative to an iPS cell.
  • the expression of a marker selected from the group consisting of: GFAP and SlOO is upregulated to by a statistically significant amount in the glial cell relative to an iPS cell.
  • the method further comprises implanting the neuron or glial cells into a subject (e.g., a subject having a neurological disorder (e.g., ALS, e.g., a familiar or sporadic ALS)).
  • a subject having a neurological disorder e.g., ALS, e.g., a familiar or sporadic ALS
  • the neurons or glial cells are from a donor different than the subject (e.g., a relative of the subject).
  • the neurons or glial cells are surgically implanted.
  • the disclosure features a kit comprising one or more of:
  • Reagents for overexpressing one or more transcription factor(s) selected from a group comprising KLF4, SOX2, OCT4 and C-MYC in a somatic cell selected from a group comprising KLF4, SOX2, OCT4 and C-MYC in a somatic cell.
  • RA retinoic acid
  • SHH sonic hedgehog
  • the somatic cell is from a subject.
  • the subject is suffering from a neurological disorder (e.g., ALS).
  • a neurological disorder e.g., ALS
  • the subject is suffering from a familiar form of neurological disorder (e.g., a familiar form of ALS).
  • a familiar form of neurological disorder e.g., a familiar form of ALS.
  • the subject has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the subject is suffering from a sporadic form of neurological disorder (e.g., a sporadic form of ALS).
  • the somatic cell has at least one unwanted SODl allel, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • the iPS cell has at least one unwanted SODl allel, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the subject is a human with advanced age (e.g., at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100).
  • advanced age e.g., at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100.
  • the expression of a marker selected from the group consisting of: AP, SSEA-3, SSEA-4, TRA1-60, TRA1-81, NANOG, REX1/ZFP42, FOXD3, TERT and CRIPTO/TDFGl is upregulated to by a statistically significant amount in the iPS cell relative to a somatic cell.
  • the expression of a marker selected from the group consisting of: TuJl, HB9, ISL1/2 and ChAT is upregulated to by a statistically significant amount in the neuron relative to an iPS cell.
  • the expression of a marker selected from the group consisting of GFAP and SlOO is upregulated to by a statistically significant amount in the glial cell relative to an iPS cell.
  • the disclosure feathers a kit comprising one or more of:
  • RA retinoic acid
  • SHH sonic hedgehog
  • the iPS cell has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the iPS cell is converted from a somatic cell.
  • the somatic cell has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the somatic cell is from a subject.
  • the subject is suffering from a neurological disorder (e.g., ALS).
  • a neurological disorder e.g., ALS
  • the subject is suffering from a familiar form of neurological disorder (e.g., a familiar form of ALS).
  • a familiar form of neurological disorder e.g., a familiar form of ALS.
  • the subject has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the subject is suffering from a sporadic form of neurological disorder (e.g., a sporadic form of ALS).
  • a sporadic form of neurological disorder e.g., a sporadic form of ALS.
  • the expression of a marker selected from the group consisting of: GFAP and SlOO is upregulated to by a statistically significant amount in the glial cell relative to an iPS cell.
  • the disclosure features a kit comprising a cell described herein.
  • the cell is an iPS cell described herein.
  • the cell is a neuron or a glial cell converted from an iPS cell described herein by a method described herein.
  • the iPS cell is converted from somatic cells.
  • the iPS cell has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the somatic cell has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the somatic cell is from a subject.
  • the subject is suffering from a neurological disorder (e.g., ALS).
  • a neurological disorder e.g., ALS
  • the subject is suffering from a familiar form of neurological disorder (e.g., a familiar form of ALS).
  • a familiar form of neurological disorder e.g., a familiar form of ALS.
  • the subject has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the subject is suffering from a sporadic form of neurological disorder (e.g., a sporadic form of ALS).
  • the disclosure features a composition, comprising a cell (e.g., an iPS cell) made from a method described herein and a culture medium described herein.
  • a cell e.g., an iPS cell
  • the disclosure feathers a method of culturing a somatic cell in culture medium, the method comprising overexpressing one or more transcription factor(s) selected from a group comprising KLF4, SOX2, OCT4 and C-MYC.
  • somatic cell can be from a subject described herein.
  • the somatic cell is cultured for a time sufficient to convert into an iPS cell.
  • the somatic cell is cultured for a time sufficient to upregulate the expression of a marker selected from the group consisting of: AP, SSEA-3, SSEA-4, TRA1-60, TRA1-81, NANOG, REX1/ZFP42, F0XD3, TERT and CRIPTO/TDFGl by a statistically significant amount.
  • a marker selected from the group consisting of: AP, SSEA-3, SSEA-4, TRA1-60, TRA1-81, NANOG, REX1/ZFP42, F0XD3, TERT and CRIPTO/TDFGl by a statistically significant amount.
  • the disclosure features a method of culturing an iPS cell in culture medium, the method comprising treating the culture medium with at least one compound selected from a group comprising retinoic acid (RA) and sonic hedgehog (SHH) agonist.
  • RA retinoic acid
  • SHH sonic hedgehog
  • the iPS cell can be from a subject described herein.
  • the iPS cell is cultured for a time sufficient to differentiate into a neuron (e.g., a motor neuron).
  • a neuron e.g., a motor neuron
  • the iPS cell is cultured for a time sufficient to upregulate the expression of a marker selected from the group consisting of: TuJl, HB9, ISL1/2 and ChAT by a statistically significant amount.
  • the iPS cell is cultured for a time sufficient to differentiate into a glial cell.
  • the iPS cell is cultured for a time sufficient to upregulate the expression of a marker selected from the group consisting of: GFAP and SlOO by a statistically significant amount.
  • a marker selected from the group consisting of: GFAP and SlOO by a statistically significant amount.
  • the disclosure feathers an isolated cell population, isolated from a method described herein.
  • the cell population comprises iPS cells described herein.
  • the cell population comprises neurons or glial cells converted from iPS cells described herein by a method described herein.
  • the iPS cells are converted from somatic cells.
  • the iPS cells have at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the somatic cells have at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the somatic cells are from a subject.
  • the subject is suffering from a neurological disorder (e.g., ALS).
  • a neurological disorder e.g., ALS
  • the subject is suffering from a familiar form of neurological disorder (e.g., a familiar form of ALS).
  • a familiar form of neurological disorder e.g., a familiar form of ALS.
  • the subject has at least one unwanted SODl allele, e.g., a disease-associated SODl allele (e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A).
  • a disease-associated SODl allele e.g., a mutation in SODl gene, e.g., SOD1L144F, SOD1G85R or SOD1D90A.
  • the subject is suffering from a sporadic form of neurological disorder (e.g., a sporadic form of ALS).
  • a sporadic form of neurological disorder e.g., a sporadic form of ALS.
  • the inventions described herein provide methods and kits for generating neuron or glial cells from somatic cells from somatic cells from a subject.
  • the inventions also provide methods and kits for generating neuron or glial cells from iPS cells.
  • the neurons or glial cells so created can be used to treat neurological disorders and to study disease mechanisms/pathology.
  • the disclosure features a method of producing a neuron (e.g., a motor neuron cell) or glial cell from a somatic cell of a subject, the method comprising:
  • a plurality of somatic cells are converted to a plurality of iPS cells.
  • the invention features, a method of producing a cell of a preselected cell type, e.g., a neuron (e.g., a motor neuron cell) or glial cell, from a somatic cell of a subject said subject having cells of said preselected cell type, e.g., neurons, which are absent (e.g., they have died or degenerated), diseased, inactive, or in general possess an unwanted phenotype.
  • the cells are absent because the subject has a disorder described herein, e.g., a neurodegenerative disorder, e.g., ALS.
  • the method comprising:
  • the method can include other embodiments described herein.
  • the invention provides, a library or panel, of isolated preparations of iPS or differentiated cells, e.g., neurons, wherein each of a plurality of the isolated preparations is from a different subject and the preparation was made by a method described herein or taken from a subject described herein.
  • each of a plurality of the subjects has or is at risk for a disorder described herein, e.g., neurodegerative disorder, e.g., ALS.
  • a disorder described herein e.g., neurodegerative disorder, e.g., ALS.
  • at least one subject has a sporadic form of ALS.
  • the disclosure features a method of providing a dedifferentiated cell, e.g., an iPS cell, having a subject-defined-genotype or -epigenome comprising: identifying a subject; providing a subject-defined disorder characteristic, e.g., severity of said disorder or age of onset of said disorder, for said subject, e.g., by evaluating the subject; obtaining a somatic cell from said subject; altering the differentiation of the cell, e.g., reverting the somatic cell to a less differentiated state, e.g., to provide a dedifferentiated cell, e.g., an iPS; optionally, forming a correlation between said defifferentiated cell (or the individual from which it was derived) and a subject-defined disorder characteristic, and optionally memorializing said correlation; optionally, forming a correlation between said defifferentiated cell and an identifier for the individual from which it was derived, and optionally memorializing said correlation; optional
  • the disorder is, e.g., a neurodegenerative disorder, a cardiac or vascular disorder, or a disorder characterized by unwanted cell proliferation.
  • the evaluating can include any of evaluating or determining, presence of said disorder, subject age, subject gender, whether a relative has presented with said disorder, age of onset, severity or other state of the disorder, or an aspect of patient genotype;
  • the disclosure features a database comprising a plurality of correlations made by the method described herein.
  • the disclosure features a panel of dedifferentiatled cell preparations made by the method of claim 100, wherein the panel includes preparations from at least 2, 4, 6, 20, or 50 individuals.
  • the panel includes a plurality of dedifferentiated cell preparations, wherein each preparation of the plurality has (and is known to have) a different value for a first parameter related to a subject-defined disorder characteristic, e.g., a plurality of preparations from individuals having different ages of onset.
  • a subject-defined disorder characteristic e.g., a plurality of preparations from individuals having different ages of onset.
  • the panel includes a plurality of dedifferentiated cell preparations, wherein each preparation of the plurality has (and is known to have) a different value for a second parameter related to a subject-defined disorder characteristic, e.g., a plurality of preparations from individuals having different severities for said disorder.
  • a subject-defined disorder characteristic e.g., a plurality of preparations from individuals having different severities for said disorder.
  • the panel further comprises a memorialization of said correlations.
  • the disclosure features a method of selecting a dedifferentiated cell, e.g., an iPS cell, having a subject-defined-genotype or -epigenome comprising: providing a dedifferentiated cell made by the method described herein; determining if, or confirming that, it has a preselected subject-defined disorder characteristic; determining if, or confirming that, it was derived from a preselected individual; and if said determinations or confirmations are both positive selecting said dedifferentiated cell, and optionally, moving an aliquot of said dedifferentiated cells from one condition, e.g., a first location or culture or storage condition (e.g., temperature) to a second condition, e.g., a second location or culture or storage condition (e.g., temperature), thereby selecting a dedifferentiated cell, e.g., an iPS cell, having a subject-defined- genotype or -epigenome.
  • a first location or culture or storage condition e
  • the disclosure features a panel of a plurality of preparations of dedifferentiated cells, e.g., iPS cells, each form a different subject, made, e.g., by a method disclosed herein, comprising: at least 2, 4, 10, 20, 50 or 100 preparations.
  • dedifferentiated cells e.g., iPS cells
  • each of said preparations is correlated with an identifier for the subject from which it was obtained.
  • each of said preparations has a different value for a first parameter related to a subject-defined disorder characteristic, e.g., the panel comprises a plurality of preparations from individuals having different ages of onset.
  • each of said preparations has a different value for a second parameter related to a subject-defined disorder characteristic, e.g., the panel comprises a plurality of preparations from individuals having different ages of onset.
  • the disclosure features a method of evaluating a compound, e.g., for the ability to modify a cell, or as a drug or therapeutic candidate, comprising: providing the panel of dedifferentiated cell preparations described herein; contacting said compound with a cell from each of said plurality of preparations from said panel; evaluating the affect of the compound on said cells, thereby evaluating a compound.
  • the disclosure features a preparation of dedifferentiated cells, e.g., iPS cells,
  • the methods herein allow the generation of pluripotent stem cells from an individual patient enables the large-scale production of the cell-types affected by that patient's disease. These cells can in turn be used for disease modeling, drug discovery, and autologous cell-replacement therapies. Methods disclosed herein allow production of induced pluripotent stem (iPS) cells from material isolated directly from patients with chronic diseases, and differentiation into the specific cell types, e.g., those needed to treat or model their condition. The description below demonstrates that iPS cells can indeed be generated by retroviral transduction of skin fibroblasts collected from patients diagnosed with a familial form of amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • PS-iPS patient-specific iPS
  • hES human embryonic stem
  • FIGURE 1 shows that iPS cells can be established from patient fibroblasts after biopsy.
  • A Primary dermal fibroblasts derived from an 82 year-old female ALS patient, A29.
  • B iPS cells produced from patient A29.
  • C iPS cells produced from a second patient, A30, sister to patient A29.
  • D Direct sequencing of a PCR product from A29 iPS cells confirming the presence of one copy of the dominant L144F SODl allele.
  • E,F SSEA-4 and NANOG protein expression in A29 iPS cells. Scale bars are all 200 ⁇ m.
  • FIGURE 2 shows that A29 iPS cells are similar to human ES cells in their expression of genes associated with pluripotency.
  • A ES cell-associated transcripts, REX1/ZFP42, F0XD3, TERT, NANOG, and CRIPTO/TDGFl, are activated in iPS cells to levels comparable to human ES cells as measured by qRT-PCR.
  • B Primers specific for either endogenously (blue) or virally (red) encoded transcripts of the four reprogramming factors the inventorsre used to measure their respective expression levels. Expression was detected from all four endogenous loci in the iPS cells at levels similar to those in the human ES cell lines HuES-3 and HuES-IO.
  • FIGURE 3 shows that patient-specific iPS cells are pluripotent stem cells.
  • A EBs formed from A29b iPS cells, five days after seeding. These EBs contained cells representative of each of the three embryonic germ layers: endoderm (B, alpha- fetoprotein), mesoderm (C, desmin; D, ⁇ -smooth muscle actin), and ectoderm (E, ⁇ - Tubulinlllb; F, glial fibrillary acidic protein). Scale bars are 200 ⁇ m (A), and 100 ⁇ m (B-F).
  • FIGURE 4 shows that iPS cells generated from ALS patients can be differentiated into motor neurons.
  • A29b iPS cell EBs the inventorsre patterned with RA and SHH, and plated on laminin either whole (A-B), or following dissociation (C-H), and allothe inventorsd to mature for 7-15 days.
  • A Neuronal-like outgrowths are visible from whole A29b patient-specific iPS cell EBs.
  • B Extensive TuJl-positive neuronal processes grow out from plated whole iPS EBs, which contain a high proportion of HB9-stained nuclei.
  • C Neuronal identity of HB9 expressing cells is confirmed by high- magnification image of HB9 and TuJl co-expression in dissociated patient-specific motor neuron cultures.
  • GFAP-expressing glial cells can be found in addition to TuJl- expressing neurons in differentiated patient-specific iPS cell cultures.
  • E-H The motor neuron identity of HB9/TuJl double positive cells is confirmed by the coexpression of HB9 and ISL.
  • HB9 (E) and ISL (F) localization is nuclear (G) and highly coincident (H). Scale bars are 100 ⁇ m (A-D) and 75 ⁇ m in (E-H).
  • SUPPLEMENTARY FIGURE Sl shows (A) Strategy for determining that the SODl genotype of the iPS cells matched that of the parental fibroblasts and the patient medical history. Primers the inventorsre designed to amplify a 347 bp region flanking the dominant L144F SNP in exon 5 of SODl. This SNP eliminates a CviKI-1 site (*) thereby generating a restriction fragment length polymorphism (RFLP). (B) Detection of the L144F CviKI-1 RFLP in A29-derived fibroblasts and iPS, as a 137 bp fragment (*).
  • A29 iPS cell lines carry retroviral integrations for each of the four reprogramming factors as detected by genomic PCR with primer pairs specific for each retroviral transgene.
  • D Patient-specific iPS cells maintain a normal 46,XX karyotype after expansion.
  • FIGURE S2 shows that A29 iPS cells share a cell cycle profile similar to human ES cells, but not their parental fibroblasts.
  • Fixed cells the inventorsre stained with propidium iodide and analyzed with a fluorescence activated cell sorter (FACS) to determine their DNA content, which is correlated to cell cycle state (proportions in different cell cycle states shown in table).
  • FACS fluorescence activated cell sorter
  • the increased proportion of 4n cells in iPS and human ES cell cultures reflects the increased proportion of cells in the active G2/M stage of the cell cycle, compared to fibroblast cultures.
  • FIGURE S3 shows that A29 iPS cells are similar to human ES cells in the expression of pluripotency associated markers.
  • A29a-, A29b-, and A29c- patient specific iPS exhibit high alkaline phosphatase activity, SSEA-3, SSEA-4, Tral- 60, Tral-81, and NANOG expression similar to human ES cells (HuES-9).
  • a fibroblast associated antigen, TE-7 is not detected on iPS cells or human ES cells.
  • Scale bars are 200 ⁇ m.
  • FIGURE S4 shows that patient- specific iPS cells are pluripotent stem cells.
  • A Schematic illustrating the spontaneous differentiation of iPS in embryoid bodies (EBs) and subsequent adherent culture.
  • iPS the inventorsre used to seed embryoid bodies that the inventorsre grown in suspension for 7-10 days before being allothe inventorsd to attach to tissue culture plastic and analyzed for the production of cell types representative of the three embryonic germ layers.
  • EBs contained cells characteristic of each of the three germ layers: endoderm (AFP), mesoderm (Desmin, ⁇ -SMA), and ectoderm (GFAP), although it has yet to be determined if any of these patient-specific iPS cell lines exhibit a bias towards a particular lineage upon differentiation. Scale bars are 100 ⁇ m.
  • FIGURE S5 shows that iPS cells generated from ALS patients can be differentiated into motor neurons.
  • A Schematic of protocol used to direct the differentiation of PS-iPS to motor neurons.
  • EBs derived from iPS cell line A29a the inventorsre grown for 10 days before treatment with retinoic acid (RA) and a small molecule sonic hedgehog (SHH) signaling agonist. After two the inventorseks of continued suspension culture in the presence of these inductive molecules, EBs the inventorsre dissociated and cells plated on laminin. After 14 days of maturation, motor neurons the inventorsre detected by immunocytochemistry for (B-D) HB9, (C) ISLETl and ISLET2, and (D) ChAT. Scale bars are 100 ⁇ m.
  • FIGURE S6 shows that PS-iPS generated motor neurons display characteristic morphology.
  • A Long TuJl positive neuronal outgrowths are visible with HB9 positive nuclei.
  • B Box in (A) magnified to show nuclear morphology. Scale bars are (A) 200 ⁇ m and (B) 100 ⁇ m.
  • HB9 encapsulated by TuJl stained ⁇ -Tubulin-IIIb expressing neuronal cell bodies and processes.
  • FIGURE S8 shows that the motor neuron markers HB9 and ISL are highly coincident.
  • A Images from A29b motor neuron cultures, as from Figure 4, depicting the efficient production of co-positive HB9/ISL motor neurons.
  • B Box in (A) magnified to demonstrate nuclear HB9 and ISL co-expression in iPS cell derived motor neurons. Scale bars are (A) 100 ⁇ m and (B) 25 ⁇ m.
  • SUPPLEMENTARY FIGURE S9 shows that progenitor cells and mature motor neurons are present in differentiated patient-specific iPS cultures.
  • A OLIG2 and PAX6 progenitor cells the inventorsre abundant in dissociated motor neuron cultures.
  • B The co-expression of HB9 and ChAT indicates the cholinergic transmitter status of matured patient-specific iPS derived motor neurons. Scale bars are 100 ⁇ m.
  • SUPPLEMENTARY FIGURE SlO shows that patient-specific A29b iPS cells also generate glial cells when induced with retinoic acid and sonic hedgehog signaling agonist. Detection of (A) GFAP and (B) SlOO positive glia. Scale bars are 100 ⁇ m.
  • SUPPLEMENTARY TABLE Sl shows that fibroblasts and iPS cells derived from patient A29 are genetically identical.
  • DNA Fingerprinting analysis at 16 independent loci indicates that both iPS cells and A29 patient-derived fibroblasts share all alleles investigated, and are different from commonly available human ES cell lines, such as HuES-I.
  • neurons and glial cells can be efficiently generated from somatic cells of a subject.
  • iPS cells were produced using skin fibroblasts collected from an 82 year-old patient diagnosed with a familial form of ALS. These patient-specific iPS cells possess a gene expression signature similar to human ES cells and can be differentiated into cell types representative of each of the three embryonic germ layers. These iPS cells were used to produce patient-specific motor neurons and glia, the cell types implicated in ALS pathology.
  • SODl superoxide dismutase
  • T familial ALS
  • SODl is also known as ALS, SOD, ALSl, IPOA and homodimer.
  • the protein encoded by this gene binds copper and zinc ions and is one of two isozymes responsible for destroying free superoxide radicals in the body.
  • the encoded isozyme is a soluble cytoplasmic protein, acting as a homodimer to convert naturally-occuring but harmful superoxide radicals to molecular oxygen and hydrogen peroxide.
  • the other isozyme is a mitochondrial protein.
  • Transgenes encoding KLF4, SOX2, OCT4, and C-MYC were introduced into patient fibroblasts using VSV-g pseudotyped Moloney-based retroviruses. Approximately 30,000 fibroblasts were transduced twice over 72 hours, cultured for 4 days in standard fibroblast medium, then passaged onto a feeder layer of irradiated mouse embryonic fibroblasts (MEFs) and grown in an ES cell-supportive medium. Within one week, hundreds of colonies composed of rapidly dividing cells with a granular morphology not characteristic of hES cells had appeared, as described previously (6). However, following 2 additional weeks of culture, a small number of colonies with an hES cell morphology (Fig. IB, C) could be identified.
  • the inventors performed DNA fingerprinting analysis for the 3 independent putative PS-iPS cell lines (A29a, A29b, and A29c) and the fibroblasts from which they were derived. Allele assignments indicated that each of the putative iPS cell lines carried the genotype of the patient fibroblasts (Table Sl). Additionally, the SODl genotype of these cell lines was compared with that of the donated fibroblasts and patient medical history using an allele-specific restriction fragment length polymorphism (RFLP) (Fig. S1A,B) and direct sequencing (Fig. ID).
  • RFLP allele-specific restriction fragment length polymorphism
  • the expected L144F polymorphism were detected in the putative A29 iPS cell lines and the fibroblasts from which they were derived, but not in fibroblasts isolated from a healthy control individual (Al 8). Furthermore, PCR analysis of genomic DNA from these three cell lines demonstrated that they all carried integrated copies of the 4 retroviral transgenes with which they were transduced (Fig. SlC).
  • the putative iPS cell lines were pluripotent, their similarity to ES cells was first evaluated. Like ES cells (8), and unlike the parental A29 fibroblasts, the A29 iPS cells displayed an active cell cycle profile with 35% of cells in S or G2/M phases (Fig. S2). The putative iPS cell lines also maintained a normal karyotype (Fig SlD).
  • iPS cell lines exhibited strong alkaline phosphatase activity, and expressed several ES- associated antigens (SSEA-3, SSEA-4, TRA1-60, TRA1-81, NANOG), but were not immunoreactive for a fibroblast-associated antigen (TE-7) (Fig. 1E,F and S3).
  • Quantitative RT-PCR showed that genes expressed in pluripotent cells (REX1/ZFP42, F0XD3, TERT, NANOG, and CRIPTO/TDGFl) were transcribed at levels comparable to hES cells in each of the three putative iPS cell lines (Fig. 2A).
  • Pluripotent cells are by definition capable of differentiating into cell types derived from each of the three embryonic germ layers (10).
  • a property of both ES cells and previously established human iPS cells is their ability, when plated in suspension culture, to form embryoid bodies (EBs) composed of differentiating cell types (Fig. S4A) (6,9,10).
  • EBs embryoid bodies
  • Fig. S4A embryoid bodies
  • Fig. S4A embryoid bodies
  • Fig. 3A immunocytochemical analyses of EBs after 13-16 days of culture showed that the resulting cells from each line had spontaneously differentiated into cell types representative of all three embryonic germ layers (Figs. 3B-F, S4B). Together, these data indicate reprogramming of primary fibroblasts isolated from an ALS patient of advanced age into iPS cells.
  • PS-iPS cells provided herein can be used for differentiation into the disease- relevant cell types required for the development of personalized regenerative medicine.
  • ALS is characterized by the progressive degeneration of spinal cord motor neurons and resultant muscle weakness (1,11), and recent studies have demonstrated that both cell autonomous and non-cell autonomous factors contribute to disease progression (12,13).
  • glia from ALS animal models have been shown to produce factors that are toxic to motor neurons (14,16).
  • These studies indicate that production of both motor neurons and glia would be essential for mechanistic studies, and perhaps eventual cell replacement therapies for ALS.
  • These cell types were generated using a directed differentiation protocol that builds on approaches developed using both mouse and human ES cells (17-20).
  • EBs formed from iPS cells were treated with two small molecules, an agonist of the sonic hedgehog (SHH) signaling pathway and retinoic acid (RA) (Fig. S5A).
  • SHH sonic hedgehog
  • RA retinoic acid
  • Fig. 4A neuronal-like outgrowths were observed.
  • Many of these processes stained positive for a neuronal form of tubulin, ⁇ -tubulin IHb (TuJl), confirming their neuronal nature (Figs. 4B, S6).
  • EBs were dissociated and plated as a single-cell suspension onto laminin-coated slides.
  • HB9 a motor neuron-specific transcription factor (1 T)
  • A29a and A29b cell lines Figs. 4C, S5B, S7.
  • the inventors were able to individually examine 3262 nuclei (from three independent differentiation experiments) and found that 651 stained for HB9, indicating that 20% of all cells express this motor neuron marker.
  • ISL1/2 ISL, transcription factors involved in motor neuron development (17, 18), Figs. 4E-H, S5C, S8).
  • HB9/ISL positive neurons Over half of these HB9/ISL positive neurons expressed choline acetyl transferase (ChAT), demonstrating an advanced degree of cholinergic motor neuron maturation (17) (Figs. S5D, S9B). In addition, cells expressing the glial marker GFAP and SlOO were identified in these cultures (Fig. 4D, SlO). Thus, PS-iPS cells respond appropriately to developmentally relevant patterning signals and neuro-morphogenetic differentiation signals, demonstrating the feasibility of producing large numbers of the cells specifically affected by ALS.
  • ChoAT choline acetyl transferase
  • PS-iPS cell lines produced using skin fibroblasts collected from patients diagnosed with familiar forms of ALS are listed in Table 1.
  • SOD1L144F allele has been described above.
  • SOD1LG85R allele is described, e.g., in Bruijn et al., Neuron 18:327-338 (1997).
  • SOD1L90A allele is described, e.g., in Robberecht et al., Neurology 47:1336-1339 (1996).
  • PS-iPS cell lines produced using skin fibroblasts collected from normal subjects are also listed in Table 1.
  • results with patient derived cells confirm the initial finding that the exogenous expression of only four factors, KLF4, SOX2, OCT4, and C-MYC, is sufficient to reprogram human fibroblasts to a pluripotent state (6).
  • Previous reports using these four genes to generate human iPS cells have required the overexpression of either a murine viral receptor (6) or additional oncogenes such as Large T Antigen and TERT (21).
  • the results using retroviruses pseudotyped to transduce human cells dispel the suggestion by a recent study that these four genes are not sufficient to induce reprogramming (21).
  • a recent report demonstrates that human iPS cells can be generated without C-MYC (27). Paired with our observation that KLF 4 is already expressed in primary human fibroblasts, these results suggest that reprogramming may be achieved by introducing only SOX2 and OCT4 into patient derived cells.
  • retroviral DNA in the host genome did not preclude our ability to terminally differentiate these cells into motor neurons, despite the presence of retroviral integrations. Nevertheless, long-term studies will be needed to compare the in vitro life-span and physiology of these iPS- derived motor neurons to those of control motor neurons derived from hES cell lines.
  • Methods disclosed herein allow the production of large numbers of motor neurons with a patient's exact genotype that would be immune-matched to that individual, a long sought-after goal of regenerative medicine.
  • the production of disease relevant cell types, motor neurons and glia, from a patient with familial ALS can provide large numbers of cells for the study of disease progression in vitro.
  • the methods described hrein will also provide materials and approaches to optimizing cell replacement therapy using iPS technology. First, among several other safety issues, iPS-derived neurons will not be optimal for transplantation until the oncogenic genes and retroviruses (24, 25) used here are replaced with more controlled methods of reprogramming.
  • PS-iPS cells will be important tools for deciphering the cellular, molecular and developmental mechanisms that drive neurodegeneration.
  • Our results demonstrate the feasibility of producing iPS cells from patients with sporadic forms of disease. These cells would carry the precise constellation of genetic information that was associated with the disease in each individual. This approach would allow study of living motor neurons generated from ALS cases with unknown genetic lesions, providing insight into their intrinsic survival properties, their interactions with other cell types, and their susceptibility to the environmental conditions that are considered to play an important role in ALS pathogenesis.
  • tissue samples from patients these PS-iPS cell lines could be propagated indefinitely while retaining the capacity to differentiate into the affected cell-types. The immortality of these cell lines would allow the influence of environmental factors on in vitro correlates of disease progression to be repeatedly tested on a single genotype, a step in understanding how interactions between genes and the environment lead to disease.
  • Patient derived fibroblasts were generated from explants of 3 mm dermal biopsies following informed consent under protocols approved both by Harvard University and Columbia University College of Physicians and Surgeons.
  • the murine leukemia retroviral vectors were engineered to express human cDNA for KLF4, SOX2, OCT4, and C-MYC.
  • Viral particles were VSV-g pseudotyped to allow for efficient transduction of human primary cells.
  • Patient derived fibroblasts were transduced twice over 72 hrs, and cultured for 4 additional days before being seeded onto irradiated MEFs. Primary colonies with hES-like morphology were observed after 3 weeks, and were picked for expansion and characterization.
  • a single cell suspension of PS-iPS cells were seeded to form embryoid bodies.
  • 7- to 10-day old EBs were allowed to adhere to a gelatin coated tissue culture dish then analyzed after an additional 3- to 6-days of culture.
  • 10 day old EBs were induced with retinoic acid and sonic hedgehog signaling agonist. After a 14 day induction period, EBs were dissociated and plated on laminin for motor neuron maturation and analysis.
  • fibroblasts were cultured in KO-DMEM supplemented with 20% Earl's salts 199 and 10% fetal calf serum, with Ix GlutaMax and penicillin/streptomycin (Invitrogen), and 100 ⁇ M 2-mercaptoethanol.
  • hES cell lines HuES-3, -9, and -10 and induced pluripotent stem (iPS) cells the inventorsre cultured in standard hES cell medium as described (Sl) on a monolayer of irradiated CF-I MEFs (GlobalStem). EBs were formed by trypsinization to a single-cell suspension and plating into low-adherence dishes in hES medium without bFGF.
  • EBs For spontaneous differentiation 7-10 day old EBs were plated onto gelatin-coated tissue culture plastics and allowed to differentiate in DMEM+10% fetal calf serum (FCS) for an additional 3-6 days before analysis. For induction of motor neuron differentiation, 10 day old embryoid bodies (EBs) were switched to a basal-neural medium (DMEM/F12 with Ix GlutaMax and penicillin/streptomycin, 1% N2 supplement (Invitrogen), 0.16% D-glucose, and 0.2mM ascorbic acid).
  • FCS fetal calf serum
  • EBs were treated with 1 ⁇ M retinoic acid (RA) and 100 nM SHH agonist for 3 days before the concentration of SHH agonist was increased to 1 ⁇ M for an additional 14 days.
  • Induced EBs were then dissociated with papain (Worthington) and plated onto laminin-coated chamber slides (BD Biosciences) with or without a primary murine glial monolayer.
  • Plated motor neuron cultures were matured in human motor neuron medium (DMEM/F12 with Ix GlutaMax and penicillin/streptomycin, 4% B27 supplement and 2% N2 supplement (Invitrogen), 0.32% D-glucose, 0.4mM ascorbic acid, and 2 ng/mL each of BDNF, GDNF, and CNTF (RScD)).
  • DMEM/F12 with Ix GlutaMax and penicillin/streptomycin 4% B27 supplement and 2% N2 supplement (Invitrogen), 0.32% D-glucose, 0.4mM ascorbic acid, and 2 ng/mL each of BDNF, GDNF, and CNTF (RScD)
  • Derivation of patient specific fibroblasts Patient derived fibroblasts were generated from explants of 3 mm dermal biopsies following informed consent under protocols approved both by Harvard University and Columbia University College of Physicians and Surgeons. After 1-2 weeks, fibroblast outgrowths from
  • Retroviral production and PS-iPS generation Human cDNAs for KLF4, SOX2, OCT4, and C-MYC (OpenBiosystems) were subcloned into the murine leukemia viral vector pMXs-Tcll (Addgene plasmid 13364) (S2,S3). Moloney gag-pol (pUMVC; Addgene plasmid 8449) and VSV-g envelope (pCMV-VSV-g; Addgene plasmid 8454) (S4) wetre obtained from Addgene.
  • Plasmids were transiently co-transfected into 293FT packaging cells (ATCC) at a 10:9:1 ratio (transgene:gag-pol:VSV-g) using SuperFect (Qiagen). Viral supernatant was harvested after 60 hours, filtered through a 0.45 ⁇ m low protein binding cellulose acetate filter, and concentrated by centrifugation.
  • PS patient specific
  • TE-7 (1:500, Chemicon), SSEA-3 (1:100, R&D), SSEA-4 (1:500, DSHB), TRA1-60 (1:500, Chemicon), TRA1-81 (1:500, Chemicon), Nanog (1:500 Abeam), AFP (1:500, DAKO), Desmin (1:100, Neomarkers), alpha-SMA (1:500, Sigma), Isletl/2 (1:200, DSHB; 1:500 Santa Cruz; 1:500 from Tom Jessell), HB9 (1:100 DSHB), ChAT (1:100, Millipore), TuJl (1:1000, Sigma), and GFAP (1:1000, DAKO; 1:500, Sigma).
  • Alkaline phosphatase activity was detected in live cultures using the alkaline phosphatase substrate kit (Vector) according to manufacture's instructions. Cell cycle analysis was performed on cells fixed overnight in cold 70% ethanol. Cells were treated with RNaseA (Qiagen) and stained with propidium iodine (Invitrogen) in 0.1% BSA and analyzed on a BD Biosystems LSRII FACS analyzer using doublet discrimination.
  • DNA fingerprinting and SODl geno typing DNA Fingerprinting analysis at 15 Codis loci plus sex chromosome assignment was performed by Cell Line Genetics (Madison, WI). Karyotyping was performed by the University of Massachusetts Memorial Hospital Cytogenetics Laboratory (Worcester, MA). Genotyping of the SODl L144F single nucleotide polymorphism was performed by PCR amplification of genomic DNA (Table S2) and either digested with CviKI-1 (NEB) or directly sequenced (Davis Sequencing, Davis, CA).
  • Genotyping of the SODl G85R single nucleotide polymorphism was performed by PCR amplification of genomic DNA and either digested with (NEB) or directly sequenced (Davis Sequencing, Davis, CA). Genotyping of the SODl D90A single nucleotide polymorphism was performed by PCR amplification of genomic DNA and either digested with (NEB) or directly sequenced (Davis Sequencing, Davis, CA).

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Abstract

L’invention concerne un procédé de production d’une cellule neuronale ou gliale à partir d’une cellule somatique d’un sujet, ledit sujet ayant des neurones qui sont absents, malades, inactifs ou qui, de manière générale, possèdent un phénotype indésirable. Le procédé selon l’invention comprend la conversion de la cellule somatique du sujet en une cellule iPS ; et la conversion de la cellule iPS en une cellule neuronale ou gliale.
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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2010008486A2 (fr) * 2008-06-24 2010-01-21 Parkinsons Institute Lignées cellulaires pluripotentes et procédés d’utilisation de celles-ci
EP2512514A1 (fr) * 2009-12-14 2012-10-24 Kyoto University Composition pharmaceutique destinée à la prévention ou au traitement de la sclérose latérale amyotrophique
US8778623B2 (en) * 2012-09-14 2014-07-15 Wisconsin Alumni Research Foundation Compositions and methods of using differentiated cells sensitized to botulinum neurotoxin

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Publication number Priority date Publication date Assignee Title
WO2010008486A2 (fr) * 2008-06-24 2010-01-21 Parkinsons Institute Lignées cellulaires pluripotentes et procédés d’utilisation de celles-ci
WO2010008486A3 (fr) * 2008-06-24 2010-04-08 Parkinsons Institute Lignées cellulaires pluripotentes et procédés d’utilisation de celles-ci
EP2512514A1 (fr) * 2009-12-14 2012-10-24 Kyoto University Composition pharmaceutique destinée à la prévention ou au traitement de la sclérose latérale amyotrophique
EP2512514A4 (fr) * 2009-12-14 2013-04-24 Univ Kyoto Composition pharmaceutique destinée à la prévention ou au traitement de la sclérose latérale amyotrophique
US8778623B2 (en) * 2012-09-14 2014-07-15 Wisconsin Alumni Research Foundation Compositions and methods of using differentiated cells sensitized to botulinum neurotoxin

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