Induced pluripotent cell-derived oligodendrocyte precursor cells for the treatment of myelin disorders

Abstract

To provide induced pluripotent cell-derived oligodendrocyte progenitor cells for treatment of myelin disorders.SOLUTION: The invention provides a preparation of CD140a / PDGFRα positive cells, where the preparation comprises oligodendrocyte progenitor cells co-expressing OLIG2 and CD140a / PDGFRα, and where the preparation of cells is derived from pluripotential cells derived from skin cells. In a preparation, the preparation of cells is derived from pluripotential cells derived from umbilical cord blood. In a preparation, the preparation of cells is derived from pluripotential cells derived from peripheral blood. In a preparation, the preparation of cells is derived from pluripotential cells derived from bone marrow.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 761,584, filed February 6, 2013, and No. 61 / 780,265, filed March 13, 2013, which are hereby incorporated by reference in their entireties.

[0002] FIELD OF THEINVENTION The present invention relates to preparations of induced pluripotent cell-derived oligodendrocyte precursor cells, and methods of making, isolating and using these cells. [Background technology]

[0003] 2. Background of the Invention A wide range of diseases, from hereditary leukodystrophies to vascular leukoencephalopathy to multiple sclerosis, result from myelin damage or loss. Particularly in childhood leukodystrophies, in the case of toxic storage disorders, compact myelin is either not developed normally or is damaged. Recent studies have focused on the use of transplanted oligodendrocytes or their progenitors for the treatment of these congenital myelin disorders. Both rodent-derived and human-derived cell implants have been evaluated in various experimental models of congenital myelin disorders. The myelinogenic potential of implanted brain cells was first recognized in shiverer mice (Lachapelle et al., "Transplantation of CNS Fragments Into the Brain of Shiverer Mutant Mice: Extensive Myelination by Implanted Oligodendrocytes," Dev. Neurosci 6:325-334 (1983)). Shiverer is a mutational deficiency in myelin basic protein (MBP) in which a premature stop codon in the MBP gene results in the loss of its last five exons (Roach et al., "Chromosomal Mapping of Mouse Myelin Basic Protein Gene and Structure and Transcription of the Partially Deleted Gene in Shiverer Mutant Mice," Cell 42:149-155 (1985)). Shiverer is an autosomal recessive mutation, and shi / shi homozygotes fail to develop central compact myelin. They die prematurely, typically at 20-22 weeks of age, from ataxia, dyscoordination, spasticity, and seizures.When fetal human brain tissue was embedded in shiverer, evidence of both oligodendrocyte differentiation and local myelination was observed (Lachapelle et al., "Transplantation of Fragments of CNS Into the Brains of Shiverer Mutant Mice: Extensive Myelination by Implanted Oligodendrocytes," Dev. Neurosci 6:326-334 (1983) (Non-Patent Document 1); Gumpel et al., "Transplantation of Human Embryonic Oligodendrocytes Into Shiverer Brain," Ann NY Acad Sci 495:71-85 (1987) (Non-Patent Document 3); and Seilhean et al., "Myelination by Transplanted Human and Mouse Central Nervous System Tissue After Long-Term Cryopreservation," Acta Neuropathol 91:82-88 (1996) (Non-Patent Document 4)). However, these unfractionated implants only result in patchy remyelination, allowing the concomitant development of other potentially undesirable phenotypes. The myelinogenic capacity of enriched glial progenitor cells was then evaluated and found to be capable of myelination of shiverer axons, albeit with low efficiency, possibly due to astrocytic differentiation by the transplanted cells (Warrington et al., "Differential Myelinogenic Capacity of Specific Development Stages of the Oligodendrocyte Lineage Upon Transplantation Into Hypomyelinating Hosts," J. Neurosci Res 34:1-13 (1993)).Yandava et al., "Global Cell Replacement is Feasible via Neural Stem Cell Transplantation: Evidence from the Dysmyelinated Shiverer Mouse Brain," Proc. Natl. Acad. Sci. 96:7029-7034 (1999) (Non-Patent Document 6), subsequently described that immortalized multipotent progenitor cells can also contribute to myelination in shiverers. Duncan et al. similarly described that oligosphere-derived cells grown from the subventricular zone of rodent neonates can be transplanted into myelin-deficient rats, another mutant for dysmyelination, during perinatal intraventricular administration (Learish et al., "Intraventricular Transplantation of Oligodendrocyte Progenitors into a Fetal Myelin Mutant Results in Widespread Formation of Myelin," Ann Neurol 46:716-722 (1999) (Non-Patent Document 7)).

[0004] Human glial precursor cells capable of oligodendrocyte maturation and myelination have been derived from both fetal and adult human brain tissues (Dietrich et al., "Characterization of A2B5+Glial Precursor Cells From Cryopreserved Human Fetal Brain Progenitor Cells," Glia 40:65-77 (2002) (Non-Patent Document 8); Roy et al., "Identification, Isolation, and Promoter-Defined Separation of Mitotic Oligodendrocyte Progenitor Cells From the Adult Human Subcortical White Matter," J. Neurosci. 19:9986-9995 (1999) (Non-Patent Document 9); Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nat. Med. 10:93-97 (2004) (Non-Patent Document 10)), and also derived from human embryonic stem cells (Hu et al., "Differentiation of Human Oligodendrocytes From Pluripotent Stem Cells," Nat. Protoc. 4:1614-1622 (2009) (Non-Patent Document 11); Izrael et al., "Human Oligodendrocytes Derived From Embryonic Stem Cells: Effect of Noggin on Phenotypic Differentiation in Vitro and on Myelination in Vivo," Mol. Cell. Neurosci. 34:310-323 (2007) (Non-Patent Document 12); and Keirstead et al."Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Transplants Remyelinate and Restore Locomotion After Spinal Cord Injury," J.Neurosci.25:4694-4705(2005) (Non-Patent Document 13)), congenital myelination disorders (Sim et al., "CD140a Identifies a Population of Highly Myelinogenic,Migration-Competent and Efficiently Engrafting Human Oligodendrocyte Progenitor Cells," Nat.Biotechnol.29:934-941(2011) (Non-Patent Document 14), Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nat.Med.10:93-97(2004) (Non-Patent Document 15), Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2: 553-565 (2008) (Non-Patent Document 16)), and adult demyelination (Windrem et al., "Progenitor Cells Derived From the Adult Human Subcortical White Matter Disperse and Differentiate as Oligodendrocytes Within Demyelinated Lesions of the Rat Brain," J. Neurosci. Res.69:966-975 (2002) (Non-Patent Document 17)) have proven effective in experimental models of both the brain and spinal cord. Despite these successes in immunodeficient mice, immune rejection has so far prevented the use of allogeneic human cells as transplantation vectors. Concerns over donor cell rejection are particularly problematic with respect to adult demyelinating diseases such as multiple sclerosis, where the inflammatory processes underlying these disorders may present an inherently hostile environment for any allogeneic graft (Keyoung and Goldman, "Glial Progenitor-Based Repair of Demyelinating Neurological Diseases," Neurosurg. Clin. N. Am. 18:93-104 (2007) (Non-Patent Document 18)).

[0005] The present invention is directed to overcoming this and other deficiencies in the art. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lachapelle et al.,"Transplantation of CNS Fragments Into the Brain of Shiverer Mutant Mice:Extensive Myelination by Implanted Oligodendrocytes,"Dev.Neurosci6:325-334(1983) [Non-Patent Document 2] Roach et al.,"Chromosomal Mapping of Mouse Myelin Basic Protein Gene and Structure and Transcription of the Partially Deleted Gene in Shiverer Mutant Mice,"Cell42:149-155(1985) [Non-Patent Document 3] Gumpel et al., "Transplantation of Human Embryonic Oligodendrocytes Into Shiverer Brain," Ann NY Acad Sci495:71-85(1987) [Non-Patent Document 4] Seilhean et al., "Myelination by Transplanted Human and Mouse Central Nervous System Tissue After Long-Term Cryopreservation," Acta Neuropathol91:82-88 (1996) [Non-Patent Document 5] Warrington et al., "Differential Myelinogenic Capacity of Specific Development Stages of the Oligodendrocyte Lineage Upon Transplantation Into Hypomyelinating Hosts," J. Neurosci Res 34:1-13 (1993) [Non-Patent Document 6] Yandava et al.,"Global Cell Replacement is Feasible via Neural Stem Cell Transplantation:Evidence from the Dysmyelinated Shiverer Mouse Brain,"Proc.Natl.Acad.Sci.96:7029-7034(1999) [Non-Patent Document 7] Learish et al., "Intraventricular Transplantation of Oligodendrocyte Progenitors into a Fetal Myelin Mutant Results in Widespread Formation of Myelin," Ann Neurol46:716-722 (1999) [Non-Patent Document 8] Dietrich et al.,"Characterization of A2B5+Glial Precursor Cells From Cryopreserved Human Fetal Brain Progenitor Cells,"Glia40:65-77(2002) [Non-Patent Document 9] Roy et al.,"Identification,Isolation,and Promoter-Defined Separation of Mitotic Oligodendrocyte Progenitor Cells From the Adult Human Subcortical White Matter,"J.Neurosci.19:9986-9995(1999) [Non-Patent Document 10] Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nat.Med.10:93-97(2004) [Non-Patent Document 11] Hu et al.,"Differentiation of Human Oligodendrocytes From Pluripotent Stem Cells,"Nat.Protoc.4:1614-1622(2009) [Non-Patent Document 12] Izrael et al.,"Human Oligodendrocytes Derived From Embryonic Stem Cells:Effect of Noggin on Phenotypic Differentiation in Vitro and on Myelination in Vivo,"Mol.Cell.Neurosci.34:310-323(2007) [Non-Patent Document 13] Keirstead et al., "Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Transplants Remyelinate and Restore Locomotion After Spinal Cord Injury," J. Neurosci. 25:4694-4705 (2005) [Non-Patent Document 14] Sim et al., "CD140a Identifies a Population of Highly Myelinogenic, Migration-Competent and Efficiently Engrafting Human Oligodendrocyte Progenitor Cells," Nat. Biotechnol. 29:934-941 (2011) [Non-Patent Document 15] Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nat. Med. 10:93-97 (2004) [Non-Patent Document 16] Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008) [Non-Patent Document 17] Windrem et al., "Progenitor Cells Derived From the Adult Human Subcortical White Matter Disperse and Differentiate as Oligodendrocytes Within Demyelinated Lesions of the Rat Brain," J. Neurosci. Res. 69:966-975 (2002) [Non-Patent Document 18] Keyoung and Goldman, "Glial Progenitor-Based Repair of Demyelinating Neurological Diseases," Neurosurg. Clin. N. Am. 18:93-104 (2007) Summary of the Invention

[0007] A first aspect of the present invention is directed to a preparation of CD140a / PDGFRα positive cells, the preparation comprising oligodendrocyte precursor cells that co-express OLIG2 and CD140a / PDGFRα, the preparation of cells being derived from pluripotent cells derived from skin cells.

[0008] A second aspect of the invention is directed to a preparation of CD140a / PDGFRα positive cells, the preparation comprising oligodendrocyte precursor cells that co-express OLIG2 and CD140a / PDGFRα, the preparation of cells being derived from pluripotent cells derived from umbilical cord blood.

[0009] Another aspect of the invention is directed to a preparation of CD140a / PDGFRα positive cells, the preparation comprising oligodendrocyte precursor cells that co-express OLIG2 and CD140a / PDGFRα, the preparation of cells being derived from peripheral blood derived pluripotent cells.

[0010] Another aspect of the invention is directed to a preparation of CD140a / PDGFRα positive cells, the preparation comprising oligodendrocyte precursor cells that co-express OLIG2 and CD140a / PDGFRα, the preparation of cells being derived from pluripotent cells derived from bone marrow.

[0011] Another aspect of the invention is directed to a preparation of cells, wherein at least 95% of the preparation are CD140a / PDGFRα positive cells, the preparation comprising oligodendrocyte progenitor cells that co-express OLIG2 and CD140a / PDGFRα, and the oligodendrocyte progenitor cells retain one or more epigenetic markers of differentiated somatic cells other than oligodendrocytes.

[0012] Another aspect of the invention is directed to a method of generating an enriched preparation of oligodendrocyte precursor cells. The method includes culturing a population of induced pluripotent stem cells under conditions effective for the cells to form embryoid bodies, and inducing differentiation of the cells of the embryoid bodies into neuroepithelial cells and formation of neuroepithelial cell colonies. The method further includes exposing the neuroepithelial cell colonies to conditions effective for inducing differentiation into oligodendrocyte precursor cells, thereby forming an enriched preparation of oligodendrocyte precursor cells that co-express OLIG2 and CD140α / PDGFRα.

[0013] Another aspect of the invention is directed to a method of treating a subject having a condition mediated by myelin loss or oligodendrocyte loss comprising administering to the subject any one of the cell preparations of the invention under conditions effective to treat the condition.

[0014] To date, no robust protocol has been reported for the scalable generation of enriched and / or purified preparations of myelinating oligodendrocytes from induced pluripotent cells (iPSCs). Pouya et al., "Human Induced Pluripotent Stem Cells Differentiation into Oligodendrocyte Progenitors and Transplantation in a Rat Model of Optic Chiasm Demyelination," PLoS ONE 6(11):e27925 (2011), which is hereby incorporated by reference in its entirety, reports the generation of human iPSC-derived oligodendrocyte progenitors, but the embryonic stem cell-based differentiation protocol utilized by Pouya was not tailored for specific and controllable direction of neural progenitor differentiation following oligodendrocyte progenitor generation. The lack of controlled differentiation in Pouya's protocol leads to irregular differentiation and the generation of heterogeneous cell preparations contaminated with pluripotent cell types (e.g., Oct4, SOX2 expressing cells), cell types that are not fully differentiated (e.g., Pax6 and Tuj1 expressing cells), and differentiated non-oligodendrocyte progenitor cell types (e.g., neurons, astrocytes, and non-neuronal cell types). Furthermore, because of the irregular differentiation, cell sorting or selection techniques based on a single marker, such as CD140a / PDGFRα, which is specific only for oligodendrocyte progenitor cells within a mixed population of brain cells but not a mixed population of non-brain cells, cannot be used to reliably enrich or purify the desired oligodendrocyte progenitor cells from Pouya's preparation. Thus, Pouya's preparation is not clinically or therapeutically useful for the treatment of conditions resulting from oligodendrocyte loss or myelin loss.

[0015] As described herein, Applicants have developed a robust and reliable protocol for the scalable generation of highly enriched preparations of myelinating oligodendrocytes from skin-derived human iPSCs. As demonstrated herein, iPSCs progress reliably through successive stages of neural progenitor, glial progenitor, oligodendrocyte, and astrocyte differentiation in vitro. Unregulated differentiation is avoided, thus allowing the population to express CD140a / PDGFRα. + The cells are oligodendrocyte precursor cells that can be further purified and enriched using CD140a / PDGFRα-based sorting prior to clinical use. The myelinating competence of the hiPSC-derived oligodendrocyte precursor cells of the present invention was evaluated in the Shiverer mouse model, a genetic model of congenital dysmyelination. These cells myelinate efficiently and robustly in the dysmyelinating Shiverer brain without evidence of either tumor formation or ectopic non-glial differentiation. Transplanted animals survived significantly longer than their untreated counterparts, and many animals otherwise apparently escaped early mortality, a remarkable clinical rescue from a fatal genetic disorder via an iPSC-based regimen. Thus, under normal conditions, human iPSCs are a feasible and effective source of oligodendrocyte precursor cells, and myelinating central oligodendrocytes derived from them are suitable for use as patient-specific therapeutic vectors for the treatment of disorders of central myelin. [The present invention 1001] A preparation of CD140a / PDGFRα positive cells, said preparation comprising oligodendrocyte precursor cells that co-express OLIG2 and CD140a / PDGFRα, said cell preparation being derived from pluripotent cells derived from skin cells. [The present invention 1002] A preparation of CD140a / PDGFRα positive cells, said preparation comprising oligodendrocyte precursor cells that co-express OLIG2 and CD140a / PDGFRα, said cell preparation being derived from pluripotent cells derived from umbilical cord blood. [The present invention 1003] A preparation of CD140a / PDGFRα positive cells, said preparation comprising oligodendrocyte precursor cells that co-express OLIG2 and CD140a / PDGFRα, said cell preparation being derived from peripheral blood derived pluripotent cells. [The present invention 1004] A preparation of CD140a / PDGFRα positive cells, said preparation comprising oligodendrocyte precursor cells that co-express OLIG2 and CD140a / PDGFRα, said cell preparation being derived from pluripotent cells derived from bone marrow. [The present invention 1005] A preparation of cells, at least 95% of which are CD140a / PDGFRα positive cells, said preparation comprising oligodendrocyte progenitor cells that co-express OLIG2 and CD140a / PDGFRα, and said oligodendrocyte progenitor cells retain one or more epigenetic markers of differentiated somatic cells other than oligodendrocytes. [The present invention 1006] The preparation of any one of claims 1001 to 1005, wherein said cells of said preparation are human cells. [The present invention 1007] The preparation of any of claims 1001 to 1005, wherein said oligodendrocyte precursor cells of said preparation further express SOX10, CD9, or a combination thereof. [The present invention 1008] 6. The preparation of any of claims 1001 to 1005, wherein said cell preparation does not contain neurons as defined by MAP2 antibodies. [The present invention 1009] 6. A preparation according to any of claims 1001 to 1005, containing less than 1% residual pluripotent cells. [The present invention 1010] A preparation of any of claims 1001-1005, wherein less than 1% of said preparation of cells express either OCT4, NANOG, or SSEA4 protein. [The present invention 1011] A2B5 + / PSA-NCAM -6. A preparation according to any of claims 1001 to 1005, having an in vivo myelination efficiency which exceeds the in vivo myelination efficiency of a preparation of sorted fetal human tissue-derived oligodendrocyte precursor cells. [The present invention 1012] At the time of transplant, A2B5 + / PSA-NCAM - 6. A preparation of any of claims 1001-1005 that is capable of achieving an in vivo myelination density that exceeds the in vivo myelination density achievable with a preparation of sorted fetal human tissue-derived oligodendrocyte precursor cells. [The present invention 1013] A2B5 + / PSA-NCAM - Any of the preparations of 1001-1005, which upon transplantation are capable of achieving improved survival in a myelination-deficient mammal as compared to the survival achievable with a preparation of sorted fetal human tissue-derived oligodendrocyte progenitor cells. [The present invention 1014] 1005. The preparation of claim 10, wherein at least 90% of said preparation comprises oligodendrocyte precursor cells. [The present invention 1015] 1005. The preparation of claim 10, wherein at least 95% of said preparation comprises oligodendrocyte precursor cells. [The present invention 1016] The preparation of the present invention 1005, wherein said one or more epigenetic markers comprise one or more epigenetic markers of somatic skin cells. [The present invention 1017] The preparation of the present invention 1005, wherein said one or more epigenetic markers comprise one or more methylation markers. [The present invention 1018] 1005. A preparation of the present invention, wherein greater than 95% of the cells in said preparation are CD140a / PDGFRα positive cells. [The present invention 1019] 1. A method of treating a subject having a condition mediated by loss of myelin or loss of oligodendrocytes, comprising: A method comprising administering to said subject any of the preparations of inventions 1001-1005 under conditions effective to treat said condition. [The present invention 1020] The method of claim 1019, wherein said cells of said preparation are human cells. [The present invention 1021] The method of claim 1019, wherein said oligodendrocyte precursor cells of said preparation further express SOX10, CD9, or a combination thereof. [The present invention 1022] The method of claim 1019, wherein said cell preparation does not contain neurons as defined by a MAP2 antibody. [The present invention 1023] The method of claim 1019, wherein said preparation contains less than 1% residual pluripotent cells. [The present invention 1024] The method of claim 1019, wherein less than 1% of said preparation of cells express either OCT4, NANOG, or SSEA4 protein. [The present invention 1025] The method of claim 1019, wherein the preparation is administered to one or more sites of the brain, brain stem, spinal cord, or a combination thereof. [The present invention 1026] The method of claim 1025, wherein said preparation is administered intracerebroventricularly, intracallosally, or intraparenchyma. [The present invention 1027] The method of claim 1019, wherein the preparation is derived from the subject. [The present invention 1028] The method of claim 1019, wherein said administering step is carried out under conditions effective to achieve engraftment of the entire neuraxis with said oligodendrocyte precursor cells of said preparation. [The present invention 1029] The method of claim 1019, wherein said condition is an autoimmune demyelination condition. [The present invention 1030] The method of claim 1029, wherein said autoimmune demyelinating condition is selected from the group consisting of multiple sclerosis, neuromyelitis optica, transverse myelitis, and optic neuritis. [The present invention 1031] The method of claim 1019, wherein said condition is vascular leukoencephalopathy. [The present invention 1032] The method of claim 1031, wherein said vascular leukoencephalopathy is selected from the group consisting of subcortical infarction, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, and spinal cord injury. [The present invention 1033] The method of claim 1019, wherein said condition is a radiation-induced demyelination condition. [The present invention 1034] The method of claim 1019, wherein said condition is childhood leukodystrophy. [The present invention 1035] The method of the present invention, wherein the childhood leukodystrophy is selected from the group consisting of Pelizaeus-Merzbacher disease, Tay-Sachs disease, Sandhoff gangliosidosis, Krabbe disease, metachromatic leukodystrophy, mucopolysaccharidoses, Niemann-Pick A disease, adrenoleukodystrophy, Canavan disease, vanishing white matter disease, and Alexander disease. [The present invention 1036] The method of claim 1019, wherein said condition is periventricular leukomalacia or cerebral palsy. [The present invention 1037] 1. A method for producing an enriched preparation of oligodendrocyte precursor cells, comprising the steps of: culturing a population of induced pluripotent stem cells under conditions effective for said cells to form embryoid bodies; inducing differentiation of cells of said embryoid bodies into neuroepithelial cells and formation of neuroepithelial cell colonies; exposing the neuroepithelial cell colonies to conditions effective to induce differentiation into oligodendrocyte progenitor cells, thereby forming an enriched preparation of oligodendrocyte progenitor cells that co-express OLIG2 and CD140α / PDGFRα. [The present invention 1038] The method of claim 1037, wherein said oligodendrocyte precursor cells of said enriched preparation further express SOX10, CD9, or a combination thereof. [The present invention 1039] separating CD140α / PDGFRα positive cells from said enriched preparation of oligodendrocyte precursor cells, thereby producing a purified preparation of oligodendrocyte precursor cells expressing CD140α / PDGFRα. The method of the present invention 1037 further comprises: [The present invention 1040] The method of claim 1037, wherein said neuroepithelial cells co-express PAX6 and SOX1. [The present invention 1041] The method of claim 1037, wherein said induced pluripotent stem cells are human induced pluripotent stem cells. [The present invention 1042] reprogramming a population of non-pluripotent cells to a pluripotent state, thereby forming said population of induced pluripotent stem cells. The method of the present invention 1037 further comprises: [The present invention 1043] The method of claim 1042, wherein said population of non-pluripotent cells comprises a population of skin cells, umbilical cord blood cells, peripheral blood cells, or bone marrow cells. [The present invention 1044] The exposing step comprises: converting said neuroepithelial cells into pre-oligodendrocyte precursor cells that are OLIG2+ / NKX2.2-; transforming said OLIG2+ / NKX2.2- pre-oligodendrocyte precursor cells into OLIG2+ / NKX2.2+ pre-oligodendrocyte precursor cells; forming said enriched population of oligodendrocyte precursor cells from said OLIG2+ / NKX2.2+ pre-oligodendrocyte precursor cells; The method of the present invention 1037, comprising: [The present invention 1045] Differentiating said enriched preparation of oligodendrocyte precursor cells into oligodendrocytes and / or astrocytes. The method of the present invention 1037 further comprises: [The present invention 1046] A preparation produced by the method of the present invention 1037. [The present invention 1047] A preparation produced by the method of the present invention 1039. [The present invention 1048] A preparation produced by the method of the present invention 1045. [The present invention 1049] 1. A method of treating a subject having a condition mediated by loss of myelin or loss of oligodendrocytes, comprising: Administering to said subject a preparation of invention 1046 or 1047 under conditions effective to treat said condition. [The present invention 1050] The method of claim 1049, wherein the preparation is administered to one or more sites of the brain, brain stem, spinal cord, or a combination thereof. [The present invention 1051] The method of claim 1050, wherein said preparation is administered intracerebroventricularly, intracallosally, or intraparenchyma. [The present invention 1052] The method of claim 1049, wherein the preparation is derived from the subject. [The present invention 1053] 1049. The method of claim 1049, wherein said administering step is carried out under conditions effective to achieve engraftment of the entire neuraxis with said oligodendrocyte precursor cells of said preparation. [The present invention 1054] The method of claim 1049, wherein said condition is an autoimmune demyelination condition. [The present invention 1055] The method of claim 1054, wherein said autoimmune demyelinating condition is selected from the group consisting of multiple sclerosis, neuromyelitis optica, transverse myelitis, and optic neuritis. [The present invention 1056] The method of claim 1049, wherein said condition is vascular leukoencephalopathy. [The present invention 1057] 1056. The method of claim 1056, wherein said vascular leukoencephalopathy is selected from the group consisting of subcortical infarction, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-associated white matter disease, and spinal cord injury. [The present invention 1058] The method of claim 1049, wherein said condition is a radiation-induced demyelination condition. [The present invention 1059] The method of claim 1049, wherein said condition is childhood leukodystrophy. [The present invention 1060] The method of the present invention, wherein the childhood leukodystrophy is selected from the group consisting of Pelizaeus-Merzbacher disease, Tay-Sachs disease, Sandhoff gangliosidosis, Krabbe disease, metachromatic leukodystrophy, mucopolysaccharidoses, Niemann-Pick A disease, adrenoleukodystrophy, Canavan disease, vanishing white matter disease, and Alexander disease. [The present invention 1061] The method of claim 1049, wherein said condition is periventricular leukomalacia or cerebral palsy. [Brief description of the drawings]

[0016] [Figure 1]We show that human iPSCs (hiPSCs) can be directed into oligodendrocyte precursor cells (OPCs). Figure 1A is a schematic protocol of directed differentiation of hiPSCs into OPCs. Embryoid bodies (EBs) were differentiated from undifferentiated hiPSCs (stage 1) from 0-5 days of in vitro culture. EBs were then differentiated as neuroepithelial (NE) cells in neural induction medium (NIM; see Materials and Methods in Examples 1-9) with bFGF. Figures 1B-1D show that undifferentiated hiPSCs (stage 1) and hiPSC colonies expressed pluripotency markers SSEA4 and OCT4. Phase contrast (Figure 1B); SSEA4 (red) (Figure 1C); DAPI (blue). OCT4 (green) (Figure 1D); DAPI (blue). Erythroid bodies (EBs) (Figure 1E) and neuroepithelial cells (Figures 1F and 1G) can be generated from hiPSCs (stages 2 and 3). hiPSC-derived neuroepithelial cells at this stage expressed neuroepithelial markers PAX6 and SOX1. Phase contrast (Fig. 1E); PAX6 (green) (Fig. 1F); SOX1 (red) (Fig. 1G). Fig. 1H and 1I demonstrate that OLIG2+ and NKX2.2- early glial progenitors emerge under the influence of retinoic acid (RA) and palmorfamine, a small molecule agonist of sonic hedgehog signaling. By stage 4, OLIG2 is expressed in early pre-OPCs, which subsequently develop NKX2.2 expression. OLIG2 (red) (Fig. 1H); NKX2.2 (green) (Fig. 1I). Fig. 1J shows that when RA is replaced by bFGF at stage 5, OLIG2+ / NKX2.2- early pre-OPCs differentiated into later stage OLIG2+ / NKX2.2+ pre-OPCs. OLIG2 (red); NKX2.2 (green). Figures 1K-1M show that pre-OPCs were further differentiated into bipotential OPCs in glial induction medium (GIM; see Materials and Methods in Examples 1-9) supplemented with PDGF-AA, T3, NT3, and IGF. Stage 6 was extended up to 3-4 months to maximize the generation of myelinating OPCs. By the time of transplantation, these cells expressed not only OLIG2 and NKX2.2 (Figure 1K), but also SOX10 (Figure 1L) and PDGFRα (Figure 1M).By the end of stage 6, hiPSC OPCs could be identified as OLIG2+ / NKX2.2+ / SOX10+ / PDGFRα+; OLIG2 (red), NKX2.2 (green) (Fig. 1K); SOX10 (red), NKX2.2 (green) (Fig. 1L); PDGFRα (red), OLIG2 (green) (Fig. 1M). Figures 1N-1P show terminal differentiation of hiPSC OPCs into hiPSC-derived oligodendrocytes (hiOLs) in vitro, identified by O4+ (Fig. 1N) and MBP+ (Fig. 10). OLs and GFAP+ astrocytes (Fig. 1P) occurred with the reduction of glial mitogen; O4 (green). (Fig. 1N); MBP (red) (Fig. 10); GFAP (green) (Fig. 1P); DAPI (blue). Scale: 100 μm (Figures 1B-1N, 1P) and 25 μm (Figure 10). [Diagram 2] Characterization of hiPSC lines. All three hiPSC lines in this study show hESC-like morphology (phase images) when compared to the hESC line WA09 / H9 (top row). Immunolabeling confirmed that hiPSCs expressed the immunoreactive NANOG, OCT4, SOX2, SSEA4 and TRA-1-60. Scale: 200 μm. [Diagram 3]Figures 3A-3J demonstrate that both astrocytes and oligodendrocytes efficiently develop from hiPSC OPCs. Figure 3A shows the expression of neural markers during induction of oligodendroglial lineage hiPSC-derived neuroepithelial cells at stage 3, during induction of pre-OPCs at stages 4 and 5, and during induction of OPCs at stage 6. Cultures were immunostained for PAX6 and SOX1, or OLIG2 and NKX2.2, respectively. The percentage of immunopositive clusters for each set of markers was recorded for each hiPSC line. At least three replicates were performed in each group, and data are provided as mean ± SEM. At stage 6, gliogenic clusters were dissociated into single cell suspensions and seeded in GIM. This led to terminal differentiation of both astrocytes and myelinating OLs. Figures 3B and 3C demonstrate that GFAP+ astrocytes were evident in cultures of hiPSC OPCs up to 95 days in vitro, showing C27 (Figure 3B) and K04 (Figure 3C) derived astrocytes. Figure 3D is a graph showing the percentage of GFAP+ astrocytes among all cultured cells at 95 days in vitro, with the remainder expressing oligodendroglial lineage markers (mean ± SEM; see Figure 4I). ICC, immunocytochemistry. Figures 3E-3J are images taken after stage 6 (160 days in vitro), where hiPSC-derived OPCs differentiated as both O4+ (Figures 3E and 3F) and MBP+ (Figure 3G) OLs. Figures 3H-3J show that when co-cultured with human fetal cortical neurons, hiPSC OPCs derived from C27hiPSCs (Figure 3H), C14hiPSCs (Figure 3I), and K04hiPSCs (Figure 3J) all generated MBP+ myelinating OLs that engaged with NF+ axons (MBP, red; NF, green). Scale: 50 μm. [Figure 4-1]Figures 4A-4M show sequential neuroepithelial and glial differentiation from hiPSCs. Figure 4A shows that neuroepithelial cells could be efficiently derived from both the keratinocyte-derived K04 hiPSC line and the fibroblast-derived C14 and C27 hiPSC lines. By stage 3, neural stem cells were evident, organized into rosette-like structures, and co-expressed proneural markers, PAX6 and SOX1 (panel of cell images labeled as Figure 4A). Figures 4B-4C show that early (stage 4; Figure 4B) pre-OPCs and late (stage 5: Figure 4C) OPCs could be derived not only from H9hESC (WA09) cells, but also from all three hiPSC (C14, C27, and K04) lines tested. By stage 4, most hiPSCs or hESC-derived pre-OPCs expressed OLIG2, and fewer expressed NKX2.2. By stage 5, many more NKX2.2+ pre-OPCs had emerged, with double-labeled OLIG2+ / NKX2.2+ cells typically constituting 70-90% of all DAPI+ cells in each line assessed. Figures 4D-4H show that neuroepithelial cells from each hiPSC and hESC line could robustly differentiate into OLIG2+ / NKX2.2+ / SOX10+ OPCs. Figures 4E-4F are separate versions of Figure 4D divided by single color. Figure 4I is a bar graph showing the percentage of OLIG2-, NKX2.2-, or SOX10-expressing OPCs quantified in K04 iPSC OPCs at stage 6. Scale: Figures 4A-4H, 50 µm. [Figure 4-2] Figures 4J-4M are bar graphs showing that oligodendrocyte precursor differentiation occurred in parallel with depletion of pluripotency-associated transcripts, including OCT4 (Figure 4J), hTERT (Figure 4K), OLIG2 (Figure 4L), or NKX2.2 (Figure 4M). mRNA from undifferentiated (stage 1) hiPSCs was compared to that extracted from differentiated (stage 6) iPSC hOPCs derived from C27, K04, and WA9 / H9 cells. Meanwhile, by stage 6, both OCT4 and hTERT transcripts were significantly downregulated in hiPSC OPCs, while pre-oligodendroglial transcripts OLIG2 and NKX2.2 were significantly upregulated. Data are shown as mean ± SEM. [Diagram 5] Figures 5A-5C show that OPCs can be isolated from mixed hiPSC cultures by CD140a and CD9 directed FACS. As shown in Figure 5A, hiPSC OPC-derived OLs were identified and isolated by FACS using monoclonal antibody O4, which distinguishes sulfatides. The incidence of O4+ oligodendroglia varied between 4% and 12% across different hiPSC lines (see Table 3; n=4-7 experiments). Figure 5B shows that OPCs derived from hiPSCs (C27 and K04) can be easily identified with cell surface marker A2B5. Figure 5C shows that OPCs derived from either hiPSCs (C27 and K04) or hESCs (WA09 / H9) can be easily identified with cell surface markers, PDGFRα (CD140a) and CD9, by FACS analysis. The relative percentages of CD140a, CD9, and CD140a / CD9 double-labeled cells varied across the different cell line-derived OPCs ( n = 4–7 experiments). [Figure 6] Figures 6A-6G demonstrate that hiPSCs migrate extensively and differentiate as astroglia and myelinating OLs. hiPSC OPCs generated from all three hiPSC lines migrated throughout Shiverer's brain and engrafted most densely in the white matter. Distribution of C27 (Figure 6A) and K04 (Figure 6B) hiPSC-derived OPCs is shown (hNA+, red, mapped with Stereo Investigator). By 13 weeks of age, C27hiPSC OPCs (Figure 6C), K04hiPSC OPCs (Figures 6E and 6G), and C14hiPSC OPCs (Figure 6H) had matured into MBP-expressing oligodendroglia (green) in the subcortical white matter, including the corpus callosum and internal capsule (Figures 6C, 6E, and 6H), as well as the striatum (Figure 6G) regions. In these 13-week-old shiverer mouse recipients, hiPSC-derived OPCs, C27 (Figure 6D), K04 (Figure 6F), and C14 (Figure 6I), also differentiated as astroglia (human-specific GFAP, green), especially fibrous astrocytes in the central white matter. Scale: 100 µm (Figure 6C-6I). [Figure 7]Figures 7A-7K show that hiPSC OPCs robustly myelinate in vivo. Confocal images of the corpus callosum and internal capsule of white matter in mice transplanted with hiPSC OPCs from all three tested hiPSC lines demonstrate dense donor-derived myelination: C27-derived (Figures 7A and 7B), K04-derived (Figures 7E-7G), and C14-derived (Figures 7I and 7J). Figures 7A, 7G, and 7J show abundant donor-derived MBP expression (green) by C27, K04, and C14 hiPSC OPCs (hNA, red), respectively. Representative Z-stacks of individual hNA+OLs are shown as asterisks (Figures 7A and 7E). By the 19-week time point evaluated here, C27 (Figure 7B), K04 (Figures 7F and 7G), and C14 (Figure 7J) hiPSC oligodendroglia had robustly myelinated axons (NF, red). hiPSC-derived oligodendroglia morphology is illustrated in Figure 7F (K04) and Figure 7I (C14), where Figure 7F shows multi-axonal myelination by a single OL in the striatum. hiPSC OPCs also generated astroglia (human-specific GFAP, green) with complex fibrous morphology typical of human astrocytes (Figure 7C, C27; Figure 7H, K04). Many cells also remained progenitor cells. Immunostaining for NG2 (Figure 7D, C27) and human-specific PDGFRα (Figure 7K, C14). Scale: 50 μm (Figures 7A-7C, 7G, 7J); 20 μm (Figures 7C-7F, 7H, 7K); and 10 μm (Figure 7I and insets into 7A and 7E). [Figure 8]Figures 8A-8E show that hiPSC OPCs myelinate extensively and significantly extend survival of hypomyelinated mice. Figure 8A is a dot map showing distribution of hiPSC-derived donor cells (C27) in the brain of a shiverer mouse at 7 months of age after neonatal transplantation. Extensive dispersion and chimerism with hiPSC OPCs is evident (hNA, red). Figure 8B shows hiPSC-OPC-derived myelination in the forebrain of a shiverer mouse at 7 months of age. Section 1 mm lateral to the section in Figure 8A. MBP immunoreactivity (green) was from all donors. Figures 8C and 8D show myelination in anterior-posterior sections taken at different mediolateral levels from two additional 7-month-old mice transplanted with C27 hiPSC OPCs at birth, respectively. Figure 8E is a Kaplan-Meier plot showing the survival rate of C27 iPSC-OPC transplanted mice (n=22) versus saline-injected control mice (n=19). The remaining transplanted mice were sacrificed for electron microscopy at 9-10 months of age (>=270 days). Scale: 2 mm (Figures 8A and 8B). [Figure 9] Extensive myelination by K04-derived hiPSC OPCs. Myelination by K04-derived hiPSC OPCs at 4.5 months of age in a coronal section of a neonatally transplanted Shiverer brain. MBP, green. Scale: 2 mm. [Figure 10]Figures 10A-10I show that hiPSC-derived oligodendrocytes generate compact myelin and induce nodes of Ranvier. Representative electron microscopy images of sections through the corpus callosum (Figures 10A and 10B) and ventral pons (Figure 10C) of 40-week-old shiverer mice neonatally transplanted with C27hiPSC OPCs show donor-derived compact myelin with prominent major dense lines ensheathing mouse axons. Figures 10D-10G show higher magnification images of donor-derived myelin in the corpus callosum, also at 40 weeks. Figures 10D and 10E show that alternating major periodic lines (arrowheads) and intraperiod lines, characteristic of mature myelin, are evident. Figures 10F and 10G show myelin sheaths within the corpus callosum ensheathing central axons, whose maturation is indicated by the parallel arrangement of tight junctions (Figure 10F, arrowheads) and main period lines (Figure 10G, arrowheads). This mature myelination allowed the organization of structurally appropriate nodes of Ranvier by hiPSC oligodendroglia. Figures 10H and 10I demonstrate the reconstitution of nodes in transplanted shiverers by immunostaining of oligodendrocytes for paranodal Caspr protein (red), where adjacent nodes of Ranvier observed are identified by βIV spectrin (green). Isolated nodes are shown in confocal cross-sections in Figure 10I. Scale: 200 nm (Figures 10A-10E); 100 nm (Figures 10F and 10G); and 5 μm (Figures 10H and 10I). [Figure 11] Figures 11A-11E show myelination in hiPSC-OPC transplanted shiverer mice but not in untreated shiverer mice. Toluidine blue stained semi-thin sections through the corpus callosum and cortical layer VI in C27 hiPSC-derived OPC transplanted (Figure 11A) or untreated (Figure 11B) shiverer brains. Figures 11C and 11D are electron micrographs of the corpus callosum of transplanted (Figure 11C) and untreated (Figure 11D) shiverer brains. Figure 11E is a low magnification view of untransplanted shiverer white matter. The same animal is shown in Figure 11D. Scale: Figures 11A-11B, 10 μm; Figures 11C-11D, 500 nm; Figure 11E, 1 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention is directed to a preparation of CD140a / PDGFRα positive cells, the preparation comprising oligodendrocyte precursor cells co-expressing OLIG2 and CD140a / PDGFRα. In one embodiment of the invention, the preparation of cells is derived from pluripotent cells derived from skin cells. In another embodiment of the invention, the preparation of cells is derived from pluripotent cells derived from umbilical cord blood. In another embodiment of the invention, the preparation of cells is derived from pluripotent cells derived from peripheral blood. In another embodiment of the invention, the preparation of cells is derived from pluripotent cells derived from bone marrow.

[0018] Oligodendrocyte precursor cells, as referred to herein, include a population of bipotential precursor cells that can give rise to both oligodendrocytes and astrocytes.

[0019] As described herein, preparations of CD140a / PDGFRα positive cells are derived from pluripotent cells using a robust and scalable protocol that directs a controlled differentiation process. In one embodiment of the present invention, the pluripotent cells are induced pluripotent stem cells (iPSCs). "Induced pluripotent stem cells" as used herein refer to pluripotent cells derived from non-pluripotent cells, such as somatic or tissue stem cells. For example, but not limited to, iPSCs, adult fibroblasts (see, e.g., Streckfuss-Bomeke et al., "Comparative Study of Human-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts," Eur. Heart J. doi:10.1093 / eurheartj / ehs203 (2012) which are hereby incorporated by reference in their entireties), umbilical cord blood (see, e.g., Cai et al., "Generation of Human Induced Pluripotent Stem Cells from Umbilical Cord Matrix and Amniotic Membrane Mesenchymal Cells," J. Biol. Chem. 285(15):112227-11234 (2110) which are hereby incorporated by reference in their entireties), and / or umbilical cord blood (see, e.g., Giorgetti et al., "Generation of Induced Pluripotent Stem Cells from Human Cord Blood Cells with only Two Factors: Oct4 and Sox2,"Nature Protocols,5(4):811-820(2010)), bone marrow (see, for example, Streckfuss-Bomeke et al., which is hereby incorporated by reference in its entirety."Comparative Study of Human-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts," Eur. Heart J. doi:10.1093 / eurheartj / ehs203 (July 12, 2012); and Hu et al., "Efficient Generation of Transgene-Free Induced Pluripotent Stem Cells from Normal and Neoplastic Bone Marrow and Cord Blood Mononuclear Cells," Blood doi:10.1182 / blood-2010-07-298331 (Feb. 4, 2011)), as well as peripheral blood (e.g., Sommer et al., "Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood using the STEMCCA Lentiviral Cells," which are hereby incorporated by reference in their entireties). Vector," J. Vis. Exp. 68:e4327doi:10.3791 / 4327 (2012). iPSCs can also be derived from keratinocytes, mature B cells, mature T cells, pancreatic beta cells, melanocytes, hepatocytes, foreskin cells, cheek cells, lung fibroblasts, bone marrow progenitor cells, hematopoietic stem cells, adipose-derived stem cells, neural stem cells, and hepatic progenitor cells. Methods for generating iPSCs from non-pluripotent cells are described in more detail herein.

[0020] In the brain, PDGFRα is highly and specifically expressed by oligodendrocyte progenitor cells (Sim et al., "CD140a Identifies a Population of Highly Myelogenic Migration-Competent and Efficiently Engrafting Human Oligodendrocyte Progenitor Cells," Nat. Biotech. 29(10):934-941 (2011), which is hereby incorporated by reference in its entirety). CD140a is the ectodomain of PDGFRα that can be easily detected and therefore serves as a reliable indicator or marker of PDGFRα expression. Thus, PDGFRα positive cells can be identified by CD140a and populations of PDGFRα cells can be enriched using CD140a. Such cells express PDGFα, but not PDGFα. + / CD140a + are called cells.

[0021] While PDGFRα is a highly specific marker of oligodendrocyte precursor cells within a population of brain-derived cells, PDGFRα is expressed by multiple cells outside the brain and therefore does not constitute an oligodendrocyte-specific marker in the context of a mixed population of cells containing both brain and non-brain cell types. Due to the pluripotent nature of iPSCs and the possible irregular differentiation of these cells, the use of two or more oligodendrocyte markers is desirable for accurate identification of oligodendrocyte precursor cells in preparations of cells derived from iPSCs. Thus, oligodendrocyte precursor cells of the various cell preparations of the present invention are identified by their co-expression of CD140a / PDGFRα and oligodendrocyte transcription factor 2 (OLIG2). In some embodiments of the invention, the oligodendrocyte progenitor cells of the preparation are additionally or alternatively identified by co-expression of one or more other oligodendrocyte progenitor markers, such as SOX10, CD9, NKX2.2, or any combination thereof (see, e.g., U.S. Patent Application No. 2011 / 0059055 to Goldman et al., which is hereby incorporated by reference in its entirety).

[0022] The CD140a / PDGFRα / OLIG2 oligodendrocyte progenitor cell fraction of the preparation of the invention may comprise more than 30% of the preparation. In another embodiment of the invention, the oligodendrocyte progenitor cell fraction of the preparation comprises more than 40% of the preparation. In other embodiments of the invention, the oligodendrocyte progenitor cell fraction of the preparation comprises more than 45% of the preparation, more than 50% of the preparation, more than 55% of the preparation, more than 60% of the preparation, more than 65% of the preparation, more than 70% of the preparation, more than 75% of the preparation, more than 80% of the preparation, or more than 90% of the preparation.

[0023] Another aspect of the invention relates to a preparation of cells enriched for oligodendrocyte precursor cells. This aspect of the invention is directed to a preparation of cells, where at least 95% of the preparation are CD140a / PDGFRα positive cells, the preparation comprises oligodendrocyte precursor cells that co-express OLIG2 and CD140a / PDGFRα, where the oligodendrocyte precursor cells retain one or more epigenetic markers of differentiated somatic cells other than oligodendrocytes. In one embodiment of the invention, more than 95% of the cells in the preparation are CD140a / PDGFRα positive cells. In another embodiment of the invention, more than 98% of the cells in the preparation are CD140a / PDGFRα positive cells. In one embodiment of this aspect of the invention, at least 90% of the cell preparation comprises oligodendrocyte precursor cells. In another embodiment of this aspect of the invention, at least 95% of the cell preparation comprises oligodendrocyte precursor cells.

[0024] The cell preparations of the invention are preferably substantially free of contaminating non-oligodendrocyte precursor cells. In particular, preparations of CD140a / PDGFRα positive cells are substantially free (e.g., contain less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of other neural cell types, such as astrocytes (e.g., cells defined by GFAP antibodies), neurons (e.g., cells defined by MAP2 and PSA-NCAM antibodies), microglia (e.g., cells defined by CD11, CD32, and CD36 antibodies), or non-brain cell types. The cell preparations of the invention containing oligodendrocyte precursor cells are also substantially free of undifferentiated, residual multipotent cell types (e.g., containing less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%), e.g., the preparations are substantially free of cells expressing either OCT4, NANOG, or SSEA4, and are substantially free of less differentiated cell lineages, e.g., neural progenitor cells identified by PAX6 and / or TUJ1 expression.

[0025] In one embodiment of the invention, the cells of the preparations of the invention are mammalian cells, including but not limited to, for example, human, monkey, rat, or mouse cells.

[0026] The cell preparations of the present invention, and in particular the oligodendrocyte precursor cell populations of the present invention, can be functionally distinguished from their tissue-derived or embryonic stem cell-derived oligodendrocyte precursor cell counterparts based on the maintenance of one or more epigenetic markers of their somatic origin. For example, when iPSCs are derived from skin cells, such as skin fibroblasts, the iPSC-derived oligodendrocyte precursor cells maintain one or more epigenetic markers of somatic skin cells. The one or more epigenetic markers can include, but are not limited to, methylation marks (e.g., DNA and / or RNA methylation markers), or histone modifications (e.g., acetylation, methylation, ubiquitination, phosphorylation, or sumoylation).

[0027] The oligodendrocyte precursor cell preparations of the invention are functionally distinguishable from their tissue-derived or embryonic stem cell-derived oligodendrocyte precursor cell counterparts. As demonstrated herein, the cell preparations of the invention are A2B5 + / PSA-NCAM - or CD140a + The cell preparations of the present invention have an in vivo myelination efficiency that exceeds that of preparations of sorted fetal human tissue-derived oligodendrocyte precursor cells, as measured by the percentage of central axons that are myelinated as a function of time after transplantation. As demonstrated herein, the cell preparations of the present invention also, upon transplantation, promote the uptake of A2B5 + / PSA-NCAM - or CD140a + It is possible to achieve in vivo myelination densities that exceed those achievable with preparations of sorted fetal human tissue-derived oligodendrocyte precursor cells. Furthermore, the cell preparations of the present invention are capable of achieving in vivo myelination densities that exceed those achievable with preparations of sorted fetal human tissue-derived oligodendrocyte precursor cells. + / PSA-NCAM - or CD140a +Improved survival rates can be achieved in myelination-deficient mammals upon transplantation, as compared to survival rates achievable with preparations of sorted fetal human tissue-derived oligodendrocyte precursor cells. In other words, the proportion of myelination-deficient mammals surviving at any given time point is greater than or equal to the proportion of otherwise similarly treated fetal human tissue-derived A2B5 cells. + / PS-NCAM - The survival rate of iPSC-derived oligodendrocyte precursor cells is greater in mammals transplanted with iPSC-derived oligodendrocyte precursor cells than in mammals transplanted with iPSC-derived oligodendrocyte precursor cells.For example, only about 25% of animals transplanted with fetal tissue-derived oligodendrocyte precursor cells survived beyond 6 months after birth, while 50% of animals transplanted with iPSC-derived oligodendrocyte precursor cells survived beyond 6 months after birth.Therefore, the oligodendrocyte precursor cell preparation of the present invention is functionally distinguishable from non-iPSC-derived oligodendrocyte precursor cell preparation.

[0028] The cell preparations of the invention, including preparations enriched for CD140a / PDGFRα, can be optionally expanded in culture to increase the total number of cells. The cells can be expanded by either continuous or intermittent exposure to PDGF-AA or AB as mitogens that support the proliferation of oligodendrocyte precursor cells. They can be exposed to fibroblast growth factors, including FGF2, FGF4, FGF8, and FGF9, which can support the mitotic proliferation of glial precursor cells but bias their differentiation towards a mixed population of oligodendrocytes and astrocytes. Cells can also be grown in medium supplemented with a combination of FGF2, PDGF, and NT3, which may optionally be supplemented with either platelet-depleted serum or whole serum (see Nunes et al., "Identification and Isolation of Multipotent Neural Progenitor Cells from the Subcortical White Matter of the Adult Human Brain," Nature Medicine 9:239-247; Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nature Medicine 10:93-97 (2004) the methods and compositions described therein are incorporated by reference).

[0029] The oligodendrocyte precursor cell population can be optionally genetically modified to express one or more proteins of interest.For example, the cells can be modified to express exogenous targeting moieties, exogenous markers (e.g., for imaging purposes), or the like.The oligodendrocyte precursor cells of the cell preparation of the present invention can be optionally modified to overexpress endogenous targeting moieties, markers, myelin basic protein, or the like.

[0030] Another aspect of the invention is directed to a method of generating an enriched preparation of oligodendrocyte precursor cells. The method includes culturing a population of induced pluripotent stem cells under conditions effective for the cells to form embryoid bodies, and inducing differentiation of the cells of the embryoid bodies into neuroepithelial cells and formation of neuroepithelial cell colonies. The method further includes exposing the neuroepithelial cell colonies to conditions effective for inducing differentiation into oligodendrocyte precursor cells, thereby forming an enriched preparation of oligodendrocyte precursor cells that co-express OLIG2 and CD140α / PDGFRα. The enriched preparation of oligodendrocyte precursor cells may further express SOX10, CD9, or a combination thereof.

[0031] iPSCs may be derived from any species, including, but not limited to, humans, non-human primates, rodents (mouse, rat), ungulates (cow, sheep, etc.), dogs, cats, rabbits, hamsters, goats, and the like. iPSCs may be obtained from embryonic, fetal, neonatal, and adult tissues from peripheral blood, umbilical cord blood, and bone marrow. Exemplary somatic cells that may be used include fibroblasts, such as skin fibroblasts obtained from skin samples or biopsies, synoviocytes from synovial tissue, keratinocytes, mature B cells, mature T cells, pancreatic beta cells, melanocytes, hepatocytes, foreskin cells, cheek cells, or lung fibroblasts. Exemplary stem or progenitor cells suitable for iPSC generation include, but are not limited to, bone marrow progenitor cells, hematopoietic stem cells, adipose-derived stem cells, neural stem cells, and hepatic progenitor cells. Although skin and cheek provide a readily available and easily attainable source of suitable cells, virtually any cell may be used.

[0032] Induced pluripotent stem cells can be generated by expressing a combination of reprogramming factors in somatic cells. Suitable reprogramming factors that promote and induce iPSC generation include one or more of Oct4, Klf4, Sox2, c-Myc, Nanog, C / EBPα, Esrrb, Lin28, and Nr5a2. In certain embodiments, at least two reprogramming factors are expressed in somatic cells to successfully reprogram the somatic cells. In other embodiments, at least three reprogramming factors are expressed in somatic cells to successfully reprogram the somatic cells. In other embodiments, at least four reprogramming factors are expressed in somatic cells to successfully reprogram the somatic cells.

[0033] iPSCs can be derived by methods known in the art, including the use of integrative viral vectors (e.g., lentiviral, inducible lentiviral, and retroviral vectors), excisable vectors (e.g., transposon and loxP-transduced lentiviral vectors), and non-integrative vectors (e.g., adenoviral and plasmid vectors) to deliver genes that promote cellular reprogramming (see, e.g., Takahashi and Yamanaka, Cell 126:663-676 (2006); Okita. et al., Nature 448:313-317 (2007); Nakagawa et al., Nat. Biotechnol. 26:101-106 (2007); Takahashi et al., Cell 131:1-12 (2007); Meissner et al., Cell 131:1-12 (2007), all of which are hereby incorporated by reference in their entireties. (see Yu et al. Nat. Biotech. 25:1177-1181 (2007), Yu et al. Science 318:1917-1920 (2007), Park et al. Nature 451:141-146 (2008), and U.S. Patent Application Publication No. 2008 / 0233610).Other methods for generating iPS cells are described in WO2007 / 069666, WO2009 / 006930, WO2009 / 006997, WO2009 / 007852, WO2008 / 118820, U.S. Patent Application Publication Nos. 2011 / 0200568 to Ikeda et al., 2010 / 0156778 to Egusa et al., 2012 / 0276070 to Music, and 2012 / 0276636 to Nakagawa, all of which are hereby incorporated by reference in their entireties; Shi et al., Cell Stem Cell 3(5):568-574 (2008); Kim et al., Nature 454:646-650 (2008); Kim et al., Nature 454:646-650 (2008); al., Cell 136(3):411-419(2009), Huangfu et al., Nature Biotechnology 26:1269-1275(2008), Zhao et al., Cell Stem Cell 3:475-479(2008), Feng et al., Nature Cell Biology 11:197-203(2009), and Hanna et al., Cell 133(2):250-264(2008).

[0034] Integration-free approaches to derive iPSCs that do not contain transgenic sequences, i.e., those using non-integrating and excisable vectors, are particularly suitable in therapeutic contexts. Suitable methods of iPSC generation utilizing non-integrating vectors include adenoviral vectors (Stadtfeld et al., "Induced Pluripotent Stem Cells Generated without Viral Integration," Science 322:945-949 (2008), and Okita et al., "Generation of Mouse Induced Pluripotent Stem Cells without Viral Vectors," Science 322:949-953 (2008), which are hereby incorporated by reference in their entireties), Sendi virus vectors (Fusaki et al., "Efficient Induction of Transgene-Free Human Pluripotent Stem Cells Using a Vector Based on Sendi Virus, an RNA Virus That Does Not Integrate into the Host Genome," Proc Jpn 123:111-112 (2008), which are hereby incorporated by reference in their entireties), and integrative vectors (Fusaki et al., "Efficient Induction of Transgene-Free Human Pluripotent Stem Cells Using a Vector Based on Sendi Virus, an RNA Virus That Does Not Integrate into the Host Genome," Proc Jpn 123:111-112 (2008), which are hereby incorporated by reference in their entireties). Acad. 85:348-362 (2009)), polycistronic minicircle vectors (Jia et al., "A Nonviral Minicircle Vector for Deriving Human iPS Cells," Nat. Methods 7:197-199 (2010) which is hereby incorporated by reference in its entirety), and self-replicating selectable episomes (Yu et al., "Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences," Science 324:797-801 (2009) which is hereby incorporated by reference in its entirety).Suitable methods for iPSC generation using excisable vectors are described in Kaji et al., "Virus-Free Induction of Pluripotency and Subsequent Excision of Reprogramming Factors," Nature 458:771-775 (2009); Soldner et al., "Parkinson's Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors," Cell 136:964-977 (2009); Woltjen et al., "PiggyBac Transposition Reprograms Fibroblasts to Induced Pluripotent Stem Cells," Nature 458:766-770 (2009); and Yusa et al., "Generation of Transgene-Free Induced Pluripotent Mouse Stem Cells by the PiggyBac Transposition Reprograms Fibroblasts to Induced Pluripotent Stem Cells," Nature 458:766-770 (2009), all of which are hereby incorporated by reference in their entireties. Transposon," Nat. Methods 6:363-369 (2009). Suitable methods for iPSC generation also include methods involving direct delivery of reprogramming factors as recombinant proteins (Zhou et al., "Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins," Cell Stem Cell 4:381-384 (2009) which is hereby incorporated by reference in its entirety) or as whole cell extracts isolated from ESCs (Cho et al., "Induction of Pluripotent Stem Cells from Adult Somatic Cells by Protein-Based Reprogramming without Genetic Manipulation," Blood 116:386-395 (2010) which is hereby incorporated by reference in its entirety).

[0035] The methods of iPSC generation described above can be modified to include small molecules that improve reprogramming efficiency or even replace reprogramming factors. These small molecules include, but are not limited to, epigenetic modulators such as the DNA methyltransferase inhibitor 5'-azacytidine, the histone deacetylase inhibitor VPA, and the G9a histone methyltransferase inhibitor BIX-01294, along with the L-type calcium channel agonist BayK8644. Other small molecule reprogramming factors include those that target signaling pathways, such as TGF-β inhibitors and kinase inhibitors (e.g., Kenpaullone) (see review by Sommer and Mostoslavsky, "Experimental Approaches for the Generation of Induced Pluripotent Stem Cells," Stem Cell Res. Ther. 1:26doi:10.1186 / scrt26 (August10, 2010), which is hereby incorporated by reference in its entirety).

[0036] Suitable iPSCs derived from adult fibroblasts can be obtained according to the procedures described in Streckfuss-Bomeke et al., "Comparative Study of Human-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts," Eur. Heart J. doi:10.1093 / eurheartj / ehs203 (2012), which is hereby incorporated by reference in its entirety. iPSCs derived from umbilical cord blood cells can be obtained as described in Cai et al., "Generation of Human Induced Pluripotent Stem Cells from Umbilical Cord Matrix and Amniotic Membrane Mesenchymal Cells," J. Biol. Chem. 285(15):112227-11234(2110), and Giorgetti et al., "Generation of Induced Pluripotent Stem Cells from Human Cord Blood Cells with only Two Factors: Oct4 and Sox2," Nature Protocols, 5(4):811-820(2010), which are hereby incorporated by reference in their entireties.Suitable iPSCs derived from bone marrow cells may be obtained using the methods described in Streckfuss-Bomeke et al., "Comparative Study of Human-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts," Eur. Heart J. doi:10.1093 / eurheartj / ehs203 (July 12, 2012), and Hu et al., "Efficient Generation of Transgene-Free Induced Pluripotent Stem Cells from Normal and Neoplastic Bone Marrow and Cord Blood Mononuclear Cells," Blood doi:10.1182 / blood-2010-07-298331 (Feb. 4, 2011), which are hereby incorporated by reference in their entireties. Peripheral blood derived iPSCs can be obtained according to the methods described in Sommer et al., "Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood using the STEMCCA Lentiviral Vector," J. Vis. Exp. 68:e4327doi:10.3791 / 4327 (2012), which is hereby incorporated by reference in its entirety. iPS cells contemplated for use in the methods of the present invention are not limited to those described in the above references, but rather include cells prepared by any method, so long as the cells are artificially derived from cells other than pluripotent stem cells.

[0037] Oligodendrocyte precursor cells can be derived from iPSCs using a protocol that directs iPSCs through successive stages of neural and glial progenitor differentiation, as described herein and shown in Figure 1 A. Each stage of lineage commitment is characterized and specified by the expression of specific cellular proteins.

[0038] Referring to FIG. 1A, stage 1 of the process involves culturing iPSCs under conditions effective to induce embryoid body formation. As described herein, iPSCs may be maintained in co-culture with other cells, such as embryonic fibroblasts, in embryonic stem cell (ESC) medium (e.g., DMEM / F12 containing suitable serum replacement and bFGF). iPSCs are passaged at, for example, 80% confluence before reaching 100% confluence, when colonies are approximately 250-300 μm in diameter. The pluripotent state of the cells may be readily assessed using SSEA4, TRA-1-60, OCT-4, NANOG, and / or SOX2 markers.

[0039] To generate embryoid bodies (EBs), which are complex three-dimensional cellular aggregates of pluripotent stem cells (Stage 2), iPSC cultures are dissociated when they achieve approximately 80% confluence with colony diameters of 250-300 μm or approximately 250-300 μm. EBs are first cultured in suspension in ESC medium without bFGF, then switched to neural induction medium with bFGF and heparin. To induce neuroepithelial differentiation (Stage 3), EBs are plated and cultured in neural induction medium supplemented with bFGF, heparin, and laminin, then switched to neural induction medium supplemented with retinoic acid. Neuroepithelial differentiation is assessed by co-expression of PAX6 and SOX1, which characterize central neural stem and progenitor cells.

[0040] To induce pre-oligodendrocyte precursor cell ("pre-OPC") differentiation, neuroepithelial cell colonies are cultured in the presence of additional factors including retinoic acid, B27 supplementation, and sonic hedgehog (shh) agonists (e.g., purmophamine). The emergence of pre-OPC colonies can be assessed by the presence of OLIG2 and / or NKX2.2 expression. While both OLIG2 and NKX2.2 are expressed by central oligodendrocyte precursor cells, NKX2.2 is a more specific indicator of oligodendroglial differentiation. Thus, the early pre-oligodendrocyte precursor cell stage is characterized by the expression of OLIG2 and / or NKX2.2.+ / NKX2.2 - Marked by cell colonies. OLIG + / NKX2.2 - Early pre-OPCs inhibit late-stage OLIG by exchanging retinoic acid for bFGF. + / NKX2.2 + Differentiate into pre-OPCs. At the end of stage 5, OLIG2 + / NKX2.2 + As shown by the expression profile, a significant percentage of the cells are pre-OPCs.

[0041] Pre-OPCs are further differentiated into bipotential oligodendrocyte progenitor cells by culture in glial induction medium supplemented with growth factors such as triiodothyronine (T3), neurotrophin 3 (NT3), insulin growth factor (IGF-1), and platelet-derived growth factor-AA (PDGF-AA) (stage 6). These culture conditions can be extended for 3-4 months or longer to maximize the generation of myelinating oligodendrocyte progenitor cells. Cell preparations suitable for therapeutic transplantation are identified using the OLIG2 + / NKX2.2 + / SOX10 + / PDGFRα + Identified as containing oligodendrocyte precursor cells. O4 + and terminal differentiation of oligodendrocyte precursor cells into oligodendrocytes in vitro, as specified by myelin basic protein (MBP), occurred in further cultures in which glial mitogens were reduced.

[0042] In some embodiments of the invention, it may be preferable to enrich for oligodendrocyte precursor cells in cell preparations generated using the methods described herein. Thus, in one embodiment, selection of CD140a / PDGFRα positive cells is used to generate a purified or enriched preparation of oligodendrocyte precursor cells. In another embodiment of the invention, selection of CD9 positive cells is used to generate a purified or enriched preparation of oligodendrocyte precursor cells. In yet another embodiment, selection of both CD140a / PDGFRα and CD9 positive cells is used to generate a purified or enriched preparation of oligodendrocyte precursor cells.

[0043] Selection for PDGFRα and / or CD9 markers can be performed sequentially or sequentially and can be performed using conventional methods known in the art, such as immunopanning. Selection methods can optionally include the use of fluorescent sorting (FACS), magnetic sorting (MACS), or any other method that allows for rapid and efficient cell sorting. Examples of methods for cell sorting are taught, for example, in U.S. Patent No. 6,692,957, which is hereby incorporated by reference in its entirety.

[0044] Generally, cell sorting methods use detectable moieties. Detectable moieties include any suitable direct or indirect label, including, but not limited to, enzymes, fluorophores, biotin, chromophores, radioisotopes, colored beads, electrochemical, chemical modifications, or chemiluminescent moieties. Common fluorescent moieties include fluorescein, cyanine dyes, coumarins, phycoerythrin, phycobiliproteins, dansyl chloride, Texas Red, and lanthanide complexes or derivatives thereof. Magnetic cell sorting can be used.

[0045] When cell sorting is performed, the marker may be an ectodomain, and no cell permeabilization or membrane disruption techniques are used. As an example, the PDGFRα marker selection step is optionally performed using an antibody or other binding moiety that binds to the ectodomain of PDGFRα (e.g., CD140a). Suitable antibodies include, but are not limited to, monoclonal and polyclonal antibodies, chimeric antibodies, antibody fragments (e.g., F(ab')2, Fab', Fab fragments) that can bind to the selected marker, and single-chain antibodies. Other binding moieties include marker ligands, cofactors, and the like that specifically bind to the marker. Thus, in the case of markers that are receptors, the receptor ligand or a binding moiety thereof can be used as the detectable moiety. Antibodies and other binding moieties are commercially available or can be made using techniques available to those skilled in the art.

[0046] Those skilled in the art will readily understand how to select according to or against a specific marker. Thus, as an example, a population of cells sorted according to a specific marker includes identifying cells that are positive for that specific marker and retaining those cells for further use or further selection steps. A population of cells sorted against a specific marker includes identifying cells that are positive for that specific marker and eliminating those cells for further use or further selection steps.

[0047] Another aspect of the invention is directed to a method of treating a subject having a condition mediated by loss of myelin or loss of oligodendrocytes. The method comprises administering to the subject any one of the oligodendrocyte precursor cell preparations of the invention under conditions effective to treat the condition. According to this aspect of the invention, any of the cell preparations described herein are suitable for therapeutic treatment.

[0048] Conditions mediated by myelin loss or oligodendrocyte loss that may be treated according to the methods of the invention include dysmyelination disorders and demyelination disorders. In one embodiment of the invention, the condition is an autoimmune demyelinating condition, such as, for example, multiple sclerosis, neuromyelitis optica, transverse myelitis, and optic neuritis. In another embodiment of the invention, the myelin-associated disorder is a vascular leukoencephalopathy, such as, for example, subcortical infarction, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, and spinal cord injury. In another embodiment of the invention, the myelin-associated condition is a radiation-induced demyelinating condition. In another embodiment of the invention, the myelin-related disorder is a childhood leukodystrophy, such as, for example, Pelizaeus-Merzbacher disease, Tay-Sachs disease, Sandhoff gangliosidosis, Krabbe disease, metachromatic leukodystrophy, mucopolysaccharidoses, Niemann-Pick A disease, adrenoleukodystrophy, Canavan disease, vanishing white matter disease, and Alexander disease. In yet another embodiment of the invention, the myelin-related condition is periventricular leukomalacia or cerebral palsy.

[0049] The number of oligodendrocyte precursor cells administered to a subject depends on the size and species of the recipient and the volume of tissue requiring myelination or replacement, ranging from approximately 10 cells per transplant (e.g., injection site). 2 ~10 8 A single transplant (e.g., injection) dose can range from 10 cells to 10 3 ~10 5 , 10 4 ~10 7 , and 10 5 ~10 8 The range may range from 1 to 20 mg / kg, or any total amount for a transplant recipient patient.

[0050] Delivery of cells to a subject may include either a single step or multiple step injection directly into the nervous system. In particular, cells may be delivered directly to one or more sites of the brain, brainstem, spinal cord, and / or any combination thereof. For localized disorders such as demyelination of the optic nerve, a single injection may be used. Although the oligodendrocyte progenitor cells of the present invention are widely distributed within the brain of the transplant recipient, for widespread demyelination or hypomyelination disorders, multiple injection sites may be performed to optimize treatment. Injection is optionally directed into regions of the central nervous system such as the corpus callosum (e.g., within the anterior and posterior annulus), the dorsal columns of the spinal cord, the cerebellar peduncle, the cerebral peduncle, and the white matter areas, via intraventricular, intracallosal, or intraparenchymal injection. Such injections may be performed unilaterally or bilaterally using precise localization methods such as stereotactic brain surgery, optionally with accompanying imaging methods (e.g., high resolution MRI imaging). Although one skilled in the art will recognize that brain regions differ across species, one skilled in the art will also recognize equivalent brain regions across mammalian species.

[0051] The cell graft is optionally injected as dissociated cells, but it can also be provided by local placement of non-dissociated cells. In either case, the cell graft optionally includes an acceptable solution. Such acceptable solutions include solutions that avoid undesirable biological activity and contamination. A suitable solution includes an appropriate amount of a pharma- ceutically acceptable salt to render the formulation isotonic. Examples of pharma- ceutically acceptable solutions include, but are not limited to, saline, Ringer's solution, dextrose solution, and culture medium. The pH of the solution is preferably about 5 to about 8, and more preferably about 7 to about 7.5.

[0052] Injection of the dissociated cell graft can be a flow injection over the inlet, outlet, or both inlet and outlet pathways of the injection device (e.g., cannula, needle, tubing). Automation can be used to provide uniform inlet and outlet velocities, as well as injection rates and volumes.

[0053] Optionally, a multi-site delivery plan, such as described in the examples, can be used. Such a multi-site delivery plan is designed to achieve widespread, dense, and cerebrospinal axis-wide donor cell transplantation through the recipient's central nervous system. Injection sites can be selected to allow the continuous infiltration of migrating donor cells into one or more of the major brain regions, brain stem, and spinal cord white matter areas, without interference (or limited interference) from intervening gray matter structures. For example, injection sites can optionally include four locations within the forebrain subcortex, particularly within the anterior and posterior of the corpus callosum anlagen (bilaterally), as well as within a fifth location within the cerebellar peduncle (dorsally).

[0054] Optionally, the methods of treatment provided herein further include directly or indirectly assessing remyelination, for example, imaging techniques, conduction velocity, or symptomatic improvement are optionally tested following transplantation.

[0055] The advantage of the oligodendrocyte precursor cells of the present invention is the ability to obtain a patient-specific preparation for autologous cell therapy (i.e., the cell preparation is derived from the subject to be treated). Autologous oligodendrocyte precursor cells can be derived, for example, from somatic skin cells, umbilical cord blood, peripheral blood, or bone marrow of the subject to be treated. Oligodendrocyte precursor cells derived from allogeneic and / or xenogeneic sources are also suitable for use in the methods of the present invention. EXAMPLES

[0056] The following examples are provided to illustrate embodiments of the invention, but they are not intended to limit its scope in any way.

[0057] Materials and Methods for Examples 1 to 9 hESC and hiPSC cultures. Four different lines of pluripotent cells were used in this study. These included hESCs (WA09 / H9; Wi Cells, Madison, WI, USA) and hiPSCs of both keratinocyte (K04; K. Hochedlinger) and fibroblast origin (C14 and C27 hiPSCs; L. Studer). The experiments described were approved by the University of Rochester Embryonic Stem Cell Research Oversight Committee.

[0058] OPC generation. OPCs were derived from hESCs and iPSCs using modifications of published protocols as outlined in FIG. 1 and described in detail below (Hu et al., "Human Oligodendrocytes From Embryonic Stem Cells: Conserved SHH Signaling Networks and Divergent FGF Effects," Development 136:1443-1452 (2009); Hu et al., "Differentiation of Human Oligodendrocytes From Pluripotent Stem Cells," Nat. Protoc. 4:1614-1622 (2009b); Izrael et al., "Human Oligodendrocytes Derived From Embryonic Stem Cells: Effect of Noggin on Phenotypic Differentiation in Vitro and on Myelination in Vivo," Mol. Cell. Neurosci. 34:310-323 (2007), which are hereby incorporated by reference in their entireties).

[0059] Directed maturation into astrocytes or oligodendrocytes. Gliogenic clumps derived from hiPSCs or hESCs at 120–170 days in vitro were cultured in suspension in GIM supplemented with platelet-derived growth factor-AA (PDGF-AA; 10 ng / ml), insulin growth factor-1 (IGF-1; 10 ng / ml), and neurotrophin-3 (NT3; 10 ng / ml). To differentiate these OPCs into mature oligodendrocytes (OLs) or astrocytes, the clumps were mechanically chopped with a Sharpoint blade (Surgimed-MLB) into small cell clusters (approximately 50–100 mm in diameter). The chopped OPC clusters were seeded onto polyornithine / laminin-coated 12-well plates and cultured in GIM for 1–2 weeks. For astrocyte induction, OPC clusters were cultured for 1–2 weeks in either GIM supplemented with PDGF-AA (10 ng / ml), IGF-1 (10 ng / ml), and NT3 (10 ng / ml) or in GIM supplemented with 10% fetal bovine serum (FBS; HyClone). To direct OL maturation, culture medium was replaced with half the volume of GIM supplemented with PDGF-AA (5 ng / ml), IGF-1 (5 ng / ml), and NT3 (5 ng / ml) plus half the volume of Neurobasal (NB) medium (Invitrogen) supplemented with B27 (1×) and brain-derived neurotrophic factor (BDNF; 10 ng / ml) and grown for 2–4 weeks. Mature astrocytes were identified by immunostaining with anti-GFAP or anti-CD44. Oligodendroglia were identified using O4 and MBP antibodies.

[0060] Isolation of fetal neural progenitor cells for co-culture. Human fetal forebrain tissue was obtained from aborted fetuses at 20 weeks gestation, second trimester. Tissue was obtained as de-identified tissue as approved by the Research Subjects Review Board of the University of Rochester Medical Center. Tissue samples were cultured in a 20-well plate using Ca 2+ / Mg 2+ (HBSS + / +) and washed 2-3 times with sterile Hank's Balanced Salt Solution. Cortical plate tissue was separated from the ventricular / subventricular zones and then dissociated with papain (Worthington Biochemical) as described below (Keyoung et al., "High-Yield Selection and Extraction of Two Promoter-Defined Phenotypes of Neural Stem Cells From the Fetal Human Brain," Nat. Biotechnol. 19:843-850 (2001); Wang et al., "Prospective Identification, Isolation, and Profiling of a Telomerase-Expressing Subpopulation of Human Neural Stem Cells, Using sox2 Enhancer-Directed Fluorescence-Activated Cell Sorting," J. Neurosci. 30:14635-14648 (2010), which are hereby incorporated by reference in their entireties). 2–4 × 10 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) / F12 supplemented with N-2 supplement (Life Technologies) and basic fibroblast growth factor (bFGF; 20 ng / ml). 6Cells were resuspended at 1000 cells / ml and seeded in suspension culture dishes. One day later, cells were harvested and neurons were isolated by magnetic activated cell sorting (Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008), which is hereby incorporated by reference in its entirety). Briefly, harvested neural progenitor cells were incubated with PSA-NCAM (Chemicon) at 1:100 for 30 minutes, then washed and labeled with rat anti-mouse immunoglobulin M microbeads (Miltenyi Biotech). Bound PSA-NCAM + Neurons were eluted, centrifuged, washed with DMEM / F12, and then cultured in DMEM / F12 with N2, 0.5% PD-FBS, and bFGF (20 ng / ml) for 4–6 days. For coculture with hiPSC OPCs, fetal cortical neurons were dissociated into single cells and then plated onto either poly-L-ornithine / laminin-coated 24-well plates or poly-L-ornithine / fibronectin-coated coverslips (50,000–100,000 cells per well or per coverslip). Re-plated neurons were then replaced with NB medium with B27 (1X) and BDNF (10 ng / ml; Invitrogen) for an additional 6–10 days before coculture.

[0061] Co-culture of hiPSC-derived OPCs with human fetal cortical neurons in vitro. Gliogenic OPC clumps derived from either K04 or C27 hiPSCs were induced for up to 130 days in vitro prior to co-culture. 3 The OPC clusters and their surrounding monolayers were then soaked in cold HBSS for 2-3 weeks. - / -Human cortical neurons were then re-harvested from culture at 4°C and manually minced into small fragments of 100–200 μm in diameter. A small aliquot was completely dissociated into single cells with Accutase (Chemicon) at room temperature for 5 min and then assessed by hemocytometry. For co-culture, hiPSC-OPCs were then seeded at 200,000 cells / ml either with or without human cortical neurons and cultured in a 1:1 mixture of NB / B27 / BDNF and GIM / NT3 / IGF-1 / PDGF-AA medium. Cultures of cortical neurons alone, hiPSC-OPCs alone, or both populations together were grown for an additional 2–4 weeks before fixation and immunolabeling for O4, MBP, GFAP, and βIII-tubulin.

[0062] RNA extraction and RT-PCR. Total RNA was extracted from undifferentiated hESCs and hiPSCs, or hESC- and hiPSC-derived OPCs, using RNeasy mini kit (QIAGEN). The first strand of complementary DNA was transcribed using TaqMan Reverse Transcription kit (Roche#N808-0234). Primer sequences are provided in Table 1 below. Relative abundance of mRNA transcript expression was measured on an ABI PRISM7000 system. The resulting expression data was normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Statistical analysis was performed using transformed data. Means and SEM were calculated according to paired t-tests.

[0063] Table 1. Primers for human genes used for qRT-PCR TIFF0007656949000001.tif129169

[0064] Neonatal xenografts into shiverer mice. shi / shi×rag2 - / -For generation of myelin-deficient, immunodeficient mice, homozygous shiverer mice (The Jackson Laboratory, Bar Harbor, ME, USA) were crossed with homozygous rag2-deficient immunodeficient mice (Shinkai et al., "RAG-2-Deficient Mice Lack Mature Lymphocytes Owing to Inability to Initiate V(D)J Rearrangement," Cell 68:855-867 (1992), which is hereby incorporated by reference in its entirety) on a C3H background (Taconic, Germantown, NY, USA). hiPSC-derived OPCs were prepared for transplantation as described for in vitro coculture. Neonatal pups were either transplanted with 100,000 cells bilaterally into the corpus callosum as described in Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nat. Med. 10:93-97 (2004), which is hereby incorporated by reference in its entirety, or transplanted with 300,000 cells using the procedure described in Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008), which is hereby incorporated by reference in its entirety. At 3 months of age, transplanted mice were anesthetized with pentobarbital and then sedated with cold HBSS. + / +Brains were extracted and further fixed in cold paraformaldehyde for 2 h. Brains processed for electron microscopy were perfused in 4% paraformaldehyde and 0.25% glutaraldehyde.

[0065] Immunohistochemistry of tissue sections. Human cells were identified with mouse anti-hNA, clone 235-1 (MAB1281 at 1:800; Millipore, Billerica, MA, USA). Phenotypes were identified with human-specific NG2 (MAB2029 at 1:200; Millipore), rat anti-MBP (Ab7349 at 1:25), rabbit anti-OLIG2 (Ab33427 at 1:1000; Abcam, Cambridge, MA, USA), human-specific mouse anti-GFAP (SMI-21 at 1:500), mouse anti-NF (SMI-311 and SMI-312 at 1:1,000; Covance, Princeton, NJ, USA), and rabbit anti-Ki67 (RM-9106 at 1:200; Thermo-Fisher, Freemont, CA, USA). Alexa Fluor secondary antibodies, including goat anti-mouse, anti-rat, and anti-rabbit antibodies conjugated with 488, 568, 594, and 647 nm fluorophores, were used at 1:400 (Invitrogen, Carlsbad, CA, USA). PAX6, NKX2.2, OCT4, NANOG, and SOX2 antibodies were used using conditions identical to those in vitro.

[0066] Counting myelinated axons. For NF and MBP staining, areas of dense implantation with human cells were selected and 1 μm stacks of 10 overlapping optical slices imaged at 0.1 μm intervals (Olympus FluoView300) were made for each of three fields within the corpus callosum. Three parallel, equidistant lines were placed over the images perpendicular to the axons. Axons were scored at the intersections with the lines as either myelinated (close apposition to MBP on both sides) or non-myelinated.

[0067] Mapping human cell transplantation. All anti-human nuclear + Cell locations were mapped on 20-μm coronal sections at 160-μm intervals from −3.2 to 1.2 anterior-posterior to bregma.

[0068] Cell counting. Three unilateral, equally spaced samples of corpus callosum, located between −0.4 and 1.2 of bregma, were counted for cells expressing hNA along with either MBP, hGFAP, OLIG2, or Ki67. White matter was also counted for any hNA co-expressing HuC / HuD, OCT4, or NANOG. + Cells were assessed for presence. All data are presented as mean±SEM.

[0069] Electron microscopy. Samples of human iPSC-derived glial chimeric white matter were taken from mice sacrificed at 22-40 weeks of age, perfused with half-strength Karnovsky's fixative, and then processed for ultrastructural analysis of myelin morphology and quality using techniques previously described (Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008) which is hereby incorporated by reference in its entirety).

[0070] Cell preparation. Both stage 1 hESCs and hiPSCs were cultured on irradiated mouse embryonic fibroblast (MEF) cells and fed daily with hESC medium consisting of DMEM / F12 containing 20% ​​KO serum replacement supplemented with bFGF (4 ng / ml, Invitrogen). Both hESCs and hiPSCs were passaged when they reached 80% confluence with colonies of 250-300 μm in diameter, typically every 3-4 days for WA09 / H9 and every 4 days (K04 cells) or 7 days (C14 and C27) for hiPSCs. Undifferentiated stem cells were immunocytochemically confirmed for their expression of human pluripotent stem cell markers, including SSEA4, TRA-1-60, OCT4, NANOG, and SOX2 (Figure 2). Of note, both OCT4 and SOX2 were also utilized as reprogramming factors in the generation of hiPSCs, the generation of which has been described previously. After 5-10 min of incubation in collagenase type IV (1 mg / ml, Invitrogen), the cells were passaged by gently scraping them off the culture dish, then dispersing them five times through a polished glass pipette, followed by centrifugation, washing, and resuspension twice. The cells were then split 1:3-1:4 onto 6-well plates pre-coated with irradiated MEF cells.

[0071] To generate stage 2 embryoid bodies (EBs), hESC or hiPSC cultures were dissociated using Dispase (0.5 mg / ml, Invitrogen) for 5–10 min at 37°C once they had achieved 80% confluence with a colony diameter of 250–300 μm and in the absence of overt differentiation. These criteria proved to be important, as it was observed that the quality of hESC and hiPSC cultures at the stage 1–2 transition crucially influenced the quality of the EBs derived from them and their subsequent differentiation into OPCs. EBs were cultured in suspension in tissue culture flasks (Nunc EasYFlasks, Thermo Scientific) in ESC medium without bFGF for 5 days, then switched to neural induction medium (NIM; DMEM / F12 supplemented with non-essential amino acids and N2) supplemented with bFGF (20 ng / ml, Sigma) and heparin (2 μg / ml, Sigma) for either 2 days (WA9 / H9hES) or 7 days (K04, C14, and C27hiPSC). EBs were then plated onto laminin / poly-ornithine-coated 6-well plates and cultured in NIM supplemented with bFGF, heparin, and laminin (10 μg / ml) for an additional 3 days, after which the medium was switched to NIM supplemented with retinoic acid (RA, 100 nM, Sigma) for 4 days.

[0072] Stage 3 At this time point, the end of stage 3, the efficiency of neuroepithelial differentiation was assessed by immunolabeling of PAX6 and SOX1. The co-expression of these markers characterizes central neural stem and progenitor cells. (PAX6 + / SOX1 + The yield of neuroepithelial colonies, defined as total number of rosette-like colonies / cells, was 52.2 ± 7.5%, 78.4 ± 4.7%, and 76.0 ± 7.0% from K04, C14, and C27 cultures, respectively (N = 3–6 cultures / strain). The efficiency of neuroepithelial colony generation from C14 and C27 hiPSCs was similar to that of WA09 / H9 (75.4 ± 8.8%) (Figures 3A and 4A).

[0073] At stage 4, day 14 (for H9) or day 19 (for K04 and C27) (at the end of stage 3, 4 days after addition of RA), palmorfamine (1 μM, Calbiochem), a sonic hedgehog (shh) agonist, and B27 (Invitrogen) were added to the medium. Nine days later, at either 23 days in vitro (WA9 / H9hES) or 28 days in vitro (K04, C14, and C27hiPSC), cultured NE colonies were mechanically detached and then cultured in suspension in 6-well ultra-low cluster plates (Figure 4B).

[0074] Stage 5. One day after seeding into ultra-low cluster plates, medium supplemented with bFGF (10 ng / ml) in addition to palmofamine and B27 was replaced with NIM. At that time, to confirm the appearance of pre-OPC colonies after RA treatment, the phenotypic composition of aliquots was assessed by staining for OLIG2 and / or NKX2.2. Both OLIG2 and NKX2.2 are expressed in central OPCs, although NKX2.2 is a more specific indicator of oligodendroglial differentiation (see refs. 16-17). At this early pre-OPC stage, the percentage of OLIG2-expressing colonies was higher than that of NKX2.2, reflecting the earlier appearance of OLIG2. + In contrast, by the end of stage 5 (35 days in vitro for WA09 / H9 or 40 days in vitro for K04, C14, and C27), under the influence of bFGF without RA, more NKX2.2 was expressed in parallel with the peak of OLIG2 expression. + A colony has appeared. By the end of this stage, OLIG2 + / NKX2.2 + The percentage of co-expressing colonies was similar among all four strains in this study (Fig. 3A ).

[0075] Stage 6 To initiate stage 6 (day 35 for WA09 / H9 or day 40 for K04, C14, and C27), OLIG2 / NKX2.2-defined pre-OPCs in suspension at stage 5 were switched to glial induction medium (GIM; DMEM / F12 at 60 ng / ml, N1, B27, T3, biotin at 100 ng / ml, dibutyryl-cAMP at 1 μM; all from Sigma) supplemented with PDGF AA (10 ng / ml), IGF-1 (10 ng / ml), and NT3 (10 ng / ml). During this long-term OPC suspension culture, 2 / 3 of the medium volume was changed every 3 days. The resulting stage 6 glial spheres were gently dispersed through a P1000 pipette tip during medium changes to prevent them from clumping.

[0076] Starting at 95 days in vitro, the efficiency of hOPC differentiation, as defined by co-expression of A2B5, CD140a, and CD140a / CD9, was assessed in vitro and in vivo using ICC at successive time points, qRT-PCR, and xenografts into neonatal shiverer mice. Gliaspheres were able to give rise to both mature oligodendrocytes and myelinating OPCs at 120 days in vitro. Between 120 and 200 days in vitro, the incidence of OPC-associated colonies steadily increased, with the percentage of OLIG2- and NKX2.2-coexpressing colonies of OPCs from K04, C14, and C27, respectively, being 73.8±8.7%, 78.9±6.1%, and 79.5±8.5%. Interestingly, the efficiency of hOPC generation by hiPSC cells was consistently higher than that exhibited by WA09 / H9 cells (45.4 ± 20.3%) (Figure 3A).

[0077] Late stage 6 / pre-transplantation By this time point (late stage 6), newly generated hOPCs also expressed other OPC markers, such as CD140a / PDGFRα and SOX10, which typically co-express with OLG2 or NKX2.2 (Figures 4D-4H). At this stage, OLG2 was expressed among all DAPI-identified cells. + , NXK2.2 + , or SOX10+ The percentages of OLIG2, NKX2.2, and GFAP were 61.9±10.3%, 63.4±7.3%, and 84.6±7.0% among hiPCS / K04-derived OPCs, while the corresponding percentages of NKX2.2 / SOX10 co-expressing OPCs were 60.6±4.4% (Figure 4I). To further confirm the efficiency of OPC differentiation, RT-PCR for OLIG2, NKX2.2, and GFAP mRNA was performed, and all genes, as well as their corresponding protein products, were substantially upregulated in stage 6 OPCs (Figures 11C-D and 3C).

[0078] Flow cytometry protocol and analysis. Flow cytometry of hESC- or hiPSC-derived OPCs was performed on a FACSAria IIIU (Becton Dickinson, San Jose, CA). Cells were gently scraped off the culture dish and then treated with Accutase (Chemicon) for 5 min at 37°C with gentle shaking. Samples were then dispersed with a narrow glass Pasteur pipette until a single cell suspension was obtained. Cells were then centrifuged and diluted to 1 × 10 in Miltenyi Washing Buffer (MWB). 6 The cells were resuspended at 1x10 cells / ml. Primary antibodies, directly conjugated antibodies, or their corresponding isotype controls were added to the cells at the concentrations listed below and then incubated on ice for 15 minutes. 5 ml of MWB was added and the cells were spun down. For unconjugated antibodies, the pelleted cells were resuspended at 1x10 cells / ml in MWB. 6 The cells were resuspended to 1–1.5 × 10 cells / ml and the appropriate secondary antibody, Alexa-488-conjugated goat anti-mouse IgM, was added at a 1:500 dilution. Samples were incubated on ice for 15 min and then washed with 5 ml MWB for 10 min. All samples were then resuspended at 1–1.5 × 10 cells / ml in phenol red-free DMEM / F-12. 6The cells were resuspended to a concentration of 1000 cells / ml and then passed through a 40 μm cell strainer (Becton Dickinson, BD). DAPI was added at 1 μg / ml. Cells were analyzed by forward and side scatter through a 582 ± 15 nm bandpass filter for PE fluorescence, a 530 ± 30 nm bandpass filter for Alexa Fluor488 / FITC fluorescence, a 695 ± 40 nm bandpass filter for PERCP-Cy5.5, and a 450 ± 50 nm bandpass filter for DAPI fluorescence. Unstained cells were used to set the background fluorescence and a false positive rate of 0.5% was accepted. Antibodies used were mouse IgM isotype control (Chemicon, PP50), mouse anti-A2B5 (IgM, Chemicon, MAB312), mouse anti-O4 (IgM, Chemicon, MAB345), PE mouse IgG 2a , κ isotype control (BD, 555574), PE mouse anti-human CD140a (IgG 2a , BD, 556002), PERCP-Cy5.5 mouse IgG1 isotype control (BD, 347212) and PERCP-Cy5.5 mouse anti-human CD9 (IgG1, BD, 341649).

[0079] In vitro immunocytochemistry. Pluripotent hESCs or hiPSCs grown on irradiated MEF cells were cultured for 3–4 days before fixation with 4% paraformaldehyde. Similarly, differentiated neurogenic or gliogenic clusters were seeded on poly-ornithine and laminin-coated 24-well plates and cultured for 3 days before fixation with 4% paraformaldehyde. Gliogenic clumps containing hOPCs at later stages were chopped into small fragments, seeded on poly-ornithine / laminin-coated 24-well plates and cultured for 2–4 weeks before fixation, depending on the experiment. Fixation was performed with 4% paraformaldehyde for 5 min at room temperature, followed by 3 washes with PBS. Immunolabeled cells were incubated with primary antibodies overnight at 4°C and secondary antibodies for 30 min at 25°C. Primary antibodies were mouse anti-OCT4, mouse anti-SSEA4, mouse anti-TRA-60 (all used at a dilution of 1:100, from Chemicon), rabbit anti-NANOG (1:500, Abcam); rabbit anti-PAX6 (1:400, Covance), goat anti-OLIG2 (1:200, R&D), mouse anti-NKX2.2 (1:100, DSHB), rabbit anti-PDGFR (1:400, Santa Cruz Biotechnology), rabbit anti-SOX10 (1:400, Advanced Bioscience Resources), goat anti-SOX1 (1:100, R&D Systems), goat anti-SOX2 (1:1000, R&D Systems), mouse anti-GFAP (1:400, Covance), rabbit anti-GFAP (1:1000, Chemicon), NESTIN (1:1000, Millipore Bioscience Research Reagents), mouse anti-III-tubulin (1:1000, Covance), oligodendrocyte sulfatide identified by MAb O4 (1:100, Millipore Bioscience), and rat anti-MBP (1:25, Abcam).

[0080] Example 1 - Human iPSCs can be efficiently directed into glial progenitor cells Four different iPSC lines from three different sources were used in this study, including WA09 / H9 hESCs (Thomson et al., "Embryonic Stem Cell Lines Derived From Human Blastocysts," Science 282:1145-1147 (1998)), keratinocyte-derived K04 hiPSCs (Maherali et al., "A High-Efficiency System for the Generation and Study of Human Induced Pluripotent Stem Cells," Cell Stem Cell 3:340-345 (2008)), and fibroblast-derived C14 and C27 hiPSCs (Chambers et al., "Highly Efficient Neural Conversion of Human ES and iPS Cells by Dual Inhibition of SMAD Cells," Cell Stem Cell 3:340-345 (2008)), which are hereby incorporated by reference in their entirety. The present invention relates to a method for generating glial progenitor cells from hESCs, including the generation of glial progenitors from hESCs using a hybrid protocol (Hu et al., "Differentiation of Human Oligodendrocytes From Pluripotent Stem Cells," Nat. Protoc. 4:1614-1622 (2009); Izrael et al., "Human Oligodendrocytes Derived From Embryonic Stem Cells: Effect of Noggin on Phenotypic Differentiation in Vitro and on Myelination in Vivo," Mol. Cell. Neurosci. 34:310-323 (2007)), which are hereby incorporated by reference in their entireties, and then optimized to generate the resulting hybrid protocol for use with WA09 / H9 hESCs.The resulting protocol was modified to further optimize its efficiency using different reprogramming protocols in three hiPSC lines derived from different cell sources and in different laboratories (Chambers et al., "Highly Efficient Neural Conversion of Human ES and iPS Cells by Dual Inhibition of SMAD Signaling," Nat. Biotechnol. 27:275-280 (2009); Maherali et al., "A High-Efficiency System for the Generation and Study of Human Induced Pluripotent Stem Cells," Cell Stem Cell 3:340-345 (2008)), which are hereby incorporated by reference in their entireties (Figure 2). The resulting six-stage OPC differentiation protocol, ranging from 110 to 150 days in vitro, described above and outlined in Figure 1, efficiently generated human OPCs (hOPCs) and their mature progeny, including both astrocytes and OLs, from hESC and hiPSC analogs (Figures 1B-1P).OLIG2+ / NKX2.2+ glia formation at stage 6 (hereby incorporated by reference in their entireties; Qi et al., "Control of Oligodendrocyte Differentiation by the Nkx2.2 Homeodomain Transcription Factor," Development 128: 2723-2733 (2001); Zhou et al., "The bHLH Transcription Factor Olig2 Promotes Oligodendrocyte Differentiation in Collaboration with Nkx2.2," Neuron 31: 791-807 (2001); Zhou et al., "Identification of a Novel Family of Oligodendrocyte Lineage-Specific Basic Helix-Loop-Helix Transcription Factor Olig2," Neuron 31: 791-807 (2001); The efficiency of OPC generation, as determined by the incidence of cell clusters, ranged from 45.4±20.3% in WA09 / H9-derived hESCs to 73.8±8.7%, 78.9±6.1%, and 79.5±8.5% in K04-, C14-, and C27-derived OPCs, respectively (all data presented as mean±SEM; Figures 3A and 4). Thus, each of the hiPSC and hESC lines could be directed towards highly enriched preparations of OLIG2+ / PDGFRa+ / NKX2.2+ / SOX10+ OPCs. Indeed, the efficiency of differentiation of OPCs from hiPSCs was always higher than that of WA09 / H9 hESCs, regardless of whether they were derived from keratinocytes (K04 cells) or fibroblasts (C14 and C27 cells).

[0081] Example 2 - Both astrocytes and OLs are efficiently derived from hiPSC-derived hOPCs Both in vitro and in vivo, hiPSC OPCs readily differentiated into astrocytes and OLs. GFAP-defined astroglia first appeared by day 70 in vitro, significantly earlier than OLs. By late stage 6, at 120 days in vitro, GFAP+ astrocytes were found to be abundant when gliogenic masses were seeded on polyornithine / laminin-coated surfaces (Figures 3B-3D). By that time, GFAP+ cells constituted 40%-50% of cells in OPC-derived cultures across all cell lines (Figure 3D). Quantitative RT-PCR confirmed the upregulation of GFAP messenger RNA (mRNA) expression during OPC differentiation in all cell lines (Table 2).

[0082] Table 2. Emergence of astrocytes during OPC induction: qPCR of GFAP TIFF0007656949000002.tif32133 Human iPSC cultures were subjected to quantitative real-time PCR (qPCR) for astrocyte glial fibrillary acidic protein (GFAP) (normalized to GAPDH) as a dual function of cell line and stage of OPC differentiation in vitro. All data are presented as mean ± SEM.

[0083] The generation of OLs from hESC- and hiPSC-derived OPCs was induced by withdrawal of glialogenic growth factors to half of normal levels (see Materials and Methods). When hiPSC OPCs were exposed to these conditions for 2 weeks, some matured into O4+ and / or myelin basic protein (MBP)+ OLs (Figures 3E-3G). By flow cytometry, O4+ OLs in C27, C14, and K04 hiPSC-derived OPC cultures constituted 11.9±3.8%, 4.1±0.9%, and 7.6±1.5% of all cells, respectively (at 194±15, 186±14, and 205±14 days in vitro, respectively; mean±SEM) (Figure 5A; Table 3). Of note, because we focused on preparing transplantable lineage-biased progenitor populations and immature oligodendroglia, rather than more mature but less transplantable processed OLs, the culture conditions favored early oligodendrocyte differentiation and not post-mitotic oligodendrocyte survival.

[0084] Table 3. Flow cytometry description of hiPSC oligodendroglial abundance in vitro. TIFF0007656949000003.tif32128 Human iPSC-derived OPCs and early oligodendroglia from different cell lines (C27, C14, and K04) were harvested and stained for oligodendrocyte sulfatide, identified by MAb O4, at late stage 6 (>120 days in vitro culture) and analyzed by flow cytometry. Results are given as percentage of O4+ cells (mean percentage ± SEM). N=4-7 replicates / cell line.

[0085] Example 3 - OPCs can be isolated from hiPSC cultures by CD140a and CD9-directed fluorescence-activated cell sorting Flow cytometry of A2B5, CD140a / PDGFaR, and tetraspanin CD9 (Berry et al., "Cytology and Lineage of NG2-Positive Glia," J. Neurocytol. 31:457-467 (2002); Terada et al., "The Tetraspanin Protein, CD9, Is Expressed by Progenitor Cells Committed to Oligodendrogenesis and Is Linked to Beta1 Integrin, CD81, and Tspan-2," Glia 40:350-359 (2002), which are hereby incorporated by reference in their entireties) was then used to identify and quantitate hiPSC OPCs in stage 6 cultures (Figures 5B and 5C). CD140a+ OPCs derived from C27, C14, and K04 hiPSCs, respectively, constituted 33.0±10.3%, 32.8±12.0%, and 41.1±6.1% of all cells compared to 37.5±10.2% of H9-derived cells (Figure 5C; Table 4). The CD9+CD140a+ fraction of cells defined in the later stage OPC pool constituted 24.0±8.0% and 12.4±2.3% of cells in stage 6 C27 and K04 hiPSC cultures, respectively. Comparable cultures of H9-derived OPCs contained 15.0±4.9% CD9+ / CD140a+ cells (Figure 5C; Table 4; n=4-7 replicates each). Thus, hiPSC OPCs can be specified and isolated at different stages of lineage restriction, sequentially represented by A2B5, CD140a, CD9, and O4. Selection based on these epitopes allows for the isolation of relatively pure populations of hiPSC OPCs while removing residual undifferentiated cells from the isolate.

[0086] Table 4. Flow cytometry of cell-selective surface markers during OPC induction. TIFF0007656949000004.tif31146OPCs derived from three different hiPSC cell lines (C27, C14, and K04), as well as hESCs (WA9 / H9) were harvested and stained for CD140a, CD9, or A2B5 at late stage 6 (>120 days in vitro culture) and then analyzed by flow cytometry. Data include the mean percentages (mean ± SEM) of CD140a+, CD9+, CD140a+ / CD9+, and A2B5+ cells (n=4-7 replicates / cell; mean ± SEM).

[0087] Example 4 - hiPSC-derived OLs generate MBP upon in vitro contact with human axons The ability of hiPSC OPCs to myelinate axons in vitro was tested. Using selection directed against polysialylated neural cell adhesion molecule (PSA-NCAM), hiPSC OPCs from each cell line were co-cultured with gestational age (ga) fetal brain (Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008), which is hereby incorporated by reference in its entirety). Neurons were cultured on laminin for 10-14 days to allow for phenotypic maturation and fiber extension, and were confirmed to be free of tissue-derived OLs by O4 immunolabeling. hiPSC OPCs were then prepared as clusters of 50-100 mm in diameter and co-cultured with fetal neurons for 4 weeks, after which cultures were immunolabeled for MBP and neurofilaments (NF). Confocal imaging revealed abundant MBP+ processes that contacted and began to ensheath axons (Figures 3H-3J), but no obvious myelination was evident at the time of imaging. Therefore, to better assess myelination by hiPSC-OPCs, we assessed their transplantation and myelination in vivo.

[0088] Example 5 - hiPSC OPCs efficiently and functionally myelinate the shiverer brain To conclusively establish the myelination competence of hiPSC OPCs, these cells were cultured in neonatal homozygous shiverer (shi / shi) × rag2 - / - The hiPSC-derived OPCs were transplanted into immunodeficient mice. For this experiment, mice were implanted with 100,000 hiPSC-derived OPCs bilaterally into the corpus callosum (n=4-7 mice per hiPSC line for K04, C27, and C14 hiPSC-derived OPCs) using methods previously described (Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008), which is hereby incorporated by reference in its entirety). At 3 or 4.5 months of age, mice were sacrificed and their brains were analyzed in terms of donor cell distribution and density, myelination and percentage of myelinated axons, and node reconstitution. All three hiPSC line-derived OPCs were able to robustly myelinate in the recipient brain, with high donor cell density and extensive dispersion throughout the forebrain white matter observed from each line (Figures 6A and 6B). Differentiation and myelination of C27, C14, and K04 hiPSC OPC-derived oligodendrocytes was similar in extent, with robust myelination of the corpus callosum and internal capsule (Figures 6C, 6E, 6H, 7B, 7G, and 7J). Higher net yields of OPCs were achieved with C27 and K04 hiPSC, a result of superior early neural induction in these two lines compared to C14. Therefore, a quantitative assessment of myelination in recipients of C27 or K04 hiPSC OPCs was performed.

[0089] Quantitative histology demonstrated that human nuclear antigen (hNA) was expressed within the corpus callosum of shiverer recipients at 3 months of age (13 weeks). + / OLIG2 +The C27 and K04 hiPSC-derived OPCs and oligodendroglia, defined as 100% endothelial cells / mm , were 29,498 ± 13,144 and 37,032 ± 8,392 cells / mm , respectively. 3 Among these, densities of 7,298 ± 2,659 (C27) and 2,328 ± 650 (K04) cells / mm 3 expressed MBP, constituting 10.9 ± 5.1% (C27) and 4.7 ± 1.1% (K04) of all donor cells within the sampled midline of the corpus callosum at 13 weeks. To assess myelination efficiency in terms of the percentage of myelinated axons, confocal analysis was used to quantify the fraction of callosal axons ensheathed by hiPSC oligodendroglia in three mice transplanted with C27 hiPSC-derived OPCs. At the 13-week time point analyzed, 17.2 ± 7.2% of host mouse axons were ensheathed within the three sampled corpora callosa (Figure 7B). Strikingly, the density of hiPSC-OPC donor-derived myelination and the percentage of ensheathed axons at 13 weeks was significantly higher than that of A2B5. + / PSA-NCAM - or CD140a +Regardless of whether they are isolated as OPCs or as gliomas, the percentages achieved by OPCs derived from second trimester fetal brain tissue have been shown to be equal to or greater than those achieved by OPCs derived from second trimester fetal brain tissue (Sim et al., "CD140a Identifies a Population of Highly Myelinogenic, Migration-Competent and Efficiently Engrafting Human Oligodendrocyte Progenitor Cells," Nat. Biotechnol. 29:934-941 (2011); Windrem et al., "Fetal and Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate the Congenitally Dysmyelinated Brain," Nat. Med. 10:93-97 (2004); Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 1:13-14 (2013), which are hereby incorporated by reference in their entireties. Cell 2:553-565(2008)).

[0090] Example 6 - hiPSC OPCs efficiently generate astrocytes and OLs in vivo In addition to the large numbers of hiPSC OPCs that differentiated into myelinating OLs in the shiverer mouse brain, many also expressed the resident OLIG2 + and NG2 +Remaining as progenitors, many OPCs from all three hiPSC lines also differentiated as astrocytes, especially fibrous astrocytes in the white matter (Figures 6D, 6F, and 6I). When hiPSC-OPC transplanted mice were evaluated 13 weeks after transplantation into neonates, most donor cells had persisted as progenitors or had initiated oligodendroglial differentiation. By that time, OLIG2, which contained both OPCs and oligodendroglia arising from all K04 and C27 transplants, had differentiated into OLIG2 cells. + The net percentage of cells was 78.7±2.4%, while the remainder was predominantly donor-derived GFAP+ astroglia (Table 5 below).

[0091] Interestingly, despite extensive infiltration of the recipient brain by hiPSC OPCs, substantial astrocytic differentiation was noted by these cells within the prospective white matter, within which donor cells differentiated as morphologically distinct fibrous astrocytes in close association with hiPSC-derived OLs. These hiPSC-derived astrocytes could have arisen by in situ differentiation into astrocytes from lineage-restricted hiPSC-derived astrogliogenic precursors or from hOPCs that still possessed bipotential differentiation potential. In either case, by 3 months after transplantation into neonatal pups, the corpus callosum and internal capsule of the white matter of shivering recipients of OPC grafts derived from all three hiPSC lines displayed a scaffold of human astrocytes with densely transplanted myelinating OLs, resulting in substantially remodeled and densely myelinated central white matter in each case (Figures 6D, 6F, and 6I).

[0092] Table 5. Phenotypic differentiation of hiPSC-OPC derivatives in vivo at 13 weeks of age. TIFF0007656949000005.tif43150Immunolabeling of transplanted mice at 13 weeks of age demonstrated that the majority of transplanted hiPSCs, OPCs, and their progeny express OLIG2 + However, the hiPSC-derived MBP +A significant complement of oligodendroglia, especially OPCs derived from the C27 line, was observed in the hGFAP + Similar to astrocytes, Ki67 indices were observed in transplanted mice at 13 weeks, which were relatively high but not higher than those of fetal tissue-derived OPCs delivered neonatally at the same postnatal age.

[0093] Example 7 - Transplantation of newborns with hiPSC OPCs can rescue shiverer mice We assessed whether the robust engraftment and myelination observed in transplanted shiverer mice was sufficient to mitigate neurological deterioration and extend survival of shiverer mice, which typically die by 20 weeks of age. To this end, a set of 22 neonatal homozygous shiverer x rag2-deficient mice were transplanted with 300,000 C27-derived hiPSC OPCs using a five-site forebrain and brainstem injection protocol that achieves engraftment throughout the neuraxis via transplanted OPCs (Figures 8A-8D) (Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008) which is hereby incorporated by reference in its entirety). A matched set of 19 littermate controls was injected with saline alone and both sets were housed without further manipulation. As expected, 19 shiverer controls without implants died before 5 months of age, with a median survival of 141 days. In contrast, 19 of the 22 implanted mice survived longer than the longest-surviving control mice. Transplanted mice exhibited significantly increased survival with reduced mortality over the 9-month observation period (Figure 8E), after which the experiment was terminated to allow surviving mice to be processed for both immunohistochemical assessment of late-stage myelination and nodal remodeling, as well as ultrastructural analysis (see Example 8). Comparison of Kaplan-Meier survival plots of transplanted and control mice revealed highly significant differences (Chi-squared=17.95 by Gehan-Breslow-Wilcoxon test; p<0.0001) (Figure 8E). These transplanted mice, which survived for more than 6 months, uniformly exhibited substantial myelination of the brain, brainstem, and cerebellum (FIGS. 8A-8D and 9).Surprisingly, the degree of consistency in cerebral myelination time points, and the percentage of shiverers surviving at any given time point, was greater in hiPSC-OPC transplanted mice than in otherwise similarly treated mice previously transplanted with A2B5-selected OPCs derived from fetal human tissue (Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008) which is hereby incorporated by reference in its entirety).

[0094] Example 8 - hiPSC OPCs generated ultrastructurally mature myelin with reconstitution of nodal regions Given the significantly extended survival of hiPSC-OPC transplanted mice, we tested whether this was related to the formation of ultrastructurally compact myelin around host axons by hiPSC OLs. To this end, we used electron microscopy on samples of corpus callosum derived from transplanted shiverers (n=3) at 22-36 weeks of age. These mice had been subjected to a five-site injection protocol and included animals that survived significantly longer than their non-transplanted controls. To allow for the assessment of the integrity and quality of their myelin, these apparently rescued mice were sacrificed after relatively long survival time points. The recipient corpus callosum was densely myelinated with mature compact myelin characterized by concentrically organized main periodic lines (Figures 10A-10E) and tight junctions between layers (Figures 10F, 10G, 11C). The transplanted corpus callosum differed significantly from that of shiverer controls lacking their transplants, and did not exhibit any other evidence of main period lines or myelin compaction (FIGS. 11B, 11D, and 11E).

[0095] Anatomical reconstitution of the nodes of Ranvier, as determined by immunolabeling for Caspr and βIV spectrin, which identify the juxtanoplegic and nodal segments of newly myelinated axons, respectively, was also observed in these mice (FIGS. 10H and 10I). In previous studies, anatomical and antigenic reconstitution of the nodal structure correlated with rapid restoration of both conduction and functional competence (Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008), which is hereby incorporated by reference in its entirety). The rapid and robust reacquisition of nodal structure in these mice indicates that hiPSC-derived OLs generate the necessary cues for the nodal organization of axonal proteins on which the formation of functional nodes of Ranvier depends.

[0096] Together, these data indicate that hiPSC-derived OPCs can efficiently generate OLs that in turn can robustly myelinate in the hypomyelinated shiverer forebrain, and that the generated myelin can restore nodal structure while ensheathing axons as efficiently as purified isolates of fetal tissue-derived OPCs.

[0097] Example 9 - hiPSC OPCs were non-tumorigenic in vivo Persistence of undifferentiated pluripotent stem cells can give rise to either teratomas or neuroepithelial tumors in graft recipients (Roy et al., "Functional Engraftment of Human ES Cell-Derived Dopaminergic Neurons Enriched by Coculture With Telomerase-Immortalized Midbrain Astrocytes," Nat. Med. 12:1259-1268 (2006), hereby incorporated by reference in its entirety). To assess whether any pluripotent or incompletely differentiated hESCs or hiPSCs remained in the nominally fully differentiated OL cultures, expression of pluripotency markers by late stage hiPSC-derived OPCs was assessed using both immunolabeling and qRT-PCR. By 100 days of in vitro culture, no detectable OCT4, NANOG, or SSEA4 protein was found in OPCs derived from any of the hESC and hiPSC lines used in this study. Similarly, qRT-PCR revealed that OCT4 and human telomerase reverse transcriptase (hTERT) transcripts were downregulated to essentially undetectable levels by 95 or more days of in vitro culture (Figures 4J and 4K). In vivo expression of OCT4, NANOG, and SSEA4 by transplanted OPCs was examined 3 months after transplantation. As observed, very few of the hNA+ donor cells stained with OLIG2, MBP, or GFAP. Although many donor-derived cells expressed nestin or SOX2, indicative of their persistence as neural progenitors, persistent expression of OCT4, NANOG, or SSEA4 was undetectable in any of these cells from any of the lines evaluated.

[0098] Therefore, 16 shi / shi × rag2 mice were tested for this purpose, including mice that were transplanted with 100,000 cells at neonatal stage and sacrificed either after 3 months (n = 11) or 4.5 months (n = 5). - / -No evidence of teratoma formation was found in any of the mice. All of them were transplanted with a total of 300,000 cells at five sites, but the mice available in the viability series, which died between 4 and 9 months of age (n=10), were also tested, and none had any evidence of teratomas, ectopic formation, or tumor formation of any kind. In addition, hiPSCs were transplanted into normally myelinated rag2-deficient mice to evaluate their tumorigenic potential in a wild-type myelin environment. None of the five mice tested showed any evidence of tumor formation, ectopic formation, or increased foci of undifferentiation 6 months after transplantation. Of note, sustained expression of SOX2, KLF4, and c-MYC mRNA was observed by qPCR in hiPSC-derived cells, reflecting some level of unrepressed expression of the reprogramming genes inserted using lentivirus. Nevertheless, expression of these transcripts was not associated with tumor formation by transplanted cells at the end of stage 6.

[0099] The lack of tumor formation in hiPSC-OPC transplanted mice was associated with a significant decrease in the mitotic fraction of implanted hiPSC-OPCs as a function of time after transplantation. In vivo hiPSC-OPC proliferation was measured as Ki67 expression by all human donor cells, which was observed to decrease linearly from 3 months (13.6 ± 0.6%) to 6 months (4.3 ± 0.04%) after transplantation (R2 = 0.9; p = 0.001; n = 7).

[0100] To establish the role of the differentiation protocol in reducing the risk of tumor formation, rag2-deficient mice were also transplanted with both C27 and K04 hiPSCs at the end of stages 1 and 3. Given the lack of tumors observed in hiPSC OPC (stage 6) transplanted mice (even up to 9 months after transplantation), this was also performed as a positive control for tumor detection. However, in contrast to hiPSC-OPC transplanted mice that did not contain any tumors, all animals transplanted with early stage hiPSCs showed histologically evident tumor formation by 3 months (n=8, mice transplanted with stage 1 hiPSCs; n=6, mice transplanted with stage 3 cells). Thus, this differentiation protocol appeared to effectively deplete the donor cell reservoir of persistently undifferentiated cells. The resulting hiPSC OPC transplants proved uniformly non-tumorigenic when studied up to 9 months after transplantation.

[0101] Considerations of Examples 1 to 9 In this study, the feasibility of using hiPSCs to generate highly enriched populations of both astrocytes and myelinating central OLs with high efficiency and yield has been established. The success of the protocol described herein with all four lines used in this study, including WA09 / H9 hESCs and K04, C14, and C27 iPSCs, demonstrates its broad applicability, and the highly efficient gliogenesis produced by this plan demonstrates its robust nature. Most importantly, the robust myelination observed in vivo, which compares favorably to that previously demonstrated by tissue-derived fetal human glial progenitor cells, indicated the near-certain functional integration and utility of these grafts. Accordingly, it was observed that myelination-deficient shiverers transplanted with hiPSC OPCs at the neonatal stage survived substantially longer than both their non-transplanted and saline-injected controls. Indeed, more than three-quarters of hiPSC-OPC transplanted mice survived beyond six months, long after all untreated control mice had died. As a result, hiPSC OPCs from a single patient skin sample can now be reliably generated in sufficient numbers to provide a myelinating autograft with little, if not complete, risk of rejection (Zhao et al., "Immunogenicity of Induced Pluripotent Stem Cells," Nature 474:212-215 (2011) which is hereby incorporated by reference in its entirety).

[0102] Importantly, the myelination efficiency of implanted iPSC-derived OPCs, defined as the percentage of myelinated central axons as a function of time after transplantation, proved to be as high as that previously achieved using tissue-derived, CD140a-selected OPCs (Sim et al., "CD140a Identifies a Population of Highly Myelinogenic, Migration-Competent and Efficiently Engrafting Human Oligodendrocyte Progenitor Cells," Nat. Biotechnol. 29:934-941 (2011), hereby incorporated by reference in its entirety). Indeed, it was notable that the percentage of ensheathed axons was found to be as high in enriched, but unsorted, hiPSC-OPC grafts as in fetal tissue-derived OPC grafts that had been sorted for CD140a+ cells prior to transplantation. Indeed, hiPSC-OPC grafts myelinate more axons and more rapidly than A2B5+ / PSA-NCAM-sorted fetal tissue-derived cells, likely reflecting a higher proportion of bipotential glial progenitor cells in the hiPSC-OPC populations by the time of their collection and transplantation.

[0103] Given the robust myelination produced by hiPSC-OPC grafts, it was asked whether transplantation of hiPSC OPCs into newborns might be sufficient to rescue the phenotype and survival of recipient shiverer homozygotes, as previously observed in a small number of shiverers transplanted with fetal human brain-derived OPCs. The hiPSC-OPC-transplanted mice indeed exhibited significantly improved survival. Over the 9-month observation period, mortality was generally delayed and reduced in the transplanted groups. Rescued mice showed gradual resolution of their neurological defects, as previously described for fetal brain tissue-derived OPC grafts (Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008) which is hereby incorporated by reference in its entirety). Surprisingly, however, the percentage of animals whose survival benefited from hiPSC-OPC transplantation was substantially higher than that previously reported using tissue-derived human OPCs. Only a quarter of shiverer mice transplanted with tissue-derived OPCs were observed to survive beyond 6 months of age (Windrem et al., "Neonatal Chimerization With Human Glial Progenitor Cells Can Both Remyelinate and Rescue the Otherwise Lethally Hypomyelinated Shiverer Mouse," Cell Stem Cell 2:553-565 (2008), which is hereby incorporated by reference in its entirety), whereas in the present study, more than half of the hiPSC-OPC transplanted mice survived beyond 6 months of age (Figure 8E). Nevertheless, some later deaths beyond 7 months of age were still observed.This may reflect the heterogeneous distribution of hiPSC OPCs observed in some animals, the characteristics of which are currently under investigation. Despite these later deaths, at least one-fifth of the mice appeared to show immediate clinical relief, but these survivors were sacrificed for histological and ultrastructural analysis at more than nine months. These exciting data demonstrate the superiority of hiPSC OPCs as therapeutic vectors, which may be a function of the prolonged differentiation state used in the present OPC induction protocol, possibly due to their more rapid myelination.

[0104] Interestingly, no evidence of tumor formation was observed from the implanted hiPSC-derived glial progenitor cells as long as 9 months after transplantation. This was surprising given that previous studies had provided ample evidence of the risk of tumor formation from residual undifferentiated cells (Pruszak et al., "CD15, CD24, and CD29 Define a Surface Biomarker Code for Neural Lineage Differentiation of Stem Cells," Stem Cells 27:2928-2940 (2009) which is hereby incorporated by reference in its entirety) or from partially differentiated neuroepithelial cells in hESC-derived grafts (Roy et al., "Functional Engraftment of Human ES Cell-Derived Dopaminergic Neurons Enriched by Coculture With Telomerase-Immortalized Midbrain Astrocytes," Nat. Med. 12:1259-1268 (2006) which is hereby incorporated by reference in its entirety). It is possible that the long-term differentiation protocol used to generate OPCs was sufficiently robust to effectively remove any residual undifferentiated cells prior to transplantation. It is equally possible that persistent epigenetic marks in the reprogrammed hiPSCs effectively reduced the later risk of tumor formation by their differentiated derivatives. In any case, further longer survival time points will be required in more animals and for a thorough search in vivo for any residual undifferentiated and / or potentially tumorigenic cells before the safety of these transplants can be declared with confidence. If tumor formation is a concern at any time, the CD9 expression of definitive pre-oligodendrocytes may be important. + / CD140a +Based on the high frequency of the cells and the ability to isolate these cells by fluorescence-activated cell sorting (FACS) based on their co-expressed epitopes, hiPSC OPCs can be selected for purification prior to transplantation (Sim et al., "CD140a Identifies a Population of Highly Myelinogenic, Migration-Competent and Efficiently Engrafting Human Oligodendrocyte Progenitor Cells," Nat. Biotechnol. 29:934-941 (2011), hereby incorporated by reference in its entirety).

[0105] These findings indicate that highly efficient in vivo oligodendrocyte differentiation and myelination can be achieved from hiPSCs, indicating the utility of iPSC-derived autografts in the treatment of acquired disorders of myelin. However, it is also important to note the efficient and context-dependent generation of both fibrous and protoplasmic astrocytes from transplanted hiPSC OPCs. In addition to the importance of astroglia in providing structural and physiological reconstitution of dysmyelinated regions, given that it has been discovered that astrocytic lysosomal enzymes can readily transfer from wild-type to deficient glia in brain tissue in a manner potentially sufficient to rescue the enzyme-deficient host, transplantation of astrocytes may be particularly important in correcting enzyme-deficient dysmyelination disorders (Lee et al., "Stem Cells Act Through Multiple Mechanisms to Benefit Mice With Neurodegenerative Metabolic Disease," Nat. Med. 13:439-447 (2007) which is hereby incorporated by reference in its entirety). Furthermore, hiPSC-derived astrocytes may prove to be critical therapeutic vectors for diseases of primarily astrocytic pathology such as Alexander disease and vanishing white matter disorders (Bugiani et al., "Defective Glial Maturation in Vanishing White Matter Disease," J. Neuropathol. Exp. Neurol. 70:69-82 (2011)), where myelin loss occurs but is secondary to astrocyte dysfunction (Krencik et al., "Specification of Transplantable Astroglial Subtypes From Human Pluripotent Stem Cells," Nat. Biotechnol. 29:528-534 (2011)), which is hereby incorporated by reference in its entirety.In any of these cases, however, the therapeutic use of iPSC-derived astroglia will need to be combined with methods for ex vivo correction of the genetic abnormalities characteristic of these disorders.

[0106] Thus, human iPSC OPCs may be an attractive vector to restore or replace glial populations in a variety of disease settings. Most crucially, the data presented herein indicate the preferential use of hiPSC-derived OPCs to restore lost myelin in disorders such as multiple sclerosis and traumatic demyelination, where genetic abnormalities may not complicate the use of the patient's own somatic cells as an iPSC source. Recognizing that the underlying genetic abnormalities must first be repaired in donor somatic cells prior to glial progenitor induction and implantation, iPSC OPCs may similarly prove therapeutically valuable in genetic disorders of myelin, such as Pelizaeus-Merzbacher disease. Thus, this study establishes the technical feasibility and efficacy of generating myelinating OLs from hiPSCs and indicates the clinical context in which this approach may be most appropriate. Clinical applications of patient-specific, somatically derived glial progenitor cell transplantation for the treatment of acquired disorders of myelin and a broader range of human glial pathologies may now be reasonably contemplated.

[0107] While preferred embodiments have been shown and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like may be made therein without departing from the spirit of the invention, and therefore are deemed to be within the scope of the invention as defined in the following claims.

[0108] Sequence information SEQUENCE LISTING <110> UNIVERSITY OF ROCHESTER <120> INDUCED PLURIPOTENT CELL-DERIVED OLIGODENDROCYTE PROGENITOR CELLS FOR THE TREATMENT OF MYELIN DISORDERS <150> US 61 / 761,584 <151> 2013-02-06 <150> US 61 / 780,265 <151> 2013-03-13 <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 21 <212> DNA <213> Artificial <220> <223> Primer <400> 1 cgtctccaca catcagcaca a 21 <210> 2 <211> 22 <212> DNA <213> Artificial <220> <223> Primer <400> 2 tcttggcagc aggatagtcc tt 22 <210> 3 <211> 18 <212> DNA <213> Artificial <220> <223> Primer <400> 3 ccacccatgg caaattcc 18 <210> 4 <211> 22 <212> DNA <213> Artificial <220> <223> Primer <400> 4 tgggatttcc attgatgaca ag 22 <210> 5 <211> 20 <212> DNA <213> Artificial <220> <223> Primer <400> 5 catcgagatc gccacctaca 20 <210> 6 <211> 19 <212> DNA <213> Artificial <220> <223> Primer <400> 6 tctgcacggg aatggtgat 19 <210> 7 <211> 17 <212> DNA <213> Artificial <220> <223> Primer <400> 7 tgcggccgat tgtgaac 17 <210> 8 <211> 21 <212> DNA <213> Artificial <220> <223> Primer <400> 8 cctcttttct ctgcggaacg t 21 <210> 9 <211> 22 <212> DNA <213> Artificial <220> <223> Primer <400> 9 accaggcact accgtaaaca ca 22 <210> 10 <211> 20 <212> DNA <213> Artificial <220> <223> Primer <400> 10 ggtccgacct ggaaaatgct 20 <210> 11 <211> 21 <212> DNA <213> Artificial <220> <223> Primer <400> 11 ccaaaggcaa acaacccact t 21 <210> 12 <211> 20 <212> DNA <213> Artificial <220> <223> Primer <400> 12 tcttgaccgg gaccttgtct 20 <210> 13 <211> 17 <212> DNA <213> Artificial <220> <223> Primer <400> 13 ggcgggcatt ccctttt 17 <210> 14 <211> 20 <212> DNA <213> Artificial <220> <223> Primer <400> 14 cgagctgtac tgggcgttgt 20 <210> 15 <211> 22 <212> DNA <213> Artificial <220> <223> Primer <400> 15 tggtccgagt gtggttctgt aa 22 <210> 16 <211> 21 <212> DNA <213> Artificial <220> <223> Primer <400> 16 tgtgcatagt cgctgcttga t 21 <210> 17 <211> 18 <212> DNA <213> Artificial <220> <223> Primer <400> 17 ggcgcgcaac tacatcct 18 <210> 18 <211> 19 <212> DNA <213> Artificial <220> <223> Primer <400> 18 cgctcaccag tcgcttcat 19 <210> 19 <211> 19 <212> DNA <213> Artificial <220> <223> Primer <400> 19 tcgggcacca cttcaacag 19 <210> 20 <211> 20 <212> DNA <213> Artificial <220> <223> Primer <400> 20 tccgggaact tgaactggaa 20 <210> 21 <211> 19 <212> DNA <213> Artificial <220> <223> Primer <400> 21 ccttggtggc accccttac 19 <210> 22 <211> 22 <212> DNA <213> Artificial <220> <223> Primer <400> 22 tccggtaccc actcttgatc tt 22 <210> 23 <211> 16 <212> DNA <213> Artificial <220> <223> Primer <400> 23 tgcgagcgct gcacat 16 <210> 24 <211> 24 <212> DNA <213> Artificial <220> <223> Primer <400> 24 gcagcgtgta cttatccttc ttca 24 <210> 25 <211> 22 <212> DNA <213> Artificial <220> <223> Primer <400> 25 ccacgaggta atgtccaaca tg 22 <210> 26 <211> 18 <212> DNA <213> Artificial <220> <223> Primer <400> 26 cattgggcgg caggtact 18

Claims

1. 1. A preparation of human oligodendrocyte precursor cells for use in the treatment of a condition mediated by loss of myelin or loss of oligodendrocytes, comprising: greater than 30% of said preparation comprises human oligodendrocyte precursor cells; the human oligodendrocyte precursor cells co-express oligodendrocyte transcription factor 2 (OLIG2), CD140a / PDGFRα and SOX10; said preparation contains less than 4% MAP2-immunoreactive neurons; the human oligodendrocyte precursor cells are capable of differentiating into human oligodendrocytes and astrocytes; and the preparation comprises more PDGFRα + cells than O4 + cells; Said preparation.

2. The preparation described in claim 1, wherein the oligodendrocyte precursor cells further express CD9.

3. A2B5 ＋ / PSA-NCAM － 3. The preparation of claim 1 or 2, having an in vivo myelination efficiency that exceeds the in vivo myelination efficiency of a preparation of sorted fetal human tissue-derived oligodendrocyte precursor cells.

4. At the time of transplantation, A2B5 ＋ / PSA-NCAM － 4. A preparation according to claim 1, which is capable of achieving an in vivo myelination density which exceeds the in vivo myelination density achievable with a preparation of sorted fetal human tissue-derived oligodendrocyte precursor cells.

5. A2B5 ＋ / PSA-NCAM － 5. A preparation according to any one of claims 1 to 4, which upon transplantation is capable of achieving improved survival in myelination deficient mammals compared to survival achievable with a preparation of sorted fetal human tissue-derived oligodendrocyte precursor cells.

6. 6. The preparation of any one of claims 1 to 5, wherein the condition is an autoimmune demyelination condition.

7. 7. The preparation of claim 6, wherein the autoimmune demyelinating condition is selected from the group consisting of multiple sclerosis, neuromyelitis optica, transverse myelitis, and optic neuritis.

8. 6. The preparation of claim 1, wherein the condition is vascular leukoencephalopathy.

9. 9. The preparation of claim 8, wherein the vascular leukoencephalopathy is selected from the group consisting of subcortical infarction, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-associated white matter disease, and spinal cord injury.

10. 6. The preparation according to any one of claims 1 to 5, wherein the condition is a radiation-induced demyelination condition.

11. 6. The preparation according to any one of claims 1 to 5, wherein the condition is childhood leukodystrophy.

12. 12. The preparation of claim 11, wherein the childhood leukodystrophy is selected from the group consisting of Pelizaeus-Merzbacher disease, Tay-Sachs disease, Sandhoff gangliosidosis, Krabbe disease, metachromatic leukodystrophy, mucopolysaccharidoses, Niemann-Pick A disease, adrenoleukodystrophy, Canavan disease, vanishing white matter disease, and Alexander disease.

13. 6. The preparation of any one of claims 1 to 5, wherein the condition is periventricular leukomalacia or cerebral palsy.

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