Method for differentiating pluripotent stem cell in dynamic suspension culture

Abstract

To provide an improved method for differentiating pluripotent stem cells first into neuroectodermal, and further into glial progenitor cells and OPCs in dynamic suspension culture.SOLUTION: Provided is a method for differentiating pluripotent stem cells into neuroectoderm using small molecule or protein inhibitors of TGFβ / actibin / nodal signaling and BMP signaling in dynamic suspension culture. Also, provided are a method and a protocol for differentiating pluripotent stem cells, such as human embryonic stem cells, first into neuroectodermal, then glial progenitor cells, and further into oligodendrocyte progenitor cells (OPCs), and a composition obtained by them. By the method of the present disclosure, neuroectoderm progenitor cells are produced reproducibly by day 7 of the differentiation process, glial progenitor cells by day 21 of the differentiation process, and OPCs by day 42 of the differentiation process.SELECTED DRAWING: None

Description

[Technical field]

[0001] Field The present disclosure relates to the field of cell biology and to the field of neuroectoderm and glial lineage cells, such as oligodendrocyte precursor cells. More specifically, the present disclosure relates to a novel method for differentiating pluripotent stem cells into neuroectoderm in dynamic suspension culture using small molecule or protein inhibitors of TGFβ / Activin / Nodal signaling and BMP signaling. The present disclosure further provides a novel method for differentiating pluripotent stem cells, such as human embryonic stem cells, first into neuroectoderm, then into glial progenitor cells, and further into oligodendrocyte precursor cells. The present disclosure further relates to the neuroectoderm cells, glial progenitor cells, and oligodendrocyte precursor cells expressing one or more markers, which are produced by the method according to the present invention. [Background technology]

[0002] background Oligodendrocyte progenitor cells (OPCs) are a subtype of glial cell in the central nervous system (CNS) that mature into myelin-forming oligodendrocytes, which form the myelin sheath that insulates neuronal axons and remyelinates CNS lesions that have lost myelin. Oligodendrocytes also contribute to neuroprotection through other mechanisms, such as the production of neurotrophic factors that promote neuronal survival (Wilkins A, Chandran S, Compston A. A role for oligodendrocyte-derived IGF-1 in trophic support of cortical neurons. 2001 Glia. 36(1):48-57 (Non-Patent Document 1); Dai X, Lercher LD, Clinton PM, Du Y, Livingston DL, Vieira C, Yang L, Shen MM, Dreyfus CF. The trophic role of oligodendrocytes in the basal forebrain. 2003 J Neurosci. 23(13):5846-53 (Non-Patent Document 2); Du Y, Dreyfus CF. Oligodendrocytes as providers of growth factors. 2002 J Neurosci Res. 68(6):647-54 (Non-Patent Document 3)). Thus, oligodendrocytes represent an important therapeutic target for demyelination and hypomyelination disorders (e.g., multiple sclerosis, adrenoleukodystrophy, adrenomedulloneuropathy, etc.), other neurodegenerative disorders (e.g., Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, etc.), as well as acute spinal cord injury (SCI).

[0003] OPCs are derived from the neuroectoderm (also called neural ectoderm or neural tube epithelium), which gives rise to neural progenitor cells that generate the various nerve cells (neurons) and glial cells that compose the CNS. Several methods have been developed to differentiate human pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), into OPCs that can be used for cell therapy. In the initial step of inducing neuroectodermal cells and neural progenitor cells, most existing protocols rely on the formation of embryoid bodies (EBs) and subsequent neurospheres in static non-adherent culture in the presence of the caudalizing agent retinoic acid (Nistor GI, Totoiu MO, Haque N, Carpenter MK, Keirstead HS. Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation. 2005 Glia. 49(3):385-96 (Non-Patent Document 4); Izrael M, Zhang P, Kaufman R, Shinder V, Ella R, Amit M, Itskovitz-Eldor J, Chebath J, Revel M. Human oligodendrocytes derived from embryonic stem cells: Effect of noggin on phenotypic differentiation in vitro and on myelination in vivo. 2007 Mol. Cell. Neurosci. 34: 310-323 (Non-Patent Document 5); Hu BY, Du ZW, Zhang SC. Differentiation of human oligodendrocytes from pluripotent stem cells. 2009 Nat. Protoc. 4:1614-1622 (Non-Patent Document 6)).Alternative differentiation protocols under adherent conditions have also been reported (Hu Z, Li T, Zhang X, Chen Y. Hepatocyte growth factor enhances the generation of high-purity oligodendrocytes from human embryonic stem cells. 2009 Differentiation. 78: 117-184). Both EB-based and adherent differentiation protocols require manual selection of neural precursor cells to optimize yield and are not easily scalable, limiting their usefulness for generating large quantities of therapeutic-grade cells. Additionally, a few cell types outside of the neuroectodermal lineage can survive the differentiation process, contributing to the contamination of the final OPC population with unwanted cell types, such as epithelial or chondrocyte precursor cells (Manley NC, Priest CA, Denham J, Wirth ED 3rd, Lebkowski JS. Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cells: Preclinical Efficacy and Safety in Cervical Spinal Cord Injury. Stem Cells Transl Med. 2017 Oct;6(10):1917-1929).

[0004] More recently, methods have been developed to enhance the efficiency of neural induction (neurectoderm formation) by using inhibitors of transforming growth factor beta (TGFβ) / activin / nodal signaling in combination with inhibitors of bone morphogenetic protein (BMP) signaling. This process, also known as dual SMAD inhibition, has been shown to promote efficient differentiation of human embryonic stem cells into neuroectodermal lineage cells (Chambers SM, Craft CA, Papapetrou EP, Tomishima M, Sadelain, M. Studer, L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. 2009 Nat Biotechnol. 27(3):275-280). In the standard dual SMAD inhibition protocol, neural induction and subsequent differentiation steps are performed in adherent monolayer cultures to eliminate the highly heterogeneous cell populations and minimize non-neuronal cells that arise in EB-based neural induction methods. This method has been successfully used to generate glial progenitor cells and OPCs from pluripotent stem cells (Douvaras P, Wang J, Zimmer M, Hanchuk S, O'Bara MA, Sadiq S, Sim FJ, Goldman J, Fossati V. Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells. 2014 Stem Cell Reports. 3(2):250-9). However, one drawback of adherent monolayer culture is its limited scalability and yield compared to three-dimensional (3D) culture methods.Furthermore, the 3D culture environment mimics the natural cellular microenvironment; cells grown using 3D cell culture techniques are believed to resemble natural tissues and organs more closely than cells grown in (2D) adherent monolayers. Finally, adherent monolayer culture relies on the use of undefined and animal-derived components, such as Matrigel® and knockout serum replacement (KSR).

[0005] To address these limitations, methods combining dual SMAD inhibition and EB formation have been recently developed and tested (Kirkeby A, Grealish S, Wolf DA, Nelander J, Wood J, Lundblad M, Lindvall O, Parmar M. Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. 2012 Cell Rep. 1(6):703-14 (Non-Patent Document 11); Crompton LA, Byrne ML, Taylor H, Kerrigan TL, Bru-Mercier G, Badger JL, Barbuti PA, Jo J, Tyler SJ, Allen SJ, Kunath T, Cho K, Caldwell MA. Stepwise, non-adherent differentiation of human pluripotent stem cells to generate basal forebrain cholinergic neurons via hedgehog signaling. 2013 Stem Cell Res. 11(3):1206-21 (Non-Patent Document 12); Pauly MG, Krajka V, Stengel F, Seibler P, Klein C, Capetian P. Adherent vs. Free-Floating Neural Induction by Dual SMAD Inhibition for Neurosphere Cultures Derived from Human Induced Pluripotent Stem Cells. 2018 Front Cell Dev Biol. 6:3 (Non-Patent Document 13). In the EB-based method, small molecule inhibitors of TGFβ / Activin / Nodal signaling and BMP signaling are applied to EBs formed in static non-adherent cultures.However, EB-based dual SMAD inhibition is not easily scalable to produce large quantities of target cells and results in high variability in the cells obtained, in part because static culture of EBs leads to the formation of aggregates of various sizes and requires frequent trituration throughout the process (Crompton LA, Byrne ML, Taylor H, Kerrigan TL, Bru-Mercier G, Badger JL, Barbuti PA, Jo J, Tyler SJ, Allen SJ, Kunath T, Cho K, Caldwell MA. Stepwise, non-adherent differentiation of human pluripotent stem cells to generate basal forebrain cholinergic neurons via hedgehog signaling. 2013 Stem Cell Res. 11(3):1206-21).

[0006] There is a need for improved methods for differentiating pluripotent stem cells into neuroectoderm and further into glial progenitor cells and OPCs. Ideally, such methods should consistently and reproducibly produce the desired cell types while also being easily scalable to produce sufficient quantities of differentiated cells for cell therapy applications. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Wilkins A, Chandran S, Compston A. A role for oligodendrocyte-derived IGF-1 in trophic support of cortical neurons. 2001 Glia. 36(1):48-57 [Non-Patent Document 2] Dai X, Lercher LD, Clinton PM, Du Y, Livingston DL, Vieira C, Yang L, Shen MM, Dreyfus CF. The trophic role of oligodendrocytes in the basal forebrain. 2003 J Neurosci. 23(13):5846-53

Table 3

Fashion 4

Wood 5

Wood 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

[0008] overview In various embodiments described herein, the present disclosure provides, inter alia, robust and reliable protocols for differentiating human pluripotent stem cells, such as ESCs and iPSCs, into neuroectoderm and glial cells in dynamic suspension culture that can be performed in bioreactors and amenable to large-scale culture. Also provided are protocols for differentiating human pluripotent stem cells into OPCs by inducing differentiation into neuroectoderm and further into glial cells in dynamic suspension culture, followed by differentiation of the glial cells into OPCs.

[0009] The present disclosure is based in part on the discovery that starting pluripotent stem cells can be aggregated in dynamic suspension into non-EB aggregates, in which the pluripotent stem cells remain undifferentiated, and then the aggregates can be induced to differentiate into neuroectoderm while in dynamic suspension by using one or more inhibitors of TGFβ / Activin / Nodal signaling and one or more inhibitors of BMP signaling. Differentiation of pluripotent stem cells in dynamic suspension according to the present disclosure provides a scalable, reproducible, and controllable process for producing large quantities of desired neuroectodermal lineage cells from starting materials, in contrast to adherent culture and EB-based methods.

[0010] The disclosed methods reproducibly produce neuroectodermal progenitor cells by day 7 of the differentiation process, glial progenitor cells by day 21 of the differentiation process, and OPCs by day 42 of the differentiation process. Day 42 OPCs generated according to the present disclosure are comparable (in terms of their overall marker expression profile) to OPCs generated by alternative methods currently in clinical trials to treat spinal cord injury (Priest CA, Manley NC, Denham J, Wirth ED 3rd, Lebkowski JS. Preclinical safety of human embryonic stem cell-derived oligodendrocyte progenitors supporting clinical trials in spinal cord injury. Regen Med. 2015 Nov;10(8):939-58; Manley NC, Priest CA, Denham J, Wirth ED 3rd, Lebkowski JS. Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cells: Preclinical Efficacy and Safety in Cervical Spinal Cord Injury. Stem Cells Transl Med. 2017 Oct;6(10):1917-1929); with the exception that OPCs generated in accordance with the present disclosure express lower levels of non-OPC markers in vitro, such as markers associated with epithelial cyst formation.

[0011] In one aspect, the disclosure provides a method of obtaining a population of cells comprising glial progenitor cells from undifferentiated human pluripotent stem cells. In certain aspects, the method comprises: (a) obtaining a suspension culture of non-embryoid body (non-EB) aggregates of undifferentiated human pluripotent stem cells, wherein the human pluripotent stem cells remain in an undifferentiated state; (b) culturing the non-EB aggregates from (a) in dynamic suspension in the presence of at least one inhibitor of transforming growth factor beta (TGFβ) / activin / nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling for a first period of time, thereby inducing differentiation into neuroectoderm; (c) culturing the non-EB aggregates from (b) in dynamic suspension in the presence of retinoic acid, and at least one agonist of the smoothened receptor, for a second period of time; and (d) culturing the aggregates from (c) in dynamic suspension in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for an additional period of time until the cells mature into glial progenitor cells.

[0012] In certain embodiments, the first period of time is about 3-4 days. In certain embodiments, the second period of time is about 4 days. In certain embodiments, steps (a) and (b) are carried out for about 7-8 days. In certain embodiments, steps (a)-(d) are carried out for about 21 days.

[0013] In certain embodiments, the method further comprises the additional step of collecting non-EB clumps from (d) and plating them on a substrate, thereby displacing cells from the clumps. In certain embodiments, the substrate is a cell adhesion peptide. In other embodiments, the substrate is an extracellular matrix protein. In certain embodiments, the substrate is recombinant human laminin 521. In other embodiments, the substrate is vitronectin or laminin 511 E8 fragment. In still other embodiments, the substrate is a synthetic substrate, such as, for example, Synthemax®-II SC Substrate.

[0014] In further embodiments, the method includes the additional step of adhering the cells migrated from the aggregates onto a substrate and culturing them in the presence of epidermal growth factor (EGF) and platelet-derived growth factor AA (PDGF-AA) for an additional period of time until the cells mature into OPCs. In certain embodiments, the substrate is a cell adhesion peptide. In other embodiments, the substrate is an extracellular matrix protein. In certain embodiments, the substrate is recombinant human laminin 521. In other embodiments, the substrate is vitronectin or laminin 511 E8 fragment. In certain embodiments, the adherent culture is performed for about 21 days.

[0015] In another aspect, the disclosure provides a method of inducing differentiation of human pluripotent stem cells into neuroectodermal cells, the method comprising: (a) obtaining a suspension culture of non-embryoid body (non-EB) aggregates of undifferentiated human pluripotent stem cells, wherein the human pluripotent stem cells remain in an undifferentiated state; (b) culturing the non-EB aggregates from (a) in dynamic suspension in the presence of at least one inhibitor of transforming growth factor beta (TGFβ) / activin / nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling for a first period of time, thereby inducing differentiation into neuroectodermal cells; and (c) culturing the non-EB aggregates from (b) in dynamic suspension in the presence of retinoic acid, and at least one agonist of the smoothened receptor, for a second period of time until the cells mature into paired box 6 (PAX6)-positive neuroectodermal cells.

[0016] In certain embodiments, the first period of time is about 3-4 days. In certain embodiments, the second period of time is about 4 days. In certain embodiments, steps (a)-(c) are carried out for about 7-8 days.

[0017] In an additional aspect, the disclosure provides a method for obtaining a population of cells comprising glial progenitor cells from undifferentiated human pluripotent stem cells, the method comprising the steps of: (a) culturing undifferentiated human pluripotent stem cells that have been disaggregated and formed a single cell suspension in dynamic suspension to obtain non-embryoid body (non-EB) aggregates, wherein the human pluripotent stem cells in the non-EB aggregates remain in an undifferentiated state; (b) culturing the non-EB aggregates from (a) in dynamic suspension with at least one inhibitor of transforming growth factor beta (TGFβ) / activin / nodal signaling and a bone morphogenetic protein (BMP). (c) culturing the non-EB aggregates from (b) in dynamic suspension in the presence of retinoic acid, and at least one agonist of a smoothened receptor, for a second period of time, thereby inducing differentiation into neuroectoderm; and (d) culturing the aggregates from (c) in dynamic suspension in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for an additional period of time until the cells mature into glial progenitor cells.

[0018] In certain embodiments, at least one inhibitor of TGFβ / activin / nodal signaling is an inhibitor of activin receptor-like kinase 5 (ALK5).In other embodiments, at least one inhibitor of TGFβ / activin / nodal signaling is selected from the group consisting of SB431542, LY2157299, GW788388, A-77-01, A-83-01, and SB505124.In still other embodiments, the inhibitor of TGFβ / activin / nodal signaling is SB431542.

[0019] In certain embodiments, at least one inhibitor of BMP signaling is an inhibitor of activin receptor-like kinase 2 (ALK2).In other embodiments, at least one inhibitor of BMP signaling is selected from the group consisting of dorsomorphin, DMH-1, K02288, ML3467, LDN193189 and noggin protein.In still other embodiments, the inhibitor of BMP signaling is dorsomorphin.

[0020] In certain embodiments, the at least one smoothened receptor agonist is selected from the group consisting of purmorphamine, smoothened agonist (SAG, CAS 364590-63-6), and Sonic Hedgehog (SHH) protein. In yet other embodiments, the smoothened receptor agonist is purmorphamine.

[0021] Another embodiment is a differentiated cell population comprising PAX6 positive neuroectodermal cells obtained according to the method of the present disclosure.In certain embodiments, the PAX6 positive neuroectodermal cells further express one or more markers selected from HES5 and ZBTB16.

[0022] Another embodiment is the differentiated cell population comprising glial progenitor cells obtained according to the method of the present disclosure.In a particular embodiment, the glial progenitor cells express one or more markers selected from calcium voltage-dependent channel auxiliary subunit gamma 4 (CACNG4), fatty acid binding protein 7 (FABP7) and sex determining region Y box 6 (SOX6).

[0023] Yet another embodiment is a differentiated cell population comprising OPCs obtained according to the disclosed method. In a preferred embodiment, the OPCs produced according to the method of the present invention express one or more markers selected from neural / glial antigen 2 (NG2), platelet derived growth factor receptor A (PDGFRα), and ganglioside GD3 (GD3) (GD3 is also known as anti-disialoganglioside and ganglioside GD3 synthase). Thus, for example, the OPCs produced according to the present invention can express NG2, PDGFRα, or GD3; a combination of NG2 and PDGFRα, NG2 and GD3, or PDGFRα and GD3; or a combination of NG2, PDGFRα, and GD3. In certain embodiments, the differentiated cell population comprises at least 60% NG2 positive cells. In certain embodiments, the differentiated cell population comprises at least 70% NG2 positive cells. In certain embodiments, the differentiated cell population comprises at least 80% NG2 positive cells. In other embodiments, the differentiated cell population comprises at least 90% NG2 positive cells. In certain embodiments, the differentiated cell population comprises at least 98% NG2 positive cells. In certain embodiments, the differentiated cell population comprises at least 60% PDGFRα positive cells. In certain embodiments, the differentiated cell population comprises at least 70% PDGFRα positive cells. In certain embodiments, the differentiated cell population comprises at least 80% PDGFRα positive cells. In other embodiments, the differentiated cell population comprises at least 90% PDGFRα positive cells. In certain embodiments, the differentiated cell population comprises at least 98% PDGFRα positive cells. In certain embodiments, the differentiated cell population comprises at least 60% GD3 positive cells. In certain embodiments, the differentiated cell population comprises at least 70% GD3 positive cells. In certain embodiments, the differentiated cell population comprises at least 80% GD3 positive cells. In other embodiments, the differentiated cell population comprises at least 90% GD3 positive cells. In certain embodiments, the differentiated cell population comprises at least 98% GD3 positive cells. [The present invention 1001] (a) obtaining a suspension culture of non-embryoid body (non-EB) aggregates of undifferentiated human pluripotent stem cells, wherein the human pluripotent stem cells remain in an undifferentiated state; (b) culturing the non-EB aggregates from (a) in dynamic suspension in the presence of at least one inhibitor of transforming growth factor beta (TGFβ) / activin / nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling for a first period of time, thereby inducing differentiation into neuroectoderm; (c) culturing the non-EB aggregates from (b) in dynamic suspension in the presence of retinoic acid and at least one agonist of the smoothened receptor for a second period of time; and (d) culturing the aggregates from (c) in dynamic suspension in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for an additional period of time until the cells mature into glial progenitor cells. 1. A method for obtaining a population of cells comprising glial progenitor cells from undifferentiated human pluripotent stem cells, comprising: [The present invention 1002] The method of claim 1001 further comprising the additional step of collecting the non-EB clumps from (d) and plating them on a substrate, thereby transferring cells from the clumps. [The present invention 1003] The method of claim 1002, wherein said substrate is recombinant human laminin-521. [The present invention 1004] 1001. The method of claim 1001, wherein said human pluripotent stem cells are human embryonic stem cells (hESCs). [The present invention 1005] 1001. The method of claim 1001, wherein said human pluripotent stem cells are human induced pluripotent stem cells (hiPSCs). [The present invention 1006] 1001. The method of claim 1001, wherein said at least one inhibitor of TGFβ / activin / nodal signaling is an inhibitor of activin receptor-like kinase 5 (ALK5). [The present invention 1007] 1001. The method of claim 1001, wherein said at least one inhibitor of TGFβ / activin / nodal signaling is selected from the group consisting of SB431542, LY2157299, GW788388, A-77-01, A-83-01, and SB505124. [The present invention 1008] 1001. The method of claim 1001, wherein said at least one inhibitor of TGFβ / activin / nodal signaling is SB431542. [The present invention 1009] 1001. The method of claim 1001, wherein said at least one inhibitor of BMP signaling is an inhibitor of activin receptor-like kinase 2 (ALK2). [The present invention 1010] 1002. The method of claim 1001, wherein said at least one inhibitor of BMP signaling is selected from the group consisting of dorsomorphin, DMH-1, K02288, ML3467, LDN193189, and Noggin protein. [The present invention 1011] 1001. The method of claim 1001, wherein said at least one inhibitor of BMP signaling is dorsomorphin. [The present invention 1012] The method of claim 1001, wherein the at least one smoothened receptor agonist is selected from the group consisting of purmorphamine, smoothened receptor agonist (SAG, CAS 364590-63-6), and Sonic Hedgehog (SHH) protein. [The present invention 1013] The method of claim 10, wherein at least one smoothened receptor agonist is purmorphamine. [The present invention 1014] 1001. The method of claim 10, wherein said first period of time is about 3 to 4 days. [The present invention 1015] 1001. The method of claim 1001, wherein said second period of time is about 3 days. [The present invention 1016] 1001. The method of claim 1001, wherein steps (a) to (d) are carried out for about 21 days. [The present invention 1017] A differentiated cell population comprising glial precursor cells, obtained according to the method of the present invention. [The present invention 1018] The differentiated cell population of the present invention 1017, wherein the glial progenitor cells express one or more markers selected from calcium voltage-dependent channel auxiliary subunit gamma 4 (CACNG4), fatty acid binding protein 7 (FABP7), and sex determining region Y box 6 (SOX6). [The present invention 1019] (a) obtaining glial progenitor cells according to the method of the present invention; (b) collecting the cells from (a) and plating them onto a substrate, thereby displacing the cells from the clumps; and (c) adherent culturing the cells from (b) in the presence of epidermal growth factor (EGF) and platelet-derived growth factor AA (PDGF-AA) for a further period of time until the cells mature into oligodendrocyte precursor cells (OPCs). 1. A method for obtaining a population of cells comprising OPCs from undifferentiated human pluripotent stem cells, comprising: The method, wherein the OPCs express one or more markers selected from neural / glial antigen 2 (NG2), platelet-derived growth factor receptor A (PDGFRα), and ganglioside GD3 (GD3). [The present invention 1020] The method of claim 1019, wherein said adherent culture is carried out for about 21 days. [The present invention 1021] The method of claim 1019, wherein the adherent culture is performed on a substrate, the substrate being selected from (i) a cell adhesion peptide, and (ii) an extracellular matrix selected from laminin and vitronectin. [The present invention 1022] The method of claim 1019, wherein said adherent culture is carried out on recombinant human laminin-521. [The present invention 1023] The method of claim 1019, wherein said adherent culture is performed on a laminin 511 E8 fragment. [The present invention 1024] 1020. The method of claim 1019, wherein said human pluripotent stem cells are hESCs. [The present invention 1025] The method of claim 10, wherein said human pluripotent stem cells are hiPSCs. [The present invention 1026] A differentiated cell population comprising OPCs obtained according to the method of the present invention. [The present invention 1027] The differentiated cell population of the present invention, wherein at least 60% of said cells are NG2 positive. [The present invention 1028] The differentiated cell population of the present invention, wherein at least 70% of said cells are NG2 positive. [The present invention 1029] The differentiated cell population of the present invention, wherein at least 80% of said cells are NG2 positive. [The present invention 1030] The differentiated cell population of the present invention, wherein at least 90% of said cells are NG2 positive. [The present invention 1031] The differentiated cell population of the present invention, wherein at least 60% of said cells are PDGFRα positive. [The present invention 1032] The differentiated cell population of the present invention, wherein at least 70% of said cells are PDGFRα positive. [The present invention 1033] The differentiated cell population of the present invention, wherein at least 80% of said cells are PDGFRα positive. [The present invention 1034] The differentiated cell population of the present invention, wherein at least 90% of said cells are PDGFRα positive. [The present invention 1035] The differentiated cell population of the present invention, wherein at least 60% of said cells are GD3 positive. [The present invention 1036] The differentiated cell population of the present invention, wherein at least 70% of said cells are GD3 positive. [The present invention 1037] The differentiated cell population of the present invention, wherein at least 80% of said cells are GD3 positive. [The present invention 1038] The differentiated cell population of the present invention, wherein at least 90% of said cells are GD3 positive. [The present invention 1039] (a) obtaining a suspension culture of non-embryoid body (non-EB) aggregates of undifferentiated human pluripotent stem cells, wherein the human pluripotent stem cells remain in an undifferentiated state; (b) culturing the non-EB aggregates from (a) in dynamic suspension in the presence of at least one inhibitor of transforming growth factor beta (TGFβ) / activin / nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling for a first period of time, thereby inducing differentiation into neuroectoderm; and (c) culturing the non-EB aggregates from (b) in dynamic suspension in the presence of retinoic acid and at least one agonist of the smoothened receptor for a second period of time until the cells mature into paired box 6 (PAX6)-positive neuroectodermal cells. 1. A method for inducing differentiation of a human pluripotent stem cell into a neuroectodermal cell, comprising: [The present invention 1040] The method of claim 1039, wherein said human pluripotent stem cells are human embryonic stem cells (hESCs). [The present invention 1041] 1039. The method of claim 1039, wherein said human pluripotent stem cells are human induced pluripotent stem cells (hiPSCs). [The present invention 1042] 1039. The method of claim 1039, wherein said at least one inhibitor of TGFβ / activin / nodal signaling is an inhibitor of activin receptor-like kinase 5 (ALK5). [The present invention 1043] 1039. The method of claim 1039, wherein said at least one inhibitor of TGFβ / activin / nodal signaling is selected from the group consisting of SB431542, LY2157299, GW788388, A-77-01, A-83-01, and SB505124. [The present invention 1044] 1039. The method of claim 1039, wherein said at least one inhibitor of TGFβ / activin / nodal signaling is SB431542. [The present invention 1045] 1039. The method of claim 1039, wherein said at least one inhibitor of BMP signaling is an inhibitor of activin receptor-like kinase 2 (ALK2). [The present invention 1046] 1039. The method of claim 1039, wherein said at least one inhibitor of BMP signaling is selected from the group consisting of dorsomorphin, DMH-1, K02288, ML3467, LDN193189, and Noggin protein. [The present invention 1047] 1039. The method of claim 1039, wherein said at least one inhibitor of BMP signaling is dorsomorphin. [The present invention 1048] 1039. The method of claim 1039, wherein the at least one smoothened receptor agonist is selected from the group consisting of purmorphamine, smoothened agonist (SAG, CAS 364590-63-6), and Sonic Hedgehog (SHH) protein. [The present invention 1049] The method of claim 1039, wherein at least one smoothened receptor agonist is purmorphamine. [The present invention 1050] The method of claim 1039, wherein said first period of time is about 3 to 4 days. [The present invention 1051] The method of claim 1039, wherein said second period of time is about 3 days. [The present invention 1052] 1039. The process of claim 10, wherein steps (a)-(c) are carried out for about 7-8 days. [The present invention 1053] A differentiated cell population comprising PAX6-positive neuroectodermal cells obtained according to the method of the present invention. [The present invention 1054] The differentiated cell population of the present invention 1053, wherein the PAX6-positive neuroectodermal cells further express one or more markers selected from Hes family BHLH transcription factor 5 (HES5) and ZBTB16 (zinc finger and BTB domain containing 16). [The present invention 1055] (a) culturing undifferentiated human pluripotent stem cells that have been disaggregated and formed a single cell suspension in dynamic suspension to obtain non-embryoid body (non-EB) aggregates, wherein the human pluripotent stem cells in the non-EB aggregates remain in an undifferentiated state; (b) culturing the non-EB aggregates from (a) in dynamic suspension in the presence of at least one inhibitor of transforming growth factor beta (TGFβ) / activin / nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling for a first period of time, thereby inducing differentiation into neuroectoderm; (c) culturing the non-EB aggregates from (b) in dynamic suspension in the presence of retinoic acid and at least one agonist of the smoothened receptor for a second period of time; and (d) culturing the aggregates from (c) in dynamic suspension in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for an additional period of time until the cells mature into glial progenitor cells. 1. A method for obtaining a population of cells comprising glial progenitor cells from undifferentiated human pluripotent stem cells, comprising: [Brief description of the drawings]

[0024] [Figure 1]Figure 1 illustrates differentiation of human embryonic stem cells into neuroectoderm (day 7) and further into glial progenitor cells (day 21) and oligodendrocyte progenitor cells (day 42) according to the present disclosure. DS = dynamic suspension culture. Several additional small molecule inhibitors of TGFβ / activin / nodal signaling (other than SB431542) and BMP signaling (other than dorsomorphin) were tested and found to work equally well in inducing differentiation into neuroectoderm (Example 7). [Diagram 2] Representative photomicrographs of undifferentiated human embryonic stem cells (uhESCs) according to the present disclosure stained by immunocytochemistry for pluripotency markers. The top and bottom photomicrographs each show a single image field of uhESCs stained for DAPI, Nanog, Oct4 (top) or DAPI, Nanog, Sox2 (bottom) and imaged on an IN Cell Analyzer 2000. The scale bar in the bottom right panel applies to all images in the figure. [Diagram 3] Representative photomicrographs of neuroectodermal progenitor cells generated as non-embryoid body (non-EB) clumps in suspension according to the present disclosure and stained by immunocytochemistry. The top and bottom photomicrographs show clumps of neuroectodermal progenitor cells generated from two representative experiments and stained by immunocytochemistry for DAPI (left panel), PAX6 (middle panel), and PSA-NCAM (right panel). Stained cell clumps were imaged on an IN Cell Analyzer 2000. The scale bar in the bottom right panel applies to all images in the figure. [Figure 4] Representative photomicrographs of oligodendrocyte precursor cells prepared according to the present disclosure and stained by immunocytochemistry are shown. The top and bottom photomicrographs show oligodendrocyte precursor cells prepared from two representative experiments and stained by immunocytochemistry for DAPI (left panel) and NG2 (right panel). Stained cells were imaged on an IN Cell Analyzer 2000. The scale bar in the bottom right panel applies to all images in the figure. [Diagram 5]Correlation plots of gene expression profiles of uhESCs before and 24 hours after forming cell aggregates in suspension. Each correlation plot shows a comparison of gene expression profiles at day -1 (before clump formation) and day 0 (24 hours after clump formation) from two separate experiments. For each plot, data points represent each of 76 genes assessed by Fluidigm qPCR and calculated as normalized ΔCT as described in Example 6. R-squared values ​​are displayed in the upper left corner of each plot and were calculated based on the best-fit line using JMP software (SAS, Cary, NC, USA). [Figure 6A] Correlation plots of day 7 gene expression profiles of uhESCs differentiated into neuroectodermal progenitors in suspension with different small molecule combinations are shown. Each correlation plot shows a comparison of day 7 gene expression profiles of cells treated with SB431542+Dorsomorphin and alternative small molecule combinations shown on the y-axis of each plot. For each plot, data points represent each of 96 genes assessed by Fluidigm qPCR and calculated as normalized ΔCT as described in Example 7. R-squared values ​​are shown in the upper left corner of each plot and were calculated based on the best fit line using JMP software (SAS, Cary, NC, USA). [Figure 6B] Correlation plots of day 7 gene expression profiles of uhESCs differentiated into neuroectodermal progenitors in suspension with different small molecule combinations are shown. Each correlation plot shows a comparison of day 7 gene expression profiles of cells treated with SB431542+Dorsomorphin and alternative small molecule combinations shown on the y-axis of each plot. For each plot, data points represent each of 96 genes assessed by Fluidigm qPCR and calculated as normalized ΔCT as described in Example 7. R-squared values ​​are shown in the upper left corner of each plot and were calculated based on the best fit line using JMP software (SAS, Cary, NC, USA). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description This description does not enumerate in detail all of the different ways in which the disclosure can be implemented or all of the features that can be added to the disclosure. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the disclosure contemplates that any feature or combination of features described herein may be excluded or omitted in some embodiments of the disclosure. Furthermore, numerous modifications and additions to the various embodiments proposed herein will be apparent to those skilled in the art in view of the disclosure, and do not depart from the disclosure. In other cases, well-known structures, interfaces, and processes have not been shown in detail so as not to unnecessarily obscure the invention. Nothing in this specification should be construed to affect the disavowal of any portion of the entire scope of the invention. Thus, the following description is intended to illustrate certain aspects of the disclosure, but does not exhaustively specify all permutations, combinations, and variations thereof.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terms used in the description of the present disclosure herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the present disclosure.

[0027] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety.

[0028] It is specifically contemplated that the various features of the present disclosure described herein may be used in any combination, unless the context dictates otherwise. Further, the present disclosure also contemplates that in some aspects of the disclosure, any feature or combination of features described herein may be excluded or omitted.

[0029] The methods disclosed herein may include one or more steps or actions to achieve the described method. The steps and / or actions of the method may be interchanged with one another without departing from the scope of the present invention. In other words, unless a particular order of steps or actions is necessary for an aspect to work properly, the order and / or use of specific steps and / or actions may be changed without departing from the scope of the present invention.

[0030] As used in the description of this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0031] As used herein, "and / or" refers to and is inclusive of any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as the alternative ("or").

[0032] As used herein, the terms "about" and "approximately" when referring to measurable values ​​such as percentages, densities, volumes, and the like, are meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, and even ±0.1% of the specified amount.

[0033] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted as including X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y", and phrases such as "about X to Y" mean "about X to about Y".

[0034] As used herein, "oligodendrocyte precursor cells" (OPCs) refer to cells of the neuroectodermal / glial lineage found in the central nervous system that express the characteristic marker neural / glial antigen 2 (NG2) and have the potential to differentiate into oligodendrocytes. OPCs generated according to the methods of the present invention can also express one or more markers selected from NG2, PDGFRα, and GD3.

[0035] The terms "glial lineage cells", "glial progenitor cells" and "glial cells" are used interchangeably herein and refer to non-neuronal CNS cells derived from neuroectoderm / neuronal progenitor cells. Glial progenitor cells can be further differentiated to form OPCs / oligodendrocytes or astrocytes. In certain aspects, the glial progenitor cells of the present disclosure express one or more markers selected from calcium voltage-dependent channel auxiliary subunit gamma 4 (CACNG4), fatty acid binding protein 7 (FABP7), and sex determining region Y box 6 (SOX6).

[0036] The terms "neurectoderm," "neurectodermal cell," "neurectodermal precursor," "neurectodermal progenitor cell," "neural precursor cell," and "neural precursor" are used interchangeably herein and refer to cells that can differentiate along a neural precursor pathway and have the capacity to form CNS neurons, oligodendrocytes, astrocytes, and ependymal cells. In certain aspects, neuroectodermal cells of the present disclosure express one or more markers selected from paired box 6 (PAX6), Hes family BHLH transcription factor 5 (HES5), and zinc finger and BTB domain containing 16 (ZBTB16).

[0037] As used herein, the term "embryoid body" (EB) refers to a three-dimensional cell aggregate derived from a pluripotent stem cell that has undergone spontaneous differentiation into all three germ layers. EBs are formed when pluripotent stem cells are removed from culture conditions that inhibit differentiation. For example, in the case of human embryonic stem cells, removal of basic fibroblast growth factor (bFGF) and transforming growth factor β (TGFβ) from the culture medium results in spontaneous differentiation into all three germ layers and the formation of EBs.

[0038] The term "non-embryoid body aggregates" as used herein refers to three-dimensional cell aggregates formed from pluripotent stem cells in which the pluripotent stem cells remain undifferentiated. In the present disclosure, non-EB aggregates are formed in dynamic suspension under cell culture conditions that maintain pluripotency and suppress spontaneous differentiation (i.e., bFGF and TGFβ are not removed from the medium). The undifferentiated cell aggregates are then induced into neuroectodermal progenitor cells by simultaneously removing bFGF and TGFβ and adding neuroectodermal differentiation factors, such as a combination of an inhibitor of the TGFβ / Activin / Nodal signaling pathway and an inhibitor of the bone morphogenetic protein signaling pathway.

[0039] As used herein, the term "inhibitor of TGFβ / activin / nodal signaling" refers to a small molecule or protein modulator that can downregulate the transmission of signals along the transforming growth factor β (TGFβ) / activin / nodal signaling pathway. In certain embodiments, the inhibitor of TGFβ / activin / nodal signaling directly targets the TGFβ type 1 receptor (TGFβR1), also known as activin receptor-like kinase 5 (ALK5). In certain embodiments, the inhibitor of TGFβ / activin / nodal signaling is selected from the group consisting of SB431542, LY2157299, GW788388, A-77-01, A-83-01, and SB505124.

[0040] The term "BMP signaling inhibitor" as used herein refers to a small molecule or protein modulator that can downregulate the transmission of signals along bone morphogenetic protein (BMP) signaling pathway.In certain embodiments, the BMP signaling inhibitor directly targets Activin A receptor type I (ACVR1), also known as Activin Receptor-Like Kinase 2 (ALK2).In certain embodiments, the BMP signaling inhibitor is selected from the group consisting of Dorsomorphin, DMH-1, K02288, ML3467, LDN193189, and Noggin protein.

[0041] As used herein, the term "Smoothened agonist" or "Smoothened receptor agonist" refers to a small molecule or protein modulator that can directly bind to and activate the G protein-coupled receptor Smoothened, which is part of the Sonic Hedgehog (SHH) signaling pathway. In certain embodiments, the Smoothened receptor agonist is selected from the group consisting of purmorphamine, Smoothened agonist (SAG, CAS 364590-63-6), and Sonic Hedgehog (SHH) protein.

[0042] As used herein, the term "undesirable cell type" refers to cells other than neuroectodermal lineage that may result in the formation of ectopic tissue upon transplantation or in the formation of one or more cysts in the cyst assay described herein. In one embodiment, "undesirable cell type" includes cells of epithelial lineage, such as cells positive for CD49f, a marker expressed in both neural progenitor cells and epithelial cells, or cells positive for CLDN6 or EpCAM, two markers expressed in both pluripotent cells and epithelial cells.

[0043] As used herein, "implantation" or "transplantation" refers to the administration of a cell population to a target tissue using an appropriate delivery technique (eg, using an injection device).

[0044] As used herein, a "subject" refers to an animal or a human.

[0045] As used herein, "subject in need thereof" refers to an animal or human suffering from central nervous system tissue damage. In one aspect, the animal or human is experiencing reduced motor function.

[0046] The terms "central nervous system" and "CNS," as used interchangeably herein, refer to the complex of nervous tissue that controls one or more activities of the body, including, but not limited to, the brain and spinal cord of a vertebrate animal.

[0047] As used herein with respect to a condition or disease, "treatment" or "treating" refers to an approach to obtain a beneficial or desired result, such as a clinical result, after a condition or disease appears in a patient. Beneficial or desired results with respect to a disease include, but are not limited to, one or more of the following: amelioration of a condition associated with the disease, cure of the disease, reduction of the severity of the disease, delay of the progression of the disease, alleviation of one or more symptoms associated with the disease, improvement of the quality of life of a person suffering from the disease, prolongation of survival, and any combination thereof. Similarly, for purposes of this disclosure, beneficial or desired results with respect to a condition include, but are not limited to, one or more of the following: amelioration of a condition, cure of the condition, reduction of the severity of the condition, delay of the progression of the condition, alleviation of one or more symptoms associated with the condition, improvement of the quality of life of a person suffering from the condition, prolongation of survival, and any combination thereof.

[0048] Proliferation and culture of undifferentiated pluripotent stem cells Differentiation of pluripotent stem cells according to the present disclosure can be performed using any suitable pluripotent stem cells as starting material. In one embodiment, a method can be performed with human embryonic stem cell (hESC) lines. In another embodiment, a method can be performed with induced pluripotent stem cells (iPSCs). In another embodiment, a method can be performed with cells derived from H1, H7, H9, H13, or H14 cell lines. In another embodiment, a method can be performed with primate pluripotent stem (pPS) cell lines. In yet another embodiment, a method can be performed with undifferentiated stem cells derived from parthenotes, which are embryos stimulated to develop hESCs without fertilization.

[0049] Methods for the growth and culture of undifferentiated pluripotent stem cells have been described previously. For the tissue and cell culture of pluripotent stem cells, please refer to any of the many publications available in the art, including, for example, the following publications: Teratocarcinomas and Embryonic Stem cells: A Practical Approach (EJ Robertson, Ed., IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (PM Wasserman et al., Eds., Academic Press 1993); Embryonic Stem Cell Differentiation in vitro (MV Wiles, Meth. Enzymol. 225:900, 1993); Properties and Uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (PD Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998; and RI Freshney, Culture of Animal Cells, Wiley-Liss, New York, 2000).

[0050] Undifferentiated pluripotent stem cells can be maintained in an undifferentiated state without the addition of feeder cells (see, e.g., (2004) Rosler et al., Dev. Dynam. 229:259). Feeder-free cultures are typically supported by a nutrient medium containing factors that promote the proliferation of cells without differentiation (see, e.g., U.S. Pat. No. 6,800,480). In one embodiment, a conditioned medium containing such factors can be used. Conditioned medium can be obtained by culturing a medium containing cells that secrete such factors. Suitable cells include, but are not limited to, irradiated (approximately 4,000 Rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells (U.S. Pat. No. 6,642,048). The medium can be conditioned by plating feeders in serum-free medium, such as knockout DMEM supplemented with 20% serum replacement and 4 ng / mL bFGF. Medium conditioned for 1-2 days can be further supplemented with bFGF and used to support pPS cell cultures for 1-2 days (see, e.g., WO 01 / 51616; Xu et al., (2001) Nat. Biotechnol. 19:971).

[0051] Alternatively, fresh or unconditioned media supplemented with additional factors (such as fibroblast growth factor or forskolin) that promote the proliferation of cells in undifferentiated morphology can be used. Non-limiting examples include basal media such as X-VIVO™ 10 (Lonza, Walkersville, Md.) or QBSF™-60 (Quality Biological Inc. Gaithersburg, Md.) supplemented with 40-80 ng / mL bFGF and optionally containing SCF (15 ng / mL) or Flt3 ligand (75 ng / mL) (see, e.g., Xu et al., (2005) Stem Cells 23(3):315). These media formulations have the advantage of supporting cell proliferation at 2-3 times the rate of other systems (see, e.g., WO 03 / 020920). In one aspect, undifferentiated pluripotent cells such as hES cells can be cultured in media containing bFGF and TGFβ. Non-limiting exemplary concentrations of bFGF include about 80 ng / ml. Non-limiting exemplary concentrations of TGFβ include about 0.5 ng / ml. In yet another embodiment, the undifferentiated pluripotent stem cells can be maintained in a commercially available complete medium, such as mTeSR™ (Stem Cell Technologies, Vancouver, Canada).

[0052] Undifferentiated pluripotent cells can be cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue (Thomson et al. (1998) Science 282:1145). Feeder cells can be from human or mouse sources. Human feeder cells can be isolated from a variety of human tissues or derived from human embryonic stem cells via differentiation into fibroblasts (see, e.g., WO 01 / 51616). Human feeder cells that can be used include, but are not limited to, placental fibroblasts (see, e.g., Genbacev et al. (2005) Fertil. Steril. 83(5):1517), fallopian tube epithelial cells (see, e.g., Richards et al. (2002) Nat. Biotechnol., 20:933), foreskin fibroblasts (see, e.g., Amit et al. (2003) Biol. Reprod. 68:2150), and endometrial cells (see, e.g., Lee et al. (2005) Biol. Reprod. 72(1):42).

[0053] A variety of solid surfaces can be used for culturing undifferentiated pluripotent cells. These include, but are not limited to, commercially available standard tissue culture flasks or cell culture plates, such as 6-well, 24-well, 96-well, 144-well plates, etc. Other solid surfaces include, but are not limited to, microcarriers and disks. Solid surfaces suitable for the growth of undifferentiated pluripotent cells can be made of a variety of materials, including, but not limited to, glass, or plastics, such as polystyrene, polyvinyl chloride, polycarbonate, polytetrafluoroethylene, melinex, thermanox, or combinations thereof. Suitable surfaces can be composed of one or more polymers, such as one or more acrylates. The solid surface can be a three-dimensional shape. Non-limiting examples of three-dimensional solid surfaces have been described, for example, in US Patent Application Publication No. 2005 / 0031598.

[0054] Undifferentiated stem cells can also be grown under feeder-free conditions on a growth substrate. The growth substrate can be Matrigel® matrix (e.g., Matrigel®, Matrigel® GFR), recombinant laminin, laminin 511 recombinant fragment E8, or vitronectin. In certain embodiments of the present disclosure, the growth substrate is recombinant human laminin 521 (Biolamina, Sweden, sold by Corning, Corning, NY). In other embodiments, the substrate is a synthetic substrate, such as, for example, Synthemax®-II SC Substrate.

[0055] Undifferentiated stem cells can be passaged or subcultured using a variety of methods, such as the use of collagenase or manual scraping. Undifferentiated stem cells can be subcultured by enzymatic means that generate a single cell suspension, for example, using Accutase® (available from Sigma Aldrich, MO) or similar trypsinases. Alternatively, undifferentiated stem cells can be subcultured using non-enzymatic means, for example, using 0.5 mM EDTA in PBS, or ReLeSR™ (Stem Cell Technologies, Vancouver, Canada).

[0056] In one embodiment, the plurality of undifferentiated stem cells are seeded or subcultured at a seeding density that allows the cells to reach confluence in about 3 to about 10 days. In one embodiment, the seeding density is about 6.0×10 3 cells / cm 2 ~Approx. 5.0×10 5 cells / cm 2 range, for example, about 1.0×10 4 cells / cm 2 , for example, about 5.0 x 10 4 cells / cm 2 , for example, about 1.0 x 10 5 cells / cm 2 , or for example, about 3.0 x 10 5 cells / cm2 In another embodiment, the seeding density is about 6.0×10 3 cells / cm 2 ~Approx. 1.0×10 4 cells / cm 2 Growth surface, e.g., about 6.0×10 3 cells / cm 2 ~Approx. 9.0×10 3 cells / cm 2 , for example, about 7.0 x 10 3 cells / cm 2 ~Approx. 1.0×10 4 cells / cm 2 , for example, about 7.0 x 10 3 cells / cm 2 ~Approx. 9.0×10 3 cells / cm 2 , or for example, about 7.0 x 10 3 cells / cm 2 ~Approx. 8.0×10 3 cells / cm 2 In yet another embodiment, the seeding density is about 1.0×10 4 cells / cm 2 ~Approx. 1.0×10 5 cells / cm 2 Growth surface, e.g., about 2.0×10 4 cells / cm 2 ~Approx. 9.0×10 4 cells / cm 2 , for example, about 3.0 x 10 4 cells / cm 2 ~Approx. 8.0×10 4 cells / cm 2 , for example, about 4.0 x 10 4 cells / cm 2 ~Approx. 7.0×10 4 cells / cm 2 , or for example, about 5.0 x 10 4 cells / cm 2 ~Approx. 6.0×10 4 cells / cm 2 The seeding density can range from about 1.0×10 5 cells / cm 2 ~Approx. 5.0×10 5 cells / cm 2Growth surface, e.g., about 1.0×10 5 cells / cm 2 ~Approx. 4.5×10 5 cells / cm 2 , for example, about 1.5 x 10 5 cells / cm 2 ~Approx. 4.0×10 5 cells / cm 2 , for example, about 2.0 x 10 5 cells / cm 2 ~Approx. 3.5×10 5 cells / cm 2 , or for example, about 2.5 x 10 5 cells / cm 2 ~Approx. 3.0×10 5 cells / cm 2 It may be a range of growth surfaces.

[0057] Any of a variety of suitable cell culture and subculture techniques can be used to culture stem cells according to the disclosed methods. For example, the medium is changed completely daily, beginning about 2 days after subculture of the cells. In one embodiment, once the culture reaches about 90% colony coverage, the cells can be detached and seeded for subsequent culture using one or more suitable reagents, such as Accutase®, which provides a single cell suspension for quantification. In one embodiment, the undifferentiated stem cells can then be subcultured and then seeded on a suitable growth substrate (e.g., recombinant human laminin 521) at a seeding density that allows the cells to reach confluence in a suitable period of time, e.g., about 3-10 days. In one embodiment, the undifferentiated stem cells are subcultured using collagenase IV and grown on recombinant laminin. In another embodiment, the undifferentiated stem cells are subcultured using collagenase IV and grown on Matrigel®. In one embodiment, undifferentiated stem cells are subcultured using ReLeSR™ and grown on recombinant human laminin 521.

[0058] When seeding undifferentiated stem cells, the seeding density is approximately 6.0 × 10 3 cells / cm 2 ~Approx. 5.0×10 5 cells / cm2 range, for example, about 1.0×10 4 cells / cm 2 , for example, about 5.0 x 10 4 cells / cm 2 , for example, about 1.0 x 10 5 cells / cm 2 , or for example, about 3.0 x 10 5 cells / cm 2 In another embodiment, the seeding density is about 6.0×10 3 cells / cm 2 ~Approx. 1.0×10 4 cells / cm 2 Growth surface, e.g., about 6.0×10 3 cells / cm 2 ~Approx. 9.0×10 3 cells / cm 2 , for example, about 7.0 x 10 3 cells / cm 2 ~Approx. 1.0×10 4 cells / cm 2 , for example, about 7.0 x 10 3 cells / cm 2 ~Approx. 9.0×10 3 cells / cm 2 , or for example, about 7.0 x 10 3 cells / cm 2 ~Approx. 8.0×10 3 cells / cm 2 In yet another embodiment, the seeding density is about 1.0×10 4 cells / cm 2 ~Approx. 1.0×10 5 cells / cm 2 Growth surface, e.g., about 2.0×10 4 cells / cm 2 ~Approx. 9.0×10 4 cells / cm 2 , for example, about 3.0 x 10 4 cells / cm 2 ~Approx. 8.0×10 4 cells / cm 2 , for example, about 4.0 x 10 4 cells / cm 2 ~Approx. 7.0×10 4 cells / cm 2, or for example, about 5.0 x 10 4 cells / cm 2 ~Approx. 6.0×10 4 cells / cm 2 The seeding density can range from about 1.0×10 5 cells / cm 2 ~Approx. 5.0×10 5 cells / cm 2 Growth surface, e.g., about 1.0×10 5 cells / cm 2 ~Approx. 4.5×10 5 cells / cm 2 , for example, about 1.5 x 10 5 cells / cm 2 ~Approx. 4.0×10 5 cells / cm 2 , for example, about 2.0 x 10 5 cells / cm 2 ~Approx. 3.5×10 5 cells / cm 2 , or for example, about 2.5 x 10 5 cells / cm 2 ~Approx. 3.0×10 5 cells / cm 2 It may be a range of growth surfaces.

[0059] Neural induction of undifferentiated pluripotent stem cells The present disclosure provides a method for differentiating pluripotent stem cells into neuroectoderm and further into glial progenitor cells and OPCs using small molecule and protein modulators of TGFβ / Activin / Nodal signaling and BMP signaling. Without being bound by any particular theory, the inventors have found that starting pluripotent stem cells are aggregated in dynamic suspension into non-EB aggregates, in which the pluripotent stem cells remain undifferentiated, and the aggregates can then be induced to differentiate into neuroectoderm during dynamic suspension by using one or more inhibitors of TGFβ / Activin / Nodal signaling and one or more inhibitors of BMP signaling (dual SMAD inhibition). Dynamic suspension culture provides a scalable, reproducible, and controllable process for producing large amounts of cells from starting materials, in contrast to adherent culture and EB-based methods. The method of dual SMAD inhibition in dynamic suspension is described in detail herein.

[0060] In one embodiment, a method comprises culturing undifferentiated stem cells that form small non-EB aggregates but remain undifferentiated in dynamic suspension in the presence of one or more inhibitors of TGFβ / activin / nodal signaling and one or more inhibitors of BMP signaling, thereby initiating neural induction. In certain embodiments, the inhibitors of TGFβ / activin / nodal signaling are small molecules. In other embodiments, the inhibitors of TGFβ / activin / nodal signaling are proteins. In some embodiments, the direct target of the inhibitors of TGFβ / activin / nodal signaling is ALK5, also known as TGFβ type 1 receptor (TGFβR1). In certain embodiments, the inhibitors of BMP signaling are small molecules. In other embodiments, the inhibitors of BMP signaling are proteins. In some embodiments, the direct target of the inhibitors of BMP signaling is ALK2, also known as activin A receptor type I (ACVR1). In certain embodiments, following dual SMAD inhibition, the resulting cells are cultured in dynamic suspension in the presence of one or more smoothened receptor agonists and retinoic acid.

[0061] In certain embodiments, the inhibitor of TGFβ / activin / nodal signaling can be selected from the group consisting of SB431542, LY2157299, GW788388, A-77-01, A-83-01, SB505124, and derivatives thereof. In certain embodiments, the inhibitor of BMP signaling can be selected from the group consisting of dorsomorphin, DMH-1, K02288, ML3467, LDN193189, and noggin protein. In certain embodiments, the smoothened agonist can be selected from the group consisting of purmorphamine, SAG (CAS 364590-63-6), SSH protein, and derivatives thereof.

[0062] In one embodiment, a method includes obtaining non-EB aggregates that contain pluripotent stem cells that remain in an undifferentiated state, as shown in Figure 1; culturing the non-EB aggregates in dynamic suspension in the presence of the small molecules SB431542 and dorsomorphin for a first period of time; and then culturing the aggregates in dynamic suspension in the presence of a smoothogonist and retinoic acid for a second period of time. In one embodiment, the first period and the second period can each be in the range of about 1 to about 4 days, e.g., about 1 day, e.g., about 2 days, e.g., about 3 days, e.g., about 4 days.

[0063] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of SB431542 at a concentration in the range of about 1 μM to about 100 μM, for example, about 5 μM, about 10 μM, for example, about 15 μM, for example, about 20 μM, for example, about 25 μM, for example, about 30 μM, for example, about 35 μM, for example, about 40 μM, for example, about 45 μM, for example, about 50 μM, for example, about 55 μM, for example, about 60 μM, for example, about 65 μM, for example, about 70 μM, for example, about 75 μM, for example, about 80 μM, for example, about 85 μM, for example, about 90 μM, or for example, about 95 μM. In another embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of SB431542 at a concentration ranging from about 1 μM to about 20 μM, for example, about 1 μM to about 13 μM, for example, about 8 μM to about 20 μM, for example, about 8 μM to about 13 μM, or for example, about 9 μM to about 11 μM. In yet another embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of SB431542 at a concentration ranging from about 20 μM to about 40 μM, for example, about 20 μM to about 33 μM, for example, about 28 μM to about 40 μM, for example, about 28 μM to about 33 μM, or for example, about 29 μM to about 31 μM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of SB431542 at a concentration ranging from about 40 μM to about 60 μM, for example, about 40 μM to about 53 μM, for example, about 48 μM to about 55 μM, for example, about 48 μM to about 53 μM, or for example, about 49 μM to about 51 μM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of SB431542 at a concentration ranging from about 60 μM to about 80 μM, for example, about 60 μM to about 73 μM, for example, about 68 μM to about 75 μM, for example, about 68 μM to about 73 μM, or for example, about 69 μM to about 71 μM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of SB431542 at a concentration ranging from about 80 μM to about 100 μM, for example, about 80 μM to about 93 μM, for example, about 88 μM to about 95 μM, for example, about 88 μM to about 93 μM, or for example, about 89 μM to about 91 μM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of SB431542 at a concentration of about 10 μM.

[0064] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of an ALK5 inhibitor at a concentration ranging from about 250 nM to about 250 μM, e.g., about 1 μM, about 10 μM, about 50 μM, about 100 μM, about 150 μM, or about 200 μM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of about 10 μM of an ALK5 inhibitor.

[0065] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of LY364947 at a concentration ranging from about 250 nM to about 250 μM, e.g., about 1 μM, about 10 μM, about 50 μM, about 100 μM, about 150 μM, or about 200 μM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of about 10 μM LY364947.

[0066] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of GW788388 at a concentration ranging from about 250 nM to about 250 μM, e.g., about 1 μM, about 10 μM, about 50 μM, about 100 μM, about 150 μM, or about 200 μM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of about 10 μM GW788388.

[0067] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of A-77-01 at a concentration ranging from about 250 nM to about 250 μM, e.g., about 1 μM, about 10 μM, about 50 μM, about 100 μM, about 150 μM, or about 200 μM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of about 10 μM A-77-01.

[0068] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of A-83-01 at a concentration ranging from about 250 nM to about 250 μM, e.g., about 1 μM, about 10 μM, about 50 μM, about 100 μM, about 150 μM, or about 200 μM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of about 10 μM A-83-01.

[0069] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of SB505124 at a concentration ranging from about 250 nM to about 250 μM, for example, about 1 μM, about 10 μM, about 50 μM, about 100 μM, about 150 μM, or about 200 μM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of about 10 μM SB505124.

[0070] In one embodiment, a method comprises: dissolving non-EB aggregates in dynamic suspension at a concentration in the range of about 0.2 μM to about 20 μM, for example, about 0.5 μM, for example, about 0.8 μM, for example, about 1 μM, for example, about 1.5 μM, for example, about 2 μM, for example, about 2.5 μM, for example, about 3 μM, for example, about 3.5 μM, for example, about 4 μM, for example, about 4.5 μM, for example, about 5 μM, for example, about 5.5 μM, for example, about 6 μM, for example, about 6. In another embodiment, a method includes culturing non-EB aggregates in the presence of dorsomorphin at a concentration of about 0.2 μM to about 1 μM, for example, about 0.2 μM to about 0.9 μM, for example, about 0.3 μM to about 0.8 μM, for example, about 0.4 μM to about 0.7 μM, or for example, about 0.5 μM to about 0.6 μM. In yet another embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of dorsomorphin at a concentration ranging from about 1 μM to about 10 μM, for example, about 1 μM to about 9 μM, for example, about 2 μM to about 8 μM, for example, about 3 μM to about 7 μM, or for example, about 4 μM to about 6 μM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of dorsomorphin at a concentration ranging from about 10 μM to about 20 μM, for example, about 10 μM to about 19 μM, for example, about 12 μM to about 18 μM, for example, about 13 μM to about 17 μM, or for example, about 14 μM to about 16 μM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of dorsomorphin at a concentration of about 2 μM.

[0071] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of an ALK2 inhibitor at a concentration ranging from about 1 nM to about 20 μM, e.g., about 10 nM, about 50 nM, about 100 nM, about 150 nM, about 200 nM, about 500 nM, about 1 μM, about 5 μM, about 10 μM, or about 15 μM.

[0072] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of DMH-1 at a concentration ranging from about 1 μM to about 10 μM.In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of about 2 μM DMH-1.

[0073] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of K02288 at a concentration ranging from about 1 μM to about 10 μM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of about 2 μM K02288.

[0074] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of ML347 at a concentration ranging from about 1 μM to about 10 μM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of about 2 μM ML347.

[0075] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of purmorphamine at a concentration ranging from about 0.05 μM to about 5 μM, for example, about 0.08 μM, for example, about 0.1 μM, for example, about 0.2 μM, for example, about 0.3 μM, for example, about 0.4 μM, for example, about 0.5 μM, for example, about 0.6 μM, for example, about 0.7 μM, for example, about 0.8 μM, for example, about 0.9 μM, for example, about 1 μM, for example, about 2 μM, for example, about 3 μM, for example, about 4 μM. In another embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of purmorphamine at a concentration ranging from about 0.05 μM to about 0.1 μM, for example, about 0.06 μM to about 0.09 μM, or for example, about 0.07 μM to about 0.08 μM. In another embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of purmorphamine at a concentration ranging from about 0.1 μM to about 1 μM, e.g., about 0.2 μM to about 0.9 μM, e.g., about 0.3 μM to about 0.8 μM, e.g., about 0.4 μM to about 0.7 μM, or e.g., about 0.5 μM to about 0.6 μM. In another embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of purmorphamine at a concentration ranging from about 1 μM to about 5 μM, e.g., about 1 μM to about 4 μM, e.g., about 2 μM to about 5 μM, or e.g., about 2 μM to about 4 μM. In one embodiment, a method includes incubating proliferated but undifferentiated ESCs with purmorphamine at a concentration of about 0.5 μM.

[0076] In one aspect, a method includes culturing non-EB aggregates in dynamic suspension in the presence of a smoothoid agonist at a concentration ranging from about 2.5 nM to about 5 μM, e.g., about 50 nM, about 100 nM, about 250 nM, about 500 nM, about 750 nM, about 1 μM, or about 2.5 μM.

[0077] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of SAG at a concentration ranging from about 10 nM to about 1 μM, e.g., about 10 nM to about 100 nM, e.g., about 100 nM to about 500 nM, or e.g., about 500 nM to about 1000 nM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of about 0.5 μM SAG.

[0078] In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of SHH protein at a concentration ranging from about 2.5 nM to about 250 nM, e.g., from about 2.5 nM to about 10 nM, e.g., from about 10 nM to about 100 nM, or e.g., from about 100 nM to about 250 nM. In one embodiment, a method includes culturing non-EB aggregates in dynamic suspension in the presence of about 25 nM SHH protein.

[0079] Any cell culture vessel or reactor suitable for dynamic suspension culture can be used in the differentiation process contemplated in the present disclosure. The vessel wall is usually inert or resistant to the adhesion of cultured cells. There is also a means to prevent cells from settling, for example, a stirring mechanism such as a magnetically or mechanically driven stirring rod or paddle, a shaking mechanism (usually attached to the vessel from the outside), or an inversion mechanism (i.e., a device that rotates the vessel to change the direction of gravity on the cells).

[0080] Suitable vessels for suspension culture for process development include a range of commercially available spinners, rocker bags, or shaker flasks. Exemplary bioreactors suitable for commercial production include the VerticalWheel™ bioreactor (PBS Biotech, Camarillo, Calif.).

[0081] OPC composition Using the methods of the present disclosure, compositions comprising oligodendrocyte precursor cells (OPCs) suitable for cell therapy can be obtained. OPCs obtained according to the present disclosure express high levels of the proteoglycan NG2, characteristic of OPCs, and do not express non-OPC markers associated with undesired cell types, such as CD49f, which is expressed in both neural progenitor cells and epithelial cells and is associated with in vitro cyst formation (Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. 2003 Methods. 3:256-68), or two markers expressed in both pluripotent and epithelial cells, CLDN6 and EpCAM (Lin D, Guo Y, Li Y, Ruan Y, Zhang M, Jin X, Yang M, Lu Y, Song P, Zhao S, Dong B, Xie Y, Dang Q, Quan C. Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions. Oncol Rep. 2017 Aug;38(2):875-885; Huang L, Yang Y, Yang F, Liu S, Zhu Z, Lei Z, Guo J. Functions of EpCAM in physiological processes and diseases (Review). Int J Mol Med. 2018 Oct;42(4):1771-1785).

[0082] In certain aspects, the OPCs produced according to the present disclosure are in vitro differentiated progeny of human pluripotent stem cells. The OPCs obtained according to the present disclosure are in vitro differentiated progeny of human embryonic stem cells. In other aspects, the OPCs obtained according to the present disclosure are in vitro differentiated progeny of induced pluripotent stem (iPS) cells.

[0083] One or more characteristics of the resulting OPC population can be determined by quantifying various cell markers using flow cytometry, for example, to ascertain what percentage of a cell population is positive for a particular marker or set of markers, or to identify undesirable cell types present in the OPC population. In one embodiment, OPCs generated according to the methods of the invention express one or more markers selected from NG2, PDGFRα, and GD3.

[0084] An OPC population obtained according to the present disclosure can comprise from about 30% to about 100% NG2 positive cells, e.g., at least about 35%, such as at least about 40%, such as at least about 45%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65%, such as at least about 70%, such as at least about 75%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95%, such as at least about 98%, such as at least about 99%, such as at least about 99.5%, such as at least about 99.8%, or such as at least about 99.9% NG2 positive cells. In certain embodiments, an OPC population obtained according to the present disclosure can comprise about 45% to about 75% NG2 positive cells, for example, about 45% to about 50%, for example, about 50% to about 55%, for example, about 55% to about 60%, for example, about 60% to about 65%, for example, about 65% to about 70%, for example, about 70% to about 75%, for example, about 50% to about 70%, for example, about 55% to about 65%, or for example, about 58% to about 63% NG2 positive cells. In other embodiments, an OPC population obtained according to the present disclosure can comprise about 60% to about 90% NG2 positive cells, for example, about 60% to about 65%, for example, about 65% to about 70% NG2 positive cells. The OPC population obtained according to the present disclosure can comprise about 30% to about 100% PDGFRα positive cells, for example, at least about 35%, such as at least about 40%, for example, at least about 45%, for example, at least about 50%, for example, at least about 55%, such as at least about 60%, for example, at least about 65%, for example, at least about 70%, for example, at least about 75%, for example, at least about 80%, for example, at least about 85%, for example, at least about 90%, for example, at least about 95%, such as at least about 98%, for example, at least about 99%, for example, at least about 99.5%, for example, at least about 99.8%, or for example, at least about 99.9% PDGFRα positive cells.In certain embodiments, an OPC population obtained according to the present disclosure can comprise about 45% to about 75% PDGFRα positive cells, for example, about 45% to about 50%, for example, about 50% to about 55%, for example, about 55% to about 60%, for example, about 60% to about 65%, for example, about 65% to about 70%, for example, about 70% to about 75%, for example, about 50% to about 70%, for example, about 55% to about 65%, or for example, about 58% to about 63% PDGFRα positive cells. In other embodiments, an OPC population obtained according to the present disclosure can comprise about 60% to about 90% PDGFRα positive cells, for example, about 60% to about 65%, for example, about 65% to about 70% positive cells. The OPC population obtained according to the present disclosure can comprise about 30% to about 100% GD3 positive cells, e.g., at least about 35%, such as at least about 40%, such as at least about 45%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65%, such as at least about 70%, such as at least about 75%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95%, such as at least about 98%, such as at least about 99%, such as at least about 99.5%, such as at least about 99.8%, or such as at least about 99.9% GD3 positive cells. In certain embodiments, an OPC population obtained according to the present disclosure can comprise about 45% to about 75% GD3 positive cells, for example, about 45% to about 50%, for example, about 50% to about 55%, for example, about 55% to about 60%, for example, about 60% to about 65%, for example, about 65% to about 70%, for example, about 70% to about 75%, for example, about 50% to about 70%, for example, about 55% to about 65%, or for example, about 58% to about 63% GD3 positive cells. In other embodiments, an OPC population obtained according to the present disclosure can comprise about 60% to about 90% GD3 positive cells, for example, about 60% to about 65%, for example, about 65% to about 70% positive cells.

[0085] In one embodiment, an OPC population obtained according to the present disclosure may be capable of forming less than or equal to 4 epithelial cysts per 100,000 cells in a cyst assay described in Example 8 of the present disclosure. In another embodiment, an OPC population obtained according to the present disclosure may be capable of forming less than or equal to 3 epithelial cysts per 100,000 cells in a cyst assay. In another embodiment, an OPC population obtained according to the present disclosure may be capable of forming less than or equal to 2 epithelial cysts per 100,000 cells in a cyst assay. In yet another embodiment, an OPC population obtained according to the present disclosure may be capable of forming less than or equal to 1 epithelial cyst per 100,000 cells in a cyst assay described in Example 8 of the present disclosure.

[0086] Undesirable cell types OPC populations obtained according to the present disclosure contain low levels of undesired cell types, for example, as measured by quantification of markers associated with undesired cell types by flow cytometry. In a non-limiting example, day 42 OPCs obtained according to the present disclosure contained 0%-4% cells expressing the epithelial cell-associated markers EpCAM, CD49f, and CLDN6 (Example 5, Table 2).

[0087] Markers associated with undesired cell types may constitute less than about 20% of the undesired cell types, such as less than about 19%, for example less than about 18%, such as less than about 17%, for example less than about 16%, for example less than about 15%, for example less than about 14%, for example less than about 13%, for example less than about 12%, for example less than about 11%, for example less than about 10%, such as less than about 9%, for example less than about 8%, for example less than about 7%, for example less than about 6%, for example less than about 5%, for example less than about 4%, for example less than about 3%, for example less than about 2%, for example less than about 1%, for example less than about 0.5%, for example less than about 0.1%, for example less than about 0.05%, or for example less than about 0.01% of the undesired cell types. In another embodiment, the cell population may comprise about 15% to about 20% of the undesired cell type, for example, about 19% to about 20%, for example, about 18% to about 20%, for example, about 17% to about 20%, for example, about 16% to about 20%, for example, about 15% to about 19%, or for example, about 16% to about 18% of the undesired cell type. In yet another embodiment, the cell population may comprise about 10% to about 15% of the undesired cell type, for example, about 14% to about 15%, for example, about 13% to about 15%, for example, about 12% to about 15%, for example, about 11% to about 15%, or for example, about 12% to about 14% of the undesired cell type. In one embodiment, the cell population may contain about 1% to about 10% of an undesired cell type, e.g., about 2% to about 10%, e.g., about 1% to about 9%, e.g., about 2% to about 8%, e.g., about 3% to about 7%, or e.g., about 4% to about 6% of an undesired cell type. In one embodiment, the cell population may contain about 0.1% to about 1% of an undesired cell type, e.g., about 0.2% to about 1%, e.g., about 0.1% to about 0.9%, e.g., about 0.2% to about 0.8%, e.g., about 0.3% to about 0.7%, or e.g., about 0.4% to about 0.6% of an undesired cell type. In one aspect, the cell population may contain from about 0.01% to about 0.1% of an undesired cell type, such as from about 0.02% to about 0.1%, such as from about 0.01% to about 0.09%, such as from about 0.02% to about 0.08%, such as from about 0.03% to about 0.07%, or such as from about 0.04% to about 0.06% of an undesired cell type.In one embodiment, a low level of an undesirable cell type can refer to the presence of less than about 15% of an undesirable cell type.

[0088] In one embodiment, the undesirable cell type may include cells that express another marker selected from CD49f, CLDN6, or EpCAM.

[0089] Formulation The OPC composition according to the present disclosure can further comprise a pharma- ceutically acceptable carrier. In one embodiment, the pharma- ceutically acceptable carrier can comprise dimethyl sulfoxide (DMSO). In one embodiment, the pharma- ceutically acceptable carrier does not comprise dimethyl sulfoxide. In one embodiment, the composition can be adapted for frozen storage at temperatures between -80°C and -195°C or lower.

[0090] An OPC composition according to the present disclosure can be formulated for administration via direct injection into the spinal cord of a subject. In one embodiment, an OPC composition according to the present disclosure can be formulated for intracerebral, intraventricular, intrathecal, intranasal, or intracapsular administration to a subject. In one embodiment, an OPC composition according to the present disclosure can be formulated for administration via injection directly into or immediately adjacent to an infarct cavity in the brain of a subject. In one embodiment, a composition according to the present disclosure can be formulated for administration via implantation. In one embodiment, a composition according to the present disclosure can be formulated as a solution.

[0091] OPC compositions according to the present disclosure have a concentration of about 1×10 per milliliter 6 ~Approx. 5×10 8 The medium can contain, for example, about 1 x 10 cells per milliliter. 6 Cells, e.g., approximately 2 x 10 per milliliter 6 Cells, e.g., approximately 3 x 10 per milliliter 6 Cells, e.g., approximately 4 x 10 per milliliter 6 Cells, e.g., approximately 5 x 10 per milliliter 6 Cells, e.g., approximately 6 x 10 per milliliter6 Cells, e.g., approximately 7 x 10 per milliliter 6 Cells, e.g., approximately 8 x 10 per milliliter 6 Cells, e.g., approximately 9 x 10 per milliliter 6 Cells, e.g., approximately 1 x 10 per milliliter 7 Cells, e.g., approximately 2 x 10 per milliliter 7 Cells, e.g., approximately 3 x 10 per milliliter 7 Cells, e.g., approximately 4 x 10 per milliliter 7 Cells, e.g., approximately 5 x 10 per milliliter 7 Cells, e.g., approximately 6 x 10 per milliliter 7 Cells, e.g., approximately 7 x 10 per milliliter 7 Cells, e.g., approximately 8 x 10 per milliliter 7 Cells, e.g., approximately 9 x 10 per milliliter 7 Cells, e.g., approximately 1 x 10 per milliliter 8 Cells, e.g., approximately 2 x 10 per milliliter 8 Cells, e.g., approximately 3 x 10 per milliliter 8 Cells, e.g., approximately 4 x 10 per milliliter 8 cells, or, for example, about 5 x 10 per milliliter 8 In another embodiment, the composition according to the present disclosure comprises about 1 x 10 cells per milliliter. 8 ~Approx. 5×10 8 The medium can contain, for example, about 1 x 10 cells per milliliter. 8 ~Approx. 4×10 8 Cells, e.g., approximately 2 x 10 per milliliter 8 ~Approx. 5×10 8 Cells, e.g., approximately 1 x 10 per milliliter 8 ~Approx. 3×10 8 Cells, e.g., approximately 2 x 10 per milliliter 8 ~Approx. 4×10 8 cells, or, for example, about 3 x 10 per milliliter 8 ~Approx. 5×10 8 In yet another embodiment, the composition according to the present disclosure comprises about 1 x 10 cells per milliliter. 7 ~Approx. 1×108 The medium can contain, for example, approximately 2 x 10 cells per milliliter. 7 ~Approx. 9×10 7 Cells, e.g., approximately 3 x 10 per milliliter 7 ~Approx. 8×10 7 Cells, e.g., approximately 4 x 10 per milliliter 7 ~Approx. 7×10 7 cells, or, for example, about 5 x 10 per milliliter 7 ~Approx. 6×10 7 In one embodiment, the composition according to the present disclosure comprises about 1 x 10 cells per milliliter. 6 ~Approx. 1×10 7 The medium can contain, for example, approximately 2 x 10 cells per milliliter. 6 ~Approx. 9×10 6 Cells, e.g., approximately 3 x 10 per milliliter 6 ~Approx. 8×10 6 Cells, e.g., approximately 4 x 10 per milliliter 6 ~Approx. 7×10 6 cells, or, for example, about 5 x 10 per milliliter 6 ~Approx. 6×10 6 In yet another embodiment, the composition according to the present disclosure comprises at least about 1 x 10 cells per milliliter. 6 The medium can contain, for example, at least about 2×10 cells per milliliter. 6 Cells, e.g., at least about 3 x 10 per milliliter 6 Cells, e.g., at least about 4 x 10 per milliliter 6 Cells, e.g., at least about 5 x 10 per milliliter 6 Cells, e.g., at least about 6 x 10 per milliliter 6 Cells, e.g., at least about 7 x 10 per milliliter 6 Cells, e.g., at least about 8 x 10 per milliliter 6 Cells, e.g., at least about 9 x 10 per milliliter 6 Cells, e.g., at least about 1 x 10 per milliliter 7 Cells, e.g., at least about 2 x 10 per milliliter 7Cells, e.g., at least about 3 x 10 per milliliter 7 Cells, e.g., at least about 4 x 10 per milliliter 7 cells, or, for example, at least about 5 x 10 per milliliter 7 In one embodiment, the composition according to the present disclosure comprises about 1 x 10 cells per milliliter. 8 Can contain up to or more than about 2 x 10 cells per milliliter, for example 8 Up to or more than about 3 x 10 cells per milliliter 8 Up to or more than about 4 x 10 cells per milliliter 8 Up to or more than about 5 x 10 cells per milliliter 8 Up to or more than about 6 x 10 cells per milliliter, for example 8 This includes cells.

[0092] In one embodiment, an OPC composition according to the present disclosure has about 4×10 7 ~about 2×10 8 It may contain cells.

[0093] In yet another embodiment, an OPC composition according to the present disclosure can have a volume ranging from about 10 microliters to about 5 milliliters, for example, about 20 microliters, such as about 30 microliters, for example, about 40 microliters, for example, about 50 microliters, for example, about 60 microliters, for example, about 70 microliters, for example, about 80 microliters, for example, about 90 microliters, for example, about 100 microliters, for example, about 200 microliters, for example, about 300 microliters, for example, about 400 microliters, for example, about 500 microliters, for example, about 600 microliters, for example, about 700 microliters, for example, about 800 microliters, for example, about 900 microliters, for example, about 1 milliliter, for example, about 1.5 milliliters, for example, about 2 milliliters, for example, about 2.5 milliliters, for example, about 3 milliliters, for example, about 3.5 milliliters, for example, about 4 milliliters, or for example, about 4.5 milliliters. In one embodiment, the composition according to the present disclosure can have a volume in the range of about 10 microliters to about 100 microliters, for example, about 20 microliters to about 90 microliters, for example, about 30 microliters to about 80 microliters, for example, about 40 microliters to about 70 microliters, or for example, about 50 microliters to about 60 microliters. In another embodiment, the composition according to the present disclosure can have a volume in the range of about 100 microliters to about 1 milliliter, for example, about 200 microliters to about 900 microliters, for example, about 300 microliters to about 800 microliters, for example, about 400 microliters to about 700 microliters, or for example, about 500 microliters to about 600 microliters.In yet another embodiment, the composition according to the present disclosure can have a volume in the range of about 1 milliliter to about 5 milliliters, e.g., about 2 milliliters to about 5 milliliters, e.g., about 1 milliliter to about 4 milliliters, e.g., about 1 milliliter to about 3 milliliters, e.g., about 2 milliliters to about 4 milliliters, or e.g., about 3 milliliters to about 5 milliliters. In one embodiment, the OPC composition according to the present disclosure can have a volume of about 20 microliters to about 500 microliters. In another embodiment, the OPC composition according to the present disclosure can have a volume of about 50 microliters to about 100 microliters. In yet another embodiment, the OPC composition according to the present disclosure can have a volume of about 50 microliters to about 200 microliters. In another embodiment, the OPC composition according to the present disclosure can have a volume of about 20 microliters to about 400 microliters. In one embodiment, the OPC composition according to the present disclosure can be present in a container configured for cryopreservation or for administration to a subject in need thereof. In one embodiment, the container can be a prefilled syringe.

[0094] How to use The OPC composition obtained according to the present disclosure can be used in cell therapy to improve one or more neurological functions in a subject in need of treatment. In one embodiment, the OPC cell population according to the present disclosure can be injected or transplanted into a subject in need thereof. In one embodiment, the cell population according to the present disclosure can be transplanted into a subject in need thereof to treat spinal cord injury, stroke, or multiple sclerosis.

[0095] In one aspect, the cell population according to the present disclosure may be capable of inducing myelination of denuded axons at the site of transplantation in a subject. In one aspect, the cell population produced according to the method of the present disclosure may exhibit improved ability for engraftment and migration. In one aspect, the cell population produced according to the method of the present disclosure may be capable of improving post-injury repair or regeneration of neural tissue in a subject.

[0096] The cell population according to the present disclosure may improve sensory function in a subject in need of treatment after transplantation of the population into the subject. Improved sensory function can be assessed using the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) test, for example, by determining the right and left sensory levels for pinprick and light touch sensation. The cell population according to the present disclosure may improve motor function in a subject in need of treatment after transplantation of the population into the subject. Improved motor function can be assessed using the ISNCSCI test, for example, by determining the right and left motor levels for complete paralysis, palpable or visible contraction, active movement, full range of motion against gravity, and sufficient resistance.

[0097] The cell population according to the present disclosure may reduce the volume of injury-induced central nervous system parenchymal cavitation in 12 months or less. In one aspect, the cell population according to the present disclosure may reduce the volume of injury-induced central nervous system parenchymal cavitation in a subject in 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, or 1 month or less.

[0098] Having now generally described the invention, it is believed the same will be more readily understood by reference to the following examples, which are provided for illustrative purposes and are not intended to limit the disclosure, unless otherwise specified. EXAMPLES

[0099] Example 1 – Culture and expansion of undifferentiated human embryonic stem cells Working cell bank (WCB)-derived undifferentiated human embryonic stem cells (uhESCs) generated from the H1 line (WA01; Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998 Nov 6; 282(5391):1145-7) were cultured in 225 cm tissue culture treated polystyrene plates coated with recombinant human laminin 521 (Corning #354224). 2 The cells were cultured in complete mTeSR™-1 medium (Stem Cell Technologies #85850) in a culture flask (Corning #431082). The medium was changed completely every day until the cells reached approximately 80-90% confluency, after which uhESCs were passaged using ReLeSR™ Reagent (Stem Cell Technologies #05872). uhESC cells detached with ReLeSR™ (ReLeSR TM -lifted uhFSC cells) were placed on fresh laminin 521-coated 225cm 2 The WCB-derived cultured uhESCs were expanded in this manner for 2-5 passages depending on the experiment and then differentiated into neuroectodermal progenitor cells as described in Example 2.

[0100] Example 2 - Method for Differentiating Human Embryonic Stem Cells into Neuroectodermal Progenitor Cells in Dynamic Suspension Culture Day -1Expanded uhESCs (~90% confluent) were detached and disaggregated with Accutase® (Stem Cell Technologies #07920) to form a single cell suspension to ensure accurate cell numbers and uniform seeding density. Disaggregated uhESCs were then added at 1 × 10 to a PBS-0.1 or PBS-0.5 mini bioreactor system (PBS Biotech) set to rotate at 35 RPM or 25 RPM, respectively. 6 Cells were seeded for dynamic suspension culture at a concentration of 100000 viable cells / mL (day -1). Cells were seeded in a 1:1 mixture of glial progenitor medium (GPM; consisting of DMEM / F12 (Gibco Cat. No. 10565-018) supplemented with 2% B27 supplement (Gibco Cat. No. 17504-044) and 0.04 μg / mL triiodothyronine (Sigma Cat. No. T5516-1MG)) and undifferentiated hESC medium (described in Example 1) supplemented with 10 μM Rho kinase inhibitor (RI, Tocris Cat. No. 1254) to support cell survival. Within the first 24 h, small and uniform aggregates of uhESCs were formed. In contrast to the formation of embryoid bodies (EBs), cells in the small aggregates did not initiate spontaneous differentiation and maintained pluripotency, as demonstrated by the expression of markers measured by qPCR (Figure 5).

[0101] Day 0-3 To initiate differentiation into neuroectoderm, small non-EB clumps were cultured in dynamic suspension in GPM supplemented with 10 μM SB431542 (Sigma-Aldrich, Catalog No. S4317) and 2 μM dorsomorphin (Sigma-Aldrich, Catalog No. P5499) for the next 4 days in PBS-0.1 or PBS-0.5 mini-bioreactors rotating at 45 or 32 RPM, respectively. Medium was replenished daily by allowing clumps to settle, removing 70-80% of the spent medium, and replacing it with an equal volume of GPM supplemented with 10 μM SB431542 and 2 μM dorsomorphin.

[0102] Days 4-6Cells were further cultured in dynamic suspension at 45 RPM (PBS-0.1 mini-bioreactors) or 32 RPM (PBS-0.5 mini-bioreactors) in GPM supplemented with 0.5 μM purmorphamine (Reprocell, Cat. No. 04-0009), 1 μM retinoic acid (Sigma-Aldrich, Cat. No. R2625), and 150 μM ascorbic acid (Sigma Aldrich, Cat. No. A4544) for an additional 3 days. Medium was replenished daily by allowing clumps to settle, removing 70-80% of the spent medium, and replacing it with an equal volume of GPM supplemented with 0.5 μM purmorphamine, 1 μM retinoic acid, and 150 μM ascorbic acid.

[0103] Day 7 A subset of differentiated cells was collected on day 7 of the differentiation process in dynamic suspension culture and subjected to analysis of marker expression by immunocytochemistry (ICC) (described in Example 5) and qPCR (described in Example 6). At day 7, the cells expressed markers characteristic of neuroectoderm (Table 2, Figure 3). The remaining day 7 cells were differentiated into glial lineage cells and further into oligodendrocyte precursor cells, as described in Examples 3 and 4, respectively.

[0104] Example 3 – Method for differentiating human embryonic stem cells into glial lineage cells in dynamic suspension culture Differentiation of uhESCs into neuroectoderm / neural progenitor cells (days 0–6) was performed as described in Example 2. On day 7, differentiation into glial progenitor cells was initiated by changing the differentiation medium to GPM supplemented with 20 ng / mL human basic fibroblast growth factor (hbFGF, Thermo Fisher, catalog #PHG0263), 10 ng / mL epidermal growth factor (EGF, Thermo Fisher, catalog #PHG0311), and 10 μM RI. Cell aggregates were maintained in dynamic suspension at 45 rpm (PBS-0.1 mini-bioreactor) or 32 rpm (PBS-0.5 mini-bioreactor) in GPM supplemented with 20 ng / mL bFGF and 10 ng / mL EGF for the next 2 weeks (days 8–20), during which the medium was replenished daily using gravity settling and 70–80% medium exchange. On day 14, 10 μM RI was also added to the fresh medium.

[0105] A subset of differentiated cells was collected on day 21 of the dynamic suspension differentiation process and subjected to analysis of marker expression by qPCR (described in Example 6). At day 21, differentiated cells expressed markers consistent with glial lineage cells (Table 2).

[0106] Example 4 – Methods for Differentiating Human Embryonic Stem Cells into Oligodendrocyte Progenitor Cells The glial progenitor cells obtained in Example 3 were further differentiated into oligodendrocyte progenitor cells. The differentiation protocol from days 0 to 20 was as described in Examples 2 and 3. On day 21, the aggregates were switched from dynamic suspension culture to adherent culture on tissue culture vessels coated with recombinant human laminin 521 (rhLN-521). For example, starting with 1×PBS-0.1L mini-bioreactor containing 60 mL of aggregate suspension, 60 mL of culture was split into 2×T75 flasks containing 30 mL of culture each. Starting on day 21 and continuing through the end of the differentiation process, the cells were cultured in GPM supplemented with 20 ng / mL EGF and 10 ng / mL platelet-derived growth factor-AA (PDGF-AA, PeproTech, Cat#AF-100-13A), during which the medium was completely changed every other day. On days 28 and 35, cell cultures were detached using TrypLE™ Select (Thermo Fisher, Cat# Al2859-01), counted, and plated at 4 × 10 cells onto rhLN-521-coated vessels. 4 live cells / cm 2 GPM was changed every other day from day 35 until harvest on day 42.

[0107] A subset of differentiated cells was harvested on day 42 of the differentiation process and subjected to analysis of marker expression by flow cytometry (as described in Example 5), immunocytochemistry (as described in Example 5) and qPCR (as described in Example 6). At day 42, differentiated cells expressed markers characteristic of oligodendrocyte precursor cells as determined by these three analytical methods (Table 1, Table 2, FIG. 4).

[0108] OPCs were harvested on day 42. Cells were detached from the vessel using TrypLE™ Select, counted, and re-formulated in CryoStorl 0 (BioLife Solutions, Cat #210102) prior to cryopreservation.

[0109] Example 5 –Characterization of differentiated cell populations by immunocytochemistry and flow cytometry Flow cytometry and immunocytochemistry (ICC) can be used to detect and characterize various aspects of protein marker expression in cell populations. While flow cytometry can be used to quantify the percentage of individual cells in a population that exhibit a given protein marker profile, ICC provides additional information about the subcellular localization of each protein marker and can be applied to single cells or cell aggregates. Using either or both of these protein profiling approaches, we have tracked the differentiation of human embryonic stem cells into neuroectodermal progenitor cells, glial progenitor cells, and oligodendrocyte progenitor cells according to the methods of the present disclosure.

[0110] For human embryonic stem cells differentiated into neuroectodermal progenitors in suspension, the expression of protein markers in the starting material (undifferentiated pluripotent cells) and in differentiated day 7 cell aggregates was characterized by ICC. Adherent pluripotent cells and cell aggregates were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature (RT). Fixed cells and aggregates were washed with phosphate-buffered saline (PBS), and then fixed aggregates were sequentially placed in increasing concentrations of sucrose solutions (10%, 20%, and 30% wt / vol) for 30 min at RT, 30 min at RT, and overnight at 4°C, respectively. After sucrose exchange, aggregates were embedded in Tissue-Tek Optimal Cutting Temperature (OCT) solution (Sakura Finetek USA #4583) and frozen at -80°C. The OCT-embedded aggregates were warmed to -20°C, cut into 30 μm sections using a cryostat (Model CM3050 S, Leica Biosystems, Buffalo Grove, IL, USA) and mounted on poly-L-lysine (Sigma-Aldrich #P4707)-coated glass slides. For immunocytochemical staining, the fixed adherent cells and slide-mounted aggregate sections were permeabilized and blocked in blocking solution consisting of 0.1% Triton™ X-100 / 2% normal goat serum / 1% bovine serum albumin in PBS for 2 h at room temperature (RT). Following permeabilization and blocking, adherent cells and clump sections were incubated overnight at 4°C in blocking solution without Triton™ X-100 but containing primary antibodies specific for protein markers of interest, such as Nanog (Abcam #ab21624), Oct4 (Millipore #MAB4401), and Sox2 (Abcam #ab92494) to detect pluripotent cells, or PAX6 (BD Pharmingen #561462) and PSA-NCAM (Invitrogen #14-9118-80) to detect neuroectodermal progenitor cells.Adherent cells and aggregate sections were then washed three times with PBS and then incubated with secondary antibodies specific for the selected primary antibodies and 4',6-diamidino-2-phenylindole (DAPI) counterstain in blocking solution without Triton™ X-100 for 1 h at RT in the dark. Adherent cells and aggregate sections were washed three times with PBS and imaged using an IN Cell Analyzer 2000 (GE Healthcare, Pittsburgh, PA, USA).

[0111] Figures 2 and 3 show representative ICC data for the starting pluripotent cell population and day 7 neuroectodermal progenitor cells, respectively. As shown in Figure 2, the starting population of undifferentiated human embryonic stem cells expressed the typical pluripotency markers Nanog, Oct4, and Sox2 (Wang Z, Oron E, Nelson B, Razis S, Ivanova N. Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell. 2012 Apr 6;10(4):440-54). After 7 days of differentiation, cell aggregates from two representative experiments expressed PAX6 and PSA-NCAM, two protein markers characteristic of neuroectodermal progenitors (Figure 3, Lippmann ES, Williams CE, Ruhl DA, Estevez-Silva MC, Chapman ER, Coon JJ, Ashton RS. Deterministic HOX patterning in human pluripotent stem cell-derived neuroectoderm. Stem Cell Reports. 2015 Apr 14;4(4):632-44; Kim DS, Lee DR, Kim HS, Yoo JE, Jung SJ, Lim BY, Jang J, Kang HC, You S, Hwang DY, Leem JW, Nam TS, Cho SR, Kim DW. Highly pure and expandable PSA-NCAM-positive neural precursors from human ESC and iPSC-derived neural rosettes. PLoS One. 2012;7(7):e39715).

[0112] For human embryonic stem cells that were differentiated into oligodendrocyte precursor cells by day 42, the expression of protein markers in the resulting single cell populations was characterized by both flow cytometry and ICC.

[0113] To characterize the expression of oligodendrocyte precursor protein markers by ICC, slide-mounted aggregate sections were stained as described above, except that permeabilization was performed with 100% methanol for 2 min at RT and the blocking solution consisted of 10% fetal bovine serum in PBS.

[0114] Figure 4 shows representative ICC data for oligodendrocyte precursor cells at day 42. Single cell populations from two representative experiments expressed the oligodendrocyte precursor cell marker NG2 (Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O'Keeffe S, Phatnani HP, Guarnieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, Daneman R, Maniatis T, Barres BA, Wu JQ. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014 Sep 3;34(36):11929-47).

[0115] To quantify cell surface markers by flow cytometry on day 42, cells were thawed in Thaw Medium (10% fetal bovine serum in DMEM medium), centrifuged, and resuspended in Stain Buffer (2% fetal bovine serum / 0.05% sodium azide in PBS). Cells were incubated with primary antibodies specific for markers of interest, such as NG2 (Invitrogen #37-2300), GD3 (Millipore #MAB2053), A2B5 (BD #563775), CD49f (Millipore #CBL458P), EpCAM (Dako #M080401-2), and CLDN6 (Thermo Fisher #MA5-24076), as well as their isotype controls, for 30 minutes on ice. Cells were washed with Stain Buffer to remove unbound antibodies. In the case of unconjugated antibodies, cells were then incubated with the appropriate fluorophore-conjugated secondary antibody for 30 minutes on ice. After washing the cells, propidium iodide was added to confirm dead cells. In some cases, to recover protein markers that showed sensitivity to the day 42 harvest procedure described in Example 4, cells were cultured overnight at 37°C / 5% CO2 in tissue culture vessels coated with Matrigel (Corning #356231) and then harvested using TrypLE™ Select (Thermo Fisher #Al2859-01) and stained for flow cytometry analysis as described above. All cells were analyzed on an Attune NxT (Thermo Fisher, Waltham, MA, USA) flow cytometer. To calculate the percentage of cells expressing a given protein marker, dead cells stained with propidium iodide were gated and the number of live cells bound to the corresponding antibody was expressed as a percentage of the total number of cells analyzed after correcting for the number of cells that showed non-specific binding to the isotype control antibody.

[0116] Table 1 shows representative flow cytometry data for day 42 oligodendrocyte precursor cells generated according to the methodology described in Example 4.As shown for two representative experimental runs, a high percentage of cells in the resulting cell population were NG2 (Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O'Keeffe S, Phatnani HP, Guarnieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, Daneman R, Maniatis T, Barres BA, Wu JQ. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014 Sep 3;34(36):11929-47), and GD3 (Gallo V, Zhou JM, McBain CJ, Wright P, Knutson PL, Armstrong RC. Oligodendrocyte progenitor cell proliferation and lineage progression are regulated by glutamate receptor-mediated K+ channel block. J Neurosci. 2014 Sep 3;34(36):11929-47). Neurosci. 1996 Apr 15;16(8):2659-70), as well as the pre-OPC marker A2B5 (Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, Sharp K, Steward O. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci. 2005 May 11;25(19):4694-705).Additionally, the neural progenitor / epithelial marker CD49f (Krebsbach PH, Villa-Diaz LG. The Role of Integrin a6 (CD49f) in Stem Cells: More than a Conserved Biomarker. Stem Cells Dev. 2017 Aug 1;26(15):1090-1099) and the epithelial marker CLDN6 (Lin D, Guo Y, Li Y, Ruan Y, Zhang M, Jin X, Yang M, Lu Y, Song P, Zhao S, Dong B, Xie Y, Dang Q, Quan C. Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions. Oncol Rep. 2017 Aug;38(2):875-885) and EpCAM (Huang L, Yang Y, Yang F, Liu S, Zhu Z, Lei Z, Guo J. Functions of EpCAM in physiological processes and diseases (Review). Int Non-OPC markers were minimally detected in the resulting population, including genomic DNA markers (JMol Med. 2018 Oct;42(4):1771-1785).

[0117] Table 1. Representative flow cytometry data for oligodendrocyte precursor cells generated by the method according to the present disclosure. TIFF2024178446000001.tif28150

[0118] Cell populations generated by the methodology described herein had a higher percentage of cells positive for the oligodendrocyte progenitor marker NG2 and lower expression of non-OPC markers CD49f, CLDN6, and EpCAM compared to OPCs generated using another method currently in clinical trials for the treatment of spinal cord injury (Priest CA, Manley NC, Denham J, Wirth ED 3rd, Lebkowski JS. Preclinical safety of human embryonic stem cell-derived oligodendrocyte progenitors supporting clinical trials in spinal cord injury. Regen Med. 2015 Nov;10(8):939-58; Manley NC, Priest CA, Denham J, Wirth ED 3rd, Lebkowski JS. Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cells: Preclinical Efficacy and Safety in Cervical Spinal Cord Injury. Stem Cells Transl Med. 2017 Oct;6(10):1917-1929).

[0119] Example 6 –Characterization of differentiated cell populations by gene expression profiling Gene expression profiling can be used to characterize the cell phenotype of each differentiation stage, including the generation of pluripotent cell starting populations as well as neuroectodermal, glial, and oligodendrocyte progenitors. Gene expression profiling includes both global transcriptome profiling using methods such as microarrays and RNA-seq, and targeted gene profiling using sensitive methods such as quantitative real-time PCR (qPCR).

[0120] To perform gene expression profiling, cells were lysed with Qiagen RLT Lysis Buffer (Qiagen #79216) and RNA was purified using the Qiagen RNeasy Mini Kit (Qiagen #74106) according to the manufacturer's guidelines. For qPCR-based analysis, purified RNA was then converted to cDNA according to standard methods using Invitrogen Superscript IV VILO Mastermix (Thermo Fisher Scientific #11756050) according to the manufacturer's guidelines. The relative expression levels of target genes and reference housekeeping genes were then quantified using gene-specific primer-probe sets (Applied Biosystems Taqman Gene Expression Assays, Thermo Fisher Scientific #4331182) according to the manufacturer's guidelines. To determine the relative expression levels of a given set of target genes, PCR reactions were performed using an ABI 7900HT Real-Time Sequence Detection System (Applied Biosystems), BioMark HD System (Fluidigm), or equivalent system. Each target gene was normalized to one or more reference genes, such as GAPDH, to determine its relative expression level.

[0121] Figure 5 shows representative qPCR analysis of uhESCs collected before the formation of 3D cell aggregates (day -1) and 24 hours later after the formation of small non-embryoid body (non-EB) aggregates (day 0, just prior to the onset of neuroectodermal differentiation). Two representative experiments were performed and RNA samples were harvested and processed for gene expression profiling by qPCR using the methods described above. Fluidigm qPCR was performed using a 76-gene panel of known markers for pluripotency and early differentiation. For each gene, normalized ΔCT values ​​were calculated relative to the average value of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1). The resulting correlation plots of all ΔCT values ​​at day -1 and day 0 are shown in Figure 5 and indicate that cells in the small non-EB aggregates at day 0 retained expression of pluripotency genes and had a similar overall expression profile of tested markers as day -1 uhESCs. This is further supported by the fold change calculations relative to baseline shown in Table 2 , which show that in both representative experiments, pluripotency markers NANOG, LIN28A, and SOX2 have high levels 24 hours after uhESC clump formation (day 0 uhESC).

[0122] Table 2 shows qPCR results from two representative experiments measuring expression of pluripotency genes, neuroectodermal progenitor genes, glial progenitor genes, and oligodendrocyte progenitor genes in cell populations generated by the method according to the present disclosure. RNA samples were taken at the following time points: 24 hours after the initiation of uhESC cell aggregate formation, before differentiation (day 0); after differentiation into neuroectodermal progenitor cells (day 7); after differentiation into glial progenitor cells (day 21); and after differentiation into oligodendrocyte progenitor cells (day 42). RNA samples were processed for qPCR using the method described above. A selected panel of genes indicative of each differentiation state was quantified, including three pluripotency genes (NANOG, LIN28A, SOX2), three neuroectodermal progenitor genes (PAX6, HES5, ZBTB16), three glial progenitor genes (CACGN4, FABP7, SOX6), and three oligodendrocyte progenitor genes (CSPG4, PDGFRα, DCN). For each gene, normalized ΔCT values ​​were calculated using the average of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1), and fold expression relative to baseline (expression below the lower limit of quantification) was calculated using the ΔΔCT method.

[0123] Table 2. qPCR analysis of pluripotency, neuroectodermal progenitor cell (NEPC), glial progenitor cell (GPC), and oligodendrocyte progenitor cell (OPC) gene markers in H1 uhESCs differentiated into OPCs according to the present disclosure. TIFF2024178446000002.tif94158

[0124] With reference to Table 2, differentiation of uhESCs for 7 days according to the methods disclosed herein resulted in a gene expression profile consistent with neuroectodermal progenitors, including downregulation of NANOG and expression of LIN28A, SOX2, PAX6, HES5, and ZBTB16 (Patterson M, Chan DN, Ha I, Case D, Cui Y, Van Handel B, Mikkola HK, Lowly WE. Defining the nature of human pluripotent stem cell progeny. Cell Res. 2012 Jan;22(1):178-93; Lippmann ES, Williams CE, Ruhl DA, Estevez-Silva MC, Chapman ER, Coon JJ, Ashton RS. Deterministic HOX patterning in human pluripotent stem cell-derived neuroectoderm. Stem Cell Reports. 2015 Apr 14;4(4):632-44; Woo SM, Kim J, Han HW, Chae JI, Son MY, Cho S, Chung HM, Han YM, Kang YK. Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells. BMC Neurosci. 2009 Aug 17;10:97; Avantaggiato V, Pandolfi PP, Ruthardt M, Hawe N, Acampora D, Pelicci PG, Simeone A. Developmental analysis of murine Promyelocyte Leukemia Zinc Finger (PLZF) gene expression: implications for the neuromeric model of the forebrain organization. J Neurosci.1995 Jul;15(7 Pt 1):4927-42). .

[0125] The cell population obtained after 21 days of suspension-based differentiation displayed a gene expression profile consistent with glial progenitors, including downregulation of pluripotent and neuroectodermal progenitor markers and induction of CACNG4, FABP7, and SOX6 (Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O'Keeffe S, Phatnani HP, Guamieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, Daneman R, Maniatis T, Barres BA, Wu JQ. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014 Sep 3;34(36):11929-47; Petit A, Sanders AD, Kennedy TE, Tetzlaff W, Glattfelder KJ, Dalley RA, Puchalski RB, Jones AR, Roskams AJ. Adult spinal cord radial glia display a unique progenitor phenotype. PLoS One. 2011;6(9):e24538; Baroti T, Zimmermann Y, Schillinger A, Liu L, Lommes P, Wegner M, Stolt CC. Transcription factors Sox5 and Sox6 exert direct and indirect influences on oligodendroglial migration in spinal cord and forebrain. Glia. 2016 Jan;64(1):122-38).

[0126] The cell population obtained after 42 days of differentiation according to the methods described herein expressed markers consistent with oligodendrocyte precursor cells, including downregulation of early lineage markers and induction of CSPG4(NG2), PDGFRα, and DCN (Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O'Keeffe S, Phatnani HP, Guamieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, Daneman R, Maniatis T, Barres BA, Wu JQ. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014 Sep 3;34(36):11929-47).

[0127] Example 7 -Differentiation of human embryonic stem cells into neuroectodermal progenitor cells using alternative small molecule inhibitors of TGFBR1 / Activin / Nodal and BMP signaling In addition to the small molecule inhibitors used in Example 2 (SB431542 and Dorsomorphin), alternative small molecule inhibitors of TGFβR1 / Activin / Nodal signaling and BMP signaling were tested for their ability to differentiate human embryonic stem cells into neuroectodermal progenitor cells in suspension. Table 3 lists the alternative small molecule inhibitors that were tested. Each condition was tested in duplicate wells of an Ultra Low Attachment 6-well tissue culture plate (Corning #3471).

[0128] Table 3. Small molecule inhibitors used to induce differentiation of human embryonic stem cells into neuroectodermal progenitor cells in suspension. TIFF2024178446000003.tif128150

[0129] On day 7 of differentiation, cells were harvested and processed for RNA extraction and gene expression profiling by qPCR as described in Example 6. For each gene, normalized ΔCT values ​​were calculated relative to the average of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1) and fold expression relative to baseline (expression below the lower limit of quantification) was calculated using the ΔΔCT method. Table 4 shows the average fold expression values ​​of biological duplicates of each small molecule combination (relative to baseline). With reference to Table 4, differentiation of uhESCs in suspension for 7 days with each of the small molecule combinations tested resulted in downregulation of the pluripotency marker NANOG and comparable maintenance or induction of expression of genes associated with neuroectodermal progenitor phenotype, such as LIN28A, SOX2, PAX6, HES5, and ZBTB16.

[0130] To obtain a more comprehensive comparison of the resulting day 7 cellular phenotypes after treatment with each small molecule combination, Fluidigm qPCR was performed using a 96-gene panel composed of known markers of pluripotency, neuroectodermal progenitors, neural tube patterning, glial progenitors, oligodendrocyte progenitors, neural crest cells, neurons, astrocytes, pericytes, Schwann cells, and epithelial cells. With reference to Figure 6, comparison of the day 7 cellular phenotypes of each alternative small molecule combination to those produced by SB431542 + Dorsomorphin treatment by regression plot of normalized ΔCT values ​​demonstrated that similar overall cellular phenotypes could be achieved by each of the small molecule combinations tested. Taken together, the results shown in Table 4 and FIG. 6 support that various combinations of (i) TGFβR1 / Activin / Nodal signaling inhibitors and (ii) BMP signaling inhibitors can be used to differentiate uhESCs in suspension into neuroectodermal progenitor cells, and further into glial and oligodendrocyte progenitor cells, using the methods of the present disclosure.

[0131] Table 4. qPCR analysis of pluripotent and neuroectodermal progenitor cell (NEPC) gene markers in H1 uhESCs differentiated into NEPCs using various combinations of small molecule inhibitors. TIFF2024178446000004.tif45150

[0132] Example 8 –Assessment of the presence of heterogeneous epithelial cells in differentiated OPC populations using an in vitro cyst assay The presence of unwanted epithelial cells in the OPC population generated according to the present disclosure was tested using an in vitro cyst assay. The cyst assay was performed essentially according to the protocol of Debnath et al. (Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. 2003 Methods. 3:256-68). Briefly, OPCs were grown in a 3D culture system for 20 days in the presence of factors known to stimulate the formation of epithelial cysts. In addition to visual detection of cysts, the presence of cystic structures, including basolateral protein expression of the epithelial marker CD49f, was also evaluated using immunocytochemistry.

[0133] OPCs were cultured at 21.9 × 10 on Matrigel® (Corning) pads using cyst support medium. 3 Cells were seeded at a density of 0.5 × 10 6Cells were seeded in 12 wells of a 24-well plate). Cells were cultured for 20 days. On day 20, viable cysts were counted, Matrigel® was dissolved using Cell Recovery Solution (Corning #354253), and cells were fixed with 4% paraformaldehyde (PFA) for 5 min on ice and permeabilized overnight with blocking buffer. Cysts were then stained for CD49f (ITGA6), phalloidin, and counterstained with DAPI. Cysts were imaged using an IN Cell Analyzer 2000 (GE Healthcare Life Sciences), and cyst frequency, size, and staining intensity were quantified using IN Cell Developer Software (GE Healthcare Life Sciences) and MATLAB™ (Mathworks).

[0134] Referring to Table 5, OPCs generated from two representative experimental runs using methods according to the present disclosure and tested in an in vitro cyst assay formed fewer cysts compared to three OPC control lots (Control A, Control B, and Control C) generated by an alternative method previously found to cause epithelial cyst formation in vivo (Manley NC, Priest CA, Denham J, Wirth ED 3rd, Lebkowski JS. Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cells: Preclinical Efficacy and Safety in Cervical Spinal Cord Injury. Stem Cells Transl Med. 2017 Oct;6(10):1917-1929). Based on these results, it is expected that OPCs generated according to the present disclosure will form few or no epithelial cysts in vivo.

[0135] Table 5. Representative cyst assay results of oligodendrocyte precursor cells generated by the method according to the present disclosure. TIFF2024178446000005.tif24128

[0136] Although the disclosure has been described with reference to specific embodiments, those skilled in the art will recognize that various modifications can be made and equivalents can be substituted for elements thereof without departing from the scope of the disclosure. In addition, many modifications can be made to adapt a particular situation or material to the teachings of the disclosure without departing from its scope.

[0137] Therefore, it is intended that this disclosure not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this disclosure, but that the disclosure will include all aspects falling within the scope and spirit of the appended claims.

Claims

1. (a) obtaining a suspension culture of non-embryoid body (non-EB) aggregates of undifferentiated human pluripotent stem cells, wherein the human pluripotent stem cells remain in an undifferentiated state; (b) culturing the non-EB aggregates from (a) in dynamic suspension in the presence of at least one inhibitor of transforming growth factor beta (TGFβ) / activin / nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling for a first period of time, thereby inducing differentiation into neuroectoderm; (c) culturing the non-EB aggregates from (b) in dynamic suspension in the presence of retinoic acid and at least one agonist of the smoothened receptor for a second period of time; and (d) culturing the aggregates from (c) in dynamic suspension in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for an additional period of time until the cells mature into glial progenitor cells.

1. A method for obtaining a population of cells comprising glial progenitor cells from undifferentiated human pluripotent stem cells, comprising:

2. 2. The method of claim 1, further comprising the additional step of collecting the non-EB clumps from (d) and plating them on a substrate, thereby displacing cells from the clumps.

3. The method of claim 2, wherein the substrate is recombinant human laminin 521.

4. The method of claim 1, wherein the human pluripotent stem cells are human embryonic stem cells (hESCs).

5. The method of claim 1, wherein the human pluripotent stem cells are human induced pluripotent stem cells (hiPSCs).

6. The method of claim 1, wherein the at least one inhibitor of TGFβ / activin / nodal signaling is an inhibitor of activin receptor-like kinase 5 (ALK5).

7. 2. The method of claim 1, wherein the at least one inhibitor of TGFβ / activin / nodal signaling is selected from the group consisting of SB431542, LY2157299, GW788388, A-77-01, A-83-01, and SB505124.

8. The method of claim 1, wherein the at least one inhibitor of TGFβ / activin / nodal signaling is SB431542.

9. The method of claim 1, wherein the at least one inhibitor of BMP signaling is an inhibitor of activin receptor-like kinase 2 (ALK2).

10. The method of claim 1, wherein the at least one inhibitor of BMP signaling is selected from the group consisting of dorsomorphin, DMH-1, K02288, ML3467, LDN193189, and Noggin protein.

11. The method of claim 1, wherein the at least one inhibitor of BMP signaling is dorsomorphin.

12. 2. The method of claim 1, wherein the at least one smoothened receptor agonist is selected from the group consisting of purmorphamine, smoothened agonist (SAG, CAS 364590-63-6), and Sonic Hedgehog (SHH) protein.

13. 2. The method of claim 1, wherein at least one smoothened receptor agonist is purmorphamine.

14. 2. The method of claim 1, wherein the first period of time is about 3 to 4 days.

15. 10. The method of claim 1, wherein the second period of time is about 3 days.

16. 2. The method of claim 1, wherein steps (a)-(d) are carried out for about 21 days.

17. A differentiated cell population comprising glial progenitor cells obtained according to the method of claim 1.

18. 18. The differentiated cell population of claim 17, wherein the glial progenitor cells express one or more markers selected from calcium voltage-dependent channel auxiliary subunit gamma 4 (CACNG4), fatty acid binding protein 7 (FABP7), and sex determining region Y box 6 (SOX6).

19. (a) obtaining glial precursor cells according to the method of claim 1; (b) collecting the cells from (a) and plating them onto a substrate, thereby displacing the cells from the clumps; and (c) culturing the cells from (b) in the presence of epidermal growth factor (EGF) and platelet-derived growth factor AA (PDGF-AA) for an additional period of time as adherent cells until the cells mature into oligodendrocyte precursor cells (OPCs).

1. A method for obtaining a population of cells comprising OPCs from undifferentiated human pluripotent stem cells, comprising: The method, wherein the OPCs express one or more markers selected from neural / glial antigen 2 (NG2), platelet-derived growth factor receptor A (PDGFRα), and ganglioside GD3 (GD3).

20. 20. The method of claim 19, wherein the adherent culture is carried out for about 21 days.

21. 20. The method of claim 19, wherein the adherent culture is performed on a substrate, the substrate being selected from (i) a cell adhesion peptide, and (ii) an extracellular matrix selected from laminin and vitronectin.

22. The method of claim 19, wherein the adherent culture is performed on recombinant human laminin 521.

23. The method of claim 19, wherein the adherent culture is performed on a laminin 511 E8 fragment.

24. 20. The method of claim 19, wherein the human pluripotent stem cells are hESCs.

25. The method of claim 19, wherein the human pluripotent stem cells are hiPSCs.

26. 20. A differentiated cell population comprising OPCs obtained according to the method of claim 19.

27. 27. The differentiated cell population of claim 26, wherein at least 60% of the cells are NG2 positive.

28. 27. The differentiated cell population of claim 26, wherein at least 70% of the cells are NG2 positive.

29. 27. The differentiated cell population of claim 26, wherein at least 80% of the cells are NG2 positive.

30. 27. The differentiated cell population of claim 26, wherein at least 90% of the cells are NG2 positive.

31. 27. The differentiated cell population of claim 26, wherein at least 60% of the cells are PDGFRα positive.

32. 27. The differentiated cell population of claim 26, wherein at least 70% of the cells are PDGFRα positive.

33. 27. The differentiated cell population of claim 26, wherein at least 80% of the cells are PDGFRα positive.

34. 27. The differentiated cell population of claim 26, wherein at least 90% of the cells are PDGFRα positive.

35. 27. The differentiated cell population of claim 26, wherein at least 60% of the cells are GD3 positive.

36. 27. The differentiated cell population of claim 26, wherein at least 70% of the cells are GD3 positive.

37. 27. The differentiated cell population of claim 26, wherein at least 80% of the cells are GD3 positive.

38. 27. The differentiated cell population of claim 26, wherein at least 90% of the cells are GD3 positive.

39. (a) obtaining a suspension culture of non-embryoid body (non-EB) aggregates of undifferentiated human pluripotent stem cells, wherein the human pluripotent stem cells remain in an undifferentiated state; (b) culturing the non-EB aggregates from (a) in dynamic suspension in the presence of at least one inhibitor of transforming growth factor beta (TGFβ) / activin / nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling for a first period of time, thereby inducing differentiation into neuroectoderm; and (c) culturing the non-EB aggregates from (b) in dynamic suspension in the presence of retinoic acid and at least one agonist of the smoothened receptor for a second period of time until the cells mature into paired box 6 (PAX6)-positive neuroectodermal cells.

1. A method for inducing differentiation of a human pluripotent stem cell into a neuroectodermal cell, comprising:

40. 40. The method of claim 39, wherein the human pluripotent stem cells are human embryonic stem cells (hESCs).

41. 40. The method of claim 39, wherein the human pluripotent stem cells are human induced pluripotent stem cells (hiPSCs).

42. 40. The method of claim 39, wherein the at least one inhibitor of TGFβ / activin / nodal signaling is an inhibitor of activin receptor-like kinase 5 (ALK5).

43. 40. The method of claim 39, wherein the at least one inhibitor of TGFβ / activin / nodal signaling is selected from the group consisting of SB431542, LY2157299, GW788388, A-77-01, A-83-01, and SB505124.

44. 40. The method of claim 39, wherein the at least one inhibitor of TGFβ / activin / nodal signaling is SB431542.

45. 40. The method of claim 39, wherein the at least one inhibitor of BMP signaling is an inhibitor of activin receptor-like kinase 2 (ALK2).

46. 40. The method of claim 39, wherein the at least one inhibitor of BMP signaling is selected from the group consisting of dorsomorphin, DMH-1, K02288, ML3467, LDN193189, and Noggin protein.

47. 40. The method of claim 39, wherein the at least one inhibitor of BMP signaling is dorsomorphin.

48. 40. The method of claim 39, wherein the at least one smoothened receptor agonist is selected from the group consisting of purmorphamine, smoothened agonist (SAG, CAS 364590-63-6), and Sonic Hedgehog (SHH) protein.

49. 40. The method of claim 39, wherein at least one smoothened receptor agonist is purmorphamine.

50. 40. The method of claim 39, wherein the first period of time is about 3 to 4 days.

51. 40. The method of claim 39, wherein the second period of time is about 3 days.

52. 40. The method of claim 39, wherein steps (a)-(c) are carried out for about 7-8 days.

53. A differentiated cell population comprising PAX6-positive neuroectodermal cells obtained according to the method of claim 39.

54. 54. The differentiated cell population of claim 53, wherein the PAX6-positive neuroectodermal cells further express one or more markers selected from Hes family BHLH transcription factor 5 (HES5) and ZBTB16 (zinc finger and BTB domain containing 16).

55. (a) culturing undifferentiated human pluripotent stem cells that have been disaggregated and formed a single cell suspension in dynamic suspension to obtain non-embryoid body (non-EB) aggregates, wherein the human pluripotent stem cells in the non-EB aggregates remain in an undifferentiated state; (b) culturing the non-EB aggregates from (a) in dynamic suspension in the presence of at least one inhibitor of transforming growth factor beta (TGFβ) / activin / nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling for a first period of time, thereby inducing differentiation into neuroectoderm; (c) culturing the non-EB aggregates from (b) in dynamic suspension in the presence of retinoic acid and at least one agonist of the smoothened receptor for a second period of time; and (d) culturing the aggregates from (c) in dynamic suspension in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for an additional period of time until the cells mature into glial progenitor cells.

1. A method for obtaining a population of cells comprising glial progenitor cells from undifferentiated human pluripotent stem cells, comprising:

Images