Method for differentiating pluripotent stem cells in dynamic suspension culture

Abstract

Provided are methods for differentiating pluripotent stem cells into neuroectoderm in a dynamic suspension culture using small molecule or protein inhibitors of TGF [beta] / activin / Nodal signaling and BMP signaling. Also provided are methods and regimens for first differentiating pluripotent stem cells, such as human embryonic stem cells, into neuroectoderm, then further differentiating into glial progenitor cells, and further differentiating into oligodendrocyte progenitor cells (OPCs), and compositions obtained thereby. The methods of the present disclosure reproducibly produce neuroectoderm progenitor cells on the 7th day of the differentiation process, glial progenitor cells on the 21st day of the differentiation process, and OPC on the 42nd day of the differentiation process.

Description

[0001] This application is a divisional application. The application number of its parent application is 201980070640.0, the application date is September 19, 2019, and the invention title is "Method for differentiating pluripotent stem cells in dynamic suspension culture". Technical Field

[0002] The present disclosure relates to the fields of cell biology and cells of the neuroectodermal and glial lineages (such as oligodendrocyte progenitor cells). More specifically, the present disclosure relates to a new method for differentiating pluripotent stem cells into the 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 new method for differentiating pluripotent stem cells (such as human embryonic stem cells) first into the neuroectoderm, then further into glial progenitor cells, and further into oligodendrocyte progenitor cells. The present disclosure further relates to neuroectodermal cells, glial progenitor cells, and oligodendrocyte progenitor cells expressing one or more markers produced by the method according to the invention. Background Art

[0003] Oligodendrocyte progenitor cells (OPCs) are a subset of glial cells in the central nervous system (CNS) that mature into myelin-producing oligodendrocytes. Oligodendrocytes produce myelin, which insulates neuronal axons and remyelinated CNS lesions upon myelin loss. Oligodendrocytes also promote neuroprotection through other mechanisms, including 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; 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; Du Y, Dreyfus CF. Oligodendrocytes as providers of growth factors. 2002 J Neurosci Res. 68(6):647-54). Thus, oligodendrocytes are important therapeutic targets for demyelinating and hypomyelinating disorders (such as multiple sclerosis, adrenoleukodystrophy, and adrenomyeloneuropathy), other neurodegenerative disorders (such as Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease), and acute spinal cord injury (SCI).

[0004] OPCs are derived from the neuroectoderm (also known as the neural ectoderm or neural tube epithelium), which gives rise to neural progenitor cells that will generate the various neurons and glial cells that make up the CNS. Several methods have been developed for differentiating 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. For the first step of inducing neuroectodermal lineage cells and neural progenitor cells, most existing protocols rely on embryoid bodies (EBs) and subsequent formation of neurospheres in static non-adherent cultures 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; 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; Hu BY, Du ZW, Zhang SC. Differentiation of human oligodendrocytes from pluripotent stem cells. 2009 Nat. Protoc. 4:1614-1622). 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 precursors to optimize yields and are not easily scalable, thus limiting their use in generating large numbers of therapeutically relevant cells. In addition, small numbers of cell types outside the neuroectodermal lineage can persist during differentiation and contribute to undesired cell types in the final OPC population, including, for example, epithelial or chondrocyte progenitors (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).

[0005] Recently, methods have been developed to improve the efficiency of neural induction (neuroectoderm formation) by simultaneously using inhibitors of transforming growth factor β (TGFβ) / activin / Nodal signaling and 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 on adherent monolayer cultures, eliminating the highly heterogeneous cell populations generated by EB-based neural induction methods and minimizing non-neural cells. 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, compared to three-dimensional (3D) culture methods, a disadvantage of adherent monolayer culture is its limited scalability and yield. In addition, the 3D culture environment mimics the natural cellular microenvironment; it is believed that cells grown using 3D cell culture techniques are more closely similar to natural tissues and organs compared to cells grown in (2D) adherent monolayers. Finally, adherent monolayer culture relies on the use of undefined components and components of animal origin, such as and knockout serum replacement (KSR).

[0006] To address these limitations, methods combining dual SMAD inhibition with EB formation have recently been 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; 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; 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). In EB-based methods, small molecule inhibitors of TGFβ / Activin / Nodal signaling and BMP signaling are applied to EBs formed in static non-adherent culture.However, EB-based dual SMAD inhibition is not easily scalable for the generation of large numbers of target cells and results in a greater degree of variability in the cells obtained, in part because static culture of EBs leads to the formation of aggregates of different sizes and requires frequent agitation 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).

[0007] Improved methods are needed for differentiating pluripotent stem cells into neuroectoderm and further into glial progenitor cells and OPCs. Ideally, such methods should be easily scalable to generate sufficient numbers of differentiated cells for cell therapy applications while consistently and reproducibly generating the target cell type. SUMMARY OF THE INVENTION

[0008] In various embodiments described herein, the present disclosure particularly provides robust and reliable protocols for differentiating human pluripotent stem cells (e.g., ESCs and iPSCs) into neuroectoderm and glial cells in dynamic suspension culture, which can be carried out in a bioreactor and are suitable for 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 and subsequently differentiating the glial cells into OPCs.

[0009] The present disclosure is in part based on the discovery that starting material pluripotent stem cells can aggregate into non-EB aggregates in dynamic suspension, where the pluripotent stem cells remain undifferentiated, and subsequently, 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. Differentiating pluripotent stem cells in dynamic suspension according to the present disclosure provides a scalable, reproducible, and controllable method for generating large numbers of target neuroectoderm lineage cells from the starting material as compared to adherent culture and EB-based methods.

[0010] The methods of the present disclosure reproducibly generate neuroectodermal progenitor cells on day 7 of the differentiation process, glial progenitor cells on day 21 of the differentiation process, and OPCs on day 42 of the differentiation process. The day 42 OPCs generated according to the present disclosure are comparable (in terms of their overall marker expression profile) to OPCs generated using alternative methods that are currently in clinical testing 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), except that the OPCs generated according to the present disclosure express lower levels of non-OPC markers, including markers associated with in vitro epithelial cyst formation.

[0011] In one embodiment, the present disclosure provides a method for obtaining a cell population comprising glial progenitor cells from undifferentiated human pluripotent stem cells. In certain embodiments, 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 a dynamic suspension for a first period of time in the presence of at least one inhibitor of transforming growth factor β (TGFβ) / activin / Nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling, thereby inducing differentiation into neuroectoderm; (c) culturing the non-EB aggregates from (b) in a dynamic suspension for a second period of time in the presence of retinoic acid and at least one Smoothened receptor agonist; and (d) culturing the aggregates from (c) in a dynamic suspension for an additional period of time in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) until the cells mature into glial progenitor cells.

[0012] In certain embodiments, the first period of time is about three to four days. In certain embodiments, the second period of time is about four days. In certain embodiments, steps (a) and (b) are carried out over a period of about seven to eight days. In certain embodiments, steps (a) to (d) are carried out over a period of about twenty-one days.

[0013] In certain embodiments, the method further comprises the additional step of harvesting the non-EB aggregates from (d) and plating them on a substrate, thereby causing the cells to migrate out of the aggregates. 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 the laminin-511E8 fragment. In other embodiments, the substrate is a synthetic substrate, such as, for example, SC substrate.

[0014] In a further embodiment, the method comprises the additional step of culturing the cells that have migrated out of the aggregates adhered to the substrate for an additional period of time in the presence of epidermal growth factor (EGF) and platelet-derived growth factor AA (PDGF-AA) 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 the laminin-511E8 fragment. In certain embodiments, the adherent culture is carried out for about 21 days.

[0015] In another embodiment, the present disclosure provides a method for inducing the 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 a dynamic suspension for a first period of time in the presence of at least one inhibitor of transforming growth factor β (TGFβ) / activin / Nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling, thereby inducing differentiation into neuroectoderm; and (c) culturing the non-EB aggregates from (b) in a dynamic suspension for a second period of time in the presence of retinoic acid and at least one Smoothened receptor agonist; until the cells mature into paired box 6 (PAX6)-positive neuroectodermal cells.

[0016] In certain embodiments, the first period of time is about three to four days. In certain embodiments, the second period of time is about four days. In certain embodiments, steps (a) to (c) are carried out over a period of about seven to eight days.

[0017] In another embodiment, the present disclosure provides a method for obtaining a cell population comprising glial progenitor cells from undifferentiated human pluripotent stem cells, the method comprising: (a) culturing undifferentiated human pluripotent stem cells that have been dissociated and formed a single cell suspension in a 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 a dynamic suspension for a first period of time in the presence of at least one inhibitor of transforming growth factor β (TGFβ) / activin / Nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling, thereby inducing differentiation into neuroectoderm; (c) culturing the non-EB aggregates from (b) in a dynamic suspension for a second period of time in the presence of retinoic acid and at least one Smoothened receptor agonist; and (d) culturing the aggregates from (c) in a dynamic suspension for an additional period of time in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF); 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 SB431542, LY2157299, GW788388, A-77-01, A-83-01, and SB505124. In 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 Dorsomorphin, DMH-1, K02288, ML3467, LDN193189, and Noggin protein. In other embodiments, the inhibitor of BMP signaling is Dorsomorphin.

[0020] In certain embodiments, at least one Smoothened receptor agonist is selected from Purmorphamine, Smoothened agonist (SAG, CAS 364590-63-6), and Sonic Hedgehog (SHH) protein. In other embodiments, the Smoothened receptor agonist is Purmorphamine.

[0021] Another embodiment is a population of differentiated cells that comprises PAX6-positive neuroectodermal cells obtained by the methods according to 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 a population of differentiated cells that comprises glial progenitor cells obtained by the methods according to the present disclosure. In certain embodiments, the glial progenitor cells express one or more markers selected from calcium voltage-gated channel auxiliary subunit gamma 4 (CACNG4), fatty acid-binding protein 7 (FABP7), and sex-determining region Y-box 6 (SOX6).

[0023] Another embodiment is a population of differentiated cells that contains OPCs obtained according to the methods of the present disclosure. In a preferred embodiment, the OPCs generated according to the methods 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 prepared 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 population of differentiated cells contains at least 60% NG2-positive cells. In certain embodiments, the population of differentiated cells contains at least 70% NG2-positive cells. In certain embodiments, the population of differentiated cells contains at least 80% NG2-positive cells. In other embodiments, the population of differentiated cells contains at least 90% NG2-positive cells. In certain embodiments, the population of differentiated cells contains at least 98% NG2-positive cells. In certain embodiments, the population of differentiated cells contains at least 60% PDGFRα-positive cells. In certain embodiments, the population of differentiated cells contains at least 70% PDGFRα-positive cells. In certain embodiments, the population of differentiated cells contains at least 80% PDGFRα-positive cells. In other embodiments, the population of differentiated cells contains at least 90% PDGFRα-positive cells. In certain embodiments, the population of differentiated cells contains at least 98% PDGFRα-positive cells. In certain embodiments, the population of differentiated cells contains at least 60% GD3-positive cells. In certain embodiments, the population of differentiated cells contains at least 70% GD3-positive cells. In certain embodiments, the population of differentiated cells contains at least 80% GD3-positive cells. In other embodiments, the population of differentiated cells contains at least 90% GD3-positive cells. In certain embodiments, the population of differentiated cells contains at least 98% GD3-positive cells. Brief Description of the Drawings

[0025] Figure 1 is a graph depicting the differentiation of human embryonic stem cells according to the present disclosure into neuroectoderm (day 7) and further into glial progenitor cells (day 21) and oligodendrocyte progenitor cells (day 42). DS = dynamic suspension. Several other small molecule inhibitors of TGFβ / activin / Nodal signaling (except SB431542) and BMP signaling (except Dorsomorphin) were tested and found to be equally effective in inducing differentiation into neuroectoderm (Example 7).

[0026] Figure 2Shows representative micrographs of undifferentiated human embryonic stem cells (uhESCs) by immunocytochemical staining for pluripotency markers according to the present disclosure. The top and bottom rows of micrographs each show individual imaging fields of uhESCs stained for DAPI, Nanog, and Oct4 (top row) or DAPI, Nanog, and Sox2 (bottom row) and imaged on an IN Cell Analyzer 2000. The scale bar in the lower right-hand figure applies to all images in the figure.

[0027] Figure 3 Shows representative micrographs of neuroectodermal progenitor cells generated as non-embryoid body (non-EB) aggregates in suspension and immunocytochemically stained according to the present disclosure. The top and bottom rows of micrographs show aggregates of neuroectodermal progenitor cells generated from two representative experiments and immunocytochemically stained for DAPI (left panel), PAX6 (middle panel), and PSA-NCAM (right panel). The stained cell aggregates were imaged on an IN Cell Analyzer 2000. The scale bar in the lower right-hand figure applies to all images in the figure.

[0028] Figure 4 Shows representative micrographs of oligodendrocyte progenitor cells generated and immunocytochemically stained according to the present disclosure. The top and bottom rows of micrographs show oligodendrocyte progenitor cells generated from two representative experiments and immunocytochemically stained for DAPI (left panel) and NG2 (right panel). The stained cells were imaged on an IN Cell Analyzer 2000. The scale bar in the lower right-hand figure applies to all images in the figure.

[0029] Figure 5 Shows correlation plots of gene expression profiles of uhESCs before and 24 hours after cell aggregate formation in suspension. Each correlation plot shows a comparison of gene expression profiles from two separate experiments on day -1 (before aggregate formation) relative to day 0 (24 hours after aggregate formation). For each plot, the data points represent each of 76 genes evaluated by Fluidigm qPCR and calculated as normalized ΔCT as described in Example 6. The R-squared value is shown in the upper left corner of each plot and was calculated using JMP software (SAS, Cary, NC, USA) based on the best-fit line.

[0030] Figure 6 shows a correlation plot of the gene expression profiles of uhESCs differentiated into neuroectodermal progenitor cells in suspension using different small molecule combinations on day 7. Each correlation plot shows a comparison of the day 7 gene expression profiles of cells treated with SB431542 plus Dorsomorphin relative to alternative small molecule combinations indicated on the y-axis of each plot. For each plot, the data points represent each of 96 genes evaluated by Fluidigm qPCR and calculated as normalized ΔCT as described in Example 7. The R-squared value is shown in the upper left corner of each plot and was calculated using JMP software (SAS, Cary, NC, USA) based on the best fit line DETAILED DESCRIPTION

[0031] This description is not intended to be an exhaustive catalog of all the different ways in which the present disclosure may be implemented or of all the features that may be added to the present disclosure. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a particular embodiment may be deleted from that embodiment. Accordingly, the present disclosure contemplates that in some embodiments of the present disclosure, any feature or combination of features set forth herein may be excluded or omitted. Additionally, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art without departing from the present disclosure. In other instances, well-known structures, interfaces, and processes have not been shown in detail so as not to unnecessarily obscure the present invention. It is intended that no part of this specification be construed as limiting any part of the full scope of the present invention. Accordingly, the following description is intended to illustrate some particular aspects of the present disclosure rather than to exhaustively specify all of its permutations, combinations, and variations.

[0032] 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 pertains. 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 limit the present disclosure.

[0033] All publications, patent applications, patents, and other references cited herein are incorporated herein by reference in their entirety.

[0034] Unless the context otherwise indicates, it is specifically intended that the various features of the present disclosure described herein may be used in any combination. Additionally, the present disclosure also contemplates that in some embodiments of the present disclosure, any feature or combination of features shown herein may be excluded or omitted.

[0035] The methods disclosed herein may include one or more steps or acts for implementing the described methods. Without departing from the scope of the present invention, the method steps and / or acts may be interchanged with one another. In other words, unless a specific order of steps or acts is required for proper operation of that aspect, the order and / or use of specific steps and / or acts may be modified without departing from the scope of the present invention.

[0036] As used in the description of the present disclosure and the appended claims, the singular forms “a,” “an,” and “the” are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0037] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in the alternative (“or”).

[0038] When referring to measurable values such as percentages, density, volume, etc., the terms “about” and “approximately” as used herein are intended to include variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0039] As used herein, phrases such as “between X and Y” and “between about X and Y” shall be interpreted to include 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”.

[0040] As used herein, “oligodendrocyte progenitor cell” (OPC) refers to a cell found in the central nervous system that has a neuroectodermal / glial lineage, expresses the characteristic marker neuron / glial antigen 2 (NG2), and is capable of differentiating into oligodendrocytes. OPCs prepared according to the methods of the present invention may also express one or more markers selected from NG2, PDGFRα, and GD3.

[0041] The terms “glial lineage cell,” “glial progenitor cell,” and “glial cell” are used interchangeably herein and refer to non-neuronal CNS cells derived from neuroectodermal / neural progenitor cells. Glial progenitor cells can further differentiate to form OPCs / oligodendrocytes or astrocytes. In certain embodiments, the glial progenitor cells of the present disclosure express one or more markers selected from calcium voltage-gated channel auxiliary subunit gamma 4 (CACNG4), fatty acid binding protein 7 (FABP7), and sex-determining region Y-box 6 (SOX6).

[0042] The terms "neuroectoderm", "neuroectodermal cell", "neuroectodermal precursor", "neuroectodermal progenitor cell", "neural progenitor cell", and "neural precursor" are used interchangeably herein and refer to cells that can differentiate along the neural precursor pathway and are capable of forming CNS neurons, oligodendrocytes, astrocytes, and ependymal cells. In certain embodiments, the 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).

[0043] As used herein, the term "embryoid body" (EB) refers to a three-dimensional cell aggregate derived from pluripotent stem cells that has undergone spontaneous differentiation towards 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 towards all three germ layers and the formation of EBs.

[0044] As used herein, the term "non-embryoid body aggregate" refers to a three-dimensional cell aggregate formed from pluripotent stem cells in which the pluripotent stem cells remain undifferentiated. In the present disclosure, non-EB aggregates are formed in a dynamic suspension under cell culture conditions that maintain pluripotency and inhibit spontaneous differentiation (i.e., bFGF and TGFβ are not removed from the culture medium). Subsequently, the undifferentiated cell aggregates are directed towards neuroectodermal progenitor cells by simultaneously removing bFGF and TGFβ and adding neuroectodermal differentiation factors (e.g., an inhibitor of the TGFβ / Activin / Nodal signaling pathway in combination with an inhibitor of the bone morphogenetic protein signaling pathway).

[0045] As used herein, the term "TGFβ / Activin / Nodal signaling inhibitor" refers to a small molecule or protein regulator capable of downregulating signaling along the transforming growth factor β (TGFβ) / Activin / Nodal signaling pathway. In certain embodiments, the TGFβ / Activin / Nodal signaling inhibitor directly targets the type I TGFβ receptor (TGFβR1), also known as activin receptor-like kinase 5 (ALK5). In certain embodiments, the TGFβ / Activin / Nodal signaling inhibitor is selected from SB431542, LY2157299, GW788388, A-77-01, A-83-01, and SB505124.

[0046] As used herein, the term "BMP signaling inhibitor" refers to a small molecule or protein modulator that can downregulate signaling along the bone morphogenetic protein (BMP) signaling pathway. In certain embodiments, the BMP signaling inhibitor directly targets type I activin A receptor (ACVR1), also known as activin receptor-like kinase 2 (ALK2). In certain embodiments, the BMP signaling inhibitor is selected from Dorsomorphin, DMH-1, K02288, ML3467, LDN193189, and Noggin protein.

[0047] 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 Purmorphamine, Smoothened agonist (SAG, CAS 364590-63-6), and Sonic Hedgehog (SHH) protein.

[0048] As used herein, the term "undesired cell type" refers to cells outside the neuroectodermal lineage that can lead to the formation of ectopic tissue upon implantation or to the formation of one or more cysts in a cyst assay, as described herein. In one embodiment, the "undesired cell type" can include epithelial lineage cells, such as cells positive for CD49f (a marker expressed by both neural progenitor cells and epithelial cells), or cells positive for CLDN6 or EpCAM (two markers expressed by both pluripotent cells and epithelial cells).

[0049] As used herein, "implantation" or "transplantation" refers to the administration of a cell population into a target tissue using a suitable delivery technique (e.g., using an injection device).

[0050] As used herein, the term "subject" refers to an animal or a human.

[0051] As used herein, the term "subject in need thereof" refers to an animal or a human having damaged tissue in the central nervous system. In one embodiment, the animal or human experiences a loss of motor function.

[0052] As used herein, the terms "central nervous system" and "CNS" are used interchangeably and refer to a complex of nerve tissue that controls one or more activities of the body, including but not limited to the brain and the spinal cord in vertebrates.

[0053] As used herein, "treatment" or "therapy" of a condition or disease is a method for obtaining a beneficial or desired result (preferably including a clinical result) after a patient has the condition or disease. Beneficial or desired results for a disease include, but are not limited to, one or more of the following: improving the condition associated with the disease, curing the disease, reducing the severity of the disease, delaying the progression of the disease, alleviating one or more symptoms associated with the disease, increasing the quality of life of a patient suffering from the disease, extending survival time, and any combination thereof. Similarly, for the purposes of the present disclosure, beneficial or desired results for a condition include, but are not limited to, one or more of the following: improving the condition, curing the condition, reducing the severity of the condition, delaying the progression of the condition, alleviating one or more symptoms associated with the condition, increasing the quality of life of a patient suffering from the condition, extending survival time, and any combination thereof.

[0054] Propagation and culture of undifferentiated pluripotent stem cells

[0055] Differentiation of pluripotent stem cells according to the present disclosure can be carried out using any suitable pluripotent stem cell as a starting material. In one embodiment, the method can be carried out on a human embryonic stem cell (hESC) line. In another embodiment, induced pluripotent stem cells (iPSCs) can be used to carry out the method. In another embodiment, cells derived from the H1, H7, H9, H13, or H14 cell lines can be used to carry out the method. In another embodiment, the method can be carried out on a primate pluripotent (pPS) cell line. In yet another embodiment, undifferentiated stem cells derived from a parthenogenetic organism, which is an embryo that is stimulated to produce hESCs without fertilization, can be used to carry out the method.

[0056] Methods for the propagation and culture of undifferentiated pluripotent stem cells have been previously described. For tissue and cell culture of pluripotent stem cells, the reader may wish to refer to any of the numerous publications available in the art, such as Teratocarcinomas and Embryonic Stem cells: A Practical Approach (E.J. Robertson, Ed., IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (P.M. Wasserman et al., Eds., Academic Press 1993); Embryonic Stem Cell Differentiation in vitro (M.V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and Uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (P.D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998; and R.I. Freshney, Culture of Animal Cells, Wiley-Liss, New York, 2000).

[0057] 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 generally supported by a nutrient medium containing factors that promote cell proliferation without differentiation (see, e.g., U.S. Patent No. 6,800,480). In one embodiment, conditioned medium containing such factors can be used. Conditioned medium can be obtained by culturing the medium together with cells that secrete such factors. Suitable cells include, but are not limited to, irradiated (~4,000 Rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells (U.S. Patent No. 6,642,048). The medium can be conditioned by plating the feeder layer in serum-free medium (e.g., knockout DMEM supplemented with 20% serum replacement and 4 ng / mL bFGF). The conditioned medium that has been conditioned for 1-2 days can be supplemented with additional bFGF and used to support pPS cell culture for 1-2 days (see, e.g., WO 01 / 51616; Xu et al., (2001) Nat. Biotechnol. 19:971).

[0058] Alternatively, fresh or unconditioned media can be used that have been supplemented with other factors that promote the proliferation of cells in an undifferentiated form (such as fibroblast growth factor or forskolin). Non-limiting examples include basal media supplemented with 40 - 80 ng / mL bFGF and optionally containing SCF (15 ng / mL) or Flt3 ligand (75 ng / mL), such as X-VIVO TM 10 (Lonza, Walkersville, MD) or QBSF TM -60 (Quality Biological Inc., Gaithersburg, Md.) (see, e.g., Xu et al., (2005) Stem Cells 23(3):315). These media formulations have the advantage of supporting cell growth at a rate 2 - 3 times that of other systems (see, e.g., WO 03 / 020920). In one embodiment, undifferentiated pluripotent cells, such as hES cells, can be cultured in a medium containing bFGF and TGFβ. Non-limiting example concentrations of bFGF include about 80 ng / ml. Non-limiting example concentrations of TGFβ include about 0.5 ng / ml. In yet another embodiment, undifferentiated pluripotent stem cells can be maintained in a commercially available complete medium, such as mTeSR TM (Stem Cell Technologies, Vancouver, Canada).

[0059] Undifferentiated pluripotent cells can be cultured on a feeder cell layer, which are typically fibroblasts derived from embryonic or fetal tissue (Thomson et al., (1998) Science 282:1145). Feeder cells can be derived from human or murine sources. Human feeder cells can be isolated from a variety of human tissues or can be derived by differentiating human embryonic stem cells 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), oviductal 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).

[0060] A variety of solid surfaces can be used to culture undifferentiated pluripotent cells. These solid surfaces include, but are not limited to, standard commercially available tissue culture flasks or cell culture plates, such as 6-well, 24-well, 96-well, or 144-well plates. Other solid surfaces include, but are not limited to, microcarriers and dishes. The solid surfaces suitable for growing undifferentiated pluripotent cells can be made of a variety of materials, including, but not limited to, glass or plastic, such as polystyrene, polyvinyl chloride, polycarbonate, polytetrafluoroethylene, polyester film (melinex), thermanox, or combinations thereof. Suitable surfaces may contain one or more polymers, such as one or more acrylates. The solid surface can be in a three-dimensional shape. Non-limiting examples of three-dimensional solid surfaces have been previously described, for example, in U.S. Patent Publication No. 2005 / 0031598.

[0061] Undifferentiated stem cells can also be grown on a growth substrate under feeder-free conditions. The growth substrate can be a matrix (e.g., 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, distributed by Corning Inc., Corning, NY). In other embodiments, the substrate is a synthetic substrate, such as, for example SC substrate.

[0062] Undifferentiated stem cells can be passaged or subcultured using a variety of methods (e.g., using collagenase, or, for example, manual scraping). Undifferentiated stem cells can be subcultured by enzymatic means that produce a single cell suspension (e.g., using (distributed by Sigma Aldrich, MO) or a similar trypsin). Alternatively, undifferentiated stem cells can be subcultured using non-enzymatic means (e.g., 0.5 mM EDTA in PBS, or, for example, using ReLeSR TM (Stem Cell Technologies, Vancouver, Canada)).

[0063] In one embodiment, a plurality of undifferentiated stem cells are seeded or subcultured at a seeding density that allows the cells to reach confluence in about three to about ten days. In one embodiment, the seeding density can be from about 6.0 x 10 3 cells / cm 2 to about 5.0 x 10 5 cells / cm 2 , for example, about 1.0 x 10 4 cells / cm 2 , for example, about 5.0 x 104 cells / cm 2 , such as about 1.0x10 5 cells / cm 2 , or for example about 3.0x 10 5 cells / cm 2 growth surface. In another embodiment, the seeding density can be about 6.0x 10 3 cells / cm 2 to about 1.0x 10 4 cells / cm 2 growth surface, such as about 6.0x 10 3 cells / cm 2 to about 9.0x 10 3 cells / cm 2 , such as about 7.0x 10 3 cells / cm 2 to about 1.0x 10 4 cells / cm 2 , such as about 7.0x 10 3 cells / cm 2 to about 9.0x 10 3 cells / cm 2 , or for example about 7.0x 10 3 cells / cm 2 to about 8.0x 10 3 cells / cm 2 growth surface. In yet another embodiment, the seeding density can be about 1.0x 10 4 cells / cm 2 to about 1.0x 10 5 cells / cm 2 growth surface, such as about 2.0x 10 4 cells / cm 2 to about 9.0x 10 4 cells / cm 2 , such as about 3.0x 10 4 cells / cm 2 to about 8.0x 10 4 cells / cm 2 , such as about 4.0x 10 4 cells / cm 2 to about 7.0x 10 4 cells / cm 2 , or for example about 5.0x 10 4 cells / cm 2 to about 6.0x 10 4 cells / cm2 Growth surface. In one embodiment, the seeding density can be from about 1.0 x 10 5 cells / cm 2 to about 5.0 x 10 5 cells / cm 2 growth surface, such as about 1.0 x 10 5 cells / cm 2 to about 4.5 x 10 5 cells / cm 2 , such as about 1.5 x 10 5 cells / cm 2 to about 4.0 x 10 5 cells / cm 2 , such as about 2.0 x 10 5 cells / cm 2 to about 3.5 x 10 5 cells / cm 2 , or such as about 2.5 x 10 5 cells / cm 2 to about 3.0 x 10 5 cells / cm 2 growth surface.

[0064] According to the methods of the present disclosure, stem cells can be cultured using any of a variety of suitable cell culture and subculture techniques. For example, the culture medium can be completely replaced daily, starting about 2 days after cell subculture. In one embodiment, when the culture reaches about 90% colony coverage, one or more suitable reagents such as (to achieve a single cell suspension for quantification) can be used to dissociate the cells and seed them for subsequent culture. In one embodiment, undifferentiated stem cells can then be subcultured and expanded on a suitable growth substrate (such as recombinant human laminin-521) at a seeding density that allows the cells to reach confluence within a suitable period of time (e.g., about three to ten days). In one embodiment, undifferentiated stem cells can be subcultured using collagenase IV and expanded on recombinant laminin. In another embodiment, undifferentiated stem cells can be subcultured using collagenase IV and expanded on. In one embodiment, undifferentiated stem cells can be subcultured using ReLeSR TM and expanded on recombinant human laminin-521.

[0065] For seeding undifferentiated stem cells, the seeding density can be from about 6.0 x 10 3 cells / cm 2 to about 5.0 x 10 5cells / cm 2 , for example, about 1.0 x 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 growth surface. In another embodiment, the seeding density can be from about 6.0 x 10 3 cells / cm 2 to about 1.0 x 10 4 cells / cm 2 growth surface, for example, about 6.0 x 10 3 cells / cm 2 to about 9.0 x 10 3 cells / cm 2 , for example, about 7.0 x 10 3 cells / cm 2 to about 1.0 x 10 4 cells / cm 2 , for example, about 7.0 x 10 3 cells / cm 2 to about 9.0 x 10 3 cells / cm 2 , or for example, about 7.0 x 10 3 cells / cm 2 to about 8.0 x 10 3 cells / cm 2 growth surface. In yet another embodiment, the seeding density can be from about 1.0 x 10 4 cells / cm 2 to about 1.0 x 10 5 cells / cm 2 growth surface, for example, about 2.0 x 10 4 cells / cm 2 to about 9.0 x 10 4 cells / cm 2 , for example, about 3.0 x 10 4 cells / cm 2 to about 8.0 x 10 4 cells / cm 2 , for example, about 4.0 x 10 4 cells / cm 2 to about 7.0 x 10 4 cells / cm 2, or for example, about 5.0 x 10 4 cells / cm 2 to about 6.0 x 10 4 cells / cm 2 growth surface. In one embodiment, the seeding density can be about 1.0 x 10 5 cells / cm 2 to about 5.0 x 10 5 cells / cm 2 growth surface, such as about 1.0 x 10 5 cells / cm 2 to about 4.5 x 10 5 cells / cm 2 , such as about 1.5 x 10 5 cells / cm 2 to about 4.0 x 10 5 cells / cm 2 , such as about 2.0 x 10 5 cells / cm 2 to about 3.5 x 10 5 cells / cm 2 , or for example, about 2.5 x 10 5 cells / cm 2 to about 3.0 x 10 5 cells / cm 2 growth surface.

[0066] Neural Induction of Undifferentiated Pluripotent Stem Cells

[0067] The present disclosure provides methods 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 the starting material, pluripotent stem cells, can aggregate into non-EB aggregates in a dynamic suspension, where the pluripotent stem cells remain undifferentiated, and subsequently, the aggregates can be induced to differentiate into neuroectoderm in the dynamic suspension by using one or more inhibitors of TGFβ / Activin / Nodal signaling and one or more inhibitors of BMP signaling (dual SMAD inhibition). Compared to adherent culture and EB-based methods, the dynamic suspension provides a scalable, reproducible, and controllable process for generating large numbers of cells from the starting material. The method of dual SMAD inhibition in a dynamic suspension is described in detail herein.

[0068] In one embodiment, a method includes culturing undifferentiated stem cells that have formed small non-EB aggregates but remain undifferentiated in a dynamic suspension in the presence of one or more inhibitors of TGFβ / Activin / Nodal signaling and one or more inhibitors of BMP signaling to initiate neural induction. In certain embodiments, the inhibitor of TGFβ / Activin / Nodal signaling is a small molecule. In other embodiments, the inhibitor of TGFβ / Activin / Nodal signaling is a protein. In some embodiments, the direct target of the inhibitor of TGFβ / Activin / Nodal signaling is ALK5, also known as type I TGFβ receptor (TGFβR1). In certain embodiments, the inhibitor of BMP signaling is a small molecule. In other embodiments, the inhibitor of BMP signaling is a protein. In some embodiments, the direct target of the inhibitor of BMP signaling is ALK2, also known as type I activin A receptor (ACVR1). In certain embodiments, after dual SMAD inhibition, the resulting cells are cultured in a dynamic suspension in the presence of one or more Smoothened receptor agonists and retinoic acid.

[0069] In certain embodiments, the inhibitor of TGFβ / Activin / Nodal signaling can be selected from SB431542, LY2157299, GW788388, A-77-01, A-83-01, and SB505124 and their derivatives. In certain embodiments, the inhibitor of BMP signaling can be selected from Dorsomorphin, DMH-1, K02288, ML3467, LDN193189, and Noggin protein. In certain embodiments, the Smoothened agonist can be selected from Purmorphamine, SAG (CAS 364590-63-6), SSH protein, and their derivatives.

[0070] In one embodiment, a method includes obtaining non-EB aggregates comprising pluripotent stem cells that remain in an undifferentiated state; culturing the non-EB aggregates in a dynamic suspension for a first period of time in the presence of the small molecules SB431542 and Dorsomorphin; and then culturing the aggregates in a dynamic suspension for a second period of time in the presence of a Smoothened agonist and retinoic acid, as Figure 1 shown. In one embodiment, the first period of time and the second period of time can each be from about 1 to about four days, such as about one day, such as about two days, such as about three days, such as about four days.

[0071] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of SB431542 at a concentration of from about 1 μM to about 100 μM, such as about 5 μM, about 10 μM, such as about 15 μM, such as about 20 μM, such as about 25 μM, such as about 30 μM, such as about 35 μM, such as about 40 μM, such as about 45 μM, such as about 50 μM, such as about 55 μM, such as about 60 μM, such as about 65 μM, such as about 70 μM, such as about 75 μM, such as about 80 μM, such as about 85 μM, such as about 90 μM, or such as 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 of from about 1 μM to about 20 μM, such as from about 1 μM to about 13 μM, such as from about 8 μM to about 20 μM, such as from about 8 μM to about 13 μM, or such as from 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 of from about 20 μM to about 40 μM, such as from about 20 μM to about 33 μM, such as from about 28 μM to about 40 μM, such as from about 28 μM to about 33 μM, or such as from 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 of from about 40 μM to about 60 μM, such as from about 40 μM to about 53 μM, such as from about 48 μM to about 55 μM, such as from about 48 μM to about 53 μM, or such as from 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 of from about 60 μM to about 80 μM, such as from about 60 μM to about 73 μM, such as from about 68 μM to about 75 μM, such as from about 68 μM to about 73 μM, or such as from about 69 μM to about 71 μM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of SB431542 at a concentration of from about 80 μM to about 100 μM, such as from about 80 μM to about 93 μM, such as from about 88 μM to about 95 μM, such as from about 88 μM to about 93 μM, or such as from about 89 μM to about 91 μM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of SB431542 at a concentration of about 10 μM.

[0072] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of an ALK5 inhibitor, the concentration of the ALK5 inhibitor being from about 250 nM to about 250 μM, such as about 1 μM, about 10 μM, 50 μM, about 100 μM, about 150 μM or about 200 μM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of about 10 μM of an ALK5 inhibitor.

[0073] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of LY364947, the concentration of LY364947 being from about 250 nM to about 250 μM, such as 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 a dynamic suspension in the presence of about 10 μM of LY364947.

[0074] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of GW788388, the concentration of GW788388 being from about 250 nM to about 250 μM, such as 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 a dynamic suspension in the presence of about 10 μM of GW788388.

[0075] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of A-77-01, the concentration of A-77-01 being from about 250 nM to about 250 μM, such as 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 a dynamic suspension in the presence of about 10 μM of A-77-01.

[0076] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of A-83-01, the concentration of A-83-01 being from about 250 nM to about 250 μM, such as 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 a dynamic suspension in the presence of about 10 μM of A-83-01.

[0077] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of SB505124 at a concentration of from about 250 nM to about 250 μM, such as 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 a dynamic suspension in the presence of about 10 μM of SB505124.

[0078] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of Dorsomorphin at a concentration of from about 0.2 μM to about 20 μM, such as about 0.5 μM, such as about 0.8 μM, such as about 1 μM, such as about 1.5 μM, such as about 2 μM, such as about 2.5 μM, such as about 3 μM, such as about 3.5 μM, such as about 4 μM, such as about 4.5 μM, such as about 5 μM, such as about 5.5 μM, such as about 6 μM, such as about 6.5 μM, such as about 7 μM, such as about 7.5 μM, such as about 8 μM, such as about 8.5 μM, such as about 9 μM, such as about 10 μM, such as about 11 μM, such as about 12 μM, such as about 13 μM, such as about 14 μM, such as about 15 μM, such as about 16 μM, such as about 17 μM, such as about 18 μM, or such as about 19 μM. In another embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of Dorsomorphin at a concentration of from about 0.2 μM to about 1 μM, such as from about 0.2 μM to about 0.9 μM, such as from about 0.3 μM to about 0.8 μM, such as from about 0.4 μM to about 0.7 μM, or such as from about 0.5 μM to about 0.6 μM. In yet another embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of Dorsomorphin at a concentration of from about 1 μM to about 10 μM, such as from about 1 μM to about 9 μM, such as from about 2 μM to about 8 μM, such as from about 3 μM to about 7 μM, or such as from about 4 μM to about 6 μM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of Dorsomorphin at a concentration of from about 10 μM to about 20 μM, such as from about 10 μM to about 19 μM, such as from about 12 μM to about 18 μM, such as from about 13 μM to about 17 μM, or such as from about 14 μM to about 16 μM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of Dorsomorphin at a concentration of about 2 μM.

[0079] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of an ALK2 inhibitor at a concentration of from about 1 nM to about 20 μM, such as 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.

[0080] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of DMH-1 at a concentration of from about 1 μM to about 10 μM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of DMH-1 at a concentration of about 2 μM.

[0081] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of K02288 at a concentration of from about 1 μM to about 10 μM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of K02288 at a concentration of about 2 μM.

[0082] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of ML347 at a concentration of from about 1 μM to about 10 μM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of ML347 at a concentration of about 2 μM.

[0083] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of Purmorphamine at a concentration of from about 0.05 μM to about 5 μM, such as about 0.08 μM, such as about 0.1 μM, such as about 0.2 μM, such as about 0.3 μM, such as about 0.4 μM, such as about 0.5 μM, such as about 0.6 μM, such as about 0.7 μM, such as about 0.8 μM, such as about 0.9 μM, such as about 1 μM, such as about 2 μM, such as about 3 μM, such as about 4 μM. In another embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of Purmorphamine at a concentration of from about 0.05 μM to about 0.1 μM, such as from about 0.06 μM to about 0.09 μM, or such as from about 0.07 μM to about 0.08 μM. In another embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of Purmorphamine at a concentration of from about 0.1 μM to about 1 μM, such as from about 0.2 μM to about 0.9 μM, such as from about 0.3 μM to about 0.8 μM, such as from about 0.4 μM to about 0.7 μM, or such as from about 0.5 μM to about 0.6 μM. In another embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of Purmorphamine at a concentration of from about 1 μM to about 5 μM, such as from about 1 μM to about 4 μM, such as from about 2 μM to about 5 μM, or such as from about 2 μM to about 4 μM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of Purmorphamine at a concentration of about 0.5 μM.

[0084] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of a Smoothened agonist at a concentration of from about 2.5 nM to about 5 μM, such as about 50 nM, about 100 nM, about 250 nM, about 500 nM, about 750 nM, about 1 μM or about 2.5 μM.

[0085] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of SAG at a concentration of from about 10 nM to about 1 μM, such as from about 10 nM to about 100 nM, such as from about 100 nM to about 500 nM, or such as from about 500 nM to about 1000 nM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of about 0.5 μM of SAG.

[0086] In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of SHH protein at a concentration of from about 2.5 nM to about 250 nM, such as from about 2.5 nM to about 10 nM, such as from about 10 nM to about 100 nM, or such as from about 100 nM to about 250 nM. In one embodiment, a method includes culturing non-EB aggregates in a dynamic suspension in the presence of SHH protein at about 25 nM.

[0087] Any cell culture vessel or reactor suitable for dynamic suspension culture can be used for the differentiation steps envisioned in the present disclosure. The vessel walls are generally inert or resistant to the adhesion of the cultured cells. There are also means to prevent cell sedimentation, such as agitation mechanisms, such as magnetic or mechanically driven stir bars or paddles, shaking mechanisms (usually externally connected to the vessel), or inversion mechanisms (i.e., devices that rotate the vessel to change the direction of gravity on the cells).

[0088] Vessels suitable for suspension culture for process development include the common range of commercially available spinner flasks, shake bags, or shake flasks. Exemplary bioreactors suitable for commercial production include the VerticalWheel TM bioreactor (PBSBiotech, Camarillo, CA).

[0089] OPC composition

[0090] The methods of the present disclosure can be used to obtain compositions comprising oligodendrocyte progenitor cells (OPCs) suitable for cell therapy. The OPCs obtained according to the present disclosure express high levels of the proteoglycan NG2 characteristic of OPCs and low levels of non-OPC markers associated with undesired cell types, such as CD49f, which can be expressed by 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 CLDN6 and EpCAM, two markers expressed by both pluripotent cells and epithelial cells (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).

[0091] In certain embodiments, the OPCs generated according to the present disclosure are the progeny of in vitro differentiation of human pluripotent stem cells. In certain embodiments, the OPCs obtained according to the present disclosure are the progeny of in vitro differentiation of human embryonic stem cells. In other embodiments, the OPCs obtained according to the present disclosure are the progeny of in vitro differentiation of induced pluripotent (iPS) cells.

[0092] One or more characteristics of the obtained OPC population can be determined by quantifying various cell markers using flow cytometry, such as to determine the percentage of cell populations that are positive for a particular marker or set of markers, or to identify undesired cell types present in the OPC population. In one embodiment, the OPCs prepared by the method according to the invention express one or more markers selected from NG2, PDGFRα, and GD3.

[0093] OPC populations obtained according to the present disclosure can comprise from about 30% to about 100% NG2-positive cells, such as 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, OPC populations obtained according to the present disclosure can comprise from about 45% to about 75% NG2-positive cells, such as from about 45% to about 50%, such as from about 50% to about 55%, such as from about 55% to about 60%, such as from about 60% to about 65%, such as from about 65% to about 70%, such as from about 70% to about 75%, such as from about 50% to about 70%, such as from about 55% to about 65%, or such as from about 58% to about 63% NG2-positive cells. In other embodiments, OPC populations obtained according to the present disclosure can comprise from about 60% to about 90% NG2-positive cells, such as from about 60% to about 65%, such as from about 65% to about 70% positive cells. OPC populations obtained according to the present disclosure can comprise from about 30% to about 100% PDGFRα-positive cells, such as 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% PDGFRα-positive cells. In certain embodiments, OPC populations obtained according to the present disclosure can comprise from about 45% to about 75% PDGFRα-positive cells, such as from about 45% to about 50%, such as from about 50% to about 55%, such as from about 55% to about 60%, such as from about 60% to about 65%, such as from about 65% to about 70%, such as from about 70% to about 75%, such as from about 50% to about 70%, such as from about 55% to about 65%, or such as from about 58% to about 63% PDGFRα-positive cells. In other embodiments, OPC populations obtained according to the present disclosure can comprise from about 60% to about 90% PDGFRα-positive cells, such as from about 60% to about 65%, such as from about 65% to about 70% positive cells.OPC populations obtained according to the present disclosure can comprise from about 30% to about 100% GD3-positive cells, such as 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, OPC populations obtained according to the present disclosure can comprise from about 45% to about 75% GD3-positive cells, such as from about 45% to about 50%, such as from about 50% to about 55%, such as from about 55% to about 60%, such as from about 60% to about 65%, such as from about 65% to about 70%, such as from about 70% to about 75%, such as from about 50% to about 70%, such as from about 55% to about 65%, or such as from about 58% to about 63% GD3-positive cells. In other embodiments, OPC populations obtained according to the present disclosure can comprise from about 60% to about 90% GD3-positive cells, such as from about 60% to about 65%, such as from about 65% to about 70% positive cells.

[0094] In one embodiment, an OPC population obtained according to the present disclosure is capable of forming fewer than or equal to four epithelial cysts per 100,000 cells in the cyst assay as described in Example 8 of the present disclosure. In another embodiment, an OPC population obtained according to the present disclosure is capable of forming fewer than or equal to three epithelial cysts per 100,000 cells in the cyst assay. In another embodiment, an OPC population obtained according to the present disclosure is capable of forming fewer than or equal to two epithelial cysts per 100,000 cells in the cyst assay. In yet another embodiment, an OPC population obtained according to the present disclosure is capable of forming fewer than or equal to one epithelial cyst per 100,000 cells in the cyst assay as described in Example 8 of the present disclosure.

[0095] undesired cell types

[0096] OPC populations obtained according to the present disclosure contain low levels of undesired cell types, as measured, for example, by quantifying markers associated with the undesired cell types by flow cytometry. In a non-limiting example, day 42 OPCs obtained according to the present disclosure comprise 0% to 4% cells expressing the epithelial cell-associated markers EpCAM, CD49f, and CLDN6 (Example 5, Table 2).

[0097] Markers associated with an undesired cell type can include less than about 20% of the undesired cell type, such as less than about 19%, such as less than about 18%, such as less than about 17%, such as less than about 16%, such as less than about 15%, such as less than about 14%, such as less than about 13%, such as less than about 12%, such as less than about 11%, such as less than about 10%, such as less than about 9%, such as less than about 8%, such as less than about 7%, such as less than about 6%, such as less than about 5%, such as less than about 4%, such as less than about 3%, such as less than about 2%, such as less than about 1%, such as less than about 0.5%, such as less than about 0.1%, such as less than about 0.05%, or such as less than about 0.01% of the undesired cell type. In another embodiment, the cell population can comprise from about 15% to about 20% of the undesired cell type, such as from about 19% to about 20%, such as from about 18% to about 20%, such as from about 17% to about 20%, such as from about 16% to about 20%, such as from about 15% to about 19%, or such as from about 16% to about 18% of the undesired cell type. In yet another embodiment, the cell population can comprise from about 10% to about 15% of the undesired cell type, such as from about 14% to about 15%, such as from about 13% to about 15%, such as from about 12% to about 15%, such as from about 11% to about 15%, or such as from about 12% to about 14% of the undesired cell type. In one embodiment, the cell population can comprise from about 1% to about 10% of the undesired cell type, such as from about 2% to about 10%, such as from about 1% to about 9%, such as from about 2% to about 8%, such as from about 3% to about 7%, or such as from about 4% to about 6% of the undesired cell type. In one embodiment, the cell population can comprise from about 0.1% to about 1% of the undesired cell type, such as from about 0.2% to about 1%, such as from about 0.1% to about 0.9%, such as from about 0.2% to about 0.8%, such as from about 0.3% to about 0.7%, or such as from about 0.4% to about 0.6% of the undesired cell type. In one embodiment, a low level of the undesired cell type can indicate the presence of less than about 15% of the undesired cell type.

[0098] In one embodiment, the undesired cell type can include cells that express one or more markers selected from CD49f, CLDN6, or EpCAM.

[0099] Formulation

[0100] The OPC composition according to the present disclosure may further comprise a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier may comprise dimethyl sulfoxide (DMSO). In one embodiment, the pharmaceutically acceptable carrier does not comprise dimethyl sulfoxide. In one embodiment, the composition may be adapted for cryopreservation at a temperature of -80°C to -195°C or below -80°C to -195°C.

[0101] The OPC composition according to the present disclosure may be formulated for administration by direct injection into the spinal cord of a subject. In one embodiment, the OPC composition according to the present disclosure may be formulated for intracerebral, intraventricular, intrathecal, intranasal or intracisternal administration to a subject. In one embodiment, the OPC composition according to the present disclosure may be formulated for administration by direct injection into or adjacent to an infarct cavity in the brain of a subject. In one embodiment, the composition according to the present disclosure may be formulated for administration by implantation. In one embodiment, the composition according to the present disclosure may be formulated as a solution.

[0102] The OPC composition according to the present disclosure may comprise from about 1 x 10 6 to about 5 x 10 8 cells / ml, such as about 1 x 10 6 cells / ml, such as about 2 x 10 6 cells / ml, such as about 3 x 10 6 cells / ml, such as about 4 x 10 6 cells / ml, such as about 5 x 10 6 cells / ml, such as about 6 x 10 6 cells / ml, such as about 7 x 10 6 cells / ml, such as about 8 x 10 6 cells / ml, such as about 9 x 10 6 cells / ml, such as about 1 x 10 7 cells / ml, such as about 2 x 10 7 cells / ml, such as about 3 x 10 7 cells / ml, such as about 4 x 10 7 cells / ml, such as about 5 x 10 7 cells / ml, such as about 6 x 10 7 cells / ml, such as about 7 x 10 7 cells / ml, such as about 8 x 10 7 cells / ml, such as about 9 x 10 7 cells / ml, such as about 1 x 10 8cells / ml, such as about 2x10 8 cells / ml, such as about 3x10 8 cells / ml, such as about 4x10 8 cells / ml, or such as about 5x10 8 cells / ml. In another embodiment, the composition according to the present disclosure may comprise from about 1x10 8 to about 5x10 8 cells / ml, such as about 1x10 8 to about 4x10 8 cells / ml, such as about 2x10 8 to about 5x10 8 cells / ml, such as about 1x10 8 to about 3x10 8 cells / ml, such as about 2x10 8 to about 4x10 8 cells / ml, or such as about 3x10 8 to about 5x10 8 cells / ml. In yet another embodiment, the composition according to the present disclosure may comprise from about 1x10 7 to about 1x10 8 cells / ml, such as about 2x10 7 to about 9x10 7 cells / ml, such as about 3x10 7 to about 8x10 7 cells / ml, such as about 4x10 7 to about 7x10 7 cells / ml, or such as about 5x10 7 to about 6x10 7 cells / ml. In one embodiment, the composition according to the present disclosure may comprise from about 1x10 6 to about 1x10 7 cells / ml, such as about 2x10 6 to about 9x10 6 cells / ml, such as about 3x10 6 to about 8x10 6 cells / ml, such as about 4x10 6 to about 7x10 6 cells / ml, or such as about 5x10 6 to about 6x10 6 cells / ml. In yet another embodiment, the composition according to the present disclosure may comprise at least about 1x10 6cells / mL, such as at least about 2x10 6 cells / mL, such as at least about 3x10 6 cells / mL, such as at least about 4x10 6 cells / mL, such as at least about 5x10 6 cells / mL, such as at least about 6x10 6 cells / mL, such as at least about 7x10 6 cells / mL, such as at least about 8x10 6 cells / mL, such as at least about 9x10 6 cells / mL, such as at least about 1x10 7 cells / mL, such as at least about 2x10 7 cells / mL, such as at least about 3x10 7 cells / mL, such as at least about 4x10 7 cells / mL, or such as at least about 5x10 7 cells / mL. In one embodiment, the composition according to the present disclosure may comprise up to about 1x10 8 cells or more, such as up to about 2x10 8 cells / mL or more, such as up to about 3x10 8 cells / mL or more, such as up to about 4x10 8 cells / mL or more, such as up to about 5x10 8 cells / mL or more, or such as up to about 6x10 8 cells / mL.

[0103] In one embodiment, the OPC composition according to the present disclosure may comprise about 4x10 7 to about 2x10 8 cells / mL.

[0104] In another embodiment, the OPC composition according to the present disclosure can have a volume of from about 10 microliters to about 5 milliliters, such as about 20 microliters, such as about 30 microliters, such as about 40 microliters, such as about 50 microliters, such as about 60 microliters, such as about 70 microliters, such as about 80 microliters, such as about 90 microliters, such as about 100 microliters, such as about 200 microliters, such as about 300 microliters, such as about 400 microliters, such as about 500 microliters, such as about 600 microliters, such as about 700 microliters, such as about 800 microliters, such as about 900 microliters, such as about 1 milliliter, such as about 1.5 milliliters, such as about 2 milliliters, such as about 2.5 milliliters, such as about 3 milliliters, such as about 3.5 milliliters, such as about 4 milliliters, or such as about 4.5 milliliters. In one embodiment, the composition according to the present disclosure can have a volume of from about 10 microliters to about 100 microliters, such as from about 20 microliters to about 90 microliters, such as from about 30 microliters to about 80 microliters, such as from about 40 microliters to about 70 microliters, or such as from about 50 microliters to about 60 microliters. In another embodiment, the composition according to the present disclosure can have a volume of from about 100 microliters to about 1 milliliter, such as from about 200 microliters to about 900 microliters, such as from about 300 microliters to about 800 microliters, such as from about 400 microliters to about 700 microliters, or such as from about 500 microliters to about 600 microliters. In yet another embodiment, the composition according to the present disclosure can have a volume of from about 1 milliliter to about 5 milliliters, such as from about 2 milliliters to about 5 milliliters, such as from about 1 milliliter to about 4 milliliters, such as from about 1 milliliter to about 3 milliliters, such as from about 2 milliliters to about 4 milliliters, or such as from about 3 milliliters to about 5 milliliters. In one embodiment, the OPC composition according to the present disclosure can have a volume of from about 20 microliters to about 500 microliters. In another embodiment, the OPC composition according to the present disclosure can have a volume of from about 50 microliters to about 100 microliters. In yet another embodiment, the OPC composition according to the present disclosure can have a volume of from about 50 microliters to about 200 microliters. In another embodiment, the OPC composition according to the present disclosure can have a volume of from about 20 microliters to about 400 microliters. In one embodiment, the OPC composition according to the present disclosure can be in a container configured for cryopreservation or for administration to a subject in need thereof. In one embodiment, the container can be a pre-filled syringe.

[0105] Method of Use

[0106] OPC compositions 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, a population of OPC cells according to the present disclosure can be injected or implanted into a subject in need thereof. In one embodiment, a population of cells according to the present disclosure can be implanted into a subject in need thereof for the treatment of spinal cord injury, stroke, or multiple sclerosis.

[0107] In one embodiment, a population of cells according to the present disclosure is capable of inducing myelination of naked axons at the implantation site in a subject. In one embodiment, a population of cells produced by the method according to the present disclosure can exhibit improved implantation and migration capabilities. In one embodiment, a population of cells produced by the method according to the present disclosure can be capable of improving post-injury repair or regeneration of neural tissue in a subject.

[0108] A population of cells according to the present disclosure can be capable of improving the sensory function of a subject in need of treatment after implantation of the population into the subject. The International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) test can be used to evaluate the improvement in sensory function, such as determining the sensory levels of pinprick and light touch on the right and left sides. A population of cells according to the present disclosure can be capable of improving the motor function of a subject in need of treatment after implantation of the population into the subject. The ISNCSCI test can be used to evaluate the improved motor function, such as determining the motor levels of complete paralysis, detectable or visible contractions, active movements, full range of motion against gravity, and sufficient resistance on the right and left sides.

[0109] A population of cells according to the present disclosure is capable of reducing the volume of injury-induced central nervous system parenchymal cavitation within 12 months or less. In one embodiment, a population of cells according to the present disclosure is capable of reducing the volume of injury-induced central nervous system parenchymal cavitation in a subject within 6 months or less, 5 months or less, 4 months or less, 3 months or less, or 2 months or less, or less than 1 month.

[0110] The invention has now been generally described. By reference to the following examples, which are provided by way of illustration, the invention will be more readily understood. These examples are not intended to limit the present disclosure, unless otherwise specified. Examples

[0111] Example 1 - Culturing and Expanding Undifferentiated Human Embryonic Stem Cells

[0112] Undifferentiated human embryonic stem cells (uhESCs) from a working cell bank (WCB) 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 on recombinant human laminin-521 (Corning #354224)-coated, tissue culture-treated polystyrene 225 cm 2 culture flasks (Corning #431082) in complete mTeSR TM -1 medium (Stem Cell Technologies #85850). The medium was completely replaced daily until the cells reached approximately 80-90% confluence, and then the uhESCs were passaged using ReLeSR TM reagent (Stem Cell Technologies #05872). The uhESC cells lifted by ReLeSR TM were seeded into new laminin-521-coated 225 cm 2 flasks, and daily medium replacement was resumed two days after seeding. Depending on the experiment, the cultured uhESCs from the WCB were amplified 2 to 5 passages in this manner before differentiation into neuroectodermal progenitor cells as described in Example 2.

[0113] Example 2 - Method for differentiating human embryonic stem cells into neuroectodermal progenitor cells in dynamic suspension culture

[0114] Day -1 : The amplified uhESCs (approximately 90% confluent) were dissociated and (Stem Cell Technologies #07920) to form a single-cell suspension, allowing accurate cell counting and uniform seeding density. Then the dissociated uhESCs were seeded at 1x 10 6Cells were seeded at a density of Figure 5 cells / mL into PBS-0.1 or PBS-0.5

[0115] Days 0 - 3 mini-bioreactor systems (PBS Biotech),

[0116] Days 4 - 6 which were set to rotate at 35 RPM or 25 RPM

[0117] (day -1), for dynamic suspension culture. Cells were seeded in a 1:1 mixture of glial progenitor cell medium (GPM; consisting of DMEM / F12 (Gibco catalog number 10565-018) supplemented with 2% B27 supplement (Gibco catalog number 17504-044) and 0.04 μg / ml triiodothyronine (Sigma catalog number T5516-1MG)) and undifferentiated hESC medium (as in Example 1) supplemented with 10 μM Rho kinase inhibitor (RI, Tocris catalog number 1254) to support cell survival. During the first 24 hours, small, homogeneous aggregates of uhESCs formed. Compared to embryoid body (EB) formation, cells in the small aggregates did not begin to spontaneously differentiate but maintained their pluripotency, as confirmed by marker expression measured by qPCR ( Figure 5 )

[0115] Days 0 - 3 : To initiate differentiation into neuroectoderm, for the next four days, the small non-EB aggregates were cultured in dynamic suspension in PBS-0.1 or PBS-0.5 mini-bioreactors rotating at 45 or 32 RPM, respectively, in GPM supplemented with 10 μM SB431542 (Sigma-Aldrich, catalog number S4317) and 2 μM Dorsomorphin (Sigma-Aldrich, catalog number P5499). The medium was replenished daily by allowing the aggregates to settle, removing 70-80% of the used medium and replacing it with an equal volume of GPM supplemented with 10 μM SB431542 and 2 μM Dorsomorphin.

[0116] Days 4 - 6 : The cells were further cultured in dynamic suspension in GPM supplemented with 0.5 μM Purmorphamine (Reprocell, catalog number 04-0009), 1 μM retinoic acid (Sigma-Aldrich, catalog number R2625) and 150 μM ascorbic acid (Sigma-Aldrich, catalog number A4544) at 45 RPM (PBS-0.1 mini-bioreactor) or 32 RPM (PBS-0.5 mini-bioreactor) for an additional three days. The medium was replenished daily by allowing the aggregates to settle, removing 70-80% of the used 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.

[0117] Day 7 : A subset of the differentiated cells was collected on day 7 of the dynamic suspension culture differentiation process and subjected to analysis of marker expression by immunocytochemistry (ICC) (as described in Example 5) and qPCR (as described in Example 6). By day 7, the cells expressed markers characteristic of the neuroectoderm (Table 2, Figure 3 ). As described in Examples 3 and 4 respectively, the remaining day 7 cells were differentiated into glial lineage cells and further into oligodendrocyte progenitor cells.

[0118] Example 3 - Method for differentiating human embryonic stem cells into glial lineage cells in dynamic suspension culture

[0119] 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 modifying the differentiation medium to GPM supplemented with 20 ng / mL human basic fibroblast growth factor (hbFGF, ThermoFisher, catalog number PHG0263), 10 ng / mL epidermal growth factor (EGF, Thermo Fisher, catalog number PHG0311), and 10 μM RI. For the next two weeks (days 8 - 20), the cell aggregates were maintained in dynamic suspension at 45 rpm (PBS - 0.1 micro - bioreactor) or 32 RPM (PBS - 0.5 micro - bioreactor) in GPM supplemented with 20 ng / mL bFGF and 10 ng / mL EGF, and the medium was replenished daily using gravity sedimentation and a 70 - 80% medium exchange. On day 14, 10 μM RI was also added to the fresh medium.

[0120] A subset of the differentiated cells was collected on day 21 of the dynamic suspension culture differentiation process and subjected to analysis of marker expression by qPCR (as described in Example 6). By day 21, the differentiated cells expressed markers consistent with glial lineage cells (Table 2).

[0121] Example 4 - Method for differentiating human embryonic stem cells into oligodendrocyte progenitor cells

[0122] The glial lineage progenitor cells obtained in Example 3 were further differentiated into oligodendrocyte progenitor cells. The differentiation protocol for days 0 - 20 was as described in Examples 2 and 3. On day 21, the aggregates were transferred from dynamic suspension culture to adherent culture on a tissue culture vessel coated with recombinant human laminin-521 (rhLN-521). For example, starting with a 1x PBS-0.1L micro-bioreactor with 60 mL of aggregate suspension, 60 mL of the culture was aliquoted into 2 x T75 flasks, each flask having 30 mL of culture. Starting on day 21 and continuing until 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, catalog number AF-100-13A), with complete medium change every other day. On days 28 and 35, the cell cultures were detached using TrypLE TM Select (Thermo Fisher, catalog number A12859-01), counted and seeded at 4 x 10 4 viable cells / cm 2 onto the rhLN-521 coated vessels. Starting on day 35 until harvest on day 42, the GPM was changed every other day.

[0123] A subset of the differentiated cells was collected 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). By day 42, the differentiated cells expressed markers characteristic of oligodendrocyte progenitor cells as measured by the three analytical methods (Table 1, Table 2, Figure 4 ).

[0124] OPCs were harvested on day 42. The cells were detached from the vessels using TrypLE TM Select, counted and reconstituted in CryoStor 10 (BioLife Solutions, catalog number 210102) and subsequently cryopreserved.

[0125] Example 5 - Characterization of the differentiated cell population by immunocytochemistry and flow cytometry

[0126] Flow cytometry and immunocytochemistry (ICC) can be used to detect and characterize different 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 display a given protein marker profile, ICC provides additional information about the subcellular localization of each protein marker and can be applied to individual cells or cell aggregates. By using one or both of these proteomic profiling methods, we tracked the differentiation of human embryonic stem cells according to the methods of the present disclosure into neuroectodermal progenitor cells, glial progenitor cells, and oligodendrocyte progenitor cells.

[0127] For human embryonic stem cells differentiating into neuroectodermal progenitor cells in suspension, protein marker expression in the starting material (undifferentiated pluripotent cells) and cell aggregates on day 7 of differentiation was characterized by ICC. At room temperature (RT), adherent pluripotent cells and cell aggregates were fixed in 4% paraformaldehyde (PFA) for 30 minutes. The fixed cells and aggregates were washed with phosphate-buffered saline (PBS), and then the fixed aggregates were sequentially placed in sucrose solutions of increasing concentration (10%, 20%, and 30% weight / volume) for 30 minutes at room temperature, 30 minutes at room temperature, and overnight at 4 °C, respectively. After sucrose replacement, the aggregates were embedded in Tissue-Tek optimal cutting temperature (OCT) compound (Sakura Finetek USA #4583) and frozen at -80 °C. The OCT-embedded aggregates were warmed to -20 °C, sectioned at 30 μm using a cryostat (model CM3050 S, Leica Biosystems, Buffalo Grove, IL, USA), and mounted onto poly-L-lysine (Sigma-Aldrich #P4707)-coated slides. For immunocytochemical staining, the fixed adherent cells and slide-mounted aggregate sections were permeabilized and blocked for 2 hours at room temperature (RT) in a blocking solution consisting of 0.1% Triton TM X-100 / 2% normal goat serum / 1% bovine serum albumin. After permeabilization and blocking, the adherent cells and aggregate sections were incubated overnight at 4 °C in a blocking solution without Triton TM X-100 and containing primary antibodies specific for the protein markers of interest (including 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). The adherent cells and aggregate sections were then washed 3 times with PBS and then in the absence of TritonTM In the blocking solution of X-100, it was incubated with a secondary antibody specific to the selected primary antibody and a 4′,6-diamidino-2-phenylindole (DAPI) counterstain for 1 hour at room temperature in the dark. The adherent cells and aggregate sections were washed 3 times with PBS and imaged using an IN Cell Analyzer 2000 (GE Healthcare, Pittsburgh, PA, USA).

[0128] Figure 2 and Figure 3 respectively show representative ICC data of the starting pluripotent cell population and neuroectodermal progenitor cells on day 7. As Figure 2 shown, the starting population of undifferentiated human embryonic stem cells expresses 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, the cell aggregates from two representative experiments express PAX6 and PSA-NCAM, which are two protein markers characteristic of neuroectodermal progenitor cells ( 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).

[0129] For human embryonic stem cells that differentiate into oligodendrocyte progenitor cells by day 42, protein marker expression in the single-cell population obtained by flow cytometry and ICC characterization was examined.

[0130] To characterize protein marker expression of oligodendrocyte progenitor cells by ICC, as described above, sections of aggregates fixed on slides were stained, except that permeabilization was carried out with 100% methanol for 2 minutes at room temperature, and the blocking solution consisted of 10% fetal bovine serum in PBS.

[0131] Figure 4 Representative ICC data for oligodendrocyte progenitor cells at day 42 are shown. The resulting single-cell populations from two representative experiments expressed the oligodendrocyte progenitor 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).

[0132] To quantitatively analyze cell surface markers by flow cytometry on day 42, cells were thawed in thawing medium (10% fetal bovine serum in DMEM medium), centrifuged, and resuspended in staining buffer (2% fetal bovine serum / 0.05% sodium azide in PBS). Cells were incubated with primary antibodies specific for the markers of interest (including NG2 (Invitrogen #37-2300), GD3 (Millipore #MAB2053), A2B5 (BD #563775), CD49f (Millipore #CBL458P), EpCAM (Dako #M080401-2), and CLDN6 (Thermo Fisher #MA5-24076)) and their isotype controls on ice for 30 minutes. Cells were washed with staining buffer to remove unbound antibodies; in the case of unconjugated antibodies, the cells were then incubated with the appropriate fluorophore-conjugated secondary antibody on ice for 30 minutes. Cells were washed and then propidium iodide was added to distinguish dead cells. In some cases, cells were cultured overnight at 37°C / 5% CO2 in tissue culture vessels coated with Matrigel (Corning #356231) to restore protein markers that were sensitive to the day 42 harvest procedure described in Example 4, and then harvested with TrypLE TM 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 fraction of the total number of cells analyzed after correction for the number of cells showing non-specific binding to the isotype control antibody.

[0133] Table 1 shows representative flow cytometry data of day 42 oligodendrocyte progenitor cells generated according to the method described in Example 4. As shown by two representative runs, a high proportion of cells in the resulting cell population express characteristic oligodendrocyte markers, including 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. 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).In addition, non-OPC markers were detected to the greatest extent in the resulting population, including the neural progenitor / epithelial marker CD49f (Krebsbach PH, Villa-Diaz LG. The Role of Integrin α6(CD49f) in Stem Cells: More than a Conserved Biomarker. Stem Cells Dev. 2017 Aug 1;26(15):1090-1099) and the epithelial markers 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 J Mol Med. 2018 Oct;42(4):1771-1785).

[0134] Table 1. Representative flow cytometry data of oligodendrocyte progenitor cells generated by the method according to the present disclosure.

[0135]

[0136] Compared to cells currently in clinical testing for the treatment of spinal cord injury and OPCs generated using another method (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), the cell population generated by the methods described in the present disclosure results in a higher proportion of cells positive for the oligodendrocyte progenitor marker NG2, and reduced expression of the non-OPC markers CD49f, CLDN6, and EpCAM.

[0137] Example 6 - Characterization of Differentiated Cell Populations by Gene Expression Profiling

[0138] Gene expression profiling can be used to characterize the starting population of pluripotent cells and the cell phenotypes at each stage of differentiation, including the generation of neuroectodermal progenitors, glial progenitors, and oligodendrocyte progenitors. Gene expression profiling includes performing global transcriptome profiling using methods such as microarrays and RNA-seq, and performing target gene profiling using more sensitive methods such as real-time quantitative PCR (qPCR).

[0139] For gene expression profiling, cells were lysed in 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 assays, the purified RNA was then converted to cDNA using Invitrogen Superscript IV VILO Mastermix (Thermo Fisher Scientific #11756050) according to the standard method and 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 GeneExpression 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 on an ABI 7900HT real-time sequence detection system (Applied Biosystems), a BioMark HD system (Fluidigm), or an equivalent system. Each target gene was normalized to one or more reference genes, such as GAPDH, to determine its relative expression level.

[0140] Figure 5 Representative qPCR analyses of uhESCs are shown at harvest, before three-dimensional cell aggregate formation (day -1) and 24 hours later, after small non-embryoid body (non-EB) aggregate formation (day 0, immediately before the onset of neuroectodermal differentiation). Two representative experiments were performed and RNA samples were collected and processed for gene expression profiling by qPCR using the methods described above. Fluidigm qPCR was performed using a set of 76 genes consisting of known markers of pluripotency and early differentiation. For each gene, normalized ΔCT values were calculated relative to the mean of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1). The resulting correlation plot of all ΔCT values at day -1 relative to day 0 is shown in Figure 5 and indicates that cells in small non-EB aggregates at day 0 retained expression of pluripotency genes and had an overall expression profile of test markers similar to that of uhESCs at day -1. This is further supported by the calculated fold change relative to baseline presented in Table 2, which shows high levels of the pluripotency markers NANOG, LIN28A, and SOX2 24 hours after aggregate formation (uhESCs at day 0) for two representative experiments.

[0141] Table 2 shows qPCR results from two representative experiments that measured the expression of pluripotency genes, neuroectodermal progenitor cell genes, glial progenitor cell genes, and oligodendrocyte progenitor cell genes in cell populations generated by the methods according to the present disclosure. RNA samples were collected at the following time points: 24 hours after the start of uhESC cell aggregate formation and prior to differentiation (day 0), followed by differentiation into neuroectodermal progenitor cells (day 7), followed by differentiation into glial progenitor cells (day 21), and followed by differentiation into oligodendrocyte progenitor cells (day 42). The RNA samples were processed for qPCR using the method described above. Quantification was performed on a selected set of genes indicative of each differentiation state, including: three pluripotency genes (NANOG, LIN28A, SOX2), three neuroectodermal progenitor cell genes (PAX6, HES5, ZBTB16), three glial progenitor cell genes (CACGN4, FABP7, SOX6), and three oligodendrocyte progenitor cell genes (CSPG4, PDGFRα, DCN). For each gene, the normalized ΔCT value was calculated using the mean of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1), and the fold expression relative to the baseline (expression below the limit of quantification) was calculated using the ΔΔCT method.

[0142] Table 2. qPCR analysis of gene markers for pluripotency, neuroectodermal progenitor cells (NEPC), glial progenitor cells (GPC), and oligodendrocyte progenitor cells (OPC) in H1 uhESCs differentiated into OPCs according to the present disclosure.

[0143]

[0144]

[0145] Referring to Table 2, differentiating uhESCs for 7 days by the method according to the present disclosure results in a gene expression profile consistent with that of neuroectodermal progenitor cells, 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 Pt1):4927 - 42).

[0146] After 21 days of suspension-based differentiation, the resulting cell population exhibited a gene expression profile consistent with that of glial progenitor cells, including downregulation of pluripotency 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).

[0147] After 42 days of differentiation according to the method described in the present disclosure, the resulting cell population expresses markers consistent with oligodendrocyte progenitor 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).

[0148] Example 7 - Differentiation of Human Embryonic Stem Cells into Neuroectodermal Progenitor Cells Using Alternative Small Molecule Inhibitors of TGFBR1 / Activin / NODAL Signaling and BMP Signaling

[0149] In addition to the small molecule inhibitors (SB431542 and Dorsomorphin) used in Example 2, the ability of alternative small molecule inhibitors of TGFβR1 / Activin / Nodal signaling and BMP signaling to differentiate human embryonic stem cells into neuroectodermal progenitor cells in suspension was tested. Table 3 lists the alternative small molecule inhibitors tested. Each condition was tested in duplicate wells of an Ultra Low Attachment 6-well tissue culture plate (Corning #3471).

[0150] Table 3. Small Molecule Inhibitors for Differentiating Human Embryonic Stem Cells into Neuroectodermal Progenitor Cells in Suspension

[0151]

[0152]

[0153] On day 7 of differentiation, cells were collected 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 mean of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1), and fold expression relative to baseline (expression below the limit of quantification) was calculated using the ΔΔCT method. Table 4 shows the mean (relative to baseline) of fold expression values for biological replicates of each small molecule combination. Referring to Table 4, differentiation of uhESCs in suspension for 7 days using each tested small molecule combination resulted in downregulation of the pluripotency marker NANOG and maintenance or induction of expression of genes associated with the neuroectodermal progenitor cell phenotype (including LIN28A, SOX2, PAX6, HES5, and ZBTB16) to a similar extent.

[0154] To obtain a more comprehensive comparison of the day 7 cell phenotypes obtained after treatment with each small molecule combination, Fluidigm qPCR was performed using a panel of 96 genes consisting 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. Referring to FIG. 6, comparison of the day 7 cell phenotypes of each alternative small molecule combination with the cell phenotype generated by treatment with SB431542 plus Dorsomorphin by regression plots of normalized ΔCT values indicates that a similar overall cell phenotype can be achieved using each tested small molecule combination. In summary, the results shown in Table 4 and FIG. 6 support that (i) various combinations of TGFβR1 / Activin / Nodal signaling inhibitors and (ii) BMP signaling inhibitors can be used to differentiate uhESCs into neuroectodermal progenitor cells in suspension using the methods of the present disclosure, and further differentiate into glial progenitor cells and differentiate into oligodendrocyte progenitor cells.

[0155] Table 4. qPCR analysis of gene markers of pluripotency and neuroectodermal progenitor cells (NEPCs) in H1 uhESCs differentiated into NEPCs using different combinations of small molecule inhibitors.

[0156]

[0157] Example 8 - Assessment of the presence of contaminating epithelial lineage cells in differentiated OPC populations using an in vitro cyst assay

[0158] The presence of undesired epithelial lineage 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 in the presence of factors known to stimulate epithelial cyst formation for 20 days. In addition to visual inspection of cysts, the presence of cystic structures with basolateral protein expression containing the epithelial marker CD49f was evaluated using immunocytochemistry.

[0159] OPCs were seeded on a 3 Matrigel (Corning) at a density of 21.9x10 2 cells / cm (a total of 0.5x10 6 cells were seeded in 12 wells of a 24-well plate). The cells were cultured for 20 days. On day 20, live cyst counts were performed and the cells were lysed using Cell Recovery Solution (Corning #354253). The cells were fixed in 4% paraformaldehyde (PFA) on ice for 5 minutes and permeabilized overnight in blocking buffer. Subsequently, the cysts were stained for CD49f (ITGA6), phalloidin, and counterstained with DAPI. The 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 TM (Mathworks).

[0160] Referring to Table 5, the OPCs generated from two representative runs using the method according to the present disclosure and tested in an in vitro cyst assay produced fewer cysts than three control batches of OPCs (Control A, Control B, and Control C) generated by an alternative method (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) that was previously found to cause epithelial cyst formation in vivo. Based on these results, it is expected that the OPCs generated according to the present disclosure will form few or no epithelial cysts in vivo.

[0161] Table 5. Representative cyst assay results of oligodendrocyte progenitor cells generated by the method according to the present disclosure.

[0162]

[0163]

[0164] Although the present disclosure has been described with reference to specific embodiments, those skilled in the art will understand that various changes can be made and elements thereof can be replaced with equivalents without departing from the scope of the present disclosure. Additionally, many modifications can be made to adapt a particular situation or material to the teachings of the present disclosure without departing from the scope of the present disclosure.

[0165] Therefore, it is intended that the present disclosure not be limited to the specific embodiments disclosed as the best mode contemplated for carrying out the present disclosure, but that the present disclosure will include all aspects falling within the scope and spirit of the appended claims.

Claims

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

2. The method of claim 1, further comprising the additional step of harvesting the non-EB aggregates from d) and plating them on a substrate, thereby causing the cells to migrate out of the aggregates.

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 (hESC).

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

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. The method of claim 1, wherein the at least one inhibitor of TGFβ / activin / Nodal signaling is selected from 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 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. The method according to claim 1, wherein the at least one Smoothened receptor agonist is selected from Purmorphamine, Smoothened agonist (SAG, CAS 364590-63-6), and Sonic Hedgehog (SHH) protein.

13. The method according to claim 1, wherein the at least one Smoothened receptor agonist is Purmorphamine.

14. The method according to claim 1, wherein the first time period is about three to four days.

15. The method according to claim 1, wherein the second time period is about three days.

16. The method according to claim 1, wherein steps a) to d) are performed over a period of about 21 days.

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

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

19. A method for obtaining a cell population comprising oligodendrocyte progenitor cells (OPCs) from undifferentiated human pluripotent stem cells, the method comprising: a) obtaining glial progenitor cells by the method according to claim 1; b) harvesting the cells from a) and plating them on a substrate, thereby causing the cells to migrate out of the aggregates; and c) culturing the cells from b) adherently for an additional period of time in the presence of epidermal growth factor (EGF) and platelet-derived growth factor AA (PDGF-AA) until the cells mature into OPCs, 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. The method according to claim 19, wherein the adherent culture is carried out for a period of about 21 days.

21. The method according to claim 19, wherein the adherent culture is carried out on a substrate selected from: (i) cell adhesion peptides and (ii) extracellular matrices selected from laminin and vitronectin.

22. The method according to claim 19, wherein the adherent culture is carried out on recombinant human laminin-521.

23. The method according to claim 19, wherein the adherent culture is carried out on the laminin-511 E8 fragment.

24. The method according to claim 19, wherein the human pluripotent stem cells are hESCs.

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

26. A population of differentiated cells comprising OPCs obtained by the method according to claim 19.

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

28. The population of differentiated cells according to claim 26, wherein at least 70% of the cells are NG2-positive.

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

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

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

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

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

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

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

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

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

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

39. A method for inducing the 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 a dynamic suspension for a first period of time in the presence of at least one inhibitor of transforming growth factor β (TGFβ) / activin / Nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling, thereby inducing differentiation into neuroectoderm; c) culturing the non-EB aggregates from b) in a dynamic suspension for a second period of time in the presence of retinoic acid and at least one Smoothened receptor agonist; until the cells mature into paired box 6 (PAX6) positive neuroectodermal cells.

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

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

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

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

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

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

46. The method according to claim 39, wherein at least one inhibitor of BMP signaling is selected from Dorsomorphin, DMH-1, K02288, ML3467, LDN193189, and Noggin protein.

47. The method according to claim 39, wherein at least one inhibitor of BMP signaling is Dorsomorphin.

48. The method according to claim 39, wherein at least one Smoothened receptor agonist is selected from Purmorphamine, Smoothened agonist (SAG, CAS 364590-63-6), and Sonic Hedgehog (SHH) protein.

49. The method according to claim 39, wherein at least one Smoothened receptor agonist is Purmorphamine.

50. The method according to claim 39, wherein the first time period is about three to four days.

51. The method according to claim 39, wherein the second time period is about three days.

52. The method according to claim 39, wherein steps a) to c) are performed over a period of about 7 to 8 days.

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

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

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

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