Method for differentiating pluripotent stem cells in dynamic suspension culture

Abstract

Provided is a method for differentiating pluripotent stem cells into neuroectoderm in dynamic suspension culture using small molecules or protein inhibitors of TGFβ / activin / Nodal signaling and BMP signaling. Also provided is a method and scheme for differentiating pluripotent stem cells, such as human embryonic stem cells, into neuroectoderm first, then further differentiated into glial progenitor cells, and further differentiated into oligodendrocyte progenitor cells (OPCs), and the compositions thus obtained. The method of the present disclosure reproducibly produces neuroectoderm progenitor cells at day 7 of the differentiation process, reproducibly produces glial progenitor cells at day 21 of the differentiation process, and reproducibly produces OPCs at day 42 of the differentiation process.

Description

Technical Field

[0001] The present disclosure relates to the field of cell biology and neuroectoderm and glial lineage cells (e.g., oligodendrocyte progenitor cells). More specifically, the present disclosure relates to a novel method for differentiating pluripotent stem cells into neuroectoderm in dynamic suspension culture using small molecules or protein inhibitors of TGFβ / activin / Nodal signaling and BMP signaling. The present disclosure further provides a novel method for differentiating pluripotent stem cells (e.g., human embryonic stem cells) first into neuroectoderm, then further differentiated into glial progenitor cells, and further differentiated into oligodendrocyte progenitor cells. The present disclosure further relates to neuroectoderm cells, glial progenitor cells, and oligodendrocyte progenitor cells expressing one or more markers produced by the method according to the present invention. Background Art

[0002] Oligodendrocyte progenitor cells (OPCs) are a subpopulation of glial cells in the central nervous system (CNS) that mature into myelin-producing oligodendrocytes. Oligodendrocytes produce myelin, which insulates neuronal axons and remyelinates CNS lesions when myelin is lost. Oligodendrocytes also promote neuroprotection through other mechanisms, including the production of neurotrophic factors that promote neuronal survival (Wilkins A, Chandran S, Compston AA 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). Oligodendrocytes are therefore important therapeutic targets for demyelinating and dysmyelinating disorders (e.g., multiple sclerosis, adrenoleukodystrophy, and adrenomedullary neuropathy), other neurodegenerative disorders (e.g., Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease), and acute spinal cord injury (SCI).

[0003] OPCs are derived from the neuroectoderm (also called neural ectoderm or neural tube epithelium), which gives rise to neural progenitor cells that will give rise to 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 cells of the neuroectodermal lineage and neural progenitors, most existing protocols rely on embryoid bodies (EBs) and subsequent neurosphere formation in static non-adherent culture in the presence of the tailing 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 spinalcord 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).Based on EB and adherent differentiation scheme all need artificial selection neural precursor to optimize yield, and be not easy to expand, thereby limit their purposes in preparing a large number of therapeutic grade cells.In addition, a small amount of cell types outside the neural ectoderm pedigree can persist in differentiation process, and facilitate undesirable cell types in final OPC colony, including, for example, epithelial cells or chondrocyte progenitor cells (Manley NC, Priest CA, Denham J, Wirth ED 3rd, Lebkowski JS.Human Embryonic Stem Cell-DerivedOligodendrocyte Progenitor Cells:Preclinical Efficacy and Safety in CervicalSpinal Cord Injury.Stem Cells Transl Med.2017Oct; 6(10):1917-1929).

[0004] Recently, a method for improving the efficiency of neural induction (neuroectoderm formation) by using an inhibitor of transforming growth factor β (TGFβ) / activin / Nodal signaling and an inhibitor of bone morphogenetic protein (BMP) signaling simultaneously has been developed. This process (also referred to as dual SMAD inhibition) has been shown to promote effective differentiation of human embryonic stem cells to neural ectoderm 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 scheme, neural induction and subsequent differentiation steps are carried out on adherent monolayer cultures, thereby eliminating the highly heterogeneous cell populations produced by the neural induction method based on EB, 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, one disadvantage of adherent monolayer culture is its limited scalability and yield compared to three-dimensional (3D) culture methods. 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 than cells grown in (2D) adherent monolayers. Finally, adherent monolayer culture relies on the use of undefined ingredients and components of animal origin, such as and knockout serum replacement (KSR).

[0005] To address these limitations, approaches 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 J. 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 the EB-based approach, small molecule inhibitors of TGFβ / Activin / Nodal signaling and BMP signaling were applied to EBs formed in static non-adherent cultures.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 varying sizes and requires frequent trituration throughout the process (Crompton LA, Byrne ML, Taylor H, Kerrigan TL, Bru-Mercier G, Badger JL, Barbuti PA, Jo J, Tyler SJ, Allen SJ, Kunath T, Cho K, Caldwell MA. Stepwise, non-adherent differentiation of human pluripotent stem cells to generate basalforebrain cholinergic neurons via hedgehog signaling. 2013 Stem Cell Res. 11(3): 1206-21).

[0006] Improved methods for differentiating pluripotent stem cells to neuroectoderm and further to glial progenitor cells and OPCs are needed. Ideally, such methods should be easily scalable to produce sufficient numbers of differentiated cells for cell therapy applications while consistently and reproducibly generating target cell types. Summary of the invention

[0007] In various embodiments described herein, the present disclosure provides, inter alia, a robust, reliable protocol for differentiating human pluripotent stem cells (e.g., ESCs and iPSCs) into neuroectoderm and glial cells in dynamic suspension culture, which can be performed in bioreactors and is suitable for large-scale culture. A protocol for differentiating human pluripotent stem cells into OPCs by inducing differentiation into neuroectoderm and further differentiation into glial cells in dynamic suspension culture and subsequently differentiating glial cells into OPCs is also provided.

[0008] The present disclosure is based in part on the following discovery: starting material pluripotent stem cells can be aggregated into non-EB aggregates in dynamic suspension, wherein the pluripotent stem cells remain undifferentiated, and subsequently, the aggregates can be induced to differentiate into neural ectoderm while in dynamic suspension by using one or more inhibitors of TGFβ / activin / Nodal signaling and one or more inhibitors of BMP signaling. Compared with adherent culture and EB-based methods, differentiating pluripotent stem cells in dynamic suspension according to the present disclosure provides a scalable, reproducible and controllable method for generating a large number of target neural ectoderm lineage cells from starting materials.

[0009] The methods of the present disclosure reproducibly produce neuroectodermal progenitor cells at day 7 of the differentiation process, reproducibly produce glial progenitor cells at day 21 of the differentiation process, and reproducibly produce OPCs at day 42 of the differentiation process. Day 42 OPCs generated according to the present disclosure are comparable to those using alternative approaches 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) are comparable (in terms of their overall marker expression profile) to OPCs produced according to the disclosure, except that OPCs produced according to the disclosure express lower levels of non-OPC markers, including markers associated with epithelial cyst formation in vitro.

[0010] 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 includes: (a) obtaining a suspension culture of non-embryoid bodies (non-EB) aggregates of undifferentiated human pluripotent stem cells, wherein the human pluripotent stem cells remain undifferentiated; (b) 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, culturing non-EB aggregates from (a) in a dynamic suspension for a first period of time, thereby inducing differentiation into neural ectoderm; (c) in the presence of retinoic acid and at least one Smoothened receptor agonist, culturing non-EB aggregates from (b) in a dynamic suspension for a second period of time; and (d) in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), culturing aggregates from (c) in a dynamic suspension for another period of time until the cells mature into glial progenitor cells.

[0011] In certain embodiments, the first time period is about three to four days. In certain embodiments, the second time period is about four days. In certain embodiments, steps (a) and (b) are performed within about seven to eight days. In certain embodiments, steps (a) to (d) are performed within about twenty-one days.

[0012] 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 move 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 laminin-511E8 fragment. In other embodiments, the substrate is a synthetic substrate, such as, for example, SC substrate.

[0013] In a further embodiment, the method includes the additional step of culturing cells removed from aggregates adhered to a 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 laminin-511E8 fragments. In certain embodiments, adherent culture is performed for about 21 days.

[0014] In another embodiment, the present disclosure provides a method for inducing human pluripotent stem cells to differentiate into neural ectoderm 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 neural ectoderm; 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 neural ectoderm cells.

[0015] In certain embodiments, the first time period is about three to four days. In certain embodiments, the second time period is about four days. In certain embodiments, steps (a) to (c) are performed over a period of about seven to eight days.

[0016] 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 disaggregated and formed into 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 neural ectoderm; (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 another 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] Another embodiment is a differentiated cell population comprising PAX6-positive neuroectodermal cells obtained according to the methods of the present disclosure. In certain embodiments, the PAX6-positive neuroectodermal cells further express one or more markers selected from HES5 and ZBTB16.

[0021] Another embodiment is a differentiated cell population comprising glial progenitor cells obtained according to the methods of 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).

[0022] Another embodiment is a differentiated cell population comprising OPCs obtained according to the methods of the present disclosure. In a preferred embodiment, the OPCs produced 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, OPCs prepared according to the present invention may express NG2, PDGFRα or GD3; a combination of NG2 and PDGFRαa, NG2 and GD3 or PDGFRα and GD3; or a combination of NG2, PDGFRα and GD3. In certain embodiments, the differentiated cell population comprises at least 60% of NG2-positive cells. In certain embodiments, the differentiated cell population comprises at least 70% of NG2-positive cells. In certain embodiments, the differentiated cell population comprises at least 80% of NG2-positive cells. In other embodiments, the differentiated cell population comprises at least 90% of NG2-positive cells. In certain embodiments, the differentiated cell population comprises at least 98% of NG2 positive cells. In certain embodiments, the differentiated cell population comprises at least 60% of PDGFRα positive cells. In certain embodiments, the differentiated cell population comprises at least 70% of PDGFRα positive cells. In certain embodiments, the differentiated cell population comprises at least 80% of PDGFRα positive cells. In other embodiments, the differentiated cell population comprises at least 90% of PDGFRα positive cells. In certain embodiments, the differentiated cell population comprises at least 98% of PDGFRα positive cells. In certain embodiments, the differentiated cell population comprises at least 60% of GD3 positive cells. In certain embodiments, the differentiated cell population comprises at least 70% of GD3 positive cells. In certain embodiments, the differentiated cell population comprises at least 80% of GD3 positive cells. In other embodiments, the differentiated cell population comprises at least 90% of GD3 positive cells. In certain embodiments, the differentiated cell population comprises at least 98% of GD3 positive cells.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a diagram depicting the differentiation of human embryonic stem cells to neuroectoderm (day 7) and further to glial progenitor cells (day 21) and oligodendrocyte progenitor cells (day 42) according to the present disclosure. 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 work equally well in inducing differentiation to neuroectoderm (Example 7).

[0025] Figure 2 Representative photomicrographs of undifferentiated human embryonic stem cells (uhESCs) stained by immunocytochemistry for pluripotency markers according to the present disclosure are shown. The upper and lower rows of photomicrographs each show a single imaging field of uhESCs stained for DAPI, Nanog, and Oct4 (upper row) or DAPI, Nanog, and Sox2 (lower row) and imaged on an IN Cell Analyzer 2000. The scale bar in the lower right figure applies to all images in the figure.

[0026] Figure 3 Shown are representative micrographs of neuroectodermal progenitor cells generated as non-embryoid bodies (non-EB) aggregates in suspension according to the present disclosure and stained by immunocytochemistry. The upper and lower rows of micrographs show aggregates of neuroectodermal progenitor cells generated from two representative experiments and stained by immunocytochemistry for DAPI (left figure), PAX6 (middle figure) and PSA-NCAM (right figure). The stained cell aggregates are imaged on IN Cell Analyzer 2000. The scale bar in the lower right figure is applicable to all images in the figure.

[0027] Figure 4 Representative micrographs of oligodendrocyte progenitor cells produced according to the present disclosure and stained by immunocytochemistry are shown. The upper and lower rows of micrographs show oligodendrocyte progenitor cells produced from two representative experiments and stained by immunocytochemistry for DAPI (left figure) and NG2 (right figure). The stained cells are imaged on IN Cell Analyzer2000. The scale bar in the lower right figure is applicable to all images in the figure.

[0028] Figure 5 The correlation diagram of the gene expression profile of uhESC before and after 24 hours of forming cell aggregates in suspension is shown. Each correlation diagram shows the comparison of the gene expression profile of day -1 (before aggregate formation) relative to day 0 (24 hours after aggregate formation) from two separate experiments. For each figure, the data point represents each of the 76 genes evaluated by Fluidigm qPCR and calculated as standardized ΔCT as described in Example 6. The R square value is shown in the upper left corner of each figure and is calculated based on the best fit line using JMP software (SAS, Cary, NC, USA).

[0029] Figure 6 shows correlation plots of the 7-day gene expression profiles of uhESCs differentiated into neuroectodermal progenitors in suspension using different small molecule combinations. Each correlation plot shows a comparison of the 7-day gene expression profiles of cells treated with SB431542 plus Dorsomorphin versus the alternative small molecule combinations indicated on the y-axis of each graph. For each graph, the data points represent each of the 96 genes evaluated by Fluidigm qPCR and calculated as normalized ΔCT as described in Example 7. R-squared values ​​are shown in the upper left corner of each graph and were calculated based on the best fit line using JMP software (SAS, Cary, NC, USA)

[0030] This description is not intended to be a detailed catalog of all different ways that the present disclosure can be realized or all features that can be added to the present disclosure. For example, the features shown in one embodiment can be incorporated into other embodiments, and the features shown in a specific embodiment can be deleted from the embodiment. Therefore, the present disclosure contemplates that in some embodiments of the present disclosure, any feature or combination of features set forth herein can be excluded or omitted. In addition, according to the present disclosure, it will be obvious to those skilled in the art for the numerous changes and additions of the various embodiments suggested herein, which do not deviate from the present disclosure. In other cases, well-known structures, interfaces and processes are not shown in detail, so as not to make the present invention unnecessarily obscure. It is intended that any part of this specification should not be interpreted as affecting the denial of any part of the full scope of the present invention. Therefore, the following description is intended to illustrate some specific aspects of the present disclosure, rather than exhaustively specifying all its permutations, combinations and variations.

[0031] 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 the present disclosure belongs. The terms used in the description of the disclosure herein are for the purpose of describing specific embodiments only and are not intended to limit the disclosure.

[0032] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety.

[0033] Unless the context indicates otherwise, it is specifically intended that the various features of the present disclosure described herein may be used in any combination. In addition, 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.

[0034] Method disclosed herein may include one or more steps or actions for realizing described method. Without departing from the scope of the present invention, method steps and / or actions may be interchangeable with each other. In other words, unless specific steps or action sequences are required for the correct operation of this aspect, the order and / or use of specific steps and / or actions may be modified without departing from the scope of the present invention.

[0035] As used in the description of the present disclosure and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0036] 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").

[0037] As used herein, the terms "about" and "approximately" when referring to measurable values ​​such as percentages, density, volume, etc., are meant to include variations of ±20%, ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the specified amount.

[0038] As used herein, phrases such as "between X and Y" and "between about X and Y" should 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."

[0039] As used herein, "oligodendrocyte progenitor cell" (OPC) refers to a cell found in the central nervous system that has a neuroectodermal / glial lineage, expresses a characteristic marker neural / glial antigen 2 (NG2), and is capable of differentiating into oligodendrocytes. The OPC prepared according to the method of the present invention may also express one or more markers selected from NG2, PDGFRα, and GD3.

[0040] The terms "glial lineage cells", "glial progenitor cells" and "glial cells" are used interchangeably herein and refer to non-neuronal CNS cells derived from neuroectoderm / 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 γ4 (CACNG4), fatty acid binding protein 7 (FABP7) and sex-determining region Y-box 6 (SOX6).

[0041] The terms "neuroectoderm", "neuroectodermal cells", "neuroectodermal precursors", "neuroectodermal progenitors", "neural progenitor cells" and "neural precursors" 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).

[0042] As used herein, the term "embryoid body" (EB) refers to a three-dimensional cell aggregate derived from a pluripotent stem cell that has undergone spontaneous differentiation toward all three germ layers. When pluripotent stem cells are removed from culture conditions that inhibit differentiation, EBs are formed. 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 toward all three germ layers and the formation of EBs.

[0043] As used herein, the term "non-embryoid aggregates" refers to three-dimensional cell aggregates formed from pluripotent stem cells, wherein 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, undifferentiated cell aggregates are directed to neuroectodermal progenitor cells by removing bFGF and TGFβ at the same time and adding neuroectodermal differentiation factors (e.g., inhibitors of TGFβ / activin / Nodal signaling pathways combined with inhibitors of bone morphogenetic protein signaling pathways).

[0044] As used herein, the term "TGFβ / activin / Nodal signaling inhibitor" refers to a small molecule or protein modulator that can downregulate 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 1 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.

[0045] 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 the 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.

[0046] 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.

[0047] As used herein, the term "undesirable cell type" refers to cells outside the neuroectodermal lineage that can result in the formation of ectopic tissue upon implantation, or in a cyst assay, the formation of one or more cysts, as described herein. In one embodiment, "undesirable cell types" may include cells of the epithelial lineage, such as cells that are positive for CD49f (a marker expressed by both neural progenitor cells and epithelial cells), or cells that are positive for CLDN6 or EpCAM (two markers expressed by both pluripotent cells and epithelial cells).

[0048] As used herein, "implantation" or "transplantation" refers to the administration of a cell population into a target tissue using an appropriate delivery technique (eg, using an injection device).

[0049] As used herein, "subject" refers to an animal or a human.

[0050] As used herein, "a subject in need thereof" refers to an animal or human having damaged tissue in the central nervous system. In one embodiment, the animal or human experiences loss of motor function.

[0051]

[0043] The terms "central nervous system" and "CNS" as used interchangeably herein refer to the complex of nervous tissue that controls one or more activities of the body including, but not limited to, the brain and, in vertebrates, the spinal cord.

[0052] As used herein, "treatment" or "treatment" of a condition or disease is a method for obtaining a beneficial or desired result (preferably including a clinical result) after a patient develops a condition or disease. Beneficial or desired results for a disease include, but are not limited to, one or more of the following: improving a 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 patients suffering from the disease, prolonging survival time, and any combination thereof. Similarly, for the purposes of this disclosure, beneficial or desired results for a condition include, but are not limited to, one or more of the following: improving a condition, curing a condition, reducing the severity of a condition, delaying the progression of a condition, alleviating one or more symptoms associated with a condition, increasing the quality of life of patients suffering from the condition, prolonging survival time, and any combination thereof.

[0053] Propagation and culture of undifferentiated pluripotent stem cells

[0054] 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) system. In another embodiment, the method can be carried out using induced pluripotent stem cells (iPSC). In another embodiment, the method can be carried out using cells derived from H1, H7, H9, H13 or H14 cell lines. In another embodiment, the method can be carried out on a primate pluripotent stem (pPS) cell line. In yet another embodiment, the method can be carried out using undifferentiated stem cells derived from a parthenogenetic organism, which is stimulated to produce an embryo of hESC in the absence of fertilization.

[0055] The propagation and culture methods of undifferentiated pluripotent stem cells have been described previously. Regarding the 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 (EJ Robertson, Ed., IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (PM Wasserman et al., Eds., Academic Press 1993); Embryonic Stem Cell Differentiation in vitro (MV Wiles, Meth. Enzymol. 225: 900, 1993); Properties and Uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (PD Rathjen et al., Reprod. Fertil. Dev. 10: 31, 1998; and RI Freshney, Culture of Animal Cells, Wiley-Liss, New York, 2000).

[0056] 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. Pat. No. 6,800,480). In one embodiment, a conditioned medium containing such factors can be used. Conditioned medium can be obtained by culturing the medium with cells that secrete such factors. Suitable cells include, but are not limited to, irradiated (~4,000Rad) primary mouse embryonic fibroblasts, telomere-derived mouse fibroblasts, or fibroblast-like cells derived from pPS cells (U.S. Pat. No. 6,642,048). The culture medium can be adapted by plating a feeder layer in a serum-free medium (e.g., knockout DMEM supplemented with 20% serum replacement and 4ng / mL bFGF). The medium that has been acclimated for 1-2 days can be supplemented with additional bFGF and used to support pPS cell culture for 1-2 days (see, eg, WO 01 / 51616; Xu et al., (2001) Nat. Biotechnol. 19:971).

[0057] Alternatively, fresh or unadapted culture medium may be used that has been supplemented with other factors that promote proliferation of cells in an undifferentiated form (e.g., fibroblast growth factor or forskolin). Non-limiting examples include basal medium 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 greater than other systems (see, e.g., WO 03 / 020920). In one embodiment, undifferentiated pluripotent cells, such as hES cells, can be cultured in a medium comprising 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).

[0058] Undifferentiated pluripotent cells can be cultured on a layer of feeder cells, 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 various 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), fallopian tube epithelial cells (see, e.g., Richards et al., (2002) Nat. Biotechnol., 20:933), foreskin fibroblasts (see, e.g., Amit et al., (2003) Biol. Reprod. 68:2150), and endometrial cells (see, e.g., Lee et al., (2005) Biol. Reprod. 72(1):42).

[0059] Various solid surfaces can be used to cultivate undifferentiated pluripotent cells. These solid surfaces include but are not limited to standard commercially available tissue culture bottles 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 plates. Solid surfaces suitable for growing undifferentiated pluripotent cells can be made of a variety of materials, including but not limited to glass or plastics, such as polystyrene, polyvinyl chloride, polycarbonate, polytetrafluoroethylene, polyester film (melinex), thermanox or a combination thereof. Suitable surfaces can include one or more polymers, such as one or more acrylates. The solid surface can be a three-dimensional shape. Non-limiting examples of three-dimensional solid surfaces have been previously described, such as in U.S. Patent Publication No. 2005 / 0031598.

[0060] Undifferentiated stem cells can also be grown on a growth substrate without a feeder layer. The growth substrate can be 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.

[0061] Undifferentiated stem cells can be passaged or subcultured using a variety of methods (e.g., using collagenase, or, for example, manually scraping). Undifferentiated stem cells can be isolated by enzymatic means to produce a single cell suspension (e.g., using (distributed by Sigma Aldrich, MO) or 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)) for subculture.

[0062] In one embodiment, a plurality of undifferentiated stem cells are seeded or subcultured at a seeding density that allows the cells to reach confluence within about three to about ten days. In one embodiment, the seeding density may be about 6.0 x 10 3 cells / cm 2 to about 5.0x10 5 cells / cm 2 , for example about 1.0x10 4 cells / cm 2 , for example about 5.0x10 4cells / cm 2 , for example about 1.0x10 5 cells / cm 2 , or for example about 3.0x10 5 cells / cm 2 In another embodiment, the seeding density can be about 6.0 x 10 3 cells / cm 2 to about 1.0x10 4 cells / cm 2 Growth surface, for example, about 6.0x10 3 cells / cm 2 to about 9.0x10 3 cells / cm 2 , for example about 7.0x10 3 cells / cm 2 to about 1.0x10 4 cells / cm 2 , for example about 7.0x10 3 cells / cm 2 to about 9.0x10 3 cells / cm 2 , or for example about 7.0x10 3 cells / cm 2 About 8.0x10 3 cells / cm 2 In yet another embodiment, the seeding density may be about 1.0 x 10 4 cells / cm 2 to about 1.0x10 5 cells / cm 2 Growth surface, for example, about 2.0x10 4 cells / cm 2 to about 9.0x10 4 cells / cm 2 , for example about 3.0x10 4 cells / cm 2 About 8.0x10 4 cells / cm 2 , for example about 4.0x10 4 cells / cm 2 to about 7.0x10 4 cells / cm 2 , or for example about 5.0x10 4 cells / cm 2 to about 6.0x10 4 cells / cm 2 In one embodiment, the seeding density can be about 1.0 x 105 cells / cm 2 to about 5.0x10 5 cells / cm 2 Growth surface, for example, about 1.0x10 5 cells / cm 2 About 4.5x10 5 cells / cm 2 , for example about 1.5x10 5 cells / cm 2 to about 4.0x10 5 cells / cm 2 , for example about 2.0x10 5 cells / cm 2 About 3.5x10 5 cells / cm 2 , or for example about 2.5x10 5 cells / cm 2 to about 3.0x10 5 cells / cm 2 Growth surface.

[0063] According to the methods of the present disclosure, stem cells may be cultured using any of a variety of suitable cell culture and subculture techniques. For example, the culture medium may be completely replaced daily, starting about 2 days after the cells are subcultured. In one embodiment, when the culture reaches about 90% colony coverage, one or more suitable reagents such as The cells are separated (to achieve a single cell suspension for quantitation) and plated for subsequent culture. In one embodiment, the undifferentiated stem cells can then be subcultured before being plated on a suitable growth substrate (e.g., 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, the undifferentiated stem cells can be subcultured using collagenase IV and expanded on recombinant laminin. In another embodiment, the undifferentiated stem cells can be subcultured using collagenase IV and expanded on recombinant laminin. In one embodiment, undifferentiated stem cells can be expanded using ReLeSR TM Subcultured and expanded on recombinant human laminin-521.

[0064] For seeding undifferentiated stem cells, the seeding density can be about 6.0x10 3 cells / cm 2 to about 5.0x10 5 cells / cm 2 , for example about 1.0x10 4 cells / cm2 , for example about 5.0x10 4 cells / cm 2 , for example about 1.0x10 5 cells / cm 2 , or for example about 3.0x10 5 cells / cm 2 In another embodiment, the seeding density can be about 6.0 x 10 3 cells / cm 2 to about 1.0x10 4 cells / cm 2 Growth surface, for example, about 6.0x10 3 cells / cm 2 to about 9.0x10 3 cells / cm 2 , for example about 7.0x10 3 cells / cm 2 to about 1.0x10 4 cells / cm 2 , for example about 7.0x10 3 cells / cm 2 to about 9.0x10 3 cells / cm 2 , or for example about 7.0x10 3 cells / cm 2 About 8.0x10 3 cells / cm 2 In yet another embodiment, the seeding density may be about 1.0 x 10 4 cells / cm 2 to about 1.0x10 5 cells / cm 2 Growth surface, for example, about 2.0x10 4 cells / cm 2 to about 9.0x10 4 cells / cm 2 , for example about 3.0x10 4 cells / cm 2 About 8.0x10 4 cells / cm 2 , for example about 4.0x10 4 cells / cm 2 to about 7.0x10 4 cells / cm 2 , or for example about 5.0x10 4 cells / cm 2 to about 6.0x10 4 cells / cm2 In one embodiment, the seeding density can be about 1.0 x 10 5 cells / cm 2 to about 5.0x10 5 cells / cm 2 Growth surface, for example, about 1.0x10 5 cells / cm 2 About 4.5x10 5 cells / cm 2 , for example about 1.5x10 5 cells / cm 2 to about 4.0x10 5 cells / cm 2 , for example about 2.0x10 5 cells / cm 2 About 3.5x10 5 cells / cm 2 , or for example about 2.5x10 5 cells / cm 2 to about 3.0x10 5 cells / cm 2 Growth surface.

[0065] Neural induction of undifferentiated pluripotent stem cells

[0066] The present disclosure provides a method for differentiating pluripotent stem cells into neural ectoderm and further differentiating into glial progenitor cells and OPCs using small molecules and protein regulators of TGFβ / activator / Nodal signaling and BMP signaling. Not bound by any particular theory, the inventors found that starting material pluripotent stem cells can be aggregated into non-EB aggregates in dynamic suspensions, wherein pluripotent stem cells remain undifferentiated, and then can be induced to differentiate into neural ectoderm in dynamic suspensions by using one or more inhibitors of TGFβ / activator / Nodal signaling and one or more inhibitors of BMP signaling (dual SMAD inhibition). Compared with adherent culture and EB-based methods, dynamic suspensions provide scalable, reproducible and controllable processes for producing a large number of cells from starting materials. The method of dual SMAD inhibition in dynamic suspensions is described in detail herein.

[0067] 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, thereby starting 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 1 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.

[0068] 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 derivatives thereof. 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 derivatives thereof.

[0069] In one embodiment, a method comprises obtaining non-EB aggregates comprising pluripotent stem cells maintained in an undifferentiated state; culturing the non-EB aggregates in a dynamic suspension for a first period of time in the presence of 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, such as Figure 1 In one embodiment, the first time period and the second time period can each be 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.

[0070] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of SB431542 at a concentration of 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 comprises culturing non-EB aggregates in dynamic suspension in the presence of SB431542, wherein the concentration of SB431542 is about 1 μM to about 20 μM, such as about 1 μM to about 13 μM, such as about 8 μM to about 20 μM, such as about 8 μM to about 13 μM, or such as about 9 μM to about 11 μM. In yet another embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of SB431542, wherein the concentration of SB431542 is about 20 μM to about 40 μM, such as about 20 μM to about 33 μM, such as about 28 μM to about 40 μM, such as about 28 μM to about 33 μM, or such as about 29 μM to about 31 μM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of SB431542 at a concentration of about 40 μM to about 60 μM, such as about 40 μM to about 53 μM, such as about 48 μM to about 55 μM, such as about 48 μM to about 53 μM, or such as about 49 μM to about 51 μM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of SB431542 at a concentration of about 60 μM to about 80 μM, such as about 60 μM to about 73 μM, such as about 68 μM to about 75 μM, such as about 68 μM to about 73 μM, or such as about 69 μM to about 71 μM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of SB431542 at a concentration of about 80 μM to about 100 μM, such as about 80 μM to about 93 μM, such as about 88 μM to about 95 μM, such as about 88 μM to about 93 μM, or such as about 89 μM to about 91 μM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of SB431542 at a concentration of about 10 μM.

[0071] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of an ALK5 inhibitor at a concentration of 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 comprises culturing non-EB aggregates in dynamic suspension in the presence of about 10 μM of an ALK5 inhibitor.

[0072] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of LY364947 at a concentration of 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 comprises culturing non-EB aggregates in dynamic suspension in the presence of about 10 μM LY364947.

[0073] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of GW788388 at a concentration of 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 comprises culturing non-EB aggregates in dynamic suspension in the presence of about 10 μM GW788388.

[0074] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of A-77-01, wherein the concentration of A-77-01 is about 250nM to about 250μM, such as about 1μM, about 10μM, about 50μM, about 100μM, about 150μM, or about 200μM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of about 10μM A-77-01.

[0075] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of A-83-01 at a concentration of 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 comprises culturing non-EB aggregates in dynamic suspension in the presence of about 10 μM A-83-01.

[0076] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of SB505124 at a concentration of 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 comprises culturing non-EB aggregates in dynamic suspension in the presence of about 10 μM SB505124.

[0077] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of Dorsomorphin at a concentration of 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. In another embodiment, a method comprises culturing non-EB aggregates in a dynamic suspension in the presence of Dorsomorphin at a concentration of about 0.2 μM to about 1 μM, for example, about 0.2 μM to about 0.9 μM, for example, about 0.3 μM to about 0.8 μM, for example, about 0.4 μM to about 0.7 μM, or for example, about 0.5 μM to about 0.6 μM. In yet another embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of Dorsomorphin, wherein the concentration of Dorsomorphin is about 1 μM to about 10 μM, such as about 1 μM to about 9 μM, such as about 2 μM to about 8 μM, such as about 3 μM to about 7 μM, or such as about 4 μM to about 6 μM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of Dorsomorphin, wherein the concentration of Dorsomorphin is about 10 μM to about 20 μM, such as about 10 μM to about 19 μM, such as about 12 μM to about 18 μM, such as about 13 μM to about 17 μM, or such as about 14 μM to about 16 μM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of Dorsomorphin, wherein the concentration of Dorsomorphin is about 2 μM.

[0078] In one embodiment, a method comprises culturing non-EB aggregates in a dynamic suspension in the presence of an ALK2 inhibitor at a concentration of about 1 nM to about 20 μM, e.g., about 10 nM, about 50 nM, about 100 nM, about 150 nM, about 200 nM, about 500 nM, about 1 μM, about 5 μM, about 10 μM, or about 15 μM.

[0079] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of DMH-1 at a concentration of about 1 μM to about 10 μM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of about 2 μM DMH-1.

[0080] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of K02288 at a concentration of about 1 μM to about 10 μM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of about 2 μM K02288.

[0081] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of ML347 at a concentration of about 1 μM to about 10 μM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of about 2 μM ML347.

[0082] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of Purmorphamine at a concentration of 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 comprises culturing non-EB aggregates in dynamic suspension in the presence of Purmorphamine at a concentration of about 0.05 μM to about 0.1 μM, such as about 0.06 μM to about 0.09 μM, or such as about 0.07 μM to about 0.08 μM. In another embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of Purmorphamine, the concentration of Purmorphamine being about 0.1 μM to about 1 μM, for example, about 0.2 μM to about 0.9 μM, for example, about 0.3 μM to about 0.8 μM, for example, about 0.4 μM to about 0.7 μM, or for example, about 0.5 μM to about 0.6 μM. In another embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of Purmorphamine, the concentration of Purmorphamine being about 1 μM to about 5 μM, for example, about 1 μM to about 4 μM, for example, about 2 μM to about 5 μM, or for example, about 2 μM to about 4 μM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of Purmorphamine at a concentration of about 0.5 μM.

[0083] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of a Smoothened agonist at a concentration of 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.

[0084] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of SAG, wherein the concentration of SAG is about 10nM to about 1μM, such as about 10nM to about 100nM, such as about 100nM to about 500nM, or such as about 500nM to about 1000nM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of about 0.5μM SAG.

[0085] In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of SHH protein at a concentration of about 2.5 nM to about 250 nM, such as about 2.5 nM to about 10 nM, such as about 10 nM to about 100 nM, or such as about 100 nM to about 250 nM. In one embodiment, a method comprises culturing non-EB aggregates in dynamic suspension in the presence of about 25 nM SHH protein.

[0086] Any cell culture vessel or reactor suitable for dynamic suspension culture may be used for the differentiation step contemplated 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 a stirring mechanism, such as a magnetic or mechanically driven stirring bar or paddle, a shaking mechanism (usually connected to the vessel externally) or an inversion mechanism (i.e., a device that rotates the vessel to change the direction of gravity on the cells).

[0087] Vessels suitable for suspension culture in process development include the common range of commercially available rotators, shake bags or shake flasks. Exemplary bioreactors suitable for commercial production include VerticalWheel TM Bioreactor (PBS Biotech, Camarillo, CA).

[0088] OPC Composition

[0089] The methods of the present disclosure can be used to obtain compositions comprising oligodendrocyte progenitor cells (OPCs) suitable for cell therapy. 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 undesirable 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.OncolRep.2017Aug;38(2):875-885;Huang L,Yang Y,Yang F,Liu S,Zhu Z,Lei Z,GuoJ.Functions of EpCAM in physiological processes and diseases(Review).Int JMol Med.2018Oct;42(4):1771-1785).

[0090] In certain embodiments, the OPCs produced according to the present disclosure are offspring of in vitro differentiation of human pluripotent stem cells. In certain embodiments, the OPCs obtained according to the present disclosure are offspring of in vitro differentiation of human embryonic stem cells. In other embodiments, the OPCs obtained according to the present disclosure are offspring of in vitro differentiation of induced pluripotent stem (iPS) cells.

[0091] One or more characteristics of the obtained OPC population can be determined by quantifying various cell markers using flow cytometry, for example, to determine the percentage of a cell population that is positive for a particular marker or a group of markers, or to identify undesirable cell types present in the OPC population. In one embodiment, the OPC prepared according to the method of the present invention expresses one or more markers selected from NG2, PDGFRα and GD3.

[0092] The OPC population obtained according to the present disclosure may contain 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, the OPC colony obtained according to the present disclosure may include about 45% to about 75% NG2-positive cells, such as about 45% to about 50%, such as about 50% to about 55%, such as about 55% to about 60%, such as about 60% to about 65%, such as about 65% to about 70%, such as about 70% to about 75%, such as about 50% to about 70%, such as about 55% to about 65%, or such as about 58% to about 63% NG2-positive cells. In other embodiments, the OPC colony obtained according to the present disclosure may include about 60% to about 90% NG2-positive cells, such as about 60% to about 65%, such as about 65% to about 70% positive cells. The OPC population obtained according to the present disclosure may 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, the OPC population obtained according to the present disclosure may include about 45% to about 75% PDGFRα-positive cells, such as about 45% to about 50%, such as about 50% to about 55%, such as about 55% to about 60%, such as about 60% to about 65%, such as about 65% to about 70%, such as about 70% to about 75%, such as about 50% to about 70%, such as about 55% to about 65%, or such as about 58% to about 63% PDGFRα-positive cells. In other embodiments, the OPC population obtained according to the present disclosure may include about 60% to about 90% PDGFRα-positive cells, such as about 60% to about 65%, such as about 65% to about 70% positive cells.The OPC population obtained according to the present disclosure may 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, the OPC population obtained according to the present disclosure may comprise about 45% to about 75% of GD3-positive cells, such as about 45% to about 50%, such as about 50% to about 55%, such as about 55% to about 60%, such as about 60% to about 65%, such as about 65% to about 70%, such as about 70% to about 75%, such as about 50% to about 70%, such as about 55% to about 65%, or such as about 58% to about 63% of GD3-positive cells. In other embodiments, the OPC population obtained according to the present disclosure may comprise about 60% to about 90% of GD3-positive cells, such as about 60% to about 65%, such as about 65% to about 70% of positive cells.

[0093] In one embodiment, the OPC population obtained according to the present disclosure is capable of forming less than or equal to four epithelial cysts per 100,000 cells in a cyst assay as described in Example 8 of the present disclosure. In another embodiment, the OPC population obtained according to the present disclosure is capable of forming less than or equal to three epithelial cysts per 100,000 cells in a cyst assay. In another embodiment, the OPC population obtained according to the present disclosure is capable of forming less than or equal to two epithelial cysts per 100,000 cells in a cyst assay. In yet another embodiment, the OPC population obtained according to the present disclosure is capable of forming less than or equal to one epithelial cyst per 100,000 cells in a cyst assay as described in Example 8 of the present disclosure.

[0094] Undesirable cell types

[0095] The OPC population obtained according to the present disclosure contains low levels of undesirable cell types, such as measured by flow cytometry quantification of markers associated with undesirable cell types. In a non-limiting example, the OPCs obtained at day 42 according to the present disclosure comprise 0% to 4% of cells expressing epithelial cell-related markers EpCAM, CD49f, and CLDN6 (Example 5, Table 2).

[0096] Markers associated with undesirable cell types may include less than about 20% of undesirable cell types, 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 undesirable cell types. In another embodiment, the cell group may include about 15% to about 20% of undesirable cell types, such as about 19% to about 20%, such as about 18% to about 20%, such as about 17% to about 20%, such as about 16% to about 20%, such as about 15% to about 19%, or such as about 16% to about 18% of undesirable cell types. In another embodiment, the cell group may include about 10% to about 15% of undesirable cell types, such as about 14% to about 15%, such as about 13% to about 15%, such as about 12% to about 15%, such as about 11% to about 15%, or such as about 12% to about 14% of undesirable cell types. In one embodiment, the cell group may include about 1% to about 10% of undesirable cell types, such as about 2% to about 10%, such as about 1% to about 9%, such as about 2% to about 8%, such as about 3% to about 7%, or such as about 4% to about 6% of undesirable cell types. In one embodiment, the cell population may include about 0.1% to about 1% of undesirable cell types, such as about 0.2% to about 1%, such as about 0.1% to about 0.9%, such as about 0.2% to about 0.8%, such as about 0.3% to about 0.7%, or such as about 0.4% to about 0.6% of undesirable cell types. In one embodiment, the cell population may include about 0.01% to about 0.1% of undesirable cell types, such as about 0.02% to about 0.1%, such as about 0.01% to about 0.09%, such as about 0.02% to about 0.08%, such as about 0.03% to about 0.07%, or such as about 0.04% to about 0.06% of undesirable cell types. In one embodiment, low levels of undesirable cell types can mean that there are less than about 15% of undesirable cell types.

[0097] In one embodiment, the undesired cell types may include cells expressing one or more markers selected from CD49f, CLDN6, or EpCAM.

[0098] preparation

[0099] 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 suitable for cryopreservation at a temperature of -80°C to -195°C or below -80°C to -195°C.

[0100] OPC compositions according to the present disclosure may be formulated for use by direct injection into the spinal cord of the experimenter. In one embodiment, OPC compositions according to the present disclosure may be formulated for use in the brain, intraventricular, intrathecal, intranasal or cisternal administration to the experimenter. In one embodiment, OPC compositions according to the present disclosure may be formulated for use by direct injection into the infarct cavity in the brain of the experimenter or the infarct cavity direct injection in the brain of the experimenter next to the experimenter. In one embodiment, compositions according to the present disclosure may be formulated for use by implantation. In one embodiment, compositions according to the present disclosure may be formulated as a solution.

[0101] The OPC composition according to the present disclosure may include about 1x10 6 About 5x10 8 cells / ml, e.g. about 1x10 6 cells / ml, e.g. about 2x10 6 cells / ml, e.g. about 3x10 6 cells / ml, e.g. about 4x10 6 cells / ml, e.g. about 5x10 6 cells / ml, e.g. about 6x10 6 cells / ml, e.g. about 7x10 6 cells / ml, e.g. about 8x10 6 cells / ml, e.g. about 9x10 6 cells / ml, e.g. about 1x10 7 cells / ml, e.g. about 2x10 7 cells / ml, e.g. about 3x10 7 cells / ml, e.g. about 4x10 7 cells / ml, e.g. about 5x10 7 cells / ml, e.g. about 6x10 7 cells / ml, e.g. about 7x10 7 cells / ml, e.g. about 8x10 7 cells / ml, e.g. about 9x10 7 cells / ml, e.g. about 1x10 8 cells / ml, e.g. about 2x108 cells / ml, e.g. about 3x10 8 cells / ml, e.g. about 4x10 8 cells / ml, or for example about 5x10 8 In another embodiment, the composition according to the present disclosure may contain about 1×10 8 About 5x10 8 cells / ml, e.g. about 1x10 8 About 4x10 8 cells / ml, e.g. about 2x10 8 About 5x10 8 cells / ml, e.g. about 1x10 8 About 3x10 8 cells / ml, e.g. about 2x10 8 About 4x10 8 cells / ml, or for example about 3x10 8 About 5x10 8 In yet another embodiment, the composition according to the present disclosure may comprise about 1×10 7 About 1x10 8 cells / ml, e.g. about 2x10 7 About 9x10 7 cells / ml, e.g. about 3x10 7 About 8x10 7 cells / ml, e.g. about 4x10 7 About 7x10 7 cells / ml, or for example about 5x10 7 About 6x10 7 In one embodiment, the composition according to the present disclosure may contain about 1×10 6 About 1x10 7 cells / ml, e.g. about 2x10 6 About 9x10 6 cells / ml, e.g. about 3x10 6 About 8x10 6 cells / ml, e.g. about 4x10 6 About 7x10 6 cells / ml, or for example about 5x10 6 About 6x10 6 In yet another embodiment, the composition according to the present disclosure may comprise at least about 1×10 6 cells / ml, for example at least about 2 x 10 6 cells / ml, such as at least about 3x106 cells / ml, for example at least about 4 x 10 6 cells / ml, for example at least about 5 x 10 6 cells / ml, for example at least about 6 x 10 6 cells / ml, for example at least about 7 x 10 6 cells / ml, for example at least about 8 x 10 6 cells / ml, for example at least about 9x10 6 cells / ml, for example at least about 1 x 10 7 cells / ml, for example at least about 2 x 10 7 cells / ml, such as at least about 3x10 7 cells / ml, for example at least about 4 x 10 7 cells / ml, or for example at least about 5 x 10 7 In one embodiment, the composition according to the present disclosure may contain up to about 1×10 8 cells or more, for example up to about 2 x 10 8 cells / ml or more, for example up to about 3x10 8 cells / ml or more, for example up to about 4 x 10 8 cells / ml or more, for example up to about 5x10 8 cells / ml or more, or for example up to about 6 x 10 8 cells / ml.

[0102] In one embodiment, an OPC composition according to the present disclosure may include about 4 x 10 7 About 2x10 8 cells / ml.

[0103] In another embodiment, the OPC composition according to the present disclosure can have a volume of about 10 microliters to about 5 milliliters, for example, about 20 microliters, for example, about 30 microliters, for example, about 40 microliters, for example, about 50 microliters, for example, about 60 microliters, for example, about 70 microliters, for example, about 80 microliters, for example, about 90 microliters, for example, about 100 microliters, for example, about 200 microliters, for example, about 300 microliters, for example, about 400 microliters, for example, about 500 microliters, for example, about 600 microliters, for example, about 700 microliters, for example, about 800 microliters, for example, about 900 microliters, for example, about 1 milliliter, for example, about 1.5 milliliters, for example, about 2 milliliters, for example, about 2.5 milliliters, for example, about 3 milliliters, for example, about 3.5 milliliters, for example, about 4 milliliters, or for example about 4.5 milliliters. In one embodiment, according to the composition of the present disclosure, there can be a volume of about 10 microlitres to about 100 microlitres, for example, about 20 microlitres to about 90 microlitres, for example, about 30 microlitres to about 80 microlitres, for example, about 40 microlitres to about 70 microlitres, or for example, about 50 microlitres to about 60 microlitres. In another embodiment, according to the composition of the present disclosure, there can be a volume of about 100 microlitres to about 1 milliliter, for example, about 200 microlitres to about 900 microlitres, for example, about 300 microlitres to about 800 microlitres, for example, about 400 microlitres to about 700 microlitres, or for example, about 500 microlitres to about 600 microlitres. In another embodiment, according to the composition of the present disclosure, there can be a volume of about 1 milliliter to about 5 milliliters, for example, about 2 milliliters to about 5 milliliters, for example, about 1 milliliter to about 4 milliliters, for example, about 1 milliliter to about 3 milliliters, for example, about 2 milliliters to about 4 milliliters, or for example, about 3 milliliters to about 5 milliliters. In one embodiment, according to the OPC composition of the present disclosure, there can be a volume of about 20 microlitres to about 500 microlitres. In another embodiment, according to the OPC composition of the present disclosure, there can be a volume of about 50 microlitres to about 100 microlitres. In another embodiment, according to the OPC composition of the present disclosure, there can be a volume of about 50 microlitres to about 200 microlitres. In another embodiment, according to the OPC composition of the present disclosure, there can be a volume of about 20 microlitres to about 400 microlitres. In one embodiment, according to the OPC composition of the present disclosure, there can be a container, and this container is configured for cryopreservation or for using to the experimenter having this need. In one embodiment, the container can be a pre-filled syringe.

[0104] How to use

[0105] The 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, the OPC cell group according to the present disclosure can be injected or implanted into a subject in need thereof. In one embodiment, the cell group 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.

[0106] In one embodiment, the cell population according to the present disclosure can induce myelination of exposed axons at the implantation site of the subject. In one embodiment, the cell population produced according to the method of the present disclosure can show improved implantation and migration ability. In one embodiment, the cell population produced according to the method of the present disclosure can be able to improve the post-injury repair or regeneration of neural tissue in the subject.

[0107] According to the cell population of the present disclosure, it is possible to improve the sensory function of the subject in need of treatment after the colony is implanted into the subject. The International Standard for Neurological Classification of Spinal Cord Injury (ISNCSCI) test can be used to assess the improvement of sensory function, such as determining the level of sensation of acupuncture and light touch on the right and left sides. According to the cell population of the present disclosure, it is possible to improve the motor function of the subject in need of treatment after the colony is implanted into the subject. The motor function improved can be assessed using the ISNCSCI test, such as determining the level of complete paralysis of the right and left sides, perceptible or visible contraction, active movement, full range of motion against gravity, and sufficient resistance.

[0108] The cell populations according to the present disclosure are capable of reducing the volume of injury-induced central nervous system parenchymal cavitations in 12 months or less. In one embodiment, the cell populations according to the present disclosure are capable of reducing the volume of injury-induced central nervous system parenchymal cavitations in a subject in 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.

[0109] Having now generally described the invention, the same will be more readily understood by reference to the following examples which are provided by way of illustration and are not intended to limit the disclosure unless otherwise specified. Example

[0110] Example 1 - Cultivation and expansion of undifferentiated human embryonic stem cells

[0111] 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 (StemCell Technologies #85850). The medium was completely replaced every day until the cells reached approximately 80-90% confluence, and then ReLeSR TM uhESCs were passaged using ReLeSR TM The uhESC cells were plated onto new laminin-521 coated 225 cm 2 flasks and resume daily media changes two days after seeding. Cultured uhESCs from WCBs were expanded in this manner for 2 to 5 passages, depending on the experiment, prior to differentiation into neuroectodermal progenitors as described in Example 2.

[0112] Example 2 - Method for Differentiating Human Embryonic Stem Cells into Neuroectodermal Progenitor Cells in Dynamic Suspension Culture

[0113] Day -1 : Expanded uhESCs (approximately 90% confluent) were isolated and used (Stem Cell Technologies #07920) to form a single cell suspension, allowing accurate cell counting and uniform seeding density. Disaggregated uhESCs were then plated at 1x10 6The concentration of viable cells / mL was inoculated into a PBS-0.1 or PBS-0.5 microbioreactor system (PBS Biotech) (set to rotate at 35RPM or 25RPM (day -1) respectively) for dynamic suspension culture. The cells were inoculated in a glial progenitor medium (GPM; DMEM / F12 (Gibco catalog number 10565-018) supplemented with 2% B27 supplement (Gibco catalog number 17504-044) and 0.04μg / ml triiodothyronine (Sigma catalog number T5516-1MG)) and supplemented with 10μM Rho kinase inhibitor (RI, Tocris catalog number 1254) to support cell survival. Undifferentiated hESC medium (such as in Example 1) 1: 1 mixture. Within the first 24 hours, small uniform aggregates of uhESC were formed. In contrast to embryoid body (EB) formation, cells in small aggregates did not initiate spontaneous differentiation but maintained their pluripotency, as confirmed by marker expression measured by qPCR ( Figure 5 ).

[0114] Day 0-3 To initiate differentiation into neuroectoderm, small non-EB aggregates were cultured in dynamic suspension in GPM supplemented with 10 μM SB431542 (Sigma-Aldrich, catalog number S4317) and 2 μM Dorsomorphin (Sigma-Aldrich, catalog number P5499) in a PBS-0.1 or PBS-0.5 microbioreactor rotating at 45 or 32 RPM, respectively, for the next four days. The medium was replenished daily by allowing the aggregates to settle, removing 70-80% of the spent medium and replacing it with an equal volume of GPM supplemented with 10 μM SB431542 and 2 μM Dorsomorphin.

[0115] Day 4-6 : The cells were further cultured for another three days 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 (SigmaAldrich, catalog number A4544) at 45 RPM (PBS-0.1 micro-bioreactor) or 32 RPM (PBS-0.5 micro-bioreactor). The medium was replenished every day by allowing the aggregates to settle, removing 70-80% of the spent medium and replacing it with an equal volume of GPM supplemented with 0.5 μM Purmorphamine, 1 μM retinoic acid and 150 μM ascorbic acid.

[0116] Day 7 : A subset of 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, cells expressed markers characteristic of the neuroectoderm (Table 2, Figure 3 ). The remaining day 7 cells were differentiated into glial lineage cells and further differentiated into oligodendrocyte progenitor cells as described in Examples 3 and 4, respectively.

[0117] Example 3 - Method for Differentiating Human Embryonic Stem Cells into Glial Lineage Cells in Dynamic Suspension Culture

[0118] As described in Example 2, uhESC is differentiated into neuroectoderm / neural progenitor cells (days 0-6). On the 7th day, differentiation medium is modified to be supplemented with 20ng / mL human basic fibroblast growth factor (hbFGF, ThermoFisher, catalog number PHG0263), 10ng / mL epidermal growth factor (EGF, Thermo Fisher, catalog number PHG0311) and 10μM RI GPM to initiate differentiation into glial progenitor cells. For the next two weeks (days 8-20), cell aggregates are maintained in dynamic suspension with 45rpm (PBS-0.1 micro-bioreactor) or 32RPM (PBS-0.5 micro-bioreactor) in GPM supplemented with 20ng / mL bFGF and 10ng / mL EGF, and culture medium is supplemented every day using gravity sedimentation and 70-80% culture medium exchange. On the 14th day, 10μM RI is also added to fresh culture medium.

[0119] A subset of 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).

[0120] Example 4 - Method for Differentiating Human Embryonic Stem Cells into Oligodendrocyte Progenitor Cells

[0121] The glial lineage precursor cells obtained in Example 3 are further differentiated into oligodendrocyte progenitor cells. The differentiation protocol for days 0-20 is as described in Examples 2 and 3. On day 21, the aggregates are transferred from dynamic suspension culture to adherent culture on a tissue culture container coated with recombinant human laminin-521 (rhLN-521). For example, starting from a 1x PBS-0.1L microbioreactor with 60mL of aggregate suspension, 60mL of culture is divided into 2x T75 flasks, each flask having 30mL of culture. Starting from day 21 and continuing until the end of the differentiation process, cells are cultured in GPM supplemented with 20ng / mL EGF and 10ng / mL platelet-derived growth factor-AA (PDGF-AA, PeproTech, catalog number AF-100-13A), and complete medium is replaced every other day. On days 28 and 35, TrypLE TM Cell cultures were detached using Select (Thermo Fisher, catalog number A12859-01), counted, and plated at 4 × 10 4 viable cells / cm 2 Inoculate onto rhLN-521 coated containers. GPM was changed every other day from day 35 until harvest on day 42.

[0122] A subset of 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 expression of markers was significantly higher than that of the control group as measured by the three analytical methods (Table 1, Table 2, Figure 4 ), the differentiated cells expressed markers characteristic of oligodendrocyte progenitor cells.

[0123] OPCs were harvested on day 42. TM Select cells were detached from the container, counted and reconstituted in CryoStor10 (BioLife Solutions, catalog number 210102) and subsequently cryopreserved.

[0124] Example 5 - Characterization of differentiated cell populations by immunocytochemistry and flow cytometry

[0125] Flow cytometry and immunocytochemistry (ICC) can be used to detect and characterize different aspects of protein marker expression in cell populations. Although flow cytometry can be used to quantify the percentage of individual cells that display a given protein marker profile in a population, ICC provides additional information about the subcellular localization of each protein marker and can be applied to single cells or cell aggregates. By using one or both of these protein profile analysis methods, we tracked the differentiation of human embryonic stem cells according to the methods of the present disclosure to neuroectodermal progenitors, glial progenitors, and oligodendrocyte progenitors.

[0126] For human embryonic stem cells that differentiate into neuroectodermal progenitor cells in suspension, protein marker expression in starting material (undifferentiated pluripotent cells) and differentiated 7th day cell aggregates was characterized by ICC. At room temperature (RT), attached pluripotent cells and cell aggregates were fixed in 4% paraformaldehyde (PFA) for 30 minutes. Fixed cells and aggregates were washed with phosphate buffered saline (PBS), and then fixed aggregates were placed in increasing concentrations of sucrose solution (10%, 20% and 30% weight / volume) at room temperature for 30 minutes, 30 minutes at room temperature and overnight at 4°C. After sucrose replacement, aggregates were embedded in Tissue-Tek optimal cutting temperature (OCT) solution (Sakura FinetekUSA#4583) and frozen at -80°C. OCT-embedded aggregates were warmed to -20°C, cut into 30 μm sections using a cryostat (model CM3050 S, Leica Biosystems, Buffalo Grove, IL, USA), and mounted on poly-L-lysine (Sigma-Aldrich #P4707)-coated slides. For immunocytochemical staining, fixed adherent cells and slide-mounted aggregate sections were permeabilized and incubated in PBS containing 0.1% Triton TM The cells were blocked in a blocking solution consisting of X-100 / 2% normal goat serum / 1% bovine serum albumin for 2 hours at room temperature (RT). After permeabilization and blocking, adherent cells and aggregates were sliced ​​and plated on a plate without Triton X-100. TMX-100 and incubated overnight at 4°C in blocking solution containing primary antibodies specific for 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 three times with PBS and then incubated in a blocking solution without Triton. TM The sections were incubated in blocking solution of X-100 with secondary antibodies specific for the selected primary antibodies and 4',6-diamidino-2-phenylindole (DAPI) counterstain for 1 h at room temperature in the dark. The adherent cells and aggregate sections were washed three times with PBS and imaged using IN Cell Analyzer 2000 (GE Healthcare, Pittsburgh, PA, USA).

[0127] Figure 2 and Figure 3 Representative ICC data for the starting pluripotent cell population and day 7 neuroectodermal progenitors are shown, respectively. Figure 2 As shown, the starting population of undifferentiated human embryonic stem cells expressed typical pluripotency markers Nanog, Oct4 and Sox2 (Wang Z, Oron E, Nelson B, Razis S, Ivanova N. Distinct lineage specific roles for NANOG, OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell. 2012 Apr 6; 10 (4): 440-54). After 7 days of differentiation, cell aggregates from two representative experiments expressed PAX6 and PSA-NCAM, two protein markers characteristic of neuroectodermal progenitor cells ( Figure 3,Lippmann ES,Williams CE,Ruhl DA,Estevez-Silva MC,Chapman ER,Coon JJ,AshtonRS.Deterministic HOX patterning in human pluripotent stem cell-derivedneuroectoderm.Stem Cell Reports.2015Apr 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, KimDW. Highly pure and expandable PSA-NCAM-positive neural precursors from humanESC and iPSC-derived neural rosettes. PLoS One. 2012;7(7):e39715).

[0128] For human embryonic stem cells differentiated into oligodendrocyte progenitor cells through day 42, protein marker expression in the resulting single cell populations was characterized by flow cytometry and ICC.

[0129] To characterize protein marker expression of oligodendrocyte progenitor cells by ICC, slide-mounted aggregate sections were stained as described above, except that permeabilization was performed with 100% methanol for 2 min at room temperature and the blocking solution consisted of 10% fetal bovine serum in PBS.

[0130] Figure 4Representative 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).

[0131] To quantify 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 on ice for 30 minutes with primary antibodies specific for 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. Cells were washed with staining buffer to remove unbound antibodies; in the case of unconjugated antibodies, cells were then incubated on ice for 30 minutes with appropriate fluorophore-conjugated secondary antibodies. Cells were washed and propidium iodide was added to distinguish dead cells. In some cases, cells were cultured overnight in tissue culture vessels coated with Matrigel (Corning #356231) at 37°C / 5% CO2 to restore protein markers that were sensitive to the day 42 harvest procedure described in Example 4 and then treated with TrypLE TMSelect (Thermo Fisher # Al2859-01) was harvested and stained as described above for flow cytometry analysis. 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 the fraction of the total number of cells analyzed after correction for the number of cells that exhibited nonspecific binding to the isotype control antibody.

[0132] Table 1 shows representative flow cytometry data of day 42 oligodendrocyte progenitor cells generated according to the methods described in Example 4. As shown in two representative runs, a high proportion of cells in the resulting cell population expressed 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+ channelblock.J Neurosci.1996Apr15;16(8):2659-70) and Pre-OPC marker A2B5 (Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, Sharp K, Steward O. Human embryonic stemcell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury.J Neurosci. 2005May 11;25(19):4694-705).In addition, non-OPC markers were detected to the greatest extent in the resulting populations, including the neural progenitor / epithelial marker CD49f (Krebsbach PH, Villa-Diaz LG. The Role of Integrinα6 (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 MolMed. 2018Oct; 42(4):1771-1785).

[0133] Table 1. Representative flow cytometry data of oligodendrocyte progenitor cells generated by methods according to the present disclosure.

[0134]

[0135] OPCs produced using another method and currently in clinical testing to treat spinal cord injury (Priest CA, Manley NC, Denham J, Wirth ED 3rd, Lebkowski JS. Preclinical safety of human embryonic stem cell-derived oligodendrocyte progenitors supporting clinical trials in spinal cord injury. Regen Med. 2015 Nov; 10(8): 939-58; Manley NC, Priest CA, Denham J, Wirth ED 3rd, Lebkowski JS. Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cells: Preclinical Efficacy and Safety in Cervical Spinal Cord Injury. Stem Cells Transl Med. 2017 Oct; 6(10): 1917-1929), the cell population produced by the method described in the present disclosure resulted in a higher proportion of cells positive for the oligodendrocyte progenitor cell marker NG2, and reduced expression of non-OPC markers CD49f, CLDN6 and EpCAM.

[0136] Example 6 - Characterization of Differentiated Cell Populations by Gene Expression Profiling

[0137] Gene expression profiling can be used to characterize the cellular phenotype of the starting multipotent cell population and each stage of differentiation, including the generation of neuroectodermal progenitors, glial progenitors, and oligodendrocyte progenitors. Gene expression profiling includes global transcriptome profiling using methods such as microarrays and RNA-seq, and targeted gene profiling using more sensitive methods such as real-time quantitative PCR (qPCR).

[0138] For gene expression profiling, cells were lysed in Qiagen RLT lysis buffer (Qiagen #79216) and RNA was purified using Qiagen RNeasy Mini Kit (Qiagen #74106) according to the manufacturer's guidelines. For qPCR-based analysis, purified RNA was then converted to cDNA using Invitrogen Superscript IV VILO Mastermix (Thermo Fisher Scientific #11756050) according to the manufacturer's guidelines. The relative expression levels of target genes and reference housekeeping genes were then quantified using gene-specific primer-probe sets (Applied Biosystems Taqman Gene Expression Assays, Thermo Fisher Scientific #4331182) according to the manufacturer's guidelines. In order to determine the relative expression levels of a given set of target genes, PCR reactions were performed on ABI 7900HT real-time sequence detection systems (Applied Biosystems), BioMark HD systems (Fluidigm), or equivalent systems. Each target gene is normalized to one or more reference genes, such as GAPDH, to determine its relative expression level.

[0139] Figure 5 Representative qPCR analysis of uhESCs is shown at harvest, before forming three-dimensional cell aggregates (day -1) and 24 hours later, after forming small non-embryoid bodies (non-EB) aggregates (day 0, immediately before the start 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 group of 76 genes consisting of known markers of pluripotency and early differentiation. For each gene, a standardized ΔCT value was calculated relative to the mean of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1). The resulting correlation graph of all ΔCT values ​​for day -1 relative to day 0 is shown in Figure 5 , and showed that cells in small non-EB aggregates at day 0 retained expression of pluripotency genes and had an overall expression profile of tested markers similar to uhESCs at day -1. This is further supported by the calculation of fold changes relative to baseline presented in Table 2, which shows high levels of pluripotency markers NANOG, LIN28A, and SOX2 24 hours after aggregate formation of uhESCs (day 0 uhESCs) for two representative experiments.

[0140] Table 2 shows the qPCR results from two representative experiments, and the experiment measures the expression of pluripotency genes, neuroectodermal progenitor cell genes, glial progenitor cell genes and oligodendrocyte progenitor cell genes in the cell mass produced according to the method of the present disclosure. RNA samples were collected at the following time points: uhESC cell aggregates formed 24 hours after the start and before differentiation (day 0), then differentiated into neuroectodermal progenitor cells (day 7), then differentiated into glial progenitor cells (day 21) and then differentiated into oligodendrocyte progenitor cells (day 42). RNA samples were processed for qPCR using the method as described above. A selected set of genes indicative of each differentiation state was quantified, including: three pluripotency genes (NANOG, LIN28A, SOX2), three neuroectodermal progenitor genes (PAX6, HES5, ZBTB16), three glial progenitor genes (CACGN4, FABP7, SOX6) and three oligodendrocyte progenitor genes (CSPG4, PDGFRα, DCN). For each gene, 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.

[0141] Table 2. qPCR analysis of gene markers of pluripotency, neuroectodermal progenitor cells (NEPCs), glial progenitor cells (GPCs), and oligodendrocyte progenitor cells (OPCs) in H1 uhESCs differentiated into OPCs according to the present disclosure.

[0142]

[0143] Referring to Table 2, differentiation of uhESC for 7 days according to the methods of the present disclosure resulted in a gene expression profile consistent with neuroectodermal progenitors, including downregulation of NANOG and expression of LIN28A, SOX2, PAX6, HES5 and ZBTB16 (Patterson M, Chan DN, Ha I, Case D, Cui Y, Van Handel B, Mikkola HK, Lowly WE. Defining the nature of human pluripotent stem cell progeny. Cell Res. 2012 Jan; 22(1): 178-93; Lippmann ES, Williams CE, Ruhl DA, Estevez-Silva MC, Chapman ER, Coon JJ, Ashton RS. Deterministic HOX patterning in human pluripotent stem cell-derived neuroectoderm. Stem Cell Reports. 2015 Apr 14; 4(4): 632-44; Woo et al., Cell Biology, 2015, 35(4): 115-116). SM,Kim J,Han HW,Chae JI,Son MY,Cho S,Chung HM,Han YM,KangYK.Notch signaling is required for maintaining stem-cell features ofneuroprogenitor cells derived from human embryonic stem cells.BMCNeurosci.2009Aug 17;10:97;Avantaggiato V,Pandolfi PP,Ruthardt M,Hawe N,Acampora D, Pelicci PG, Simeone A. Developmental analysis of murine PromyelocyteLeukemia Zinc Finger (PLZF) gene expression:implications for the neuromeric model of the forebrain organization. J Neurosci. 1995Jul; 15(7Pt1):4927-42).

[0144] After 21 days of suspension-based differentiation, the resulting cell population displayed a gene expression profile consistent with 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 JQ. RA, Puchalski RB, Jones AR, Roskams AJ. Adult spinal cord radial glia displays a unique progenitor phenotype. PLoS One. 2011;6(9):e24538; Baroti T, Zimmermann Y, Schillinger A, Liu L, Lommes P, Wegner M, Stolt CC. Transcriptionfactors Sox5 and Sox6 exert direct and indirect influences onoligodendroglial migration in spinal cord and forebrain.Glia.2016Jan;64(1):122-38).

[0145] After 42 days of differentiation according to the methods described in the present disclosure, the resulting cell population expressed 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).

[0146] 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

[0147] In addition to the small molecule inhibitors used in Example 2 (SB431542 and Dorsomorphin), alternative small molecule inhibitors of TGFβR1 / Activin / Nodal signaling and BMP signaling were tested for their ability to differentiate human embryonic stem cells into neuroectodermal progenitors in suspension. Table 3 lists the alternative small molecule inhibitors tested. Each condition was tested in duplicate wells of Ultra Low Attachment 6-well tissue culture plates (Corning #3471).

[0148] Table 3. Small molecule inhibitors for differentiating human embryonic stem cells into neuroectodermal progenitors in suspension.

[0149]

[0150]

[0151] At the 7th day of differentiation, cells were collected and processed for RNA extraction and gene expression profiling by qPCR as described in Example 6. For each gene, the standardized ΔCT value was calculated relative to the mean value of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1), and the fold expression relative to the baseline (expression below the quantitative limit) was calculated using the ΔΔCT method. Table 4 shows the mean value (relative to the baseline) of the fold expression values ​​of the biological replicates of each small molecule combination. Referring to Table 4, the use of each small molecule combination tested to differentiate uhESC in suspension for 7 days resulted in the downregulation of the pluripotency marker NANOG and the expression or induction of a similar degree of maintenance of genes (including LIN28A, SOX2, PAX6, HES5 and ZBTB16) associated with the phenotype of neuroectodermal progenitors.

[0152] 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 96-gene panel consisting of known markers for pluripotency, neuroectodermal progenitors, neural tube patterning, glial progenitors, oligodendrocyte progenitors, neural crest cells, neurons, astrocytes, pericytes, Schwann cells, and epithelial cells. Referring to Figure 6, comparison of the day 7 cell phenotypes of each alternative small molecule combination with the cell phenotype produced by treatment with SB431542 plus Dorsomorphin by regression plots of normalized ΔCT values ​​indicated that similar overall cell phenotypes could be achieved using each small molecule combination tested. In summary, the results shown in Table 4 and Figure 6 support that various combinations of (i) TGFβR1 / Activin / Nodal signaling inhibitors and (ii) BMP signaling inhibitors can be used to differentiate uhESCs into neuroectodermal progenitors, and further into glial progenitors and into oligodendrocyte progenitors in suspension using the methods of the present disclosure.

[0153] 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.

[0154]

[0155] Example 8 - Evaluation of the Presence of Extraneous Epithelial Lineage Cells in Differentiated OPC Populations Using an In Vitro Cyst Assay

[0156] The presence of undesirable epithelial lineage cells in the OPC colony produced according to the present disclosure was tested using in vitro cyst assay. The cyst assay was performed substantially according to the scheme 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). In short, OPC was grown 20 days in the presence of a factor known to stimulate epithelial cyst formation in a 3D culture system. In addition to visual detection cysts, the presence of cystic structures containing the basolateral protein expression of epithelial marker CD49f was assessed using immunocytochemistry.

[0157] OPCs were cultured in cyst-supporting medium at 21.9 x 10 3 cells / cm 2 The density of inoculation was Corning pad (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 cysts were counted and lysed using Cell Recovery Solution (Corning #354253). Cells were fixed in 4% paraformaldehyde (PFA) on ice for 5 min and permeabilized overnight in blocking buffer. Cysts were then stained for CD49f (ITGA6), phalloidin, and counterstained with DAPI. Cysts were imaged using IN Cell Analyzer2000 (GE Healthcare Life Sciences) and analyzed using IN Cell Developer software (GE Healthcare Life Sciences) and MATLAB. TM (Mathworks) quantified cyst frequency, size, and staining intensity.

[0158] With reference to Table 5, OPCs produced from two representative runs and tested in an in vitro cyst assay using methods according to the present disclosure produced fewer cysts than three control batches of OPCs (control A, control B, and control C) produced by an alternative method previously found to cause epithelial cyst formation in vivo (Manley NC, Priest CA, Denham J, Wirth ED 3rd, Lebkowski JS. Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cells: Preclinical Efficacy and Safety in Cervical Spinal Cord Injury. Stem Cells Transl Med. 2017 Oct; 6(10): 1917-1929). Based on these results, it is expected that OPCs produced according to the present disclosure will form little or no epithelial cysts in vivo.

[0159] Table 5. Representative cyst assay results of oligodendrocyte precursor cells generated by methods according to the present disclosure.

[0160]

[0161] Although the present disclosure has been described with reference to specific embodiments, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted for its elements without departing from the scope of the present disclosure. In addition, many modifications may be made to adapt specific situations or materials to the teachings of the present disclosure without departing from the scope of the present disclosure.

[0162] Therefore, it is intended that the disclosure not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this disclosure, but that the disclosure will include all aspects falling within the scope and spirit of the appended claims.

Claims

1. A 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 aggregates of undifferentiated human pluripotent stem cells, wherein the human pluripotent stem cells remain in an undifferentiated state; b) culturing the non-embryoid body aggregates from a) in a dynamic suspension for a first period of time in the presence of at least one transforming growth factor β / activin / Nodal signaling inhibitor and at least one bone morphogenetic protein signaling inhibitor selected from the group consisting of inhibitors of activin receptor-like kinase 5, SB431542, LY2157299, GW788388, A-77-01, A-83-01 and SB505124, and inducing differentiation into neural ectoderm, wherein the first period of time is one to four days; c) culturing the non-embryoid body aggregates from b) in dynamic suspension in the presence of retinoic acid and at least one Smoothened receptor agonist for a second period of time, wherein the second period of time is one to four days; d) culturing the aggregates from c) in dynamic suspension in the presence of basic fibroblast growth factor and epidermal growth factor for an additional period of time until the cells mature into glial progenitor cells; and e) Harvesting cells from d) and plating them on a substrate, resulting in cell dislodgement from the aggregates, Wherein steps a) to d) are performed within a period of 21 days.

2. The method of claim 1, wherein the substrate is recombinant human laminin-521.

3. The method of claim 1, wherein the human pluripotent stem cells are human embryonic stem cells.

4. The method of claim 1, wherein the human pluripotent stem cells are human induced pluripotent stem cells.

5. The method of claim 1, wherein the at least one transforming growth factor beta / activin / Nodal signaling inhibitor is SB431542.

6. The method of claim 1, wherein the at least one bone morphogenetic protein signaling inhibitor is Dorsomorphin.

7. The method of claim 1, wherein the at least one Smoothened receptor agonist is selected from the group consisting of Purmorphamine, Smoothened agonists, and Sonic Hedgehog proteins.

8. The method of claim 1, wherein the at least one Smoothened receptor agonist is Purmorphamine.

9. A method for obtaining a cell population comprising oligodendrocyte progenitor cells from undifferentiated human pluripotent stem cells, the method comprising: a) obtaining glial progenitor cells according to the method of claim 1; b) culturing the cells from a) for an additional period of time in the presence of epidermal growth factor and platelet-derived growth factor AA until the cells mature into oligodendrocyte progenitor cells, wherein the adherent culture is performed for a period of 21 days, and The oligodendrocyte progenitor cells express one or more markers selected from the group consisting of neural / glial antigen 2, platelet-derived growth factor receptor A and ganglioside GD3.

10. The method of claim 9, wherein the adherent culture is performed on a substrate selected from the group consisting of: (i) a cell adhesion peptide and (ii) an extracellular matrix selected from the group consisting of laminin and vitronectin. The method of claim 9 , wherein the adherent culture is performed on recombinant human laminin-521.

12. The method of claim 9, wherein the adherent culture is performed on laminin-511 E8 fragment.

13. The method of claim 9, wherein the human pluripotent stem cells are human embryonic stem cells.

14. The method of claim 9, wherein the human pluripotent stem cells are human induced pluripotent stem cells.

15. The method of claim 9, wherein at least 60% of the cells are neural / glial antigen 2 positive.

16. The method of claim 9, wherein at least 70% of the cells are neural / glial antigen 2 positive.

17. The method of claim 9, wherein at least 80% of the cells are neural / glial antigen 2 positive.

18. The method of claim 9, wherein at least 90% of the cells are neural / glial antigen 2 positive.

19. The method of claim 9, wherein at least 60% of the cells are platelet-derived growth factor receptor A positive.

20. The method of claim 9, wherein at least 70% of the cells are platelet-derived growth factor receptor A positive.

21. The method of claim 9, wherein at least 80% of the cells are platelet-derived growth factor receptor A positive.

22. The method of claim 9, wherein at least 90% of the cells are platelet-derived growth factor receptor A positive.

23. The method of claim 9, wherein at least 60% of the cells are positive for ganglioside GD3.

24. The method of claim 9, wherein at least 70% of the cells are positive for ganglioside GD3.

25. The method of claim 9, wherein at least 80% of the cells are positive for ganglioside GD3.

26. The method of claim 9, wherein at least 90% of the cells are positive for ganglioside GD3.

27. 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 disaggregated and formed into a single-cell suspension in a dynamic suspension to obtain non-embryoid body aggregates, wherein the human pluripotent stem cells in the non-embryoid body aggregates remain in an undifferentiated state; b) culturing the non-embryoid body aggregates from a) in a dynamic suspension for a first period of time in the presence of at least one transforming growth factor β / activin / Nodal signaling inhibitor and at least one bone morphogenetic protein signaling inhibitor selected from the group consisting of inhibitors of activin receptor-like kinase 5, SB431542, LY2157299, GW788388, A-77-01, A-83-01 and SB505124, and inducing differentiation into neural ectoderm, wherein the first period of time is one to four days; c) culturing the non-embryoid body aggregates from b) in dynamic suspension in the presence of retinoic acid and at least one Smoothened receptor agonist for a second period of time, wherein the second period of time is one to four days; d) culturing the aggregates from c) in dynamic suspension in the presence of basic fibroblast growth factor and epidermal growth factor for an additional period of time until the cells mature into glial progenitor cells; and e) Harvesting cells from d) and plating them on a substrate, resulting in cell dislodgement from the aggregates, Wherein steps a) to d) are performed within a period of 21 days.

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