Dorsally-derived oligodendrocyte progenitor cells from human pluripotent stem cells

Abstract

Methods are provided for differentiating human pluripotent stem cells into dorsal neuroectodermal progenitor cells and further into glial progenitor cells and oligodendrocyte progenitor cells (OPCs) using inhibitors of BMP signaling and MAPK / ERK signaling. Also provided are cells and cell compositions obtained by such methods, and uses of such cells. Further provided are methods and protocols for effectively differentiating human pluripotent stem cells into OPCs in the absence of the ventral morphogen SHH or an activator of SHH signaling. The methods of the present disclosure reproducibly generate dorsal neuroectodermal progenitor cells at day 7 of the differentiation process, glial progenitor cells at day 21 of the differentiation process, and OPCs at day 42 of the differentiation process.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 796,077, filed on Jan. 23, 2019, the entire content of which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to new methods of differentiating pluripotent stem cells, such as human embryonic stem cells, first into neuroectodermal progenitor cells with a dorsal spinal cord progenitor cell phenotype, then further into glial progenitor cells, and further into oligodendrocyte progenitor cells. Also provided are cells and cell compositions obtained by such methods, and uses of such cells. The present disclosure further relates to cells expressing one or more markers produced by the methods according to the invention. Background Art

[0004] Oligodendrocyte progenitor cells (OPCs) are a subtype of glial cells in the central nervous system (CNS) that arise in the ventricular zones of the brain and spinal cord and migrate throughout the developing CNS before maturing into oligodendrocytes. Mature oligodendrocytes produce myelin, which insulates neuronal axons and remyelinates CNS lesions where myelin has been lost. Oligodendrocytes also contribute to neuroprotection by other mechanisms, including the production of neurotrophic factors that promote neuronal survival (Wilkins et al., 2001 Glia 36(1):48 - 57; Dai et al., 2003 J Neurosci. 23(13):5846 - 53; Du and Dreyfus, 2002 J Neurosci Res. 68(6):647 - 54). Unlike most progenitor cells, OPCs remain abundant in the adult CNS, where they retain the ability to generate new oligodendrocytes. Thus, OPCs and mature oligodendrocytes derived from OPCs are important therapeutic targets for demyelinating and myelin - forming disorders (such as multiple sclerosis, adrenoleukodystrophy, and adrenomyeloneuropathy), other neurodegenerative disorders (such as Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease), and acute neurological injuries (such as stroke and spinal cord injury (SCI)).

[0005] Several protocols have been developed for differentiating human pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) into oligodendrocyte progenitor cells (OPCs) that can be used for cell therapy. To date, protocols for generating oligodendrocyte progenitor cells from human pluripotent stem cells have recapitulated the ventral motor neuron progenitor (pMN) domain of the developing spinal cord, which is known to give rise to most spinal cord OPCs in vivo (Rowitch, 2004 Nat Rev Neurosci. 5(5):409-19; Ravanelli and Appel, 2015 Genes Dev. 29(23):2504-15). Induction of ventral neural progenitors that give rise to ventrally derived OPCs requires activation of sonic hedgehog (SHH) signaling. (Ericson et al., 1996 Cell 87:661-673; Orentas et al., 1999 Development 126(11):2419-29). Thus, existing in vitro protocols for generating OPCs from pluripotent stem cells rely on embryoid body formation as a means to stimulate endogenous SHH activation (Nistor et al., 2005 Glia 49(3):385-96) or the direct addition of SHH or activators of SHH signaling (Stacpoole et al., 2013 Stem Cell Reports 1(5):437-50; Douvaras and Fossati, 2015 Nat Protoc. 10(8):1143-54; Piao et al., 2015 Cell Stem Cell 16(2):198-210; Wang et al., 2013 Cell Stem Cell 12(2):252-64; Rodrigues et al., 2017 Stem Cell Reports 8(6):1770-1783; and Yamashita et al., 2017 PLoS One 12(2):e0171947). The former approach can be problematic because it relies on spontaneous differentiation within embryoid bodies and may result in unwanted cell types at the end of the differentiation process (Priest et al., 2015 Regen Med. 10(8):939-58; Manley et al., 2017 Stem Cells Transl Med. 6(10):1917-1929). The latter directed differentiation represents the current approach for generating pluripotent stem cell-derived OPCs. Although these methods have successfully generated OPCs from human pluripotent stem cells for research purposes, challenges still remain in terms of the quality, scalability, and cost of translating existing protocols into products relevant for clinical commercial-scale production.

[0006] In mice, a smaller secondary wave of OPCs is generated in the dorsal spinal cord independent of SHH signaling (Cai et al., 2005 Neuron 45(1):41-53; Vallstedt et al., 2005 Neuron 45(1):55-67). These dorsally-derived murine OPCs mature into oligodendrocytes that contribute to axonal myelination in response to focal myelinogenic injury during development and remyelination (Zhu et al., 2011 Glia 59(11):1612-21). Little is known about the putative dorsally-derived OPC population in humans. Recently, it has been reported that human brain region-specific forebrain organoids (dorsal forebrain organoids and ventral forebrain organoids) composed of multiple cell types and generated using an OLIG2-GFP knock-in hPSC reporter line can differentiate into functional neurons and oligodendrocytes. (Kim et al., available at https: / / www.biorxiv.org / content / biorxiv / early / 2018 / 11 / 04 / 460907.full.pdf). However, to date, there has been no report of dorsally-derived OPCs obtained by directed differentiation of human pluripotent stem cells that can generate a target lineage-specific cell population suitable for downstream cell therapy applications.

[0007] There is a need for improved methods of differentiating pluripotent stem cells into OPCs. Ideally, such methods should be easily scalable to produce sufficient numbers of OPCs for cell therapy applications while consistently and reproducibly generating target cell OPCs with the desired quality attributes. SUMMARY OF THE INVENTION

[0008] In various embodiments described herein, the present disclosure particularly provides robust and reliable protocols for differentiating human pluripotent stem cells such as ESCs and iPSCs into dorsal neuroectodermal progenitor cells (dNPCs) and further into glial progenitor cells and OPCs.

[0009] The present disclosure is based in part on the discovery that human pluripotent stem cells can be readily and efficiently differentiated into spinal cord OPCs in the absence of ventralization of neuroectodermal-restricted progenitors mediated by SHH signaling.

[0010] In certain aspects of the present disclosure, neuroectodermal progenitor cells with a dorsal spinal cord phenotype are obtained by exposing human pluripotent stem cells to one or more inhibitors of bone morphogenetic protein (BMP) signaling and one or more inhibitors of mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) signaling in combination with retinoic acid. This method contrasts with current methods of inducing neuroectoderm, which rely on the combinatorial addition of transforming growth factor β (TGFβ) / activin / Nodal signaling inhibitors and BMP signaling inhibitors (also known as dual SMAD inhibition) (Chambers et al., 2009 Nat. Biotechnol 27(3):275-280; Douvaras and Fossati, 2015 Nat Protoc. 10(8):1143-54; Piao et al., 2015 Cell Stem Cell 16(2)).

[0011] Surprisingly, it was found that dorsal neuroectodermal progenitor cells obtained according to the above protocol and not exposed to the ventral morphogen SHH or an activator of SHH signaling can be readily differentiated into spinal cord OPCs. It was also found that the method of the present disclosure generates a significantly greater number of differentiated cells compared to differentiation protocols that activate SHH signaling. Due to the significant increase in cell expansion and cell yield, the method of the present disclosure provides a scalable and reproducible process for generating large numbers of OPCs and other neuroectodermal lineage cells for cell therapy and other applications.

[0012] The method of the present disclosure reproducibly generates neuroectodermal progenitor cells with a dorsal spinal cord phenotype on day 7 of the differentiation process, glial progenitor cells on day 21 of the differentiation process, and OPCs on day 42 of the differentiation process. The day 42 OPCs generated according to the present disclosure (in terms of their overall marker expression profile) are comparable to OPCs generated using alternative methods currently in clinical testing for the treatment of spinal cord injury (Priest et al., 2015 RegenMed. 10(8):939-58; Manley et al., 2017 Stem Cells Transl Med. 6(10):1917-1929), except that the OPCs generated according to the present disclosure express lower levels of non-OPC markers, including markers associated with in vitro epithelial cyst formation.

[0013] In a preferred embodiment, the OPCs generated according to the method of the present invention express one or more markers selected from the group consisting of neural / glial antigen 2 (NG2), platelet-derived growth factor receptor A (PDGFRα), and ganglioside GD3 (GD3). In a further preferred embodiment, the cells can be characterized by the expression of a single marker or a combination of markers. For example, the OPCs generated according to the method of the present invention can be characterized by NG2, PDGFRα, or GD3 alone, or a marker combination having two or three of the markers NG2, PDGFRα, and GD3. In a preferred embodiment, at least 90% of the cells generated according to the method of the present invention express NG2 and / or PDGFRα. In a further preferred embodiment, at least 50% of the cells further express GD3.

[0014] In a further preferred embodiment, the process of generating the cells includes one or more steps of cryopreserving the cells in an intermediate bank and then thawing the cells to continue the differentiation process until the final product is obtained. For example, the intermediate cell bank can be cryopreserved on day 14, day 28, and / or day 35 of the process.

[0015] In another embodiment, the OPCs generated according to the present invention are prepared as a ready-to-administer (RTA) OPC cell therapy composition for treating a patient. Also provided are methods of formulating human OPCs for direct administration to a subject after thawing and formulating an OPC cell therapy composition for cryopreservation and administration of the cryopreserved composition to a subject after thawing. In another aspect, the RTA composition can be formulated as a thaw-and-inject (TAI) composition, whereby the composition is administered by injection after thawing without further processing of the OPCs.

[0016] In one embodiment, the present disclosure provides a method for obtaining a cell population comprising dorsal neural progenitor cells (dNPCs) from undifferentiated human pluripotent stem cells. In certain embodiments, the method comprises: a) obtaining a culture of undifferentiated human pluripotent stem cells; b) adherently culturing the undifferentiated human pluripotent stem cells for a first period of time in the presence of at least one inhibitor of mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK), at least one inhibitor of bone morphogenetic protein (BMP) signaling, and retinoic acid, thereby inducing differentiation into the neuroectoderm; and c) adherently culturing the cells from b) for a second period of time in the presence of retinoic acid and in the absence of sonic hedgehog (SHH) or an SHH signaling activator, thereby obtaining dorsal neural progenitor cells.

[0017] In certain embodiments, the first period of time is about 3 to 4 days. In certain embodiments, the second period of time is about 3 to 4 days.

[0018] In certain embodiments, the method further comprises the additional steps of harvesting the cells from c), re-plating the harvested cells on a substrate and culturing the cells in adherence for an additional period of time in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) to thereby expand the cells. In certain embodiments, the substrate is a cell adhesion peptide. In other embodiments, the substrate is an extracellular matrix protein. In certain embodiments, the substrate is recombinant human laminin-521. In other embodiments, the substrate is vitronectin or the laminin-511E8 fragment. In other embodiments, the substrate is a synthetic substrate, e.g., SC substrate.

[0019] In a further embodiment, the method comprises the additional steps of harvesting the expanded cells and culturing them as aggregates in suspension for an additional period of time in the presence of bFGF and EGF until the cells mature into glial progenitor cells. In certain embodiments, the culturing is performed in a dynamic suspension. In certain embodiments, the cells are cultured in suspension for about five to ten days. In one embodiment, the cells are cultured in suspension for about 7 days.

[0020] In an even further embodiment, the method comprises the additional steps of plating the aggregates containing glial progenitor cells on a substrate and culturing the cells in adherence for an additional period of time in the presence of epidermal growth factor (EGF), optionally separating the cells from time to time, until the cells mature into oligodendrocyte progenitor cells (OPCs). In certain embodiments, the culture medium further comprises platelet-derived growth factor AA (PDGF-AA). In certain embodiments, after plating the aggregates containing glial progenitor cells, the cells are cultured for about 2 to 4 weeks. In one embodiment, after plating the aggregates containing glial progenitor cells, the cells are cultured for 21 days. In certain embodiments, the substrate is a cell adhesion peptide. In other embodiments, the substrate is an extracellular matrix protein. In certain embodiments, the substrate is recombinant human laminin-521. In other embodiments, the substrate is vitronectin or the laminin-511E8 fragment. In other embodiments, the substrate is a synthetic substrate, e.g., SC substrate.

[0021] In another embodiment, the cells are cultured without coating in some parts of the process. In another embodiment, coated or uncoated microcarriers of the suspension are used instead of culture dishes.

[0022] In certain embodiments, at least one inhibitor of MAPK / ERK is selected from PD0325901, AZD6244, GSK1120212, PD184352, and Cobimetinib. In other embodiments, the MAPK / ERK inhibitor is PD0325901.

[0023] In certain embodiments, at least one inhibitor of BMP signaling is an inhibitor of activin receptor-like kinase 2 (ALK2). In certain embodiments, at least one inhibitor of BMP signaling is selected from Dorsomorphin, DMH-1, K02288, ML347, LDN193189, and Noggin protein. In other embodiments, the BMP signaling inhibitor is Dorsomorphin.

[0024] Another embodiment is a population of differentiated cells that comprises paired box 6 (PAX6)-positive dorsal neural progenitor cells (dNPCs) obtained according to the present disclosure. In certain embodiments, the PAX6-positive dNPCs further express one or more markers selected from paired box 3 (PAX3), paired box 7 (PAX7), and activating protein 2 (AP2).

[0025] In another embodiment, the present disclosure provides a method for obtaining a cell population comprising neural / glial antigen 2 (NG2)-positive oligodendrocyte progenitor cells (OPCs) from undifferentiated human pluripotent stem cells, the method comprising: a) obtaining a culture of undifferentiated human pluripotent stem cells; b) adherently culturing the undifferentiated human pluripotent stem cells for a first period of time in the presence of at least one inhibitor of mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK), at least one inhibitor of bone morphogenetic protein (BMP) signaling, and retinoic acid, thereby inducing differentiation into neuroectoderm; c) adherently culturing the cells from b) for a second period of time in the presence of retinoic acid and in the absence of sonic hedgehog (SHH) or an SHH signaling activator, thereby obtaining dorsal neural progenitor cells; d) harvesting the cells from c), re-plating the harvested cells on a substrate and adherently culturing the cells for an additional period of time in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), thereby expanding neural progenitor cells; e) harvesting the cells from d) and culturing the cells as aggregates in suspension for an additional period of time in the presence of bFGF and EGF until the cells mature into dorsal neural glial progenitor cells; and f) plating the aggregates from e) on a substrate and adherently culturing the cells for an additional period of time in the presence of epidermal growth factor (EGF), optionally separating the cells from time to time, until the cells mature into OPCs. In certain embodiments, the medium in step f) further comprises platelet-derived growth factor AA (PDGF-AA). In certain embodiments, the cells are cultured for about 2 to 4 weeks after the aggregates are plated. In one embodiment, the cells are cultured for about twenty-one days after the aggregates are plated. In certain embodiments, the substrate is a cell adhesion peptide. In other embodiments, the substrate is an extracellular matrix protein. In certain embodiments, the substrate is recombinant human laminin-521. In other embodiments, the substrate is vitronectin or the laminin-511E8 fragment. In other embodiments, the substrate is a synthetic substrate, e.g., SC substrate.

[0026] Another embodiment is a differentiated cell population comprising NG2-positive OPCs obtained according to the present disclosure. In certain embodiments, the differentiated cell population comprises at least 60% NG2-positive cells. In certain embodiments, the differentiated cell population comprises at least 70% NG2-positive cells. In certain embodiments, the differentiated cell population comprises at least 80% NG2-positive cells. In other embodiments, the differentiated cell population comprises at least 90% NG2-positive cells. In certain embodiments, the differentiated cell population comprises at least 98% NG2-positive cells.

[0027] In certain embodiments, the differentiated cell population comprises at least 60% PDGFRα-positive cells. In certain embodiments, the differentiated cell population comprises at least 70% PDGFRα-positive cells. In certain embodiments, the differentiated cell population comprises at least 80% PDGFRα-positive cells. In other embodiments, the differentiated cell population comprises at least 90% PDGFRα-positive cells. In certain embodiments, the differentiated cell population comprises at least 98% PDGFRα-positive cells.

[0028] In certain embodiments, the differentiated cell population comprises at least 50% GD3-positive cells. In certain embodiments, the differentiated cell population comprises at least 60% GD3-positive cells. In certain embodiments, the differentiated cell population comprises at least 70% GD3-positive cells. In certain embodiments, the differentiated cell population comprises at least 80% GD3-positive cells. In other embodiments, the differentiated cell population comprises at least 90% GD3-positive cells. In certain embodiments, the differentiated cell population comprises at least 98% GD3-positive cells.

[0029] In another embodiment, the differentiated cell population comprises NG2 and PDGFRα-positive cells within the above percentages. In another embodiment, the differentiated cell population comprises NG2 and GD3-positive cells within the above percentages. In another embodiment, the differentiated cell population comprises PDGFRα and GD3-positive cells within the above percentages. In another embodiment, the differentiated cell population comprises NG2, PDGFRα, and GD3-positive cells within the above percentages.

[0030] In certain embodiments, the human pluripotent stem cells are human embryonic stem cells. In other embodiments, the human pluripotent stem cells are human induced pluripotent stem cells.

[0031] In another embodiment, the cells prepared according to the present invention are cryopreserved and then thawed for delivery to a patient. In a preferred embodiment, the cells do not require further processing prior to delivery to the patient. Once thawed, the cells prepared according to the present invention can be immediately delivered to the patient. In a preferred embodiment, the cells can be delivered by injection. The injection volume can be, for example, about 100 microliters, wherein the cell concentration is 100,000,000 viable cells per milliliter.

[0032] Brief Description of the Drawings

[0033] Figure 1It is a figure depicting the differentiation of human pluripotent stem cells according to the present disclosure into dorsal neuroectodermal progenitor cells (dNPCs) and further into glial progenitor cells (GPCs) and oligodendrocyte progenitor cells (OPCs). Neuroectodermal progenitor cells with a dorsal spinal cord phenotype are obtained around day 7, and they can be easily differentiated into GPCs (day 21) and further into OPCs (day 42). Several additional small molecule inhibitors of MAPK / ERK signaling (other than PD0325901) and BMP signaling (other than Dorsomorphin) were tested and found to be equally effective in inducing differentiation into dorsal neuroectoderm (Example 7).

[0034] Figures 2A - 2C Representative data showing the effect on cell yield in the absence of SHH or SHH signaling agonists are presented. Figure 2A The yields from day 7 to day 14 in various different combinations of small molecule treatments tested are shown. “+PMA” and “-PMA” refer to the presence or absence of the SHH agonist Purmorphamine during days 4 - 6 of the differentiation process, respectively. The black bars for conditions A, B, and E correspond to neuroectodermal cells derived using a differentiation mixture containing the MAPK / ERK inhibitor PD0325901, the BMP inhibitor Dorsomorphin, and retinoic acid, while the gray bars for conditions C, D, and F correspond to neuroectodermal cells derived using other signaling modulators. Figure 2B Shows the step - wise yields at different time points for two representative runs (Run 1 and Run 2) of the differentiation protocol disclosed in the present disclosure compared to a differentiation process including PMA (previous process (+PMA)). Figure 2C Depicts the overall theoretical yield of one uhESC for two representative runs (Run 1 and Run 2) according to the present disclosure and compares it with a differentiation process including PMA (previous process (+PMA)).

[0035] Figure 3 Shows representative micrographs of dorsal neuroectodermal progenitor cells generated according to the present disclosure and stained by immunocytochemistry. The dorsal neuroectodermal progenitor cells were stained for DAPI, PAX7, PAX3, and PAX6 (upper row, left to right) and DAPI and AP2 (lower row, left to right). The stained cells were imaged on an IN Cell Analyzer 2000. The scale bar represents 200 μm and applies to all images in the figure.

[0036] Figure 4Shows representative micrographs of aggregates of glial progenitor cells generated according to the present disclosure and immunocytochemically stained. Glial progenitor cells were stained for DAPI, PAX7, PAX3, and PAX6 (top row, left to right) and DAPI, AP2, and NG2 (bottom row, left to right). The stained cells were imaged on an IN Cell Analyzer 2000. The scale bar represents 200 μm and applies to all images in the figure.

[0037] Figure 5 Shows representative micrographs of oligodendrocyte progenitor cells generated according to the present disclosure and immunocytochemically stained. Oligodendrocyte progenitor cells were immunocytochemically stained for DAPI and NG2 (top row, left to right) and DAPI and AP2 (bottom row, left to right). The stained cells were imaged on an IN Cell Analyzer 2000. The scale bar represents 200 μm and applies to all images in the figure.

[0038] Figure 6 Shows correlation plots of the day 7 gene expression profiles of uhESCs differentiated into dorsal neuroectodermal progenitor cells (dNPCs) using different small molecule combinations. Each correlation plot shows a comparison of the day 7 gene expression profile of cells treated with PD0325901 plus Dorsomorphin to alternative small molecule combinations indicated on the y-axis of each plot. For each plot, the data points represent each of 96 genes evaluated by Fluidigm qPCR and calculated as normalized ΔCT as described in Example 7. The R-squared value is shown in the upper left corner of each plot and was calculated using JMP software (SAS, Cary, NC, USA) based on the best fit line. Detailed Description of the Invention

[0040] This description is not intended to be an exhaustive catalog of all the different ways in which the present disclosure may be implemented or of all the features that may be added to the present disclosure. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the present disclosure contemplates that in some embodiments of the present disclosure, any feature or combination of features set forth herein may be excluded or omitted. Additionally, many variations and additions to the various embodiments presented herein will be apparent to those skilled in the art in accordance with the present disclosure without departing from the present disclosure. In other instances, well-known structures, interfaces, and processes have not been shown in detail in order to avoid unnecessarily obscuring the invention. It is intended that no part of this specification be construed as limiting any part of the full scope of the invention. Accordingly, the following description is intended to illustrate some particular aspects of the present disclosure rather than to specify exhaustively all of its permutations, combinations, and variations.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terms used in the description herein are for the purpose of describing particular embodiments only and are not intended to limit the disclosure.

[0042] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety.

[0043] Unless the context dictates otherwise, it is specifically intended that the various features of the disclosure described herein can be used in any combination. Additionally, the disclosure contemplates that in some embodiments of the disclosure, any feature or combination of features set forth herein can be excluded or omitted.

[0044] The methods disclosed herein may include one or more steps or acts for implementing the methods. Without departing from the scope of the invention, the method steps and / or acts may be interchanged with one another. In other words, unless a particular order of steps or acts is required for proper operation of that aspect, the order and / or use of specific steps and / or acts may be modified without departing from the scope of the invention.

[0045] As used in the description of the disclosure and the appended claims, the singular forms "a", "an", and "the" are also intended to include the plural forms unless the context clearly dictates otherwise.

[0046] As used herein, "and / or" means and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0047] As used herein, the terms "about" and "approximately" when referring to a measurable value such as a percentage, density, volume, etc., are intended to include variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0048] As used herein, phrases such as "between X and Y" and "between about X and Y" shall be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y" and phrases such as "from about X to Y" mean "from about X to about Y".

[0049] As used herein, "oligodendrocyte progenitor cell" (OPC) refers to a cell found in the central nervous system that belongs to the neuroectodermal / glial lineage, expresses the characteristic marker neural / glial antigen 2 (NG2), and is capable of differentiating into oligodendrocytes.

[0050] The terms "glial lineage cells", "glial progenitor cells", and "glial cells" are used interchangeably herein and refer to non-neuronal CNS cells derived from the neuroectoderm / neural progenitor cells. Glial progenitor cells can further differentiate into OPC / 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 gamma4 (CACNG4), fatty acid binding protein 7 (FABP7), and netrin-1 receptor (DCC).

[0051] The terms "neuroectoderm", "neuroectodermal cells", "neuroectodermal precursors", "neuroectodermal progenitor cells", "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 central nervous system 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).

[0052] As used herein, the terms "dorsal" and "ventral" refer to different subtypes of neural cells that arise from progenitors arranged in spatially discrete domains along the dorsoventral axis of the neural tube in the developing spinal cord. This process (referred to as dorsoventral patterning) is controlled by secreted signals that partition neural progenitor cells. BMP and Wnt signaling initiate patterning from the dorsal neural tube (Lee and Jessell, 1999 Annu. Rev. Neurosci. 22:261-294), while secretion of SHH plays a key role in establishing ventral neuronal cell fates (Chiang et al., 1996 Nature 383:407-413; Ericson et al., 1996 Cell 87:661-683; Briscoe et al., 2001 Mol. Cell 7:1279-1291).

[0053] The terms "dorsal neuroectodermal progenitor cells", "dorsal neural progenitor cells", and "dNPC" are used interchangeably herein and refer to neural progenitor cells that have a dorsal spinal cord phenotype and have been obtained by differentiating pluripotent stem cells into neuroectoderm-restricted precursors in the absence of exogenous SHH or SHH signaling activators. In certain embodiments, dNPCs express one or more markers selected from paired box 3 (PAX3), paired box 7 (PAX7), and activator protein 2 (AP2).

[0054] As used herein, the term "embryoid body" (EB) refers to a three-dimensional cell aggregate derived from pluripotent stem cells that has undergone spontaneous differentiation into all three germ layers. EBs are formed when pluripotent stem cells are removed from culture conditions that inhibit differentiation. For example, in the case of human embryonic stem cells, removal of basic fibroblast growth factor (bFGF) and transforming growth factor β (TGFβ) from the culture medium results in spontaneous differentiation into all three germ layers and the formation of EBs.

[0055] As used herein, the term "BMP signaling inhibitor" refers to a small molecule or protein regulator that is capable of downregulating signaling along the bone morphogenetic protein (BMP) signaling pathway. In certain embodiments, the BMP signaling inhibitor directly targets activin A receptor type I (ACVR1), also known as activin receptor-like kinase 2 (ALK2). In certain embodiments, the BMP signaling inhibitor is selected from Dorsomorphin, DMH-1, K02288, ML347, LDN193189, and Noggin protein.

[0056] As used herein, the term "MAPK / ERK inhibitor" refers to a small molecule or protein regulator that inhibits the MAPK / ERK kinase. In certain embodiments, the MAPK / ERK inhibitor is selected from PD0325901, AZD6244, GSK1120212, PD184352, and cobimetinib.

[0057] The terms "SHH signaling activator", "SHH signaling agonist", "SHH activator", and "SHH agonist" are used interchangeably herein and refer to a small molecule or protein regulator that is capable of activating the Sonic Hedgehog (SHH) signaling pathway. Non-limiting examples of SHH signaling activators include Purmorphamine (PMA), Smoothened Agonist (SAG, CAS 364590-63-6), and Sonic Hedgehog (SHH) protein.

[0058] As used herein, the term "undesired cell type" refers to cells outside the neuroectodermal lineage that can lead to the formation of ectopic tissue upon implantation or to the formation of one or more cysts in the cyst assay, as described herein. In one embodiment, the "undesired cell type" can include cells of the epithelial lineage, such as cells that are positive for CD49f (a marker expressed by neural progenitors and epithelial cells) or positive for CLDN6 or EpCAM (two markers expressed by pluripotent and epithelial cells).

[0059] As used herein, "implantation" or "transplantation" refers to the administration of a cell population into a target tissue using a suitable delivery technique (e.g., using an injection device).

[0060] As used herein, "subject" refers to an animal or a human being.

[0061] As used herein, "subject in need thereof" refers to an animal or a human being with damaged central nervous system tissue. In one embodiment, the animal or human being is experiencing a loss of motor function.

[0062] The terms "central nervous system" and "CNS" are used interchangeably herein and refer to a complex of nerve tissues that control one or more activities of the body (including but not limited to the brain and spinal cord of vertebrates).

[0063] As used herein, "treatment" or "therapy" of a condition or disease is a method for obtaining a beneficial or desired result (preferably including a clinical outcome after the occurrence of the condition or disease in a patient). Beneficial or desired results for a disease include, but are not limited to, one or more of the following: improvement of a condition associated with the disease, cure of the disease, reduction in the severity of the disease, delay in the progression of the disease, alleviation of one or more symptoms associated with the disease, improvement in the quality of life of a person suffering from the disease, prolongation of survival, and any combination thereof. Similarly, for the purposes of the present disclosure, beneficial or desired results for a condition include, but are not limited to, one or more of the following: improvement of the condition, cure of the condition, reduction in the severity of the condition, delay in the progression of the condition, alleviation of one or more symptoms associated with the condition, improvement in the quality of life of a person suffering from the condition, prolongation of survival, and any combination thereof.

[0064] Proliferation and culture of undifferentiated pluripotent stem cells

[0065] Any suitable pluripotent stem cell can be used as a starting material for the differentiation of pluripotent stem cells according to the present disclosure. In one embodiment, a method can be performed on a human embryonic stem cell (hESC) line. In another embodiment, a method can be performed using induced pluripotent stem cells (iPSCs). In another embodiment, a method can be performed using cells derived from the H1, H7, H9, H13, or H14 cell lines. In another embodiment, a method can be performed on a primate pluripotent (pPS) cell line. In yet another embodiment, a method can be performed using undifferentiated stem cells derived from parthenotes (embryos that are stimulated to produce hESCs without fertilization).

[0066] Methods for the proliferation and culture of undifferentiated pluripotent stem cells have been described previously. For tissue and cell culture of pluripotent stem cells, the reader may wish to refer to any of the numerous publications available in the art, such as Teratocarcinomas and Embryonic Stem cells: A Practical Approach (E.J. Robertson, Ed., IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (P.M. Wasserman et al., Eds., Academic Press 1993); Embryonic Stem Cell Differentiation in Vitro (M.V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and Uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (P.D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998; and R.I. Freshney, Culture of Animal Cells, Wiley-Liss, New York, 2000).

[0067] Undifferentiated pluripotent stem cells can be maintained in an undifferentiated state without the addition of feeder cells (see, e.g., (2004) Rosler et al., Dev. Dynam. 229:259). Feeder-free cultures are generally supported by a nutrient medium containing factors that promote cell proliferation without differentiation (see, e.g., U.S. Patent No. 6,800,480). In one embodiment, conditioned medium containing these factors can be used. Conditioned medium can be obtained by culturing cells that secrete these factors with the medium. Suitable cells include, but are not limited to, irradiated (~4,000 Rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells (U.S. Patent No. 6,642,048). The medium can be conditioned by plating the feeder cells in serum-free medium (e.g., Knockout DMEM supplemented with 20% serum replacement and 4 ng / mL bFGF). Medium that has been conditioned for 1-2 days can be supplemented with additional bFGF and used to support pPS cell cultures for 1-2 days (see, e.g., WO01 / 51616; Xu et al., (2001) Nat. Biotechnol. 19:971).

[0068] Alternatively, fresh or conditioned media can be used that have been supplemented with added factors (such as fibroblast growth factor or forskolin) that promote the proliferation of cells in an undifferentiated form. Non-limiting examples include basal media 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). The advantage of these media formulations is that they support cell growth at a rate 2-3 times that of other systems (see, e.g., WO03 / 020920). In one embodiment, undifferentiated pluripotent cells such as hES cells can be cultured in a medium containing bFGF and TGFβ. Non-limiting example concentrations of bFGF include about 80 ng / ml. Non-limiting example concentrations of TGFβ include about 0.5 ng / ml. In yet another embodiment, undifferentiated pluripotent stem cells can be maintained in a commercially available complete medium such as mTeSR TM (Stem Cell Technologies, Vancouver, Canada).

[0069] Undifferentiated pluripotent cells can be cultured on a feeder cell layer (usually fibroblasts from embryonic or fetal tissue) (Thomson et al., (1998) Science 282:1145). Feeder cells can be of human or murine origin. Human feeder cells can be isolated from a variety of human tissues or can be derived by differentiating human embryonic stem cells into fibroblasts (see, e.g., WO01 / 51616). Human feeder cells that can be used include, but are not limited to, placental fibroblasts (see, e.g., Genbacev et al., (2005) Fertil. Steril. 83(5):1517), oviductal epithelial cells (see, e.g., Richards et al., (2002) Nat. Biotechnol., 20:933), foreskin fibroblasts (see, e.g., Amit et al., (2003) Biol. Reprod. 68:2150), and endometrial cells (see, e.g., Lee et al., (2005) Biol. Reprod. 72(1):42).

[0070] A variety of solid surfaces can be used for culturing undifferentiated pluripotent cells. These solid surfaces include, but are not limited to, standard commercially available tissue culture flasks or cell culture plates, such as 6-well, 24-well, 96-well, or 144-well plates. Other solid surfaces include, but are not limited to, microcarriers and discs. The solid surfaces suitable for culturing undifferentiated pluripotent cells can be made of a variety of materials, including, but not limited to, glass or plastic, such as polystyrene, polyvinyl chloride, polycarbonate, polytetrafluoroethylene, melinex, thermanox, or combinations thereof. Suitable surfaces can contain one or more polymers, such as one or more acrylates. The shape of the solid surface can be three-dimensional. Non-limiting examples of three-dimensional solid surfaces have been previously described, for example, in U.S. Patent Publication No. 2005 / 0031598.

[0071] Undifferentiated stem cells can also be grown on a growth substrate under feeder-free conditions. The growth substrate can be a matrix (such as GFR), recombinant laminin, recombinant fragment E8 of laminin-511, 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 SC substrate.

[0072] Undifferentiated stem cells can be passaged or subcultured using various methods (such as using collagenase, or for example, scraping by hand). Undifferentiated stem cells can be subcultured by enzymatic means that produce a single cell suspension (such as using (distributed by Sigma Aldrich, MO) or a similar trypsin). Alternatively, undifferentiated stem cells can be subcultured using non-enzymatic means (such as 0.5 mM EDTA in PBS, or for example, using ReLeSR TM (Stem Cell Technologies, Vancouver, Canada)).

[0073] In one embodiment, multiple undifferentiated stem cells are seeded or subcultured at a seeding density that allows the cells to reach confluence in about three to about ten days. In one embodiment, the seeding density can range from about 6.0x10 3 cells / cm 2 to about 5.0x10 5 cells / cm 2 , such as about 1.0x10 4 cells / cm 2 , such as about 5.0x104 cells / cm 2 , such as about 1.0x10 5 cells / cm 2 , or for example about 3.0x10 5 cells / cm 2 of the growth surface. In another embodiment, the seeding density can range from about 6.0x10 3 cells / cm 2 to about 1.0x10 4 cells / cm 2 of the growth surface, such as about 6.0x10 3 cells / cm 2 to about 9.0x10 3 cells / cm 2 , such as about 7.0x10 3 cells / cm 2 to about 1.0x10 4 cells / cm 2 , such as 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 to about 8.0x10 3 cells / cm 2 of the growth surface. In yet another embodiment, the seeding density can range from about 1.0x10 4 cells / cm 2 to about 1.0x10 5 cells / cm 2 of the growth surface, such as about 2.0x10 4 cells / cm 2 to about 9.0x10 4 cells / cm 2 , such as about 3.0x10 4 cells / cm 2 to about 8.0x10 4 cells / cm 2 , such as 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 2Growth surface. In one embodiment, the seeding density can range from about 1.0x10 5 cells / cm 2 to about 5.0x10 5 cells / cm 2 of the growth surface, such as about 1.0x10 5 cells / cm 2 to about 4.5x10 5 cells / cm 2 such as about 1.5x10 5 cells / cm 2 to about 4.0x10 5 cells / cm 2 such as about 2.0x10 5 cells / cm 2 to about 3.5x10 5 cells / cm 2 or such as about 2.5x10 5 cells / cm 2 to about 3.0x10 5 cells / cm 2 of the growth surface.

[0074] According to the methods of the present disclosure, stem cells can be cultured using any of a variety of suitable cell culture and subculture techniques. For example, the culture medium can be completely replaced daily, starting about 2 days after subculture of the cells. In one embodiment, when the culture reaches about 90% colony coverage, one or more suitable reagents (e.g., ) can be used to dissociate the cells to obtain a single-cell suspension for quantification and seeded for subsequent culture. In one embodiment, undifferentiated stem cells can then be subcultured before seeding the cells at a seeding density that allows the cells to reach confluence within a suitable period of time (e.g., within about three to ten days) on a suitable growth substrate (e.g., recombinant human laminin-521). In one embodiment, undifferentiated stem cells can be subcultured using collagenase IV and expanded on recombinant laminin. In another embodiment, undifferentiated stem cells can be subcultured using collagenase IV and expanded on . In one embodiment, undifferentiated stem cells can be subcultured using ReLeSR TM and expanded on recombinant human laminin-521.

[0075] For seeding undifferentiated stem cells, the seeding density can range from about 6.0x10 3 cells / cm 2 to about 5.0x10 5 cells / cm2 , such as about 1.0x10 4 cells / cm 2 , such as about 5.0x10 4 cells / cm 2 , such as about 1.0x10 5 cells / cm 2 , or for example about 3.0x10 5 cells / cm 2 of the growth surface. In another embodiment, the seeding density can range from about 6.0x10 3 cells / cm 2 to about 1.0x10 4 cells / cm 2 , such as about 6.0x10 3 cells / cm 2 to about 9.0x10 3 cells / cm 2 , such as about 7.0x10 3 cells / cm 2 to about 1.0x10 4 cells / cm 2 , such as 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 to about 8.0x10 3 cells / cm 2 of the growth surface. In yet another embodiment, the seeding density can range from about 1.0x10 4 cells / cm 2 to about 1.0x10 5 cells / cm 2 , such as about 2.0x10 4 cells / cm 2 to about 9.0x10 4 cells / cm 2 , such as about 3.0x10 4 cells / cm 2 to about 8.0x10 4 cells / cm 2 , such as about 4.0x10 4 cells / cm 2 to about 7.0x10 4 cells / cm 2 , or for example about 5.0x10 4cells / cm 2 to about 6.0x10 4 cells / cm 2 of the growth surface. In one embodiment, the seeding density can range from about 1.0x10 5 cells / cm 2 to about 5.0x10 5 cells / cm 2 of the growth surface, such as about 1.0x10 5 cells / cm 2 to about 4.5x10 5 cells / cm 2 such as about 1.5x10 5 cells / cm 2 to about 4.0x10 5 cells / cm 2 such as about 2.0x10 5 cells / cm 2 to about 3.5x10 5 cells / cm 2 or such as about 2.5x10 5 cells / cm 2 to about 3.0x10 5 cells / cm 2 of the growth surface.

[0076] Human pluripotent stem cells are differentiated into dorsal neuroectodermal progenitor cells and further differentiated into dorsal-derived glial progenitor cells and oligodendrocyte progenitor cells

[0077] The present disclosure provides methods for differentiating human pluripotent stem cells into neuroectoderm with a dorsal spinal cord phenotype and further into glial progenitor cells and oligodendrocyte progenitor cells using a combination of small molecule and protein regulators of BMP signaling and inhibitors of MAPK / ERK kinases. Without being bound by any particular theory, the inventors have found that human dorsal neuroectodermal progenitor cells obtained by the methods according to the present disclosure can be readily and efficiently differentiated into spinal cord OPCs in the absence of activation of SHH signaling. Surprisingly, this early dorsal phenotype (although not the region of early OPC generation in vivo) gives rise to glial progenitor cells on day 21 of the differentiation process and OPCs on day 42 of the differentiation process. The day 42 OPCs generated according to the present disclosure express the classical OPC markers NG2 and PDGFRα and are comparable (in terms of their overall marker expression profiles) to OPCs generated using alternative methods currently in clinical testing for the treatment of spinal cord injury.

[0078] In addition, the inventors have found that the methods of the present disclosure produce a significantly greater number of differentiated cells compared to differentiation protocols in which SHH signaling is activated, thereby providing a scalable process for generating large numbers of dorsal neuroectodermal progenitor cells and their progeny such as glial progenitor cells or OPCs for downstream applications.

[0079] In one embodiment, a method includes contacting human pluripotent stem cells with one or more inhibitors of mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) in combination with one or more inhibitors of bone morphogenetic protein (BMP) signaling. In certain embodiments, the MAPK / ERK inhibitor is a small molecule. In other embodiments, the MAPK / ERK inhibitor is a protein, such as a phosphatase that dephosphorylates MAPK / ERK kinases. 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 activin A receptor type I (ACVR1). In certain embodiments, after inhibition of MAPK / ERK and BMP signaling, the cells are cultured in the presence of the caudalizing agent retinoic acid to obtain neuroectoderm-restricted progenitor cells with a dorsal spinal cord phenotype.

[0080] In certain embodiments, the MAPK / ERK inhibitor can be selected from PD0325901, AZD6244, GSK1120212, PD184352, and cobimetinib and their derivatives. In certain embodiments, the inhibitor of BMP signaling can be selected from Dorsomorphin, DMH-1, K02288, ML347, LDN193189, and Noggin protein.

[0081] In one embodiment, a method includes obtaining undifferentiated human pluripotent stem cells maintained in an undifferentiated state; adherently culturing the undifferentiated human pluripotent stem cells in the presence of the small molecules PD0325901, Dorsomorphin, and retinoic acid for a first period of time; and then adherently culturing the cells for a second period of time in the presence of retinoic acid and in the absence of an SHH signaling activator to obtain dorsal neuroectodermal cells, as Figure 1 shown. In one embodiment, the first period of time and the second period of time can each be from about one to about six days, such as about one day, such as about two days, such as about three days, such as about four days, such as about five days, or such as about six days.

[0082] In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of PD0325901 at a concentration range of from about 1 μM to about 100 μM, such as about 2 μM, such as about 3 μM, such as about 4 μM, such as about 5 μM, such as about 6 μM, such as about 7 μM, such as about 8 μM, such as about 9 μM, such as about 10 μM, such as about 11 μM, such as about 12 μM, such as about 13 μM, such as about 14 μM, such as about 15 μM, such as about 20 μM, such as about 30 μM, such as about 40 μM, such as about 50 μM, or such as about 60 μM, 70 μM, 80 μM or 90 μM. In another embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of PD0325901 at a concentration of about 10 μM.

[0083] In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of AZD6244 at a concentration range of from about 1 μM to about 100 μM, such as about 2 μM, such as about 3 μM, such as about 4 μM, such as about 5 μM, such as about 6 μM, such as about 7 μM, such as about 8 μM, such as about 9 μM, such as about 10 μM, such as about 11 μM, such as about 12 μM, such as about 13 μM, such as about 14 μM, such as about 15 μM, such as about 20 μM, such as about 30 μM, such as about 40 μM, such as about 50 μM, or such as about 60 μM, 70 μM, 80 μM or 90 μM. In another embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of AZD6244 at a concentration of about 10 μM.

[0084] In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of GSK1120212 or PD0325901 at a concentration range of from about 1 μM to about 100 μM, such as about 2 μM, such as about 3 μM, such as about 4 μM, such as about 5 μM, such as about 6 μM, such as about 7 μM, such as about 8 μM, such as about 9 μM, such as about 10 μM, such as about 11 μM, such as about 12 μM, such as about 13 μM, such as about 14 μM, such as about 15 μM, such as about 20 μM, such as about 30 μM, such as about 40 μM, such as about 50 μM, or such as about 60 μM, 70 μM, 80 μM or 90 μM. In another embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of GSK1120212 at a concentration of about 10 μM.

[0085] In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of PD184352 at a concentration range of from about 1 μM to about 100 μM, such as about 2 μM, such as about 3 μM, such as about 4 μM, such as about 5 μM, such as about 6 μM, such as about 7 μM, such as about 8 μM, such as about 9 μM, such as about 10 μM, such as about 11 μM, such as about 12 μM, such as about 13 μM, such as about 14 μM, such as about 15 μM, such as about 20 μM, such as about 30 μM, such as about 40 μM, such as about 50 μM, or such as about 60 μM, 70 μM, 80 μM or 90 μM. In another embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of PD184352 at a concentration of about 10 μM.

[0086] In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of cobimetinib at a concentration range of from about 1 μM to about 100 μM, such as about 2 μM, such as about 3 μM, such as about 4 μM, such as about 5 μM, such as about 6 μM, such as about 7 μM, such as about 8 μM, such as about 9 μM, such as about 10 μM, such as about 11 μM, such as about 12 μM, such as about 13 μM, such as about 14 μM, such as about 15 μM, such as about 20 μM, such as about 30 μM, such as about 40 μM, such as about 50 μM, or such as about 60 μM, 70 μM, 80 μM or 90 μM. In another embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of cobimetinib at a concentration of about 10 μM.

[0087] In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of Dorsomorphin at a concentration range of from about 0.2 μM to about 20 μM, such as about 0.5 μM, such as about 0.8 μM, such as about 1 μM, such as about 1.5 μM, such as about 2 μM, such as about 2.5 μM, such as about 3 μM, such as about 3.5 μM, such as about 4 μM, such as about 4.5 μM, such as about 5 μM, such as about 5.5 μM, such as about 6 μM, such as about 6.5 μM, such as about 7 μM, such as about 7.5 μM, such as about 8 μM, such as about 8.5 μM, such as about 9 μM, such as about 10 μM, such as about 11 μM, such as about 12 μM, such as about 13 μM, such as about 14 μM, such as about 15 μM, such as about 16 μM, such as about 17 μM, such as about 18 μM, or such as about 19 μM. In another embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of Dorsomorphin at a concentration range of from about 0.2 μM to about 1 μM, such as from about 0.2 μM to about 0.9 μM, such as from about 0.3 μM to about 0.8 μM, such as from about 0.4 μM to about 0.7 μM, or such as from about 0.5 μM to about 0.6 μM. In yet another embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of Dorsomorphin at a concentration range of from about 1 μM to about 10 μM, such as from about 1 μM to about 9 μM, such as from about 2 μM to about 8 μM, such as from about 3 μM to about 7 μM, or such as from about 4 μM to about 6 μM. In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of Dorsomorphin at a concentration range of from about 10 μM to about 20 μM, such as from about 10 μM to about 19 μM, such as from about 12 μM to about 18 μM, such as from about 13 μM to about 17 μM, or such as from about 14 μM to about 16 μM. In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of Dorsomorphin at a concentration of about 2 μM.

[0088] In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of an ALK2 inhibitor at a concentration range of from about 1 nM to about 20 μM, such as about 10 nM, about 50 nM, about 100 nM, about 150 nM, about 200 nM, about 500 nM, about 1 μM, about 5 μM, about 10 μM or about 15 μM.

[0089] In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of DMH-1 at a concentration range of from about 1 μM to about 10 μM. In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of DMH-1 at a concentration of about 2 μM.

[0090] In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of K02288 at a concentration range of about 1 μM to about 10 μM. In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of about 2 μM of K02288.

[0091] In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of ML347 at a concentration range of about 1 μM to about 10 μM. In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of about 2 μM of ML347.

[0092] In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of LDN193189 at a concentration range of about 1 μM to about 10 μM. In one embodiment, a method includes culturing undifferentiated human pluripotent stem cells in the presence of about 2 μM of LDN193189.

[0093] According to the present disclosure, any tissue culture vessel suitable for adherent cell culture can be used to obtain dorsal neuroectodermal progenitor cells. Suitable growth substrates include, for example, recombinant laminin, vitronectin, the recombinant fragment E8 of laminin-511, or a matrix (such as GFR). 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 SC substrate.

[0094] In one embodiment, the dorsal neuroectodermal progenitor cells obtained according to the present disclosure can be harvested and further cultured as aggregates in suspension culture in the presence of bFGF and EGF until the cells mature into glial progenitor cells. In one embodiment, the further culture period can range from about five days to fifteen days, such as about five days, about six days, about seven days, about eight days, about nine days, about ten days, about eleven days, about twelve days, about thirteen days, about fourteen days, or about fifteen days. In one embodiment, the further culture period is about 7 days.

[0095] In one embodiment, adherently cultured dorsal neuroectodermal progenitor cells can be harvested by enzymatic means, such as using TrypLE TM Select (Thermo Fisher Scientific, Waltham, MA), (SigmaAldrich, MO) or a similar trypsin. Alternatively, adherently cultured dorsal neural ectoderm progenitor cells can be harvested using non-enzymatic means, such as using 0.5 mM EDTA in PBS, or for example using ReLeSR TM (Stem Cell Technologies, Vancouver, Canada).

[0096] Any cell culture vessel or reactor suitable for suspension culture can be used for the non-adherent culture steps contemplated in the present disclosure. The vessel walls are typically inert or resistant to the adhesion of the cultured cells. For dynamic suspension, there are also means to prevent cell sedimentation, such as stirring mechanisms, such as magnetic or mechanically driven stir bars or paddles, shaking mechanisms (usually externally connected to the vessel), or inversion mechanisms (i.e., devices that rotate the vessel to change the direction of gravity on the cells).

[0097] Vessels suitable for suspension culture for process development include the generally available commercially available spinner flasks, roller bottles, shake bags, or shake flasks. Exemplary bioreactors suitable for commercial production include VerticalWheel TM bioreactor (PBS Biotech, Camarillo, CA).

[0098] Aggregates can also be formed prior to dynamic suspension. For example, cells can be placed on an AggreWell TM plate to generate uniform cell aggregates. After approximately three days, the cell aggregates can be transferred to a dynamic suspension.

[0099] In one embodiment, the glial progenitor cells obtained according to the present disclosure can be harvested, plated, and adherently cultured for an additional period of time in the presence of epidermal growth factor (EGF) until the cells mature into oligodendrocyte progenitor cells. In certain embodiments, the culture medium additionally contains platelet-derived growth factor AA (PDGF-AA). In one embodiment, the range of the further culture period can be from about ten days to thirty days, such as about ten days, about fifteen days, about twenty days, about twenty-five days, or about thirty days. In one embodiment, the range of the further culture period is from about fifteen to twenty-five days, such as about fifteen days, about sixteen days, about seventeen days, about nineteen days, about twenty days, about twenty-one days, about twenty-two days, about twenty-three days, about twenty-four days, or about twenty-five days. In one embodiment, the further culture period is about twenty-one days.

[0100] OPC composition

[0101] The methods of the present disclosure can be used to obtain compositions comprising oligodendrocyte progenitor cells (OPCs) suitable for cell therapy. The OPCs obtained according to the present disclosure express high levels of the OPC characteristic proteoglycans NG2, PDGFRα, and / or GD3 and low levels of non-OPC related markers associated with unwanted cell types, such as CD49f, which can be expressed by 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, which are two markers expressed by pluripotent cells and epithelial cells (Lin D, Guo Y, Li Y, Ruan Y, Zhang M, Jin X, Yang M, Lu Y, Song P, Zhao S, Dong B, Xie Y, Dang Q, Quan C. Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions. Oncol Rep. 2017 Aug;38(2):875-885; Huang L, Yang Y, Yang F, Liu S, Zhu Z, Lei Z, Guo J. Functions of EpCAM in physiological processes and diseases (Review). Int J Mol Med. 2018 Oct;42(4):1771-1785).

[0102] In certain embodiments, the OPCs generated according to the present disclosure are in vitro differentiated progeny of human pluripotent stem cells. In certain embodiments, the OPCs obtained according to the present disclosure are in vitro differentiated progeny of human embryonic stem cells. In other embodiments, the OPCs obtained according to the present disclosure are in vitro differentiated progeny of induced pluripotent stem (iPS) cells.

[0103] One or more characteristics of the obtained OPC population can be determined by using flow cytometry to quantify various cell markers, for example, to determine the percentage of the cell population positive for a specific marker or group of markers or to identify unwanted cell types present in the OPC population.

[0104] OPC populations obtained according to the present disclosure may comprise from about 30% to about 100% NG2-positive cells, such as at least about 35%, such as at least about 40%, such as at least about 45%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65%, such as at least about 70%, such as at least about 75%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95%, such as at least about 98%, such as at least about 99%, such as at least about 99.5%, such as at least about 99.8%, or such as at least about 99.9% NG2-positive cells. In certain embodiments, OPC populations obtained according to the present disclosure may comprise from about 45% to about 75% NG2-positive cells, such as from about 45% to about 50%, such as from about 50% to about 55%, such as from about 55% to about 60%, such as from about 60% to about 65%, such as from about 65% to about 70%, such as from about 70% to about 75%, such as from about 50% to about 70%, such as from about 55% to about 65%, or such as from about 58% to about 63% NG2-positive cells. In other embodiments, OPC populations obtained according to the present disclosure may comprise from about 60% to about 90% NG2-positive cells, such as from about 60% to about 65%, such as from about 65% to about 70% positive cells.

[0105] OPC populations 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, OPC populations obtained according to the present disclosure may comprise from about 45% to about 75% PDGFRα-positive cells, such as from about 45% to about 50%, such as from about 50% to about 55%, such as from about 55% to about 60%, such as from about 60% to about 65%, such as from about 65% to about 70%, such as from about 70% to about 75%, such as from about 50% to about 70%, such as from about 55% to about 65%, or such as from about 58% to about 63% PDGFRα-positive cells. In other embodiments, OPC populations obtained according to the present disclosure may comprise from about 60% to about 90% PDGFRα-positive cells, such as from about 60% to about 65%, such as from about 65% to about 70% positive cells.

[0106] OPC populations obtained according to the present disclosure can comprise from about 30% to about 100% GD3-positive cells, such as at least about 35%, such as at least about 40%, such as at least about 45%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65%, such as at least about 70%, such as at least about 75%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95%, such as at least about 98%, such as at least about 99%, such as at least about 99.5%, such as at least about 99.8%, or such as at least about 99.9% GD3-positive cells. In certain embodiments, OPC populations obtained according to the present disclosure can comprise from about 45% to about 75% GD3-positive cells, such as from about 45% to about 50%, such as from about 50% to about 55%, such as from about 55% to about 60%, such as from about 60% to about 65%, such as from about 65% to about 70%, such as from about 70% to about 75%, such as from about 50% to about 70%, such as from about 55% to about 65%, or such as from about 58% to about 63% GD3-positive cells. In other embodiments, OPC populations obtained according to the present disclosure can comprise from about 60% to about 90% GD3-positive cells, such as from about 60% to about 65%, such as from about 65% to about 70% positive cells.

[0107] In one embodiment, OPC populations obtained according to the present disclosure are capable of forming less than or equal to four epithelial cysts per 100,000 cells in the cyst assay as described in Example 8 of the present disclosure. In another embodiment, OPC populations obtained according to the present disclosure are capable of forming less than or equal to three epithelial cysts per 100,000 cells in the cyst assay. In another embodiment, OPC populations obtained according to the present disclosure are capable of forming less than or equal to two epithelial cysts per 100,000 cells in the cyst assay. In yet another embodiment, OPC populations obtained according to the present disclosure are capable of forming less than or equal to one epithelial cyst per 100,000 cells in the cyst assay as described in Example 8 of the present disclosure.

[0108] Unwanted cell types

[0109] OPC populations obtained according to the present disclosure contain low levels of unwanted cell types, as measured, for example, by quantifying markers associated with the unwanted cell types by flow cytometry. In one non-limiting example, day 42 OPCs obtained according to the present disclosure can contain low levels of cells expressing the epithelial cell-associated markers EpCAM, CD49f, and CLDN6.

[0110] Markers associated with unwanted cell types may comprise less than about 20% unwanted 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% unwanted cell types. In another embodiment, the cell population may comprise from about 15% to about 20% unwanted cell types, such as from about 19% to about 20%, such as from about 18% to about 20%, such as from about 17% to about 20%, such as from about 16% to about 20%, such as from about 15% to about 19%, or such as from about 16% to about 18% unwanted cell types. In yet another embodiment, the cell population may comprise from about 10% to about 15% unwanted cell types, such as from about 14% to about 15%, such as from about 13% to about 15%, such as from about 12% to about 15%, such as from about 11% to about 15%, or such as from about 12% to about 14% unwanted cell types. In one embodiment, the cell population may comprise from about 1% to about 10% unwanted cell types, such as from about 2% to about 10%, such as from about 1% to about 9%, such as from about 2% to about 8%, such as from about 3% to about 7%, or such as from about 4% to about 6% unwanted cell types. In one embodiment, the cell population may comprise from about 0.1% to about 1% unwanted cell types, such as from about 0.2% to about 1%, such as from about 0.1% to about 0.9%, such as from about 0.2% to about 0.8%, such as from about 0.3% to about 0.7%, or such as from about 0.4% to about 0.6% unwanted cell types. In one embodiment, a low level of unwanted cell types may indicate the presence of less than about 15% unwanted cell types.

[0111] In one embodiment, unwanted cell types may include cells expressing one or more markers selected from CD49f, CLDN6, or EpCAM.

[0112] Cryopreservation

[0113] After harvest, an expanded population of OPCs can be formulated at a specific therapeutic dose (e.g., cell number) and cryopreserved for shipment to a clinic. Then, a ready-to-administer (RTA) OPC therapeutic composition can be administered directly after thawing without further processing. Examples of media suitable for cryopreservation include, but are not limited to, 90% human serum / 10% DMSO, Medium 310% (CS10), Medium 2 5% (CS5), and Medium 1 2% (CS2), Stem Cell Banker, PRIME FREEZIS, CSB, trehalose, and the like.

[0114] In some embodiments, the percentage of viable filtered cells stored in the cryopreservation medium for about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments, the percentage of recovered filtered cells stored in the cryopreservation medium for about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0115] In further embodiments, the percentage of viable filtered cells stored in the neutralization medium for about 0 to about 8 hours and then in the cryopreservation medium for about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments, the percentage of recovered filtered cells stored in the neutralization medium for about 0 to about 8 hours and then in the cryopreservation medium for about 0 to about 8 hours is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0116] In other embodiments, the percentage of viable filtered cells stored in neutralization medium for about 0 to about 8 hours and then in cryopreservation medium for about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% after thawing of the cryopreserved composition. In other embodiments, the percentage of recovered filtered cells stored in neutralization medium for about 0 to about 8 hours and then in cryopreservation medium for about 0 to about 8 hours is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% after thawing of the cryopreserved composition.

[0117] In some embodiments, filtered OPCs stored in neutralization medium for about 0 to about 8 hours and then in cryopreservation medium for about 0 to about 8 hours are capable of secreting versican after thawing of the cryopreserved composition. In other embodiments, filtered OPCs stored in neutralization medium for about 0 to about 8 hours and then in cryopreservation medium for about 0 to about 8 hours are capable of being expanded after thawing of the cryopreserved composition.

[0118] In some embodiments, the percentage of viable filtered OPCs stored in neutralization medium for about 0 to about 8 hours at room temperature is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the percentage of viable filtered OPCs stored in cryopreservation medium for about 0 to about 8 hours at room temperature is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In further embodiments, the percentage of viable filtered cells stored in neutralization solution for about 0 to about 8 hours at room temperature and then in cryopreservation medium for about 0 to about 8 hours at room temperature is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In still further embodiments, the percentage of recovered filtered cells stored in neutralization solution for about 0 to about 8 hours at room temperature and then in cryopreservation medium for about 0 to about 8 hours at room temperature is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 140%, 150%.

[0119] OPCs formulated in cryopreservation media suitable for ready-to-administer-after-thawing (RTA) applications can comprise OPCs suspended in adenosine, dextran-40, lactobionic acid, HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate, potassium phosphate, glucose, sucrose, mannitol, calcium chloride, magnesium chloride, potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), and water. An example of such a cryopreservation media can be obtained commercially under the trade name and is manufactured by BioLife Solutions, Inc.

[0120] DMSO can be used as a cryoprotectant to prevent the formation of ice crystals that can kill cells during cryopreservation. In some embodiments, the cryopreservable OPC therapeutic composition comprises from about 0.1% to about 2% DMSO (v / v). In some embodiments, the RTA OPC therapeutic composition comprises from about 1% to about 20% DMSO. In some embodiments, the RTA OPC therapeutic composition comprises about 10% DMSO. In some embodiments, the RTA OPC cell therapeutic composition comprises about 5% DMSO.

[0121] In some embodiments, an OPC therapy formulated in a cryopreservation media suitable for ready-to-administer-after-thawing (RTA) applications can comprise OPCs suspended in a cryopreservation media without DMSO. For example, the RTA OPC therapeutic cell composition can comprise OPCs suspended in a cryopreservation media without DMSO (dimethyl sulfoxide, (CH 3 ) 2 SO) or any other dipolar aprotic solvent, Trolox, Na+, K+, Ca2+, Mg2+, Cl-, H2PO4-, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, glutathione. An example of such a cryopreservation media can be obtained commercially under the trade name or and is also manufactured by BioLife Solutions, Inc. In other embodiments, an OPC composition formulated in a cryopreservation media suitable for ready-to-administer-after-thawing applications can comprise OPCs suspended in trehalose.

[0122] The RTA OPC therapeutic composition can optionally comprise additional factors that support OPC engraftment, integration, survival, efficacy, etc. In some embodiments, the RTA OPC therapeutic composition comprises a functional activator of the OPC formulation described herein.

[0123] In some embodiments, the RTA OPC therapeutic composition can be formulated in a culture medium comprising components that reduce molecular and cellular stress during freezing and thawing by scavenging free radicals, pH buffering, swelling / osmotic support, and maintaining ionic concentration balance.

[0124] In some embodiments, an OPC therapy formulated in a cryopreservation medium suitable for ready-to-use applications after thawing can comprise one or more immunosuppressive compounds. In certain embodiments, an OPC therapy formulated in a cryopreservation medium suitable for ready-to-use applications after thawing can comprise one or more immunosuppressive compounds that are formulated for slow release of one or more immunosuppressive compounds. Immunosuppressive compounds used in conjunction with the formulations described herein may belong to the following classes of immunosuppressive drugs: glucocorticoids, cytostatic agents (e.g., alkylating agents or antimetabolites), antibodies (polyclonal or monoclonal), drugs acting on immunophilins (e.g., cyclosporine, tacrolimus, or sirolimus). Other drugs include interferons, opioids, TNF-binding proteins, mycophenolate mofetil, and small biologics. Examples of immunosuppressive drugs include: mesenchymal stem cells, antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, BAS 1L1 (anti-IL-2Ra receptor antibody), cyclosporine (cyclosporine A), (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, (anti-CD20 antibody), sirolimus, tacrolimus, Tacrolimus, and / or mycophenolate mofetil.

[0125] Formulations

[0126] The OPC composition according to the present disclosure can further comprise a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier can comprise dimethyl sulfoxide (DMSO). In one embodiment, the pharmaceutically acceptable carrier does not comprise dimethyl sulfoxide. As described above, the composition can be further adjusted to be suitable for cryopreservation at -80°C to -195°C or below -80°C to -195°C.

[0127] OPC compositions according to the present disclosure can be formulated for administration by direct injection into the spinal cord of a subject. In one embodiment, an OPC composition according to the present disclosure can be formulated for administration intracranially, intraventricularly, intrathecally, intranasally, or intracisternally to a subject. In one embodiment, an OPC composition according to the present disclosure can be formulated for administration by direct injection into the infarct cavity in the brain of a subject or by injection adjacent to the infarct cavity in the brain of a subject. In one embodiment, a composition according to the present disclosure can be formulated for administration by implantation. In one embodiment, a composition according to the present disclosure can be formulated as a solution.

[0128] The OPC composition according to the present disclosure may comprise from about 1x10 6 to about 5x10 8 cells per milliliter, such as about 1x10 6 cells per milliliter, such as about 2x10 6 cells per milliliter, such as about 3x10 6 cells per milliliter, such as about 4x10 6 cells per milliliter, such as about 5x10 6 cells per milliliter, such as about 6x10 6 cells per milliliter, such as about 7x10 6 cells per milliliter, such as about 8x10 6 cells per milliliter, such as about 9x10 6 cells per milliliter, such as about 1x10 7 cells per milliliter, such as about 2x10 7 cells per milliliter, such as about 3x10 7 cells per milliliter, such as about 4x10 7 cells per milliliter, such as about 5x10 7 cells per milliliter, such as about 6x10 7 cells per milliliter, such as about 7x10 7 cells per milliliter, such as about 8x10 7 cells per milliliter, such as about 9x10 7 cells per milliliter, such as about 1x10 8 cells per milliliter, such as about 2x10 8 cells per milliliter, such as about 3x10 8 cells per milliliter, such as about 4x10 8 cells per milliliter, or such as about 5x10 8 cells per milliliter. In another embodiment, a composition according to the present disclosure may comprise from about 1x10 8 to about 5x10 8cells / ml, such as about 1x10 8 to about 4x10 8 cells / ml, such as about 2x10 8 to about 5x10 8 cells / ml, such as about 1x10 8 to about 3x10 8 cells / ml, such as about 2x10 8 to about 4x10 8 cells / ml, or such as about 3x10 8 to about 5x10 8 cells / ml. In yet another embodiment, the composition according to the present disclosure may comprise about 1x10 7 to about 1x10 8 cells / ml, such as about 2x10 7 to about 9x10 7 cells / ml, such as about 3x10 7 to about 8x10 7 cells / ml, such as about 4x10 7 to about 7x10 7 cells / ml, or such as about 5x10 7 to about 6x10 7 cells / ml. In one embodiment, the composition according to the present disclosure may comprise about 1x10 6 to about 1x10 7 cells / ml, such as about 2x10 6 to about 9x10 6 cells / ml, such as about 3x10 6 to about 8x10 6 cells / ml, such as about 4x10 6 to about 7x10 6 cells / ml, or such as about 5x10 6 to about 6x10 6 cells / ml. In yet another embodiment, the composition according to the present disclosure may comprise at least about 1x10 6 cells / ml, such as at least about 2x10 6 cells / ml, such as at least about 3x10 6 cells / ml, such as at least about 4x10 6 cells / ml, such as at least about 5x10 6 cells / ml, such as at least about 6x10 6 cells / ml, such as at least about 7x10 6 cells / ml, such as at least about 8x10 6cells / mL, such as at least about 9x10 6 cells / mL, such as at least about 1x10 7 cells / mL, such as at least about 2x10 7 cells / mL, such as at least about 3x10 7 cells / mL, such as at least about 4x10 7 cells / mL, or such as at least about 5x10 7 cells / mL. In one embodiment, the composition according to the present disclosure may comprise up to about 1x10 8 cells or more, such as up to about 2x10 8 cells / mL or more, such as up to about 3x10 8 cells / mL or more, such as up to about 4x10 8 cells / mL or more, such as up to about 5x10 8 cells / mL or more, or such as up to about 6x10 8 cells / mL.

[0129] In one embodiment, the OPC composition according to the present disclosure may comprise from about 4×10 7 to about 2×10 8 cells / mL.

[0130] In yet another embodiment, the volume of the OPC composition according to the present disclosure can be from about 10 microliters to about 5 milliliters, such as about 20 microliters, such as about 30 microliters, such as about 40 microliters, such as about 50 microliters, such as about 60 microliters, such as about 70 microliters, such as about 80 microliters, such as about 90 microliters, such as about 100 microliters, such as about 200 microliters, such as about 300 microliters, such as about 400 microliters, such as about 500 microliters, such as about 600 microliters, such as about 700 microliters, such as about 800 microliters, such as about 900 microliters, such as about 1 milliliter, such as about 1.5 milliliters, such as about 2 milliliters, such as about 2.5 milliliters, such as about 3 milliliters, such as about 3.5 milliliters, such as about 4 milliliters, or such as about 4.5 milliliters. In one embodiment, the volume of the composition according to the present disclosure can be from about 10 microliters to about 100 microliters, such as from about 20 microliters to about 90 microliters, such as from about 30 microliters to about 80 microliters, such as from about 40 microliters to about 70 microliters, or such as from about 50 microliters to about 60 microliters. In another embodiment, the volume of the composition according to the present disclosure can be from about 100 microliters to about 1 milliliter, such as from about 200 microliters to about 900 microliters, such as from about 300 microliters to about 800 microliters, such as from about 400 microliters to about 700 microliters, or such as from about 500 microliters to about 600 microliters. In yet another embodiment, the volume of the composition according to the present disclosure can be from about 1 milliliter to about 5 milliliters, such as from about 2 milliliters to about 5 milliliters, such as from about 1 milliliter to about 4 milliliters, such as from about 1 milliliter to about 3 milliliters, such as from about 2 milliliters to about 4 milliliters, or such as from about 3 milliliters to about 5 milliliters. In one embodiment, the OPC composition according to the present disclosure can have a volume of from about 20 microliters to about 500 microliters. In another embodiment, the OPC composition according to the present disclosure can have a volume of from about 50 microliters to about 100 microliters. In yet another embodiment, the OPC composition according to the present disclosure can have a volume of from about 50 microliters to about 200 microliters. In another embodiment, the OPC composition according to the present disclosure can have a volume of from about 20 microliters to about 400 microliters. In one embodiment, the OPC composition according to the present disclosure can be in a container configured for cryopreservation or administration to a subject in need thereof. In one embodiment, the container can be a prefilled syringe.

[0131] Method of Use

[0132] The OPC composition obtained according to the present disclosure can be used in cell therapy to improve one or more neurological functions of a subject in need of treatment. In one embodiment, a population of OPC cells according to the present disclosure can be injected or implanted into a subject in need thereof. In one embodiment, a population of cells according to the present disclosure can be implanted into a subject in need thereof for the treatment of spinal cord injury, stroke, or multiple sclerosis.

[0133] In one embodiment, a cell population according to the present disclosure is capable of inducing myelination of naked axons at an implantation site in a subject. In one embodiment, a cell population produced by the method according to the present disclosure may exhibit improved transplantation and migration capabilities. In one embodiment, a cell population produced by the method according to the present disclosure is capable of improving post-injury repair or regeneration of neural tissue in a subject.

[0134] A cell population according to the present disclosure is capable of improving the sensory function of a subject in need of treatment after implanting the cell population into the subject. The improvement in sensory function can be evaluated using the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) examination, for example, to determine the sensory levels of pinprick and light touch on the right and left sides. A cell population according to the present disclosure is capable of improving the motor function of a subject in need of treatment after implanting the cell population into the subject. The improved motor function can be evaluated using the ISNCSCI examination, for example, to determine the motor levels on the right and left sides for complete paralysis, palpable or visible contraction, active movement, full movement against gravity, and sufficient resistance.

[0135] A cell population according to the present disclosure is capable of reducing the volume of injury-induced central nervous system parenchymal cavitation within 12 months or less. In one embodiment, a cell population according to the present disclosure is capable of reducing the volume of injury-induced central nervous system parenchymal cavitation in a subject within 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, or less than 1 month.

[0136] The present invention has now been generally described, and these 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 present disclosure unless otherwise specified. Examples

[0137] Example 1 - Culture and Expansion of Undifferentiated Human Embryonic Stem Cells

[0138] Undifferentiated human embryonic stem cells (uhESC) 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 tissue culture-treated polystyrene T-75 flasks (Corning #431082) coated with recombinant human laminin-521 (rhLn-521, Corning #354224) in complete mTeSR TM -1 medium (Stem Cell Technologies #85850). The medium was completely replaced daily until the cells reached approximately 80-90% confluence, and then the uhESC were passaged using ReLeSR TM reagent (Stem Cell Technologies #05872). The ReLeSR TM lifted uhESC cells were seeded in new rhLn-521-coated 225 cm 2 flasks, and daily medium replacement was resumed two days after seeding. Depending on the experiment, the cultured uhESC from the WCB were expanded in this manner for 2 to 5 passages before differentiating into neuroectodermal progenitor cells as described in Example 2.

[0139] Example 2 - Method for differentiating human embryonic stem cells into neuroectodermal progenitor cells with a dorsal spinal cord progenitor cell phenotype

[0140] The expanded uhESC were seeded on rhLn-521-coated containers and cultured until 40-70% confluence was reached, at which point differentiation was initiated.

[0141] Days 0-3: By completely removing mTeSR TM-1 Medium and initiate differentiation by adding glial progenitor cell medium (GPM; consisting of DMEM / F12 (Gibco catalog number 10565-018) supplemented with 2% B27 supplement (Gibco catalog number 17504-044) and 0.04 μg / mL triiodothyronine (Sigma-Aldrich catalog number T5516-1MG)) supplemented with 10 μM MAPK / ERK inhibitor PD0325901 (PD; Sigma-Aldrich catalog number PZ0162), 2 μM BMP signaling inhibitor Dorsomorphin (Dorso; Sigma-Aldrich catalog number P5499), and 1 μM retinoic acid (RA; Sigma-Aldrich catalog number R2625). The medium was replenished daily.

[0142] Days 4 - 6: On day 4, switch the medium to GPM supplemented with 1 μM RA and 150 μM ascorbic acid (Sigma-Aldrich catalog number A4544) and replenish daily.

[0143] Day 7: On day 7, harvest the cells for expansion and further differentiation into glial progenitor cells as described in Example 3. A subset of the cells was collected for analysis by quantitative PCR (qPCR; as described in Example 6), flow cytometry (as described in Example 5), and when available, individual well plates were set up for analysis for immunocytochemistry (ICC) (as described in Example 5). On day 7, these cells exhibited marker expression consistent with dorsal spinal cord progenitors (Table 2, Figure 3 ).

[0144] Example 3 - Method for differentiating human embryonic stem cells into glial lineage cells

[0145] Days 7 - 13: Differentiate uhESCs into neuroectodermal progenitor cells (specifically with a dorsal phenotype) as described in Example 2. On day 7, lift the cells using TrypLE TM Select (Thermo Fisher, catalog number A12859-01), count, and seed at 2.7x10 4 cells / cm 2The seeding density was seeded onto rhLn-521-coated containers in GPM supplemented with 20 ng / mL human basic fibroblast growth factor (hbFGF, Thermo Fisher, catalog number PHG0263), 10 ng / mL epidermal growth factor (EGF, Thermo Fisher, catalog number PHG0311), and 10 μM Rho kinase inhibitor (RI, Tocris catalog number 1254). The medium was replenished daily by aspirating the used medium and replacing it with fresh GPM+hbFGF+EGF.

[0146] Days 14 - 21: On day 14, cells were lifted using TrypLE TM Select, counted, resuspended in GPM+hbFGF+EGF+RI, and re-seeded into a dynamic suspension culture in a PBS-0.1L or PBS-0.5L mini-bioreactor system (PBS Biotech) at a density of 1.83x10 6 viable cells / mL. A subset of cells from day 14 was collected for flow cytometry (Example 5), ICC (Example 5), and qPCR (Example 6) analysis. The PBS0.1L and PBS0.5L mini-bioreactors were set to rotate at 35 RPM and 28 RPM, respectively. The medium was replenished daily by allowing the aggregates to settle, removing 70 - 80% of the used medium, and replacing it with an equal volume of GPM+bhFGF+EGF. On day 15, the rotation speeds of the PBS0.1L and PBS0.5L mini-bioreactors were increased to 45 RPM and 32 RPM, respectively.

[0147] On day 21, a subset of aggregates was collected for ICC (Example 5) and qPCR (Example 6). By day 21, the differentiated cells expressed markers consistent with glial-restricted cells (Table 2, Figure 4 ).

[0148] Example 4 - Method for differentiating human embryonic stem cells into oligodendrocyte progenitor cells

[0149] Days 21 - 42: The glial-restricted progenitor cells obtained in Example 3 were further differentiated into oligodendrocyte progenitor cells (OPCs). The differentiation protocol for days 0 - 20 was carried out as described in Examples 2 and 3. On day 21, the aggregates were transferred from the dynamic suspension to a culture vessel coated with rhLn-521. For example, starting with a total volume of 60 mL in a lxPBS-0.1L micro-bioreactor, 60 mL of the culture was separated into 2 x T75 culture flasks, each with a volume of 30 mL. Subsequently, the cells were fed every other day with GPM supplemented with 20 ng / mL EGF and 10 ng / mL platelet-derived growth factor AA (PDGFAA; PeproTech, catalog number AF-100-13A). Every 7 days (i.e., days 28 and 35), the cells were lifted, counted, and re-seeded at an inoculation density of 4 x 10 TM viable cells / cm 4 into fresh culture vessels coated with rhLn-521. 2

[0150] On day 42, the differentiated cells were harvested. The cells were detached from the vessels, counted, and re-formulated in CryoStor 10 (BioLife Solutions, catalog number 210102) before cryopreservation using TrypLE TM Select. A subset of the cells was collected for analysis by flow cytometry (Example 5), ICC (Example 5), and qPCR (Example 6). By day 42, the differentiated cells expressed markers characteristic of OPCs, as measured by the three analytical methods (Tables 1, 2, Figure 5 ).

[0151] Example 5 - Characterization of the Differentiated Cell Populations by Immunocytochemistry and Flow Cytometry

[0152] Flow cytometry and immunocytochemistry (ICC) can be used to detect and characterize different aspects of protein marker expression in cell populations. While flow cytometry can be used to quantify the percentage of individual cells in a population exhibiting a given protein marker profile, ICC provides additional information about the subcellular localization of each protein marker and can be applied to single cells or cell aggregates. By using one or both of these protein analysis methods, we tracked the differentiation of human embryonic stem cells into neuroectodermal progenitor cells, glial progenitor cells, and oligodendrocyte progenitor cells according to the methods of the present disclosure.

[0153] ​For human embryonic stem cells differentiated into neuroectodermal progenitors and glial progenitors, protein marker expression in cells at day 7 and day 21 of differentiation was characterized by ICC. At room temperature (RT), adherent 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 the fixed aggregates were sequentially placed in sucrose solutions of increasing concentration (10%, 20%, and 30% weight / volume) for 30 minutes at RT, 30 minutes at RT, and overnight at 4 °C, respectively. After sucrose replacement, the aggregates were embedded in Tissue-Tek Optimal Cutting Temperature (OCT) compound (Sakura Finetek USA #4583) and frozen at -80 °C. The OCT-embedded aggregates were heated to -20 °C, sectioned into 30-μm sections using a cryostat (model CM3050 S, Leica Biosystems, Buffalo Grove, IL, USA), and mounted on poly-L-lysine (Sigma-Aldrich #P4707)-coated glass slides. For immunocytochemical staining, fixed adherent cells and aggregate sections mounted on slides were permeabilized and blocked in a blocking solution consisting of 0.1% Triton TM X-100 / 2% normal goat serum / 1% bovine serum albumin for 2 hours at room temperature (RT). After permeabilization and blocking, adherent cells and aggregate sections were incubated overnight at 4 °C in a blocking solution without Triton TM X-100 and containing primary antibodies specific for the protein markers of interest, including PAX6 (BD Pharmingen #561462 or BioLegend #901301) to detect neuroectodermal progenitors and AP2 (Developmental Studies Hybridoma Bank - DSHB #3B5), PAX3 (DSHB #Pax3), and PAX7 (DSHB #Pax7) to detect dorsal spinal cord progenitors. Then, adherent cells and aggregate sections were washed 3 times with PBS and then incubated in the dark for 1 hour at room temperature in a blocking solution without Triton TM X-100 with a secondary antibody specific for the selected primary antibody and a 4’,6-diamidino-2-phenylindole (DAPI) counterstain. Adherent cells and aggregate sections were washed 3 times with PBS and imaged using an IN Cell Analyzer 2000 (GE Healthcare, Pittsburgh, PA, USA).

[0154] Figure 3Representative ICC data of day 7 neuroectodermal progenitors showing a dorsal spinal cord progenitor phenotype are shown. After 7 days of differentiation, adherent cell populations from two representative experiments expressed PAX6, a protein marker characteristic of neuroectodermal progenitors (Lippmann ES, Williams CE, Ruhl DA, Estevez-Silva MC, Chapman ER, Coon JJ, Ashton RS. Deterministic HOX patterning in human pluripotent stemcell-derived neuroectoderm. Stem Cell Reports. 2015 Apr14;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, NamTS, Cho SR, Kim DW. Highly pure and expandable PSA-NCAM-positive neuralprecursors from human ESC and iPSC-derived neural rosettes. PLoS One. 2012;7(7):e39715), and also expressed the dorsal spinal cord progenitor markers AP2, PAX3, and PAX7 (Le Dréau G, MartíE. Dorsal-ventral patterning of the neural tube: a tale of three signals. DevNeurobiol. 2012Dec;72(12):1471-81; Marklund U, Alekseenko Z, Andersson E, Falci S, Westgren M, Perlmann T, Graham A, E, Ericson J. Detailed expressionanalysis of regulatory genes in the early developing human neural tube. StemCells Dev. 2014 Jan1;23(1):5-15).

[0155] Figure 4Shows representative ICC data of glial progenitor cells on day 21. Aggregates were sectioned and stained for dorsal progenitor markers AP2, PAX3, and PAX7, as well as the pan-neural progenitor marker PAX6. Although these early progenitors were still present on day 21, there was also a distinct glial cell population expressing the oligodendrocyte progenitor 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).

[0156] For human embryonic stem cells that differentiated into oligodendrocyte progenitor cells by day 42, the expression of protein markers in the resulting single-cell population was characterized by both flow cytometry and ICC.

[0157] To characterize the protein marker expression of oligodendrocyte progenitor cells by ICC, the slide-mounted aggregate sections were stained as described above, except that permeabilization was performed with 100% methanol for 2 minutes at room temperature, and the blocking solution consisted of 10% fetal bovine serum in PBS.

[0158] Figure 5Shows representative ICC data of oligodendrocyte progenitor cells on day 42. The resulting single-cell populations from two representative experiments expressed the oligodendrocyte progenitor 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) and showed reduced expression of the dorsal spinal cord progenitor marker AP2 (compare Figure 3 and 5 ).

[0159] 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 the markers of interest (including NG2 (Invitrogen #37-2300), PDGFRα (BD Biosciences #563575), GD3 (Millipore #MAB2053), A2B5 (BD #563775), CD49f (Millipore #CBL458P), EpCAM (Dako #M080401-2) and CLDN6 (ThermoFisher #MA5-24076)) and their isotype controls. Cells were washed with staining buffer to remove unbound antibodies; in the case of unconjugated antibodies, the cells were then incubated on ice for 30 minutes with the appropriate fluorophore-conjugated secondary antibody. Cells were washed and then propidium iodide was added to distinguish dead cells. In some cases, cells were cultured overnight at 37°C / 5% CO 2 in tissue culture vessels coated with Matrigel (Corning #356231) to recover protein markers that showed sensitivity to the day 42 harvest procedure described in Example 4, and then TrypLE TMHarvested cells (Thermo Fisher #A12859-01) were stained as described above for flow cytometry analysis. All cells were analyzed on an Attune NxT flow cytometer (Thermo Fisher, Waltham, MA, USA). To calculate the percentage of cells expressing a given protein marker, dead cells stained with propidium iodide were gated, and the number of live cells bound to the corresponding antibody was expressed as a fraction of the total number of cells analyzed after correcting for cells showing non-specific binding to the isotype control antibody.

[0160] Table 1 shows representative flow cytometry data of oligodendrocyte progenitor cells on day 42 generated according to the method described in Example 5. As shown in two representative runs, a high proportion of cells in the resulting cell population express characteristic oligodendrocyte markers, including NG2 and PDGFRα (Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O'Keeffe S, Phatnani HP, Guarnieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, Daneman R, Maniatis T, Barres BA, Wu JQ. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014 Sep 3;34(36):11929-47) and GD3 (Gallo V, Zhou JM, McBain CJ, Wright P, Knutson PL, Armstrong RC. Oligodendrocyte progenitor cell proliferation and lineage progression are regulated by glutamate receptor-mediated K+ channel block. J Neurosci. 1996 Apr 15;16(8):2659-70), as well as the OPC precursor (pre-OPC) marker A2B5 (Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, Sharp K, Steward O. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci. 2005 May 11;25(19):4694-705).In addition, minimal non-OPC markers were detected in the resulting population, including the neural progenitor / epithelial marker CD49f (Krebsbach PH, Villa-Diaz LG. The Role of Integrin α6(CD49f) in Stem Cells: More than a Conserved Biomarker. Stem Cells Dev. 2017 Aug 1;26(15):1090-1099) and the epithelial markers CLDN6 (Lin D, Guo Y, Li Y, Ruan Y, Zhang M, Jin X, Yang M, Lu Y, Song P, Zhao S, Dong B, Xie Y, Dang Q, Quan C. Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions. Oncol Rep. 2017 Aug;38(2):875-885) and EpCAM (Huang L, Yang Y, Yang F, Liu S, Zhu Z, Lei Z, Guo J. Functions of EpCAM in physiological processes and diseases(Review). Int J Mol Med. 2018 Oct;42(4):1771-1785).

[0161] Table 1. Representative flow cytometry data of oligodendrocyte progenitor cells generated by the method according to the present disclosure.

[0162]

[0163] Compared with OPCs generated by another method and currently used in clinical trials for the treatment of spinal cord injury (Priest CA, Manley NC, Denham J, Wirth ED 3rd, Lebkowski JS. Preclinical safety of human embryonic stem cell-derived oligodendrocyte progenitors supporting clinical trials in spinal cord injury. Regen Med. 2015 Nov;10(8):939-58; Manley NC, Priest CA, Denham J, Wirth ED 3rd, Lebkowski JS. Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cells: Preclinical Efficacy and Safety in Cervical Spinal Cord Injury. Stem Cells Transl Med. 2017 Oct;6(10):1917-1929), the cell population generated by the methods described in the present disclosure results in a higher proportion of oligodendrocyte progenitor marker NG2-positive cells and reduced expression of non-OPC markers CD49f, CLDN6, and EpCAM.

[0164] Example 6 - Characterization of Differentiated Cell Populations by Gene Expression Profiling

[0165] Gene expression profiling can be used to characterize the starting pluripotent cell population and the cell phenotypes at each differentiation stage, 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, as well as targeted gene profiling using methods with increased sensitivity such as quantitative real-time PCR (qPCR).

[0166] For gene expression profiling, cells were lysed in Qiagen RLT lysis buffer (Qiagen #79216) and RNA was purified using the Qiagen RNeasy Mini Kit (Qiagen #74106) according to the manufacturer's guidelines. For qPCR-based analysis, the purified RNA was then reverse-transcribed into cDNA using Invitrogen Superscript IV VILO Mastermix (Thermo Fisher Scientific #11756050) according to the standard method and the manufacturer's guidelines. The relative expression levels of target genes and reference housekeeping genes were then quantified using gene-specific primer-probe sets (Applied Biosystems Taqman GeneExpression Assays, Thermo Fisher Scientific #4331182) according to the manufacturer's guidelines. To determine the relative expression levels of a given set of target genes, PCR reactions were performed on an ABI 7900HT real-time sequence detection system (Applied Biosystems), a BioMark HD system (Fluidigm), or an equivalent. Each target gene was normalized to one or more reference genes, such as GAPDH, to determine its relative expression level.

[0167] Table 2 shows qPCR results from two representative experiments measuring the expression of pluripotency genes, neuroectodermal progenitor genes, glial progenitor genes, dorsal spinal cord progenitor genes, ventral spinal cord progenitor genes, and oligodendrocyte progenitor genes in cell populations generated by the methods according to the present disclosure. RNA samples were collected at the following time points: before differentiation (day 0), after differentiation into neuroectodermal progenitors (day 7), after differentiation into glial progenitors (day 21), and after differentiation into oligodendrocyte progenitors (day 42). The RNA samples were processed for qPCR using the methods described above. The selected gene sets indicative of each differentiation state were quantified, including: three pluripotency genes (NANOG, LIN28A, SOX2), three neuroectodermal progenitor genes (PAX6, HES5, ZBTB16), three glial progenitor genes (CACGN4, DCC, FABP7), 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.

[0168] Table 2. qPCR analysis of gene markers for pluripotency, neural ectodermal progenitor cells (NPCs), dorsal spinal cord progenitor cells, ventral spinal cord progenitor cells, glial progenitor cells (GPCs), and oligodendrocyte progenitor cells (OPCs) in H1 uhESCs differentiated into OPCs according to the present disclosure.

[0169]

[0170] Referring to Table 2, differentiating uhESCs for 7 days by the method according to the present disclosure results in a gene expression profile consistent with that of neuroectodermal progenitor cells, including downregulation of NANOG and expression of LIN28A, SOX2, PAX6, HES5, and ZBTB16 (Patterson M, Chan DN, Ha I, Case D, Cui Y, Van Handel B, Mikkola HK, Lowry WE. Defining the nature of human pluripotent stem cell progeny. Cell Res. 2012 Jan;22(1):178-93; Lippmann ES, Williams CE, Ruhl DA, Estevez-Silva MC, Chapman ER, Coon JJ, Ashton RS. Deterministic HOX patterning in human pluripotent stem cell-derived neuroectoderm. Stem Cell Reports. 2015 Apr 14;4(4):632-44; Woo SM, Kim J, Han HW, Chae JI, Son MY, Cho S, Chung HM, Han YM, Kang YK. Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells. BMC Neurosci. 2009 Aug 17;10:97; Avantaggiato V, Pandolfi PP, Ruthardt M, Hawe N, Acampora D, Pelicci PG, Simeone A. Developmental analysis of murine Promyelocyte Leukemia Zinc Finger (PLZF) gene expression: implications for the neuromeric model of the forebrain organization. J Neurosci. 1995 Jul;15(7Pt1):4927-42).

[0171] In addition, the neuroectodermal progenitor cells generated after 7 days of differentiation exhibited a phenotype consistent with that of dorsal spinal cord progenitor cells based on the expression of the dorsal markers TFAP2A (also known as AP2), PAX3, and PAX7 (Le Dréau G, Martí E. Dorsal-ventral patterning of the neural tube: a tale of three signals. Dev Neurobiol. 2012 Dec;72(12):1471-81; Marklund U, Alekseenko Z, Andersson E, Falci S, Westgren M, Perlmann T, Graham A, E, Ericson J. Detailed expression analysis of regulatory genes in the early developing human neural tube. Stem Cells Dev. 2014 Jan 1;23(1):5-15). As further evidence of the dorsal spinal cord progenitor cell phenotype, the resulting neuroectodermal progenitor cells did not express the ventral spinal cord progenitor cell markers OLIG2 or NKX2-2, the expression of which requires activation of the sonic hedgehog signaling pathway (Le Dréau G, Martí E. Dorsal-ventral patterning of the neural tube: a tale of three signals. Dev Neurobiol. 2012 Dec;72(12):1471-81; Marklund U, Alekseenko Z, Andersson E, Falci S, Westgren M, Perlmann T, Graham A, E, Ericson J. Detailed expression analysis of regulatory genes in the early developing human neural tube. Stem Cells Dev. 2014 Jan 1;23(1):5-15).

[0172] After 21 days of differentiation, the resulting cell population exhibited a gene expression profile consistent with that of glial progenitor cells, including downregulation of pluripotency and neuroectodermal progenitor markers and induction of CACNG4, DCC (also known as the axon guidance factor receptor), and FABP7 (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; Fitzgerald DP, Cole SJ, Hammond A, Seaman C, Cooper HM. Characterization of neogenin-expressing neural progenitor populations and migrating neuroblasts in the embryonic mouse forebrain. Neuroscience. 2006 Oct 27;142(3):703-16; Rosenzweig S, Carmichael ST. The axon-glia unit in white matter stroke: mechanisms of damage and recovery. Brain Res. 2015 Oct 14;1623:123-34; Petit A, Sanders AD, Kennedy TE, Tetzlaff W, Glattfelder KJ, Dalley RA, Puchalski RB, Jones AR, Roskams AJ. Adult spinal cord radial glia display a unique progenitor phenotype. PLoS One. 2011;6(9):e24538).As further evidence of the glial progenitor cell phenotype, in addition to its expression in neuroectodermal progenitors / neural progenitor cells (Woo SM, Kim J, Han HW, Chae JI, Son MY, Cho S, Chung HM, Han YM, Kang YK. Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells. BMC Neurosci. 2009 Aug 17;10:97), the resulting day 21 cells also showed persistent expression of HES5, which has also been shown to promote the neuro-to-glial progenitor cell transition in the developing central nervous system of mammals (Bansod S, Kageyama R, Ohtsuka T. Hes5 regulates the transition timing of neurogenesis and gliogenesis in mammalian neocortical development. Development. 2017 Sep 1;144(17):3156-3167). In addition, the resulting day 21 glial progenitor cells showed persistent expression of the dorsal spinal cord progenitor markers TFAP2A, PAX3, and PAX7, providing further evidence of derivation from dorsally patterned neural progenitors.

[0173] After 42 days of differentiation according to the method described in the present disclosure, the resulting cell population expresses markers consistent with oligodendrocyte progenitor cells, including downregulation of early lineage markers and dorsal spinal cord progenitor cell markers, and induction of CSPG4 (also known as NG2), PDGFRα, and DCN (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).

[0174] Example 7 - Differentiation of Human Embryonic Stem Cells into Dorsal Neuroectodermal Progenitor Cells Using Alternative Small Molecule Inhibitors of MAPK / ERK and BMP Signaling

[0175] In addition to the small molecule inhibitors (PD0325901 and Dorsomorphin) used in Example 2, the ability of alternative small molecule inhibitors of MAPK / ERK and BMP signaling to differentiate human embryonic stem cells into dorsal neuroectodermal progenitor cells was tested. Table 3 lists the alternative small molecule inhibitors tested. Each condition was tested in duplicate wells of a 6-well tissue culture plate.

[0176] Table 3. Small Molecule Inhibitors for Differentiating Human Embryonic Stem Cells into Dorsal Neuroectodermal Progenitor Cells.

[0177]

[0178]

[0179] On day 7 of differentiation, cells were collected and processed for RNA extraction and gene expression profiling by qPCR as described in Example 6. For each gene, the normalized ΔCT value relative to the mean of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1) was calculated and the fold expression relative to the baseline (expression below the limit of quantification) was calculated using the ΔΔCT method. Table 4 shows the mean fold expression values (relative to the baseline) of biological replicates for each small molecule combination. Referring to Table 4, differentiation of uhESCs for 7 days with each of the tested small molecule combinations resulted in downregulation of the pluripotency marker NANOG and maintenance or induction of a similar degree of expression of genes associated with the neuroectodermal progenitor cell phenotype (including LIN28A, SOX2, PAX6, HES5, and ZBTB16). In addition, based on the expression of dorsal markers TFAP2A, PAX3, and PAX7 and the absence of expression of ventral markers OLIG2 and NKX2-2, each of the tested small molecule combinations resulted in a dorsal spinal cord progenitor cell phenotype.

[0180] 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 progenitor cells, neural tube patterning, glial progenitor cells, oligodendrocyte progenitor cells, neural crest cells, neurons, astrocytes, pericytes, Schwann cells, and epithelial cells. Referring to Figure 6 , the day 7 cell phenotypes of each alternative small molecule combination were compared to the cell phenotype generated by treatment with PD0325901 plus Dorsomorphin by regression plots of the normalized ΔCT values, indicating that each of the tested small molecule combinations could achieve a similar overall cell phenotype. In summary, the results shown in Table 4 and Figure 6 support the following various combinations: (i) MAPK / ERK inhibitors, and (ii) BMP signaling inhibitors, (iii) in the absence of an SHH signaling activator, can be used to differentiate uhESCs into dorsal neuroectodermal progenitor cells and further into glial progenitor cells and oligodendrocyte progenitor cells using the methods of the present disclosure.

[0181] Table 4. qPCR analysis of gene markers for pluripotency and neuroectodermal progenitor cells (NPCs) in H1 uhESCs differentiated into NPCs using different combinations of small molecule inhibitors.

[0182]

[0183] Example 8 - Assessment of the presence of foreign epithelial lineage cells in differentiated OPC populations using an in vitro encapsulation assay

[0184] The presence of unwanted epithelial lineage cells in the OPC population generated according to the present disclosure was tested using an in vitro cyst assay. The cyst assay was performed essentially according to the protocol of Debnath et al. (Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. 2003 Methods. 3:256-68). Briefly, OPCs were grown in a 3D culture system for 20 days in the presence of factors known to stimulate epithelial cyst formation. In addition to visual detection of cysts, the presence of cystic structures expressing basolateral proteins containing the epithelial marker CD49f was evaluated using immunocytochemistry.

[0185] OPCs were seeded at a density of 21.9x10 3 cells / cm 2 onto (Corning) pads (a total of 0.5x10 6 cells were seeded in 12 wells of a 24-well plate). The cells were cultured for 20 days. On day 20, live cyst counts were performed, and the cells were lysed using Cell Recovery Solution (Corning #354253) The cells were fixed in 4% paraformaldehyde (PFA) on ice for 5 minutes and permeabilized overnight in blocking buffer. Subsequently, the cysts were stained for CD49f (ITGA6), phalloidin, and counterstained with DAPI. The cysts were imaged using an IN Cell Analyzer 2000 (GE Healthcare Life Sciences) and analyzed using IN Cell Developer Software (GE Healthcare Life Sciences) and MATLAB TM(Mathworks) Quantify the frequency, size, and staining intensity of the cysts. Referring to Table 5, no detectable cysts were produced per 100,000 cells of OPCs generated from two representative runs using the method according to the present disclosure and tested in an in vitro cyst assay. In contrast, three control batches of OPCs (Control A, Control B, and Control C) generated by an alternative method previously found to cause epithelial cyst formation in vivo (Manley NC, Priest CA, Denham J, Wirth ED 3rd, Lebkowski JS. Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cells: Preclinical Efficacy and Safety in Cervical Spinal Cord Injury. Stem Cells Transl Med. 2017 Oct;6(10):1917-1929) did produce cysts in the assay.

[0186] Table 5. Representative cyst assay results of oligodendrocyte progenitor cells generated by the method according to the present disclosure.

[0187]

[0188] Example 9 - Comparison of cell yields of cells differentiated in the presence versus absence of an SHH signaling activator

[0189] When testing various small molecule differentiation protocols, it was found that removing an SHH signaling activator such as Purmorphamine (PMA) from the differentiation process consistently led to an increased stepwise yield of cells between day 7 and day 14 ( Figure 2A ). Since PMA and other SHH signaling agonists drive the ventralization of early spinal cord progenitors (Kutejova E, Sasai N, Shah A, Gouti M, Briscoe J. Neural Progenitors Adopt Specific Identities by Directly Repressing All Alternative Progenitor Transcriptional Programs. Dev Cell. 2016 Mar 21;36(6):639-53), this suggests that the culture conditions present from day 7 to day 14 are favorable for the expansion of neural progenitors with a more dorsal phenotype. Surprisingly, it was found that this early dorsal phenotype (although not the region of early OPC generation in vivo) still produced OPC cells at day 42 (Figure 5 )。

[0190] The effects of removing SHH signaling activators from the differentiation process according to the present disclosure were further tested and quantified. In two representative experiments (Run 1 and Run 2) where the SHH agonist PMA was removed from the differentiation process, the stepwise yield increased from day 7 to day 14 compared to runs containing PMA ( Figure 2B ). This led to a substantial increase in the overall theoretical cell yield ( Figure 2C )。

[0191] Example 10 - In Vitro Functional Bioassays (Decorin Secretion and Migration Assays)

[0192] Decorin is a naturally occurring small leucine-rich extracellular proteoglycan TGF-β1 / 2 antagonist that regulates multiple cellular functions by interacting with components of the extracellular matrix (ECM). Decorin expressed by neurons and astrocytes in the central nervous system attenuates scar tissue, blocks cavitation, and promotes wound healing through its anti-scarring effect (significantly reducing the accumulation of scar-derived axon growth inhibitor titers by degrading and inhibiting its synthesis) [Ahmed, Z., et al., Decorin blocks scarring and cystic cavitation in acute and induces scar dissolution in chronic spinal cord wounds. Neurobiol Dis, 2014. 64: p. 163 - 76]. When added exogenously, the human recombinant protein itself has been shown to have beneficial effects in animal models of spinal cord injury [Wu, L., et al., Combined transplantation of GDAs (BMP) and hr-decorin in spinal cord contusion repair. Neural Regen Res, 2013. 8(24): p. 2236 - 48].

[0193] Long-term stability data for three manufactured GPOR-OPC1 clinical batches indicated secretion levels of ~15 - 30 ng / ml.

[0194] In this in vitro assay, decorin produced by OPC1 cells was measured using a commercially manufactured solid-phase sandwich ELISA kit. The test material for potency assay was supernatant generated by thawing one or more vials of OPC1 drug product cells, culturing the cells for 48 hours, harvesting the conditioned medium (CM), and cryostoring these CM supernatants until tested by ELISA. Ongoing OPC1 process development studies have shown that the level of decorin secretion after 48 hours of in vitro culture of the improved process OPC1 cells is similar to that of the GPOR-OPC1 batch (as described above) and is in the range of ~20 - 35 ng / ml.

[0195] As exemplified by the rat cervical SCI model, one of the hallmarks of in vivo OPC1 activity is the in vivo migration of OPC1 cells from one or more injection sites to the cavitated lesion and the surrounding area of the spinal cord. The migration assay is based on the in vitro measurement of the migration of OPC1 cells in response to different chemokines (such as PDGFαα and PDGFββ) [Armstrong, R.C., L. Harvath and M.E. Dubois-Dalcq, Type 1 astrocytes and oligodendrocyte-type 2 astrocyte glial progenitors migrate toward distinct molecules. J Neurosci Res, 1990. 27(3): p. 400 - 7; Milner, R., et al., Contrasting effects of mitogenic growth factors on oligodendrocyte precursor cell migration. Glia, 1997. 19(1): p. 85 - 90; Sanchez-Rodriguez, M.A. et al., The endocannabinoid 2-arachidonoylglycerol regulates oligodendrocyte progenitor cell migration. Biochem Pharmacol, 2018. 157: p. 180 - 188].

[0196] In this in vitro assay, OPC-1 cells were seeded in a transwell system (Corning TM Transwell TMIndividual pores of a polycarbonate membrane insert (8 μm pore size). Cells were exposed to the medium in the lower well, which contained either no or the chemoattractant (PDGFββ). After overnight incubation, cells that had migrated through the transwell were collected and counted to determine the percentage of input cells that had migrated, which was then reported as the percentage migrated. This method is currently in the research stage and further research is ongoing to evaluate and improve the performance of the method. Ongoing OPC1 process development studies have shown that, in response to PDGFββ chemoattractant stimulation for 16 - 24 hours, the in vitro migration percentage of OPC1 cells ranges from ~15 - 50%.

[0197] Assessment of OPC1 in vitro function using decorin secretion and migration bioassays showed that for improved process OPC1 batches exhibiting higher purity (flow cytometry analysis of biomarker expression), better yield, and excellent morphological assessment, higher decorin secretion and higher migration percentages were observed, and vice versa; for OPC1 batches exhibiting lower purity, lower yield, and poorer morphological assessment, lower decorin secretion and lower migration percentages were observed.

[0198] For example, Table 6 provides a summary of the day 42 final product characterized by biomarker data, decorin secretion, and migration bioassays.

[0199]

[0200] Although the present disclosure has been described with reference to specific embodiments, those skilled in the art will understand that various changes can be made and elements thereof can be replaced with equivalents without departing from the scope of the present disclosure. In addition, many modifications can be made to adapt a particular situation or material to the teachings of the present disclosure without departing from the scope of the present disclosure.

[0201] Accordingly, the present disclosure is not intended to be limited to the specific embodiments disclosed as the best mode contemplated for carrying out the present disclosure, but rather the present disclosure will include all aspects falling within the scope and spirit of the appended claims.

Claims

1. A method for obtaining a cell population comprising dorsal neural progenitor cells (dNPCs) from undifferentiated human pluripotent stem cells, the method comprising: a) obtaining a culture of undifferentiated human pluripotent stem cells; b) culturing the undifferentiated human pluripotent stem cells adherently for a first period of time in the presence of at least one inhibitor of mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK), at least one inhibitor of bone morphogenetic protein (BMP) signaling, and retinoic acid, thereby inducing differentiation into the neuroectoderm; and c) culturing the cells from b) adherently for a second period of time in the presence of retinoic acid and in the absence of sonic hedgehog (SHH) or an SHH signaling activator, thereby obtaining dorsal neural progenitor cells.

2. The method of claim 1, further comprising the additional steps of: harvesting the cells from c), re-plating the harvested cells on a substrate and culturing the cells adherently for an additional period of time in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), thereby expanding the neural progenitor cells.

3. The method of claim 2, further comprising the additional steps of: harvesting the expanded cells and culturing them as aggregates in suspension for an additional period of time in the presence of bFGF and EGF until the cells mature into glial progenitor cells.

4. The method of claim 3, further comprising the additional steps of: plating the aggregates comprising glial progenitor cells on a substrate and culturing the cells adherently for an additional period of time in the presence of epidermal growth factor (EGF), optionally separating the cells from time to time, until the cells mature into oligodendrocyte progenitor cells (OPCs).

5. The method of claim 3, further comprising the additional steps of: plating the aggregates comprising glial progenitor cells on a substrate and culturing the cells adherently for an additional period of time in the presence of platelet-derived growth factor AA (PDGF-AA) and EGF, optionally separating the cells from time to time, until the cells mature into oligodendrocyte progenitor cells (OPCs).

6. The method of claim 3, wherein the cells are cryopreserved at one stage of the method, and subsequently the cells are thawed and the method is continued.

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

8. The method of claim 1, wherein the human pluripotent stem cells are derived from the H1, H7, H9, H13 or H14 cell line; or wherein the human pluripotent stem cells are derived from a parthenogenote.

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

10. The method of claim 1, wherein at least one inhibitor of the MAPK / ERK kinase is selected from PD0325901, AZD6244, GSK1120212, PD184352, and cobimetinib.

11. The method of claim 1, wherein at least one inhibitor of the MAPK / ERK kinase is PD0325901.

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

13. The method of claim 1, wherein at least one inhibitor of BMP signaling is selected from Dorsomorphin, DMH-1, K02288, ML347, LDN193189, and Noggin protein.

14. The method of claim 1, wherein at least one inhibitor of BMP signaling is Dorsomorphin.

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

16. The method of claim 1, wherein the second time period is 3 to 4 days.

17. The method of claim 3, wherein the aggregates are cultured in suspension for 7 days.

18. The method of claim 4, wherein the cells are cultured adherently for 21 days after plating of the aggregates.

19. A method for obtaining a cell population comprising oligodendrocyte progenitor cells (OPCs) from undifferentiated human pluripotent stem cells, the method comprising: a) obtaining dorsal neural progenitor cells (dNPCs) according to the method of claim 1; b) harvesting the cells from a), re-plating them on a substrate, and culturing the cells adherently for an additional period of time in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) to expand the neural progenitor cells; c) harvesting the cells from b), and culturing the cells as aggregates in suspension for an additional period of time in the presence of bFGF and EGF until the cells mature into dorsal neural glial progenitor cells; and d) plating the aggregates from c) on a substrate and culturing the cells adherently for an additional period of time in the presence of epidermal growth factor (EGF), optionally separating the cells from time to time, until the cells mature into OPCs; wherein the OPCs express one or more markers selected from neural / glial antigen 2 (NG2), platelet-derived growth factor receptor A (PDGFRα), and ganglioside GD3 (GD3).

20. The method of claim 19, wherein the adherent culture is performed on a substrate selected from: (i) cell adhesion peptides and (ii) extracellular matrices selected from laminin and vitronectin.

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

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

23. The method of claim 19, wherein step c) is performed in a dynamic suspension.

24. The method of claim 19, wherein during step d), the culture medium further comprises platelet-derived growth factor AA (PDGF-AA).

25. The method of claim 19, wherein the human pluripotent stem cells are derived from the H1, H7, H9, H13, or H14 cell lines; or wherein the human pluripotent stem cells are derived from parthenotes.

26. The method of claim 19, wherein the human pluripotent stem cells are hiPSCs. The method of claim 19, wherein the OPC is cryopreserved and ready for administration to a subject after thawing.

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