Dorsal-derived oligodendrocyte progenitor cells from human pluripotent stem cells

Abstract

This invention provides a novel method for differentiating pluripotent stem cells into neuroectoderm progenitor cells possessing a dorsal spinal cord progenitor cell phenotype, and further into glial progenitor cells and oligodendrocyte progenitor cells. [Solution] A method is provided for differentiating human pluripotent stem cells into dorsal neuroectoderm progenitor cells, and further into glial progenitor cells and oligodendrocyte progenitor cells (OPCs), using inhibitors of BMP signaling and MAPK / ERK signaling. The resulting cells and cell compositions, as well as the use of such cells, are also provided. Furthermore, a method and protocol are provided for efficiently differentiating human pluripotent stem cells into OPCs in the absence of the ventralizing morphogen SHH or SHH signaling activators. The method of this disclosure reliably generates dorsal neuroectoderm progenitor cells by day 7 of the differentiation process, glial progenitor cells by day 21 of the differentiation process, and OPCs by day 42 of the differentiation process.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 796,077, filed on 23 July 2019, the entire contents of which are incorporated herein by reference.

[0002] Field of Invention This disclosure relates to a novel method for differentiating pluripotent stem cells, such as human embryonic stem cells, first into neuroectoderm progenitor cells having a dorsal spinal cord progenitor cell phenotype, then further into glial progenitor cells, and then further into oligodendrocyte progenitor cells. Cells and cell compositions obtained by such a method, as well as uses of such cells, are also provided. This disclosure further relates to cells produced by the method according to the present invention that express one or more markers. [Background technology]

[0003] Background of the Invention Oligodendrocyte progenitor cells (OPCs) are a subtype of glial cells in the central nervous system (CNS) that originate in the ventricular zone of the brain and spinal cord, migrate throughout the developing CNS, and mature into oligodendrocytes. Mature oligodendrocytes produce myelin sheaths that insulate neuronal axons and remyelinate CNS lesions where myelin sheaths have been lost. Oligodendrocytes also contribute to neuroprotection through other mechanisms, including the production of neurotrophic factors that promote neuronal survival (Wilkins et al., 2001 Glia 36(l):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 and retain the ability to generate new oligodendrocytes. Therefore, OPCs and mature oligodendrocytes derived from OPCs are important therapeutic targets for demyelinating disorders and myelin sheathing disorders (such as multiple sclerosis, adrenoleukodystrophy, and adrenal spinal neuropathy), other neurodegenerative disorders (such as Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease), and acute nerve injuries (such as stroke and spinal cord injury (SCI)).

[0004] Several protocols have been developed to differentiate human pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), into oligodendrocytes (OPCs) that can be used in cell therapy. To date, protocols for generating oligodendrocyte progenitor cells from human pluripotent stem cells have replicated the ventral motor neuron progenitor (pMN) domain of the developing spinal cord, which is known to generate the majority of 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 progenitor cells that generate ventral-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). As a result, existing in vitro protocols for generating OPCs from pluripotent stem cells have been found to involve embryoid body formation as a means of stimulating endogenous SHH activation (Nistor et al., 2005 Glia 49(3):385-96), or direct addition of SHH or direct addition of SHH signaling activators (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., It depends on one of the following: 2017 PLoS One 12(2):e0171947).The former approach relies on spontaneous differentiation within embryoid bodies, which can be problematic because it may result in the generation of 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, corresponds to the current approach for generating OPCs from pluripotent stem cells. While these methods have successfully produced OPCs from human pluripotent stem cells for research purposes, challenges remain regarding the quality, scalability, and cost of the product in relation to translating existing protocols into clinical-scale commercial production processes.

[0005] In mice, smaller second-wave OPCs are generated in the dorsal spinal cord independently of SHH signaling (Cai et al., 2005 Neuron 45(1): 41-53; Vallstedt et al., 2005 Neuron 45(1): 55-67). These dorsal-derived mouse OPCs mature into oligodendrocytes and contribute to axonal myelination during development and remyelination in response to localized myelin formation damage (Zhu et al., 2011 Glia 59(11):1612-21). Little is known about the putative dorsal-derived OPC population in humans. Recently, it was reported that human brain region-specific forebrain organoids (dorsal and ventral forebrain organoids) consisting of multiple cell types, generated using an OLIG2-GFP knock-in hPSC reporter strain, can be differentiated into both 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 have been no reports of dorsal-derived OPCs obtained by targeted differentiation of human pluripotent stem cells that can yield target-lineage-specific cell populations suitable for downstream cell therapy applications.

[0006] An improved method is needed for differentiating pluripotent stem cells into OPCs (Optical Potential Cells). Ideally, such a method should be easily scalable to generate sufficient quantities of OPCs for cell therapy applications, while consistently and reproducibly producing target cell OPCs with desired quality characteristics. [Overview of the project]

[0007] In various embodiments described herein, this disclosure provides, among other things, robust and reliable protocols for differentiating human pluripotent stem cells, such as ESCs and iPSCs, into dorsal neuroectoderm progenitor cells (dNPCs), and further into glial progenitor cells and OPCs.

[0008] This disclosure is partly based on the discovery that human pluripotent stem cells can be easily and efficiently differentiated into spinal cord OPCs in the absence of ventralization of neuroectoderm-restricted progenitor cells mediated by SHH signaling.

[0009] In certain embodiments of this disclosure, neuroectoderm progenitor cells having a dorsal spinal cord phenotype are obtained by contacting human pluripotent stem cells with 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 approach contrasts with current methods for inducing neuroectoderm that rely on the combined addition of a transforming growth factor β (TGFβ) / activin / Nodal signaling inhibitor and a BMP signaling inhibitor, also known as dual SMAD inhibitors (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)).

[0010] Surprisingly, it was discovered that dorsal neuroectoderm progenitor cells obtained according to the above protocol and not exposed to either the ventralizing morphogen SHH or SHH signaling activators could be readily differentiated into spinal cord OPCs. It was also found that the method disclosed yielded significantly more differentiated cells compared to differentiation protocols in which SHH signaling is activated. Due to the substantial increase in cell proliferation and cell yield, the method disclosed provides a scalable and reproducible process for generating large quantities of OPCs and other neuroectoderm cells for cell therapy and other applications.

[0011] The method of this disclosure reliably generates neuroectoderm progenitor cells with a dorsal spinal cord phenotype by day 7 of differentiation, glial progenitor cells by day 21 of differentiation, and OPCs by day 42 of differentiation. The day 42 OPCs generated according to this disclosure are equivalent (in terms of their overall marker expression profile) to OPCs produced by other methods currently in clinical trials to treat spinal cord injury (Priest et al., 2015 Regen Med. 10(8):939-58; Manley et al., 2017 Stem Cells Transl Med. 6(10):1917-1929), except that the OPCs generated according to this disclosure express lower levels of non-OPC markers, including markers associated with in vitro epithelial cyst formation.

[0012] In a preferred embodiment, OPCs produced according to the method of the present invention express one or more markers selected from neuronal / glial antigen 2 (NG2), platelet-derived growth factor receptor A (PDGFRα), and ganglioside GD3 (GD3). In a more preferred embodiment, cells can be characterized by the expression of a single marker or a combination of markers. For example, OPCs produced according to the method of the present invention can be characterized simply by NG2, PDGFRα, or GD3, or by a combination of markers two or three of the markers NG2, PDGFRα, and GD3. In one preferred embodiment, at least 90% of the cells produced according to the method of the present invention express NG2 and / or PDGFRα. In a more preferred embodiment, at least 50% of the cells further express GD3.

[0013] In a more preferred embodiment, the cell generation process involves one or more steps of cryopreserving cells in an intermediate bank, then thawing the cells, and continuing the differentiation process to the final product. For example, the intermediate cell bank may be cryopreserved on day 14, day 28, and / or day 35 of the process.

[0014] In another embodiment, OPCs produced according to the present invention are prepared as ready-to-administer (RTA) OPC cell therapy compositions for patient treatment. Methods for formulating human OPCs for administration to a subject immediately after thawing, and methods for formulating OPC cell therapy compositions for cryopreservation and administration to a subject after thawing of the cryopreserved composition are also presented. In another embodiment, the RTA composition can be formulated as a thaw-and-inject (TAI) composition, thereby the composition is administered by injection after thawing without further processing of the OPCs.

[0015] In one embodiment, the present disclosure provides a method for obtaining a cell population containing dorsal neural progenitor cells (dNPCs) from undifferentiated human pluripotent stem cells. In a particular embodiment, the method comprises: a) obtaining a culture of undifferentiated human pluripotent stem cells; b) adhering culture the undifferentiated human pluripotent stem cells for a first period in the presence of at least one mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) inhibitor, at least one bone morphogenetic protein (BMP) signaling inhibitor, and retinoic acid, thereby inducing differentiation into neuroectoderm; and c) adhering culture the cells from b) for a second period in the presence of retinoic acid and in the absence of sonic hedgehog (SHH) and SHH signaling activators, thereby obtaining dorsal neural progenitor cells.

[0016] In a particular embodiment, the first period is approximately 3 to 4 days. In a particular embodiment, the second period is approximately 3 to 4 days.

[0017] In a particular embodiment, the method further comprises the additional steps of collecting cells from step c), replating the collected cells onto a substrate, and further adhering and culturing the cells for a further period in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) to thereby promote cell proliferation. In a particular embodiment, the substrate is a cell adhesion peptide. In another embodiment, the substrate is an extracellular matrix protein. In a particular embodiment, the substrate is recombinant human laminin-521. In another embodiment, the substrate is vitronectin or laminin-511 E8 fragment. In yet another embodiment, the substrate is a synthetic substrate, such as Synthemax®-II SC substrate.

[0018] In a further embodiment, the method includes the additional step of collecting the proliferated cells and culturing these cells as aggregates in a suspension for a further period in the presence of bFGF and EGF until the cells mature into glial progenitor cells. In a particular embodiment, the culturing step is carried out in a dynamic suspension. In a particular embodiment, the cells are cultured in the suspension for about 5 to 10 days. In one embodiment, the cells are cultured in the suspension for about 7 days.

[0019] In further embodiments, the method includes the additional step of plating aggregates containing glial progenitor cells onto a substrate and adhering the cells to epidermal growth factor (EGF) for a further period, optionally dividing the cells from time to time, until the cells mature into oligodendrocyte progenitor cells (OPCs). In certain embodiments, the medium further comprises platelet-derived growth factor AA (PDGF-AA). In certain embodiments, the cells are cultured for approximately 2–4 weeks after plating aggregates containing glial progenitor cells. In one embodiment, the cells are cultured for 21 days after plating aggregates containing glial progenitor 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 laminin-511 E8 fragment. In yet another embodiment, the substrate is a synthetic substrate, such as Synthemax®-II SC substrate.

[0020] In another embodiment, cells are cultured without coating for part of the process. In yet another embodiment, coated or uncoated microcarriers in suspension are used instead of culture dishes.

[0021] In one particular embodiment, at least one MAPK / ERK inhibitor is selected from the group consisting of PD0325901, AZD6244, GSK1120212, PD184352, and cobimetinib. In yet another embodiment, the MAPK / ERK inhibitor is PD0325901.

[0022] In a particular embodiment, at least one inhibitor of BMP signaling is an inhibitor of activin receptor-like kinase 2 (ALK2). In a particular embodiment, at least one inhibitor of BMP signaling is selected from the group consisting of dorsomorphine, DMH-1, K02288, ML347, LDN193189, and noggin protein. In yet another embodiment, the inhibitor of BMP signaling is dorsomorphine.

[0023] An additional embodiment is a differentiated cell population comprising paired box 6 (PAX6)-positive dorsal neural progenitor cells (dNPCs) obtained in accordance with 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).

[0024] In another embodiment, the present disclosure provides a method for obtaining a cell population containing 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) adhering culture the undifferentiated human pluripotent stem cells for a first period in the presence of at least one mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) inhibitor, at least one bone morphogenetic protein (BMP) signaling inhibitor, and retinoic acid, thereby inducing differentiation into neuroectoderm; c) adhering culture the cells from b) for a second period in the presence of retinoic acid and in the absence of sonic hedgehog (SHH) and SHH signaling activators, thereby obtaining dorsal neural progenitor cells; d) collecting the cells from c), replating the cells onto a substrate, and fermenting them with basic fibroblast growth factor (bFGF) and epidermal growth factor The process includes the steps of: e) further adhering and culturing the cells for a further period in the presence of (EGF) to promote the growth of neural progenitor cells; e) collecting the cells from d) and further culturing the cells as aggregates in suspension for a further period in the presence of bFGF and EGF until the cells mature into dorsal glial progenitor cells; and f) plating the aggregates from e) onto a substrate and further adhering and culturing the cells for a further period in the presence of epidermal growth factor (EGF), optionally dividing 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 2-4 weeks after plating the aggregates. In one embodiment, the cells are cultured for 21 days after plating the aggregates. In certain embodiments, the substrate is a cell adhesion peptide. In other embodiments, the substrate is an extracellular matrix protein. In certain embodiments, the substrate is recombinant human laminin-521. In another embodiment, the substrate is vitronectin or laminin-511 E8 fragment. In yet another embodiment, the substrate is a synthetic substrate such as Synthemax®-II SC substrate.

[0025] An additional embodiment is a differentiated cell population containing NG2-positive OPCs obtained in accordance with this disclosure. In certain embodiments, the differentiated cell population contains at least 60% NG2-positive cells. In certain embodiments, the differentiated cell population contains at least 70% NG2-positive cells. In certain embodiments, the differentiated cell population contains at least 80% NG2-positive cells. In other embodiments, the differentiated cell population contains at least 90% NG2-positive cells. In certain embodiments, the differentiated cell population contains at least 98% NG2-positive cells.

[0026] In one particular embodiment, the differentiated cell population contains at least 60% PDGFRα-positive cells. In one particular embodiment, the differentiated cell population contains at least 70% PDGFRα-positive cells. In one particular embodiment, the differentiated cell population contains at least 80% PDGFRα-positive cells. In another embodiment, the differentiated cell population contains at least 90% PDGFRα-positive cells. In one particular embodiment, the differentiated cell population contains at least 98% PDGFRα-positive cells.

[0027] In one particular embodiment, the differentiated cell population contains at least 50% GD3-positive cells. In one particular embodiment, the differentiated cell population contains at least 60% GD3-positive cells. In one particular embodiment, the differentiated cell population contains at least 70% GD3-positive cells. In one particular embodiment, the differentiated cell population contains at least 80% GD3-positive cells. In another embodiment, the differentiated cell population contains at least 90% GD3-positive cells. In one particular embodiment, the differentiated cell population contains at least 98% GD3-positive cells.

[0028] In another embodiment, the differentiated cell population includes cells that are NG2 and PDGFRα positive within the proportions described above. In another embodiment, the differentiated cell population includes cells that are NG2 and GD3 positive within the proportions described above. In another embodiment, the differentiated cell population includes cells that are PDGFRα and GD3 positive within the proportions described above. In another embodiment, the differentiated cell population includes cells that are NG2, PDGFRα, and GD3 positive within the proportions described above.

[0029] In certain embodiments, human pluripotent stem cells are human embryonic stem cells. In other embodiments, human pluripotent stem cells are human induced pluripotent stem cells.

[0030] In another embodiment, cells prepared according to the present invention are cryopreserved and then thawed and delivered to the patient. In a preferred embodiment, the cells do not require further processing before delivery to the patient. Once thawed, the cells prepared according to the present invention can be delivered to the patient immediately. In a preferred embodiment, the cells can be delivered by injection. The volume of the injection is, for example, about 100 microliters at a cell concentration of 100,000,000 live cells / ml. [Invention 1001] A method for obtaining a cell population containing dorsal neural progenitor cells (dNPCs) from undifferentiated human pluripotent stem cells, a) A step to obtain cultures of undifferentiated human pluripotent stem cells; b) Adhering culture of undifferentiated human pluripotent stem cells for a first period 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; and c) Adhering culture of cells from b) for a second period in the presence of retinoic acid and in the absence of sonic hedgehog (SHH) and SHH signaling activators, thereby obtaining dorsal neural progenitor cells. The method, including the method. [Invention 1002] The method of the present invention 1001, further comprising the additional steps of collecting cells from step c), replating the collected cells onto a substrate, and further adhering and culturing the cells for a further period of time in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) to thereby increase the neural progenitor cells. [Invention 1003] The method of the present invention 1002 further comprises the additional step of collecting the proliferated cells and culturing the cells as aggregates in a suspension for a further period of time in the presence of bFGF and EGF until the cells mature into glial progenitor cells. [Invention 1004] The method of the present invention 1003, further comprising the additional step of plating the aggregate containing glial progenitor cells onto a substrate and adhering the cells to epidermal growth factor (EGF) for a further period of time, optionally dividing the cells from time to time, until the cells mature into oligodendrocyte progenitor cells (OPCs). [Invention 1005] The method of the present invention 1003, further comprising the additional step of plating the aggregate containing glial progenitor cells onto a substrate and adhering the cells for a further period of time in the presence of platelet-derived growth factor AA (PDGF-AA) and EGF, with the cells optionally being divided from time to time until they mature into oligodendrocyte progenitor cells (OPCs). [Invention 1006] The method of the present invention 1003, wherein the cells are cryopreserved at a certain stage in the method described above, and then the cells are thawed and the method is continued. [Invention 1007] The method of the present invention 1001, wherein the substrate is recombinant human laminin-521. [Invention 1008] The method of the present invention 1001, wherein the human pluripotent stem cells are human embryonic stem cells (hESCs). [Invention 1009] The method of the present invention 1001, wherein the human pluripotent stem cells are human induced pluripotent stem cells (hiPSCs). [Invention 1010] The method of the present invention 1001, wherein the at least one inhibitor of MAPK / ERK kinase is selected from the group consisting of PD0325901, AZD6244, GSK1120212, PD184352, and cobimetinib. [Invention 1011] The method of the present invention 1001, wherein the at least one inhibitor of MAPK / ERK kinase is PD0325901. [Invention 1012] The method of the present invention 1001, wherein the at least one inhibitor of BMP signaling is an inhibitor of activin receptor-like kinase 2 (ALK2). [Invention 1013] The method of the present invention 1001, wherein the at least one inhibitor of BMP signaling is selected from the group consisting of dolsomorphin, DMH-1, K02288, ML347, LDN193189, and noggin protein. [Invention 1014] The method of the present invention 1001, wherein the at least one inhibitor of BMP signaling is dorsomorphine. [Invention 1015] The method of the present invention 1001, wherein the first period is approximately 3 to 4 days. [Invention 1016] The method of the present invention 1001, wherein the second period is approximately 3 to 4 days. [Invention 1017] The method of the present invention 1003, wherein the aggregate is cultured in a suspension for about 7 days. [Invention 1018] The method of the present invention 1004, wherein the cells are adherently cultured for approximately 21 days after plating of aggregates. [Invention 1019] A differentiated cell population containing paired box 6 (PAX6)-positive dNPCs obtained according to the method of Invention 1001. [Invention 1020] A differentiated cell population according to the present invention 1018, wherein the dNPC expresses one or more markers selected from paired box 3 (PAX3), paired box 7 (PAX7), and activating protein 2 (AP2). [Invention 1021] A method for obtaining a cell population containing oligodendrocyte progenitor cells (OPCs) from undifferentiated human pluripotent stem cells, The method is a) A step of obtaining dorsal neural progenitor cells (dNPCs) according to the method of the present invention 1001; b) The step of collecting cells from a), replating the cells onto a substrate, and adhering the cells to a culture for a further period in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) to promote the growth of the neural progenitor cells; c) The step of collecting cells from b) and further culturing the cells as aggregates in suspension for a further period of time in the presence of bFGF and EGF until the cells mature into dorsal glial progenitor cells; and d) Plating aggregates from c) onto a substrate, and adhering the cells to the substrate for a further period of time in the presence of epidermal growth factor (EGF), optionally dividing the cells from time to time until the cells mature into OPCs. Includes, The OPC expresses one or more markers selected from neuronal / glial antigen 2 (NG2), platelet-derived growth factor receptor A (PDGFRα), and ganglioside GD3 (GD3). The method. [Invention 1022] The method of the present invention 1021, wherein the above-mentioned adhesion culture step is performed on a substrate, and the substrate is selected from (i) cell adhesion peptides and (ii) an extracellular matrix selected from laminin and vitronectin. [Invention 1023] The method of the present invention 1021, wherein the above-mentioned adhesion culture step is performed on recombinant human laminin-521. [Invention 1024] The method of the present invention 1021, wherein the aforementioned adhesion culture step is performed on laminin-511 E8 fragments. [Invention 1025] The method of the present invention 1021, wherein step c) is performed in a dynamic suspension. [Invention 1026] The method of the present invention 1021, wherein during step d), the culture medium further contains platelet-derived growth factor AA (PDGF-AA). [Invention 1027] The method of the present invention 1021, wherein the human pluripotent stem cells are hESCs. [Invention 1028] The method of the present invention 1021, wherein the human pluripotent stem cells are hiPSCs. [Invention 1029] The method of the present invention 1021, wherein the OPC can be frozen and stored, and administered to the subject immediately after thawing. [Invention 1030] A differentiated cell population containing OPCs obtained according to the method of Invention 1021. [Brief explanation of the drawing]

[0031] [Figure 1] This figure shows the differentiation of human pluripotent stem cells into dorsal neuroectoderm progenitor cells (dNPCs), and further into glial progenitor cells (GPCs) and oligodendrocyte progenitor cells (OPCs), according to this disclosure. Neuroectoderm progenitor cells with a dorsal spinal cord phenotype were obtained around day 7 and could be readily 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 dolsomorphin) were tested and found to function equally well in inducing differentiation into dorsal neuroectoderm (Example 7). [Figure 2A]Representative data demonstrating the effect of the absence of both SHH and SHH signaling agonists on cell yield are shown. Figure 2A shows yields from day 7 to day 14 across several different combinations of small molecule treatments tested. "+PMA" and "-PMA" refer to the presence or absence of the SHH agonist plumorphamine during days 4 to 6 of the differentiation process, respectively. The black bars for conditions A, B, and E correspond to neuroectoderm cells derived using a differentiation cocktail containing the MAPK / ERK inhibitor PD0325901, the BMP inhibitor dorsomorphine, and retinoic acid, while the gray bars for conditions C, D, and F correspond to neuroectoderm cells derived using other signaling modulators. [Figure 2B] Representative data demonstrating the effect of the absence of SHH and SHH signaling agonists on cell yield are shown. Figure 2B shows step yields at various time points for two representative runs (run 1 and run 2) following the differentiation protocol disclosed herein, compared to a differentiation process including PMA (previous process (+PMA)). [Figure 2C] Representative data demonstrating the effect of the absence of SHH and SHH signaling agonists on cell yield are shown. Figure 2C shows the overall theoretical yield from one uhESC for two representative runs (run 1 and run 2) following this disclosure, compared to a differentiation process including PMA (previous process (+PMA)). [Figure 3] Representative micrographs of dorsal neuroectoderm progenitor cells prepared in accordance with this disclosure and stained by immunocytochemistry are shown. Dorsal neuroectoderm progenitor cells were stained for DAPI, PAX7, PAX3, and PAX6 (upper panel, left to right), as well as DAPI and AP2 (lower panel, left to right). Stained cells were imaged using an IN Cell Analyzer 2000. The scale bar represents 200 μm and applies to all images in the figure. [Figure 4]Representative micrographs of glial progenitor cell aggregates prepared in accordance with this disclosure and stained by immunocytochemistry are shown. Glial progenitor cells were stained for DAPI, PAX7, PAX3, and PAX6 (upper panel, left to right), and DAPI, AP2, and NG2 (lower panel, left to right). Stained cells were imaged using an IN Cell Analyzer 2000. The scale bar represents 200 μm and applies to all images in the figure. [Figure 5] Representative micrographs of oligodendrocyte progenitor cells prepared in accordance with this disclosure and stained by immunocytochemistry are shown. Oligodendrocyte progenitor cells were stained by immunocytochemistry for DAPI and NG2 (upper panel, left to right) and DAPI and AP2 (lower panel, left to right). Stained cells were imaged using an IN Cell Analyzer 2000. The scale bar represents 200 μm and applies to all images in the figure. [Figure 6] This shows correlation plots of gene expression profiles at day 7 of uhESCs differentiated into dorsal neuroectoderm progenitor cells (dNPCs) using different small molecule combinations. Each correlation plot shows a comparison of the gene expression profile at day 7 of cells treated with PD0325901 + dolsomorphin with the alternative small molecule combination shown on the Y axis of each plot. For each plot, the data points represent each of the 96 genes evaluated by Fluidigm qPCR as described in Example 7 and calculated as normalized ΔCT. The R-squared value is shown in the upper left corner of each plot and was calculated based on the best-fitted line using JMP software (SAS, Cary, NC, USA). [Modes for carrying out the invention]

[0032] Detailed description of the invention This description is not intended to be a detailed enumeration of all different ways in which the Disclosure may be carried out, or of all features that may be added to the Disclosure. For example, a feature described in relation to one aspect may be incorporated into another aspect, and a feature described in relation to a particular aspect may be omitted from that aspect. Accordingly, the Disclosure is intended to show that in some aspects of the Disclosure, any feature or combination of features described herein may be excluded or omitted. In addition, numerous variations and additions to the various aspects proposed herein, which do not deviate from the Disclosure, will be obvious to those skilled in the art in light of the Disclosure. In other examples, well-known structures, interfaces, and processes are not described in detail so as not to unnecessarily obscure the Invention. Nothing in this Specification is intended to be construed as negating any part of the entire scope of the Invention. Accordingly, the following description is intended to illustrate some specific aspects of the Disclosure and is not intended to exhaustively specify all sorts, combinations, and variations thereof.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. The technical terms used in describing this disclosure are for the sole purpose of describing specific aspects and are not intended to limit this disclosure.

[0034] All publications, patent applications, patents, and other references cited herein are incorporated by reference as a whole.

[0035] Unless the context indicates otherwise, the various features of the Disclosure described herein are particularly intended to be used in any combination. Furthermore, the Disclosure also intends that in some aspects of the Disclosure, any feature or combination of features described herein may be excluded or omitted.

[0036] The methods disclosed herein may include one or more steps or actions to achieve the described methods. The steps and / or actions of the methods may be interchangeable without departing from the scope of the invention. In other words, the order and / or use of a particular set of steps and / or actions may be changed without departing from the scope of the invention, unless a particular order of steps or actions is required for proper operation of the situation.

[0037] Where used in the description of this disclosure and the attached claims, the singular forms “a,” “an,” and “the” also include the plural forms unless the context clearly indicates otherwise.

[0038] As used herein, “and / or” means and encompasses any possible combination of one or more of the related enumerated items, as well as the absence of any combination when interpreted as choosing one of them ("or").

[0039] As used herein, the terms “about” and “approximately” when referring to measurable values ​​such as proportions, densities, volumes, and the like mean that the specified quantity will include variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1%.

[0040] As used herein, phrases such as "between X and Y" and "approximately between X and Y" should be interpreted as including X and Y. As used herein, phrases such as "approximately between X and Y" mean "approximately between X and approximately Y," and phrases such as "approximately from X to Y" mean "approximately from X to approximately Y."

[0041] As used herein, “oligodendrocyte precursor cells” (OPCs) refer to cells found in the central nervous system that belong to the neuroectoderm / glial lineage, express the characteristic marker neuronal / glial antigen 2 (NG2), and are capable of differentiating into oligodendrocytes.

[0042] The terms “glial cells,” “glial progenitor cells,” and “glial cells” are used interchangeably herein and refer to non-neuronal CNS cells derived from neuroectoderm / neural progenitor cells. Glial progenitor cells may further differentiate to form OPCs / oligodendrocytes or astrocytes. In certain embodiments, the glial progenitor cells of this disclosure express one or more markers selected from calcium voltage-gated channel co-subunit γ4 (CACNG4), fatty acid-binding protein 7 (FABP7), and netrin-1 receptor (DCC).

[0043] The terms “neuroectoderm,” “neuroectoderm cell,” “neuroectoderm precursor,” “neuroectoderm progenitor cell,” “neural progenitor cell,” and “neural precursor” are used interchangeably herein and refer to cells that can differentiate along the neural precursor pathway and form CNS neurons, oligodendrocytes, astrocytes, and ependymal cells. In certain embodiments, the neuroectoderm cells of this 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).

[0044] As used herein, the terms “dorsal” and “ventral” refer to distinct neuronal subtypes arising from progenitor cells located in spatially separated domains along the dorsal-ventral axis of the neural tube of the developing spinal cord. This process, known as dorsal-ventral patterning, is controlled by secretory signals that divide 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 SHH secretion plays a crucial role in establishing the fate of ventral neurons (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).

[0045] The terms “dorsal neuroectoderm progenitor cells,” “dorsal neural progenitor cells,” and “dNPC” are used interchangeably herein and refer to neural progenitor cells having a dorsal spinal cord phenotype and obtained by differentiating pluripotent stem cells into neuroectoderm restraint precursors in the absence of exogenous SHH and SHH signaling activators. In certain embodiments, dNPCs express one or more markers selected from paired box 3 (PAX3), paired box 7 (PAX7), and activating protein 2 (AP2).

[0046] As used herein, the term “embryoid body” (EB) refers to a three-dimensional cell aggregate derived from pluripotent stem cells that have spontaneously differentiated 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, removing basic fibroblast growth factor (bFGF) and transforming growth factor β (TGFβ) from the culture medium causes them to spontaneously differentiate into all three germ layers, leading to the formation of EBs.

[0047] As used herein, the term “BMP signaling inhibitor” refers to a small molecule or protein regulator that can downregulate 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 the group consisting of dolsomorphine, DMH-1, K02288, ML347, LDN193189, and noggin proteins.

[0048] As used herein, the term "MAPK / ERK inhibitor" refers to a small molecule or protein regulator that inhibits MAPK / ERK kinase. In certain embodiments, the MAPK / ERK inhibitor is selected from the group consisting of PD0325901, AZD6244, GSK1120212, PD184352, and cobimetinib.

[0049] The terms “SHH signaling activator,” “SHH signaling agonist,” “SHH activator,” and “SHH agonist” are used interchangeably herein and refer to small molecules or protein regulators that can activate the Sonic Hedgehog (SHH) signaling pathway. Non-exclusive examples of SHH signaling activators include purmorphamine (PMA), smoothed agonist (SAG, CAS 364590-63-6), and the Sonic Hedgehog (SHH) protein.

[0050] As used herein, the term “undesirable cell type” refers to non-neuroectoderm cells that may cause the formation of ectopic tissue at transplantation or cause the formation of one or more cysts in the cyst assay described herein. In one embodiment, “undesirable cell type” may include epithelial cells such as cells that are positive for CD49f, a marker expressed by both neural progenitor cells and epithelial cells, or cells that are positive for CLDN6 or EpCAM, two markers expressed by both pluripotent cells and epithelial cells.

[0051] As used herein, “implantation” or “transplantation” refers to the administration of a population of cells to a target tissue using an appropriate delivery technique (e.g., using an injection device).

[0052] As used herein, “subject” refers to an animal or a human being.

[0053] As used herein, “the subject requiring it” refers to an animal or human being in which the tissues of the central nervous system are damaged. In one embodiment, the animal or human being is experiencing a loss of motor function.

[0054] The terms “central nervous system” and “CNS” are used interchangeably herein and refer to a complex of nerve tissue that controls one or more activities of the body, including, but not limited to, the brain and spinal cord of vertebrates.

[0055] As used herein, “treatment” or “to treat” a condition or disease is an approach taken after the condition or disease has appeared in a patient to obtain a beneficial or desired outcome, preferably including a clinical outcome. Beneficial or desired outcomes with respect to a disease include, but are not limited to, one or more of the following: improvement of the condition associated with the disease, cure of the disease, reduction of the severity of the disease, delay of the progression of the disease, relief of one or more symptoms associated with the disease, improvement of the quality of life of the person suffering from the disease, extension of survival, and any combination thereof. Similarly, for the purposes of this disclosure, beneficial or desired outcomes with respect to a condition include, but are not limited to, one or more of the following: improvement of the condition, cure of the condition, reduction of the severity of the condition, delay of the progression of the condition, relief of one or more symptoms associated with the condition, improvement of the quality of life of the person suffering from the condition, extension of survival, and any combination thereof.

[0056] Proliferation and culture of undifferentiated pluripotent stem cells The differentiation of pluripotent stem cells in accordance with this disclosure can be carried out using any suitable pluripotent stem cells as starting material. In one embodiment, the method can be carried out in human embryonic stem cells (hESCs). In another embodiment, the method can be carried out in induced pluripotent stem cells (iPSCs). In another embodiment, the method can be carried out using cells derived from H1, H7, H9, H13, or H14 cell lines. In yet another embodiment, the method can be carried out in primate pluripotent stem (pPS) cell lines. In yet another embodiment, the method can be carried out using undifferentiated stem cells derived from parthenogenetic organisms, which are embryos stimulated to produce hESCs without fertilization.

[0057] Methods for the proliferation and culture of undifferentiated pluripotent stem cells have been previously described. Regarding tissue and cell culture of pluripotent stem cells, readers may wish to refer to any of the numerous publications available in this art, such as *Teratocarcinomas and Embryonic Stem Cells: A Practical Approach* (EJ Robertson, Ed., IRL Press Ltd. 1987); *Guide to Techniques in Mouse Development* (PM Wasserman et al., Eds., Academic Press 1993); *Embryonic Stem Cell Differentiation in Vitro* (MV Wiles, Meth. Enzymol. 225:900, 1993); *Properties and Uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy* (PD Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998; and RI Freshney, *Culture of Animal Cells*, Wiley-Liss, New York, 2000).

[0058] Undifferentiated pluripotent stem cells can be maintained in an undifferentiated state without the addition of feeder cells (see, e.g., (2004) Rosier et al., Dev. Dynam. 229:259). Feeder-free cultures are typically supported by nutrient media containing factors that promote cell proliferation without differentiation (see, e.g., U.S. Patent No. 6,800,480). In one embodiment, a conditioned medium containing such factors can be used. The conditioned medium can be obtained by culturing the medium with cells that secrete such factors. Suitable cells include, but are not limited to, irradiated (approximately 4,000 rad) primary mouse embryonic fibroblasts, telomereated mouse fibroblasts, or fibroblast-like cells derived from pPS cells (U.S. Patent No. 6,642,048). The medium can be conditioned by plating feeders in serum-free medium such as knockout DMEM supplemented with 20% serum substitute and 4 ng / ml bFGF. The culture medium, after being acclimatized for 1-2 days, can be used for 1-2 days to supplement with further bFGF and support the culture of pPS cells (see, for example, WO 01 / 51616; Xu et al., (2001) Nat. Biotechnol. 19:971).

[0059] Alternatively, fresh or unadapted media can be used, supplemented with additional factors that promote the proliferation of undifferentiated cells (such as fibroblast growth factor or forskolin). Non-limiting examples include basic media such as X-VIVO® 10 (Lonza, Walkersville, Md.) or QBSF®-60 (Quality Biological Inc. Gaithersburg, Md.) supplemented with 40–80 ng / mL bFGF and optionally containing SCF (15 ng / mL) or Flt3 ligand (75 ng / mL) (see, e.g., Xu et al., (2005) Stem Cells 23(3):315). These media formulations have the advantage of supporting cell proliferation at 2–3 times the rate of other systems (see, e.g., WO 03 / 020920). In one embodiment, undifferentiated pluripotent cells such as hES can be cultured in a medium containing bFGF and TGFβ. A non-limiting exemplary concentration of bFGF is approximately 80 ng / ml. A non-limiting exemplary concentration of TGFβ is approximately 0.5 ng / ml. In yet another embodiment, undifferentiated pluripotent stem cells can be maintained in a commercially available complete medium such as mTeSR® (Stem Cell Technologies, Vancouver, Canada).

[0060] Undifferentiated pluripotent cells can be cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue (Thomson et al. (1998) Science 282:1145). Feeder cells can be derived from human or mouse sources. Human feeder cells can be isolated from various human tissues or derived via the differentiation of human embryonic stem cells into fibroblasts (see, e.g., WO 01 / 51616). Possible human feeder cells include, but are not limited to, placental fibroblasts (e.g., Genbacev et al. (2005) Fertil. Steril. 83(5):1517), fallopian tube epithelial cells (e.g., see Richards et al. (2002) Nat. Biotechnol, 20:933), prepuce fibroblasts (e.g., see Amit et al. (2003) Biol. Reprod. 68:2150), and endometrial cells (e.g., see Lee et al. (2005) Biol. Reprod. 72(1):42).

[0061] Various solid surfaces can be used in the culture of 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 disks. Solid surfaces suitable for growing undifferentiated pluripotent cells can be made from a variety of materials, including, but not limited to, glass or plastics such as polystyrene, polyvinyl chloride, polycarbonate, polytetrafluoroethylene, melinex, thermanox, or combinations thereof. A suitable surface may contain one or more polymers, such as one or more acrylates. The solid surface may have a three-dimensional shape. Non-limiting examples of three-dimensional solid surfaces are previously described, for example, in U.S. Patent Application Publication No. 2005 / 0031598.

[0062] Undifferentiated stem cells can also be grown on a growth substrate under feeder-free conditions. The growth substrate may be Matrigel® matrix (e.g., Matrigel® or Matrigel® GFR), recombinant laminin, laminin-511 recombinant fragment E8, or vitronectin. In certain embodiments of the present disclosure, the growth substrate is recombinant human laminin-521 (Biolamina, Sweden, sold by Corning Inc., Corning, NY). In other embodiments, the substrate is a synthetic substrate such as, for example, Synthemax®-II SC substrate.

[0063] Undifferentiated stem cells can be passaged or subcultured using various methods such as using collagenase or scraping by hand. Undifferentiated stem cells can be subcultured by enzymatic means to generate a single cell suspension, such as using Accutase® (sold by Sigma Aldrich, MO) or a similar trypsinase. Alternatively, undifferentiated stem cells can be subcultured using non-enzymatic means such as 0.5 mM EDTA in PBS or using ReLeSR™ (Stem Cell Technologies, Vancouver, Canada).

[0064] In one embodiment, a plurality of undifferentiated stem cells are seeded or subcultured at a seeding density that allows the cells to reach confluence in about 3 to about 10 days. In one embodiment, the seeding density is in the range of about 6.0×10 3 cells / cm 2 ~ about 5.0×10 5 cells / cm 2 For example, about 1.0×10 4 cells / cm 2 such as, about 5.0×10 4 cells / cm 2 such as, about 1.0×10 5 cells / cm 2 such as, or about 3.0×10 5 cells / cm 2And so on. In another embodiment, the seeding density is approximately 6.0 × 10 cells per growth surface. 3 pieces / cm 2 ~Cells approx. 1.0×10 4 pieces / cm 2 For example, the range of cells per growth surface is approximately 6.0 × 10 3 pieces / cm 2 ~Cells approx. 9.0×10 3 pieces / cm 2 For example, cells approximately 7.0 × 10 3 pieces / cm 2 ~Cells approx. 1.0×10 4 pieces / cm 2 For example, cells approximately 7.0 × 10 3 pieces / cm 2 ~Cells approx. 9.0×10 3 pieces / cm 2 For example, or cells approximately 7.0 × 10 3 pieces / cm 2 ~Cells approx. 8.0×10 3 pieces / cm 2 And so on. In yet another embodiment, the seeding density is approximately 1.0 × 10 cells per growth surface. 4 pieces / cm 2 ~Cells approx. 1.0×10 5 pieces / cm 2 For example, the range of cells per growth surface is approximately 2.0 × 10 4 pieces / cm 2 ~Cells approx. 9.0×10 4 pieces / cm 2 For example, cells approximately 3.0 × 10 4 pieces / cm 2 ~Cells approx. 8.0×10 4 pieces / cm 2 For example, cells approximately 4.0 × 10 4 pieces / cm 2 ~Cells approx. 7.0×10 4 pieces / cm 2 For example, or cells approximately 5.0 × 10 4 pieces / cm 2 ~cells approx. 6.0×10 4 pieces / cm 2 For example, the seeding density is approximately 1.0 × 10 cells per growth surface. 5 pieces / cm 2 ~Cells approx. 5.0×10 5pieces / cm 2 For example, the range of cells per growth surface is approximately 1.0 × 10 5 pieces / cm 2 ~cells approx. 4.5×10 5 pieces / cm 2 For example, cells approximately 1.5 × 10 5 pieces / cm 2 ~Cells approx. 4.0×10 5 pieces / cm 2 For example, cells approximately 2.0 × 10 5 pieces / cm 2 ~Cells approx. 3.5×10 5 pieces / cm 2 For example, or cells approximately 2.5 × 10 5 pieces / cm 2 ~Cells approx. 3.0×10 5 pieces / cm 2 It could be something like that.

[0065] Stem cells can be cultured according to the method of this disclosure using any of a variety of suitable cell culture and subculture techniques. For example, the culture medium can be completely replaced daily, starting about two days after the subculture of the cells. In one embodiment, when the culture reaches a colony coverage of about 90%, the cells can be detached using one or more suitable reagents, such as Accutase®, to obtain a single-cell suspension for quantification, and seeded for subsequent culture. In one embodiment, after subculturing the undifferentiated stem cells, the cells can be seeded on a suitable growth substrate (e.g., recombinant human laminin-521) at a seeding density that allows the cells to reach confluence for a suitable period, for example, about 3 to 10 days. In one embodiment, undifferentiated stem cells can be subcultured using collagenase IV and grown on recombinant laminin. In another embodiment, undifferentiated stem cells can be subcultured using collagenase IV and grown on Matrigel®. In one embodiment, undifferentiated stem cells can be subcultured using ReLeSR® and grown on recombinant human laminin-521.

[0066] Regarding the seeding of undifferentiated stem cells, the seeding density is approximately 6.0 × 10⁶ cells.3 cells / cm 2 ~ about 5.0×10 5 cells / cm 2 in the range of, for example, about 1.0×10 4 cells / cm 2 such as, about 5.0×10 4 cells / cm 2 such as, about 1.0×10 5 cells / cm 2 such as, or about 3.0×10 5 cells / cm 2 etc. Another aspect, the seeding density is about 6.0×10 3 cells / cm 2 ~ about 1.0×10 4 cells / cm 2 in the range of, for example, about 6.0×10 3 cells / cm 2 ~ about 9.0×10 3 cells / cm 2 such as, about 7.0×10 3 cells / cm 2 ~ about 1.0×10 4 cells / cm 2 such as, about 7.0×10 3 cells / cm 2 ~ about 9.0×10 3 cells / cm 2 such as, or about 7.0×10 3 cells / cm 2 ~ about 8.0×10 3 cells / cm 2 etc. In yet another aspect, the seeding density is about 1.0×10 4 cells / cm 2 ~ about 1.0×10 5 cells / cm 2 in the range of, for example, about 2.0×10 4 cells / cm 2 ~ about 9.0×10 4 cells / cm<000​​​​​​​​For example, cells approximately 4.0 × 10 4 pieces / cm 2 ~Cells approx. 7.0×10 4 pieces / cm 2 For example, or cells approximately 5.0 × 10 4 pieces / cm 2 ~cells approx. 6.0×10 4 pieces / cm 2 For example, the seeding density is approximately 1.0 × 10 cells per growth surface. 5 pieces / cm 2 ~Cells approx. 5.0×10 5 pieces / cm 2 For example, the range of cells per growth surface is approximately 1.0 × 10 5 pieces / cm 2 ~cells approx. 4.5×10 5 pieces / cm 2 For example, cells approximately 1.5 × 10 5 pieces / cm 2 ~Cells approx. 4.0×10 5 pieces / cm 2 For example, cells approximately 2.0 × 10 5 pieces / cm 2 ~Cells approx. 3.5×10 5 pieces / cm 2 For example, or cells approximately 2.5 × 10 5 pieces / cm 2 ~Cells approx. 3.0×10 5 pieces / cm 2 It could be something like that.

[0067] Differentiation of human pluripotent stem cells into dorsal neuroectoderm progenitor cells, and further into dorsal-derived glial progenitor cells and oligodendrocyte progenitor cells. This disclosure provides a method 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 molecules and protein regulators of BMP signaling, as well as MAPK / ERK kinase inhibitors. Without being bound by any particular theory, the inventors have found that human dorsal neuroectoderm progenitor cells obtained according to the method of this disclosure can be readily and efficiently differentiated into spinal cord OPCs in the absence of SHH signaling activation. Surprisingly, this early dorsal phenotype, despite not being a region of early OPC generation in vivo, produces glial progenitor cells by day 21 of the differentiation process and OPCs by day 42 of the differentiation process. The day 42 OPCs produced according to this disclosure express the reference OPC markers NG2 and PDGFRα and are equivalent (in terms of their overall marker expression profile) to OPCs produced by other methods, which are currently in clinical trials to treat spinal cord injury.

[0068] Furthermore, the inventors have found that the method of this disclosure yields significantly more differentiated cells compared to differentiation protocols in which SHH signaling is activated, and provides an extensible process for generating a large number of dorsal neuroectoderm progenitor cells and their offspring for downstream applications, such as glial progenitor cells or OPCs.

[0069] In one embodiment, the method comprises 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 kinase. In certain embodiments, the BMP signaling inhibitor is a small molecule. In other embodiments, the BMP signaling inhibitor is a protein. In some embodiments, the direct target of the BMP signaling inhibitor is ALK2, also known as activin A receptor type I (ACVR1). In certain embodiments, following inhibition of MAPK / ERK and BMP signaling, cells are cultured in the presence of the caudalizing agent retinoic acid to obtain neuroectoderm-restricted progenitor cells having a dorsal spinal cord phenotype.

[0070] In a particular embodiment, the MAPK / ERK inhibitor may be selected from the group consisting of PD0325901, AZD6244, GSK1120212, PD184352, and cobimetinib, as well as their derivatives. In a particular embodiment, the BMP signaling inhibitor may be selected from the group consisting of dorsomorphine, DMH-1, K02288, ML347, LDN193189, and noggin proteins.

[0071] In one embodiment, the method comprises the steps of: obtaining undifferentiated human pluripotent stem cells that remain in an undifferentiated state, as shown in Figure 1; adhering culture of the undifferentiated human pluripotent stem cells for a first period in the presence of the small molecule PD0325901, dorsomorphin, and retinoic acid; and subsequently adhering culture of the cells for a second period in the presence of retinoic acid and in the absence of an SHH signaling activator, thereby obtaining dorsal neuroectoderm cells. In one embodiment, the first and second periods may each be in the range of about 1 to about 6 days, for example, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days.

[0072] In one embodiment, the method includes culturing undifferentiated human pluripotent stem cells in the presence of PD0325901 at a concentration ranging from approximately 1 μM to approximately 100 μM, for example, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 11 μM, approximately 12 μM, approximately 13 μM, approximately 14 μM, approximately 15 μM, approximately 20 μM, approximately 30 μM, approximately 40 μM, approximately 50 μM, or approximately 60 μM, 70 μM, 80 μM, or 90 μM. In another embodiment, the method includes culturing undifferentiated human pluripotent stem cells in the presence of PD0325901 at a concentration of approximately 10 μM.

[0073] In one embodiment, the method includes culturing undifferentiated human pluripotent stem cells in the presence of AZD6244 at a concentration ranging from approximately 1 μM to approximately 100 μM, for example, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 11 μM, approximately 12 μM, approximately 13 μM, approximately 14 μM, approximately 15 μM, approximately 20 μM, approximately 30 μM, approximately 40 μM, approximately 50 μM, or approximately 60 μM, 70 μM, 80 μM, or 90 μM. In another embodiment, the method includes culturing undifferentiated human pluripotent stem cells in the presence of AZD6244 at a concentration of approximately 10 μM.

[0074] In one embodiment, the method includes culturing undifferentiated human pluripotent stem cells in the presence of GSK1120212 of PD0325901 at a concentration ranging from approximately 1 μM to approximately 100 μM, for example, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 11 μM, approximately 12 μM, approximately 13 μM, approximately 14 μM, approximately 15 μM, approximately 20 μM, approximately 30 μM, approximately 40 μM, approximately 50 μM, or approximately 60 μM, 70 μM, 80 μM, or 90 μM. In another embodiment, the method includes culturing undifferentiated human pluripotent stem cells in the presence of GSK1120212 at a concentration of approximately 10 μM.

[0075] In one embodiment, the method includes culturing undifferentiated human pluripotent stem cells in the presence of PD184352 at a concentration ranging from approximately 1 μM to approximately 100 μM, for example, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 11 μM, approximately 12 μM, approximately 13 μM, approximately 14 μM, approximately 15 μM, approximately 20 μM, approximately 30 μM, approximately 40 μM, approximately 50 μM, or approximately 60 μM, 70 μM, 80 μM, or 90 μM. In another embodiment, the method includes culturing undifferentiated human pluripotent stem cells in the presence of PD184352 at a concentration of approximately 10 μM.

[0076] In one embodiment, the method includes culturing undifferentiated human pluripotent stem cells in the presence of cobimetinib at a concentration ranging from approximately 1 μM to approximately 100 μM, for example, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 11 μM, approximately 12 μM, approximately 13 μM, approximately 14 μM, approximately 15 μM, approximately 20 μM, approximately 30 μM, approximately 40 μM, approximately 50 μM, or approximately 60 μM, 70 μM, 80 μM, or 90 μM. In another embodiment, the method includes culturing undifferentiated human pluripotent stem cells in the presence of cobimetinib at a concentration of approximately 10 μM.

[0077] In one embodiment, the method uses a range of approximately 0.2 μM to approximately 20 μM, for example, approximately 0.5 μM, approximately 0.8 μM, approximately 1 μM, approximately 1.5 μM, approximately 2 μM, approximately 2.5 μM, approximately 3 μM, approximately 3.5 μM, approximately 4 μM, approximately 4.5 μM, approximately 5 μM, approximately 5.5 μM, approximately 6 μM, approximately 6.5 μM, approximately 7 μM, etc. The method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of dorsomorphin at concentrations such as approximately 7.5 μM, approximately 8 μM, approximately 8.5 μM, approximately 9 μM, approximately 10 μM, approximately 11 μM, approximately 12 μM, approximately 13 μM, approximately 14 μM, approximately 15 μM, approximately 16 μM, approximately 17 μM, approximately 18 μM, or approximately 19 μM. In another embodiment, the method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of dorsomorphin at concentrations in the range of approximately 0.2 μM to approximately 1 μM, for example, approximately 0.2 μM to approximately 0.9 μM, approximately 0.3 μM to approximately 0.8 μM, approximately 0.4 μM to approximately 0.7 μM, or approximately 0.5 μM to approximately 0.6 μM. In yet another embodiment, the method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of dorsomorphin at a concentration in the range of about 1 μM to about 10 μM, for example, about 1 μM to about 9 μM, about 2 μM to about 8 μM, about 3 μM to about 7 μM, or about 4 μM to about 6 μM. In one embodiment, the method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of dorsomorphin at a concentration in the range of about 10 μM to about 20 μM, for example, about 10 μM to about 19 μM, about 12 μM to about 18 μM, about 13 μM to about 17 μM, or about 14 μM to about 16 μM. In one embodiment, the method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of dorsomorphin at a concentration of about 2 μM.

[0078] In one embodiment, the method includes culturing undifferentiated human pluripotent stem cells in the presence of an ALK2 inhibitor at a concentration ranging from about 1 nM to about 20 μM, for example, about 10 nM, about 50 nM, about 100 nM, about 150 nM, about 200 nM, about 500 nM, about 1 μM, about 5 μM, about 10 μM, or about 15 μM.

[0079] In one embodiment, the method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of DMH-1 at a concentration in the range of about 1 μM to about 10 μM. In one embodiment, the method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of about 2 μM of DMH-1.

[0080] In one embodiment, the method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of K02288 at a concentration ranging from about 1 μM to about 10 μM. In one embodiment, the method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of K02288 at a concentration of about 2 μM.

[0081] In one embodiment, the method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of ML347 at a concentration ranging from about 1 μM to about 10 μM. In one embodiment, the method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of about 2 μM of ML347.

[0082] In one embodiment, the method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of LDN193189 at a concentration ranging from about 1 μM to about 10 μM. In one embodiment, the method includes the step of culturing undifferentiated human pluripotent stem cells in the presence of LDN193189 at a concentration of about 2 μM.

[0083] Any tissue culture vessel suitable for adherent cell culture can be used to obtain dorsal neuroectoderm progenitor cells according to this disclosure. Suitable growth substrates include, for example, recombinant laminin, vitronectin, laminin-511 recombinant fragment E8, or Matrigel® matrix (e.g., Matrigel®, Matrigel® GFR). In certain embodiments of this disclosure, the growth substrate is recombinant human laminin-521 (Biolamina, Sweden, distributed by Corning Inc., Corning, NY). In other embodiments, the substrate is a synthetic substrate such as, for example, Synthemax®-II SC substrate.

[0084] In one embodiment, dorsal neuroectoderm progenitor cells obtained according to this disclosure can be collected and further cultured in suspension as aggregates in the presence of bFGF and EGF until the cells mature into glial progenitor cells. In one embodiment, the further culture period may range from about 5 to 15 days, for example, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 days. In one embodiment, the further culture period is about 7 days.

[0085] In one embodiment, adherent cultured dorsal neuroectoderm progenitor cells can be collected by enzymatic means, such as using TrypLE® Select (Thermo Fisher Scientific, Waltham, MA), Accutase® (Sigma Aldrich, MO), or a similar trypsinase. Alternatively, adherent cultured dorsal neuroectoderm progenitor cells can be collected by non-enzymatic means, such as using 0.5 mM EDTA in PBS, or ReLeSR® (Stem Cell Technologies, Vancouver, Canada).

[0086] Any cell culture vessel or reactor suitable for suspension culture can be used for the non-adherent culture process envisioned in this disclosure. The walls of the vessel are typically inert to or resistant to adhesion of cultured cells. With respect to dynamic suspensions, there are also means for preventing cells from settling, such as stirring mechanisms like magnetically or mechanically driven stirrs or paddles, shaking mechanisms (typically attached to the vessel externally), or inversion mechanisms (i.e., devices that rotate the vessel to change the direction of gravity acting on the cells).

[0087] Suitable vessels for suspension cultures in process development include a standard range of commercially available spinners, spinner flasks, rocker bags, or shaking flasks. An example of a bioreactor suitable for commercial production is the VerticalWheel® bioreactor (PBS Biotech, Camarillo, CA).

[0088] It is also possible to induce the formation of aggregates before transferring to a dynamic suspension. For example, cells can be placed on an AggreWell® plate to create uniform cell aggregates. After approximately 3 days, the cell aggregates can be transferred to a dynamic suspension.

[0089] In one embodiment, glial progenitor cells obtained according to this disclosure can be collected, plated, and adherently cultured for a further period in the presence of epidermal growth factor (EGF) until the cells mature into oligodendrocyte progenitor cells. In a particular embodiment, the culture medium further comprises platelet-derived growth factor AA (PDGF-AA). In one embodiment, the further culture period may range from about 10 to 30 days, e.g., about 10 days, about 15 days, about 20 days, about 25 days, or about 30 days. In one embodiment, the further culture period may range from about 15 to 25 days, e.g., about 15 days, about 16 days, about 17 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, or about 25 days. In one embodiment, the further culture period is about 21 days.

[0090] OPC composition Using the method disclosed herein, a composition containing oligodendrocyte progenitor cells (OPCs) suitable for cell therapy can be obtained. OPCs obtained in accordance with this disclosure express high levels of OPC-specific proteoglycans NG2, PDGFRα, and / or GD3, and non-OPC markers associated with undesirable cell types, such as CD49f (Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis and oncogenesis of MCF-1OA mammary epithelial acini grown in three-dimensional basement membrane cultures. 2003 Methods. 3:256-68), which can be expressed by both neural progenitor cells and epithelial cells and is associated with in vitro cyst formation, or two markers expressed by both pluripotent and epithelial cells, CLDN6 and EpCAM (Lin D, Guo Y, Li Y, Ruan Y, Zhang M, Jin X, Yang M, Lu Y, Song P, Zhao S, Dong B, Xie Y, Dang Q, Quan C. Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions). It expresses EpCAM at low levels (as reported in 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 JMol Med. 2018 Oct;42(4): 1771-1785).

[0091] In certain embodiments, OPCs produced according to this disclosure are in vitro differentiated offspring of human pluripotent stem cells. In certain embodiments, OPCs obtained according to this disclosure are in vitro differentiated offspring of human embryonic stem cells. In other embodiments, OPCs obtained according to this disclosure are in vitro differentiated offspring of induced pluripotent stem (iPS) cells.

[0092] One or more characteristics of the obtained OPC population can be determined by quantifying various cell markers using flow cytometry, for example, determining what percentage of the cell population is positive for a particular marker or set of markers, or identifying undesirable cell types present in the OPC population.

[0093] The OPC population obtained in accordance with this disclosure may include approximately 30% to approximately 100% NG2-positive cells, for example, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, at least approximately 99.5%, at least approximately 99.8%, or at least approximately 99.9% NG2-positive cells. In a particular embodiment, the OPC population obtained in accordance with this disclosure may contain approximately 45% to approximately 75% NG2-positive cells, for example, approximately 45% to approximately 50%, approximately 50% to approximately 55%, approximately 55% to approximately 60%, approximately 60% to approximately 65%, approximately 65% ​​to approximately 70%, approximately 70% to approximately 75%, approximately 50% to approximately 70%, approximately 55% to approximately 65%, or approximately 58% to approximately 63% NG2-positive cells. In another embodiment, the OPC population obtained in accordance with this disclosure may contain approximately 60% to approximately 90% NG2-positive cells, for example, approximately 60% to approximately 65%, approximately 65% ​​to approximately 70%, or other positive cells.

[0094] The OPC population obtained in accordance with this disclosure may include approximately 30% to approximately 100% PDGFRα-positive cells, for example, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, at least approximately 99.5%, at least approximately 99.8%, or at least approximately 99.9% PDGFRα-positive cells. In a particular embodiment, the OPC population obtained in accordance with this disclosure may include approximately 45% to approximately 75% PDGFRα-positive cells, for example, approximately 45% to approximately 50%, approximately 50% to approximately 55%, approximately 55% to approximately 60%, approximately 60% to approximately 65%, approximately 65% ​​to approximately 70%, approximately 70% to approximately 75%, approximately 50% to approximately 70%, approximately 55% to approximately 65%, or approximately 58% to approximately 63% PDGFRα-positive cells. In another embodiment, the OPC population obtained in accordance with this disclosure may include approximately 60% to approximately 90% PDGFRα-positive cells, for example, approximately 60% to approximately 65%, approximately 65% ​​to approximately 70%, or similar positive cells.

[0095] The OPC population obtained in accordance with this disclosure may include approximately 30% to approximately 100% GD3-positive cells, for example, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, at least approximately 99.5%, at least approximately 99.8%, or at least approximately 99.9%. In a particular embodiment, the OPC population obtained in accordance with this disclosure may contain approximately 45% to approximately 75% GD3-positive cells, for example, approximately 45% to approximately 50%, approximately 50% to approximately 55%, approximately 55% to approximately 60%, approximately 60% to approximately 65%, approximately 65% ​​to approximately 70%, approximately 70% to approximately 75%, approximately 50% to approximately 70%, approximately 55% to approximately 65%, or approximately 58% to approximately 63% GD3-positive cells. In another embodiment, the OPC population obtained in accordance with this disclosure may contain approximately 60% to approximately 90% GD3-positive cells, for example, approximately 60% to approximately 65%, approximately 65% ​​to approximately 70%, or similar positive cells.

[0096] In one embodiment, an OPC population obtained according to this disclosure may form no more than 4 epithelial cysts per 100,000 cells in the cyst assay described in Example 8 of this disclosure. In another embodiment, an OPC population obtained according to this disclosure may form no more than 3 epithelial cysts per 100,000 cells in the cyst assay. In yet another embodiment, an OPC population obtained according to this disclosure may form no more than 2 epithelial cysts per 100,000 cells in the cyst assay. In yet another embodiment, an OPC population obtained according to this disclosure may form no more than 1 epithelial cyst per 100,000 cells in the cyst assay described in Example 8 of this disclosure.

[0097] Undesirable cell type The OPC population obtained in accordance with this disclosure may contain low levels of undesirable cell types, for example, by quantification of markers associated with undesirable cell types using flow cytometry. In non-limiting examples, 42-day OPCs obtained in accordance with this disclosure may contain low levels of cells expressing the epithelial cell-related markers EpCAM, CD49f, and CLDN6.

[0098] Markers associated with undesirable cell types may include undesirable cell types representing less than approximately 20%, such as less than approximately 19%, less than approximately 18%, less than approximately 17%, less than approximately 16%, less than approximately 15%, less than approximately 14%, less than approximately 13%, less than approximately 12%, less than approximately 11%, less than approximately 10%, less than approximately 9%, less than approximately 8%, less than approximately 7%, less than approximately 6%, less than approximately 5%, less than approximately 4%, less than approximately 3%, less than approximately 2%, less than approximately 1%, less than approximately 0.5%, less than approximately 0.1%, less than approximately 0.05%, or less than approximately 0.01%. In another embodiment, the cell population may contain about 15% to about 20% of undesirable cell types, for example, about 19% to about 20%, about 18% to about 20%, about 17% to about 20%, about 16% to about 20%, about 15% to about 19%, or about 16% to about 18%. In yet another embodiment, the cell population may contain about 10% to about 15% of undesirable cell types, for example, about 14% to about 15%, about 13% to about 15%, about 12% to about 15%, about 11% to about 15%, or about 12% to about 14%. In one embodiment, the cell population may contain about 1% to about 10% of undesirable cell types, e.g., about 2% to about 10%, about 1% to about 9%, about 2% to about 8%, about 3% to about 7%, or about 4% to about 6%. In one embodiment, the cell population may contain about 0.1% to about 1% of undesirable cell types, e.g., about 0.2% to about 1%, about 0.1% to about 0.9%, about 0.2% to about 0.8%, about 0.3% to about 0.7%, or about 0.4% to about 0.6%. In one embodiment, the cell population may contain about 0.01% to about 0.1% of undesirable cell types, for example, about 0.02% to about 0.1%, about 0.01% to about 0.09%, about 0.02% to about 0.08%, about 0.03% to about 0.07%, or about 0.04% to about 0.06%. In one embodiment, a low level of undesirable cell types may mean the presence of less than about 15% of undesirable cell types.

[0099] In one embodiment, the undesirable cell type may include cells expressing one or more markers selected from CD49f, CLDN6, or EpCAM.

[0100] Cryopreservation After collection, the OPC proliferation population can be formulated at a specific therapeutic dose (e.g., cell count) and cryopreserved for shipment to clinics. The ready-to-administer (RTA) OPC therapeutic composition can then be administered immediately after thawing without further processing. Examples of culture media suitable for cryopreservation include, but are not limited to, 90% human serum / 10% DMSO, medium 3 10% (CS10), medium 2 5% (CS5), and medium 1 2% (CS2), Stem Cell Banker, PRIME XV® FREEZIS, HYPOTHERMASOL®, CSB, and trehalose.

[0101] In some embodiments, the viability percentage of post-filtered cells stored in cryopreservation medium for approximately 0 to approximately 8 hours is at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments, the recovery percentage of post-filtered cells stored in cryopreservation medium for approximately 0 to approximately 8 hours is at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0102] In a further embodiment, the viability percentage of post-filtered cells stored in neutralizing medium for about 0 to about 8 hours, followed by storage 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%. In another embodiment, the recovery percentage of post-filtered cells stored in neutralizing medium for about 0 to about 8 hours, followed by storage 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%.

[0103] In yet another embodiment, the composition is stored in a neutralizing medium for about 0 to about 8 hours, then in a cryopreservation medium for about 0 to about 8 hours, and the viability percentage of cells after filtration following thawing of the cryopreserved composition is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In yet another embodiment, the composition is stored in a neutralizing medium for about 0 to about 8 hours, then in a cryopreservation medium for about 0 to about 8 hours, and the recovery percentage of cells after filtration following thawing of the cryopreserved composition is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0104] In some embodiments, the OPC, after being stored in a neutralizing medium for about 0 to about 8 hours and then in a cryopreservation medium for about 0 to about 8 hours, may secrete decorin. In other embodiments, the OPC, after being stored in a neutralizing medium for about 0 to about 8 hours and then in a cryopreservation medium for about 0 to about 8 hours, may be able to grow after being filtered following the thawing of the cryopreservation medium.

[0105] In some embodiments, the survival percentage of filtered OPCs stored in neutralizing 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 survival percentage of filtered OPCs stored in cryopreserving 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 a further embodiment, the viability percentage of filtered cells stored in a neutralizing solution for about 0 to about 8 hours at room temperature, and then stored in a 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 a further embodiment, the recovery percentage of filtered cells stored in a neutralizing solution at room temperature for approximately 0 to 8 hours, followed by storage in a cryopreservation medium at room temperature for approximately 0 to 8 hours, is at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 140%, and 150%.

[0106] OPC formulated in cryopreservation media suitable for immediate administration after thawing (RTA) applications may contain adenosine, dextran-40, lactobionic acid, HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate, potassium phosphate, dextrose, sucrose, mannitol, calcium chloride, magnesium chloride, potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), and OPC suspended in water. An example of this cryopreservation medium is commercially available under the trademark CRYOSTOR® and manufactured by BioLife Solutions, Inc.

[0107] DMSO can be used as a cryoprotectant to prevent the formation of ice crystals that could kill cells during the cryopreservation process. In some embodiments, the cryopreservable OPC therapeutic composition contains about 0.1% to about 2% DMSO (v / v). In some embodiments, the RTA OPC therapeutic composition contains about 1% to about 20% DMSO. In some embodiments, the RTA OPC therapeutic composition contains about 10% DMSO. In some embodiments, the RTA OPC cell therapeutic composition contains about 5% DMSO.

[0108] In some embodiments, OPC therapeutic agents formulated in cryopreserved media suitable for immediate-to-administer (RTA) applications may include OPC suspended in DMSO-free cryopreserved media. For example, an RTA OPC therapeutic cell composition may include OPC suspended in torox, Na+, K+, Ca2+, Mg2+, c1-, H2P04-, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, and glutathione, without DMSO (dimethyl sulfoxide, (CH3)2SO) or any other bipolar aprotic solvent. Examples of these cryopreserved media are commercially available under the trademark HYPOTHERMOSOL® or HYPOTHERMOSOL®-FRS and are manufactured by BioLife Solutions, Inc. In other embodiments, an OPC composition formulated in cryopreserved media suitable for immediate-to-administer (RTA) applications may include OPC suspended in trehalose.

[0109] The RTA OPC therapeutic composition may optionally contain additional factors that support the engraftment, integration, survival, and efficacy of OPCs. In some embodiments, the RTA OPC therapeutic composition contains an activator of the function of the OPC preparation described herein.

[0110] In some embodiments, RTA OPC therapeutic compositions can be formulated in a culture medium containing components that reduce molecular and cellular stress during the freezing and thawing process by removing free radicals, buffering pH, supporting expansion / osmosis, and maintaining ion concentration balance.

[0111] In some embodiments, an OPC therapeutic formulation prepared in a cryopreserved medium suitable for immediate administration after thawing may comprise one or more immunosuppressive compounds. In certain embodiments, an OPC therapeutic formulation prepared in a cryopreserved medium suitable for immediate administration after thawing may comprise one or more immunosuppressive compounds formulated for sustained release of one or more immunosuppressive compounds. Immunosuppressive compounds for use with the formulations described herein may belong to the following classes of immunosuppressants: glucocorticoids, cell proliferation inhibitors (e.g., alkylating agents or antimetabolites), antibodies (polyclonal or monoclonal), and immunophilin-acting drugs (e.g., cyclosporine, tacrolimus, or sirolimus). Additional drugs include interferons, opioids, TNF-binding proteins, mycophenolates, and minor biological agents. Examples of immunosuppressants include mesenchymal stem cells, anti-lymphocyte globulin (ALG) polyclonal antibodies, anti-thymocyte globulin (ATG) polyclonal antibodies, azathioprine, BAS 1L1 X lMAB® (anti-I L-2Ra receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB® (anti-I L-2Ra receptor antibody), everolimus, mycophenolate, RITUX lMAB® (anti-CD20 antibody), sirolimus, tacrolimus, tacrolimus, and / or mycophenolate mofetil.

[0112] formulation The OPC compositions according to this disclosure may further comprise a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier may comprise dimethyl sulfoxide (DMSO). In one embodiment, the pharmaceutically acceptable carrier may not comprise dimethyl sulfoxide. As described above, the compositions can be further adapted for cryopreservation at temperatures below -80°C to -195°C.

[0113] OPC compositions according to this disclosure can be formulated for administration by direct injection into the spinal cord of a subject. In one embodiment, OPC compositions according to this disclosure can be formulated for intracerebral, intraventricular, intrathecal, intranasal, or intracisional administration to a subject. In one embodiment, OPC compositions according to this disclosure can be formulated for administration by direct or near-direct injection into an infarct cavity in the brain of a subject. In one embodiment, compositions according to this disclosure can be formulated for administration by implantation. In one embodiment, compositions according to this disclosure can be formulated as a solution.

[0114] The OPC composition according to this disclosure contains approximately 1 × 10 cells per milliliter. 6 ~Approx. 5×10 8 For example, approximately 1 × 10 cells per milliliter. 6 Approximately 2 x 10 cells per milliliter 6 Approximately 3 x 10 cells per milliliter 6 Approximately 4 x 10 cells per milliliter 6 Approximately 5 x 10 cells per milliliter 6 Approximately 6 x 10 cells per milliliter 6 Approximately 7 x 10 cells per milliliter 6 Approximately 8 x 10 cells per milliliter 6 Approximately 9 x 10 cells per milliliter 6 Approximately 1 x 10 cells per milliliter 7 Approximately 2 x 10 cells per milliliter 7 Approximately 3 x 10 cells per milliliter 7Approximately 4 x 10 cells per milliliter 7 Approximately 5 x 10 cells per milliliter 7 Approximately 6 x 10 cells per milliliter 7 Approximately 7 x 10 cells per milliliter 7 Approximately 8 x 10 cells per milliliter 7 Approximately 9 x 10 cells per milliliter 7 Approximately 1 x 10 cells per milliliter 8 Approximately 2 x 10 cells per milliliter 8 Approximately 3 x 10 cells per milliliter 8 Approximately 4 x 10 cells per milliliter 8 For example, one cell, or approximately 5 x 10 cells per milliliter. 8 It may include, for example, 1 x 10 cells per milliliter. In another embodiment, a composition according to this disclosure contains approximately 1 x 10 cells per milliliter. 8 ~Approx. 5×10 8 For example, approximately 1 × 10 cells per milliliter. 8 ~Approx. 4×10 8 Approximately 2 x 10 cells per milliliter 8 ~Approx. 5×10 8 Approximately 1 x 10 cells per milliliter 8 ~Approx. 3×10 8 Approximately 2 x 10 cells per milliliter 8 ~Approx. 4×10 8 For example, individual cells, or approximately 3 x 10 cells per milliliter. 8 ~Approx. 5×10 8 It may include, for example, 1 x 10 cells per milliliter. In another embodiment, a composition according to this disclosure contains approximately 1 x 10 cells per milliliter. 7 ~Approx. 1×10 8 For example, approximately 2 × 10 cells per milliliter. 7 ~Approx. 9×10 7 Approximately 3 x 10 cells per milliliter 7 ~Approx. 8×10 7 Approximately 4 x 10 cells per milliliter 7 ~Approx. 7×10 7 For example, one cell, or approximately 5 x 10 cells per milliliter. 7~about 6×10 7 It may include, for example, 1 x 10 cells per milliliter. In one embodiment, a composition according to this disclosure contains approximately 1 x 10 cells per milliliter. 6 ~Approx. 1×10 7 For example, approximately 2 × 10 cells per milliliter. 6 ~Approx. 9×10 6 Approximately 3 x 10 cells per milliliter 6 ~Approx. 8×10 6 Approximately 4 x 10 cells per milliliter 6 ~Approx. 7×10 6 For example, one cell, or approximately 5 x 10 cells per milliliter. 6 ~about 6×10 6 It may include, for example, a number of cells. In yet another embodiment, a composition according to this disclosure contains at least about 1 × 10 cells per milliliter. 6 For example, at least 2 × 10 cells per milliliter. 6 Each cell contains at least approximately 3 x 10 cells per milliliter. 6 Each milliliter contains at least 4 x 10 cells. 6 Each milliliter contains at least 5 x 10 cells. 6 Each cell contains at least approximately 6 x 10 cells per milliliter. 6 Each milliliter contains at least 7 x 10 cells. 6 Each milliliter contains at least 8 x 10 cells. 6 Each milliliter contains at least 9 x 10 cells. 6 At least 1 x 10 cells per milliliter 7 Each milliliter contains at least 2 x 10 cells. 7 Each cell contains at least approximately 3 x 10 cells per milliliter. 7 Each milliliter contains at least 4 x 10 cells. 7 For example, individual cells, or at least about 5 x 10 cells per milliliter. 7 This may include, for example, a cell. In one embodiment, a composition according to this disclosure contains up to approximately 1 × 10 cells. 8 One or more cells, for example, up to approximately 2 x 10 cells per milliliter. 8One or more cells, up to approximately 3 x 10 cells per milliliter. 8 One or more cells, up to approximately 4 x 10 cells per milliliter. 8 One or more cells, up to approximately 5 x 10 cells per milliliter. 8 One or more cells, or up to approximately 6 x 10 cells per milliliter. 8 This may include one or more individuals.

[0115] In one embodiment, an OPC composition according to this disclosure contains approximately 4 × 10 cells per milliliter. 7 ~Approx. 2×10 8 It may include individuals.

[0116] In yet another embodiment, an OPC composition according to this disclosure may have a volume in the range of about 10 microliters to about 5 milliliters, for example, about 20 microliters, about 30 microliters, about 40 microliters, about 50 microliters, about 60 microliters, about 70 microliters, about 80 microliters, about 90 microliters, about 100 microliters, about 200 microliters, about 300 microliters, about 400 microliters, about 500 microliters, about 600 microliters, about 700 microliters, about 800 microliters, about 900 microliters, about 1 milliliter, about 1.5 milliliters, about 2 milliliters, about 2.5 milliliters, about 3 milliliters, about 3.5 milliliters, about 4 milliliters, or about 4.5 milliliters. In one embodiment, a composition according to the Disclosure may have a volume in the range of about 10 microliters to about 100 microliters, for example, about 20 microliters to about 90 microliters, about 30 microliters to about 80 microliters, about 40 microliters to about 70 microliters, or about 50 microliters to about 60 microliters. In another embodiment, a composition according to the Disclosure may have a volume in the range of about 100 microliters to about 1 milliliter, for example, about 200 microliters to about 900 microliters, about 300 microliters to about 800 microliters, about 400 microliters to about 700 microliters, or about 500 microliters to about 600 microliters. In yet another embodiment, compositions according to the Disclosure may have a volume in the range of about 1 milliliter to about 5 milliliters, for example, about 2 milliliters to about 5 milliliters, about 1 milliliter to about 4 milliliters, about 1 milliliter to about 3 milliliters, about 2 milliliters to about 4 milliliters, or about 3 milliliters to about 5 milliliters. In one embodiment, an OPC composition according to the Disclosure may have a volume of about 20 microliters to about 500 microliters.In another embodiment, an OPC composition according to this disclosure may have a volume of about 50 microliters to about 100 microliters. In yet another embodiment, an OPC composition according to this disclosure may have a volume of about 50 microliters to about 200 microliters. In yet another embodiment, an OPC composition according to this disclosure may have a volume of about 20 microliters to about 400 microliters. In one embodiment, an OPC composition according to this disclosure may be contained in a container configured for cryopreservation or administration to subjects requiring it. In one embodiment, the container may be a pre-filled syringe.

[0117] How to use The OPC compositions obtained in accordance with this disclosure can be used in cell therapy to improve one or more neurological functions in subjects requiring treatment. In one embodiment, an OPC cell population according to this disclosure can be injected or transplanted into a subject requiring it. In one embodiment, a cell population according to this disclosure can be transplanted into a subject requiring it to treat spinal cord injury, stroke, or multiple sclerosis.

[0118] In one embodiment, a cell population prepared according to the present disclosure may induce myelin formation of demyelinated axons at the transplantation site in a subject. In one embodiment, a cell population prepared according to the method of the present disclosure may exhibit improved engraftment and migration capabilities. In one embodiment, a cell population prepared according to the method of the present disclosure may improve the repair or regeneration of nerve tissue after injury in a subject.

[0119] Cell populations according to this disclosure may improve sensory function in subjects in need of treatment after transplantation of the population. Improvement in sensory function can be assessed using the International Standard for Neurological Classification of Spinal Cord Injuries (ISNCSCI) test, for example, by determining right and left sensory levels for puncture and light touch. Cell populations according to this disclosure may improve motor function in subjects in need of treatment after transplantation of the population. Improvement in motor function can be assessed using the ISNCSCI test, for example, by determining right and left motor levels for complete paralysis, palpable or observable contraction, active movement, full range of motion against gravity, and sufficient resistance.

[0120] Cell populations conforming to this disclosure may reduce the volume of injury-induced central nervous system parenchyma cavitation in 12 months or less. In one embodiment, cell populations conforming to this disclosure may reduce the volume of injury-induced central nervous system parenchyma cavitation in a subject in 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, or 1 month or less.

[0121] Having outlined the present invention to this point, it will be more readily understood by referring to the following examples. The examples are provided as illustrative examples and are not intended to limit the present disclosure unless otherwise specified. [Examples]

[0122] Example 1 - Culture and proliferation of undifferentiated human embryonic stem cells Undifferentiated human embryonic stem cells (uhESCs) (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) from a working cell bank (WCB) created from the H1 strain were coated with recombinant human laminin-521 (rhLn-521, Corning # 354224) and cultured in complete mTeSR®-1 medium (Stem Cell Technologies # 85850) on tissue culture-treated polystyrene T-75 culture flasks (Corning # 431082). The culture medium was completely changed daily until the cells reached a concentration of approximately 80-90%, and then the uhESCs were subcultured using ReLeSR® reagent (Stem Cell Technologies # 05872). The uhESCs that were subcultured with ReLeSR® were coated with rhLn-521 and then subcultured in new 225 cm⁻¹ containers. 2 The cells were seeded in flasks, and daily culture medium changes were resumed two days after seeding. The uhESCs cultured from whole-body cell borders were grown in this manner for 2 to 5 passages, depending on the experiment, and then differentiated into neuroectoderm progenitor cells as described in Example 2.

[0123] Example 2 - Method for differentiating human embryonic stem cells into neuroectoderm progenitor cells having a dorsal spinal cord progenitor cell phenotype The propagated uhESCs were seeded in containers coated with rhLn-521 and cultured until a concentration of 40-70% was reached, at which point differentiation began.

[0124] Days 0-3Differentiation was initiated by completely removing mTeSR(trademark)-1 medium and adding glial progenitor cell medium (GPM; consisting of DMEM / F12 (Gibco catalog number 10565-018) supplemented with 10 μM MAPK / ERK inhibitor, PD0325901 (PD; Sigma-Aldrich catalog number PZ0162), 2 μM BMP signaling inhibitor, dorsomorphine (Dorso; Sigma-Aldrich catalog number P5499), and 1 μM retinoic acid (RA; Sigma-Aldrich catalog number R2625)). This medium was replenished daily.

[0125] Days 4-6 On day 4, the culture medium was switched to GPM supplemented with 1 μM RA and 150 μM ascorbic acid (Sigma-Aldrich catalog number A4544), and supplemented daily.

[0126] Day 7 On day 7, cells were collected for proliferation and further differentiation into glial progenitor cells as described in Example 3. A subset of cells was collected and analyzed by quantitative PCR (qPCR; described in Example 6) and flow cytometry (described in Example 5), and, where available, immunocytochemical testing (ICC) (described in Example 5) was performed on separate well plates set aside for analysis. At day 7, these cells showed marker expression consistent with dorsal spinal cord progenitor cells (Table 2, Figure 3).

[0127] Example 3 - Method for differentiating human embryonic stem cells into glial cells Days 7-13The differentiation of uhESCs into neuroectoderm progenitor cells, particularly for the dorsal phenotype, was performed as described in Example 2. On day 7, cells were fished out using TrypLE® Select (Thermo Fisher, cat# A12859-01), counted, and placed on rhLn-521-coated containers in GPM supplemented with 20 ng / mL human basic fibroblast growth factor (bFGF, Thermo Fisher, cat# PHG0263), 10 ng / mL epidermal growth factor (hEGF, Thermo Fisher, cat# PHG0311), and 10 μM Rho kinase inhibitor (RI, Tocris catalog number 1254). 2.7 × 10⁶ cells 4 pieces / cm 2 Seeds were sown at the following seeding density. The culture medium was replenished daily by aspirating the used medium and replacing it with fresh GPM+hbFGF+EGF.

[0128] Days 14-21 On day 14, cells were harvested using TrypLE® Select, counted, resuspended in GPM+hbFGF+EGF+RI, and placed in either a PBS-0.1L or PBS-0.5L mini bioreactor system (PBS Biotech) with 1.83 × 10⁶ live cells. 6 The cells were re-seed as dynamic suspension culture at a density of cells / mL. A subset of cells was harvested on day 14 and analyzed by flow cytometry (Example 5), ICC (Example 5), and qPCR (Example 6). 0.1L and 0.5L PBS mini bioreactors were set to rotate at 35 RPM and 28 RPM, respectively. The medium was replenished daily by allowing 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 were increased to 45 RPM and 32 RPM for the 0.1L and 0.5L PBS mini bioreactors, respectively.

[0129] On day 21, a subset of aggregates was collected and subjected to ICC (Example 5) and qPCR (Example 6). By day 21, differentiated cells expressed markers consistent with glial-bound cells (Table 2, Figure 4).

[0130] Example 4 - Method for differentiating human embryonic stem cells into oligodendrocyte progenitor cells 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 culture vessels coated with rhLn-521. For example, starting with a 60 mL total volume 1 × PBS-0.1 L mini bioreactor, 60 mL of culture was divided into 30 mL volumes 2 × T75 flasks. Subsequently, GPM supplemented with 20 ng / mL EGF and 10 ng / mL platelet-derived growth factor AA (PDGFAA; PeproTech, cat# AF-100-13A) was supplied to the cells every other day. Every 7 days (i.e., on days 28 and 35), cells were fished out and counted using TrypLE® Select, and 4 × 10⁶ viable cells were counted. 4 pieces / cm 2 The seeds were reseeded at the specified seeding density into new culture vessels coated with rhLn-521.

[0131] Differentiated cells were collected on day 42. Cells were detached from the container using TrypLE® Select, counted, re-formulated in CryoStor10 (BioLife Solutions, cat# 210102), and then cryopreserved. Subsets of cells were recovered and analyzed by flow cytometry (Example 5), ICC (Example 5), and qPCR (Example 6). By day 42, differentiated cells expressed markers characteristic of OPCs, as measured by the three analytical methods (Table 1, Table 2, Figure 5).

[0132] Example 5 - Characterization of differentiated cell populations by immunocytochemical analysis and flow cytometry Flow cytometry and immunocytochemical (ICC) analysis can be used to detect and characterize different aspects of protein marker expression in cell populations. Flow cytometry can be used to quantify the proportion of individual cells in a population exhibiting a given protein marker profile, while ICC provides additional information about the intracellular localization of each protein marker and can be applied to single cells or cell aggregates. By using one or both of these protein profiling approaches, the inventors tracked the differentiation of human embryonic stem cells into neuroectoderm progenitor cells, glial progenitor cells, and oligodendrocyte progenitor cells by the methods of this disclosure.

[0133] Human embryonic stem cells differentiated into neuroectoderm progenitor cells and glial progenitor cells were characterized by ICC expression of protein markers in differentiated cells at days 7 and 21. Adherent cells and cell aggregates were fixed with 4% paraformaldehyde (PFA) at room temperature (RT) for 30 minutes. The fixed cells and aggregates were washed with phosphate-buffered saline (PBS), and the fixed aggregates were then sequentially placed in increasingly concentrated sucrose solutions (10%, 20%, and 30% by weight / volume) at RT for 30 minutes, RT for 30 minutes, and overnight at 4°C, respectively. After sucrose replacement, the aggregates were embedded in Tissue-Tek optimal cleavage temperature (OCT) solution (Sakura Finetek USA # 4583) and frozen at -80°C. OCT-embedded aggregates were warmed to -20°C, cut into 30 μm sections using a cryostat (model CM3050 S, Leica Biosystems, Buffalo Grove, IL, USA), and mounted on slides coated with poly-L-lysine (Sigma-Aldrich # P4707). For immunocytochemical staining, fixed adherent cells and slide-mounted aggregate sections were permeabilized and blocked at room temperature (RT) for 2 hours in a blocking solution consisting of 0.1% Triton® X-100 / 2% normal goat serum / 1% bovine serum albumin in PBS. After permeabilization and blocking, adherent cell and aggregate sections were incubated overnight at 4°C in a Triton® X-100-free blocking solution containing primary antibodies specific to the protein marker of interest, including PAX6 (BD Pharmingen # 561462 or BioLegend # 901301) for detecting neuroectoderm progenitor cells, and AP2 (Developmental Studies Hybridoma Bank - DSHB #3B5), PAX3 (DSHB #Pax3), and PAX7 (DSHB #Pax7) for detecting dorsal spinal cord progenitor cells.Next, adherent cell and aggregate sections were washed three times with PBS and incubated for 1 hour at RT in a blocking solution free of Triton® X-100 with a secondary antibody specific to the selected primary antibody and 4',6-diamidino-2-phenylindole (DAPI) counterstain, while shielded from light. The adherent cell and aggregate sections were washed three times with PBS and imaged using an IN Cell Analyzer 2000 (GE Healthcare, Pittsburgh, PA, USA).

[0134] Figure 3 shows representative ICC data for neuroectoderm progenitor cells on day 7, illustrating the phenotype of dorsal spinal cord progenitor cells. Seven days after differentiation, adherent cell populations from two representative experiments showed PAX6, a protein marker specific to neuroectoderm progenitor cells. (Lippmann ES, Williams CE, Ruhl DA, Estevez-Silva MC, Chapman ER, Coon JJ, Ashton RS. Deterministic HOX patterning in human pluripotent stem cell-derived neuroectoderm. Stem Cell Reports. 2015 Apr 14;4(4):632-44; Kim DS, Lee DR, Kim HS, Yoo JE, Jung SJ, Lim BY, Jang J, Kang HC, You S, Hwang DY, Leem JW, Nam TS, Cho SR, Kim DW. Highly pure and expandable PSA-NCAM-positive neural precursors from human ESC and iPSC-derived neural rosetes. PLoS One.) It expressed AP2, PAX3, and PAX7, which are dorsal spinal cord progenitor cell markers (2012;7(7):e39715).

[0135] Figure 4 shows representative ICC data for glial progenitor cells on day 21. Sections of aggregates were prepared and stained for the dorsal progenitor cell markers AP2, PAX3, and PAX7, as well as the panneurial progenitor cell marker PAX6. These early progenitor cells were still present on day 21, but a distinct glial population expressing the oligodendrocyte progenitor cell marker NG2 was also present (Zhang Y, Chen K, Sloan SA, Bennet ML, Scholze AR, O'Keeffe S, Phatnani HP, Guamieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, Daneman R, Maniatis T, Barres BA, Wu JQ. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014 Sep 3;34(36):11929-47).

[0136] For human embryonic stem cells that differentiated into oligodendrocyte progenitor cells by day 42, protein marker expression in the resulting single-cell population was characterized by both flow cytometry and ICC.

[0137] To characterize the expression of protein markers in oligodendrocyte progenitor cells by ICC, the aggregate sections mounted on slides were stained as described above, except that permeabilization was performed with 100% methanol at RT for 2 minutes, and the blocking solution consisted of 10% fetal bovine serum in PBS.

[0138] Figure 5 shows representative ICC data for oligodendrocyte progenitor cells on day 42. Single-cell populations obtained from two representative experiments expressed the oligodendrocyte progenitor cell marker NG2 (Zhang Y, Chen K, Sloan SA, Bennet ML, Scholze AR, O'Keeffe S, Phatnani HP, Guamieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, Daneman R, Maniatis T, Barres BA, Wu JQ. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014 Sep 3;34(36):11929-47), and showed decreased expression of the dorsal spinal cord progenitor cell marker AP2 (compare Figures 3 and 5).

[0139] To quantify cell surface markers on day 42 by flow cytometry, 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 to 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 (Thermo Fisher # MA5-24076), as well as 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 a suitable fluorophore-conjugated secondary antibody. The cells were washed, and then propidium iodide was added to distinguish dead cells. In some cases, cells were cultured overnight at 37°C / 5% CO2 in tissue culture vessels coated with Matrigel (Corning # 356231) to recover protein markers sensitive to the 42-day collection procedure described in Example 4, and then collected using TrypLE® Select (Thermo Fisher # A12859-01) and stained for flow cytometry analysis as described above. All cells were analyzed using an Attune NxT (Thermo Fisher, Waltham, MA, USA) flow cytometer. To calculate the proportion of cells expressing a given protein marker, dead cells stained with propidium iodide were gated, and after correcting for the number of cells showing nonspecific binding to the isotype control antibody, the number of living cells bound to the corresponding antibody was expressed as a percentage of the total number of cells analyzed.

[0140] Table 1 shows representative flow cytometry data of oligodendrocyte precursor cells prepared on day 42 according to the methodology described in Example 5.As shown in two representative runs, a high proportion of cells in the resulting cell population were NG2 and PDGFRα (Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O'Keeffe S, Phatnani HP, Guamieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, Daneman R, Maniatis T, Barres BA, Wu JQ. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014 Sep 3;34(36):11929-47), 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 The cells expressed characteristic oligodendrocyte markers, including Neurosci. 1996 Apr 15; 16(8):2659-70), as well as the pre-OPC marker A2B5 (Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, Sharp K, Steward O. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci. 2005 May 11;25(19):4694-705).In addition, the obtained population showed evidence of the neural progenitor cell / epithelial marker CD49f (Krebsbach PH, Villa-Diaz LG. The Role of Integrin a6 (CD49f) in Stem Cells: More than a Conserved Biomarker. Stem Cells Dev. 2017 Aug 1;26(15):1090-1099), as well as 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). Non-OPC markers, including (Review). Int J Mol Med. 2018 Oct;42(4):1771-1785), were detected minimally.

[0141] (Table 1) Representative flow cytometry data for oligodendrocyte progenitor cells generated by the method according to this disclosure. TIFF2026131657000002.tif29161

[0142] Cell populations produced by the methodology described herein are currently undergoing clinical trials to treat spinal cord injury, and are comparable to OPCs produced by other methods (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). In comparison, the proportion of cells positive for the oligodendrocyte progenitor cell marker NG2 was higher, and the expression of non-OPC markers CD49f, CLDN6, and EpCAM was reduced.

[0143] Example 6 - Characterization of differentiated cell populations by gene expression profiling Gene expression profiling can be used to characterize the cellular phenotypes of the starting pluripotent cell population, as well as at each stage of differentiation, including the generation of neuroectoderm progenitor cells, glial progenitor cells, and oligodendrocyte progenitor cells. Gene expression profiling includes both comprehensive transcriptome profiling using methods such as microarrays and RNA-seq, and targeted gene profiling using more sensitive methods such as quantitative real-time PCR (qPCR).

[0144] To perform 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 instructions. For qPCR-based analysis, the purified RNA was then converted to cDNA according to the standard method using the Invitrogen Superscript IV VILO master mix (Thermo Fisher Scientific # 11756050) according to the manufacturer's instructions. Subsequently, the relative expression levels of target genes and reference housekeeping genes were quantified using a gene-specific primer-probe set (Applied Biosystems Taqman gene expression assay, Thermo Fisher Scientific # 4331182) according to the manufacturer's instructions. To determine the relative expression levels of a given set of target genes, PCR reactions were performed using the ABI 7900HT real-time sequence detection system (Applied Biosystems), BioMark HD system (Fluidigm), or equivalent. Each target gene was normalized against one or more reference genes, such as GAPDH, to determine its relative expression level.

[0145] Table 2 shows the results of qPCR from two representative experiments measuring the expression of pluripotency genes, neuroectoderm progenitor cell genes, glial progenitor cell genes, dorsal spinal cord progenitor cell genes, ventral spinal cord progenitor cell genes, and oligodendrocyte progenitor cell genes in cell populations prepared by the method according to this disclosure. RNA samples were collected at the following time points: pre-differentiation (day 0), after differentiation into neuroectoderm progenitor cells (day 7), after differentiation into glial progenitor cells (day 21), and after differentiation into oligodendrocyte progenitor cells (day 42). RNA samples were processed for qPCR using the method described above. A selected panel of genes representing each differentiation state was quantified, including three pluripotency genes (NANOG, LIN28A, SOX2), three neuroectoderm progenitor cell genes (PAX6, HES5, ZBTB16), three glial progenitor cell genes (CACGN4, DCC, FABP7), and three oligodendrocyte progenitor cell genes (CSPG4, PDGFRα, DCN). For each gene, a normalized ΔCT value was calculated using the mean values ​​of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1), and the expression ratio relative to baseline (expression below the limit of quantification) was calculated using the ΔΔCT method.

[0146] (Table 2) qPCR analysis of genetic markers for pluripotent, neuroectoderm progenitor cells (NPCs), dorsal spinal progenitor cells, ventral spinal progenitor cells, glial progenitor cells (GPCs), and oligodendrocyte progenitor cells (OPCs) in H1 uhESCs differentiated into OPCs according to this disclosure. TIFF2026131657000003.tif136168

[0147] Referring to Table 2, 7-day differentiation of uhESCs by the method according to this disclosure yielded a gene expression profile consistent with neuroectoderm 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(7 Pt 1):4927-42).

[0148] In addition, neuroectoderm progenitor cells generated 7 days after differentiation showed a phenotype consistent with dorsal spinal cord progenitor cells based on the expression of the dorsal markers TFAP2A (also known as AP2), PAX3, and PAX7 (Le Dreau G, Marti 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, Sundstrom 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). Further evidence of the dorsal spinal cord progenitor cell phenotype was found that the obtained neuroectoderm progenitor cells did not express ventral spinal cord progenitor cell markers OLIG2 or NKX2-2, whose expression requires activation of the Sonic Hedgehog signaling pathway (Le Dreau G, Marti 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, Sundstrom 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).

[0149] After 21 days of differentiation, the resulting cell population exhibited a gene expression profile consistent with glial progenitor cells, including downregulation of pluripotency and neuroectoderm progenitor cell markers, as well as induction of CACNG4, DCC (also known as the Netrin receptor), and FABP7 (Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O'Keeffe S, Phatnani HP, Guamieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, Daneman R, Maniatis T, Barres BA, Wu JQ. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014 Sep 3;34(36):11929-47; 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, Dailey 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, the obtained day 21 cells showed sustained expression of HES5, in addition to its expression in neuroectoderm progenitor cells / 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). It has also been shown that HES5 promotes the transition from neural progenitor cells to glial progenitor cells 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 glial progenitor cells obtained on day 21 showed sustained expression of TFAP2A, PAX3, and PAX7, which are dorsal spinal cord progenitor cell markers, providing further evidence of derivation from dorsally patterned neural progenitor cells.

[0150] Following a 42-day differentiation period according to the method described herein, the resulting cell population expressed oligodendrocyte progenitor cell-specific markers, including downregulation of both early lineage markers and dorsal spinal cord progenitor cell markers, as well as 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, Guamieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, Daneman R, Maniatis T, Barres BA, Wu JQ. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014 Sep 3;34(36):11929-47).

[0151] Example 7 - Differentiation of human embryonic stem cells into dorsal neuroectoderm progenitor cells using alternative small molecule inhibitors of MAPK / ERK and BMP signaling. In addition to the small molecule inhibitors used in Example 2 (PD0325901 and dolsomorphine), alternative small molecule inhibitors of MAPK / ERK and BMP signaling were tested for their ability to differentiate human embryonic stem cells into dorsal neural ectoderm progenitor cells. Table 3 lists the alternative small molecule inhibitors that were tested. Each condition was tested in a pair of wells on a 6-well tissue culture plate.

[0152] (Table 3) Small molecule inhibitors used to differentiate human embryonic stem cells into dorsal neuroectoderm progenitor cells TIFF2026131657000004.tif96164

[0153] On day 7 of differentiation, cells were harvested and processed, and gene expression profiling was performed by RNA extraction and qPCR as described in Example 6. For each gene, the normalized ΔCT value was calculated relative to the mean of five housekeeping genes (ACTB, GAPDH, EP300, PGK1, SMAD1), and the expression fold ratio relative to baseline (expression below the limit of quantification) was calculated using the ΔΔCT method. Table 4 shows the mean values ​​(relative to baseline) of the biological 2-pair expression fold ratios for each small molecule combination. Referring to Table 4, 7-day differentiation of uhESCs using each of the small molecule combinations tested resulted in downregulation of the pluripotency marker NANOG, as well as similar levels of maintenance or induction of expression of genes associated with the neuroectoderm progenitor cell phenotype, including LIN28A, SOX2, PAX6, HES5, and ZBTB16. In addition, each of the small molecule combinations tested resulted in a dorsal spinal cord progenitor cell phenotype based on the expression of the dorsal markers TFAP2A, PAX3, and PAX7, as well as the absence of the expression of the ventral markers OLIG2 and NKX2-2.

[0154] To more comprehensively compare the cell phenotypes obtained on day 7 after treatment with each small molecule combination, Fluidigm qPCR was performed using a 96-gene panel consisting of known markers for pluripotency, neuroectoderm 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, regression plots of normalized ΔCT values ​​comparing the cell phenotypes on day 7 for each alternative small molecule combination with those produced by treatment with PD0325901 + dorsomorphin showed that similar overall cell phenotypes could be achieved with each of the small molecule combinations tested. In summary, the results shown in Table 4 and Figure 6 support the use of various combinations of (i) a MAPK / ERK inhibitor and (ii) a BMP signaling inhibitor, in the absence of (iii) an SHH signaling activator, to differentiate uhESCs into dorsal neuroectoderm progenitor cells, and further into glial progenitor cells and oligodendrocyte progenitor cells using the method of this disclosure.

[0155] (Table 4) Pluripotency in H1 uhESCs differentiated into NPCs using different combinations of small molecule inhibitors and qPCR analysis of gene markers in neuroectoderm progenitor cells (NPCs) TIFF2026131657000005.tif96166

[0156] Example 8 - Evaluation of the presence of heterogeneous epithelial cells in a differentiated OPC population using an in vitro cyst assay. The presence of undesirable epithelial cells in the OPC population prepared in accordance with this disclosure was tested using an in vitro cyst assay. The cyst assay was essentially performed according to the protocol of Debnath et al. (Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis and oncogenesis of MCF-1OA 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, including basal extrabasal protein expression of the epithelial marker CD49f, was also evaluated using immunocytochemical tests.

[0157] OPCs were placed on a pad of Matrigel® (Corning) in cyst support medium, with cells measuring 21.9 × 10⁶. 3 Seeds were seeded at a density of cells / cm² (total of 0.5 × 10¹⁶ cells in 12 wells of a 24-well plate). 6(Cells were seeded). The cells were cultured for 20 days. On day 20, viable cysts were counted, Matrigel® was lysed with cell recovery solution (Corning # 354253), cells were fixed on ice with 4% paraformaldehyde (PFA) for 5 minutes, and permeabilized overnight in blocking buffer. Subsequently, cysts were stained with CD49f (ITGA6) and phalloidin, and counterstained with DAPI. Cysts were imaged using IN Cell Analyzer 2000 (GE Healthcare Life Sciences), and cyst frequency, size, and staining intensity were quantified using IN Cell Developer software (GE Healthcare Life Sciences) and MATLAB® (Mathworks). Referring to Table 5, OPCs prepared from two representative runs using the method according to this disclosure and tested in the in vitro cyst assay did not produce any detectable cysts per 100,000 cells. In contrast, three control lots of OPCs (control A, control B, and control C) prepared by an alternative method previously found to induce 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) developed cysts in the assay.

[0158] (Table 5) Representative cyst assay results for oligodendrocyte progenitor cells generated by the method according to this disclosure TIFF2026131657000006.tif26128

[0159] Example 9 - Comparison of differentiated cell yield in the presence and absence of SHH signaling activators In testing various small molecule differentiation regimens, it was discovered that removing SHH signaling activators such as purmorphamine (PMA) from the differentiation process consistently increased the step 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 progenitor cells (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 between day 7 and day 14 are favorable for the proliferation of more dorsal phenotypic neural progenitor cells. Surprisingly, this early dorsal phenotype, despite not being the region where early OPCs are generated in vivo, still resulted in OPC cells at day 42 (Figure 5).

[0160] The effects of removing SHH signaling activators from the differentiation process in accordance with this disclosure were further tested and quantified. In two representative experiments (Run 1 and Run 2) in which the SHH agonist PMA was removed from the differentiation process, the step yield from day 7 to day 14 increased compared to runs containing PMA (Figure 2B). This resulted in a substantial increase in the overall theoretical cell yield (Figure 2C).

[0161] Example 10 - In vitro functional bioassay (decolin secretion and migration assay) Decorin is a naturally occurring, small extracellular, leucine-rich proteoglycan and TGF-β1 / 2 antagonist that regulates diverse cellular functions through interactions with components of the extracellular matrix (ECM). Expressed by neurons and astrocytes in the central nervous system, decorin reduces scar tissue, prevents cavitation, and promotes wound healing through its anti-scarring effect, which significantly reduces the accumulation of titers of scar-derived axon growth inhibitors by inhibiting their degradation and 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]. Human recombinant proteins have been shown to have beneficial effects in animal models of spinal cord injury when added exogenously [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].

[0162] Long-term stability data from three manufactured GPOR-OPC1 clinical batches indicate secretion levels of approximately 15–30 ng / ml.

[0163] In this in vitro assay, decorin production by OPC1 cells is measured using a commercially available solid-phase sandwich ELISA kit. The test material for the efficacy assay is the supernatant produced by thawing vials of OPC1 pharmaceutical cells, culturing the cells for 48 hours, collecting the conditioned medium (CM), and freezing the CM supernatant until ELISA testing. Ongoing OPC1 process development research has shown that the level of decorin secretion after culturing improved OPC1 cells in vitro for 48 hours is similar to that of the GPOR-OPC1 batch (described above), and is in the range of approximately 20–35 ng / ml.

[0164] One of the characteristic features of OPC1 activity in vivo, as exemplified by the rat cervical SCI model, is the in vivo migration of OPC1 cells from the injection site to the site of cavitation injury and surrounding areas in the spinal cord. The migration assay is based on in vitro measurement of OPC1 cell migration in response to different chemotactic factors such as PDGFαα and PDGFββ [Armstrong, RC, L. Harvath, and ME 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, MA, et al., The endocannabinoid 2-arachidonoylglycerol regulates oligodendrocyte progenitor cell migration. Biochem Pharmacol, 2018. 157: p. 180-188].

[0165] In this in vitro assay, OPC-1 cells are seeded into individual wells of a transwell system (Corning® Transwell® polycarbonate membrane insert, pore size 8 μm). The cells are exposed to culture medium in the lower wells with or without a chemoattractant (PDGFββ). After incubation overnight, cells that have migrated through the transwells are collected and counted to determine the percentage of migrated input cells, which is then reported as the % of migration. This method is currently in the research phase, and further research is being conducted to evaluate and improve its performance. Ongoing OPC1 process development studies have shown that the % of in vitro migration of OPC1 cells in response to 16–24 hours of stimulation with the PDGFββ chemoattractant is in the range of approximately 15–50%.

[0166] Higher decorin secretion and migration rates were observed in OPC1 batches of the improved process, which showed higher purity (flow cytometry analysis of biomarker expression), better yield, and better morphological evaluation, and vice versa; lower decorin secretion and migration rates were observed in OPC1 batches, which showed lower purity, lower yield, and inferior morphological evaluation, as demonstrated by evaluation of OPC1 function in vitro using decorin secretion and migration bioassays.

[0167] For example, Table 6 provides an overview of the final product at day 42, characterized by marker data, decorin secretion, and migratory bioassays.

[0168] (Table 6) Day 42 - Final Product TIFF2026131657000007.tif20170

[0169] While this disclosure has been described with reference to specific embodiments, it will be understood by those skilled in the art that various modifications can be made and equivalents can be substituted for elements without departing from the scope of this disclosure. In addition, many modifications can be made to adapt specific circumstances or materials to the teachings of this disclosure without departing from the scope of this disclosure.

[0170] Therefore, this disclosure is not limited to any particular form disclosed as the best form intended for carrying out this disclosure, and is intended to include all aspects within the scope and spirit of the attached claims.

Claims

1. A method for obtaining a cell population containing dorsal neural progenitor cells (dNPCs) from undifferentiated human pluripotent stem cells, a) A step to obtain cultures of undifferentiated human pluripotent stem cells; b) Adhering culture of undifferentiated human pluripotent stem cells for a first period 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; and c) Adhering culture of cells from b) for a second period in the presence of retinoic acid and in the absence of sonic hedgehog (SHH) and SHH signaling activators, thereby obtaining dorsal neural progenitor cells. The method, including the method.

2. The method according to claim 1, further comprising the additional steps of collecting cells from step c), replating the collected cells onto a substrate, and further adhering and culturing the cells for a further period in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) to thereby increase the neural progenitor cells.

3. The method according to claim 2, further comprising the additional step of collecting the proliferated cells and culturing the cells as aggregates in a suspension for a further period of time in the presence of bFGF and EGF until the cells mature into glial progenitor cells.

4. The method according to claim 3, further comprising the additional step of plating the aggregate containing glial progenitor cells onto a substrate and adhering the cells to epidermal growth factor (EGF) for a further period of time, optionally dividing the cells from time to time, until the cells mature into oligodendrocyte progenitor cells (OPCs).

5. The method according to claim 3, further comprising the additional step of plating the aggregates containing glial progenitor cells onto a substrate and adhering the cells for a further period of time in the presence of platelet-derived growth factor AA (PDGF-AA) and EGF, with the cells optionally being divided from time to time until they mature into oligodendrocyte progenitor cells (OPCs).

6. The method according to claim 3, wherein the cells are cryopreserved at a certain stage in the method, and then the cells are thawed and the method is continued.

7. The method according to claim 1, wherein the substrate is recombinant human laminin-521.

8. The method according to claim 1, wherein the human pluripotent stem cell is a human embryonic stem cell (hESC).

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

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

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

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

13. The method according to claim 1, wherein the at least one inhibitor of BMP signaling is selected from the group consisting of dorsomorphin, DMH-1, K02288, ML347, LDN193189, and noggin protein.

14. The method according to claim 1, wherein the at least one inhibitor of BMP signaling is dorsomorphine.

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

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

17. The method according to claim 3, wherein the aggregate is cultured in a suspension for about 7 days.

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

19. A differentiated cell population containing paired box 6 (PAX6)-positive dNPCs obtained according to the method described in claim 1.

20. The differentiated cell population according to claim 18, wherein the dNPC expresses one or more markers selected from paired box 3 (PAX3), paired box 7 (PAX7), and activating protein 2 (AP2).

21. A method for obtaining a cell population containing oligodendrocyte progenitor cells (OPCs) from undifferentiated human pluripotent stem cells, The method is a) A step of obtaining dorsal neural progenitor cells (dNPCs) according to the method described in claim 1; b) The step of collecting cells from a), replating the cells onto a substrate, and adhering the cells to a culture for a further period in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) to promote the growth of the neural progenitor cells; c) The step of collecting cells from b) and further culturing the cells as aggregates in suspension for a further period of time in the presence of bFGF and EGF until the cells mature into dorsal glial progenitor cells; and d) Plating aggregates from c) onto a substrate, and adhering the cells to the substrate for a further period of time in the presence of epidermal growth factor (EGF), optionally dividing the cells from time to time until the cells mature into OPCs. Includes, The OPC expresses one or more markers selected from neuronal / glial antigen 2 (NG2), platelet-derived growth factor receptor A (PDGFRα), and ganglioside GD3 (GD3). The method.

22. The method according to claim 21, wherein the adhesion culture step is performed on a substrate, and the substrate is selected from (i) a cell adhesion peptide and (ii) an extracellular matrix selected from laminin and vitronectin.

23. The method according to claim 21, wherein the step of adhering culture is performed on recombinant human laminin-521.

24. The method according to claim 21, wherein the step of adhering culture is performed on a laminin-511 E8 fragment.

25. The method according to claim 21, wherein step c) is performed in a dynamic suspension.

26. The method according to claim 21, wherein during step d), the culture medium further comprises platelet-derived growth factor AA (PDGF-AA).

27. The method according to claim 21, wherein the human pluripotent stem cell is an hESC.

28. The method according to claim 21, wherein the human pluripotent stem cell is hiPSC.

29. The method according to claim 21, wherein the OPC is frozen and stored, and can be administered to the subject immediately after thawing.

30. A differentiated cell population containing OPCs obtained according to the method of claim 21.

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