Methods for generating oligodendrocyte precursor cells and uses thereof

Abstract

Methods are provided for differentiating human pluripotent stem cells into oligodendrocyte precursor cells (OPCs). Cells and cell compositions obtained by such methods, as well as uses of such cells, are also provided. Methods and protocols are further provided for efficiently differentiating human pluripotent stem cells into OPCs.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 159,350, filed March 10, 2021, the entirety of which is incorporated by reference herein for all purposes.

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

[0003] background Oligodendrocyte precursor 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 and mature into oligodendrocytes after migrating through the developing CNS. Mature oligodendrocytes produce myelin that insulates nerve axons and remyelinates demyelinated CNS lesions. Oligodendrocytes also contribute to neuroprotection through other mechanisms, including the production of neurotrophic factors that promote neuronal survival (Wilkins et al., 2001 Glia 36(1):48-57 (Non-Patent Document 1); Dai et al., 2003 JNeurosci.23(13):5846-53 (Non-Patent Document 2); Du and Dreyfus, 2002 JNeurosci Res.68(6):647-54 (Non-Patent Document 3)). Unlike most progenitor cells, OPCs remain abundant in the adult CNS and retain the ability to generate new oligodendrocytes. Thus, OPCs, and mature oligodendrocytes derived from OPCs, are important therapeutic targets for demyelinating and myelinating disorders (such as multiple sclerosis, adrenoleukodystrophy, and adrenal myeloneuropathy), other neurodegenerative disorders (such as Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease), and acute neurological injuries (such as stroke and spinal cord injury (SCI)).

[0004] Several protocols have been developed for the differentiation of human pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), into OPCs that can be used for cell therapy. Although these methods have been successful in generating OPCs from human pluripotent stem cells for research purposes, challenges remain regarding the quality of goods, scalability, and costs associated with translating existing protocols into clinical commercial-scale production processes.

[0005] There is a need for improved methods for differentiating pluripotent stem cells into OPCs. Ideally, such methods should be easily scalable to produce sufficient quantities of OPCs for cell therapy applications while consistently and reproducibly producing target cell OPCs with desired quality characteristics. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Wilkins et al.,2001 Glia 36(1):48-57 [Non-Patent Document 2] Dai et al.,2003 JNeurosci.23(13):5846-53 [Non-Patent Document 3] Du and Dreyfus,2002 JNeurosci Res.68(6):647-54 Summary of the Invention

[0007] overview In various embodiments described herein, the present disclosure provides, inter alia, robust and reliable protocols for differentiating human pluripotent stem cells, such as ESCs and iPSCs, into oligodendrocyte precursor cells (OPCs).

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

[0009] In one aspect, provided herein is a method for obtaining a population of oligodendrocyte precursor cells (OPCs) from undifferentiated pluripotent stem cells, the method comprising: a) obtaining a culture of undifferentiated pluripotent human embryonic stem cells (hESCs); b) culturing the undifferentiated pluripotent hESCs for a first period of time under culture conditions sufficient to induce differentiation of the hESCs into neuroectodermal cells and neural progenitor cells; and c) culturing the neural progenitor cells from step b) for a second period of time under culture conditions sufficient to differentiate the neural progenitor cells into OPCs.

[0010] In an embodiment, the method, wherein the pluripotent cells of step b) are cultured on laminin in adherent tissue culture vessels, or in suspension complexes, or both.

[0011] In an embodiment, the method, wherein the pluripotent stem cells are human embryonic stem cells (hESCs). In an embodiment, the pluripotent stem cells are human induced pluripotent stem cells (hiPSCs).

[0012] In an embodiment, the undifferentiated hESCs of step b) are cultured in the presence of Dorsomorphin, PD0325901, and RA (retinoic acid). In an embodiment, the culturing step further comprises in the presence of AA (ascorbic acid) and RA (retinoic acid).

[0013] In an embodiment, the method, wherein the neuroectodermal cells from step b) are cultured in the presence of EGF and hsbFGF (thermostable basic fibroblast growth factor). In an embodiment, the method further comprises culturing in the presence of EGF (epidermal growth factor) and PDGF-AA (platelet derived growth factor AA).

[0014] In an embodiment, the method, wherein the first period of time in step b) is from about 3 days to about 60 days. In an embodiment, the first period of time is from about 10 days to about 15 days. In an embodiment, the first period of time is about 14 days.

[0015] In an embodiment, the method, wherein the second period of time from step c) is from about 10 days to about 60 days. In an embodiment, the second period of time is from about 20 days to about 40 days. In an embodiment, the second period of time is about 28 days.

[0016] In an embodiment, the method, wherein the differentiated hESCs in step b) are cryopreserved at about day 14.

[0017] In an embodiment, the method wherein the cryopreserved cells are thawed and the thawed cells are subsequently cultured in any remaining steps of the method.

[0018] In an embodiment, the method comprises the step of cryopreserving the neural progenitor cells from step b) at or near the completion of the first period of time.

[0019] In an embodiment, a method wherein cryopreserved neuroectodermal cells are thawed and cultured according to step c).

[0020] In an embodiment, the method wherein the OPCs of step c) are cryopreserved.

[0021] In an embodiment, the method wherein cryopreserved OPCs are thawed.

[0022] In an embodiment, the method, wherein the OPCs express one or more markers selected from neural / glial antigen 2 (NG2), platelet-derived growth factor receptor A (PDGFRα), and platelet-derived growth factor receptor B (PDGFRβ).

[0023] In one aspect, provided herein is a method of formulating an oligodendrocyte precursor cell (OPC) composition for administration to a subject immediately after thawing, the method comprising: (a) suspending OPCs from the methods described above in a cryopreservation medium to form a cell suspension; (b) storing the cell suspension at a cryopreservation temperature; and (c) thawing the cryopreserved suspension.

[0024] In an embodiment, the method, wherein the cryopreservation medium comprises one or more of 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, dimethylsulfoxide (DMSO), and water.

[0025] In one aspect, provided herein is a pharmaceutical composition for administration to a subject, the composition comprising OPCs according to the methods described above and a cryopreservation medium.

[0026] In an embodiment, the pharmaceutical composition, wherein the cryopreservation medium comprises one or more of 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 water.

[0027] In one aspect, provided herein is a method for treating spinal cord injury in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described above.

[0028] In an embodiment, a method for treating spinal cord injury in a subject, wherein administering comprises administering the composition within or adjacent to the site of spinal cord injury.

[0029] In an embodiment, a method for treating spinal cord injury in a subject, wherein administering is by injection.

[0030] In an embodiment, a method for treating spinal cord injury in a subject, wherein administering is by implantation.

[0031] In an embodiment, a method for treating spinal cord injury in a subject, wherein administering is by implantation.

[0032] In embodiments, the concentration of cells is about 1×10 per mL 6 ~100 x 10 cells per mL 6 The pharmaceutical composition is a cell.

[0033] In an embodiment, the pharmaceutical composition is stored in a volume of about 100 microliters to about 1 milliliter.

[0034] In an embodiment, the concentration of cells is 100×10 per mL 6 The pharmaceutical composition is a cell.

[0035] In an embodiment, the pharmaceutical composition is stored in a volume of 250 microliters.

[0036] In an embodiment, the pharmaceutical composition is stored in a volume of 300 microliters.

[0037] In an embodiment, the pharmaceutical composition, wherein the cryopreservation medium is a freezing solution.

[0038] In an embodiment, the pharmaceutical composition, wherein the freezing solution is CryoStor10 (CS10).

[0039] In an embodiment, the pharmaceutical composition, wherein the OPCs express one or more markers selected from neural / glial antigen 2 (NG2), platelet-derived growth factor receptor A (PDGFRα), and platelet-derived growth factor receptor B (PDGFRβ). [Brief description of the drawings]

[0040] [Figure 1] 1 is a flow chart outlining the OPC thaw and injection (TAI) harvesting and cryopreservation process of the present disclosure. [Diagram 2] 1 is an illustration of the Geron Process of Records (GPOR process) (bottom) compared to the improved OPC manufacturing process of the present disclosure (top). [Diagram 3] 1 is a flow chart outlining the pluripotent stem cell to oligodendrocyte precursor signaling sequence used to establish the OPC differentiation process of the present disclosure. [Figure 4] 1 is a flowchart illustrating the flow and timeline of the OPC manufacturing process of the present disclosure. [Diagram 5] 1 is a flow chart illustrating a good manufacturing process (GMP) for producing a 100 TAI vial of OPC according to the present disclosure. [Figure 6] Shown at 10x magnification (left) and 20x magnification (right) are OPC morphology for cells cultured in control culture conditions and glial precursor medium (GPM / E) at day 10 (time = 0, day 7). [Figure 7] Shown are OPC morphology of cells cultured in 50% GPM / E and 50% N2.1 (no IBMX) at 3 days after treatment at 10x magnification (left) and 20x magnification (right). [Figure 8] OPC morphology of cells cultured in control culture conditions, GPM / E on day 12, at 10x magnification (left) and 20x magnification (right). [Figure 9] Shown is OPC morphology of cells cultured in 100% N2.1 (with IBMX for 2 days) at 5 days after treatment at 10x magnification (left) and 20x magnification (right). [Figure 10] Confocal immunostaining of Hoechst (blue) and MBP (green) on day 13 (fixed cells) for a control line cultured in GPM / E (left) and cells cultured in 100% N2.1 (right) are shown. [Figure 11] FIG. 1 is a flow chart of the large-scale batch OPC differentiation protocol. [Figure 12] Photographs of cell morphology starting from day 35 to day 42 are shown. Successful and unsuccessful cultures differ by different morphologies. The ER-ICBRD-02 batch of cells presented an organized, small, dense cell morphology and did not acquire the spindle-shaped "failed" morphology. [Figure 13] FIG. 1 is a flow chart of the thawed cell OPC differentiation protocol. [Figure 14] Photographs of the morphology of ongoing runs are shown and presented in comparison to ICB-thawed cells, presenting the similarities in aggregate morphology between the initial runs and their derived ICBs at day 21. [Figure 15-1] Photographs of the morphology of ongoing runs are presented in comparison to ICB-thawed cells at days 35 and 42. Morphological similarities between the initial runs and their derived ICBs are presented, with cells organized as small, dense cells. [Figure 15-2] See description of Figure 15-1. [Figure 15-3] See description of Figure 15-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description This specification is not intended to be a detailed listing of all the different ways in which the disclosure may be implemented or all the features that may be added to the disclosure. For example, features illustrated with respect to one embodiment may be incorporated in other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the disclosure contemplates that in some embodiments of the disclosure, any feature or combination of features described herein may be excluded or omitted. In addition, numerous modifications and additions to the various embodiments suggested herein will become apparent to those skilled in the art in view of the present disclosure, which do not depart from the disclosure. In other instances, well-known structures, interfaces, and processes have not been shown in detail so as not to unnecessarily obscure the invention. No part of this specification is intended to result in a denial of any portion of the full scope of the invention. Thus, the following description is intended to illustrate certain aspects of the disclosure, and is not intended to exhaustively specify all permutations, combinations, and variations thereof.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terms used in the description of the present disclosure herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the present disclosure.

[0043] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety.

[0044] Unless the context indicates otherwise, it is specifically contemplated that the various features of the present disclosure described herein can be used in any combination. Further, the present disclosure also contemplates that in some embodiments of the present disclosure, any feature or combination of features described herein can be excluded or omitted.

[0045] The methods disclosed herein may include one or more steps or actions for achieving the described method. Method steps and / or actions may be interchanged with one another without departing from the scope of the invention. In other words, unless a specific order of steps or actions is required for proper operation of an aspect, the order and / or use of specific steps and / or actions may be modified without departing from the scope of the invention.

[0046] I. Definition As used in the description of this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0047] As used herein, "and / or" refers to and includes any and all possible combinations with one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0048] As used herein, the terms "about" and "approximately" when referring to measurable values ​​such as percentages, densities, volumes, and the like, are meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0049] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y," and phrases such as "about X to Y" mean "about X to about Y."

[0050] As used herein, "oligodendrocyte precursor cells" (OPCs) refer to cells found in the central nervous system that are of the neuroectodermal / glial lineage, express the characteristic marker neural / glial antigen 2 (NG2), and can differentiate into oligodendrocytes.

[0051] The terms "glial lineage cells," "glial progenitor cells," and "glial cells" are used interchangeably herein and refer to non-neuronal CNS cells derived from neuroectoderm / neuronal progenitor cells. Glial progenitor cells can be further differentiated to form OPCs / oligodendrocytes or astrocytes. In certain embodiments, the glial progenitor cells of the present disclosure express one or more markers selected from calcium voltage-gated channel auxiliary subunit gamma 4 (CACNG4), fatty acid binding protein 7 (FABP7), and netrin-1 receptor (DCC).

[0052] The terms "neurectoderm," "neurectodermal cell," "neurectodermal progenitor cell," "neurectodermal precursor," "neural precursor," and "neural precursor cell" are used interchangeably herein to refer to cells that can differentiate along a neural progenitor pathway and form CNS neurons, oligodendrocytes, astrocytes, and ependymal cells. In certain embodiments, neuroectodermal cells of the present disclosure express one or more markers selected from paired box 6 (PAX6), Hes family BHLH transcription factor 5 (HESS), and zinc finger and BTB domain containing 16 (ZBTB16).

[0053] As used herein, the terms "dorsal" and "ventral" refer to different neural cell subtypes that emerge from precursor cells arranged in spatially distinct domains along the dorsal-ventral axis of the neural tube in the developing spinal cord. This process, known as dorsal-ventral patterning, is controlled by secreted signals that compartmentalize neural precursor cells. BMP and Wnt signaling initiate patterning from the dorsal neural tube (Lee and Jessell, 1999 Annu.Rev.Neurosci.22:261-294), while secretion of SHH plays a key role in establishing the fate of ventral neural cells (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).

[0054] The terms "dorsal neuroectodermal progenitor cells", "dorsal neural progenitor cells", and "dNPCs" are used interchangeably herein and refer to neural progenitor cells that have a dorsal spinal cord phenotype and are obtained by differentiating pluripotent stem cells into neuroectodermal restricted progenitor cells in the absence of exogenous SHH or SHH signaling activators. In certain embodiments, dNPCs express one or more markers selected from paired box 3 (PAX3), paired box 7 (PAX7), and activator protein 2 (AP2).

[0055] As used herein, the term "embryoid body" (EB) refers to a three-dimensional cell aggregate derived from a pluripotent stem cell that has undergone spontaneous differentiation toward all three germ layers. EBs are formed when pluripotent stem cells are removed from culture conditions that inhibit differentiation. For example, in the case of human embryonic stem cells, removal of basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGE13) from the culture medium results in spontaneous differentiation into all three germ layers and the formation of EBs.

[0056] As used herein, the term "BMP signaling inhibitor" refers to a small molecule or protein modulator that can downregulate signaling along the bone morphogenetic protein (BMP) signaling pathway. In certain embodiments, the BMP signaling inhibitor directly targets Activin A receptor, type I (ACVR1), also known as Activin Receptor-Like Kinase 2 (ALK2). In certain embodiments, the BMP signaling inhibitor is selected from the group consisting of Dorsomorphin, DMH-1, K02288, ML347, LDN193189, and Noggin protein.

[0057] As used herein, the term "MAPK / ERK inhibitor" refers to a small molecule or protein modulator 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.

[0058] The terms "SHH signaling activator", "SHH signaling agonist", "SHH activator" and "SHH agonist" are used interchangeably herein and refer to small molecule or protein modulators that can activate the Sonic Hedgehog (SHE) signaling pathway. Non-limiting examples of SHE signaling activators include purmorphamine (PMA), smoothon agonist (SAG, CAS 364590-63-6), and Sonic Hedgehog (SHH) protein.

[0059] As used herein, the terms "FGF" or "bFGF" refer to human basic fibroblast growth factor, or FGF-2, a growth factor and signaling protein encoded by the FGF2 gene. These terms are also used interchangeably to refer to any sequence variants or conjugates thereof, including commercially available "thermostable" FGFs (hs-FGF, hs-bFGF, hs-hbFGF, and / or hsbFGF).

[0060] As used herein, the term "undesirable cell type" refers to cells other than the neuroectodermal lineage that may cause the formation of ectopic tissue upon implantation or cause the formation of one or more cysts in the cyst assay described herein. In one embodiment, "undesirable cell type" may include cells of epithelial lineage, such as cells positive for CD49f, a marker expressed by both neural progenitor cells and epithelial cells, or cells positive for CLDN6 or EpCAM, two markers expressed by both pluripotent cells and epithelial cells.

[0061] As used herein, "implantation" or "implantation" refers to the administration of a cell population to a target tissue using an appropriate delivery technique (eg, using an injection device).

[0062] As used herein, a "subject" refers to an animal or a human.

[0063] As used herein, a "subject in need thereof" refers to an animal or human having damaged tissue in the central nervous system. In one embodiment, the animal or human is experiencing loss of motor function.

[0064] The terms "central nervous system" and "CNS" are used interchangeably herein and refer to the complex of nervous tissue that controls one or more activities of the body, including, but not limited to, the brain and spinal cord of a vertebrate animal.

[0065] As used herein, "treatment" or "treating" with respect to a condition or disease is an approach to obtain beneficial or desired results, preferably including clinical results, after the condition or disease has manifested in a patient. Beneficial or desired results with respect to a disease include, but are not limited to, one or more of the following: amelioration of the condition associated with the disease, cure of the disease, reduction of the severity of the disease, delay in the progression of the disease, alleviation of one or more symptoms associated with the disease, improvement of the quality of life of a person suffering from the disease, prolongation of survival, and any combination thereof. Similarly, for purposes of this disclosure, beneficial or desired results with respect to a condition include, but are not limited to, one or more of the following: amelioration of the condition, cure of the condition, reduction of the severity of the condition, delay in the progression of the condition, alleviation of one or more symptoms associated with the condition, improvement of the quality of life of a person suffering from the condition, prolongation of survival, and any combination thereof.

[0066] I.OPC composition The methods of the present disclosure can be used to obtain compositions comprising oligodendrocyte precursor cells (OPCs) suitable for cell therapy. OPCs obtained according to the present disclosure express high levels of the proteoglycans NG2, PDGFRa, and / or PDGFRβ, characteristic of OPCs, as well as low levels of non-OPC markers associated with unwanted cell types, such as CD49f, which can be expressed by both neural progenitor cells and epithelial cells and has been shown to mediate cyst formation in vitro (Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. 2003 Methods. 3:256-68), cytokeratin 7 (CK7), or CLDN6 and EpCAM, two markers expressed by both pluripotent and epithelial cells (Lin D, Guo Y, Li Y, Ruan Y, Zhang M, Jin X, Yang M, Lu Y, Song P, Zhao S, Dong B, Xie Y, Dang Q, Quan C.Bioinformatic analysis reveals potential properties of human Claudin-6 regulation and functions.Oncol Rep.2017 Aug;38(2):875-885, Huang L,Yang Y,Yang F,Liu S,Zhu Z,Lei Z,Guo J.Functions of EpCAM in physiological processes and diseases(Review).Int JMol Med.2018 Oct;42(4):1771-1785).

[0067] In certain embodiments, the OPCs generated according to the present disclosure are in vitro differentiated progeny of human pluripotent stem cells. In certain embodiments, the OPCs obtained according to the present disclosure are in vitro differentiated progeny of human embryonic stem cells. In other embodiments, the OPCs obtained according to the present disclosure are in vitro differentiated progeny of induced pluripotent stem (iPS) cells.

[0068] One or more characteristics of the resulting OPC population can be determined by quantifying various cell markers using flow cytometry, for example to determine what percentage of the cell population is positive for a particular marker or set of markers, or to identify undesirable cell types present in the OPC population.

[0069] The OPC population obtained according to the present disclosure can comprise about 30% to about 100% OPCs, such as at least about 35%, such as at least about 40%, such as at least about 45%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65%, such as at least about 70%, such as at least about 75%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95%, such as at least about 98%, such as at least about 99%, such as at least about 99.5%, such as at least about 99.8%, or such as at least about 99.9% OPCs. The percentages can be any value or subrange within the recited ranges, including the endpoints. The percentage of OPCs in a population can be determined, for example, by the presence of a positive marker for OPCs and / or the absence of a negative marker for OPCs. Examples of OPC positive markers include, but are not limited to, NG2, PDGFRa, and / or PDGFRβ. For example, further examples of OPC markers, including markers for OPC and other cell types, methods of differentiating cells, methods of using differentiated cells, cell types, reagents, and all other methods, compositions, and disclosures can be found in WO2020 / 154533, which is incorporated by reference herein for all that is disclosed therein.

[0070] The OPC population obtained according to the present disclosure may comprise from about 30% to about 100% NG2 positive cells, such as at least about 35%, such as at least about 40%, such as at least about 45%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65%, such as at least about 70%, such as at least about 75%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95%, such as at least about 98%, such as at least about 99%, such as at least about 99.5%, such as at least about 99.8%, or such as at least about 99.9% NG2 positive cells. In certain embodiments, an OPC population obtained according to the present disclosure can comprise about 45% to about 75% NG2 positive cells, such as about 45% to about 50%, such as about 50% to about 55%, such as about 55% to about 60%, such as about 60% to about 65%, such as about 65% to about 70%, such as about 70% to about 75%, such as about 50% to about 70%, such as about 55% to about 65%, or such as about 58% to about 63% NG2 positive cells. In other embodiments, an OPC population obtained according to the present disclosure can comprise about 60% to about 90% NG2 positive cells, such as about 60% to about 65%, such as about 65% to about 70% positive cells. The percentages can be any value or subrange within the recited ranges, including the endpoints.

[0071] The OPC population obtained according to the present disclosure may comprise from about 30% to about 100% PDGFRa positive cells, such as at least about 35%, such as at least about 40%, such as at least about 45%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65%, such as at least about 70%, such as at least about 75%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95%, such as at least about 98%, such as at least about 99%, such as at least about 99.5%, such as at least about 99.8%, or such as at least about 99.9% PDGFRa positive cells. In certain embodiments, an OPC population obtained according to the present disclosure can comprise about 45% to about 75% PDGFRa positive cells, such as about 45% to about 50%, such as about 50% to about 55%, such as about 55% to about 60%, such as about 60% to about 65%, such as about 65% to about 70%, such as about 70% to about 75%, such as about 50% to about 70%, such as about 55% to about 65%, or such as about 58% to about 63% PDGFRa positive cells. In other embodiments, an OPC population obtained according to the present disclosure can comprise about 60% to about 90% PDGFRa positive cells, such as about 60% to about 65%, such as about 65% to about 70% positive cells. The percentages can be any value or subrange within the recited ranges, including the endpoints.

[0072] The OPC population obtained according to the present disclosure may comprise from about 30% to about 100% PDGFRβ positive cells, such as at least about 35%, such as at least about 40%, such as at least about 45%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65%, such as at least about 70%, such as at least about 75%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95%, such as at least about 98%, such as at least about 99%, such as at least about 99.5%, such as at least about 99.8%, or such as at least about 99.9% PDGFRβ positive cells. In certain embodiments, an OPC population obtained according to the present disclosure can comprise about 45% to about 75% PDGFRβ positive cells, such as about 45% to about 50%, such as about 50% to about 55%, such as about 55% to about 60%, such as about 60% to about 65%, such as about 65% to about 70%, such as about 70% to about 75%, such as about 50% to about 70%, such as about 55% to about 65%, or such as about 58% to about 63% PDGFRβ positive cells. In other embodiments, an OPC population obtained according to the present disclosure can comprise about 60% to about 90% PDGFRβ positive cells, such as about 60% to about 65%, such as about 65% to about 70% positive cells. The percentages can be any value or subrange within the recited ranges, including the endpoints.

[0073] In one embodiment, an OPC population obtained according to the present disclosure may be capable of forming no more than four epithelial cysts per 100,000 cells in a cyst assay, as described in Example 8 of the present disclosure. In another embodiment, an OPC population obtained according to the present disclosure may be capable of forming no more than three epithelial cysts per 100,000 cells in a cyst assay. In another embodiment, an OPC population obtained according to the present disclosure may be capable of forming no more than two epithelial cysts per 100,000 cells in a cyst assay. In yet another embodiment, an OPC population obtained according to the present disclosure may be capable of forming no more than one epithelial cyst per 100,000 cells in a cyst assay, as described in Example 8 of the present disclosure.

[0074] II. Pharmaceutical Preparations The OPC composition according to the present disclosure may further comprise a pharma- ceutically acceptable carrier. In one embodiment, the pharma- ceutically acceptable carrier may comprise dimethyl sulfoxide (DMSO). In one embodiment, the pharma- ceutically acceptable carrier does not comprise dimethyl sulfoxide. As described above, the composition may be further adapted for cryopreservation at -80°C to -195°C or lower. In an embodiment, the composition may be formulated to thaw and may be administered directly to a subject, for example, by injection, without additional manipulation prior to administration. In an embodiment, the composition may be formulated to include a freezing solution, such as CryoStor® 10 (CS10), as a cryopreservation medium. In one embodiment, the composition may be filtered using a 60um filter kit prior to cryopreservation.

[0075] The OPC composition according to the present disclosure may be formulated for administration via direct injection into the spinal cord of a subject. In one embodiment, the OPC composition according to the present disclosure may be formulated for intracerebral, intraventricular, intrathecal, intranasal, or intracisternal administration to a subject. In one embodiment, the OPC composition according to the present disclosure may be formulated for administration via injection directly into or directly adjacent to an infarct cavity in the brain of a subject. In one embodiment, the composition according to the present disclosure may be formulated for administration by implantation. In one embodiment, the composition according to the present disclosure may be formulated as a solution.

[0076] OPC compositions according to the present disclosure have a concentration of about 1×10 per milliliter 6 ~About 200×10 6 cells per milliliter, e.g., approximately 1 x 10 6 cells per milliliter, e.g., approximately 2 x 10 6 cells per milliliter, e.g., about 3 x 10 6 cells per milliliter, e.g., about 4 x 10 6 cells per milliliter, e.g., about 5 x 10 6 cells per milliliter, e.g., about 6 x 10 6 cells, e.g., about 7 x 10 per milliliter 6 cells per milliliter, e.g., about 8 x 10 6 cells per milliliter, e.g., about 9 x 10 6 cells per milliliter, e.g., about 10 x 10 6 cells per milliliter, e.g., about 20 x 10 6 cells per milliliter, e.g., about 30 x 10 6 cells per milliliter, e.g., about 40 x 10 6 cells per milliliter, e.g., about 50 x 10 6 cells per milliliter, e.g., about 60 x 6 cells per milliliter, e.g., about 70 x 10 6 cells per milliliter, e.g., about 80 x 10 6cells per milliliter, e.g., about 90 x 10 6 cells per milliliter, e.g., about 100 x 10 6 cells per milliliter, e.g., about 101 x 10 6 cells per milliliter, e.g., about 102 x 10 6 cells per milliliter, e.g., about 103 x 10 6 cells per milliliter, e.g., about 104 x 10 6 cells per milliliter, e.g., about 10 x 10 6 cells per milliliter, e.g., about 10 x 10 6 cells per milliliter, e.g., about 107 x 10 6 cells per milliliter, e.g., about 10 x 10 6 cells per milliliter, e.g., about 109 x 10 6 cells per milliliter, e.g., about 110 x 10 6 cells per milliliter, e.g., about 120 x 10 6 cells per milliliter, e.g., about 130 x 10 6 cells per milliliter, e.g., about 140 x 10 6 cells per milliliter, e.g., about 150 x 10 6 cells per milliliter, e.g., about 160 x 10 6 cells per milliliter, e.g., about 170 x 10 6 cells per milliliter, e.g., about 180 x 10 6 cells per milliliter, e.g., about 190 x 10 6 cells per milliliter, e.g., about 200 x 10 6 The number of cells can be any value or subrange within the recited range.

[0077] In yet another embodiment, an OPC composition according to the present disclosure can have a volume in the range of about 250 microliters to about 1 milliliter, such as about 300 microliters, such as about 400 microliters, such as about 500 microliters, such as about 600 microliters, such as about 700 microliters, such as about 800 microliters, such as about 900 microliters, or such as about 1 milliliter. In one embodiment, a composition according to the present disclosure can have a volume in the range of about 250 microliters to about 1 milliliter, such as about 250 microliters to about 900 microliters, such as about 300 microliters to about 800 microliters, such as about 400 microliters to about 700 microliters, or such as about 500 microliters to about 600 microliters. In another embodiment, a composition according to the present disclosure can have a volume ranging from about 100 microliters to about 1 milliliter, e.g., from about 200 microliters to about 900 microliters, e.g., from about 300 microliters to about 800 microliters, e.g., from about 400 microliters to about 700 microliters, or e.g., from about 500 microliters to about 600 microliters. The volume can be any value or subrange within the recited range. In one embodiment, an OPC composition according to the present disclosure can be in a container configured for cryopreservation or administration to a subject in need thereof. In one embodiment, the container can be a prefilled syringe.

[0078] In one embodiment, an OPC composition according to the present disclosure may be administered in a volume ranging from about 50 microliters to about 100 microliters, such as about 55 microliters, such as about 60 microliters, such as about 65 microliters, such as about 70 microliters, such as about 75 microliters, such as about 80 microliters, such as about 85 microliters, such as about 90 microliters, such as about 95 microliters, or such as about 100 microliters. In one embodiment, an OPC composition according to the present disclosure may be administered in a volume ranging from about 50 microliters to about 100 microliters, such as about 55 microliters to about 95 microliters, such as about 60 microliters to about 90 microliters, such as about 65 microliters to about 85 microliters, or such as about 70 microliters to about 80 microliters. The volume may be any value or subrange within the recited range.

[0079] In one embodiment, an OPC composition according to the present disclosure comprises about 5.0×10 6 ~ approx. 10.0 x 10 cells 6 A volume in the range of 5.5 x 10 cells, e.g. 6 cells, e.g., about 6.0 x 10 6 cells, e.g., about 6.5 x 10 6 cells, e.g., about 7.0 x 10 6 cells, e.g., about 7.5 x 10 6 cells, e.g., about 8.0 x 10 6 cells, e.g., about 8.5 x 10 6 cells, e.g., about 9.0 x 10 6 cells, e.g., about 9.5 x 10 6 cells, or for example, about 10.0×10 6 In one embodiment, the OPC composition according to the present disclosure may be administered in an amount of about 5.0×10 6 ~ approx. 10.0 x 10 cells 6 cells, e.g., about 5.5 x 10 6 ~ approx. 9.5 x 10 cells 6 cells, e.g., about 6.0 x 10 6 ~ approx. 9.0 x 10 cells 6cells, e.g., about 6.5 x 10 6 ~ approx. 8.5 x 10 cells 6 cells, or for example, about 7.0×10 6 ~ approx. 8.0 x 10 cells 6 The number of cells can be any value or subrange within the recited range.

[0080] III.How to use The OPC compositions obtained according to the present disclosure may be used in cell therapy to improve one or more neurological functions in a subject in need of treatment. In one embodiment, the OPC cell population according to the present disclosure may be injected or implanted into a subject in need thereof. In one embodiment, the cell population according to the present disclosure may be implanted into a subject in need thereof for the treatment of spinal cord injury, stroke, or multiple sclerosis.

[0081] In one embodiment, the cell population according to the present disclosure may be capable of inducing myelination of denuded axons at an implantation site in a subject. In one embodiment, the cell population generated according to the method of the present disclosure may exhibit improved engraftment and migration capabilities. In one embodiment, the cell population generated according to the method of the present disclosure may be capable of improving post-injury repair or regeneration of neural tissue in a subject.

[0082] The cell population according to the present disclosure may be capable of improving sensory function in a subject in need of treatment after implantation of the population into the subject. Improved sensory function may be assessed using the International Standard for Neurological Classification of Spinal Cord Injury (ISNCSCI) test, such as determining right and left sensory levels for pin prick and light touch. The cell population according to the present disclosure may be capable of improving motor function in a subject in need of treatment after implantation of the population into the subject. Improved motor function may be assessed using the ISNCSCI test, such as determining right and left motor levels for complete paralysis, palpable or visible contraction, active movement, full range of motion against gravity, and sufficient resistance.

[0083] The cell population according to the present disclosure may be capable of reducing the volume of injury-induced central nervous system parenchymal cavitation within 12 months. In one embodiment, the cell population according to the present disclosure may be capable of reducing the volume of injury-induced central nervous system parenchymal cavitation in a subject in 6 months or less, 5 months or less, 4 months or less, or 3 months or less, or 2 months or less, or less than 1 month.

[0084] Use in treating CNS trauma In certain embodiments, the cell populations according to the present disclosure may be capable of engrafting at the site of spinal cord injury following implantation of a composition comprising the cell population at the site of spinal cord injury.

[0085] In certain embodiments, the cell population according to the present disclosure can remain within the spinal cord injury site of the subject for a period of about 90 days or more after implantation of a dose of the composition at the spinal cord injury site. In other embodiments, the cell population according to the present disclosure can remain within the spinal cord injury site of the subject for a period of about 1 year or more after implantation of a dose of the composition at the spinal cord injury site. In further embodiments, the cell population according to the present disclosure can remain within the spinal cord injury site of the subject for a period of about 2 years or more after implantation of a dose of the composition at the spinal cord injury site. In further embodiments, the cell population according to the present disclosure can remain within the spinal cord injury site of the subject for a period of about 3 years or more after implantation of a dose of the composition at the spinal cord injury site. In still further embodiments, the cell population according to the present disclosure can remain within the spinal cord injury site of the subject for a period of about 4 years or more after implantation of a dose of the composition at the spinal cord injury site. In still further embodiments, the cell population according to the present disclosure can remain within the spinal cord injury site of the subject for a period of about 5 years or more after implantation of a dose of the composition at the spinal cord injury site.

[0086] In certain embodiments, the cell composition according to the present disclosure can improve upper limb motor function in a human subject having a spinal cord injury when administered to the subject. In certain embodiments, the subject has a cervical spinal cord injury. In other embodiments, the subject has a thoracic spinal cord injury.

[0087] In one embodiment, the present disclosure provides a method of improving upper limb motor function in a human subject with spinal cord injury, comprising administering to the subject a composition comprising a population of allogeneic human oligodendrocytes capable of engrafting at the site of spinal cord injury. In certain embodiments, administering the composition comprises injecting the composition at the site of spinal cord injury. In some embodiments, the composition is injected about 2-10 mm posterior to the site of spinal cord injury. In further embodiments, the composition is injected about 5 mm posterior to the site of spinal cord injury. In some embodiments, the subject has a cervical spinal cord injury. In other embodiments, the subject has a thoracic spinal cord injury.

[0088] In certain embodiments, a subject to whom a composition comprising an allogeneic human oligodendrocyte population is administered according to the methods of the present disclosure obtains an improvement in upper limb motor function equivalent to at least one motor level (as defined according to the International Standard for Neurological Classification of Spinal Cord Injury [ISNCSCI]). The improvement in function can be unilateral or bilateral. In other embodiments, a subject to whom a composition comprising an allogeneic human oligodendrocyte population is administered according to the methods of the present disclosure obtains an improvement in upper limb motor function equivalent to at least two motor levels, either unilaterally or bilaterally. In certain embodiments, a subject obtains an improvement in upper limb motor function equivalent to at least one motor level on one side and at least two motor levels on the other side. In certain embodiments, a subject exhibits an improved upper limb motor score (UEMS) compared to the subject's baseline score prior to administration of an allogeneic human oligodendrocyte population according to the methods of the present disclosure.

[0089] In certain embodiments, cell compositions according to the present disclosure are formulated for delivery or administration to the spinal parenchyma, or adjacent to or via peripheral perfusion at the site of injury beneath the dura, arachnoid, or pia mater.

[0090] IV. Proliferation and culture of undifferentiated pluripotent stem cells Differentiation of pluripotent stem cells according to the present disclosure may be performed using any suitable pluripotent stem cell as the starting material. In one embodiment, the method may be performed on a human embryonic stem cell (hESC) line. In another embodiment, the method may be performed using induced pluripotent stem cells (iPSCs). In another embodiment, the method may be performed using cells derived from H1, H7, H9, H13, or H14 cell lines. In another embodiment, the method may be performed on a primate pluripotent stem (pPS) cell line. In yet another embodiment, the method may be performed using undifferentiated stem cells derived from a parthenogenetic organism, an embryo that has been stimulated to produce hESCs without fertilization.

[0091] Methods for expanding and culturing undifferentiated pluripotent stem cells have been previously described. With regard to tissue and cell culture of pluripotent stem cells, the reader may wish to refer to any of the numerous publications available in the art, such as Teratocarcinomas and Embryonic Stem cells: A Practical Approach (E. J. Robertson, Ed., IRL Press Ltd. 1987), Guide to Techniques in Mouse Development (P. M. Wasserman et al., Eds., Academic Press 1993), Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993), Properties and Uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (P. D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998, and R. I. Freshney, Culture of Animal Cells, Wiley-Liss, New York, 2000). each of which is incorporated herein by reference in its entirety.

[0092] Undifferentiated pluripotent stem cells can be maintained in an undifferentiated state without added feeder cells (see, e.g., (2004) Rosler et al., Dev. Dynam. 229:259). Feeder-free cultures are typically supported by a nutrient medium containing factors that promote the proliferation of cells without differentiation (see, e.g., U.S. Pat. No. 6,800,480). In one embodiment, a conditioned medium containing such factors can be used. 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 (4,000 Rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells (U.S. Pat. No. 6,642,048). The medium can be conditioned by plating feeders in a serum-free medium such as knockout DMEM supplemented with 20% serum replacement and 4 ng / mL bFGF. The 1-2 day conditioned medium can be supplemented with additional bFGF and used to support pPS cell cultures for 1-2 days (see, e.g., WO01 / 51616, Xu et al., (2001) Nat. Biotechnol. 19:971).

[0093] Alternatively, fresh or unconditioned media can be used that are supplemented with additional factors (such as fibroblast growth factor or forskolin) that promote the proliferation of cells in an undifferentiated form. Non-limiting examples include basal media such as X-VIVO™ 10 (Lonza, Walkersville, MD.), or QBSF™-60 (Quality Biological Inc. Gaithersburg, MD.), or mTeSR1, mTeSR Plus, or NutriSTem (Biological Industries, Cromwell, CT) 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 in other systems (see, e.g., WO03 / 020920). In one embodiment, undifferentiated pluripotent cells, such as hES cells, can be cultured in a medium containing bFGF and TGFβ. Non-limiting example concentrations of bFGF include about 80 ng / ml. Non-limiting example concentrations of TGFβ include about 0.5 ng / ml. In yet another embodiment, undifferentiated pluripotent stem cells can be cultured in a medium containing mTeSk rm The cells can be maintained in commercially available complete medium, such as that available from Stem Cell Technologies, Vancouver, Canada.

[0094] Undifferentiated pluripotent cells can be cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue (Thomson et al. (1998) Science 282:1145). Feeder cells can be from human or mouse sources. Human feeder cells can be isolated from various human tissues or derived via differentiation of human embryonic stem cells into fibroblasts (see, e.g., WO01 / 51616). Human feeder cells that may be used include, but are not limited to, placental fibroblasts (see, e.g., Genbacev et al. (2005) Fertil. Steril. 83(5):1517), fallopian tube epithelial cells (see, e.g., Richards et al. (2002) Nat. Biotechnol., 20:933), foreskin fibroblasts (see, e.g., Amit et al. (2003) Biol. Reprod. 68:2150), and endometrial cells (see, e.g., Lee et al. (2005) Biol. Reprod. 72(1):42).

[0095] A variety of solid surfaces can be used for culturing undifferentiated pluripotent cells. These solid surfaces include, but are not limited to, standard commercially available tissue culture flasks or cell culture plates, such as 6-well, 24-well, 96-well, or 144-well plates. Other solid surfaces include, but are not limited to, microcarriers and disks. Solid surfaces suitable for the growth of undifferentiated pluripotent cells can be made from a variety of materials, including, but not limited to, glass or plastic, such as polystyrene, polyvinyl chloride, polycarbonate, polytetrafluoroethylene, Melinex, Thermanox, or combinations thereof. Suitable surfaces can include, for example, one or more polymers, such as one or more acrylates. The solid surface can be three-dimensional in shape. Non-limiting examples of three-dimensional solid surfaces have been previously described, for example, in U.S. Patent Publication No. 2005 / 0031598.

[0096] Undifferentiated stem cells can also be grown under feeder-free conditions on a growth substrate. The growth substrate can be Matrigel® matrix (e.g., Matrigel®, Matrigel® GFR), recombinant laminin, laminin-511 recombinant fragment E8, or vitronectin. In certain embodiments of the present disclosure, the growth substrate is recombinant human laminin-521 (Biolamina, Sweden, distributed by Corning Inc., Corning, NY). In other embodiments, the substrate is a synthetic substrate, such as, for example, Synthemax®-II SC substrate.

[0097] Undifferentiated stem cells can be passaged or subcultured using various methods, such as using collagenase, or manual detachment. Undifferentiated stem cells can be subcultured by enzymatic means that generate a single cell suspension, such as using Accutase® (distributed by Sigma Aldrich, MO) or similar trypsinase. Alternatively, undifferentiated stem cells can be subcultured using non-enzymatic means, such as 0.5 mM EDTA in PBS, or ReLeSR (Stem Cell Technologies, Vancouver, Canada).

[0098] In one embodiment, the plurality of undifferentiated stem cells are seeded or subcultured at a seeding density that allows the cells to reach confluence in about 3 to about 10 days. In one embodiment, the seeding density is about 6.0×10 of the growth surface. 3 Cells / cm 2 ~Approx. 5.0×10 5 Cells / cm 2 range, for example, about 1.0×10 4 Cells / cm 2 , for example, about 5.0 x 10 4 Cells / cm 2 , for example, about 1.0 x 10 5 Cells / cm 2 , or for example, about 3.0×10 5 Cells / cm 2In another embodiment, the seeding density is about 6.0×10 of the growth surface. 3 Cells / cm 2 ~Approx. 1.0×10 4 Cells / cm 2 for example, about 6.0 x 10 of the growth surface 3 Cells / cm 2 ~Approx. 9.0×10 3 Cells / cm 2 , for example, about 7.0 x 10 3 Cells / cm 2 ~Approx. 1.0×10 4 Cells / cm 2 , for example, about 7.0 x 10 3 Cells / cm 2 ~Approx. 9.0×10 3 Cells / cm 2 , or, for example, about 7.0×10 3 Cells / cm 2 ~Approx. 8.0×10 3 Cells / cm 2 In yet another embodiment, the seeding density is about 1.0×10 of the growth surface. 4 Cells / cm 2 ~Approx. 1.0×10 5 Cells / cm 2 for example, about 2.0 x 10 of the growth surface 4 Cells / cm 2 ~Approx. 9.0×10 4 Cells / cm 2 , for example, about 3.0 x 10 4 Cells / cm 2 ~Approx. 8.0×10 4 Cells / cm 2 , for example, about 4.0 x 10 4 Cells / cm 2 ~Approx. 7.0×10 4 Cells / cm 2 , or for example, about 5.0×10 4 Cells / cm 2 ~Approx. 6.0×10 4 Cells / cm 2 In one embodiment, the seeding density is about 1.0×10 of the growth surface. 5 Cells / cm 2~Approx. 5.0×10 5 Cells / cm 2 for example, about 1.0×10 of the growth surface 5 Cells / cm 2 ~Approx. 4.5×10 5 Cells / cm 2 , for example, about 1.5 x 10 5 Cells / cm 2 ~Approx. 4.0×10 5 Cells / cm 2 , for example, about 2.0 x 10 5 Cells / cm 2 ~Approx. 3.5×10 5 Cells / cm 2 , or, for example, about 2.5×10 5 Cells / cm 2 ~Approx. 3.0×10 5 Cells / cm 2 The seeding density can be any value or subrange within the recited range.

[0099] Any of a variety of suitable cell culture and subculture techniques can be used to culture stem cells according to the methods of the present disclosure. For example, the culture medium can be changed completely every day, beginning about 2 days after subculture of the cells. In one embodiment, once the culture reaches about 90% colony coverage, the cells can be dissociated using one or more suitable reagents, such as, for example, Accutase®, to achieve a single cell suspension for quantification and seeded for subsequent culture. In one embodiment, the undifferentiated stem cells can then be subcultured at a seeding density that allows the cells to reach confluence in a suitable period of time, for example, about 3-10 days, before seeding the cells onto a suitable growth substrate (e.g., recombinant human laminin-521). In one embodiment, the undifferentiated stem cells can be subcultured using collagenase IV and grown on recombinant laminin. In another embodiment, the 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.

[0100] To seed undifferentiated stem cells, the seeding density is approximately 6.0 x 10 cells per growth surface. 3 Cells / cm 2 ~Approx. 5.0×10 5 Cells / cm 2 range, for example, about 1.0×10 4 Cells / cm 2 , for example, about 5.0 x 10 4 Cells / cm 2 , for example, about 1.0 x 10 5 Cells / cm 2 , or for example, about 3.0×10 5 Cells / cm 2 In another embodiment, the seeding density is about 6.0×10 of the growth surface. 3 Cells / cm 2 ~Approx. 1.0×10 4 Cells / cm 2 for example, about 6.0 x 10 of the growth surface 3Cells / cm 2 ~Approx. 9.0×10 3 Cells / cm 2 , for example, about 7.0 x 10 3 Cells / cm 2 ~Approx. 1.0×10 4 Cells / cm 2 , for example, about 7.0 x 10 3 Cells / cm 2 ~Approx. 9.0×10 3 Cells / cm 2 , or, for example, about 7.0×10 3 Cells / cm 2 ~Approx. 8.0×10 3 Cells / cm 2 In yet another embodiment, the seeding density is about 1.0×10 of the growth surface. 4 Cells / cm 2 ~Approx. 1.0×10 5 Cells / cm 2 for example, about 2.0 x 10 of the growth surface 4 Cells / cm 2 ~Approx. 9.0×10 4 Cells / cm 2 , for example, about 3.0 x 10 4 Cells / cm 2 ~Approx. 8.0×10 4 Cells / cm 2 , for example, about 4.0 x 10 4 Cells / cm 2 ~Approx. 7.0×10 4 Cells / cm 2 , or for example, about 5.0×10 4 Cells / cm 2 ~Approx. 6.0×10 4 Cells / cm 2 In one embodiment, the seeding density is about 1.0×10 of the growth surface. 5 Cells / cm 2 ~Approx. 5.0×10 5 Cells / cm 2 for example, about 1.0×10 of the growth surface 5 Cells / cm 2 ~Approx. 4.5×10 5 Cells / cm 2 , for example, about 1.5 x 105 Cells / cm 2 ~Approx. 4.0×10 5 Cells / cm 2 , for example, about 2.0 x 10 5 Cells / cm 2 ~Approx. 3.5×10 5 Cells / cm 2 , or, for example, about 2.5×10 5 Cells / cm 2 ~Approx. 3.0×10 5 Cells / cm 2 The seeding density can be any value or subrange within the recited range.

[0101] V. Differentiation of human pluripotent stem cells into dorsal neuroectodermal progenitor cells and further into dorsal-derived glial and oligodendrocyte progenitor cells The present disclosure provides a method for differentiating human pluripotent stem cells into neuroectoderm with a dorsal spinal cord phenotype, as well as into glial and oligodendrocyte precursor cells, using a combination of small molecule and protein modulators of BMP signaling and inhibitors of MAPK / ERK kinases. Without being bound to any particular theory, the inventors have discovered that human dorsal neuroectoderm precursor cells obtained according to the methods of the present disclosure can be easily and efficiently differentiated into spinal cord OPCs in the absence of activation of SHH signaling. Surprisingly, this early dorsal phenotype gives rise to glial precursor cells by day 21 of the differentiation process and to OPCs by day 28 of the differentiation process, even though this is not the initial OPC generating region in vivo. Day 28 OPCs generated according to the present disclosure express the canonical OPC markers NG2 and PDGFRa and are comparable (in terms of their overall marker expression profile) to OPCs generated using alternative methods currently in clinical trials to treat spinal cord injury.

[0102] In one embodiment, the method includes contacting human pluripotent stem cells with one or more inhibitors of mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) in combination with one or more inhibitors of bone morphogenetic protein (BMP) signaling. In certain embodiments, the MAPK / ERK inhibitor is a small molecule. In other embodiments, the MAPK / ERK inhibitor is a protein, such as a phosphatase, that dephosphorylates MAPK / ERK kinase. In certain embodiments, the inhibitor of BMP signaling is a small molecule. In other embodiments, the inhibitor of BMP signaling is a protein. In some embodiments, the direct target of the inhibitor of BMP signaling is ALK2, also known as activin A receptor type I (ACVR1). In certain embodiments, following MAPK / ERK and BMP signaling inhibition, the cells are cultured in the presence of the caudalizing agent retinoic acid, thereby obtaining neuroectodermal restricted progenitor cells with a dorsal spinal cord phenotype.

[0103] In certain embodiments, the inhibitor of MAPK / ERK can be selected from the group consisting of PD0325901, AZD6244, GSK1120212, PD184352, and cobimetinib, and derivatives thereof. In certain embodiments, the inhibitor of BMP signaling can be selected from the group consisting of dorsomorphin, DMH-1, K02288, ML347, LDN193189, and noggin protein.

[0104] In one embodiment, the method comprises obtaining undifferentiated human pluripotent stem cells that remain in an undifferentiated state, adherently culturing the undifferentiated human pluripotent stem cells in the presence of small molecules PD0325901 and dorsomorphin and retinoic acid for a first period of time, and then adherently culturing the cells in the presence of retinoic acid and in the absence of an SHH signaling activator for a second period of time, thereby obtaining dorsal neuroectodermal cells. In one embodiment, the first period of time and the second period of time can each be in the range of about 1 day to about 6 days, such as about 1 day, such as about 2 days, such as about 3 days, such as about 4 days, such as about 5 days, or such as about 6 days.

[0105] In one embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of PD0325901 at a concentration in the range of about 1 μM to about 100 μM, such as about 2 μM, such as about 3 μM, such as about 4 μM, such as about 5 μM, such as about 6 μM, such as about 7 μM, such as about 8 μM, such as about 9 μM, such as about 10 μM, such as about 11 μM, such as about 12 μM, such as about 13 μM, such as about 14 μM, such as about 15 μM, such as about 20 μM, such as about 30 μM, such as about 40 μM, such as about 50 μM, or such as about 60 μM, 70 μM, 80 μM or 90 μM. In another embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of PD0325901 at a concentration of about 10 μM. The concentration can be any value or subrange within the recited range.

[0106] In one embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of AZD6244 at a concentration in the range of about 1 μM to about 100 μM, such as about 2 μM, such as about 3 μM, such as about 4 μM, such as about 5 μM, such as about 6 μM, such as about 7 μM, such as about 8 μM, such as about 9 μM, such as about 10 μM, such as about 11 μM, such as about 12 μM, such as about 13 μM, such as about 14 μM, such as about 15 μM, such as about 20 μM, such as about 30 μM, such as about 40 μM, such as about 50 μM, or such as about 60 μM, 70 μM, 80 μM or 90 μM. In another embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of AZD6244 at a concentration of about 10 μM. The concentration can be any value or subrange within the recited range.

[0107] In one embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of GSK1120212 at a concentration of PD0325901 in the range of about 1 μM to about 100 μM, such as about 2 μM, for example about 3 μM, such as about 4 μM, for example about 5 μM, such as about 6 μM, for example about 7 μM, for example about 8 μM, for example about 9 μM, such as about 10 μM, for example about 11 μM, such as about 12 μM, for example about 13 μM, such as about 14 μM, for example about 15 μM, such as about 20 μM, for example about 30 μM, such as about 40 μM, for example about 50 μM, or for example about 60 μM, 70 μM, 80 μM or 90 μM. In another embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of GSK1120212 at a concentration of about 10 μM. The concentration can be any value or subrange within the recited range.

[0108] In one embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of PD184352 at a concentration in the range of about 1 μM to about 100 μM, such as about 2 μM, such as about 3 μM, such as about 4 μM, such as about 5 μM, such as about 6 μM, such as about 7 μM, such as about 8 μM, such as about 9 μM, such as about 10 μM, such as about 11 μM, such as about 12 μM, such as about 13 μM, such as about 14 μM, such as about 15 μM, such as about 20 μM, such as about 30 μM, such as about 40 μM, such as about 50 μM, or such as about 60 μM, 70 μM, 80 μM or 90 μM. In another embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of PD184352 at a concentration of about 10 μM. The concentration can be any value or subrange within the recited range.

[0109] In one embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of cobimetinib at a concentration in the range of about 1 μM to about 100 μM, such as about 2 μM, such as about 3 μM, such as about 4 μM, such as about 5 μM, such as about 6 μM, such as about 7 μM, such as about 8 μM, such as about 9 μM, such as about 10 μM, such as about 11 μM, such as about 12 μM, such as about 13 μM, such as about 14 μM, such as about 15 μM, such as about 20 μM, such as about 30 μM, such as about 40 μM, such as about 50 μM, or such as about 60 μM, 70 μM, 80 μM, or 90 μM. In another embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of cobimetinib at a concentration of about 10 μM. The concentration can be any value or subrange within the recited range.

[0110] In one embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of dorsomorphin at a concentration in the range of about 0.2 μM to about 20 μM, such as about 0.5 μM, for example about 0.8 μM, such as about 1 μM, for example about 1.5 μM, such as about 2 μM, for example about 2.5 μM, for example about 3 μM, such as about 3.5 μM, for example about 4 μM, such as about 4.5 μM, for example about 5 μM, such as about 5.5 μM, for example about 6 μM, such as about 6.5 μM, for example about 7 μM, such as about 7.5 μM, for example about 8 μM, such as about 8.5 μM, for example about 9 μM, such as about 10 μM, for example about 11 μM, such as about 12 μM, for example about 13 μM, such as about 14 μM, for example about 15 μM, for example about 16 μM, such as about 17 μM, for example about 18 μM, or for example about 19 μM. In another embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of dorsomorphin at a concentration in the range of about 0.2 μM to about 1 μM, such as about 0.2 μM to about 0.9 μM, such as about 0.3 μM to about 0.8 μM, such as about 0.4 μM to about 0.7 μM, or for example about 0.5 μM to about 0.6 μM. In yet another embodiment, the method comprises 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, such as about 1 μM to about 9 μM, such as about 2 μM to about 8 μM, such as about 3 μM to about 7 μM, or for example about 4 μM to about 6 μM. In one embodiment, the method comprises 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, such as about 10 μM to about 19 μM, such as about 12 μM to about 18 μM, such as about 13 μM to about 17 μM, or such as about 14 μM to about 16 μM. In one embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of dorsomorphin at a concentration of about 2 μM. The concentration can be any value or subrange within the recited range.

[0111] In one embodiment, the method comprises 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, e.g., about 10 nM, about 50 nM, about 100 nM, about 150 nM, about 200 nM, about 500 nM, about 1 μM, about 5 μM, about 10 μM, or about 15 μM. The concentration can be any value or subrange within the recited range.

[0112] In one embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of DMH-1 at a concentration ranging from about 1 μM to about 10 μM. In one embodiment, the method comprises culturing undifferentiated human pluripotent stem cells in the presence of DMH-1 at about 2 μM. The concentration can be any value or subrange within the recited range.

[0113] In one embodiment, the method comprises 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 comprises culturing undifferentiated human pluripotent stem cells in the presence of K02288 at about 2 μM. The concentration can be any value or subrange within the recited range.

[0114] In one embodiment, the method comprises 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 comprises culturing undifferentiated human pluripotent stem cells in the presence of ML347 at about 2 μM. The concentration can be any value or subrange within the recited range.

[0115] In one embodiment, the method comprises 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 comprises culturing undifferentiated human pluripotent stem cells in the presence of LDN193189 at about 2 μM. The concentration can be any value or subrange within the recited range.

[0116] Any tissue culture vessel suitable for adherent cell culture can be used to obtain dorsal neuroectodermal progenitor cells according to the present disclosure. Suitable growth substrates include, for example, recombinant laminin, laminin-511 recombinant fragment E8 or Matrigel® matrix (e.g., Matrigel®, Matrigel® GFR), dorsomorphin, PD0325901, retinoic acid (RA), ascorbic acid (AA), recombinant human EGF (rhEGF), heat stable (recombinant human basic FGF (rhbFGF), ROCK inhibitor, rhLN521 or PDGF-A.

[0117] In one embodiment, dorsal neuroectodermal progenitor cells obtained according to the present disclosure may be further harvested and cultured as aggregates in suspension culture in the presence of thermostable rhbFGF and EGF until the cells mature into glial progenitor cells. In one embodiment, the further culture period may be in the range of about 5 to 15 days, such as about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days. In one embodiment, the further culture period is about 7 days.

[0118] In one embodiment, the adherent cultured dorsal neuroectodermal progenitor cells can be harvested by enzymatic means, including but not limited to TrypLE™ Select (Thermo Fisher Scientific, Waltham, MA), Accutase® (Sigma Aldrich, MO) or similar trypsinases. Alternatively, the adherent cultured dorsal neuroectodermal progenitor cells can be harvested using non-enzymatic means, including but not limited to using 0.5 mM EDTA in PBS, or ReLeSR™ (Stem Cell Technologies, Vancouver, Canada).

[0119] Any cell culture vessel or reactor suitable for suspension culture can be used for the non-adherent culture step contemplated in this disclosure. The vessel walls are typically inert or resistant to adhesion of cultured cells. In the case of dynamic suspension, there is also a means to prevent cells from settling, such as a stirring mechanism such as a magnetically or mechanically driven stirring bar or paddle, a vibration mechanism (typically attached to the vessel by the outside), or an inversion mechanism (i.e., a device that rotates the vessel to change the direction of gravity on the cells).

[0120] Suitable vessels for suspension culture for process development include the usual range of commercially available spinners, spinner flasks, rocker bags, or shaker flasks. An example of a bioreactor suitable for commercial production is the VerticalWheel™ bioreactor (PBS Biotech, Camarillo, Calif.).

[0121] Aggregates can also be formed prior to dynamic suspension. For example, cells can be placed on AggreWell™ plates to generate uniform cell aggregates. After about 3 days, the cell aggregates can be transferred to dynamic suspension.

[0122] In one embodiment, glial progenitor cells obtained according to the present disclosure may be harvested, plated down, and adherently cultured for an additional period in the presence of epidermal growth factor (EGF) until the cells mature into oligodendrocyte progenitor cells. In certain embodiments, the culture medium further comprises platelet-derived growth factor AA (PDGF-AA). In one embodiment, the additional culture period may be in the range of about 7 to 14 days, such as about 7 days, about 10 days, about 12 days, or about 14 days. In one embodiment, the additional culture period may be in the range of about 14 to 25 days, such as 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. The culture period may be any value or subrange within the recited range, including the endpoint.

[0123] VI. Undesirable Cell Types OPC populations obtained according to the present disclosure contain low levels of undesired cell types, as measured by quantification of markers associated with undesired cell types, for example, by flow cytometry. In a non-limiting example, day 35 OPCs obtained according to the present disclosure may contain low levels of cells expressing epithelial cell associated markers cytokeratin 7 (CK7), EpCAM, CD49f, and CLDN6. In another non-limiting example, day 42 OPCs obtained according to the present disclosure may contain low levels of cells expressing epithelial cell associated markers ck7, EpCAM, CD49f, and CLDN6, without hESC remnants associated with markers TRA-1-60 and OCT4.

[0124] Markers associated with an undesired cell type may include less than about 20% of the undesired cell type, such as less than about 19%, such as less than about 18%, for example less than about 17%, such as less than about 16%, for example less than about 15%, such as less than about 14%, for example less than about 13%, such as less than about 12%, for example less than about 11%, such as less than about 10%, for example less than about 9%, such as less than about 8%, for example less than about 7%, such as less than about 6%, for example less than about 5%, such as less than about 4%, for example less than about 3%, such as less than about 2%, for example less than about 1%, such as less than about 0.5%, for example less than about 0.1%, such as less than about 0.05%, or such as less than about 0.01% of the undesired cell type. In another embodiment, the cell population may comprise about 15% to about 20% of an undesired cell type, such as about 19% to about 20%, for example about 18% to about 20%, for example about 17% to about 20%, for example about 16% to about 20%, for example about 15% to about 19%, or for example about 16% to about 18% of an undesired cell type. In yet another embodiment, the cell population may comprise about 10% to about 15% of an undesired cell type, such as about 14% to about 15%, for example about 13% to about 15%, for example about 12% to about 15%, for example about 11% to about 15%, or for example about 12% to about 14% of an undesired cell type. In one embodiment, the cell population may contain about 1% to about 10% of an undesired cell type, such as about 2% to about 10%, for example about 1% to about 9%, for example about 2% to about 8%, for example about 3% to about 7%, or about 4% to about 6% of an undesired cell type. In one embodiment, the cell population may contain about 0.1% to about 1% of an undesired cell type, such as about 0.2% to about 1%, for example about 0.1% to about 0.9%, for example about 0.2% to about 0.8%, for example about 0.3% to about 0.7%, or about 0.4% to about 0.6% of an undesired cell type. In one embodiment, the cell population may contain about 0.01% to about 0.1% of an undesired cell type, such as about 0.02% to about 0.1%, such as about 0.01% to about 0.09%, such as about 0.02% to about 0.08%, such as about 0.03% to about 0.07%, or about 0.04% to about 0.06% of an undesired cell type. In one embodiment, a low level of an undesired cell type may refer to the presence of less than about 15% of an undesired cell type.

[0125] In one embodiment, the undesirable cell type may include cells expressing one or more markers selected from ck7, CD49f, CLDN6, or EpCAM.

[0126] VII. Cryopreservation After harvesting, the expanded population of OPCs can be formulated at a specific therapeutic dose (e.g., cell number) and cryopreserved for transport to the clinic. Ready-to-administer (RTA) OPC therapy compositions can then be administered directly after thawing without further processing. Examples of media suitable for cryopreservation include, but are not limited to, 90% human serum / 10% DMSO, medium 3 10% (CS10), medium 2 5% (CS5) and medium 1 2% (CS2), Stem Cell Banker, PRIME XV® FREEZIS, HYPOTHERMASOL®, CSB, trehalose, and the like.

[0127] In some embodiments, the percent viability of cells after filtration stored 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 other embodiments, the percent recovery of cells after filtration stored 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%. Viability can be any value or subrange within the recited range.

[0128] In further embodiments, the percent viability of cells after filtration 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 other embodiments, the percent recovery of cells after filtration 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%. Viability can be any value or subrange within the recited range.

[0129] In yet other embodiments, the percent viability of cells after filtration, stored in neutralizing medium for about 0 to about 8 hours after thawing of the cryopreserved composition, 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 yet other embodiments, the percent recovery of cells after filtration, stored in neutralizing medium for about 0 to about 8 hours after thawing of the cryopreserved composition, 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%. Viability can be any value or subrange within the recited range.

[0130] In some embodiments, filtered OPCs that have been stored in neutralizing medium for about 0 to about 8 hours after thawing of the cryopreserved composition and subsequently stored in cryopreservation medium for about 0 to about 8 hours are capable of secreting decorin. In other embodiments, filtered OPCs that have been stored in neutralizing medium for about 0 to about 8 hours after thawing of the cryopreserved composition and subsequently stored in cryopreservation medium for about 0 to about 8 hours are capable of proliferating.

[0131] In some embodiments, the percent viability of filtered OPCs stored in neutralizing medium at room temperature 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%. In some embodiments, the percent viability of filtered OPCs stored in cryopreservation medium at room temperature 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%. In further embodiments, the percent viability of cells after filtration stored in neutralizing solution at room temperature for about 0 to about 8 hours followed by storage in cryopreservation medium at room temperature 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 still further embodiments, the percent recovery of cells after filtration stored in neutralizing solution at room temperature for about 0 to about 8 hours followed by storage in cryopreservation medium at room temperature for about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 140%, 150%. Survival can be any value or subrange within the ranges listed.

[0132] OPCs formulated in a cryopreservation medium suitable for ready-to-administer (RTA) applications can include 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, dimethylsulfoxide (DMSO), and OPCs suspended in water. An example of this cryopreservation medium is commercially available under the trade name CRYOSTOR® and is manufactured by BioLife Solutions, Inc.

[0133] DMSO may be used as a cryoprotectant to prevent the formation of ice crystals that can kill cells during the cryopreservation process. In some embodiments, the cryopreservable OPC therapy composition comprises about 0.1% to about 2% DMSO (v / v). In some embodiments, the RTA OPC therapy composition comprises about 1% to about 20% DMSO. In some embodiments, the RTA OPC therapy composition comprises about 10% DMSO. In some embodiments, the RTA OPC cell therapy composition comprises about 5% DMSO. The concentrations can be any value or subrange within the recited ranges.

[0134] In some embodiments, an OPC therapy formulated in a cryopreservation medium suitable for post-thaw ready-to-administer (RTA) applications may include OPCs suspended in a cryopreservation medium that does not contain DMSO. For example, an RTA OPC therapeutic cell composition may include OPCs suspended in Trolox, Na+, K+, Ca2+, Mg2+, c1-, H2P04-, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, glutathione without DMSO (dimethylsulfoxide, (CH3)2S0) or any other dipolar aprotic solvent. An example of this cryopreservation medium is commercially available under the trade name HYPOTHERMOSOL® or HYPOTHERMOSOL®-FRS, and is also manufactured by BioLife Solutions, Inc. In other embodiments, an OPC composition formulated in a cryopreservation medium suitable for post-thaw ready-to-administer applications may include OPCs suspended in trehalose.

[0135] An RTA OPC therapeutic composition may optionally include additional factors that support OPC engraftment, integration, survival, efficacy, etc. In some embodiments, an RTA OPC therapeutic composition includes an activator of function of an OPC preparation as described herein.

[0136] In some embodiments, the RTA OPC therapy composition may be formulated in a medium containing components that reduce molecular cellular stress during the freezing and thawing process by scavenging free radicals, pH buffering, swelling / osmotic support, and maintaining ion concentration balance.

[0137] In some embodiments, the OPC therapy formulated in a cryopreservation medium suitable for use in preparation for administration after thawing may include one or more immunosuppressant compounds. In certain embodiments, the OPC therapy formulated in a cryopreservation medium suitable for use in preparation for administration after thawing may include one or more immunosuppressant compounds formulated for sustained release of the one or more immunosuppressant compounds. Immunosuppressant compounds for use with the formulations described herein may belong to the following classes of immunosuppressants: glucocorticoids, cytostatics (e.g., alkylating agents or antimetabolites), antibodies (polyclonal or monoclonal), drugs acting on immunophilins (e.g., cyclosporine, tacrolimus, or sirolimus). Additional drugs include interferons, opioids, TNF binding proteins, mycophenolic acid, and small biological agents. Examples of immunosuppressants include mesenchymal stem cells, antilymphocyte globulin (ALG) polyclonal antibodies, antithymocyte globulin (ATG) polyclonal antibodies, azathioprine, BAS 1L1 X 1MABO (anti-IL-2Ra receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB® (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, RITUX1MABO (anti-CD20 antibody), sirolimus, tacrolimus, and / or mycophenolate mofetil.

[0138] Having now generally described the present invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limitations of the present disclosure unless otherwise specified. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or alterations thereto will be suggested to those skilled in the art and are within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein in their entirety by reference for all purposes. EXAMPLES

[0139] Example 1: Establishment of a Drug Product (DP) Formulation for Thawing and Injection (TAI) of OPC1 The purpose of this example is to demonstrate the establishment of a new oligodendrocyte precursor cell (OPC1) thaw and injection (TAI) pharmaceutical formulation. An OPC1 drug product (DP) for TAI was developed to be thawed and applied directly to the clinical site. Previously, a series of studies were carried out to develop an improved scale-up production and harvest process for OPC1 cells. The focus of this example is on the development of 100 x 10 cells per ml with a final volume of cell suspension of 300 μl per vial. 6 The objective of this study was to establish a method for thawing TAI DP preparations, and OPC1 TAI vials using CryoStor 10 (CS10) as the cryopreservation medium to generate clinical doses of viable cells.

[0140] The following aspects are specifically established in this example: 100×10 per ml in CS10 with a final volume of cell suspension of 300 μl per vial 6 establishing an OPC1 TAI preparation as individual viable cells, establishing a thawing procedure for OPC1 TAI vials containing 300 μl, evaluating the comparability of OPC1 TAI derived from LN521-expanded cells with iMatrix511-E8-expanded cells, evaluating the optimal cell dissociation enzyme neutralization solution, and demonstrating the ability of the optimized scale-up OPC differentiation process to generate OPC1 TAI that meet all acceptance criteria.

[0141] The following abbreviations and definitions are used in this example: CF - Cell Factory (cell culture vessel). CS10 - CryoStor 10 (cell cryopreservation agent). DMEM / F12 - cell culture medium. DP - Drug Product. DS - Drug Substance. GF - Growth Factor. HSA - Human Serum Albumin. ICB - Intermediate Cell Bank. iMatrix511 - E8 - Fragmented Laminin-511 Coated Matrix. LN521 - Laminin-521 Coated Matrix. Min. - Minutes. NutriStem (cell culture medium) containing NUTS(-)-Human Serum Albumin with HSA. OPC1 TAI - OPC1® "Thaw and Inject" formulation. RT - Room Temperature. Sec. - Seconds. TS - TrypLE Select (cell dissociation enzyme).

[0142] Tables 1 and 2 below list the materials and equipment used in this example.

[0143] (Table 1) Test materials TIFF2024512385000002.tif72128

[0144] (Table 2) Equipment TIFF2024512385000003.tif60128

[0145] Figure 1 shows an overview of the experimental procedure for harvesting and cryopreserving OPC1 TAI. As indicated, OPC1 cells are cultured from days 28-35 or 35-42. OPC1 cells are then harvested using TS, followed by neutralization of cell dissociation enzymes, filtration through a 70um cell strainer, and centrifugation and cell resuspension in CS10. OPC1 TAI preparations are prepared at 100 x 10 per mL in CS10. 6 Concentrated at 30 x 10 viable cells per 300uL in a 0.5mL vial 6 The cells are stored at a concentration of 1000 x 1000 viable cells. Finally, OPC1 TAI is cryopreserved and finally, the OPC1 TAI vial is thawed.

[0146] Table 3 below lists details and objectives for some TAI experiments.

[0147] (Table 3) Test conditions TIFF2024512385000004.tif250150 2 1% HSA based on previous dosing of GPOR dose preparation of 1% HSA in Isolyte S

[0148] In Table 3 above, experiments TAI #9 and TAI #10 were not included because bilayer CFs were found not to support OPC1 proliferation.

[0149] OPC1 culture (stage II) OPC1 cells were cultured on CFs (either directly coated with iMatrix511-E8 or pre-coated with LN521) for 7 days. Initial qualification of the final DP volume and cell concentration was performed using OPC1 cells derived from H1 cells cultured in LN521-coated vessels using mTeSR1 (TAI no. 3 and no. 4) or mTeSR Plus medium (TAI no. 5-13) and differentiated using GPM(+) as culture medium. TAI no. 11 and TAI no. 12 were later established DP formulations from cells cultured when applying the direct coating method using seeding medium supplemented with iMatrix511-E8 rather than LN521-coated flasks.

[0150] Harvesting of OPCs Cells were harvested on day 35 or 42 using TS. Three cell dissociation enzyme (TS) neutralizing solutions were tested (TAI #6 and #12) to evaluate their effect on TAI cell quality attributes and to upgrade the GMP grade of raw materials: GPM+ ​​(without GF), DMEM / F12 as is or supplemented with 1% HSA, which was the solution originally established for OPC culture, and NUT(-) with HSA. The selected cell dissociation enzyme (TS) neutralizing solution, NUT(-) with HSA, was applied in study TAI #13 and DEVOPC_study #25.

[0151] Filtration and centrifugation of OPC1 DS OPC1 TAI is intended to be administered as a single cell suspension. To obtain single cell DS, the DS obtained from cell harvest had to be passed through a 70 μm cell strainer. After filtration, the cell suspension was centrifuged (supernatant was discarded) and immediately continued to the final formulation of DP.

[0152] OPC1 TAI DP preparation The cell pellet obtained after DS centrifugation was diluted with 100 × 10 6 The cells were resuspended in CS10 to produce a final DP cell concentration of 10 viable cells / ml.

[0153] Dispensing DP into vials The final formulated DP was immediately dispensed into vials and 30 x 10 per 300 μl per vial using an automated dispenser and capper / decapper. 6 The viable cells are then kept cryopreserved.

[0154] Thawing an OPC1 TAI vial The need to thaw the TAI vials generated during the course of this study for cell analysis mandated conducting a comparability study evaluating a dry-bath-based thawing method using an MD-Mini dry bath equipped with a customized heating block to accommodate the vials compared to the standard thawing method using a 37 °C water bath.

[0155] QC Assays Thawed TAI vials were assayed for biomarker expression on days 35 or 42. Thawed cells were assayed for decorin secretion.

[0156] Acceptance Criteria Purity marker (PDGFRα) >95.00%. Impurity markers (Claudin 6, EpCAM, CK7) <2.00%. Decorin secretion (day 42 cells) ≥15ng / ml. Morphology: small compact cells, absence of elongated spindle morphology.

[0157] Payback calculation The recovery percentage is 100 x 10 6 Calculations were based on a target final DP concentration of viable cells / ml.

[0158] result Establishing a thawing method for OPC1 TAI vials. Because OPC1 TAI was developed to be thawed and applied directly at the clinical site, a robust, transportable, and simple method for thawing cryopreserved, vapor-phase LN2-stored vials needed to be established. This mandated a comparability study to be performed evaluating a dry-bath-based thawing method using a MD-Mini dry bath with a customized heating block to accommodate the vials compared to the common thawing method using a 37°C water bath. A preliminary study using 0.5 ml cryovials filled with 300 μl of CS10 to simulate the actual volume of clinical vials established the optimal thawing duration as 2 min in the MD-Mini dry bath and 1 min 25 s in the 37°C water bath. In addition, it was established that thawed cells were incubated in GPM+ ​​for 2 min before assaying for % recovery and % viability using an NC-200 cell counter (TAI number 3 / 4). All TAI vials were then thawed with one of the aforementioned methods for both R&D and QC analysis.

[0159] Establishing OPC1 TAI DP cell concentration and volume OPC1 TAI DP, total 30 x 10 per vial 6 For viable cells, 100 x 10 6 The CS10 cells were incubated in a 300 μl suspension with a concentration of 100 viable cells / ml. Table 4 below summarizes the % recovery and % viability values ​​after thawing TAI vials from the relevant study.

[0160] Table 4. Recovery and survival percentage results after thawing at TAI TIFF2024512385000005.tif111136 NOTE: Due to the large cell volumes required to produce the desired TAI DP, depending on the size of the vessel, obtaining two TAI vials was not always achievable.

[0161] The % viability range of thawed DP TAI cells obtained from GPM(+) neutralized DS was 88%-98% in all tests, with the % recovery range in these tests being 90%-117% (Table 6). This relatively wide range of % recovery is due to the cumulative error when working with high cell concentrations in combination with the very small working volumes of DP (usually a few hundred μl) used in each of these tests to generate the final DP formulations of the desired concentrations. TAI #7 showed that cells harvested at day 35 from LN521-coated flasks produced TAI vials that were as good as day 42 TAI vials in terms of % viability and % recovery. However, slightly lower PDGFRα marker expression values ​​(Table 6) necessitated that these results be corroborated with TAI #13, using cells harvested from LN521-coated 1-layer CFs. Indeed, the results of TAI #13 met all acceptance criteria. Demonstrating the ability to generate TAI DPs from cells derived from LN521-coated flasks at day 35, TAI#11, TAI#12 and DEVOPC-study#25 demonstrated the feasibility of generating TAI DPs when using iMatrix511-E8 supplemented medium, with recovery% and viability% results as good as DP cells derived from LN521-coated flasks. TAI#6 and TAI#12 were aimed at evaluating the feasibility of replacing the enzyme neutralization solution with either DMEM / F12 (as in TAI#6 or supplemented with 1% HSA in TAI#12), or NUT(-) with HSA.

[0162] The results show that both GPM(+) and NUT(-) with HSA gave very similar survival and recovery results. Marker expression analysis also showed that NUT(-) with HSA was comparable to the currently established GPM(+) enzyme neutralizing solution, with all relevant markers meeting the current acceptance criteria (Table 6). Furthermore, it is evident that the use of DMEM / F12 (as is or supplemented with 1% HSA in group B of TAI#6 and TAI#12) as enzyme neutralizing solution reduces the post-thaw survival% of cells by about 8%, but does not significantly affect the recovery%. DEVOPC-study no. 25, in which NUT(-) with HSA was also applied as TS neutralizing solution, further supported this data.

[0163] Tables 5 and 6 below show the marker expression and decorin secretion analysis results.

[0164] Table 5. Marker expression analysis results TIFF2024512385000006.tif255147Note: GFAP, CK7 and beta tubulin III marker expression values ​​in studies TAI #12 and TAI #13 were derived from a relatively small number of FACS events, as detailed herein below. 1 low viable cell count (>2000, <5000); 2 low viable cell count (>1000, <2000); 3 Low viable cell count (>400, <1000) 4 Group G: cells filtered using a 60 μm filter kit and cryopreserved in either CryoMed or CoolCell.

[0165] Marker expression values, including impurity markers, met their respective acceptance criteria in all studies, except for PDGFRα expression at TAI number 7, which was slightly below 95.00% (Table 5). These results correlate with the original RD run, and therefore this slight decrease is not attributable to the OPC1 TAI cryopreservation method.

[0166] Although NG2 expression appeared to differ between studies, it has been determined that its expression is highly influenced by the harvesting procedure, and thus a decrease in NG2 positive cells does not necessarily indicate a potential loss of OPC identity, particularly as it has been established that NG2 expression is fully restored following an overnight culture period and GMP batches are assayed for NG2 expression following an overnight culture period.

[0167] Table 6. Decorin secretion assay results TIFF2024512385000007.tif132128

[0168] Decorin secretion in all tests and conditions met the acceptance criteria (>20.00 ng / ml, Table 8). It is noted that the decorin secretion values ​​for TAI #6 were relatively low in all three conditions compared to the other tests, leading to the assumption that this may be due to the cellular source rather than a direct result of the test conditions, as further evident from TAI #12, which applied identical test conditions as TAI #6.

[0169] Consideration Establishing the final formulation of the OPC TAI DP generated in the optimized OPC differentiation process stage II was the focus of this study, along with a method for thawing the TAI vials.

[0170] To engage OPC-DP in clinical trials, the OPC-1 TAI DP should be formulated while maintaining its target viable cell concentration at 100 × 10 6 This requires maintaining 10 x 10 cells / ml in 100 µl. 6 Each clinical vial therefore contains 30 x 10 cells in 300 μl of CS10 cell suspension. 6 It contains viable cells.

[0171] A robust and simple method for thawing OPC1 TAI vials for performing clinical studies was a prerequisite to allow further analysis of OPC1 TAI cells during the course of this study. Therefore, a standard thawing method using a 37 °C water bath was compared to a 37 °C dry bath using the MD-Mini device.

[0172] A 0.5 ml NUNC vial filled with 300 μl CS10 was used to simulate clinical TAI vials, and thawing using the MD-Mini dry bath was established as a 2 min procedure, which was shown to be equivalent to a 1 min 25 s thawing procedure in a 37°C water bath (with vials showing visible ice flakes at the end of the thawing time). All TAI vials in this study were then thawed in the MD-mini dry bath. In addition, the incubation time of thawed cells in the diluent required for NC-200 analysis was established as 2 min at RT in GPM(+).

[0173] Initial qualification of the OPC TAI DP was established using cells differentiated and cultured using LN521-coated vessels with mTeSR1 and GPM(+) as culture medium in the OPC differentiation process step I according to the OPC differentiation process step II (tests TAI #3 and TAI #4, Table 3). These demonstrated the feasibility of generating TAI cells that met all the acceptance criteria, including marker expression and efficacy (decorin secretion), in the newly optimized OPC differentiation process step II, thus paving the way for further process scale-up.

[0174] The objective of TAI #6 and TAI #12 was to demonstrate the feasibility of using a cell dissociation enzyme neutralization solution that would not only maintain OPC1 TAI DP quality attributes but also allow for the removal of non-clinical grade and animal derived media components (B27 and T3 supplements) on the day of harvest (Table 3). Comparing the three solution types showed that using NUT(-) with HSA was as good as standard GPM(+) for neutralizing cell dissociation enzymes, with cell quality attributes meeting all acceptance criteria (Tables 4 and 5) as well as viability and recovery % results that were as good as those obtained when using GPM(+) for that purpose (Table 4).

[0175] The objective of TAI number 11 was to evaluate the feasibility of producing OPC1 TAI DP generated from OPC cells derived from day 14 ICB cultures and harvested on day 42. Results showed that cell quality attributes were comparable to those of cells obtained from ongoing cultures harvested on day 42 (Tables 4 and 5).

[0176] Studies were also conducted to evaluate TAI DPs formulated with cells harvested on day 35 and compare their quality attributes to those of OPC1 TAI DPs formulated with cells harvested on day 42. Results from TAI #7 and TAI #13 showed that these cells met all acceptance criteria (although TAI #7 showed slightly lower PDGFRα marker expression compared to its source culture). Furthermore, the day 35 TAI DP generated in TAI #13 not only confirmed the quality of cells obtained from the Cell Factory vessels on day 35, but also the benefit of using NUT(-)HSA as a cell dissociation enzyme neutralizing solution during harvest.

[0177] Finally, since this study encompassed the cell expansion process in CFs applying the direct coating method using iMatrix511-E8 together with both the optimized scaled up filtration procedure using 60μm filter kit and the OPC1 TAI DP cryopreservation process using CryoMed, study DEVOPC_study no.25 demonstrated the robustness and reproducibility of the scaled up OPC differentiation process step II as a complete process overall. Both marker expression and decorin secretion values ​​of OPC1 TAI generated from 60μm filtered cells cryopreserved in CryoMed met the acceptance criteria.

[0178] Overall, the data presented in this study indicate that 100 × 10 6 The OPC1 TAI DP as a cell suspension of viable cells is supported and established in a final volume of 300 μl per 0.5 ml NUNC vial. Furthermore, this study supports the ability of the OPC scaled-up manufacturing process to produce OPC1 TAI DP from cells cultured in a one-layer CF directly coated using iMatrix511-E8 supplemented medium, harvested on either day 35 or day 42 using TS and NUT(-) with HSA as a TS neutralizer, filtered through a 60 μm filter kit, and cryopreserved at CryoMed. The cells obtained from this optimized scaled-up manufacturing process met the OPC1 acceptance criteria and demonstrated functional biological activity of OPC1 TAI.

[0179] OPC TAI is 100x10 in CS10 6 Viable cells / ml suspension; 30 x 10 per 300 µl in a 0.5 ml vial 61000 TAI vials containing viable cells. Thawing OPC TAI vials for 2 min in a 37°C MD-Mini dry bath is as good and robust as thawing in a 37°C water bath. OPC1 TAI DP generated from cells cultured in 1-layer CFs directly coated with iMatrix511-E8 supplemented medium is as good as OPC1 TAI DP obtained from cells cultured in LN521 pre-coated vessels. Harvesting cells at day 35 of the OPC differentiation process stage II to generate TAI DP is feasible under the aforementioned process conditions. NUT(-) with HSA as cell dissociation enzyme neutralizing solution is as good as GPM(+) for generating OPC1 TAI DP from cells harvested at either day 35 or day 42. Day 14 ICB has been shown to generate OPC1 TAI DP that exhibits comparable quality attributes to DP obtained from ongoing cell cultures. OPC1 TAI DP produced using an optimized scaled-up manufacturing process using 60 μm filter kits and cryopreserved at CryoMed met all acceptance criteria and exhibited functional biological activity.

[0180] Example 2: OPC1 for one-time administration for the treatment of subacute spinal cord injury (SCI) The purpose of this example is to present the scientific data generated during the development of the OPC1 (also referred to as LTCOPC1) CMC program. Information provided includes preliminary comparability results based on R&D implementation of an improved manufacturing process, comparability between GPOR and LCT R&D manufactured materials, introduction of a new proposed efficacy assay, review of OPC1 safety status based on GPOR in vivo data, and development plans for LCTOPC1 regarding reanalysis of GPOR lots utilizing improved analytical methods.

[0181] LCTOPC1 (OPC1), previously termed GRNOPC1 and AST-OPC1, is an oligodendrocyte precursor cell population derived from the H1 hESC line intended for one-time administration to treat subacute spinal cord injury (SCI). OPC1 has been shown in preclinical studies to produce neurotrophic factors, migrate within the spinal cord parenchyma, stimulate angiogenesis, and induce remyelination of denuded axons, all of which are essential functions of oligodendrocyte precursors and important for axonal survival, regrowth, and function.

[0182] Clinical evaluation of LCTOPC1 began in 2010. The first clinical trials included a low dose of 2 × 10 6 OPC1 cells were injected into the lesion site in subjects with subacute neurologically complete thoracic spinal cord T3-T11 injuries in a Phase 1 safety study (NCT01217008). A total of five subjects out of a planned eight received OPC1 as part of the initial Phase 1 CP35A007 safety study from October 2010 to November 2011. Enrollment into this trial was halted in November 2011.

[0183] In 2014, a Phase 1 / 2a dose escalation study of OPC1 in subjects with subacute sensorimotor complete (American Spinal Cord Injury Association Impairment Scale A (Asian Impairment Scale A)) single neurological level (SNL) cervical spinal cord injury from C5 to C7 was initiated (NCT02302157), and subsequently extended the study to patients with C4 neurological level injury (NLI) if upper extremity motor score (UEMS) ≥ 1, changing the dosing window from 14–30 days to 21–42 days after spinal cord injury. A total of 25 subjects across five cohorts were enrolled in the study and received a single dose of OPC1 cells delivered by intraparenchymal injection within the spinal cord injury site using a syringe positioning device during a dedicated surgical procedure. Enrollment into the study was completed in December 2017.

[0184] In November 2018, a randomized, controlled, single-blind Phase 2 study was proposed to the FDA, and the agency had no objection to the use of OPC1 drug lot M26D1A in the proposed Phase 2 clinical study.

[0185] Briefly, the origin of the new Master Cell Bank (MCB) is H1 bank lot number MCBH101. MCBH101 was produced by Geron Corporation (Geron; Foster City, CA) directly from the H1 Original Cell Bank (OCB) in 2009. The new MCB originated directly from H1 bank-lot number MCBH101. It was produced in feeder-free conditions using well-defined raw materials, a new culture system, and harvesting procedures, and cryopreserved by a cryopreservation process customized with hESCs. In addition, a method for assessing H1 hESC pluripotency was optimized. The new WCB was derived from the new MCB and expanded in tissue culture for four passages while maintaining hESC characteristics, and then cryopreserved. The starting material for LCTOPC1 production is provided by the WCB. New master and working cell banks were released in accordance with predefined release criteria and tested for undetermined drug substance using an approved laboratory in accordance with the approved study protocol, prevailing industry standards (ICH Q5A(R1), Q5D), and current manufacturing practices ("Regulations") and legislation applicable to the study being performed, as amended from time to time.

[0186] OPC1 is an investigational drug that was studied in Phase 1 and Phase 1 / 2a spinal cord injury clinical studies using Geron-produced OPC1 clinical lots manufactured between 2008 and 2011. Geron's manufacturing process (GPOR) was initially developed in the early 2000s. At that time, well-defined cell therapy grade reagents and materials were not widely available. Thus, stage 1 of the GPOR manufacturing process included the growth of H1 embryonic cells on Matrigel™, an animal-derived extracellular matrix (ECM), collagenase, and manual peeling of the culture dish surface for harvesting, passaging, and expansion of the H1 embryonic stem cells.

[0187] Moreover, the GPOR manufacturing process was based on a differentiation process that occurred in cell aggregates starting directly from pluripotent H1 cells, in the form of embryoid bodies (day 0-26, Figure 2) with a strong susceptibility to spontaneous differentiation. From day 27, differentiation was completed on an adhesive surface coated with Matrigel™ for the proliferation and maturation of oligodendrocyte precursors. Thus, the GPOR manufacturing process had low yields and the main quality attributes defined by purity / impurity / non-target cell population markers showed limited reproducibility. In addition, the final cryopreserved product required thawing, washing, and formulation preparation before administration.

[0188] The development of an improved manufacturing process according to the present disclosure is a more controlled directed differentiation of H1 towards OPCs, induced by a specific sequence of growth factors and small molecules to inhibit or direct the differentiation pathway, possibly using cell therapy grade materials (as detailed in FIG. 3). Furthermore, the new process shortens the long aggregation phase used by Geron to 7 days, from 26 days directly from a pluripotent cell state prone to spontaneous aberrant differentiation, after 14 days of monolayer directed differentiation of H1 cells into neuroectoderm, reducing the possibility of spontaneous differentiation in the aggregation phase. The differentiation process of GPOR vs. LCTOPC1 is summarized in FIG. 2. The biological basis for the signaling sequences of inducers and inhibitors of the improved differentiation process is described in FIG. 4. In addition, new in-process controls (IPCs) were added to better monitor and characterize the differentiation process, as detailed in FIG. 4.

[0189] Finally, the product is cryopreserved as an injection-ready OPC1 formulation, referred to herein as "Thaw and Inject (TAI)". The materials used to manufacture OPC1 cells (both the original GPOR and modified process) are summarized in Table 7 below.

[0190] Table 7: Materials used during the production of OPC cells (GPOR and LCTOPC1 processes. TIFF2024512385000008.tif72151

[0191] Stage I-H1 proliferation Pluripotent H1 cells are thawed and cultured on laminin-coated vessels in mTeSR plus medium for 12-15 days. The cells are passaged and expanded using the non-enzymatic reagent ReLeSR. During expansion, cells are assessed morphologically and at the end of three passages (before the start of the differentiation process), hESC pluripotency is assessed by a flow cytometry-based method.

[0192] Stage II-H1 differentiation to OPC1 From day 0 of differentiation until the end of the process, cells are cultured in Glial Progenitor Medium (GPM), which is DMEM / F-12 supplemented with B27 and T3.

[0193] Days 0-7 - On day 0, when H1 cultures reach the required criteria defined by lactate concentration and cell morphology, the differentiation process is initiated by changing the medium for expanded pluripotent hESCs cultured on laminin-coated vessels as follows: On days 0-3, GPM medium is supplemented with retinoic acid (RA), dorsomorphin and PD0325901 to direct the differentiation process towards the neuroectoderm pathway (Kudoh, Wilson et al. 2002). Dorsomorphin inhibits bone morphogenetic protein (BMP) signaling (SMAD pathway), thus inhibiting mesoderm and trophoblast differentiation (Li and Parast 2014). PD0325901 inhibits downstream bFGF signaling in MEK1 and MEK2, inhibiting pluripotency and endoderm differentiation (Sui, Bouwens et al. 2013). In summary, inhibition of pluripotency, endoderm, mesoderm and trophoblast formation, as well as activation of the RA signaling pathway, promotes neural tube (neurectoderm) formation (Watabe and Miyazono 2009; Sui, Bouwens et al. 2013; Li and Parast 2014; Patthey and Gunhaga 2014; Janesick, Wu et al. 2015). On days 4-7, cultures are supplemented with retinoic acid and ascorbic acid to continue the induction of neuroectoderm differentiation (Duester 2008).

[0194] Days 7–14 - On day 7, cells are enzymatically harvested using TrypLE Select and then plated as monolayer cultures on laminin-coated vessels from day 7 to day 14 and cultured in GPM supplemented with rhEGF, hsFGF, and ROCK inhibitor (ROCK inhibitor for the first 48 hours only) (Hu, Du et al. 2009, Patthey and Gunhaga 2014, Zheng, Li et al. 2018).

[0195] Days 14-21 - On day 14, cells are enzymatically harvested using TrypLE Select to promote neurobody (NB) aggregate formation and cultured for 1 week as dynamic suspension cultures in GPM supplemented with ROCK inhibitor (first 48 hours), as well as rhEGF and hs-rhFGF throughout.

[0196] Days 21-42 - On day 21, aggregates were plated back as adherent cultures on laminin-coated vessels in GPM supplemented with rhEGF and PDGF (Ota and Ito 2006; Koch, Lehal et al. 2013), then on day 28, cells were enzymatically harvested using TrypLE Select and grown as adherent cultures on laminin-coated vessels in GPM supplemented with rhEGF and PDGF until days 35-42 for terminal expansion and maturation, with enzymatic passaging using TrypLE Select approximately every 7 days.

[0197] At the end of the expansion process, OPC1 cells are harvested using TrypLE Select and cryopreserved as a Thaw-and-Inject (TAI) formulation in CryoStor® 10 (CS10; BioLife Solutions, Inc.) cryopreservation solution. The LCTOPC1 manufacturing process flow is shown in Figure 4.

[0198] In-process control tests are performed at all major steps during the hESC to OPC1 differentiation process, as shown in Figure 4. Multicolor flow cytometry (FCM) and qPCR methods (respectively) are used to evaluate biomarker protein and mRNA expression. Cells are tested for expression of OPC1, epithelial, mesodermal, astrocytic and neuronal biomarkers, as well as residual hESC. In addition, viability, cell yield and metabolic activity (e.g., lactate) are evaluated during the process. Lactate concentration is used as an indicator to initiate differentiation starting on day 0 and as a surrogate for cell counting on day 21 to determine the surface area required for aggregate plating for pre-OPC generation and proliferation.

[0199] Thawing and cryopreservation of LCTOPC1 DP as an injectable formulation The improved LCTOPC1 Drug Product (DP) has been developed as a Thaw and Inject (TAI) formulation, which is thawed and immediately loaded into a delivery device prior to spinal injection. This improvement significantly reduces the risks associated with further manipulations at the clinical site, such as washing and resuspension for final formulation prior to administration, and increases control over product consistency, safety and quality.

[0200] Development of the TAI DP formulation was performed using 100 × 10 cells per mL in a final volume of cell suspension of 300 µL per vial. 6 The study included using CryoStor® 10 (CS10) as the cryopreservation medium to generate a clinical dose targeting 100 x 10 viable cells, evaluating an optimal cell dissociation enzyme neutralization solution prior to cryopreservation and adding a filtration step, and obtaining 100 x 10 cells per mL in CS10 in a final volume of 300 µL of cell suspension per vial. 6 The focus will be on establishing an OPC1 TAI formulation targeting 100 viable cells, establishing a thawing procedure for OPC1 TAI vials containing 300 µL, as well as verifying that the optimized scaled-up OPC1 improved manufacturing process produces OPC1 TAI that meets all acceptance criteria.

[0201] Development studies were performed using 100 x 10 per mL of CS10 in a final volume of 300 µL in 0.5 mL NUNC vials. 6 This study supports and establishes the LCT OPC1 TAI DP as a cell suspension targeting viable cells. Furthermore, this study demonstrates the ability of the scaled-up manufacturing process of LCT OPC1 to produce OPC1 TAI DP harvested in NUT(-) with TrypLE Select and HSA as TrypLE Select neutralizer, filtered through a 60 μm filter kit, and then cryopreserved in a CryoMed controlled rate freezer. The cells obtained from this optimized and scaled-up manufacturing process exhibit acceptable quality attributes, viability and recovery, as well as functional biological activity.

[0202] Material Management Control of auxiliary materials. Each auxiliary material used in the manufacture of LCTOPC1 is subject to a risk level assessment (USP NF <1043> The risk level (RL) is determined by the risk assessment procedures to evaluate the manufacturer's quality system, safety risks (material origin and manufacturing process related risks) and material grade (quality criteria). The process / product criticality is determined by the evaluation of the production procedure and / or the impact of the material on the product quality.

[0203] Noncellular impurities. Calculation of the estimated amount of potential noncellular impurities in the LCTOPC1 DP is based on the procedures performed prior to manufacturing of the final DP drug product and the DP dose. A risk assessment is performed based on theoretical calculations of the noncellular impurity levels, the biosafety of the material, and the pharmacological risk. Based on the risk assessment results, further mitigation is performed, such as analytical testing of the DP for detection and measurement of specific noncellular impurities, removal / reduction of materials from the DP, and expanded biosafety testing as necessary.

[0204] Proposed CMC Comparability Test OPC1 will be manufactured in a GMP facility according to the improved process, released according to revised release parameters, and stored frozen in the developed TAI formulation. LCTOPC1 DP will be compared to Geron's manufactured representative batches and characterized with the updated analytical methods used for the release of OPC1 manufactured via the new process. This plan includes testing of attributes used as release criteria for GPOR as well as additional markers identified. The sponsor's proposal for comparison is based on the quality attributes that characterize the drug product, as described in Table 8.

[0205] A side-by-side comparison between the LCTOPC1 batch and the GPOR OPC1 batch is based on statistical analysis to calculate expected ranges for quantitative measurements of quality attributes from the GPOR OPC1 batch. The values ​​of these quality attributes measured in the LCTOPC1 batch are evaluated in relation to these expected ranges for the quality attributes tested. The comparability data analysis establishes reproducible release criteria for the LCTOPC1 process and is expected to demonstrate that LCTOPC1 has low batch-to-batch variability.

[0206] Quality attributes tested included viability, identity / purity, impurity populations, and functional / potency assays for 3-4 representative GPOR and LCT OPC1 batches each.

[0207] The proposed quality attributes for comparability are: Viability - a critical quality attribute for any live cell medicine; Identity / Purity - assessment of characteristic oligodendrocyte precursor cell protein markers: NG2, PDGFRα and PDGRFβ; Non-target cell populations / impurities; and in vitro cell function / potency.

[0208] For impurity populations of non-target cells. Residual H1 hESC from starting material - human embryonic stem cell protein markers TRA-1-60 and Oct-4 as potential sources for teratogenic drugs combined in multicolor flow cytometry testing. TRA-1-60 and Oct-4 are commonly known and used markers for embryonic stem cells and were previously used as OPC1 release criteria. Evaluation of potential aberrant differentiation pathways. Epithelial cell protein markers: Keratin 7, Claudin-6, EpCAM and CD49f as known epithelial markers. Mesodermal cell protein markers CXCR4 and CD56 as known mesoderm and cartilage markers. Astrocyte protein marker GFAP as known astrocyte marker. Mesenchymal cell mRNA OLR1 inducing epithelial-mesenchymal transition. Endodermal cell mRNA marker FOXA2, SOX17 as known endoderm markers. At this time, it is not planned to include the previously used nestin and α-actinin attributes, since new data show that nestin is not specific for OPCs, but rather for NSCs and other cell types (see website ncbi.nlm.nih.gov / pmc / articles / PMC5948302 / ) and that α-actinin can be effectively replaced by the CXCR4 / CD56 combination. CXCR4 is expressed in definitive endoderm and mesoderm: see website pubmed.ncbi.nlm.nih.gov / 16258519 / and website pnas.org / content / 107 / 31 / 13742.

[0209] Regarding cell function / efficacy in vitro. Decorin secretion - a small secreted cell matrix proteoglycan as efficacy indicator for OPC1. Decorin expressed by neurons and astrocytes in the central nervous system attenuates scar tissue formation, inhibits cavity formation and promotes wound healing. OPC1 cell migration in response to platelet-derived growth factor-BB (PDGF-BB) is important for cell motility at the injury site to ensure the widest anatomical coverage from a single injection site in the spine. Development of a new efficacy test for the evaluation of OPC1 maturation and myelination potential. This assay is based on the remyelination of denuded axons, which is an essential function of OPC1 cells. In this assay, OPC1 cells are thawed and placed in a specific medium in a 3D tissue culture environment (e.g. Matrigel or nanofiber tubes) that should induce maturation of OPC1 into oligodendrocytes (OLs). The 3D environment nanotubes mimics denuded axons to induce myelination activity by OPC1 cells in a simple in vitro setting. Assays will measure expression of proteins associated with OL function and maturation status, such as MBP, O4, MAG, MOG, PLP, and CNPaseby immunocytochemistry, flow cytometry, and qPCR.

[0210] The proposed test panels to be used to test GPOR OPC1 and LCTOPC1, along with the proposed emission standards for LCTOPC1, can be seen in Table 8 below.

[0211] (Table 8) Proposed test panels and the rationale used for process development, release, and comparability. TIFF2024512385000009.tif173151

[0212] Preliminary comparability of R&D LCTOPC1 batches from a representative run is presented in Tables 9-13 below.

[0213] Table 9. Comparability data from representative GPOR OPC1 and LCTOPC1 R&D runs - OPC1 identity profile by flow cytometry TIFF2024512385000010.tif79151*MG-cells are seeded on Matrigel overnight prior to FCM analysis. GPOR OPC1 preparation and freezing reduces NG-2 and PDGFR-α expression. Overnight culture on Matrigel as previously shown and with the present these data. 1 Clinical batch

[0214] Table 10. Comparability data from representative GPOR and LCTOPC1 R&D runs - Non-target / impurity cell population profiles by flow cytometry TIFF2024512385000011.tif76151 1 Clinical batch

[0215] Table 11. Comparability data from representative GPOR OPC1 and LCTOPC1 R&D runs - hESC residues by flow cytometry TIFF2024512385000012.tif33151 1 Clinical batch

[0216] Table 12. Comparability data from representative GPOR OPC1 and LCTOPC1 R&D runs - Non-targeted / impurity cell population gene profile by qPCR (vs. GPOR OPC1 M08) TIFF2024512385000013.tif46151*ND - Not Detected 1 Clinical batch

[0217] Table 13. Comparability data from representative GPOR OPC1 and LCT OPC1 R&D runs - in vitro function determined by decorin secretion and translocation assays TIFF2024512385000014.tif38151 1 Clinical batch

[0218] A review of the evaluation of ectopic tissue formation as a release criterion for OPC1 drugs Historical approach of the OPC1 program to address ectopic tissue (cyst) formation risk. Toxicology studies conducted and provided as part of the initial OPC1 IND proposal revealed ectopic tissue formation of cystic structures and granular hyperplasia (AGS) in animals receiving certain OPC1 batches. The significant lot-to-lot variability and FDA concerns that the proposed release criteria were not sufficient to predict the safety of the intended clinical lots led to FDA's request to conduct a 9-month toxicity study for each intended clinical lot as a release requirement. The in vitro cyst assay was presented as the research tool used to screen OPC1 lots. The hypothesis stated that lots with a strong tendency to form cysts in vitro could be easily identified and that all of the in vitro cyst positive lots would demonstrate cyst formation in vivo. In addition, it stated that steps were being taken to refine the assay by improving cyst identification through more consistent staining and evaluation of in vitro cyst size as a predictor for in vivo cyst formation. Further studies conducted with the in vitro cyst assay revealed the following conclusions: Despite several years of development efforts, the cyst assay does not meet the fundamental requirements for a QC release method, and there is no linear correlation or accurate prediction between in vitro cyst formation and in vivo cyst frequency.

[0219] A review of OPC1 safety status based on GPOR data and preliminary results from comparability testing, correlations between pass / fail GPOR batches, and new / developing markers. The inventors have been continuously working on improving the OPC1 manufacturing process, including new in-process controls, release, and efficacy testing for the OPC1 product. The rationale for the process improvements and comparability plans for the manufactured material was presented in the previous section of this example. As part of the development efforts, the inventors performed a comprehensive analysis and retesting of relevant GPOR OPC1 GMP batches using an improved product attribute panel based on newly developed methods. This analysis revealed specific correlations between available data from GLP tox studies of Geron GMP batches and the purity and expression levels of impurity / non-target cell population specific markers. The retest results and analysis show a correlation between impurities and batch failure in vivo, indicating that LCTOPC1, characterized by significantly lower levels of impurities and a high purity profile, significantly reduces the likelihood of cyst formation in vivo than GPOR OPC1.

[0220] summary Preliminary comparability data of representative GPOR and LCTOPC1 batches demonstrates the following: GPOR OPC1 and R&D LCTOPC1 demonstrate similar OPC1 identity / purity profile profiles, with the exception of the PDGFR-α biomarker, which is higher in R&D LCTOPC1 and may be due to the improved OPC1 manufacturing process (directed differentiation). R&D LCTOPC1 has lower levels of impurities from epithelial cells, astrocytes and neural non-target cells compared to GPOR OPC1. Both LCTOPC1 and GPOR OPC1 have very rarely detectable residual hESC (detected by %TRA-1-60+ / Oct4+), however GPOR OPC1 has a higher percentage of pluripotent cells compared to LCTOPC1, as demonstrated by cell populations exhibiting TRA-1-60+ / Oct4- and CD49f+. LCTOPC1 has lower levels of endodermal and mesenchymal non-target cell gene expression compared to GPOR OPC1. LCTOPC1 and GPOR OPC1 demonstrate similar decorin secretion and translocation capabilities.

[0221] Conclusion: The data accumulated so far indicate that LCTOPC1 drug products, generated from a defined cell bank system with a highly reproducible and tightly controlled differentiation process and manufactured with an improved process monitored with updated analytical methods, present similar essential quality attributes as GPOR OPC1, but benefit from an overall higher expression of purity markers and a lower expression of impurity / non-target population markers, thus potentially increasing the safety profile for LCTOPC1 drug products.

[0222] Example 3: Proof-of-concept study The aim of this study is to provide experimental data for a proof-of-concept study of OPC1 myelination and maturation.

[0223] LCT OPC1 cells were cultured in Matrigel (MG) 1:30 in 96 Vision plates in different media components as listed in Table 14. Each culture condition constitutes a "line". Cells were cultured in GPM / E for the first 5-6 days of culture, and then for mature lines, the medium was replaced according to the steps listed in Table 14. Line number 2 showed morphological changes to oligodendrocyte-like cells by immunostaining, and MBP expression.

[0224] (Table 14) Research design TIFF2024512385000015.tif117151

[0225] Figures 6-9 show OPC morphology for different lines. Figure 6 shows control line #1, GPM / E at day 10 (time = 0, day 7). OPC morphology is typical for DCN ELISA efficacy assay. Figure 7 shows OPC morphology for line #2, 50% GPM / E, and 50% N2.1 (without IBMX) at day 3 after treatment. Cells show morphological changes compared to control. Figure 8 shows control line #1 at day 12, and OPC morphology typical for DCN ELISA efficacy assay. Figure 9 shows line #2, 100% N2.1 (with IBMX for 2 days) at day 5 after treatment. Cells show morphological changes.

[0226] Figure 10 shows confocal immunostaining (fixed cells) for Hoechst and MBP on day 13. Control line no. 1 in GPM / E is shown on the left and line no. 2 with 100% N2.1 on the right.

[0227] Example 4: OPC Differentiation Protocol

[0228] Table 15. Volume of stock solution to add per ml of GPM to reach the desired final concentration TIFF2024512385000016.tif132170

[0229] The purpose of this example is to demonstrate the establishment of GMP conditions for the differentiation of hESCs into OPCs.

[0230] Day 0 - Culture Assessment. Day 0 IPC: Before starting stage II, perform hESC colony assessment. Differentiation can only be initiated if the following criteria are met: Confluency in culture is 95% or higher. % Differentiation in culture is less than 5%. % Confluent colonies is greater than 50%. Lactate concentration in medium is greater than 17.40 mM.

[0231] Medium change, days 0-6: Medium should be changed every 24 hours ± 4 hours. On days 0 and 4, observe and photograph the cultures using 5x and 10x magnification. On days 3 and 4, measure lactate. Thaw small molecules according to Table 15 and warm up the required GPM volume. Add GF volume to the warmed GPM according to Table 15. Change medium. Starting on day 4, change GF according to Table 15.

[0232] Harvest, Day 7: 1 day to 1 week prior to harvest, if necessary, determine the number of flasks required and coat with 10 μg / ml rh LN521. Observe and photograph cultures using 5x and 10x objectives. Measure lactate concentration. Divide 2 ml of CM into 2 cryovials (1 ml each) and store at -80°C. Thaw GFs and warm required GPM volume according to Table 15 (for GPM+GF and GPM w / o GF final solutions). Add GF volume to warmed GPM according to Table 15.

[0233] Harvesting protocol: Aspirate medium. Add 3ml PBS(-) to each T25 flask. Tilt flask 2-3 times and incubate 1 min at RT. Use timer. Aspirate and add 1ml / T25 TrypLE Select. NOTE: Open a new bottle of TS. Incubate 6 min at 37°C and 5% CO2. Use timer. Tap flask very firmly on one side, tilt flask to wash cells, tap flask very firmly on other side. Add 5ml GPM w / o GF. Add medium directly to cells and gently wash surface 1-2 times. Collect cells in conical tube. Add 5ml / T25 GPM w / o GF and gently rinse 2-3 times. Collect cells. Centrifuge: 200g, 5 min, RT. Use timer. OPTIONAL: After quenching, take an image of each treated flask using a 2.5x objective and record the % of cells remaining. Aspirate the supernatant, firmly tap the pellet, and gently resuspend in 5 ml of GPM+GFS for each T25 flask taken using a 5 ml pipette. Be careful not to pipette over the top.

[0234] Cell counting protocol: Prepare two marked Eppendorf tubes and transfer approximately 100 μl of cell suspension to each separate Eppendorf tube. For 5x dilutions: Prepare two additional Eppendorf tubes with 200 μl GPM+GF and transfer to each medium containing tube of cell suspension prepared in section 0, 50 μl, for a final volume of 250 μl. Mix thoroughly. Count each diluted Eppendorf tube using an NC-200 cell counter.

[0235] Cell seeding protocol: According to Table 16, determine the volume of medium and the number of cells required for seeding (2.67 x 10 4 Viable cells / cm 2 Calculate the density of

[0236] Table 16. Volumes and cell numbers for seeding TIFF2024512385000017.tif11130

[0237] Retrieve LN521 coated flasks from refrigerator if applicable. For LN521 coated flasks: Add 6ml GPM+GF to each T225 LN521 coated flask. Incubate flasks for 1 minute at RT. Use timer. Aspirate coating solution and GPM+GF medium from flask. Wash with 24ml PBS(+) / T225. Add calculated GPM+GF to flask. Gently pipette to mix cell suspension and seed to calculated cell volume. Transfer vessel to incubator and gently agitate vessel to evenly distribute cells over vessel surface. IPC Day 7: Cryopreserve cells for samples.

[0238] Medium change, days 9-13: On days 9, 11, and 13, observe cultures, take pictures using 2.5x, 5x, and 10x objectives, and measure lactate. Thaw GFs according to Table 15. Determine required GPM volume and warm in a 37 °C water bath. Add GFs to GPM according to Table 15. Aspirate medium and add 90 ml of GPM+GF medium to each T225 flask.

[0239] Harvesting, Day 14: Preparation of PBS Wheel: On day 13, label and pre-fill a PBS wheel with 70 ml of GPM medium, tilt the PBS and wash the walls of the wheel. Incubate overnight at 37 °C and 5% CO2 in a humidified CO2 incubator at 35 RPM. Observe the cultures on day 14 and take pictures using 2.5x, 5x and 10x objectives. Measure lactate. Divide 2 ml of CM into two cryovials (1 ml each) and store at -80 °C. Thaw GFs according to Table 15 and warm up the required GPM volume (for final solutions of GPM + GFs and GPM w / o GFs). Add GF volume to the warmed GPM according to Table 15.

[0240] Harvest: Aspirate medium. Add 24 ml PBS(-) to each T225 flask. Tilt flask 2-3 times and incubate 1 min at RT. Aspirate and add 9 ml / T225 TrypLE Select. Incubate 8 min at 37°C and 5% CO2. Firmly tap flask on one side, tilt flask to wash cells, and firmly tap flask on other side. Add 18 ml GPM w / o GF. Add medium directly to cells. Collect cells in a conical tube. Add 18 ml / T225 GPM w / o GF and rinse gently 2-3 times. Collect cells. Centrifuge: 170-180g, 5 min, RT. Optional: After quenching, take an image of each treated flask using a 2.5x objective and record the % of cells remaining. Aspirate the supernatant, tap the pellet firmly, resuspend in 30 ml (first resuspend in 5 ml with a 5 ml pipette, then add 25 ml) of GPM+GFS per T225 flask, and collect. Be careful not to do this over the pipette.

[0241] Cell counting: Prepare two marked Eppendorf tubes and transfer approximately 100 μl of cell suspension to each separate Eppendorf tube. For 4-fold dilutions: Prepare two additional Eppendorf tubes with 150 μl GPM+GF and transfer 50 μl of cell suspension to each medium containing tube, for a final volume of 200 μl. Mix thoroughly. Count each diluted Eppendorf tube using an NC-200 cell counter.

[0242] Cell seeding: According to Table 17, the volume of medium and the number of cells required for seeding (128 x 10 6 Calculate the number of live cells (number of viable cells / 0.1 PBS wheel).

[0243] Table 17. Volumes and cell numbers for seeding TIFF2024512385000018.tif11130

[0244] Aspirate pre-filled media from wheel. Add calculated GPM+GF to flask. Gently pipette to mix cell suspension and seed to calculated cell volume. Place PBS wheel container on PBSmini in 37°C and 5% humidified CO2 incubator. Set PBSmini speed to 35 RPM for 24±4 hours. IPC Day 14: Cryopreserve cells.

[0245] Day 15: Increase the speed of the PBSmini to 45 RPM. Turn on the light on the PBSmini and carefully check that no clumps are observed while the PBS wheel is still spinning. Only if clumps are observed, remove them quickly to avoid settling. Do not remove the PBS wheel from the PBSmini unless necessary.

[0246] Medium change: Days 16, 18, 20: On days 16, 18, and 20, observe the cultures, take pictures using 2.5x and 5x objectives, and measure lactate. Thaw GF according to Table 15. Determine the required GPM volume and warm in a 37°C water bath. Add GF to GPM according to Table 15. Remove the PBS wheel from the PBSmini and place it on a rack in the incubator for the settling time indicated per day, as per Table 18.

[0247] (Table 18) Settling time per day TIFF2024512385000019.tif11128

[0248] Check for sedimentation before changing the medium. If aggregates have not settled, extend the sedimentation time for another 5 minutes. Using a 10 ml pipette, remove 80% (56 ml) of the volume in the PBS wheel and keep it for lactate measurement. Add 56 ml (same volume of GPM+GF as removed in the section above) to the side of the PBS wheel. Inspect the PBS wheel for clump formation and if clumps are observed, remove them as investigation and removal of clumps is critical to the process. Place the PBS wheel back into the PBSmini in a humidified CO2 incubator at 37°C and 5% CO2, 45 RPM. Measure lactate.

[0249] Smoothing of aggregates, day 21: 1 day to 1 week prior to harvest, determine the number of T75 / T150 flasks and coat with the appropriate coating solution (rhLN521 or iMatrix-511-E8 at 10 μg / ml). Measure lactate approximately 24 hours after medium change to determine which vessels to seed. If lactate concentration is 15.00 mM or less, seed the aggregates into one T75. Otherwise, seed the aggregates into one T150. Divide 2 ml of CM into two cryovials (1 ml each) and store at -80°C. Observe the cultures and photograph the aggregates. Thaw GFs and warm the required GPM volume according to Table 15 (for final solution of GPM+GF). Add GF volume to warmed GPM according to Table 15.

[0250] Preparation of coated vessels for seeding: Retrieve coated flasks from refrigerator. Label each flask. Add 2 / 4 ml of GPM+GF to each T75 / T150 coated flask. Incubate flasks for 1 min at RT. Use timer. Aspirate coating solution and GPM+GF medium from flasks. For LN521 coated flasks only, wash with 10 / 20 ml of PBS(+) for each T75 / T150 coated vessel. Add 20 / 40 ml of GPM+GF to T75 / T150. Transfer vessels to humidified CO2 incubator at 37°C and 5% CO2 until plating. Using a 25 ml pipette, transfer 35 ml of PBS-wheel content to a 50 ml conical tube. Allow aggregates to settle for 4 min. Using a 25 ml pipette, manually aspirate and remove 30 ml of GPM to another 50 ml tube. Repeat the step (25 ml pipette transfer 35 ml of PBS-wheel contents to a 50 ml conical tube, allow aggregates to settle for 4 min) and transfer the remainder of the aggregates from the PBS-wheel to the same 50 ml conical tube labeled "seeded aggregates". During the settling of the aggregates, use a 2 ml pipette to collect the remainder of the aggregates left on the PBS-wheel and slowly transfer them to the bottom of the "seeded aggregates" tube. While the aggregates are settling in the 50 ml tube, add 20 ml of GPM+GF to the PBS-wheel. Repeat the step one more time (25 ml pipette transfer 35 ml of the PBS-wheel content into a 50 ml conical tube, allow the aggregates to settle for 4 min, transfer the rest of the aggregates from the PBS-wheel into the same 50 ml conical tube labeled "seeded aggregates", during aggregate sedimentation, use a 2 ml pipette to collect the rest of the aggregates remaining in the PBS-wheel, slowly transfer it to the bottom of the "seeded aggregates" tube while the aggregates are settling into the 50 ml tube, add 20 ml of GPM+GF to the PBS-wheel). Collect the washing solution from the PBS-wheel into the 50 ml tube. Tilt the PBS-wheel and collect the remaining medium from the PBS-wheel using a 2 ml pipette. Centrifuge the 50 ml conical tube: 140 G, 3 min, RT.Transfer the prepared vessel into the BSC. Verify that the top surface of the flask is clear. If necessary, wash the surface with medium before seeding. Gently aspirate the supernatant. Using a 25 ml pipette, resuspend the pellet with 5 / 10 ml of GPM+GF per T75 / T150 seeding (do not tap before resuspension). Measure the volume of the suspension. Quickly seed the total volume into the designated vessel (keep the flask vertical to avoid non-homogeneous attachment of aggregates). Using a 25 ml pipette, wash the tube with excess volume (75 / 150 cm, respectively). 2 Add 10 ml of PBS (complete 30 / 60 ml per seeding) to the flask. Incubate the flask in a humidified incubator at 37 °C and 5% CO2. While in the incubator, ensure that the aggregates are evenly distributed in the flask.

[0251] Medium Change Days 23, 25, 27: On days 23, 25, and 27, observe cultures, take pictures using 5x and 10x objectives, and measure lactate. Thaw GFs according to Table 15. Determine required GPM volume and warm in 37°C water bath. Add GFs to GPM according to Table 15. Aspirate medium and add 30 / 60ml of GPM+GF medium to each T75 / T150 flask.

[0252] Day 28: If necessary, 1 day to 1 week before harvest, determine the number of flasks required and coat with 10 µg / ml rhLN521. Observe and photograph the cultures using 5x and 10x objectives. Measure lactate concentration. Divide 2 ml of CM into 2 cryovials (1 ml each) and store at -80 °C. Thaw GFs according to Table 15 and warm up the required GPM volume (for final solutions of GPM + GF and GPM w / o GF). Add GF volume to warmed GPM according to Table 15. Harvest: Aspirate medium. Add 8 / 16 ml of PBS(-) to each T75 / T150 flask. Tilt flask 2-3 times and incubate at RT for 1 min. Aspirate and add 3 / 6 ml per T75 / T150 TrypLE Select. Incubate at 37 °C and 5% CO2 for 7 min. Gently tap the flask on one side, tilt the flask to wash the cells, and gently tap the other side of the flask. Using a 25 ml pipette, gently add 6 / 12 ml of GPM w / o GF on the surface of the cells. Collect the cells in a conical tube. Do not wash the surface of the cells. Centrifuge: 260-270 g, 10 min, RT. Aspirate the supernatant, gently tap the pellet until it dissociates from the tube, and resuspend in 15 / 30 ml of GPM+GFS for each T75 / T150 flask harvested. First, resuspend in 5 ml using a 25 ml pipette, pipette up and down 2-3 times, then add the remaining volume using a 25 ml pipette. Be careful not to do it over the pipette.

[0253] Cell counting: Prepare two marked Eppendorf tubes and transfer approximately 150 μl of cell suspension to each separate Eppendorf tube, being careful not to transfer aggregates for counting samples. For 2-fold dilutions: Prepare two additional Eppendorf tubes with 100 μl of GPM+GF and transfer 100 μl of cell suspension to each medium containing tube, bringing to a final volume of 200 μl. Mix thoroughly. Count each diluted Eppendorf tube using an NC-200 cell counter.

[0254] Cell seeding: According to Table 16, determine the volume of medium and the number of cells required for seeding (4.0 x 10 4 Viable cells / cm 2 Calculate the density of

[0255] Table 19. Volumes and cell numbers for seeding TIFF2024512385000020.tif11130

[0256] Retrieve LN521 coated flasks from the refrigerator, if applicable. Label each flask. For LN521 coated flasks: Add 6ml GPM+GF to each T225 LN521 coated flask. Incubate flasks for 1 minute at RT. Use a timer. Aspirate coating solution and GPM+GF medium from flask. Wash with 24ml PBS(+) / T225. Add calculated GPM+GF to flask. Prepare medium+E8 pool, if applicable. Gently pipette to mix cell suspension and seed to calculated cell volume. Transfer flask to incubator and gently agitate flask to evenly distribute cells over the surface of vessel.

[0257] IPC Day 28: Cryopreserve cells. Medium Change Days 30, 32, 34: On days 30, 32, and 34, observe cultures, take pictures using 5x and 10x objectives, and measure lactate. Thaw GFs according to Table 15. Determine required GPM volume and warm in 37°C water bath. Add GFs to GPM according to Table 15. Aspirate medium and add 90ml GPM+GF medium to each T225 flask.

[0258] Day 35: On day 35, observe the culture and take pictures using 5x and 10x objectives. Measure lactate. Divide 2ml of CM into two cryovials (1ml each) and store at -80°C. Thaw GFs and warm up the required GPM volume according to Table 15 (for final solutions of GPM+GF and GPM w / o GF). If cells are not seeded for further culture, do not thaw GFs and do not prepare GPM+GF, only GPM w / o GF is needed. Add GF volume to warmed GPM according to Table 15.

[0259] Harvesting: Aspirate medium. Add 24 ml PBS(-) to each T225 flask. Tilt vessel 2-3 times and incubate 1 min at RT. Aspirate and add 9 ml / T225 TrypLE Select. Incubate 7 min at 37°C and 5% CO2. Gently tap vessel on one side, tilt flask to wash cells, and gently tap vessel on the other side. Add 18 ml GPM w / o GF. Add medium directly to cells. Collect cells into conical tube. Add 18 ml / T225 GPM w / o GF and rinse gently 2-3 times. Harvest cells. If only harvesting cells without seeding, proceed to cell count. If seeding cells for further culture, centrifuge: 260-270 g, 10 min, RT. Aspirate supernatant, firmly tap pellet and resuspend in 30 ml GPM+GF per harvested T225 vessel. First, resuspend in 5 ml and pipette up and down 2-3 times, then use a 25 ml pipette to add the remaining volume. Be careful not to over pipette.

[0260] Cell counting: Prepare two additional Eppendorf tubes with 200 μl of cell suspension and transfer to each tube. Count each tube using an NC-200 cell counter.

[0261] Cell seeding (if applicable): Determine the volume of medium and number of cells (4.0 x 10) required for seeding according to Table 16. 4 Viable cells / cm 2 Calculate the density of

[0262] Table 20. Volumes and cell numbers for seeding TIFF2024512385000021.tif11130

[0263] Retrieve LN521 coated flasks from refrigerator, if applicable. Label each flask. For LN521 coated flasks: Add 6ml GPM+GF to each T225 LN521 coated flask. Incubate flasks for 1 minute at RT. Use timer. Aspirate coating solution and GPM+GF medium from flask. Add calculated GPM+GF to flask. Prepare medium+E8 pool, if applicable. Gently pipette to mix cell suspension and seed to calculated cell volume. Transfer flask to incubator and gently agitate flask to evenly distribute cells over flask surface. IPC Day 35: Cryopreserve cells.

[0264] Medium Change Days 37, 39, 41: On days 37, 39, and 41, observe cultures, take pictures using 5x and 10x objectives, and measure lactate. Thaw GFs according to Table 15. Determine required GPM volume and warm in a 37°C water bath. Add GFs to GPM according to Table 15. Aspirate medium and add 90ml of GPM+GF medium to each T225 flask.

[0265] Day 42: On day 42, observe the culture and take pictures using 5x and 10x objectives. Measure lactate. Divide 2ml of CM into 2 cryovials (1ml each) and store at -80°C. Warm the required amount of GPM.

[0266] Harvest: Aspirate media. Add 24ml PBS(-) to each T225 flask. Tilt flask 2-3 times and incubate 1 min at RT. Aspirate and add 9ml / T225 TrypLE Select. Incubate 7 min at 37°C and 5% CO2. Gently tap flask on one side, tilt flask to wash cells, and gently tap flask on other side. Add 18ml GPM w / o GF. Note: Add media directly to cells. Collect cells in a conical tube. Add 18ml / T225 GPM w / o GF and rinse gently 2-3 times. Cell Count: Prepare 2 additional Eppendorf tubes with 200μl of cell suspension and transfer to each tube. Count each tube using an NC-200 cell counter. IPC Day 42: Cryopreserve cells.

[0267] Example 5: Large scale OPC batch production: The purpose of this example is to demonstrate the establishment of large-scale conditions for the generation of OPCs.

[0268] Experimental procedure: Figure 13 shows the steps of the OPC differentiation protocol.

[0269] Culture of hESCs (stage 1) - H1 hESCs (PILOT-LCT-H1-002) were cultured on LN521 coated vessels for 3 passages according to ERI numbers ERI-H1-01 and ERI-H1-02. At the end of p30+6+4+2, cells were seeded for 4 days for initiation of OPC differentiation.

[0270] OPC Differentiation - On day 4 of stage 1 p30+6+4+3, OPC differentiation was initiated in six T25 vessels according to Figure 13. (Differentiation protocol from day 0).

[0271] Days 0-3 - Culture as above. Change GPM medium daily, supplemented with 2 μM dorsomorphin, 10 μM PD0325901, and 1 μM RA.

[0272] Days 4-6 - Culture according to the protocol of Example 5. GPM medium supplemented with 150 μM AA and 1 μM RA was changed daily.

[0273] Day 7 - Cells were harvested using TS according to the protocol in Example 5. Eleven T225 vessels were cultured at 26,667 viable cells / cm in GPM supplemented with 10 ng / ml EGF, 10 ng / ml hs-FGF, and 10 μM RI. 2 Then, they were seeded into containers coated with LN521.

[0274] Days 9-13 - Culture according to the protocol of Example 5. GPM medium supplemented with 10 ng / ml EGF and 10 ng / ml hs-FGF was replaced every 2 days.

[0275] Day 14 - 128 x 10 6 TS and 12 PBS wheels were seeded for aggregate formation in GPM supplemented with 10 ng / ml EGF, 10 ng / ml hs-FGF, and 10 μM RI at 1 viable cell / PBS wheel (for ongoing run no. 20-EROPCRD-02). Cells were harvested according to the protocol in Example 5. Additionally, all other cells were cryopreserved as ICB in CS10.

[0276] Day 14 Thawing - For ICB thawing, cryopreserved cells were thawed, counted and seeded in PBS wheels according to study design no. ER-ICBRD-02. 128 × 10 6 10 x 10 live cells / PBS wheel were seeded in six vessels according to the protocol in Example 5. 6 10 viable cells / PBS wheel were seeded in one vessel.

[0277] Days 16-20 - Culture according to the protocol of Example 5. Change 80% of GPM medium supplemented with 10 ng / ml EGF and 10 ng / ml hs-FGF every 2 days.

[0278] Day 21 - Aggregates were blunted on day 21 and seeded in seven T75 LN521 coated vessels in GPM supplemented with 20 ng / ml EGF and 10 ng / ml PDGF-AA.

[0279] Days 23-27 - Culture as above. Change GPM medium supplemented with 20 ng / ml EGF and 10 ng / ml PDGF-AA every 2 days.

[0280] Day 28 - Cells were harvested with TS and plated at 40,000 viable cells / cm on 36 T225 E8 directly coated vessels in GPM supplemented with 10 ng / ml EGF and 10 ng / ml PDGF-AA. 2 The seeds were sown in.

[0281] Days 29-34 - Culture as above. Change GPM medium supplemented with 20 ng / ml EGF and 10 ng / ml PDGF-AA every 2 days.

[0282] Day 35 - Cells were harvested with TS and plated at 40,000 viable cells / cm in 45 Cell-factories E8 directly coated vessels in GPM supplemented with 10 ng / ml EGF and 10 ng / ml PDGF-AA. 2 The deviation of CO2 levels in the incubator on day 35 was reported. Since no differences were found between the affected vessel and all the other vessels, the morphological and lactate level evaluations showed that the deviation entailed a small risk for the cells, and therefore it was decided to proceed with all vessels.

[0283] Days 35-41 - Culture as above. Change GPM medium supplemented with 20 ng / ml EGF and 10 ng / ml PDGF-AA every 2 days.

[0284] Day 42 - Harvesting and cryopreservation of cells with TS in TAI formulation according to number ERI-OPC-07.

[0285] QC Testing - Flow Cytometry - Analysis of markers by FACS was performed on cryopreserved cells on days 7, 14, 28, 35, and 42 according to Table 21.

[0286] (Table 21) FACS staining TIFF2024512385000022.tif98170

[0287] Batch Release Acceptance Criteria - The proposed acceptance criteria for batch release testing of cells at Day 42 are presented in Table 22.

[0288] Table 22: Batch release proposed acceptance criteria. TIFF2024512385000023.tif128142

[0289] Results: Passage parameters All passaging parameters for number 20-EROPCRD-02 and number ER-ICBRD-02 are presented in Table 23 and Table 24. At each passage, yield and PDL values ​​were calculated according to the following formula: TIFF2024512385000024.tif44141

[0290] Table 23. Passage parameters Days 0 to 28 TIFF2024512385000025.tif22940

[0291] Table 24. Passage parameters Days 35-42 TIFF2024512385000026.tif19725

[0292] Up to day 14, all passaging parameters were within the range of the previous successful run (report no. OPC-REP-03). On the other hand, between days 14-21, extensive clump formation was observed in the ongoing run, thus reducing the number of clumps and lactates on day 21 to an average of 6.57 mM. As a result, the ongoing run was stopped and cryopreserved cells were thawed and seeded as described above. In ICB-thawed cells, no clump formation was observed and, as a result, lactates on day 21 were higher (average 7.58 mM). Passaging parameters from days 28-42 were within the expected acceptable ranges.

[0293] Marker Expression - Marker expression of No. 20-EROPCRD-02 and No. ER-ICBRD-02 is presented in Table 25.

[0294] Table 25. Marker expression on days 7 to 28 TIFF2024512385000027.tif23722

[0295] Table 26. Marker expression at day 35 TIFF2024512385000028.tif19629

[0296] As described in the passaging parameters, marker expression up to day 35 did not indicate any major discrepancies from previous successful runs.

[0297] Morphology - Starting from day 35, successful and unsuccessful cultures differ by different morphologies. In Figure 12, the photograph of number ER-ICBRD-02 shows that the cells were organized as small dense cells and did not acquire the spindle-shaped "failed" morphology.

[0298] Batch Release - A summary of the No. ER-ICBRD-02 batch release test according to Table 23 is presented in Table 27.

[0299] Table 27: Batch release test of No. ER-ICBRD-02 TIFF2024512385000029.tif180170

[0300] Batch ER-ICBRD-02 cells met all the acceptance criteria proposed for batch release.

[0301] Discussion: The ongoing run of batch no. 20-EROPCRD-02 and run no. ER-ICBRD-02 thawed from an ICB day 14 operational run were manufactured in the R&D laboratory as part of the proof of feasibility of full-scale LCTOPC1 batch production, and their products are designated for comparability and animal study purposes.

[0302] In the ongoing run of number 20-EROPCRD-02, it became clear that processing multiple vessels simultaneously resulted in the formation of an abnormally high number of clumps in all PBS wheels, which led to the termination of this run. Clump formation in the PBS wheels between days 14-21 impairs the differentiation process with aggregate loss and non-indicative lactate readout on day 21. In addition, cryopreservation and thawing of cells harvested on day 14 (LCTOPC1 ICB) can reduce the number of clumps formed on days 14-21 compared to ongoing runs of the same cells. Indeed, in run number ER-ICBRD-02, which was initiated from ICBs generated during number 20-EROPCRD-02, no clumps were observed in the PBS wheels. These differences between the ongoing run and the ICB run led us to conclude that cryopreservation and thawing of ICBs on day 14 is beneficial and necessary for the OPC1 process in GMP facilities.

[0303] Batch number ER-ICBRD-02 passed all proposed IPC and batch release criteria in assays tested and was approved as viable for full scale LCTOPC1 batch manufacturing with cryopreservation of ICB at day 14. This batch, number ER-ICBRD-02, represents the LCTOPC1 GMP process and release cells.

[0304] Conclusion: No. ER-ICBRD-02 is a representative batch of GMP process, releasing LCTOPC1 cells and passed all the proposed criteria for batch release. Full-scale manufacturing of OPC1 cells meeting the proposed criteria for batch release is feasible. Cryopreserving the ICB on day 14 and thawing before seeding for aggregate formation is beneficial for the cells and prevents clump formation.

[0305] Example 6: Differentiation of day 14 thawed cells to produce OPCs. The purpose of this example is to demonstrate the establishment of conditions for differentiation of thawed cells into OPCs.

[0306] Experimental procedure: Figure 13 shows the steps of the OPC differentiation protocol.

[0307] (Table 28) Test conditions TIFF2024512385000030.tif95170** Pre-clean with anti-adhesion fluid - The anti-adhesion fluid was thought to prevent the aggregates from sticking to the bottom of the wheel on days 14-16. It was later found that the main reason for the loss of aggregates was their tendency to stick to each other and form large clumps.

[0308] HESC culture (stage 1) - H1 hESCs (PILOT-LCT-H1-002) were cultured for three passages on LN521-coated vessels. At the end of p30+6+4+2, cells were seeded for 4 days for initiation of OPC differentiation.

[0309] OPC Differentiation - OPC differentiation was initiated on day 4 of p30+6+4+3 in stage 1. All variables tested are detailed in Table 28.

[0310] Days 0-3 - Cells were cultured in GPM medium supplemented with 2 μM dorsomorphin, 10 μM PD0325901, and 1 μM RA, and replaced daily.

[0311] Days 4-6 - Cultures were replaced daily with GPM medium supplemented with 150 μM AA and 1 μM RA.

[0312] Day 7 - Cells were harvested with TS and plated at 26,667 viable cells / cm on LN521-coated vessels in GPM supplemented with 10 ng / ml EGF, 10 ng / ml hs-FGF, and 10 μM RI. 2 The seeds were sown in.

[0313] Days 9-13 - Culture in GPM medium supplemented with 10 ng / ml EGF and 10 ng / ml hs-FGF, changed every 2 days.

[0314] Day 14 - Cells were harvested in TS. For ongoing cultures, unseeded cells were cryopreserved in CS10.

[0315] ICB day 14 thaw - cells were thawed, resuspended, counted, and 128 x 10 6 Viable cells were seeded in 70 ml of GPM medium supplemented with 10 ng / ml EGF, 10 ng / ml hs-FGF, and 10 μM RI.

[0316] Days 16-20: Culture in 80% GPM medium supplemented with 10 ng / ml EGF and 10 ng / ml hs-FGF, and replaced every 2 days.

[0317] Day 21 - Aggregates were assayed for lactate on day 21 in GPM supplemented with 20 ng / ml EGF and 10 ng / ml PDGF-AA on LN521-coated vessels (75 cm if lactate concentration was less than 15.00 mM). 2 or 150 cm if lactate concentration is 15.00 mM or more. 2 The data were smoothed according to the following formula:

[0318] Days 23-27 - Culture in GPM medium supplemented with 20 ng / ml EGF and 10 ng / ml PDGF-AA, changed every 2 days.

[0319] Day 28 - Cells were harvested with TS and plated on LN521-coated vessels at 40,000 viable cells / cm.2 The cells were seeded in GPM supplemented with 10 ng / ml EGF and 10 ng / ml PDGF-AA at 37°C.

[0320] Days 29-34 - Culture in GPM medium supplemented with 20 ng / ml EGF and 10 ng / ml PDGF-AA, changed every 2 days.

[0321] Day 35 - Cells were harvested with TS and plated on LN521-coated vessels at 40,000 viable cells / cm. 2 The cells were seeded in GPM supplemented with 10 ng / ml EGF and 10 ng / ml PDGF-AA at 37°C.

[0322] Days 36-41 - Culture as above. Change GPM medium supplemented with 20 ng / ml EGF and 10 ng / ml PDGF-AA every 2 days.

[0323] Day 42 - Harvesting and cryopreservation of cells by TS.

[0324] QC Testing - Flow Cytometry - Analysis of markers by FACS was performed on cryopreserved cells on days 7, 14, 28, 35, and 42 according to Table 29.

[0325] (Table 29) FACS staining TIFF2024512385000031.tif75163

[0326] Decorin Secretion - Cryopreserved cells from days 35 and 42 were tested for decorin secretion.

[0327] Acceptance criteria - Assays to evaluate OPC batches were developed along with the process so that successful batches were defined according to the following criteria: purity markers (PDGFRα) >95.00%, impurity markers (Claudin, EpCAM, CK7) <2.00%, decorin secretion (cells on day 42) >25.00ng / ml, morphology - small and compact cells, absence of elongated spindle-shaped morphology.

[0328] Results: Passage parameters At each passage, yield and PDL values ​​were calculated according to the following formula: TIFF2024512385000032.tif44128

[0329] Table 30: Passage parameters Days 21-35. All ICB14 runs are as follows: TIFF2024512385000033.tif243110

[0330] Table 31: Passage parameters Day 42. All ICB14 runs are as follows: TIFF2024512385000034.tif22069

[0331] Some similarities can be found between the passage parameters of the RD run and its derivative, the ICB run, mainly in the lactate concentrations on days 21 and 28, all other parameters showed acceptable values. The low survival in run number 10.1 on day 28 is related to the manual handling.

[0332] Marker Expression - At each passage, yield and PDL values ​​were calculated according to the following formula:

[0333] (Table 32) Marker expression on day 28. All ICB14 runs are below. TIFF2024512385000035.tif20169

[0334] (Table 33) Marker expression on day 35. All ICB14 runs are below. TIFF2024512385000036.tif24383

[0335] (Table 34) Marker expression on day 42. All ICB14 runs are below. TIFF2024512385000037.tif24397*Marker expression of failed runs is outside of acceptance criteria.

[0336] All ICB groups except for group B of run 10.1 met the proposed acceptance criteria for marker expression at day 42, indicating that the cryopreservation and thawing procedures at day 14 did not harm the cells or the OPC1 differentiation process. For group B of run 10.1, the PBS wheel treatment with anti-adhesion solution did not improve the process and is not eligible for final OPC1 processing in a GMP facility.

[0337] Efficacy Assay—Decorin secretion on days 35 and 42 is presented in Table 35.

[0338] Table 35: Decorin secretion. All ICB14 runs are as follows: TIFF2024512385000038.tif54128 Decorin secretion in all groups exceeded 25 ng / ml on day 42, meeting expected criteria.

[0339] Morphology - Starting from day 35, successful and failed cultures differ by different morphologies. In Figures 14 and 15, pictures of the morphology of ongoing runs are presented in comparison to ICB-thawed cells. At days 35 and 42, cells were organized as small dense cells and did not acquire the spindle-shaped "failed" morphology.

[0340] The morphological similarities of the clear day 21 aggregates between the origin runs and their derived ICBs are presented in Figure 14 .

[0341] Discussion: Cryopreservation of day 14 ICBs during the OPC1 differentiation process retains significant potential advantages such as reduction of clump formation in the PBS wheel that resulted in run failure, production of multiple batches from the same ICB, and allows flexibility for the 9 week long process. Results showed that OPC1 cells produced from both ongoing full runs and those day 14 ICB-derived runs acquired the expected baseline for an OPC population.

[0342] In this report, we summarize the differentiation results of thawed cells on day 14 compared to ongoing groups of origin. Passage parameters, marker expression and efficacy assays (decorin secretion) showed that all cells thawed on day 14 met the proposed acceptance criteria for OPC1 cells, except for group B of run 10.1. For group B of run 10.1, the PBS wheels were pretreated with anti-adhesion solution prior to seeding the cells on day 14. This treatment did not show a clear advantage to the process compared to pre-filling with GPM, so it was decided not to use this material in the process. In addition, it was found that implementing the ICB cryopreservation procedure described herein improved the recovery % from 75% to 92% (see Table 28). The procedure will be implemented in the CCN OPC1 process.

[0343] Conclusion: Day 14 may be a breakpoint for the OPC1 differentiation protocol. Day 14 ICB cells can be thawed directly onto a PBS wheel and continue the established process of the ongoing protocol. Day 14 ICB show clear advantages in reducing deposits in the aggregation step from days 14 to 21. Post-thaw recovery at day 14 was better obtained when cryopreservation was performed according to the methods described herein.

[0344] References Duester, G. (2008).”Retinoic acid synthesis and signaling during early organogenesis.”Cell 134(6):921-931. Hu, BY, ZWDu, XJLi, M. Ayala and SCZhang (2009).”Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects.”Development 136(9):1443-1452. Janesick,A.,S.C.Wu and B.Blumberg(2015).”Retinoic acid signaling and neuronal differentiation.”Cell Mol Life Sci 72(8):1559-1576. Koch,U.,R.Lehal and F.Radtke(2013).”Stem cells living with a Notch.”Development 140(4):689-704. Kudoh,T.,S.W.Wilson and I.B.Dawid(2002).”Distinct roles for Fgf,Wnt and retinoic acid in posteriorizing the neural ectoderm.”Development 129(18):4335-4346. Li,Y.and M.M.Parast(2014).”BMP4 regulation of human trophoblast development.”Int J Dev Biol 58(2-4):239-246. Ota,M.and K.Ito(2006).”BMP and FGF-2 regulate neurogenin-2 expression and the differentiation of sensory neurons and glia.”Dev Dyn 235(3):646-655. Patthey,C.and L.Gunhaga(2014).”Signaling pathways regulating ectodermal cell fate choices.”Exp Cell Res 321(1):11-16. Sui,L.,L.Bouwens and J.K.Mfopou(2013).”Signaling pathways during maintenance and definitive endoderm differentiation of embryonic stem cells.”Int J Dev Biol 57(1):1-12. Watabe, T. and K. Miyazono (2009). “Roles of TGF-beta family signaling in stem cell renewal and differentiation.” Cell Res 19(1):103-115. Zheng, W., Q. Li, C. Zhao, Y. Da, HLZhang and Z. Chen (2018). “Differentiation of Glial Cells From hiPSCs: Potential Applications in Neurological Diseases and Cell Replacement Therapy.” Front Cell Neurosci 12:239.

[0345] All references provided herein (including all non-patent literature, patents, and patent publications) are hereby incorporated by reference in their entirety.

Claims

1. 1. A method for obtaining a population of oligodendrocyte precursor cells (OPCs) from undifferentiated pluripotent stem cells, comprising: a) obtaining a culture of undifferentiated pluripotent human embryonic stem cells (hESCs); b) culturing the undifferentiated pluripotent hESCs for a first period of time under culture conditions sufficient to induce differentiation of the hESCs into neuroectodermal cells and neural progenitor cells; c) culturing the neural progenitor cells from step b) for a second period of time under culture conditions sufficient to differentiate the neural progenitor cells into OPCs; A method comprising:

2. 2. The method of claim 1, wherein the pluripotent cells in step b) are cultured on laminin in adherent tissue culture vessels, or in suspension complexes, or both.

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

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

5. 2. The method of claim 1, wherein the undifferentiated hESCs of step b) are cultured in the presence of dorsomorphin, PD0325901, and RA.

6. The method of claim 5, further comprising culturing in the presence of AA and RA.

7. The method of claim 1, wherein the neuroectodermal cells from step b) are cultured in the presence of EGF and hsbFGF.

8. The method of claim 7, further comprising culturing in the presence of EGF and PDGF-AA.

9. 10. The method of claim 1, wherein the first period of time is from about 3 days to about 60 days.

10. 10. The method of claim 9, wherein the first period of time is from about 10 days to about 15 days.

11. 10. The method of claim 9, wherein the first period of time is about 14 days.

12. The method of claim 1 , wherein the second period of time is from about 10 days to about 60 days.

13. The method of claim 12, wherein the second period of time is from about 20 days to about 40 days.

14. 13. The method of claim 12, wherein the second period of time is about 28 days.

15. The method of claim 1, wherein the differentiated hESCs in step b) are cryopreserved at about day 14.

16. 16. The method of claim 15, wherein the cryopreserved cells are thawed and the thawed cells are subsequently cultured in any remaining steps of the method.

17. 16. The method of claim 15, comprising cryopreserving the neural precursor cells from step b) at or near the completion of the first period of time.

18. 17. The method of claim 16, wherein the cryopreserved neuroectodermal cells are thawed and cultured according to step c).

19. 15. The method of claim 14, wherein the OPCs of step c) are cryopreserved.

20. 20. The method of claim 19, wherein cryopreserved OPCs are thawed.

21. 2. The method of claim 1, wherein the OPCs express one or more markers selected from neural / glial antigen 2 (NG2), platelet-derived growth factor receptor A (PDGFRα), and platelet-derived growth factor receptor B (PDGFRβ).

22. 1. A method of formulating an oligodendrocyte precursor cell (OPC) composition for administration to a subject immediately after thawing, comprising: (a) suspending the OPCs of claim 1 in a cryopreservation medium to form a cell suspension; (b) storing the cell suspension at a cryopreservation temperature; and (c) thawing the cryopreserved suspension; A method comprising:

23. 23. The method of claim 22, wherein the cryopreservation medium comprises one or more of 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, dimethylsulfoxide (DMSO), and water.

24. 13. A pharmaceutical composition for administration to a subject, comprising the OPCs of claim 1 and a cryopreservation medium.

25. 25. The pharmaceutical composition of claim 24, wherein the cryopreservation medium comprises one or more of 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, dimethylsulfoxide (DMSO), and water.

26. A composition for use in a method for treating spinal cord injury in a subject, comprising oligodendrocyte precursor cells (OPCs), the method comprising administering a therapeutically effective amount of the composition to the subject.

27. 27. The composition for use of claim 26, wherein said administering comprises administering said composition within or adjacent to a site of spinal cord injury.

28. 27. The composition for use according to claim 26, wherein administering is by injection.

29. 27. The composition for use according to claim 26, wherein said administering is by implantation.

30. 27. The composition for use according to claim 26, wherein said administering is by implantation.

31. The cell concentration was approximately 1 x 10 per mL. ６ ~100 x 10 cells per mL ６ 27. The composition for use according to claim 26, wherein the cell is a cell.

32. 27. The composition for use according to claim 26, stored in a volume of about 100 microliters to about 1 milliliter.

33. The cell concentration was 100 x 10 per mL. ６ 27. The composition for use according to claim 26, wherein the cell is a cell.

34. 27. The composition for use according to claim 26, stored in a volume of 250 microliters.

35. 27. The composition for use according to claim 26, stored in a volume of 300 microliters.

36. 27. The composition for use according to claim 26, wherein the cryopreservation medium is a freezing solution.

37. 27. The composition for use according to claim 26, wherein the freezing solution is CryoStor 10 (CS10).

38. 27. The composition for use of claim 26, wherein the OPCs express one or more markers selected from neural / glial antigen 2 (NG2), platelet-derived growth factor receptor A (PDGFRα), and platelet-derived growth factor receptor B (PDGFRβ).

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