Oligodendrocyte precursor cell composition

Abstract

To provide compositions comprising a population of oligodendrocyte progenitor cells (OPCs), and to provide methods of making and using the same.SOLUTION: In one aspect, provided is a container comprising a composition, where the composition comprises a population of cells comprising a plurality of OPCs, and where the population of cells comprises less than 15% undesirable cell types. In another aspect, the population of cells comprises less than 15% undesirable epithelial lineage cells. In yet another aspect, the population of cells comprises less than 2% K7 positive cells. In an aspect, provided is a population of cells comprising a plurality of oligodendrocyte progenitor cells that forms less than one epithelial cyst per 100,000 cells in a cyst assay. An even further aspect of the present disclosure is a container comprising a composition, where the composition comprising a plurality of oligodendrocyte progenitor cells is useful in treating stroke, spinal cord injury, and multiple sclerosis.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to the filing date of U.S. Provisional Patent Application No. 62 / 315,454, filed March 30, 2016, entitled "Oligodendrocyte Progenitor Cell Compositions," the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to the field of cell biology of oligodendrocyte precursor cells. More particularly, the present disclosure relates to compositions comprising oligodendrocyte precursor cells, and methods of making and using the same. [Background technology]

[0003] Pluripotent cells, including human embryonic stem cells (hESCs), can undergo varying levels of spontaneous differentiation during expansion, which impairs the ability of hESCs to subsequently undergo directed differentiation into specific cell types, including oligodendrocyte progenitor cells (OPCs). For example, the H1 lineage of hESCs has been shown to spontaneously differentiate when grown under conventional culture conditions and to have a propensity for differentiation into trophectoderm lineages (Drukker M, Tang C, Ardehali R, Rinkevich Y, Seita J, Lee AS, Mosley AR, Weissman IL, Soen Y. Isolation of primitive endoderm, mesoderm, vascular endothelial, and trophoblast progenitors from human pluripotent stem cells. Nat Biotechnol. 2012 May 27;30(6):531-42; Wang Z, Oron E, Nelson B, Razis S, Ivanova N. Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell. 2012 April 6;10(4):440-54). This spontaneous differentiation is regulated in part by endogenous WNT signaling in hESCs (Kumar RM, Cahan P, Shalek AK, Satija R, Daley-Keyser AJ, Li H, Zhang J, Pardee K, Gennert D, Trombetta JJ, Ferrante TC, Regev A, Daley GQ, Collins JJ. Deconstructing transcriptional heterogeneity in pluripotent stem cells. Nature. 2014 Dec 4;516(7529):56-61). However, the field is divided on whether activating or inhibiting the WNT pathway is beneficial in controlling differentiation.Thus, Kurek et al. suggest that inhibition of the WNT pathway reduces spontaneous differentiation (Kurek D, Neagu A, Tastemel M, Tuysuz N, Lehmann J, van de Werken HJ, Philipsen S, van der Linden R, Maas A, van IJcken WF, Drukker M, ten Berge D. Endogenous WNT signals mediate BMP-induced and spontaneous differentiation of epiblast stem cells and human embryonic stem cells. Stem Cell Reports 2015 Jan 13;4(1):114-28).

[0004] When H1 hESCs are expanded and then subjected to directed differentiation into OPCs, the differentiated cells show increased expression of trophectoderm-associated genes, including transcription factors, suggesting that cell types other than the neuroectodermal lineage can still persist during differentiation. This persistence of non-neurectodermal lineage markers during differentiation is then associated with the presence of unwanted cell types within the final OPC population, including, for example, epithelial cells, chondroprogenitor cells, and retinal pigment epithelial cells.

[0005] Several markers have been used to quantify the levels of unwanted cells within OPC populations. Detection of such markers can be achieved by cell phenotyping methods, such as flow cytometry and gene expression profiling. For example, flow cytometry analysis can be used to detect various levels of epithelial cells based on the presence of markers associated with epithelial cells, including EpCAM, CD49f / ITGA6, E-cadherin, cytokeratin 7 (K7), pan-cytokeratin (PCK), CA125 / MUC16, endorepellin / perlecan, and other markers listed in Table 1. Furthermore, gene expression profiling by quantitative polymerase chain reaction (qPCR) can be used to indicate various levels of chondroprogenitor cells and retinal pigment epithelial cells based on the expression of OLR1 and RPE65, respectively.

[0006] Although efforts have been made to produce populations of OPCs with reduced levels of undesired cell types, techniques such as cell sorting and antibody depletion are complex and often require the generation of customized reagents that are expensive and not amenable to scale-up. Summary of the Invention [Problem to be solved by the invention]

[0007] Aspects of the present disclosure include compositions comprising a plurality of oligodendrocyte progenitor cells (OPCs), as well as methods of making and using the same, for improving one or more neurological functions in a subject in need of treatment. In one aspect, the present disclosure includes a composition comprising a plurality of OPCs and a cell population comprising less than 15% of an undesired cell type. In one aspect, the present disclosure further includes a method of achieving a cell population comprising a plurality of OPCs and less than 15% of an undesired cell type. In one aspect, the present disclosure also includes a method of characterizing a cell population comprising a plurality of OPCs and less than 15% of an undesired cell type. In one aspect, the present disclosure also includes a method of using a cell population comprising a plurality of OPCs and less than 15% of an undesired cell type. In one aspect, the present disclosure further includes a method of generating a cell population comprising a plurality of OPCs and less than 15% of an undesired cell type. [Means for solving the problem]

[0008] More specifically, the present disclosure includes, in certain embodiments, a container containing a composition, the composition comprising a cell population comprising a plurality of oligodendrocyte progenitor cells, the cell population comprising less than 15% of undesired cell types. In certain embodiments, the cell population comprises less than 15% epithelial lineage cells. In certain embodiments, the cell population comprises less than 2% K7-positive cells. In certain embodiments, the cell population comprises less than 5% PCK-positive cells.

[0009] An aspect of the disclosure is a container containing a composition, the composition comprising a cell population comprising a plurality of oligodendrocyte progenitor cells (OPCs), the plurality of OPCs comprising in vitro derived progeny of one or more stem cell sources selected from the group consisting of human embryonic stem cells, primate pluripotent stem cells, and induced pluripotent stem cells.

[0010] A further aspect of the present disclosure is a container containing a composition, the composition comprising a cell population comprising a plurality of oligodendrocyte precursor cells, the cell population capable of forming no more than 1 epithelial cyst per 100,000 cells in a cyst assay. In yet another aspect, the cell population comprises up to 20 x 10 6 When transplanted into the site of central nervous system injury, these cells can produce ectopic tissue in less than 2% of subjects.

[0011] A still further aspect of the present disclosure is a container comprising a composition, said composition comprising a cell population comprising a plurality of oligodendrocyte progenitor cells, said cell population being useful for treating stroke, spinal cord injury, and / or multiple sclerosis.

[0012] The patent or application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the chemical structure of SB431542 according to the present disclosure. [Figure 2] FIG. 1 shows the chemical structure of dorsomorphin according to the present disclosure. [Figure 3] FIG. 1 shows the chemical structure of CHIR99021 according to the present disclosure. [Figure 4] FIG. 1 shows the chemical structure of palmorfamine according to the present disclosure. [Figure 5] FIG. 1 shows a representative comparison of changes in NG2, K7, and PCK marker levels expressed in OPCs generated by methods according to embodiments of the present disclosure compared to matched controls generated by methods without prior treatment of undifferentiated cells. [Figure 6A]The left and middle panels show representative pictographs of H1 hESC-derived OPCs generated by a method according to the present disclosure that does not pre-treat undifferentiated cells, demonstrating varying levels of cyst formation in an in vitro cyst assay according to the present disclosure. The right panel shows representative pictographs of H1 hESC-derived OPCs generated by a method according to the present disclosure that includes pre-treating undifferentiated cells, demonstrating no cyst formation in an in vitro cyst assay according to the present disclosure. [Figure 6B] FIG. 1 shows a series of representative histology images from an adult female rat 9 months after cervical spinal cord injury and administration of H1 hESC-derived OPCs generated according to the present disclosure without prior treatment of undifferentiated cells. [Figure 6C] FIG. 1 shows a linear regression plot of in vivo cyst formation frequency against in vitro cyst counts in several populations of OPCs generated by a method without prior treatment of undifferentiated cells. [Figure 7] FIG. 1 shows a representative photomicrograph of a rat cervical spinal cord engrafted with OPCs generated using a method comprising pre-treatment of undifferentiated cells according to the present disclosure. [Figure 8] FIG. 1 shows a non-limiting example of an experimental timeline for testing the effects of transplanted OPCs in a mouse model of white matter stroke according to the present disclosure. [Figure 9] FIG. 1 shows a non-limiting example of expected results from a grid walking test, demonstrating improved performance in stroke-injured mice transplanted with OPCs generated by methods according to embodiments of the present disclosure. [Figure 10] FIG. 1 shows a non-limiting example of the results expected from the cylinder test, demonstrating improved performance in stroke-injured mice transplanted with OPCs generated by methods according to embodiments of the present disclosure. [Figure 11] FIG. 1 shows a representative first principal component analysis of gait parameters demonstrating improved locomotor activity in injured rats transplanted with OPCs generated by a method according to the present disclosure without prior treatment with undifferentiated cells. [Figure 12] FIG. 1 shows a representative plot of average running speed, demonstrating improved running speed in spinal cord injured rats transplanted with OPCs generated by a method according to the present disclosure without prior treatment with undifferentiated cells. [Figure 13] FIG. 1 shows a representative plot of stride frequency of the right hind limb, demonstrating the reduction of motor deficits in spinal cord injured rats transplanted with OPCs generated by a method according to the present disclosure without prior treatment with undifferentiated cells. [Figure 14] FIG. 1 shows a representative plot of the mean maximum longitudinal deviation of the right forepaw, demonstrating recovery of longitudinal displacement of the affected limb in spinal cord injured rats transplanted with OPCs generated by a method according to the present disclosure without prior treatment with undifferentiated cells. [Figure 15] FIG. 1 shows a representative section of an OPC graft 4 months after injection of OPCs generated by a method according to the present disclosure without prior treatment with undifferentiated cells. [Figure 16] FIG. 1 shows a non-limiting example of an experimental timeline for testing the effects of transplanted OPCs in a rat model of multiple sclerosis according to the present disclosure. [Figure 17] FIG. 1 shows a non-limiting example plot of the Encephalomyelitis (EAE) score expected to be generated when transplanted with OPCs generated by methods according to embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0014] This description is not intended to be a detailed catalog of all the different ways in which the present disclosure may be implemented or all the features that may be added to the present disclosure. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be omitted from that embodiment. Accordingly, the present disclosure contemplates that some embodiments of the present disclosure may exclude or omit any of the features or combinations of features described herein. Moreover, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the present disclosure without departing from the present disclosure. In other instances, well-known structures, interfaces, and processes have not been shown in detail so as not to unnecessarily obscure the present invention. No part of this specification is intended to invalidate any portion of the entire scope of the present invention. Therefore, the following description is intended to describe some particular embodiments of the present disclosure, and is not intended to exhaustively specify all permutations, combinations, and modifications thereof.

[0015] 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 describing this disclosure herein are limited to the purpose of describing particular embodiments and are not intended to limit the disclosure.

[0016] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety.

[0017] Unless the context suggests otherwise, it is specifically intended that the various features of the present disclosure described herein can be used in any combination. Furthermore, the present disclosure contemplates that in some aspects of the present disclosure, any of the features or combinations of features described herein can be excluded or omitted.

[0018] The methods disclosed herein may include one or more steps or actions for achieving the described method. Method steps and / or actions may be interchangeable without departing from the scope of the present invention. In other words, unless a specific order of steps or actions is required for the proper operation of the present embodiment, the order and / or use of specific steps and / or actions may be modified without departing from the scope of the present invention.

[0019] 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 dictates otherwise.

[0020] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0021] The terms "about" and "approximately," as used herein, when referring to measurable numbers, such as percentages (%), densities, volumes, etc., are meant to include variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the stated amount.

[0022] As used herein, idiomatic phrases such as "between X and Y" and "approximately between X and Y" should be interpreted as including X and Y. As used herein, idiomatic phrases such as "approximately between X and Y" mean "approximately between X and approximately Y," and idiomatic phrases such as "approximately X to Y" mean "approximately X to approximately Y."

[0023] As used herein, "oligodendrocyte progenitor cells" (OPCs) refer to cells of the neuroectodermal / glial lineage that have characteristics of cell types found in the central nervous system and can differentiate into oligodendrocytes. Such cells typically express the characteristic markers nestin, NG2, and PDGF-Rα.

[0024] As used herein, the phrase "ectopic tissue" refers to tissue that has unwanted or unexpected characteristics. When used in the context of defining a composition comprising OPCs, "ectopic tissue" refers to tissue that is not derived from a neuroectodermal lineage. More specifically, "ectopic tissue" is tissue that resembles epithelium, epithelial cysts, tissue derived from mesothelial cells, cartilage, bone, or other tissue not typically found in the central nervous system.

[0025] As used herein, the term "undesirable cell type" refers to cells other than the neuroectodermal lineage that, when transplanted, result in ectopic tissue formation or, as described herein, result in the formation of one or more cysts in a cyst assay. More specifically, "undesirable cell types" include cells that express the K7 and / or PCK markers. In some embodiments, "undesirable cell types" can include cells of the epithelial lineage. In some embodiments, "undesirable cell types" can include cells that express markers characteristic of the trophoblast lineage, e.g., Hand1.

[0026] The terms "K7," "keratin 7," and "cytokeratin 7" are used interchangeably herein and refer to the protein encoded by the KRT7 gene or any variant thereof.

[0027] The terms "PCK" and "pan-cytokeratin" are used interchangeably herein and refer to any member of the cytokeratin protein family, including, but not limited to, the acidic and basic (type I and type II) subfamilies of cytokeratins and variants thereof. Non-limiting examples of PCK proteins include those bound by the AE1 / AE3 monoclonal antibody cocktail (Millipore catalog number MAB3412).

[0028] As used herein, "epithelial-lineage cells" refer to cells derived from developmental precursors that are ectodermal but derived from neuroectodermal / glial lineages. In certain embodiments, "epithelial-lineage cells" can be distinguished by one or more markers listed in Table 1. Such cells include developmental progeny that have undergone epithelial-mesenchymal transition (EMT) and epithelial-like cells derived from trophoblasts.

[0029] [Table 1-1]

[0030] [Table 1-2]

[0031] As used herein, the phrase "cell enrichment" refers to the purification of a desired cell type, in this case OPCs, away from undesired cell types. Purification can be by positive selection (targeting OPCs) or negative selection (targeting undesired cell types). Antibodies can be used to bind to and physically remove the targeted cell type. Antibodies can be linked to a solid surface to achieve a physical separation effect. The solid surface is typically a magnetic bead, column, or flask.

[0032] As used herein, "implantation" or "transplantation" refers to the administration of a cell population into a target tissue using a suitable delivery technique (e.g., using an injection device).

[0033] As used herein, a "subject" refers to an animal or a human.

[0034] As used herein, a "subject in need thereof" refers to an animal or human having damaged tissue within the central nervous system. In one embodiment, the animal or human is experiencing loss of motor function.

[0035] 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.

[0036] As used herein, "treatment" or "treating" with respect to a condition or disease is an approach to obtaining 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, curing the disease, reducing the severity of the disease, slowing the progression of the disease, alleviating one or more symptoms associated with the disease, improving the quality of life of the person suffering from the disease, prolonging 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: improvement of the condition, curing the condition, reducing the severity of the condition, slowing the progression of the condition, alleviating one or more symptoms associated with the condition, improving the quality of life of the person suffering from the condition, prolonging survival, and any combination thereof.

[0037] Methods for differentiating pluripotent stem cells Aspects of the present disclosure include methods of differentiating pluripotent stem cells. In certain aspects, methods are provided for producing a cell population comprising a plurality of neural cells. In certain aspects, methods are provided for producing a cell population comprising a plurality of OPCs. In certain aspects, methods are provided for producing a cell population comprising a plurality of OPCs that are the in vitro differentiated progeny of human embryonic stem cells. In one aspect, methods are provided for producing a cell population comprising a plurality of OPCs that are the in vitro differentiated progeny of primate pluripotent stem cells. In certain aspects, methods are provided for producing a cell population comprising a plurality of OPCs that are the in vitro differentiated progeny of induced pluripotent stem cells. In certain aspects, methods of differentiating pluripotent stem cells include one or more steps associated with pre-treating a plurality of undifferentiated stem cells. In certain aspects, methods including one or more pre-treatment steps can produce cell populations comprising low levels of undesired cell types. In certain aspects, methods including one or more pre-treatment steps can produce cell populations comprising a plurality of OPCs with less than about 15% undesired cell types. In one aspect, a method comprising one or more pre-treatment steps is capable of producing a cell population comprising a plurality of OPCs, said cell population being capable of forming no more than 1 epithelial cyst per 100,000 cells in a cyst assay according to the present disclosure.

[0038] Proliferation and culture of undifferentiated pluripotent stem cells In one aspect, the method can be performed in a pluripotent stem cell line. In another aspect, the method can be performed in an embryonic stem cell line. In one aspect, the method can be performed on a plurality of undifferentiated stem cells derived from an H1, H7, H9, H13, or H14 cell line. In another aspect, the undifferentiated stem cells can be derived from an induced pluripotent stem (iPS) line. In one aspect, the method can be performed on a primate pluripotent stem (pPS) cell line. In yet another aspect, the undifferentiated stem cells can be derived from a parthenogenetic organism, an embryo stimulated to produce hESCs without fertilization.

[0039] Methods for the proliferation and culture of undifferentiated pluripotent stem cells have been described. Regarding the tissue and cell culture of pluripotent stem cells, numerous published documents are available in the art, and some of the references include, for example, "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., ed., 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).

[0040] In one embodiment, undifferentiated pluripotent stem cells can be maintained in an undifferentiated state without the addition of feeder cells (see, e.g., (2004) Rosler et al., Dev. Dynam. 229:259). Feeder-free cultures are generally maintained in a nutrient medium containing factors that promote cell proliferation without differentiation (see, e.g., U.S. Patent No. 6,800,480). In one embodiment, conditioned media containing such factors are available. Conditioned media can be obtained by culturing media containing cells that secrete such factors. Suitable cells include, but are not limited to, irradiated (approximately 4,000 Rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells (U.S. Patent No. 6,642,048). Media can be conditioned by plating feeders in serum-free media such as knockout DMEM supplemented with 20% serum replacement and 4 ng / mL bFGF. Medium conditioned for 1-2 days can be further supplemented with bFGF and used to maintain pPS cell cultures for 1-2 days (see, e.g., WO 01 / 51616; Xu et al. (2001) Nat. Biotechnol. 19:971).

[0041] Alternatively, fresh or unconditioned media can be used, supplemented with additional factors (e.g., fibroblast growth factor or forskolin) that promote cell proliferation 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.), 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). Such media formulations have the advantage of maintaining cell proliferation at rates two to three times higher than other systems (see, e.g., WO 03 / 020920). In one embodiment, undifferentiated pluripotent cells, such as hES cells, can be cultured in a medium containing bFGF and TGFβ. Non-limiting examples of bFGF concentrations include about 80 ng / ml. Non-limiting examples of TGFβ concentrations include about 0.5 ng / ml.

[0042] In one embodiment, 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). In one embodiment, the feeder cells can be derived from humans or mice. Human feeder cells can be isolated from various human tissues or can be derived from the differentiation of human embryonic stem cells into fibroblasts (see, e.g., WO 01 / 51616). In one embodiment, available human feeder cells 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 (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).

[0043] A variety of solid surfaces are available for culturing undifferentiated pluripotent cells. Such solid surfaces include, but are not limited to, standard commercially available cell culture plates, such as 6-well, 24-well, 96-well, or 144-well plates. Other solid surfaces include, but are not limited to, microcarriers and discs. Solid surfaces suitable for growing undifferentiated pluripotent cells can be made of a variety of materials, including, but not limited to, glass or plastics, such as polystyrene, polyvinyl chloride, polycarbonate, polytetrafluoroethylene, melinex, thermanox, etc., or combinations thereof. In one embodiment, a suitable surface can include one or more polymers, such as one or more acrylates. In one embodiment, the solid surface can be three-dimensional. Non-limiting examples of three-dimensional solid surfaces are described, for example, in U.S. Patent Publication No. 2005 / 0031598.

[0044] In some embodiments, undifferentiated stem cells can be grown under feeder-free conditions on a growth substrate. In some embodiments, the growth substrate can be Matrigel® matrix (e.g., Matrigel®, Matrigel® GFR), recombinant laminin, or vitronectin. In some embodiments, undifferentiated stem cells can be passaged using various methods, such as using collagenase, or by manual scraping. In other embodiments, undifferentiated stem cells can be passaged using non-enzymatic means, such as PBS containing 0.5 mM EDTA, or by using ReLeSR™. In some embodiments, a plurality of undifferentiated stem cells are seeded or passaged at a seeding density that allows the cells to reach confluence within about 3 to about 10 days. In some embodiments, the seeding density is about 6.0 x 10 cells per growth surface. 3 cells / cm 2 ~Approx. 5.0×10 5 cells / cm 2 , for example, about 1.0 × 10 4 cells / cm 2 etc., for example, about 5.0 × 104 cells / cm 2 etc., for example, about 1.0 × 10 5 cells / cm 2 etc., or for example about 3.0 x 10 5 cells / cm 2 In another embodiment, the seeding density can range from about 6.0 x 10 cells per growth surface. 3 cells / cm 2 ~Approx. 1.0×10 4 cells / cm 2 , e.g., about 6.0 x 10 3 cells / cm 2 ~Approx. 9.0×10 3 cells / cm 2 etc., for example, about 7.0 × 10 3 cells / cm 2 ~Approx. 1.0×10 4 cells / cm 2 etc., for example, about 7.0 × 10 3 cells / cm 2 ~Approx. 9.0×10 3 cells / cm 2 etc., or for example about 7.0 x 10 3 cells / cm 2 ~Approx. 8.0×10 3 cells / cm 2 In yet another aspect, the seeding density can range from about 1.0 x 10 cells per growth surface. 4 cells / cm 2 ~Approx. 1.0×10 5 cells / cm 2 , approximately 2.0 × 10 4 cells / cm 2 ~Approx. 9.0×10 4 cells / cm 2 etc., for example, about 3.0 × 10 4 cells / cm 2 ~Approx. 8.0×10 4 cells / cm 2 etc., for example, about 4.0 × 10 4 cells / cm 2 ~Approx. 7.0×10 4 cells / cm 2 etc., or for example about 5.0 x 10 4 cells / cm 2~Approx. 6.0×10 4 cells / cm 2 etc. In one embodiment, the seeding density is about 1.0 x 10 cells per growth surface. 5 cells / cm 2 ~Approx. 5.0×10 5 cells / cm 2 , e.g., about 1.0 × 10 5 cells / cm 2 ~Approx. 4.5×10 5 cells / cm 2 etc., for example, about 1.5 x 10 5 cells / cm 2 ~Approx. 4.0×10 5 cells / cm 2 etc., for example, about 2.0 × 10 5 cells / cm 2 ~Approx. 3.5×10 5 cells / cm 2 etc., or for example about 2.5 x 10 5 cells / cm 2 ~Approx. 3.0×10 5 cells / cm 2 etc.

[0045] Any of a variety of suitable cell culture and subculture techniques can be used to culture cells according to embodiments of the subject method. For example, in certain embodiments, the culture medium can be changed at suitable time intervals. In one embodiment, the culture medium can be completely changed daily, beginning about two days after subculturing the cells. In certain embodiments, once the culture reaches about 90% colony coverage, the surrogate flask can be sacrificed and counted using one or more suitable reagents, such as collagenase IV and 0.05% trypsin-EDTA, sequentially to achieve a single-cell suspension for quantification. In certain embodiments, the plurality of undifferentiated stem cells can then be subcultured and then plated onto a suitable growth substrate (e.g., Matrigel® matrix) at a seeding density that allows the cells to reach confluence in a suitable period of time, such as about 3-10 days. In one embodiment, the undifferentiated stem cells can be subcultured using collagenase IV and expanded on a recombinant laminin matrix. In one embodiment, the undifferentiated stem cells can be subcultured using collagenase IV and expanded on a Matrigel® matrix. In one embodiment, the undifferentiated stem cells can be subcultured using ReLeSR™ and expanded on a vitronectin matrix.

[0046] In one embodiment, the seeding density is about 6.0 x 10 cells per growth surface. 3 cells / cm 2 ~Approx. 5.0×10 5 cells / cm 2 , for example, about 1.0 × 10 4 cells / cm 2 etc., for example, about 5.0 × 10 4 cells / cm 2 etc., for example, about 1.0 × 10 5 cells / cm 2 etc., or for example about 3.0 x 10 5 cells / cm 2 In another embodiment, the seeding density can range from about 6.0 x 10 cells per growth surface. 3 cells / cm 2 ~Approx. 1.0×10 4 cells / cm2 , e.g., about 6.0 × 10 3 cells / cm 2 ~Approx. 9.0×10 3 cells / cm 2 etc., for example, about 7.0 × 10 3 pieces / cm 2 ~Approx. 1.0×10 4 cells / cm 2 etc., for example, about 7.0 × 10 3 cells / cm 2 ~Approx. 9.0×10 3 cells / cm 2 etc., or for example, about 7.0 x 10 3 cells / cm 2 ~Approx. 8.0×10 3 cells / cm 2 In yet another aspect, the seeding density can range from about 1.0 x 10 cells per growth surface. 4 cells / cm 2 ~Approx. 1.0×10 5 cells / cm 2 , e.g., about 2.0 × 10 4 cells / cm 2 ~Approx. 9.0×10 4 cells / cm 2 etc., for example, about 3.0 × 10 4 cells / cm 2 ~Approx. 8.0×10 4 cells / cm 2 etc., for example, about 4.0 × 10 4 cells / cm 2 ~Approx. 7.0×10 4 cells / cm 2 etc., or for example about 5.0 x 10 4 cells / cm 2 ~Approx. 6.0×10 4 cells / cm 2 etc. In one embodiment, the seeding density is about 1.0 x 10 of the growth surface 5 cells / cm 2 ~Approx. 5.0×10 5 cells / cm 2 , e.g., about 1.0 × 10 5 cells / cm 2 ~Approx. 4.5×105 cells / cm 2 etc., for example, about 1.5 x 10 5 cells / cm 2 ~Approx. 4.0×10 5 cells / cm 2 etc., for example, about 2.0 × 10 5 cells / cm 2 ~Approx. 3.5×10 5 cells / cm 2 etc., or for example about 2.5 x 10 5 cells / cm 2 ~Approx. 3.0×10 5 cells / cm 2 etc.

[0047] Pre-treatment of undifferentiated pluripotent stem cells In another aspect, methods are provided that include pre-treating undifferentiated stem cells prior to differentiation. Without being bound by any particular theory, the inventors have identified various pre-treatment steps that can facilitate a further reduction in spontaneous differentiation of pluripotent stem cells, thereby providing further improvements over differentiation methods that do not include a pre-treatment step. In one aspect, methods involving one or more pre-treatment steps described herein can produce cell populations that contain low levels of undesired trophoblast lineage cells. In another aspect, methods including one or more pre-treatment steps described herein can produce cell populations that contain low levels of undesired epithelial lineage cells. Furthermore, the inventors have identified various pre-treatment steps that can facilitate the production of cell populations that form no more than one epithelial cyst per 100,000 cells in a cyst assay according to the present disclosure. Pre-treatment steps are described in detail herein.

[0048] In one embodiment, the method comprises incubating the expanded but undifferentiated stem cells with one or more stem cell differentiation-regulating molecules for a period of time. In one embodiment, the stem cell differentiation-regulating molecule can be a molecule that promotes subsequent cell differentiation separate from the epithelial lineage. Without being bound by theory, the inventors have identified that incubating stem cells with one or more stem cell differentiation-regulating molecules can result in a decrease in epithelial lineage cells. In one embodiment, the method comprises incubating the expanded but undifferentiated stem cells for a period of time with one or more of the following four stem cell differentiation-regulating molecules: (1) an inhibitor of ALK5, which is part of the SMAD / TGFβ-RII signaling pathway; (2) an inhibitor of ALK2, which is part of the BMPRI signaling pathway; (3) a GSK3 inhibitor that activates the WNT signaling pathway; and (4) a smoothened agonist that activates the SHH pathway.

[0049] In one embodiment, the method comprises incubating the expanded but undifferentiated stem cells with one or more stem cell differentiation regulators. In one embodiment, the stem cell differentiation regulator can be a small molecule. In one embodiment, the small molecule stem cell differentiation regulator is an inhibitor of ALK5, ALK2, or GSK3, or a smoothening agonist. In one embodiment, the ALK5 inhibitor can be selected from the group consisting of SB431542, LY364947, RepSox, and derivatives thereof. In another embodiment, the ALK2 inhibitor can be selected from the group consisting of dorsomorphin, LDN193189, noggin protein, and derivatives thereof. In yet another embodiment, the GSK3 inhibitor can be selected from the group consisting of CHIR99021, 6-bromoindirubin-3'-oxime (BIO), kempaullone, SB216762, Wnt protein, and derivatives thereof. In one embodiment, the smoothening agonist can be selected from the group consisting of palmorphamin, SAG (CAS364590-63-6), SSH proteins, and derivatives thereof. In one embodiment, the method comprises incubating expanded but undifferentiated stem cells with four small molecules: SB431542, dorsomorphin, CHIR99021, and palmorphamin for a period of time. The chemical structures of SB431542, dorsomorphin, CHIR99021, and palmorphamin are shown in Figures 1-4, respectively.

[0050] In some embodiments, the method comprises incubating expanded but undifferentiated stem cells, as described herein, with one or more stem cell differentiation regulators for a period of time. In some embodiments, the method comprises incubating expanded but undifferentiated stem cells with two or more stem cell differentiation regulators for a period of time. In some embodiments, the method comprises incubating expanded but undifferentiated stem cells with three or more stem cell differentiation regulators for a period of time. In some embodiments, the method comprises incubating expanded but undifferentiated stem cells with four or more stem cell differentiation regulators for a period of time. In some embodiments, the incubation period can range from about 1 to about 10 days, such as about 2 days, such as about 3 days, such as about 4 days, such as about 5 days, such as about 6 days, such as about 7 days, such as about 8 days, or such as about 9 days. In some embodiments, the incubation period can range from about 1 to about 5 days, such as about 1 to about 3 days, or such as about 2 to about 5 days. In another aspect, the incubation period may be in the range of about 6 to about 10 days, for example, about 6 to about 8 days, or for example, about 7 to about 10 days, etc. In yet another aspect, the incubation period may be in the range of about 3 to about 8 days, for example, about 3 to about 5 days, for example, about 4 to about 6 days, for example, about 5 to about 7 days, or for example, about 6 to about 8 days, etc.

[0051] In one embodiment, the method comprises incubating an expanded but undifferentiated stem cell population with two or more first stem cell differentiation regulators for a first incubation period, and incubating the population with two or more second stem cell differentiation regulators for a second incubation period. In one embodiment, the first incubation period can be in the range of about 1 to about 7 days, 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. In one embodiment, the second incubation period can be in the range of about 1 to about 7 days, 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.

[0052] In some embodiments, the two or more first stem cell differentiation regulators are different from the two or more second stem cell differentiation regulators. In some embodiments, the two or more first stem cell regulators are identical to the two or more second stem cell regulators. In some embodiments, the first and second stem cell regulators share at least one common regulator. In some embodiments, the first and second stem cell regulators share two or more common regulators, such as three or more common regulators, such as four or more common regulators.

[0053] In one embodiment, the method comprises incubating the expanded but undifferentiated stem cells with one or more of the following differentiation regulators: SB431542, dorsomorphin, CHIR99021, and palmorphamin for about 1-10 days. In one embodiment, the method comprises incubating the expanded but undifferentiated stem cells with SB431542, dorsomorphin, CHIR99021, and palmorphamin for about 1-5 days, followed by incubation with CHIR99021 and palmorphamin for about 1-5 days.

[0054] In one embodiment, the method comprises incubating the expanded but undifferentiated stem cells with SB431542, dorsomorphin, CHIR99021, and palmorphamin for about four days, followed by incubation with CHIR99021 and palmorphamin for three days.

[0055] In one aspect, the method comprises incubating expanded but undifferentiated ESCs with SB431542 at a concentration ranging from about 1 μM to about 100 μM, such as about 5 μM, such as about 10 μM, such as about 15 μM, for example, about 20 μM, such as about 25 μM, such as about 30 μM, for example, about 35 μM, such as about 40 μM, such as about 45 μM, such as about 50 μM, for example, about 55 μM, such as about 60 μM, for example, about 65 μM, such as about 70 μM, for example, about 75 μM, such as about 80 μM, for example, about 85 μM, such as about 90 μM, or such as about 95 μM. In another aspect, the method comprises incubating the expanded but undifferentiated ESCs with SB431542 at a concentration ranging from about 1 μM to about 20 μM, such as about 1 μM to about 13 μM, such as about 8 μM to about 20 μM, such as about 8 μM to about 13 μM, or such as about 9 μM to about 11 μM. In yet another aspect, the method comprises incubating the expanded but undifferentiated ESCs with SB431542 at a concentration ranging from about 20 μM to about 40 μM, such as about 20 μM to about 33 μM, such as about 28 μM to about 40 μM, such as about 28 μM to about 33 μM, or such as about 29 μM to about 31 μM. In one aspect, the method comprises incubating expanded but undifferentiated ESCs with SB431542 at a concentration ranging from about 40 μM to about 60 μM, such as about 40 μM to about 53 μM, such as about 48 μM to about 55 μM, such as about 48 μM to about 53 μM, or such as about 49 μM to about 51 μM. In one aspect, the method comprises incubating expanded but undifferentiated ESCs with SB431542 at a concentration ranging from about 60 μM to about 80 μM, such as about 60 μM to about 73 μM, such as about 68 μM to about 75 μM, such as about 68 μM to about 73 μM, or such as about 69 μM to about 71 μM. In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with SB431542 at a concentration ranging from about 80 μM to about 100 μM, such as about 80 μM to about 93 μM, such as about 88 μM to about 95 μM, such as about 88 μM to about 93 μM, or such as about 89 μM to about 91 μM, etc. In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with SB431542 at a concentration of about 10 μM.

[0056] In one embodiment, the method comprises incubating the expanded but undifferentiated ESCs with an ALK5 inhibitor at a concentration ranging from about 250 nM to about 250 μM, e.g., about 1 μM, about 10 μM, about 50 μM, about 100 μM, about 150 μM, or about 200 μM, etc. In one embodiment, the method comprises incubating the expanded but undifferentiated ESCs with LY364947 at a concentration ranging from about 250 nM to about 25 μM, e.g., about 250 nM to about 1 μM, e.g., about 1 μM to about 10 μM, etc., or e.g., about 10 μM to about 25 μM, etc. In one embodiment, the method comprises incubating the expanded but undifferentiated ESCs with about 2.5 μM of LY364947. In another aspect, the method comprises incubating the expanded but undifferentiated ESCs with RepSox at a concentration ranging from about 2.5 μM to about 250 μM, such as from about 2.5 μM to about 10 μM, such as from about 10 μM to about 100 μM, or such as from about 100 μM to about 250 μM, etc. In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with RepSox at about 25 μM.

[0057] In one aspect, the method comprises treating the expanded but undifferentiated ESCs with a soluble form of ATP at a concentration of between about 0.2 μM and 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, such as about 3 μM, for example, about 3.5 μM, such as about 4 μM, for example, about 4.5 μM, such as about 5 μM, for example, about 5.5 μM, such as about 6 μM, for example, about 6.5 μM, In another aspect, the method comprises incubating the expanded but undifferentiated ESCs with dorsomorphin at a concentration ranging from 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 such as about 0.5 μM to about 0.6 μM. In yet another aspect, the method comprises incubating the expanded but undifferentiated ESCs with dorsomorphin at a concentration ranging from about 1 μM to about 10 μM, such as about 1 μM to about 9 μM, such as about 2 μM to about 8 μM, such as about 3 μM to about 7 μM, or such as about 4 μM to about 6 μM. In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with dorsomorphin at a concentration ranging from 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 aspect, the method comprises incubating the expanded but undifferentiated ESCs with dorsomorphin at a concentration of about 2 μM.

[0058] In one embodiment, the method comprises incubating the expanded but undifferentiated ESCs with 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, etc. In one embodiment, the method comprises incubating the expanded but undifferentiated ESCs with LDN193189 at a concentration ranging from about 1 nM to about 100 nM, e.g., about 1 nM to about 10 nM, e.g., about 10 nM to about 50 nM, or e.g., about 50 nM to about 100 nM, etc. In one embodiment, the method comprises incubating the expanded but undifferentiated ESCs with about 10 nM of LDN193189. In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with Noggin protein at a concentration ranging from about 2 nM to about 200 nM, such as about 2 nM to about 10 nM, such as about 10 nM to about 100 nM, or such as about 100 nM to about 200 nM, etc. In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with about 20 nM Noggin protein.

[0059] In one aspect, the method comprises treating the expanded but undifferentiated ESCs with a soluble form of ATP at a concentration of between about 0.3 μM and about 30 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, such as about 3 μM, for example, about 3.5 μM, such as about 4 μM, for example, about 4.5 μM, such as about 5 μM, for example, about 5.5 μM, such as about 6 μM, for example In another aspect, the method comprises incubating the expanded but undifferentiated ESCs with CHIR99021 at a concentration ranging from about 0.3 μM to about 1 μM, such as about 0.3 μM to about 0.9 μM, such as about 0.4 μM to about 0.8 μM, or such as about 0.5 μM to about 0.7 μM. In yet another aspect, the method comprises incubating the expanded but undifferentiated ESCs with CHIR99021 at a concentration ranging from about 1 μM to about 10 μM, such as about 1 μM to about 9 μM, such as about 2 μM to about 8 μM, such as about 3 μM to about 7 μM, or such as about 4 μM to about 6 μM. In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with CHIR99021 at a concentration ranging from about 10 μM to about 20 μM, such as about 11 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 aspect, the method comprises incubating the expanded but undifferentiated ESCs with CHIR99021 at a concentration ranging from about 20 μM to about 30 μM, such as about 21 μM to about 29 μM, such as about 22 μM to about 28 μM, such as about 23 μM to about 27 μM, or such as about 24 μM to about 26 μM, etc. In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with CHIR99021 at a concentration of about 3 μM.

[0060] In one embodiment, the method comprises incubating expanded but undifferentiated ESCs with a GSK3 inhibitor at a concentration ranging from about 25 nM to about 100 μM, e.g., about 100 nM, about 250 nM, about 500 nM, about 750 nM, about 1 μM, about 10 μM, or about 50 μM. In one embodiment, the method comprises incubating expanded but undifferentiated ESCs with BIO(6-bromoindirubin-3'-oxime) at a concentration ranging from about 500 nM to about 50 μM, e.g., about 500 nM to about 1 μM, e.g., about 1 μM to about 10 μM, or e.g., about 10 μM to about 50 μM. In one embodiment, the method comprises incubating expanded but undifferentiated ESCs with about 5 μM BIO(6-bromoindirubin-3'-oxime). In another aspect, the method comprises incubating expanded but undifferentiated ESCs with Kenpaullone at a concentration ranging from about 1 μM to about 100 μM, e.g., from about 1 μM to about 10 μM, e.g., from about 10 μM to about 50 μM, or e.g., from about 50 μM to about 100 μM. In one aspect, the method comprises incubating expanded but undifferentiated ESCs with Kenpaullone at about 10 μM. In another aspect, the method comprises incubating expanded but undifferentiated ESCs with SB216763 at a concentration ranging from about 500 nM to about 50 μM, e.g., from about 500 nM to about 1 μM, e.g., from about 1 μM to about 10 μM, or e.g., from about 10 μM to about 50 μM. In one aspect, the method comprises incubating expanded but undifferentiated ESCs with SB216763 at about 5 μM. In yet another aspect, the method comprises incubating the expanded but undifferentiated ESCs with a Wnt protein at a concentration ranging from about 25 nM to about 2.5 μM, such as about 25 nM to about 100 nM, such as about 100 nM to about 1 μM, or such as about 1 μM to about 2.5 μM, etc. In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with about 250 nM of Wnt protein.

[0061] In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with palmorphamin at a concentration ranging from about 0.05 μM to about 5 μM, such as about 0.08 μM, for example, about 0.1 μM, such as about 0.2 μM, for example, about 0.3 μM, such as about 0.4 μM, for example, about 0.5 μM, such as about 0.6 μM, such as about 0.7 μM, for example, about 0.8 μM, such as about 0.9 μM, for example, about 1 μM, such as about 2 μM, for example, about 3 μM, for example, about 4 μM, etc. In another aspect, the method comprises incubating the expanded but undifferentiated ESCs with palmorphamin at a concentration ranging from about 0.05 μM to about 0.1 μM, for example, about 0.06 μM to about 0.09 μM, or for example, about 0.07 μM to about 0.08 μM. In another aspect, the method comprises incubating the expanded but undifferentiated ESCs with palmorphamin at a concentration ranging from about 0.1 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 such as about 0.5 μM to about 0.6 μM. In another aspect, the method comprises incubating the expanded but undifferentiated ESCs with palmorphamin at a concentration ranging from about 1 μM to about 5 μM, such as about 1 μM to about 4 μM, such as about 2 μM to about 5 μM, or such as about 2 μM to about 4 μM. In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with palmorphamin at a concentration of about 0.5 μM.

[0062] In one embodiment, the method comprises incubating the expanded but undifferentiated ESCs with a smoothening agonist at a concentration ranging from about 2.5 nM to about 5 μM, e.g., about 50 nM, about 100 nM, about 250 nM, about 500 nM, about 750 nM, about 1 μM, or about 2.5 μM. In one embodiment, the method comprises incubating the expanded but undifferentiated ESCs with SAG at a concentration ranging from about 10 nM to about 1 μM, e.g., about 10 nM to about 100 nM, e.g., about 100 nM to about 500 nM, or e.g., about 500 nM to about 1000 nM. In one embodiment, the method comprises incubating the expanded but undifferentiated ESCs with about 100 nM of SAG. In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with SHH protein at a concentration ranging from about 2.5 nM to about 250 nM, such as about 2.5 nM to about 10 nM, such as about 10 nM to about 100 nM, or such as about 100 nM to about 250 nM, etc. In one aspect, the method comprises incubating the expanded but undifferentiated ESCs with about 25 nM SHH protein.

[0063] In some embodiments, undifferentiated stem cells can be expanded on a growth substrate in preparation for pre-treatment. Non-limiting examples of growth substrates include Matrigel® matrix, recombinant laminin (e.g., recombinant laminin 521), and vitronectin. In some embodiments, the medium can be completely changed daily starting about two days after subculturing the undifferentiated stem cells. In some embodiments, undifferentiated stem cells can be subcultured using collagenase and manual scraping, or other non-enzymatic means, such as PBS or ReLeSR™ containing 0.5 mM EDTA. In some embodiments, 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 some embodiments, the seeding density is about 6.0 x 10 cells on the growth surface for subculturing before the initiation of differentiation. 3 pieces / cm 2 ~Cells approx. 5.0×10 5 pieces / cm 2 , e.g., approximately 1.0 x 10 cells 4 pieces / cm 2 For example, approximately 5.0 x 10 cells4 pieces / cm 2 etc., for example, about 1.0 x 10 cells 5 pieces / cm 2 etc., or for example about 3.0 x 10 cells 5 pieces / cm 2 In another embodiment, the seeding density can range from about 6.0 x 10 cells to the growth surface. 3 pieces~cells approx. 1.0×10 4 pieces / cm 2 , e.g., about 6.0 × 10 3 pieces / cm 2 ~Cells approx. 9.0×10 3 pieces / cm 2 For example, approximately 7.0 x 10 cells 3 pieces~cells approx. 1.0×10 4 pieces / cm 2 For example, approximately 7.0 x 10 cells 3 pieces~cells approx. 9.0×10 3 pieces / cm 2 etc., or for example about 7.0 x 10 cells 3 pieces / cm 2 ~Cells approx. 8.0×10 3 pieces / cm 2 In yet another aspect, the seeding density can range from about 1.0 x 10 cells to the growth surface. 4 pieces / cm 2 ~Approx. 1.0×10 5 pieces / cm 2 For example, approximately 2.0 x 10 cells on the growth surface 4 pieces / cm 2 ~Cells approx. 9.0×10 4 For example, approximately 3.0 x 10 cells 4 pieces / cm 2 ~Cells approx. 8.0×10 4 pieces / cm 2 For example, approximately 4.0 x 10 cells 4 pieces / cm 2 ~Cells approx. 7.0×10 4 pieces / cm 2 etc., or for example about 5.0 x 10 cells 4 pieces / cm 2 ~cells approx. 6.0×10 4 pieces / cm 2In one aspect, the seeding density is about 1.0 x 10 cells on the growth surface in subculture before the initiation of differentiation. 5 pieces / cm 2 ~Cells approx. 5.0×10 5 pieces / cm 2 , e.g., approximately 1.0 x 10 cells 5 pieces / cm 2 ~cells approx. 4.5×10 5 pieces / cm 2 etc., for example, about 1.5 x 10 cells 5 pieces / cm 2 ~Cells approx. 4.0×10 5 pieces / cm 2 For example, approximately 2.0 x 10 cells 5 pieces / cm 2 ~Cells approx. 3.5×10 5 pieces / cm 2 etc., or for example about 2.5 x 10 cells 5 pieces / cm 2 ~Cells approx. 3.0×10 5 pieces / cm 2 etc. In one embodiment, once the undifferentiated stem cells reach 30-50% confluence, the medium is changed to glial progenitor medium containing one or more stem cell differentiation regulators as described herein. In one embodiment, the glial progenitor medium containing one or more stem cell differentiation regulators as described herein is changed daily for about four days. In one embodiment, the medium containing one or more stem cell differentiation regulators as described herein is changed daily for the next three days before initiation of differentiation.

[0064] In one aspect, differentiation of undifferentiated stem cells, with or without prior treatment, can be initiated by counting cells using surrogate flasks sequentially with collagenase IV and 0.05% trypsin-EDTA to achieve a single cell suspension for quantification, and harvesting the remaining undifferentiated stem cell culture using collagenase IV and manual scraping. In one aspect, undifferentiated stem cell cultures can be cultured at approximately 1 x 10 5 cells / cm 2 ~Approx. 14×10 5 cells / cm 2 , e.g., about 2 × 10 5cells / cm 2 ~Approx. 13×10 5 cells / cm 2 etc., for example, about 3 x 10 5 cells / cm 2 ~Approx. 12×10 5 cells / cm 2 etc., for example, about 4 x 10 5 cells / cm 2 ~Approx. 11×10 5 cells / cm 2 etc., for example, about 5 x 10 5 cells / cm 2 ~About 10×10 5 cells / cm 2 etc., for example, about 6 × 10 5 cells / cm 2 ~Approx. 9×10 5 cells / cm 2 etc., for example, about 7 x 10 5 cells / cm 2 ~Approx. 8×10 5 cells / cm 2 In one embodiment, undifferentiated stem cells can be seeded in ultra-low attachment (ULA) vessels to form embryoid bodies (EBs) at densities ranging from about 8×10 5 cells / cm 2 It is sown in.

[0065] In some embodiments, the day after differentiation, a complete medium change can be performed using a 1:1 mixture of X-VIVO10 and glial progenitor medium (GPM), where GPM is composed of DMEM / F12 (Gibco catalog no. 10565-018), 2% B27 supplement (Gibco catalog no. 17504-044), 0.04 μg triiodothyronine (Sigma catalog no. T5516-1MG) supplemented with 4 ng / mL hbFGF and 20 ng / mL EGF (Life Technologies catalog no. PHG0311). In some embodiments, GPM can be supplemented with hbFGF, EGF, and retinoic acid (RA). In some embodiments, starting on day 2 of differentiation, the medium can be 100% GPM supplemented only with EGF and RA. In some embodiments, this medium is replaced daily until day 9. In one embodiment, the EGF-supplemented GPM medium is changed approximately every other day from day 9 to day 27. In one embodiment, on day 27, EBs are cultured on 1 cm ULAs seeded at the initiation of differentiation. 2 Approximately 2cm per 2 In some embodiments, the coated vessels may be coated with Matrigel® matrix, recombinant laminin, or vitronectin. In some embodiments, the culture medium used throughout the remainder of differentiation is PM supplemented with 20 ng / mL EGF. In some embodiments, from about day 27 onward, the cell cultures undergo complete medium changes every other day. In some embodiments, cells can be harvested and counted at about day 34 using TrypLE Select supplemented with trypsin-EDTA or Benzonase and 0.01% Pluronic-F68, and plated at approximately 5 x 10 cells on Matrigel® matrix-coated vessels. 4 viable cells / cm 2In one embodiment, the GPM medium is replaced every other day from about day 34 until final harvest about 7 days later. In one embodiment, OPCs are harvested at about day 41 using 0.05% trypsin-EDTA or TrypLE Select supplemented with Benzonase and 0.01% Pluronic-F68. In one embodiment, detached cells are pooled in a mixture of DMEM-F12 medium and HypoThermosol FRS supplemented with Benzonase and 0.01% Pluronic-F68, then counted and reconstituted in CryoStor5 prior to cryopreservation.

[0066] OPC composition As noted above, aspects of the present disclosure include compositions comprising a plurality of oligodendrocyte progenitor cells (OPCs), as well as methods for making and using the same to improve one or more neurological functions in a subject in need of treatment. In certain aspects, the plurality of OPCs are in vitro differentiated progeny of primate pluripotent stem (pPS) cells. In certain aspects, the plurality of OPCs are in vitro differentiated progeny of human embryonic stem cells. In other aspects, the plurality of OPCs are in vitro differentiated progeny of induced pluripotent stem (iPS) cells. In some aspects, compositions are provided that comprise a plurality of OPCs and a cell population comprising low levels of an undesirable cell type.

[0067] In certain embodiments, the cell population may have a common genetic background. In certain embodiments, the cell population may be derived from a single host. In certain embodiments, the cell population may be derived from a pluripotent stem cell line. In other embodiments, the cell population may be derived from an embryonic stem cell line. In certain embodiments, the cell population may be derived from an hESC line. In certain embodiments, the hESC line may be an H1, H7, H9, H13, or H14 cell line. In other embodiments, the cell population may be derived from an induced pluripotent stem cell (iPS) line. In certain embodiments, the cell population may be derived from a subject in need thereof (e.g., the cell population may be derived from a subject in need of treatment). In yet other embodiments, the hESC line may be derived from a parthenogenetic organism, an embryo stimulated to produce hESCs without fertilization.

[0068] In some embodiments, the cell population has not undergone cell enrichment. In some embodiments, the cell population has not undergone positive selection for OPCs. In some embodiments, positive selection of OPCs can be antibody selection for nestin, NG2, or PDGF-Rα, e.g., using flow cytometry or magnetic beads. In some embodiments, the cell population may not undergo negative selection for undesired cell types. In some embodiments, negative selection of OPCs can be antibody selection for EpCAM or CD49f, e.g., using flow cytometry or magnetic beads.

[0069] In certain embodiments, one or more characteristics of a cell population can be determined by quantifying various cell markers using flow cytometry, e.g., determining what percentage of a cell population is positive for a particular marker or set of markers. In certain embodiments, a cell population can comprise between about 30% and 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 another aspect, the cell population may contain about 30% to about 60% NG2-positive cells, for example, about 30% to about 35%, such as about 35% to about 40%, such as about 40% to about 45%, such as about 45% to about 50%, such as about 35% to about 55%, such as about 40% to 50%, or for example, about 43% to about 48% NG2-positive cells. In yet another aspect, the cell population may contain about 45% to about 75% NG2-positive cells, for example, 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 for example, about 58% to about 63% NG2-positive cells. In one aspect, the cell population may contain about 60% to about 90% NG2-positive cells, for example, about 60% to about 65%, such as about 65% to about 70%, such as about 75% to about 80%, such as about 85% to about 90%, such as about 65% to about 85%, such as about 70% to about 80%, or for example, about 73% to about 78% NG2-positive cells.In one embodiment, the cell population may contain about 75% to about 100% NG2-positive cells, such as about 75% to about 80%, such as about 85% to about 90%, such as about 90% to about 95%, such as about 95% to about 100%, such as about 80% to about 95%, such as about 85% to about 90%, or for example, about 73% to about 78% NG2-positive cells. In another embodiment, the cell population may contain at least about 30% NG2-positive cells. In another embodiment, the cell population may contain at least about 40% NG2-positive cells. In another embodiment, the cell population may contain at least about 50% NG2-positive cells.

[0070] In one aspect, the cell population may comprise about 80% to about 100% nestin-positive cells, for example, at least about 85%, such as at least about 90%, such as at least about 95%, such as about 99%, such as at least about 99.5%, such as at least about 99.8%, or for example, at least about 99.9%, etc. In another aspect, the cell population may comprise about 80% to about 90% nestin-positive cells, for example, about 80% to about 85%, or for example, about 83% to about 88% nestin-positive cells. In yet another embodiment, the cell population may comprise about 90% to about 100% nestin-positive cells, for example, about 90% to about 95%, such as, for example, about 95% to about 98%, such as, for example, about 98% to about 99%, such as, for example, about 99% to about 99.5%, such as, for example, about 99.5% to about 99.8%, such as, for example, about 99.8% to about 99.9%, such as, for example, about 99.9% to about 100%, such as, for example, about 90% to about 99.9%, such as, for example, about 95% to about 99.8%, or for example, about 98% to about 99.5%, etc. In one embodiment, the cell population may comprise at least about 99% nestin-positive cells.

[0071] In one embodiment, the cell population may contain about 80% to about 100% PDGF-Rα-positive cells, for example, at least about 85%, such as at least about 90%, such as at least about 95%, such as about 99%, such as about 99.5%, such as about 99.9%, or for example, at least about 100%, etc. In another embodiment, the cell population may contain about 80% to about 90% PDGF-Rα-positive cells, for example, about 80% to about 85%, such as about 85% to about 90%, or for example, about 83% to about 88%, etc. In yet another embodiment, the cell population may comprise about 90% to 100% PDGF-Rα-positive cells, for example, about 90% to about 95%, such as, for example, about 95% to about 98%, such as, for example, about 98% to about 99%, such as, for example, about 99% to about 99.5%, such as, for example, about 99.5% to about 99.8%, such as, for example, about 99.8% to about 99.9%, such as, for example, about 99.9% to about 100%, such as, for example, about 90% to about 99.9%, such as, for example, about 95% to about 99.8%, or for example, about 98% to about 99.5%, etc. In one embodiment, the cell population may comprise at least about 80% PDGF-Rα-positive cells.

[0072] In one embodiment, the cell population may comprise about 50% NG2-positive cells and about 81% PDGF-Rα-positive cells, hi another embodiment, the cell population may comprise about 50% NG2-positive cells, about 81% PDGF-Rα-positive cells, and about 99% nestin-positive cells.

[0073] In one embodiment, at least about 90% of the cell population can express at least one of the markers selected from the group consisting of nestin, PDGF-Rα, Nkx2.2, Olig1, and IGF2. In another embodiment, at least about 90% of the cell population can express at least two of the markers selected from the group consisting of nestin, PDGF-Rα, Nkx2.2, Olig1, and IGF2. In yet another embodiment, at least about 90% of the cell population can express at least three of the markers selected from the group consisting of nestin, PDGF-Rα, Nkx2.2, Olig1, and IGF2. In another embodiment, at least about 90% of the cell population can express at least four of the markers selected from the group consisting of nestin, PDGF-Rα, Nkx2.2, Olig1, and IGF2. In one embodiment, at least about 90% of the cell population can express all of nestin, PDGF-Rα, Nkx2.2, Olig1, and IGF2. In one embodiment, about 90% to about 100% of the cell population can express at least one marker selected from the group consisting of nestin, PDGF-Rα, Nkx2.2, Olig1, and IGF2. In one embodiment, about 95% to about 100%, for example, about 98% to about 100%, for example, about 99% to about 100%, for example, about 99.5% to about 100%, for example, about 99.8% to about 100%, or for example, about 99.9% to about 100% of the cell population can express at least one marker selected from the group consisting of nestin, PDGF-Rα, Nkx2.2, Olig1, and IGF2.

[0074] In certain embodiments, the cell population may be capable of producing one or more biological signaling factors. In certain embodiments, the cell population may be capable of producing one or more angiogenic signaling factors. In certain embodiments, the angiogenic signaling factor may be thrombospondin-1, serpine 1, or serpine 2. In certain embodiments, the cell population may be capable of producing one or more neurotrophic signaling factors. In certain embodiments, the neurotrophic signaling factor may be NGF, netrin 4, tenascin C, thrombospondin 1, thrombospondin 3, SLIT 1, or SLIT 3. In certain embodiments, the neurotrophic signaling factor is detectable by ELISA. In certain embodiments, the biological signaling factor may be glial-derived nexin 1, lumican, TIMP2, IGF2, MMP15, or VEGF. In certain embodiments, the biological signaling factor may be decorin. In certain embodiments, the biological signaling factor may be midkine.

[0075] In certain aspects, the biological signaling factor may be secreted by the composition comprising a cell population comprising OPCs at a concentration of greater than about 50 pg / ml, such as greater than about 100 pg / ml, such as greater than about 200 pg / ml, such as greater than about 300 pg / ml, such as greater than about 400 pg / ml, such as greater than about 500 pg / ml, such as greater than about 1,000 pg / ml, such as greater than about 2,000 pg / ml, such as greater than about 3,000 pg / ml, such as greater than about 4,000 pg / ml, such as greater than about 5,000 pg / ml, such as greater than about 6,000 pg / ml, or such as greater than about 7,000 pg / ml.In one embodiment, the biological signaling factor is about 50 pg / ml to about 100,000 pg / ml, such as about 100 pg / ml, for example, about 150 pg / ml, for example, about 200 pg / ml, for example, about 250 pg / ml, for example, about 300 pg / ml, for example, about 350 pg / ml, for example, about 400 pg / ml, for example, about 450 pg / ml, for example, about 500 pg / ml, for example, about 550 pg / ml, for example, about 600 pg / ml, for example, about 650 pg / ml, for example, about 700 pg / ml l, for example, about 750 pg / ml, for example, about 800 pg / ml, for example, about 850 pg / ml, for example, about 900 pg / ml, for example, about 1,000 pg / ml, for example, about 1,500 pg / ml, for example, about 2,000 pg / ml, for example, about 2,500 pg / ml, for example, about 3,000 pg / ml, for example, about 3,500 pg / ml, for example, about 4,000 pg / ml, for example, about 4,500 pg / ml, for example, about 5,000 pg / ml, for example, about 5,500 pg / ml, 6,000 pg / ml etc., for example, about 6,500 pg / ml etc., for example, about 7,000 pg / ml etc., for example, about 7,500 pg / ml etc., for example, about 8,000 pg / ml etc., for example, about 8,500 pg / ml etc., for example, about 9,000 pg / ml etc., for example, about 10,000 pg / ml etc., for example, about 15,000 pg / ml etc., for example, about 20,000 pg / ml etc., for example, about 25,000 pg / ml etc., for example, about 30,000 pg / ml etc., for example, about 35,000 pg / ml etc., for example, about 40,000 pg / ml The OPC-containing composition may be secreted by the composition comprising a cell population at a concentration in the range of about 45,000 pg / ml, such as about 50,000 pg / ml, for example, about 55,000 pg / ml, such as about 60,000 pg / ml, for example, about 65,000 pg / ml, such as about 70,000 pg / ml, for example, about 75,000 pg / ml, such as about 80,000 pg / ml, for example, about 85,000 pg / ml, such as about 90,000 pg / ml, for example, about 95,000 pg / ml.In one aspect, the biological signaling factor may be secreted by the composition comprising a cell population comprising OPCs at a concentration ranging from about 50 pg / ml to about 1,000 pg / ml, such as from about 50 pg / ml to about 100 pg / ml, for example, from about 100 pg / ml to about 200 pg / ml, for example, from about 200 pg / ml to about 300 pg / ml, for example, from about 300 pg / ml to about 400 pg / ml, for example, from about 400 pg / ml to about 500 pg / ml, for example, from about 500 pg / ml to about 600 pg / ml, for example, from about 600 pg / ml to about 700 pg / ml, for example, from about 700 pg / ml to about 800 pg / ml, for example, from about 800 pg / ml to about 900 pg / ml, or for example, from about 900 pg / ml to about 1,000 pg / ml. In one embodiment, the biological signaling factor is about 1,000 pg / ml to about 10,000 pg / ml, for example, about 1,000 pg / ml to about 2,000 pg / ml, for example, about 2,000 pg / ml to about 3,000 pg / ml, for example, about 3,000 pg / ml to about 4,000 pg / ml, for example, about 4,000 pg / ml to about 5,000 pg / ml, for example, about 5,000 pg / ml The OPC-containing composition may be secreted by a composition comprising a cell population containing OPCs at a concentration ranging from about 6,000 pg / ml to about 6,000 pg / ml, for example, from about 6,000 pg / ml to about 7,000 pg / ml, for example, from about 7,000 pg / ml to about 8,000 pg / ml, for example, from about 8,000 pg / ml to about 9,000 pg / ml, or for example, from about 9,000 pg / ml to about 10,000 pg / ml.In one embodiment, the biological signaling factor is about 10,000 pg / ml to about 100,000 pg / ml, for example, about 10,000 pg / ml to about 20,000 pg / ml, for example, about 20,000 pg / ml to about 30,000 pg / ml, for example, about 30,000 pg / ml to about 40,000 pg / ml, for example, about 40,000 pg / ml to about 50,000 pg / ml, for example, about 50,000 pg / ml The OPC-containing composition may be secreted from a cell population containing OPCs at a concentration ranging from 0 pg / ml to about 60,000 pg / ml, for example, from about 60,000 pg / ml to about 70,000 pg / ml, for example, from about 70,000 pg / ml to about 80,000 pg / ml, for example, from about 80,000 pg / ml to about 90,000 pg / ml, or for example, from about 90,000 pg / ml to about 100,000 pg / ml.

[0076] In one embodiment, midkine can be secreted by a composition comprising a cell population comprising OPCs at a concentration ranging from about 100 pg / ml to about 10,000 pg / ml. In another embodiment, decorin can be secreted by a composition comprising a cell population comprising OPCs at a concentration ranging from about 500 pg / ml to about 50,000 pg / ml. In yet another embodiment, netrin-4 can be secreted by a composition comprising a cell population comprising OPCs at a concentration ranging from about 500 pg / ml to about 50,000 pg / ml. In one embodiment, glial-derived nexin-1 can be secreted by a composition comprising a cell population comprising OPCs at a concentration ranging from about 500 pg / ml to about 50,000 pg / ml. In one embodiment, lumican can be secreted by a composition comprising a cell population comprising OPCs at a concentration ranging from about 500 pg / ml to about 50,000 pg / ml. In one embodiment, TIMP2 may be secreted by a composition comprising a cell population comprising OPCs at a concentration ranging from about 500 pg / ml to about 50,000 pg / ml. In one embodiment, IGF2 may be secreted by a composition comprising a cell population comprising OPCs at a concentration ranging from about 100 pg / ml to about 10,000 pg / ml. In one embodiment, MMP15 may be secreted by a composition comprising a cell population comprising OPCs at a concentration ranging from about 50 pg / ml to about 5000 pg / ml. In one embodiment, VEGF may be secreted by a composition comprising a cell population comprising OPCs at a concentration ranging from about 50 pg / ml to about 5000 pg / ml. In one embodiment, NGF may be secreted by a composition comprising a cell population comprising OPCs at a concentration ranging from about 50 pg / ml to about 5000 pg / ml.

[0077] In certain embodiments, the cell population may have the ability to form no more than 1 epithelial cyst per 100,000 cells in a cyst assay such as that described in Example 5 of the present disclosure. In certain embodiments, the cell population may have the ability to form no more than 1 epithelial cyst per about 200,000 cells, per about 300,000 cells, per about 400,000 cells, or per about 500,000 cells in a cyst assay such as that described in Example 5 of the present disclosure.

[0078] Undesired cell types In one aspect, the cell population may comprise less than about 20% of an undesired cell type, such as less than about 19%, such as less than about 18%, such as less than about 17%, such as less than about 16%, such as less than about 15%, such as less than about 14%, such as less than about 13%, such as less than about 12%, such as less than about 11%, such as less than about 10%, such as less than about 9%, such as less than about 8%, such as less than about 7%, such as less than about 6%, such as less than about 5%, such as less than about 4%, such as less than about 3%, such as less than about 2%, such as less than about 1%, such as less than about 0.5%, such as less than about 0.1%, such as less than about 0.05%, or such as less than about 0.01% of an undesired cell type. In another aspect, the cell population may contain about 15% to about 20% of the undesired cell type, such as about 19% to about 20%, such as about 18% to about 20%, such as about 17% to about 20%, such as about 16% to about 20%, such as about 15% to about 19%, or such as about 16% to about 18%. In yet another aspect, the cell population may contain about 10% to about 15% of the undesired cell type, such as about 14% to about 15%, such as about 13% to about 15%, such as about 12% to about 15%, such as about 11% to about 15%, or such as about 12% to about 14%. In certain embodiments, the cell population may contain about 1% to about 10% of an undesired cell type, such as about 2% to about 10%, such as about 1% to about 9%, such as about 2% to about 8%, such as about 3% to about 7%, or such as about 4% to about 6% of an undesired cell type. In certain embodiments, the cell population may contain about 0.1% to about 1% of an undesired cell type, such as about 0.2% to about 1%, such as about 0.1% to about 0.9%, such as about 0.2% to about 0.8%, such as about 0.3% to about 0.7%, or such as about 0.4% to about 0.6% of an undesired cell type. In some embodiments, the cell population can 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 such as about 0.04% to about 0.06%, of an undesired cell type. In some embodiments, a low level of an undesired cell type can refer to the presence of less than about 15% of an undesired cell type.

[0079] In some embodiments, the undesired cell type may include epithelial lineage cells. In some embodiments, epithelial lineage cells may be characterized by the presence of one of K7 or PCK. In some embodiments, epithelial lineage cells may be characterized by the presence of both K7 and PCK.

[0080] In certain embodiments, the undesired cell type may comprise K7-positive cells. In certain embodiments, the cell population may comprise less than about 5% K7-positive cells, such as less than about 4%, such as less than about 3%, such as less than about 2%, such as less than about 1.5%, such as less than about 1%, such as less than about 0.9%, such as less than about 0.8%, such as less than about 0.7%, such as less than about 0.6%, such as less than about 0.5%, such as less than about 0.4%, such as less than about 0.3%, such as less than about 0.2%, such as less than about 0.1%, such as less than about 0.09%, such as less than about 0.08%, such as less than about 0.07%, such as less than about 0.06%, such as less than about 0.05%, or such as less than about 0.01% K7-positive cells. In one embodiment, the cell population may contain about 0.01% to about 5% K7-positive cells, for example, about 0.05%, such as, for example, about 0.1%, such as, for example, about 0.2%, such as, for example, about 0.3%, such as, for example, about 0.4%, such as, for example, about 0.5%, such as, for example, about 0.6%, such as, for example, about 0.7%, such as, for example, about 0.8%, such as, for example, about 0.9%, such as, for example, about 1%, such as, for example, about 1.5%, such as, for example, about 2%, such as, for example, about 2.5%, such as, for example, about 3%, such as, for example, about 3.5%, such as, for example, about 4%, or for example, about 4.5% K7-positive cells. In one embodiment, the cell population may contain about 1% to about 5% K7-positive cells, for example, about 2% to about 4%, such as, for example, about 1% to about 3%, or for example, about 3% to about 5% K7-positive cells. In certain embodiments, the cell population may contain about 0.1% to about 1%, such as about 0.2% to about 0.9%, such as about 0.3% to about 0.8%, such as about 0.4% to about 0.7%, or for example, about 0.5% to about 0.6% K7-positive cells. In certain embodiments, the cell population may contain about 0.01% to about 0.1% K7-positive cells, such as about 0.02% to about 0.09%, such as about 0.03% to about 0.08%, such as about 0.04% to about 0.07%, or for example, about 0.05% to about 0.06% K7-positive cells. In certain embodiments, the cell population may contain less than about 2% K7-positive cells. In other embodiments, the cell population may contain less than about 0.2% K7-positive cells.

[0081] In some embodiments, the undesired cell type may comprise PCK-positive cells. In some embodiments, the cell population may comprise less than about 10% PCK-positive cells, such as less than about 9%, such as less than about 8%, such as less than about 7%, such as less than about 6%, such as less than about 5%, such as less than about 4.5%, such as less than about 4%, such as less than about 3.5%, such as less than about 3%, such as less than about 2.5%, such as less than about 2%, such as less than about 1.5%, such as less than about 1%, such as less than about 0.5%, such as less than about 0.1%, such as less than about 0.05%, or for example less than about 0.01% PCK-positive cells. In one embodiment, the cell population may contain about 0.01% to about 10% PCK-positive cells, for example, about 0.05%, such as, for example, about 0.1%, such as, for example, about 0.2%, such as, for example, about 0.3%, such as, for example, about 0.4%, such as, for example, about 0.5%, such as, for example, about 0.6%, such as, for example, about 0.7%, such as, for example, about 0.8%, such as, for example, about 0.9%, such as, for example, about 1%, such as, for example, about 2%, such as, for example, about 3%, such as, for example, about 4%, such as, for example, about 5%, such as, for example, about 6%, such as, for example, about 7%, such as, for example, about 8%, or such as, for example, about 9% PCK-positive cells. In one embodiment, the cell population may contain about 1% to about 10% PCK-positive cells, for example, about 2% to about 9%, such as, for example, about 3% to about 8%, such as, for example, about 4% to about 7%, or for example, about 5% to about 6% PCK-positive cells. In some embodiments, the cell population may contain about 0.1% to about 1% PCK-positive cells, such as about 0.2% to about 0.9%, such as about 0.3% to about 0.8%, such as about 0.4% to about 0.7%, or for example, about 0.5% to about 0.6% PCK-positive cells. In some embodiments, the cell population may contain about 0.01% to about 0.1% PCK-positive cells, such as about 0.02% to about 0.09%, such as about 0.03% to about 0.08%, such as about 0.04% to about 0.07%, or for example, about 0.05% to about 0.06% PCK-positive cells. In some embodiments, the cell population may contain less than about 5% PCK-positive cells.

[0082] In some embodiments, a population of OPCs may comprise about 2% K7-positive cells and about 4% PCK-positive cells.

[0083] compound In some embodiments, the compositions disclosed herein may further comprise a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier may comprise dimethyl sulfoxide (DMSO). In some embodiments, the pharmaceutically acceptable carrier does not comprise dimethyl sulfoxide. In some embodiments, the compositions may be adapted for cryopreservation.

[0084] In some aspects, compositions of the present disclosure may be formulated to be administered by direct injection into the spinal cord of a subject. In some aspects, compositions of the present disclosure may be formulated for intracerebral, intraventricular, intrathecal, intranasal, or intracisternal administration to a subject. In some aspects, compositions of the present disclosure may be formulated to be administered by direct injection into or immediately adjacent to the infarct cavity in the brain of a subject. In some aspects, compositions of the present disclosure may be formulated to be administered via implantation. In some aspects, compositions of the present disclosure may be formulated as a solution.

[0085] In some embodiments, compositions according to the present disclosure contain about 1 x 10 per milliliter 6 ~Approx. 5×10 8 cells, e.g., about 1 x 10 per milliliter 6 cells, e.g., about 2 x 10 cells per milliliter 6 cells, e.g., about 3 x 10 cells per milliliter 6 cells, e.g., about 4 x 10 cells per milliliter 6 cells, e.g., about 5 x 10 cells per milliliter 6 cells, e.g., about 6 x 10 cells per milliliter 6 cells, e.g., about 7 x 10 cells per milliliter 6 cells, e.g., about 8 x 10 cells per milliliter 6 cells, e.g., about 9 x 10 cells per milliliter 6 cells, e.g., about 1 x 10 cells per milliliter 7 cells, e.g., about 2 x 10 cells per milliliter 7 cells, e.g., about 3 x 10 cells per milliliter 7 cells, e.g., about 4 x 10 cells per milliliter 7cells, e.g., about 5 x 10 cells per milliliter 7 cells, e.g., about 6 x 10 cells per milliliter 7 cells, e.g., about 7 x 10 cells per milliliter 7 cells, e.g., about 8 x 10 cells per milliliter 7 cells, e.g., about 9 x 10 cells per milliliter 7 cells, e.g., about 1 x 10 cells per milliliter 8 cells, e.g., about 2 x 10 cells per milliliter 8 cells, e.g., about 3 x 10 cells per milliliter 8 cells, e.g., about 4 x 10 cells per milliliter 8 cells, or for example, about 5 x 10 cells per milliliter 8 In another aspect, the compositions of the present disclosure may contain about 1 x 10 cells per milliliter. 8 ~Approx. 5×10 8 cells, e.g., about 1 x 10 per milliliter 8 ~Approx. 4×10 8 cells, e.g., about 2 x 10 cells per milliliter 8 ~Approx. 5×10 8 cells, e.g., about 1 x 10 cells per milliliter 8 ~Approx. 3×10 8 cells, e.g., about 2 x 10 cells per milliliter 8 ~Approx. 4×10 8 cells, or for example, about 3 x 10 cells per milliliter 8 ~Approx. 5×10 8 In yet another aspect, the compositions of the present disclosure may contain about 1 x 10 cells per milliliter. 7 ~Approx. 1×10 8 cells, e.g., about 2 x 10 per milliliter 7 ~Approx. 9×10 7 cells, e.g., about 3 x 10 cells per milliliter 7 ~Approx. 8×10 7 cells, e.g., about 4 x 10 cells per milliliter 7 ~Approx. 7×10 7 cells, or for example, about 5 x 10 cells per milliliter7 ~about 6×10 7 In one embodiment, the compositions of the present disclosure may contain about 1 x 10 cells per milliliter. 6 ~Approx. 1×10 7 cells, e.g., about 2 x 10 per milliliter 6 ~Approx. 9×10 6 cells, e.g., about 3 x 10 cells per milliliter 6 ~Approx. 8×10 6 cells, e.g., about 4 x 10 cells per milliliter 6 ~Approx. 7×10 6 cells, or for example, about 5 x 10 cells per milliliter 6 ~about 6×10 6 In yet another aspect, the compositions of the present disclosure may contain at least about 1 x 10 cells per milliliter. 6 cells, e.g., at least about 2 x 10 cells per milliliter 6 cells, e.g., at least about 3 x 10 cells per milliliter 6 cells, e.g., at least about 4 x 10 cells per milliliter 6 cells, e.g., at least about 5 x 10 cells per milliliter 6 cells, e.g., at least about 6 x 10 cells per milliliter 6 cells, e.g., at least about 7 x 10 cells per milliliter 6 cells, e.g., at least about 8 x 10 cells per milliliter 6 cells, e.g., at least about 9 x 10 cells per milliliter 6 cells, e.g., at least about 1 x 10 cells per milliliter 7 cells, e.g., at least about 2 x 10 cells per milliliter 7 cells, e.g., at least about 3 x 10 cells per milliliter 7 cells, e.g., at least about 4 x 10 cells per milliliter 7 cells, or for example, at least about 5 x 10 cells per milliliter 7 In some embodiments, the compositions of the present disclosure may contain up to about 1 x 10 cells. 8or more cells, e.g., up to about 2 x 10 cells per milliliter 8 or more cells, e.g., up to about 3 x 10 cells per milliliter 8 or more cells, e.g., up to about 4 x 10 cells per milliliter 8 or more cells, e.g., up to about 5 x 10 cells per milliliter 8 or more cells, or, for example, up to about 6 x 10 cells per milliliter 8 It may include cells, etc.

[0086] In some embodiments, compositions according to the present disclosure contain about 4 x 10 per milliliter 7 pieces ~ approx. 2×10 8 The cell may contain cells.

[0087] In yet another embodiment, the composition according to the present disclosure may have a volume in the range of about 10 microliters to about 5 milliliters, such as about 20 microliters, such as about 30 microliters, such as about 40 microliters, for example, about 50 microliters, such as about 60 microliters, for example, about 70 microliters, such as about 80 microliters, for example, about 90 microliters, such as about 100 microliters, for example, about 200 microliters, such as about 300 microliters, for example, about 400 microliters, such as about 500 microliters, for example, about 600 microliters, such as about 700 microliters, for example, about 800 microliters, such as about 900 microliters, such as about 1 milliliter, for example, about 1.5 milliliters, such as about 2 milliliters, for example, about 2.5 milliliters, such as about 3 milliliters, for example, about 3.5 milliliters, such as about 4 milliliters, or for example, about 4.5 milliliters. In one embodiment, the composition according to the present disclosure may have a volume in the range of about 10 microliters to about 100 microliters, for example, about 20 microliters to about 90 microliters, for example, about 30 microliters to about 80 microliters, for example, about 40 microliters to about 70 microliters, or for example, about 50 microliters to about 60 microliters. In another embodiment, the composition according to the present disclosure may have a volume in the range of about 100 microliters to about 1 milliliter, for example, about 200 microliters to about 900 microliters, for example, about 300 microliters to about 800 microliters, for example, about 400 microliters to about 700 microliters, or for example, about 500 microliters to about 600 microliters. In yet another embodiment, the composition according to the present disclosure may have a volume in the range of about 1 milliliter to about 5 milliliters, such as about 2 milliliters to about 5 milliliters, such as about 1 milliliter to about 4 milliliters, such as about 1 milliliter to about 3 milliliters, such as about 2 milliliters to about 4 milliliters, or such as about 3 milliliters to about 5 milliliters.In one embodiment, a composition according to the present disclosure may have a volume of about 20 microliters to about 500 microliters. In another embodiment, a composition according to the present disclosure may have a volume of about 50 microliters to about 100 microliters. In yet another embodiment, a composition according to the present disclosure may have a volume of about 50 microliters to about 200 microliters. In another embodiment, a composition according to the present disclosure may have a volume of about 20 microliters to about 400 microliters.

[0088] container In some embodiments, the container may contain a composition comprising a cell population according to the present disclosure. In some embodiments, the container may be configured for cryopreservation. In some embodiments, the container may be configured for administration to a subject in need thereof. In some embodiments, the container may be a pre-filled syringe.

[0089] How to use Aspects of the present disclosure include methods of using a cell population comprising a plurality of OPCs as described herein to improve one or more neurological functions in a subject in need of treatment. In certain aspects, a cell population according to the present disclosure can be injected or implanted into a subject in need thereof. In certain aspects, the subject may be in need of functional improvement of the central nervous system. In certain aspects, a cell population according to the present disclosure can be implanted into a subject in need thereof to treat spinal cord injury, stroke, or multiple sclerosis.

[0090] In one aspect, the cell populations of the present disclosure comprise up to about 1 x 10 9 cells, e.g., up to about 1 x 10 6 cells, for example, up to approximately 25 x 10 6 cells, for example, up to about 50 × 10 6 cells, for example, up to approximately 75 x 10 6 cells, for example, up to about 100 × 10 6 cells, for example, up to approximately 200 × 10 6 cells, for example, up to approximately 300 × 10 6 cells, for example, up to approximately 400 × 10 6 cells, for example, up to approximately 500 × 106 cells, for example, up to approximately 600 × 10 6 cells, for example, up to approximately 700 × 10 6 cells, for example, up to approximately 800 × 10 6 cells, or for example up to about 900 x 10 6 When transplanted into an injured site of the central nervous system, the cell population of the present disclosure may have the ability to produce ectopic tissue in less than about 2% of subjects. In one aspect, the cell population of the present disclosure may have a cell population of about 1 x 10 6 cells ~ approx. 1 x 10 9 cells, e.g., about 50 x 10 6 cells ~ approx. 900 x 10 6 cells, e.g., about 100 × 10 6 cells ~ approx. 800 x 10 6 cells, e.g., about 200 × 10 6 cells ~ approx. 700 x 10 6 cells, e.g., about 300 × 10 6 cells ~ approx. 600 x 10 6 cells, or for example, about 400 x 10 6 cells ~ approx. 500 x 10 6 When transplanted into a site of central nervous system injury, the cell populations disclosed herein may have the ability to produce ectopic tissue in less than about 2% of subjects. In one aspect, the cell populations disclosed herein may have a maximum of about 1 x 10 9 When transplanted into a site of central nervous system injury, the cells may be capable of producing ectopic tissue in less than about 2% of subjects. In one embodiment, the cell populations of the present disclosure comprise up to about 1 x 10 9 When transplanted into the site of central nervous system injury, these cells may have the capacity to produce ectopic tissue in less than about 1% of subjects.

[0091] In certain aspects, cell populations according to the present disclosure may be capable of inducing myelination of demyelinated axons at the site of transplantation in a subject. In certain aspects, cell populations generated with pre-treatment according to the methods of the present disclosure may exhibit improved engraftment and migration capabilities compared to cell populations generated without pre-treatment. In certain aspects, cell populations generated with pre-treatment according to the methods of the present disclosure may exhibit improved repair or regeneration of neural tissue following injury compared to cell populations generated without pre-treatment.

[0092] In certain embodiments, cell populations of the present disclosure may be capable of improving sensory function in a subject in need of therapy after transplantation thereof into the subject. In certain embodiments, improved sensory function may be assessed using the International Standard for Neurological Classification of Spinal Cord Injury (ISNCSCI) test, e.g., by determining right and left side sensory levels for pinprick and instantaneous touch sensation. In certain embodiments, cell populations of the present disclosure may be capable of improving motor function in a subject in need of therapy after transplantation thereof into the subject. In certain embodiments, cell populations may be capable of sustainably improving a subject's motor function for at least two months. In certain embodiments, cell populations may be capable of sustainably improving a subject's motor function for at least six months, at least one year, at least two years, or at least three years. In certain embodiments, improved motor function may be improved standing or weight bearing, improved limb function or limb strength, improved walking distance, improved walking speed, improved bowel or bladder function, improved arm or hand movement, or improved grasping, grasping, or prehension. In some embodiments, improvement in motor function can be assessed using, for example, the ISNCSCI test to determine right and left side movement levels relative to total paralysis, palpable or visible contractions, active movement, maximum range of motion against gravity, and sufficient resistance.

[0093] In certain aspects, cell populations of the present disclosure may be capable of reducing the volume of injury-induced central nervous system parenchymal cavities within 12 months, hi certain aspects, cell populations of the present disclosure may be capable of reducing the volume of injury-induced central nervous system parenchymal cavities within 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, or less than 1 month.

[0094] In certain embodiments, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location in the central nervous system of a subject in need thereof to one or more second locations. In certain embodiments, one or more cells from a cell population according to the present disclosure may be capable of migrating from a subject's spinal cord to a diseased tissue in the subject's brain. In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location in a subject's spinal cord to a second location located in a diseased tissue in the subject's spinal cord. In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location in a subject's brain to a second location located in a diseased tissue in the subject's brain. In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location in a subject's brain to a diseased tissue in the subject's spinal cord. In one embodiment, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location in a subject's spinal cord to a second location located in a diseased tissue in the subject's spinal cord, as well as to one or more locations located in one or more diseased tissues in the subject's brain. In one embodiment, one or more cells from a cell population of the present disclosure may have the ability to migrate from a first location within the brain of a subject to a second location located in a diseased tissue within the brain of the subject, as well as to one or more locations located in one or more diseased tissues within the spinal cord of the subject.

[0095] In certain aspects, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location within a subject's central nervous system to one or more second locations located in one or more diseased tissues in less than about 150 days, such as less than about 100 days, such as less than about 50 days, or such as less than about 10 days. In certain aspects, one or more cells from a cell population according to the present disclosure may be capable of migrating from a first location within a subject's central nervous system to one or more second locations located in one or more diseased tissues in about 180 days or less.

[0096] Additional Embodiments An embodiment of the present disclosure includes a container containing a composition, the composition comprising a cell population comprising a plurality of oligodendrocyte progenitor cells (OPCs), the cell population comprising less than 15% of an undesired cell type. In one embodiment, the undesired cell type comprises an epithelial lineage cell. In one embodiment, the epithelial lineage cell is characterized by the presence of one or more markers selected from the group consisting of K7 and PCK. In one embodiment, the cell population comprises less than 2% K7-positive cells. In one embodiment, the cell population comprises less than 0.2% K7-positive cells. In one embodiment, the cell population comprises less than 5% PCK-positive cells. In one embodiment, the cell population has a common genetic background. In one embodiment, the cell population has not undergone cell enrichment. In one embodiment, the container is configured for cryopreservation.

[0097] Embodiments of the present disclosure also include a container containing a composition, the composition comprising a cell population comprising a plurality of oligodendrocyte progenitor cells (OPCs), the cell population capable of forming no more than one epithelial cyst per 100,000 cells in a cyst assay. In certain embodiments, the cell population is capable of reducing the volume of injury-induced central nervous system parenchymal cavities in 12 months or less. In certain embodiments, the cell population is capable of migrating from a first location within a subject's central nervous system tissue to one or more second locations located in one or more diseased tissues in 180 days or less. In certain embodiments, the central nervous system tissue is spinal cord tissue. In certain embodiments, the central nervous system tissue is brain tissue. In certain embodiments, the cell population is capable of improving motor function in a subject in need of therapy after transplantation of the population into the subject. In certain embodiments, the cell population is capable of sustainably improving motor function for at least two months. In certain embodiments, the motor function is improved standing ability or weight bearing. In some aspects, the motor function is improved limb function or limb strength. In some aspects, the motor function is improved walking distance. In some aspects, the motor function is improved walking speed. In some aspects, the motor function is improved bowel or bladder function. In some aspects, the motor function is improved arm or hand movement. In some aspects, the motor function is improved grasping, grasping, or grasping.

[0098] Embodiments of the present disclosure also include a container containing a composition, the composition comprising a cell population comprising a plurality of oligodendrocyte progenitor cells (OPCs), the cell population capable of forming no more than one epithelial cyst per 100,000 cells in a cyst assay. In certain embodiments, the cell population is capable of producing one or more biological signaling factors. In certain embodiments, the cell population is capable of producing an angiogenic signaling factor. In certain embodiments, the angiogenic signaling factor is selected from the group consisting of thrombospondin-1, serpine1, serpine2, and combinations thereof. In certain embodiments, the cell population is capable of producing a neurotrophic signaling factor. In certain embodiments, the neurotrophic factor is selected from the group consisting of NGF, netrin-4, tenascin-C, thrombospondin-1, thrombospondin-3, SLIT1, SLIT3, and combinations thereof. In certain embodiments, the one or more biological signaling factors comprise decorin. In certain embodiments, the one or more biological signaling factors comprise midkine. In some embodiments, the cell population is capable of inducing myelination of demyelinated axons at the site of implantation within a subject.

[0099] An aspect of the disclosure is a container containing a composition, the composition comprising a cell population comprising a plurality of oligodendrocyte progenitor cells (OPCs), the cell population comprising up to 1 x 10 9 The present invention also includes a vessel in which the cells, when transplanted into a site of central nervous system injury, are capable of producing ectopic tissue in less than 2% of subjects. In one embodiment, the cell population comprises up to 1 x 10 9 When transplanted into the site of central nervous system injury, these cells can produce ectopic tissue in less than 1% of subjects.

[0100] The above aspects of the present disclosure can further include a composition comprising a cell population, wherein at least 30% of the cell population are NG2-positive cells. In some aspects, at least 40% of the cell population are NG2-positive cells. In some aspects, at least 50% of the cell population are NG2-positive cells.

[0101] An embodiment of the present disclosure is a container containing a composition, the composition comprising a population of oligodendrocyte progenitor cells (OPCs), wherein at least 95% of the OPCs express a marker selected from the group consisting of PDGF-Rα, IGF2, Nkx2.2, Olig1, and nestin. In some embodiments, at least 98% of the OPCs express a marker selected from the group consisting of PDGF-Rα, IGF2, Nkx2.2, Olig1, and nestin. In some embodiments, at least 99% of the OPCs express a marker selected from the group consisting of PDGF-Rα, IGF2, Nkx2.2, Olig1, and nestin. In some embodiments, at least 99.5% of the OPCs express a marker selected from the group consisting of PDGF-Rα, IGF2, Nkx2.2, Olig1, and nestin. In some embodiments, 100% of the OPCs express a marker selected from the group consisting of PDGF-Ra, IGF2, Nkx2.2, Oligl, and nestin. In some embodiments, at least 95% of the OPCs express a second marker selected from the group consisting of PDGF-Ra, IGF2, Nkx2.2, Oligl, and nestin. In some embodiments, at least 98% of the OPCs express a second marker selected from the group consisting of PDGF-Ra, IGF2, Nkx2.2, Oligl, and nestin. In some embodiments, at least 99% of the OPCs express a second marker selected from the group consisting of PDGF-Ra, IGF2, Nkx2.2, Oligl, and nestin. In some embodiments, at least 99.5% of the OPCs express a second marker selected from the group consisting of PDGF-Ra, IGF2, Nkx2.2, Oligl, and nestin. In one embodiment, 100% of the OPCs express a second marker selected from the group consisting of PDGF-Rα, IGF2, Nkx2.2, Olig1, and nestin.

[0102] The above aspects of the present disclosure can further include a pharmaceutically acceptable carrier. In some aspects, the pharmaceutically acceptable carrier includes dimethyl sulfoxide. In some aspects, the pharmaceutically acceptable carrier does not include dimethyl sulfoxide.

[0103] The above aspects of the present disclosure can further include a composition comprising a cell population, said composition comprising at least 1 x 10 cells per milliliter. 6 In one embodiment, the composition contains 1 x 10 cells per milliliter. 6 ~5×10 8 In one embodiment, the composition contains 4 x 10 cells per milliliter. 7 ~2×10 8 In some embodiments, the composition has a volume of 20 to 500 microliters. In some embodiments, the composition has a volume of 50 to 200 microliters. In some embodiments, the composition has a volume of about 100 microliters. In some embodiments, the composition is an injectable solution. In some embodiments, the composition is adapted for cryopreservation.

[0104] Further embodiments of the present disclosure include methods of differentiating pluripotent stem cells, the methods comprising one or more steps associated with pre-treating a plurality of undifferentiated stem cells. In certain embodiments, the one or more steps associated with pre-treating comprise incubating the expanded but undifferentiated stem cells for a period of time, during which the undifferentiated stem cells are contacted with one or more stem cell differentiation-regulating molecules. In certain embodiments, the one or more stem cell differentiation-regulating molecules are selected from the group consisting of: (1) an inhibitor of ALK5, which is part of the SMAD / TGFβ-RII signaling pathway; (2) an inhibitor of ALK2, which is part of the BMPRI signaling pathway; (3) a GSK3 inhibitor that activates the WNT signaling pathway; and (4) a smoothened agonist that activates the SHH pathway. In certain embodiments, the method comprises incubating the expanded but undifferentiated stem cells for a period of time with four small molecules: SB431542, dorsomorphin, CHIR99021, and parmorphamine. In certain embodiments, the method comprises incubating an expanded but undifferentiated stem cell population for a first period of time, during which the undifferentiated stem cells are contacted with two or more first stem cell differentiation regulators, and incubating the population for a second period of time, during which the cells are contacted with two or more second stem cell differentiation regulators. In certain embodiments, the two or more first stem cell differentiation regulators are different from the two or more second stem cell differentiation regulators. In certain embodiments, the two or more first stem cell regulators are identical to the two or more second stem cell regulators. In certain embodiments, the first and second stem cell regulators share at least one common regulator. In certain embodiments, the first and second stem cell regulators share two or more common regulators, such as three or more common regulators, such as four or more common regulators.

[0105] Having now generally described the invention, the same will be more readily understood by reference to the following examples, which are offered by way of illustration and are not intended to limit the disclosure unless otherwise specified. [Example]

[0106] Example 1 - Method of generating a population of OPCs containing a pre-treatment Undifferentiated human embryonic stem cells (uhESCs) derived from the H1 line (WA01; Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998 Nov 6;282(5391):1145-7) were cultured on Matrigel® GFR in KO-DMEM diluted 1:30 with X-VIVO10 medium supplemented with 80 ng / mL bFGF (ThermoFisher, PHG0263) and 0.5 ng / mL TGF-β1 (R&D System, Cat. No. 240-B). The medium was completely changed daily, starting 2 days after subculture. Undifferentiated human embryonic stem cells were subcultured using collagenase and manual scraping.

[0107] uhESCs were exposed to a mixture of small molecules to stimulate the cells and then induce differentiation. For subculture before the initiation of differentiation, cells were cultured at approximately 0.8–1.0 × 10 5 cells / cm 2The uhESCs were seeded at 100°C and cultured as previously described. When the uhESCs reached 30-50% confluence, the medium was changed to glial progenitor medium supplemented with the following small molecules: SB431542 (Sigma-Aldrich, Catalog No. S4317) at final concentrations of 10 μM, dorsomorphin (Sigma-Aldrich, Catalog No. p5499) at final concentrations of 2 μM, CHIR99021 (Stemgent, Catalog No. 04-0004) at final concentrations of 3 μM, and palmorfamine (Stemgent, Catalog No. 04-0009) at final concentrations of 0.5 μM. The glial progenitor medium containing the small molecules was changed daily for 4 days. On day 4, the glial progenitor medium was changed with the further modification of small molecule addition: 3 μM CHIR99021, 0.5 μM palmorfamine, and 150 μM ascorbic acid (Sigma-Aldrich, Cat. No. A4544). The medium containing added small molecules was changed daily for the next 3 days before initiating differentiation.

[0108] Differentiation of pre-treated uhESCs into OPCs was initiated by sequentially using collagenase IV (Life Technologies, catalog no. 17104-019) and 0.05% trypsin-EDTA (Life Technologies, catalog no. 25300-054) to count cells in surrogate flasks to achieve a single-cell suspension for quantification, and harvesting the remaining pre-treated cell culture using PBS containing 0.5 mM EDTA. uhESC cultures were seeded in ultra-low attachment (ULA) vessels at 7.33 × 10 cells in a 1:1 mixture of X-VIVO10 and glial progenitor medium (GPM). 5 cells / cm 2Embryoid bodies (EBs) were formed at a density of 1000 x g / mL. The day after differentiation, a complete medium change was performed using a 1:1 mixture of X-VIVO10 and GPM. On this day, GPM was supplemented with 2 ng / mL hbFGF, 20 ng / mL EGF, and 10 μM retinoic acid in DMSO (RA, Sigma-Aldrich, Cat. No. R-2625). From day 2 of differentiation onward, the medium was 100% GPM supplemented only with 20 ng / mL EGF and 10 μM RA. This medium was replaced daily until day 9. From day 9 to day 27, GPM medium supplemented with 20 ng / mL EGF was replaced every other day. On day 27, the 1 cm ULA surface seeded at the initiation of differentiation was cultured. 2 2cm per 2 EBs were plated onto Matrigel® GFR-coated vessels at a ratio of 1:10. The culture medium used throughout the remainder of differentiation was GPM supplemented with 20 ng / mL EGF. From day 27, cell cultures were subjected to complete medium changes every other day. On day 34, cell cultures detached using 0.05% trypsin-EDTA were counted and plated at 5 x 10 on Matrigel® GFR-coated vessels. 4 viable cells / cm 2 GPM was replaced every other day from day 34 until the final harvest at 7 days old.

[0109] Day 41 OPC harvest contained a mixture of 0.05% trypsin-EDTA (EMD Millipore, catalog no. P24-5513P3) supplemented with 20 U / mL Benzonase and 0.01% Pluronic-F68 (Life Technologies, catalog no. 24040-032). Detached cells were pooled in a 1:1 mixture of DMEM-F12 medium and HypoThermosol FRS (BioLife Solutions, catalog no. 101104) supplemented with 20 U / mL Benzonase and 0.01% Pluronic-F68, then counted and reconstituted in CryoStorage prior to cryopreservation.

[0110] 5A shows the changes in the levels of NG2, K7, and PCK markers expressed in OPCs generated by an example method compared to OPCs generated without prior treatment with a small molecule described herein. OPCs generated with a prior treatment step express higher levels of NG2 and lower levels of K7 and PCK.

[0111] Example 2 - Comparison of alternative methods for generating OPC populations including pre-treatment Undifferentiated human embryonic stem cells (uhESCs) derived from the H1 line (WA01; Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science 1998 Nov 6;282(5391):1145-7) were cultured on one of the following matrices: Matrigel® GFR diluted 1:30 in KO-DMEM, recombinant laminin 521 (Corning SelfCoat, Cat. No. 354221 or PureCoat), or 80 ng / mL bFGF (ThermoFisher, PHG0263) and 0.5 ng / mL TGF-β1 (R&D Vitronectin (StemCell Technologies, Catalog No. 07180) dissolved in CellAdhere Dilution Buffer (StemCell Technologies, Catalog No. 07183) was used with X-VIVO10 medium supplemented with PBS (StemCell Systems, Catalog No. 240-B). Starting two days after subculture of the cells, the medium was completely changed daily. Undifferentiated human embryonic stem cells were subcultured using collagenase and manual scraping or other non-enzymatic means, such as 0.5 mM EDTA (Life Technologies, Catalog No. 15575-020) dissolved in PBS or ReLeSR™ (StemCell Technologies, Catalog No. 5872).

[0112] uhESCs were exposed to a mixture of small molecules to stimulate the cells and then induce differentiation. For subculture before the initiation of differentiation, cells were cultured at approximately 0.2–1.3 × 10 5 cells / cm 2 The uhESCs were seeded at 100°C and cultured as in previous subcultures. When the uhESCs reached 30-50% confluence, the medium was changed to glial progenitor medium (GPM, as described herein) containing small molecules at final concentrations of 10 μM SB431542 (Sigma-Aldrich, Catalog No. S4317), 2 μM dorsomorphin (Sigma-Aldrich, Catalog No. p5499), 3 μM CHIR99021 (Stemgent, Catalog No. 04-0004), and 0.5 μM palmorfamine (Stemgent, Catalog No. 04-0009). The glial progenitor medium containing small molecules was changed daily for 4 days. On day 4, the glial progenitor medium was changed with the further modification of small molecule addition: 3 μM CHIR99021, 0.5 μM palmorfamine, and 150 μM ascorbic acid (Sigma-Aldrich, Cat. No. A4544). The medium with added small molecules was changed daily for the next 3 days before initiating differentiation.

[0113] Differentiation of the pre-treated uhESCs into OPCs was then initiated by sequentially using collagenase IV (Life Technologies, catalog no. 17104-019) and 0.05% trypsin-EDTA (Life Technologies, 25300-054) to count the cells using the surrogate flask to achieve a single-cell suspension for quantification, and harvesting the remaining pre-treated culture using PBS containing 0.5 mM EDTA. Pre-treated uhESC cultures were seeded in ultra-low attachment (ULA) vessels at 7.33 × 10 cells in a 1:1 mixture of X-VIVO10 and glial progenitor medium (GPM) composed of DMEM / F12, Gibco catalog number 10565-018, 2% B27 supplement, Gibco catalog number 17504-044, 0.04 μg triiodothyronine, Sigma catalog number T5516-1MG supplemented with 4 ng / mL hbFGF and 20 ng / mL EGF (Life Technologies, catalog number PHG0311). 5 cells / cm 2 Alternatively, rather than using ULA tissue culture vessels, pre-treated cultures were grown in a stirred suspension system, e.g., at a density of 1.8 x 10 6 EBs were formed by culturing the cells at a concentration of 100 cells / mL in 0.1 L disposable PBS spinner flasks agitated at 25 rpm. The day after differentiation, a complete medium change was performed using a 1:1 mixture of X-VIVO10 and glial progenitor medium or GPM. On this day, GPM was supplemented with DMSO containing 2 ng / mL hbFGF, 20 ng / mL EGF, and 10 μM retinoic acid (RA, Sigma-Aldrich, Cat. No. R-2625). Starting on day 2 of differentiation, the medium was 100% GPM supplemented only with 20 ng / mL EGF and 10 μM RA. This medium was replaced daily until day 9. Starting on day 9 and continuing until day 27, GPM medium supplemented with 20 ng / mL EGF was replaced every other day. On day 27, 1 cm of ULAs seeded at the initiation of differentiation were cultured. 2 , or 2 cm per 30 mL of suspension 2EBs were plated onto vessels coated with Matrigel® GFR, recombinant laminin, or vitronectin at a ratio of 1:10. The culture medium used throughout the remainder of differentiation was GPM supplemented with 20 ng / mL EGF. Starting on day 27, cell cultures were subjected to complete medium changes every other day. On day 34, cell cultures detached using 0.05% trypsin-EDTA or TrypLE Select (Life Technologies, Cat. No. A12859) were counted and plated at a ratio of 5 x 10 onto vessels coated with Matrigel® GFR, recombinant laminin, or vitronectin. 4 viable cells / cm 2 GPM was replaced every other day from day 34 until the final harvest at 7 days old.

[0114] Day 41 OPC harvesting was performed using a mixture of 0.05% trypsin-EDTA or TrypLE Select supplemented with 20 U / mL Benzonase (EMD Millipore, catalog no. P24-5513P3) and 0.01% Pluronic-F68 (Life Technologies, catalog no. 24040-032). Detached cells were pooled in a 1:1 mixture of DMEM-F12 medium and HypoThermosol FRS (BioLife Solutions, catalog no. 101104) supplemented with 20 U / mL Benzonase and 0.01% Pluronic-F68, then counted and reconstituted in CryoStor5 prior to cryopreservation.

[0115] According to the examples, the effect of pre-treatment of uhESCs with small molecules on the expression of NG2, K7, and PCK marker levels in OPCs was evaluated across different culture conditions (see Figures 5B-5F). OPCs generated after the pre-treatment step express higher levels of NG2 and lower levels of K7 and PCK. During the uhESC expansion phase, OPCs were generated under the following conditions: XVIVO10 culture medium and a recombinant laminin matrix, with collagenase IV used to harvest the expanded ES cells (see Figure 5B1-A). During the uhESC expansion phase, OPCs were generated under the following conditions: XVIVO10 culture medium and a recombinant laminin matrix, with collagenase IV used to harvest the expanded ES cells; the pre-treated cultures were then cultured in disposable PBS spinner flasks, while the control cultures were cultured in ULA vessels (see Figure 5B2-A). During the uhESC expansion phase, OPCs were generated under the following conditions: XVIVO10 culture medium and Matrigel® GFR, with collagenase IV used to harvest the expanded ES cells; both the pre-treated and control cultures were then cultured in disposable PBS spinner flasks (see Figure 5C). During the uhESC expansion phase, OPCs were generated under the following conditions: XVIVO10 culture medium and Matrigel® GFR, with collagenase IV used to harvest the expanded ES cells (see Figures 5D and 5E). During the uhESC expansion phase, OPCs were generated under the following conditions: XVIVO10 culture medium and a vitronectin matrix with ReLeSR™ used to harvest the expanded ES cells; cultures were then subjected to a short pre-treatment while cultured on the vitronectin matrix and harvested prior to differentiation with EDTA (see Figure 5F).

[0116] Example 3 - Characterization of cell populations by flow cytometry Flow cytometry was used to quantify the relative proportion of OPCs present in the differentiated populations through the identification of specific markers, such as NG2, nestin, and PDGF-Rα, which were quantified for each population.

[0117] To detect the expression of cell surface markers, cells were washed with PBS and incubated in staining buffer (PBS containing 2% FBS and 0.5% sodium azide) containing 10% goat serum to block nonspecific binding sites. Next, antibodies specifically recognizing the marker of interest (e.g., NG2, nestin, PDGF-Rα, K7, pan-cytokeratin, etc.) and their isotype controls were incubated with the cells. Unbound antibodies were removed by washing with staining buffer, and if the antibody was not conjugated with a fluorophore, cell-bound antibodies were detected using an anti-antibody conjugated with a fluorophore. The cells were washed, and propidium iodide was then added to distinguish dead cells. The cells were acquired on a flow cytometer, such as a FACSCalibur, and only viable cells were analyzed. The percentage of cells expressing a given marker was calculated by subtracting the percentage of cells nonspecifically bound to the isotype control antibody from the percentage of cells bound to the specific antibody. The degree of marker expression per cell was calculated as the ratio of the fluorescence intensity of the marker local population to that of isotype control stained cells.

[0118] Table 2 compares representative marker expression between OPCs generated by methods according to embodiments of the present disclosure with and without pre-treatment of undifferentiated cells. Methods including pre-treatment as described herein produced OPCs with significantly higher expression of OPC markers NG2 and PDGF-Rα, and lower expression of non-OPC markers such as Oct4, Tra-1-60, K7, and PCK, compared to differentiation methods without pre-treatment.

[0119] [Table 2]

[0120] Table 3 compares representative marker expression between OPCs generated by variations of methods according to embodiments of the present disclosure. Column headings "Stage I Medium" and "Stage I Process" refer to conditions applied during the uhESC expansion phase, while column headings "Stage II Process" refer to the type of pretreatment step applied: (1) control—cultures not pretreated in flasks or spinner flasks; (2) pretreatment; (3) pretreatment in spinner flasks; or (4) a short pretreatment performed in parallel with culture on a vitronectin matrix. In all conditions tested, expression of NG2 was increased in OPCs generated with pretreatment compared to the control, while the epithelial markers K7 and PCK were decreased in OPCs generated with pretreatment compared to the control.

[0121] [Table 3]

[0122] Example 4 - Characterization of cell populations by gene expression profiling Gene expression profiling can be used to further characterize the cell types present in both the final OPC population and during derivation from the starting hESC population, including both global transcriptome profiling using methods such as microarrays and RNA-seq, and targeted gene profiling using sensitive methods such as quantitative real-time PCR (qPCR).

[0123] To perform gene expression profiling, cells were lysed in a nucleic acid stabilizing solution, such as Qiagen's RLT lysis buffer (Qiagen No. 79216), and RNA was purified using a standard extraction kit, such as Qiagen's RNeasy Mini Kit (Qiagen No. 74106), according to the manufacturer's guidelines. For qPCR-based analysis, the purified RNA was then converted to cDNA using standard methods, such as Qiagen's RT2 Easy First Strand Kit (Qiagen No. 330421), according to the manufacturer's guidelines. The relative expression levels of target genes and reference housekeeping genes were then quantified using pre-made qPCR arrays, such as Qiagen's RT2 Profiler PCR Arrays (Qiagen No. 330231), or individual probes, such as Qiagen's RT2 qPCR Primer Assays (Qiagen No. 330001), according to the manufacturer's guidelines. To determine the relative expression levels of a given target gene, PCR reactions were performed on a standard real-time PCR instrument, such as an ABI 7900HT Real-Time Sequence Detection System (Applied Biosystems) or equivalent. Each target gene was normalized to one or more reference genes, such as GAPDH, to determine its relative expression level.

[0124] For microarray analysis, purified RNA can be used to construct cDNA libraries using standard methods, such as the Affymetrix GeneChip WT PLUS Reagent Kit (Affymetrix No. 902281), per manufacturer's guidelines, hybridized to whole transcriptome arrays, such as the Affymetrix HUGENE2.0ST array (Affymetrix No. 902113), and analyzed using standard equipment, such as the Affymetrix GeneChip Scanner3000 7G System (Affymetrix No. 00-0213). The resulting microarray data can then be normalized, and subsequent analysis of relative gene expression can be performed using the Affymetrix Expression Console software package (Affymetrix) or equivalent.

[0125] Table 4 shows representative qPCR analysis results for neural / glial progenitor genes, ectodermal / neurectodermal lineage genes, and non-neurectodermal lineage genes in OPCs generated with or without prior treatment of undifferentiated cells by methods according to embodiments of the present disclosure. Here, RNA samples were collected on day 9 of differentiation and processed for qPCR using the methods described above. Four neural / glial progenitor genes were quantified: FABP7, NEUROG2, NKX2.2, and OLIG2. Four early ectodermal / neurectodermal lineage genes were quantified: FGF5, FOXA1, GAD1, and GAD2. Five early non-neurectodermal lineage genes were quantified: HAND1, HAND2, MYL3, NPPA, and OTX2. Folds relative to H1 hESC6 OPC d9 were calculated using the ΔΔC method with GAPDH as the housekeeping gene.

[0126] Referring to Table 4, OPCs generated by methods involving pretreatment according to embodiments of the present disclosure exhibited significantly increased neuronal / glial progenitor gene expression compared to OPCs generated by methods without pretreatment. Specifically, the neuronal / glial precursor genes FABP7, NEUROG2, NKX2.2, and OLIG2 were highly expressed in OPCs generated by methods with pretreatment. Expression of the early ectodermal / neuroectodermal lineage gene GAD2 was also significantly reduced in OPCs generated by methods with pretreatment compared to OPCs generated without pretreatment. Furthermore, compared to methods without pretreatment, methods involving pretreatment according to the present disclosure resulted in reduced expression of non-neuroectodermal lineage genes during the differentiation process, including reduced expression of the trophoblast lineage gene, Hand1.

[0127] [Table 4]

[0128] When gene expression profiling was used to compare OPCs generated using a method without prior treatment in accordance with the present disclosure with OPCs generated using a method with prior treatment, both similarities and differences were observed. Both types of methods produced populations of NG2-positive OPCs that also expressed markers such as PDGF-Rα, DCN, and IGF2. However, when OPCs were generated using a method including prior treatment in accordance with the present disclosure, expression of genes associated with other cell types, including epithelial-related genes such as K7 and CDH1 / E-cadherin, was reduced.

[0129] Example 5 - Evaluation of Unwanted Epithelial Lineage Cells Using an In Vitro Cyst Assay The presence of unwanted epithelial-lineage cells in hESC-derived OPC populations was tested using an in vitro cyst assay. Cyst assays were performed as described by Debnath et al., with the following modifications: (1) cells were cultured in 24-well plates at an input of 40,000 cells per well; (2) cells were cultured for up to 35 days to allow more time for epithelial cell proliferation and epithelial structure expansion; (3) cyst structures were detected by immunofluorescence staining and whole-well image acquisition using an IN Cell Analyzer 2000 (GE Healthcare Life Sciences) or similar automated imaging system; and (4) cyst frequency and size were quantified using analysis software, such as IN Cell Developer Software (GE Healthcare Life Sciences). (Debnath J, Muthuswamy SK, Brugge JS, Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods, June 2003;30(3):256~68).

[0130] Specifically, to test for cyst-forming ability, cells were grown in a 3D culture system for 20–35 days in the presence of factors known to stimulate epithelial cyst formation. In addition to visual detection of cysts, the presence of cystic structures, including basal protein expression of the epithelial marker CD49f, was also assessed using immunocytochemistry. Furthermore, to confirm the correlation between activity levels measured by the cyst assay and in vivo cyst activity, flow cytometry was used to detect epithelial markers and cyst formation in injured rodents.

[0131] When OPCs generated by the method without prior treatment were tested in an in vitro cyst assay, varying levels of epithelial cyst formation were observed. Referring to Figure 6A, the left and center panels are representative pictographs of H1 hESC-derived OPCs generated by the method without prior treatment, showing large and small cystic structures, respectively. In contrast, when OPCs generated by the method with prior treatment according to the present disclosure were tested in an in vitro cyst assay, different results were observed. The right panel of Figure 6A is a representative pictograph of H1 hESC-derived OPCs generated by the method with prior treatment according to the present disclosure, showing the absence of cystic structures.

[0132] When OPC lots that formed cysts in the cyst assay were administered to the injured spinal cord of adult rodents, epithelial-like cystic structures formed in vivo. Figure 6B shows representative histology images of an adult female rat 9 months after cervical spinal cord injury and administration of H1 hESC-derived OPCs generated without prior treatment, at different magnifications and with different histological stains. The top panel of Figure 6B shows a low-magnification image of hematoxylin / eosin (H&E)-stained spinal cord tissue containing the injury / OPC graft site. The black box indicates the location of the epithelial cystic structures. The bottom panel of Figure 6B shows a high-magnification image of the epithelial cystic structures stained with various histological stains. The bottom left panel shows H&E staining. The bottom center panel shows staining with eosin and the human-specific probe, hALU, confirming the presence of human cells within the structures. The bottom right panel shows staining with eosin and the proliferation marker Ki67, demonstrating minimal intracystic cell proliferation. Finally, Figure 6C shows a linear regression plot of in vitro cyst counts versus in vivo cyst formation frequency in several populations of OPCs generated without prior treatment, confirming that the number of cysts formed in vitro correlates with cyst formation frequency in this animal model.

[0133] In contrast, OPCs generated using the methods of the present disclosure incorporating pretreatment expressed low levels of epithelial markers by flow cytometry and did not form epithelial cysts in the cyst assay (Figure 6A, right panel). See Table 5, OPCs generated using the method without pretreatment produced significantly more cysts in vitro compared to OPCs produced using the method incorporating pretreatment according to the present disclosure.

[0134] [Table 5]

[0135] Because the frequency of cyst formation and size of cysts formed in cyst assays positively correlates with the relative ability of a population to form ectopic cystic structures in injured rodent spinal cords, OPCs generated using the pre-treatment methods according to the present disclosure are not expected to form epithelial cysts in vivo.

[0136] Example 6 - Engraftment of OPCs The ability of OPC populations to engraft within the mammalian central nervous system and migrate toward the site of injury is an important indicator of their biological activity and potential efficacy. Furthermore, the presence of injury-induced cavities and myelinated axons within the injury site can be measured as potential surrogate indicators of repair / regeneration after injury.

[0137] To evaluate engraftment / migration and post-injury repair / regeneration in a rodent model of spinal cord injury, adult female athymic nude rats were used. Prior to OPC transplantation, rats underwent laminectomy at the desired injury site (e.g., C5-C6 for cervical spine injury), and a semi-contusion / crush injury was induced using an Infinite Horizons Impactor (Precision Systems and Instrumentation No. IH-0400 or equivalent) according to the manufacturer's guidelines. Approximately 1 week to 1 month after injury, OPCs were transplanted at a concentration of 2.4 × 10 5 ~2.4×10 6The cells were directly implanted into the spinal cord adjacent to the injury site at a dose of 0.01 mg / kg / day. Between 2 weeks and 12 months after implantation, animals were sacrificed, and tissues were processed using standard histological methods. To assess engraftment and migration, fixed spinal cord tissue sections were stained with a human-specific probe, such as an anti-human nuclei antibody (Millipore no. MAB1281), followed by an appropriate colorimetric or fluorescently conjugated secondary antibody, according to the manufacturer's guidelines. To assess injury-induced cavities, fixed spinal cord tissue sections were stained with hematoxylin and eosin solution using standard histological methods. Images collected by brightfield imaging were used to measure the area of ​​cavities using standard imaging software, such as ImageJ (NIH). To assess the relative abundance of myelinated fibers within the injury site, fixed spinal cord tissue sections were stained with eriochrome cyanine using standard histological methods and imaged by brightfield imaging.

[0138] When OPCs were generated without prior treatment and evaluated as described above, several observations were made. First, the transplanted cells demonstrated robust engraftment for up to 12 months and migrated to the injury site as early as 2 weeks after transplantation. Compared with rats subjected to spinal cord injury and transplanted with vehicle, rats transplanted with such OPCs demonstrated a reduction in injury-induced cavities as early as 2 weeks after transplantation, and this effect persisted for up to 12 months after transplantation. Furthermore, although myelinated axons were visible within the injury site, beginning approximately 3 months after transplantation and increasing up to 12 months after transplantation, myelinated axons failed to cross the cavitary sites typically seen in vehicle-treated injured rats (see, for an example of such observations, Priest CA, Manley NC, Denham J, Wirth ED 3rd, Lebkowski JS, Preclinical safety of human embryonic stem cell-derived oligodendrocyte progenitors supporting clinical trials in spinal cord injury, Regen Med. 2015 Nov;10(8):939-58).

[0139] Referring to Figure 7, adult female rats were subjected to a C5 cervical spinal cord contusion injury and transplanted with OPCs generated using the pretreatment method according to the present disclosure. Two weeks after transplantation, longitudinal spinal cord sections were immunofluorescently stained with anti-human nuclear antibody (hNUC) to label engrafted cells and DAPI to label all cell nuclei. Figure 7 shows the injury site (white arrow) labeled with DAPI (left panel) and hNUC (right panel) and imaged on a Zeiss Axioskop2. OPCs generated using the pretreatment method according to the present disclosure demonstrated robust engraftment and migration toward the injury site two weeks after transplantation.

[0140] Example 7 - Use of OPC populations to treat stroke OPCs generated using the methods of the present disclosure can be used to treat stroke. To demonstrate functional improvement, previously established mouse models of subcortical white matter stroke (Sozmen et al. (2009), J. Neurosci Methods 180(2):261; Hinman et al. (2013), Stroke 44(1):182) can be adapted to immunodeficient NSG mice (Shultz et al. (2007), Nat Rev Immunol. 7(20:118; jaxmice.jax.org / nod-scid-gamma).

[0141] Specifically, to induce a focal ischemic lesion, N5-(1-iminoethyl)-L-ornithine dihydrochloride (L-Nio, Calbiochem) is injected directly into the corpus callosum of each mouse brain. Subsequently, 100,000 OPCs / mouse (high dose) or 10,000 OPCs / mouse (low dose) are injected as a single 1 µL dose adjacent to the stroke lesion core on day 7 after stroke. An example experimental timeline is shown in Figure 8. Neurological recovery is assessed by monthly behavioral testing.

[0142] Two types of behavioral tests can be performed in this study: the grid walking test and the cylinder test.

[0143] In the grid walking test, animals are placed on an elevated, leveled grid with openings. Animals without brain damage typically place their paws precisely on the wire frame to support themselves while moving along the grid. A "foot fault" is recorded each time a paw slips onto the open grid. The number of both contralateral and ipsilateral faults per paw is compared to the total number of steps taken and then scored using a foot fault index.

[0144] In the cylinder test, animals are placed in a transparent Plexiglas cylinder and observed. Mice actively explore a vertical surface by standing on their hind limbs and exploring the surface with their forepaws and vibrissae. Behavior within the cylinder is assessed by recording the number of independent wall placements observed with the right forepaw, the left forepaw, and both forepaws simultaneously.

[0145] Test groups can be constructed using a total of 72 mice, with 12 mice each assigned to the following subgroups: (1) control (sham operation), (2) stroke alone, (3) OPC transplantation into non-stroke animals (half transplanted with low-dose OPCs and the other half transplanted with high-dose OPCs), (4) low-dose OPCs transplanted adjacent to the stroke lesion into stroke animals, (5) high-dose OPCs transplanted adjacent to the stroke lesion into stroke animals, and (6) high-dose OPCs transplanted into the stroke lesion of stroke animals.

[0146] Expected data that may be generated from experiments performed in accordance with the examples is shown in FIGS.

[0147] Referring to FIG. 9, performance in the grid walking test is expected to demonstrate better motor control and improved gait 4 months after stroke and transplantation of OPCs generated according to embodiments of the present disclosure.

[0148] Referring to FIG. 10, performance in the cylinder test is expected to show improvement at 4 months after implantation of OPCs generated according to one or more aspects of the present disclosure.

[0149] Example 8 - Use of OPC populations to treat spinal cord injury OPCs generated using the methods of the present disclosure can be used to treat spinal cord injury. An efficacy study was conducted to test OPCs in a rat model of cervical spinal cord injury using both behavioral and histological assessments. This study modeled the most common human spinal cord injury, cervical contusion, and was limited to a hemi-contusion injury to ensure animal survival and maintain reasonable animal care requirements. Initial studies were conducted using OPCs generated by the methods of the present disclosure without prior treatment.

[0150] A unilateral (right-sided) spinal cord contusion injury at cervical level C-6 was administered to female athymic RNU rats using an Infinite Horizons Impactor set to apply a contusion force of 250 kdynes. One week after the contusion, the animals received 2.4 x 10 5 The animals were treated with 100 OPCs, vehicle (HBSS), or a sham operation. All animals were immunosuppressed with anti-asialoGM1 antibody. Behavioral and motor testing was performed at four time points during the study (baseline and 1, 2, or 4 months after transplantation), after which spinal cords were harvested for histological evaluation.

[0151] To administer OPCs or vehicle, a stereotactic manipulator arm was used to advance a syringe into the spinal cord. Vehicle or OPCs were administered as a single 2.4 μL injection into the dorsal spinal cord parenchyma posterior to the injury site.

[0152] To assess behavioral recovery after injury and treatment, the study incorporated the TreadScan gait analysis system (Clever Sys Inc., Reston, VA), which allows quantitative measurements of 90 different movement characteristics and gait mechanics.

[0153] For TreadScan analysis, animals were placed on a motorized, transparent treadmill and imaged from the ventral side using a high-speed camera for 20 seconds during a period of unrestrained exercise. TreadScan Analyzing System software was then used to calculate gait characteristics derived from each of the four legs.

[0154] Using these gait parameters, the entire data set was statistically analyzed using a multifactorial approach. A first principal component (PC) analysis was performed to combine the 90 individual TreadScan measurements into a single value for each of the 43 animals at each of the four time points. Plotted from baseline, the first PC (Figure 11) shows the difference between injured animals and the uninjured sham-operated group during the first month after injury. After the first month, animals receiving OPCs showed some recovery in locomotor activity, while animals receiving HBSS vehicle showed little recovery (p ≤ 0.05). Functional improvement began to appear at 2 months after transplantation and continued to increase through 4 months.

[0155] The multivariate analysis used to generate the first principal component also identified the individual parameters most related to the overall score. The top three parameters were running speed, right hind paw stride frequency, and maximum right front paw longitudinal deviation. These three parameters are plotted in Figures 12, 13, and 14, respectively, for injured rats administered OPCs generated by the non-pretreatment method.

[0156] Referring to Figure 12, the average running speeds of uninjured sham-operated rats, vehicle-treated injured rats, and OPC-treated injured rats were plotted at the four designated time points. At baseline, animals ran at similar speeds across all groups. One month after injury, animals treated with OPCs or vehicle (HBSS) ran slower than sham-operated animals, suggesting an effect of injury. At two and four months, running speeds increased in both the sham-operated and OPC-treated groups, and the degree of improvement was similar. In contrast, the HBSS vehicle group showed little increase in running speed throughout the study. OPC-transplanted animals had statistically significantly faster running speeds than animals in the vehicle control group at both two and four months after treatment (p < 0.05).

[0157] Referring to Figure 13, stride frequency of the right hind paw of uninjured sham-operated rats, vehicle-treated injured rats, and OPC-treated injured rats is plotted at the four indicated time points. Animals receiving OPCs generated by the no-pretreatment method exhibited less severe impairment after transplantation. Notably, OPC-treated animals had stride frequencies for the right hind paw that were closer to those of sham-operated animals than to those of HBSS vehicle-treated injured rats (Figure 13), and were significantly different from the stride frequency of the vehicle group (p < 0.05).

[0158] Referring to Figure 14, the maximum longitudinal displacement of the right forepaw was plotted at four designated time points for uninjured sham-operated rats, vehicle-administered injured rats, and injured rats administered OPCs generated by the non-pretreatment method. This measures the maximum distance of the right forepaw (injured side) relative to the short body axis (waist axis). In this case, animals administered OPCs showed longitudinal displacement of the affected right forepaw that was more similar to that of sham-operated animals and statistically different from that of animals administered HBSS vehicle (Figure 14).

[0159] Post-mortem histological analysis from efficacy studies revealed OPC survival, reduced parenchymal cavity formation, and the presence of myelinated fibers traversing the injury site in OPC-treated rats (Figure 15). Cell nuclei stained with human nuclear antigen, and myelin stained with eriochrome cyanin. Nerve pathway tracing identified axonal fibers within the OPC graft and from the rubrospinal and reticulospinal tracts posterior to the injury site. The left panel of Figure 15 demonstrates complete filling of the lesion cavity by transplanted OPCs. The right panel of Figure 15 demonstrates the presence of myelinated axons (arrows) within the OPC graft.

[0160] Overall, TreadScan analysis demonstrated that OPCs generated without prior treatment were capable of reversing motor deficits caused by contusion injury to the rat cervical spinal cord, resulting in improvements in both kinetic measurements and spinal cord histology in injured animals transplanted with OPCs generated without prior treatment.

[0161] Transplantation of OPCs generated by methods involving prior treatment is expected to provide at least the same level of functional improvement as that provided by transplantation of OPCs generated by methods involving no prior treatment.

[0162] Example 9 - Use of OPC populations to treat multiple sclerosis OPCs generated using the methods of the present disclosure can be used to treat multiple sclerosis (MS), an autoimmune disease that causes demyelination of axons in the central nervous system. To test the ability of OPCs to remyelinate damaged axons and restore motor function, a rodent model of MS, essentially as described by Stosic-Grujicic et al. (Stosic-Grujicic S, Ramic Z, Bumbasirevic V, Harhaji L, Mostarica-Stojkovic M. Induction of experimental autoimmune encephalonmyelitis in Dark Agouti rats without adjuvant. Clin. Exp. Immunol. 2004. 136:49-55), was employed. As a model, myelin oligodendrocyte glycoprotein MOG was used. 1-125 Dark Agouti rats were immunized with IgG4-dependent immunization (IgG4-dependent immunization) to develop experimental autoimmune encephalomyelitis (EAE). A non-limiting example of the experimental outline for transplanting OPCs 30 days after immunization is shown in Figure 16.

[0163] To determine the effect of OPC transplantation on the behavior of test animals, a clinical score is determined for each animal using the scoring system described in Stosic-Grujicic et al., 2004. Expected data that may be generated from experiments performed according to this example and that may be obtained from comparing control groups with animals transplanted with OPCs generated according to embodiments of the present disclosure is shown in Figure 17. Histological analysis and electron microscopy performed on transplants of OPCs generated without prior treatment in a demyelinated animal model suggest that the transplanted OPCs engraft and produce myelin.

[0164] Collectively, these results demonstrate that transplantation of OPCs generated without prior treatment has the potential to reverse motor dysfunction and hypomyelination in an EAE model.

[0165] Transplantation of OPCs generated by the pre-treatment approach is expected to provide at least similar levels of remyelination and functional improvement in the EAE model of MS.

[0166] Although the present disclosure has been described with reference to particular embodiments, those skilled in the art will recognize that various modifications can be made and equivalents substituted for elements thereof without departing from the scope of the disclosure. In addition, many modifications can be made to adapt to a particular situation or material to the teachings of the disclosure without departing from the scope of the disclosure.

[0167] Therefore, this disclosure is not limited to the particular embodiment disclosed as the best mode contemplated for carrying out this disclosure, but rather, this disclosure is intended to include all embodiments falling within the scope and spirit of the appended claims.

Claims

1. 1. A composition comprising a cell population comprising a plurality of oligodendrocyte progenitor cells (OPCs), wherein the OPCs express nestin and one or more markers selected from PDGF-Rα, IGF2, Nkx2.2, Olig1, NG2, Olig2, FABP7, and NEUROG2, and wherein the cell population is expanded but undifferentiated stem cells are treated with an inhibitor of ALK5 selected from SB431542, LY364947, and RepSox; 1. A composition comprising: an inhibitor of ALK2 selected from lusomorphin, LDN193189, and Noggin protein; an inhibitor of GSK3 selected from CHIR99021, 6-bromoindirubin-3'-oxime (BIO), kempauron, SB216762, and Wnt protein; and one or more stem cell differentiation regulators comprising a smoothened agonist selected from palmorfamine, SAG (CAS 364590-63-6), and SSH protein, wherein the expanded but undifferentiated stem cells produced by incubating the cell population with one or more stem cell differentiation regulators comprising palmorfamine, SAG (CAS 364590-63-6), and SSH protein, wherein the expanded but undifferentiated stem cells comprise human embryonic stem cells, primate pluripotent stem cells, or induced pluripotent stem cells, and the cell population contains less than 15% of an undesired cell type.

2. The composition described in claim 1, wherein one or more stem cell differentiation regulatory factors include SB431542, dorsomorphin, CHIR99021 and palmorfamine.

3. 2. The composition of claim 1, wherein the undesired cell type comprises an epithelial lineage cell characterized by the presence of one or more markers selected from the group consisting of K7 and PCK.

4. The composition of claim 3 , wherein the cell population has a content of PCK-positive cells of less than 5%.

5. The composition of claim 1 , wherein the cell populations have a common genetic background.

6. The composition of claim 5 , wherein the cell population has not undergone cell enrichment.

7. 1. A composition comprising a cell population comprising a plurality of oligodendrocyte progenitor cells (OPCs), wherein the OPCs express nestin and one or more markers selected from PDGF-Rα, IGF2, Nkx2.2, Olig1, NG2, Olig2, FABP7, and NEUROG2, and wherein the cell population is expanded but undifferentiated stem cells are treated with an inhibitor of ALK5 selected from SB431542, LY364947, and RepSox; a composition comprising: an inhibitor of ALK2 selected from lusomorphin, LDN193189, and noggin protein; an inhibitor of GSK3 selected from CHIR99021, 6-bromoindirubin-3'-oxime (BIO), kempauron, SB216762, and Wnt protein; and one or more stem cell differentiation regulators comprising a smoothened agonist selected from palmorfamine, SAG (CAS 364590-63-6), and SSH protein, wherein the expanded but undifferentiated stem cells produced by incubating the cell population with one or more stem cell differentiation regulators comprising palmorfamine, SAG (CAS 364590-63-6), and SSH protein are capable of forming no more than one epithelial cyst per 100,000 cells in a cyst assay.

8. 8. The composition of claim 7, wherein the cell population is capable of reducing the volume of an injury-induced central nervous system parenchymal cavity within 12 months.

9. 8. The composition of claim 7, wherein the cell population is capable of migrating from a first location within a central nervous system tissue of a subject to one or more diseased tissues located at one or more second locations within 180 days.

10. The composition of claim 9, wherein the central nervous system tissue is spinal cord tissue or brain tissue.

11. 8. The composition of claim 7, wherein the cell population is capable of improving motor function in a subject in need of treatment after transplantation of the population into the subject.

12. The composition of claim 11, wherein the cell population is capable of sustained improvement of motor function for at least two months.

13. 12. The composition of claim 11, wherein the motor function is selected from improved standing ability, improved weight bearing, improved limb function, improved limb strength, improved walking distance, improved walking speed, improved bowel function, improved bladder function, improved arm movement, improved hand movement, improved grasping, improved prehension, and / or improved grasping.

14. The composition of claim 7 , wherein the cell population is capable of producing one or more biological signaling factors.

15. 15. The composition of claim 14, wherein the cell population is capable of producing an angiogenic signaling factor selected from the group consisting of thrombospondin 1, serpine 1, serpine 2, and combinations thereof.

16. 15. The composition of claim 14, wherein the cell population is capable of producing a neurotrophic signaling factor selected from the group consisting of NGF, netrin 4, tenascin C, thrombospondin 1, thrombospondin 3, SLIT1, SLIT3, and combinations thereof.

17. 15. The composition of claim 14, wherein the one or more biological signaling factors comprise decorin or midkine.

18. 8. The composition of claim 7, wherein the cell population is capable of inducing myelination of demyelinated axons at a site of implantation within a subject.

19. 1. A composition comprising a cell population comprising a plurality of oligodendrocyte progenitor cells (OPCs), wherein the OPCs express nestin and one or more markers selected from PDGF-Rα, IGF2, Nkx2.2, Olig1, NG2, Olig2, FABP7, and NEUROG2, and wherein the cell population is expanded but undifferentiated stem cells are treated with an inhibitor of ALK5 selected from SB431542, LY364947, and RepSox; and one or more stem cell differentiation regulators, including an inhibitor of ALK2 selected from lusomorphin, LDN193189, and Noggin protein, an inhibitor of GSK3 selected from CHIR99021, 6-bromoindirubin-3'-oxime (BIO), kempauron, SB216762, and Wnt protein, and a smoothened agonist selected from palmorfamine, SAG (CAS 364590-63-6), and SSH protein, wherein the expanded but undifferentiated stem cells produced by incubating the cell population with one or more stem cell differentiation regulators, including palmorfamine, SAG (CAS 364590-63-6), and SSH protein, comprise human embryonic stem cells, primate pluripotent stem cells, or induced pluripotent stem cells, and the cell population is at most 1 x 10 ９ A composition, wherein the cells, when transplanted into a site of central nervous system injury, are capable of producing ectopic tissue in less than 2% of subjects.

20. The cell population is up to 1 x 10 ９ 20. The composition of claim 19, wherein the cells, when transplanted into a site of central nervous system injury, are capable of producing ectopic tissue in less than 1% of subjects.

21. 20. The composition of claim 1, 7 or 19, wherein at least 30% of the cell population are NG2 positive cells.

22. 1. A composition comprising a cell population comprising a plurality of oligodendrocyte progenitor cells (OPCs), wherein the OPCs express nestin and one or more markers selected from PDGF-Rα, IGF2, Nkx2.2, Olig1, NG2, Olig2, FABP7, and NEUROG2, and wherein the cell population is expanded but undifferentiated stem cells are treated with an inhibitor of ALK5 selected from SB431542, LY364947, and RepSox; 1. A composition comprising: an inhibitor of ALK2 selected from lusomorphin, LDN193189, and Noggin protein; an inhibitor of GSK3 selected from CHIR99021, 6-bromoindirubin-3'-oxime (BIO), kempauron, SB216762, and Wnt protein; and one or more stem cell differentiation regulators comprising a smoothened agonist selected from palmorfamine, SAG (CAS 364590-63-6), and SSH protein, wherein the expanded but undifferentiated stem cells produced by incubating the OPCs with one or more stem cell differentiation regulators comprising palmorfamine, SAG (CAS 364590-63-6), and SSH protein, wherein at least 95% of the OPCs express a marker selected from the group consisting of PDGF-Rα, IGF2, Nkx2.2, Olig1, and nestin.

23. 23. The composition of claim 22, wherein at least 98% of the OPCs express a marker selected from the group consisting of PDGF-Rα, IGF2, Nkx2.2, Olig1, and nestin.

24. 23. The composition of claim 22, wherein at least 95% of the OPCs express a second marker selected from the group consisting of PDGF-Rα, IGF2, Nkx2.2, Olig1, and nestin.

25. 25. The composition of any one of claims 1 to 24, wherein the composition further comprises a pharmaceutically acceptable carrier.

26. 26. The composition of claim 25, wherein the pharmaceutically acceptable carrier comprises dimethyl sulfoxide.

27. 26. The composition of claim 25, wherein the pharmaceutically acceptable carrier does not include dimethyl sulfoxide.

28. The composition has a concentration of at least 1 x 10 per milliliter ６ 28. The composition of any one of claims 25 to 27, comprising cells.

29. 28. The composition of any one of claims 25 to 27, wherein the composition has a volume of 20 to 500 microliters.

30. 30. The composition of any one of claims 25 to 29, wherein the composition is an injectable solution.

31. 23. The composition of claim 1, 7, 19 or 22, wherein the composition is adapted for cryopreservation.

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