Compositions and methods for determination of pluripotent stem cell differentiability

Abstract

Described herein are methods for testing the differentiability of pluripotent stem cells (PSCs). The methods comprise assaying PSCs or PSC populations for differentiability marker mutation(s) and / or expression and determining the suitability of the PSCs or PSC populations for differentiation, e.g., into a product suitable for cell therapy. The methods can be used in cell banking and manufacturing cell therapies. Also provided herein are kits suitable for performing the PSC differentiability assessment methods disclosed herein.

Description

COMPOSITIONS AND METHODS FOR DETERMINATION OF PLURIPOTENT STEM CELL DIFFERENTIABILITY1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U. S. provisional application nos.63 / 712,882 filed October 28, 2024, 63 / 810,998, filed May 23, 2025, and 63 / 827,544, filed June 20, 2025, the contents of each of which are incorporated herein in their entireties by reference thereto.2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on October 22, 2025, is named BRT-004WO_SL.xml and is 173,326 bytes in size.3. BACKGROUND

[0003] Cell therapy provides great promise for the treatment of a variety of diseases and conditions. In cell therapy, autologous or allogeneic cells are transplanted into a patient to replace or repair defective or damaged tissue or cells that may have arisen from any of a multitude of medical conditions including genetic disorders, cancer, neurologic disorders, cardiac disorders, or eye-related issues. Pluripotent stem cells (PSCs) can serve as a renewable source to make differentiated cells and tissues due to their capacity for extensive expansion and commitment to various somatic cell fates and thus are especially useful for cell therapy.

[0004] Manufacture of cell therapy products from PSCs has been hindered by low yields and limited scalability due to PSC heterogeneity, bank-to-bank variability, and activity changes in culture. Thus, there is a need for determining the suitability of a PSC population to differentiate and produce a differentiated cell population for therapy.4. SUMMARY

[0005] Despite similarities in various indications of cell health, different populations of PSCs demonstrate varying degrees of ability to differentiate. This disclosure is based, at least in part, on the discovery that the expression of one or more genes in a population of PSCs can be indicative of their ability to differentiate, e.g., towards ectodermal cells like dopaminergic neurons. The present disclosure addresses the need for improved methods of manufacturing cell therapy products containing differentiated cells from pluripotent stem cells (PSCs).

[0006] The present disclosure provides methods of characterizing PSCs or a population of PSCs fortheir differentiability or ability to differentiate based on the expression of markers whose expression varies between the naive and primed pluripotency states (referred to herein as “differentiability markers” for convenience). PSCs in the primed state can be more efficiently differentiated, e.g., differentiated into cells useful in cell therapy. A spectrum of PSC differentiability, originating from naive to primed pluripotency, is illustrated in FIG. 1. For clarity and avoidance of doubt, the use of the term “primed” is not intended to convey that a PSC having a differentiability marker profile indicative of primeness is primed to differentiate into all other cell types, but rather is indicative that the PSC is more predisposed to differentiate into at least one other cell type (e.g., a dopaminergic neuron) using a defined process (e.g., as described in Example 1) than another PSC having differentiability marker profile more indicative of naiveness. Further, while a differentiability marker profile indicative of primeness provides an increased likelihood of successful differentiation (e.g., into at least one cell type such as a dopaminergic neuron), a less suitable differentiability marker profile is indicative of a likelihood of failed differentiation (e.g., into at least one cell type such as a dopaminergic neuron).

[0007] The methods typically entail detecting the expression levels of one or more differentiability markers and determining if the expression levels are indicative of the suitability of the PSCs for differentiation.

[0008] The methods of the disclosure have applicability at a variety of steps involved in cell banking of PSCs and in manufacturing differentiated cells for cell therapy.5. BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1 is a schematic depicting the continuum of pluripotency in pluripotent stem cells (PSCs), ranging from naive on the left to primed on the right. Primed cells are desirable for manufacturing differentiated cells, e.g., for cell therapy applications. The primed vs. naive status of PSCs can be determined by measuring the expression levels of and / or presence of mutations in correlated genes, referred to herein as differentiability markers. A composite “score” of differentiability, referred to as a “PSC score” can be generated based on the expression levels of and / or presence of mutations in multiple differentiability markers. FIG. 1 depicts an exemplary PSC score ranging from approximately 1.5 for naive cells to 0.8 as the cells transitioned to a primed state based on the gene expression profiles shown in Examples 1 and 2.

[0010] FIG. 2 is a graph that shows the normalized Cq values of 96 genes, ordered by Random-Forest (RF) regression model importance. RF regression model was trained on 29 samples of PSCs with differentiation success data, wherein successful differentiations are shown in green, and failed differentiations are shown in red. The first 17 genes displayed themost significant differences between samples that pass differentiation compared to those that fail differentiation.

[0011] FIG. 3 shows PSC scores in successful differentiation (pass) samples and failed differentiation (fail) samples. PSC scores were calculated using dCq values normalized to the geometric mean of the four housekeeping genes p-actin, 18S rRNA, GAPDH and TBP using the formula: PSC score =Mean Cq values (PRTG, FAS, RAB17)Mean Cq values NR5A2, HHEXIGBX2, GDF3, NANOGINODALIHMX1, UTF1ISIX6, MT2A, TBXTINFIA, ID2ISP8)

[0012] A different threshold of 1.34 was calculated using the following formula: median (fails) -3 * median absolute deviation (fails). The calculated differentiation threshold is marked by the arrow. Therefore, a sample with a PSC score below 1.34 according to the formula above would be predicted to successfully differentiate into DA neurons, whereas a sample with a PSC score above 1.34 for the according to the formula above would be predicted to fail differentiation.

[0013] FIG. 4 shows the PSC scores of various clones of PSC line 1 with either wild-type (WT) BCOR allele, BCOR SNP1, or BCOR SNP2.

[0014] FIGS. 5A-5F show the results of flow cytometry data for on and off-target genes respective of dopaminergic neurons for PSC line 1 clones with WT BCOR allele, BCOR SNP1, or BCOR SNP2, after they were differentiated to dopaminergic neurons and thawed and matured for 5 days (DIV5). FIG.5A shows the percentage of cells positive for FOXA2. FIG. 5B shows the percentage of cells positive for OTX2. FIG. 5C shows the percentage of cells positive for CRABP1. FIG. 5D shows the percentage of cells positive for PAX6. FIG.5E shows the percentage of cells positive for CD36. FIG. 5F shows the percentage of cells positive for TH.

[0015] FIGS. 6A-6F show the results of immunocytochemistry assessments for on and off-target genes respective of dopaminergic neurons for PSC line 1 clones with WT BCOR allele, BCOR SNP1, or BCOR SNP2, after they were differentiated to dopaminergic neurons and thawed and matured for 1 day (DIV1) or 5 days (DIV5). FIG. 6A shows representative FOXA2 immunofluorescence (IF) images alone or with nuclear (DAPI) IF in the same sections on DIV1.FIG. 6B shows representative PAX6 IF images alone or with DAPI IF in the same sections on DIV1. FIG. 6C shows representative CRABP1 IF images alone or with DAPI IF in the same sections on DIV1. FIG. 6D shows representative TH IF images alone, together with FOXA2 IF, or together with FOXA2 IF and DAPI IF in the same sections on DIV5. FIG. 6E shows representative PAX6 IF images alone or with DAPI IF in the same sections on DIV5. FIG. 6F shows representative CRABP1 IF images alone or with DAPI IF in the same sections on DIV5.

[0016] FIGS. 7A-7B show results of qPCR displaying differentially expressed genes (DEGs) in cells differentiated from PSCs with BCOR SNP1 or BCOR SNP2, relative to the expression ofthe same genes in cells differentiated from PSCs with WT BCOR allele. FIG. 7A shows DEGs in cells differentiated from PSCs with BCOR SNP1 or BCOR SNP2 on DIV0. FIG. 7B shows DEGs in cells differentiated from PSCs with BCOR SNP1 or BCOR SNP2 on DIV5.

[0017] FIGS. 8A-8D show the results of functional assessments of dopaminergic cells differentiated from PSCs with BCOR SNP1 or BCOR SNP2, PSCs with WT BCOR allele, and DA01 control cells. FIG. 8A shows the count of neural firing in each condition assessed from DIV10 to DIV19. FIG. 8B shows the synchronous firing among neurons in each condition, assessed from DIV10 to DIV19. FIG. 8C shows neurite length associated with each condition, assessed from DIV3 to DIV7. FIG. 8D shows the amount of dopamine released in each condition, assessed from DIV14 to DIV28.

[0018] FIGS. 9A-9C show HERV-K expression in PSC line 1 clones. FIG. 9A shows the normalized Cq values of HERV-K genes in parental PSC line 1, a “naive” PSC line 1 clone (c34), and a “primed” PSC line 1 clone (c46). FIG. 9B shows the experimental setup of sorting of PSC line 1 c34 cells by IFITM1 cell surface expression and subsequent QPCR analysis. PSC scores are shown inside the boxes under each cell group. FIG. 9C shows the normalized Cq values of HERV-K genes in PSC line 1 c34 cells that were sorted by I FITM 1 expression and unsorted PSC line 1 c34 (presort) cells.

[0019] FIGS. 10A-10B show the results of evaluations of PSC clones with different % EP300 mut values. FIG. 10A shows the correlation between % EP300 mut values and PSC score. FIG.10B shows differentially expressed genes in cells differentiated from PSCs with different %EP300 mut values, relative to the expression of genes in cells differentiated from a PSC clone with an % EP300 mut value close to 0.6. DETAILED DESCRIPTION6.1. Definitions

[0020] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0021] The practice of some methods disclosed herein employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M. J.MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.l. Freshney, ed. (2010)); Barbare Detrick, Robert Hamilton, John L Schmitz, Manual of Molecular and Clinical Lab Immunology (2016).

[0022] A, An, The: As used herein, the articles “a,” “an,” and “the” are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

[0023] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0024] Differentiation: As used herein, the term “differentiation”, and its grammatical equivalents, refers to a process by which a stem cell or progenitor cell alters from one cell type to a more specialized cell type. Each specialized cell type in an organism can express a subset of all the genes that constitute the genome of the cell. Each cell type can be defined by its particular pattern of regulated gene expression. Cell differentiation can thus be described as a transition of a cell from one cell type to another cell type coincident with a switch from one pattern of gene expression to another.

[0025] Differentiability marker: As used herein, the term “differentiability marker” refers to a gene whose expression correlates with the differentiation potential of a PSC, e.g., into an ectodermal cell type such as a dopaminergic neuron. The methods of the disclosure utilize NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, BCOR, EP300, IFITM1, FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, and SFRP1 as differentiability markers. A “positive differentiability marker” is a marker whose increased expression is correlated to a PSC’s increased potential for differentiation. A“negative differentiability marker” is a marker in which the presence of mutations and / or whose increased expression is correlated to a PSC’s reduced potential for differentiation. For clarity and avoidance of doubt, the presence of mutations in, or expression of, a ‘‘differentiability marker” (whether positive or negative) need not be indicative of a PSC’s potential to differentiate into all other cell types, but rather be indicative of a PSC’s predisposition to differentiate into at least one other cell type (e.g., a dopaminergic neuron) using at least one process (e.g., as described in Example 1). Further, while a differentiability marker profile indicative of primeness shows an increased likelihood of successful differentiation (e.g., into at least one cell type such as a dopaminergic neuron), a less suitable differentiability marker profile shows a likelihood of failed differentiation (e.g., into at least one cell type such as a dopaminergic neuron).

[0026] Exemplary differentiability markers are set forth in Section 6.3. The differentiability markers disclosed herein are particularly useful for identifying PSCs that are primed to differentiate into ectodermal cells such as dopaminergic neurons, e.g., using the processes described in Example 1.

[0027] Expanding, Expansion: The methods of the present disclosure may include a step of culturing cells, e.g., from a cell bank in order to expand a population, e.g., prior to differentiation, selection and / or banking. As used herein, the terms “expanding” and “expansion” refers to culturing cells under proliferation conditions to increase the cell number. A cell culture undergoing expansion might be subject to one or more rounds of passaging, e.g., to maintain suitable conditions for expansion and / or for the health of dividing cells.

[0028] Isogenic: As used herein, the term “isogenic” refers to cells having the same or closely similar genotypes. Cells that are isogenic share the same genetic background but may have a small number of genetic differences (e.g., 2, 3, 4, 5, 10 or 20 genetic differences), for example differences that spontaneously arise during cell culture. The term “isogenic” encompasses the term “monogenic,” which refers to cells of the same genotype.

[0029] Passaging, Passage: As used herein in the context of a cell culture, the terms “passaging” and “passage” shall refer to the transfer of all or a portion cells, e.g., from a previous cell culture, into a new cell culture. The transfer can be done into a new cell culture container, such as a dish, bottle or flask, having fresh cell culture medium that allows for further culturing and / or expansion of the cells.

[0030] Pluripotent Stem Cell, PSC: As used herein, the term “pluripotent stem cell”, and its grammatical equivalents, refers to a stem cell that has the capacity for self-renewal and the potential to differentiate into a more specified cell type. Examples of pluripotent stem cells include embryonic stem cells, fetal stem cells, embryonic germ cells, induced pluripotent stem cells, and the like. A stem cell established by cultivating an early embryo generated bytransplantation of the nucleus of a somatic cell is also a pluripotent stem cell. The pluripotent stem cell may be an animal cell, a vertebrate cell, a mammalian cell, a primate cell, or human cell.

[0031] As shown in FIG. 1, PSCs exist along a naive-to-primed pluripotency spectrum. In the context of a developing human embryo, “naive” stem cells are those that derive from the inner cell mass and “primed” stem cells are those stem cells that derive from the later epiblast. Thus, cells from the inner cell mass can be referred to as naive, while cells from the later differentiated epiblast can be referred to as primed. “Naive” stem cells are thought to have greater possibilities of differentiation in vivo because they have higher pluripotency potential, whereas “primed” stem cells are considered to have lesser number of possibilities of differentiation to other cell types because they have lower pluripotency potential. In another context, “naive” stem cells are thought to have infinite possibilities of differentiation in vivo because they have higher pluripotency potential and can differentiate into almost any type of cell in vivo, whereas “primed” stem cells are considered to have lesser number of possibilities of differentiation to other cell types in vivo because they have lower pluripotency potential and can differentiate into fewer types of cells in vivo. In some contexts, “naive” cells are considered to need more time and / or changes to differentiate into a downstream cell, whether a terminally differentiated or intermediate cell, whereas “primed” stem cells are considered to need less time and / or fewer changes to differentiate into the same downstream cell. In certain aspects of the present disclosure, a primed PSC can more readily differentiate into a more specialized cell type in vitro than a naive PSC, and thus is more suitable for manufacturing cell therapy products, e.g., cell therapy products of an ectodermal cell lineage such as DA neurons. In some embodiments, a primed PSC can more readily (than a naive PSC) differentiate into at least one more specialized cell type in vitro. In some embodiments, the more specialized cell type is a dopaminergic neuron.6.2. PSC Differentiability Determination and its Applications

[0032] The present disclosure provides methods of evaluating pluripotent stem cells (PSCs) for their differentiability, e.g., into cells of an ectodermal cell lineage such as DA neurons, based on the expression of markers whose expression varies between the naive and primed state (referred to herein as “differentiability markers” for convenience). PSCs in the primed state can be more efficiently differentiated into more mature cells, e.g., cell useful in cell therapy. A spectrum of differentiability, from naive to primed, is illustrated in FIG. 1.

[0033] The methods typically entail detecting the expression levels of one or more differentiability markers and determining if the expression levels are indicative of the suitability of the PSCs for differentiation. The PSCs can be scored for their differentiability based on the expression levels of one or more differentiability markers.

[0034] In some embodiments, the suitability of PSCs for differentiation is based on determining if the expression of a differentiability marker is above or below a cutoff or threshold value.

[0035] In some embodiments, a PSC score is based on determining if a ratio of the expression of two differentiability markers is above or below a cutoff or threshold value.

[0036] In some embodiments, the PSC score is a composite score based on the expression of a plurality of PSC differentiability markers, e.g., at least 5 or at least 10 differentiability markers.

[0037] Prior to scoring PSCs for differentiability, their expression levels can be normalized against the expression levels of one or more control genes, e.g., against the expression levels of one or more housekeeping genes in the PSCs. Examples of housekeeping genes include actin (e.g., p-actin), GAPDH, 18S and TBP and any housekeeping gene disclosed in WO2024 / 167814A1. For example, when expression of one or more differentiability markers and one or more housekeeping genes are determined using real-time PCR, Cq values for the expression of the one or more differentiability markers is then normalized to average Cq of reference genes. To arrive at a PSC score for a collection of differentiability markers, the geometric or arithmetic mean of normalized Cq values of genes increased in samples that passed differentiation (“positive differentiability markers”) into a desired cell type (e.g., cells of an ectodermal lineage such as DA neurons) divided by the Cq values of genes decreased in samples that passed differentiation (“negative differentiability markers”) into a desired cell type (e.g., cells of an ectodermal lineage such as DA neurons).

[0038] In certain aspects, PSC scoring is a computer-implemented method performed by a computerized device having a processor. An exemplary computer-implemented method comprises: (a) receiving data on the Cq value(s) of one or more positive differentiability markers; (b) receiving data on the Cq value(s) of one or more negative differentiability markers; and (c) calculating the ratio of the geometric or arithmetic mean of the Cq value(s) of one or more positive differentiability markers and the Cq value(s) of one or more negative differentiability markers. In some embodiments, the Cq value(s) are normalized against one or more (e.g., one, two, three, four or more) control genes, for example housekeeping genes, prior to step (c). In some embodiments, the one or more housekeeping genes comprise at least one of p-actin, 18S rRNA, GAPDH and TBP. In some embodiments, the one or more housekeeping genes comprise any combination of two, three, or all four of p-actin, 18S rRNA, GAPDH and TBP.

[0039] In certain aspects, PSC scoring is a computer-implemented method performed by a computerized device having a processor. An exemplary computer-implemented method comprises: (a) receiving data on the Cq value(s) of one or more positive differentiability markers; (b) receiving data on the Cq value(s) of one or more negative differentiability markers; and (c) calculating the ratio of the arithmetic mean of the Cq value(s) of one or more positivedifferentiability markers and the Cq value(s) of one or more negative differentiability markers. In some embodiments, the Cq value(s) are normalized against one or more (e.g., one, two, three, four or more) control genes, for example housekeeping genes, prior to step (c). In some embodiments, the one or more housekeeping genes comprise at least one of p-actin, 18S rRNA, GAPDH and TBP. In some embodiments, the one or more housekeeping genes comprise any combination of two, three, or all four of p-actin, 18S rRNA, GAPDH and TBP. In some embodiments, step (c) comprising calculating a PSC score according to the formula:Mean Cq values of genes upregulated in samples that pass DA differentiation PSC score = - Mean Cq values of genes upregulated in samples that fail DA differentiation

[0040] In some embodiments, the computer-implement method comprises: (a) receiving data on the Cq value(s) of one or more positive differentiability markers; (b) receiving data on the Cq value(s) of one or more negative differentiability markers; (b) receiving data on the Cq value(s) of one or more control genes; (d) normalizing the Cq value(s) of the differentiability markers against the Cq value(s) of the one or more control gene(s); and (d) calculating the ratio of the arithmetic mean of the normalized Cq value(s) of one or more positive differentiability markers and the normalized Cq value(s) of one or more negative differentiability markers according to the formula above. In some embodiments, the Cq value(s) are normalized against one or more (e.g., one, two, three, four or more) control genes, for example housekeeping genes, prior to step (c). In some embodiments, the one or more housekeeping genes comprise at least one of p-actin, 18S rRNA, GAPDH and TBP. In some embodiments, the one or more housekeeping genes comprise any combination of two, three, or all four of p-actin, 18S rRNA, GAPDH and TBP.

[0041] In some embodiments, the computer-implement method comprises: (a) receiving data on the Cq value(s) of one or more positive differentiability markers; (b) receiving data on the Cq value(s) of one or more negative differentiability markers; (b) receiving data on the Cq value(s) of one or more control genes; (d) normalizing the Cq value(s) of the differentiability markers against the Cq value(s) of the one or more control gene(s); and (d) calculating the ratio of the geometric mean of the normalized Cq value(s) of one or more positive differentiability markers and the normalized Cq value(s) of one or more negative differentiability markers, e.g., according to the formula above. In some embodiments, the control genes are housekeeping genes. In some embodiments, the one or more housekeeping genes comprise at least one of p-actin, 18S rRNA, GAPDH and TBP. In some embodiments, the one or more housekeeping genes comprise any combination of two, three, or all four of p-actin, 18S rRNA, GAPDH and TBP.

[0042] A threshold or cutoff score can be utilized to categorize the PSCs as suitable or unsuitable for differentiation, e.g., as set forth in Example 2 or Example 6.

[0043] The methods of the disclosure have applicability at a variety of steps involved in cell banking and manufacturing differentiated cells for cell therapy.

[0044] For example, the methods of the disclosure can be used in the preparation of a PSC bank. In some embodiments, the disclosure provides a method of preparing a pluripotent stem cell (PSC) stem cell bank suitable for differentiation, comprising (a) detecting the expression levels of one or more differentiability markers in a population of PSCs; (b) determining if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation (e.g., if a PSC score based on the expression levels is above or below a threshold); and, if expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation, (c) generating a PSC stem cell bank from the population of PSCs. In various embodiments, the PSCs are expanded prior to and / or following determining their differentiability (e.g., via PSC scoring).

[0045] The methods of the disclosure can also be used to qualify a population of PSCs for manufacturing differentiated cells, e.g., cells suitable for cell therapy. In some embodiments, the disclosure provides a method of qualifying a population of PSCs for manufacturing differentiated cells, comprising (a) detecting the expression levels of one or more differentiability markers in a population of PSCs; (b) determining if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation (e.g., if a PSC score based on the expression levels is above or below a threshold), wherein a determination that the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation qualifies the population of PSCs for manufacturing differentiated cells.

[0046] The methods of the disclosure can also be used in the manufacture of differentiated cells suitable for cell therapy. In some embodiments, the disclosure provides a method of identifying PSCs suitable for differentiation, comprising (a) detecting the expression levels of one or more differentiability markers in a population of PSCs; (b) determining if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation (e.g., if a PSC score based on the expression levels is above or below a threshold); and, if expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation, (c) differentiating PSCs from the population and optionally formulating them into a pharmaceutical preparation suitable for cell therapy.

[0047] The methods of the disclosure can also be used in quality control in manufacturing methods involving PSCs intended for differentiation, e.g., into differentiated cells suitable for cell therapy. In some embodiments, the disclosure provides a method of monitoring a clonal population of pluripotent stem cells (PSCs) for its suitability for manufacturing differentiated cells, comprising: (a) detecting the expression levels of one or more differentiability markers in PSCs of the clonal population; (b) determining if the expression levels of the one or moredifferentiability markers are indicative of the suitability of the PSCs for differentiation (e.g., if a PSC score based on the expression levels is above or below a threshold); and if the expression levels of the one or more differentiability markers in step (b) are indicative of the suitability of the PSCs for differentiation, maintaining the clonal population; and (d) repeating steps (a)-(c) one or more times. In some embodiments, steps (a) and (b) are repeated after introducing a transgene, e.g, a transgene encoding a therapeutic protein, into a PSC line, to confirm that it retains its suitability for differentiation, e.g., into cells useful for cell therapy.

[0048] Exemplary differentiability markers are disclosed in Section 6.3.

[0049] Exemplary differentiability marker assays are disclosed in Section 6.4.

[0050] Exemplary sources and populations of PSCs are disclosed in Section 6.7.

[0051] Exemplary differentiated cell types and compositions comprising differentiated cell types are disclosed in Section 6.8.6.3. PSC Differentiability Markers

[0052] The methods of the disclosure generally relate to measuring one or more PSC differentiability markers in order to assess whether a PSC is suitable for differentiation.

[0053] In some embodiments, the one or more differentiability markers comprise one or more negative differentiability markers and / or one or more positive differentiability markers.

[0054] Exemplary differentiability markers based on expression levels (e.g., using Cq values) are set forth in the table below:

[0055] Additionally, BOOR and EP300 are useful as differentiability markers based on the presence of mutations (e.g., that can be measured as a percentage of the mutant gene or allele in the population) that correlate with negative differentiability. Accordingly, “mutant” or “mut” forms of BCOR and EP300 referred to herein as negative differentiability markers.

[0056] In some embodiments, the one or more negative differentiability markers comprise one or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, FGF4, mutant EP300, and mutant BCOR. In some embodiments, the one or more negative differentiability markers comprise a combination of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or all of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, FGF4, mutant EP300, and mutant BCOR.

[0057] In some embodiments, the one or more positive differentiability markers comprise one or more of RAB17, PRTG, FAS, SFRP1, COL2A1, FAM129A, LCK, FZD7, and IDO1. In some embodiments, the one or more positive differentiability markers comprise a combination of two or more, three or more, four or more, or all of RAB17, PRTG, FAS, SFRP1, COL2A1, FAM129A, LCK, FZD7, and IDO1. In some embodiments, the one or more positive differentiability markers comprise RAB17, PRTG, and FAS.

[0058] In some embodiments, expression levels of up to 30, up to 20, or up to 10 total genes are measured. In some embodiments, expression levels of 60 or fewer, 50 or fewer, 40 or fewer, 35 or fewer, 30 or fewer, 25 or fewer, or 20 or fewer total genes are measured. In some embodiments, expression levels of 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, 30 or fewer, 25 or fewer, or 20 or fewer differentiability marker genes are measured. In some embodiments, up to 10 or up to 5 control genes (e.g., housekeeping genes) are measured.

[0059] Details of these differentiability markers, their sequences and suitable methods fortheir detection as disclosed in Sections 6.3.1 through 6.3.30 below.6.3.1. NR5A2

[0060] Nuclear receptor subfamily 5 group Ah member 2 (NR5A2), which is also known as liver receptor homolog-1 (LRH-1), is a DNA-binding zinc finger transcription factor and is a member of the fushi tarazu factor-1 subfamily of orphan nuclear receptors. The encoded protein acts as a key metabolic sensor by regulating the expression of genes involved in bile acid synthesis, cholesterol homeostasis and steroidogenesis (Fayard et al., 2004, Trends Cell Biol. 14(5):250-60). The protein product of NR5A2 is also important for maintaining pluripotency of stem cells during embryonic development (Gu et al., 2005, Mol Cell Biol. 25 (9):3492-505). An exemplaryhuman NR5A2 transcript corresponds to GenBank # NM_205860.3 and has the nucleotide sequence set forth in SEQ ID NO:1.

[0061] In some embodiments, the expression levels of NR5A2 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.2. HHEX

[0062] Hematopoietically-expressed homeobox (HHEX) is a transcriptional repressor, which is a member of the homeobox family of transcription factors. The protein encoded by HHEX is involved in fundamental stages of embryogenesis in the foregut endoderm, regulates the development of the pancreas, bile duct and liver, and mediates hematopoiesis and lymphopoiesis (Jackson et al., Front. Immunol., 2023; 14: 1197490). An exemplary human HHEX transcript corresponds to GenBank # NM_002729.5 and has the nucleotide sequence set forth in SEQ ID NO:2.

[0063] In some embodiments, the expression levels of HHEX are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.3. GBX2

[0064] Gastrulation brain homeobox 2 (GBX2) is a transcription factor involved in the regulation of nervous system development. In the embryo, GBX2 is expressed in the inner cell mass of the preimplantation embryo before primitive streak formation. In vitro, GBX2 is expressed in undifferentiated embryonic stem cells but was shown to be downregulated in differentiated cell populations (Chapman et al., Genomics. 46:223-233). An exemplary human GBX2 transcript corresponds to Genbank# NM_001485.4 and has the nucleotide sequence set forth in SEQ ID NO:3.

[0065] In some embodiments, the expression levels of GBX2 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.4. GDF3

[0066] Growth differentiation factor 3 (GDF3) gene encodes a protein that is a member of the bone morphogenetic protein (BMP) family within the transforming growth factor beta (TGFp) superfamily. The protein product of GDF3 is involved in early embryonic development and adipose-tissue homeostasis (Chen et al., 2006. Development 133(2):319-29). An exemplary human GDF3 transcript corresponds to GenBank # NM_020634.3 and has the nucleotide sequence set forth in SEQ ID NO:4.

[0067] In some embodiments, the expression levels of GDF3 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.5. PRTG

[0068] Protogenin (PRTG) encodes a protein that is a member of the immunoglobulin superfamily expressed in the early stages of organogenesis, which is thought to be involved in anteroposterior axis elongation and has been associated with the development of various tissues, especially neurogenesis. Protogenin has been suggested to play a critical role in suppressing premature neuronal differentiation during early neural development (Wong et al., J. Neurosci. 2010, 30(12):4428-4439). An exemplary human PRTG transcript corresponds to GenBank # NM_173814.6 and has the nucleotide sequence set forth in SEQ ID NO:5.

[0069] In some embodiments, the expression levels of PRTG are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.6. NANOG

[0070] Nanog homeobox (NANOG) is a homeobox transcription factor involved in embryonic stem (ES) cell proliferation, renewal, and pluripotency. The encoded protein can block ES cell differentiation and can also auto-repress its own expression in differentiating cells. NANOG along with OCT4, SOX2 and LIN28 was shown to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem cells (Yu et al., 2007, Science. 318:1917-1929). An exemplary human NANOG transcript corresponds to GenBank# NM_024865.4 and has the nucleotide sequence set forth in SEQ ID NO:6.

[0071] In some embodiments, the expression levels of NANOG are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.7. NODAL

[0072] Nodal growth differentiation factor (NODAL) encodes a secretory protein that is a member of the transforming growth factor beta (TGFp) superfamily. NODAL is thought to be involved in the maintenance of stem cell self-renewal and pluripotency and overexpression of NODAL in hESCs has been associated with the repression of cell differentiation (Shen, 2007, Development. 134:1023-1034). An exemplary human NODAL transcript corresponds to GenBank # NM_018055.5 and has the nucleotide sequence set forth in SEQ ID NO:7.

[0073] In some embodiments, the expression levels of NODAL are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.8. HMX1

[0074] H6 family homeobox 1 (HMX1) is a homeobox transcription factor prominently expressed in developing eye, trigeminal ganglion, second branchial arch, and dorsal root ganglia and was shown to be involved in the development of craniofacial structures (Munroe et al., 2009, BMC Dev. Biol. 9:27). An exemplary human HMX1 transcript corresponds to GenBank # NM_018942.3 and has the nucleotide sequence set forth in SEQ ID NO:8.

[0075] In some embodiments, the expression levels of HMX1 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.9. UTF1

[0076] Undifferentiated embryonic cell transcription factor 1 (UTF1) is a leucine zippercontaining transcriptional coactivator that links link the transcriptional activator ATF2 with the basal transcription complex. UTF1 is expressed in undifferentiated embryonic and / or pluripotent cells and are downregulated during differentiation (Okuda et al., 1998, EMBO J. 17(7):2019-32). An exemplary human UTF 1 transcript corresponds to GenBank # NM_003577.3 and has the nucleotide sequence set forth in SEQ ID NO:9.

[0077] In some embodiments, the expression levels of UTF1 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.10. SIX6

[0078] Sine oculis homeobox homolog 6 (SIX6) is a homeobox transcription factor homologous to Drosophila “sine oculis” gene, which is thought to play a role in embryonic development, promote self-renewal of progenitor cell populations and govern mechanisms of cell differentiation (Meurer et al., 2021. Front. Cell Dev. Biol. 9:707854). An exemplary human SIX6 transcript corresponds to GenBank # NM_007374.3 and has the nucleotide sequence set forth in SEQ ID NO:10.

[0079] In some embodiments, the expression levels of SIX6 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.11. MT2A

[0080] Metallothionein 2A (MT2A) is a member of the metallothionein family of genes. MT2A is upregulated during oxidative stress and hypoxia / reoxygenation with the increasing levels of reactive oxygen species (ROS) and metal ions (e.g., cadmium, copper and zinc) and is thought to play a protective role against oxidative stress and inflammation, and is associated with the regulation of intracellular levels of metal ions and regulation of apoptotic pathways (Ling et al., 2016, Int. J. Mol. Sci. 17(9):1483). An exemplary human MT2A transcript corresponds to GenBank # NM_005953.5 and has the nucleotide sequence set forth in SEQ ID NO:11.

[0081] In some embodiments, the expression levels of MT2A are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.12. TBXT

[0082] T-box transcription factor T (“T” or “TBXT”) gene encodes a protein called brachyury or T protein, which is an embryonic transcription factor. During development, expression of T is localized to notochord-derived cells and the expression of T has been implicated in the establishment of the anterior-posterior axis of the embryo, mesoderm formation, and differentiation (Bulger et al., 2023, bioRxiv (preprint) doi: 10.1101 / 2023.11.06.565933). Anexemplary human TBXT transcript corresponds to GenBank# NM_001366285.2 and has the nucleotide sequence set forth in SEQ ID NO: 12.

[0083] In some embodiments, the expression levels of TBXT are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.13. NFIA

[0084] Nuclear factor 1A (NFIA) is a member of the nuclear factor I (NFI) transcription factors and is involved in glial cell fate specification (Daneen et al., 2006, Neuron. 52:953-968). NFIA might repress differentiation by inhibiting the expression of differentiation-specific genes (Rosa etal., 2007, PNAS. 104:19849-19854). An exemplary human NFIA transcript corresponds to GenBank # NM_001134673.4 and has the nucleotide sequence set forth in SEQ ID NO: 13.

[0085] In some embodiments, the expression levels of NFIA are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.14. FAS

[0086] Fas cell surface death receptor (FAS) is a member of the TNF-receptor superfamily and has been shown to play a central role in the physiological regulation of programmed cell death through formation of the death-inducing signaling complex (DISC) upon ligand binding. An exemplary human FAS transcript corresponds to GenBank # NM_000043.6 and has the nucleotide sequence set forth in SEQ ID NO: 14.

[0087] In some embodiments, the expression levels of FAS are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.15. ID2

[0088] Inhibitor of DNA binding 2 (ID2) is a member of the inhibitor of DNA binding family, which is thought to promote cell proliferation and suppress cell differentiation (Roschger and Cabrele, 2017, Cell Commun. Signal. 15(1):7). An exemplary human ID2 transcript corresponds to GenBank # NM_002166.5 and has the nucleotide sequence set forth in SEQ ID NO: 15.

[0089] In some embodiments, the expression levels of ID2 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.16. RAB17

[0090] Ras-related protein Rab-17 (RAB17) is an epithelial cell-specific GTPase and is a key regulator of intracellular membrane trafficking. It has been shown to be absent from the mesenchymal precursors but to be induced upon differentiation of the mesenchymal precursors into polarized epithelial cells (Lutcke et al., 1993, J. Cell Biol. 121:553-564). An exemplary human RAB17 transcript corresponds to GenBank # NM_022449.4 and has the nucleotide sequence set forth in SEQ ID NO:16.

[0091] In some embodiments, the expression levels of RAB17 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.17. SP8

[0092] Specificity protein 8 (SP8) is an SP family zinc-finger transcription factor. During development, SP8 has been shown to be expressed in neurogenic regions, which give rise to olfactory bulb interneurons and is thought to contribute to olfactory bulb interneuron diversity by regulating the survival, migration, and molecular specification of neuroblasts and interneurons (Waclaw et al., 2006. Neuron 49(4):503-516). An exemplary human SP8 transcript corresponds to GenBank# NM_182700.6 and has the nucleotide sequence set forth in SEQ ID NO: 17.

[0093] In some embodiments, the expression levels of SP8 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.18. IFITM1

[0094] Interferon-induced transmembrane protein 1 (IFITM1) is a member of the interferon-induced antiviral protein superfamily. The protein encoded by IFITM 1 is expressed on the cellular surface and inhibits the entry of a variety of viruses to the cytoplasm. IFITM-1 has been shown to play a key role in the antiproliferative action of IFN-y (Yang et al., 2007, Oncogene.26(4):594-603). An exemplary human IFITM1 transcript corresponds to GenBank# NM_003641.5 and has the nucleotide sequence set forth in SEQ ID NO:18.

[0095] In some embodiments, the expression levels of IFITM 1 are measuring at the protein level (e.g., using flow-cytometry) and / or at the nucleotide levels (e.g., using qPCR-based methods), e.g., as disclosed in Section 6.4.6 and Section 8.6.6.3.19. FAM129A

[0096] Family with Sequence Similarity 129 Member A (FAM 129A), also known as Niban apoptosis regulator 1 (NIBAN1), is involved in regulation of stress response and apoptosis (Diana and Carvalheira, 2022, Front Cell Dev Biol. 10:867003). An exemplary human FAM129A transcript corresponds to GenBank # NMJ352966.4 and has the nucleotide sequence set forth in SEQ ID NO:19.

[0097] In some embodiments, the expression levels of FAM129A are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.20. LCK

[0098] Lymphocyte protein tyrosine kinase (LCK) is a member of the Src family protein tyrosine kinases. LCK plays a key role in the activation of T-cell receptor signaling and regulates cell proliferation and survival (Bommhardt et al., 2019, Int J Mol Sci 29(14):3500). An exemplary human LCK transcript corresponds to GenBank # NM_005356.5 and has the nucleotide sequence set forth in SEQ ID NQ:20.

[0099] In some embodiments, the expression levels of LCK are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.21. FZD7

[0100] Frizzled class receptor? (FZD7) is a member of the frizzled gene family and encodes a 7-transmembrane domain proteins that serves a receptor for Wnt signaling proteins. FZD7 was shown to be involved in the maintenance of pluripotency on human ESCs (Fernandez et al., 2014, PNAS, 111(4)1409-1414. An exemplary human FZD7 transcript corresponds to GenBank # NM_003507.2 and has the nucleotide sequence set forth in SEQ ID NO:21.

[0101] In some embodiments, the expression levels of FZD7 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.22. IDO1

[0102] Indoleamine 2, 3-dioxygenase 1 (IDO1) is a rate-limiting metabolic enzyme that catalyzes the conversion of the amino acid tryptophan into kynurenine. IDO1 is an immune regulator and has been implicated in autoimmune diseases, neurodegeneration, and cancer (Pallotta et al., 2022, FEBS J. 289(2):6099-6118). An exemplary human IDO1 transcript corresponds to GenBank # NM_002164.6 and has the nucleotide sequence set forth in SEQ ID NO:22.

[0103] In some embodiments, the expression levels of IDO1 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.23. TRIML2

[0104] Tripartite motif family like 2 (TRIML2) has been implicated in p53 SUMOylation and transactivation of proapoptotic genes and functions as a regulatory inhibitor of the proinflammatory cytokine production pathways in trophoblasts (Zhang et al., 2020. Mol Biol Evol. 37:507-523). An exemplary human TRIML2 transcript corresponds to GenBank # NM_173553.4 and has the nucleotide sequence set forth in SEQ ID NO:23.

[0105] In some embodiments, the expression levels of TRIML2 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.24. FGF4

[0106] Fibroblast growth factor 4 (FGF4) facilitates the survival and growth of the inner cell mass during the post-implantation phase of embryonic development and FGF5 knockout mice have been shown to display various developmental defects (Feldman et al., 1995, Science. 267(5195):246-9). An exemplary human FGF4 transcript corresponds to GenBank# NM_002007.4 and has the nucleotide sequence set forth in SEQ ID NO:24.

[0107] In some embodiments, the expression levels of FGF4 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.25. C0L2A1

[0108] Collagen type II alpha 1 chain (COL2A1) is a structural protein component of the extracellular matrix and is the major collagen in the cartilage. An exemplary human COL2A1 transcript corresponds to GenBank # NM_001844.5 and has the nucleotide sequence set forth in SEQ ID NO:25.

[0109] In some embodiments, the expression levels of COL2A1 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.26. EOMES

[0110] Eomesodermin (EOMES) belongs to the TBR1 (T-box brain protein 1) sub-family of T-box genes that share the common DNA-binding T-box domain. The encoded protein is a transcription factor which is crucial for embryonic development of mesoderm and the central nervous system in vertebrates. The protein may also be necessary for the differentiation of effector CD8+ T cells (Banerjee et al., 2010, J Immunol. 185(9): 4988-4992). An exemplary human EOMES transcript corresponds to GenBank # NM_001278182.2 and has the nucleotide sequence set forth in SEQ ID NO:26.

[0111] In some embodiments, the expression levels of EOMES are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.27. SFRP1

[0112] Secreted frizzled related protein 1 (SFRP1) is a member of the SFRP family, members of which serve as soluble modulators of Wnt signaling. SFRP1 is considered to be a putative tumor suppressor gene and loss of SFRP1 expression has been implicated in various cancers (Caldwell et al., 2004, Cancer Res. 64(3):883-888). An exemplary human SRFP1 transcript corresponds to GenBank # NM_003012.5 and has the nucleotide sequence set forth in SEQ ID NO:27.

[0113] In some embodiments, the expression levels of SFRP1 are quantified using qPCR-based methods, e.g., as disclosed in Section 6.4.6.6.3.28. HERV-K

[0114] Human endogenous retrovirus K (HERV-K) is a family of human endogenous retroviruses present in the human genome, among which HERV-K subgroup HML-2 (also referred to as endogenous retrovirus group K member 6 (ERVK-6)) is the youngest and most transcriptionally active and has been shown to be transcribed during embryogenesis from the eight-cell stage up to the stem cell derivation. The activity of HERV-K (HML-2) was linked to increased IFITM1 levels on the cell surface and inhibition of viral infections (Grow et al., 2015, Nature. 522(7555): 221-5). An exemplary HERV-K (HML-2) complete transcript corresponds to GenBank # AF074086.2 and has the nucleotide sequence set forth in SEQ ID NO:28.

[0115] In some embodiments, the expression levels of HERV-K (e.g., HERV-K REC, HERV-K POL, HERV-K ENV, HERV-K GAG) are measuring at the protein level (e.g., using flowcytometry) and / or at the nucleotide levels (e.g., using qPCR-based methods), e.g., as disclosed in Section 6.4.6 and Section 8.6.6.3.29. BCOR

[0116] BCL6 co-repressor (BCOR) is a transcriptional repressor that is associated with normal germinal center formation in B cells. BCOR is a chromatin regulatory protein that functions in a variety of non-canonical epigenetic repressive complexes and binds the transcriptional repressor BCL6. Depletion of BCOR is associated with a loss of Polycomb protein binding at target genes, which is important for maintaining repressed chromatin. An exemplary BCOR gene is available as Gene ID: 54880 (e.g., available at the website www.ncbi.nlm.nih.gov / gene / 54880). An exemplary nucleotide sequence of human wildtype BCOR transcript is set forth in SEQ ID NO:29.

[0117] As used herein, the term “mutant BCOR” refers to a BCOR gene that has one or more mutations that are associated with reduced PSC differentiability. Nonlimiting examples of mutant BCORs are a mutant BCOR gene, “BCOR SNP1”, that has a c.572G> A mutation relative to the nucleotide sequence of SEQ ID NO:29 (e.g., resulting in a transcript comprising the nucleotide sequence of SEQ ID NO:30), and a mutant BCOR gene, “BCOR SNP2”, that has a c.4819+1G> A mutation relative to the nucleotide sequence of SEQ ID NO:29 (e.g., resulting in a transcript comprising the nucleotide sequence of SEQ ID NO:31).

[0118] The presence of mutant BCOR (e.g., as a percentage of all BCOR genes or BCOR alleles in a cell population) can be determined as a measure of differentiability, for example as disclosed in Section 8.5.6.3.30. EP300

[0119] The E1A-associated protein (EP300) gene encodes a histone acetyltransferase, p300, that regulates transcription via chromatin remodeling and is important in the processes of cell proliferation and differentiation (Goodman and Smolik, 2000, Genes Dev. 14(13):1553-77). An exemplary full-length human EP300 sequence corresponds to GenBank# NM_001429 and has the nucleotide sequence set forth in SEQ ID NO:32. An exemplary EP300 coding sequence has the nucleotide sequence set forth in SEQ ID NO:33.

[0120] The presence of mutant EP300 (e.g., as a percentage of all EP300 genes or EP300 alleles in a cell population) can be determined as a measure of differentiability, for example as disclosed in Section 8.7. In some embodiments, mutant EP300 has a c.4540G> A (p. Glu1514Lys, aka p. E1514K) mutation. In some embodiments, mutant EP30 has a c.1317del mutation. In some embodiments, mutant EP300 has a c.1474C> T mutation. In some embodiments, mutant EP300 has a EP300 c.2528dup mutation.6.4. Differentiability Marker Expression Assays

[0121] Expression of differentiability markers in the methods of the disclosure can be detected and quantified using any suitable means.

[0122] In some embodiments, the expression of one or more differentiability markers is measured at the protein level. Exemplary methods of measuring protein expression levels include Western blotting (e.g., as disclosed in Section 6.4.1), an enzyme-linked immunosorbent assay (ELISA) (e.g., as disclosed in Section 6.4.2), a radioimmunoassay (RIA) (e.g., as disclosed in Section 6.4.3), flow cytometry (e.g., as disclosed in Section 6.4.4), and an immunohistochemical assay (IHC) (e.g., as disclosed in Section 6.4.5).

[0123] In some embodiments, the expression of one or more differentiability markers is measured at the nucleic acid level, e.g., mRNA levels. Exemplary methods of measuring mRNA expression levels include polymerase chain reaction (PCR) (e.g., as disclosed in Section 6.4.6) and microarray-based methods (e.g., as disclosed in Section 6.4.7).6.4.1. Western Blotting

[0124] In some embodiments, the expression of one or more differentiability markers is assayed using Western blotting.

[0125] Western blotting, also known as immunoblotting, is an analytical technique used to detect specific proteins or other antigens (e.g, peptides, polypeptides, glycoproteins, etc.) in a given sample of a cell lysate, cell or tissue homogenate, or other protein containing samples. Proteins or other antigens in the sample are separated via gel electrophoresis under nondenaturing (native) or denaturing conditions and detected via an antibody. For quantification purposes, the proteins are transferred from the gel (e.g. a polyacrylamide gel) onto a suitable surface (e.g., a nitrocellulose membrane or a PVDF membrane) and quantified using suitable image analysis software (e.g., ImageJ software). Western blot methods are described in U. S. Patent. No. 8,592,141 and PCT Publication No. 2012 / 057689 A1, the disclosures of which are incorporated by reference herein.6.4.2. ELISA

[0126] In some embodiments, the expression of one or more differentiability markers is assayed using enzyme-linked immunosorbent assay (ELISA).

[0127] ELISA is an immunological assay commonly used to detect the presence and measure the level of expression of a protein or another antigen (e.g., a peptide, a polypeptide, a glycoprotein, etc.) in a sample. Typically, ELISA is performed using antibodies, but they can also be performed with any non-antibody capture agents that bind specifically to a target protein or antigen and that can be detected.

[0128] Various formats of ELISA (e.g., direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, multiplex ELISA, ELISPOT, etc.) have been developed and principles ofthese ELISA formats are known in the art, see for example John R. Crowther, 2009, The ELISA Guidebook. 2ndEd., Humana Press and Hayrapetyan et al., 2023, Methods Mol. Biol. 2612:1-17.6.4.3. Radioimmunoassay (RIA)

[0129] In some embodiments, the expression of one or more differentiability markers is assayed using radioimmunoassay (RIA).

[0130] RIA is a competition-based assay that is well known in the art and involves mixing known quantities of radioactively-labelled (e.g.,125l or tritium-labelled) target analyte (e.g., a target protein, peptide, polypeptide, etc.) with antibody specific for the analyte (e.g., protein, peptide, polypeptide, etc.), then adding non-labeled analyte from a sample and measuring the amount of labeled analyte that is displaced (see, e.g., Chard T, ed., 1995, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science).6.4.4. Flow Cytometry

[0131] In some embodiments, the expression of one or more differentiability markers is assayed using flow cytometry.

[0132] Flow cytometry is a technology that rapidly analyzes single particles (e.g., individual cells) suspended in a buffered salt-based solution as they flow past one or more lasers. Each particle is analyzed for visible light scatter and one or more fluorescence parameters. Visible light scatter is measured in two different directions. Samples can be prepared for fluorescence measurement through staining with fluorescently conjugated antibodies (e.g., FITC-conjugated antibodies). Flow cytometry principles and methods are known in the art, see McKinnon, 2018, Curr. Protoc. Immunol. 120:5.1.1-5.1.11).6.4.5. Immunohistochemical assay (IHC)

[0133] In some embodiments, the expression of one or more differentiability markers is assayed using an immunohistochemical assay (IHC).

[0134] IHC is an immunostaining technique, which involves specific binding of an antibody to a target antigen (e.g., a target protein, peptide, polypeptide etc.) and allows detection, localization, and quantification of one or more target antigens (e.g., target proteins, peptides, polypeptides etc.) in cells and tissues. Direct IHC, which uses labeled antibodies against one or more target antigens, or indirect IHC, which uses non-labeled primary antibodies against one or more target antigens and labeled secondary antibodies that bind to the primary antibodies. Antibodies can be labeled with different types of labels or tags (e.g., a fluorescent, a chemiluminescent, or a radioactive label or tag). Principles and methods of IHC are known in the art and further information can be found in Magaki et al., 2019, Methods Mol. Biol. 1897: 289-298.6.4.6. Polymerase Chain Reaction (PCR)

[0135] Polymerase Chain Reaction (PCR) is a technique that can quickly generate a large number of copies of a target nucleotide sequence (e.g., a genomic DNA sequence, a cDNA sequence, an RNA sequence, etc.) in a sample.

[0136] One variation of PCR, which is referred to as quantitative PCR (qPCR) or real-time PCR, couples amplification of a target DNA sequence with quantification of the concentration of that DNA species in the reaction. This method enables calculation of the starting template concentration and is therefore used to evaluate DNA copy number. Two common methods for the detection of PCR products in qPCR are non-specific fluorescent dyes that intercalate with any double-stranded DNA and sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence. When preceded by reverse-transcription PCR, qPCR is a powerful tool to measure mRNA expression levels. See Dymond, 2013 Methods Enzymol. 2013:529:279-89. Other PCR methods, such as droplet digital PCR (ddPCR), can also be used to measure mRNA expression levels. ddPCR is based on the isolated amplification of a multitude of individual DNA molecules simultaneously, with each molecule compartmentalized in a droplet, in which a fluorescent signal indicates the presence of amplified product in each droplet and the proportion of positive droplets allows the precise quantification of a given sequence. See, e.g., Vossen and White, 2017, Methods Mol Biol. 1492:167-177.6.4.7. Microarrays

[0137] A microarray is a type of assay that uses a surface (e.g., a glass slide) with a solid support (e.g., cellulose, agarose, etc.) comprising an array of spatially defined nonoverlapping regions or sites, each of which contain an immobilized nucleic acid (e.g., a fluorescently labeled hybridization probe). The different immobilized nucleic acid fragments are arranged in rows and columns such that the identity of each fragment is known through its location on the array. Microarrays can be used to analyze the expression profiles of multiple target sequences in a sample simultaneously.6.5. Differentiability Marker Kits

[0138] The present disclosure provides kits comprising reagents for performing the PSC differentiability assays and / or generating a PSC score for a sample.

[0139] In some embodiments, the kit comprises PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of one or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, HERV-K (e.g., HERV-K REC, HERV-K POL, HERV-K ENV, HERV-K GAG), mutant EP300, mutant BCOR (e.g., BCOR SNP1, BCOR SNP2), FAM129A,LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, and SFRP1. In some embodiments, the kit comprises PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of GBX2 and optionally one or more additional differentiability markers, e.g., one or more of NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, HERV-K (e.g., HERV-K REC, HERV-K POL, HERV-K ENV, HERV-K GAG), mutant EP300, mutant BCOR (e.g., BCOR SNP1, BCOR SNP2), FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, and SFRP1.

[0140] In some embodiments, the kit comprises PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of two or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, HERV-K (e.g., HERV-K REC, HERV-K POL, HERV-K ENV, HERV-K GAG), mutant EP300, mutant BCOR (e.g., BCOR SNP1, BCOR SNP2), FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, and SFRP1. In some embodiments, the kit comprises PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of GBX2 and SFRP1 and optionally one or more additional differentiability markers, e.g., one or more of NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, HERV-K (e.g., HERV-K REC, HERV-K POL, HERV-K ENV, HERV-K GAG), mutant EP300, mutant BCOR (e.g., BCOR SNP1, BCOR SNP2), FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, and EOMES.

[0141] In some embodiments, the kit comprises PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of three or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, HERV-K (e.g., HERV-K REC, HERV-K POL, HERV-K ENV, HERV-K GAG), mutant EP300, mutant BCOR (e.g., BCOR SNP1, BCOR SNP2), FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, and SFRP1.

[0142] In some embodiments, the kit comprises PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of four or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, HERV-K (e.g., HERV-K REC, HERV-K POL, HERV-K ENV, HERV-K GAG), mutant EP300, mutant BCOR (e.g., BCOR SNP1, BCOR SNP2), FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, and SFRP1.

[0143] In some embodiments, the kit comprises PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of at least five of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG and FAS. The kit can further include PCR primers and optionally one ormore probes suitable for performing PCR (e.g., qPCR) to measure expression levels of one, two, three, four, five or more (e.g., all) of HERV-K (e.g., HERV-K REC, HERV-K POL, HERV-K ENV, HERV-K GAG), mutant EP300, mutant BCOR (e.g., BCOR SNP1, BCOR SNP2), FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, and SFRP1.

[0144] In some embodiments, the kit comprises PCR primers and probes suitable for performing PCR (e.g., qPCR) to measure expression levels of at least seven of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG and FAS. The kit can further include PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of one, two, three, four, five or more (e.g., all) of HERV-K (e.g., HERV-K REC, HERV-K POL, HERV-K ENV, HERV-K GAG), mutant EP300, mutant BCOR (e.g., BCOR SNP1, BCOR SNP2), FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, and SFRP1.

[0145] In some embodiments, the kit comprises PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of at least ten of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG and FAS. The kit can further include PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of one, two, three, four, five or more (e.g., all) of HERV-K (e.g., HERV-K REC, HERV-K POL, HERV-K ENV, HERV-K GAG), mutant EP300, mutant BCOR (e.g., BCOR SNP1, BCOR SNP2), FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, and SFRP1.

[0146] In some embodiments, the kit comprises PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of at fifteen of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG and FAS. The kit can further include PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of one, two, three, four, five or more (e.g., all) of HERV-K (e.g., HERV-K REC, HERV-K POL, HERV-K ENV, HERV-K GAG), mutant EP300, mutant BCOR (e.g., BCOR SNP1, BCOR SNP2), FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, and SFRP1.

[0147] In some embodiments, the kit comprises PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG and FAS. The kit can further include PCR primers and optionally one or more probes suitable for performing PCR (e.g., qPCR) to measure expression levels of one, two, three, four, five or more (e.g., all) of HERV-K (e.g., HERV-K REC, HERV-K POL, HERV-K ENV, HERV-K GAG), mutant EP300, mutant BCOR (e.g., BCOR SNP1, BCOR SNP2), FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, and SFRP1.

[0148] In each of the foregoing cases, the primers and / or probes can be labeled. In some embodiments, the kit includes a primer that has a fluorescent reporter on its 5' end and a fluorescent quencher of the reporter at the 3' end. So long as the primer is intact, the fluorescent signal from the reporter is quenched. However, when the primer is digested during the elongation process, the fluorescent reporter no longer is in proximity to the quencher and emits a fluorescent signal. In a quantitative PCR reaction, the relative accumulation of free fluorescent reporter for a given amplicon may be compared to the accumulation of the same amplicons for a control, such as p-actin, 18S rRNA, GAPDH and / or TBP, to determine the relative abundance of a differentiability marker in a PSC sample to the control. Thus, in some embodiments, the kit can comprise PCR primers and optionally one or more probes to measure expression levels of one or more controls against which differentiability marker expression can be normalized.

[0149] In each of the foregoing cases, the kit can further comprise additional reagents, such as polymerase and / or nucleotide mixtures for performing PCR.

[0150] In some embodiments, the kit further comprises an antibody (e.g., a labeled antibody) suitable for detection of IFITM1 protein, e.g., via flow cytometry.6.6. Computer Implementation

[0151] Certain aspects of the disclosure relate to calculation of a PSC score on a computerized device having a processor capable of executing a readable program code for calculating a PSC score according to the methods described herein.

[0152] Accordingly, the disclosure provides computerized device can include means for receiving expression level data for one or more differentiability markers and optionally one or more control genes. In some embodiments, expression level data are Cq values. Thus, the computerized device may be connected to real-time PCR instrumentation.

[0153] In some embodiments, a non-transitory storage medium stores instructions that are executable by a digital processing device to perform a method according to the present invention as described herein. The non-transitory storage medium may be a computer-readable storage medium, such as a hard drive or other magnetic storage medium, an optical disk or other optical storage medium, a random access memory (RAM), read only memory (ROM), flash memory, or other electronic storage medium, a network server, or so forth. The digital processing device may be a handheld device (e.g., a personal data assistant or smartphone), a notebook computer, a desktop computer, a tablet computer or device, a remote network server, or so forth.

[0154] In some embodiments, an apparatus comprises a digital processor configured to perform a method according to the present invention as described herein.

[0155] In some embodiments, a computer program comprises program code means for causing a digital processing device to perform a method according to the present invention as described herein. The digital processing device may be a handheld device (e.g., a personal data assistant or smartphone), a notebook computer, a desktop computer, a tablet computer or device, a remote network server, or so forth.

[0156] In some embodiments, the computer program or system includes a means for displaying the TGF-β pathway signaling status in a sample from a host. In some embodiments, a means for displaying can include a computer monitor, a visual display, a paper print out, a liquid crystal display (LCD), a cathode ray tube (CRT), a graphical keyboard, a character recognizer, a plasma display, an organic light-emitting diode (OLED) display, or a light emitting diode (LED) display, or a physical print out.6.7. PSCs

[0157] The methods of the disclosure entail assaying PSCs for differentiability, e.g., by measuring expression of one or more differentiability markers and / or by calculating PSC scores in PSC samples.

[0158] In some embodiments, the PSCs assayed for differentiability are embryonic stem cells (ESCs). In some embodiments, the PSCs assayed for differentiability are induced pluripotent stem cells (iPSCs). Methods of isolating and maintaining PSCs, including ESCs and iPSCs, are well known in the art. See, e.g., Thomson et al., 1998, Science 282(5391 ):1145-7; Hovatta et al., 2003, Human Reprod. 18(7): 1404-09; Ludwig et al., 2006, Nat Methods 3:637-46; Kennedy et al., 2007, Blood 109:2679-87; Chen et al., 2011, Nat Methods 8:424-9; and Wang et al., 2013, Stem Cell Res. 11(3): 1103-16.

[0159] In some embodiments, the PSC cells are assayed following preparation of a PSC population, e.g., an iPSC population as described in Section 6.7.1. If a PSC sample is determined to be differentiable (e.g., achieves a suitable PSC score), then the PSC sample can be cryopreserved, e.g., as described in Section 6.7.2. In some embodiments, it is expanded, e.g, under conditions that maintain pluripotency, prior to cryopreservation so as to store as a cell bank. The expanded cells may be assayed a gain for differentiability prior to cryopreservation.

[0160] In some embodiments, cells from a PSC population assayed by the methods of the disclosure, for example following a determination that the PSCs are differentiable (e.g., achieving a suitable PSC score), are cryopreserved, e.g., as disclosed in Section 6.7.2.

[0161] In some embodiments, a PSC line, e.g., a PSC line determined to be differentiable (e.g., having a suitable PSC score), is subcloned, and the one or more subclone lines are assayed for differentiability according to the methods disclosed herein.

[0162] In some embodiments, the PSCs assayed are from a population of cryopreserved cells, e.g., PSCs that have been cryopreserved as described in Section 6.7.2. In some embodiments, the PSCs are reactivated prior to assaying for differentiability, e.g., as described in Section 6.7.3.6.7.1. Methods of preparing iPSCs

[0163] Methods of preparing iPSCs are known in the art and include, for example, inducing expression of one or more genes (e.g., POU5F1 / OCT4 (Gene ID: 5460) in combination with, but not restricted to, SOX2 (Gene ID: 6657), KLF4 (Gene ID: 9314), c-MYC (Gene ID: 4609, NANOG (Gene ID: 79923), and / or LIN28 / LIN28A (Gene ID: 79727)). Reprogramming factors may be delivered by various means (e.g., viral, non-viral, RNA, DNA, or protein delivery); alternatively, endogenous genes may be activated by using, e.g., CRISPR and other gene editing tools, to reprogram non-pluripotent cells into PSCs.

[0164] In some embodiments, the PSCs are generated from somatic cells (e.g., hematopoietic cells) using a non-integrating vector or episomal vector, e.g., an alphavirus vector, encoding reprogramming factors. In some embodiments, the iPSCs are generated by reprogramming a hematopoietic cell using any combination of the reprogramming factors, alphavirus vectors, and / or reprogramming methods described in WO2022 / 204567 A1 and / or WO2013 / 177133 A2, the contents of each of which are incorporated by reference herein in their entireties. In some embodiments, the hematopoietic cells are erythroid progenitor cells.6.7.2. Cryopreservation of PSCs

[0165] The freezing of cells is ordinarily destructive, such as due to ice crystal formation, osmotic dehydration and increased intracellular solute concentration during cooling and / or freezing. These injurious effects can be circumvented by (a) use of a cryoprotective agent, (b) control of the freezing rate, and (c) storage at a temperature sufficiently low to minimize degradative reactions. Cells can be stored using conventional equipment and methods, which include, but are not limited to, placing the container containing the cells in a freezer (e.g., ultralow temperature freezer), and bringing the cells into contact with a low temperature medium (e.g., liquid nitrogen, etc.) and preserving them in a freezer or cryopreservation system).

[0166] In some embodiments, a program cooling method may be used. Cryopreserving cells with a program cooling method can be achieved by providing a freezing means set to a desired temperature in contact with a container containing cells directly or after accommodating the container in a freezing treatment container, e.g., a freezing treatment container such as a BICELL™, Mr. Frosty or CoolCell container. The freezing temperature is typically not more than 0° C, preferably not more than -20°C, more preferably not more than -40°C, and in some embodiments not more than -80°C. The freezing treatment container may have a function of controlling the rate of temperature decrease inside the container to a predetermined speed. For instance, the cooling rate can be achieved by using a freezer whose cooling rate can becontrolled by setting a program, such as a program freezer (e.g., KryoMed (Thermo Fisher), PDF-2000G (Strex), KRYO-560-16 (Asahi Life Science)). The cooling rate in the freezing operation is typically a cooling rate of 1-5 hours, for example 2-4 hours, particularly about 3 hours, from the start of cooling at 4°C to reach -80°C. A typical cooling program comprises a cooling rate of -1°C / min to -2°C / min; when the temperature reaches below -25°C, the cooling rate can be adjusted to -5°C / min to -10°C / min; when the temperature reaches -100°C, the cell container can be quickly immersed in liquid nitrogen. Cells can be stored in liquid nitrogen for decades or longer.

[0167] The freezing operation can be performed using a culture medium in which colonies were immersed, a physiological buffer solution, or the like as a cryoprotective solution, and treating the solution by adding a cryoprotective agent, or replacing the culture medium with a cryoprotective solution containing a cryoprotective agent. When replacing the culture medium with a cryoprotective solution, the cryoprotective solution may be added after substantially removing all the culture medium, or the cryoprotective solution may be added while leaving a part of the culture medium. The cryoprotective solution may be commercially available and, for example, CryoStor™ CS10, and STEM-CELLBANKER™ (ZENOAQ) can be used.

[0168] The nonlimiting examples of cryoprotective agents include dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), 1,2-propanediol (1,2-PD), 1,3-propanediol (1,3-PD), butyleneglycol (BG), isopreneglycol (IPG), dipropylene glycol (DPG), and glycerol. The cryoprotective agent may be used alone or two, three or more cryoprotective agents may be used in combination. Moreover, the cryoprotective agent may be used in combination with an extracellular cryoprotective agent. Nonlimiting examples of the extracellular cryoprotective agent include polyethylene glycol, carboxymethylcellulose sodium, polyvinylpyrrolidone, hydroxyethyl starch (HES), dextran, and albumin.

[0169] The concentration of the cryoprotective agent added to the culture medium, or the concentration of the cryoprotective agent in the cryoprotective solution, is typically 2%-20% (v / v), for example 5%-15% and in some embodiments 8%-13%, of the total culture medium or cryoprotective solution. The above-mentioned concentration ranges can be appropriately adjusted by the type of cryoprotective agent. For example, when DMSO is used as the cryoprotective agent, concentration of DMSO is typically 2%-20% (v / v), typically 2.5%-12.5%, for example 5%-10%, of the total culture medium or cryoprotective solution.

[0170] A cryopreservation container can be used. Specific examples of cryopreservation containers include but are not limited to aseptic vials (e.g., glass ampoule, polymer vial (AT-closed vial) etc.).

[0171] In some embodiments, cryopreserved cells are maintained as a cell bank. The term “cell bank” refers to a repository of cells, typically isogenic cells from a parental clone, in storage.Typically, the cells are preserved, e.g., in a cryopreservation solution. Cells in a cell bank can be kept for years, decades or longer.6.7.3. Reactivation

[0172] In some embodiments, the PSCs are prepared by reactivating cryopreserved cells prior. The terms "cell reactivation” and “reactivation” refer to the process of reactivation of dormant cells, e.g., cryopreserved cells in a cell bank.

[0173] In some embodiments, a cryopreservation container containing one or more cryopreserved cells is collected from a cell bank and then contacted with a solid, liquid or gaseous medium (e.g., water, culture medium) having a temperature higher than the cryopreservation temperature, e.g., by using a water bath, an incubator, or the like. The temperature of the medium is typically 4°C-50°C, preferably 30°C-40°C, more preferably 36°C-38°C. The thawing time is typically within 2 minutes, and in some embodiments about 20 seconds, whereby a decrease in the survival rate of the cell can be drastically suppressed. The thawing time can be adjusted by, for example, changing the thawing means, temperature of immersion medium, volume or composition of the culture medium or cryoprotective solution during freezing, and the like.

[0174] Following thawing, the thawed cells can be washed by a known method. For example, the method includes, but is not limited to, suspending the cells in a cell washing solution (e.g., culture medium or biological buffer etc. containing serum, serum components (serum albumin and the like)), centrifuging, discarding the supernatant, and collecting the precipitated cells. In the process of washing the cells, the cycle of suspending, centrifugation, and recovery may be performed once or plural times (e.g., 2, 3, 4, 5 or more). In one embodiment, the process of washing the cells is performed immediately after the process of thawing banked cells.Examples of commercially available cell washing solutions that can be used include CELLOTION (Japan ZENYAKU KOGYO) and the like.

[0175] In some embodiments, the thawed cells are pre-cultured. The pre-culture and maintenance culture can be performed for an appropriate culture period according to the purpose. The culture period is preferably not less than 1 day (e.g., 5, 6, 7 days) and not more than 10 days (e.g., 7, 8, 9, 10 days). The culture temperature is not particularly limited and in some embodiment ranges between 30°C and 40°C, and is preferably 37°C. Culture can be performed in the presence of CC>2-containing air, e.g., at a CO2 concentration of about 2%-5%. In some embodiments, the pre-culturing is performed on a surface treated for increased PSC adherence, optionally wherein the surface is treated for increased hydrophilicity.6.8. Use of Primed PSCs to Produce Cell Therapy

[0176] PSCs and PSC populations identified as primed by the methods disclosed herein can advantageously be used to generate and / or maintain differentiated cells useful for cell therapy.

[0177] Accordingly, in some embodiments, a PSC or PSC population identified as primed is differentiated and optionally formulated as a cell therapy.

[0178] In some embodiments, a primed PSC or PSC population is differentiated into a cell population comprising or consisting of ectodermal cells. In some embodiments, the ectodermal cells are dopaminergic (DA) neurons.

[0179] Differentiation of human PSCs into ectoderm provides neurons and glia. Ectodermal cell differentiation of PSCs or embryonic stem cells is influenced by specific signaling pathways, such as bone morphogenetic proteins (BMPs), Wnt / p-catenin, fibroblast growth factor (FGF), and TGFp signaling, and environmental factors, such as cell density (Walsh et al., 2020, Stem Cells. 38(11): 1400-1408; Tchieu et al., 2017, Cell Stem Cell 21 (3): 399-410).

[0180] Ectoderm lineage cells (e.g., DA neurons) can be differentiated from PSCs by exposing the PSCs to ectodermal cell differentiation conditions. For example, PSCs can be contacted with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling; and subsequently contacting the cells with at least one activator of fibroblast growth factor (FGF) signaling and at least one inhibitor of Wnt signaling to obtain a population of differentiated cells expressing at least one marker indicating a DA neuron or a precursor thereof. See WO 2021 / 203009 A1.

[0181] An exemplary protocol that can be used to generate DA neurons is described in Piao et al., 2021, Cell Stem Cell 28(2):217-229. e7, the contents of which are incorporated herein by reference in their entireties. In the protocol, stem cells are expanded in E8 basal medium for 10-14 days, with cell splittings performed every 3-5 days at ratios between 1:4 and 1:6. Next, cell are washed and plated on a suitable substrate at 400,000 cells / cm2in Neurobasal (NB) medium supplemented with N2 (which supports neural differentiation) and B27 (which supports neuron growth and survival) without vitamin A containing 2mM L-glutamine, 10.8pM of TGFp inhibitor SB431542, 250nM of BNP inhibitor LDN193189 (LDN), 0.7pM of GSK-3 inhibitor CHIR99021, and 500ng / mL SHH with 10pM of ROCK inhibitor Y-27632. Media is replaced daily, except without Y-27632. On day 4, the same media is used except with 7.5pM CHIR99021. Same media is replaced on day 6. On day 7 SB, LDN and SHH are withdrawn from the medium. Same media is changed on day 9. On day 10, NB (with B27) with 2mM L-glutamine, 20ng / ml BDNF, 20ng / ml GDNF, 200pM Ascorbic acid (AA), 500pM Dibutyryl-cAMP, 1 ng / ml TGF 3 and 3pM CHIR99021 is added. On day 11, cells are dissociated and replated. On day 12, the medium is switched to include 10pM DAPT and CHIR99021 is withdrawn. Complete media changes are performed daily until harvest at day 16.

[0182] In some embodiments, the differentiated cells are formulated with a pharmaceutically acceptable carrier or excipient for use in cell therapy.

[0183] Accordingly, the disclosure provides methods of administering to a subject in need thereof a differentiated cell produced from a PSC identified as a primed PSC by the methods disclosed herein.

[0184] Where the PSCs are differentiated into DA neurons, the PSCs can be transplanted into, including, without limitation, a patient having a neurodegenerative disease. Examples of neurodegenerative diseases are Parkinson's Disease, Alzheimer’s Disease, dementia, epilepsy, Lewy Body syndrome, Huntington’s Disease, Spinal Muscular Atrophy, Friedreich’s Ataxia, Amyotrophic Lateral Sclerosis, Batten Disease, Multiple System Atrophy, among others.7. SPECIFIC EMBODIMENTS

[0185] The present disclosure is exemplified by the specific embodiments below.

[0186] Group A Embodiments:1. A method of measuring expression levels of differentiability markers in pluripotent stem cells (PSCs), comprising:(a) measuring one or more differentiability markers in a sample prepared from the PSCs; and(b) determining the expression levels of the differentiability markers.2. The method of embodiment 1, wherein measuring the one or more differentiability markers comprises performing a polymerase chain reaction (PCR) on nucleic acids extracted from PSCs using primers capable of amplifying the one or more differentiability markers.3. The method of embodiment 2, wherein the PCR is a real-time PCR, and the method further comprises quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the one or more differentiability markers.4. The method of any one of embodiments 1 to 3, which further comprises measuring the expression levels of one or more control genes in the sample.5. The method of embodiment 4, which further comprises normalizing the expression levels of the one or more differentiability markers against the one or more control genes.6. An assay method comprising:(a) performing a polymerase chain reaction (PCR) on one or more reaction mixtures comprising:(i) RNA extracted from PSCs or cDNA reverse transcribed therefrom;(ii) primers suitable for amplifying one or more differentiability markers;(iii) labeled probes for detecting the one or more differentiability markers; and(iv) a DNA polymerase;(b) detecting expression levels of the one or more differentiability markers based on signals emitted by the labeled probes.7. The method of embodiment 6, wherein the PCR is performed in parallel on a plurality of reaction mixtures, each comprising a primer pair for amplifying a differentiability marker and a labeled probe suitable for detecting the differentiability marker.8. The method of embodiment 6 or embodiment 7, wherein the PCR is performed on a solid or flat surface.9. The method of any one of embodiments 6 to 8, wherein the PCR is performed in a well or on a tape.10. The method of embodiment 9, wherein the PCR is performed in a 96-well plate.11. The method of embodiment 9, wherein the PCR is performed in a 384-well plate.12. The method of embodiment 9, wherein the PCR is performed on a tape.13. The method of any one of embodiments 6 to 12, wherein the reaction mixture further comprises primers suitable for amplifying one or more control genes and one or more labeled probes for detecting the one or more control genes.14. A method of determining PSC differentiability, comprising:(a) performing a Taqman polymerase chain reaction (PCR) on one or more reaction mixtures comprising:(i) RNA extracted from iPSCs or cDNA reverse transcribed therefrom;(ii) primers suitable for amplifying one or more differentiability markers, e.g., one or more positive differentiability markers and one or more negative differentiability markers;(iii) optionally, primers suitable for amplifying one or more control genes; and(iv) a DNA polymerase;(b) determining the cycle count (Cq) to attain the cycle threshold for the one or more differentiability markers and optionally one or more control genes;(c) calculating mean Cq values for the one or more differentiability markers (“mean negative Cq”); and(d) determining the mean positive Cq: mean negative Cq ratio, wherein the ratio is indicative of PSC differentiability.15. The method of embodiment 14, wherein the Cq values for the one or more differentiability markers are normalized against the expression levels of one or more control genes.16. A method of evaluating pluripotent stem cells (PSCs) for their differentiability, comprising:(a) detecting the expression levels of one or more differentiability markers in the PSCs; and(b) determining if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation.17. The method of embodiment 16, wherein the PSCs have been cryopreserved prior to step (a).18. The method of embodiment 17, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).19. The method of embodiment 16, wherein the PSCs have not been cryopreserved prior to step (a).20. The method of any one of embodiments 16 to 19, which further comprises cryopreserving the PSCs after step (b) if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation.21. The method of any one of embodiments 16 to 20, which further comprises, prior to step (a), generating the PSCs.22. The method of embodiment 21, wherein the PSCs are generated by reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells.23. A method of preparing a pluripotent stem cell (PSC) stem cell bank suitable for differentiation, comprising:(a) detecting the expression levels of one or more differentiability markers in a population of PSCs;(b) determining if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation; (c) if expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation, forming a PSC stem cell bank from the population of PSCs.24. The method of embodiment 23, wherein the PSCs have been cryopreserved prior to step (a).25. The method of embodiment 24, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).26. The method of embodiment 23, wherein the PSCs have not been cryopreserved prior to step (a).27. The method of any one of embodiments 23 to 26, which further comprises cryopreserving the PSCs after step (b) if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation.28. The method of any one of embodiments 23 to 27, which further comprises, prior to step (a), generating the PSCs.29. The method of embodiment 28, wherein the PSCs are generated by reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells.30. A method for qualifying a population of pluripotent stem cells (PSCs) for manufacturing differentiated cells, comprising:(a) detecting the expression levels of one or more differentiability markers in the PSCs; and(b) determining if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation, wherein a determination that the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation qualifies the population of PSCs for manufacturing differentiated cells.31. The method of embodiment 30, wherein the population of PSCs is a clonal population.32. The method of embodiment 30 or embodiment 31, wherein the PSCs have been cryopreserved prior to step (a).33. The method of embodiment 32, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).34. The method of embodiment 30 or embodiment 31, wherein the PSCs have not been cryopreserved prior to step (a).35. The method of any one of embodiments 30 to 34, which further comprises cryopreserving the PSCs after step (b) if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation.36. The method of any one of embodiments 30 to 35, which further comprises, prior to step (a), generating the PSCs.37. The method of embodiment 36, wherein the PSCs are generated by reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells.38. A method for selecting a population of pluripotent stem cells (PSCs) for manufacturing differentiated cells, comprising:(a) detecting the expression levels of one or more differentiability markers in one or more PSC populations;(b) determining if the expression levels of the one or more differentiability markers are indicative of the suitability of the one or more PSC populations for differentiation; and(c) selecting a PSC population for manufacturing differentiated cells if the expression levels of the one or more differentiability markers areindicative of the suitability of the one or more PSC populations for differentiation.39. The method of embodiment 38, which comprises differentiating iPSCs determined to have differentiability markers expression levels indicative of the suitability for differentiation.40. The method of embodiment 38, which comprises discarding PSCs determined to have differentiability markers expression levels indicative of unsuitability for differentiation.41. The method of any one of embodiments 38 to 40, wherein the one or more PSC populations are subclones of a parental PSC population.42. The method of embodiment 38, wherein the one or more PSC populations have been cryopreserved prior to step (a).43. The method of embodiment 41, wherein the one or more PSC populations have been reactivated following cryopreservation and prior to step (a).44. The method of embodiment 38, wherein the one or more PSC populations have not been cryopreserved prior to step (a).45. The method of any one of embodiments 38 to 44, which further comprises cryopreserving one or more PSC populations after step (b) if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation.46. The method of any one of embodiments 38 to 45, which further comprises, prior to step (a), generating the one or more PSC populations.47. The method of embodiment 46, wherein the one or more PSC populations are generated by (a) generating a parental PSC population reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells and (b) culturing subclones of the parental PSC population, thereby generating the one or more PSC populations.48. A method for monitoring a population of pluripotent stem cells (PSCs) for its suitability manufacturing differentiated cells, comprising:(a) detecting the expression levels of one or more differentiability markers in PSCs of the clonal population;(b) determining if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation; and (c) if the expression levels of the one or more differentiability markers in step (b) are indicative of the suitability of the PSCs for differentiation, maintaining the clonal population; and(d) repeating steps (a)-(c) one or more times.49. The method of embodiment 48, wherein the PSCs are a clonal (e.g., isogenic) population of PSCs.50. The method of embodiment 48 or embodiment 49, wherein the PSCs have been cryopreserved prior to step (a).51. The method of embodiment 49, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).52. The method of embodiment 48 or embodiment 49, wherein the PSCs have not been cryopreserved prior to step (a).53. The method of any one of embodiments 48 to 52, which further comprises cryopreserving the PSCs after step (b) if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation.54. The method of any one of embodiments 48 to 53, which further comprises, prior to step (a), generating the PSCs.55. The method of embodiment 54, wherein the PSCs are generated by reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells.56. The method of any one of embodiments 1 to 55, wherein the one or more differentiability markers comprise GBX2.57. The method of embodiment 56, wherein the one or more differentiability markers comprise SFRP1.58. The method of embodiment 56 or embodiment 57, which further comprises determining the relative expression levels of GBX2 and SFRP1, for example as described in Example 3.59. The method of any one of embodiments 1 to 58, wherein the one or more differentiability markers comprise at least one negative differentiability marker and / or at least one positive differentiability marker.60. The method of embodiment 59, wherein one or more negative differentiability markers are markers of pluripotency.61. The method of embodiment 59 or embodiment 60, wherein one or more negative differentiability markers comprise NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, or a combination of two or more of the foregoing.62. The method of embodiment 61, wherein one or more negative differentiability markers comprise a combination of three or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, and SP8.63. The method of embodiment 61, wherein one or more negative differentiability markers comprise a combination of four or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, and SP8.64. The method of embodiment 61, wherein one or more negative differentiability markers comprise a combination of five or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, and SP8.65. The method of embodiment 61, wherein one or more negative differentiability markers comprise a combination of six or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, and SP8.66. The method of embodiment 61, wherein one or more negative differentiability markers comprise a combination of seven or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, and SP8.67. The method of embodiment 61, wherein one or more negative differentiability markers comprise a combination of eight or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, and SP8.68. The method of embodiment 61, wherein one or more negative differentiability markers comprise a combination of nine or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, and SP8.69. The method of embodiment 61, wherein one or more negative differentiability markers comprise a combination of ten or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, and SP8.70. The method of embodiment 61, wherein one or more negative differentiability markers comprise a combination of eleven or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, and SP8.71. The method of embodiment 61, wherein one or more negative differentiability markers comprise a combination of twelve or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, and SP8.72. The method of embodiment 61, wherein one or more negative differentiability markers comprise a combination of thirteen or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2 and SP8.73. The method of any one of embodiments 59 to 72, wherein one or more positive differentiability markers comprise one or more of RAB17, PRTG, FAS, and IFITM1.74. The method of any one of embodiments 59 to 73, wherein one or more positive differentiability markers are markers of pluripotent stem cell (PSC) variability.75. The method of embodiment 74, wherein the one or more one or more markers of pluripotent stem cell (PSC) variability comprise RAB17.76. The method of any one of embodiments 59 to 75, wherein one or more positive differentiability markers are markers of early differentiation.77. The method of embodiment 76, wherein the one or more one or more markers of early differentiation comprise PRTG.78. The method of any one of embodiments 59 to 77, wherein one or more positive differentiability markers are markers of autophagy.79. The method of embodiment 76, wherein the one or more one or more markers of autophagy comprise FAS.80. The method of any one of embodiments 16 to 79, which comprises detecting the expression levels of one or more differentiability markers by a method comprising:(a) measuring protein expression levels using an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), flow cytometry, a Western blot, an immunohistochemical assay (IHC), or a combination of two or more thereof; and(b) determining the measured protein expression levels.81. The method of any one of embodiments 16 to 80, which comprises detecting the expression levels of one or more differentiability markers by a method comprising:(a) measuring nucleic acid (e.g., mRNA) expression levels using a polymerase chain reaction and / or a microarray-based method; and (b) determining the measured nucleic acid expression levels.82. The method of any one of embodiments 1 to 81, wherein determining if the expression levels of the differentiability markers are indicative of the suitability of the PSCs for differentiation by:(a) assigning the PSCs a PSC score based on the expression of the differentiability markers; and(b) determining if the PSC score falls above or below a cutoff value.83. The method of embodiment 82, wherein the PSC score is calculated using the formula' PSC > Mean Cq values of genes upregulated in samples that pass DA differentiation Mean Cq values of genes upregulated in samples that fail DA differentiation '84. The method of embodiment 83, wherein the PSC score formula for the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS is: PSC score =Mean Cq values PRTG, FAS, RAB17)Mean Cq values NR5A2, HHEX BX2, GDF3 AIWG WDAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP&)85. The method of any one of embodiments 82 to 84, wherein the cutoff value is 1.34.86. The method of any one of embodiments 1 to 85, wherein the PSCs are embryonic stem cells.87. The method of any one of embodiments 1 to 85, wherein the PSCs are induced pluripotent stem cells (iPSCs).88. The method of embodiment 87, which further comprises producing the PSCs.89. The method of embodiment 87 or embodiment 88, which further comprises forming a clonal (e.g., isogenic) population of PSCs.90. The method of embodiment 89, wherein the PSCs tested for differentiability markers are from the clonal population.91. The method of any one of embodiments 1 to 90, which further comprises differentiating a PSC identified as a suitable precursor of a differentiated cell into the differentiated cell.92. The method of embodiment 91, wherein the differentiated cell is of the ectoderm lineage.93. The method of embodiment 92, wherein the differentiated cell is a dopaminergic neuron.94. The method of any one of embodiments 91 to 93, herein the differentiated cell is engineered to express a transgene.95. The method of embodiment 94, wherein the transgene is introduced into the PSC prior to differentiation.96. The method of embodiment 95, which further comprises evaluating the PSCs following introduction of the transgene for their suitability as precursors of differentiated cells, comprising:(a) detecting the expression levels one or more differentiability markers in the PSCs; and(b) determining if the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation.97. The method of embodiment 96, wherein the evaluating the PSCs following introduction of the transgene for their suitability as precursors of differentiated cells is performed as defined in any one of embodiments 59 to 85.98. The method of embodiment 94, wherein the transgene is introduced into the differentiated cell following differentiation.99. The method of embodiment 94, wherein the transgene is introduced into a precursor of the differentiated cell.100. The method of any one of embodiments 91 to 99, which further comprises administering the differentiated cell to a subject in need thereof.101. A method of treating a subject, comprising administering to a subject in need thereof a differentiated cell produced by the method of any one of embodiments 91 to 99.102. A method for determining the suitability of pluripotent stem cells (PSCs) for differentiation into differentiated cells following introduction of a transgene, comprising:(a) introducing a transgene into PSCs to produce recombinant PSCs; (b) the evaluating the recombinant PSCs for their suitability as precursors of differentiated cells by a method comprising:(i) detecting the expression levels of one or more differentiability markers in the recombinant PSCs; and(ii) determining if the expression levels of the one or more differentiability markers are indicative of the suitability of the recombinant PSCs for differentiation.103. The method of embodiment 102, wherein evaluating the recombinant PSCs for their suitability as precursors of differentiated cells is performed as defined in any one of embodiments 59 to 85.104. The method of embodiment 102 or embodiment 103, wherein the transgene encodes a therapeutic protein.105. The method of any one of embodiments 102 to 104, which further comprises differentiating a PSC identified as a suitable precursor of a differentiated cell into the differentiated cell.106. The method of embodiment 105, wherein the differentiated cell is a cell of the ectodermal lineage.107. The method of embodiment 106, wherein the differentiated cell is a dopaminergic neuron.108. A method of treating a subject, comprising administering to a subject in need thereof a differentiated cell population produced by the method of embodiment 105 or embodiment 106.109. A method of treating a subject suffering from a neurodegenerative disease, comprising administering to a subject in need thereof a dopaminergic neuron population produced by the method of embodiment 107.110. The method of claim 109, wherein the neurodegenerative diseases is Parkinson’s Disease, Alzheimer's Disease, dementia, epilepsy, Lewy Body syndrome, Huntington’s Disease, Spinal Muscular Atrophy, Friedreich’s Ataxia, Amyotrophic Lateral Sclerosis, Batten Disease, or Multiple System Atrophy.111. A kit comprising one or more probes and / or one or more PCR primer pairs for at least five, at least ten, at least fifteen or all of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG and FAS.112. The kit of embodiment 111, which further comprises one or more probes and / or one or more PCR primer pairs for one, two, three four, five or more (e.g., all) of FAM129A, LCK, FZD7, IDO1. TRIML2, FGF4, COL2A1, EOMES, SFRP1.113. The kit of embodiment 111 or embodiment 112, which comprises one or more probes and / or one or more PCR primer pair for one or more control genes.114. The kit of embodiment 113, wherein the one or more control gene comprise housekeeping genes.115. The kit of embodiment 114, optionally wherein the housekeeping genes comprise one, two, three or all four of p-actin, 18S rRNA, GAPDH and TBP.116. The kit of any one of embodiments 111 to 115 in which at least one primer for each differentiability marker is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end and, if present, at least one primer for each control gene is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end.117. The kit of any one of embodiments 111 to 116, which further comprises polymerase and / or a nucleotide mixture.118. The kit of any one of embodiments 111 to 117, further comprising an anti-IFITM 1 antibody, optionally wherein the antibody is fluorescently labeled.119. The kit of any one of embodiments 111 to 118, wherein the probes are in a 96-well plate.

[0187] Group B Embodiments:1. A method of measuring expression levels of differentiability markers in pluripotent stem cells (PSCs), comprising:(a) measuring expression of at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS in a sample prepared from the PSCs; and(b) determining the expression levels of the at least five differentiability markers.2. The method of embodiment 1, wherein measuring the at least five differentiability markers comprises performing a polymerase chain reaction (PCR) on nucleic acids extracted from the PSCs using primers capable of amplifying the at least five differentiability markers.3. The method of embodiment 2, wherein the PCR is a real-time PCR, and the method further comprises quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the at least five differentiability markers.4. The method of embodiment 3, which comprises:(a) measuring expression of at least 10 of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS in a sample prepared from the PSCs;(b) determining the expression levels of the at least 10 differentiability markers by a method comprising:(i) performing real-time PCR on nucleic acids extracted from the PSCs using primers capable of amplifying the at least 10 differentiability markers; and(ii) quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the at least 10 differentiability markers.The method of embodiment 3, which comprises:(a) measuring expression of at least 15 of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS in a sample prepared from the PSCs;(b) determining the expression levels of the at least 15 differentiability markers by a method comprising:(i) performing real-time PCR on nucleic acids extracted from the PSCs using primers capable of amplifying the at least 15 differentiability markers; and(ii) quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the at least 15 differentiability markers.The method of embodiment 3, which comprises:(a) measuring expression of all 17 of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS in a sample prepared from the PSCs;(b) determining the expression levels of the all 17 differentiability markers by a method comprising:(i) performing real-time PCR on nucleic acids extracted from the PSCs using primers capable of amplifying the all 17 differentiability markers; and(ii) quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the all 17 differentiability markers.An assay method comprising:(a) performing a polymerase chain reaction (PCR) on one or more reaction mixtures comprising:(i) RNA extracted from PSCs or cDNA reverse transcribed therefrom;(ii) primers suitable for amplifying at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1,UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS;(iii) labeled probes for detecting the at least five differentiability markers; and(iv) a DNA polymerase;(b) detecting expression levels of the at least five differentiability markers based on signals emitted by the labeled probes.8. The method of embodiment 7, wherein the PCR is performed in a well or on a tape.9. A method of determining PSC differentiability, comprising:(a) performing a TaqMan polymerase chain reaction (PCR) on one or more reaction mixtures comprising:(i) RNA extracted from iPSCs or cDNA reverse transcribed therefrom;(ii) primers suitable for amplifying at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS;(iii) optionally, primers suitable for amplifying one or more control genes; and(iv) a DNA polymerase;(b) determining the cycle count (Cq) to attain the cycle threshold for the at least five differentiability markers and optionally one or more control genes;(c) calculating mean Cq values for the at least five differentiability markers (“mean negative Cq”); and(d) determining the mean positive Cq: mean negative Cq ratio, wherein the ratio is indicative of PSC differentiability.10. A method of evaluating pluripotent stem cells (PSCs) for their differentiability, comprising:(a) detecting the expression levels of at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS in the PSCs; and(b) determining if the expression levels of the at least five differentiability markers are indicative of the suitability of the PSCs for differentiation.11. The method of embodiment 10, wherein the PSCs have been cryopreserved prior to step (a).12. The method of embodiment 11, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).13. A method of preparing a pluripotent stem cell (PSC) stem cell bank suitable for differentiation, comprising:(a) detecting the expression levels of the at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS in a population of PSCs;(b) determining if the expression levels of the at least five differentiability markers are indicative of the suitability of the PSCs for differentiation; (c) if expression levels of the at least five differentiability markers are indicative of the suitability of the PSCs for differentiation, forming a PSC stem cell bank from the population of PSCs.14. The method of embodiment 13, wherein the PSCs have been cryopreserved prior to step (a).15. The method of embodiment 14, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).16. The method of any one of embodiments 13 to 15, which further comprises cryopreserving the PSCs after step (b) if the expression levels of the at least five differentiability markers are indicative of the suitability of the PSCs for differentiation.17. A method for qualifying a population of pluripotent stem cells (PSCs) for manufacturing differentiated cells, comprising:(a) detecting the expression levels of at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS in the PSCs; and(b) determining if the expression levels of the at least five differentiability markers are indicative of the suitability of the PSCs for differentiation, wherein a determination that the expression levels of the at least five differentiability markers are indicative of the suitability of the PSCs for differentiation qualifies the population of PSCs for manufacturing differentiated cells.18. The method of embodiment 17, wherein the population of PSCs is a clonal population.19. The method of embodiment 17 or embodiment 18, wherein the PSCs have been cryopreserved prior to step (a).20. The method of embodiment 19, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).21. A method for selecting a population of pluripotent stem cells (PSCs) for manufacturing differentiated cells, comprising:(a) detecting the expression levels of at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS in one or more PSC populations;(b) determining if the expression levels of the at least five differentiability markers are indicative of the suitability of the one or more PSC populations for differentiation; and(c) selecting a PSC population for manufacturing differentiated cells if the expression levels of the at least five differentiability markers are indicative of the suitability of the one or more PSC populations for differentiation.22. A method for monitoring a population of pluripotent stem cells (PSCs) for its suitability manufacturing differentiated cells, comprising:(a) detecting the expression levels of at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS in PSCs of the clonal population;(b) determining if the expression levels of the at least five differentiability markers are indicative of the suitability of the PSCs for differentiation; and(c) if the expression levels of the at least five differentiability markers in step (b) are indicative of the suitability of the PSCs for differentiation, maintaining the clonal population; and(d) repeating steps (a)-(c) one or more times.23. The method of embodiment 22, wherein the PSCs have been cryopreserved prior to step (a).24. The method of embodiment 23, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).25. The method of any one of embodiments 1 to 24, wherein the differentiability markers comprise at least one negative differentiability marker and at least one positive differentiability marker.26. The method of embodiment 25, wherein one or more negative differentiability markers are markers of pluripotency.27. The method of embodiment 25 or embodiment 26, wherein one or more negative differentiability markers comprise NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, or a combination of two or more of the foregoing.28. The method of any one of embodiments 25 to 27, wherein one or more positive differentiability markers comprise one or more of RAB17, PRTG, FAS, and IFITM1.29. The method of any one of embodiments 10 to 28, which comprises detecting the expression levels of one or more differentiability markers by a method comprising:(a) measuring protein expression levels using an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), flow cytometry, a Western blot, an immunohistochemical assay (IHC), or a combination of two or more thereof; and(b) determining the measured protein expression levels.30. The method of any one of embodiments 10 to 29, which comprises detecting the expression levels of one or more differentiability markers by a method comprising:(a) measuring nucleic acid (e.g., mRNA) expression levels using a polymerase chain reaction and / or a microarray-based method; and (b) determining the measured nucleic acid expression levels.31. The method of any one of embodiments 1 to 30, which comprises determining if the expression levels of the differentiability markers are indicative of the suitability of the PSCs for differentiation by:(a) assigning the PSCs a PSC score based on the expression of the differentiability markers; and(b) determining if the PSC score falls above or below a cutoff value.32. The method of embodiment 31, wherein the PSC score is calculated using the33. The method of embodiment 32, wherein the PSC score formula for the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS is: PSC score =Mean Cq values (PRTG, FAS, RAB17)Mean Cq values NR5A2 / HHEXlGBX2lGDF3lNANOGlNODALlHMXllUTFllSiX6, MT2AlTBXTlNFIAlID2lSPS)34. The method of any one of embodiments 31 to 33, wherein the cutoff value is 1.34.35. The method of any one of embodiments 1 to 34, wherein the PSCs are induced pluripotent stem cells (iPSCs).36. The method of any one of embodiments 1 to 35, which further comprises differentiating a PSC identified as a suitable precursor of a differentiated cell into the differentiated cell.37. A kit comprising one or more probes and / or one or more PCR primer pairs for at least five, at least ten, at least fifteen or all of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG and FAS.38. The kit of embodiment 37, which further comprises one or more probes and / or one or more PCR primer pairs for one, two, three four, five or more (e.g., all) of FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, SFRP1.39. The kit of embodiment 37 or embodiment 38, which comprises one or more probes and / or one or more PCR primer pairs for GBX2 and SFRP1.40. The kit of any one of embodiments 37 to 39 which comprises one or more probes and / or one or more PCR primer pair for one or more control genes.41. The kit of any one of embodiments 37 to 40 in which at least one primer for each differentiability marker is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end and, if present, at least one primer for each control gene is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end.

[0188] Group C Embodiments:1. A method of measuring expression levels of differentiability markers in pluripotent stem cells (PSCs), comprising:(a) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs) in the PSCs, optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR;(b) optionally, measuring the expression of one or more additional differentiability markers in a sample prepared from the PSCs; and (c) determining the presence of mutations in the differentiability markers of (a) and optionally expression levels of the differentiability markers of (b).2. The method of embodiment 1, which comprises:(a) measuring the presence of one or mutations in one or more differentiability marker genes (e.g., as a percentage of the alleles or the genes in a population of PSCs), optionally wherein mutant EP300 and / or mutant BCOR is measured; and(b) optionally, performing polymerase chain reaction (PCR) on nucleic acids extracted from PSCs using primers capable of amplifying one or more additional differentiability markers.3. The method of embodiment 2, wherein the PCR is a real-time PCR, and the method further comprises quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the one or more additional differentiability markers.4. The method of any one of embodiments 1 to 3, which further comprises measuring the expression levels of one or more control (e.g., housekeeping) genes in thesample, optionally wherein the one more control genes comprise one, two, three, or all four of p-actin, 18S rRNA, GAPDH and TBP.5. The method of embodiment 4, which further comprises normalizing the expression levels of the one or more additional differentiability markers against the one or more control genes.6. An assay method comprising:(a) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR;(b) optionally, performing a polymerase chain reaction (PCR) on one or more reaction mixtures comprising:(i) RNA extracted from PSCs or cDNA reverse transcribed therefrom; (ii) primers suitable for amplifying one or more additional differentiability markers;(iii) labeled probes for detecting the one or more additional differentiability markers; and(iv) a DNA polymerase; and(c) optionally, detecting expression levels of the one or more additional differentiability markers based on signals emitted by the labeled probes.7. The method of embodiment 6, wherein the PCR is performed in parallel on a plurality of reaction mixtures, each comprising a primer pair for amplifying a differentiability marker and a labeled probe suitable for detecting the differentiability marker.8. The method of embodiment 6 or embodiment 7, wherein the PCR is performed on a solid or flat surface.9. The method of any one of embodiments 6 to 8, wherein the PCR is performed in a well or on a tape.10. The method of embodiment 9, wherein the PCR is performed in a 96-well plate.11. The method of embodiment 9, wherein the PCR is performed in a 384-well plate.12. The method of embodiment 9, wherein the PCR is performed on a tape.13. The method of any one of embodiments 6 to 12, wherein the reaction mixture further comprises primers suitable for amplifying one or more control genes and one or more labeled probes for detecting the one or more control (e.g., housekeeping) genes, optionally wherein the one more control genes comprise one, two, three, or all four of p-actin, 18S rRNA, GAPDH and TBP genes.14. A method of determining PSC differentiability, comprising:(a) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR;(b) performing a Taqman polymerase chain reaction (PCR) on one or more reaction mixtures comprising:(v) RNA extracted from PSCs or cDNA reverse transcribed therefrom;(vi) primers suitable for amplifying one or more differentiability markers, e.g., one or more positive differentiability markers and one or more negative differentiability markers;(vii) optionally, primers suitable for amplifying one or more control genes; and(viii) a DNA polymerase;(c) determining the cycle count (Cq) to attain the cycle threshold for the one or more differentiability markers and optionally one or more control genes;(d) calculating mean Cq values for the one or more negative differentiability markers (“mean negative Cq”) and one or more positive differentiability markers (“mean positive Cq”); and(e) determining the mean positive Cq: mean negative Cq ratio, wherein the ratio and / or presence of mutations are indicative of PSC differentiability.15. The method of embodiment 14, wherein the Cq values for the one or more differentiability markers are normalized against the expression levels of one or more control (e.g., housekeeping) genes, optionally wherein the one more control genes comprise one, two, three, or all four of p-actin, 18S rRNA, GAPDH and TBP.16. A method of evaluating pluripotent stem cells (PSCs) for their differentiability, comprising:(a) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in the PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR;(b) optionally, detecting the expression levels of one or more additional differentiability markers in the PSCs; and(c) determining if the presence of mutations and optionally the expression levels of the one or more additional differentiability markers are indicative of the suitability of the PSCs for differentiation.17. The method of embodiment 16, wherein the PSCs have been cryopreserved prior to step (a).18. The method of embodiment 17, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).19. The method of embodiment 16, wherein the PSCs have not been cryopreserved prior to step (a).20. The method of any one of embodiments 16 to 19, which further comprises cryopreserving the PSCs after step (c) if the presence of mutations in and / or expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation.21. The method of any one of embodiments 16 to 20, which further comprises, prior to step (a), generating the PSCs.22. The method of embodiment 21, wherein the PSCs are generated by reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells.23. A method of preparing a pluripotent stem cell (PSC) stem cell bank suitable for differentiation, comprising:(a) detecting the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR;(b) optionally detecting the expression levels of one or more additional differentiability markers in a population of PSCs;(c) determining if the presence of mutations in the differentiability markers of (a) and optionally the expression levels of the differentiability markers of (b) are indicative of the suitability of the PSCs for differentiation;(d) if the presence of mutations in and optionally the expression levels of the differentiability markers are indicative of the suitability of the PSCs for differentiation, forming a PSC stem cell bank from the population of PSCs.24. The method of embodiment 23, wherein the PSCs have been cryopreserved prior to step (a).25. The method of embodiment 24, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).26. The method of embodiment 23, wherein the PSCs have not been cryopreserved prior to step (a).27. The method of any one of embodiments 23 to 26, which further comprises cryopreserving the PSCs after step (c) if the presence of mutations in and optionally the expression levels of the differentiability markers of (a) and (b) are indicative of the suitability of the PSCs for differentiation.28. The method of any one of embodiments 23 to 27, which further comprises, prior to step (a), generating the PSCs.29. The method of embodiment 28, wherein the PSCs are generated by reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells.30. A method for qualifying a population of pluripotent stem cells (PSCs) for manufacturing differentiated cells, comprising:(a) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR;(b) optionally, detecting the expression levels of one or more additional differentiability markers in the PSCs; and(c) determining if the presence of mutations in and optionally the expression levels of the differentiability markers of (a) and (b) are indicative of the suitability of the PSCs for differentiation,wherein a determination that presence of mutations in and optionally the expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation qualifies the population of PSCs for manufacturing differentiated cells.31. The method of embodiment 38, wherein the population of PSCs is a clonal population.32. The method of embodiment 30 or embodiment 31, wherein the PSCs have been cryopreserved prior to step (a).33. The method of embodiment 32, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).34. The method of embodiment 30 or embodiment 31, wherein the PSCs have not been cryopreserved prior to step (a).35. The method of any one of embodiments 30 to 34, which further comprises cryopreserving the PSCs after step (c) if the presence of the presence of mutations in the differentiability markers of (a) and optionally and the expression levels of the differentiability markers of (b) are indicative of the suitability of the PSCs for differentiation.36. The method of any one of embodiments 30 to 35, which further comprises, prior to step (a), generating the PSCs.37. The method of embodiment 36, wherein the PSCs are generated by reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells.38. A method for selecting a population of pluripotent stem cells (PSCs) for manufacturing differentiated cells, comprising:(a) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutantgene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR;(b) optionally, detecting the expression levels of one or more additional differentiability markers in one or more PSC populations;(c) determining if the presence of mutations in the differentiability markers of (a) and optionally the expression levels of the one or more differentiability markers of (b) are indicative of the suitability of the one or more PSC populations for differentiation; and(d) selecting a PSC population for manufacturing differentiated cells if the presence of mutations in the differentiability markers of (a) and optionally the expression levels of the one or more differentiability markers of (b) are indicative of the suitability of the one or more PSC populations for differentiation.39. The method of embodiment 38, which comprises differentiating PSCs determined to have differentiability marker mutation levels and optionally expression levels that are indicative of the suitability for differentiation.40. The method of embodiment 38, which comprises discarding PSCs determined to have differentiability marker mutation levels and optionally expression levels that are indicative of unsuitability for differentiation.41. The method of any one of embodiments 38 to 40, wherein the one or more PSC populations are subclones of a parental PSC population.42. The method of embodiment 38, wherein the one or more PSC populations have been cryopreserved prior to step (a).43. The method of embodiment 41, wherein the one or more PSC populations have been reactivated following cryopreservation and prior to step (a).44. The method of embodiment 38, wherein the one or more PSC populations have not been cryopreserved prior to step (a).45. The method of any one of embodiments 38 to 44, which further comprises cryopreserving one or more PSC populations after step (c) if the presence of mutations in the differentiability markers of (a) and optionally the expression levels of the one or more differentiability markers of (b) are indicative of the suitability of the PSCs for differentiation.46. The method of any one of embodiments 38 to 45, which further comprises, prior to step (a), generating the one or more PSC populations.47. The method of embodiment 46, wherein the one or more PSC populations are generated by (a) generating a parental PSC population reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells and (b) culturing subclones of the parental PSC population, thereby generating the one or more PSC populations.48. A method for monitoring a population of pluripotent stem cells (PSCs) for its suitability manufacturing differentiated cells, comprising:(a) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR;(b) detecting the expression levels of one or more additional differentiability markers in PSCs of the clonal population;(c) determining if the presence of mutations in the differentiability markers of (a) and optionally the expression levels of the one or more differentiability markers of (b) are indicative of the suitability of the PSCs for differentiation; and(d) if the presence of mutations in the differentiability markers of (a) and optionally the expression levels of the one or more differentiability markers of (b) are indicative of the suitability of the PSCs for differentiation, maintaining the clonal population; and (e) repeating steps (a)-(d) one or more times.49. The method of embodiment 48, wherein the PSCs are a clonal (e.g., isogenic) population of PSCs.50. The method of embodiment 48 or embodiment 49, wherein the PSCs have been cryopreserved prior to step (a).51. The method of embodiment 49, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).52. The method of embodiment 48 or embodiment 49, wherein the PSCs have not been cryopreserved prior to step (a).53. The method of any one of embodiments 48 to 52, which further comprises cryopreserving the PSCs after step (c) if the presence of mutations in the differentiability markers of (a) and optionally the expression levels of the one or more differentiability markers of (b) are indicative of the suitability of the PSCs for differentiation.54. The method of any one of embodiments 48 to 53, which further comprises, prior to step (a), generating the PSCs.55. The method of embodiment 54, wherein the PSCs are generated by reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells.56. The method of any one of embodiments 1 to 55, wherein the one or more differentiability markers comprise GBX2.57. The method of embodiment 56, wherein the one or more differentiability markers comprise SFRP1.58. The method of embodiment 56 or embodiment 57, which further comprises determining the relative expression levels of GBX2 and SFRP1, for example as described in Example 3.59. The method of any one of embodiments 1 to 58, wherein the one or more differentiability markers comprise HERV-K.60. The method of embodiment 59, wherein HERV-K is a HERV-K REC, HERV-K POL, HERV-K ENV, or a HERV-K GAG transcript.61. The method of embodiment 59 or embodiment 60, wherein the one or more differentiability markers comprise IFITM1.62. The method of any one of embodiments 59 to 61, which further comprises determining the expression levels of IFITM1 and expression levels of one, two, three, or all four HERV-K transcripts, for example as described in Example 6.63. The method of any one of embodiments 1 to 62, wherein the one of more differentiability markers comprise a mutant BCOR.64. The method of embodiment 63, wherein the mutant BOOR comprises one or more loss of function (LOF) mutations.65. The method of embodiment 63 or embodiment 64, wherein the mutant BOOR comprises a single nucleotide polymorphism (BOOR SNP).66. The method of embodiment 65, wherein the BOOR SNP is a c.572G> A mutation relative to the nucleotide sequence of SEQ ID NO:29 (e.g., resulting in a transcript of SEQ ID NO:30 (BOOR SNP1)),67. The method of embodiment 65, wherein the BOOR SNP is a c.4819+1 G> A mutation relative to the nucleotide sequence of SEQ ID NO:29 (e.g., resulting in a transcript comprising the nucleotide sequence of SEQ ID NO:31 (BCOR SNP2))68. The method of any one of embodiments 1 to 67, wherein the one or more differentiability markers comprise EP300.69. The method of embodiment 68, which further comprises determining the percent of mutant EP300 (for example as described in Example 7).70. The method of any one of embodiments 1 to 69, wherein the one or more differentiability markers comprise at least one negative differentiability marker and / or at least one positive differentiability marker.71. The method of embodiment 70, wherein one or more negative differentiability markers are markers of pluripotency.72. The method of embodiment 70 or embodiment 71, wherein one or more negative differentiability markers comprise one or more (or two or more) of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.73. The method of embodiment 72, wherein one or more negative differentiability markers comprise a combination of three or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.74. The method of embodiment 72, wherein one or more negative differentiability markers comprise a combination of four or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.75. The method of embodiment 72, wherein one or more negative differentiability markers comprise a combination of five or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.76. The method of embodiment 72, wherein one or more negative differentiability markers comprise a combination of six or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.77. The method of embodiment 72, wherein one or more negative differentiability markers comprise a combination of seven or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.78. The method of embodiment 72, wherein one or more negative differentiability markers comprise a combination of eight or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.79. The method of embodiment 72, wherein one or more negative differentiability markers comprise a combination of nine or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.80. The method of embodiment 72, wherein one or more negative differentiability markers comprise a combination of ten or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.81. The method of embodiment 72, wherein one or more negative differentiability markers comprise a combination of eleven or more of NR5A2, HHEX, GBX2, GDF3, NANOG,NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.82. The method of embodiment 72, wherein one or more negative differentiability markers comprise a combination of twelve or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.83. The method of embodiment 72, wherein one or more negative differentiability markers comprise a combination of thirteen or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.84. The method of any one of embodiments 70 to 83, wherein one or more positive differentiability markers comprise one or more of RAB17, PRTG, FAS, SFRP1, COL2A1, FAM129A, LCK, FCD7, and IDO1.85. The method of any one of embodiments 70 to 84, wherein one or more positive differentiability markers are markers of pluripotent stem cell (PSC) variability.86. The method of embodiment 85, wherein the one or more markers of pluripotent stem cell (PSC) variability comprise RAB17.87. The method of any one of embodiments 70 to 86, wherein one or more positive differentiability markers are markers of early differentiation.88. The method of embodiment 87, wherein the one or more markers of early differentiation comprise PRTG.89. The method of any one of embodiments 70 to 88, wherein one or more positive differentiability markers are markers of autophagy.90. The method of embodiment 87, wherein the one or more markers of autophagy comprise FAS.91. The method of any one of embodiments 16 to 90, which comprises detecting the expression levels of one or more differentiability markers by a method comprising:(a) measuring protein expression levels using an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), flow cytometry, a Western blot, an immunohistochemical assay (IHC), or a combination of two or more thereof; and(b) determining the measured protein expression levels.92. The method of any one of embodiments 16 to 91, which comprises detecting the expression levels of one or more differentiability markers by a method comprising:(a) measuring nucleic acid (e.g., mRNA) expression levels using a polymerase chain reaction and / or a microarray-based method; and (b) determining the measured nucleic acid expression levels.93. The method of any one of embodiments 1 to 92, wherein determining if the expression levels of the differentiability markers are indicative of the suitability of the PSCs for differentiation by:(a) assigning the PSCs a PSC score based on the expression of the differentiability markers; and(b) determining if the PSC score falls above or below a cutoff value.94. The method of embodiment 93, wherein the PSC score is calculated using the96. The method of any one of embodiments 93 to 95, wherein the cutoff value is 1.34.97. The method of any one of embodiments 1 to 93, wherein the PSCs are embryonic stem cells.98. The method of any one of embodiments 1 to 93, wherein the PSCs are induced pluripotent stem cells (iPSCs).99. The method of embodiment 98, which further comprises producing the PSCs.100. The method of embodiment 98 or embodiment 99, which further comprises forming a clonal (e.g., isogenic) population of PSCs.101. The method of embodiment 100, wherein the PSCs tested for differentiability markers are from the clonal population.102. The method of any one of embodiments 1 to 101, which further comprises differentiating a PSC identified as a suitable precursor of a differentiated cell into the differentiated cell.103. The method of embodiment 102, wherein the differentiated cell is of the ectoderm lineage.104. The method of embodiment 103, wherein the differentiated cell is a dopaminergic neuron.105. The method of any one of embodiments 102 to 104, herein the differentiated cell is engineered to express a transgene.106. The method of embodiment 105, wherein the transgene is introduced into the PSC prior to differentiation.107. The method of embodiment 106, which further comprises evaluating the PSCs following introduction of the transgene for their suitability as precursors of differentiated cells, comprising:(a) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR;(b) optionally detecting the expression levels one or more additional differentiability markers in the PSCs; and(c) determining if the presence of mutations in the differentiability markers of (a) and optionally the expression levels of the one or more differentiability markers of (b) are indicative of the suitability of the PSCs fordifferentiation.108. The method of embodiment 107, wherein the evaluating the PSCs following introduction of the transgene for their suitability as precursors of differentiated cells is performed as defined in any one of embodiments 70 to 93.109. The method of embodiment 105, wherein the transgene is introduced into the differentiated cell following differentiation.110. The method of embodiment 105, wherein the transgene is introduced into a precursor of the differentiated cell.111. The method of any one of embodiments 102 to 110, which further comprises administering the differentiated cell to a subject in need thereof.112. A method of treating a subject, comprising administering to a subject in need thereof a differentiated cell produced by the method of any one of embodiments 102 to 110.113. A method for determining the suitability of pluripotent stem cells (PSCs) for differentiation into differentiated cells following introduction of a transgene, comprising:(a) introducing a transgene into PSCs to produce recombinant PSCs;(b) evaluating the recombinant PSCs for their suitability as precursors of differentiated cells by a method comprising:(i) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR; (ii) optionally, detecting the expression levels of one or more additional differentiability markers in the recombinant PSCs; and (iii) determining if the presence of mutations in the differentiability markers of (i) and optionally the expression levels of the one or more differentiability markers of (ii) are indicative of the suitability of the recombinant PSCs for differentiation.114. The method of embodiment 113, wherein evaluating the recombinant PSCs for their suitability as precursors of differentiated cells is performed as defined in any one of embodiments 70 to 93.115. The method of embodiment 113 or embodiment 114, wherein the transgene encodes a therapeutic protein.116. The method of any one of embodiments 113 to 115, which further comprises differentiating a PSC identified as a suitable precursor of a differentiated cell into the differentiated cell.117. The method of embodiment 116, wherein the differentiated cell is a cell of the ectodermal lineage.118. The method of embodiment 118, wherein the differentiated cell is a dopaminergic neuron.119. A method of treating a subject, comprising administering to a subject in need thereof a differentiated cell population produced by the method of embodiment 116 or embodiment 117.120. A method of treating a subject suffering from a neurodegenerative disease, comprising administering to a subject in need thereof a dopaminergic neuron population produced by the method of embodiment 118.121. The method of claim 120, wherein the neurodegenerative diseases is Parkinson’s Disease, Alzheimer's Disease, dementia, epilepsy, Lewy Body syndrome, Huntington’s Disease, Spinal Muscular Atrophy, Friedreich’s Ataxia, Amyotrophic Lateral Sclerosis, Batten Disease, or Multiple System Atrophy.122. A kit comprising one or more probes and / or one or more PCR primer pairs for mutant EP300 and / or mutant BCOR.123. The kit of embodiment 122, which further comprises one or more probes and / or one or more PCR primer pairs for at least five, at least ten, at least fifteen or all of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, SFRP1, IFITM1, and HERV-K.124. The kit of embodiment 122 or embodiment 123, which comprises one or more probes and / or one or more PCR primer pair for one or more control genes.125. The kit of embodiment 124, wherein the one or more control genes comprise housekeeping genes.126. The kit of embodiment 125, optionally wherein the housekeeping genes comprise one, two, three or all four of p-actin, 18S rRNA, GAPDH and TBP.127. The kit of any one of embodiments 122 to 126 in which at least one primer for each differentiability marker is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end and, if present, at least one primer for each control gene is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end.128. The kit of any one of embodiments 122 to 127, which further comprises polymerase and / or a nucleotide mixture.129. The kit of any one of embodiments 122 to 128, further comprising an anti-IFITM 1 antibody, optionally wherein the antibody is fluorescently labeled.130. The kit of any one of embodiments 122 to 129, wherein the probes are in a 96-well plate.

[0189] Group D Embodiments:1. A method of measuring expression levels of differentiability markers in pluripotent stem cells (PSCs), comprising:(a) measuring the presence of mutant EP300 and / or mutant BOOR and optionally expression levels of at least three, at least four, or at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in a sample prepared from the PSCs; and(b) determining the presence (e.g., percentage in the population) of mutant EP300 and / or mutant BCOR and optionally the expression levels of the at least three, at least four, or at least five of the differentiability.2. The method of embodiment 1, wherein measuring the at least three, at least four, or at least five differentiability markers comprises performing a polymerase chain reaction (PCR) on nucleic acids extracted from the PSCs using primers capable of amplifying the at least five differentiability markers.3. The method of embodiment 2, wherein the PCR is a real-time PCR, and the method further comprises quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the at least three, at least four, or at least five differentiability markers.4. The method of embodiment 3, which comprises:(a) measuring expression of at least 10 of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV- K in a sample prepared from the PSCs;(b) determining the expression levels of the at least 10 differentiability markers by a method comprising:(i) performing real-time PCR on nucleic acids extracted from the PSCs using primers capable of amplifying the at least 10 differentiability markers; and(ii) quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the at least 10 differentiability markers.5. The method of embodiment 3, which comprises:(a) measuring expression of at least 15 of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV- K, in a sample prepared from the PSCs;(b) determining the expression levels of the at least 15 differentiability markers by a method comprising:(i) performing real-time PCR on nucleic acids extracted from the PSCs using primers capable of amplifying the at least 15 differentiability markers; and(ii) quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the at least 15 differentiability markers.6. The method of embodiment 3, which comprises:(a) measuring expression of all 17 of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS in a sample prepared from the PSCs;(b) determining the expression levels of the all 17 differentiability markers by a method comprising:(i) performing real-time PCR on nucleic acids extracted from the PSCs using primers capable of amplifying the all 17 differentiability markers; and(ii) quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the all 17 differentiability markers.7. An assay method comprising:(a) assaying for mutant EP300 and / or mutant BCOR in a PSC population (e.g., as a percentage of EP300 and / or BCOR in the PSC population); and(b) optionally performing a polymerase chain reaction (PCR) on one or more reaction mixtures comprising:(i) RNA extracted from the PSC population or cDNA reverse transcribed therefrom;(ii) primers suitable for amplifying at least three, at least four, or at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K;(iii) labeled probes for detecting the at least five differentiability markers; and(iv) a DNA polymerase;(c) detecting the presence mutant EP300 and / or mutant BCOR (e.g., as a percentage of EP300 and / or BCOR in the PSC population) and optionally of expression levels of the at least three, at least four, or at least five differentiability markers based on signals emitted by the labeled probes.8. The method of embodiment 7, wherein the PCR is performed in a well or on a tape.9. A method of evaluating pluripotent stem cells (PSCs) for their differentiability, comprising:(a) detecting presence of mutant EP300 and / or mutant BCOR and optionally expression levels of at least three, at least four, or at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in the PSCs; and(b) determining if the presence of mutant EP300 and / or mutant BCOR and optionally expression levels of at least three, at least four, or at least five of the differentiability markers are indicative of the suitability of the PSCs for differentiation.10. The method of embodiment 9, wherein the PSCs have been cryopreserved prior to step (a).11. The method of embodiment 10, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).12. A method of preparing a pluripotent stem cell (PSC) stem cell bank suitable for differentiation, comprising:(a) detecting the presence of mutant EP300 and / or mutant BCOR and optionally expression levels of at least three, at least four, or at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in a population of PSCs;(b) determining if the presence of mutant EP300 and / or mutant BCOR (e.g., as a percentage of EP300 and / or BCOR in the PSC population) and optionally expression levels of at least three, at least four, or at least five of the differentiability markers are indicative of the suitability of the PSCs for differentiation;(c) if the presence of mutant EP300 and / or mutant BCOR and optionally expression levels of the at least three, at least four, or at least five differentiability markers are indicative of the suitability of the PSCs for differentiation, forming a PSC stem cell bank from the population of PSCs.13. The method of embodiment 12, wherein the PSCs have been cryopreserved prior to step (a).14. The method of embodiment 13, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).15. The method of any one of embodiments 12 to 14, which further comprises cryopreserving the PSCs after step (b) if the presence of mutant EP300 and / or mutant BCORand optionally the expression levels of at least three, at least four, or at least five of the differentiability markers are indicative of the suitability of the PSCs for differentiation.16. A method for qualifying a population of pluripotent stem cells (PSCs) for manufacturing differentiated cells, comprising:(a) detecting the presence of mutant EP300 and / or mutant BCOR and optionally expression levels of at least three, at least four, or the expression levels of at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K; and (b) determining if the presence of mutant EP300 and / or mutant BCOR and optionally the expression levels of the at least three, at least four, or at least five differentiability markers are indicative of the suitability of the PSCs for differentiation,wherein a determination that presence of mutant EP300 and / or mutant BCOR and optionally expression levels of the at least three, at least four, or at least five differentiability markers are indicative of the suitability of the PSCs for differentiation qualifies the population of PSCs for manufacturing differentiated cells.17. The method of embodiment 16, wherein the population of PSCs is a clonal population.18. The method of embodiment 16 or embodiment 17, wherein the PSCs have been cryopreserved prior to step (a).19. The method of embodiment 18, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).20. A method for selecting a population of pluripotent stem cells (PSCs) for manufacturing differentiated cells, comprising:(a) detecting the presence of mutant EP300 and / or mutant BCOR and optionally the expression levels of at least three, at least four, or at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in one or more PSC populations;(b) determining if the presence of mutant EP300 and / or mutant BCOR and optionally expression levels of the at least three, at least four, or at least five differentiability markers are indicative of the suitability of the one or more PSC populations for differentiation; and(c) selecting a PSC population for manufacturing differentiated cells if the presence of mutant EP300 and / or mutant BCOR and optionally expression levels of the at least three, at least four, or at least five differentiability markers are indicative of the suitability of the one or more PSC populations for differentiation.21. A method for monitoring a population of pluripotent stem cells (PSCs) for its suitability manufacturing differentiated cells, comprising:(a) detecting the presence of mutant EP300 and / or mutant BCOR and optionally expression levels of at least three, at least four, or at least five of the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in PSCs of the clonal population; (b) determining if the presence of mutant EP300 and / or mutant BCOR and optionally expression levels of the at least three, at least four, or least five differentiability markers are indicative of the suitability of the PSCs for differentiation; and(c) if the presence of mutant EP300 and / or mutant BCOR and optionally expression levels of the at least three, at least four, or at least five differentiability markers in step (b) are indicative of the suitability of the PSCs for differentiation, maintaining the clonal population; and(d) repeating steps (a)-(c) one or more times.22. The method of embodiment 21, wherein the PSCs have been cryopreserved prior to step (a).23. The method of embodiment 22, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).24. The method of any one of embodiments 1 to 23, wherein the differentiability markers comprise at least one negative differentiability marker and at least one positive differentiability marker.25. The method of embodiment 24, wherein one or more negative differentiability markers are markers of pluripotency.26. The method of embodiment 24 or embodiment 25, wherein one or more negative differentiability markers comprise one or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, IFITM1, and HERV-K.27. The method of any one of embodiments 24 to 26, wherein one or more positive differentiability markers comprise one or more of RAB17, PRTG, FAS, FAM129A, LCK, FZD7, IDO1, COL2A1, and SFRP1.28. The method of any one of embodiments 9 to 27, which comprises detecting the expression levels of one or more differentiability markers by a method comprising:(a) measuring protein expression levels using an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), flow cytometry, a Western blot, an immunohistochemical assay (IHC), or a combination of two or more thereof; and(b) determining the measured protein expression levels.29. The method of any one of embodiments 9 to 28, which comprises detecting the expression levels of one or more differentiability markers by a method comprising:(a) measuring nucleic acid (e.g., mRNA) expression levels (e.g., using Cq value) using a polymerase chain reaction and / or a microarray-based method; and(b) determining the measured nucleic acid expression levels.30. The method of any one of embodiments 1 to 29, which comprises determining if the presence of mutant EP300 and / or mutant BCOR and optionally expression levels of at least three, at least four, or at least five of the differentiability markers are indicative of the suitability of the PSCs for differentiation by:(a) determining percentage of mutant EP300 and / or mutant BCOR in the PSCs;(b) assigning the PSCs a PSC score based on the expression of the differentiability markers; and(c) determining if the percentage of mutant EP300 and / or mutant BCOR and / or the PSC score falls above or below a cutoff value.31. The method of embodiment 30, wherein the PSC score is calculated using the Mean Cq values of genes upregulated in samples that pass DA differentiation formula: PSC score - Mean Cq values of genes upregulated in samples that fail DA differentiation ‘32. The method of embodiment 31, wherein the PSC score formula for the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS is: PSC score =Mean Cq values (PRTG, FAS, RAB17)Mean Cq values (NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8)33. The method of any one of embodiments 30 to 32, wherein the cutoff value is 1.34.34. The method of any one of embodiments 1 to 33, wherein the PSCs are induced pluripotent stem cells (iPSCs).35. The method of any one of embodiments 1 to 34, which further comprises differentiating a PSC identified as a suitable precursor of a differentiated cell into the differentiated cell.36. A kit comprising one or more probes and / or one or more PCR primer pairs for mutant EP300 and / or mutant BCOR and optionally at least five, at least ten, at least fifteen or all of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS.37. The kit of embodiment 36, which further comprises one or more probes and / or one or more PCR primer pairs for one, two, three four, five or more (e.g., all) of FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, SFRP1, IFITM1, and HERV-K.38. The kit of embodiment 36 or embodiment 37, which comprises one or more probes and / or one or more PCR primer pairs for GBX2 and SFRP1.39. The kit of any one of embodiments 36 to 38, which comprises one or more probes and / or one or more PCR primer pairs for IFITM1.40. The kit of any one of embodiments 36 to 39, which comprises one or more probes and / or one or more PCR primer pairs for HERV-K.41. The kit of any one of embodiments 36 to 40, which comprises one or more probes and / or one or more PCR primer pairs for mutant EP300.42. The kit of any one of embodiments 36 to 41, which comprises one or more probes and / or one or more PCR primer pairs for mutant BCOR.43. The kit of any one of embodiments 36 to 42, which comprises one or more probes and / or one or more PCR primer pair for one or more control genes, optionally wherein the one more control genes comprise one, two, three, or all four of β-actin, 18S rRNA, GAPDH and TBP.44. The kit of any one of embodiments 36 to 43, in which at least one primer for each differentiability marker is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end and, if present, at least one primer for each control gene is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end.

[0190] Group E Embodiments:1. A method of measuring expression levels of differentiability markers in pluripotent stem cells (PSCs), comprising:(a) (i) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs) in the PSCs, optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR; and / or (ii) measuring the expression of one or more additional differentiability markers in a sample prepared from the PSCs; and (b) determining the presence of mutations in the differentiability markers of (a)(i) and / or expression levels of the differentiability markers of (a)(ii).2. The method of embodiment 1, which comprises (a)(i).3. The method of embodiment 1, which comprises (a)(ii).4. The method of embodiment 1, which comprises (a)(i) and (a)(ii).5. The method of any one of embodiments 1 to 4, which comprises:(a) measuring the presence of one or mutations in one or more differentiability marker genes (e.g., as a percentage of the alleles or the genes in a population of PSCs), optionally wherein mutant EP300 and / or mutant BCOR is measured; and(b) optionally, performing polymerase chain reaction (PCR) on nucleic acids extracted from PSCs using primers capable of amplifying one or more additional differentiability markers.6. The method of embodiment 5, wherein the PCR is a real-time PCR, and the method further comprises quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the one or more additional differentiability markers.7. The method of any one of embodiments 1 to 6, which further comprises measuring the expression levels of one or more control (e.g., housekeeping) genes in the sample, optionally wherein the one more control genes comprise one, two, three, or all four of P-actin, 18S rRNA, GAPDH and TBP.8. The method of embodiment 7, wherein the one or more control genes comprise P-actin.9. The method of embodiment 7 or embodiment 8, wherein the one or more control genes comprise 18S rRNA.10. The method of any one of embodiments 7 to 9, wherein the one or more control genes comprise GAPDH.11. The method of any one of embodiments 7 to 10, wherein the one or more control genes comprise TBP.12. The method of any one of embodiments 7 to 11, which further comprises normalizing the expression levels of the one or more additional differentiability markers against the one or more control genes.13. An assay method comprising:(a) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR; and / or(b) (A) performing a polymerase chain reaction (PCR) on one or more reaction mixtures comprising:(i) RNA extracted from PSCs or cDNA reverse transcribed therefrom; (ii) primers suitable for amplifying one or more additional differentiability markers;(iii) labeled probes for detecting the one or more additional differentiability markers; and(iv) a DNA polymerase; and(B) detecting expression levels of the one or more additional differentiability markers based on signals emitted by the labeled probes.14. The assay method of embodiment 13, which comprises (a).15. The assay method of embodiment 13, which comprises (b).16. The assay method of embodiment 13, which comprises (a) and (b).17. The method of any one of embodiments 13 to 16, wherein the PCR is performed in parallel on a plurality of reaction mixtures, each comprising a primer pair for amplifying a differentiability marker and a labeled probe suitable for detecting the differentiability marker.18. The method of any one of embodiments 13 to 17, wherein the PCR is performed on a solid or flat surface.19. The method of any one of embodiments 13 to 18, wherein the PCR is performed in a well or on a tape.20. The method of embodiment 19, wherein the PCR is performed in a 96-well plate.21. The method of embodiment 19, wherein the PCR is performed in a 384-well plate.22. The method of embodiment 19, wherein the PCR is performed on a tape.23. The method of any one of embodiments 13 to 22, wherein the reaction mixture further comprises primers suitable for amplifying one or more control genes and one or more labeled probes for detecting the one or more control (e.g., housekeeping) genes, optionallywherein the one more control genes comprise one, two, three, or all four of p-actin, 18S rRNA, GAPDH and TBP genes.24. The method of embodiment 23, wherein the one or more control genes comprise P-actin.25. The method of embodiment 23 or embodiment 24, wherein the one or more control genes comprise 18S rRNA.26. The method of any one of embodiments 23 to 25, wherein the one or more control genes comprise GAPDH.27. The method of any one of embodiments 23 to 26, wherein the one or more control genes comprise TBP.28. A method of determining PSC differentiability, comprising:(a) performing a Taqman polymerase chain reaction (PCR) on one or more reaction mixtures comprising:(i) RNA extracted from PSCs or cDNA reverse transcribed therefrom; (ii) primers suitable for amplifying one or more differentiability markers, e.g., one or more positive differentiability markers and one or more negative differentiability markers;(iii) optionally, primers suitable for amplifying one or more control genes; and(iv) a DNA polymerase;(b) determining the cycle count (Cq) to attain the cycle threshold for the one or more differentiability markers and optionally one or more control genes;(c) calculating mean Cq values for the one or more negative differentiability markers (“mean negative Cq”) and one or more positive differentiability markers (“mean positive Cq”);(d) determining the mean positive Cq: mean negative Cq ratio, wherein the ratio and / or presence of mutations are indicative of PSC differentiability; and(e) optionally, testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR.29. The method of embodiment 28, which comprises (e).30. The method of embodiment 28 or embodiment 29, wherein the Cq values for the one or more differentiability markers are normalized against the expression levels of one or more control (e.g., housekeeping) genes, optionally wherein the one more control genes comprise one, two, three, or all four of p-actin, 18S rRNA, GAPDH and TBP.31. The method of embodiment 30, wherein the one or more control genes comprise P-actin.32. The method of embodiment 30 or embodiment 31, wherein the one or more control genes comprise 18S rRNA.33. The method of any one of embodiments 30 to 32, wherein the one or more control genes comprise GAPDH.34. The method of any one of embodiments 30 to 33, wherein the one or more control genes comprise TBP.35. A method of evaluating pluripotent stem cells (PSCs) for their differentiability, comprising:(a) (i) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in the PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR; and / or(ii) detecting the expression levels of one or more additional differentiability markers in the PSCs; and(b) determining if the presence of mutations and optionally the expression levels of the one or more additional differentiability markers are indicative of the suitability of the PSCs for differentiation.36. The method of embodiment 35, which comprises (a)(i).37. The method of embodiment 35, which comprises (a) (ii).38. The method of embodiment 35, which comprises (a)(i) and (a)(ii).39. The method of any one of embodiments 35 to 38, wherein the PSCs have been cryopreserved prior to step (a).40. The method of embodiment 39, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).41. The method of any one of embodiments 35 to 38, wherein the PSCs have not been cryopreserved prior to step (a).42. The method of any one of embodiments 35 to 41, which further comprises cryopreserving the PSCs after step (b) if the presence of mutations in and / or expression levels of the one or more differentiability markers are indicative of the suitability of the PSCs for differentiation.43. The method of any one of embodiments 35 to 42, which further comprises, prior to step (a), generating the PSCs.44. The method of embodiment 43, wherein the PSCs are generated by reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells.45. A method of preparing a pluripotent stem cell (PSC) stem cell bank suitable for differentiation, comprising:(a) (i) detecting the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR; and / or(ii) detecting the expression levels of one or more additional differentiability markers in a population of PSCs;(b) determining if the presence of mutations in the differentiability markers of (a)(i) and / or the expression levels of the differentiability markers of (a)(ii) are indicative of the suitability of the PSCs for differentiation;(c) if the presence of mutations in and / or the expression levels of the differentiability markers are indicative of the suitability of the PSCs for differentiation, forming a PSC stem cell bank from the population of PSCs.46. The method of embodiment 45, which comprises (a)(i).47. The method of embodiment 45, which comprises (a) (ii).48. The method of embodiment 45, which comprises (a)(i) and (a)(ii).49. The method of any one of embodiments 45 to 48, wherein the PSCs have been cryopreserved prior to step (a).50. The method of embodiment 49, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).51. The method of any one of embodiments 45 to 48, wherein the PSCs have not been cryopreserved prior to step (a).52. The method of any one of embodiments 45 to 51, which further comprises cryopreserving the PSCs after step (b) if the presence of mutations in and / or the expression levels of the differentiability markers of (a)(i) and (a)(ii) are indicative of the suitability of the PSCs for differentiation.53. The method of any one of embodiments 45 to 52, which further comprises, prior to step (a), generating the PSCs.54. The method of embodiment 53, wherein the PSCs are generated by reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells.55. A method for qualifying a population of pluripotent stem cells (PSCs) for manufacturing differentiated cells, comprising:(a) (i) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR; and / or(ii) detecting the expression levels of one or more additional differentiability markers in the PSCs; and(b) determining if the presence of mutations in the differentiability markers of (a)(i) and / or the expression levels of the one or more differentiability markers of (a)(ii) are indicative of the suitability of the PSCs for differentiation,wherein a determination that presence of mutations in the differentiability markers and / or the expression levels of the one or more additional differentiability markers are indicative of the suitability of the PSCs for differentiation qualifies the population of PSCs for manufacturing differentiated cells.56. The method of embodiment 55, which comprises (a)(i).57. The method of embodiment 55, which comprises (a) (ii).58. The method of embodiment 55, which comprises (a)(i) and (a)(ii).59. The method of any one of embodiments 55 to 58, wherein the population of PSCs is a clonal population.60. The method of any one of embodiments 55 to 59, wherein the PSCs have been cryopreserved prior to step (a).61. The method of embodiment 60, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).62. The method of any one of embodiments 55 to 59, wherein the PSCs have not been cryopreserved prior to step (a).63. The method of any one of embodiments 55 to 62, which further comprises cryopreserving the PSCs after step (b) if the presence of the presence of mutations in the differentiability markers of (a)(i) and / or and the expression levels of the differentiability markers of (a)(ii) are indicative of the suitability of the PSCs for differentiation.64. The method of any one of embodiments 55 to 63, which further comprises, prior to step (a), generating the PSCs.65. The method of embodiment 64, wherein the PSCs are generated by reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells.66. A method for selecting a population of pluripotent stem cells (PSCs) for manufacturing differentiated cells, comprising:(a) (i) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR; and / or(ii) detecting the expression levels of one or more additional differentiability markers in one or more PSC populations;(b) determining if the presence of mutations in the differentiability markers of (a)(i) and / or the expression levels of the one or more differentiability markers of (a)(ii) are indicative of the suitability of the one or more PSC populations for differentiation; and(c) selecting a PSC population for manufacturing differentiated cells if the presence of mutations in the differentiability markers of (a)(i) and / or the expression levels of the one or more differentiability markers of (a)(ii) are indicative of the suitability of the one or more PSC populations for differentiation.67. The method of embodiment 66, which comprises (a)(i).68. The method of embodiment 66, which comprises (a) (ii).69. The method of embodiment 66, which comprises (a)(i) and (a)(ii).70. The method of any one of embodiments 66 to 68, which comprises differentiating PSCs determined to have differentiability marker mutation levels and / or expression levels that are indicative of the suitability for differentiation.71. The method of any one of embodiments 66 to 68, which comprises discarding PSCs determined to have differentiability marker mutation levels and / or expression levels that are indicative of unsuitability for differentiation.72. The method of any one of embodiments 66 to 71, wherein the one or more PSC populations are subclones of a parental PSC population.73. The method of any one of embodiments 66 to 69, wherein the one or more PSC populations have been cryopreserved prior to step (a).74. The method of embodiment 72, wherein the one or more PSC populations have been reactivated following cryopreservation and prior to step (a).75. The method of any one of embodiment 66 to 69, wherein the one or more PSC populations have not been cryopreserved prior to step (a).76. The method of any one of embodiments 66 to 75, which further comprises cryopreserving one or more PSC populations after step (b) if the presence of mutations in the differentiability markers of (a)(i) and optionally the expression levels of the one or more differentiability markers of (a)(ii) are indicative of the suitability of the PSCs for differentiation.77. The method of any one of embodiments 66 to 76, which further comprises, prior to step (a), generating the one or more PSC populations.78. The method of embodiment 77, wherein the one or more PSC populations are generated by (a) generating a parental PSC population reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells and (b) culturing subclones of the parental PSC population, thereby generating the one or more PSC populations.79. A method for monitoring a population of pluripotent stem cells (PSCs) for its suitability manufacturing differentiated cells, comprising:(a) (i) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR; and / or(ii) detecting the expression levels of one or more additional differentiability markers in PSCs of the clonal population;(b) determining if the presence of mutations in the differentiability markers of (a)(i) and / or the expression levels of the one or more differentiability markers of (a)(ii) are indicative of the suitability of the PSCs for differentiation; and(c) if the presence of mutations in the differentiability markers of (a)(i) and / or the expression levels of the one or more differentiability markers of (a)(ii) are indicative of the suitability of the PSCs for differentiation, maintaining the clonal population; and(d) repeating steps (a)-(c) one or more times.80. The method of embodiment 79, which comprises (a)(i).81. The method of embodiment 79, which comprises (a) (ii).82. The method of embodiment 79, which comprises (a)(i) and a(ii).83. The method of any one of embodiments 79 to 82, wherein the PSCs are a clonal (e.g., isogenic) population of PSCs.84. The method of any one of embodiments 79 to 83, wherein the PSCs have been cryopreserved prior to step (a).85. The method of embodiment 83, wherein the PSCs have been reactivated following cryopreservation and prior to step (a).86. The method of any one of embodiments 79 to 85, wherein the PSCs have not been cryopreserved prior to step (a).87. The method of any one of embodiments 79 to 86, which further comprises cryopreserving the PSCs after step (b) if the presence of mutations in the differentiability markers of (a)(i) and / or the expression levels of the one or more differentiability markers of (a) (ii) are indicative of the suitability of the PSCs for differentiation.88. The method of any one of embodiments 79 to 87, which further comprises, prior to step (a), generating the PSCs.89. The method of embodiment 88, wherein the PSCs are generated by reprogramming hematopoietic cells, optionally wherein the hematopoietic cells are erythroid progenitor cells.90. A method for generating differentiated cells of an ectodermal lineage comprising exposing pluripotent stem cells (PSCs) of a PSC population to ectodermal cell differentiation conditions, wherein the PSC population has been identified as suitable for differentiation by a process comprising:(a) (i) testing PSCs from the population or a parental PSC population for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR; and / or(ii) detecting the expression levels of one or more additional differentiability markers in PSCs from the population or a parental PSC population;(b) determining if the presence of mutations in the differentiability markers of (a)(i) and / or the expression levels of the one or more differentiability markers of (a)(ii) are indicative of the suitability of the PSC population for differentiation.91. The method of embodiment 90, wherein the process comprises (a)(i).92. The method of embodiment 90, wherein the process comprises (a)(ii).93. The method of embodiment 90, wherein the process comprises (a)(i) and (a)(ii).94. The method of any one of embodiments 90 to 93, further comprising identifying the PSC population as suitable for differentiation.95. The method of any one of embodiments 90 to 94, wherein ectodermal differentiation conditions comprise contacting the PSCs with an inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, an activator of Sonic hedgehog (SHH) signaling, an activator of wingless (Wnt) signaling; and subsequently, an activator of fibroblast growth factor (FGF) signaling and an inhibitor of Wnt signaling.96. The method of any one of embodiments 90 to 95, wherein the differentiated cells comprise dopaminergic neurons.97. The method of any one of embodiments 90 to 96, wherein the PSC population has been cryopreserved after being identified as suitable for differentiation.98. The method of embodiment 97, wherein the PSCs have been reactivated following cryopreservation and before differentiation.99. The method of any one of embodiments 90 to 96, wherein the PSC population has not been cryopreserved before differentiation.100. A method of determining the differentiation potential of a pluripotent stem cell (PSC) population comprising:(a) detecting the expression levels of one or more differentiability markers in the population;(b) assigning the population a PSC score based on the expression of the differentiability markers; and(c) determining if the PSC score falls above or below a cutoff value.101. The method of any one of embodiments 1 to 100, wherein the one or more differentiability markers comprise GBX2.102. The method of embodiment 101, wherein the one or more differentiability markers comprise SFRP1.103. The method of embodiment 101 or embodiment 102, which further comprises determining the relative expression levels of GBX2 and SFRP1, for example as described in Example 3.104. The method of any one of embodiments 1 to 103, wherein the one or more differentiability markers comprise HERV-K.105. The method of embodiment 104, wherein HERV-K is a HERV-K REC, HERV-K POL, HERV-K ENV, or a HERV-K GAG transcript.106. The method of embodiment 105, wherein HERV-K is a HERV-K REC107. The method of embodiment 105, wherein HERV-K is a HERV-K POL transcript.108. The method of embodiment 105, wherein HERV-K is a HERV-K ENV transcript.109. The method of embodiment 105, wherein HERV-K is a HERV-K GAG transcript.110. The method of any one of embodiments 104 to 109, wherein the one or more differentiability markers comprise IFITM1.111. The method of any one of embodiments 104 to 110, which further comprises determining the expression levels of IFITM1 and expression levels of one, two, three, or all four HERV-K transcripts, for example as described in Example 6.112. The method of any one of embodiments 1 to 111, wherein the one of more differentiability markers comprise a mutant BCOR.113. The method of embodiment 112, wherein the mutant BCOR comprises one or more loss of function (LOF) mutations.114. The method of embodiment 112 or embodiment 113, wherein the mutant BCOR comprises a single nucleotide polymorphism (BCOR SNP).115. The method of embodiment 114, wherein the BCOR SNP is a c.572G> A mutation relative to the nucleotide sequence of SEQ ID NO:29 (e.g., resulting in a transcript of SEQ ID NO:30 (BCOR SNP1)),116. The method of embodiment 114, wherein the BCOR SNP is a c.4819+1G> A mutation relative to the nucleotide sequence of SEQ ID NO:29 (e.g., resulting in a transcript comprising the nucleotide sequence of SEQ ID NO:31 (BCOR SNP2))117. The method of any one of embodiments 1 to 116, wherein the one or more differentiability markers comprise EP300.118. The method of embodiment 117, which further comprises determining the percent of mutant EP300 (for example as described in Example 7).119. The method of any one of embodiments 1 to 118, wherein the one or more differentiability markers comprise at least one negative differentiability marker and / or at least one positive differentiability marker.120. The method of embodiment 119, wherein one or more negative differentiability markers are markers of pluripotency.121. The method of embodiment 119 or embodiment 120, wherein one or more negative differentiability markers comprise one or more (or two or more) of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.122. The method of embodiment 121, wherein one or more negative differentiability markers comprise a combination of three or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.123. The method of embodiment 121, wherein one or more negative differentiability markers comprise a combination of four or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.124. The method of embodiment 121, wherein one or more negative differentiability markers comprise a combination of five or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.125. The method of embodiment 121, wherein one or more negative differentiability markers comprise a combination of six or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.126. The method of embodiment 121, wherein one or more negative differentiability markers comprise a combination of seven or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.127. The method of embodiment 121, wherein one or more negative differentiability markers comprise a combination of eight or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.128. The method of embodiment 121, wherein one or more negative differentiability markers comprise a combination of nine or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.129. The method of embodiment 121, wherein one or more negative differentiability markers comprise a combination of ten or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.130. The method of embodiment 121, wherein one or more negative differentiability markers comprise a combination of eleven or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.131. The method of embodiment 121, wherein one or more negative differentiability markers comprise a combination of twelve or more of NR5A2, HHEX, GBX2, GDF3, NANOG,NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.132. The method of embodiment 121, wherein one or more negative differentiability markers comprise a combination of thirteen or more of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, HERV-K, IFITM1, TRIML2, EOMES, and FGF4.133. The method of any one of embodiments 119 to 132, wherein the negative differentiability marker(s) comprise NR5A2.134. The method of any one of embodiments 119 to 133, wherein the negative differentiability marker(s) comprise HHEX.135. The method of any one of embodiments 119 to 134, wherein the negative differentiability marker(s) comprise GBX2.136. The method of any one of embodiments 119 to 135, wherein the negative differentiability marker(s) comprise GDF3.137. The method of any one of embodiments 119 to 136, wherein the negative differentiability marker(s) comprise NANOG.138. The method of any one of embodiments 119 to 137, wherein the negative differentiability marker(s) comprise NODAL.139. The method of any one of embodiments 119 to 138, wherein the negative differentiability marker(s) comprise HMX1.140. The method of any one of embodiments 119 to 139, wherein the negative differentiability marker(s) comprise UTF1.141. The method of any one of embodiments 119 to 140, wherein the negative differentiability marker(s) comprise SIX6.142. The method of any one of embodiments 119 to 141, wherein the negative differentiability marker(s) comprise MT2A.143. The method of any one of embodiments 119 to 142, wherein the negative differentiability marker(s) comprise TBXT.144. The method of any one of embodiments 119 to 143, wherein the negative differentiability marker(s) comprise NFIA.145. The method of any one of embodiments 119 to 144, wherein the negative differentiability marker(s) comprise ID2.146. The method of any one of embodiments 119 to 145, wherein the negative differentiability marker(s) comprise SP8.147. The method of any one of embodiments 119 to 146, wherein the negative differentiability marker(s) comprise HERV-K.148. The method of any one of embodiments 119 to 147, wherein the negative differentiability marker(s) comprise IFITM1.149. The method of any one of embodiments 119 to 148, wherein the negative differentiability marker(s) comprise TRIML2.150. The method of any one of embodiments 119 to 149, wherein the negative differentiability marker(s) comprise EOMES.151. The method of any one of embodiments 119 to 150, wherein the negative differentiability marker(s) comprise FGF4.152. The method of any one of embodiments 119 to 151, wherein one or more positive differentiability markers comprise one or more of RAB17, PRTG, FAS, SFRP1, COL2A1, FAM129A, LCK, FCD7, and IDO1.153. The method of any one of embodiments 119 to 151, wherein one or more positive differentiability markers comprise one or more of RAB17, PRTG, and FAS.154. The method of any one of embodiments 119 to 153, wherein one or more positive differentiability markers are markers of pluripotent stem cell (PSC) variability.155. The method of embodiment 154, wherein the one or more markers of pluripotent stem cell (PSC) variability comprise RAB17.156. The method of any one of embodiments 119 to 155, wherein one or more positive differentiability markers are markers of early differentiation.157. The method of embodiment 156, wherein the one or more markers of early differentiation comprise PRTG.158. The method of any one of embodiments 119 to 157, wherein one or more positive differentiability markers are markers of autophagy.159. The method of embodiment 156, wherein the one or more markers of autophagy comprise FAS.160. The method of any one of embodiments 119 to 159, wherein the positive differentiability marker(s) comprise SFRP1.161. The method of any one of embodiments 119 to 160, wherein the positive differentiability marker(s) comprise COL2A1.162. The method of any one of embodiments 119 to 161, wherein the positive differentiability marker(s) comprise FAM129A.163. The method of any one of embodiments 119 to 162, wherein the positive differentiability marker(s) comprise LCK.164. The method of any one of embodiments 119 to 163, wherein the positive differentiability marker(s) comprise FCD7.165. The method of any one of embodiments 119 to 164, wherein the positive differentiability marker(s) comprise IDO1.166. The method of any one of embodiments 35 to 165, which comprises detecting the expression levels of one or more differentiability markers by a method comprising:(a) measuring protein expression levels using an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), flowcytometry, a Western blot, an immunohistochemical assay (IHC), or a combination of two or more thereof; and(b) determining the measured protein expression levels.167. The method of any one of embodiments 35 to 166, which comprises detecting the expression levels of one or more differentiability markers by a method comprising:(a) measuring nucleic acid (e.g., mRNA) expression levels using a polymerase chain reaction and / or a microarray-based method; and (b) determining the measured nucleic acid expression levels.168. The method of any one of embodiments 1 to 167, which comprises determining if the expression levels of the differentiability markers are indicative of the suitability of the PSCs for differentiation by:(a) assigning the PSCs a PSC score based on the expression of the differentiability markers; and(b) determining if the PSC score falls above or below a cutoff value.169. The method of embodiment 168, wherein the PSC score is calculated using the formula' PSC score = Mean Cq values of genes upregulated in samples that pass DA differentiation / Mean Cq values of genes upregulated in samples that fail DA differentiation170. The method of embodiment 169, wherein the PSC score formula for the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS is: PSC score =Mean Cq values (PRTG, FAS, RAB17~)Mean Cq values NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8)171. The method of any one of embodiments 168 to 170, wherein the cutoff value is 1.34.172. The method of any one of embodiments 1 to 168, wherein the PSCs are embryonic stem cells.173. The method of any one of embodiments 1 to 168, wherein the PSCs are induced pluripotent stem cells (iPSCs).174. The method of embodiment 173, which further comprises producing the PSCs.175. The method of embodiment 173 or embodiment 174, which further comprises forming a clonal (e.g., isogenic) population of PSCs.176. The method of embodiment 175, wherein the PSCs tested for differentiability markers are from the clonal population.177. The method of any one of embodiments 1 to 176, which comprises measuring the expression of up to 30 total genes.178. The method of embodiment 177, which comprises measuring the expression of up to 20 total genes.179. The method of embodiment 177, which comprises measuring the expression of up to 10 total genes.180. The method of any one of embodiments 1 to 176, which comprises measuring the expression of 60 or fewer total genes.181. The method of embodiment 180, which comprises measuring the expression of 50 or fewer total genes.182. The method of embodiment 180, which comprises measuring the expression of 40 or fewer total genes.183. The method of embodiment 180, which comprises measuring the expression of 35 or fewer total genes.184. The method of embodiment 180, which comprises measuring the expression of 30 or fewer total genes.185. The method of embodiment 180, which comprises measuring the expression of 25 or fewer total genes.186. The method of embodiment 180, which comprises measuring the expression of 20 or fewer total genes.187. The method of embodiment 180, which comprises measuring the expression of 50 or fewer differentiability marker genes.188. The method of 180 or embodiment 181, which comprises measuring the expression of 45 or fewer differentiability marker genes.189. The method of 180 or embodiment 181, which comprises measuring the expression of 40 or fewer differentiability marker genes.190. The method of any one of embodiments 180 to 182, which comprises measuring the expression of 35 or fewer differentiability marker genes.191. The method of any one of embodiments 180 to 183, which comprises measuring the expression of 30 or fewer differentiability marker genes.192. The method of any one of embodiments 180 to 184, which comprises measuring the expression of 25 or fewer differentiability marker genes.193. The method of any one of embodiments 180 to 185, which comprises measuring the expression of 20 or fewer differentiability marker genes.194. The method of any one of embodiments 180 to 193, which comprises measuring the expression of up to 10 control genes.195. The method of any one of embodiments 180 to 193, which comprises measuring the expression of up to 5 control genes.196. The method of any one of embodiments 1 to 195, which further comprises differentiating a PSC identified as a suitable precursor of a differentiated cell into the differentiated cell.197. The method of embodiment 196, wherein the differentiated cell is of the ectoderm lineage.198. The method of embodiment 197, wherein the differentiated cell is a dopaminergic neuron.199. The method of any one of embodiments 196 to 198, herein the differentiated cell is engineered to express a transgene.200. The method of embodiment 199, wherein the transgene is introduced into the PSC prior to differentiation.201. The method of embodiment 200, which further comprises evaluating the PSCs following introduction of the transgene for their suitability as precursors of differentiated cells, comprising:(a) (i) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR; and / or(ii) detecting the expression levels one or more additional differentiability markers in the PSCs; and(b) determining if the presence of mutations in the differentiability markers of (a)(i) and / or the expression levels of the one or more differentiability markers of (a)(ii) are indicative of the suitability of the PSCs for differentiation.202. The method of embodiment 201, which comprises (a)(i).203. The method of embodiment 201, which comprises (a)(ii).204. The method of embodiment 201, which comprises (a)(i) and (a)(ii).205. The method of any one of embodiments 201 to 204, wherein the evaluating the PSCs following introduction of the transgene fortheir suitability as precursors of differentiated cells is performed as defined in any one of embodiments 119 to 168.206. The method of embodiment 199, wherein the transgene is introduced into the differentiated cell following differentiation.207. The method of embodiment 199, wherein the transgene is introduced into a precursor of the differentiated cell.208. The method of any one of embodiments 196 to 207, which further comprises administering the differentiated cell to a subject in need thereof.209. A method of treating a subject, comprising administering to a subject in need thereof a differentiated cell produced by the method of any one of embodiments 196 to 207.210. A method for determining the suitability of pluripotent stem cells (PSCs) for differentiation into differentiated cells following introduction of a transgene, comprising:(a) introducing a transgene into PSCs to produce recombinant PSCs;(b) evaluating the recombinant PSCs for their suitability as precursors of differentiated cells by a method comprising:(i) (A) testing for the presence of mutations in one or more differentiability markers (e.g., to determine the prevalence of a mutant allele or mutant gene in PSCs), optionally wherein the one or more differentiability markers comprise EP300 and / or BCOR; and / or(B) detecting the expression levels of one or more additional differentiability markers in the recombinant PSCs; and(ii) determining if the presence of mutations in the differentiability markers of (i) and / or the expression levels of the one or more differentiability markers of (ii) are indicative of the suitability of the recombinant PSCs for differentiation.211. The method of embodiment 210, which comprises (A).212. The method of embodiment 210, which comprises (B).213. The method of embodiment 210, which comprises (A) and (B).214. The method of any one of embodiments 210 to 213, wherein evaluating the recombinant PSCs fortheir suitability as precursors of differentiated cells is performed as defined in any one of embodiments 119 to 168.215. The method of any one of embodiments 210 to 214, wherein the transgene encodes a therapeutic protein.216. The method of any one of embodiments 210 to 215, which further comprises differentiating a PSC identified as a suitable precursor of a differentiated cell into the differentiated cell.217. The method of embodiment 216, wherein the differentiated cell is a cell of the ectodermal lineage.218. The method of embodiment 218, wherein the differentiated cell is a dopaminergic neuron.219. A method of treating a subject, comprising administering to a subject in need thereof a differentiated cell population produced by the method of embodiment 216 or embodiment 217.220. A method of treating a subject suffering from a neurodegenerative disease, comprising administering to a subject in need thereof a dopaminergic neuron population produced by the method of embodiment 218.221. The method of claim 220, wherein the neurodegenerative diseases is Parkinson’s Disease, Alzheimer's Disease, dementia, epilepsy, Lewy Body syndrome, Huntington’s Disease, Spinal Muscular Atrophy, Friedreich’s Ataxia, Amyotrophic Lateral Sclerosis, Batten Disease, or Multiple System Atrophy.222. A kit comprising one or more probes and / or one or more PCR primer pairs for mutant EP300 and / or mutant BCOR.223. The kit of embodiment 222, which comprises one or more probes and / or one or more PCR primer pairs for mutant EP300.224. The kit of embodiment 222 or embodiment 223, which comprises one or more probes and / or one or more PCR primer pairs for mutant BCOR.225. The kit of any one of embodiments 222 to 224, which further comprises one or more probes and / or one or more PCR primer pairs for at least five, at least ten, at least fifteen or all of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, SFRP1, IFITM1, and HERV-K.226. A kit comprising one or more probes and / or one or more PCR primer pairs for at least two, at least three, at least four, at least five, at least ten, at least fifteen or all of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, SFRP1, IFITM1, and HERV-K227. The kit of any one of embodiments 222 to 226, which comprises one or more probes and / or one or more PCR primer pairs for NR5A2.228. The kit of any one of embodiments 222 to 227, which comprises one or more probes and / or one or more PCR primer pairs for HHEX.229. The kit of any one of embodiments 222 to 228, which comprises one or more probes and / or one or more PCR primer pairs for GBX2.230. The kit of any one of embodiments 222 to 229, which comprises one or more probes and / or one or more PCR primer pairs for GDF3.231. The kit of any one of embodiments 222 to 230, which comprises one or more probes and / or one or more PCR primer pairs for NANOG.232. The kit of any one of embodiments 222 to 231, which comprises one or more probes and / or one or more PCR primer pairs for NODAL.233. The kit of any one of embodiments 222 to 232, which comprises one or more probes and / or one or more PCR primer pairs for HMX1.234. The kit of any one of embodiments 222 to 233, which comprises one or more probes and / or one or more PCR primer pairs for UTF1.235. The kit of any one of embodiments 222 to 234, which comprises one or more probes and / or one or more PCR primer pairs for SIX6.236. The kit of any one of embodiments 222 to 235, which comprises one or more probes and / or one or more PCR primer pairs for MT2A.237. The kit of any one of embodiments 222 to 236, which comprises one or more probes and / or one or more PCR primer pairs for TBXT.238. The kit of any one of embodiments 222 to 237, which comprises one or more probes and / or one or more PCR primer pairs for NFIA.239. The kit of any one of embodiments 222 to 238, which comprises one or more probes and / or one or more PCR primer pairs for ID2.240. The kit of any one of embodiments 222 to 239, which comprises one or more probes and / or one or more PCR primer pairs for SP8.241. The kit of any one of embodiments 222 to 240, which comprises one or more probes and / or one or more PCR primer pairs for RAB17.242. The kit of any one of embodiments 222 to 241, which comprises one or more probes and / or one or more PCR primer pairs for PRTG.243. The kit of any one of embodiments 222 to 242, which comprises one or more probes and / or one or more PCR primer pairs for FAS.244. The kit of any one of embodiments 222 to 243, which comprises one or more probes and / or one or more PCR primer pairs for FAM129A.245. The kit of any one of embodiments 222 to 244, which comprises one or more probes and / or one or more PCR primer pairs for LCK.246. The kit of any one of embodiments 222 to 245, which comprises one or more probes and / or one or more PCR primer pairs for FZD7.247. The kit of any one of embodiments 222 to 246, which comprises one or more probes and / or one or more PCR primer pairs for IDO1.248. The kit of any one of embodiments 222 to 247, which comprises one or more probes and / or one or more PCR primer pairs for TRIML2.249. The kit of any one of embodiments 222 to 248, which comprises one or more probes and / or one or more PCR primer pairs for FGF4.250. The kit of any one of embodiments 222 to 249, which comprises one or more probes and / or one or more PCR primer pairs for COL2A1.251. The kit of any one of embodiments 222 to 250, which comprises one or more probes and / or one or more PCR primer pairs for EOMES.252. The kit of any one of embodiments 222 to 251, which comprises one or more probes and / or one or more PCR primer pairs for SFRP1.253. The kit of any one of embodiments 222 to 252, which comprises one or more probes and / or one or more PCR primer pairs for IFITM1.254. The kit of any one of embodiments 222 to 253, which comprises one or more probes and / or one or more PCR primer pairs for HERV-K.255. The kit of any one of embodiments 222 to 254, which comprises one or more probes and / or one or more PCR primer pair for one or more control genes.256. The kit of embodiment 255, wherein the one or more control genes comprise housekeeping genes.257. The kit of embodiment 256, wherein the housekeeping genes comprise one, two, three or all four of p-actin, 18S rRNA, GAPDH and TBP.258. The kit of embodiment 256, wherein the housekeeping genes comprise p-actin.259. The kit of embodiment 256 or embodiment 257, wherein the housekeeping genes comprise 18S rRNA.260. The kit of any one of embodiments 256 to 259, wherein the housekeeping genes comprise GAPDH.261. The kit of any one of embodiments 256 to 260, wherein the housekeeping genes comprise TBP.262. The kit of any one of embodiments 222 to 261 in which at least one primer for each differentiability marker is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end and, if present, at least one primer for each control gene is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end.263. The kit of any one of embodiments 222 to 262, which further comprises polymerase and / or a nucleotide mixture.264. The kit of any one of embodiments 222 to 263, further comprising an anti-IFITM 1 antibody, optionally wherein the antibody is fluorescently labeled.265. The kit of any one of embodiments 222 to 264, wherein the probes are in a 96-well plate.266. A PCR (e.g., quantitative PCR (qPCR)) reaction mixture comprising:(a) RNA extracted from PSCs or cDNA reverse transcribed therefrom;(b) primers suitable for amplifying one or more differentiability markers, e.g., one or more positive differentiability markers and one or more negative differentiability markers;(c) optionally, primers suitable for amplifying one or more control genes; and (d) a DNA polymerase; and(e) a nucleotide mixture, optionally comprising labeled nucleotides, e.g., fluorescently labeled nucleotides.267. The PCR reaction mixture of embodiment 266, which comprises one or more PCR primer pairs for mutant EP300 and / or mutant BCOR.268. The PCR reaction mixture of embodiment 267, which comprises one or more PCR primer pairs for mutant EP300.269. The PCR reaction mixture of embodiment 266 or embodiment 267, which comprises one or more PCR primer pairs for mutant BCOR.270. The PCR reaction mixture of any one of embodiments 266 to 269, which further comprises one or more PCR primer pairs for at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen or all of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, SFRP1, IFITM1, and HERV-K.271. The PCR reaction mixture of embodiment 266, which comprises one or more PCR primer pairs for at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen or all of NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, SFRP1, IFITM1, and HERV-K272. The PCR reaction mixture of any one of embodiments 266 to 271, which comprises one or more PCR primer pairs for NR5A2.273. The PCR reaction mixture of any one of embodiments 266 to 272, which comprises one or more PCR primer pairs for HHEX.274. The PCR reaction mixture of any one of embodiments 266 to 273, which comprises one or more PCR primer pairs for GBX2.275. The PCR reaction mixture of any one of embodiments 266 to 274, which comprises one or more PCR primer pairs for GDF3.276. The PCR reaction mixture of any one of embodiments 266 to 275, which comprises one or more PCR primer pairs for NANOG.277. The PCR reaction mixture of any one of embodiments 266 to 276, which comprises one or more PCR primer pairs for NODAL.278. The PCR reaction mixture of any one of embodiments 266 to 277, which comprises one or more PCR primer pairs for HMX1.279. The PCR reaction mixture of any one of embodiments 266 to 278, which comprises one or more PCR primer pairs for UTF1.280. The PCR reaction mixture of any one of embodiments 266 to 279, which comprises one or more PCR primer pairs for SIX6.281. The PCR reaction mixture of any one of embodiments 266 to 280, which comprises one or more PCR primer pairs for MT2A.282. The PCR reaction mixture of any one of embodiments 266 to 281, which comprises one or more PCR primer pairs for TBXT.283. The PCR reaction mixture of any one of embodiments 266 to 282, which comprises one or more PCR primer pairs for NFIA.284. The PCR reaction mixture of any one of embodiments 266 to 283, which comprises one or more PCR primer pairs for ID2.285. The PCR reaction mixture of any one of embodiments 266 to 284, which comprises one or more PCR primer pairs for SP8.286. The PCR reaction mixture of any one of embodiments 266 to 285, which comprises one or more PCR primer pairs for RAB17.287. The PCR reaction mixture of any one of embodiments 266 to 286, which comprises one or more PCR primer pairs for PRTG.288. The PCR reaction mixture of any one of embodiments 266 to 287, which comprises one or more PCR primer pairs for FAS.289. The PCR reaction mixture of any one of embodiments 266 to 288, which comprises one or more PCR primer pairs for FAM129A.290. The PCR reaction mixture of any one of embodiments 266 to 289, which comprises one or more PCR primer pairs for LCK.291. The PCR reaction mixture of any one of embodiments 266 to 290, which comprises one or more PCR primer pairs for FZD7.292. The PCR reaction mixture of any one of embodiments 266 to 291, which comprises one or more PCR primer pairs for IDO1.293. The PCR reaction mixture of any one of embodiments 266 to 292, which comprises one or more PCR primer pairs for TRIML2.294. The PCR reaction mixture of any one of embodiments 266 to 293, which comprises one or more PCR primer pairs for FGF4.295. The PCR reaction mixture of any one of embodiments 266 to 294, which comprises one or more PCR primer pairs for COL2A1.296. The PCR reaction mixture of any one of embodiments 266 to 295, which comprises one or more PCR primer pairs for EOMES.297. The PCR reaction mixture of any one of embodiments 266 to 296, which comprises one or more PCR primer pairs for SFRP1.298. The PCR reaction mixture of any one of embodiments 266 to 297, which comprises one or more PCR primer pairs for IFITM1.299. The PCR reaction mixture of any one of embodiments 266 to 298, which comprises one or more PCR primer pairs for HERV-K.300. The PCR reaction mixture of any one of embodiments 266 to 299, which comprises one or more PCR primer pair for one or more control genes.301. The PCR reaction mixture of embodiment 300, wherein the one or more control genes comprise housekeeping genes.302. The PCR reaction mixture of embodiment 301, wherein the housekeeping genes comprise one, two, three or all four of p-actin, 18S rRNA, GAPDH and TBP.303. The PCR reaction mixture of embodiment 302, wherein the housekeeping genes comprise p-actin.304. The PCR reaction mixture of embodiment 302 or embodiment 302, wherein the housekeeping genes comprise 18S rRNA.305. The PCR reaction mixture of any one of embodiments 302 to 304, wherein the housekeeping genes comprise GAPDH.306. The PCR reaction mixture of any one of embodiments 302 to 305, wherein the housekeeping genes comprise TBP.307. The PCR reaction mixture of any one of embodiments 266 to 261 in which at least one primer for each differentiability marker is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end and, if present, at least one primer for each control gene is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end.8. EXAMPLES8.1. Example 1: Identification of Differentiability Markers8.1.1. Overview

[0191] PSCs from 29 samples of a PSC line referred to herein as PSC line 1 were differentiated into dopaminergic (“DA”) neurons as described in Section 8.1.2.1.

[0192] Differentiation success was determined by flow cytometry according to the method of Section 8.1.2.2. A DA neuron pass / fail cutoff was used, whereby passing PSC samples were associated

[0193] An initial qPCR panel with 96 genes was generated to evaluate whether gene expression in PSC samples correlated with differentiation success. Raw qPCR data for the 96 genes in the initial qPCR panel were measured at the PSC stage (day 0 of differentiation) for the 29 samples and normalized to the geometric mean of four housekeeping genes ACTS, TBP, 18S, and GAPDH.

[0194] A random forest (RF) classifier was trained on the dataset, which allowed the ranking of genes based on the segregation of the gene expression for each gene between successfully differentiated (pass) and failed differentiation (fail) samples (FIG. 2).8.1.2. Methods8.1.2.1. Differentiation of PSCs into DA Neurons

[0195] PSCs were differentiated into DA neurons for 16 days (D1-D16) using a differentiation protocol adapted from Piao et al., 2021, Cell Stem Cell 28(2):217-229. e7, PCT Publication Nos. WO 2016,196661 A1, WO 2010 / 096496 A2, WO 2013 / 067362 A1, WO 2021 / 042027 A1, WO 2021 / 203009 A1, and US Patent No. 10,711,243, which are hereby incorporated by reference in their entireties.

[0196] After completion of DA differentiation at D16 the cells were cryopreserved until further use.8.1.2.2. Flow Cytometry

[0197] Differentiated cells were allowed to mature for five days post-differentiation (DIV5) before assessment of differentiation success with flow cytometry by determining expression of the on-target marker FOXA2, and off-target markers PAX6 and CRABP1 in cells.8.1.2.3. qPCR

[0198] Frozen cell pellets with a total of 3x106to 4x106cells were slightly thawed until the cell pellets could be dislodged and loosened by flicking the tube containing the cell pellets. Cell lysates were generated by resuspending the pellets in 350 pL buffer by pipetting up and down at least five times with a P1000 pipette. RNA was extracted from the samples and RNA concentration was determined. 500 ng of RNA was reverse transcribed using the parameters in Table E1 with in a thermal cycler. Resulting cDNA was assessed with a TaqMan qPCR assay using the parameters in Table E2.8.1.3. Results

[0199] Normalized qPCR values were sorted by Random-Forest (RF) regression model in order of segregation of expression between successfully differentiated samples (pass samples) and samples that failed differentiation (fail samples) (FIG. 2). To predict whether a PSC sample would succeed or fail in differentiating into DA neurons, differential expression of genes upregulated in pass samples and fail samples was used to develop a formula, herein referred to as PSC score formula, as set forth below, to be used with any combination of differentially expressed genes and determine a suitable threshold value for differentiability based on those differentially expressed genes.PSC Score-8.2. Example 2: PSC Score Development

[0200] An exemplary PSC score that incorporates the differential expression of the top 17 genes associated with the most significant segregation identified in Example 1 was developed. Among these genes, PRTG, FAS, and RAB17 displayed increased expression in Pass samples relative to Fail samples, and thus are classified as positive differentiability markers. In contrast, NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8 displayed decreased expression in Pass samples relative to Fail samples, and thus are classified as negative differentiability markers.

[0201] Raw qPCR data for each of the 17 genes were normalized to the geometric mean of four housekeeping genes ACTB, TBP, 18S, and GAPDH. Arithmetic mean of normalized Cq values of genes that had increased gene expression in Pass samples (PRTG, FAS, RAB17) was divided by the arithmetic mean of normalized Cq values of genes that had decreased gene expression in Pass samples (NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8) to determine the PSC score for each of the 29 samples tested in Example 1 as follows:PSC Score ~ Mean Cq values (PRTG, FAS, RAB17Mean Cq values (NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8)

[0202] Pass samples were associated with lower PSC scores than Fail samples (FIG. 3). To predict whether a PSC sample would succeed or fail in differentiating into DA neurons, a differentiation threshold value of 1.34 was determined by subtracting 3 mean absolute deviations from the median (FIG. 3). Therefore, a sample with a PSC score below 1.34 for the17 genes would be predicted to successfully differentiate into DA neurons, whereas a sample with a PSC score above 1.34 for the 17 genes would be predicted to fail differentiation.8.3. Example 3: Prediction of Differentiation Success using SFRP1 and / or GBX2 Ratio

[0203] This time, the utility of a ratio of two genes, SFRP1 and GBX2, to predict PSC differentiability was evaluated in comparison to the PSC score. Given that GBX2 was shown to be expressed at different levels between differentiated cell populations and undifferentiated stem cells, the utility of GBX2 ACq to predict differentiability was also assessed.

[0204] A total of six samples (three PSC samples grown on two different substates, substrate 1 (S1) and substrate 2 (S2)) were used. Among these, two PSC samples failed differentiation, whereas the rest successfully differentiated into DA neurons (Table E3, last column).

[0205] Both PSC samples that failed to differentiate were associated with PSC scores greater than 1.34 (Table E3, second column), which was consistent with the predictions of Example 2.

[0206] A differentiability threshold of 0.450 for the SFRP1 / GBX2 ratio was determined by normalizing against the geometric mean of the housekeeping gene ACTB, to predict whether a PSC would successfully differentiate into DA neurons. The samples that failed to differentiate were associated with scores near or at 0.500, whereas the highest SFRP1 / GBX2 ratio for successfully differentiated PSC samples was below 0.420 (Table E3, third column). Therefore, the differentiability threshold of the SFRP1 / GBX2 ratio predicted PSC sample differentiability similar to the differentiability threshold of the 17-gene PSC score.

[0207] Generally, GBX2 ACq values were relatively higher for samples that successfully differentiated into DA neurons than values obtained with samples that failed to differentiate (Table E3, fourth column). Thus, GBX2 ACq values are correlated with successful differentiation and can be used to predict to PSC differentiability. However, additional data (not shown) indicates that a lower GBX2 ACq cutoff value can be used successfully to select PSCs for differentiation into DA neurons. Using a lower GBX2 ACq cutoff value is potentially a more stringent approach for selection of PSCs to differentiate into DA neurons, but can result in some PSC samples that could be successfully differentiated being overlooked.8.4. Example 4: Correlation of PSC Score with BCOR SNP Variants8.4.1. Overview

[0208] Genetic mutations accumulating in extended PSC cultures can affect differentiation. For instance, single nucleotide polymorphisms (SNPs) of BCL6 corepressor (BCOR) gene impair differentiation of PSCs (Rouhani et al., 2022, Nat. Genet. 54:1406-1416). To determine whether the PSC score differs between PSCs that comprise a BCOR SNP or PSCs that comprise a wild-type BCOR allele, PSC scores of twelve PSC line 1 clones that comprise either a wild-type BCOR (see SEQ ID NO:29) or a BCOR SNP (SNP1 (see SEQ ID NO:30) or SNP2 (see SEQ ID NO:31)) were determined using the formula in Section 8.2.8.4.2. Results

[0209] PSC line 1 clones with wild-type BCOR alleles had an average PSC score of 0.88, whereas PSC line 1 clones with BCOR SNP1 had an average PSC score of 1.42 and PSC line 1 clones with BCOR SNP2 had an average PSC score of 1.50 (FIG. 4). Hence, the average PSC scores for the clones with BCOR SNPs were above the 1.34 differentiation threshold determined in Section 8.2 but the average PSC scores for the clones with wild-type BCOR alleles were below the differentiation threshold. These results indicate that the PSC score correlates with the observation that BCOR mutants were more likely to fail differentiation.8.5. Example 5: Correlation of PSC Score and BCOR status with BCOR SNP Variants8.5.1. Overview

[0210] Three clones (PSC line 1 clone 65 (“c.65”) comprising BCOR SNP1, PSC line 1 clone 43 (“c.43”) comprising BCOR SNP2, and PSC line 1 clone 57 (“c.57”) comprising WT BCOR) were selected for the next set of assessments to determine whether PSC scores correlate with differentiation success.

[0211] Cells of the three clones were differentiated into DA neurons for 16 days as described in Section 8.1.2.1. Following differentiation, the DA neurons were thawed and matured for 5 days (DIV5) and assessed with flow cytometry for off-target (CRABP1 and PAX6) and on-target (FOXA2, OTX2, and TH) differentiation markers as described in Section 8.1.2.2.Immunohistochemical assessments of on-target (FOXA2 and TH) and off-target (PAX6 and CRABP1) markers were performed using standard procedures on DIV1 and DIV5. Profiling of differentially expressed genes in cells differentiated from PSCs with BCOR SNP1 or SNP2 relative to cells differentiated from BCOR WT cells was performed using qPCR on DIV0 and DIV5 as described in Section 8.1.2.3. DA neuron activity and connectivity was assessed with live cell calcium imaging using Incucyte® Neuroburst, following the manufacturer’s protocol. DA neuron functionality was further evaluated with neurite outgrowth image analysis and extracellular DA concentration (ECDC) assessment.8.5.2. Results

[0212] Flow cytometry results showed high expression of on-target midbrain floorplate DA identity markers FOXA2, OTX and TH and low expression of off-target markers CRABP1 and PAX6 for cells differentiated from BCOR WT cells, which was similar to the expression of these genes in control DA neurons (FIGS. 5A-5G). In contrast, PSCswith BCOR SNP1 orSNP2 resulted in cells with low levels of on-target markers and high levels of off-target markers (FIGS.5A-5G). Similarly, cells differentiated from BCOR WT cells showed high levels of FOXA2 and low levels of PAX6 and CRABP1 immunofluorescence on DIV1 (FIGS. 6A-6C) and DIV5 (FIGS.6D-6F), whereas cells differentiated from PSCs with BCOR SNP1 or SNP2 were associated with low levels of FOXA2 and high levels of PAX6 and CRABP1 immunofluorescence on DIV1 (FIGS. 6A-6C) and DIV5 (FIGS. 6D-6F).

[0213] Assessment of differentially expressed genes revealed high expression of off-target genes such as CRABP1, GBX2, HOXB2, PAX6, ZIC1&3, SP8 and RSPO3 in cells differentiated from BCOR SNP1 and BCOR SNP2 cell lines relative to those differentiated from BCOR WT cells (FIGS. 7A-7B).

[0214] Functional analyses showed that cells differentiated from BCOR SNP1 or BCOR SNP2 cell lines behaved differently than those differentiated from BCOR WT PSCs and control DA cells (hESC-derived DA neurons). Neuroburst assessment showed that cells differentiated from PSCs with BCOR SNP1 or SNP2 had higher firing neuron counts between DIV10 and DIV14 relative to DA control cells or cells differentiated from BCOR WT cells (FIG. 8A). The highest correlation of firing, which is associated with the degree of synchronization of neurons, was associated with DA control cells, followed by cells differentiated from BCOR WT cells and cells differentiated from PSCs with SNP2, which displayed comparable levels of correlation of firing, in turn followed by cells differentiated from PSCs with SNP1, which showed limited correlation of firing (FIG. 8B). Starting neurite lengths differed among cells of distinct lineages, but all cells were associated with similar lengths of neurite growth for the duration they were monitored (FIG. 8C). Although all cells released dopamine, the highest levels of release was associated with control DA cells and cells differentiated from BCOR WT PSCs (FIG. 8D). The lowest level of dopamine release was observed with PSCs with SNP1 (FIG. 8D).8.6. Example 6: Correlation of HERV-K Expression and PSC Score in PSCs 8.6.1. Overview

[0215] Human endogenous retroviruses comprise different families including human endogenous retrovirus K (HERV-K). Expression of HERV-K (HML-2) has been shown to be transiently expressed during development (Grow etal., 2015, Nature 522(7555):221-5). To determine whether PSC scores correlate with HERV-K transcription, expression of HERV-K genes was assessed in parental PSC line 1 cells, a PSC line 1 clone with a high PSC score(PSC line 1 clone 34 (”c.34”) and a PSC line 1 clone with a low PSC score (PSC line 1 clone 46, “c.46”)) using qPCR as described in Section 8.1.2.3.

[0216] Interferon-induced transmembrane protein 1 (IFITM 1 ) is a cell-surface protein that is activated in response to HERV-K and HERV-K transcription correlates with IFITM-1 cell surface staining. To determine whether PSC scores and HERV-K transcription differed between cells that were positive for IFITM 1, PSC line 1 c.34 cells were sorted for IFITM 1 expression with flow cytometry.8.6.2. Results

[0217] HERV-K genes were typically expressed at relatively higher levels in PSC line 1 c.34 cells and at relatively lower levels in PSC line 1 c.46 cells (FIG. 9A), suggesting that HERV-K transcription and PSC scores were correlated.

[0218] Before cell sorting, the average PSC score was calculated to be 1.47 for PSC line 1 c.34 cells and 0.92 for c.46 cells. Cells that were positive for IFITM 1 had an average PSC score of 1.70 and had the highest levels of HERV-K gene expression, whereas cells that were negative for IFITM1 had an average PSC score of 1.29 and were associated with the lowest HERV-K gene expression (FIG. 9B and 9C).

[0219] Thus, HERV-K and IFITM 1 are categorized as negative differentiability markers.8.7. Example 7: Correlation of PSC Score with %EP300 mut8.7.1. Overview

[0220] Five clones with different % mutant EP300 (EP300 c.4540G> A) values (EP300 mut) were evaluated. The % mutant EP300 values and PSC ratios for each clone are presented in Table E4 below.

[0221] Cells of each clone were differentiated into DA neurons for 16 days as described in Section 8.1.2.1. Following differentiation, the DA neurons were thawed and matured for five days. Profiling of differentially expressed genes in cells differentiated from each clone was performed using qPCR as described in Section 8.1.2.3. The resulting values were normalized by the values obtained with cells differentiated from Line 2-I. A similar gene expression profiling was performed with additional clones, resulting values of which were normalized by the values obtained with cells differentiated from BCOR WT PSCs.8.7.2. Results

[0222] There was a strong correlation (r = 0.989) between % EP300 mut and PSC score (FIG.10A). Relative expression of genes in cells differentiated from Line 2-F, which had the lowest % EP300 mut value and PSC ratio, remained close to zero (FIG. 10B), which meant that the expression of genes in those cells was similar to the expression of genes in cells differentiated from Line 2-I. Largest relative deviations were observed with SIX6, GBX2, HMX1, FGF5 in cells differentiated from the other clones, which had higher % EP300 mut and PSC ratios (FIG. 10B).9. SEQUENCES

[0223] Exemplary sequences of the disclosure are set forth in Table S below:10. CITATION OF REFERENCES

[0224] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event that there is an inconsistency between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended.

Claims

WHAT IS CLAIMED IS:

1. A method for generating differentiated cells of an ectodermal lineage comprising exposing pluripotent stem cells (PSCs) of a PSC population to ectodermal cell differentiation conditions, wherein the PSC population has been identified as suitable for differentiation by a process comprising:(a) measuring the expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in PSCs of the PSC population or a parental PSC population;(b) determining if the expression levels of the at least one of the differentiability markers are indicative of the suitability of the PSC population for differentiation.

2. The method of claim 1, wherein measuring the expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers comprises measuring the expression level of GBX2 and optionally SFRP1.

3. The method of claim 2, wherein measuring the expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers comprises measuring the expression levels of GBX2 and SFRP1, and wherein determining if the expression levels of the at least one of the differentiability markers are indicative of the suitability of the PSC population for differentiation comprises determining that the PSC population is suitable for differentiation when the SFRP1 / GBX2 expression level ratio is less than 0.450.

4. The method of any one of claims 1 to 3, wherein the differentiated cells comprise dopaminergic neurons.

5. The method of any one of claims 1 to 4, wherein the PSC population has been cryopreserved and reactivated following cryopreservation and prior to exposing the PSCs to ectodermal cell differentiation conditions.

6. A method of determining the differentiation potential of a pluripotent stem cell (PSC) population comprising:(a) measuring the expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in the population;(b) assigning the population a PSC score based on the expression of the differentiability markers; and(c) determining if the PSC score falls above or below a cutoff value.

7. A method of measuring expression levels of differentiability markers in pluripotent stem cells (PSCs), comprising:(a) measuring (i) the presence of mutant EP300 and / or mutant BCOR and / or (ii) expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TEXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in a sample prepared from the PSCs; and(b) determining the presence (e.g., percentage in the population) of mutant EP300 and / or mutant BCOR and / or the expression levels of the at least three, at least four, or at least five of the differentiability markers.

8. The method of claim 7, which comprises (a)(i).

9. The method of claim 7, which comprises (a)(ii).

10. The method of claim 7, which comprises (a)(i) and (a)(ii).

11. The method of any one of claims 1 to 10, wherein measuring the at least one, at least two, at least three, at least four, or at least five differentiability markers comprises performing a polymerase chain reaction (PCR) on nucleic acids extracted from the PSCs using primers capable of amplifying the at least one, at least two, at least three, at least four, or at least five differentiability markers.

12. The method of claim 11, wherein the PCR is a real-time PCR, and the measuring further comprises quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the at least one, at least two, at least three, at least four, or at least five differentiability markers.

13. The method of any one of claims 1 to 12, wherein measuring the at least one, at least two, at least three, at least four, or at least five differentiability markers comprises measuring at least three, at least four, or at least five differentiability markers.

14. The method of claim 12, which comprises:(a) measuring expression of at least five of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TEXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV- K in a sample prepared from the PSCs;(b) determining the expression levels of the at least 10 differentiability markers by a method comprising:(i) performing real-time PCR on nucleic acids extracted from the PSCs using primers capable of amplifying the at least 10 differentiability markers; and(ii) quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the at least 10 differentiability markers.

15. The method of claim 12, which comprises:(a) measuring expression of at least 10 of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in a sample prepared from the PSCs;(b) determining the expression levels of the at least 10 differentiability markers by a method comprising:(i) performing real-time PCR on nucleic acids extracted from the PSCs using primers capable of amplifying the at least 10 differentiability markers; and(ii) quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the at least 10 differentiability markers.

16. The method of claim 12, which comprises:(a) measuring expression of at least 15 of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K, in a sample prepared from the PSCs;(b) determining the expression levels of the at least 15 differentiability markers by a method comprising:(i) performing real-time PCR on nucleic acids extracted from the PSCs using primers capable of amplifying the at least 15 differentiability markers; and(ii) quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the at least 15 differentiability markers.

17. The method of claim 12, which comprises:(a) measuring expression of all 17 of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS in a sample prepared from the PSCs;(b) determining the expression levels of the all 17 differentiability markers by a method comprising:(i) performing real-time PCR on nucleic acids extracted from the PSCs using primers capable of amplifying the all 17 differentiability markers; and(ii) quantifying the PCR products by hybridizing the PCR products using probes that hybridize to the all 17 differentiability markers.

18. An assay method comprising:(a) assaying for mutant EP300 and / or mutant BCOR in a PSC population (e.g., as a percentage of EP300 and / or BCOR in the PSC population); and / or(b) performing a polymerase chain reaction (PCR) on one or more reaction mixtures comprising:(i) RNA extracted from the PSC population or cDNA reverse transcribed therefrom;(ii) primers suitable for amplifying at least one, at least two, at least three, at least four, or at least five of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K;(iii) labeled probes for detecting the at least one, at least two, at least three, at least four, or at least five differentiability markers; and (iv) a DNA polymerase; and(c) detecting the presence of mutant EP300 and / or mutant BCOR (e.g., as a percentage of EP300 and / or BCOR in the PSC population), and / or detecting expression levels of the at least one, at least two, at least three,at least four, or at least five differentiability markers based on signals emitted by the labeled probes.

19. A method of evaluating pluripotent stem cells (PSCs) for their differentiability, comprising:(a) detecting (i) presence of mutant EP300 and / or mutant BCOR, and / or (ii) expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TEXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in the PSCs; and (b) determining if the presence of mutant EP300 and / or mutant BCOR, and / or expression levels of the at least one, at least two, at least three, at least four, or at least five of the differentiability markers are indicative of the suitability of the PSCs for differentiation.

20. The method of claim 19, wherein the PSCs have been cryopreserved prior to step (a), and optionally wherein the PSCs have been reactivated following cryopreservation and prior to step (a).

21. A method of preparing a pluripotent stem cell (PSC) stem cell bank suitable for differentiation, comprising:(a) detecting (i) the presence of mutant EP300 and / or mutant BCOR, and / or (ii) expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in a population of PSCs; (b) determining if the presence of mutant EP300 and / or mutant BCOR (e.g., as a percentage of EP300 and / or BCOR in the PSC population) and / or expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers are indicative of the suitability of the PSCs for differentiation;(c) if the presence of mutant EP300 and / or mutant BCOR and / or expression levels of the at least one, at least two, at least three, at least four, or at least five differentiability markers are indicative of the suitability of the PSCs for differentiation, forming a PSC stem cell bank from the population of PSCs.

22. The method of claim 21, wherein the PSCs have been cryopreserved prior to step (a), and optionally wherein the PSCs have been reactivated following cryopreservation and prior to step (a).

23. The method of any one of claims 21 to 22, which further comprises cryopreserving the PSCs after step (b) if the presence of mutant EP300 and / or mutant BCOR and / or the expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers are indicative of the suitability of the PSCs for differentiation.

24. A method for qualifying a population of pluripotent stem cells (PSCs) for manufacturing differentiated cells, comprising:(a) detecting (i) the presence of mutant EP300 and / or mutant BCOR, and / or (ii) expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TEXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K; and (b) determining if the presence of mutant EP300 and / or mutant BCOR, and / or the expression levels of the at least one, at least two, at least three, at least four, or at least five differentiability markers are indicative of the suitability of the PSCs for differentiation,wherein a determination that presence of mutant EP300 and / or mutant BCOR, and / or expression levels of the at least one, at least two, at least three, at least four, or at least five differentiability markers are indicative of the suitability of the PSCs for differentiation qualifies the population of PSCs for manufacturing differentiated cells.

25. The method of claim 24, wherein the population of PSCs is a clonal population.

26. The method of claim 24 or claim 25, wherein the PSCs have been cryopreserved prior to step (a), and optionally wherein the PSCs have been reactivated following cryopreservation and prior to step (a).

27. A method for selecting a population of pluripotent stem cells (PSCs) for manufacturing differentiated cells, comprising:(a) detecting (i) the presence of mutant EP300 and / or mutant BCOR, and / or the expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TEXT, NFIA, ID2,SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in one or more PSC populations;(b) determining if the presence of mutant EP300 and / or mutant BCOR, and / or expression levels of the at least three, at least four, or at least five differentiability markers are indicative of the suitability of the one or more PSC populations for differentiation; and(c) selecting a PSC population for manufacturing differentiated cells if the presence of mutant EP300 and / or mutant BCOR, and / or expression levels of the at least one, at least two, at least three, at least four, or at least five differentiability markers are indicative of the suitability of the one or more PSC populations for differentiation.

28. A method for monitoring a population of pluripotent stem cells (PSCs) for its suitability manufacturing differentiated cells, comprising:(a) detecting (i) the presence of mutant EP300 and / or mutant BCOR, and / or expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K in PSCs of the clonal population;(b) determining if the presence of mutant EP300 and / or mutant BCOR, and / or expression levels of the at least one, at least two, at least three, at least four, or least five differentiability markers are indicative of the suitability of the PSCs for differentiation; and(c) if the presence of mutant EP300 and / or mutant BCOR, and / or expression levels of the at least one, at least two, at least three, at least four, or at least five differentiability markers in step (b) are indicative of the suitability of the PSCs for differentiation, maintaining the clonal population; and(d) repeating steps (a)-(c) one or more times.

29. The method of claim 28, wherein the PSCs have been cryopreserved prior to step (a), and optionally wherein the PSCs have been reactivated following cryopreservation and prior to step (a).

30. The method of any one of claims 1 to 29, wherein the differentiability markers comprise at least one negative differentiability marker and at least one positive differentiability marker.

31. The method of claim 30, wherein one or more negative differentiability markers are markers of pluripotency.

32. The method of claim 30 or claim 31, wherein one or more negative differentiability markers comprise one or more of GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, IFITM1, and HERV-K.

33. The method of any one of claims 30 to 32, wherein one or more positive differentiability markers comprise one or more of SFRP1, RAB17, PRTG, FAS, FAM129A, LCK, FZD7, IDO1, and COL2A1.

34. The method of any one of embodiments 27 to 30, wherein the differentiability markers comprise GBX2 and SFRP1.

35. The method of any one of claims 19 to 34, which comprises detecting the expression levels of one or more differentiability markers by a method comprising:(a) measuring protein expression levels using an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), flow cytometry, a Western blot, an immunohistochemical assay (IHC), or a combination of two or more thereof; and(b) determining the measured protein expression levels.

36. The method of any one of claims 19 to 34, which comprises detecting the expression levels of one or more differentiability markers by a method comprising:(a) measuring nucleic acid (e.g., mRNA) expression levels (e.g., using Cq value) using a polymerase chain reaction and / or a microarray-based method; and(b) determining the measured nucleic acid expression levels.

37. The method of any one of claims 1 to 36, which comprises determining if the presence of mutant EP300 and / or mutant BCOR, and / or expression levels of at least one, at least two, at least three, at least four, or at least five of the differentiability markers are indicative of the suitability of the PSCs for differentiation by:(a) (i) determining percentage of mutant EP300 and / or mutant BCOR in the PSCs; and / or(ii) assigning the PSCs a PSC score based on the expression of the differentiability markers; and(b) determining if the percentage of mutant EP300 and / or mutant BCOR and / or the PSC score falls above or below a cutoff value.

38. The method of claim 37, wherein the PSC score is calculated using the formula:Mean Cq values of genes unregulated in samples that pass DA differentiationrS SCOT'S — - Mean Cq values of genes upregulated in samples that fail DA differentiation39. The method of claim 38, wherein the PSC score formula for the differentiability markers NR5A2, HHEX, GBX2, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS is: PSC score =Mean Cq values PRTG, FAS, RAB17)Mean Cq values (NR5A2, HHEX, GBX2. GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A,‘ TBXT, NFIA, ID2, SP8)40. The method of any one of claims 37 to 39, wherein the cutoff value is 1.34.

41. The method of any one of claims 1 to 40, wherein the PSCs are induced pluripotent stem cells (iPSCs).

42. The method of any one of claims 6 to 41, which further comprises differentiating a PSC identified as a suitable precursor of a differentiated cell into the differentiated cell.

43. The method of any one of claims 1 to 42, which comprises measuring the expression of 60 or fewer total genes.

44. The method of claim 43, which comprises measuring the expression of 50 or fewer differentiability markers.

45. The method of claim 43 or claim 44, which comprises measuring the expression of up to 10 control genes.

46. A kit comprising one or more probes and / or one or more PCR primer pairs for mutant EP300 and / or mutant BCOR and optionally at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen or all of GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS.

47. A kit comprising one or more probes and / or one or more PCR primer pairs for at least two, at least three, at least four, least five, at least ten, at least fifteen or all of GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, and FAS.

48. The kit of claim 46 or claim 47, which further comprises one or more probes and / or one or more PCR primer pairs for one, two, three four, five or more (e.g., all) of FAM129A, LCK, FZD7, IDO1, TRIML2, FGF4, COL2A1, EOMES, SFRP1, IFITM1, and HERV-K.

49. The kit of any one of claims 46 to 48, which comprises one or more probes and / or one or more PCR primer pairs for GBX2 and SFRP1.

50. The kit of any one of claims 46 to 49, which comprises one or more probes and / or one or more PCR primer pairs for IFITM1.

51. The kit of any one of claims 46 to 50, which comprises one or more probes and / or one or more PCR primer pairs for HERV-K.

52. The kit of any one of claims 46 to 51, which comprises one or more probes and / or one or more PCR primer pairs for mutant EP300.

53. The kit of any one of claims 46 to 52, which comprises one or more probes and / or one or more PCR primer pairs for mutant BCOR.

54. The kit of any one of claims 46 to 53, which comprises one or more probes and / or one or more PCR primer pair for one or more control genes, optionally wherein the one more control genes comprise one, two, three, or all four of p-actin, 18S rRNA, GAPDH and TBP.

55. The kit of any one of claims 46 to 54, in which at least one primer for each differentiability marker is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end and, if present, at least one primer for each control gene is labeled with a fluorescent reporter on its 5' end and optionally a fluorescent quencher of the reporter at the 3' end.

56. A PCR (e.g., quantitative PCR (qPCR)) reaction mixture comprising:(a) RNA extracted from PSCs or cDNA reverse transcribed therefrom;(b) primers suitable for amplifying at least one, at least two, at least three, at least four, or at least five of the differentiability markers GBX2, NR5A2, HHEX, GDF3, NANOG, NODAL, HMX1, UTF1, SIX6, MT2A, TBXT, NFIA, ID2, SP8, RAB17, PRTG, FAS, IFITM1, and HERV-K;(c) optionally, primers suitable for amplifying one or more control genes; and(d) a DNA polymerase; and(e) a nucleotide mixture, optionally comprising labeled nucleotides, e.g., fluorescently labeled nucleotides.

57. The PCR reaction mixture of claim 56, which comprises one or more primer pairs for GBX2 and optionally one or more primer pairs for SFRP1.

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