Markers specific to pluripotent stem cells and methods of using same

Abstract

The present disclosure provides markers specific for pluripotent stem cells. In particular, the present disclosure relates to nucleic acid and polypeptide markers that are selectively expressed by pluripotent stem cells, and methods for detecting the presence and / or absence of one or more pluripotent stem cells by detecting such markers.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to pluripotent stem cell specific markers. In particular, the present disclosure relates to nucleic acid and polypeptide markers selectively expressed by pluripotent stem cells, and methods for detecting the presence and / or absence of one or more pluripotent stem cells by detecting the presence and / or absence of one or more such markers. [Background technology]

[0003] Pluripotent stem cells (PSCs) are generally recognized to exhibit unlimited self-renewal potential in vitro (i.e., the ability to divide indefinitely in an undifferentiated state) and to have the potential to differentiate into cell types that represent the three primary germ cell layers of the early embryo (i.e., ectoderm, endoderm, and mesoderm). By virtue of their capacity to become these three germ layers, PSCs can thereby give rise to all cell types of the adult organism. See Thomson et al. (Science, 1998; 282: 1145-1147). Under certain circumstances, such as changes in environmental conditions and / or cell signaling pathways, PSCs exit the self-renewal cycle and differentiate into specialized cell types derived from the three germ layers.

[0004] Initially, PSCs were derived from embryos (embryonic stem cells, ESCs), but more recently, it has been shown that PSCs can be generated from adult (somatic) cells by expression of reprogramming factors. See Takahashi et al. (Cell, 2006, 126(4):663-76). These PSCs, termed induced PSCs (iPSCs), can generally be generated by delivering genetic material encoding reprogramming factors to somatic cells to trigger the reversion of somatic cells to a pluripotent state. As such, iPSCs represent an important source of PSCs that is free of the ethical and technical constraints (e.g., production of patient-matched cell lines) that limit the utility of ESCs.

[0005] As a result of their pluripotency, PSCs, such as iPSCs, are important, for example, in the fields of medical and scientific research, drug discovery, cell therapy, regenerative medicine, and tissue engineering. In applications in which PSCs are subsequently differentiated into specialized cells, for example, in tissue engineering, it is desirable to be able to determine whether any PSCs remain after the differentiation process. By way of example, in cell therapy products and engineered tissues, the presence of residual undifferentiated PSCs poses a quality control challenge, as such cells can form teratomas in vivo (i.e., are tumorigenic).

[0006] Established techniques for detecting the presence of PSCs have included phenotypic pluripotency assays (e.g., in vivo embryoid body formation, teratoma formation) as well as molecular pluripotency assays, including detection of molecular markers associated with PSCs. However, many of the markers differentially expressed in PSCs used for this purpose also show some significant expression level in differentiated cells (albeit at different expression levels and / or patterns), making detection of small numbers of residual PSCs within a larger population of derived tissue cells problematic and difficult to address. Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure addresses the technical limitations described above by providing a subset of marker genes that are uniquely expressed in PSCs. These marker genes are therefore useful as molecular markers for detecting the presence of even a small number of PSCs, including a small number of residual undifferentiated PSCs in a population of cells derived from, for example, primarily differentiated PSCs. Non-limiting embodiments of the present disclosure include: [Means for solving the problem]

[0008] [1] A method for detecting pluripotent stem cells, the method comprising: (a) obtaining a target sample containing a plurality of cells; (b) analyzing the sample of interest to detect expression of at least one marker gene, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 5; (c) determining that the sample of interest contains pluripotent stem cells when expression of the at least one marker gene is detected in the sample of interest; and A method comprising:

[0009] [2] The method according to [1], wherein the target sample comprises induced pluripotent stem cells.

[0010] [3] The method according to [1], wherein the target sample comprises embryonic pluripotent stem cells.

[0011] [4] The method according to [1], wherein the plurality of cells in the target sample is in the form of a cell mixture, a cell aggregate, or a tissue.

[0012] [5] The method according to [1], wherein in step (b), the expression of at least one marker gene is detected by measuring the level of mRNA expression of the marker gene.

[0013] [6] The method according to [1], wherein in step (b), the expression of at least one marker gene is detected by measuring the level of an expressed protein encoded by the marker gene.

[0014] [7] The method of [5], wherein the expression of the at least one marker gene is detected by a process comprising a technique selected from the group consisting of droplet digital polymerase chain reaction (dd-PCR), polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next-generation sequencing.

[0015] [8] The method according to [7], wherein the technique comprises microarray analysis or next-generation sequencing.

[0016] [9] A method for detecting pluripotent stem cells, the method comprising: (a) obtaining a target sample containing a plurality of cells; (b) measuring the expression level of at least one marker gene in the target sample, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 5; (c) comparing the expression level detected in step (b) with a reference expression level, wherein the reference expression level is obtained by measuring the expression level of the at least one marker gene in at least one reference sample, wherein the at least one reference sample comprises a plurality of cells, and wherein the at least one reference sample is substantially free of pluripotent stem cells; (d) determining that the target sample contains pluripotent stem cells when the expression level detected in step (b) is higher than the reference expression level, or determining that the target sample does not substantially contain pluripotent stem cells when the expression level detected in step (b) is equal to or lower than the reference expression level; A method comprising:

[0017]

[10] A method for quantifying the number of pluripotent stem cells in a sample, the method comprising: (a) obtaining a target sample containing a plurality of cells; (b) measuring the expression level of at least one marker gene in the target sample, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 5; (c) comparing the expression level detected in step (b) with a reference expression level, wherein the reference expression level is obtained by measuring the expression level of the at least one marker gene in at least one reference sample, the at least one reference sample comprising a plurality of cells, the at least one reference sample comprising pluripotent stem cells, and substantially all of the cells in the at least one reference sample are pluripotent stem cells; and (d) calculating the amount of pluripotent stem cells in the target sample based on the comparison in step (c); A method comprising:

[0018]

[11] The method according to [9], wherein the target sample comprises induced pluripotent stem cells.

[0019]

[12] The method according to [9], wherein the target sample comprises embryonic pluripotent stem cells.

[0020]

[13] The method according to [9], wherein the plurality of cells in the target sample is in the form of a cell mixture, a cell aggregate, or a tissue.

[0021]

[14] The method according to [9], wherein in step (b), the expression of at least one marker gene is detected by measuring the level of mRNA expression of the marker gene.

[0022]

[15] The method according to [9], wherein in step (b), the expression of at least one marker gene is detected by measuring the level of an expressed protein encoded by the marker gene.

[0023]

[16] The method according to

[14] , wherein the expression of at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next-generation sequencing.

[0024]

[17] The method according to

[10] , wherein the target sample comprises induced pluripotent stem cells.

[0025]

[18] The method according to

[10] , wherein the target sample comprises embryonic pluripotent stem cells.

[0026]

[19] The method according to

[10] , wherein the plurality of cells in the target sample is in the form of a cell mixture, a cell aggregate, or a tissue.

[0027]

[20] The method according to

[10] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of mRNA expression of the marker gene.

[0028]

[21] The method according to

[10] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of an expressed protein encoded by the marker gene.

[0029]

[22] The method according to

[20] , wherein the expression of at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next-generation sequencing.

[0030]

[23] An assay for detecting residual iPSCs in a therapeutic product, the assay comprising: (a) obtaining a sample of a therapeutic product produced from iPSCs, said sample comprising a plurality of cells; (b) analyzing the sample to detect expression of at least one marker gene, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; (c) determining that the therapeutic product contains residual iPSCs when expression of the at least one marker gene is detected in the sample; and The assay comprises:

[0031]

[24] The assay of

[23] , wherein the plurality of cells in the sample is in the form of a cell mixture, a cell aggregate, or a tissue.

[0032]

[25] The assay according to

[23] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of mRNA expression of the marker gene.

[0033]

[26] The assay described in

[23] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of an expressed protein encoded by the marker gene.

[0034]

[27] The assay of

[25] , wherein expression of at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next-generation sequencing.

[0035]

[28] An assay for quantifying the number of residual iPSCs in a therapeutic product, the assay comprising: (a) obtaining a sample of a therapeutic product produced from iPSCs, said sample comprising a plurality of cells; (b) measuring the expression level of at least one marker gene in the sample, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; (c) comparing the expression level detected in step (b) with a reference expression level, wherein the reference expression level is obtained by measuring an expression level of the at least one marker gene in at least one reference sample, wherein the at least one reference sample comprises a plurality of cells, wherein the at least one reference sample comprises iPSCs, and wherein substantially all of the cells in the at least one reference sample are iPSCs; and (d) quantifying the number of remaining iPSCs in the therapeutic product based on the comparison in step (c); The assay comprises:

[0036]

[29] The assay of

[28] , wherein the plurality of cells in the sample is in the form of a cell mixture, a cell aggregate, or a tissue.

[0037]

[30] The assay described in

[28] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of mRNA expression of the marker gene.

[0038]

[31] The assay described in

[28] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of an expressed protein encoded by the marker gene.

[0039]

[32] The assay described in

[30] , wherein expression of at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next-generation sequencing.

[0040]

[33] An assay for detecting residual iPSCs in a therapeutic product, the assay comprising: (a) obtaining a sample of a therapeutic product produced from iPSCs, said sample comprising a plurality of cells; (b) measuring the expression level of at least one marker gene in the sample, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; (c) comparing the expression level detected in step (b) with a reference expression level, wherein the reference expression level is obtained by measuring the expression level of the at least one marker gene in at least one reference sample, wherein the at least one reference sample comprises a plurality of cells, and wherein the at least one reference sample is substantially free of pluripotent stem cells; (d) determining that the therapeutic product contains residual iPSCs when the expression level detected in step (b) is higher than the reference expression level, or determining that the therapeutic product is substantially free of residual iPSCs when the expression level detected in step (b) is equal to or lower than the reference expression level; The assay comprises:

[0041]

[34] The assay of

[33] , wherein the plurality of cells in the sample is in the form of a cell mixture, a cell aggregate, or a tissue.

[0042]

[35] The assay according to

[33] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of mRNA expression of the marker gene.

[0043]

[36] The assay described in

[33] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of an expressed protein encoded by the marker gene.

[0044]

[37] The assay of

[35] , wherein expression of at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next-generation sequencing.

[0045]

[38] An assay for quantifying the number of remaining iPSCs in a therapeutic product at different differentiation times, the assay comprising: (a) obtaining a first sample of a therapeutic product, said first sample being obtained at a first differentiation time or prior to the initiation of differentiation, said first sample containing a plurality of cells; (b) obtaining at least one additional sample of the therapeutic product at a different differentiation time, said at least one additional sample containing a plurality of cells; and (c) measuring the expression level of at least one marker gene in the samples obtained in (a) and (b), wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 5; (d) quantifying the number of iPSCs in the therapeutic product at different differentiation times based on the measurements in step (c); The assay comprises:

[0046]

[39] The assay of

[38] , wherein the plurality of cells in the sample is in the form of a cell mixture, a cell aggregate, or a tissue.

[0047]

[40] The assay described in

[38] , wherein in step (c), the expression of at least one marker gene is detected by measuring the level of mRNA expression of the marker gene.

[0048]

[41] The assay described in

[38] , wherein in step (c), the expression of at least one marker gene is detected by measuring the level of an expressed protein encoded by the marker gene.

[0049]

[42] The assay of

[40] , wherein expression of at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next-generation sequencing.

[0050]

[43] An assay for determining purity of a differentiation product, comprising: (a) obtaining a sample of a differentiation product, said sample containing a plurality of cells; (b) measuring the expression level of at least one marker gene in the sample, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; (c) comparing the expression level detected in step (b) with a reference expression level, wherein the reference expression level is obtained by measuring an expression level of the at least one marker gene in at least one reference sample, wherein the at least one reference sample comprises a plurality of cells, wherein the at least one reference sample comprises iPSCs, and wherein substantially all of the cells in the at least one reference sample are iPSCs; and (d) determining the purity of the differentiation product by calculating the amount of iPSCs in the sample based on the comparison in step (c); The assay comprises:

[0051]

[44] The assay of

[43] , wherein the plurality of cells in the sample is in the form of a cell mixture, a cell aggregate, or a tissue.

[0052]

[45] The assay according to

[43] , wherein in step (b), expression of at least one marker gene is detected by measuring the level of mRNA expression of the marker gene.

[0053]

[46] The assay described in

[43] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of an expressed protein encoded by the marker gene.

[0054]

[47] The assay of

[45] , wherein expression of at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next-generation sequencing.

[0055]

[48] ​​An assay for determining purity of a differentiation product, comprising: (a) obtaining a sample of a differentiation product, said sample containing a plurality of cells; (b) measuring the expression level of at least one marker gene in the sample, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; (c) comparing the expression level detected in step (b) with a reference expression level, wherein the reference expression level is obtained by measuring the expression level of the at least one marker gene in at least one reference sample, wherein the at least one reference sample comprises a plurality of cells, and wherein the at least one reference sample is substantially free of pluripotent stem cells; (d) determining the purity of the differentiation product by calculating the amount of iPSCs in the sample based on the comparison in step (c); The assay comprises:

[0056]

[49] The assay of

[48] , wherein the plurality of cells in the sample is in the form of a cell mixture, a cell aggregate, or a tissue.

[0057]

[50] The assay described in

[48] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of mRNA expression of the marker gene.

[0058]

[51] The assay described in

[48] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of an expressed protein encoded by the marker gene.

[0059]

[52] The assay of

[50] , wherein expression of at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next-generation sequencing.

[0060]

[53] A method for assessing the differentiation potential of an iPSC population, the method comprising: (a) obtaining a first sample of an iPSC population, said first sample being obtained before differentiation of said iPSCs is induced, said first sample comprising a plurality of cells; (b) obtaining at least one additional sample of the population of iPSCs, said at least one additional sample being obtained after differentiation of said iPSCs has been induced, said at least one additional sample containing a plurality of cells; and (c) measuring the expression level of at least one marker gene in the samples obtained in (a) and (b), wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 5; (d) assessing the differentiation potential of iPSCs in said population of iPSCs by comparing the expression levels from the different samples detected in step (c) with each other and / or with a reference expression level; A method comprising:

[0061]

[54] The method according to

[53] , wherein the plurality of cells in the sample is in the form of a cell mixture, a cell aggregate, or a tissue.

[0062]

[55] The method according to

[53] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of mRNA expression of the marker gene.

[0063]

[56] The method according to

[53] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of an expressed protein encoded by the marker gene.

[0064]

[57] The method of

[55] , wherein the expression of at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next-generation sequencing.

[0065]

[58] A method for assessing pluripotency of an iPSC population, the method comprising: (a) obtaining a sample of an iPSC population, said sample containing a plurality of cells; (b) measuring the expression level of at least one marker gene in the sample, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; (c) comparing the expression level detected in step (b) with a reference expression level, wherein the reference expression level is obtained by measuring an expression level of the at least one marker gene in at least one reference sample, the at least one reference sample comprising a plurality of cells, the at least one reference sample comprising iPSCs, and substantially all of the cells in the at least one reference sample are iPSCs capable of differentiation; and (d) assessing the pluripotency of iPSCs in the population of iPSCs based on the comparison in step (c); A method comprising:

[0066]

[59] The method according to

[58] , wherein the plurality of cells in the sample is in the form of a cell mixture, a cell aggregate, or a tissue.

[0067]

[60] The method according to

[58] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of mRNA expression of the marker gene.

[0068]

[61] The method according to

[58] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of an expressed protein encoded by the marker gene.

[0069]

[62] The method according to

[60] , wherein the expression of at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next-generation sequencing.

[0070]

[63] A method for assessing pluripotency of an iPSC population, the method comprising: (a) obtaining a first sample of an iPSC population, said first sample being obtained before differentiation of said iPSCs is induced, said first sample comprising a plurality of cells; (b) obtaining at least one additional sample of the population of iPSCs, said at least one additional sample being obtained after differentiation of said iPSCs has been induced, said at least one additional sample containing a plurality of cells; and (c) measuring the expression level of at least one marker gene in the samples obtained in (a) and (b), wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 5; (c) assessing the pluripotency of iPSC cells in said iPSC population by comparing the expression levels from the different samples detected in step (c) with each other and / or with a reference expression level; A method comprising:

[0071]

[64] The method according to

[63] , wherein the plurality of cells in the sample is in the form of a cell mixture, a cell aggregate, or a tissue.

[0072]

[65] The method according to

[63] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of mRNA expression of the marker gene.

[0073]

[66] The method according to

[63] , wherein in step (b), the expression of at least one marker gene is detected by measuring the level of an expressed protein encoded by the marker gene.

[0074]

[67] The method of

[65] , wherein the expression of at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next-generation sequencing.

[0075]

[68] An assay kit for detecting residual iPSCs in a therapeutic product, the assay kit comprising at least one reagent suitable for specifically detecting the expression level of at least one marker gene, the at least one marker gene expressing a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5.

[0076]

[69] The assay kit described in

[68] , wherein the reagent comprises a nucleic acid, a probe, or one or more primers capable of specifically detecting the expression level of at least one marker gene, which is the at least one marker gene.

[0077]

[70] An assay reagent for detecting residual iPSCs in a therapeutic product, the reagent being capable of specifically detecting the expression level of at least one marker gene, the at least one marker gene expressing a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; The reagents include a nucleic acid, a probe, or one or more primers.

[0078]

[71] An assay kit for quantifying the number of iPSCs in a therapeutic product, the assay kit comprising at least one reagent suitable for specifically detecting the expression level of at least one marker gene, the at least one marker gene expressing a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5.

[0079]

[72] The assay kit described in

[71] , wherein the reagent comprises a nucleic acid, a probe, or one or more primers capable of specifically detecting the expression level of at least one marker gene, which is the at least one marker gene.

[0080]

[73] An assay reagent for quantifying the number of iPSCs in a therapeutic product, the reagent being capable of specifically detecting the expression level of at least one marker gene, the at least one marker gene expressing a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; The reagents include a nucleic acid, a probe, or one or more primers.

[0081]

[74] An assay kit for determining the purity of a differentiation product, the assay kit comprising at least one reagent suitable for specifically detecting the expression level of at least one marker gene, the at least one marker gene expressing a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5.

[0082]

[75] The assay kit described in

[74] , wherein the reagent comprises a nucleic acid, a probe, or one or more primers capable of specifically detecting the expression level of at least one marker gene, which is the at least one marker gene.

[0083]

[76] An assay reagent for determining the purity of a differentiation product, the reagent being capable of specifically detecting the expression level of at least one marker gene, the at least one marker gene expressing a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; The reagents include a nucleic acid, a probe, or one or more primers.

[0084]

[77] An assay kit for assessing the differentiation potential of an iPSC population, the assay kit comprising at least one reagent suitable for specifically detecting the expression level of at least one marker gene, the at least one marker gene expressing a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5.

[0085]

[78] The assay kit described in

[77] , wherein the reagent comprises a nucleic acid, a probe, or one or more primers capable of specifically detecting the expression level of at least one marker gene, which is the at least one marker gene.

[0086]

[79] An assay reagent for assessing the differentiation potential of an iPSC population, the reagent being capable of specifically detecting the expression level of at least one marker gene, the at least one marker gene expressing a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; The reagents include a nucleic acid, a probe, or one or more primers.

[0087]

[80] An assay kit for assessing pluripotency of an iPSC population, the assay kit comprising at least one reagent suitable for specifically detecting the expression level of at least one marker gene, the at least one marker gene expressing a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5.

[0088]

[81] The assay kit described in

[80] , wherein the reagent comprises a nucleic acid, a probe, or one or more primers capable of specifically detecting the expression level of at least one marker gene, which is the at least one marker gene.

[0089]

[82] An assay reagent for assessing pluripotency of an iPSC population, the reagent being capable of specifically detecting the expression level of at least one marker gene, the at least one marker gene expressing a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; The reagents include a nucleic acid, a probe, or one or more primers.

[0090] Incorporation by Reference All patents, publications, and patent applications cited in this specification are herein incorporated by reference for all purposes to the same extent as if each individual patent, publication, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0091] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0092] [Figure 1] 1 depicts detection of expression of marker genes LNCPRESS2, AC009446.1, and AL117378.1 in pure iPSC samples, pure photoreceptor precursor (PRP) cell samples, and iPSC / PRP mixtures containing different iPSC:PRP ratios. Expression of marker genes was measured by preparing cDNA from cell samples followed by droplet digital PCR (dd-PCR).

[0093] [Diagram 2] 1 depicts detection of expression of marker genes LNCPRESS2, AC009446.1, and AL117378.1 in pure iPSC samples, pure retinal pigment epithelial (RPE) cell samples, and iPSC / RPE mixtures containing different iPSC:RPE ratios. Expression of marker genes was measured by preparing cDNA from cell samples followed by droplet digital PCR (dd-PCR).

[0094] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0095] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes one or more cells.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although other methods and materials similar or equivalent to those described herein can be useful in the present invention, the preferred materials and methods are described herein.

[0097] As used herein, "subject", "individual", or "patient" are used interchangeably herein and refer to any member of the phylum Chordata, including, but not limited to, humans and other primates, including non-human primates, such as rhesus monkeys, chimpanzees, and other caudate and anauta species, farm animals, such as cows, sheep, pigs, goats, and horses, domestic mammals, such as dogs and cats, laboratory animals, such as rabbits, mice, rats, and guinea pigs, birds, including domestic, wild, and game birds, such as chickens, turkeys, and other poultry, ducks and geese, and others. The term does not denote a particular age or sex. Thus, the term includes adult, juvenile, and newborn individuals, as well as males and females. In some embodiments, the cells (e.g., stem cells, including pluripotent stem cells, progenitor cells, or tissue-specific cells) are derived from a subject. In some embodiments, the subject is a non-human subject.

[0098] As used herein, "cell culture" refers to cells grown under controlled conditions. Primary cell cultures are cultures of cells, tissues, or organs taken directly from an organism before the first subculture. Cells are expanded in culture when placed in a growth medium under conditions that promote cell growth and / or division, resulting in a larger population of cells. When cells are expanded in culture, the rate of cell proliferation is often measured by the amount of time required to double the number of cells (also known as doubling time).

[0099] As used herein, "feeder layer" refers to a non-proliferative cell layer that can be used to support the growth of stem cells. Protocols for the generation of feeder layers are known in the art and available on the Internet, for example, at the National Stem Cell Resource website maintained by the American Type Culture Collection (ATCC).

[0100] As used herein, "differentiation" refers to the process by which unspecialized cells, such as pluripotent stem cells or other stem cells, acquire specialized structural and / or functional characteristics characteristic of mature cells. As used herein, "expanding" or "proliferating" can refer to the process of increasing the number of cells in a cell culture by cell division. In certain embodiments herein, during the expansion or proliferation phase, cells do not differentiate to form mature cells, but rather divide to form more (undifferentiated) cells.

[0101] As used herein, "embryoid bodies" refer to three-dimensional aggregates of pluripotent stem cells. These cells are capable of undergoing differentiation into cells of the three germ layers: endoderm, mesoderm, and ectoderm. The three-dimensional structure allows for differentiation and morphogenesis, including the establishment of complex cell adhesion and paracrine signaling within the embryoid body microenvironment.

[0102] As used herein, "stem cell" refers to a cell that has the ability to self-renew, i.e., to undergo numerous cell division cycles while maintaining its non-terminally differentiated state. Stem cells can be totipotent, pluripotent, multipotent, oligopotent, or unipotent. Stem cells can be, for example, embryonic, fetal, amniotic, adult, or induced pluripotent stem cells.

[0103] As used herein, "pluripotent stem cells" (PSCs) refer to cells that have the capacity to self-renew indefinitely and differentiate into any other cell type of an adult organism. Generally, pluripotent stem cells are stem cells that are capable of inducing teratomas when transplanted into immune-deficient (SCID) mice, capable of differentiating into cell types of all three germ layers (e.g., capable of differentiating into ectodermal, mesodermal, and endodermal cell types), and express one or more markers characteristic of PSCs. Examples of such markers expressed by PSCs, such as embryonic stem cells (ESCs) and iPSCs, include Oct4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, and REX1. PSCs within the scope of the present disclosure can be, for example, embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0104] As used herein, "induced pluripotent stem cells" (iPSCs) refer to a pluripotent stem cell type artificially derived from a non-pluripotent cell, typically a somatic cell. In some embodiments, the somatic cell is a human somatic cell. Examples of somatic cells include, but are not limited to, dermal fibroblasts, bone marrow-derived mesenchymal cells, cardiomyocytes, keratinocytes, hepatocytes, gastric cells, neural stem cells, lung cells, kidney cells, splenocytes, and pancreatic cells. Other examples of somatic cells include cells of the immune system, including, but not limited to, B cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes / macrophages, myeloid-derived suppressor cells, natural killer (NK) cells, T cells, thymocytes, and hematopoietic stem cells.

[0105] iPSCs can be generated by expressing or inducing the expression of one or a combination of factors (referred to herein as reprogramming factors) in somatic cells to reprogram the somatic cells. iPSCs can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In some cases, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (Oct3 / 4), Sox2, c-Myc, and Klf4, Nanog, and Lin28. In some embodiments, somatic cells can be reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or at least four reprogramming factors to reprogram the somatic cells into pluripotent stem cells. Cells can be reprogrammed by introducing the reprogramming factors using vectors, including, for example, lentivirus, retrovirus, adenovirus, and Sendai virus vectors. Alternatively, non-viral techniques for introducing reprogramming factors include, for example, mRNA transfection, miRNA infection / transfection, PiggyBac, minicircle vectors, and episomal plasmids. iPSCs can also be generated by introducing reprogramming factors or activating endogenous programming genes, for example, using CRISPR-Cas9-based technology.

[0106] As used herein, an "embryonic stem cell" is an embryonic cell derived from embryonic tissue, preferably the inner cell mass of a blastocyst or morula, optionally serially passaged as a cell line. The term includes cells isolated from one or more blastomeres of an embryo, preferably without destroying the remainder of the embryo. The term also includes cells produced by somatic cell nuclear transfer. ESCs can be produced or derived from mammalian embryos at the zygote, blastomere, or blastocyst stage, for example, produced by fusion of a sperm cell with an egg cell, nuclear transfer, or parthenogenesis. Human ESCs include, but are not limited to, MAO1, MAO9, ACT-4, No.3, H1, H7, H9, H14, and ACT30 embryonic stem cells. Exemplary pluripotent stem cells include embryonic stem cells derived from the inner cell mass (ICM) of a blastocyst stage embryo, as well as embryonic stem cells derived from one or more blastomeres of a cleavage stage embryo or a morula stage embryo. These embryonic stem cells can develop from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. PSCs alone are unable to develop into a fetus or an adult animal when implanted in the uterus, as they lack the potential to contribute to all extraembryonic tissues (e.g., placenta in vivo or trophoblast in vitro).

[0107] As used herein, the terms "wild-type," "naturally occurring," and "unmodified" are used herein to mean the typical (or most common) form, appearance, phenotype, or strain that occurs in nature, e.g., the typical form of a cell, organism, polynucleotide, protein, macromolecular complex, gene, RNA, DNA, or genome that is present in a natural source and isolatable therefrom. A wild-type form, appearance, phenotype, or strain serves as the parent of origin prior to intentional modification. As such, mutated, variant, engineered, recombinant, and modified forms are not wild-type forms.

[0108] "Isolated," when referring to a cell, polynucleotide, or polypeptide molecule, including, for example, a marker molecule, means that the indicated molecule is discrete and separated from the organism in which it is found in nature and / or exists in the substantial absence of other biological macromolecules of the same type. The term "isolated" with respect to a polynucleotide refers to a nucleic acid molecule that is devoid, in whole or in part, of sequences that are normally associated with it in nature, or a sequence that exists in nature but has heterologous sequences associated with it, or a molecule that is dissociated from the chromosome.

[0109] As used herein, the term "purified" means that preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the molecule is present.

[0110] The terms "engineering," "genetic engineering," "genetically modified," "recombinant," "modified," "non-naturally occurring," and "non-native" refer to the deliberate human manipulation of the genome of an organism or cell. These terms encompass methods of genome modification, including genome editing as defined herein, as well as techniques that alter gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, codon optimization, and the like. Methods of genetic engineering are known in the art.

[0111] As used herein, the terms "nucleic acid sequence," "nucleotide sequence," and "oligonucleotide" all refer to a polymeric form of nucleotides. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides that, when in linear form, has one 5' end and one 3' end and can contain one or more nucleic acid sequences. The nucleotides can be deoxyribonucleotides (DNA), ribonucleotides (RNA), analogs thereof, or combinations thereof, and can be of any length. Polynucleotides can perform any function and can have a variety of secondary and tertiary structures. These terms encompass known analogs of natural nucleotides as well as nucleotides modified at the base, sugar, and / or phosphate moieties. Analogs of a particular nucleotide have the same base-pairing specificity (e.g., analogs of A and T base pairs). A polynucleotide can contain one modified nucleotide or multiple modified nucleotides. Examples of modified nucleotides include fluorinated nucleotides, methylated nucleotides, and nucleotide analogs. The nucleotide structure can be modified before or after assembling the polymer. Following polymerization, the polynucleotide may be additionally modified, for example, via conjugation with a labeling moiety or a target binding moiety. The nucleotide sequence may incorporate non-nucleotide moieties. These terms also encompass synthetic, naturally occurring, and / or non-naturally occurring nucleic acids that contain modified backbone residues or linkages that have similar binding properties to a reference polynucleotide (e.g., DNA or RNA). Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs), locked nucleic acids (LNA™) (Exiqon, Inc., Woburn, Mass.) nucleosides, glycol nucleic acids, bridged nucleic acids, and morpholino structures. Peptide nucleic acids (PNAs) are synthetic homologs of nucleic acids in which the polynucleotide phosphate-sugar backbone is replaced with a flexible pseudopeptide polymer. Nucleobases are linked to the polymer.PNAs have the ability to hybridize with high affinity and specificity to complementary sequences of RNA and DNA. Polynucleotide sequences are presented herein in the conventional 5' to 3' orientation unless otherwise indicated.

[0112] As used herein, "sequence identity" generally refers to the percent identity of nucleotide bases or amino acids when comparing a first polynucleotide or polypeptide to a second polynucleotide or polypeptide using algorithms with various weighting parameters. Sequence identity between two polynucleotides or two polypeptides can be determined using sequence alignment by a variety of methods and computer programs (e.g., Exonerate, BLAST, CS-BLAST, FASTA, HMMER, L-ALIGN, etc.) available through the World Wide Web at sites including, but not limited to, GENBANK (www.ncbi.nlm.nih.gov / genbank / ) and EMBL-EBI (www.ebi.ac.uk.). Sequence identity between two polynucleotide or two polypeptide sequences is generally calculated using standard default parameters of the various methods or computer programs. A high degree of sequence identity between two polynucleotides or two polypeptides is often about 90% to 100% identity over the length of the reference polynucleotide or polypeptide or query sequence, for example, about 90% or more identity, about 91% or more identity, about 92% or more identity, about 93% or more identity, about 94% or more identity, about 95% or more identity, about 96% or more identity, about 97% or more identity, about 98% or more identity, or about 99% or more identity over the length of the reference polynucleotide or polypeptide or query sequence. Sequence identity can also be calculated over the overlapping regions of two sequences when only a portion of the two sequences are alignable.

[0113] Moderate sequence identity between two polynucleotides or two polypeptides is often about 80% identity to about 90% identity over the length of a reference polynucleotide or polypeptide or query sequence, for example, about 80% or more identity, about 81% or more identity, about 82% or more identity, about 83% or more identity, about 84% or more identity, about 85% or more identity, about 86% or more identity, about 87% or more identity, about 88% or more identity, or about 89% or more identity, but less than 90%, over the length of a reference polynucleotide or polypeptide or query sequence.

[0114] A low degree of sequence identity between two polynucleotides or two polypeptides is often between about 50% identity and 75% identity over the length of a reference polynucleotide or polypeptide or query sequence, for example, about 50% or more identity, about 60% or more identity, about 70% or more identity, but less than 75% identity over the length of a reference polynucleotide or polypeptide or query sequence.

[0115] The present disclosure contemplates polynucleotides and polypeptides, e.g., molecular markers, that may have less than 100% identity to a known or reference polynucleotide or polypeptide sequence (e.g., a known or reference marker polynucleotide sequence or a known or reference marker polypeptide sequence) or a query sequence.

[0116] As used herein, "hybridization," "hybridize," or "hybridizing" is the process of combining two complementary single-stranded DNA or RNA molecules to form a single double-stranded molecule (DNA / DNA, DNA / RNA, RNA / RNA) through hydrogen base pairing. Hybridization stringency is typically determined by the hybridization temperature and the salt concentration of the hybridization buffer, e.g., high temperature and low salt provide high stringency hybridization conditions. Examples of salt concentration ranges and temperature ranges for different hybridization conditions are as follows: high stringency, approximately 0.01 M to approximately 0.05 M salt, T m Hybridization temperature 5°C to 10°C lower than m hybridization temperature 20° C. to 29° C. lower than 0.33 M to 0.82 M salt; m The hybridization temperature is 40°C to 48°C lower than the T m is calculated by standard methods known in the art (see, for example, Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: New York (1982); Casey, J., et al., Nucleic Acids Research 4:1539-1552 (1977); Bodkin, DK, et al., Journal of Virological Methods 10(1):45-52 (1985); Wallace, RB, et al., Nucleic Acids Research 9(4):879-894 (1981)). mAlgorithm prediction tools that estimate are also widely available. High stringency hybridization conditions typically refer to conditions under which a polynucleotide complementary to a target sequence hybridizes primarily to the target sequence and does not substantially hybridize to non-target sequences. Typically, hybridization conditions are medium stringency, preferably high stringency.

[0117] As used herein, "complementarity" refers to the ability of a nucleic acid sequence to form hydrogen bonds with another nucleic acid sequence (e.g., via canonical Watson-Crick base pairing). The percentage of complementarity represents the percentage of residues in a nucleic acid sequence that can form hydrogen bonds with a second nucleic acid sequence. If two nucleic acid sequences have 100% complementarity, the two sequences are fully complementary; that is, every contiguous residue in a first polynucleotide will hydrogen bond with the same number of contiguous residues in the second polynucleotide.

[0118] As used herein, "binding" refers to a non-covalent interaction between macromolecules (e.g., between a protein and a polynucleotide, between a polynucleotide and a polynucleotide, between a protein and a protein, etc.). Such non-covalent interactions are also referred to as "associations" or "interactions" (e.g., when a first macromolecule interacts with a second macromolecule, the first macromolecule binds non-covalently to the second macromolecule). Some portions of the binding interaction may be sequence specific (the terms "sequence-specific binding," "sequence-specific binding," "site-specific binding," and "site-specific binding" are used interchangeably herein). Binding interactions can be characterized by a dissociation constant (Kd). "Binding affinity" refers to the strength of the binding interaction. Increased binding affinity correlates to a lower Kd.

[0119] As used herein, "gene" refers to a polynucleotide sequence comprising exons and associated regulatory sequences. A gene may further comprise introns and / or untranslated regions (UTRs).

[0120] As used herein, the term "operably linked" refers to polynucleotide sequences or amino acid sequences placed in a functional relationship with each other. For example, a regulatory sequence (e.g., a promoter or enhancer) is "operably linked" to a polynucleotide if it regulates or contributes to the modulation of the transcription of a polynucleotide encoding a gene product. An operably linked regulatory element is typically adjacent to the coding sequence. However, an enhancer can function even if separated from the promoter by more than a few kilobases. Thus, some regulatory elements can be operably linked to a polynucleotide sequence without being adjacent to the polynucleotide sequence. Similarly, a translational regulatory element contributes to the modulation of protein expression from a polynucleotide. In some embodiments, operably linked elements can be heterologous to each other.

[0121] As used herein, "expression" refers to the transcription of a polynucleotide from a DNA template, resulting in, for example, messenger RNA (mRNA) or other RNA transcripts (e.g., non-coding, such as structural or scaffolding RNA). The term further refers to the process by which transcribed mRNA is translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as the "gene product." Expression may include splicing of the mRNA in a eukaryotic cell, if the polynucleotide is derived from genomic DNA.

[0122] A "coding sequence" or a sequence "encoding" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences in vitro or in vivo. The boundaries of the coding sequence are determined by a start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0123] As used herein, "complementary DNA (cDNA) sequence" refers to a DNA sequence synthesized from an RNA template, generally via reverse transcription, and may include first strand cDNA molecules, second strand cDNA molecules, or double stranded cDNA molecules containing both first and second strand cDNA. A "corresponding complementary DNA (cDNA) sequence" for a given RNA template (e.g., an mRNA transcript) refers to a DNA sequence that may result from reverse transcribing the RNA template.

[0124] As used herein, the term "modulate" refers to a change in the quantity, degree, or amount of a property, activity, function, or physical molecule. "Modulation" of gene expression includes both gene activation and gene repression. Modulation can be assayed by determining any property that is directly or indirectly affected by expression of the target gene. Such properties include, for example, changes in RNA or protein levels, protein activity, product levels, expression of a gene, or activity levels of a reporter gene.

[0125] As used herein, a "different" or "altered" expression level of, for example, a marker gene of the present disclosure is a difference that is measurably different, preferably a statistically significant difference (e.g., not attributable to the standard error of the assay employed to assess expression). In some embodiments, for example, the difference in gene expression level of a marker gene of the present disclosure in a sample of interest compared to a control or reference sample may be, for example, greater than a 2-fold difference, greater than a 5-fold difference, greater than a 10-fold difference, greater than a 20-fold difference, greater than a 50-fold difference, greater than a 75-fold difference, greater than a 100-fold difference, greater than a 250-fold difference, greater than a 500-fold difference, greater than a 750-fold difference, greater than a 1,000-fold difference, greater than a 5,000-fold difference, greater than a 10,000-fold difference, greater than a 25,000-fold difference, greater than a 50,000-fold difference, greater than a 75,000-fold difference, greater than a 100,000-fold difference, depending, for example, on the gene expression being analyzed and the analysis technique. the fold difference may be greater than 250,000-fold, greater than 500,000-fold, greater than 750,000-fold, greater than 1,000,000-fold, greater than 2,500,000-fold, greater than 5,000,000-fold, greater than 7,500,000-fold, greater than 10,000,000-fold, greater than 50,000,000-fold, greater than 100,000,000-fold, greater than 250,000,000-fold, greater than 500,000,000-fold, greater than 750,000,000-fold, greater than 1,000,000,000-fold, greater than 5,000,000,000-fold, or greater than 10,000,000,000-fold.

[0126] As used herein, "vector" and "plasmid" refer to a polynucleotide vehicle for introducing genetic material into a cell. A vector can be linear or circular. A vector can contain a replication sequence (e.g., an origin of replication) capable of replicating the vector in a suitable host cell. Upon transformation of a suitable host, the vector can replicate and function independently of the host genome or can integrate into the host genome. Vector design depends, among other things, on the intended use and host cell for the vector, and design of the vector of the invention for a particular use and host cell is within the level of skill in the art. Four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. In many cases, vectors contain an origin of replication, a multiple cloning site, and / or a selectable marker. An expression vector can contain an expression cassette. "Recombinant virus" refers to a virus that has been genetically modified, for example, by the addition or insertion of a heterologous nucleic acid construct into the viral genome or a portion thereof.

[0127] As used herein, "expression cassette" refers to a polynucleotide construct produced using recombinant methods or by synthetic means that includes a regulatory sequence operably linked to a selected polynucleotide to facilitate expression of the selected polynucleotide in a host cell. For example, the regulatory sequence can facilitate transcription of the selected polynucleotide in a host cell or transcription and translation of the selected polynucleotide in a host cell. The expression cassette can, for example, be integrated into the genome of the host cell or can be present in a vector to form an expression vector.

[0128] As used herein, the term "to" includes the ends of a given range (e.g., a length of about 1 to about 50 nucleotides includes 1 nucleotide and 50 nucleotides).

[0129] As used herein, the term "amino acid" refers to natural and synthetic (non-natural) amino acids, including amino acid analogs, modified amino acids, peptidomimetics, glycine, and D or L optical isomers.

[0130] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acids. A polypeptide may be of any length. It may be branched or linear, it may be interrupted by non-amino acids, and it may contain modified amino acids. These terms also refer to amino acid polymers that have been modified, for example, through acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, pegylation, biotinylation, cross-linking, and / or conjugation (e.g., with a labeling moiety or ligand). Polypeptide sequences are presented herein in the conventional N-terminal to C-terminal orientation unless otherwise indicated. Polypeptides and polynucleotides can be produced using routine techniques in the field of molecular biology.

[0131] As used herein, the terms "fusion protein" and "chimeric protein" refer to a single protein created by joining two or more proteins, protein domains, or protein fragments that do not naturally occur together in a single protein. Fusion proteins can include epitope tags (e.g., histidine tags, FLAG® (Sigma Aldrich, St. Louis, Mo.) tags, Myc tags), reporter protein sequences (e.g., glutathione-S-transferase, beta-galactosidase, luciferase, green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein), and / or nucleic acid sequence binding domains (e.g., DNA binding domains or RNA binding domains).

[0132] As used herein, a "moiety" refers to a portion of a molecule. A moiety can be a functional group or can describe a portion of a molecule that has multiple functional groups (e.g., that share a common structural aspect). The terms "moiety" and "functional group" are typically used interchangeably, although "functional group" more specifically refers to a portion of a molecule that includes some common chemical behavior. "Moiety" is often used as a structural description.

[0133] The term "effective amount" or "therapeutically effective amount" of a composition or agent, e.g., an engineered tissue, as provided herein, refers to an amount of a composition or agent sufficient to provide a desired response. Such response will depend on the particular disease involved. By way of example, in some embodiments, it is desirable to produce differentiated cells or tissues from PSCs that are administered to a subject, e.g., to treat one or more symptoms, diseases, or defects.

[0134] As used herein, "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, regardless of the method used for the insertion. For example, transformation can be by direct uptake, transfection, infection, etc. The exogenous polynucleotide may be maintained as a non-integrated vector, such as an episome, or alternatively, may be integrated into the host genome.

[0135] Methods for Producing iPSCs

[0136] The present disclosure relates, in part, to methods for detecting the presence of PSCs. In certain embodiments herein, the PSCs are primarily residual undifferentiated PSCs present in a population of PSC-derived differentiated cells.

[0137] The present disclosure contemplates the generation of iPSC cells from somatic cells, including, for example, human somatic cells. The somatic cells may be derived from humans or non-human animals, including, for example, humans and other primates, including non-human primates, such as rhesus monkeys, chimpanzees, and other caudate and anauta species, farm animals, such as cows, sheep, pigs, goats, and horses, domestic mammals, such as dogs and cats, laboratory animals, such as rabbits, mice, rats, and guinea pigs, birds, including domestic, wild, and game birds, such as chickens, turkeys, and other poultry, ducks and geese, and others. In some embodiments, the somatic cells are selected from keratinizing epithelial cells, mucosal epithelial cells, exocrine epithelial cells, endocrine cells, hepatocytes, epithelial cells, endothelial cells, fibroblasts, muscle cells, cells of the blood and immune systems, cells of the nervous system, including neurons and glial cells, pigment cells, and progenitor cells, including hematopoietic stem cells. Somatic cells may be fully differentiated (specialized) or may not be fully differentiated. By way of example, undifferentiated progenitor cells and terminally differentiated mature cells that are not PSCs, including somatic stem cells, can be used. Somatic cells may be from animals of any age, including adult and fetal cells.

[0138] The somatic cells may be of mammalian origin. Allogeneic cells may be used, for example, if the cells are to be used for transplantation in vivo. In some embodiments, the iPSCs are not MHC / HLA matched to the subject. In some embodiments, the iPSCs are MHC / HLA matched to the subject. For example, in embodiments where iPSCs are used to produce PSC-derived cells for use in regenerative medicine, the somatic cells may be obtained from the subject to be treated or from another subject having the same or substantially the same HLA type as that of the subject. The somatic cells may be cultured, for example, prior to nuclear reprogramming, or may be reprogrammed without culture after isolation.

[0139] For example, to introduce reprogramming factors into somatic cells, viral vectors can be used, including vectors derived from viruses such as SV40, adenovirus, vaccinia virus, adeno-associated virus, herpes virus (including HSV and EBV), Sindbis virus, alphavirus, human herpes virus vectors (HHV) (e.g., HHV-6 and HHV-7), retroviruses, etc. Lentiviruses include, but are not limited to, human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), bovine immunodeficiency virus (BIV), ovine visna virus (VISNA), and caprine arthritis-encephalitis virus (CAEV). Lentiviral vectors are capable of infecting non-dividing cells and can be used for gene transfer and expression of nucleic acid sequences both in vivo and in vitro. Viral vectors can be targeted to specific cell types by linking viral proteins, such as envelope proteins, to binding agents, such as antibodies, or specific ligands (e.g., to target receptors or proteins on or within specific cell types).

[0140] In some embodiments, viral vectors, such as lentiviral vectors, can integrate into the genome of the host cell. The genetic material thus transferred is then transcribed and possibly translated into proteins within the host cell. In other embodiments, viral vectors are used that do not integrate into the genome of the host cell.

[0141] Viral gene delivery systems can be RNA-based or DNA-based viral vectors. Episomal gene delivery systems can be, for example, plasmids, Epstein-Barr Virus (EBV)-based episomal vectors, yeast-based vectors, adenovirus-based vectors, Simian Virus 40 (SV40)-based episomal vectors, bovine papilloma virus (BPV)-based vectors, or lentiviral vectors.

[0142] Somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to those skilled in the art. Those skilled in the art can easily produce induced pluripotent stem cells. See, for example, US Patent Application Publication No. 2009 / 0246875, US Patent Application Publication No. 2010 / 0210014, US Patent Application Publication No. 2012 / 0276636, US Patent Nos. 8,058,065, 8,129,187, and 8,268,620 (all of which are incorporated herein by reference).

[0143] In general, reprogramming factors that can be used to generate induced pluripotent stem cells, either alone, in combination, or in fusion with a transactivation domain, include, but are not limited to, one or more of the following genes: Oct4 (Oct3 / 4, Pou5f1), Sox (e.g., Sox1, Sox2, Sox3, Sox18, or Sox15), Klf (e.g., Klf4, Klf1, Klf3, Klf2, or Klf5), Myc (e.g., c-myc, N-myc, or L-myc), nanog, or LIN28. Examples of these gene and protein sequences include the following accession numbers: mouse MyoD, M84918, NM_010866; mouse Oct4 (POU5F1), NM_013633; mouse Sox2, NM_011443; mouse Klf4, NM_010637; mouse c-Myc, NM_001177352, NM_001177353, NM_001177354; mouse Nanog, NM_0280 16, mouse Lin28, NM_145833, human MyoD, NM_002478, human Oct4(POU5F1), NM_002701, NM_203289, NM_001173531, human Sox2, NM_003106, human Klf4, NM_004235, human c-Myc, NM_002467, human Nanog, NM_024865, and / or human Lin28, NM_024674 are provided. Also contemplated are sequences similar thereto, including those with at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are utilized.

[0144] Exemplary reprogramming factors for the generation of iPSCs include: (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17, or Sox18, and Klf4 can be replaced with Klf1, Klf2, or Klf5); (2) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, SV40 large T antigen (SV40LT); (3) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, human papillomavirus (HPV) 16 E6; (4) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E7; (5) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7, (6)Oct3 / 4, Klf4, Sox2, L-Myc, TERT, Bmi1, (7)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, (8)Oct3 / 4 , Klf4, Sox2, L-Myc, Lin28, SV40LT, (9)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, TERT, SV40LT, (10)Oct 3 / 4, Klf4, Sox2, L-Myc, SV40LT, (11)Oct3 / 4, Esrrb, Sox2, L-Myc (Esrrb can be replaced by Esrrg), (12)Oct3 / 4, Klf4, Sox2, (13)Oct3 / 4, Klf4, Sox2, TERT, SV40LT, (14)Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, (15) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E7, (16) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7, (17) Oct3 / 4, Klf4, Sox2, TERT, Bmi1, (18) Oct3 / 4, Klf4, Sox2, Lin28, (19) Oct3 / 4, Klf4, Sox2, Lin28, SV40LT, (20) Oct3 / 4, Klf4, Sox2, Lin28, TERT, SV40LT, (21) Oct3 / 4, Klf4, Sox2, SV40LT, or (22) Oct3 / 4, Esrrb, Sox2 (Esrrb can be replaced by Esrrg).

[0145] During and after preparation of iPSCs, cells can be cultured in a suitable culture medium depending on the cell type, including, for example, Dulbecco's Modified Eagle Medium (DMEM), DMEM F12 medium, Eagle's Minimum Essential Medium, F-12K medium, Iscove's Modified Dulbecco's Medium, Knockout DMEM, or RPMI-1640 medium. Also contemplated is supplementation of cell culture medium with mammalian serum. Examples of such serum include fetal bovine serum (FBS), bovine serum (BS), calf serum (CS), fetal calf serum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum (HS), human serum, chicken serum, porcine serum, sheep serum, rabbit serum, rat serum (RS), serum replacement, and bovine embryo fluid. For example, cells can be isolated and / or expanded in a total serum (e.g., FBS) or serum replacement concentration of about 0.5% to about 5% or more, including about 5% to about 15% or more, e.g., about 20%, about 25%, or about 30%.

[0146] Additional supplements can also be used to provide cells with trace elements for optimal growth and expansion. Such supplements include insulin, transferrin, sodium selenium, and combinations thereof. These components can be included in salt solutions, such as, but not limited to, Hank's Balanced Salt Solution, Earle's Salt Solution, antioxidant supplements, MCDB-201, phosphate buffered saline (PBS), N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES), nicotinamide, ascorbic acid and / or ascorbic acid-2-phosphate, as well as additional amino acids. Such amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-inositol, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

[0147] Antibiotics are also typically used in cell culture to reduce bacterial, mycoplasmal, and fungal contamination. Such antibiotic or antifungal compounds may include, for example, penicillin / streptomycin, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin.

[0148] Optionally, hormones can also be used in the cell culture, including, but not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, beta estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine, and L-thyronine. Beta-mercaptoethanol can also be supplemented to the cell culture medium.

[0149] Lipids and lipid carriers can also be used to supplement cell culture media, depending on the cell type and the fate of the differentiated cells. Such lipids and carriers can include, but are not limited to, cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic acid-oleic acid-arachidonic acid conjugated to albumin, unconjugated and oleic acid conjugated to albumin, among others. Albumin can also be used in fatty acid free formulations.

[0150] Cells in culture can be maintained either in suspension or attached to a solid support, e.g., attached to extracellular matrix components and synthetic or biopolymers. Additional factors useful for enhancing attachment to solid supports include, for example, types I, II, and IV collagen, concanavalin A, chondroitin sulfate, fibronectin, fibronectin-like polymers, gelatin, laminin, poly-D and poly-L-lysine, matrigel, thrombospondin, and / or vitronectin.

[0151] Cultures of iPSCs may also contain cellular factors that cause the iPSCs to remain in an undifferentiated form, including, for example, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and combinations thereof.

[0152] iPSCs typically display the characteristic morphology of human embryonic stem cells (hESCs) and express the pluripotency factor NANOG. Embryonic stem cell-specific surface antigens (SSEA-3, SSEA-4, TRA1-60, TRA1-81) can also be used to identify fully reprogrammed human cells. Moreover, at the functional level, PSCs, such as ESCs and iPSCs, also demonstrate the ability to differentiate into lineages from all three embryonic germ layers and form teratomas in vivo (e.g., in SCID mice).

[0153] Methods for differentiating PSCs

[0154] The present disclosure further contemplates differentiating PSCs, including ESCs and iPSCs, into differentiated (specialized) cells and then distinguishing the remaining PSCs from the differentiated cells, for example, using the marker genes described herein.

[0155] PSCs may be partially or fully specialized cells, such as hematopoietic progenitors, erythrocytes, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, and platelets, neural progenitors, neurons, such as adrenergic or dopaminergic neurons, motor neurons, peripheral neurons, astrocytes and oligodendrocytes, microglia, pigment epithelial cells, skin cells, and inner ear cells, absorptive cells, goblet cells, Paneth cells, and enteroendocrine cells, hepatocytes, and other cells. The pluripotent stem cells can be differentiated into any desired cell type, including alveolar spleen, pancreatic progenitor cells, insulin producing cells, bile duct cells, alveolar epithelial cells, and intestinal epithelial cells, keratinocytes that reside at the base of hair follicles and give rise to both hair follicles and epidermis, cardiomyocytes, cardiac progenitor, epicardial cells, skeletal muscle cells, skeletal myoblasts, endothelial cells, including vascular endothelial cells, hepatic cells, bone cells, chondrocytes, renal progenitor and renal epithelial cells, mesenchymal stem cells, cells of endoderm-related tissues, retinal pigment epithelial cells, and photoreceptor precursor cells. Methods for differentiating pluripotent stem cells are known in the art.

[0156] Markers for detecting PSC

[0157] The present disclosure contemplates the detection of markers specific for pluripotent stem cells, which can be used, for example, to confirm the presence and / or absence of one or more pluripotent stem cells in a sample. In particular, the present disclosure relates to nucleic acid and polypeptide markers selectively expressed by pluripotent stem cells, and methods for detecting the presence and / or absence of one or more pluripotent stem cells by detecting the presence and / or absence of one or more such markers.

[0158] In some embodiments of the present invention, the expression of at least one marker gene is detected and / or measured. In some embodiments, the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence selected from the following: AC009446.1 (SEQ ID NO: 1), AL117378.1a (SEQ ID NO: 2), AL117378.1b (SEQ ID NO: 3), AL117378.1c (SEQ ID NO: 4), and LNCPRESS2 (SEQ ID NO: 5). In some embodiments, at least two, at least three, at least four, or five of the above markers may be detected.

[0159] In some embodiments, at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having less than 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5. By way of example, contemplated herein is the detection of at least one marker gene that expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5. In some embodiments, at least two, at least three, at least four, or at least five of the above variant sequences may be detected.

[0160] The markers of the present disclosure may be detected individually, in combination, sequentially, in parallel, simultaneously, and / or in conjunction with other markers that may or may not be specific to PSCs, such as markers that are also expressed in differentiated cells but in different amounts or with different expression patterns compared to PSCs.

[0161] In some embodiments, in addition to measuring and / or detecting the expression of at least one marker gene expressing a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5 (and / or in addition to measuring and / or detecting the expression of at least one marker gene expressing a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5), at least one additional marker specific for PSCs may be detected and / or measured.

[0162] In some embodiments, the at least one additional marker gene that is specific for PSCs and can be detected and / or measured is selected from the group consisting of AC106875.1a (SEQ ID NO: 6), AC106875.1b (SEQ ID NO: 7), AL138720.1a (SEQ ID NO: 8), AL138720.1b (SEQ ID NO: 9), AL353052.1 (SEQ ID NO: 10), AL392023.1 (SEQ ID NO: 11), AL591742.2 (SEQ ID NO: 12), AP002856.2 (SEQ ID NO: 13), C11orf86 (SEQ ID NO: 14), CCDC172a (SEQ ID NO: 15), CCDC172b (SEQ ID NO: 16), and / or AC106875.1b (SEQ ID NO: 17). No. 16), CCDC172c (SEQ ID NO: 17), CCDC172d (SEQ ID NO: 18), DPPA5 (SEQ ID NO: 19), FOXI2 (SEQ ID NO: 20), LINC01194a (SEQ ID NO: 21), LINC01194b (SEQ ID NO: 22), LINC01194c (SEQ ID NO: 23), LINC01194d (SEQ ID NO: 24), SLC52A3a (SEQ ID NO: 25), SLC52A3b (SEQ ID NO: 26), SLC52A3c (SEQ ID NO: 27), and TCL1B (SEQ ID NO: 28).

[0163] In some embodiments, at least one additional marker gene that is specific for PSCs and can be detected and / or measured expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having less than 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-28. By way of example, contemplated herein is the detection of at least one additional marker gene that expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6-28.

[0164] In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, or at least twenty-three of the additional or more markers or variant sequences thereof may be detected.

[0165] In some embodiments, the expression of one or more of the markers listed above is measured to determine the presence of PSCs, for example, in a composition, solution, cell aggregate, cell suspension, or tissue.

[0166] In certain embodiments, expression of one or more markers in a plurality of cells, such as a composition, solution, cell aggregate, cell suspension, tissue, etc., is compared to expression of the same one or more markers by a control or reference sample. The control or reference sample may contain a plurality of cells, such as a composition, solution, cell aggregate, cell suspension, tissue, etc. The control or reference sample may, for example, contain a plurality of PSCs, such as ESCs, iPSCs, or both (e.g., a positive control). Additionally or alternatively, the control or reference sample may, for example, contain a plurality of non-PSCs, such as specialized cells or non-terminally differentiated non-PSCs (e.g., a negative control). In some embodiments, the control or reference sample is substantially free of PSCs. In some embodiments, the control or reference sample is free of PSCs. In some embodiments, the only cells or substantially the only cells in the control or reference sample are PSCs. "Substantially free of PSCs" means that the amount of PSCs in the sample is below the limit of detection under the assay conditions. By "substantially the only cells in a control or reference sample are PSCs" it is meant that the amount of non-PSCs in the sample is undetectable under the assay conditions.

[0167] In some embodiments, the expression level of one or more markers of the present disclosure in a sample of interest is compared to a control or reference value using normalized data. Normalization can be based, for example, on the expression levels of one or more reference genes or gene products, e.g., genes understood to be consistently expressed between undifferentiated and differentiated cells and / or under different conditions. Normalization of expression data can also be based on global gene expression patterns. Other data normalization methods contemplated herein include, for example, Global Rank Invariant Set Normalization (GRSN), Cross-Correlation Normalization (Xcorr), Nonparametric Variable Selection and Approximation (NVSA), Kernel Density Weighted Loess Normalization (KWDL), Kernel Density Quantile Normalization (KDQ), Iterative Rank Order Normalization (IRON), Minimal Variant Set Normalization (LVS), Modified Minimal Variant Set Normalization (LVSmiR), Invariant Normalization, HMM-Assisted Normalization (HMM), Biological Sequence Normalization (BSR), and Normalization (MSR). These include Kehring normalization (BSN), support vector regression (SVR), invariant set normalization (ISN), spike-in normalization, weighted lowess normalization (wlowess), weighted cyclic loess normalization (wcloess), subset quantile normalization (SQN), loessm, generalized Procrustes analysis (GPA) analysis, robust multi-array average (RMA), cross normalization (CrossNorm), informative cross normalization (ICN), global median, and dChip.

[0168] In high-throughput RNA / cDNA sequencing applications, such as RNA-seq, normalization can be based on gene length, sequencing depth, or both. For example, normalized expression data can be expressed as RPM (reads per million mapped reads), RPKM (reads per kilobase per million mapped reads), TPM (transcripts per million), or FPKM (fragments per kilobase per million mapped reads).

[0169] Marker expression can be assessed by any of a wide variety of well-known methods for detecting expression of a transcription molecule (e.g., mRNA) or protein, including, but not limited to, immunological methods for detection of secreted, cell surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

[0170] In certain embodiments, expression of a marker of the present disclosure can be measured by determining the level of messenger RNA (mRNA) expression of the marker gene. Such mRNA molecules can be isolated, derived, or amplified from a biological sample, such as a cell-containing composition, a cell-containing solution, a cell aggregate, a cell suspension, a tissue, etc. Such mRNA can be analyzed directly, or, for example, a corresponding cDNA molecule can be synthesized and used for further analysis.

[0171] When isolating RNA from tissues or cells, it may be important to prevent any further changes in gene expression after the tissues or cells are removed from culture. Changes in expression levels are known to change rapidly after perturbation, such as heat shock or activation by lipopolysaccharide (LPS) or other reagents. In addition, RNA from tissues and cells can become degraded quickly. Therefore, in some embodiments, the obtained tissues or cells can be stored or, for example, quickly frozen before analysis.

[0172] RNA can be extracted from tissue or cell samples by various methods, for example, guanidinium thiocyanate lysis followed by CsCl centrifugation (Chirgwin et al., 1979, Biochemistry 18:5294-5299). RNA from single cells can be obtained as described in methods for preparing cDNA libraries from single cells, for example, Dulac, C. (1998) Curr. Top. Dev. Biol. 36, 245, and Jena et al. (1996) J. Immunol. Methods 190:199. Other methods and kits for RNA extraction are well known in the art.

[0173] The isolated RNA sample can also be enriched for a particular species. For example, poly(A)+RNA can be isolated from an RNA sample. Typically, such purification takes advantage of the poly-A tail on the mRNA. As an example, poly-T oligonucleotides can be immobilized to act as affinity ligands for the mRNA. Kits for this purpose are commercially available, for example, the MessageMaker kit (Life Technologies, Grand Island, NY).

[0174] In some embodiments, an RNA population can be specifically enriched for certain sequences, including, for example, one or more marker sequences. Enrichment can be achieved, for example, by primer-directed cDNA synthesis or multiple rounds of linear amplification based on cDNA synthesis and template-directed in vitro transcription (see, e.g., Wang et al. (1989) PNAS 86,9717; Dulac et al., supra; and Jena et al., supra).

[0175] The isolated RNA, whether enriched for a particular species or sequence, can be further amplified. For example, if the RNA is mRNA, an amplification process such as RT-PCR can be used to amplify the mRNA so that the signal is detectable or the detection is enhanced. Such an amplification process is particularly beneficial, for example, when the tissue or cell sample is small in size or volume.

[0176] Various amplification and detection methods can be used to aid in the detection of the expression of marker genes. For example, it is within the scope of the present disclosure to reverse transcribe mRNA into cDNA prior to further analysis (e.g., polymerase chain reaction, RT-PCR). Other known amplification methods that can be used herein include, but are not limited to, "NASBA" or "3SR" technology, Q-beta amplification, strand displacement amplification, target-mediated amplification, ligase chain reaction (LCR), self-sustained sequence replication (SSR), and transcription amplification.

[0177] Methods for detecting, characterizing, and / or quantifying nucleic acid sequences, as well as methods for detecting, characterizing, and / or quantifying mRNA expression, are known to those of skill in the art and include, but are not limited to, PCR procedures, RT-PCR, quantitative PCR or RT-PCR, Northern blot analysis, differential gene expression, RNA protection assays, microarray analysis, hybridization methods, sequential analysis of gene expression (SAGE), hybridization based on digital barcode quantitative assays, multiplex RT-PCR, digital droplet PCR (ddPCR), qRT-PCR, qPCR, UV spectroscopy, DNA sequencing, RNA sequencing, next generation sequencing, e.g., RNAseq, lysate-based hybridization assays utilizing branched DNA signal amplification, e.g., QuantiGene 2.0 Single Plex, and branched DNA analysis methods.

[0178] For example, non-limiting examples of nucleic acid sequencing techniques for DNA sequencing and RNA sequencing include Maxam-Gilbert sequencing, Sanger sequencing (i.e., chain termination), sequencing by synthesis (SBS), sequencing by ligation, pyrosequencing, single molecule real-time sequencing, MiSeq sequencing, massively parallel signature sequencing (MPSS), polony sequencing, 454 sequencing, nanopore sequencing, etc. The present disclosure also encompasses, but is not limited to, next generation sequencing techniques.

[0179] Non-limiting examples of next generation sequencing technologies include, for example, Ion Torrent, Illumina, SOLiD, 454, solid-phase massively parallel signature sequencing, reversible dye terminator sequencing, and DNA nanoball sequencing. Digital barcode quantification assays can include BeadArray (Illumina), xMAP system (Luminex), nCounter (Nanostring), Molecular High Throughput Genomics (HTG), BioMark (Fluidigm), or Wafergen microarrays. Assays can include DASL (Illumina), RNA-Seq (Illumina), TruSeq (Illumina), SureSelect (Agilent), Bioanalyzer (Agilent), and TaqMan (ThermoFisher).

[0180] In general, PCR describes a method of gene amplification that consists of (i) sequence-specific hybridization of primers to specific genes or sequences in a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, extension, and denaturation using a thermostable DNA polymerase, and (iii) screening of the PCR products for bands of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization. That is, each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified. The mRNA levels of genes can be determined by reverse transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art. The nucleic acid sequences of exemplary marker genes are set forth herein. Thus, one skilled in the art can design appropriate primers based on the sequences of the present disclosure to determine the mRNA levels of each marker gene.

[0181] Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a sample using any of a number of procedures well known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample.

[0182] In some embodiments, one or more of the reagents described herein (e.g., nucleic acid probes) may include a detectable label and / or may include the ability to generate a detectable signal (e.g., by catalyzing a reaction that converts a compound into a detectable product). Detectable labels may include, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods for detecting them, and methods for incorporating them into reagents (e.g., antibodies and nucleic acid probes) are well known in the art.

[0183] In some embodiments, detectable labels may include labels detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemiluminescence, or chemiluminescence, or any other suitable means. Detectable labels may be primary labels (which include or generate a directly detectable moiety) or secondary labels (which bind to another moiety to generate a detectable signal, as is commonly done, for example, in immunological labeling using secondary and tertiary antibodies). Detectable labels may be linked to reagents by covalent or non-covalent means. Alternatively, detectable labels may be linked by directly labeling a molecule that achieves binding to the reagent, for example, via a ligand-receptor binding pair arrangement, or other such specific recognition molecule. Detectable labels may include, but are not limited to, radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

[0184] In some embodiments, the expression levels of multiple markers of the present disclosure can be determined simultaneously (eg, in a multiplex assay) or in parallel.

[0185] In other embodiments, the presence of PSCs may be determined by detecting gene expression products (proteins) associated with the marker genes of the present disclosure. Such detection techniques are known to those of skill in the art and include, for example, ELISA (enzyme-linked immunosorbent assay), Western blot, FACS, radioimmunoassay (RIA), sandwich assay, fluorescent in situ hybridization (FISH), immunohistological staining, immunoelectrophoresis, immunoprecipitation, and immunofluorescence (using detection reagents such as antibodies and protein binding agents).

[0186] In some embodiments, the disclosure relates to a method for detecting the presence of PSCs, e.g., ESCs or iPSCs, or both, in a sample. In some embodiments, the sample is a cell-containing composition, a cell-containing solution, a cell aggregate, a cell suspension, or a tissue. In certain embodiments, the sample contains primarily non-PSC cells. In yet further embodiments, the cells in the sample are primarily cells in a more differentiated state than PSCs, such as specialized cells, and the method determines the presence and / or amount of PSCs present in the composition. In further embodiments, the cells in the sample are primarily cells in a more differentiated state than PSCs produced by differentiation of PSCs. By way of example, the more differentiated state of cells than PSCs can be, for example, photoreceptor precursor (PRP) cells or retinal pigment epithelium (RPE) cells. Thus, the methods described herein can be used to detect and / or quantify PSCs in a composition, including residual PSCs following the process of differentiation of PSCs (e.g., production of specialized cells).

[0187] In some embodiments, a sample of interest (e.g., a cell-containing composition, a cell-containing solution, a cell aggregate, a cell suspension, or a tissue) is tested to determine the presence of PSCs therein by detecting expression of one or more of the markers of the present disclosure in cells within the sample of interest. In some embodiments, the determined expression levels of the one or more markers in the sample of interest are also compared, e.g., to the expression levels of one or more markers from a reference sample known not to contain PSCs, to confirm the presence or absence of PSCs in the sample of interest.

[0188] In other embodiments, the number of PSCs in a sample of interest (e.g., a cell-containing composition, a cell-containing solution, a cell aggregate, a cell suspension, or a tissue) is quantified by initially measuring the expression level of one or more of the markers of the present disclosure in the sample of interest. The detected expression level of one or more markers in the sample of interest can then be compared to the expression level of one or more markers from a reference sample, for example, where all of the cells in the reference sample are PSCs. In other embodiments, the detected expression level of one or more markers in the sample of interest is then compared to the expression level of one or more markers from a reference sample that contains a known percentage of PSC cells (relative to the total cells in the sample). By way of example, the percentage of PSCs to total cells can be, for example, 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less, 0.01% or less, 0.001% or less, 0.0001% or less, 0.00001% or less, 0.000001% or less, 0.0000001% or less, 0.00000001% or less, or 0.000000001% or less.

[0189] Therefore, by comparing the measured expression level of one or more of the markers disclosed herein in a sample of interest with the expression level of one or more markers from a reference sample containing only PSCs (or containing a known proportion of PSCs relative to the total cells in the sample), it is possible to determine the proportion of PSC cells in the sample of interest.

[0190] If the target sample is determined to not contain PSCs according to the above methods, then that determination can be made. Additionally, if the target sample is obtained from a cell or tissue culture, then based on this determination, the cells or tissue in the culture can be further expanded or grown.

[0191] Additionally, where the sample of interest relates, for example, to a cellular composition or engineered tissue for administration to a subject, the cells or tissue may be administered with or without further expansion or proliferation steps prior to administration based on this determination.

[0192] In other embodiments, expression of one or more of the markers of the disclosure can be detected and / or quantified at the single cell level. That is, expression of one or more of the markers of the disclosure can be detected and / or quantified in a single PSC (e.g., single cell analysis). Single cell analysis can be performed, for example, after isolation of a single cell (e.g., by methods known in the art, such as serial dilution, micromanipulation, laser capture microdissection, FACS, microfluidics, etc.). Additionally, the disclosure encompasses embodiments in which a sample of interest may contain only a single PSC among a plurality of other (non-PSC) cells, and expression of one or more of the markers of the disclosure can be detected and / or quantified in that single PSC.

[0193] Another aspect of the present disclosure relates to a kit, composition, or device for the analysis of the expression of at least one marker gene described herein. In one embodiment, contemplated is a kit, composition, or device for the analysis of the expression of at least one marker gene, comprising at least one primer and / or probe selected to determine the expression level of at least one marker gene. Some embodiments relate to a kit, composition, or device comprising at least 10 primers and / or probes, at least 30 primers and / or probes, at least 50 primers and / or probes, or at least 100 primers and / or probes selected to determine the expression level of a plurality of marker genes.

[0194] The term "probe" refers to a nucleotide fragment such as RNA or DNA that can specifically bind to a nucleotide such as an mRNA and has a length of several to several hundred bases. The probe may be labeled with a radioisotope so that the presence or absence or expression level of a specific mRNA can be determined. The probe may be constructed in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like.

[0195] The term "primer" refers to a short nucleotide sequence with a free 3' hydroxyl group that is capable of undergoing base-pairing interactions with a complementary template and serving as a starting point for replicating the template strand. A primer is capable of initiating DNA synthesis in the presence of an agent for polymerization (e.g., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in a suitable buffer and at a suitable temperature.

[0196] The term "nucleotide" means deoxyribonucleotides or ribonucleotides, and, unless otherwise specified, nucleotides can include analogs of naturally occurring nucleotides and analogs containing modified sugars or bases.

[0197] Probes or primers can be chemically synthesized using phosphoramidite solid support method or other widely known methods. These nucleotide sequences can also be modified using various methods known in the art. Examples of such modifications include methylation, encapsulation, replacement of one or more native nucleotides with their analogs, and internucleotide modifications, such as modification to uncharged conjugates (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged conjugates (e.g., phosphorothioates, phosphorodithioates, etc.).

[0198] A probe or primer can have a length of 10 or more nucleotides, or can have a length of 20 or more nucleotides. A probe or primer can have a length of 100 or less nucleotides, 90 or less nucleotides, 80 or less nucleotides, 70 or less nucleotides, 60 or less nucleotides, 50 or less nucleotides, 40 or less nucleotides, 30 or less nucleotides, or 25 or less nucleotides.

[0199] The kit may also include instructions for practicing the subject methods using the components of the kit. The instructions for practicing the subject methods are typically recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be present in the kit as a package insert or on the labeling of the container of the kit or its components (e.g., in association with the packaging or subpackaging). EXAMPLES

[0200] experiment Non-limiting embodiments of the present invention are illustrated in the following examples. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, concentrations, percent changes, etc.), but some experimental error and deviation should be accounted for. It should be understood that these examples are provided for illustrative purposes only, and are not intended to limit the scope of what the inventors consider to be various embodiments of the present invention. Not all of the following steps defined in each example are required, nor do the steps in each example have to be in the exact order presented.

[0201] Example 1 Validation of PSC-specific markers Three marker genes (LNCPRESS2, AC009446.1, and AL117378.1) were analyzed to determine whether they were differentially expressed in PSCs. To this end, sequence-specific probes and primers for these three marker genes were designed and synthesized to specifically detect complementary DNA (cDNA) corresponding to each marker gene. Using these probes and primers, the expression of these three marker genes was measured by droplet digital polymerase chain reaction (ddPCR) as follows.

[0202] Total RNA was extracted from either iPSCs, retinal pigment epithelial (RPE) cells, or photoreceptor precursor (PRP) cells. cDNA was then prepared by reverse transcription, and the resulting cDNA samples were subjected to ddPCR using a BioRad® QX200 digital PCR platform.

[0203] In one set of experiments, cDNA samples from iPSC and PRP cells were analyzed individually and also mixed together at different ratios (1:100, 1:1000, 1:10000, 1:100000, and 1:1000000) and analyzed after serial dilution at various ratios of iPSC:PRP cDNA. For the pure iPSC sample, a 1 / 100 dilution of the cDNA sample was used compared to the other samples tested due to signal saturation. The results are shown in Figure 1. As shown in Figure 1, each of the three marker genes tested (LNCPRESS2, AC009446.1, and AL117378.1) was differentially expressed in iPSC cells but not in PRP cells, as confirmed by the serial dilution results.

[0204] In a second set of experiments, cDNA samples derived from iPSC and RPE cells were analyzed individually and also mixed together at different ratios (1:100, 1:1000, 1:10000, 1:100000, and 1:1000000) and analyzed after serial dilution at various ratios of iPSC:RPE cDNA. For pure iPSC samples, a 1 / 100 dilution of the cDNA sample was used compared to the other samples tested due to signal saturation. The results are shown in Figure 2. As shown in Figure 2, each of the three marker genes tested (LNCPRESS2, AC009446.1, and AL117378.1) was differentially expressed in iPSC cells but not in RPE cells, as confirmed by the serial dilution results.

[0205] These results demonstrate that the three marker genes (LNCPRESS2, AC009446.1, and AL117378.1) can be used to specifically detect the presence of PSCs in samples, such as samples that contain primarily RPE or PRP cells (generated from iPSC cells).

[0206]

Claims

1. 1. A method for detecting pluripotent stem cells, the method comprising: (a) obtaining a target sample containing a plurality of cells; (b) analyzing the sample of interest to detect expression of at least one marker gene, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; (c) determining that the sample of interest contains pluripotent stem cells when the expression of the at least one marker gene is detected in the sample of interest; and A method comprising:

2. The method of claim 1 , wherein the sample of interest comprises induced pluripotent stem cells or embryonic pluripotent stem cells.

3. 2. The method of claim 1, wherein in step (b), the expression of the at least one marker gene is detected by measuring the level of mRNA expression of the marker gene or by measuring the level of expressed protein encoded by the marker gene.

4. 4. The method of claim 3, wherein the expression of the at least one marker gene is detected by a process comprising a technique selected from the group consisting of droplet digital polymerase chain reaction (dd-PCR), polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next generation sequencing.

5. 1. A method for detecting pluripotent stem cells, the method comprising: (a) obtaining a target sample containing a plurality of cells; (b) measuring the expression level of at least one marker gene in the sample of interest, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; (c) comparing the expression level detected in step (b) with a reference expression level, wherein the reference expression level is obtained by measuring the expression level of the at least one marker gene in at least one reference sample, wherein the at least one reference sample comprises a plurality of cells, and wherein the at least one reference sample is substantially free of pluripotent stem cells; (d) determining that the target sample contains pluripotent stem cells when the expression level detected in step (b) is higher than the reference expression level, or determining that the target sample does not substantially contain pluripotent stem cells when the expression level detected in step (b) is equal to or lower than the reference expression level; A method comprising:

6. 1. A method for quantifying the number of pluripotent stem cells in a sample, the method comprising: (a) obtaining a target sample containing a plurality of cells; (b) measuring the expression level of at least one marker gene in the sample of interest, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; (c) comparing the expression level detected in step (b) with a reference expression level, wherein the reference expression level is obtained by measuring the expression level of the at least one marker gene in at least one reference sample, the at least one reference sample comprising a plurality of cells, the at least one reference sample comprising pluripotent stem cells, and substantially all of the cells in the at least one reference sample are pluripotent stem cells; and (d) calculating the amount of pluripotent stem cells in the target sample based on the comparison in step (c); and A method comprising:

7. The method of claim 5 , wherein the sample of interest comprises induced pluripotent stem cells or embryonic pluripotent stem cells.

8. 6. The method of claim 5, wherein in step (b), the expression of the at least one marker gene is detected by measuring the level of mRNA expression of the marker gene or by measuring the level of expressed protein encoded by the marker gene.

9. 9. The method of claim 8, wherein the expression of the at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next generation sequencing.

10. The method of claim 6 , wherein the sample of interest comprises induced pluripotent stem cells or embryonic pluripotent stem cells.

11. 1. An assay for detecting residual iPSCs in a therapeutic product, the assay comprising: (a) obtaining a sample of a therapeutic product produced from iPSCs, said sample containing a plurality of cells; (b) analyzing the sample to detect expression of at least one marker gene, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; (c) determining that the therapeutic product contains residual iPSCs when the expression of the at least one marker gene is detected in the sample; and The assay comprises:

12. 12. The assay of claim 11 , wherein in step (b), the expression of the at least one marker gene is detected by measuring the level of mRNA expression of the marker gene or by measuring the level of expressed protein encoded by the marker gene.

13. 13. The assay of claim 12, wherein the expression of the at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next generation sequencing.

14. 1. A method for assessing pluripotency of an iPSC population, the method comprising: (a) obtaining a sample of an iPSC population, said sample containing a plurality of cells; (b) measuring an expression level of at least one marker gene in the sample, wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; (c) comparing the expression level detected in step (b) with a reference expression level, wherein the reference expression level is obtained by measuring an expression level of the at least one marker gene in at least one reference sample, the at least one reference sample comprising a plurality of cells, the at least one reference sample comprising iPSCs, and substantially all of the cells in the at least one reference sample are iPSCs capable of differentiation; and (d) assessing pluripotency of iPSCs in said population of iPSCs based on said comparison in step (c); and A method comprising:

15. 15. The method of claim 14, wherein in step (b), the expression of the at least one marker gene is detected by measuring the level of mRNA expression of the marker gene or by measuring the level of expressed protein encoded by the marker gene.

16. 16. The method of claim 15, wherein the expression of the at least one marker gene is detected by a process comprising a technique selected from the group consisting of dd-PCR, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative PCR, quantitative RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization assay, and next generation sequencing.

17. 1. A method for assessing pluripotency of an iPSC population, the method comprising: (a) obtaining a first sample of an iPSC population, said first sample being obtained before differentiation of said iPSCs is induced, said first sample containing a plurality of cells; (b) obtaining at least one additional sample of the population of iPSCs, the at least one additional sample being obtained after differentiation of the iPSCs has been induced, the at least one additional sample containing a plurality of cells; and (c) measuring the expression level of at least one marker gene in the samples obtained in (a) and (b), wherein the at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; (d) assessing the pluripotency of iPSC cells in the iPSC population by comparing the expression levels from the different samples detected in step (c) with each other and / or with a reference expression level; A method comprising:

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