Markers specific for pluripotent stem cells, and methods of using the same
AssigneeFUJIFILM Cellular Dynamics, Inc.
InventorsThomas BURKE, Steven SMITH, Anne STROUSE
Life of this patent
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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 to methods for detecting the presence and/or absence of one or a plurality of pluripotent stem cells, by detecting such markers.
Claims82
A method for detecting pluripotent stem cells, said method comprising:
- (a) obtaining a sample of interest containing a plurality of cells;
- (b) analyzing said sample of interest to detect expression of at least one marker gene, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5;
- and
- (c) determining that said sample of interest contains pluripotent stem cells when the expression of said at least one marker gene is detected in said sample of interest.
A method for detecting pluripotent stem cells, said method comprising:
- (a) obtaining a sample of interest containing a plurality of cells;
- (b) measuring the expression level of at least one marker gene in said sample of interest, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises 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) to a reference expression level, said reference expression level having been obtained by measuring the expression level of said at least one marker gene in at least one reference sample, wherein said at least one reference sample comprises a plurality of cells, and wherein said at least one reference sample contains substantially no pluripotent stem cells;
- and
- (d) determining that said sample of interest contains pluripotent stem cells when the expression level detected in step (b) is greater than said reference expression level, or
- determining that said sample of interest contains substantially no pluripotent stem cells when the expression level detected in step (b) is equal to or less than said reference expression level.
A method for quantifying the number of pluripotent stem cells in a sample, said method comprising:
- (a) obtaining a sample of interest containing a plurality of cells;
- (b) measuring the level of expression of at least one marker gene in said sample of interest, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises 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) to a reference expression level, said reference expression level having been obtained by measuring the expression level of said at least one marker gene in at least one reference sample, wherein said at least one reference sample comprises a plurality of cells, wherein said at least one reference sample comprises pluripotent stem cells, and wherein substantially all of the cells in said at least one reference sample are pluripotent stem cells;
- and
- (d) calculating the amount of pluripotent stem cells in said sample of interest based on the comparison in step (c).
An assay for detecting residual iPSCs in a therapeutic product, said assay comprising:
- (a) obtaining a sample of a therapeutic product produced from iPSCs, said sample containing a plurality of cells;
- (b) analyzing said sample to detect expression of at least one marker gene, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5;
- and
- (c) determining that said therapeutic product contains residual iPSCs when the expression of said at least one marker gene is detected in said sample.
An assay for quantifying the number of residual iPSCs in a therapeutic product, said assay comprising:
- (a) obtaining a sample of a therapeutic product produced from iPSCs, said sample containing a plurality of cells;
- (b) measuring the level of expression of at least one marker gene in said sample, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises 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) to a reference expression level, said reference expression level having been obtained by measuring the expression level of said at least one marker gene in at least one reference sample, wherein said at least one reference sample comprises a plurality of cells, wherein said at least one reference sample comprises iPSCs, and wherein substantially all of the cells in said at least one reference sample are iPSCs;
- and
- (d) quantifying the number of residual iPSCs in said therapeutic product based on the comparison in step (c).
An assay for detecting residual iPSCs in a therapeutic product, said assay comprising:
- (a) obtaining a sample of a therapeutic product produced from iPSCs, said sample containing a plurality of cells;
- (b) measuring the expression level of at least one marker gene in said sample, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises 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) to a reference expression level, said reference expression level having been obtained by measuring the expression level of said at least one marker gene in at least one reference sample, wherein said at least one reference sample comprises a plurality of cells, and wherein said at least one reference sample contains substantially no pluripotent stem cells;
- and
- (d) determining that said therapeutic product contains residual iPSCs when the expression level detected in step (b) is greater than said reference expression level, or
- determining that said therapeutic product contains substantially no residual iPSCs when the expression level detected in step (b) is equal to or less than said reference expression level.
An assay for quantifying the number of residual iPSCs in a therapeutic product at different differentiation times, said 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 onset 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;
- (c) measuring the level of expression of at least one marker gene in the samples obtained in (a) and (b), wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5;
- and
- (d) quantifying the number of iPSCs in said therapeutic product at different differentiation times, based on the measurements in step (c).
An assay for determining the purity of a differentiated product, said assay comprising:
- (a) obtaining a sample of a differentiated product, said sample containing a plurality of cells;
- (b) measuring the level of expression of at least one marker gene in said sample, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises 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) to a reference expression level, said reference expression level having been obtained by measuring the expression level of said at least one marker gene in at least one reference sample, wherein said at least one reference sample comprises a plurality of cells, wherein said at least one reference sample comprises iPSCs, and wherein substantially all of the cells in said at least one reference sample are iPSCs;
- and
- (d) calculating the amount of iPSCs in said sample based on the comparison in step (c), to thereby determine the purity of the differentiated product.
An assay for determining the purity of a differentiated product, said assay comprising:
- (a) obtaining a sample of a differentiated product, said sample containing a plurality of cells,
- (b) measuring the expression level of at least one marker gene in said sample, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises 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) to a reference expression level, said reference expression level having been obtained by measuring the expression level of said at least one marker gene in at least one reference sample, wherein said at least one reference sample comprises a plurality of cells, and wherein said at least one reference sample contains substantially no pluripotent stem cells;
- and
- (d) calculating the amount of iPSCs in said sample based on the comparison in step (c), to thereby determine the purity of the differentiated product.
A method for evaluating the capacity of an iPSC population to differentiate, said method comprising:
- (a) obtaining a first sample of a population of iPSCs, 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, said at least one additional sample being obtained after differentiation of said iPSCs is induced, said at least one additional sample containing a plurality of cells;
- (c) measuring the level of expression of at least one marker gene in the samples obtained in (a) and (b), wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5;
- and
- (d) comparing the expression levels from the different samples detected in step (c) to each other, and/or to a reference expression level, to thereby evaluate the capacity of the iPSCs in said iPSCs population to differentiate.
A method for evaluating the pluripotency of a iPSC population, said method comprising:
- (a) obtaining a sample of an iPSC population, said sample containing a plurality of cells;
- (b) measuring the level of expression of at least one marker gene in said sample, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises 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) to a reference expression level, said reference expression level having been obtained by measuring the expression level of said at least one marker gene in at least one reference sample, wherein said at least one reference sample comprises a plurality of cells, wherein said at least one reference sample comprises iPSCs, and wherein substantially all of the cells in said at least one reference sample are iPSCs capable of differentiation;
- and
- (d) evaluating the pluripotency of the iPSCs in said iPSC population based on the comparison in step (c).
A method for evaluating the pluripotency of a iPSC population, said method comprising:
- (a) obtaining a first sample of a population of iPSCs, 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, said at least one additional sample being obtained after differentiation of said iPSCs is induced, said at least one additional sample containing a plurality of cells;
- (c) measuring the level of expression of at least one marker gene in the samples obtained in (a) and (b), wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises 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 levels from the different samples detected in step (c) to each other, and/or to a reference expression level, to thereby evaluate the pluripotency of the iPSC cells in said iPSC population.
An assay kit for detecting residual iPSCs in a therapeutic product, said assay kit comprising at least one reagent suitable for specifically detecting the level of expression of at least one marker gene, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5.
An assay reagent for detecting residual iPSCs in a therapeutic product, wherein said reagent is able to specifically detect the level of expression of at least one marker gene, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5, and
- wherein said reagent comprises a nucleic acid, a probe, or one or more primers.
An assay kit for quantifying the number of iPSCs in a therapeutic product, said assay kit comprising at least one reagent suitable for specifically detecting the level of expression of at least one marker gene, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5.
An assay reagent for quantifying the number of iPSCs in a therapeutic product, wherein said reagent is able to specifically detect the level of expression of at least one marker gene, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5, and
- wherein said reagent comprises a nucleic acid, a probe, or one or more primers.
An assay kit for determining the purity of a differentiated product, said assay kit comprising at least one reagent suitable for specifically detecting the level of expression of at least one marker gene, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5.
An assay reagent for determining the purity of a differentiated product, wherein said reagent is able to specifically detect the level of expression of at least one marker gene, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5, and
- wherein said reagent comprises a nucleic acid, a probe, or one or more primers.
An assay kit for evaluating the capacity of an iPSC population to differentiate, said assay kit comprising at least one reagent suitable for specifically detecting the level of expression of at least one marker gene, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5.
An assay reagent for evaluating the capacity of an iPSC population to differentiate, wherein said reagent is able to specifically detect the level of expression of at least one marker gene, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5, and
- wherein said reagent comprises a nucleic acid, a probe, or one or more primers.
An assay kit for evaluating the pluripotency of an iPSC population, said assay kit comprising at least one reagent suitable for specifically detecting the level of expression of at least one marker gene, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5.
An assay reagent for evaluating the pluripotency of an iPSC population, wherein said reagent is able to specifically detect the level of expression of at least one marker gene, wherein said at least one marker gene expresses a transcript having a corresponding complementary DNA (cDNA) sequence that comprises a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-5, and
- wherein said reagent comprises a nucleic acid, a probe, or one or more primers.
Dependencies are inferred from the claim text.