Methods for telomere length and genomic dna quality control and analysis in pluripotent stem cells

Abstract

The generation of clinical-grade cell-based therapies from human embryonic stem cells or cells reprogrammed to pluripotency from somatic cells, requires stringent quality controls to insure that the cells have long enough telomeres and resulting cellular lifespan to be clinically useful, and normal gene expression and genomic integrity so as to insure cells with a desired and reproducible phenotype and to reduce the risk of the malignant transformation of cells. Assays useful in identifying human embryonic stem cell lines and pluripotent cells resulting from the transcriptional reprogramming of somatic cells that have embryonic telomere length are described as well as quality control assays for screening genomic integrity in cells expanded and banked for therapeutic use, as well as assays to identify cells capable of abnormal immortalization,

1 . A method of identifying pluripotent stem cell lines suitable for clinical use, comprising: evaluating telomere length of a pluripotent stem cell line; and identifying said pluripotent stem cell line as suitable for clinical use if said evaluated telomere length is restored to near-embryonic telomere length. 
     
     
         2 . The method of  claims 1 , further comprising evaluating telomere length of said pluripotent stem cell line in at least two passages, wherein said pluripotent stem cell line is identified as suitable for clinical use when the evaluated telomere length in a later of the at least two passages is longer than the evaluated telomere length in an earlier of the at least two passages. 
     
     
         3 . The method of  claim 2 , wherein telomere length is evaluated in three or more passages, wherein the pluripotent stem cell line is identified as capable of restoring telomere length when the evaluated telomere length in each later passage is longer than the evaluated telomere length in each earlier passage. 
     
     
         4 . The method of  claim 3 , wherein said evaluated telomere length of said later passage is at least 12 kb. 
     
     
         5 . The method of  claim 3 , wherein said evaluated telomere length of said later passage is at least 14 kb. 
     
     
         6 . The method of  claim 3 , wherein said evaluated telomere length of said later passage is at least 17 kb. 
     
     
         7 . The method of  claim 1 , wherein the pluripotent stem cell line is an induced pluripotent stem cell line (iPS cell line). 
     
     
         8 . The method of  claim 1 , wherein said evaluating step comprises performing one or more of: single telomere length analysis (STELA), fluorescence in-situ hybridization (FISH), flow-FISH, and Southern blot analysis. 
     
     
         9 . The method of  claim 1 , further comprising: evaluating the expression level of at least one gene in said pluripotent stem cell line to obtain a gene expression level result, wherein the at least one gene is selected from one or more of: PCNA, CDC2, MSH2, ZNF146, TERF1 transcript variant 2, VENTX and PRKDC; and
 identifying the pluripotent stem cell line as suitable for clinical use based on the gene expression level result.   
     
     
         10 . The method of  claim 9 , wherein the evaluating step comprises comparing the gene expression level result to a reference gene expression result. 
     
     
         11 . The method of  claim 10 , wherein the reference gene expression level result is from embryonic stem cells and wherein the expression of the at least one gene in the pluripotent stem cells is equivalent to the expression of the at least one gene in the embryonic stem cells. 
     
     
         12 . The method of  claim 1 , further comprising:
 assessing the genomic integrity of said pluripotent stem cell line to obtain a genomic integrity result, wherein said assessing comprises one or more of: karyotyping; analysis of variable number tandem repeats (VNTRs), short tandem repeats (STRs), single nucleotide polymorphisms (SNP), and/or copy number variations (CNVs); analysis of culture mosaicism; analysis of DNA sequences related to genetic diseases; and complete genome sequencing and analysis; and   identifying the pluripotent stem cell lines as suitable for clinical use based on the genomic integrity result.   
     
     
         13 . A method of identifying pluripotent stem cells capable of restoring telomere length comprising:
 evaluating telomerase activity in the pluripotent stem cells to obtain a telomerase activity result; and   identifying the pluripotent stem cells as capable of restoring telomere restriction fragment length based on the telomerase activity result.   
     
     
         14 . The method of  claim 13 , wherein:
 the evaluating step further comprises evaluating expression level of at least one gene in the pluripotent stem cells to obtain a gene expression level result, wherein the at least one gene is selected from one or more of: PCNA, CDC2, MSH2, ZNF146, TERF1 transcript variant 2, VENTX and PRKDC; and   the identifying step further comprises identifying the pluripotent stern cells as capable of restoring telomere restriction fragment length based on the gene expression level result.   
     
     
         15 . An induced pluripotent stem cell (iPS cell) identified according to the method of  claim 1 . 
     
     
         16 . The iPS cell of  claim 15 , wherein said pluripotent cell line displays a normal karyotype. 
     
     
         17 . A system for identifying a pluripotent stem cell capable of capable of restoring telomere length comprising:
 a gene expression level evaluation element configured for evaluating the level of expression of at least one gene in a pluripotent stem cell to obtain a gene expression level result, wherein the at least one gene is selected from one or more of: PCNA, CDC2, MSH2, ZNF146, TERF1 transcript variant 2, VENTX and PRKDC; and   a phenotype determination element configured for employing the gene expression level result to identify a pluripotent stem cell capable of capable of restoring telomere length.   
     
     
         18 . The system of  claim 17 , wherein the phenotype determination element comprises a reference gene expression level result. 
     
     
         19 . The system of  claim 18 , wherein the reference gene expression level result is from embryonic stem cells. 
     
     
         20 . The system of  claim 19 , wherein the embryonic stem cells are human embryonic stem cells with telomere restriction fragment lengths of 12 to 18 kilobases (kb).