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Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q.

ABSTRACT: Pluripotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source for basic and applied research as well as in therapeutic medicine. The introduction of human induced pluripotent cells (hiPSCs) holds great promise for patient-tailored regenerative medicine therapies. However, for hESCs and hiPSCs to be applied for therapeutic purposes, long-term genomic stability in culture must be maintained. Until recently, G-banding analysis was considered as the default approach for detecting chromosomal abnormalities in stem cells. Our goal in this study was to apply fluorescence in-situ hybridization (FISH) and comparative genomic hybridization (CGH) for the screening of pluripotent stem cells, which will enable us identifying chromosomal abnormalities in stem cells genome with a better resolution. We studied three hESC lines and two hiPSC lines over long-term culture. Aneuploidy rates were evaluated at different passages, using FISH probes (12,13,16,17,18,21,X,Y). Genomic integrity was shown to be maintained at early passages of hESCs and hiPSCs but, at late passages, we observed low rates mosaiciam in hESCs, which implies a direct correlation between number of passages and increased aneuploidy rate. In addition, CGH analysis revealed a recurrent genomic instability, involving the gain of chromosome 1q. This finding was detected in two unrelated cell lines of different origin and implies that gains of chromosome 1q may endow a clonal advantage in culture. These findings, which could only partially be detected by conventional cytogenetic methods, emphasize the importance of using molecular cytogenetic methods for tracking genomic instability in stem cells.

SUBMITTER: Dekel-Naftali M

PROVIDER: S-EPMC3499752 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q.

Dekel-Naftali Michal M Aviram-Goldring Ayala A Litmanovitch Talia T Shamash Jana J Reznik-Wolf Haike H Laevsky Ilana I Amit Michal M Itskovitz-Eldor Joseph J Yung Yuval Y Hourvitz Ariel A Schiff Eyal E Rienstein Shlomit S

European journal of human genetics : EJHG 20120620 12

Pluripotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source for basic and applied research as well as in therapeutic medicine. The introduction of human induced pluripotent cells (hiPSCs) holds great promise for patient-tailored regenerative medicine therapies. However, for hESCs and hiPSCs to be applied for therapeutic purposes, long-term genomic stability in culture must be maintained. Until recently, G-banding analysis was considered as the defau ...[more]

PMID: 22713809

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Project description:A subset of preimplantation embryos identified as euploid may in fact possess both whole and sub-chromosomal mosaicism, raising concerns regarding the predictive value of current comprehensive chromosome screening (CCS) methods utilizing a single biopsy. Current CCS methods may be capable of detecting sub-chromosomal mosaicism in a trophectoderm biopsy by examining intermediate levels of segmental aneuploidy within a biopsy. This study evaluates the sensitivity and specificity of segmental aneuploidy detection by three commercially available CCS platforms utilizing a cell line mixture model of segmental mosaicism in a six-cell trophectoderm biopsy.Two cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female cell line containing a 16.2 Mb deletion on chromosome 5 and a male cell line containing a 25.5 Mb deletion on chromosome 4 were used to create mixtures at each level. Six replicates of each mixture were prepared, randomized, and blinded for analysis by one of the three CCS platforms (SNP-array, VeriSeq NGS, or NexCCS). Sensitivity and specificity of segmental aneuploidy at each level of mosaicism was determined and compared between each platform. Additionally, an alternative VeriSeq NGS analysis method utilizing previously published criteria was evaluated.Examination of the default settings of each platform revealed that the sensitivity was significantly different between NexCCS and SNP up to 50% mosaicism, custom VeriSeq, and SNP-array up to 66% mosaicism, and between NexCCS and custom VeriSeq up to 50% mosaicism. However, no statistical difference was observed in mixtures with >50% mosaicism with any platform. No comparison was made between default VeriSeq, as it does not report segmental imbalances. Furthermore, while the use of previously published criteria for VeriSeq NGS significantly increased sensitivity at low levels of mosaicism, a significant decrease in specificity was observed (66% false positive prediction of segmental aneuploidy).These results demonstrate the potential of NGS-based detection methods to detect segmental mosaicism within a biopsy. However, these data also demonstrate that a balance between sensitivity and specificity should be more carefully considered. These results emphasize the importance of vigorous preclinical evaluation of new testing criteria prior to clinical implementation providing a point of departure for further algorithm development and improved detection of mosaicism within preimplantation embryos.

Dataset Information

Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q.

Publications

Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q.

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