Anti-apoptotic Mutations Desensitize Human Pluripotent Stem Cells to Mitotic Stress and Enable Aneuploid Cell Survival

摘要

class="head no_bottom_margin" id="sec1title">IntroductionFor the safe use of human pluripotent stem cells (hPSCs) in regenerative medicine, they should be devoid of mutations that could render them or their differentiated progeny malignant upon transplantation into a patient (). One important question is whether genetic or epigenetic changes acquired during hPSC in vitro culture will affect the safety and efficacy of derivatives of hPSCs produced for therapeutic application (). While at low passage, most of the hPSC lines have normal diploid karyotype, the incidence of aneuploidy increases significantly with passage number, and gains of the whole or parts of chromosomes 1, 12, 17, and 20 are substantially more common than other changes (, ). Most likely, these genetic changes are selected because they confer a growth advantage (), which may be attributed to their ability to evade the bottlenecks that restrict the expansion of wild-type cells in culture, including mass cell death following plating, failure to re-enter the cell cycle, and the high death rate of daughter cells in incipient colonies ().The frequent appearance of hPSCs with gains of whole chromosomes suggests their susceptibility to chromosome segregation errors during mitosis. In somatic cells a key regulatory mechanism controlling accurate chromosome segregation is the mitotic checkpoint, which delays the onset of anaphase and arrests cells in prometaphase to correct the defects (). After prolonged prometaphase arrest, cells may either die or exit mitosis without proper chromosome separation, thereby forming tetraploid or aneuploid cells in G1 phase, a process termed mitotic slippage (). Cell fates following mitotic slippage include apoptosis, senescence, or re-entry into the cell cycle, with the latter often resulting in highly aberrant genomes (). The frequency of aberrant divisions in hPSCs and their behavior following the mitotic checkpoint activation is poorly characterized. High rates of death in hPSC cultures () suggest a reliance of cells on apoptosis for clearing genetically damaged cells. For example, hPSCs subjected to DNA-replication stress in S phase rapidly commit to apoptosis rather than initiate DNA repair mechanisms ().Given the important role of apoptosis in protecting the genome stability of a cell population, an increase in apoptotic threshold through overexpression of anti-apoptotic genes could provide a mechanism for survival of cells with genetic damage. This phenomenon, previously observed in cancer cells (), may be particularly pertinent to hPSCs. In a large-scale study of karyotype and copy-number variation (CNV) in hPSCs by the International Stem Cell Initiative (ISCI), 26% of karyotypically normal hPSC lines examined contained amplifications of a small region of the long arm of chromosome 20 (20q11.21) including the BCL2L1 gene. Subsequent studies identified increased expression levels of BCL-XL, the BCL2L1 anti-apoptotic isoform from the amplified chromosome 20q11.21 region, as an underlying cause for the enhanced survival of the CNV cells (, ). However, it remains unknown how acquired overexpression of BCL2L1 may affect the subsequent genetic stability of hPSCs.Here we show that hPSCs commit to apoptosis rapidly in response to nocodazole-induced prometaphase arrest or following a highly aberrant cell division due to high mitochondrial priming. After differentiation, hPSCs are no longer sensitive to prometaphase arrest. The proapoptotic gene NOXA is responsible for the highly sensitive mitochondrial apoptosis present in hPSCs. Knockout of NOXA by CRISPR in hPSCs or overexpression of the anti-apoptotic protein, BCL-XL, significantly reduced cell death caused by defective mitosis. BCL-XL overexpression or the presence of the BCL2L1 CNV had enhanced survival ability, altered mitochondrial morphology, and aneuploidy formation after perturbing mitosis. Our study reveals the vulnerability of hPSC mitosis and comprehensively assesses the biological consequence of gaining anti-apoptotic mutations in hPSC. These findings reveal an important roadmap that drives hPSC culture adaptation.