Derivation of normal diploid human embryonic stem cells from tripronuclear zygotes with analysis of their copy number variation and loss of heterozygosity

Abstract Genetic alterations have been reported in most human embryonic stem cells (hESCs) for decades. ‘Survival advantage,’ a typical trait acquired during long-term in vitro culture, results from induction of BCL2L1 upon frequent copy number variation (CNV) at locus 20q11.21 and is one of the strongest candidates associated with genetic alteration via escape from mitotic stress. However, the underlying mechanisms for BCL2L1 induction remain undefined. Furthermore, abnormal mitosis and ‘survival advantage’ frequently occurring in the late passage are respectively associated with the expression of TPX2 and BCL2L1, which are located in locus 20q11.21. In this study, we observed that 20q11.21 CNV was not sufficient for BCL2L1 induction and consequent survival traits in pairs of hESCs and human induced pluripotent stem cells (iPSCs) with normal and 20q11.21 CNVs. Inducible expression of TPX2 and basal TPX2 expression due to leakage of the inducible system in hESCs with normal copy number was sufficient to promote BCL2L1 expression and promoted high tolerance to mitotic stress. High Aurora A kinase activity by TPX2 stabilized YAP1 protein to promote YAP1 dependent BCL2L1 expression. Thus, a chemical inhibitor of Aurora A kinase and knockdown of YAP/TAZ significantly abrogated the aforementioned high tolerance to mitotic stress through BCL2L1 suppression. These results suggest that the collective expression of TPX2 and BCL2L1 from CNV at loci 20q11.21 and a consequent increase in YAP1 signaling would promote genome instability during long-term in vitro hESC culture.

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Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions

10.1101/2020.11.06.360685 ◽

2020 ◽

Author(s):

Liselot M. Mus ◽

Stéphane Van Haver ◽

Mina Popovic ◽

Wim Trypsteen ◽

Steve Lefever ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Copy Number ◽

Selection Process ◽

Embryonic Stem ◽

Selective Advantage ◽

Dna Copy Number ◽

Chromosomal Imbalances ◽

Replicative Stress

ABSTRACTHuman embryonic stem cells (hESCs) and embryonal tumors share a number of common features including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress which is known to induce genomic instability causing chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea induced replicative stress versus normal growth conditions in an equally-mixed cell population of isogenic euploid and 17q+ hESCs. We could show that 17q+ hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.

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