(T1265) HPSC WITH A GAIN OF 1Q OR 20Q11.21 RETAIN THEIR GROWTH ADVANTAGE DURING NEUROECTODERM DIFFERENTIATION AND MIS-SPECIFY TOWARDS NON-NEURAL LINEAGES
Abstract: hPSC frequently acquire chromosomal abnormalities during culture. While these abnormalities provide cells with a selective growth advantage, their impact on differentiation and lineage specification remains poorly understood. This knowledge is critical, as it has significant implications for our understanding of embryonic development, for the use of cells as research tools and for their clinical application. Here we studied 6 aneuploid hPSC lines with gains of 1q or 20q11.21 and 4 isogenic genetically balanced control lines. Competition assays were done by co-culturing fluorescently labelled hPSC with different karyotypes during directed neuroectoderm differentiation in monolayer cultures and embryoid bodies. We found that both aneuploidies retain their selective advantage, with mutant cell loads tripling in 8 days of culture. Bulk and single-cell RNA sequencing showed that both aneuploidies result in cell mis-specification. While 1q cells specified to cranial placode, 20q11.21 cells progressed to surface ectoderm and, remarkably, to amnion, showing that both mutants bypassed the neural lineage and mis-specified to other ectodermal fates. To explore the mechanisms underlying their growth advantage, we performed competition assays with siRNA-treated cells and studied the apoptosis rates of 1q cells over time after bleomycin-induced DNA damage. We found that the 1q gene MDM4 mediates a reduced sensitivity to p53-driven apoptosis in 1q gain cells. Additionally, quantification of γH2AX foci and tracking the ratio of aneuploid cells in culture over time, revealed that high cell density in culture lead to increased levels of DNA damage, promoting the takeover by the mutant cells. The higher BCL2L1 expression in 20q cells not only confers resistance to apoptosis, but also is sufficient to drive the alternative cell fates via TGFβ and BMP signaling. Our work shows that aneuploid cells not only retain their growth advantage during differentiation, but also consistently mis-specify during directed neuroectoderm differentiation, suggesting that this lineage is particularly vulnerable to genetic aberrations. Further, we identify MDM4 as the key driver gene of the selective advantage of gains of 1q, and uncover a novel role for BCL2L1 in modulating TGFβ superfamily pathways to promote alternative cell fates.
Funding Source: Fonds Wetenschappelijk Onderzoek (FWO) and Methusalem Grant
