Fig. 1: hESC lines with a gain of 1q show impaired neuroectoderm differentiation.

A Lentiviral transduction was used to induce stable expression of fluorescent proteins in hESC^1q. Working cell banks were established comprising mutant-labeled cell lines and their isogenic counterparts. Pure wild-type and 1q lines were utilized, along with mixes containing 10% of 1q cells, for differentiation to three lineages: neuroectoderm, hepatoblasts and cardiac progenitors. Differentiation capacity was assessed through immunostaining, qPCR, and RNA sequencing. Cell competition results were measured by analyzing the ratio of mutant and wild-type cells before and after differentiation using flow cytometry and immunostaining. To study the role of MDM4 in the growth advantage of hESC^1q, MDM4 was downregulated by siRNA prior to the competition assays. To study the mechanisms by which MDM4 provides the selective advantage, DNA damage was induced by Bleomycin, and apoptosis levels were evaluated using Annexin V staining and flow cytometry, while DNA damage levels were studied through immunostaining for gamma-H2AX. B Breakpoints for the minimal region of gain in chromosome 1 are 202,475,000 and 205,800,000. C Shallow DNA sequencing–based karyotyping of the hESC lines included in the study. Gain in 1q chromosome is visible for VUB03^1q32.1, VUB03^1q21.1qter and VUB19^1q21.1qter. D Representative images of immunostainings for POU5F1 (turquoise, 1st panel), PAX6 (magenta, 2nd panel), HNF4A (magenta, 3rd panel) and GATA4 (magenta 4th panel) of hESC^wt and hESC^1q before and after the completion of induction of differentiation to neuroectoderm (NE, day 8), hepatoblast (HEP, day 8), cardiac progenitors (CP, day 5) of VUB19^1q21.1qter. Figure S1 shows the images for cell lines VUB03^1q21.1qter and VUB03^1q32.1 included in the study. E Quantification of the percentage of PAX6, HNF4A and GATA4-positive cells in the immunostainings shown in panel D and in Fig. S1. F mRNA quantification by quantitative real-time PCR of PAX6 and SOX1 in NE^wt and NE^1q, HNF4A and GATA4 in HEP^wt and HEP^1q, and GATA4 and NKX2.5 in CP^wt and CP^1q. Results for POU5F1 and NANOG are shown in Fig. S1. Each differentiation was carried out in three technical replicates at the same time. Different hESC lines and independent experimental replicates (rep) are coded in different colors. *p < 0.05, **p < 0.01, ****p < 0.0001. ns = non-significant.