Sex-biased Gene Expression During Neural Differentiation of Human Embryonic Stem Cells
Sex differences in the developing human brain are primarily attributed to hormonal influence. Recently however, genetic differences and their impact on the developing nervous system have attracted increased attention. To understand genetically driven sexual dimorphisms in neurodevelopment, we investigated genome-wide gene expression in an in vitro differentiation model of male and female human embryonic stem cell lines (hESC), independent of the effects of human sex hormones. Four male and four female-derived hESC lines were differentiated into a population of mixed neurons over 37 days. Differential gene expression and gene set enrichment analyses were conducted on bulk RNA sequencing data. While similar differentiation tendencies in all cell lines demonstrated the robustness and reproducibility of our differentiation protocol, we found sex-biased gene expression already in undifferentiated ESCs at day 0, but most profoundly after 37 days of differentiation. Male and female cell lines exhibited sex-biased expression of genes involved in neurodevelopment, suggesting that sex influences the differentiation trajectory. Interestingly, the highest contribution to sex differences was found to arise from the male transcriptome, involving both Y chromosome and autosomal genes. We propose 13 sex-biased candidate genes (10 upregulated in male cell lines and 3 in female lines) that are likely to affect neuronal development. Additionally, we confirmed gene dosage compensation of X/Y homologs escaping X chromosome inactivation through their Y homologs and identified a significant overexpression of the Y-linked demethylase UTY and KDM5D in male hESC during neuron development, confirming previous results in neural stem cells. Our results suggest that genetic sex differences affect neuronal differentiation trajectories, which could ultimately contribute to sex biases during human brain development. Overall design: Neural differentiation of 4 male and 4 female human embryonic stem cell lines over 37 days. Six cell lines (WA14, 983a, 401, LT2e, 980, 975)  and four timepoints were submitted for bulk RNA sequencing (T0, D4, D9, D37). Cell culture experiments were performed in triplicates (A, B, C). Thus, a total of 72 samples were submitted for sequencing on the NovaSeq instrument with an S4 lane (Illumina) resulting in approximately 27.7 million 150 bp paired-end read-pairs per sample. Then, gene expression was compared between male and female cell lines using DEseq2 and GSEA analysis (MSigDB).