Transcriptomic analysis of iPSC-derived endothelium reveals adaptations to high altitude hypoxia in energy metabolism and inflammation

Tibetan adaptation to high-altitude hypoxia remains a classic example of Darwinian selection in humans. Amongst Tibetans, alleles in the EPAS1 gene - whose protein product, Hif-2a, is a central regulator of the hypoxia response - have repeatedly been shown to carry some of the strongest signals of positive selection in humans and to influence several adaptive phenotypes. We recently showed that the selected haplotype at this locus spans a hypoxia-dependent enhancer (ENH5) that contributes to the regulation of EPAS1 in a variety of cell types. However, selective sweep signals alone may account for only part of the phenotypes that differentiate Tibetans from closely related lowlanders. Therefore, there is a pressing need to functionally probe adaptive alleles and their impact at the genome-wide level and across cell types to uncover the full range of beneficial traits. To cast a wider net, we established a library of induced pluripotent stem cells (iPSCs) derived from Tibetan and Han Chinese individuals, a robust model system allowing precise exploration of both locus-specific and genome-wide effects on transcriptional responses. We harness this system by differentiating the iPSC library into vascular endothelium and investigating the locus-specific effects of the ENH5 enhancer in this cellular context. In addition, we use it to explore Tibetan-specific transcriptome-wide responses and find evidence that energy metabolism and immune pathways have been shaped by natural selection in Tibetans. Finally, to aid with the interpretation of the transcriptional differences between populations, we test for polygenic adaptations as a complementary in silico approach for the identification of beneficial Tibetan phenotypes. Overall design: We developed a panel of sex-matched Tibetan and Han Chinese iPSCs using lymphoblastoid cell lines (LCLs) from 10 unrelated individuals of Tibetan origin and 10 Han Chinese (CHB) from the 1000 Genome Project.These 20 LCLs were reprogrammed into iPSCs in batches, balanced by population and sex and then successfully differentiated into vascular endothelium. We used CRISPR-CAS9 editing to delete the region (chr2: 46578867 to 46579857; hg19) spanning the ENH5 enhancer region of the iPSC line derived from Han individual CHB633 in order to create knockout cells carrying a low-altitude haplotype across the entire EPAS1 locus, but with complete loss of ENH5 activity. These DENH5 iPS cells (subsequently referred to as knockout, KO) and the original CHB633 (wild type, WT) iPS cells were differentiated into vascular endothelium in five independent replicates and cultured in hypoxic conditions (1% O2) for 48 hours. In order to assess the impact of the ENH5 deletion in vascular endothelial cells, we performed single cell RNA-sequencing in a targeted 10,000 cells per KO and WT replicate. We assessed the expression of 25 endothelial markers (in the 10 UMAP clusters identified by Seurat) and identified two cell clusters as being robustly enriched in all endothelial markers. Transcript data from the cells in these two clusters were pooled to compare expression differences between KO and WT in a cell-type specific manner. We utilized our complete panel of 20 cell lines to compare transcript levels between TAC and CHB iPSC-derived vascular endothelium under both normoxic and hypoxic conditions. Given the larger number of cell lines and treatment conditions, we performed bulk RNA-sequencing rather than single cell RNA-sequencing.