Isogenic hiPSC models of Turner syndrome development reveal shared roles of inactive X and Y in the human cranial neural crest network

Modeling the developmental etiology of viable human aneuploidy can be challenging in rodents due to syntenic boundaries, or primate-specific biology. In humans, monosomy-X (45,X) causes Turner syndrome (TS), altering craniofacial, skeletal, endocrine, and cardiovascular development, which in contrast remain unaffected in 39,X-mice. To learn how human monosomy-X may impact early embryonic development, we turned to human 45,X and isogenic euploid induced pluripotent stem cells (hiPSCs) from male and female mosaic donors. Because neural crest (NC) derived cell types are hypothesized to underpin craniofacial and cardiovascular changes in TS, we performed a highly-powered differential expression study on hiPSC-derived anterior neural crest cells (NCCs). Across three independent isogenic panels, 45,X NCCs show impaired acquisition of PAX7+SOX10+ markers, and disrupted expression of other NCC-specific genes, relative to their isogenic euploid controls. In particular, 45,X NCCs increase cholesterol biosynthesis genes while reducing transcripts that feature 5' terminal oligopyrimidine (TOP) motifs, including those of ribosomal protein and nuclear-encoded mitochondrial genes. Such metabolic pathways are also over-represented in weighted co-expression gene modules that are preserved in monogenic neurocristopathy. Importantly, these gene modules are also significantly enriched in 28% of all TS-associated terms of the human phenotype ontology. Our analysis identifies specific sex-linked genes that are expressed from two copies in euploid males and females alike and qualify as candidate haploinsufficient drivers of TS phenotypes in NC-derived lineages. This study demonstrates that isogenic hiPSC-derived NCC panels representing monosomy-X can serve as a powerful model of early NC development in TS and inform new hypotheses towards its etiology. Overall design: We differentiated human iPSCs to neural crest cells (NCCs) via moderate WNT and BMP activation, reported to enable reproducible derivation of anterior NCCs (PMID: 30095409). We applied this method to a total of 11 distinct human iPSC lines from all three of our cytogenomically validated isogenic hiPSC panels (PMID: 36161909). We next performed a highly-powered RNA-seq study, with a minimum of 4 replicates per cell line, grouped by a combination of donor and karyotype (“condition”), comprising 7-12 replicates each. This enables our analysis to assess monosomy-X driven changes within each isogenic context (or donor). RNA was extracted from NCCs using the PureLink RNA Mini Kit (ThermoFisher Scientific). Libraries were prepared at the UConn Center for Genome Innovation using the Illumina Strand mRNA Kit and 100bp paired-ends reads were sequenced to an average depth of 40 million reads/replicate on the NovaSeq 6000 (Illumina).