A dynamic in vitro model of Down syndrome neurogenesis with trisomy 21 gene dosage correction.

Excess gene dosage from chromosome 21 (chr21) causes Down syndrome (DS), spanning developmental and acute phenotypes in terminal cell types. Which phenotypes remain amenable to intervention after development is unknown. To address this question in a model of DS neurogenesis, we derived trisomy 21 (T21) human induced pluripotent stem cells (iPSCs) alongside, otherwise, isogenic euploid controls from mosaic DS fibroblasts and equipped one chr21 copy with an inducible XIST transgene. Monoallelic chr21 silencing by XIST is near-complete and irreversible in iPSCs. Differential expression reveals that T21 neural lineages and iPSCs share suppressed translation and mitochondrial pathways and activate cellular stress responses. When XIST is induced before the neural progenitor stage, T21 dosage correction suppresses a pronounced skew toward astrogenesis in neural differentiation. Because our transgene remains inducible in postmitotic T21 neurons and astrocytes, we demonstrate that XIST efficiently represses genes even after terminal differentiation, which will empower exploration of cell type-specific T21 phenotypes that remain responsive to chr21 dosage.

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.

Altered T cell infiltration and enrichment of leukocyte regulating pathways within aged skeletal muscle are associated impaired muscle function following influenza infection.

Older adults have diminished immune responses that increase susceptibility to infectious diseases, such as influenza (flu). In older adults, flu infection can lead to hospitalization, catastrophic disability, and mortality. We previously demonstrated severe and prolonged muscle degradation and atrophy in aged mice during flu infection. Here, we utilized an unbiased transcriptomic analysis to elucidate mechanisms of flu-induced muscular declines in a mouse model. Our results showed age-related gene expression differences including downregulation of genes associated with muscle regeneration and organization and upregulation of genes associated with pro-inflammatory cytokines and migratory immune pathways in aged mice when compared to young. Pathway analysis revealed significant enrichment of leukocyte migration and T cell activation pathways in the aged muscle during infection. Intramuscular CD4 T cells increased in both young and aged mice during infection, while intramuscular CD8 T cells increased exclusively in aged muscle. CD4 T cells in young muscle were regulatory T cells (Treg), while those in aged were T follicular helper (Tfh) and Th2 cells. Correspondingly, IL-33, an important cytokine for Treg accumulation within tissue, increased only in young flu-infected muscle. Conversely, CXCL10 (IP-10) increased only in aged muscle suggesting a continued recruitment of CD8 T cells into the aged muscle during flu infection. Overall, our findings elucidate a link between flu-induced disability and dysregulated intracellular T cell recruitment into flu-injured muscle with aging. Furthermore, we uncovered potential pathways involved that can be targeted to develop preventative and therapeutic interventions to avert disability and maintain independence following infection.

Monosomy X in isogenic human iPSC-derived trophoblast model impacts expression modules preserved in human placenta.

Mammalian sex chromosomes encode homologous X/Y gene pairs that were retained on the Y chromosome in males and escape X chromosome inactivation (XCI) in females. Inferred to reflect X/Y pair dosage sensitivity, monosomy X is a leading cause of miscarriage in humans with near full penetrance. This phenotype is shared with many other mammals but not the mouse, which offers sophisticated genetic tools to generate sex chromosomal aneuploidy but also tolerates its developmental impact. To address this critical gap, we generated X-monosomic human induced pluripotent stem cells (hiPSCs) alongside otherwise isogenic euploid controls from male and female mosaic samples. Phased genomic variants in these hiPSC panels enable systematic investigation of X/Y dosage-sensitive features using in vitro models of human development. Here, we demonstrate the utility of these validated hiPSC lines to test how X/Y-linked gene dosage impacts a widely used model for human syncytiotrophoblast development. While these isogenic panels trigger a GATA2/3- and TFAP2A/C-driven trophoblast gene circuit irrespective of karyotype, differential expression implicates monosomy X in altered levels of placental genes and in secretion of placental growth factor (PlGF) and human chorionic gonadotropin (hCG). Remarkably, weighted gene coexpression network modules that significantly reflect these changes are also preserved in first-trimester chorionic villi and term placenta. Our results suggest monosomy X may skew trophoblast cell type composition and function, and that the combined haploinsufficiency of the pseudoautosomal region likely plays a key role in these changes.

Contiguous erosion of the inactive X in human pluripotency concludes with global DNA hypomethylation.

Female human pluripotent stem cells (hPSCs) routinely undergo inactive X (Xi) erosion. This progressive loss of key repressive features follows the loss of XIST expression, the long non-coding RNA driving X inactivation, and causes reactivation of silenced genes across the eroding X (Xe). To date, the sporadic and progressive nature of erosion has obscured its scale, dynamics, and key transition events. To address this problem, we perform an integrated analysis of DNA methylation (DNAme), chromatin accessibility, and gene expression across hundreds of hPSC samples. Differential DNAme orders female hPSCs across a trajectory from initiation to terminal Xi erosion. Our results identify a cis-regulatory element crucial for XIST expression, trace contiguously growing reactivated domains to a few euchromatic origins, and indicate that the late-stage Xe impairs DNAme genome-wide. Surprisingly, from this altered regulatory landscape emerge select features of naive pluripotency, suggesting that its link to X dosage may be partially conserved in human embryonic development.