Supplementing culture medium with the weak acid, 5,5-dimethyl-2,4-oxazolidinedione (DMO) limits the development of aneuploid mouse embryos through a Trp53-dependent mechanism.

PURPOSE: This study was designed to determine if DMO limits in vitro development of aneuploid-enriched mouse embryos by activating a Trp53-dependent mechanism. METHODS: Mouse cleavage-stage embryos were treated with reversine to induce aneuploidy or vehicle to generate controls, and then cultured in media supplemented with DMO to reduce the pH of the culture media. Embryo morphology was assessed by phase microscopy. Cell number, mitotic figures, and apoptotic bodies were revealed by staining fixed embryos with DAPI. mRNA levels of Trp53, Oct-4, and Cdx2 were monitored by quantitative polymerase chain reactions (qPCRs). The effect of Trp53 on the expression of Oct-4 and Cdx2 was assessed by depleting Trp53 using Trp53 siRNA. RESULTS: Aneuploid-enriched late-stage blastocysts were morphologically indistinguishable from control blastocysts but had fewer cells and reduced mRNA levels of Oct-4 and Cdx2. Adding 1 mM DMO to the culture media during the 8-cell to blastocyst transition reduced the formation of aneuploid-enriched late-stage blastocysts but not control blastocysts and further suppressed the levels of Oct-4 and Cdx2 mRNA. Trp53 RNA levels in aneuploid-enriched embryos that were exposed to DMO were > twofold higher than controls, and Trp53 siRNA levels reduced the levels of Trp53 and increased levels of Oct-4 and Cdx2 mRNA by > twofold. CONCLUSION: These studies suggest that the development of morphologically normal aneuploid-enriched mouse blastocysts can be inhibited by adding low amounts of DMO to the culture media, which results in elevated levels of Trp53 mRNA that suppresses Oct-4 and Cdx2 expression.

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.

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.