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Impaired neurogenesis in Down syndrome (DS) is characterized by reduced neurons, increased glial cells, and delayed cortical lamination. However, the underlying cause for impaired neurogenesis in DS is not clear. Using both human and mouse iPSCs, we demonstrate that DS impaired neurogenesis is due to biphasic cell cycle dysregulation during the generation of neural progenitors from iPSCs named the “neurogenic stage” of neurogenesis. Upon neural induction, DS cells showed reduced proliferation during the early phase followed by increased proliferation in the late phase of the neurogenic stage compared to control cells. While reduced proliferation in the early phase causes reduced neural progenitor pool, increased proliferation in the late phase leads to delayed post mitotic neuron generation in DS. RNAseq analysis of late-phase DS progenitor cells revealed upregulation of S phase-promoting regulators, Notch, Wnt, Interferon pathways, and REST, and downregulation of several genes of the BAF chromatin remodeling complex. NFIB and POU3F4, neurogenic genes activated by the interaction of PAX6 and the BAF complex, were downregulated in DS cells. ChIPseq analysis of late-phase neural progenitors revealed aberrant PAX6 binding with reduced promoter occupancy in DS cells. Together, these data indicate that impaired neurogenesis in DS is due to biphasic cell cycle dysregulation during the neurogenic stage of neurogenesis.

Human mouse chimeric models are valuable tools to develop in-vivo disease models. However, in-vivo detection of human cells limits their analysis. To facilitate in-vivo modeling of Down syndrome (DS), we generated a stable AAVS1-EGFP isogenic pair of DS human iPSCs by zinc finger mediated genetic engineering of the AAVS1 locus. These lines overcome the limitation of reporter human iPSCs generated using random integration, which may not express reporter gene in all tissues due to heterochromatin-induced gene silencing. These reporter cell lines provide a valuable tool to facilitate in-vivo tracking of the graft cell integration, differentiation, and distinction from host cells.

Human-induced pluripotent stem cells (hiPSCs) clones NSi001-A, NSi001-B, and NSi001-C were generated from a female individual of Indian origin having Robertsonian translocation down syndrome (DS) by reprogramming peripheral blood mononuclear cells (PBMCs) using integration-free Sendai viral vectors. The established hiPSCs clones had karyotype similar to the patient sample with Robertsonian translocation [46, XX rob (14;21)], normal ES-like morphology, expression of pluripotency markers, and potential for three germ layer differentiation, i.e., ectoderm, mesoderm, and endoderm.