Project description:We report on a comprehensive time-course study of transcription factor (TF)-induced iPSC neurons cultured in vitro through an intrinsic maturation program following neurogenesis. Combining the present RNA-seq study with immunocytochemistry and electrophysiological analyses, we determined the transcriptional and morphological sequences of key developmental events associated with postnatal brain-like transition and spinogenesis. TF-induced iPSC neurons successfully acquired structural and functional synaptic maturity, which will critically expand their utility in modeling higher brain functions and disorders.
Project description:Purpose: The transcriptional differences between iPSC-derived cortical neurons from patients with idiopathic ASD and unaffected controls was examined over a 135-day course of neuronal differentiation using RNAseq analysis Methods: Induced pluripotent stem cells (5 control iPSC and 6 ASD-specific iPSC) were differentiated into cortical neurons and RNA samples were obtained at two different time points day 35 post differentiation and day 135 post differentiation. RNAseq was performed from the RNA isolated at the different time points and the differentially expressed genes identified. Pathway analysis for gene ontology and biological processes, as well as, weighted gene co-expression network analysis was performed. Results: The results of this analysis show ASD-specific misregulation of genes involved in neuronal differentiation, axon guidance, cell migration, DNA and RNA metabolism, and neural region patterning. Furthermore, functional analysis revealed defects in neuronal migration and electrophysiological activity, providing compelling support for the transcriptome analysis data. Conclusions: Transcriptional analyses of the ASD and control neurons showed ASD-specific molecular phenotypes affecting networks involved in neuronal differentiation, the cytoskeletal matrix structure formation, patterning, DNA and RNA metabolism.
Project description:To comprehensively profile early neurodevelopmental alterations in individuals with ASD, we harnessed a time series approach to monitor patient-derived induced pluripotent stem cells (iPSCs) throughout the recapitulation of cortical development. This dataset consists of patient derived neurons that go through all consecutive developmental stages (NSC-derived neurons) as well as a comparative set of iPSC-iNs (neurons generated from the same patients that bypass early NSC-like stages using an Ngn2-transgene approach). For this, we first used fluorescence-activated cell sorting (FACS) to purify a homogeneous population of NSCs based on the expression of the cell-surface markers CD184+/CD271-/CD44-/CD24-/CD15+. To trace ASD and control neurons over time, we performed a series of retroviral lineage-tracing experiments to trace the progenies of dividing NSCs using a retroviral vector expressing a membrane-tagged enhanced green fluorescent protein (eGFP) (CAG::LckN-eGFP). As differentiating neurons express PSA-NCAM on the cell surface, we established a FACS-based protocol for purification of defined subpopulations of retrovirally labeled eGFP+/PSA-NCAM+ double-positive neurons after 2, 4, 7 and 14 days of differentiation. IPSCs were sorted based on the expression of SSEA-4 and TRA1-81 and maturing iPSC-iNs were collected at the indicated days after induction by sorting for eGFP (indicative for the Ngn2 transgene)- and PSA-NCAM-positive cells.