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NFIB induces functional astrocytes from human pluripotent stem cell-derived neural precursor cells mimicking in vivo astrogliogenesis

ABSTRACT: The ability to generate astrocytes from human pluripotent stem cells (hPSCs) offers a promising cellular model to study the development and physiology of human astrocytes. However, the extant methods for generating functional astrocytes required long culture periods and remained much ambiguity whether their paradigm follows the innate developmental program. In this report, we provided an efficient and rapid method for generating physiologically functional astrocytes from hPSCs. Overexpressing the nuclear factor IB (NFIB) in hPSC-derived neural precursor cells (NPCs) induced a highly enriched astrocyte population in 2 weeks. RNA sequencing and functional analyses demonstrated progressive transcriptomic and physiological changes in the cells, resembling in vivo astrocyte development. Further analyses substantiated previous results and established the MAPK pathway necessary for astrocyte differentiation. Hence, this differentiation paradigm provides a prospective in vitro model for human astrogliogenesis studies and pathophysiology of neurological diseases concerning astrocytes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE157768 | GEO | 2020/09/11

REPOSITORIES: GEO

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Project description:The gene regulatory mechanisms that steer neural stem cells towards astrocytes during brain development are largely unknown. Here we set out to comprehensively identify transcription factors that drive the developmental trajectory of astrogliogenesis from mouse embryonic stem cells. To attain this we performed a genomewide analysis of gene expression and epigenetic signature hallmarks of primed and active enhancers through the distinct stages of astrogliogenesis. We revealed distal regulatory elements that become activated in a stage-specific manner which confer the transcriptional program underlying generation and maturation of astrocytes. By searching for lineage-specific transcription factors functioning at these elements, we identified ATF3 and NFIA as drivers of differentiation of neural precursor cells to astrocytes, while RUNX2 promotes their maturation. These transcription factors are crucial for switching the chromatin state of these elements from primed to active, thereby driving the gene expression program underlying astrocyte differentiation and maturation.Method: RNA was extracted from embryonic stem cells (ES) differentiated into neural progenitor cells (aNPC) and subsequently differentiated for 1 day into early astrocytes (eA) and further progressively for 5 and 21 days into mature astrocytes (lA_1 and lA_2). These samples are further subjected for RNA-sequencing to reveal the genome wide changes at transcriptional level at each stage. aNPC, eA and lA_1 (further called as lA) were subjected for H3K27ac and H3K4me1 ChIP and sequenced genome wide (ChIP-seq) to reveal the active and primed enhancers regions during astrogliogenesis, respectively. In quest to identify the TF required for early and late astrogliogeneis, we depleted Nfia (SiNfia) and Atf3 (siAtf3) in eA and Stat3 (siStat3), Prdm9 (siPrdm9) and Runx2 (siRunx2) at lA by using siRNAs and perfomed H3K27ac ChIP and sequence them. We also extracted RNA after depletion (only after siNfia, siAtf3 and siRunx2) and perform the RNA-seq.

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NFIB induces functional astrocytes from human pluripotent stem cell-derived neural precursor cells mimicking in vivo astrogliogenesis

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