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Endothelial cell-conditioned medium enhanced astrocyte differentiation in cerebral organoids

ABSTRACT: Human brain organoids (hBOs) are self-organizing, three-dimensional structures that recapitulate aspects of early brain development, including astrogliogenesis. However, astrocyte differentiation does not occur until months into the differentiation process, and astrocytes continue to mature for years after that. Astrogliogenesis and astrocyte maturation could be further delayed by innate shortcomings of the model such as a lack of endothelial cells (ECs). Indeed, studies suggest that ECs or EC-derived factors promote astrogliogenesis and astrocyte maturation in vitro. Here, we exposed unguided hBOs to conditioned medium of immortalized human cerebral microvascular ECs (hCMEC/D3 cells) and determined their expression profile on the single-cell level at two time points. We found that EC-secreted proteins do not induce premature astrogliogenesis in hBOs but do affect hBO cellular composition and the maturation of radial glia, which are the precursors to astrocytes. These findings indicate the potential of EC-secreted factors to enhance astrocyte (precursor) maturation in hBOs and emphasize the importance of considering EC effects when aiming to accurately study developing astrocytes using hBOs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE299777 | GEO | 2026/02/02

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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Endothelial cell-conditioned medium enhanced astrocyte differentiation in cerebral organoids

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