A single-cell transcriptomic dataset of pluripotent stem cell-derived astrocytes via NFIB/SOX9 overexpression
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ABSTRACT: Astrocytes, the predominant glial cells in the central nervous system, play essential roles in maintaining brain function. Reprogramming induced pluripotent stem cells (iPSCs) to become astrocytes through overexpression of the transcription factors, NFIB and SOX9, is a rapid and efficient approach for studying human neurological diseases and identifying therapeutic targets. However, the precise differentiation path and molecular signatures of induced astrocytes remain incompletely understood. Accordingly, we performed single-cell RNA sequencing analysis on 64,736 cells to establish a comprehensive atlas of NFIB/SOX9-directed astrocyte differentiation from human iPSCs. Our dataset provides detailed information about the path of astrocyte differentiation, highlighting the stepwise molecular changes that occur throughout the differentiation process. This dataset serves as a valuable reference for dissecting uncharacterized transcriptomic features of NFIB/SOX9-induced astrocytes and investigating lineage progression during astrocyte differentiation. Moreover, these findings pave the way for future studies on neurological diseases using the NFIB/SOX9-induced astrocyte model.
Project description: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.
Project description:Astrocytes are key regulators of CNS homeostasis and their dysfunction is implicated in neurological and neurodegenerative disorders. Here, we describe a two-step protocol to generate astrocytes from iPSCs using a bankable neural progenitor cell (NPC) intermediate, followed by low-density passaging and overexpression of the gliogenic transcription factor NFIA. A bankable NPC intermediates allows for facile differentiation into both purified neuronal and astrocyte cell types in parallel from the same genetic background, depending on the experimental needs. This article presents a protocol to generate NPCs from iPSCs (Basic Protocol 1), which are then differentiated into iPSC-derived astrocytes, termed iAstrocytes (Basic Protocol 2). The resulting iAstrocytes express key markers of astrocyte identity at transcript and protein levels by bulk RNA-seq and immunocytochemistry respectively. Additionally, they respond to the inflammatory stimuli poly(I:C) and generate waves of calcium activity in response to either physical activity or addition of ATP. Our approach offers a simple and robust method to generate and characterize human astrocytes which can be used to model human disease affecting this cell type.
Project description:Growing evidence implicates the importance of glia, particularly astrocytes, in neurological and psychiatric diseases. Here, we describe a rapid and robust method for the differentiation of highly pure populations of astrocytes from human induced pluripotent stem cells (hiPSCs), via a neural progenitor cell (NPC) intermediate. Using this method, we generated hiPSC-derived astrocyte populations (hiPSC-astrocytes) from 42 NPC lines (derived from 30 individuals) with an average of ~90% S100β-positive cells. Transcriptomic analysis demonstrated that the hiPSC-astrocytes are highly similar to primary human fetal astrocytes and characteristic of a non-reactive state. hiPSC-astrocytes respond to inflammatory stimulants, display phagocytic capacity and enhance microglial phagocytosis. hiPSC-astrocytes also possess spontaneous calcium transient activity. Our novel protocol is a reproducible, straightforward (single media) and rapid (<30 days) method to generate homogenous populations of hiPSC-astrocytes that can be used for neuron-astrocyte and microglia-astrocyte co-cultures for the study of neuropsychiatric disorders.
Project description:Differentiation of astrocytes from human pluripotent stem cells (hPSCs) is a tedious and variable process. This hampers the study of hPSC-generated astrocytes in disease processes and drug development. By using CRISPR/Cas9-mediated inducible expression of NFIA or NFIA plus SOX9 in hPSCs, we developed a method to efficiently generate astrocytes in 4-7 weeks. The astrocytic identity of the induced cells was verified by their characteristic molecular and functional properties as well as after transplantation. Furthermore, we developed a strategy to generate region-specific astrocyte subtypes by combining differentiation of regional progenitors and transgenic induction of astrocytes. This simple and efficient method offers a new opportunity to study the fundamental biology of human astrocytes and their roles in disease processes.
2018-08-11 | GSE118384 | GEO
Project description:A single-cell transcriptomic dataset of pluripotent stem cell-derived astrocytes via NFIB/SOX9 overexpression
Project description:Direct cell reprogramming has enabled the direct conversion of skin fibroblasts into functional neurons and oligodendrocytes using a minimal set of cell lineage-specific transcription factors. This approach has substantial advantages since it is rapid and simple, generating the cell type of interest in a single step. However, it remains unknown whether this technology can be applied for directly reprogramming skin cells into astrocytes, the third neural lineage. Astrocytes play crucial roles in neuronal homeostasis and their dysfunctions contribute to the origin and progression of multiple human diseases. Herein, we carried out a screening using several transcription factors involved in defining the astroglial cell fate and identified NFIA, NFIB and SOX9 to be sufficient to convert with high efficiency embryonic and post-natal mouse fibroblasts into astrocytes (iAstrocytes). We proved both by gene expression profiling and functional tests that iAstrocytes are comparable to native brain astrocytes. This protocol can be then employed to generate functional iAstrocytes for a wide range of experimental applications. Induced astrocytes (iAstro) were compared to Fibroblasts (Fibro) as negative control and to primary astrocytes (astro) as positive control. Three biological replicates were analyzed for each experimental group.
Project description:Reactive astrocytes play a pivotal role in the pathogenesis of neurological diseases; however, their functional phenotype and the downstream molecules by which they modify disease pathogenesis remain unclear. Here, we genetically increase P2Y1 receptor (P2Y1R) expression, which is upregulated in reactive astrocytes in several neurological diseases, in astrocytes of male mice to explore its function and the downstream molecule. This astrocyte-specific P2Y1R overexpression causes neuronal hyperexcitability by increasing both astrocytic and neuronal Ca2+ signals. We identify insulin-like growth factor-binding protein 2 (IGFBP2) as a downstream molecule of P2Y1R in astrocytes; IGFBP2 acts as an excitatory signal to cause neuronal excitation. In neurological disease models of epilepsy and stroke, reactive astrocytes upregulate P2Y1R and increase IGFBP2. The present findings identify a mechanism underlying astrocyte-driven neuronal hyperexcitability, which is likely to be shared by several neurological disorders, providing insights that might be relevant for intervention in diverse neurological disorders.
Project description:Astrocytes play key roles in brain function but it is still poorly understood how these are orchestrated by pan-astrocyte transcriptions factors (TFs). Here we examined the function of the well-known pan-astrocyte TF Sox9 and the novel astrocyte TF Trps1 (Transcriptional Repressor GATA Binding 1) by Cas9-mediated in vivo deletion using Mokola-pseudotyped lentiviral delivery into the adult cerebral cortex. The consequences of deleting either Sox9 or Trps1 alone or simultaneously were explored at single cell level (by patch-based single cell transcriptomics) and tissue level (by spatial transcriptomics). This revealed TF-specific functions in astrocytes, like synapse maintenance and immune response, as well as non-cell-autonomous effects on the surrounding cells such as on oligodendrocytes and other immune cells. Interestingly, deleting both factors cancels out some effects, suggesting that they antagonize each other’s function to some extent. Taken together, this study reveals a novel role of Trps1 and Sox9 in adult astrocytes and their communication with other glial and immune cells.
Project description:APOE4 genotype is the strongest risk factor for the pathogenesis of sporadic Alzheimer’s disease (AD), but the detailed molecular mechanism of APOE4-mediated synaptic impairment remains to be determined in human cellular context. In this study, we generated human astrocyte model carrying APOE3 or APOE4 genotype using human induced pluripotent stem cells (iPSCs), in which isogenic APOE4 iPSCs were genome-edited from healthy control APOE3 iPSCs. By transcriptome analysis of human astrocytes between APOE genotypes, we showed the upregulation of an extracellular matrix glycoprotein in human APOE4 astrocytes, which may cause synaptic degeneration in concert with the equivocal reactive character and lipid change. Together, these results demonstrate novel negative impact of human APOE4 astrocyte on synaptic integrity and lead to a promising therapeutic intervention into APOE4-carriers.