CROP-seq of hiPSC-derived astrocytes (scRNA-Seq)

In response to central nervous system injury or disease, astrocytes become reactive, adopting context-dependent states with altered functions. Certain inflammatory insults induce reactive astrocyte states that lose homeostatic functions and gain neurotoxicity, and likely contribute to neuroinflammatory and neurodegenerative diseases. However, the cellular pathways controlling these states are not fully understood. Here, we combined single-cell transcriptomics with CRISPRi screening in human iPSC-derived astrocytes to systematically interrogate inflammatory reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-kB activation drove two distinct inflammatory reactive states dependent on STAT3 and IRF1, respectively. Furthermore, these states corresponded with those observed in other experimental contexts, including in vivo, and their markers were upregulated in the human brain in Alzheimer's disease and ischemic-hypoxic encephalopathy. These results and the platform we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity. hiPSC-derived astrocytes (iAstrocytes) with stable integration of CRISPRi machinery were transduced with a lentiviral sgRNA library in the CROP-seq vector (Datlinger et al, PMID: 28099430) to knockdown a set of genes hypothesized to control inflammatory reactivity. iAstrocytes were then treated with vehilce (Veh) or IL1a+TNF+C1q (ITC) to induce inflammatory reactivity, and then processed for single-cell RNA-sequencing using 10x v3. The sgRNA identities of each cell was recovered by targeted PCR enrichment of poly-adenylated transcripts containing the sgRNA sequence from the 10x cDNA (see Datlinger et al). This dataset includes both the 10x RNA libraries as well as the CROP-seq PCR enrichment libraries.