Profiling of Lineage, Connectivity, and Gene Regulatory Networks During Human Cortical Development and Cellular Heterogeneity of Human Neurovasculature

We sought to characterize the cell types produced by individual human cortical progenitors and describe their lineage relationships. We labeled individual human cortical cells with a massively complex lentivirus library encoding a GFP reporter gene and a molecular barcode that could be captured by standard 3' scRNA-seq methodologies. We then cultured the labeled human cells for 6 weeks in either a 1) mouse cortical astrocyte co-culture system or 2) in the mouse cortex as a xenograft. In a separate experiment, we maintained human cortical tissue slices in organotypic slice culture for 12 days. GFP+ cells were then isolated by FACS and then captured on the 10x chromium scRNA-seq platform. An additional sample was labeled by lentivirus and briefly cultured for 3 days prior to being mixed with mouse 3T3 cells in order to perform a cell mixing experiment ("barnyard") to benchmark our methodologies. We found that human individual cortical progenitors gave rise to both excitatory and inhibitory neurons as well as glia. (PMID: 34912114; 41193842) We also sought to characterize the adult brain vasculature in both vascular malformations and non-malformed tissue. We use the 10x chromium scRNA-seq platform across 10 individuals (5 control and 5 arteriovenous malformations). We find broad disregulation across multiple cell types including the immune cells and find a specific monocyte subtype to contribute to a rupture phenotype. We also find a novel population of fibromyocytes in both conditions. (PMID: 35084939) In addition, we confirmed the genotype status of iPS lines that were derived from either karyotypically normal control individuals or from DiGeorge Syndrome patients carrying the 22q11.2 deletion using the Illumina Infinium Global Screening Array v3.0. (PMID: 38382530) Furthermore, we characterized the neuronal connectivity in the human cerebral cortex during the second trimester of development by performing barcoded rabies tracing using single-cell RNA sequencing or single-nucleus RNA sequencing readout using the Fluent PIP-seq platform (PMID: 39713304). Finally, we performed single cell CRISPRi screening on 44 transcriptional factors in primary human 2D and organotypic culture, and combined repression of 5 transcription factors with lineage tracing, revealing mechanisms governing radial glia lineage progression. We also performed dual repression of ARX and LMO1 to partially rescue an "ectopic" inhibitory neurons status induced by ARX repression. (PMID: 41040362)]]>