Single-cell RNA sequencing of vascularized brain organoids

Organoids have been widely used as unique models of human brain development and disorders. However, the lack of vasculatures in brain orgnoids limits their application in the study of brain vasculature development and diseases. Here, we described to the generation of vascularized brain organoids (VBOrs) with different brain regions from human embryonic stem cells. The goals of this study are to analyze the cell populations of the new model of vasularized brain organoids cultured from human embryonic stem cells (H9). We found that VBOrs contain variant brain cells inculding neural progenitors, neuronal cells, astrocytes, sparse endothelial cells, and pericytes. The new model of VBOrs should be valuable for addressing questions between brain vasculatures and neural cells. Overall design: VBOrs were generated by two methods distinct in the step of vascular progenitor induction: method 1 (group BMPi), EBs were treated with bFGF, VEGF and SB431542 for two days; method 2 (group BMPa), EBs were treated with bFGF, VEGF and BMP4 for two days. Single-cell suspensions from two groups of VBOrs (BMPa and BMPi) were prepared by 20-min digestion in 0.25 mg/ml trypsin. About 10,000-12,000 cells for each sample (2 independent repeats) were loaded for soating, barcoding and library construction. Sequencing was performed with HiSeq X.