Enhancing human kidney organoid differentiation from pluripotent stem cells with high-throughput automation

Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. In this study, single-cell RNA sequencing identify within organoids previously-undetected parietal and interstitial compartments. We discovered that addition of vascular endothelial growth factor (VEGF) during the differentiation process resulted in an approximately ten-fold increase in endothelial cells, without compromising the formation of the organoids. Although VEGF clearly increased the number of endothelial cells by immunofluorescence, relatively few endothelial cells were captured by scRNA-seq and only a modest increase in endothelial cells was observed. This suggested that either a substantial number of endothelial cells were lost or destroyed before sequencing, or that a spectrum of maturation states was present in the cultures. Overall design: We have used DropSeq to perform single cell sequencing on human kidney organoids.Organoids included in this analysis were generated by treatment with and without VEGF. We did a bulk RNA analysis on the organoids treated with and without VEGF源自人类多能干细胞的类器官(organoids)是一类极具应用潜力的高通量筛选(high-throughput screening, HTS)工具，但类器官培养的复杂性为其小型化与自动化带来了显著挑战。本研究通过单细胞RNA测序(single-cell RNA sequencing, scRNA-seq)，在类器官中鉴定出了此前未被检测到的壁细胞区室与间质区室。我们发现，在分化过程中添加血管内皮生长因子(vascular endothelial growth factor, VEGF)可使内皮细胞数量提升约10倍，且不会干扰类器官的正常形成。尽管通过免疫荧光实验可明确观测到VEGF显著增加了内皮细胞的数量，但scRNA-seq捕获到的内皮细胞数量相对较少，仅能观察到内皮细胞数量出现小幅增长。这提示，要么有大量内皮细胞在测序前便已丢失或被破坏，要么培养体系中存在一系列处于不同成熟状态的内皮细胞。整体实验设计：本研究采用DropSeq技术对人类肾脏类器官开展单细胞测序。本次分析纳入的类器官均通过添加或不添加VEGF进行培养处理构建获得。我们还对经VEGF处理与未处理的类器官进行了批量RNA分析。