scRNA sequencing analysis for human ureters and patient derived ureter oragnoid

Tissue engineering offers a promising treatment strategy for ureteral strictures. Successful ureter engineering is necessitated by detailed understanding of the tissue architecture, cellular heterogeneity, and signaling pathways underlying regeneration. We define and spatially map cell populations within the human ureter by using a combinatorial approach: single-cell RNA sequencing, 10X Visium spatial transciptomics, and immunofluorescence. The stromal and urothelial cell populations are analyzed in detail, and we infer potential cell-cell communication networks underpinning the bi-directional crosstalk between these compartments. Specifically, we analyze and experimentally validate the importance of Sonic Hedgehog (SHH) signaling pathway in adult stem cell maintenance. The SHH-expressing basal cells support organoid generation in vitro, and accurately predict the differentiation trajectory of basal stem cells, to terminally differentiated umbrella cells, in vivo. Our results highlight essential processes involved in adult ureter tissue homeostasis, and provide a toolkit for guiding ureter tissue engineering. 10 normal human ureters each were subjected to RNA sequencing at single cell resolution on Novaseq using 10X Gemonics library preparation platform. Data was processed using Cellranger 4.0.0 and anayzed using Seurat v3.2.1 to characterize various cell types and understand finer details of the regenrative pathways in the ureter urothelium. This was further explored by analyzing single cell RNA sequencing data from human ureter derived organoid (generated in the same manner as the 10 ureter samples). Freshly processed and thawed tissue sample from the same human subject was used as control to further understand the urothelial regenerative pathways.