Single-Cell Transcriptomic Map Of Human And Mouse Bladders Based On Single-cell RNA Sequencing Technology

Background:Having a comprehensive map of the cellular anatomy of the normal bladder is vital to understanding the cellular origins of benign bladder disease and bladder cancer.Previous studies of bladder cell classification mostly relied on the cell’s morphology,location,electrophysiology,and function.However,these traditional methods may be difficult to fully classify bladder cells accurately.m RNA,as the messenger of genetic material,plays an important physiological role in translating and guiding protein synthesis.Bulk RNA sequencing can be used to determine the mean RNA expression in bladder tissues,but it is not possible to estimate the RNA expression in individual cells precisely.In recent years,with the development of high-throughput single-cell RNA sequencing(sc RNA-seq)and The Human Cell Atlas,we can perform sc RNA-seq on bladder tissues.Objective:We aim to understand the unsupervised classification and function of both human and mouse bladder cells precisely.The homology and heterogeneity of human and mouse bladder cells can be revealed by sc RNA-seq.We hope to provide important information for studying the relationship between different bladder cell types and bladder diseases.Part I.The establishment of a single-cell transcriptomic map of human bladderMethods:We obtained normal bladder tissues far from the tumor from three patients who underwent radical cystectomy or partial cystectomy,and prepared single-cell suspensions by digestive enzymes.We used the 10x Genomics to prepare single-cell suspension into water-in-oil structures,then established c DNA library and sequencing.We used Cellranger software provided by 10x Genomics for preliminary analysis of sc RNA-seq data,and then used R package Seurat,Monocle2 and Velocity for secondary analysis.At the same time,we used immunohistochemistry,immunofluorescence and Western blot to verify the novel cell types of human bladder found by sc RNA-seq.Results:We created a single-cell transcriptomic map of human bladder,including 12423 human bladder cells.These cells can be classified into 16different cell types by unbiased clustering.We constructed the developmental trajectory of human bladder interstitial cells and analyzed the RNA velocity of bladder epithelial and interstitial cells.We also discovered two novel types of human bladder cells.One type is ADRA2A~+and HRH2~+interstitial cells which may be associated with nerve conduction and allergic reactions.The other type is TNNT1~+epithelial cells that may be involved with bladder emptying.The results of this part showed the abundant single-cell transcriptomic information of human bladder.By clustering analysis,bladder cells were accurately classified into 16 different cell types,including two new cell types.However,as one of the most common animal models,the homology and heterogeneity of human and mouse bladder are unclear.To address this problem,we subsequently performed sc RNA-seq on mouse bladder.Part II.The establishment of a single-cell transcriptomic map of mouse bladderMethods:We selected normal bladder tissues from 10 C57BL/6 mice and prepared single-cell suspension with digestive enzymes.We also used the 10x Genomics to prepare single-cell suspension into water-in-oil structures,then established c DNA library and sequencing.Mouse sc RNA-seq data was analyzed using the method mentioned above.We calculated the Pearson correlation coefficient to determine the correlation between human and mouse cell types.Meanwhile,we performed immunohistochemistry and Western blot to verify the species heterogeneity between human and mouse.Results:We created a single-cell transcriptomic map of mouse bladder,including 12884 human bladder cells.These cells can be classified into 15different cell types by unbiased clustering.The homology and heterogeneity of human and mouse bladder cell types were compared and both conservative and heterogeneous aspects of human and mouse bladder evolution were identified.Conclusion:Our study provides the most comprehensive information on the cell types found in the normal human and mouse bladders thus far.These findings provide insights into the discovery of the novel cell type,specific cell markers,signaling receptors,and gene data set which will aid in studying the relationship between cell types and bladder diseases.