| Part I Construction of human ureteral single cell transcriptome mapPurpose and Significance:The ureter connects the kidney and the bladder,which is an important bridge for the urinary system to transport urine.Understanding the cell types and functions of the ureter is of great importance for the exploration of the occurrence and development of benign ureteral diseases and ureteral cancer.Previous studies of ureteral cells have relied on the identification of cell morphology and location under the microscope and mouse experiments to further understand their possible physiological and pathological functions.However,these methods are difficult to accurately identify and classify human ureteral cells.At the same time,whether there are differences in the cell composition of different parts of the ureter also needs to be further explored.Total RNA sequencing technology can understand the average RNA expression in the ureteral tissue,but it is not accurate to the RNA expression in individual cells,so it is not possible to accurately classify the heterogeneous cell population.In recent years,the rise of high-throughput single-cell RNA sequencing(sc RNA-seq)technology can not only detect the RNA expression level from the single-cell level,but also carry out cluster analysis based on it to find out new cell subgroups or new cell groups.At the same time,it can also analyze the development trajectory of different cell populations,and analyze the relationship between them by ligand receptor pairs,which provides a reliable research method for us to understand the cell composition,biological characteristics and mutual relations of human ureter.The aim of this study was to construct a map of the human ureteral single-cell transcriptome at different anatomical sites,to accurately classify various cells of the ureter at the single-cell resolution,to reveal the differences between the cells of the ureter at different anatomical sites,and to further explore their potential physiological functions.At the same time,the ligand-receptor pair analysis revealed the relationship between human ureteral non-immune cells and immune cells.Methods:The samples in this study included 4 human ureteral tissues,which were obtained from 2 patients undergoing radical renal carcinoma resection and2 normal ureteral tissues from donor patients undergoing renal transplantation,respectively,and were digested by the enzymatic hydrolysis method of digestive enzymes and prepared into the suspension of single cells.We used 10X Genomics instrument to prepare the obtained single-cell suspension into oil-in-water structure,then performed c DNA purification and amplification,and finally established single-cell library and sequenced it.We used 10X Genomics to provide Cellranger software for preliminary processing and analysis of the obtained single cell sequencing data,and then used R software packages Seurat and Monocle2 for integration,clustering and other secondary analysis of the single cell sequencing data.At the same time,we used immunohistochemistry and immunofluorescence to verify the results of single cell sequencing.Results:We obtained the transcriptome information of 14,837 normal ureteral cells from 2 samples of the upper ureter,and 17396 normal ureteral cells from 2 samples of the lower ureter,and constructed a transcriptome map of tissue cells in different anatomical parts of the human ureter.Through unbiased clustering between cells,the cells in the upper segment of the human ureter can be divided into 15 different cell types,and the cells in the lower segment can be divided into 16 different cell types.The cell types and functions of urothelial cells,fibroblasts and immune cells in the upper and lower segments were analyzed and compared.The composition and function of fibroblasts and immune cells in the upper and lower segments of the ureter are significantly different.Compared with bladder cells,it was found that the transcriptome characteristics of ADRA2A~+ICs cells in the lower ureter were more similar to those of bladder cells.We found a large number of immune cells infiltrated in the upper part of the ureter,among which CD8+T cells have strong cytotoxicity.However,only a single immune cell composed of CD8+T cells in the lower part of the ureter had a weak cytotoxic effect.Conclusions:our study provides a comprehensive,human ureteral single-cell transcriptome mapping in different anatomical site,reveals the human lower ureter,heterogeneity and similarity between the cells,to find the cell types of ureter and its potential function provides new evidence,for the further research of ureteral cell types and the relationship between the disease lay a good foundation.Partâ…¡Comparative study of human ureteral and bladder singlecell transcriptome profilesPurpose and Significance:The ureter and the bladder belong to the upper urinary tract and the lower urinary tract respectively,and the structure and function of the two have many similarities,but there are also many differences.However,previous studies only compared the cell groups defined based on cell morphology,function and a few biomarkers,and there is no single cell level comparative study reported so far.We are in the second part of this paper to the ureter and bladder epithelial and mesenchymal cells,immune cells and their mutual relationship between a comprehensive comparative analysis,trying to explain cell classification and functional characteristics of ureter,further explore its physiological and pathological significance,to understand the pathogenesis of ureteral diseases provide new clues and understanding.Methods:Our research team has previously conducted s RNA-seq analysis on human and mouse bladders,and mapped the cell map of human and mouse bladders,and the results have been published in JASN.Next,we extracted the sequencing data of human bladder single cell transcriptome obtained in previous studies and integrated them with the sequencing data of human ureteral single cell transcriptome obtained in the first part of this study for reanalysis using the R package cluster Profiler for gene function enrichment analysis."GSVA"analysis package ureter and bladder tissues,under paragraphs on epithelial cells and immune cells in the data path concentration variance analysis,the key of ureter and bladder epithelial and mesenchymal cells,immune cells and their mutual relationship between a comprehensive comparative analysis,to explain the cell classification of ureter and functional characteristics,to deepen our understanding of ureteral-related diseases and their physiological mechanisms.Results:Bladder epithelial cells and upper and lower ureteral epithelial cells have their own unique marker genes.Bladder epithelial characteristic genes mainly include LCN2,CXCL2,UCA1,DKK1,PAX8,etc.The expression level of KRT5 gene in ureteral epithelial cells(upper and lower segments)is higher than that in urinary bladder.There was no significant difference in the expression of KRT8,KRT13 and KRT17 in ureter and bladder.Bladder stromal cells specifically expressed ACTA1,FGL2,S100A13,DES and PCP4.Tob1,ID1 and JUND were specifically expressed in the interstitial cells of the upper segment of ureter.SLC7A2,MCAM and IGFBP6 were specifically expressed in the interstitial cells of the lower segment of the ureter.Bladder immune cells significantly expressed C5AR1,CXCL8 and CXCL3.In the lower segment of ureter,the immune cells mainly expressed KLRD1,CCl4,S100A2,PSCA,SNCG and IL7R.In the upper part of the ureter,the immune cells mainly expressed GZMK,HSPH1 and IL7R.Conclusion:This study provides a comprehensive comparative analysis of human ureteral single-cell transcriptome and bladder single-cell transcriptome,revealing the cellular differences between the ureter and bladder,and providing new evidence for further exploration of the cell types and potential functions of the ureter and bladder.It will lay a good foundation for further study on the relationship between ureteral cell types and diseases. |
