Identification of cell types in human and rat bladders
After strict filtering and quality control, a total of 35,510 cells from two human (one male and one female) and 19,946 cells from two rat (one male and one female) bladder tissues were obtained for further clustering (Fig. 1, Fig. S1 and Supplementary Table 1). The cells were separated from zones of the bladder anterior wall in the human bladder and from the overall bladder in rats, and was then digested to a single cell suspension, which was subjected to scRNA-seq on the 10x Genomics platform version 3.0 (Fig. 1a). On average, we detected 2,340 genes from human and 1,996 genes from rat single cells (Supplementary Table 1). Subsequently, unsupervised clustering analysis showed 26 and 23 clusters in all filtered human and rat cells, separately (Fig. S1a and Fig. S1f). Cells from different sexes were distributed evenly in the same cluster and showed minimal differences (Fig. S1b and Fig. S1g). According to the characterization of the bladder histological structure and marker genes from reference (Supplementary Table 2), we identified 13 major cell clusters in human and 14 major cell clusters in rat (Fig. 1b-1e). The number and type of cells show minimal differences between human and rat (Fig. 1f, Fig. S1d-S1e, Fig. S1i-S1j).
To further analyze the features of major cell clusters from human and rat bladders, we performed pseudotime trajectory, CD markers, cell junctions, ion channels, cell surface receptors, and transcription factor distribution among different clusters. To clarify the states and relationships between the major cell clusters in humans and rats, 3 major states were identified using pseudotime trajectory (Fig. 2a-2b, Fig. S2a and S3a), indicating the cells undergoing different developmental and differentiation stages. Pearson correlation analysis showed high consistency between homologous cell types of human and rat bladder tissues (Fig. 2c). In addition, CD molecular markers can specifically label cell populations in human or rat bladder tissues, such as CD24, CD138, and CD358 for epithelium stratum cells; CD364 and CD91 for fibroblasts; CD362 and CD121a for myofibroblasts; and CD146 for smooth muscle cells (Fig. 2d and 2g). The intercellular junction complex between bladder cells plays a key role in adhesion and barrier function and consists of adherent junctions, gap junctions, desmosomes, and tight junctions. We found that homologous genes of CLDN4, CLDN7, F11R, GJB2, GJB6, and OCLN were expressed in epithelial stratum cells; GJC1, GJA4, and JAM3 were expressed in smooth muscle cells; PANX1 was expressed in fibroblasts, and JAM2 was expressed in endothelial cells of both humans and rats (Fig. 2e and 2h). One of the main functions of epithelial and smooth muscle cells in the bladder is ion transport and sensory transduction. We also identified the water transporter AQP3, epithelial sodium channel SCNN1A, potassium two pore domain channel KCNK1, chloride voltage-gated channel CLCN3 expressed in epithelial stratum cells, CACNA1H, P2RX1, KCNJ8, and ITPR1 expressed in smooth muscle cells, TRPA1 expressed in myofibroblasts, PKD2 expressed in fibroblasts, AQP1, and ORAI3 expressed in endothelial cells (Fig. 2f and 2i). Regarding transcription factors, the expression profile among the major cell clusters in humans and rats were revealed to be similar to each other (Fig. 2j and 2n). Comparison of expression profiles between different genders in human and rat samples showed that the Pearson coefficient was 0.98 (Fig. 2K, P < 0.001) in human and 0.98 (Fig. 2O, P < 0.001) in rat. Gene Ontology (GO) enrichment analysis indicated that genes with higher expression in the human male bladder were associated with muscle contraction (Fig. 2l), genes with higher expression in the human female bladder were associated with extracellular structure organization (Fig. 2m), genes with higher expression in the rat male bladder were associated with wound healing (Fig. 2p), and genes with higher expression in the rat female bladder were associated with metabolic processes (Fig. 2q).
Characterization of epithelial stratum cells in the human and rat bladder
To identify the subpopulations of epithelial stratum cells in human and rat, second-level clustering was performed, which distinguished the epithelial stratum cells into three clusters including umbrella, intermediate, and basal cells (Fig. 3a and 3b, Fig. S4). Umbrella cells in human (n = 1139) and rat (n = 3380) bladder highly express uroplakin proteins (UPK1A, UPK1B, and UPK2), which cover the apical plasma membrane and reducing membrane permeability (Fig. 3c and 3e). GO pathway enrichment indicated that the functions of signal transduction and protein secretion were enriched in umbrella cells (Fig. 3g and 3i). Basal cells in human (n = 686) and rat (n = 5075) bladder tissues highly expressed the protein members of the keratin gene family (KRT5 and KRT17), which are widely distributed during the differentiation of simple and stratified epithelial tissues (Fig. S4). GO pathway enrichment indicated that the basal cells interacted with neurons, responding to ion and hormone signaling (Fig. 3h and 3k). Pseudotime trajectory analysis demonstrated that umbrella and basal cells appeared at the end of the branch and intermediate cells at the continuations (Fig. 3d and 3f), which may indicate development and differentiation direction among the three types of cells. However, this assumption needs further validation. There is no clear boundary regarding CD markers, ion channels and receptors, cell junctions, and transcription factors among the three types of epithelial stratum cells (Fig. S16f- S16l).
Characterization of fibroblasts in human and rat bladder
The marker genes for distinguishing fibroblasts are SFRP2, MMP2, COL1A, and PDGFRA (Fig. S5a-S5b). In addition, algorithmically identified marker genes for bladder fibroblast include GSN, DCN, PI16 (CD364), and LRP1 (CD91) (Fig. S5c-S5d) in both human or rat bladder tissues. Second-level clustering showed 5 subpopulations of fibroblasts in human and 4 in rat (Fig. 4a and 4b). The marker gene expression levels among subpopulations of fibroblast showed ambiguous boundaries, but high expression level of A2M gene in fibroblast_3 of human and fibroblast_1 of rat and high expression level of FOS gene in fibroblast_2 of human and fibroblast_3 of rat (Fig. 4c and 4e) were observed simultaneously. We did not find any significant difference of states in the subpopulations of fibroblasts in the following pseudotime trajectory analysis (Fig. 4d and 4f). Interestingly, GO enrichment analysis of special marker genes in the subgroups of fibroblasts showed cell response stimuli for hormones (Fig. S14a), chemokines (Fig. S14b, S14e), calcium ions (Fig. S14a, S14g), leukocytes (Fig. S14c, S14f), zinc ions (Fig. S14c), TGFβ (Fig. S14d, S14h), and so on. It thus seems like fibroblasts can respond to different stimuli, but their role in bladder signaling transduction still needs further clarity. We did not find any special CD markers, ion channels and receptors, cell junctions, and transcript factors to distinguish the subpopulations of fibroblasts in human and rat samples (Fig. S14i- S14n).
Fibroblasts are one of the main interstitial cells in the bladder. Therefore, it is important to determine their location in bladder tissue. Double immunofluorescence staining performed with anti-THY1 (only expressed in fibroblast) and anti-DES (only expressed in smooth muscle cells) antibody in human bladder tissue indicated that fibroblasts are located under the epithelial stratum and among muscular bundle cells (Fig. 4g). Immunohistochemical staining performed with anti-THY1 antibody confirmed this finding (Fig. 4h). Flow cytometric sorting of cells from a single cell suspension of human bladder cells using anti-THY1 (CD90) antibody showed a fibroblast-like cell type presentation as a sheet-like body with prominent cytoplasm, abundance of mitochondria and prominent rough and smooth endoplasmic reticulum (Fig. 4i). Similarly, rat bladder tissues stained with double immunofluorescence (anti-LBP and anti-Des) indicated the same location as that determined in human bladder tissues (Fig. 4j).
Characterization of smooth muscle cells in human and rat bladder
Marker gene (DES, MYH11, MYL9, TAGLN, TPM2, ACTA2, ACTG2, and CNN1) expression levels showed consistency in human and rat smooth muscle cells (SMCs) (Fig. S6). Five subpopulations of SMCs were present after second-level clustering in both human and rat SMCs (Fig. 5a and 5b). SMCs in the bladder may exist in the artery, vein, lymphatic vessels, detrusor muscle, and even in the muscularis mucosae located in the lamina propria. All sub-clusters of SMCs appeared at the end of the branch in the state of pseudotime trajectory analysis (Fig. 5d and 5f), indicating that they are mature terminally differentiated cells. However, these cannot be distinguished based only on algorithmically identified marker genes (Fig. 5c and 5e). GO enrichment pathway analysis may provide some clues (Fig. 15s). SMC_1 in human tissues shows higher Pearson correlation coefficients with SMC_1 in rat (Fig. S3d), cells in this cluster are associated with artery/vein vascular development and also on zinc ion response (Fig. S15a and S15f). SMC_2 in human tissues correlated with SMC_2 in rat tissues (Fig. S3d), cells in SMC_2 are associated with muscle contraction and striated muscle tissue development (Fig. S15b and S15g). SMC_3 in human is related with SMC_3 in rat (Fig. S3d) and cells in SMC_3 are associated with leukocyte chemotaxis and vascular development (Fig. S15c and S15h), which indicated that the sources of SMC_3 may originate from lymphatic vessels. SMC_4 in human parallels with SMC_4 in rat (Fig. S3d); cells in SMC_4 are associated with response to type I interferon and endopeptidase activity (Fig. S15d and S15i). SMC_5 in human and rat function with extracellular structure organization, cell-substrate adhesion, and so on (Fig. S15e and S15j). It most likely that SMC_5 are the kind of cells located in the muscularis mucosae, which participate in wound healing and tissue repair. CD markers, ion channels and receptors, cell junctions, and transcription factor classification analysis showed that SMC_5 was significantly different from other clusters (Fig. S15k-o). Therefore, functions of this cluster in the bladder need further investigation.
Characterization of endothelial cells in human and rat bladder
Common marker genes (SELE, VCAM1, CDH5, and ENG) are expressed in endothelial cells consistently within human and rat bladder tissues (Fig. S8a and S8b). Algorithmically identified marker genes in human (ACKR1, TM4SF1, IFI27 and STC1) and rat (Tm4sf1, Plvap, Selp and Cst3) show differences between each other. Three subpopulations of endothelial cells were present after second-level clustering in both human and rat (Fig. 6a and 6b), which may correspond with the artery, vein, and lymphatic vessels. The difference among these three subpopulations of endothelial cells is obscure (Fig. 6c and 6e). However, pseudotime trajectory analysis (Fig. 6d and 6f) indicated that they are mature terminally differentiated cells distributed at the end of the branch, respectively. Interestingly, GO analysis indicated that endothelial cells_1 in human and rat are majorly associated with leukocyte migration and response to lipopolysaccharide, which demonstrates that these clusters may include a kind of endothelial cell in lymphatic vessels (Fig. 6g and 6j). Endothelial cells_2 in human and endothelial cells_3 in rat showed high correlation (Fig. S3d) and GO analysis showed that these clusters were associated with notch signaling pathway and angiogenesis (Fig. 6h and 6l). Endothelial cells_3 in human and endothelial cells_2 in rat were correlated with each other (Fig. S3d) and GO analysis showed these clusters were associated with wound healing and extracellular structure organization (Fig. 6i and 6k). CD markers, ion channels and receptors, cell junctions, and transcription factor classification analysis showed that endothelial cells_3 in human and endothelial cells_2 in rat showed a significant difference compared with other clusters (Fig. S16a-S16e), indicating that these clusters may be a kind of endothelial cells in the artery.
Characterization of myofibroblasts in human and rat bladder
There is an ongoing debate about the occurrence of true myofibroblasts in the bladder. The key feature of these essentially reactive cells is the fibronexus. Myofibroblasts express marker genes (ACTA2, ACTG2, TAGLN and PDGFRB) that exist in both muscle cells and fibroblasts (Fig. S7A-S7B). Algorithmically identified marker genes in human (STC1, AREG, PLAT and TBX3) and rat (Cxcl14, Tgfbi, Crayb and Car3) showed a special relation between each other (Fig. 7d, 7e, Fig. S7c-S7d). The gene expression Pearson coefficient between human and rat myofibroblasts was 0.77 (Fig. 7A, P < 0.001), which denotes the difference in the two kinds of myofibroblasts. However, GO analysis of marker genes in human (Fig. 7b) and rat (Fig. 7c) myofibroblasts demonstrated simultaneous enrichment in the extracellular matrix organization, which suggests that they have the same function in the bladder. To locate myofibroblasts in the bladder, antibodies against TRPA1 (specifically expressed in human myofibroblast), DES, and Tgfbi were used for double immunofluorescence staining. The results showed that myofibroblasts were located mainly between the epithelial stratum and muscle stratum. However, the function of myofibroblasts in the process of bladder contraction needs further validation. Myofibroblasts showed similar features between human and rat, i.e. common CD markers (CD362 and CD121a) as mentioned before (Fig. 2d and 2g), common junction proteins of GJC1, JAM3, and CLDN11 (Fig. 2e and 2h). However, considering the receptor and ion channel, the expression level of TRPA1 and KCNF1 was higher in the human myofibroblast; in contrast, the expression level of Grin2c, Kcnq5, and Kcnma1 was enriched in rat myofibroblasts (Fig. 2f and 2i).
Characterization of Mki67+ cells in human and rat bladder
Surprisingly, a new cell type, Mki67+ cells, were found both in the human and rat bladder tissues (Fig.1b and 1d). These cells demonstrated a clear boundary distinguished from other clusters and also showed a reliable mean UMI value (Fig. S1a, S1c, S1f and S1h). The Pearson correlation of gene expression for Mki67+ cells between human and rat was 0.74 (Fig. 8a). However, GO analysis of marker genes demonstrated enrichment mainly in the pathway of chromosome segregation and cell division in human and rat, separately (Fig. 8b and 8c) which coincides with the feature of Ki-67 protein, which is widely used as a proliferation marker. Algorithmically identified marker genes in human (MKI67 and BIRC5) and rat (Mki67 and Clca5) tissues shows a difference between each other (Fig. 8d and 8e). We also performed immunofluorescence and immunohistochemical staining with anti-MKI67 antibody to locate the position of Mki67+ cells in bladder tissues. The results showed that Mki67+ cells lie in the mucosal layer and also within the muscle bundle (Fig. 8f and 8g). Despite this long-standing characterization of Ki-67 protein, to our knowledge, this is the first report showing that Mki67+ cells exist in the bladder, and these should be investigated for their cellular functions.
Characterization of other types of cells in human and rat bladder
In addition to well-known cells in bladder tissues, we also found neurons and clusters of immune cells such as T cells, B cells, monocytes, mast cells, macrophages, and granulocytess (Fig.1b and 1d). The marker genes used to distinguish neurons are GPM6A and RELN, and the algorithmically identified marker genes are CCL21, MMRN1, CLDN5, and LYVE1 which coincide with each other in human and rat (Fig. S9a-S9d). The Pearson correlation coefficient between human and rat neuron is 0.77 (Fig. S9e), and GO analysis shows that these neuronal cells are mainly focus on the regulation epithelial cell proliferation, ion homeostasis, vascular and muscle development (Fig. S9f and S9g). The B cells marker genes include CD79A, MZB1, MS4A1, and CD19 (Fig. S10a), and the algorithmically identified marker genes are IGLC2, IGKC, IGLC3, and IGHA1 (Fig. S10b). T cell marker genes include CD3D, CD3E, CD3Gm and CD8A both in human and rat (Fig. S12a and S12b). Three genes (CD14, CD163, and MS4A7) were selected as markers of monocytes both in human and rat (Fig. S13a and S13b). Two genes (Cd68 and Cd74) from reference (Fig. S13c) and two specific marker genes (RT1-Da and RT1-Db1) were selected to mark rat macrophages (Fig. S13d). Four specific marker genes (S100A9, G0S2, SRGN and S100A8) were selected as markers for human granulocytes (Fig. S13e). Four genes (Mt-atp6, Mt-co1, Mt-co2, and Mt-co3) were selected to mark rat adipose tissue cells (Fig. S13f).
The special marker gene KIT must be given more attention because c-kit+ interstitial cells of cajal are reported distributed in the bladder interstitial region. We luckily found a cluster of c-kit+ cells (Fig. S11a) in the human sample. However, further bioinformatics analysis showed that these clusters of cells express marker genes (ENPP3, FCER1A and SLC18A2) similar to the markers of mast cells (Fig. S11a). GO and KEGG pathway analysis suggested that these are cells associated with mast cell migration, activity, and mast cell mediated immunity (Fig. S11c and S11d). So, we considered these clusters of cells as mast cells.