INTRODUCTION AND OBJECTIVE: Prune Belly Syndrome (PBS) is characterized by bladder dysmyogenesis, yielding a dysfunctional compliant thick wall with excess collagen deposition. To dissect the cellular heterogeneity and gene expression networks altered in PBS, we report the cell type composition and transcriptional activity of PBS human bladder by using single cell RNA sequencing (scRNA-seq). METHODS: Using IRB-approved methods, bladder dome from 2 PBS and 6 non-PBS control (CO) males underwent fresh single-cell digestion. scRNA-seq was performed and 5277 and 31828 bladder cells from PBS and CO patients was detected, respectively. Cell type clusters were graphically displayed by Uniform Manifold Approximation and Projection (UMap) plot and differentially expressed genes (DEGs) were generated to assign each cluster identity. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was performed for PBS affected genes. RESULTS: We identified 17 distinct bladder cell clusters, including 6 fibroblast (1, 2, 3, and 4, immunofibroblast, myofibroblast), 1 smooth muscle (SM), and 2 urothelial (umbrella and basal+intermediate) clusters (Fig 1A-B). Counts of individual cell types were expressed as relative proportions, identifying significant PBS fibroblast enrichment, (67% PBS vs 40% CO). Five of 6 PBS fibroblast sub-types are proportionately fewer in number than in CO. The exception is a dominant fibroblast sub-type we label as fibroblast 4, (61% of all PBS fibroblasts vs <10% CO fibroblast subtypes). SM and urothelial cell populations are dramatically reduced in PBS (SM: 5% PBS vs 11% CO and urothelial: <1% PBS vs 7% CO) (Fig 1C-E). PBS fibroblast DEGs, but not SM cells, are enriched in collagen genes. Fibroblast markers (DCN and PLA2G2A) and SM genes (DES, TPM2 and TAGLN) are reduced (by 4, 13, 2, 4, and 2x respectively) in PBS cells (Fig 1G). KEGG pathways analysis for fibroblasts and SM showed a highly similar enrichment for neurodegenerative disease pathways (Fig 1H-I). CONCLUSIONS: Using scRNA-seq, we identified and characterized the disarrayed cell type populations in PBS bladders, generating their unbiased transcriptomic signatures which highlight commonality with neurodegenerative diseases. This PBS transcriptomic map is a step toward potential markers for diagnosis and therapeutic intervention.[Figure: see text]Source of Funding:NIH DK100483, DK127589 PI: Baker, L.
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