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Single prostate stem cells can generate stem and progenitor cells to form prostaspheres in 3D culture. Using a prostasphere-based label retention assay, we recently identified keratin 13 ( KRT13 )-enriched prostate stem cells at single-cell resolution, distinguishing them from daughter progenitors. Herein, we characterized the epithelial cell lineage hierarchy in prostaspheres using single-cell RNA-seq analysis. Keratin profiling revealed three clusters of label-retaining prostate stem cells; cluster I represents quiescent stem cells ( PSCA , CD36 , SPINK1 , and KRT13/23/80/78/4 enriched), while clusters II and III represent active stem and bipotent progenitor cells ( KRT16/17/6 enriched). Gene set enrichment analysis revealed enrichment of stem and cancer-related pathways in cluster I . In non-label-retaining daughter progenitor cells, three clusters were identified; cluster IV represents basal progenitors ( KRT5/14/6/16 enriched), while clusters V and VI represent early and late-stage luminal progenitors, respectively ( KRT8/18/10 enriched). Furthermore, MetaCore analysis showed enrichment of the "cytoskeleton remodeling-keratin filaments" pathway in cancer stem-like cells from human prostate cancer specimens. Along with common keratins ( KRT13/23/80/78/4 ) in normal stem cells, unique keratins ( KRT10/19/6C/16 ) were enriched in cancer stem-like cells. Clarification of these keratin profiles in human prostate stem cell lineage hierarchy and cancer stem-like cells can facilitate the identification and therapeutic targeting of prostate cancer stem-like cells.

Keratin Profiling by Single-Cell RNA-Sequencing Identifies Human Prostate Stem Cell Lineage Hierarchy and Cancer Stem-Like Cells