![]() ![]() With this aim, we referred to the gene expression profile of human colonic organoids and nominated cyclin-dependent kinase inhibitors, p21, p27, and p57, as potential markers for nondividing cells. Mean cell position of Lgr5 +Ki67 + and Lgr5 +Ki67 – cells in mouse colonic crypts.Īlthough scRNA-seq illuminated the presence of nondividing LGR5 + cells in the human colon, their isolation and functional assessment require identification of their positive markers. ( F) Ki67 staining and the expression of Lgr5-GFP reporter in a mouse colon. Mean cell position of LGR5 +Ki67 + and LGR5 +Ki67 – cells in human colonic crypts. ( E) Co-staining of Ki67 with LGR5 in situ hybridization (ISH) in a human colon. ( D) Distribution of the S and G2/M phase scores in human and mouse colonic stem cells. A UMAP plot of human colonic epithelium cells using human-specific classification used in Figure 1 A is shown. Cells with a top 0.5% ISC score are highlighted in red. ( C) Integrated scRNA-seq analysis of the mouse and human colonic epithelium. The expressions of single genes are shown in log 2-transformed normalized counts. ![]() The levels of ISC, LRC, and Mex3a signature scores refer to the abundance of signature gene transcripts in percents. The expression of cell cycle genes and differentiation-related genes in each cluster. ( B) The expression of ISC-related genes (ISC score, LGR5, and OLFM4), secretory precursor gene ( NEUROG3), mouse LRC, and Mex3a signature scores. ![]() Transit amplifying (TA) cells are classified into 3 clusters based on cycling status. ( A) A uniform manifold approximation and projection ( UMAP) plot of single cell transcriptomes derived from 7103 human colonic epithelium cells. Methods Human Tissuesįigure 1 Nondividing LGR5 + stem cells in the human colonic epithelium. Our findings not only highlight the difference in ISC biology between mice and humans, but they also lay a foundation for future research that aspires to control or facilitate regenerative response in the human gut. By leveraging genetic lineage tracing and orthotopic xenotransplantation, we demonstrated the stemness of slow-cycling human LGR5 + cells in homeostasis and during regeneration. To address these questions, we obtained a panorama of human colonic epithelial cells using single-cell RNA sequencing (scRNA-seq) and identified a nondividing subpopulation in human LGR5 + colonic cells. The expression of Ki67, however, provides only snapshot information on cell cycle states, and it remains unclear whether the low Ki67 positivity among hCoSCs reflects the G 1 transition of cycling cells or cell-intrinsic quiescence. Contrary to Lgr5 + cells in the surrounding mouse tissue, most human LGR5 + cells were Ki67-negative, suggesting that hCoSCs are slowly cycling. ![]()
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