(P41-29-26) Single-Cell Transcriptomics Identifies an Fgfbp1-Associated Transit-Amplifying Cell Population Linked to Enterocyte Differentiation in Mice

Objectives: To investigate the role of β-carotene oxygenase 2 (BCO2) in regulating intestinal epithelial renewal dynamics at the single-cell level.

Methods: Single-cell suspensions were prepared from ileum of 8-week-old male BCO2 knockout (KO) and C57BL/6J (WT) mice. Ileal tissues were incubated with HBSS containing EDTA, followed by digestion with collagenase type VIII. The yield cells were enriched by 40%-80% Percoll gradient centrifugation. Samples with >80% viable cells were fixed and stored at −80°C until sequencing. scRNA-seq data analysis and visualization were performed using Seurat (v5.1.0)in R. CytoTRACE and pseudotime trajectory analyses were conducted using cytoTRACE2 (v1.1.0) and monocle 2 (v2.32.0).

Results: A total of 9,847 stem cells were identified and classified into: crypt base columnar cells, transit amplifying (TA) cells, and progenitors of enterocytes, Paneth cells, enteroendocrine cells, tuft cells, and goblet cells. TA cells were further divided into three subtypes based on their top marker genes: high-Mki67 TA, high-Fgfbp1 TA, and high-Mcm TA. Cell cycle and CytoTRACE analyses indicated that the high-Mcm TA cluster had the highest differentiation potential, with most cells in the G2/M or S phase, whereas cells in both high-Mki67 TA and high-Fgfbp1 TA clusters were mainly in the G1 phase.

Intestinal epithelial cell fate signature scores were subsequently evaluated in stem cells. Enterocyte fate scores were significantly increased in KO stem cells, suggesting that loss of BCO2 may influence absorptive enterocyte dynamics. Notably, the high-Fgfbp1 TA cluster was transcriptionally closer to enterocyte progenitors and co-localized with them along the developmental trajectory, suggesting a role for Fgfbp1 in enterocyte differentiation. Enterocyte progenitors showed the highest enterocyte fate scores among stem cell subtypes, followed by cells in the high-Fgfbp1 TA cluster. Consistently, Fgfbp1 expression was decreased in KO mice across high-Fgfbp1 TA cells and the three enterocyte subtypes.

Conclusions: These findings reveal heterogeneity within TA cells and identify Fgfbp1 as a potential mediator of enterocyte differentiation.

Funding Sources: NIH and USDA