Fig. 5

Immunofluorescence and gene expression analysis of 2D rectal monolayers cultured in expansion (MEM) or differentiation (MDM) media. (A) Immunofluorescent staining of 2D rectal monolayers cultured in both MEM and MDM revealed diverse cell populations, including actively proliferating cells (EdU, cyan), stem or transit-amplifying cells (SOX9, yellow), mucin-producing Goblet cells (SNA, green), epithelial adherens junctions (E-cadherin, green), and intestinal epithelial cells (EpCAM, yellow). Phalloidin (red) was used to stain F-actin, highlighting cellular borders where applicable, and DAPI (blue) was used to label nuclei. Top-down and z-stack images are shown. Scale bar = 25 μm. (B) Quantification of cells positive for EdU, SOX9, and SNA per field of view, normalized to the total number of nuclei. Ten independent fields of view were analyzed from three biological replicates. Data are shown as mean ± SE. No statistically significant difference was observed between MEM and MDM-cultured monolayers. (C) Gene expression analysis of marker genes for stem cells (LGR5), enteroendocrine cells (CHGA), Paneth cells (LYZC), goblet cells (MUC2), and intestinal epithelial cells (FABP2) was performed using RT-qPCR. Reference genes (ACTB, GAPDH, and RPL0) were used for normalization. Rectal monolayers were cultured for 3 days in MEM, followed by an additional 3 days in either MEM or MDM. Two technical replicates from three biological replicates were evaluated. Data are shown as mean ± SE. Statistical significance was assessed using the Mann-Whitney U test or unpaired t-test, as appropriate (* p < 0.05, and ** p < 0.01)