FIG 7 .
LPSs purified from CSCM members induce the differentiation/maturation of PC epithelial cells and act on Lgr5+ cells. (A) LPSs purified from CSCM strains induce mRNA abundance associated with maturated colonocyte transcripts. The CSCM members are A. modestus (Am), A. radioresistens (Ar), D. tsuruhatensis (Dt), and S. maltophilia (Sm). RT-PCR showing the expression of Alpi, Klf4, Tff, and keratin 20 (krt20) as mature epithelial cell markers and Car1, Slc9a2, and Slc9a3 as markers of colonoids cultured from WT crypts. There were three mice in each group. *, P < 0.05 versus the nonstimulated control (Ctrl) group. (B) Representative micrographs of alkaline phosphatase (Alpi) immunostaining (red) of PC colonoids stimulated with LPSs from CSCM members. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue). Quantitative analysis of the ratio of Alpi-stained area to DAPI-stained area were measured using the ImageJ software from day 5 PC colonoid stimulated with LPS (1.0 μg/ml). In the graph in the middle of panel B, the red bars indicate the average ratios of Alpi-positive area/DAPI-positive area of the PC organoid for the group. In the graph to the right in panel B, values are means plus standard errors (SE) (error bars). (C) Effects of LPSs from CSCM members on terminal differentiation of goblet cells (GC) in PC organoids. Fluorescence-activated cell sorting (FACS) analysis showing the ratios of WGA+ UEA-1+ cells, which are located in the middle of the PC crypt, WGA-1low UEA-1+ cells, which are located at the top of the PC crypt, and c-kit+ WGA+ goblet-like cells (GLC) in doublet and dead cells depleted of EpCam+ CD45.2− cells from day 5 PC organoids. The organoids were from mice given A. modestus LPS (1.0 μg/ml), A. radioresistens LPS (1.0 μg/ml), D. tsuruhatensis LPS (1.0 μg/ml), or S. maltophilia LPS (1.0 μg/ml). There were six mice in each group. *, P < 0.05 versus nonstimulated control group. (D) Gene expression level of colonoids treated with CHIR99021 and valproic acid showing the upregulation of Lgr5 expression and downregulation of genes involved in differentiation. (E) Ratio of PC colonoids during stimulation of organoids derived from C57BL/6 mice cultured with CHIR99021 and vaproic acid with LPSs from CSCM members (1.0 μg/ml).