Fig 2: Intestinal apoptotic and proliferating cell responses following low-dose T. gondii infection of human microbiota-associated mice. Human microbiota-associated (hma) mice were perorally infected with one cyst of T. gondii strain ME49 to induce non-lethal subacute ileitis as described in Materials and Methods (ILE; filled symbols). Noninfected hma mice served as controls (N, open symbols). The average numbers of (A, B) ileal and (C, D) colonic apoptotic cells (positive for caspase3 [Casp3]; A, C) and proliferating cells (positive for Ki67; B, D) from six high power fields (HPF, 400× magnification) per animal were determined microscopically in immunohistochemically stained intestinal paraffin sections at day 9 post-ileitis induction. Numbers of animals (in parentheses), means, and significance levels (p-values) determined by Mann–Whitney U test are indicated. Data shown were pooled from three independent experiments.

Fig 3: Intestinal microbiota composition in low-dose T. gondii-infected human microbiota-associated mice. Human microbiota-associated (hma) mice were perorally infected with one cyst of T. gondii strain ME49 to induce non-lethal subacute ileitis as described in Materials and Methods. Main intestinal bacterial groups abundant in the ileum (A, B) and the colon lumen (C, D) of hma mice were quantitatively assessed applying both culture (A, C) and culture-independent (i.e., molecular 16S rRNA based; B, D) methods 9 days following ileitis induction (ILE, filled circles; n = 17). Noninfected hma mice served as controls (N, open circles; n = 9). Total bacterial loads (TLs) as well as numbers of enterobacteria (EB), enterococci (EC), Gram-positive rods (GPR), Bacteroides/Prevotella species (B/P), and Clostridium/Eubacterium species (C/E) are expressed as colony-forming units per gram feces (CFU/g). 16S rRNAs of the total eubacterial loads (TL), as well as of the main intestinal bacterial groups including enterobacteria (EB), enterococci (EC), lactobacilli (LB), bifidobacteria (Bif), Bacteroides/Prevotella species (B/P), Clostridium coccoides group (Clocc), Clostridium leptum group (Clept), and Mouse Intestinal Bacteroides (MIB), are expressed as gene numbers per ng DNA. Medians (black bars) and significance levels (p-values) determined by Mann–Whitney U test are indicated. Data shown are pooled from three independent experiments.

Fig 4: Intestinal microbiota composition following multidrug-resistant P. aeruginosa infection of mice suffering from chronic colitis. IL-10-/- mice with chronic colitis (n = 19) were perorally infected with a multidrug-resistant P. aeruginosa strain on day (d) 0. Before (N, naive; white circles) and six weeks thereafter (d42 postinfection; black circles) a comprehensive survey of the intestinal microbiota composition was performed on fecal samples applying both (a) culture (expressed as colony forming units per gram, CFU/g) and (b) 16S rRNA based molecular analyses (expressed as gene numbers per ng DNA) of the total bacterial load (TL) and main intestinal bacterial groups including enterobacteria (EB), enterococci (EC), lactobacilli (LB), Bacteroides/Prevotella spp. (BP), Clostridium/Eubacterium spp. (CE), bifidobacteria (BB), Clostridium coccoides group, Clostridium leptum group (CL) and Mouse Intestinal Bacteroides (MIB). Medians are indicated. Data shown were pooled from three independent experiments.

Fig 5: Generation of TesLCs from ESCs.a Schematic outline of TesLC differentiation with media containing CHIR99021, BMP7 and FGF9 (CBF), CBF + IGF1 + GDNF (CBF + IG) or CBF + DHH (CBF + D). b Bright-field images and those superimposed with CFP-fluorescent images of cells treated with CBF at different time-points during differentiation. c Molecular characterisation during TesLC differentiation in CBF-treated culture. The expression of an IM marker (OSR1) was detected at day 5. CFP expression and Sertoli cell markers (SF1/NR5A1 and SOX9) colocalized at day 8. A steroidogenic marker (HSD3B) also appeared at day 8. d Quantitative RT-PCR analysis for the expression of ESC markers (Sox2 and Nanog), renal markers (Foxd1 and Hoxd11), a steroidogenic marker Hsd3b1, and 5 key transcription factors required for Sertoli cell differentiation (Sox9, Gata4, Wt1, Dmrt1 and Sf1/Nr5a1), over 12 days of differentiation. Fold expression changes relative to day 0 were calculated by ??Ct method with standard error. Three or four biological replicates, each of which had three technical replicates, were used for two-sided t-test. **P < 0.01, *P < 0.05. e Bright-field and CFP-superimposed f images of cells treated with CBF + IG at day 12 and 20. Arrows indicate ridge-like structures. f Immunohistochemistry detected an increase of Ki67-positive proliferative cells (red) treated with CBF + IG (bottom row), compared with only CBF treatment (top row) at day 8. g A vast majority of CBF + IG-treated cells were SOX9-positive (green). The dotted line demarcates a ridge-like structure. GATA4-positive cells (red) were surrounded by basal membrane denoted by laminin expression (green) at day 8. DAPI (blue) for nuclear staining. Scale bar, 50 µm.