Paneth cells inhibit intestinal stem cell proliferation through the bone morphogenic protein 7 pathway under rotavirus-mediated intestinal injury

Figure 6 Paneth cell-specific modulation of bone morphogenic protein 7 regulates intestinal stem cell activity in organoids. A: Schematic workflow illustrating the generation of intestinal organoids. Crypts were isolated from lysozyme 1-Cre mice and embedded in Matrigel to establish three-dimensional organoid cultures; B: Design of conditional lentiviral vectors enabling Paneth cell-specific knockdown (KD) or overexpression (OE) of bone morphogenic protein 7 (Bmp7). Both vectors contain a cytomegalovirus promoter driving a LoxP-STOP-LoxP cassette, followed by either a short hairpin RNA targeting Bmp7 (blue) or Bmp7 open reading frame (red) linked to an internal ribosome entry site-fluorescent reporter. Cre-mediated recombination in Paneth cells excises the stop cassette, permitting expression of the short hairpin RNA or transgene; C: Reverse transcription quantitative polymerase chain reaction analysis of Bmp7 mRNA levels in organoids from four groups (KD-Ctrl, OE-Ctrl, Bmp7-KD, Bmp7-OE; n = 5 organoid lines per group). Expression is normalized to glyceraldehyde-3-phosphate dehydrogenase and presented as mean ± SD; D: Lysozyme 1 expression is comparable across all groups, indicating that Bmp7 manipulation does not affect Paneth cell abundance; E: Representative bright-field images of organoids. Bmp7-KD organoids exhibit enlarged, highly branched morphologies, whereas Bmp7-OE organoids appear compact and spherical with markedly reduced budding; F: Quantitative polymerase chain reaction analysis of Id1; G: Quantitative polymerase chain reaction analysis of intestinal stem cell-associated markers leucine-rich repeat-containing G protein-coupled receptor 5; H: Quantitative polymerase chain reaction analysis of olfactomedin 4; I: Quantitative polymerase chain reaction analysis of marker of proliferation Ki-67. Statistical significance was determined using the non-parametric Mann-Whitney test between groups. ^bP < 0.01. Lyz: Lysozyme; Bmp: Bone morphogenic protein; KD: Knockdown; OE: Overexpression; CMV: Cytomegalovirus; IRES: Internal ribosome entry site; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; Lgr5: Leucine-rich repeat-containing G protein-coupled receptor 5; Olfm4: Olfactomedin 4; Mki67: Marker of proliferation Ki-67.