Fig. 2: Differential role of OGDH in cell fate.

a, Diagram of Ogdh suppression experiments in ISC-enriched organoids derived from TRE-shRen^Cag-rtTA3, TRE-shOgdh^Cag-rtTA3 or wild-type mice. ISC-enriched organoids were grown in ENR-CV medium (C, CHIR2099; ENR, EGF, Noggin and R-spondin; V, valproic acid) for six days, then treated with doxycycline (Dox) (TRE-shRen^Cag-rtTA3 and TRE-shOgdh^Cag-rtTA3) or DM-αKG (wild type) while switching to ENR medium to facilitate differentiation into all intestinal lineages. Created in BioRender. Chaves-perez, A. (2025) (https://BioRender.com/dimozin). b, Heat map showing early changes in late-TA and secretory-lineage signatures from RNA-seq analysis of organoids from TRE-shRen^Cag-rtTA3 and TRE-shOgdh^Cag-rtTA3 mice treated with doxycycline or DM-αKG for 72 h. Two different hairpins were used for TRE-shOgdh^Cag-rtTA3 mice (346 and 2081). Each column represents organoids derived from three mice. c, Immunofluorescence and staining in organoids from b, showing cell proliferation (Ki67), cell death (cleaved caspase 3; CC3) and lineage-specific markers (ACE2, enterocytes; ABP, goblet cells; lysozyme, Paneth cells) after six days of the indicated treatments. d, Experimental schematic for Ogdh suppression and exogenous αKG supplementation studies in progenitor-enriched organoids. ISC-enriched organoids were cultured for six days in ENR-CV medium, then differentiated for three days in the pertinent lineage-specific medium (C, CHIR2099; D, DAPT; ENR, EGF, Noggin and R-spondin; I, IWP2; V, valproic acid). Subsequently, they were treated with either doxycycline (TRE-shRen^Cag-rtTA3 and TRE-shOgdh^Cag-rtTA3 organoids) or DM-αKG (TRE-shRen^Cag-rtTA3 organoids). Organoids were cultured in the appropriate lineage-specific differentiation medium for six days to direct differentiation into the desired cell lineage. Created in BioRender. Chaves-perez, A. (2025) (https://BioRender.com/dimozin). e, Immunofluorescence in progenitor-enriched control (TRE-shRen^Cag-rtTA3), OGDH-depleted (TRE-shOgdh^Cag-rtTA3) or αKG-treated (TRE-shRen^Cag-rtTA3) organoids showing cell death (cleaved caspase 3) after eight days in culture. This figure is adapted from our published patent (WO2024229094A1)⁵⁰. Scale bars, 10 μm (c,e).