Extended Data Fig. 1: Optimizing conditions to induce insulin-expressing cells from cultured human gastric stem cells.
From: Stomach-derived human insulin-secreting organoids restore glucose homeostasis
a, Top: stomach samples from 3 different donors; middle, primary hGSC colonies derived from the stomach samples; bottom, immunofluorescence of passage-10 hGSC colony staining for SOX9 and KI67. b, Growth kinetics of hGSCs from three different donors. c, Co-expression of Ngn3, Pdx1, and Mafa using a polycistronic inducible construct in hGSCs yielded low levels of insulin expression (n=3 biological independent samples). Ubc: ubiquitin promoter. d, To optimize the relative timing of Ngn3 and Pdx1- MafA expression, we expressed a Ngn3ER fusion protein in which Ngn3 activity was induced by 4-OH Tamoxifen (4-OH-TAM). Polycistronic PDX1 and MAFA co-expression was controlled by rtTA-TetO and activated by the addition of Doxycycline in the culture medium. Higher INS expression was achieved by sequential activation of the transcription factors NGN3 and PDX1-MAFA. n=3 independent experiments. e, Comparison of PDX1-MAFA with the other transcription factor combinations in insulin induction. 2-day Ngn3ER induction (by 4-OH-TAM) preceded the other TFs, or alternatively, co-expression cassettes were used. ND: not detected. n=3 independent experiments. c, d, e, Data presented as mean ± s.d.; two-tailed unpaired t-test (c), or one-way ANOVA with Dunnett multiple comparisons test (d, e).
