Fig. 1: Niche factor deprivation strategy successfully selected AXIN1-mutant and AXIN2-mutant organoids from MLH1^KO human colon organoids.

a, Strategy to select the survived organoids with deprivation of Wnt pathway factors. Organoids were dissociated into single cells and then 200,000 single cells were cultured in niche-factor-deprived medium to select mutated stem cells (red) out from WT stem cells (blue) and differentiated cells (white). W, Wnt3a; R, R-spondin 1; N, Noggin; E, EGF. Surviving clone organoids were expanded briefly for WGS and WES analysis. Accumulated mutations were determined by subtracting the mutations in the original clone (blue) from the mutations in the selected subclone (red). b, WT organoids and MLH1^KO-1 organoids grew in complete medium (WRNE) but some of organoids grew out only from MLH1^KO-1 organoids in −WR medium (representative pictures from n = 3 clonal organoid lines). Scale bars, 500 µm. (c) Quantification of survived organoids in −WR medium. Mean and s.d. (error bars) of n = 4 clonal organoid lines. P values (two-sided Welch’s t-test): MLH1^KO-1 versus MLH1^KO-2, P = 0.0001; MLH1^KO-1 versus MLH1^KO-3, P = 0.0002. d, Single survived organoids (MLH1^KO-1−WR-1 and MLH1^KO-1−WR-2) were expanded and passaged in −WR medium (representative pictures from n = 3 independent replicates). Scale bars, 500 µm. e, The Venn diagrams illustrate the number of base substitutions and indels found in WGS data of survived clones (MLH1^KO-1−WR-1 and MLH1^KO-1−WR-2). AXIN1 and AXIN2 candidate gene mutations are listed in the overlapping section. The numbers indicate nonsynonymous mutations and the numbers in brackets indicate all mutations found in WGS. f, RT–qPCR analysis of AXIN2 mRNA levels. AXIN2 transcripts were compared between each clone cultured in the medium with or without Wnt3a and R-spondin 1. Mean and s.d. (error bars) of n = 3 biologically independent experiments. P values (two-sided Welch’s t-test): MLH1^KO-1 versus MLH1^KO-1−WR-1 in −WR medium, P = 0.0048; MLH1^KO-1 versus MLH1^KO-1−WR-2 in −WR medium, P = 0.0037. g, Representative images of Sanger DNA sequencing of AXIN1 and AXIN2 mutations in Wnt-independent clones (MLH1^KO-1−WR-1 and MLH1^KO-1−WR-2).

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