Figure 2: Hmga1 enhances ISC function in gut organoid cultures.

(a) Typical 3D organoids cultured from crypts are shown from WT or Hmga1 transgenic mice. (b) Bud numbers were ascertained in 3D organoids from isolated crypts (n>100 organoids per mouse; 3 mice per genotype). Bars show mean percentage of organoids±s.d. with different bud numbers. (Yellow≥3, grey=2, pink=1, blue=0). The mean percentage of organoids with ≥ 3 buds was increased in Hmga1 organoids compared to WT organoids. *P<0.05; two-tailed Student’s t-test. (c) Organoid projected surface area (PSA) is shown (mean±s.d.) from WT or Hmga1 mice (n>20 organoids/genotype). **P<0.01; two-tailed Student’s t-test. (d) Representative confocal imaging of crypt cells (day 0) and organoids (day 6) with GFP+ staining for Lgr5+ ISCs, Phalloidin (F-actin; red) for cell clusters and organoid structure, and Hoechst (blue) for nuclei are shown. (e) Relative expansion rate of ISCs was calculated by the ratio of the PSA of GFP+ ISCs on day 6 (n≥42 organoids per group) over the PSA on day 0 (n≥72 crypt cell clusters/group). **P<0.01; Mann–Whitney test. (f) ISC PSA (y axis) and bud number (x axis) at days 0 and 6 are shown. **P<0.01, *P<0.05; Mann–Whitney test. (g) Lgr5-GFP+ ISCs were purified by flow cytometry and cultured in matrigel. (h) Colony (organoid)-forming efficiency (mean±s.d.) was calculated from purified GFP+ ISCs isolated as single cells (n=3,000 cells per group) from WT and Hmga1 mice in three experiments; **P<0.01; two-tailed Student’s t-test. (i) Re-plating efficiency (mean±s.d.; n=3,000 cells per group) was assessed after dissociating colonies from purified GFP+ ISCs isolated as single cells from WT and Hmga1 mice using flow cytometry. **P<0.01; two-tailed Student’s t-test. Scale bars, 50 μm.