Extended Data Fig. 2. Wnt3a/RSPO1 activated growth and expansion of CD24⁺ cells.

a-d, Representative images and quantification of cell-cluster sizes of Wnt3a conditioned medium (CM) treated CD24⁺ cells compared to CD13⁺ cells at 72 hours. Statistical analysis in n = 7 mean ± s.e.m., **P = 0.0045 by two-tailed t-test with Welch’s correction(c); CD24⁺ cells formed large cell clusters before Wnt3a was removed at day 4 (d). Scale bars 100 μm-a and b; 50 μm-d. e-f, Expansion of CD24⁺ cells into early tubuloids at day 6, which subjected to 4-phase protocol under Wnt3a/RSPO1 conditioned medium (e); CD13⁺ cells that subjected to 4-phase protocol under Wnt3a/RSPO1 conditioned medium, did not produce tubuloids and presented as loose cell-aggregates with vacuole formation (f). Scale bar 50 μm: e and f. g, Representative images of CD24⁺ cell derived organoid formation at day 7, 12, 17, 21 as well as organoid growth maintenance at day 30 and day 69, which were subjected to the 1-phase protocol published by Schutgens et al. Scale bar, 200 μm: day 21, 30, 69; 100 μm: day 0, 7, 17; 50 μm: day 12. h, Representative image of CD24⁺ cell derived organoid formation at day 21 that was subjected to the 4-phase protocol. Scare bar 50μm. i-j, Schematic (I) and representative images of CD24⁺ cells subjected to a tubuloid protocol without WNT3a/RSPO1(j). In the schematic, a FGF2, FGF10 stability factor (SF), heparin was used in this protocol, stability factor (SF) represents heparin (i). Scale bars 200 μm-m at day 30, 73; 100 μm-m at day 0, day 14; 50 μm-m at day 5, 8, 12 (j). k, Organoid formation rate comparison between CD24⁺ cell subjected to the 4-phase protocol without Wnt3a/RSPO1 (i-j) and the original 4-phase protocol, n = 4 mean ± s.e.m with two-tailed unpaired t-test with Welch’s correction, ***P = 0.0003. Arrows mark borders of tubuloids. Wnt-Wnt3a; RS-RSPO1. For details on statistics and reproducibility, see Methods.