Supplementary Figure 4: Spontaneous differentiation of SIX2+ cells into nephrons and growth factor screening in 3D culture

(a) Staining with PAX8 and LHX1 in hESCs on day 10 of the differentiation. hESCs were differentiated with CHIR 8 μM for 4 days, activn 10 ng/ml for 3 days, and FGF9 10 ng/ml for 3 days. Sporadic expression of LHX1 was observed in PAX8+ cells. Scale bar: 50 μm. (b) Brighfield imaging for hESCs on day 10 and 21 of the differentiation. FGF9 was withdrawn on day 10, and cells were cultured in the basic medium by day 21. Scale bars: 50 μm. (c) Brightfield and immunocytochemistry for LTL and NPHS1 in hESCs on day 28 of the differentiation. Scale bar: 50 μm. (d) The protocol for growth factor screening. Cells were replated to 3D culture on day 10, and the listed growth factors and small molecules were tested. (e) The number of LTL+ tubules in the organoids. HGF showed a tendency to increase LTL+ tubules. n=2. (f) Brightfield imaging of 3D co-culture.with an ureteric bud cell sphere. Scale bar: 100 μm. (g) Whole-mountstaining for LTL in the organoids on day 16. Scale bar: 100 μm. (h) Immunohistochemistry of the organoids on day 16. Scale bars: 50 μm. LTL: lotus tetragonolobus lectin. NPHS1: Nephrin.