Supplementary Figure 2: Adjustment of the dose and CHIR treatment time

(a) A schematic illustration of primitive streak and subsequent differentiation into each mesoderm lineage. (b) Pluripotency was evaluated by staining with OCT4 and SOX2 before the differentiation. hESCs differentiated with CHIR 5 μM were positive for T and TBX6 on day 1.5 of differentiation, but cells did not stain for HOXD11. Scale bars: 200 μm. (c) Immunocytochemistry on day 4 of the differentiation with CHIR (3 to 10 μM). Sustained TBX6 expression was observed when the cells were differentiated with high doses of CHIR (7-10 μM). Scale bar: 100 μm. (d) Real-time PCR for MIXL1 in hESCs from day 0 to 7. hESCs were differentiated with CHIR 8 μM for 4 days, and activin 10 ng/ml for 3 days. MIXL1, another marker for primitive streak also showed sustained expression at least until day 4 of the differentiation. Expression returned to very low levels by day 7. n=2. (e) Staining with WT1 and HOXD11 on day 7 of the differentiation. hESCs were differentiated with CHIR 8 μM for 4 days and subsequently with the basic medium (ARPMI) for 3 days. Cells expressed WT1, but not HOXD11. Scale bar 100 μm.