Fig. 1: Robust differentiation of human iPSC-derived syndetome.

a Modeling step-wise narrowing fate decisions of syndetome in the embryo with human iPSCs. iPSCs were differentiated toward presomitic mesoderm in medium supplemented with SB, CHIR, DMH1, and FGF2 for four days. Subsequently, day-4 presomitic mesoderm cells were passaged and differentiated toward somites in medium supplemented with SB and CHIR for another four days. Day-8 somites were then cultured in a medium containing SAG and LDN for sclerotome differentiation. Day-11 sclerotome cells were passaged and subsequently differentiated toward syndetome in medium supplemented with FGF8 and TGFβ3 for the first two days, and in medium supplemented with TGFβ3 and BMP7 for the following six days. b, c Differentiation toward syndetome fate was assessed by RT-qPCR (b), bright-field imaging, and immunocytochemistry (c). The expression of markers for sclerotome (PAX1) and syndetome (SCX, MKX, TNMD, COL1A1, COL1A2, FMOD) was assessed by RT-qPCR. Cells were stained with anti-SCX, MKX, COL1A1, COL1A2 antibodies (red) and co-stained with DAPI (blue). Positive rates to DAPI were calculated using Image J. Representative data are shown. Scale bars: 50 μm. d EdU staining of iPSC-syndetome. iPSC-syndetome were stained with EdU (green) and co-stained with anti-MKX antibody (red) and DAPI (blue). EdU-positive rates to MKX were calculated using Image J. Representative data are shown. Scale bars: 100 μm. Data represent mean ± SE (n = 3: biologically independent samples). iPSC, induced pluripotent stem cell; SB, SB431542; CHIR, CHIR99021; LDN, LDN193189. Source data are provided as a Source Data file.