Fig. 1.

A Schematic presentation of the SOX9 HDR vector and the human SOX9-locus before and after homologous recombination. B Schematic presentation of the 4-stage experimental 2D differentiation protocol for the generation of SOX9⁺ progenitors. C GFP2 expression during differentiation of the SC30 and NSC20 cells. Data are means ± SEM, n = 4–8. Arrows mark developmental stages. D Flow cytometry analysis of GFP2 and H2-K^k expression in GFP2^± cells at d12. E RT-qPCR analysis of sorted SC30 derived GFP2⁺ and GFP2⁻ cells (d11-d12). Depicted is the relative gene expression of HNF6, NKX6-1, PDX1, SOX9 and NEUROG3. Data are means ± SEM, n = 3–4. Two-tailed Student’s t-test. F IF staining of SOX9 (red) in SC30 derived GFP2⁺ and GFP2⁻ cells (d12). Nuclei were counterstained with DAPI. G IF staining of PDX1, HNF-6 and NKX6-1 (red) and FOXA2 (green) of sorted SC30 derived GFP2⁺ cells (d12). F/G Scale bar = 100 µm. H Flow cytometry dot plot and histogram of SC30 GFP2 and CD200, CD142 and GP2 expression at d12 and d15 (end of stage 4). Bifurcated gates were set according to unstained controls, values are means ± SEM, n = 3–4