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. 2019 Mar 7;12(4):647–656. doi: 10.1016/j.stemcr.2019.02.003

Figure 1.

Figure 1

Differentiation of NCCs and NC-SMCs from hiPSCs

(A) Schematic illustration of the NCC differentiation protocol.

(B) Phase-contrast images of hiPSCs and NCCs at different stages of differentiation. Scale bar, 200 μm.

(C) Representative FACS plots showing NGFR, HNK1, TRA-1-60, and SOX2 expression in NCCs at passage 3 (red) and non-differentiated hiPSCs (black).

(D) Representative immunofluorescent images showing expression of TFAP2A, SOX9, SOX10, and SOX2 (green), and DAPI (blue) in NCCs. Scale bars, 200 μm.

(E) Percentages of NGFR+/TRA-1-60− NCCs at passage 3 (P3), differentiated from three hiPSC lines (FLB243, LUMC054, and NCRM1). Error bars are shown as mean ± SD from three independent differentiation experiments.

(F) Schematic representation of NC-SMC differentiation. Five conditions are depicted: protocol A (TGF-β3, 2 ng/mL and PDGF-BB, 10 ng/mL for 12 days) (A, yellow), protocol B (TGF-β3, 2 ng/mL and PDGF-BB, 10 ng/mL for 6 days followed by TGF-β3, 1 ng/mL and 0.5% fetal bovine serum [FBS]) (B, light blue), protocol C (TGF-β3, 2 ng/mL and PDGF-BB, 10 ng/mL for 30 days) (C, dark blue), protocol D (TGF-β3, 2 ng/mL and PDGF-BB, 10 ng/mL for 12 days followed by TGF-β3, 1 ng/mL and 0.5% FBS for 18 days) (D, red), and protocol E (TGF-β3, 2 ng/mL and PDGF-BB, 10 ng/mL for 12 days followed by 10% FBS for 18 days) (E, green).

(G) RT-PCR analysis of relative gene expression of NC (TFAP2A, SOX9, SOX10, PAX3) and SMC (ACTA2, CNN1, SM22, MYH11) markers in NCCs, NC-SMCs differentiated using protocols A to E, HBVPs, and HBVSMCs. Error bars represent mean ± SD from three independent experiments and normalized to housekeeping gene RPL37A (×1,000).

(H) Representative immunofluorescent images showing expression of ACTA2, SM22, and CNN1 (green) and DAPI (blue) in NC-SMCs (differentiated using protocols A to E), HBVPs, and HBVSMCs. Scale bars, 200 μm.