PNF-Derived iPSCs Correctly Differentiate into NCs
(A) Schematic representation of the protocol used for differentiating iPSCs into NCs. Control (ES4 and FiPS) and PNF-derived iPSCs were seeded on Matrigel and cultured in NC induction medium for 20 days (see Supplemental Experimental Procedures). Representative bright-field images during the differentiation process over time (in days, D) are shown. PSC, pluripotent stem cell. Scale bar, 50 μm.
(B) Flow cytometry analysis for p75 and Hnk1 before and after NC differentiation. The percentage of double p75 and Hnk1-positive cells is shown inside the graph. P1, passage 1; P4-5, passages 4–5.
(C) Immunocytochemistry analysis showing that both control (ES4 and FiPS) and PNF-derived iPSCs differentiated to NCs (passage 5) express p75 (green), AP2 (green), and SOX10 (red). DAPI was used to stain cell nuclei. Scale bar, 50 μm.
(D) qRT-PCR expression analysis of pluripotent (POU5F1), NC (NGFR, SOX10, AP2), and SC (S100B) markers, in pluripotent cells (PSCs), PSCs differentiated to NCs and PNF-derived SCs. qRT-PCR values are expressed as the mean normalized relative expression (NRE) ± SEM from three independent differentiation experiments.