FIG. 2.
Increased purity and maturation of HESC derived NC cells with serial passages. (A) Immunostaining of the NC markers SOX9, P75, and HNK1 and the NE marker SOX1 with DAPI counterstain, in H9s HESC-derived NC after two passages (NC P2) in FSB media. Blue scale bar: 50 μm, white scale bar: 100 μm. (B) Flow cytometric analysis of P75 and HNK1 in H9s HESC after 4 days of differentiation in FSB media before passage (H9s 4D IN FSB) and H9s-derived NC after three passages maintained in FSB as single cells (H9s NC P3). Red contour plots represent HNK1+P75 double stained populations, blue contour plots represent IgG controls. (C) Flow cytometric analysis of TFAP2A and P75 in the differentiation of undifferentiated H9s and H9s-derived NC after three passages maintained in FSB as single cells (H9s NC P3). Colonies were passaged at day 4 of differentiation. Red contour plots represent TFAP2A+P75 double stained populations, blue contour plots represent IgG controls. (D) Flow cytometric analysis of SOX17 and SOX10 in H9s HESC, endoderm cells differentiated from H9s (H9s END), and H9s HESC at day 4 of differentiation in FSB without splitting (H9s 4D IN FSB). SOX10 induction was observed following 4 days of FSB treatment, whereas the population was negative for expression of the endoderm marker SOX17. Red contour plots represent SOX17+SOX10 double stained populations, and blue contour plots represent IgG controls. (E) Top panel: qRT-PCR expression analysis of NC markers PAX3, ZIC1, and SOX9. Bottom panel: qRT-PCR expression analysis of NC markers P75, TFAP2A, and TWIST1. mRNA of HESC-derived NC cells at different passages was used to perform the qRT-PCR. The relative mRNA levels were relative to NC passage 1 levels (NC P1). The relative mRNA level was normalized to the housekeeping gene PBGD. P1, P3, P5, P7, P8, and P13: number of passages of the NC cells as single cells. Results presented as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Two-sided Student's t-test. (F) Representative images of in vitro scratch wound assay performed in H9s HESC derived NC cells (NC P7). Pictures were obtained from 0 to 24 h postscratch. White scale bar: 100 μm. (G) Flow cytometric analysis of TFAP2A and P75 in H9s HESC and H9s HESC-derived NC passage 12 in FSB (NC P12). Red contour plots represent TFAP2A+P75 double stained populations, and blue contour plots represent IgG controls. (H) Chemotaxis potential and migration in representative H9s-derived NC P7 was assessed using a CytoSelect™ Transwell Cell Migration Assay Kit. The NC chemoattractant FGF8B was added to FSB media to assess migration. DMEM/F12 + 10% fetal bovine serum (SERUM) was used as a positive control of migration. FSB migration without any chemoattractant was set as 1, and data are expressed relative to this. Equal cell numbers were used in each condition, and results are presented as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01. DMEM, Dulbecco's modified Eagle's medium. Color images available online at www.liebertpub.com/scd