To investigate the role of nicotinamide (NIC) in the differentiation of neural crest cells from human embryonic stem cells (hESCs), and lay the foundation for the induction of hESC-derived corneal endothelial cells.

hESCs line H1 cultured for 5-7 days was used for induction.According to the different components of the neural crest induction medium, cells were assigned into different groups for 7-days induction, including group treated without NIC cultured in induction medium only, group treated with NIC cultured in induction medium containing 10 mmol/L NIC, NIC+ resveratrol (Res) group cultured in induction medium containing 10 mmol/L NIC and 10 μmol/L Res and Sirtinol group cultured in induction medium containing 10 μmol/L Sirtinol.Res and Sirtinol were used as SIRT1 activity agonist and inhibitor, respectively.The relative mRNA expression levels of hESCs and neural crest cell markers were detected by real-time fluorescence quantitative PCR at 1, 3, 5 and 7 days during the induction.The expression of neural crest cells markers after 7 days of induction was assayed by immunofluorescence staining.The induction efficiency of NIC and the effect of SIRT1 regulation on human natural killer 1 (HNK-1) positive cells expression were evaluated through flow cytometry analysis of percentages of nerve growth factor receptor (P75) and HNK-1⁺ cells.

Compared with the group treated without NIC, the mRNA expressions of totipotent genes octamer transcription factor 4 (OCT4) and homeodomain proteins (NANOG) were significantly decreased, and the mRNA expression levels of neural crest cell markers P75, HNK-1, SRY-related HMG box (SOX) 9 and SOX10 were significantly increased in the group treated with NIC after 5 days of induction (all at P<0.05). In the group treated without NIC, P75 was weakly expressed, and HNK-1 was sporadically expressed, and transcription factor AP-2β (AP-2β) and paired-like homeodomain transcription factor 2 (PITX2) were not detected.In the group treated with NIC, P75, HNK-1, AP-2β and PITX2 were strongly expressed.The proportion of P75⁺ HNK-1⁺ cells and P75⁺ cells were both significantly higher in the group treated with NIC than without NIC (t=8.481, P=0.001; t=2.987, P=0.041). The percentage of HNK-1⁺ cells in groups treated without and with NIC, NIC+ Res group and Sirtinol group were (34.267±12.522)%, (89.633±1.358)%, (64.667±6.429)% and (86.300±3.460)%, respectively, with a statistically significant overall difference (F=36.799, P<0.001). The proportion of HNK-1⁺ cells in NIC+ Res group was significantly lower than that in the groups treated with NIC and Sirtinol (all at P<0.05).

NIC promotes the differentiation of hESCs-derived neural crest cells by inhibiting the activity of SIRT1 to enhance the expression of HNK-1.NIC treatment may provide a new strategy for source of seed cells in the treatment of neural crest cell-related diseases, such as corneal endothelial transplantation.

Neural crest; Nicotinamide; Cell differentiation; Human embryonic stem cells