Promoting effect of nicotinamide on generation of neural crest stem cells derived from human embryonic stem cells

Authors: Duan Haoyun,  Li Wenjing,  Jia Yanni,  Zhao Can,  Zhou Qingjun,  Li Zongyi
DOI: 10.3760/cma.j.cn115989-20200624-00450
Published 2022-12-10
Cite as Chin J Exp Ophthalmol, 2022, 40(12): 1141-1148.

Abstract                              [View PDF] [Read Full Text]

Objective

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.

Methods

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.

Results

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).

Conclusions

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.

Key words:

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

Contributor Information

Duan Haoyun

Eye Institute of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao 266071, China

Li Wenjing

Eye Institute of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao 266071, China

Jia Yanni

Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University (Shandong Eye Hospital), Jinan 250000, China

Zhao Can

Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University (Shandong Eye Hospital), Jinan 250000, China

Zhou Qingjun

Eye Institute of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao 266071, China

Li Zongyi

Eye Institute of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao 266071, China

(Read 7 times, 1 visits today)