Inhibition of forkhead box O1 expression promotes the differentiation of human embryonic stem cells into insulin producing cells

Abstract

Objective To investigate the regulatory role of forkhead box O1 (FoxO1) on differentiation of human embryonic stem cells (hESCs) into insulin producing cells (IPCs). Methods The expression levels of FoxO1 during IPC differentiation of hESCs in vitro were determined by realtime PCR and Western blotting. The dynamic expression of octamer-binding transcription factor 4 (Oct4), forkhead box a2 (Foxa2), pancreatic and duodenal homeobox 1 (Pdx1) and insulin during the differentiation was analyzed by realtime PCR. The stage-3 cells (pancreatic progenitors) of the differentiation were infected with FoxO1-knockdown and FoxO1-overexprssion lentiviral vectors. The expression levels of Foxa2, Pdx1 and insulin were detected by realtime PCR. The infected cells were induced to differentiate into IPCs, and glucose stimulated insulin secretion was measured by using an ELISA kit. The t test was used to compare the difference between two groups, and one-way ANOVA was used among the groups. Results FoxO1 mRNA and protein were invariably expressed during the differentiation of hESCs to IPCs. The expression level of Oct4 dropped significantly with the differentiation progress. The expression level of Foxa2 increased during the differentiation and peaked at stage 3 of the differentiation. The expression levels of Pdx1 and insulin were significantly upregulated during the differentiation. In the infected pancreatic progenitors, the mRNA levels of Foxa2 (4.31±0.74 vs 1.00±0.34, t=7.08, P<0.01), Pdx1 (2.91±0.24 vs 1.00±0.15, t=11.62, P<0.01) and insulin in the FoxO1-knockdown group were significantly increased compared with those in its control group. By contrast, overexpression of FoxO1 downregulated the mRNA level of insulin (0.81±0.07 vs 1.00±0.03, t=4.19, P<0.05), but had no effect on the mRNA levels of Foxa2 and Pdx1. In addition, knockdown of FoxO1 promoted insulin secretion stimulated by both low glucose (2 mmol/L) (7.53±2.02 vs 1.00±0.05, t=3.23, P<0.05) and high glucose (20 mmol/L) (17.39±3.03 vs 6.45±1.34, t=3.30, P<0.05). Overexpression of FoxO1 had little effect on insulin secretion under low glucose, but inhibited high glucose stimulated insulin secretion (2.02±0.50 vs 4.85±1.42, t=2.61, P<0.05), thus resulting in loss of responsibility to glucose. Conclusion FoxO1 negatively regulates the IPC differentiation of hESCs. Inhibition of FoxO1 expression may be a potential strategy for promoting the differentiation and maturation of IPCs from hESCs. Key words: Forkhead transcription factors; Embryonic stem cells; Islets of Langerhans