Differentiation of induced pluripotent stem cells into neural stem cells induced by brain-derived neurotrophic factor via Wnt/β-catenin and extracellular signal-regulated kinase/mitogen-activated protein kinases signal pathway

Abstract

Objective: To investigate the mechanism of brain-derived neurotrophic factor (BDNF) promoting induced pluripotent stem cells (iPSCs) to differentiate into neural stem cells (NSCs) via Wnt/β-catenin and extracellular signal-regulated kinase/mitogen-activated protein kinases (ERK/MAPK) signal pathways. Methods: iPSCs were cultured and identified. The iPSCs were induced to differentiate into NSCs by BDNF and retinoic acid (RA). Nestin was detected by immunofluorescence and flow cytometry after iPSCs differentiated. The technique of small interfering RNA (siRNA) was used to silence the gene expression of β-catenin and ERK, and iPSCs were divided into control group, BDNF group (adding 10 μg/L BDNF), siRNA-ERK/BDNF group (transfected with siRNA-ERK and adding 10 μg/L BDNF) and siRNA-β-catenin/BDNF group (transfected with siRNA-β-catenin and adding 10 μg/L BDNF). Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of key elements of Wnt/β-catenin and ERK/MAPK signaling pathways, included β-catenin, ERK1/2, c-fos, c-jun, and c-myc. The least significant difference test was used when data were compared between groups. Results: The immunofluorescence showed that iPSCs expressed octamer-binding transcription factor-4 (Oct4), SRY-related HMG box protein-2 (Sox2) and Nanog genes. The flow cytometry showed that Nestin-positive cells were 78.7% for BDNF and 43.5% for RA, and it was only 7.8% for routine medium. Compared with those in the control group, the mRNA expression of β-catenin, ERK1/2, c-fos, c-jun, and c-myc in the BDNF group were upregulated significantly (t=2.80, 2.318, 2.255, 1.799, 1.582, 1.663, all P<0.05), and the same results were acquired with the protein expression (t=2.805, 2.318, 2.255, 1.799, 1.582, 1.663, all P<0.050). Compared with those in BDNF group, the mRNA and protein expression of ERK1/2 in siRNA-ERK/BDNF group down-regulated obviously (t=1.917, 2.042, 1.673, 1.540, all P<0.05), and the mRNA and protein expression of c-fos and c-jun were down-regulated (t=1.022, 0.907, 0.848, 0.801, all P<0.05). However, the mRNA and protein expression of β-catenin and c-myc were not suppressed by siRNA-ERK (t=0.216, 0.185, 0.097, 0.112, all P>0.05). In siRNA-β-catenin/BDNF group, the mRNA and protein expression of β-catenin and c-myc was obviously down-regulated when compared with those in BDNF group (t=3.104, 2.774, 2.235, 1.911, all P<0.05), and expression of ERK1/2, c-fos and c-jun were down-regulated too (t=0.776-1.192, all P<0.05). Conclusion: BDNF promotes the differentiation of iPSCs by activating Wnt/β-catenin and ERK/MAPK signal pathway, there should be cross-talk between the two signal pathways, and c-fos and c-jun may be common nuclear transcription factors.