Hepatocyte growth factor enhances the neural differentiation from human embryonic stem cells / 中国病理生理杂志

ABSTRACT

AIM: To investigate the possibility that hepatocyte growth factor (HGF) directly induces differentiation of human embryonic stem cells (hESCs) into neural progenitors (NPs). METHODS: hESCs colonies were induced to form the embryoid body (EB). Four-day-old EBs were randomly divided into 4 groups control group (EBs were cultured in neural induction medium);G5 supplement group (EBs were cultured in neural induction medium supplied with G5 supplement);HGF group (EBs were cultured in neural induction medium supplied with 10 mg/L HGF), and HGF+G5 group (EBs were cultured in neural induction medium supplied with 10 mg/L HGF and G5 supplement). After induced in suspension system for 7 days, EBs with various treatments were cultured in poly-D-lysine/laminin-coated plates for 7-10 days for selection of NPs. NPs were gathered by 0.3 g/L dispase treatment and characterized by immunofluorescence staining. The percentages of the nestin+ cells in NPs in various groups were detected by fluorescent activated cell sorter (FACS). The multipotency of NPs was determined by immunofluorescence staining after the NPs were cultured without G5 and HGF for 7 days. The expression of region markers of neural progenitors treated with sonic hedgehog (Shh) protein (one of the neural inductive signals), was detected by RT-PCR. RESULTS: HGF+G5 supplement induced hESCs differentiation into neural progenitors. Immunofluorescence staining indicated that NPs differentiated from hESCs expressed NP markers including nestin, Pax6 and musashi-1. FACS data showed that the proportion of nestin positive cells in HGF+G5 supplement group (87.3%±3.9%) was the highest in all treatment groups. The time of HGF and G5 supplement treatment was important to differentiate into NPs, the maximal effect was observed at 7th day. After treated with Shh, the expression of ventral forebrain/hindbrain marker genes (Nkk2.1, and Nkk2.2) and hindbrain progenitor marker gene Gbx2 in NPs were upregulated, while the forebrain progenitor marker genes Otx2 and Bf1 were downregulated. CONCLUSION: The neural induction system containing HGF and G5 supplement effectively induces the differentiation of hESCs into NPs, which might be a potent model for investigating the mechanism of neural development and differentiation.