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Study on Electrophysiological Function of Neuron-like Cells Induced from Rat Mesenchymal Stem Cells by Salvia miltorrhiza with Optimized Proposal
Lin-Yan Hu1, Jia-Lin Yu1*, Guan-Xin Liu1,2, Bo-Man Chen3, Fang Li1, Lu-Quan Li1, Hai-Ying Zhong1,2
1Department of Neonatology, 2Pediatrics Research Institute, Children's Hospital, Chongqing Medical University, Chongqing 400014, China; 3Department of Neonatology, Women's Hospital, School of Medicine, Zhejiang University,
Abstract: Mesenchymal stem cells (MSCs) of rat were isolated and cultured， MSCs of passage 4? were induced to differentiate into neuron-like cells and dedifferentiate repeatedly. The process of differentiation was observed. Nestin， neurofilament (NF) and synaptophysin were detected by immunofluorescence cytochemistry staining to identify the differentiated cells and to calculate the differentiated rate. MSCs and the neuron-like cells were labeled with voltage-sensitive fluorescent dye DiBAC4(3)， 50 mmol/L KCl acted as stimuli to evoke action potential， fluorescence excitation of DiBAC4(3) was challenged by laser-scanning confocal microscope (LSCM) for the measurement of membrane potential (MP). The change of fluorescence intensity was observed and analyzed. In this study， MSCs began contracting and extending processes， changing its appearance into neurons?after 2 h of the first induction. However most of cells were monopole and dipolar. After 6 h of the 5th induction， the cells became multipolar and the processes stretched out and extended to form complicated net. Immunofluorescence cytochemistry presented that after 2 h of the first induction， the cells expressed nestin only [with ratio of (95.1±2.1)% (x±s， n=3)]， but NF hardly. After 1 h of the 4th induction， the ratio of NF expression rose up to (95.3±1.6)% (x±s， n=3) and synaptophysin was expressed， after 5 h of the 4th induction， synaptophysin expression was more extensive. The study of MP demonstrated the voltage-sensitive fluorescence of cells enhanced as soon as the cells were stimulated by high concentration KCl solution， and physiology chart showed the curve rose shapely， but MSCs which were not induced had no any response and the physiology chart showed the curve kept at horizon. It suggests Salvia miltorrhiza can induce MSCs to differentiate into neuron-like cells with electrophysiological properties efficiently and rapidly through optimized proposal.
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