Electrophysiological changes of bone marrow mesenchymal stem cells following the differentiation into neuron-like cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Previous in vitro studies mainly focused on morphological and nerve marker aspects in the study of bone marrow mesenchymal stem cells (BMSCs)-derived neuron-like cells,but less focused on the olectrophysiological properties of neuron-like cells following differentiation.OBJECTIVE:To study the electrophysiological changes of differentiation from BMSCs into neuron-like cells after induction by brain-derived neurotrophic factor (BDNF)/basic fibroblast growth factor (bFGF)/alltrans-retinolc acid (AT-RA).DESIGN,TIME AND SETTING:The cytological comparative in vitro study was performed at the Department of Neural Cell Laboratory,Tianjin Huanhu Hospital and College of life Science of Nankai University from June 2005 to October 2007.MATERIALS:Totally 3 male Wistar rats (6-week old,weighing about 160 g) were used in this study.METHODS:BMSCs were cultured and purified by their characteristic of plastic adhesion in vitro,and then induced by BDNF,bFGF and AT-RA,and differentiate into neuron-like cells.Whole-cell patch damp technique was used to detect cell membrane current prior to and 3 days following induction.MAIN OUTCOME MEASURES:MSC phenotype was determined by flow cytometry.Cell morphology was observed under the inverted microscope before and after differentiation.Neuron specific enolase expression was assessed by immunocytochemistry.Whole-cell current results were measured.RESULTS:Flow cytometry results demonstrated that CD90 positive rate (99±3)%,CD31 (3.4±0.8)%,and CD34 (0.3±0.1)%.This indicated that these cells were undifferentiated stem cells,with purity of 95%.Undifferentiated MSCs under the optical microscope were mostly fiat cells with processes,similar to fibroblast-like cells.Neuron-like cells appeared 3 days following induction.Immunocytochemistry results showed that MSCs before induction were weakly positive for neuron specific enolase,but strongly positive for neuron specific enolase.At 72 hours,the differentiated rate was (24.01±3.76)%.The peak currents of outward currents in neuron-like cells were significantly higher in the induction group compared with the control group (P ＜ 0.05),but no inward Na~+ current was detected.CONCLUSION:(bFGF & BDNF)/AT-RA could induce the differentiation of MSCs into neuron-like cells.These cells showed the tendency to differentiate into mature neurons,though having no electrophysiological properties of mature neurons.