Electrophysiological characteristics of bone marrow stem cells following in vitro induction / 中国组织工程研究

ABSTRACT

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can grow in host myocardium, differentiate under myocardial condition, improve cardiac function. However, biological characteristics of BMSC differentiation are still unclear presently.OBJECTIVE: To study the expression and electrophysiological characteristics of BMSC/n vitro connexin-43 following 5-azacitidine (5-aza) treatment.DESIGN, TIME AND SETTING: The cytological in vitro controlled study was perormed at the Heart Center, Hebei Provincial People's Hospital from July 2007 to February 2009.MATERIALS A total of 24 male pigs aged 2 months were purchased from Exparimental Animal Center, Hebei Medical University.METHODS: Bilateral femoral bone marrow was obtained from pigs under sterile condition. BMSCs were harvested by Percoll density gradient in vitro. At passage 2, BMSCs were treated with 10 μmol/L 5-aza, and incubated in DMeM without inductor 24 hours later. Indices were measured I, 2, 3 weeks following induction. A control group was set up, which was not treated with 5-azacitidine. Following bone marrow extraction, experimental pigs were anesthetized to obtain ventricular muscle. Normal ventricular muscle cells were isolated and cultured by tissue block enzyme digestion method.MAIN OUTCOME MEASURES: Expression of connexin-43 was measured by immunohistochemical staining (ABC method). Ito current density and action potential were determined by patch clamp technique.RESULTS: At 1 and 2 weeks following 5-aza induction, some BMSCs were positive for connexin-43, with the presence of brown particles surrounding nuclei. At 3 weeks, positive rate of connexin-43 was 95%. The area with large cell density was presented with similar structure to normal myocardium. At +80 mV, compared with normal myocardial cells, Ito current density was significantly reduced in BMSCs following 1 and 2 weeks and in the control group (P < 0.05). Ito current density was significantly increased to a normal levels in BMSCs 3 weeks following induction (P > 0.05). No action potential was detected in BMSCs following 1 and 2 weeks of 5-aza, and action potential could be determined 3 weeks following induction, which was identical to normal myocardial cells.CONCLUSION: Through induced by 5-aza for three weeks, BMSCs have the similar expression of connexin-43 and electrophysiological characteristics as normal myocardium.