Isolation and culture of human marrow mesenchymal stem cells and their voltage-operated Ca2+ channels on the membrane surface

Abstract

Objective To isolate and culture human marrow mesenchymal stem cells (hMSCs),and to detect the voltage-operated Ca2+ channels (VOCCs) on the membrane surface of hMSCs using patch clamp technique. Methods Human bone marrow mononuclear cells were collected by gradient centrifugation on Percoll at a density of 1.073 g/ml combined with wall adherence method. The hMSCs were further cultured and expanded in vitro. Trypan blue exclusion staining method was used to count the number of living cells. The cellular phenotypes and cell cycle of hMSCs were identified by fluorescent activated cell sorting (FACS), and the expression of VOCCs on the membrane surface of hMSCs was observed with patch clamp technique. Results The typical morphology of passaged hMSCs featured a dense array of fibroblastlike structure figure. Up to 99% of hMSCs was viable. Flow cytometry demonstrated that hMSCs expressed CD44 and CD105 but not CD34, CD45 and CD31. Cell cycles of hMSCs showed that 81.52 % of hMSCs was in G1 and G0 stages, and 18.48 % in G2, M and S stages, with a G2/G1 ratio of 1.67. Patch clamp examination displayed nifedipine-sensitive calcium influx in 6 out of 20 hMSCs. The peak calcium influx (ICa-Peak) was (96.67±13.50) pA at -0 mV when activated voltage was about -40 mV. However, the Ica-Peak was restrained to (47.75± 11.24) pA with 5 μ mol/L nifedipine (P＜0.05). Conclusion hMSCs isolated and cultured in vitro were highly viable and mostly were at G0 or G1 stages. VOCCs were shown to be present on the membrane surface of hMSCs. Key words: Human mesenchymal stem cells; Patch clamp; Voltage-operated Ca2+ channels; Calcium ion current; Cell cycle