We used the patch-clamp technique and RT-PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L-type Ca²⁺ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L-type Ca²⁺ channel function.

Introduction: During osteogenic differentiation, mesenchymal stem cells from human bone marrow (hMSCs) must adopt the calcium handling of terminally differentiated osteoblasts. There is evidence that voltage-operated calcium channels (VOCCs), including L-type calcium channels, are involved in regulation of osteoblast function. We therefore studied whether VOCCs play a critical role during osteogenic differentiation of hMSCs.

Materials and Methods: Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, β-glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca²⁺ channel α1 subunits was shown by semiquantitative or single cell RT-PCR. Voltage-activated calcium currents of hMSCs were measured with the whole cell voltage-clamp technique.

Results: mRNA for the pore-forming α1C and α1G subunits of the L-type and T-type Ca²⁺ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L-type Ca²⁺ currents to a subpopulation of hMSCs was confirmed by single cell RT-PCR, where mRNA for the α1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine-sensitive L-type Ca²⁺ currents were found in 13% of cells cultured in MM and in 12% of the cells cultured in ODM. Under MM and ODM culture conditions, the cells positive for L-type Ca²⁺ currents were significantly larger than cells without Ca²⁺ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L-type Ca²⁺ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization.

Conclusion: These results suggest that, in the majority of hMSCs, Ca²⁺ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L-type Ca²⁺ channels does not affect early osteogenic differentiation of hMSCs.