Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia
Fig 4
Measurement of L-type Ca2+ channels during differentiation process and intracellular Ca2+ concentration in IPCs in response to glucose stimulation.
The whole-cell patch-clamp experiments were performed on hMSCs, HD-MSCs and IPCs to determine the amount and functional change of L-type Ca2+ channel during the differentiation process. Nifedipine at 10 μM suppressed the inward currents. Nifedipine-sensitive inward Ca2+ currents (ICa.L) were recorded by 300-ms voltage step between +10 and +60 mV from a holding potential of –50 mV (to inactivate INa). A: The ICa.L was recorded among the hMSCs, HD-MSCs and IPCs. B: The I-V relationship of ICa.L displayed the maximum current peak appeared at 10 mV in hMSCs, HD-MSCs and IPCs (left), and the mean peak current amplitudes in IPCs is higher than that in hMSCs or HD-MSCs (right, *P<0.05 compared to hMSCs). C: The changes of intracellular Ca2+ concentration in IPCs in response to glucose stimulation were measured by labeling with Fluo-3/AM under laser confocal scanning microscopy. The results were expressed as respective fluorescence intensity of 10 of IPCs (left) and average fluorescence intensity of 30 of IPCs (right). After stimulated with 30mmol/L glucose at 240 s, the intracellular Ca2+ concentration rapidly increased in IPCs due to significant Ca2+ influx when extracellular Ca2+ existing. When Ca2+ channels were blocked by 25 mg/ml of Verapamil at 480 s and then stimulated with 3.5 mM glucose at 720 s, intracellular Ca2+ rapidly increased again in IPCs due to the release of Ca2+ from calcium stores. D: The images of intracellular Ca2+ before (left) and after 2 times of glucose stimulation (middle and right) in IPCs, reflected by green fluorescence. Scale bars: 50μm for D.