Objective To test if the pacemaker current can be elicited from mesenchymal stem cells (MSCs) transfected with HCN4 genes by LentiV.
Methods 1. MSCs of rabbit from the posterior iliac crest of rabbit were cultured at 37°C in a humidified atmosphere of 5% CO2. 2. The self-inactivating HIV-based lentiviral vector (LentiV) was used as transgene delivery, which was constructed with plasmid hHCN4/pcDNA3. 3. Total RNA was extracted from control MSCs and those transfected with hHCN4, and RT-PCR was performed. 4. Whole-cell patch clamp was used to study membrane currents. If were elicited in the whole cell configuration by holding cells at −40 mV for 50 ms followed by 10 -mV steps (2 s) to −130 mV and returned to −40 mV (50 ms) after each step. After the If was recorded, cells were superfused with extracellular solution containing 4 mM caesium chloride and the currents were measured accordingly.
Results 1. In addition to expressing characteristic hHCN4 protein, mHCN4-transfected hMSCs also express an anticipated high level of hHCN4 gene by RT-PCR and Western blot analysis. And immunofluorescence image is shown for GFP. Control MSCs were negative. 2. With the use of the whole cell configuration of the patch-clamp technique, If was elicited using hyperpolarizing steps in 10-mV increments from −40 mV to −140 mV from a holding potential of −40 mV and was voltage-dependent. The threshold of voltage for activation of If is around −80 mV. Remarkably, all hHCN4 positive cells exhibit a large caesium-sensitive If and it was significantly inhibited by 4 mM caesium chloride.
Conclusions The pacemaker current of If can be elicited from the mesenchymal stem cells transfected with HCN4 genes by LentiV. The genetically engineered MSC expressing hHCN4 is a demonstration of feasibility of preparing MSC-based biological pacemaker cells.