Transcription factor Tbx18 induces the differentiation of c-kit canine mesenchymal stem cells (cMSCs) into SAN-like pacemaker cells in a co-culture model in vitro.

Bone mesenchymal stem cells (MSCs), as well as cardiomyocytes, are derived from early mesoderm, becoming committed to their fate under the influence of different differentiation factors. We examined whether the overexpression of Tbx18 can induce the differentiation of c-kit cMSCs into a phenotype similar to that of native pacemaker cells and whether these transfected cells can couple to adjacent atrial cells with functional consequences. The c-kit cMSCs were first sorted, then transfected with different lentiviral vectors. Tbx18-c-kit cMSCs represented the experimental group, while EYFP-c-kit cMSCs and canine sinoatrial node (SAN) cells were used as controls. Within days of transfection, the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel HCN4 protein and gap junction protein Connexin 45 (Cx45) expression in Tbx18-c-kit cMSCs were 12-fold and 5.6-fold higher, respectively, than that in EYFP-c-kit cMSCs. After co-culture with canine atrial cells in vitro for three days, the funny currents (I) were recorded in the Tbx18-c-kit cMSCs, but not in EYFP-c-kit cMSCs. The trend of these I currents was highly similar to that of SAN cells, although the current density was smaller. The Tbx18-EYFP-c-kit cMSCs showed responsiveness to β-adrenergic stimulation, and the intracellular cyclic adenosine monophosphate (cAMP) level was higher than that in EYFP-c-kit cMSCs. The Tbx18-EYFP-c-kit cMSCs delivered fluorescent dye to neighboring atrial cells via gap junctions, thus these cell pairs could communicate as a pacemaker unit. We propose that the overexpression of Tbx18 in c-kit cMSCs induces their differentiation to SAN-like pacemaker cells.