Transcription factor TBX18 promotes adult rat bone mesenchymal stem cell differentiation to biological pacemaker cells

Abstract

Bone mesenchymal stem cells (BMSCs) are currently considered the optimal stem cells for biological pacemaker cell transformation. The cardiac‑specific transcription factor T‑Box protein 18 (TBX18) is essential for sinoatrial node (SAN) formation, particularly formation of the head region that generates the electrical impulses that induce heart contraction. The present study aimed to confirm the effects of TBX18 on biological pacemaker differentiation of rat BMSCs. Flow cytometry was used to identify the surface markers of BMSCs, in order to acquire pure mesenchymal stem cells. Subsequently, BMSCs were transduced with TBX18 or green fluorescent protein adenovirus vectors. The effects of TBX18 were evaluated using SAN‑specific makers including TBX18, α‑actin, cardiac troponin I, hyperpolarization‑activated cyclic nucleotide‑gated channel 4 and connexin 43 by reverse transcription‑quantitative polymerase chain reaction, western blotting and immunofluorescence. The findings demonstrated that direct conversion of BMSCs to biological pacemaker cells via TBX18 is a feasible method in the field of cardiology.

Figure 1

Characterization of BMSCs. (A) BMSCs at passage 3 exhibited unified spindle cell morphology, and tightly adhered to the bottom of the culture dish (magnification, ×100). (B) Cultured BMSCs, 80-90% confluent (magnification, ×40), were applied for subsequent experiments. (C) Cell surface markers of BMSCs. BMSCs, bone mesenchymal stem cells; CD, cluster of differentiation.

Figure 2

Green fluorescence of BMSCs (passage 3) transduced with TBX18 or GFP for 24 and 48 h under an Olympus IX51 inverted fluorescence microscope. (A) TBX18-transduced BMSCs at 24 h (magnification, ×40). (B) GFP-transduced BMSCs at 24 h (magnification, ×40). (C) TBX18-transduced BMSCs at 48 h (magnification, ×100). (D) GFP-transduced BMSCs at 48 h (magnification, ×100). BMSCs, bone mesenchymal stem cells; GFP, green fluorescent protein; TBX18, T-box protein 18.

Figure 3

Western blotting and reverse transcription-quantitative polymerase chain reaction analysis of target protein and mRNA expression post-transduction. (A) pHBAd-MCMV-GFP-TBX18-transduced BMSCs displayed exogenous TBX18 expression, as determined by western blotting. (B) Protein expression levels of TBX18 were verified in differentiated cells by western blotting. (C) Alterations in the relative mRNA expression levels of TBX18 were compared between the T group and the G and B groups. (D) Quantitative analysis of the mRNA expression levels of α-actin, HCN4 and CX43. (E) Western blotting detected increased α-actin, cTnI and HCN4 protein expression, and reduced CX43 protein expression, in the T group compared with in the G and B groups. ^※P<0.05, T group vs. G group; ^△P<0.05, T group vs. B group. BMSCs, bone mesenchymal stem cells; B, blank group; cTnI, cadiac troponin I; CX43, connexin 43; G, GFP group/empty plasmid group; GFP, green fluorescent protein; HCN4, hyperpolarization-activated cyclic nucleotide-gated channel 4; hTBX18, human TBX18; T, TBX18 group; TBX18, T-box protein 18.

Figure 4

Fluorescence images of BMSCs post-transduction (magnification, ×200). (A) BMSCs were transduced with pHBAd-MCMV-GFP-TBX18, and HCN4 and GFP expression were detected. Nuclei were stained blue with DAPI. (B) BMSCs transduced with the pHBAd-MCMV-GFP control vector expressed GFP but not HCN4. BMSCs, bone mesenchymal stem cells; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; HCN4, hyperpolarization-activated cyclic nucleotide-gated channel 4; TBX18, T-box protein 18.