A new combination of transcription factors increases the harvesting efficiency of pacemaker‑like cells

Figures

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  Figure 1.. Transfection rate, transfected gene expression and culture conditions. (A) ISL-1-ADSCs observed at 48 h under a fluorescence microscope (magnification, ×40). (B) Tbx18-ADSCs observed at 48 h under a fluorescence microscope (magnification, ×40). (C) ISL-1+Tbx18-ADSCs observed at 48 h under a fluorescence microscope (magnification, ×40). (D) Transfection rate of different MOI values. (E) Expression of human ISL-1mRNA and Tbx18mRNA in the transfected groups. Scale bar=200 µm. *P<0.05 vs. GFP group. Tbx18, T-box 18; ISL-1, insulin gene enhancer binding protein 1; ADSCs, adipose tissue-derived stem cells; MOI, multiplicity of infection; GFP, green fluorescent protein.

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  Figure 2.. Expression of associated genes by RT-qPCR and western blotting after co-culture for 7 days. (A) HCN4, cTnT, Tbx3, Nkx2.5, Cx43 and Cx45 gene expression was examined using RT-qPCR. (B) HCN4, Cx43 and Cx45 protein expression examined using western blotting. (C) Quantitative assessment of HCN4, Cx43 and Cx45 protein levels by integrated optical density analyses. Similar results were obtained in three independent experiments. GAPDH was used as the protein control. *P<0.05 and **P<0.01 vs. GFP group. HCN4, hyperpolarization-activated cyclic nucleotide-gated cation channel 4; cTnT, cardiac troponin T; Tbx3, T-box 3; Nkx2.5, homeobox protein Nkx-2.5; Cx45, connexin 45; Cx43, connexin 43; GFP, green fluorescent protein; Tbx18, T-box 18; ISL-1, insulin gene enhancer binding protein 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction

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  Figure 3.. Cardiac-specific proteins examined by immunofluorescence staining in differentiated ADSCs after co-culture for 7 days (magnification, ×400). Grey, nuclei stained with DAPI; red, ISL-1-ADSCs; green, Tbx18-ADSCs; and blue, representative positive staining of HCN4 and cTnT. ADSCs, adipose tissue-derived stem cells; Tbx18, T-box 18; ISL-1, insulin gene enhancer binding protein 1; HCN4, hyperpolarization-activated cyclic nucleotide-gated cation channel; cTnT, cardiac troponin T. Scale bar, 50 µm.

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  Figure 4.. Single-cell electrophysiology of ADSCs. (A) Spindle-shaped cells were used for all electrophysiological recordings (magnification, ×200). (B) Hyperpolarization-activated inward currents recorded from ISL-1-ADSCs using the patch clamp technique. (C) Hyperpolarization-activated inward currents recorded from Tbx18-ADSCs using the patch clamp technique. (D) Hyperpolarization-activated inward currents recorded from ISL-1+Tbx18-ADSCs using the patch clamp technique. (E) Hyperpolarization-activated inward currents (If) were blocked by CsCl (4 mmol/l). (F) Current density-voltage association of ISL-1 (red, n=6), Tbx18 (green, n=6) and ISL-1+Tbx18 (yellow, n=6) groups. Tbx18, T-box 18; ISL-1, insulin gene enhancer binding protein 1; ADSCs, adipose tissue-derived stem cells; If, funny current. Scale bar, 50 µm.