Insulin gene enhancer binding protein 1 induces adipose tissue‑derived stem cells to differentiate into pacemaker‑like cells

Figures

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  Figure 1. Transfection rate and the expression of ISL‑1. (A) ADSCs observed following culture for 48 h and 5‑7 days under a light microscope (magnification, ×100). (B) ISL‑1‑ADSCs observed under a fluorescence microscope at different MOI values (magnification, ×200). (C) Numbers of positive cells at MOI values detected by flow cytometric analysis. (D) Transfection rate at different MOI values. In group A, cells were transfected with lentivirus overexpressing mCherry or ISL-1-mCherry. In group B, cells were transfected with a mix of lentivirus overexpressing mCherry or ISL-1-mCherry, 5-10 μg/ml polybrene and enhanced infection solution. (E) mRNA expression of human ISL-1 in the transfected groups. *P<0.05, vs. mCherry-ADSCs. ADSCs, adipose tissue-derived stem cells; ISL-1, insulin gene enhancer binding protein 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MOI, multiplicity of infection.

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  Figure 2. Cell morphology and beating rate in co-culture systems. (A) Morphology of ADSCs following co-culture under a light microscope (magnification, ×100). (B) Morphology of ADSCs following co‑culture under a fluorescence microscope (magnification, ×400). (C) Beating rate of ADSCs and NRVMs on different days in the co-culture system. Green represents NRVMs on different days in the NRVMs only group; black represents the beating rate of mCherry-ADSCs in the co-culture system; red represents the beating rate of ISL-1-ADSCs in the co-culture system, *P<0.05. ADSCs, adipose tissue-derived stem cells; NRVMs, neonatal rat ventricular cardiomyocytes; ISL-1, insulin gene enhancer binding protein 1.

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  Figure 3. Expression of related genes by western blot and RT-qPCR analyses following co-culture for 5-7 days. (A) Gene expression levels of HCN4, Cx45 and Cx43 were examined by RT-qPCR analysis. (B) Protein expression of HCN4, Cx45 and Cx43 was examined using western blotting. (C) Quantitative assessment of protein levels of HCN4, Cx45 and Cx43 by integrated optical density analyses. Similar results were obtained in three independent experiments. GAPDH was used as the protein control. *P<0.05 vs. other groups, **P<0.01, vs. other groups. HCN4, hyperpolarization-activated cyclic nucleotide-gated cation channel 4; Cx45, connexin 45; Cx43, connexin 43; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ADSCs, adipose tissue-derived stem cells; NRVMs, neonatal rat ventricular cardiomyocytes; ISL-1, insulin gene enhancer binding protein 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

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  Figure 4. Cardiac‑specific proteins examined by immunofluorescence staining in differentiated ADSCs following co‑culture for 5‑7 days with NRVMs (magnification, ×400). (A) Clear positive staining for cTnT was observed in ADSCs transfected with Ubi-MCS-ISL-1-3FLAG-SV40-mCherry and Ubi-MCS-3FLAG-SV40-mCherry control vector. (B) Clear positive staining for HCN4 was observed in ADSCs transfected with Ubi-MCS-ISL-1-3FLAG -SV40-mCherry and Ubi-MCS-3FLAG-SV40-mCherry control vector. Scale bar, 25 μm. The nuclei were stained with DAPI (grey), mCherry-ADSCs were detected by immunofluorescence (red). Representative positive staining of HCN4 and cTnT is shown in blue. ADSCs, adipose tissue‑derived stem cells; ISL‑1, insulin gene enhancer binding protein 1; HCN4, hyperpolarization-activated cyclic nucleotide-gated cation channel; DAPI, 4′,6-diamidino-2-phenylindole.

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  Figure 5. Spontaneous electrical activity in differentiated ADSCs. (A) Spindle‑shaped cells were used for electrophysiological recordings (magnification, ×200). (B) If current was detected in ISL-1-ADSCs using the patch clamp technique and was blocked by CsCl (4 mM/l). (C) Current density-voltage relationships of ISL-1-ADSCs (black, n=6) and ISL-1-ADSCs+NRVMs (red, n=6) groups. ADSCs, adipose tissue-derived stem cells; ISL-1, insulin gene enhancer binding protein 1; NRVMs, neonatal rat ventricular cardiomyocytes; If, hyperpolarization-activated inward current.