RA signaling pathway combined with Wnt signaling pathway regulates human-induced pluripotent stem cells (hiPSCs) differentiation to sinus node-like cells

Figures

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  Fig. 1. Differentiation of hiPSCS into CMs and exploration of the optimal RA intervention time. Data points present as means ± SEM (n = 1). a Schematic of the experimental protocol for the differentiations of cardiomyocybytes from hiPCs with small molecule modulators of Wnt signaling. CHIR (CHIR99021) and IWRI, agonists, and inhibitors of the Wnt pathway, respectively. b Schematic of the experimental protocol for RA = 02.4/0.5/1.0umol at days D3-D5 to include hiPSCs differentiation, c–h Samples of mRNA were collected and measured from differentiating cells between day0–9 using qRT-PCR and normalized to TBP expression. i–t The4 transcriptional level of the central atrial muscle cell marker and pacemaker markers. Data points present as means ± SEM (n = 1). *p < 0.05 compared to control group (CHIR = 0 group). RT-qPCR, quantitative reverse transcriptase-polymerase chain reaction

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  Fig. 2. The expression of genes and proteins related to cardiac pacemaker cells in four groups. a Schematic of the experimental protocol for RA in four groups. b–i The mRNA level of the pacemaker markers. j–o The protein expressions of the pacemaker markers. Data points present as means ± SEM (n = 1). *p < 0.05 compared to CHIR = 0 group. #p < 0.05 compared to CHIR = 3 group. &p < 0.05 compared to RA + CHIR = 0 group

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  Fig. 3. Ra promoted the differentiation of hiPSCs inti pacemaker cells. a The pacemaker cells rate marked by both CTNT positive and Nkx2.5 negative (cTNT+Nkx2.5−) in four groups detected by flow cytometry (n = 4). b The pacemaker cells rate marked by both Shox2 positive and Nkx2.5 negative (Shox2+Nkx2.5.−) in four groups detected by flow cytometry (n = 4). c Cells differentiated from four groups were stained for cTNT (green), Shox2 (red) and DAPI (blue). Magnification, × 200, n = 4

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  Fig. 4. The frequency of beats and the expression of ionic channels in four groups. a The frequency of beats in four groups. b–h The mRNA level of ionic channels in four groups. Data points present as means ± SEM (n = 5). *p < 0.05 compared to CHIR = 0 group. #p < 0.05 compared to CHIR = 3 groups. &p < 0.05 compared to RA + CHIR = 0 group

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  Fig. 5. If current and typical action potential (AP) of four groups. a–d If Current was detected in four groups using the patch clamp technique (n = 28). e Current density–voltage relationships in four groups (n = 28). f AP was detected in four groups. g APA, MDP level, 0-phase depolarization rate (dV/dt) and APD of four groups.*p < 0.05 compared to CHIR = 0 groups. APA, action potential amplitude; action potential duration; MDP, maximum diastolic potential

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  Fig. 6. The biological mechanism by which RA promotes pacing cell differentiation. a–e The mRNA level of the pacemaker markers on D20. f The mRNA level of TBX5 on D0–D19. g–h The protein expression of TBX5. Data points present as means ± SEM (n = 5). *p < 0.05 compared to CHIR = 0 group. #p < 0.05 compared to CHIR = 3 group. &p < 0.005 compared to RA + CHIR = 0 group

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  Fig. 7. The changes of cardiac pacing-related factors during D0-D19 dynamically

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  Fig. 8. The potential biological of combining Wnt signaling and RA inducing sinus node-like cells