Enrichment differentiation of human induced pluripotent stem cells into sinoatrial node-like cells by combined modulation of BMP, FGF, and RA signaling pathways

Figures

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  Fig. 1. Characterization of the hiPSC. a hiPSC expressed high levels of the pluoripotency marker genes, OCT3/4, SOX2, and NANOG, which were comparable to hESC, a human embryonic stem cell (t test; NS, not significant; ND, not detectable; n = 3). b Ki67, pluoripotency markers of hiPSC were identified using IF method. c–e Pluoripotency markers of hiPSC (NANOG, OCT4, SSEA4, and TRA-1-60) were confirmed by immunoflorescence (IF) assay. g, h Representative flow cytometry analysis further confirmed the pluoripotency of the hiPSC. Scale bars, 100 μm (× 400). Expression values of all PCR analyses were normalized to the housekeeping gene GAPDH. Data are presented as “mean ± SD”

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  Fig. 2. Characterization of the hiPSC-derived cardiomyocytes. a, b CHIR concentration-dependently increased the expression of BRACHYURY (a) and MESP1 (b) in differentiated cells at day 1 and day 3 (t test, *p < 0.05, **p < 0.01, and *** p < 0.001 versus 0 μM or day 1; n = 3). c The hiPSC-induced cardiomyocytes expressed cTNT and α-actinin indicated by IF assay in induced cardiomyocytes from day 21 differentiation. Scale bars, 100 μm (× 400). d Representative plots of flow cytometry displaying high yields of cardiomyocytes derived from hiPSC at day 21. Expression values of all PCR analyses were normalized to the housekeeping gene GAPDH. Data are presented as “mean ± SD’”

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  Fig. 3. Optimization of the timing and concentration of small molecule chemicals targeting FGF, RA, and BMP signaling pathways for enriched SANLC differentiation. a The expression of cardiac mesoderm marker (MESP1), cardiac progenitor marker (NKX2-5), and cardiomyocyte marker (TNNT2) at days 3, 4, 5, 7, and 10, respectively, by qRT-PCR (t test, *p < 0.05, **p < 0.01, and *** p < 0.001 versus day 3; n = 3). b The expression of SHOX2, TBX18, TBX3, HCN4, and TNNT2 was evaluated by qRT-PCR at day 16 after the differentiation of hiPSC-induced cardiomyocytes was treated with BMP4 at the indicated concentrations at day 5–7 (t test, *p < 0.05, **p < 0.01, and NS, not significant versus 0 μM; n = 3). c The expression of SHOX2, TBX18, TBX3, HCN4, and TNNT2 was analyzed by qRT-PCR at day 16 of the differentiation after the induced cardiomyocytes were treated with PD at the indicated concentrations at day 5–7 (t test, *p < 0.05, **p < 0.01, ***p < 0.001, and NS, not significant versus 0 μM; n = 3).d The expression of SHOX2, TBX18, TBX3, HCN4, and TNNT2 was analyzed by qRT-PCR at day 16 of the differentiation after the induced cardiomyocytes were treated with BMS at the indicated concentrations at…

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  Fig. 4. The BMP4/PD/BMS (BPM) method promotes the differentiation of hiPSC toward SANLC. a Scheme of the GiWi-based protocol used for enriched differentiation of SANLC from hPSC via treatment of BMP4, PD, and BMS. Gi, GSK inhibitor (CHIR); Wi, Wnt inhibitor (IWP2); iCM, induced cardiomyocytes. b Compared to the GiWi control, the levels of the expression of SHOX2, TBX18, TBX3, HCN4, and TNNT2 (from left to right) generally displayed the gradual increasing tendency from the individual treatment of BMP4 (B), PD (P), and BMS (M) to the two combination of BMP4 + PD (BP), BMP4 + BMS (BM), and PD + BMS (PM), and BMP4 + PD + BMS (BPM) caused the highest expression of the markers above as shown by qPCR analysis at day 16 (t test, *p < 0.05, **p < 0.01, ***p < 0.001, and NS, not significant versus GiWi control; n = 3). Expression values of all PCR analyses were normalized to the housekeeping gene GAPDH. Data are presented as “mean ± SD”

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  Fig. 5. Validation of the enriched differentiation of SANLC by the BMP4/PD/BMS (BPM). a–c Representative IF analysis showed that SANLCs expressed SAN-specific transcription factor, SHOX2, TBX3, and TBX18. Scale bars, 100 μm (× 400). d Representative flow cytometry analysis showed that BPM significantly increased the percentage of CTNT+/NKX2.5− cells as compared to the GiWi group (t test, ***p < 0.001 versus GiWi control; n = 5). e Representative flow cytometry analysis showed that BPM significantly increased the percentage of CTNT+/SHOX2+ cells as compared to the GiWi group (t test, ***p < 0.001 versus GiWi control; n = 5). Data are presented as “mean ± SD”

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  Fig. 6. SANLCs induced by the BMP4/PD/BMS (BPM) possess typical electrophysiological characteristics of SAN. a Spontaneous beating frequency of SANLCs was significantly higher in the BPM (n = 6) than in the GiWi groups (n = 6) (t test, ***p < 0.001 versus GiWi control followed by Bonferroni’s post hoc test). b HCN4 channel inhibitor treatment caused significantly decreased beating rate in the BPM (n = 7) compared to the GiWi groups (n = 7) (t test, **p < 0.01 versus GiWi control followed by Bonferroni’s post hoc test). c Representative ventricule-like, atria-like, and sinus node-like action potential (AP) curves were recorded by whole cell patch clamp (left panel). The percentage of cells with pacemaker-like AP was remarkably increased in the BPM compared to the GiWi group (6/18 versus 2/15, BPM versus GiWi) (t test, ***p < 0.001 versus GiWi control). Data are presented as “mean ± SD”

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  Fig. 7. Graphic abstract. Working model of enriched differentiation of SANLC from hiPSC by the BMP4 + PD + BMS (BPM) treatment versus the GiWi method alone. Simultaneous activation of BMP and inhibition of RA and FGF signaling pathways during cardiac mesoderm stage of the GiWi-induced hiPSC differentiation strongly favor a SAN-specific gene transcriptional landscape leading to enhanced pacemaker cell specification