The Biphasic Effect of Retinoic Acid Signaling Pathway on the Biased Differentiation of Atrial-like and Sinoatrial Node-like Cells from hiPSC

Abstract

Background and objectives: Although human-induced pluripotent stem cells (hiPSC) can be efficiently differentiated into cardiomyocytes (CMs), the heterogeneity of the hiPSC-CMs hampers their applications in research and regenerative medicine. Retinoic acid (RA)-mediated signaling pathway has been proved indispensable in cardiac development and differentiation of hiPSC toward atrial CMs. This study was aimed to test whether RA signaling pathway can be manipulated to direct the differentiation into sinoatrial node (SAN) CMs.

Methods and results: Using the well-characterized GiWi protocol that cardiomyocytes are generated from hiPSC via temporal modulation of Wnt signaling pathway by small molecules, RA signaling pathway was manipulated during the differentiation of hiPSC-CMs on day 5 post-differentiation, a crucial time point equivalent to the transition from cardiac mesoderm to cardiac progenitor cells in cardiac development. The resultant CMs were characterized at mRNA, protein and electrophysiology levels by a combination of qPCR, immunofluorescence, flow cytometry, and whole-cell patch clamp. The results showed that activation of the RA signaling pathway biased the differentiation of atrial CMs, whereas inhibition of the signaling pathway biased the differentiation of sinoatrial node-like cells (SANLCs).

Conclusions: Our study not only provides a novel and simple strategy to enrich SANLCs but also improves our understanding of the importance of RA signaling in the differentiation of hiPSC-CMs.

Keywords: Atrial-like cells; Biased differentiation; Human-induced pluripotent stem cell (hiPSC); Retinoic acid (RA) signaling; Sinoatrial node-like cells (SANLCs).

Fig. 1

Characterization of the hiPSC. hiPSC expresses high levels of OCT4 (A, E, I), NANOG (B, F, J), TRA-1-60 (C, K), and Ki67 (D, H, L) by IF, qPCR and flow cytometry respectively. In addition, pluripotent marker SOX2 was also highly expressed in hiPSC using the qPCR data (G). For qPCR analysis (E∼G), the terminal differentiated human fibroblast (hF) cell line (BJ) served as negative control and human embryonic stem cell (hESC) line (HN4) as positive control. The expression was normalized to that of GAPDH. Data are presented as ‘Mean±SD’ from at least 3 independent experiments with duplicate on each sample, with ns denoting non-significant and nd, not detectable. Scale bars=100 μm (400× magnification).

Fig. 2

Characterization of the cardiomyocytes derived from hiPSC. Representative images of immunofluorescence and FACS plots showing that cardiomyocytes derived from hiPSC on day 35 after differentiation express α-actinin by IF (A) and cTNT by IF (B) and flow cytometry (C). Scale bars=100 μm (400× magnification).

Fig. 3

Activation of RA signaling pathway by RA biases the differentiation of hiPSC toward atrial cardiomyocytes. (A) RA was introduced on day 5 at the concentrations indicated, the effect on the expression of NPPA, MYL7, COUPTFII, KCNJ5, and CX40 was analyzed by qPCR. (B) 2 μM RA was introduced on day 5 after the differentiation, and the expression of SHOX2, TBX18, TBX3, HCN4, ISL1, CX30.2, CACNB1, CACNA1A, KCNN4, KCNK2, KCND2, and SCN3B was quantitated by qPCR on day 21. The expression was normalized to that of GAPDH. Data are presented as ‘Mean±SD’ from at least 3 indepen-dent experiments with duplicate on each sample, with ns denoting non -significant, *denoting p<0.05, and **denoting p<0.01.

Fig. 4

Inhibition of RA signaling pathway by BMS biases the differentiation of hiPSC toward Sinoatrial node-like cells. (A) BMS was introduced on day 5 at the concentrations indicated, the effect on the expression of NPPA, MYL7, COUPTFII, KCNJ5, and CX40 was analyzed by qPCR. (B) 5 μM BMS was introduced on day 5 after the differentiation, and the expression of SHOX2, TBX18, TBX3, HCN4, ISL1, CX30.2, CACNB1, CACNA1A, KCNN4, KCNK2, KCND2, and SCN3B was quantitated by qPCR on day 21. The expression was normalized to that of GAPDH. Data are presented as ‘Mean±SD’ from at least 3 independent experiments with duplicate on each sample, with ns denoting non-significant, *denoting p<0.05, **denoting p<0.01, and ***denoting p<0.001.

Fig. 5

Inhibition of RA signaling pathway by BMS biases the differentiation of hiPSC to Sinoatrial node-like cells. BMS at 5 μM was introduced on day 5 after the differentiation, and the expression of COUPTFII (A, B), TBX18 (C, D), and TBX3 (E, F) was evaluated by IF. Representative images and corresponding quantitations showing that inhibition of RA signaling pathway by BMS decreases the percentage of COUPTFII (A, B) positive cardio-myocytes and increases the percentage of TBX18-(C, D) and TBX3-positive (E, F) cardiomyocytes. Scale bars=100 μm (400× magnification).

Fig. 6

Inhibition of RA signaling pathway by BMS biases the differentiation of hiPSC to Sinoatrial node-like cells. BMS at 5 μM was introduced on day 5 after the differentiation, and the expression of COUPTFII, cTNT^＋/SHOX2^＋, and cTNT^＋/NKX2.5⁻ was evaluated by flow cytometry. Representative FACS plots showing that inhibition of RA signaling pathway by BMS decreases the percentage of COUPTFII positive cardiomyocytes (A) and increases the percentage of SHOX2^＋/cTNT^＋ (B) and cTNT^＋/NKX2.5⁻ (C) cardio-myocytes.

Fig. 7

Inhibition of RA signaling pathway by BMS biases the differentiation of cardiomyocytes possessing typical electrophysiological features of Sinoatrial node-like cells. BMS at 5 μM was introduced on day 5 after the differentiation, and the beating frequency and AP were analyzed after the differentiation on day 21 and 60 respectively. BMS significantly increased the beating frequency of cardiomyocytes (A), sensitized cardiomyocytes to TCN ion channel inhibitor (B), decreased the ratio of cardiomyocytes displaying typical atrial-like AP (C, middle panel; D, blue bar, 5/15), and increased the ratio of cardiomyocytes displaying typical pacemaker-like AP (C, bottom panel; D, green bar, 4/15), compared to RA group (10/15 and 2/15 respectively). Data in (A) and (B) are presented as ‘Mean±SD’ from recording of 7 cardiomyocytes, with *denoting p<0.05 and ****denoting p<0.0001.

Fig. 8

Graphic abstract. Working model of biphasic effect of modulating RA signaling on the enrichment differentiation of atrial-like and sinoatrial node-like cells based on the GiWi method. During the cardiac mesoderm stage of pan cardio-myocytes differentiation from hiPSCS, activation of RA signaling pathway promotes biased differentiation of atrial-like cells. In contrast, enrichment differentiation of sinoatrial node-like cells could be enabled by RA inhibition.