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

Abstract

Background The source of SAN is debated among researchers. Many studies have shown that RA and Wnt signaling are involved in heart development. In this study, we investigated the role of retinoic acid (RA) and Wnt signaling in the induction of sinus node-like cells. Methods The experimental samples were divided into four groups: control group (CHIR = 0), CHIR = 3, RA + CHIR = 0 andRA + CHIR = 3. After 20 days of differentiation, Western blot, RT-qPCR, immunofluorescence and flow cytometry were performed to identify sinus node-like cells. Finally, whole-cell patch clamp technique was used to record pacing funny current and action potential (AP) in four groups. Results The best intervention method used in our experiment was RA = 0.25 µmol/L D5-D9 + CHIR = 3 µmol/L D5-D7. Results showed that CHIR can increase the expression of ISL-1 and TBX3, while RA mainly elevated Shox2. Immunofluorescence assay and flow cytometry further illustrated that combining RA with CHIR can induce sinus node-like cells (CTNT+Shox2+Nkx2.5−). Moreover, CHIR might reduce the frequency of cell beats, but in conjunction with RA could partly compensate for this side effect. Whole cell patch clamps were able to record funny current and the typical sinus node AP in the experimental group, which did not appear in the control group. Conclusions Combining RA with Wnt signaling within a specific period can induce sinus node-like cells.

Cadherin‐5 facilitated the differentiation of human induced pluripotent stem cells into sinoatrial node‐like pacemaker cells by regulating β‐catenin

“…VSNL1 exhibited a high level in the CDH5 group compared with that of in CON group, further identifying that CDH5 favored the differentiation of hiPSCs into SANLPCs. Consistent with previous studies (Liu et al, 2020; Protze et al, 2017; Yin et al, 2022; Zhao, Wang, et al, 2020), these results suggest that CDH5 promoted hiPSC‐CMs to develop key gene expression patterns similar to cardiac pacemaker cells, highlighting the potential regulatory role of CDH5 in modulating the expression of these key transcriptional factors involved in SANPCs development and function.…”

“…In contrast to prior investigations (Gharibeh et al, 2021; Liu et al, 2020; Protze et al, 2017; Wang et al, 2023; Yin et al, 2022; Zhang et al, 2022; Zhao, Wang, et al, 2020), our approach to procuring SANLPCs exhibits superior attributes in terms of simplicity, controllability, and cost‐effectiveness. However, in relation to the final proportion of acquired SANLPCs, our study did not present a notable advantage compared with previous research (Liang et al, 2020; Ren et al, 2019).…”

“…Consistent with a previous study (Protze et al, 2017), compared with CON cardiomyocytes, hiPSC-CMs in the CDH5 group exhibited higher levels of pacemaker channel genes (HCN1, HCN4, and KCNJ3). Suppressing the endogenous inward rectifier potassium current (I K1 ) was first used to create biological pacemaker via gene therapy (Cingolani et al, 2018) In contrast to prior investigations (Gharibeh et al, 2021;Liu et al, 2020;Protze et al, 2017;Wang et al, 2023;Yin et al, 2022;Zhang et al, 2022;Zhao, Wang, et al, 2020), our approach to procuring SANLPCs exhibits superior attributes in terms of simplicity, controllability, and cost-effectiveness. However, in relation to the final proportion of acquired SANLPCs, our study did not present a notable advantage compared with previous research (Liang et al, 2020;Ren et al, 2019).…”

“…U1 cell line was cultured on Matrigel (#354277; Corning)-coated plates using mTeSR1 medium (#85852; STEMCELL Technologies), and passaged with Versene digestion solution (#15040066; Gibco) at a 1:4 to 1:6 ratio upon reaching 80%-90% confluency. According to previous studies (Lian et al, 2012;Yin et al, 2022;Zhang et al, 2022), directed cardiac differentiation of hiPSCs was induced by modulation of canonical Wnt/β-catenin signaling. Briefly, RPMI1640/B27 minus insulin (#A189560; Gibco) containing 10 μM CHIR99021 (Wnt activator, #2052; STEMCELL Technologies) were used to activate Wnt signaling on Day (D) 0-D2 for 48 h. After 2 days, the medium was refreshed with RPMI1640/B27 minus insulin for 24 h. On D3-D5, cells were treated with fresh RPMI1640/B27 minus insulin containing 5 μM IWR1 (Wnt inhibitor, #72562; STEMCELL Technologies) for another 48 h. Two days later, medium was refreshed with RPMI1640/B27 minus insulin medium.…”

Retinoic acid modulation guides human-induced pluripotent stem cell differentiation towards left or right ventricle-like cardiomyocytes

“…TBX5 alone or in combination with GATA4, NKX2.5 and MEF2C regulates the expression of multiple target genes that are critical to the structure and function of the heart, including MYH6, MYH7 and NPPA [ 11 , 65 ]. As RA has been shown to operate upstream of the transcription factor TBX5, indirectly modulating its expression [ 18 , 61 ], RA concentrations may govern gene and protein expression crucially involved in heart morphogenesis (Fig. 1 B).…”