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 under-standing of the importance of RA signaling in the differentiation of hiPSC-CMs.
ABSTRACT
Background and Objectives@#Manipulating different signaling pathways via small molecules could efficiently inducecardiomyocytes from human induced pluripotent stem cells (hiPSC). However, the effect of transcription factors on the hiPSC-directed cardiomyocytes differentiation remains unclear. Transcription factor, p53 has been demonstrated indispensable for the early embryonic development and mesendodermal differentiation of embryonic stem cells (ESC).We tested the hypothesis that p53 promotes cardiomyocytes differentiation from human hiPSC. @*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, we demonstrated that forced expression of p53 in hiPSC remarkably improved the differentiation efficiency of cardiomyocytes from hiPSC, whereas knockdown endogenous p53 decreased the yield of cardiomyocytes. This p53-mediated increased cardiomyocyte differentiation was mediated through WNT3, as evidenced by that overexpression of p53 upregulated the expression of WNT3, and knockdown of p53 decreased the WNT3 expression. Mechanistic analysis showed that the increased cardiomyocyte differentiation partially depended on the amplified mesendodermal specification resulted from p53-mediated activation of WNT3-mediated Wnt signaling. Consistently, endogenous WNT3 knockdown significantly ameliorated mesendodermal specification and subsequent cardiomyocyte differentiation. @*Conclusions@#These results provide a novel insight into the potential effect of p53 on the development and differentiation of cardiomyocyte during embryogenesis.