Promoting differentiation of human induced pluripotent stem cells into sinoatrial nodelike cells through programmed regulation of AMPK signaling pathway

Background and aims: The differentiation of human induced pluripotent stem cells (hiPSCs) into sinoatrial node-like cells (SANLCs), which can provide a new cell resource for study or treatment of sick sinus syndrome, remains challenged by complex differentiation protocols and low efficiency.  This study aims to reveal the role of AMPK signaling in the differentiation process of hiPSCs into SANLCs, providing new strategies for obtaining SANLCs in vitro.

Methods: Cells from different stages during differentiation of hiPSCs into cardiomyocytes (hiPSC-CMs) were taken for transcriptome sequencing and analyses using PCR, immunostaining, FACS and patch clamp techniques.  
Results KEGG analysis identified that the AMPK signaling pathway is significantly enriched with the expression of NKX2.5 (sinus node cell development-related transcription factor). The early activation (on day 4-6 of differentiation) and late inhibition (on day 6-8) of AMPK signaling were both effective in upregulating SANLCs markers. The combined manipulation of both stages (early activation and late inhibition) further enhanced the differentiation efficiency reflected by higher SANLCs marker expression, which was also confirmed at the protein level by immunofluorescence and flow cytometry analyses. SANLCs obtained from the differentiation with combined modulation of AMPK signaling displayed typical features of pacemaker cells in the heart, including receptors, ion channels, action potentials and the funny current (If).

Conclusion: Early activation and then inhibition of the AMPK signaling pathway during the differentiation process can promote hiPSCs differentiation to SANLCs. This will provide a novel strategy for obtaining SANLCs in vitro and for mechanistic or therapeutic studies on sinoatrial node diseases.