4.7 Article

Chemically defined and small molecules-based generation of sinoatrial node-like cells

Journal

STEM CELL RESEARCH & THERAPY
Volume 13, Issue 1, Pages -

Publisher

BMC
DOI: 10.1186/s13287-022-02834-y

Keywords

Human pluripotent stem cells; Sinoatrial node-like cells; Differentiation; Chemically defined medium; Small molecule; Signaling pathway

Funding

  1. Sichuan Province science and technology projects [2021YFH0148, 2018JY0405]
  2. Science and Technology Strategic Cooperation Program of Luzhou Municipal People's Government [2019LZXNYDJ30, 2020LZXNYDF01]
  3. National Natural Science Foundation of China [81970205, 82070277, 82170325]
  4. Beijing Natural Science Foundation [Z190013]
  5. Science and Technology Strategic Cooperation Program of Southwest Medical University [2019LZXNYDJ30, 2020LZXNYDF01]
  6. nonprofit Central Research Institute Fund of Chinese Academy of Medical Sciences [2019PT320026, 2021-F02]
  7. Shenzhen Fundamental Research Program [ZDSYS20200923172000001]

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In this study, a chemically defined method for generating SANLCs was established through combined modulation of WNT, RA, and FGF signaling pathways and metabolic selection. This method may serve as a platform for disease modeling, drug discovery, predictive toxicology, and biological pacemaker construction.
Background Existing methods for in vitro differentiation of human pluripotent stem cells (hPSCs) into sinoatrial node-like cells (SANLCs) require complex and undefined medium constituents. This might hinder the elucidation of the molecular mechanisms involved in cardiac subtype specification and prevent translational application. In our study, we aimed to establish a chemically defined differentiation methods to generate SANLCs effectively and stably. Methods We induced human embryonic stem cells (hESCs)/induced PSCs (hiPSCs) to pan-cardiomyocytes by temporal modulation of the WNT/beta-catenin (WNT) signaling pathway with GSK3 inhibitor and WNT inhibitor. During cardiac mesoderm stage of the differentiation process, signaling of WNT, retinoid acid (RA), and fibroblast growth factor (FGF) was manipulated by three specific molecules. Moreover, metabolic selection was designed to improve the enrichment of SANLCs. Finally, RT-PCR, immunofluorescence, flow cytometry, and whole cell patch clamp were used to identify the SANLCs. Results WNT, RA, and FGF signaling promote the differentiation of hPSCs into SANLCs in a concentration- and time window-sensitive manner, respectively. Synergetic modulation of WNT, FGF, and RA signaling pathways enhance the pacemaker phenotype and improve the differentiation efficiency of SANLCs (up to 45%). Moreover, the purification based on lactate metabolism and glucose starvation further reached approximately 50% of SANLCs. Finally, the electrophysiological data demonstrate that cells differentiated with the proposed protocol produce a considerable number of SANLCs that display typical electrophysiological characteristics of pacemaker cells in vitro. Conclusion We provide an optimized and chemically defined protocol to generate SANLCs by combined modulation of WNT, RA, and FGF signaling pathways and metabolic selection by lactate enrichment and glucose starvation. This chemically defined method for generating SANLCs might provide a platform for disease modeling, drug discovery, predictive toxicology, and biological pacemaker construction.

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