Abstract
Long non-coding RNA (lncRNA) is receiving increasing attention in embryonic stem cells (ESCs) research. However, the roles of lncRNA in the differentiation of ESCs into pacemaker-like cells are still unclear. Therefore, the present study aims to explore the roles and mechanisms of lncRNA in the differentiation of ESCs into pacemaker-like cells. ESCs were cultured and induced differentiation to pacemaker-like cells. RNA sequencing was used to identify the differential expression lncRNAs during the differentiation of ESCs into pacemaker-like cells. Cell morphology observation, flow cytometry, quantitative real-time polymerase chain reaction, western blot, and immunofluorescence were used to detect the differentiation of ESCs into pacemaker-like cells. LncRNA and genes overexpression or knockdown through transfected adenovirus in the differentiation process. The fluorescence in situ hybridization (FISH) detected the lncRNA location in the differentiated ESCs. Luciferase reporter gene assay, methylation-specific PCR, chromatin immunoprecipitation assay, and RNA immunoprecipitation assay were performed to reveal the mechanism of lncRNA-regulating HCN4 expression. Rescue experiments were used to confirm that lncRNA regulates the differentiation of ESCs into pacemaker-like cells through HCN4. We cultured the ESCs and induced the differentiation of ESCs into pacemaker-like cells successfully. The expression of lncRNA RCPCD was significantly decreased in the differentiation of ESCs into pacemaker-like cells. Overexpression of RCPCD inhibited the differentiation of ESCs into pacemaker-like cells. RCPCD inhibited the expression of HCN4 by increasing HCN4 methylation at the promoter region through DNMT1, DNMT2, and DNMT3. RCPCD inhibited the differentiation of ESCs into pacemaker-like cells by inhibiting the expression of HCN4. Our results confirm the roles and mechanism of lncRNA RCPCD in the differentiation of ESCs into pacemaker-like cells, which could pave the path for the development of a cell-based biological pacemaker.
Highlights
The sinoatrial node (SAN) is the natural pacemaker of the heart that is composed of pacemaker cells [1]
embryoid bodies (EBs) were observed at d6 + 1, d6 + 5, and d6 + 10 through a microscope, and the morphology results confirmed that embryonic stem cells (ESCs) differentiated into pacemaker‐like cells (Fig. 1B)
Cell morphology was observed through microscopic and the results showed overexpression of RCPCD inhibited the differentiation of ESCs to pacemaker‐like cells (Fig. 3B)
Summary
The sinoatrial node (SAN) is the natural pacemaker of the heart that is composed of pacemaker cells [1]. It is necessary to develop a biological pacemaker that overcomes the limitations of electronic pacemakers as well as ethical and immunogenic issues, which may be generated from pluripotent stem cells. Angela Scavone et al have explored the possibility of using CD166 expression for isolating SAN progenitors from differentiating embryonic stem cells (ESCs). They demonstrated that the isolation of cardiac precursors from non-teratogenic populations was able to mature and form a fully functional SAN-like tissue [6]. Stephanie et al described a transgene-independent method for the generation of SAN-like pacemaker cells from human pluripotent stem cells, with their function as a biological pacemaker in vitro and in mice [7]. Ionta et al [5] found that overexpression of Received: November 2020 Revised: June 2021 Accepted: 16 June 2021
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