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Canonical Wnt signaling promotes pacemaker cell specification of cardiac mesodermal cells derived from mouse and human embryonic stem cells.

ABSTRACT: Cardiac differentiation of embryonic stem cells (ESCs) can give rise to de novo chamber cardiomyocytes and nodal pacemaker cells. Compared with our understanding of direct differentiation toward atrial and ventricular myocytes, the mechanisms for nodal pacemaker cell commitment are not well understood. Taking a cue from the prominence of canonical Wnt signaling during cardiac pacemaker tissue development in chick embryos, we asked if modulations of Wnt signaling influence cardiac progenitors to bifurcate to either chamber cardiomyocytes or pacemaker cells. Omitting an exogenous Wnt inhibitor, which is routinely added to maximize cardiac myocyte yield during differentiation of mouse and human ESCs, led to increased yield of spontaneously beating cardiomyocytes with action potential properties similar to those of native sinoatrial node pacemaker cells. The pacemaker phenotype was accompanied by enhanced expression of genes and gene products that mark nodal pacemaker cells such as Hcn4, Tbx18, Tbx3, and Shox2. Addition of exogenous Wnt3a ligand, which activates canonical Wnt/?-catenin signaling, increased the yield of pacemaker-like myocytes while reducing cTNT-positive pan-cardiac differentiation. Conversely, addition of inhibitors of Wnt/?-catenin signaling led to increased chamber myocyte lineage development at the expense of pacemaker cell specification. The positive impact of canonical Wnt signaling on nodal pacemaker cell differentiation was evidenced in direct differentiation of two human ESC lines and human induced pluripotent stem cells. Our data identify the Wnt/?-catenin pathway as a critical determinant of cardiac myocyte subtype commitment during ESC differentiation: endogenous Wnt signaling favors the pacemaker lineage, whereas its suppression promotes the chamber cardiomyocyte lineage.

SUBMITTER: Liang W

PROVIDER: S-EPMC7932048 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Canonical Wnt signaling promotes pacemaker cell specification of cardiac mesodermal cells derived from mouse and human embryonic stem cells.

Liang Wenbin W Han Pengcheng P Kim Elizabeth H EH Mak Jordan J Zhang Rui R Torrente Angelo G AG Goldhaber Joshua I JI Marbán Eduardo E Cho Hee Cheol HC

Stem cells (Dayton, Ohio) 20191230 3

Cardiac differentiation of embryonic stem cells (ESCs) can give rise to de novo chamber cardiomyocytes and nodal pacemaker cells. Compared with our understanding of direct differentiation toward atrial and ventricular myocytes, the mechanisms for nodal pacemaker cell commitment are not well understood. Taking a cue from the prominence of canonical Wnt signaling during cardiac pacemaker tissue development in chick embryos, we asked if modulations of Wnt signaling influence cardiac progenitors to ...[more]

PMID: 31648393

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Project description: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.

Dataset Information

Canonical Wnt signaling promotes pacemaker cell specification of cardiac mesodermal cells derived from mouse and human embryonic stem cells.

Publications

Canonical Wnt signaling promotes pacemaker cell specification of cardiac mesodermal cells derived from mouse and human embryonic stem cells.

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