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Genetic isolation of stem cell-derived pacemaker-nodal cardiac myocytes

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Abstract

Dysfunction of the cardiac pacemaker tissues due to genetic defects, acquired diseases, or aging results in arrhythmias. When arrhythmias occur, artificial pacemaker implants are used for treatment. However, the numerous limitations of electronic implants have prompted studies of biological pacemakers that can integrate into the myocardium providing a permanent cure. Embryonic stem (ES) cells cultured as three-dimensional (3D) spheroid aggregates termed embryoid bodies possess the ability to generate all cardiac myocyte subtypes. Here, we report the use of a SHOX2 promoter and a Cx30.2 enhancer to genetically identify and isolate ES cell-derived sinoatrial node (SAN) and atrioventricular node (AVN) cells, respectively. The ES cell-derived Shox2 and Cx30.2 cardiac myocytes exhibit a spider cell morphology and high intracellular calcium loading characteristic of pacemaker-nodal myocytes. These cells express abundant levels of pacemaker genes such as endogenous HCN4, Cx45, Cx30.2, Tbx2, and Tbx3. These cells were passaged, frozen, and thawed multiple times while maintaining their pacemaker-nodal phenotype. When cultured as 3D aggregates in an attempt to create a critical mass that simulates in vivo architecture, these cell lines exhibited an increase in the expression level of key regulators of cardiovascular development, such as GATA4 and GATA6 transcription factors. In addition, the aggregate culture system resulted in an increase in the expression level of several ion channels that play a major role in the spontaneous diastolic depolarization characteristic of pacemaker cells. We have isolated pure populations of SAN and AVN cells that will be useful tools for generating biological pacemakers.

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Acknowledgments

We thank Dr. Gudrun Rappold, University of Heidelberg, Germany for the SHOX2 promoter; Dr. Eric Olson, UT Southwestern for the Cx30.2 enhancer, and Dr. YiPing Chen, Tulane University for CJ-7 (wild type) and Shox2lacZ/+ ES cells.

Author information

Correspondence to William C. Claycomb.

Electronic supplementary material

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Supplementary material 3 (AVI 13080 kb). Supplemental online video 1 A representative contracting Shox2lacZ/+ EB at day 14 of differentiation. Fluorescent image of this representative EB is shown in Fig. 1A delineating the area of Shox2 expression (green). The movie was acquired following live fluorescein digalactoside staining. The contracting region is directly adjacent to the Shox2 cell cluster located at the bottom left of this video

Supplementary material 4 (AVI 13080 kb). Supplemental online video 2 A representative contracting Cx30.2-RFP EB at day 14 of differentiation. Fluorescent image of this representative EB is shown in Fig. 1B delineating the area of Cx30.2 expression (red). The contracting region is located at the center of this video

Supplementary material 1 (TIFF 2102 kb). Supplemental Fig. 1 Fold change in gene expression in 3D aggregates versus 2D monolayer cultures. (A) A histogram plot of the fold change in gene expression in Shox2 cells passage 5 cultured as 3D aggregates compared to Shox2 cells passage 5 cultured as 2D monolayers. (B) A histogram plot of the fold change in gene expression in Cx30.2 cells passage 7 cultured as 3D aggregates compared to Cx30.2 cells passage 7 cultured as 2D monolayers. All cultures were allowed to grow for 10 days before RNA was collected. Please note the difference in the Y-axis in (A) and (B)

Supplementary material 2 (DOC 47 kb)

Supplementary material 3 (AVI 13080 kb). Supplemental online video 1 A representative contracting Shox2lacZ/+ EB at day 14 of differentiation. Fluorescent image of this representative EB is shown in Fig. 1A delineating the area of Shox2 expression (green). The movie was acquired following live fluorescein digalactoside staining. The contracting region is directly adjacent to the Shox2 cell cluster located at the bottom left of this video

Supplementary material 4 (AVI 13080 kb). Supplemental online video 2 A representative contracting Cx30.2-RFP EB at day 14 of differentiation. Fluorescent image of this representative EB is shown in Fig. 1B delineating the area of Cx30.2 expression (red). The contracting region is located at the center of this video

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Hashem, S.I., Claycomb, W.C. Genetic isolation of stem cell-derived pacemaker-nodal cardiac myocytes. Mol Cell Biochem 383, 161–171 (2013) doi:10.1007/s11010-013-1764-x

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Keywords

  • Atrioventricular
  • Embryoid body
  • Embryonic stem cell
  • Sinoatrial
  • Pacemaker