Comparative expression analysis of Shox2-deficient embryonic stem cell-derived sinoatrial node-like cells

Sandra Hoffmann; Stefanie Schmitteckert; Anne Griesbeck; Hannes Preiss; Simon Sumer; Alexandra Rolletschek; Martin Granzow; Volker Eckstein; Beate Niesler; Gudrun A Rappold

doi:10.1016/j.scr.2017.03.018

Comparative expression analysis of Shox2-deficient embryonic stem cell-derived sinoatrial node-like cells

Stem Cell Res. 2017 May:21:51-57. doi: 10.1016/j.scr.2017.03.018. Epub 2017 Mar 29.

Authors

Affiliations

¹ Department of Human Molecular Genetics, Institute of Human Genetics, University Heidelberg, Germany; DZHK, German Centre for Cardiovascular Research, Partner site Heidelberg/Mannheim, Germany.
² Department of Human Molecular Genetics, Institute of Human Genetics, University Heidelberg, Germany.
³ Institute for Biological Interfaces, Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany.
⁴ Department of Human Genetics, Institute of Human Genetics, University Heidelberg, Germany.
⁵ FACS Core Facility, Department of Medicine V, University Hospital Heidelberg, Germany.
⁶ Department of Human Molecular Genetics, Institute of Human Genetics, University Heidelberg, Germany; DZHK, German Centre for Cardiovascular Research, Partner site Heidelberg/Mannheim, Germany; nCounter Core Facility, Department of Human Molecular Genetics, Institute of Human Genetics, University Heidelberg, Germany.
⁷ Department of Human Molecular Genetics, Institute of Human Genetics, University Heidelberg, Germany; DZHK, German Centre for Cardiovascular Research, Partner site Heidelberg/Mannheim, Germany. Electronic address: Gudrun.Rappold@med.uni-heidelberg.de.

PMID: 28390247
DOI: 10.1016/j.scr.2017.03.018

Abstract

The homeodomain transcription factor Shox2 controls the development and function of the native cardiac pacemaker, the sinoatrial node (SAN). Moreover, SHOX2 mutations have been associated with cardiac arrhythmias in humans. For detailed examination of Shox2-dependent developmental mechanisms in SAN cells, we established a murine embryonic stem cell (ESC)-based model using Shox2 as a molecular tool. Shox2^+/+ and Shox2^-/- ESC clones were isolated and differentiated according to five different protocols in order to evaluate the most efficient enrichment of SAN-like cells. Expression analysis of cell subtype-specific marker genes revealed most efficient enrichment after CD166-based cell sorting. Comparative cardiac expression profiles of Shox2^+/+ and Shox2^-/- ESCs were examined by nCounter technology. Among other genes, we identified Nppb as a novel putative Shox2 target during differentiation in ESCs. Differential expression of Nppb could be confirmed in heart tissue of Shox2^-/- embryos. Taken together, we established an ESC-based cardiac differentiation model and successfully purified Shox2^+/+ and Shox2^-/- SAN-like cells. This now provides an excellent basis for the investigation of molecular mechanisms under physiological and pathophysiological conditions for evaluating novel therapeutic approaches.

Keywords: Differentiation model; Mouse embryonic stem cells; Shox2; Sinoatrial node-like cells; Transgenic cells.

Publication types

Comparative Study
Research Support, Non-U.S. Gov't

MeSH terms

Activated-Leukocyte Cell Adhesion Molecule / metabolism
Animals
Cell Separation
Female
Gene Expression Regulation, Developmental*
Homeodomain Proteins / genetics
Homeodomain Proteins / metabolism*
Mice
Mice, Inbred C57BL
Mouse Embryonic Stem Cells / cytology*
Mouse Embryonic Stem Cells / metabolism*
Myocytes, Cardiac / cytology
Myocytes, Cardiac / metabolism
Sinoatrial Node / cytology*

Substances

Activated-Leukocyte Cell Adhesion Molecule
Homeodomain Proteins
Shox2 protein, mouse