title: FACS-based isolation, propagation and characterization of mouse embryonic cardiomyocytes based on VCAM-1 surface marker expression.
abstract: Purification of cardiomyocytes from the embryonic mouse heart, embryonic stem (ES) or induced pluripotent stem cells (iPS) is a challenging task and will require specific isolation procedures. Lately the significance of surface markers for the isolation of cardiac cell populations with fluorescence activated cell sorting (FACS) has been acknowledged, and the hunt for cardiac specific markers has intensified. As cardiomyocytes have traditionally been characterized by their expression of specific transcription factors and structural proteins, and not by specific surface markers, this constitutes a significant bottleneck. Lately, Flk-1, c-kit and the cellular prion protein have been reported to specify cardiac progenitors, however, no surface markers have so far been reported to specify a committed cardiomyocyte. Herein show for the first time, that embryonic cardiomyocytes can be isolated with 98% purity, based on their expression of vascular cell adhesion molecule-1 (VCAM-1). The FACS-isolated cells express phenotypic markers for embryonic committed cardiomyocytes but not cardiac progenitors. An important aspect of FACS is to provide viable cells with retention of functionality. We show that VCAM-1 positive cardiomyocytes can be isolated with 95% viability suitable for in vitro culture, functional assays or expression analysis. In patch-clamp experiments we provide evidence of functionally intact cardiomyocytes of both atrial and ventricular subtypes. This work establishes that cardiomyocytes can be isolated with a high degree of purity and viability through FACS, based on specific surface marker expression as has been done in the hematopoietic field for decades. Our FACS protocol represents a significant advance in which purified populations of cardiomyocytes may be isolated and utilized for downstream applications, such as purification of ES-cell derived cardiomyocytes.
authors.affiliation: Lund Strategic Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden.
authors.forename: Annica
authors.initials: A
authors.lastname: Pontén
authors.affiliation: Lund Strategic Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden.
authors.forename: Stuart
authors.initials: S
authors.lastname: Walsh
authors.affiliation: Institute of Physiology I, Life & Brain Center, University of Bonn, Bonn, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Malan
authors.affiliation: Lund Strategic Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden.
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Xian
authors.affiliation: Lund Strategic Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden.
authors.forename: Susanne
authors.initials: S
authors.lastname: Schéele
authors.affiliation: Lund Strategic Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden.
authors.forename: Laura
authors.initials: L
authors.lastname: Tarnawski
authors.affiliation: Institute of Physiology I, Life & Brain Center, University of Bonn, Bonn, Germany.
authors.forename: Bernd K
authors.initials: BK
authors.lastname: Fleischmann
authors.affiliation: Lund Strategic Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden ; Department of Cardiology Scania University Hospital, Lund, Sweden.
authors.forename: Stefan
authors.initials: S
authors.lastname: Jovinge
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology; D005434:Flow Cytometry; D051379:Mice; D008810:Mice, Inbred C57BL; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D019010:Vascular Cell Adhesion Molecule-1