title: Non-genetic Purification of Ventricular Cardiomyocytes from Differentiating Embryonic Stem Cells through Molecular Beacons Targeting IRX-4.
abstract: Isolation of ventricular cardiomyocytes (vCMs) has been challenging due to the lack of specific surface markers. Here we show that vCMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. We designed MBs (IRX4 MBs) to target mRNA encoding Iroquois homeobox protein 4 (Irx4), a transcription factor specific for vCMs. To purify mESC vCMs, IRX4 MBs were delivered into cardiomyogenically differentiating mESCs, and IRX4 MBs-positive cells were FACS-sorted. We found that, of the cells isolated, ~98% displayed vCM-like action potentials by electrophysiological analyses. These MB-purified vCMs continuously maintained their CM characteristics as verified by spontaneous beating, Ca(2+) transient, and expression of vCM-specific proteins. Our study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes. The homogeneous and functional ventricular CMs generated via the MB-based method can be useful for disease investigation, drug discovery, and cell-based therapies.
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Kiwon
authors.initials: K
authors.lastname: Ban
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Wile
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Kyu-Won
authors.initials: KW
authors.lastname: Cho
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Sangsung
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Ming-Ke
authors.initials: MK
authors.lastname: Song
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Sang Yoon
authors.initials: SY
authors.lastname: Kim
authors.affiliation: Department of Pediatrics, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA 30322, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Singer
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA.
authors.forename: Anum
authors.initials: A
authors.lastname: Syed
authors.affiliation: Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Shan Ping
authors.initials: SP
authors.lastname: Yu
authors.affiliation: Department of Pediatrics, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA 30322, USA.
authors.forename: Mary
authors.initials: M
authors.lastname: Wagner
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA. Electronic address: gang.bao@rice.edu.
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, GA 30322, USA; Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 120-752, Korea. Electronic address: yyoon5@emory.edu.
authors.forename: Young-Sup
authors.initials: YS
authors.lastname: Yoon
mesh_terms: D000200:Action Potentials; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D005434:Flow Cytometry; D006352:Heart Ventricles / Q000166:cytology*; D018398:Homeodomain Proteins / Q000235:genetics*; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology*; D015345:Oligonucleotide Probes / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D018398:Homeodomain Proteins; C485534:Irx4 protein, mouse; D015345:Oligonucleotide Probes; D012333:RNA, Messenger
keywords: IRX4; cell selection; molecular beacons; pluripotent stem cell; ventricular cardiomyocyte