title: Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA.
abstract: BACKGROUND
  Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs.
  
  
  RESULTS
  Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors.
  
  
  CONCLUSIONS
  We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.
authors.affiliation: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA.
authors.forename: Kiwon
authors.initials: K
authors.lastname: Ban
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA.
authors.forename: Brian
authors.initials: B
authors.lastname: Wile
authors.affiliation: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA.
authors.forename: Sangsung
authors.initials: S
authors.lastname: Kim
authors.affiliation: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA.|Division of Cardiology, Department of Internal Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea.
authors.forename: Hun-Jun
authors.initials: HJ
authors.lastname: Park
authors.affiliation: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA.
authors.forename: Jaemin
authors.initials: J
authors.lastname: Byun
authors.affiliation: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA.
authors.forename: Kyu-Won
authors.initials: KW
authors.lastname: Cho
authors.affiliation: Department of Pediatrics, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA.
authors.forename: Talib
authors.initials: T
authors.lastname: Saafir
authors.affiliation: Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA.
authors.forename: Ming-Ke
authors.initials: MK
authors.lastname: Song
authors.affiliation: Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA.
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.
authors.forename: Mary
authors.initials: M
authors.lastname: Wagner
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA.
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
authors.affiliation: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA.
authors.forename: Young-Sup
authors.initials: YS
authors.lastname: Yoon
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000818:Animals; D015415:Biomarkers; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D017690:Cell Transplantation / Q000379:methods*; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D051379:Mice; D009203:Myocardial Infarction / Q000628:therapy*; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000502:physiology; D018995:Myosin Heavy Chains / Q000235:genetics; D036103:Nanotechnology; D009690:Nucleic Acid Conformation; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000502:physiology; D015347:RNA Probes / Q000737:chemistry / Q000302:isolation & purification; D012333:RNA, Messenger / Q000737:chemistry / Q000302:isolation & purification*; D019210:Troponin I / Q000235:genetics; D020107:Troponin T / Q000235:genetics
chemical_list: D015415:Biomarkers; D015347:RNA Probes; D012333:RNA, Messenger; D019210:Troponin I; D020107:Troponin T; D018995:Myosin Heavy Chains
keywords: myocytes, cardiac; oligonucleotide probes; pluripotent stem cells; regeneration