Molecular beacon-based detection and isolation of working-type cardiomyocytes derived from human pluripotent stem cells.
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) provide a potential source of cells to repair injured ventricular myocardium. CM differentiation cultures contain non-cardiac cells and CMs of both nodal and working subtypes. Direct application of such cultures in clinical studies could induce arrhythmias; thus, further purification of working-type CMs from heterogeneous cultures is desirable. Here, we designed 10 molecular beacons (MBs) targeting NPPA mRNA, a marker associated with working-type CMs and highly up-regulated during differentiation. We examined these MBs by solution assays and established their specificity using NPPA-overexpressing CHO cells as well as hPSC-CMs. We selected one MB for subsequent CM subtype isolation using fluorescence-activated cell sorting because the signal-to-background ratio was the highest for this MB in solution assays and a linear correlation was observed between MB signals and the CM purity in differentiation cultures. Compared with cells with low MB signals, cells positively selected based on MB signal had higher expression levels of genes associated with working-type CMs and lower expression levels of genes associated with nodal-type CMs. Therefore, the MB-based method is capable of separating working-type CMs from nodal-type CMs with high specificity and throughput, potentially providing working-type CMs for biomedical applications.
Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA; Children's Healthcare of Atlanta, Atlanta, GA 30322, USA.
Rajneesh
R
Jha
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Brian
B
Wile
Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA; Children's Healthcare of Atlanta, Atlanta, GA 30322, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Qingling
Q
Wu
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Aaron H
AH
Morris
Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA; Children's Healthcare of Atlanta, Atlanta, GA 30322, USA.
Kevin O
KO
Maher
Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA; Children's Healthcare of Atlanta, Atlanta, GA 30322, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Mary B
MB
Wagner
Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA; Children's Healthcare of Atlanta, Atlanta, GA 30322, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA. Electronic address: gang.bao@bme.gatech.edu.
Gang
G
Bao
Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA; Children's Healthcare of Atlanta, Atlanta, GA 30322, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA. Electronic address: chunhui.xu@emory.edu.
Chunhui
C
Xu
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D002454:Cell Differentiation / Q000235:genetics; D002469:Cell Separation / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D015335:Molecular Probes / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology*; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
D015335:Molecular Probes; D012333:RNA, Messenger; D009320:Atrial Natriuretic Factor
Cardiomyocyte; Flow cytometry; Gene expression; Molecular imaging; Stem cell