Molecular beacon-based detection and isolation of working-type cardiomyocytes derived from human pluripotent stem cells

• Type of Material: Article
• Authors: Rajneesh Jha, Emory University; Brian Wile, Georgia Institute of Technology; Qingling Wu, Emory University; Aaron H. Morris, Georgia Institute of Technology; Kevin Maher, Emory University; Mary Wagner, Emory University; Gang Bao, Emory University; Chunhui Xu, Emory University
• Language: English
• Date: 2015-05-01
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2015 Elsevier Ltd.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1016/j.biomaterials.2015.01.043
• Title of Journal or Parent Work: Biomaterials
• ISSN: 0142-9612
• Volume: 50
• Issue: 1
• Start Page: 176
• End Page: 185
• Grant/Funding Information: This study was supported in part by an NIH-NHLBI grant R21HL118454 (C.X. and G.B.) and in part by the NIH-NHLBI Program of Excellence in Nanotechnology award under Contract No. HHSN268201000043C (G.B. and C.X.).
• Keywords: LIVING CELLS; MYOCYTES; Materials Science, Biomaterials; Technology; Materials Science; Molecular imaging; Gene expression; HEART; DIFFERENTIATION; Engineering; Cardiomyocyte; Stem cell; Engineering, Biomedical; MESSENGER-RNA; Flow cytometry
• Research Categories: Engineering, Biomedical; Health Sciences, General; Engineering, Materials Science

Abstract

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.