title: Molecular beacon-enabled purification of living cells by targeting cell type-specific mRNAs.
abstract: Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics, the MB-based method enables the isolation of a wide variety of cells. For example, the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design, validation and nucleofection into cells, we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol, we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with ∼ 97% purity, as confirmed by electrophysiology and immunocytochemistry. After designing MBs, their ordering and validation requires 2 weeks, and the isolation process requires 3 h.
authors.affiliation: 1] Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA. [2].
authors.forename: Brian M
authors.initials: BM
authors.lastname: Wile
authors.affiliation: 1] Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. [2].
authors.forename: Kiwon
authors.initials: K
authors.lastname: Ban
authors.affiliation: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.
authors.forename: Young-Sup
authors.initials: YS
authors.lastname: Yoon
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D004594:Electrophysiology / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D051379:Mice; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods*; D015335:Molecular Probes / Q000737:chemistry; D032383:Myocytes, Cardiac / Q000166:cytology; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D009928:Organ Specificity; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D015335:Molecular Probes; D009841:Oligonucleotides; D012333:RNA, Messenger