Abstract 17986: Molecular Beacon-based Purification of Ventricular Cardiomyocytes From Differentiating Embryonic Stem Cells by Targeting Intracellular Mrna

Background: Ventricular cardiomyocytes (CMs) are an ideal cell type for cardiac cell therapy since they are the main cells generating cardiac forces. However, isolating them from differentiating pluripotent stem cells (PSCs) has been challenging due to the lack of specific surface markers. Here we show that ventricular CMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. MBs are dual-labeled oligonucleotide hairpin probes that emit a fluorescence signal when hybridized to target mRNAs, allowing isolation of specific target cells by fluorescence activated cell sorting (FACS) with high specificity and sensitivity. Methods and Results: We generated three different MBs (IRX4-1, -2, -3) designed to target specific regions of mRNAs of iroquois homeobox protein 4 (Irx4), a specific transcription factor for ventricular CMs. Among three IRX4 MBs, IRX4-2 MB demonstrated the highest sensitivity and specificity, thus IRX4-2 MB was selected to purify mESC-derived ventricular CMs. Subsequently, IRX4-2 MBs were delivered into cardiomyogenically differentiating mESC cultures and cells showing strong signals from IRX4-2 MBs were FACS-sorted. Flow cytometry demonstrated that 92~97% of IRX4-2 MB-positive cells expressed a marker for ventricular CMs myosin light chain 2 ventricular isoform (Myl2) as well as cardiac troponin 2 (Tnnt2). Importantly, higher than 98% of IRX4-2 MB-positive cells displayed ventricular CM-like action potentials during electrophysiological analyses. These IRX4-2 MB-based purified ventricular CMs continuously maintained their CM characteristics verified by synchronous beating, Ca2+ transient, and expression of ventricular CM-specific proteins. Conclusions: We established a novel MB-based cell sorting system targeting a transcription factor that is specific for ventricular CM to generate homogeneous and functional ventricular CMs. This is the first report to show the feasibility of isolating pure ventricular CMs without modifying host genes, and this platform will be useful for therapeutic applications, disease modeling, and drug discovery.