Abstract 154: Generation of Purified Cardiomyocytes from Pluripotent Stem Cells Using Molecular Beacons

Abstract

Background: While various methods for generating cardiomyocytes (CMs) from pluripotent stem cells (PSCs) including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been reported, all available methods are only allowed to produce heterogeneous population of CM mixed with non-CM cells. Therefore, strategies to enrich pure CMs for scientific and clinical applications have been highly required. Hence, we developed a novel system in which CMs can be purified by cardiac specific molecular beacons (MBs). MBs are dual-labeled antisense nano-scale probes that emit a fluorescence signal when hybridized to target mRNAs. We hypothesized that MBs targeted to CM specific mRNAs can identify CMs and allow isolation of purified CMs by fluorescence-activated cell sorting (FACS). Methods and results: Five MBs targeting distinct sites on either cardiac troponin T (cTNT) or α/β myosin heavy chain (α/βMHC) were designed and characterized in various cell types. To find the optimal MB that can selectively identifying CMs, each MB was delivered into HL-1 CMs, an immortalized mouse CM cell line, smooth muscle cells, endothelial cells, mouse ESCs and fibroblasts and its specificity was determined by flow cytometry. As a result, two MBs identified MB+ cells up to 98% from HL-1 CMs but lower than 10% of the non-CM cells suggesting these MBs are CM specific. Subsequently, the selected MBs were delivered into both mouse and human PSCs derived CMs and 41 to 49% of the cells were identified as an MB+ population. Interestingly, the rate of MB+ cells was similar to CM quantification determined by cTNT intracellular flow cytometry. Finally, we determined whether cell sorting with cardiac-specific MBs can enrich CMs from the heterogeneous mouse and human PSC cultures and found that ∼97% of MB-based sorted CMs expressed cTNT. These enriched cells were further cultured and their CM identity was verified by immunocytochemistry and qRT-PCR analysis. Ca2+ transient analysis further confirmed that these purified CMs displayed functional CM characteristics Conclusion: Using cardiac specific MBs, we were able to obtain highly purified CMs. These purified CMs and the system can be highly useful for clinical applications as well as drug discovery.