Abstract 18585: Purification of Cardiomyocytes from Differentiating Human Pluripotent Stem Cells Using Molecular Beacons Targeting mRNA of a Cardiomyocyte-specific Gene

Abstract

Background: While various methods for generating cardiomyocytes (CMs) from pluripotent stem cells (PSCs) including both embryonic stem cells (ESCs) and recently identified induced pluripotent stem cells (iPSCs) have been reported, all available methods are only able to produce heterogeneous populations of CMs mixed with non-CM cells. Therefore, strategies to enrich pure CMs for scientific and clinical applications are urgently required. In the present study, we developed an entirely novel system in which PSC-derived CMs can be purified by cardiac-specific molecular beacons (MBs). MBs are 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 CMs by FACS. Method and Results: Five MBs targeting distinct sites on mRNA of either cardiac troponin T or myosin heavy chain 6 and -7 were designed and characterized in HL-1 CMs, an immortalized mouse CM cell line and several non-CM cell types such as mouse smooth muscle cells and fibroblasts. Among five MBs in flow cytometry analysis, MHC1-MB identified up to 98% of HL-1 CMs but less than 5% of non-CM cell types, indicating that MHC1-MB is specific for identifying CMs. Subsequently, MHC1-MBs delivered into heterogeneous mouse and human PSC differentiation cultures identified 41 to 49% of the MB + cells. Interestingly, the rates of MB + cells were highly correlated with the rate of cTnT/I + cells, supporting their specificity for detecting PSC-derived CMs. Finally, we determined whether cell sorting with MHC1-MBs can enrich CMs from mouse and human PSC differentiating cultures and found that ∼97% of MB-based sorted CMs expressed cTnT/I. Importantly, these MB-based sorted cells continuously maintained their CM characteristics, which were verified by observation of spontaneous beating and significant expression of cardiac specific genes and proteins in qRT-PCR analysis and immunocytochemistry. Furthermore, Ca 2+ transient analysis confirmed that these enriched cells displayed functional CM characteristic. Conclusion: Using cardiac-specific MBs, we were able to obtain highly purified CMs. These purified CMs and this system can be highly useful for cell therapy as well as drug discovery.