Abstract 14396: Purification of Cardiomyocytes From Differentiating Pluripotent Stem Cells Using Molecular Beacons Targeting Cardiomyocyte-specific mRNA
Background: Pluripotent stem cells (PSCs) offer unprecedented hopes for myocardial repair; however, there are numerous technical hurdles for their clinical uses. While methods for generating cardiomyocytes (CMs) from PSCs have been reported, currently available methods produce only heterogeneous mixtures of CMs and non-CM cells may cause negative outcomes. Therefore, it has been highly required to develop strategies to purify homogeneous populations of CM for their further clinical applications. Here, we report an entirely novel system in which PSC-derived CMs can be purified by CM-specific molecular beacons (MBs). MBs are nano-scale probes that emit a fluorescence signal when hybridized to target mRNAs. We hypothesized that MBs targeting CM-specific mRNA can specifically bind to CMs and allow to sort MB positive CM population by FACS.
Method and Results: Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6 and -7 were generated. Among five MBs, a MB targeting myosin heavy chain 6 and -7 mRNA (MHC1-MB) identified up to 99% of HL-1 CMs, an immortalized mouse CM cell line, but less than 3% of four non-CM cell types in flow cytometry analysis, indicating that MHC1-MB is specific for identifying CMs. We delivered MHC1-MB into cardiomyogenically differentiated PSCs through nucleofection and found that the detection rate of CMs was similar to the percentages of cardiac troponin T (TNNT2) or cardiac troponin I (TNNI3)-positive CMs, supporting the specificity of MBs. Finally, ~97% of CMs from mouse and human PSC differentiating cultures FACS-sorted with MHC1-MBs expressed TNNT2- or TNNI3 determined by flow cytometry. These MB-based sorted cells maintained their CM characteristics verified by spontaneous beating, electrophysiologic studies, and expression of cardiac proteins by immunocytochemistry. When transplanted in a myocardial infarction model, while unpurified CMs derived from mESCs developed tumors, MB-based purified CMs improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors.
Conclusion: We developed a novel CM selection system that allows production of highly purified CMs. These purified CMs and this system can be valuable for cell therapy and drug discovery.
- Cardiac regeneration
- Regenerative medicine stem cells
- Stem cell biology
- Stem cell therapy
- New technology
- © 2013 by American Heart Association, Inc.