Abstract
Non-viral transfection of mammalian cardiomyocytes (CMs) is challenging. The current study aims to characterize and determine the non-viral vector based gene transfection efficiency with human induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (hiPSC-CMs). hiPSC-CMs differentiated from PCBC hiPSCs were used as a cell model to be transfected with plasmids carrying green fluorescence protein (pGFP) using polyethylenimine (PEI), including Transporter 5 Transfection Reagent (TR5) and PEI25, and liposome, including lipofectamine-2000 (Lipo2K), lipofectamine-3000 (Lipo3K), and Lipofectamine STEM (LipoSTEM). The gene transfection efficiency and cell viability were quantified by flow cytometry. We found that the highest gene transfection efficiency in hiPSC-CMs on day 14 of contraction can be achieved by LipoSTEM which was about 32.5 ± 6.7%. However, it also cuased poor cell viability (60.1 ± 4.5%). Furthermore, a prolonged culture of (transfection on day 23 of contraction) hiPSC-CMs not only improved gene transfection (54.5 ± 8.9%), but also enhanced cell viability (74 ± 4.9%) by LipoSTEM. Based on this optimized gene transfection condition, the highest gene transfection efficiency was 55.6 ± 7.8% or 34.1 ± 4%, respectively, for P1C1 or DP3 hiPSC line that was derived from healthy donor (P1C1) or patient with diabetes (DP3). The cell viability was 80.8 ± 5.2% or 92.9 ± 2.24%, respectively, for P1C1 or DP3. LipoSTEM is a better non-viral vector for gene transfection of hiPSC-CMs. The highest pGFP gene transfection efficiency can reach >50% for normal hiPSC-CMs or >30% for diabetic hiPSC-CMs.
Highlights
Cardiovascular disease is a major cause of mortality throughout the world
We found that Lipofectamine-STEM (LipoSTEM) based non-viral vector transfection achieved a higher gene transfection efficiency than PEI using plasmids carrying green fluorescence protein
The ex vivo delivery of gene into mammalian CMs is mainly successful through viral vectors, including adenovirus, adeno-associated virus, and lentivirus, which yield high gene transduction efficiencies[3,5,6,7,8,9]
Summary
Cardiovascular disease is a major cause of mortality throughout the world. In addition to pharmacologic drugs and device therapies, gene-based angiogenic therapies for treatment of heart failure are tested in hopes of being translated to the clinical setting[1]. Our previous study showed that polyethyleminine (PEI) or liposome based non-viral vector can efficiently transfect human skeletal myoblasts which transiently expressed VEGF protein for 14 days[10,11,12]. We aim to characterize non-viral vector based gene transfer with human induced pluripotent stem cells (hiPSCs) derived CMs (hiPSC-hiPSC-CMs). HiPSCs, which are reprogrammed from adult human somatic cells by defined transcription factors, have emerged as a better alternative for deriving autologous hiPSC-CMs. Using established differentiation protocols[13,14,15], a large amount of CMs can be generated from hiPSCs and are being tested as cell transfer therapy for cardiac repair in animal models[16]. We found that Lipofectamine-STEM (LipoSTEM) based non-viral vector transfection achieved a higher gene transfection efficiency than PEI using plasmids carrying green fluorescence protein (pGFP)
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