Non-viral vector based gene transfection with human induced pluripotent stem cells derived cardiomyocytes

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.

Fluorescent indicators for continuous and lineage‐specific reporting of cell‐cycle phases in human pluripotent stem cells

“…As expected, CCND2 overexpression significantly increased the Clover expression in the TNNT2‐FUCCI hPSC‐CMs (Figure 5a,b). Consistent with a previous report (Tan et al, 2019), transfection efficiency of ~70% was achieved in wild‐type H9 CMs with an eGFP plasmid (Figure S5F), and the propidium iodide (PI) staining showed the CM cycle profile changed significantly with CCND2 transfection (Figure S5G). In summary, our TNNT2‐FUCCI reporter works in a lineage‐specific manner and allows for the sensitive spatiotemporal detection of cycling CMs during hPSC differentiation, enabling the identification of novel regulators for heart regeneration.…”

“…When cells were more than 80% confluent, drug selection was performed with 1 μg/ml puromycin (Puro) for approximately 1 week, and individual clones were picked using a microscope inside a tissue culture hood and expanded for 2–5 days in each well of a 96‐well plate pre‐coated with Matrigel, followed by a PCR genotyping. The plasmid transfection of hPSC‐CMs was performed according to a published method (Tan et al, 2019). Briefly, 1 μg cyclin D2 (CCND2) plasmid (Addgene; #8958) was added to 100 μl DMEM/F12 medium containing 1 μl Lipofectamine Stem Transfection Reagent, mixed well, and incubated for 10 min.…”

Transfection of hPSC-Cardiomyocytes Using Viafect™ Transfection Reagent

“…Nevertheless, Viafect transfection resulted in ~66% cardiomyocyte viability. While a previous method using liposomes [ 20 ] achieved higher cardiomyocyte viabilities upon transfection (80.8–92.8%), the transfection efficiency reported was much lower (~56%) than when using Viafect (~95%). Importantly, the cell loss upon transfection did not prevent further phenotypic assays, such as the identification of hypertrophy markers (such as Brain Natriuretic Peptide, BNP) ( Supplementary Figure S2d ).…”

“…While high efficiency transfection (>80%) of undifferentiated hiPSCs was achieved long ago [ 34 ], the same is not true of hiPSC-CMs, which have proved refractory to simple approaches for gene transfer. For example, other chemical and physical methods using Lipofectamine and magnetic nanoparticles achieved a maximum TE of up to ~56% and ~20%, respectively, in hiPSC-CMs [ 19 , 20 ]. Viral gene transfer methods have shown more success, with adeno associated virus (AAV), adenovirus and lentivirus achieving 90–95% in hiPSC-CMs [ 35 ], but often causing cell toxicity, and entailing technical complexity of viral engineering and/or biosafety risks.…”

“…This is a critical requirement, as a PubMed search reflects an exponential increase in publications on the subject of human induced pluripotent stem cells (hiPSC) and also hiPSC-CMs, but displays a severe deficiency in the number of papers on the transfection of hiPSC-CMs ( Figure 1 ). In comparison to the 2133 publications on the topic of hiPSC-CMs since 2000, there are only 85 publications referring to transfection with even best-in-class physical or chemical methods achieving between 20% and 56%, respectively [ 19 , 20 ]. The importance of obtaining a high transfection efficiency (TE) for enhanced mechanistic understandings of disease compels us to explore better transfection methods for hPSC-CMs.…”