Dataset Information

Transfection of hPSC-Cardiomyocytes Using Viafect™ Transfection Reagent.

ABSTRACT: Twenty years since their first derivation, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have shown promise in disease modelling research, while their potential for cardiac repair is being investigated. However, low transfection efficiency is a barrier to wider realisation of the potential this model system has to offer. We endeavoured to produce a protocol for improved transfection of hPSC-CMs using the ViafectTM reagent by Promega. Through optimisation of four essential parameters: (i) serum supplementation, (ii) time between replating and transfection, (iii) reagent to DNA ratio and (iv) cell density, we were able to successfully transfect hPSC-CMs to ~95% efficiencies. Transfected hPSC-CMs retained high purity and structural integrity despite a mild reduction in viability, and preserved compatibility with phenotyping assays of hypertrophy. This protocol greatly adds value to the field by overcoming limited transfection efficiencies of hPSC-CMs in a simple and quick approach that ensures sustained expression of transfected genes for at least 14 days, opening new opportunities in mechanistic discovery for cardiac-related diseases.

SUBMITTER: Bodbin SE

PROVIDER: S-EPMC7564709 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Transfection of hPSC-Cardiomyocytes Using Viafect™ Transfection Reagent.

Bodbin Sara E SE Denning Chris C Mosqueira Diogo D

Methods and protocols 20200809 3

Twenty years since their first derivation, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have shown promise in disease modelling research, while their potential for cardiac repair is being investigated. However, low transfection efficiency is a barrier to wider realisation of the potential this model system has to offer. We endeavoured to produce a protocol for improved transfection of hPSC-CMs using the Viafect<sup>TM</sup> reagent by Promega. Through optimisation of four essent ...[more]

PMID: 32784848

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Project description:BackgroundDeoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown.ResultsIn the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment. In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups. In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis.ConclusionTreatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10. The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated.

Dataset Information

Transfection of hPSC-Cardiomyocytes Using Viafect™ Transfection Reagent.

Publications

Transfection of hPSC-Cardiomyocytes Using Viafect™ Transfection Reagent.

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