mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

Abstract

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

Optimization of the Linker Length of Mannose-Cholesterol Conjugates for Enhanced mRNA Delivery to Dendritic Cells by Liposomes

“…Taken the transfection efficiency and MFI of the GFP positive cells into consideration, the ratio of N/P of 5 was selected for future studies. The difference between the percentage of transfected cells and the MFI of transfected cells was consistent with previously reported results ( McLenachan et al, 2013 ; Avci-Adali et al, 2014 ; Lee et al, 2015 ; Li et al, 2017a ). Transfection using the same transfection reagent led to similar transfection efficiency but not the MFI ( Figures 4D,E ) with the increase of the incubation time, which was consistent with previously report ( Avci-Adali et al, 2014 ).…”

Opportunities and Challenges in the Delivery of mRNA-Based Vaccines

“…However, the outermost stratum corneum layer of the epidermis serves as a tight barrier to the absorption of topically administered drugs [97]. Heretofore, various approaches have been adopted to overcome this barrier, including physical (e.g., microporation [98], microneedles [99], and jet injection [100,101]), active (e.g., electroporation [102], iontophoresis [103], and sonophoresis [104]), and passive methods (e.g., nanoparticles [105] and liposomes [106]). Electroporation and sonoporation can transiently permeabilize the skin by means of electric pulses and low-frequency ultrasound, respectively, for efficient delivery of genes into the skin [107].…”

Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy

“…Therefore, for subsequent experiments, this copy number was used for double transfection (multifection) in order to yield maximal gene transfer. Double transduction/transfection is routinely used for many viral as well as non-viral genetic modification approaches such as lipid-mediated gene delivery and electroporation in order to enhance transfection efficiency and to prolong gene expression [37][38][39]. We previously showed that multifection of NSCs (cultured as 3-D neurospheres) resulted in higher transfection levels (~27%) compared to single transfection (~13%) with no effect on cell physiology and health [22].…”