Professor University of Waterloo Waterloo, Ontario, Canada
Abstract: Transfection and transdifferentiation strategies capable of producing clinically applicable products could have a significant impact on tissue engineering. Non-viral vectors have the potential to reduce immunogenicity and have added benefits, such as ease of production, but come at the cost of efficiency. Substrate topographies are known to be able to modulate cell behaviors including efficiency of non-viral transfection and transdifferentiation. We hypothesized that nanotopographical substrates would be able to increase non-viral transfection efficiency and/or enhance non-viral neuronal transdifferentiation of human mesenchymal stem cells (hMSCs). We screened the effects of 16 different topographies, with different geometries. We found that five of the topographical patterns (nano- and micro-gratings, micro-lenses, micro-holes, and nano-pillars) increased the efficiency of Lipofectamine-mediated transfection. Convex and concave micro-lenses showed opposite effects on transfection efficiency. Neuronal transdifferentiation of hMSCs was induced by non-viral delivery of BAM (Brn2, Ascl1, and Myt1l) transcription factors, using poly(N,N-cystaminebisacrylamide-4-amino-1-butanol) (pABOL) polyplex. The transdifferentiation study showed weak immunofluorescent staining for the neuronal lineage marker, microtubule-associated protein 2 (MAP2) in all samples. We found that the pABOL transfection reagent used during the transdifferentiation procedure was more toxic to hMSCs than to mouse embryonic fibroblasts or COS-7 cells. Together, our observations suggest that optimization of the specific non-viral transfection reagent used as well as factors affecting cell confluence, such as seeding density or length of transfection phase, might enable the use of nanotopography to enhance non-viral neuronal transdifferentiation of hMSCs for clinically applicable products.
Funding Source: Natural Sciences and Engineering Research Council (NSERC) Discovery (RGPIN-2016-04043, RGPIN-2021-03200)
