Effects of Gene Delivery Approaches on Differentiation Potential and Gene Function of Mesenchymal Stem Cells
- 8 June 2021
- journal article
- research article
- Published by Institute of Electrical and Electronics Engineers (IEEE) in IEEE Transactions on Biomedical Engineering
- Vol. 69 (1), 83-95
- https://doi.org/10.1109/tbme.2021.3087129
Abstract
Introduction of a gene to mesenchymal stem cells (MSCs) is a well-known strategy to purposely manipulate the cell fate and further enhance therapeutic performance in cell-based therapy. Viral and chemical approaches for gene delivery interfere with differentiation potential. Although microinjection as a physical delivery method is commonly used for transfection, its influence on MSC cell fate is not fully understood. The current study aimed to evaluate the effects of four nonviral gene delivery methods on stem cell multi-potency. The four delivery methods are robotic microinjection, polyethylenimine (PEI), cationic liposome (cLipo), and calcium phosphate nanoparticles (CaP). Among the four methods, microinjection has exhibited the highest transfection efficiency of ~60%, while the three others showed lower efficiency of 10-25%. Robotic microinjection preserved fibroblast-like cell morphology, stress fibre intactness, and mature focal adhesion complex, while PEI caused severe cytotoxicity. No marked differentiation bias was observed after microinjection and cLipo treatment. By contrast, CaP-treated MSCs exhibited excessive osteogenesis, while PEI-treated MSCs showed excessive adipogenesis. Robotic microinjection system was used to inject the CRISPR/Cas9-encoding plasmid to knock out PPAR gene in MSCs, and the robotic microinjection did not interfere with PPAR function in differentiation commitment. Meanwhile, the bias in osteo-adipogenic differentiation exhibited in CaP and PEI-treated MSCs after PPAR knockout via chemical carriers. Our results indicate that gene delivery vehicles variously disturb MSCs differentiation and interfere with exogenous gene function. Our findings further suggest that robotic microinjection offers a promise of generating genetically modified MSCs without disrupting stem cell multi-potency and therapeutic gene function.Keywords
Funding Information
- Research Grant Council of Hong Kong SAR (11209917, C1134-20G)
- National Natural Science Foundation of China (U20A20194)
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