Torben Helledie, Victor Nurcombe, Simon M Cool
Stem Cells and Development
Adult human mesenchymal stem cells (hMSCs) are able to differentiate into a range of specific cell types in vitro and in vivo, and thus hold tremendous potential for use in regenerative medicine. Despite this promise, deficient understanding of the mechanisms that regulate their differentiation has precluded their widespread use. Genetic manipulation of hMSCs by introduction of transgenes is an indispensable tool for gaining insight into these mechanisms. Like most primary cultures, hMSCs are difficult to transfect with conventional techniques, and although some viral transduction techniques are highly efficient, the protocols require extensive optimization and contain significant health risks. We were generally unable to achieve high transfection efficiencies with lipofection-based reagents that we found, in contrast to electroporation, adversely affected hMSC proliferation and differentiation. Here we report a simple and reliable electroporation protocol that results in transfection efficiencies up to 90% that are comparable to most viral methods while maintaining hMSC stemness. Most importantly, our protocol does not rely on a specific electroporator with preset programs and unique buffers, and is thus much simpler, cheaper, and easier to optimize. Furthermore, we show sustained transgene expression lasting several weeks that was useful for assessing the effects on hMSC function and in transient expression gene therapy.
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