Lipofectamine® 3000: A new transfection reagent for iPSC generation and stem cell genomic engineering

Stem cells, specifically induced pluripotent stem cells (iPSCs), hold immense promise for the future of regenerative medicine and personalized therapeutics. However, the lack of advanced technologies has been hindering the current pace of research and discovery. One of the greatest challenges lies in the manipulation of stem cells. Lipofectamine® 3000, a new transfection reagent, has been developed for nucleic acid delivery to enable the use of new technologies for stem cell applications. In the present study, we first demonstrated that Lipofectamine® 3000 can be used to efficiently deliver the EPi5™ episomal reprogramming vectors to BJ skin fibroblasts for the generation of iPSCs. This method allows researchers to perform efficient in-situ reprogramming at lower cost, providing a great alternative over electroporation techniques typically used for iPSC generation. Furthermore, it was discovered that Lipofectamine® 3000 can achieve optimal transfection efficiency of various sizes of plasmid DNA and low toxicity in both embryonic stem cells (ESCs) and iPSCs which have been traditionally proven to be hard to transfect. More importantly, manipulation of stem cells can be achieved utilizing TALs and CRISPRs for genome engineering purposes. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) allow for editing and engineering of DNA at specific loci. However, the effectiveness of these tools depends on efficient delivery and the intrinsic properties of the locus of interest. Lipofectamine® 3000 demonstrated improved delivery of CRISPRs into iPSCs for targeted genome engineering. Taken together these advancements in delivery greatly improve downstream workflows, enable easier stem cell manipulation, and enhance site-specific insertion or deletion of transgenes for the generation of knock-in or knock-out cell models and transgenic small animal models.