Different Transfection Methods for Human Bone and Dental Stem Cells

Adult bone-marrow stem cell (BMSC) and dental pulp derived stem cells (DPSC) present unique resources for tissue engineering strategies aimed at regenerating specific mineralized matrices. Hence, manipulation of these cell lines by introducing genes that control terminal differentiation is central to successful tissue regeneration. Objectives: Our aim was to compare and contrast the efficiencies of three different transfection methods that can be used for gene overexpression studies. Methods: Human BMSC and DPSC were cultured with Alpha-MEM containing 15% FBS. Three different transfection systems were tested to transiently transfect the vector with Green Fluorescent Protein (GFP) and the vector with GFP plus DMP1. These included: liposome of LipofectaminTM2000 (Invitrogen); FuGENE HD (Roche) and, CMV-mediated adenoviral plasmid. Transfection efficiency was detected by green fluorescent expression, gene expression levels were determined by quantitative RT-PCR, real-time PCR and Western blot. Results: Both LipofectamineTM2000 and FuGENE HD had less than a 5% transfection rate in our human stem cells that showed signs of toxicity. The CMV-mediated adenoviral had about 80% infection rate of GFP expression. Real-time PCR confirmed overexpression of DMP1 by this method. Conclusions: The adenovirus transfection method is the best way for gene delivery into bone and tooth-derived stem cells and will be useful to study the effects of various molecules on terminal differentiation. Further studies will now explore ultrasound enhanced gene delivery and small peptide gene delivery. Supported by Baylor Oral Health Foundation to SD and NIH grant RO1-DE013368 to RDS