The transfection efficiency of photosensitizer-induced gene delivery to human MSCs and internalization rates of EGFP and Runx2 genes.
To improve the transfection efficiency of non-viral gene vectors to human mesenchymal stem cells (hMSCs), a photosensitizer (PS)-induced gene delivery system was designed by using pheophorbide-a (pheo-a) as a PS. In FACS results, this system showed excellent gene transfection efficiency depending on irradiation power. The result was strongly supported by western blot and real-time quantitative PCR (RT-qPCR) assays. The protein and mRNA expression of enhanced green fluorescent protein (EGFP) in hMSCs treated with 0.9 J/cm(2) irradiation increased 9.8- and 8.7-fold compared with non-irradiated hMSCs, respectively. Furthermore, the internalization of PEI/pDNA complexes in hMSCs was enhanced by light irradiation even under conditions that inhibited endocytosis. The hemolytic activity of PS with irradiation (0.9 J/cm(2)) significantly increased to 55%. Thus, PS with light irradiation facilitated both the internalization and endosomal escape of gene complexes. For osteogenic induction, the Runt-related transcription factor 2 (Runx2) gene was transferred to hMSCs via PS-induced transfection. Von Kossa staining indicated that Runx2 overexpression significantly enhanced the osteogenesis of hMSCs. Therefore, this PS-induced gene delivery method has potential value for stem cell therapy via gene delivery.
Department of Biotechnology, The Catholic University of Korea, Wonmi-gu, Gyeonggi-do 420-743, South Korea.
Sin-jung
SJ
Park
Kun
K
Na
D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects* / Q000528:radiation effects; D005786:Gene Expression Regulation / Q000187:drug effects / Q000528:radiation effects; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008027:Light; D059630:Mesenchymal Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D018613:Microscopy, Confocal; D010012:Osteogenesis / Q000187:drug effects / Q000528:radiation effects; D017319:Photosensitizing Agents / Q000494:pharmacology*; D010957:Plasmids / Q000378:metabolism; D011094:Polyethyleneimine / Q000737:chemistry; D016133:Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D050659:Core Binding Factor Alpha 1 Subunit; D017319:Photosensitizing Agents; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; D004247:DNA