Osteoblast differentiation of rabbit adipose-derived stem cells by polyethylenimine-mediated BMP-2 gene transfection in vitro.

ABSTRACT

The aim of this study was to examine the efficiency of polyethylenimine-mediated transfection of the human bone morphogenetic protein-2 (BMP-2) gene into rabbit adipose-derived stem cells (ADSCs), and its effect on osteoblast differentiation. Adipose tissue was isolated from the necks of adult Japanese white rabbits and cultured in vitro to obtain ADSCs. Gene delivery of BMP-2 was mediated by polyethylenimine and stable transformants were selected by G-418. The expression of BMP-2 mRNA was confirmed by reverse transcription-polymerase chain reaction, and of the BMP-2 protein by ELISA. Osteocalcin and collagen type I were detected by western blot and by an alkaline phosphatase kit. Alizarin red S stain was also utilized to examine osteogenesis. The non-transfected group was considered as a control. In this study, we successfully derived ADSCs from rabbit adipose tissue. Through passages 3-6, the expression of CD29 and CD44 gradually increased, whereas the expression of CD34 and CD45 gradually decreased. Both mRNA and protein expression of BMP-2 were confirmed following polyethylenimine-mediated BMP-2 gene delivery. In addition, the expression of alkaline phosphatase, osteocalcin, and collagen type I was found to be upregulated and alizarin red S staining was positive in transfected ADSCs, indicating BMP-2-induced osteogenesis. Therefore, this study determined that polyethylenimine was able to mediate BMP-2 gene delivery and induce osteogenic differentiation of ADSCs.