Objective: Production and characterization of mesenchymal stem cells (C3H10T1/2) that express human amelogenin.
Materials and Methods: Amelogenin-Adenovirus(+) (experimental) and amelogenin(-) adenovirus (lacking amelogenin coding region) were propagated in HEK293 cells, harvested and purified. HEK293 and C3H10T1/2 cells were then infected by both types of adenovirus. RT-PCR, cDNA sequencing, Western blot and immunohistochemistry were performed two and six days after infection.
Results: HEK293 and C3H10T1/2 cells infected by amelogenin adenovirus(+) expressed high levels of amelogenin mRNA both at two days and six days after infection. In non-infected cells and infected by amelogenin(-) adenovirus, no amelogenin mRNA was detected. Two human amelogenin isoforms were identified in both cell types, infected by amelogenin(+) adenovirus; One- corresponding to mouse M180 amelogenin isoform, and LRAP (M59 in mouse, suggested to be signaling molecule), indicating splicing in HEK293 cells or in both. Immonuhistochemistry revealed strong staining of C3H10T1/2 cells infected by amelogenin(+) adenovirus at 2 days and gradually decreasing at 4 and 6 days after infection.
Conclusions: The infection of C3H10T1/2 and HEK293 cells by amelogenin(+) adenovirus induced the expression of two amelogenin splice products (corresponding to mouse M180 and M59 amelogenin isoforms). More recently, it was shown that both M180 and LRAP bind to cell-surface receptors LAMP-1, LAMP-2 and CD63, rapidly entering the cells and localizing in the perinuclear region. Currently the changes in expression of osteogenic genes associated with over-expression of amelogenin in this C3H10T1/2 cells is being investigated. The results will serve as baseline for better understanding the role of amelogenin in bone formation and regeneration.
Supported by BSF grant number 2003239