Direct conversion of mouse embryonic fibroblast to osteoblast cells using hLMP-3 with Yamanaka factors

Abstract

Large bone defects and bone loss after fractures remain significant challenges for orthopedic surgeons. Our study aims to find an available, applicable and biological treatment for bone regeneration overcoming the limitations in ESC/iPSC technology. We directly reprogrammed the mouse embryonic fibroblast (MEF) into osteoblast cells using different combinations of Yamanaka factors with human lim mineralization protein-3 (hLMP-3). LMP is an intracellular LIM-domain protein acting as an effective positive regulator of the osteoblast differentiation. After transduction, cells were cultured in osteogenic medium, and then examined for osteoblast formation. The expression of osteogenic markers (BMP2, Runx2 and Osterix) during reprogramming and in vitro mineralization assay revealed that the best reprogramming cocktail was (c-Myc - Oct4) with hLMP-3. In addition, both immunofluorescent staining and western blot analysis confirmed that osteocalcin (OCN) expression increased in the cells treated with the c-Myc/Oct4/hLMP3 cocktail than using hLMP-3 alone. Furthermore, this reprogramming cocktail showed efficient healing in an induced femoral bone defect in rat animal model one month after transplantation. In the present study, we reported for the first time the effect of combining Yamanaka factors with hLMP-3 to induce osteoblast cells from MEF both in vitro and in vivo.

Keywords: Bone regeneration; Direct reprogramming; MEF; Yamanaka factors; hLMP-3.