Effect of Recombinant ApoE on Gene Expression During Osteoblast Differentiation of Mouse Bone Marrow Stromal Cells (BMSCs)
Advanced age impairs bone fracture healing; however, the underlying mechanisms remain unclear. We found that circulating levels of apolipoprotein E (ApoE) increase with age and contribute to impaired fracture healing. In vitro, ApoE treatment of aged bone marrow stromal cells (BMSCs) inhibited osteoblast differentiation. To investigate the mechanism by which ApoE inhibits osteoblast differentiation, we treated primary BMSCs isolated from the femurs and tibiae of 24-month-old C57BL6/J mice with recombinant ApoE.  For osteoblast differentiation, BMSCs were cultured in osteogenic medium. Recombinant ApoE (rApoE) was added at a concentration of 100 ng/mL starting two days after induction of differentiation and continued throughout the 10-day differentiation period. BMSCs treated with vehicle or rApoE were harvested on day 10. Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer's protocol for downstream transcriptomic analysis. Overall design: RNA-seq profiling of bone marrow stromal cells (BMSCs) derived from 24-month-old wild-type (C57BL/6) mice during osteoblast differentiation. After 2 days of differentiation, cells were treated with either vehicle (n = 3) or recombinant ApoE (n = 3) for 8 additional days (total 10 days of differentiation) before RNA extraction and sequencing.