Control animals at all stages displayed a normal glial population with few or no apoptotic nuclei

ative control, cells were cultured in cell culture medium containing 10% FCS. To osteogenic induction medium, 0.1, 1 and 10 mM resveratrol and/or 1, 10 and 100 mM nicotinamide were added for the indicated time periods. Cells were nurtured through diffusion at the filter medium interface and evaluated after indicated time periods. Light microscopy Monolayer PP 242 site cultures were stained with von Kossa for mineralized matrix deposition or stained with Oil Red O solution to visualize the formation of fat vacuoles as previously described. Antisense and lipofectin-mediated transfection The Sirt-1 antisense sequences used in these experiments were designed using a computational neural network mode. MSCs were plated in 3 cm2 tissue culture dishes or in a four-well glass plate at a concentration of 36105 cells/dish or 16104 cells/well and were grown to confluence. All transfection experiments were carried out on 50% confluent monolayer cultures. Antisense oligonucleotide sequence was derived from the nucleotide at position 844 to 864 lying in upstream region of the nucleotide sequences coding for the catalytic domain of Sirt-1 mRNA registered under accession number NM012238 in GenBank. To overcome the rapid degradation of antisense sequence by intracellular endo- and exonucleases, the non-bridging oxygen on the phosphate linkage was replaced with a sulfur atom. The phosphothioate modified sense oligonucleotide sequence, complementary to the antisense sequence, was used as control. The modified oligonucleotides were purchased from MWG. To Resveratrol Promotes Osteogenesis of MSCs fixation for 10 min at ambient temperature, and rinsing with PBS. Cell membranes were permeabilized by treatment with 0.1% Triton X-100 for 1 min on ice. Cells were overlaid with proteasefree bovine serum albumin for 10 min at AT, rinsed with PBS and incubated with primary antibodies in a humid chamber overnight at 4uC. They were gently washed several times with PBS before incubation with rhodamine-red conjugated secondary antibody for 2 h at AT and finally washed again three times with Aqua Dest laboratory water. Counterstaining was performed with DAPI to visualize the cell nuclei. Samples were evaluated under light microscope and photomicrographs were digitally captured and stored. Immunoprecipitation and Immunoblotting provide enhanced transfection of oligonucleotides to the cytoplasm of the target cells, lipofectin reagent was used according to the manufacturer’s instructions. Briefly, 10 ml lipofectin was mixed with 1, 0.5 and 0.2 mM of sense or antisense oligonucleotide respectively for 30 min at AT and subsequently the mixture was added to 990 ml serum-free medium to obtain a working medium with 1, 0.5 and 0.2 mM of the corresponding oligonucleotide. The medium was then added to the already prepared cells and incubated for 24 h at 37uC. After 24 h of incubation, transfection media was replaced by the regular culture or osteogenic induction media and evaluated after 21 days. Electron microscopy Transmission electron microscopy was performed as previously described. Briefly, high-density cultures were fixed for one hour in Karnovsky’s fixative and then post-fixed in 1% OsO4 solution. After dehydration, pellets were embedded in Epon, ultrathin cuts made on a Reichert-Ultracut E. and contrasted with a mixture of 2% uranyl acetate/lead citrate. A transmission electron microscope was used to examine the cultures. To quantify adipocyte formation, the number of cells exhibiting