2 for 2.5 hours to form 3D cell aggregates. Then chondrocyte differentiation media was gently added 2901691 to each chamber. Chondrocyte differentiation media consisted of: a-MEM, Lonza with 0% FBS, 100 nM dexamethasone, 10 ng/ml TGF b-3, R&D Systems & Cell Signaling, SITE liquid media supplement, Sigma-Aldrich, 100 mg/ml Sodium Pyruvate, and 1.5 mg/ml BSA. Media was replenished every 3 days, and cells were harvested after 28 days. Materials and Methods Reagents and Antibodies All chemicals were purchased from Sigma-Aldrich MedChemExpress PCI-32765 unless otherwise noted. Polyclonal Rabbit anti-YKL-40 antibody was purchased from Quidel. Polyclonal chicken anti-Neuron Specific Enolase, Polyclonal chicken anti-bTubulin III, Polyclonal rabbit anti-NeuN, Polyclonal rabbit antiCollagen II, and rabbit anti-chicken HRP antibodies were purchased from Millipore. Monoclonal mouse anti-CD44 antibodies were purchased from R&D Systems. Monoclonal mouse anti-OC4-30 antibodies were purchased from Abcam. Goat anti-rabbit HRP conjugate antibody was purchased from Bio-Rad Laboratories. Horse anti-mouse HRP antibodies were purchased from Cell Signaling Technology. Mouse monoclonal anti-b-actin HRP conjugate antibody was purchased from Sigma-Aldrich. Osteoblast Differentiation MSC’s of passage 25 were grown in TCC until 90% confluent. Then a-MEM with 10% FBS, 50 mg/ml ascorbic acid was added on day `09. After 7 days, 3 mM b-glycerol phosphate was added to the existing protocol and the cells exposed for 23 days, with media changes every 3 to 4 days. Isolation of RNA The initial RNA isolation utilized the RNeasy Minikit from Qiagen, following the manufacturer’s instructions. For all subsequent RNA isolations, Trizol was added and cells were lysed directly in the flask. The resulting lysate was transferred to 1.5 ml eppi tubes. Samples were then either used or stored at 80uC. 1 ml of each sample 9504387 of Trizol lysate was then vortexed, and centrifuged at 12,000 g for 10 minutes at 4uC. Cleared supernatants were then moved to new 1.5 ml eppi tubes. Samples were then incubated for 5 minutes at room temperature to allow nucleoprotein dissociation. For phase separation, 0.2 ml of chloroform was added and the tube shook vigorously for 15 seconds, then incubated at room temperature for 3 minutes. Samples were centrifuged at 12,000 g for 15 minutes at 4uC. The clear aqueous phase was moved to a new 1.5 ml eppi tube and RNA precipitation was performed. For RNA precipitation, 5 mg of RNase free glycogen was added to act as an RNA carrier. Then 0.5 ml of 100% isopropanol was added and the sample was incubated at room temperature for 10 minutes. The samples were then centrifuged at 12,000 g for 10 minutes at 4uC. The supernatant was discarded and the resulting RNA pellet was Cell Culture Mesenchymal stem cells were purchased from Lonza. All other cell lines were purchased from the American Type and Culture Collection and cultured in typical culture conditions “TCC”of DMEM media Mediatech Inc. containing 10% FBS, Hyclone, and 0.292 mg/ml L-Glutamine, Mediatech unless otherwise stated. All cell cultures were grown in vented tissue culture flasks from CorningH,, or tissue culture chamber slides from Lab-Tek/Nalge Nunc for staining. Neuronal Differentiation Protocol 1 MSCs of passage 25 were differentiated into neurons by first growing them in TCC to 20% confluence, approximately 100,000 cells per tissue culture dish. Then MSCs were exposed for 7, 14 and 30 days to neuronal induction media: Ham’s DMEM/F12,, 2%