All experiments were approved by the Research Ethics Board at the University of Western Ontario, London, Ontario, Canada. Fresh bone marrow samples (1M-125, Lonza, Inc., Walkersville, MD, www.lonza.com) were obtained and mononuclear cell fraction was prepared as shown by us previously [10, 16]. Bone marrow samples were cultured on fibronectin-coated (FN; 1 µg/cm2; FC010-10MG, Millipore, Temecula, CA, www.millipore.com) plates in Dulbecco's modified Eagle's medium (DMEM) low glucose with pyruvate and l-glutamine (10–014-CV, Mediatech, Manassas, VA, www.cellgro.com) media, supplemented with 20% fetal bovine serum (FBS; Life Technologies, Burlington, Canada, www.lifetechnologies.com), 1× penicillin-streptomycin-amphotericin (PSF; Mediatech), and no additional growth factors. We have shown that in the presence of serum, bone marrow cells lose the ability to produce clonal populations and differentiate into endothelial cells and neuroglial cells . Therefore, we refer to these cells as MPCs as they retain the ability to produce mesenchymal lineages: adipocytes, chondrocytes, and osteoblasts [10, 18]. All experiments using bone marrow-derived MPCs (bmMPCs) were conducted on passage 2–6 cells with at least three technical and three to five biological replicates.
To induce differentiation, we seeded MPCs at 40,000 cells per square centimeter on 12-well or 24-well plates in specific differentiation media but without FN coating (StemPro Adipogenesis; Life Technologies). The components of the STEMpro media are proprietary. However, it does not contain any PPARγ agonists and we are able to reproduce our results with DMEM/10% FBS, 5 µg/ml insulin, 1 µM dexamethasone (D2915; Sigma-Aldrich, Oakville, Canada, www.sigmaaldrich.com), 0.5 mM isobutylmethylxanthine (I7018; Sigma-Aldrich), 60 µM indomethacin (I7378; Sigma-Aldrich), and 1× PSF. However, for the ease of use and low intra-assay variability, we used StemPro for our studies. We also plated cells at high density to study mechanisms specifically at play during differentiation without the confounding effect of early proliferation, which takes place in most differentiation assays. Control or differentiation media were supplemented with HG (25 mmol/l final concentration). Control media formulation contains approximately 5.5 mM glucose (100 mg/dl), which approximates to normal blood glucose levels in vivo. In cell culture studies, concentrations of glucose approaching 10 mM (180 mg/dl) are considered prediabetic and concentrations above 10 mM are analogous to diabetic condition. To study the effect of HG, conditions mimicking diabetes, we have used 25 mM glucose levels (450 mg/dl). We also tested the effect of recombinant Wnt5a (50 ng/ml; 645-WN-010, R&D Systems, Minneapolis, MN, www.rndsystems.com), Wnt5b (50 ng/ml; 7347-WN-025, R&D Systems), and Wnt11 (50 ng/ml; 6179-WN-010, R&D Systems) in adipogenic differentiation media. IWR-1-endo (13659-10, Cayman Chemical, Ann Arbor, MI, www.caymanchem.com) and PNU74654 (3534/10, R&D Systems), both were added at concentrations of 5, 10, and 20 µM. A Wnt signaling agonist (Calbiochem CAS 853220-52-7; Millipore) was also used and added at concentrations varying from 100 nM to 5 µM. In Solution Rac1 Inhibitor II Z62954982 (25 µM, 553512; Millipore), Rho Kinase Inhibitor VII (10 nM; 555556; EMD Millipore), pan-protein kinase C (PKC) inhibitor chelerythrine chloride (700 nM, 1278; Cayman Chemicals), specific epsilon-PKC Inhibitor (5 µM; 62187 [AN]; Anaspec, Inc., Fremont, CA, www.anaspec.com), and calmodulin-dependent kinase inhibitor KN93 (500 nM, 1278; Tocris Biosciences; Bristol, U.K., www.tocris.com) were also used depending on the experiment. Media were changed every other day. RNA was isolated from cells in order to perform qRT-PCR. For oil red O staining, cells were fixed in 10% neutral buffered formalin and placed in 100% propylene for 5 minutes before applying 0.5% oil red O solution (O0625-25G, Sigma-Aldrich).