This project was supported by grants from the National Institutes of Health 1R21AG036963 and the AO Craniomaxillofacial Foundation (C10-39L) to JKL, and the National Institutes of Health R03 AR057547 to DCG. with biocompatible scaffolds and additional delivery systems,5,6 but successful translation to environment is definitely in the beginning hampered from the harsh environment, including serum deprivation and reductions in local oxygen pressure Obtusifolin (hypoxia) (SD/H) in the defect or fracture site. Indeed, 99% of MSC do not survive tradition under ischemia after 3 Obtusifolin days7 and 99% of MSC implanted into ischemic heart cells pass away within 96?h,8 severely limiting the therapeutic potential of such treatments. Without overcoming such poor conditions, considerable apoptosis can significantly impede or prevent cells formation, regardless of the cell transplantation method.9C11 Although growth factors such as angiopoietin-1 have been shown to protect MSC against ischemia-induced apoptosis,12 the high cost of producing and purifying recombinant proteins and the difficulty of accurate delivery render large-scale implementation impractical. Lysophosphatidic acid (LPA) is definitely a glycerophospholipid signaling molecule that binds to cognate G-protein-coupled receptors and has HVH3 a wide range of effects on many different cell types.13C16 Naturally present in serum at low micromolar concentrations,15 LPA plays regulatory roles in the adhesion, migration, and proliferation of endothelial cells as well as neurons.14,17,18 Additionally, LPA affects actin polymerization in fibroblasts, osteoblasts, and other cell types to modulate cytoskeletal tension and contractile forces.13,15 Of particular interest for tissue engineering applications is the capacity for LPA to reduce apoptosis in MSC. Earlier studies have shown that LPA rescues rat MSC from SD/H-induced apoptosis over 4 weeks. Materials and Methods Cell tradition For studies, human bone marrow-derived MSC (Lonza, Walkersville, MD) were expanded without further characterization in a growth medium (GM) consisting of the minimum essential alpha medium (-MEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; JR Scientific, Woodland, CA) and Obtusifolin 1% penicillinCstreptomycin (P/S; Mediatech, Manassas, VA). MSC were transduced to express firefly luciferase (MSC-Luc) for studies as previously explained.25,26 Cells were cultured under standard conditions inside a humidified incubator and utilized at passages 5C6. To induce osteogenic differentiation, cells were cultured in either osteogenic press (OM: GM supplemented with 10?mM -glycerophosphate and 50?g/mL ascorbate-2-phosphate; Sigma-Aldrich, St. Louis, MO) or in OM supplemented with dexamethasone (OM+: OM with 10?nM dexamethasone, Sigma-Aldrich).4 All press were replaced every 3 days. For all experiments examining the effects of SD/H, MSC were preconditioned in GM, OM, or OM+ for 7 days in T-225 cells tradition flasks and consequently seeded on six-well cells tradition plates at 30,000 cells/cm2. After attaching over night, cells were washed 3with PBS to remove all traces of serum. To induce apoptosis, media were replaced with serum-free GM, OM, or OM+ supplemented with 0.1% (w/v) fatty acid-free BSA, and cells were incubated in hypoxia for 24?h ((HS00204173_m1), (Hs00231692_m1), (Hs00173500_m1), (Hs01113287_m1), (Hs00173857_m1), (Hs00271072_s1), and (Hs00252675_s1) were purchased from Applied Biosystems (Foster City, CA). Amplification conditions were 95C for 3?min, followed by 40 cycles at 95C for 3?s and 60C for 30?s. Quantitative PCR results were normalized to transcript levels to yield Ct, and collapse change in manifestation relative to the housekeeping gene was determined using 2?Ct.30 Visual and quantitative assessment of MSC exposed to SD/H MSC conditioned in GM were exposed to SD/H as explained above, and the morphological characteristics of MSC in each condition were observed Obtusifolin and recorded at 100magnification. DNA from MSC in each condition (BLI at 1, 3, 7, 14, 21, and 28 days on an IVIS Spectrum (Perkin Elmer, Waltham, MA) as previously explained.25,26 Briefly, mice were injected with D-Luciferin, Firefly (Caliper, Hopkinton, MA; 10?L/g body weight), and luminescence was measured using Living Image software (Perkin Elmer). Total photons per second per centimeter were recorded from each bioluminescent region of interest. Data are normalized to luminescence from gels comprising undifferentiated cells within each animal at each time point. Animals were euthanized 7 and 28 days postsurgery (compared with cells cultured in GM or OM (Fig. 1B). Based on these and earlier data,6.