[PubMed] [CrossRef] [Google Scholar] 47. cells, an observation that was supported by biomechanical testing of bone samples from and WT mice. We conclude that loss of increases the magnitude of glycolysis upon the metabolic switch, which fuels the conversion of the osteoblast into a super-secretor of matrix resulting in more bone with improvements in intrinsic quality. mice exhibit more bone marrow osteoprogenitors than their wild-type (WT) littermates (16, 41, 95). Expanded cultures of mesenchymal stem/progenitor cells (MSPCs) induced with osteogenic Alisol B 23-acetate medium exhibit elevated mRNA expression of the bone matrix proteins type I collagen ((expanded MSPCs during osteogenesis. Network analyses of the RNA-seq output were used for driving hypothesis testing, i.e., select pathways that were significantly altered in the transcriptome were evaluated experimentally. The results phenotypically anchored bioinformatic predictions to changes in metabolic and biochemical properties of the osteogenic cells. Based on the RNA-seq data, we hypothesized that osteoblasts elaborate a matrix that improves bone material Alisol B 23-acetate and structural Alisol B 23-acetate characteristics. Therefore we examined these bone properties from experimental WT and mice that had undergone various osteoporosis therapies. These data reveal new aspects of how loss of alters bone matrix secretion as well as the impact of this single gene on bone quality. MATERIALS AND Alisol B 23-acetate METHODS Cell culture. MSPCs were derived from individual mice as previously described (16, 109). Briefly, long bone marrow was harvested from euthanized mice 6C8 wk of age, and a Ficoll gradient was used to isolate the mononuclear cells. These cells were seeded in Mesencult Media + Mesencult Stimulatory Supplement (StemCell Technologies, Vancouver, Canada) and sustained for 3C4 wk without passage while being fed every 5C7 days by removing 50% of the aged media and adding 50% fresh media, so as not to disturb the cells. Upon reaching 80% confluence, the cells were passaged at 1:3 dilution for 2 additional passages before use in experiments or were frozen for stock vials. Cells were used for study between passages 5C10. To assess the mineralization phenotype of each MSPC preparation, cells were seeded in -MEM supplemented with 100 IU/ml penicillin, 100 g/ml streptomycin, 25 g/ml amphotericin, 2 mM l-glutamine (GIBCO-BRL, Life Technologies, Grand Island, NY), and 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO). At 48 h postseeding, the medium was refreshed and further supplemented with ascorbic acid (5 g/ml; Sigma-Aldrich), dexamethasone (10 nM; Sigma-Aldrich), and 10 mM glycerol 2-phosphate disodium salt hydrate (BGP; Sigma-Aldrich). To visualize the mineralization in culture, cells were stained with alizarin red as previously described (16). RNA-seq analysis. To compare transcriptome profiles of nondifferentiating and osteogenic-differentiating WT and MSPCs, cells were seeded into 12-well plates at either 10,000 cells/well (25 cells/mm2) or 25,000 cells/well (62 cells/mm2). The cells seeded at the lower density were maintained in Mesencult Media + Mesencult Stimulatory Supplement (nondifferentiating medium) for 3 days postseeding and then harvested for total RNA. Cells plated at the higher density were maintained in -MEM complete medium throughout the experiment. At 48 h postseeding, the medium was refreshed with the ascorbic acid, dexamethasone, and BGP supplement. These cells were harvested at 7 days postseeding as early osteogenic cells. Total RNA was harvested using RNeasy (Qiagen, Valencia, CA) and measured for quality using the Agilent 2100 Bioanalyzer and Qubit 2.0 Fluorometer. High RNA integrity is critical for evaluating the transcriptome. The RNA integrity number is an algorithm for assigning integrity values to RNA measurements and assigns an electropherogram a value of 1 1 to 10, with 10 being the least degraded. All RNA integrity number numbers Rabbit polyclonal to ADI1 for our samples ranged between 8.2 and 9.7. A conservative cut-off value in the context of RNA degradation lies between 6.4 and.
