title: Isolation of Mouse Bone Marrow Mesenchymal Stem Cells.
abstract: Mesenchymal stem cells (MSCs) were initially characterized as connective tissue progenitors resident in bone marrow, but have now been isolated from a variety of tissues and organs and shown to also exhibit potent tissue regenerative properties mediated largely via paracrine actions. These findings have spurred the development of MSC-based therapies for treating a diverse array of nonskeletal diseases. Although genetic and experimental rodent models of disease represent important tools for developing efficacious MSC-based therapies, development of reliable methods to isolate MSCs from mouse bone marrow has been hampered by the unique biological properties of these cells. Indeed, few isolation schemes afford high yields and purity while maintaining the genomic integrity of cells. We recently demonstrated that mouse MSCs are highly sensitive to oxidative stress, and long-term expansion of these cells in atmospheric oxygen selects for immortalized clones that lack a functional p53 protein. Herein, we describe a protocol for the isolation of primary MSCs from mouse bone marrow that couples immunodepletion with culture in a low-oxygen environment and affords high purity and yield while preserving p53 function.
authors.affiliation: Department of Molecular Therapeutics, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL, 33458, USA.
authors.forename: Siddaraju V
authors.initials: SV
authors.lastname: Boregowda
authors.affiliation: Department of Molecular Therapeutics, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL, 33458, USA.
authors.forename: Veena
authors.initials: V
authors.lastname: Krishnappa
authors.affiliation: Department of Molecular Therapeutics, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL, 33458, USA. dphinne@scripps.edu.
authors.forename: Donald G
authors.initials: DG
authors.lastname: Phinney
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D016159:Tumor Suppressor Protein p53; D010100:Oxygen
keywords: Immunodepletion; Low oxygen; Marrow stromal cells; Mesenchymal stem cells; Oxidative stress; p53