title: Immunodepletion and Hypoxia Preconditioning of Mouse Compact Bone Cells as a Novel Protocol to Isolate Highly Immunosuppressive Mesenchymal Stem Cells.
abstract: Compact bones (CB) are major reservoirs of mouse mesenchymal stem cells (mMSC). Here, we established a protocol to isolate MSC from CB and tested their immunosuppressive potential. Collagenase type II digestion of BM-flushed CB from C57B/6 mice was performed to liberate mMSC precursors from bone surfaces to establish nondepleted mMSC. CB cells were also immunodepleted based on the expression of CD45 (leukocytes) and TER119 (erythroid cells) to eliminate hematopoietic cells. CD45-TER119-CB cells were subsequently used to generate depleted mMSC. CB nondepleted and depleted mMSC progenitors were cultured under hypoxic conditions to establish primary mMSC cultures. CB depleted mMSC compared to nondepleted mMSC showed greater cell numbers at subculturing and had increased functional ability to differentiate into adipocytes and osteoblasts. CB depleted mMSC had high purity and expressed key mMSC markers (>85% Sca-1, CD29, CD90) with no mature hematopoietic contaminating cells (<5% CD45, CD11b) when subcultured to passage 5 (P5). Nondepleted mMSC cultures, however, were less pure and heterogenous with <72% Sca-1+, CD29+, and CD90+cells at early passages (P1 or P2), along with high percentages of contaminating CD11b+(35.6%) and CD45+(39.2%) cells that persisted in culture long term. Depleted and nondepleted mMSC nevertheless exhibited similar potency to suppress total (CD3+), CD4+and CD8+T cell proliferation, in a dendritic cell allostimulatory one-way mixed lymphocyte reaction. CB depleted mMSC, pretreated with proinflammatory cytokines IFN-γ, TNF-α, and IL-17A, showed superior suppression of CD8+T cell, but not CD4+T cell proliferation, relative to untreated-mMSC. In conclusion, CB depleted mMSC established under hypoxic conditions and treated with selective cytokines represent a novel source of potent immunosuppressive MSC. As these cells have enhanced immune modulatory function, they may represent a superior product for use in clinical allotransplantation.
authors.affiliation: 1 School of Medicine, Faculty of Health Sciences, University of Adelaide , Adelaide, Australia .|2 Centre for Clinical and Experimental Transplantation, Royal Adelaide Hospital , Adelaide, Australia .
authors.forename: Kisha Nandini
authors.initials: KN
authors.lastname: Sivanathan
authors.affiliation: 3 South Australian Health and Medical Research Institute , Adelaide, Australia .|4 Mesenchymal Stem Cell Laboratory, School of Medicine, Faculty of Health Sciences, University of Adelaide , Adelaide, Australia .
authors.forename: Stan
authors.initials: S
authors.lastname: Gronthos
authors.affiliation: 5 Transplantation Immunology Group, Garvan Institute of Medical Research , Sydney, Australia .
authors.forename: Shane T
authors.initials: ST
authors.lastname: Grey
authors.affiliation: 1 School of Medicine, Faculty of Health Sciences, University of Adelaide , Adelaide, Australia .|2 Centre for Clinical and Experimental Transplantation, Royal Adelaide Hospital , Adelaide, Australia .
authors.forename: Darling
authors.initials: D
authors.lastname: Rojas-Canales
authors.affiliation: 1 School of Medicine, Faculty of Health Sciences, University of Adelaide , Adelaide, Australia .|2 Centre for Clinical and Experimental Transplantation, Royal Adelaide Hospital , Adelaide, Australia .|6 Central Northern Adelaide Renal Transplantation Service, Royal Adelaide Hospital , Adelaide, Australia .
authors.forename: Patrick T
authors.initials: PT
authors.lastname: Coates
mesh_terms: D017667:Adipocytes / Q000166:cytology; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology*; D002469:Cell Separation; D020381:Interleukin-17 / Q000378:metabolism; D008213:Lymphocyte Activation / Q000502:physiology; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000276:immunology*; D051379:Mice; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D020381:Interleukin-17; D014409:Tumor Necrosis Factor-alpha
keywords: T cells; compact bone mesenchymal stem cells; hypoxia; immunodepletion; immunosuppression; proinflammatory cytokines