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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 (&gt;85% Sca-1, CD29, CD90) with no mature hematopoietic contaminating cells (&lt;5% CD45, CD11b) when subcultured to passage 5 (P5). Nondepleted mMSC cultures, however, were less pure and heterogenous with &lt;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.

Immunodepletion and Hypoxia Preconditioning of Mouse Compact Bone Cells as a Novel Protocol to Isolate Highly Immunosuppressive Mesenchymal Stem Cells