The Mesenchymal Stem Cell Core (MSC Core) will be responsible for the production and characterization of Cynomolgus MSCs that are essential for carrying out this multi-center grant. Bone marrow aspirates will be drawn from spuma-negative donor animals and sent to the MSC Core. The aspirates will be processed and MSCs will be expanded over several passages to provide enough cells to perform the experiments. When cell-dose has been achieved, MSCs will be cryopreserved in liquid nitrogen in preparation for shipping to the research sites. A sample of the cells will be evaluated by flow cytometry to evaluate characteristics and homogeneity of the population. MSCs will also be evaluated for suppression of the mixed lymphocyte reaction since this function is critical for several experiments proposed in this application. The MSC Core will also be responsible for tracking MSCs after they are administered to recipient animal. Because these cells are going to be used to induce tolerance or mediate suppression, it will be important to correlate their appearance (or disappearance) from critical tissues with the immunological status of the recipient. We plan to use a combination of histological and molecular techniques that have proven successful at Osiris in tracking MSCs in baboons for at least 12 weeks after implantation. MSCs from male donors will be labeled with a fluorescent dye for subsequent detection in biopsy or necropsy specimens obtained from female recipients. The presence and tissue location of dye-labeled MSCs will be determined using confocal laser scanning microscopy. Estimates of MSC cell number in tissues will be deterIniI1ed using real time PCR and primers specific for the Y chromosome in male cells. Immunocytochemistry will be used to characterize host cell infiltrates surrounding transplanted cells.
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