Previous work from our laboratory has established models of stem engraftment in syngeneic mice with no or minimal myeloablative host treatment and strongly pointed to stem cell competition as being the key determinant of engraftment. This work has been extended to allogeneic transplants in H2 mice, and has shown that with costimulator blockade, antigen preexposure and 100 cGy total body irradiation, long-term, stable nontoxic allochimerism can be obtained. The present grant is to continue studies of engraftment in non or minimally myeloablated syngeneic (inbred) mice, focusing on engraftment of highly purified stem cells and the role of accessory cells in such engraftment, alternate host treatment with myleran and regional engraftment persistence. We will also evaluate whether mobilization of host stem cells could enhance engraftment. We also plan to continue studies on models of non-toxic allochimerism extending our knowledge on antigen preexposure and costimulators. We will evaluate the impact of sequence and number of marrow cells transplanted, and delayed transplant and the effect of selected stem cell subpopulations and mega stem cell approaches. We will apply effective approaches developed in the syngeneic models to engraftment in the allogeneic transplant models. Techniques utilized will include fluorescence activated cell sorting and cytometry, immunofluorescent cell tracking, long-term stromal cultures, antibody labeling of cells for cell separation, both syngeneic and allogeneic murine transplant models, skin grafting, fluorescent in situ hybridization, Southern blots and cytokine injections in vivo to mobilize host stem cells. Mice will be subjected to no or varying levels of irradiation from a gamma cell source and homing assays with PKH26, PKH2 or CFSE will be employed. These studies should shed light on the stem cell/accessory cell mechanism involved in both syngeneic and allogeneic transplantation and provide approaches to minimize toxicity in both types of transplantation.
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