The long term objective of this project is to characterize the most primative adult stem cells - those with the most repopulating ability over the longest time. Such cells continuously populate the hemopoietic and immune systems and are the essential element in clinical marrow transplantation. They are assayed by directly measuring repopulating ability in competition with a standard dose of simultaneously grafted, genetically marked stem cells. Optimal conditions for this assay will be defined, using congenic mouse strains that are identical except for the genetic markers, defining optimal cell numbers, calibrating the assay with standard frozen cell types, and repeating proliferative stimuli over the recipient's life to make the assay as rigorous as possible. Deleterious effects of transplanting stem cells are of major concern in clinical marrow grafts. The degree to which these effects are caused by proliferative exhaustion, irradiation, stem cell displacement, excessive stimuli to differentiate, and handling will be determined by removing possible causes. Mutant mice defective in either erythropoietic or immune stem cells will be studied to determine whether the same stem cell produces both. Other mutants with hemolytic anemias requiring over 50 times normal rates of erythrocyte production will be tested for proliferative exhaustion from fetal life through old age. Tetraparental mice will be used to determine whether newly synthesized erythrocytes arise from few or many stem cells. Variabilities among irradiated recipients of identical marrow mixtures and among recipients with a constant portion of bone shielded, will be used to estimate stem cell numbers with the binomial equation. Both stimulatory and inhibitory effects of immune responses on stem cell repopulation will be studied, and antigens causing each will be characterized with monoclonal antibodies. Stem cells will be purified, or treated with antibody, to remove regulatory cells and analyze their effects. Long-term repopulation assays will be used to assess stem cell growth in vitro and to evaluate assays for stem cells in vitro. Currently used tissue culture procedures will be tested, and improved procedures will be developed for stem cell growth and analysis. The ability to assay, or even to grow, the most primative human stem cells in vitro might have clinical importance.
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