Hematopoiesis is the process by which functional red cells, white cells, and platelets develop. The behavior and characteristics of the earliest stem cells contributing to blood cell production are unknown, In part because such cells are difficult to define, label, arid, thus, analyze. Furthermore, it is not clear if the regulation of stem cell reserve in a mouse, an animal with a small blood volume and limited lifespan and, therefore, significantly less demand for hematopoiesis, will be comparable to that In man. For these reasons, we developed a large animal system of G-6-PD cellular mosaicism. Geoffroy cats and domestic cats, animals with electrophoretically-distinct G-6-PD types, were bred to produce Fl female offspring who were obligate G-6PD heterozygotes. Because of X-inactivation during embryogenesis, each stem cell and its progeny contain either domestic (d) or Geoffroy (G) -type G-6-PD. Through analyses of Individual BFU-E and CFU-GM over time, we can determine the relative contribution of the d or G -type stem cells from which these progenitors derive. We have used this system to demonstrate clonal succession following autologous transplantation and after damage or depletion of the marrow reserve with chemotherapy. In addition, a state space Markov model was developed to estimate the number of active stem cell clones and the mean lifetimes of active clones for each data set. In this proposal, we extend this approach to derive principles that may govern stem cell kinetics In normal cats, In elderly cats, and after transplantation of fetal liver cells or of autologous marrow cells (either fresh or passaged in long term marrow culture). Also we propose alternate schemes for stochastic modeling and for computer simulation to provide insights Into the data. Such studies, which can not be done In man, should provide Information about the dynamics of stem cells In a large animal system and thus provide a physiologic basis for strategies for gene therapy, transplantation, and aggressive chemotherapies.
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