The present study demonstrates a decrease in self-renewal capacity with serial transfer of murine hematopoietic stem cells. Production of differentiated cell progeny is maintained longer than stem cell self-renewal. In normal animals the capacity for self-renewal is not decreased with increasing donor age. The stem cell compartment in normal animals, both young and old, appears to be proliferatively quiescent. After apparent recovery from the alkylating agent busulfan, the probability of stem cell self-renewal is decreased, there is a permanent defect in the capacity of the bone marrow for serial transplantation, and the stem cells are proliferatively active. These findings support a model of the hematopoietic stem cell compartment as a continuum of cells with decreasing capacities for self-renewal, increasing likelihood for differentiation, and increasing proliferative activity. Cells progress in the continuum in one direction and such progression is not reversible.

Bone marrow pluripotent stem cells (CFUs) demonstrate capacity for both proliferation and differentiation. The proliferative capacity of CFUs has been measured by serial transplantability and by the Rs, a measurement of CFU production in a single 14-day transfer. In the present study, the self-renewal capacity of both adherent and nonadherent CFUs from long-term bone marrow cultures was measured. Culture conditions were established such that nonadherent cells were derived from the adherent cell layer. Both adherent and nonadherent cells produced spleen colonies, demonstrating that significant proliferative potential was present in both locations; however, at all times in culture, the CFUs within the adherent stromal cell layer had a significantly greater self-renewal capacity than did the nonadherent CFUs. During the initial establishment of the cultures, the self-renewal capacity of the adherent CFUs decreased as the total number of CFUs per flask increased. After 3 weeks in culture, the self-renewal potential of the adherent CFUs stabilized and was maintained. These results suggest two different mechanisms of stem cell proliferation. In order to increase the most primitive stem cell pool size, there was initial proliferation of early stem cells with a concomitant decrease in self-renewal capacity. Once this pool was established, the self-renewal capacity of the adherent CFUs maintained for 13 weeks in culture suggests that CFU production and cell maintenance were achieved by clonal succession.The pluripotent hematopoietic stem cell (CFU) is the most primitive of the known hematopoietic progenitor cells. A standard assay for this cell described by Till and McCulloch (1) measures the ability of donor marrow cells to form colonies in the spleens of lethally irradiated syngeneic mice. The selfrenewal capacity of the CFUs has been measured by both the RS, a measurement of CFU production over 14 days of serial marrow transplantation, and by the maximal transplantation time in a series of passages in irradiated animals (2). The standard CFU assay cannot distinguish stem cells with different self-renewal capacities because all produce spleen colonies. By assaying marrow from mice long after the administration of certain alkylating agents, a decrease in the self-renewal potential of the CFU compartment has been demonstrated in vivo (2-5). These experiments are consistent with the notion that the CFU compartment is heterogeneous and consists of a continuum of cells varying in self-renewal capacity. Within this continuum, early CFUs would be less committed to differentiation and have a high self-renewal capacity whereas CFUs further along in the continuum would be more committed to differentiation and have a lower self-renewal potential. Busulfan and certain other alkylating agents would preferentially destroy cells early in the continuum (2, 3). Heterogeneity within the CFU compartment has also been measured in vitro (6, 7). With selective inactivation of the CFUs by rabbit anti-mouse brain antiserum, Monette...

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Bone marrow from old (greater than 2 yr of age) and young (8-10 wk of age) male C3H/HeJ mice was grown in long-term bone marrow cultures and assayed for weekly hematopoietic stem cell (CFUs) production and interval self-renewal capacity (R5). Greater numbers of CFUs were noted in old marrow as compared to young marrow cultures over the first 4 wk duration, but subsequently, greater numbers of CFUs were seen weekly in the young marrow cultures. Over 1 3 wk in culture, the total CFUs determination was greater in young marrow cultures. Similarly. self-renewal capacity (R1) was greater in old as compared to young marrow-adherent CFUs over the first From the Joint Center for Radiation Therapy.