To study the behavior of hematopoietic stem cells in vivo, we transplanted glucose-6-phosphate dehydrogenase (G6PD) heterozygous (female Safari) cats with small amounts of autologous marrow. The G6PD phenotypes of erythroid burst-forming units and granulocyte/macrophage colony-forming units were repeatedly assayed for 3.5-6 years after transplantation to track contributions of stem cell clones to the progenitor cell compartment. Two phases of stem cell kinetics were observed, which were similar to the pattern reported in comparable murine studies. Initially there were significant fluctuations in contributions of stem cell clones. Later clonal contributions to hematopoiesis stabilized. The
Females are natural mosaics for X chromosome-linked genes. As X chromosome inactivation occurs randomly, the ratio of parental phenotypes among blood cells is approximately 1:1. Recently, however, ratios of greater than 3:1 have been observed in 38-56% of women over age 60. This could result from a depletion of hematopoietic stem cells (HSCs) with aging (and the maintenance of hematopoiesis by a few residual clones) or from myelodysplasia (the dominance of a neoplastic clone). Each possibility has major implications for chemotherapy and for transplantation in elderly patients. We report similar findings in longitudinal studies of female Safari cats and demonstrate that the excessive skewing that develops with aging results from a third mechanism that has no pathologic consequence, hemizygous selection. We show that there is a competitive advantage for all HSCs with a specific X chromosome phenotype and, thus, demonstrate that an X chromosome gene (or genes) regulates HSC replication, differentiation, and͞or survival.
To test if hematopoiesis can be maintained by the sequential activation of stem-cell clones, we performed autologous marrow transplantations with limited numbers of cells in cats heterozygous for the X chromosome-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) and observed the G6PD phenotypes of erythroid and granulocyte/macrophage progenitors over time. The animals were the female offspring of Geoffroy male and domestic female cats. In repeated studies of marrow from control animals (n = 5) or experimental animals prior to transplantation (n = 3), the percent of progenitors with domestic-type G6PD did not vary. After transplantation, the peripheral blood counts, marrow morphologies, frequencies of progenitors, and progenitor cell cycle kinetics returned to normal. However, abrupt and significant fluctuations were seen in the G6PD type of progenitors from each cat during the 1-1.5 years of observation. These data cannot be explained if there were either a large or constant population ofactive stem cells and thus imply, in a large-animal system, that hematopoiesis was maintained through clonal succession. A stochastic model was developed to estimate the numbers of active clones and their mean lifetimes.Two theories describe early hematopoietic stem-cell differentiation. The first states that stem cells may contribute indefinitely to hematopoiesis. The second theory, the theory of clonal succession, initially proposed by Kay (1), states that hematopoiesis is maintained by a subset of active stem cells and a dormant reserve. As clones are depleted, perhaps through terminal differentiation, reserve cells become active. These theories have been difficult to test experimentally and transplantation studies in mice with enzymatically or retrovirally marked cells present evidence both for (2-5) or against (6-8) clonal succession. Many murine experiments are limited by difficulties obtaining samples for repeated analysis over time. Also, when the infection of marrow cells with retroviral vectors is used to mark clonal origin, it is difficult to distinguish the contribution of the earliest stem cells from that of cells with a more restricted proliferative potential. In addition, the growth characteristics of a stem cell may change during in vitro incubation (9). For these reasons and to extend the observations to a large-animal system, we studied cats heterozygous for isotypes of the X chromosome-linked enzyme glucose-6-phosphate dehydrogenase (G6PD).Geoffroy cats (Leopardus geoffroyi, South American origin) and domestic cats (Felis catus, Euroasian origin) have evolved independently for 12 million years (10) and have electrophoretically distinct G6PD types (11). These animals were bred to produce female F1 (Safari) cats that were obligate G6PD heterozygotes. In previous studies, we demonstrated that individual colonies derived from erythroid progenitors (burst-forming units-erythroid; BFU-E) or granulocyte/macrophage progenitors (colony-forming unitsgranulocyte/macrophage, CFU-GM) contained domestictype G6P...
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