No abstract

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

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...

Neoplasms result from the uncontrolled clonal proliferation of abnormal or transformed cells. The early stages of this process are difficult to study because of the lack of sensitive and specific markers of clonal evolution in an experimental system. We have developed a cat model using cellular mosaicism for glucose-6-phosphate dehydrogenase (G-6PD). Our findings confirm that the structural locus for feline G-6-PD is on the X-chromosome and demonstrate that it is randomly inactivated in somatic cells. Heterozygous cats have balanced ratios of G-6-PD enzyme types in peripheral blood cells and hematopoietic progenitors that remain stable over time. In our initial studies, we used the model to analyze the events surrounding marrow failure experimentally induced by selected strains of feline leukemia virus (FeLV). Two G-6-PD heterozygous cats, one F1 male hybrid and one domestic cat were infected with FeLV (C or KT) and developed pure red cell aplasia (PRCA). Colonies arising from the more mature erythroid colony-forming cell were not detected in marrow culture of anemic animals although erythroid bursts persisted, suggesting that the differentiation of early erythroid progenitors (BFU-E) was inhibited in vivo. The ratio of G-6-PD types in hematopoietic progenitors and peripheral blood cells from the heterozygous cats did not change when the animals developed PRCA. Thus, the anemia did not result from the clonal expansion of a transformed myeloid stem cell. With this experimental approach, one may prospectively assess clonal evolution and cellular interactions in other FeLV-induced diseases.

I NTROD UCTI 0 N The coronaviral nature of feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) has been well documented by morphological. physicochemical and antigenic studies 1 -10 • However. biochemical and detailed immunochemical analyses of FIPV and FECV have been difficult due to the inability to prepare sufficient quantities of viral material. Recently. we have been able to propagate FIPV and FECV in continuous cell culture of feline origin8.11-13.The purpose of this report is to describe the purification of fel ine coronavi ruses from infected cell culture and to compare five strains with respect to: 1) plaque characteristics. 2) viral structural polypeptide composition and 3) serologic reactivity of experimentally infected cats against the structural polypeptides of homologous and heterologous strains. MATERIALS AND METHODS Cells and vi rusFetal cat \'kIole fetus (fcwf-4) cells were used to propagate all feline coronavirus strains. Cells \\ere cultured with Eagle's 133 P. J. M. Rottier et al. (eds.), Molecular Biology and Pathogenesis of Coronaviruses