Simulation of hematopoiesis: implications for the gene therapy of lysosomal enzyme disorders.

Although the hematopoietic stem cell is an attractive target for gene transfer, little is known about its biology in vivo in large animals (including humans). We have studied the in vivo behavior of hematopoietic stem cells in glucose-6-phosphate dehydrogenase heterozygous (female Safari) cats, and demonstrated that clonal instability persists for up to 4.5 years after autologous marrow transplantation. This contrasts with the 2-6 months of clonal disequilibrium reported in comparable murine studies. Our data also suggest that hematopoietic stem cells do not self-renew more than once every 3 weeks. These data may have relevance for strategies to optimize gene therapy in large animals and, by extension, in humans.

Prospective hematologic and clinicopathologic study of asymptomatic cats with naturally acquired feline immunodeficiency virus infection.

Prospective studies were performed over a 28- to 77-month period (median, 66 months) on 5 cats with naturally acquired feline immunodeficiency virus (FIV) infection in an attempt to correlate hematologic and clinicopathologic changes with the emergence of clinical disease. On presentation, all cats were asymptomatic; free of opportunistic infections; and had normal complete blood counts, bone marrow morphologies, marrow progenitor frequencies, and progenitor in vitro growth characteristics. During study, 2 cats remained healthy, 2 cats showed mild clinical signs, and 1 cat developed a malignant neoplasm (ie, bronchiolar-alveolar adenocarcinoma). Although persistent hematologic abnormalities were not observed, intermittent peripheral leukopenias were common. In 3 of 5 FIV-seropositive cats, lymphopenia (< 1,500 lymphs/microL; normal reference range, 1,500 to 7,000 lymphs/microL) was a frequent finding and the absolute lymphocyte counts had a tendency to progressively decline. One of the other 2 cats had consistently low to low-normal absolute neutrophil counts (1,300 to 4,800 segs/microL; mean, 2,730 segs/microL; normal reference range, 2,500 to 12,500 segs/microL), and the remaining cat had consistently normal leukograms, except for a transient period (ie, 11 months) of benign lymphocytosis (7,200 to 13,430 lymphs/microL) early in the study. Periodic examinations of bone marrow aspirates revealed normal to slightly depressed myeloid-to-erythroid ratios with normal cellular morphology and maturation. Bone marrow abnormalities observed late in the study included mild dysmorphic changes (ie, megaloblastic features) in 2 cats, and a significant decrease (60% of controls, P < .001) in the frequencies of burst-forming units erythroid (BFU-E) in marrow cultures of FIV-seropositive cats compared with uninfected control cats.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavior of hematopoietic stem cells in a large animal.

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 initial phase of clonal disequilibrium, however, extended for 1-4.5 years (and not 2-6 months as seen in murine experiments). After this subsided, all progenitor cells from some animals expressed a single parental G6PD phenotype, suggesting that blood cell production could be stably maintained by the progeny of one (or a few) cells. As the hematopoietic demand of a cat (i.e., number of blood cells produced per lifetime) is over 600 times that of a mouse, this provides evidence that an individual hematopoietic stem cell has a vast self-renewal and/or proliferative capacity. The long phase of clonal instability may reflect the time required for stem cells to replicate sufficiently to reconstitute a large stem cell reserve.

Hematopoiesis in asymptomatic cats infected with feline immunodeficiency virus.

Anemia and neutropenia often develop in cats that are infected with the feline immunodeficiency virus (FIV), a lentivirus biologically similar to the human immunodeficiency virus (HIV). To assess the role of FIV in the pathogenesis of these abnormalities, marrow culture studies were performed on nine asymptomatic, hematologically normal cats that were chronically infected with FIV. In these experiments, the frequencies of granulocyte/macrophage progenitors (CFU-GM) and early and late erythroid progenitors (CFU-E and BFU-E, respectively) were equivalent to progenitor frequencies in simultaneously studied uninfected control cats. Asymptomatic FIV infection was not associated with a change in the cell-cycle kinetics of CFU-E, BFU-E, or CFU-GM, nor was there an alteration in the dose-response of BFU-E or CFU-GM to hematopoietic growth factors present in fibroblast-derived conditioned medium. Sera from FIV-infected cats supported progenitor growth in vitro as well as normal cat sera. Furthermore, there was no evidence that these sera contained complement-fixing antibodies that recognized hematopoietic progenitors. Therefore, these data show that the in vitro behavior of hematopoietic progenitors is not affected by FIV infection alone, and they are in agreement with recent evidence that human progenitors are not a major target of HIV infection. It is likely that factors associated with progressive immunodeficiency, opportunistic infections, nutritional deficiencies, or malignancies play significant roles in the cytopenias that develop during the symptomatic disease induced by FIV, and by analogy, HIV. Prospective marrow culture studies of FIV-infected cats that develop hematologic abnormalities should provide a valuable animal model of acquired immunodeficiency syndrome-associated hematologic disorders.