Growth of human hematopoietic cells in immunodeficient mice conditioned with cyclophosphamide and busulfan.

Human hematopoietic cells survive and proliferate for at least 10 weeks in severe combined immunodeficient mice prepared with the cytotoxic drugs busulfan and cyclophosphamide. The human cells growing in the mice can be detected by in situ hybridization using a probe detecting human repetitive DNA or by staining the cells with antihuman antibodies (anti-CD45 and anti-HLA I). Busulfan/cyclophosphamide-treated mice were injected with a wide range of cell doses, ranging from 5 to 50 million unfractionated bone marrow cells and 2 to 40 million low density bone marrow cells. Animals were killed at 1, 3, 5, 7 and 10 weeks after transplantation. Human cells were found in many animals and could be detected as early as one week after transplantation. The peak of repopulation was at two to five weeks, but in some animals human cells could be detected for as long as 10 weeks. Many of the human cells expressed high levels of glycophorin, but mature human erythrocytes were not found. The human cells were not uniformly distributed throughout the marrow. They grew in small clusters in the subepiphyseal region. The extent of human hematopoietic repopulation in the mouse was extremely variable. At no time and at no dose was repopulation achieved in all of the animals. Treatment with human growth factors is not necessary for the survival of the human hematopoietic cells but, in their absence, normal hematopoiesis does not occur.

Effects of fibroblast growth factor-4 (k-FGF) on long-term cultures of human bone marrow cells.

Fibroblast growth factor-4 (FGF-4), a highly mitogenic protein encoded by the k-fgf/hst oncogene, stimulates the growth of a variety of cells of mesenchymal and neuroectodermal origin. Addition of FGF-4 to human long-term bone marrow cultures increased both the cell density of the stromal layer and the number of hematopoietic colony forming cells in the cultures in a dose-dependent manner. Hematopoiesis in the stromal layer persisted for up to 8 months. Erythropoiesis was maintained for up to 4 weeks, but granulocytes were the predominant nonadherent cell type. Cultures treated with FGF had increased numbers of monocytes compared with control cultures and some CD14+, CD45+ monocytes could still be detected after 8 months of continuous culture. The addition of the growth factor increased the rate of growth of the stromal layer and appeared to delay its senescence. Subcultures made in the presence of FGF-4 had up to 10-fold increases in plating efficiency and grew as relatively uniform monolayers. These subcultures retained the capacity to support hematopoiesis for several months, while untreated subcultures, made without FGF-4, grew erratically and generally lost the capacity to support hematopoiesis within 4 to 6 weeks. The improved growth after subculture greatly enhanced the reliability of limit-dilution assays of multipotential hematopoietic stem cells that use stromal cell monolayers. The primary effect of FGF-4 appeared to be on the stromal cells of the long-term bone marrow cultures, but a direct effect on hematopoietic progenitors could not be ruled out.