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Unseparated or Ficoll-Hypaque (Pharmacia, Piscataway, NJ)--fractionated human cord blood cells were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice. High levels of multilineage engraftment, including myeloid and lymphoid lineages, were obtained with 80% of the donor samples as assessed by DNA analysis, fluorescence-activated cell sorting (FACS), and morphology. In contrast to previous and concurrent studies with adult human bone marrow (BM), treatment with human cytokines was not required to establish high-level human cell engraftment, suggesting that neonatal cells either respond differently to the murine microenvironment or they provide their own cytokines in a paracrine fashion. Committed and multipotential myelo- erythroid progenitors were detected using in vitro colony assays and FACS analysis of the murine BM showed the presence of immature CD34+ cells. In addition, human hematopoiesis was maintained for at least 14 weeks providing further evidence that immature hematopoietic precursors had engrafted the murine BM. This in vivo model for human cord blood- derived hematopoiesis will be useful to gain new insights into the biology of neonatal hematopoietic cells and to evaluate their role in gene therapy. There is growing evidence that there are ontogeny-related changes in immature human hematopoietic cells, and therefore, the animal models we have developed for adult and neonatal human hematopoiesis provide useful tools to evaluate these changes in vivo.

Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony- forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst- forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood- derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.

Retroviral vectors containing the selectable bacterial gene for G418 resistance (neo) were used to demonstrate gene transfer into primary human bone-marrow progenitor cells. To obtain populations of cells in which a high proportion of cells were expressing the neo gene, several important modifications were made to earlier procedures. Cells from normal donors were infected in vitro, were exposed to high concentrations of G418 for two days in liquid culture to enrich for cells expressing the neo gene, and were plated in semisolid medium. Gene transfer and expression were detected in colonies arising from progenitors of granulocyte-macrophage and erythroid lineages. Survival curves indicated that a high proportion of progenitor cells, approaching 100%, were G418 resistant. Furthermore, addition of growth factors contained in 5637-conditioned medium to the bone marrow improved the recovery of G418-resistant progenitors twofold to threefold. In addition to these biological measurements of gene expression in progenitor cells, significant levels of neo-specific RNA, similar to the levels of RNA expression in the virus-producing fibroblast cell line, were detected in the bone marrow cells after preselection. These results demonstrate that retrovirus vectors can be used successfully to transfer genes at high efficiency into progenitor cells in the human blood-forming system.

Progress in understanding the abnormal regulation of hematopoiesis in chronic myelogenous leukemia (CML) would be facilitated if neoplastic cells, at all stages of the disease, could be studied in an animal model. In this report, we show that irradiated severe combined immunodeficient (SCID) mice can be transplanted with both normal (Philadelphia chromosome [Ph]-negative) and neoplastic (Ph+) cells from CML patients with either chronic or blast phase disease. Mice transplanted with peripheral blood (PB) or bone marrow (BM) cells from 9 of 12 chronic phase CML patients were well engrafted with human cells including multilineage colony-forming progenitors and CD34+ cells for at least 90 days posttransplantation. Repeated posttransplant injections of cytokines did not enhance the number of engrafted human cells. Interestingly, approximately 70% of the human progenitors found in the engrafted SCID BM were Ph-, suggesting that the growth of primitive normal cells is favored in this in vivo transplant model. A similar number of normal cells were found in mice transplanted with either PB or BM cells, suggesting that elevated numbers of primitive normal cells are present in CML PB. When cells from patients with CML in either myeloid or lymphoid blast crisis were transplanted into SCID mice, the BM of these mice was more rapidly repopulated and to a higher level than that observed with transplants of chronic phase cells. Moreover, all human colony-forming progenitors present in the BM of mice transplanted with blast crisis cells were Ph+, and the majority of cells showed the same morphological features of the blast crisis cells originally transplanted. These experiments provide a starting point for the creation of an animal model of CML and establish the feasibility of using this model for the future characterization of transplantable CML stem cells during disease progression.

The ability to transfer new genetic material into human hematopoietic cells provides the foundation for characterizing the organization and developmental program of human hematopoietic stem cells. It also provides a valuable model in which to test gene transfer and long-term expression in human hematopoietic cells as a prelude to human gene therapy. At the present time such studies are limited by the absence of in vivo assays for human stem cells, although recent descriptions of the engraftment of human hematopoietic cells in immune-deficient mice may provide the basis for such an assay. This study focuses on the establishment of conditions required for high efficiency retrovirus- mediated gene transfer into human hematopoietic progenitors that can be assayed in vitro in short-term colony assays and in vivo in immune- deficient mice. Here we report that a 24-hour preincubation of human bone marrow in 5637-conditioned medium, before infection, increases gene transfer efficiency into in vitro colony-forming cells by sixfold; interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) provide the same magnitude increase as 5637-conditioned medium. In contrast, incubation in recombinant growth factors IL-1, IL-3, and granulocyte- macrophage colony-stimulating factor increases gene transfer efficiency by 1.5- to 3-fold. Furthermore, preselection in high concentrations of G418 results in a population of cells significantly enriched for G418- resistant progenitors (up to 100%). These results, obtained using detailed survival curves based on colony formation in G418, have been substantiated by directly detecting the neo gene in individual colonies using the polymerase chain reaction. Using these optimized protocols, human bone marrow cells were genetically manipulated with a neo retrovirus vector and transplanted into immune-deficient bg/nu/xid mice. At 1 month and 4 months after the transplant, the hematopoietic tissues of these animals remained engrafted with genetically manipulated human cells. More importantly, G418-resistant progenitors that contained the neo gene were recovered from the bone marrow and spleen of engrafted animals after 4 months. These experiments establish the feasibility of characterizing human stem cells using the unique retrovirus integration site as a clonal marker, similar to techniques developed to elucidate the murine stem cell hierarchy.

The introduction of a retrovirus vector expressing p210bcr-abl (P210) into the human factor-dependent cell line M07E resulted in the rapid outgrowth of factor-independent cells. Early after infection, four factor-independent clones were isolated and analyzed in greater detail along with mass populations obtained from separate infections. High levels of P210 tyrosine kinase activity were measured in the factor- independent cells. The mass populations and three of the four clones remained responsive to exogenous growth factors. Concentrated conditioned media isolated from the factor-independent populations and from all clones contained biologically active granulocyte-macrophage colony-stimulating factor (GM-CSF); interleukin-3 (IL-3) was detected at low levels in the mass population and in two of the clones. Neutralizing antibodies to IL-3, GM-CSF, and mast cell growth factor inhibited proliferation of the factor responsive clones by 60% to 90%. These results indicate that the growth autonomy of the P210-expressing M07E cells was acquired via an autocrine mechanism. In addition to factor-independent growth, P210-expressing M07E cells readily acquired a more mature megakaryocytic phenotype compared with control M07E cells. These data provide experimental evidence that expression of P210 tyrosine kinase in human hematopoietic cells induced growth factor secretion resulting in a pleiotropic effect on growth factor dependence and differentiation.