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Biomedical subjects

J E Dick

Publications and source records attributed to J E Dick.

At least 73 records · Page 4Linked to original sources
PubMed1991

Gene transfer into normal human hematopoietic cells using in vitro and in vivo assays.

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.

Adult↗
PubMed1991

Immune-deficient mice as models for human hematopoietic disease.

The growth of human hematopoietic cells in immune-deficient mice promises to revolutionize our ability to study the normal developmental program of human hematopoiesis and the biological consequences of aberrant proliferation and differentiation. Advances in stem cell purification will require assays to test for function, and the identification and the characterization of novel hematopoietic growth factors will be aided by in vivo experiments. The engraftment of hematopoietic cells directly from patients with disease should ultimately lead to animal models for many human hemopathies and leukemias. Already important preliminary experiments have established the feasibility of such models for leukemia, cancer, infectious diseases, and autoimmunity. The production of human antibodies directed against toxic agents for which humans cannot be immunized could provide the basis for improved pharmaceuticals. Although an important foundation has been laid, much work remains to explore the full potential of this mouse transplantation system.

Animals↗
PubMed1991

Transplantation of normal and leukemic human bone marrow into immune-deficient mice: development of animal models for human hematopoiesis.

The successful engraftment of human hematopoietic cells into immune-deficient mice offers a novel approach to characterize the developmental program of human hematopoiesis. While it is not yet possible to achieve high-level engraftment of all human lineages, several methods have been developed to successfully engraft human lymphoid cells and reconstitute partial immune function. In addition to mature cell types, there is evidence that progenitors and perhaps stem cells can engraft the murine bone marrow. Recent work suggests that provision of exogenous human cytokines significantly increases the level of human cell engraftment and stimulates the development of multiple lineages. Progress has also been made to establish animal models of human hematopoietic diseases such as leukemia, autoimmunity, and infectious diseases.

Animals↗
PubMed1991

Mouse models for human hematopoiesis.

The successful engraftment of human hematopoietic cells into immune-deficient mice offers a novel approach to characterize the developmental program of human hematopoiesis. While it is not yet possible to achieve high level engraftment of all human lineages, several methods have been developed to successfully engraft human lymphoid cells and reconstitute partial immune function. In addition to mature cell types, there is evidence that progenitors and perhaps stem cells can engraft the murine bone marrow. Recent work suggests that provision of exogenous human cytokines significantly increases the level of human cell engraftment and stimulates the development of multiple lineages. Progress has been made to establish animal models of human hematopoietic disease such as leukemia, autoimmunity, and infectious diseases. One major challenge for the future will be reconstitution of mice with the entire human hematopoietic system.

Animals↗
PubMed1991

Immune-deficient mice as models of normal and leukemic human hematopoiesis.

A complete understanding of the organization of the human hematopoietic stem cell hierarchy and of the molecular events regulating the stem cell developmental program has been hampered by the absence of suitable in vivo stem cell assays. Perturbations in the normal stem cell program that lead to neoplastic growth are also difficult to study because leukemic cells do not grow readily in culture. Recent advances in the transplantation of human cells into immune-deficient mice provide an unprecedented opportunity to study human hematopoiesis--both normal and abnormal--in the context of a small animal model. Here I review several of these new animal models and the basic concepts of murine and human hematopoiesis which led to their development.

Animals↗
PubMed1989

A model of human acute lymphoblastic leukemia in immune-deficient SCID mice.

A human acute lymphoblastic leukemia (ALL) cell line that was transplanted into immune-deficient SCID mice proliferated in the hematopoietic tissues, invaded various organs, and led to the death of the mice. The distribution of leukemic cells in SCID mice was similar to the course of the disease in children. A-1 cells marked with a retrovirus vector showed clonal evolution after the transplant. SCID mice that were injected with bone marrow from three patients with non-T ALL had leukemic cells in their bone marrow and spleen. This in vivo model of human leukemia is an approach to understanding leukemic growth and progression and is a novel system for testing new treatment strategies.

Animals↗
PubMed1989

Establishment of assays for human hematopoietic cells in immune deficient mice.

Our knowledge of the organization and regulation of the murine hematopoietic stem cell hierarchy has, in large measure, been the result of the development of in vivo reconstitution assays for cells of various levels of differentiation. For example, many basic stem cell concepts were established using CFU-S as a paradigm. Although there are no clonal assays for the earliest pluripotential cells, they can nevertheless be quantitated based on their ability to reconstitute animals with a deficient hematopoietic system. A similar understanding of the human hematopoietic system has lagged because of the lack of a suitable assay for human stem cells. Using several different approaches it has become possible to transplant human hematopoietic cells into immune-deficient mice. Since both lymphoid and myeloid cells have been engrafted, these experiments may lay the foundation for an assay for human pluripotent stem cells. Moreover it should now become possible to establish experimental animal models of a large number of human hemopathies including leukemia.

Animals↗
PubMed1988

Engraftment of immune-deficient mice with human hematopoietic stem cells.

A system in which immune-deficient mice are repopulated with cells from the human myeloid lineage, and that provides an in vivo stem cell assay for human hematopoietic cells is described. Generation of the chimeric human/immune-deficient (HID) mice was dependent on the use of immune-deficient bg/nu/xid mice. Infusion of these mice with human bone marrow gave rise to increases in human macrophage progenitors during more than 5 weeks of in vivo growth, indicating the seeding, proliferation, and differentiation of human stem cells. The human identity of the progenitors was confirmed by sequence analysis and their dependence on human growth factors. The creation of HID mice lays the foundation for establishing animal models for a wide variety of human hemopathies, from leukemia to infectious disease.

Animals↗
PubMed1988

High-efficiency gene transfer and expression in normal human hematopoietic cells with retrovirus vectors.

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.

Drug Resistance, Microbial↗
PubMed1987

High efficiency gene transfer and expression in normal murine B lymphocytes.

The introduction of new genetic information into hematopoietic cells offers a new approach for investigating the molecular events controlling differentiation. Retrovirus vectors have been used to transfer new genes with high efficiency into murine hematopoietic cells, primarily of the myeloid lineage. In this report, we show that vectors carrying the dominant, selectable gene for neomycin resistance (neo gene) can successfully infect normal murine B lymphocytes (CFU-B). The infected CFU-B formed colonies in vitro in high concentrations (750 micrograms/ml) of G418, a neomycin analogue. That B lymphocytes contained the neo gene was confirmed by the findings that the putative B cell colonies growing in G418 contained antibody-producing cells and that the cells responding to the B cell mitogen, LPS, were resistant to G418. Infection of normal spleen cells with different vectors containing a variety of transcriptional regulatory sequences resulted in 7-40% of the CFU-B becoming G418 resistant. Introduction of the immunoglobulin heavy chain enhancer into NEO vectors appeared to augment the expression of the neo gene, since the level of G418 resistance was higher in B cells infected with a NEO vector containing the enhancer than in cells infected with a vector lacking the enhancer.

Animals↗
PubMed1987

Generation of drug-resistant variants in metastatic B16 mouse melanoma cell lines.

Genetic instability is recognized as an important aspect of the development of tumor heterogeneity and malignancy. In a previous study [Hill et al. Science (Wash. DC), 244:998-1001, 1984], we demonstrated that metastatic variants are generated at a more rapid rate in the highly metastatic B16F10 mouse melanoma cell line than in the less metastatic B16F1 cell line. The metastatic variants were phenotypically unstable, being generated and lost at high rates; consequently, we proposed a dynamic heterogeneity model of tumor metastasis which describes these properties quantitatively. As an extension of this work, we have examined the ability of these two melanoma cell lines to generate variants resistant to the drugs methotrexate and N-(phosphonacetyl)-L-aspartate. We observed that the highly metastatic B16F10 cell line generated variants resistant to a given concentration of methotrexate or N-(phosphonacetyl)-L-aspartate at higher rates than the B16F1 cell line. We conclude that B16F10 cells are genetically less stable than B16F1 cells and since resistance to methotrexate and N-(phosphonacetyl)-L-asparate usually results from gene amplification that B16F10 cells possess increased ability to amplify DNA. This higher rate of generation of drug-resistant variants corresponds to the higher rate of generation of metastatic variants we observed previously and suggests that a gene amplification mechanism may be involved in the generation of a metastic phenotype in B16 melanoma cells.

Animals↗
PubMed1987

Modulation of gene expression in multiple hematopoietic cell lineages following retroviral vector gene transfer.

Retrovirus vectors offer a simple and highly efficient method for introducing new genes into mammalian cells. Here, we have examined the efficiency of gene transfer into hematopoietic cells with retrovirus vectors carrying the neomycin (neo) resistance gene expressed from different transcriptional regulatory regions. Direct infection of mouse bone marrow cells resulted in high efficiencies of gene transfer into a variety of myeloid progenitor cells, including pluripotent, erythroid, and granulocyte-macrophage colony-forming cells with all the vectors examined. However, the progeny derived from individual pluripotent progenitor cells infected with different vectors differed markedly in the proportion of G418-resistant progenitor cells, as judged by their ability to survive selection in the drug G418. This biological assay suggests that the highest level of expression was observed when the neo gene was expressed from constructs that contained the herpes thymidine kinase promoter rather than the viral long terminal repeat or the simian virus 40 early region promoter. In contrast, neo gene expression was highest in fibroblasts infected with the vector containing the simian virus 40 early region promoter. These results show that high and sustainable levels of gene expression in hematopoietic cells can be obtained with retrovirus vectors containing appropriate transcriptional regulatory regions.

Animals↗
PubMed1987

Retrovirus-mediated gene transfer into hematopoietic stem cells.

The ability to transfer new genetic information into murine and human hematopoietic cells with retrovirus vectors offers a powerful approach to identifying the molecular mechanisms governing stem cell development. The newly integrated provirus also serves as a clonal marker for elucidating the lineage relationships of the cells in the stem cell hierarchy. High efficiency gene transfer into human cells opens the way to developing new therapy for treating genetic diseases by introducing functional genes into deficient cells. Present transplantation technology would only permit gene therapy with bone marrow cells. However, as gene transfer and transplantation technologies improve it may be possible to consider treating diseases that affect other organs and tissues. I would like to acknowledge the support of Alan Bernstein and R. Phillips in whose labs the murine work was carried out during a post-doctoral fellowship. I also thank Sharon Kerbel for excellent assistance in the preparation of this manuscript.

Animals↗
PubMed1985

Ribonucleotide reductase activity during the senescence of normal human diploid fibroblasts in culture.

Changes in ribonucleotide reductase occur during the senescence of normal human diploid fibroblasts in culture. Enzyme activity is significantly lower in cells and extracts at high mean population doublings (MPD) as compared to fibroblasts at low MPD. Although many of the kinetic properties of the enzyme remain unaltered in cells at high MPD, changes in the extent and kinetic mechanism of inhibition of CDP and ADP reductase activity by dATP, the overall negative effector of ribonucleotide reductase, were observed. These results and a previous observation that the four deoxyribonucleotide pools are markedly altered during in vitro senescence of human diploid fibroblasts, provide evidence for a link between ribonucleotide reduction, deoxyribonucleotide pools, and the establishment of the non-proliferative or senescent state.

Cell Division↗
PubMed1985

Membrane glycoprotein changes during the senescence of normal human diploid fibroblasts in culture.

The plasma membrane is intimately involved in a variety of cellular phenomena and may play an important role in the aging of human cells in culture. Significant differences in human diploid fibroblast surface glycoproteins were observed with in vitro aging. Senescent cells bound more concanavalin A (Con A) than young cells and exhibited two distinct classes of binding sites. Cell surfaces of senescent cells incorporated less labelled mannose and more labelled fucose and glucosamine than young cell surfaces. Membranes prepared from older cells were also less effective than membrane preparations from young cells in incorporating mannose from GDP-mannose into a group of oligolipid intermediates, required for the synthesis of glycoproteins with asparagine-linked oligosaccharides. These results demonstrate novel quantitative changes in membrane structure and lectin reactivity in aging human diploid fibroblasts, which must reflect the fundamental physiological modifications in the cell that occur during senescence.

Cell Membrane↗
PubMed1985

On the importance of deoxyribonucleotide pools in the senescence of cultured human diploid fibroblasts.

Previous studies have indicated that ribonucleotide reductase may participate in a mechanism of cellular senescence, which involves modifications in deoxyribonucleotide pools, the products of the reductase reaction. Since very little information about the levels of these pools in senescing cells is currently available, an analysis of deoxyribonucleotide levels was carried out in young, old and very old normal human diploid fibroblasts, as well as in a variant human fibroblast strain with an altered replicative life span. These studies indicated that there are marked age-related perturbations in these pools which imply that they may be fundamentally important in a process determining the nonproliferative or senescent state.

Cell Line↗
PubMed1985

Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice.

We have used the random chromosomal integration sites of retrovirus vectors as unique clonal markers to analyze cell lineage relationships within the hemopoietic stem cell hierarchy. Using a high efficiency protocol for retrovirus-mediated gene transfer, anemic W/Wv mutant mice were reconstituted with bone marrow cells infected with a NEO vector. Analysis of the DNA from bone marrow, thymus, and spleen of these reconstituted W/Wv mice indicated insertion of the vector into primitive pluripotent stem cells capable of producing both myeloid and lymphoid progeny as well as into more committed stem cells apparently restricted to either the myeloid or lymphoid lineages. The neo gene was also expressed in these mice, as they contained a variety of G418 resistant in vitro colony-forming cells. These results demonstrate high-efficiency gene transfer and expression in primitive hemopoietic stem cells and provide a direct approach for analyzing the hemopoietic stem cell hierarchy.

Animals↗