Search PubMed⌕ Search

Biomedical subjects

J E Dick

Publications and source records attributed to J E Dick.

At least 55 records · Page 3Linked to original sources
PubMed1996

Human stem cell assays in immune-deficient mice.

Much of our understanding of the organization of the cells that comprise the hematopoietic system and the cellular and molecular mechanisms that regulate their development is derived from mouse models. However, knowledge of the human hematopoietic system and identification of human stem cells have, until recently, been hampered by the absence of in vivo assays that measure their repopulation capacity. The development of methods to transplant normal and leukemic human hematopoietic cells into immune-deficient SCID mice provides the foundation for human stem cell assays. This review will focus on recent evidence that normal and leukemic human stem cells can be assayed in these systems.

Animals↗
PubMed1995

A recombinant bcl-x s adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells.

Many cancers overexpress a member of the bcl-2 family of inhibitors of apoptosis. To determine the role of these proteins in maintaining cancer cell viability, an adenovirus vector that expresses bcl-xs, a functional inhibitor of these proteins, was constructed. Even in the absence of an exogenous apoptotic signal such as x-irradiation, this virus specifically and efficiently kills carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating severe combined immunodeficient mice were refractory to killing by the bcl-xs adenovirus. These results suggest that Bcl-2 family members are required for survival of cancer cells derived from solid tissues. The bcl-xs adenovirus vector may prove useful in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation.

Adenoviruses, Human↗
PubMed1995

Engraftment of immune-deficient mice with primitive hematopoietic cells from beta-thalassemia and sickle cell anemia patients: implications for evaluating human gene therapy protocols.

Permanent correction of genetic deficiencies of the hematopoietic system requires gene transfer into stem cells and long-term lineage specific expression after autologous transplantation. However, progress to develop gene therapy protocols has been hampered by the absence of in vivo assays that detect genetically deficient human hematopoietic stem cells and their diseased differentiated progeny. The establishment of systems to transplant human cells into immune-deficient SCID mice provides such an assay. We report that primitive bone marrow cells from beta-thalassemia major and sickle cell anemia patients engraft immune-deficient mice, giving rise to high levels of human erythroid and myeloid cells in response to treatment with human cytokines. The bone marrow of transplanted mice contained the entire erythroid lineage from BFU-E to mature erythrocytes expressing human gamma, beta or beta s-globin. Moreover, human erythroid cells from mice transplanted with sickle cell anemia bone marrow showed characteristic sickling under reducing conditions in an in vitro assay. This model provides a powerful in vivo system that can be used to evaluate the efficiency of globin gene transfer into primitive human hematopoietic cells, lineage-specific expression in mature erythrocytes, and ultimately correction of the cellular defect found in the erythroid lineage.

Anemia, Sickle Cell↗
PubMed1994

Rescue of T cell-specific V(D)J recombination in SCID mice by DNA-damaging agents.

Assembly of antigen receptor V (variable), D (diversity), and J (joining) gene segments requires lymphocyte-specific genes and ubiquitous DNA repair activities. Severe combined immunodeficient (SCID) mice are defective in general double-strand (ds) DNA break repair and V(D)J coding joint formation, resulting in arrested lymphocyte development. A single treatment of newborn SCID mice with DNA-damaging agents restored functional, diverse, T cell receptor beta chain coding joints, as well as development and expansion of thymocytes expressing both CD4 and CD8 coreceptors, but did not promote B cell development. Thymic lymphoma developed in all mice treated with DNA-damaging agents, suggesting an interrelation between V(D)J recombination, dsDNA break repair, and lymphomagenesis.

Animals↗
PubMed1994

Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice.

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.

Animals↗
PubMed1994

Expression of bcr-abl abrogates factor-dependent growth of human hematopoietic M07E cells by an autocrine mechanism.

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.

Cell Division↗
PubMed1994

A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.

Most human acute myeloid leukaemia (AML) cells have limited proliferative capacity, suggesting that the leukaemic clone may be maintained by a rare population of stem cells. This putative leukaemic stem cell has not been characterized because the available in vitro assays can only detect progenitors with limited proliferative and replating potential. We have now identified an AML-initiating cell by transplantation into severe combined immune-deficient (SCID) mice. These cells homed to the bone marrow and proliferated extensively in response to in vivo cytokine treatment, resulting in a pattern of dissemination and leukaemic cell morphology similar to that seen in the original patients. Limiting dilution analysis showed that the frequency of these leukaemia-initiating cells in the peripheral blood of AML patients was one engraftment unit in 250,000 cells. We fractionated AML cells on the basis of cell-surface-marker expression and found that the leukaemia-initiating cells that could engraft SCID mice to produce large numbers of colony-forming progenitors were CD34+ CD38-; however, the CD34+ CD38+ and CD34- fractions contained no cells with these properties. This in vivo model replicates many aspects of human AML and defines a new leukaemia-initiating cell which is less mature than colony-forming cells.

ADP-ribosyl Cyclase↗
PubMed1994

Gene transfer into hematopoietic stem cells: long-term maintenance of in vitro activated progenitors without marrow ablation.

Adoptive transfer of genetically modified somatic cells will play an increasingly important role in the management of a wide spectrum of human diseases. Among the most appealing somatic cells as potential gene transfer vehicles are hematopoietic cells, because of their wide distribution and their well-characterized capacities for proliferation, differentiation, and self-renewal. Genes can be readily transferred into short-lived and lineage-restricted hematopoietic cells, but there remains a need to develop reliable methods for gene transfer into hematopoietic stem cells in large animals. In this work, we used a gene transfer approach in which hematopoietic cells in long-term marrow cultures were exposed to the replication-defective retrovirus N2, bearing the reporter gene neo, on multiple occasions during 21 days of culture. Genetically marked cultured autologous cells were infused into 18 canine recipients in the absence of marrow-ablative conditioning. neo was detected by Southern blotting and/or the polymerase chain reaction in the marrow, blood, marrow-derived granulocyte/macrophage and erythroid progenitors, and cultured T cells in dogs after infusion. In most dogs, the proportion of long-term marrow culture cells contributing to hematopoiesis rose during the first 3 months after infusion and peaked within the first 6. The maximal levels attained were between 10% and 30% G418-resistant (neo-positive) granulocyte/macrophage progenitors. At 12 months, five dogs maintained greater than 10% G418-resistant progenitors, and for two of them this level exceeded 20%. Two dogs had greater than 5% G418-resistant hematopoietic progenitors at 24 months after infusion. Our data suggest that very primitive hematopoietic progenitors are maintained in long-term marrow cultures, where they can be triggered into entering the cell cycle. In vivo, these activated cells likely continue normal programs of proliferation, differentiation, and self-renewal. Their progeny can be maintained at clinically relevant levels for up to 2 years without the requirement that endogenous hematopoiesis be suppressed through chemo- or radiotherapy prior to adoptive transfer. Long-term marrow culture cells may thus be ideal targets for gene therapy involving adoptive transfer of transduced hematopoietic cells.

Animals↗
PubMed1994

SCID mice as an in vivo model of human cord blood hematopoiesis.

Cord blood is increasingly used as an alternative stem cell source for autologous and allogeneic transplantation, particularly in pediatric patients. We therefore adopted our protocol for transplanting human adult bone marrow cells into severe combined immunodeficient (SCID) mice [1] to develop an in vivo model for cord blood hematopoiesis. Intravenous injection of unfractionated or Ficoll-separated cord blood cells into sublethally irradiated SCID mice led to high levels of human hematopoiesis in the majority of the recipients [2]. Multilineage human hematopoiesis including committed and multipotential myeloerythroid progenitors as well as CD19+ B-lymphoid cells were observed in the murine bone marrow for at least 18 weeks. Together, these data indicate that the SCID mice were engrafted with an immature cell that was able to maintain multiple progenitor lineages in vivo. In contrast to our experiences with adult bone marrow, high levels of human cell engraftment in the mouse could be achieved without exogenous cytokine treatment, suggesting that the cord blood cells respond differently to the murine microenvironment. Alternatively, the cord blood cells might have been able to provide themselves with the necessary growth factors in a paracrine fashion. This model will be useful in gaining new insights into the biology of immature human cord blood progenitors and cord blood transplantation.

Animals↗
PubMed1993

Engraftment of human lymphoid cells into newborn SCID mice leads to graft-versus-host disease.

Although there has been considerable progress in transplanting normal human hematopoietic cells into immune-deficient mice, the establishment of a functional human immune system has proved to be difficult. Current methods of introducing mature human lymphoid cells into adult SCID mice lead to oligoclonal engraftment with restricted dissemination to various organs. We have attempted to improve human lymphoid cell engraftment in mice, both qualitatively and quantitatively, by injecting human bone marrow cells and peripheral blood leukocytes intraperitoneally into newborn SCID mice. Newborn mice were used as recipients because certain immune functions such as natural killer cell activity do not develop until several weeks after birth and the numerous growth factors secreted in young mice may facilitate the engraftment and proliferation of transplanted human cells. At various times after transplantation, the presence of human cells in different organs was determined by Southern blot analysis using a human specific probe. Within 4 weeks, 70% of the mice were engrafted with human cells. Human cell engraftment of the bone marrow, spleen, lungs, kidneys, liver, and thymus exceeded 10% in at least 40% of the transplanted mice; most of these highly engrafted mice were sick. Flow cytometry and immunocytochemistry indicated these organs were heavily infiltrated with mature T and B lymphocytes. Histologic and molecular analysis showed massive human cell infiltrates within the liver, lung and spleen. The presence of human IgG and IgM antibodies against mouse red blood cells provided evidence that the engrafted human cells retained some immune function. Mice transplanted with peripheral blood leukocytes from donors that were allergic to mouse antigens engrafted to the same extent as normal cells but in addition developed the classical symptoms of acute allogeneic graft-versus-host disease (GVHD) including infiltrates of the skin, gut, and liver. The newborn SCID system provides a new in vivo model to study human xenoreactivity and GVHD.

Animals↗
PubMed1993

Clinical and pathological findings in dogs following supralethal total body irradiation with and without infusion of autologous long-term marrow culture cells.

We developed a canine model for autologous bone marrow transplantation (AuBMT) with long-term marrow culture (LTMC) cells. Marrow was harvested from nine normal dogs. Harvests from dogs 2-7 were placed into 21 day LTMC. Cells in LTMC from dogs 4-7 were labelled with the neomycin phosphotransferase gene neo. Dogs were given 60Co total body irradiation (TBI) and then infused with LTMC cells: dog 1 received 500 cGy TBI and 2.08 x 10(8)/kg uncultured marrow cells. Dogs 2-7 received 600-800 cGy TBI and 0.07-0.45 x 10(8)/kg LTMC cells. Dogs 8 and 9 received 600 and 800 cGy TBI, respectively, but no infusion of marrow or LTMC cells. For all dogs, profound myelosuppression developed during week 1 and pyrexia developed during week 2. Enrofloxacin was given from one day before TBI until a peripheral neutrophil count > 1.0 x 10(9)/L was achieved, which eliminated Escherichia coli from feces. Dogs 1, 2 and 5-9 also received gentamicin and/or combination beta-lactam antibiotics. Numerous platelet transfusions were needed to control hemorrhages in all dogs except dog 1. Dog 1 achieved neutrophils > 1.0 x 10(9)/L on day 15, while dogs 2 and 5-9 achieved this count on days 33-48. Dogs 3 and 4 died on days 17 and 18, respectively, of beta-hemolytic streptococcal sepsis and hemorrhage, with no evidence of hematopoiesis at necropsy. The marker gene, neo, was documented in lymphoid and myeloid cells of dogs 5-7 up to 21 months post-AuBMT. Our studies indicate that dogs can recover following supralethal TBI and can survive the delayed engraftment associated with AuBMT using LTMC cells, if they receive intensive platelet and antimicrobial therapy. Used prophylactically for such therapy, enrofloxacin achieved selective intestinal decontamination, but did not prevent sepsis when used as the sole antimicrobial agent during myelosuppression. Furthermore, our studies indicate that infused LTMC cells, at the above doses, can contribute to hematopoietic recovery, but are not essential for recovery following TBI, and do not shorten the period of prolonged profound myelosuppression induced by TBI.

Animals↗
PubMed1992

Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice.

Severe combined immunodeficient (SCID) mice transplanted with human bone marrow were treated with human mast cell growth factor, a fusion of interleukin-3 and granulocyte-macrophage colony-stimulating factor (PIXY321), or both, starting immediately or 1 month later. Immature human cells repopulated the mouse bone marrow with differentiated human cells of multiple myeloid and lymphoid lineages; inclusion of erythropoietin resulted in human red cells in the peripheral blood. The bone marrow of growth factor-treated mice contained both multipotential and committed myeloid and erythroid progenitors, whereas mice not given growth factors had few human cells and only granulocyte-macrophage progenitors. Thus, this system allows the detection of immature human cells, identification of the growth factors that regulate them, and the establishment of animal models of human hematopoietic diseases.

Animals↗
PubMed1992

Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors.

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.

Animals↗
PubMed1992

Murine models of normal and neoplastic human haematopoiesis.

The ability to transplant human haematopoietic cells into immune deficient mice provides a unique opportunity for studying the organization and regulation of the human stem cell developmental program. One challenge for the future will be to reconstitute mice with functional cells of all lineages. The creation of animal models of many haematopoietic diseases should revolutionize the development and testing of novel therapeutic strategies. Significant progress has been made in establishing models of human neoplastic diseases such as leukaemia and lymphoma. Although the number of patients examined is still small, it appears that there may be a correlation between growth in immune deficient mice and clinical outcome. Future studies should examine the range of diseases that grow in mice and whether in vivo assays have prognostic value clinically. These leukaemia models, in conjunction with high efficiency gene transfer techniques, offer a powerful approach to examine the biological consequences of expressing oncogenes or other key regulatory genes on human leukaemic transformation and progression.

Animals↗
PubMed1992

Differential kinetics of engraftment and induction of CD10 on human pre-B leukemia cell lines in immune deficient scid mice.

The sensitivity of the scid mouse model was assessed by comparing the growth of two pre-B acute lymphoblastic leukemia (ALL) cell lines, A1 and G2, established from patients at relapse. When cell numbers varying from 10(4) to 10(7) were injected intravenously into scid mice, advanced growth and dissemination of leukemia was observed at 10-12 weeks with the G2 cells. Bone marrow, spleen and thymus contained high levels of human leukemic cells and infiltration into lung, kidney, liver, and brain was observed. Two of three mice grafted with only 100 cells showed high levels of infiltration at 15 weeks, suggesting that 100 G2 cells was near the limiting cell number that could produce disseminated leukemia. With the A1 line, a minimum of 10(5) cells was needed to obtain dissemination to liver, lung, brain, and kidney; a low level of spleen infiltration occurred and thymus invasion was not observed. In vitro, both lines showed a density dependent growth in clonogenic assays but the cloning efficiency of the A1 line was 10-fold higher than for G2 cells. These results indicate that G2 and A1 lines have a dissimilar aggressiveness in vivo which does not correlate with clonogenic assay in vitro. Neither G2 nor A1 lines, growing in vitro, expressed CD10/CALLA on their surface, despite low levels of antigen on the freshly obtained relapse samples. Although A1 cells remained CD10-negative in the scid mice, G2 cells showed detectable levels of CD10, particularly on those cells found in the thymus. Several subclones of the G2 line were derived from isolated colonies in vitro; they were found to be CD10- in vitro, but to become CD10+ when proliferating into scid mouse thymus, suggesting the induction of CD10 by the murine microenvironment.

Animals↗