Johanne Cashman

Sustained proliferation, multi‐lineage differentiation and maintenance of primitive human haemopoietic cells in NOD/SCID mice transplanted with human cord blood

Time course studies of sublethally irradiated non‐obese mice with severe combined immunodeficiency (NOD/SCID mice) transplanted intravenously with 107 human cord blood cells showed a rapid and parallel regeneration of human erythroid, granulopoietic, megakaryopoietic and B‐lymphoid progenitors, as well as more primitive subpopulations of CD34+ cells (defined by their multi‐lineage in vitro colony‐forming ability, coexpression of Thy‐1, or functional activity in long‐term culture‐initiating cell [LTC‐IC] assays), in the marrow, spleen and blood. Maximum numbers of human cells were reached within 6 weeks and were then sustained for another 18–20 weeks. 3H‐thymidine suicide studies showed all types of in vitro clonogenic human progenitors tested and the human LTC‐IC to be proliferating in vitro throughout this period. A 2‐week course of injections of human Steel factor, interleukin‐3, granulocyte‐macrophage colony‐stimulating factor and erythropoietin given just prior to assessment of the mice had no effect on any of these human engraftment parameters. 4–6 weeks post‐transplant, the marrow of primary NOD/SCID recipients contained human cells that were able to regenerate lymphopoiesis and/or myelopoiesis in secondary irradiated NOD/SCID mice. These findings establish a baseline for the kinetics of engraftment, multi‐lineage differentiation and self‐renewal of human cord blood stem cells in this xenogeneic transplant model and thus set the stage for future studies of their regulation in vivo.

Methodology of long-term culture of human hemopoietic cells

Long-term culture (LTC) of hemopoietic cells has become a relatively specific operational term that is used to refer to a system in which very primitive hemopoietic cells can survive, proliferate, and differentiate into precursors of many lineages in the absence of exogenously added growth factors, but in the presence of other “stromal” cells. The latter are also of marrow origin but are developmentally unrelated to hemopoietic cells. Not surprisingly, therefore, a knowledge of the essential components of the system is required to initiate and maintain cultures in which sustained hemopoiesis will be reproducibly obtained. A considerable body of empirical data has been accumulated to define procedures that achieve this with normal human marrow, and analysis of the system itself has provided an understanding of some of the cellular and molecular dynamics that take place. This review surveys some of the more important features of LTC of human hemopoietic cells, provides associated methodologic information, and summarizes some current and anticipated applications.

Defective regulation of leukemic hematopoiesis in chronic myeloid leukemia

Over the last two decades considerable knowledge has been acquired about the distribution of cell types within the dominant leukemic (Ph+/BCR-ABL+) clone that results in human chronic myeloid leukemia (CML). Evidence is now growing to indicate that three key biological changes affecting the development of such clones are: (1) an increased probability of differentiation at the level of the most primitive leukemic stem cells; (2) an increased turnover rate of the leukemic progenitors at all stages of differentiation: and (3) their increased ability to survive under conditions of factor-deprivation. Such a model explains the long latent period for the development of CML as well as why normal stem cells may persist in large numbers but still fail to compete in contributing to the daily output of mature blood cells in patients with disease. The recent development of new genetic and transplant models of human CML may now allow the molecular basis of these biological disturbances to be delineated and more effective therapeutic strategies developed.

Clinical signficance of long-term cultures of myeloid blood cells

The long-term maintenance of primitive hemopoietic precursor populations in cultures of human marrow was first described in 1981. This system, which was developed following previous work with murine marrow, appears to establish conditions that reproducibly allow the continuous turnover of a number of primitive progenitor cells, detected by their capacity upon transfer into semisolid assay cultures to generate limited numbers and types of mature blood cells. If not transferred, only those hemopoietic cells that are committed to the granulopoietic pathway are able to undergo terminal maturation. The demonstrated localization of the most primitive hemopoietic cells within the adherent fraction, primarily composed of nonhemopoietic mesenchymal elements expressing markers of fibroblasts, adipocytes, endothelial cells, and smooth-muscle cells has provided indirect evidence that interactions between these cells may be key to the survival and functional integrity of normal stem cells in this system. Such a concept has received additional support from recent studies on the cell cycle control of primitive hemopoietic cells located in and dependent on this adherent network of nonhemopoietic elements. Applications of this culture system to neoplastic populations of hemopoietic cells has revealed a number of intriguing differences in their behavior. Under conditions where maintenance of neoplastic hemopoiesis can be achieved, the most primitive progenitor classes remain continuously in cycle as they do in vivo. Thus the same inability to respond to signals that induce a noncycling state in their normal counterparts appears to be reproduced in the long-term culture system. For some populations, e.g., most CML marrows and many AML marrows, neoplastic hemopoiesis fails to become established. Although the reasons for this are not yet clear, this behavior is of interest, not only because it offers a sensitive method for detecting residual normal cells, but also as a practical approach to purging marrows of leukemic cells for autologous marrow transplantation.

Variable expression of features of normal and neoplastic stem cells in patients with thrombocytosis.

Summary Essential thrombocytosis (ET) is currently diagnosed by histopathologic assessment of the marrow after exclusion of a secondary cause or another myeloproliferative disorder. To evaluate the potential of more direct diagnostic methods, we compared the frequency and association of several abnormal features characteristic of neoplastic precursors in 32 patients presenting with platelet counts >500.109/1. Assays for erythropoietin (Ep)‐independent erythroid progenitors were performed on all patients, determination of the cycling status of circulating progenitors on 27. and assessment of granulocyte clonality on 15. In most, but not all, patients deregulated progenitor turnover. Ep‐independent progenitors and clonal granulocytes were concordant findings. The presence of polyclonal granulocytes and lack of evidence of abnormalities in Ep‐dependence or progenitor cycling were also concordant findings in most, but not all patients. Thus, normal (i.e. polyclonal) granulocytes may be produced in occasional patients in spite of the presence of a neoplastic clone. Interestingly, one third of patients thought to have ET on the basis of blood and marrow histopathology showed no abnormalities previously associated with neoplastic progenitors. These findings suggest variability in dominance of the neoplastic clone in some ET patients and the potential utility of a multifaceted laboratory approach to investigate the underlying pathology in patients with thrombocytosis.

3 Cytokines acting early in human haematopoiesis

Summary In long-term cultures (LTC) of human haematopoietic cells, primitive progenitors termed LTC-initiating cells can be maintained for several months and will differentiate to produce clonogenic cells and mature granulocytes and macrophages when provided with a supportive feeder layer of adherent mesenchymal cells. Primitive haematopoietic cells become associated with this feeder layer and their proliferative status and differentiation are regulated by their interaction with these feeder cells and the growth factors they produce. Both positive and negative regulators are generated in LTC and the balance between these diverse factors is readily manipulated by both direct and indirect mechanisms which appear to operate in a localized fashion. These features parallel those believed to characterize the mechanisms that regulate haematopoiesis in the bone marrow microenvironment in vivo and suggest that further analysis of the LTC system will be useful in delineating the full mystery of this process.