Thesis (Ph. D.)--University of Adelaide, Dept of Science, 1987. / Includes bibliographical references (leaves 154-185).

Thesis (Ph. D.)--University of Adelaide, Dept. of Medicine, 1998. / Includes bibliographical references (17 leaves ).

Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 145-153).

Thesis (doctoral)--Freie Universität Berlin, 1980.

Thesis (Ph. D.)--University of Chicago, Committee on Genetics, August 1999. / Includes bibliographical references. Also available on the Internet.

The hematopoietic system supplies short-lived functional end cells of multiple lineages from a common pool of pluripotent stem cells throughout life. In man neoplastic transformation of these stem cells results in the development of the acute and chronic myeloid leukemias. An in vitro system where functional murine stem cells can be maintained for several months has recently become available. This system has been a powerful tool to analyze mechanisms regulating normal hematopoiesis and leukemogenesis in mice. The purpose of this work was to develop an analogous culture system applicable to human marrow and to evaluate its ability to support leukemic hematopoiesis. Long-term cultures were established with normal human marrow cells. In these, the more primitive progenitors were found to be almost exclusively located in the adherent layer for the duration of the culture (i.e., at least 2 months). A prerequisite for these studies was the development of a procedure for detaching adherent cells so that various colony-forming progenitors could be assayed in semi-solid media. The consistent finding of adherent layer-associated hematopoiesis suggests that cell-cell interactions between primitive hematopoietic cells and adherent layer elements may be essential for the maintenance of the former. Long-term cultures were also initiated with marrow from 11 CML patients and 13 AML patients (all untreated) and maintenance of normal and neoplastic progenitor cell populations assessed. A common finding was the rapid disappearance of neoplastic progenitor cells (recognized either by the presence of chromosomal abnormalities or by their abnormal differentiation capacity) in most cultures, even though cytogenetically normal precursors were often maintained. These differences between the behaviour of normal and neoplastic cells in long-term cultures may reflect changes at the stem cell level that are related to the acquisition of abnormal growth properties. In a minority of patients a different result was obtained. Clonal dominance persisted in vitro and normal hematopoiesis was not detected. Thus long-term cultures have also allowed differences in the behaviour of primitive neoplastic cells from different patients to be revealed. Future investigation of the basis for these differences may provide new insights into the biological heterogeneity that characterizes these disorders / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Hemopoiesis is thought to be regulated in part by specific, but as yet undefined, interactions between primitive hemopoietic cells and fixed, non-hemopoietic marrow elements collectively referred to as the stroma. Recently, a marrow culture system has been described that allows the maintenance of primitive human hemopoietic progenitor cells for many weeks in the absence of exogenously added hemopoietic growth factors. The formation of a heterogeneous adherent layer in which many stromal elements are found appears to be important to the maintenance of hemopoiesis in this system. As part of the overall goal of delineating the cellular and molecular interactions involved, my first objective was to develop an experimental system for assessing the hemopoiesis-sustaining function of the adherent layer of long-term human marrow cultures. This required the identification of a suitable procedure for separating the hemopoietic and non-hemopoietic regulatory components so that the former could be used to quantitate the function of the latter. This was achieved using irradiation to selectively inactivate residual hemopoietic cells in long-term culture adherent layers, and using a medium containing cis-4-hydroxy-L-proline to selectively inactivate stromal cells and their precursors present in suspensions of unseparated human marrow which were then added back in co-culture experiments. My second objective was to develop a strategy for obtaining purified populations of cells corresponding to the various mesenchymal cell types in long-term adherent layers. I therefore prepared a high titre SV-40 virus stock and used it to establish permanent, cloned lines from human marrow "fibroblast" colonies, long-term culture adherent layers, and umbilical cord endothelial cells. Characterization of the transformants generated showed that they were all positive for SV-40, and in general expressed the phenotypic characteristics of the cells originally infected. Functional studies showed that these transformants, like their normal counterparts, respond to activation by producing two types of hemopoietic growth factors. These studies suggest that marrow mesenchymal cells may regulate the growth and maintenance of primitive hemopoietic cells by producing hemopoietic growth factors in response to appropriate perturbation. The availability of permanent cloned lines of human marrow stromal cells should facilitate future analysis of these events at the molecular level. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Oviposited eggs of Eumeces fasciatus contain embryos in the limb bud stage. Amniogenesis is complete and two yolk sac membranes, vascular trilaminar omphalopleure (choriovitelline membrane) and bilaminar omphalopleure, enclose the yolk vesicle. A small allantoic vesicle contacts the chorion. The choriovitelline membrane is the primary vascular system. Blood islands, sites of hematopoiesis, are associated with omphalomesenteric vessels of the choriovitelline membrane. The bilaminar omphalopleure, which contacts the eggshell over the abembryonic hemisphere of the egg, lies external to an isolated yolk mass and yolk cleft and is not vascularized. The definitive yolk sac (splanchnopleure) is formed when the extraembryonic coelom and allantoic vesicle intrude into the choriovitelline membrane. Omphalomesenteric vessels are retained with the yolk sac splanchnopleure and the associated hematopoietic sites are present throughout incubation. The chorioallantoic membrane reaches the equator of the egg, entirely supplanting the choriovitelline membrane, after 25% of incubation is completed. Further growth of the allantois is stalled until 65% of incubation is completed when rapid expansion of the allantoic vesicle, in conjunction with resorption of the isolated yolk mass, supplants the bilaminar omphalopleure. As a result, the chorioallantoic membrane completely envelops the egg for the final 35% of incubation. This developmental event is coincident with published reports for the timing of increased growth and metabolism of embryos. As the isolated yolk mass regresses, intravitelline cells associated with the yolk cleft invade and resorb the yolk to form a large cavity. The wall of this cavity is a germinal epithelium that produces cells that fill the cavity. This structure appears to be a site of hematopoiesis previously undescribed in vertebrates.

Indiana University-Purdue University Indianapolis (IUPUI) / Maintenance of hematopoietic stem cell (HSC) function is an orchestrated event requiring the participation of multiple cell types within the hematopoietic niche. Among the key cellular components of the niche are a group of specialized bone-resident macrophages known as osteomacs (OM). Reported here is a detailed characterization of OM and description of discriminating phenotypic and functional properties that clearly distinguish OM from bone marrow-derived macrophages (BM Mφ). Furthermore, it was established that OM support hematopoiesis enhancing activity of osteoblasts and that this activity was augmented by megakaryocytes. Serial transplantation demonstrated that HSC repopulating potential was best maintained by in vitro cultures containing OM, osteoblasts and megakaryocytes. Interestingly, BM Mφ were unable to mediate the same hematopoiesis enhancing activity regardless of whether megakaryocytes were present in co-culture or not. Subsequently, to understand the importance of networking between the residents of the niche, 3D tissue cytometry was performed on fixed and stained unperturbed bone marrow sections. This approach identified the spatial relationships between OM, osteoblasts, megakaryocytes and HSC within the niche and defined parameters, under which these cell types coexist in undamaged bone marrow. In addition, single cell mRNAseq and CyTOF was performed to assess genetic and proteomic expression changes in OM following their interaction with megakaryocytes. These studies revealed the upregulation of CD166 and embigin on OM via osteoblast and megakaryocyte interactions. Clonogenic assays were conducted to examine the impact of these molecules in hematopoietic function. When these assays were initiated with CD166 KO OM or shRNA-mediated embigin knockdown OM, it was established that loss of these surface molecules on OM caused a decline in the normal OM-mediated hematopoietic enhancing activity. Conversely, recombinant CD166 and embigin partially substituted for OM activity thus identifying potential mediators through which OM maintain hematopoietic function. This data, for the first time, reveal intimate spatial interactions between OM, osteoblasts, megakaryocytes and HSC in the hematopoietic niche. They also illustrate the importance of crosstalk between OM, osteoblasts and megakaryocytes and reveal novel mediators such as CD166 and embigin that cooperate with other elements of the niche to support HSC function. / 2020-09-10

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