Ulm, Univ., Diss., 2008.

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

Vertebrate wnt8a occupies a position at a crossroads linking anteroposterior and dorsoventral axis patterning. While functional aspects of wnt8a are beginning to be understood, the regulation of wnt8a expression and its relationship to mesoderm induction and maintenance pathways are unclear. Three inputs that control wnt8a expression in the zebrafish embryonic margin have been identified: the Brachyury-related T-box transcription factors No tail a (Ntla) and No tail b (Ntlb, previously called Bra) and the maternal zinc-finger transcription factor Zbtb4 (previously called Kzp) are known direct regulators, and Nodal signaling is genetically upstream of wnt8a expression. The transcriptional mechanisms by which the wnt8a locus integrates these diverse temporal inputs are not yet known. We have generated zebrafish transgenic for a modified genomic PAC clone that expresses EGFP from the wnt8a locus. The EGFP reporter transgene is expressed in a pattern nearly identical to wnt8a, including maternal deposition, expression in the ventrolateral mesoderm and in the yolk syncytial layer. Using this transgenic line, we identified two phases of wnt8a transcriptional regulation in zebrafish: phase I comprises Nodal-dependent activation during early gastrulation and phase II comprises No tail (Ntl)-dependent regulation from mid to late gastrula stages onwards. These phases mirror the transition from Nodal-dependent mesoderm induction to Ntl-dependent mesoderm maintenance. To further understand how the wnt8a locus integrates these signals to achieve its transcriptional output, we analyzed upstream cis-regulatory regions through transgenic reporter assays. We identified three promoters in the bicistronic wnt8a locus, two of which drive expression of the upstream coding region (wnt8a.1). We identified two regulatory regions, proximal and distal: the proximal regulatory region contains a mesodermal enhancer with potential binding sites for FoxH1 and Ntl that is required for both the Nodal and Ntl responses. Phase I expression requires Nodal signaling through the mesoderm enhancer in combination with the distal regulatory region, which bears a Zbtb4 consensus binding site. Phase II expression requires Ntl regulation of the mesoderm enhancer in the context of the proximal regulatory region. The distal regulatory region negatively impacts phase II expression driven by the proximal regulatory region, indicating a complex relationship of regulatory elements.

No description available.

Thesis (M.S.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 29-33.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 207-242) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 207-242) Also available online.

Zugl.: Berlin, Freie Universiẗat, Diss., 2009.

Mesoderm is one of the three primary germ layers from which the cardiovascular system, muscle and bone originate and derivatives of the mesoderm lineage are affected in a number of pathologies. Therefore, understanding the mechanisms regulating formation of mesoderm is interesting for a diversity of diseases and clinical application. In vivo study of human development beyond gastrulation is technically challenging and the mechanisms controlling mesoderm specification are difficult to study since the maximum number of days allowed to grow human embryos is 14 days. Thus, in this dissertation I use human pluripotent stem cells (hPSCs) as a simplified model of human development. Studies have shown that the cell cycle machinery plays a direct role in the differentiation of endoderm and ectoderm lineages but its role in guiding mesoderm subtype formation remains elusive. In this dissertation, I provide new insights of the importance of the cell cycle regulators in mesoderm specification. I first developed tools such as the FUCCI2A reporter line to isolate cells in the different cell cycle phases and to investigate propensity of mesoderm differentiation. I have shown that the propensity of differentiation into the three mesoderm subtypes lateral plate mesoderm, cardiac mesoderm and presomitic mesoderm varies during the cell cycle phases, with differentiation being more efficient in the G1 and to a lesser extend in G2/M phase. Furthermore, I developed a protocol where cells can be efficiently synchronised in the different cell cycle phases using the G2/M inhibitor nocodazole. Using this tool, I showed that developmental signalling pathways such as BMP and WNT are active in all cell cycle phases indicating that alternative mechanisms are involved in the differentiation process. In order to further explore these mechanisms, I investigated the role of cell cycle regulators controlling the G1 and G2 checkpoint. I have shown that the cell cycle regulators CDK4/6, CDK2, Retinoblastoma phosphorylation and CDK1 are essential for mesoderm subtype formation. Furthermore, I have shown that CDK1 regulates the activity of ERK1/2 signalling, an important pathway for the differentiation process confirming the existence of complex interplays between cell cycle machinery, signalling pathways and transcription factors in mesoderm subtype formation. This knowledge will be useful to further improve protocols for generating mesoderm subtypes from hPSCs for clinical applications such as drug screening, disease modelling and cell based therapy.

No description available.