Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (doctoral)--Københavns universitet.

Thesis (M. D.)--University of Hong Kong, 2001. / Also available in print.

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The ability for hematopoietic stem cells (HSCs) to regenerate the vascular and blood systems following injury suggests great potential for future therapies. Unfortunately, the various signaling pathways that regulate the regeneration of the adult HSC population in the bone marrow are not clearly understood. One of the proposed regulators for the regeneration of the hematopoietic system is an extracellular secreted protein R Spondin-2 (Rspo2), also known as roof plate-specific spondin-2. The novel interaction between the Rspo2 protein and c-kit+sca-1+lineage– (KSL) HSCs shows an increased number of KSL and of more differentiated hematopoietic stem and progenitor cells (HSPCs) in vivo during both the steady and injured states. To determine the most efficient concentration of Rspo2 for such an interaction to occur, various doses of Rspo2 recombinant protein are plated with the KSL cells. They are then examined through flow cytometry and colony forming cell (CFC) assay. Rspo2 is widely known to interact with the canonical Wnt3a protein to activate the beta-catenin pathway. However, when various concentrations of Rspo2 recombinant proteins are plated with the Wnt3a protein, the results show the opposite effect of plating the cells only with the Rspo2 protein. The overall increase in the total number of cells and KSL cells was concluded to be not significant. This study nonetheless provides the scientific community with a greater foundation for the usage of Rspo2 concentration for future experiments.

Indiana University-Purdue University Indianapolis (IUPUI) / Cell cycle checkpoints guarantee movement through the cell cycle in an appropriate manner. The spindle assembly checkpoint (SAC) ensures the proper segregation of chromosomes into daughter cells during mitosis. Mitotic arrest deficiency 2 (Mad2), a member of the mitotic checkpoint proteins, appears to be crucial for generating the wait anaphase signal to prevent onset of anaphase. We first studied the SAC in hematopoietic stem cells (HSC) to ensure that it was functional. Our previous studies found that prolonged SAC activation was uncoupled from apoptosis initiation in mouse and human embryonic stem cells (ESC). We found that upon treatment with a microtubule-destabilizing agent, HSC arrested in M-phase and subsequently initiated apoptosis. Thus unlike ESC, HSC exhibit coupling of prolonged SAC activation with apoptosis. We studied the effects of Mad2+/- on in vivo recovery of bone marrow HPC from cytotoxic effects and also effects of cytostatic agents on HPC growth in vitro using Mad2-haploinsufficient (Mad2+/-) mice. We found that Mad2+/- HPCs were protected from the cytotoxic effects of cytarabine (Ara-C), a cycle specific agent, consistent with Mad2+/- HPCs being in a slow or non-cycling state. Mad2 haploinsufficiency did not affect recovery of functional HPC after treatment with cyclophosphamide or high sub-lethal dose irradiation, both non-cycle specific agents. There were no differences in immunophenotype defined HSCs in Mad2+/- and Mad2+/+ mice, data confirmed by functional HSC competitive repopulation assays. To better understand the role of Mad2 in HPC, E3330, a cytostatic agent, was used to assess the redox function of Ape1/Ref-1, and colony formation in vitro was examined under normoxic and lowered O2 tension. Mad2+/- HPCs were less responsive to E3330 than Mad2+/+ HPCs, and E3330 was more effective under lowered O2 tension. Mad2+/- HPCs did not exhibit enhanced growth in lowered oxygen tension, in contrast to Mad2+/+ HPCs. Our studies have unexpectedly found that Mad2 haploinsufficiency is protective from the cytotoxic effects of a cycle specific DNA synthesis agent in vivo, and Ape1/Ref-1 inhibitor in vitro.

No description available.

published_or_final_version / abstract / Medicine / Master / Doctor of Medicine

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

Hematopoietic stem cells (HSCs) take on the extraordinary role of sustaining life-long production of blood cells. Despite their indisputable therapeutic potential, HSC biology is poorly understood, and the field remains limited by the inability to maintain, expand, or generate HSCs in vitro. The aim of this study was to elucidate a particular gap in our understanding of the organellar cell biology of HSCs, specifically the role and function of the mitochondria. Several signaling pathways and biological processes converge onto the mitochondria, yet these organelles were found to be largely dispensable in HSCs on the basis of their predominantly glycolytic metabolism and reports of low mitochondrial content. Our studies show that MitoTracker Green (MTG), a frequently used fluorescent dye to measure mitochondrial mass in hematopoietic populations, is effluxed by HSCs resulting in their systematic and deceptive enrichment in the subset of cells with the lowest MTG fluorescence. Using dye-independent methods we discovered that HSCs have elevated mitochondrial content despite their reliance on glycolysis for ATP production. Moreover, mechanisms of mitochondrial quality control and clearance by autophagy appear to be comparatively lower in HSCs than in any other hematopoietic population we analyzed, suggesting HSCs maintain their mitochondria over time. To investigate the function of mitochondria in HSCs we generated mice with disruption of mitofusins (MFN) 1 and 2. These proteins are key mediators of mitochondrial fusion, a process that in coordination with mitochondrial fission regulates mitochondrial size, number, and function. Mice with deletion of Mfn1 and Mfn2 (DKO) die perinatally, are pale in appearance and their HSCs show complete loss of regenerative capacity. Several processes linked to dysfunctional mitochondrial fusion and known to be tightly regulated in HSCs are altered in these mutants, including mitochondrial morphology, mitochondrial mass, proliferation, and altered metabolism. Interestingly, one allele of Mfn1 is sufficient to rescue the hematopoietic function and lethality of DKO mice, while one allele of Mfn2 only rescues myeloid reconstitution. Taken together, our findings highlight the importance and complexity of mitochondrial function and dynamics in HSCs and have contributed to the recently increased appreciation of a vital role for mitochondria in HSCs.