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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.

The first transplantable hematopoietic stem cells (HSC) arise in the aorta-gonad mesonephros region (AGM) during early stages of embryo development. Specifically, ventral aspect of embryonic dorsal aorta (DA) contains HSC that upon transplantation into irradiated recipients can reconstitute all lineages of the haematopoietic system [Medvinsky et al. 1993; Muller and Medvinsky, 1994; Medvinsky and Dzierzak, 1996; Cumano et al., 1996; Tavian et al., 1996; Peault and Tavian, 2003; Taoudi and Medvinsky, 2007; Ivanovs et al., 2011, 2014]. The ventral aspect of DA bears so-called intra-aortic cell clusters (IAC), the appearance of which coincides with the emergence of HSC [Babovic and Eaves, 2014; Bhatia, 2007; Boisset et al., 2010, 2011; Bollerot et al., 2005; de Bruijin et al., 2002; Bertrand et al., 2010]. According to recent reports, HSC are a heterogeneous population of cells [Dykstra et al., 2007; Seita and Weissman, 2010; Muller-Sieburg et al., 2012]. It is unclear whether all HSC precursors originate from the same location, for example, DA lining, IAC or sub-aortic tissues; or HSC precursors migrate into DA lining from other parts of the embryo [Tavian et al., 1999; Yoder et al., 1997; Oberlin et al., 2002; Peault and Tavian, 2003; Dzierzak, 2003; Samokhvalov et al., 2007; Medvinsky et al., 2011]. To elucidate ontogeny of early HSC precursors (pro-HSC), two approaches were applied in this PhD project. First, we mapped potential pro-HSC in pre-circulation mouse embryos (embryonic day 6-8.5, E6-E8.5). We defined potential pro-HSC as cells co-expressing the transcription factor Runx1, endothelial markers (VE-Cad or CD31) and/or haematopoietic markers (CD45, CD41) [Oberlin et al., 2002; de Bruijn and Dzierzak, 2012; Liakhovitskaia et al., 2009, 2014]. In E6-E8 mouse embryo, prospective pro-HSC were found to be located in chorionic plate, yolk sac and in allantoic core domain. In early somitic mouse embryo (E8-8.5) cells with pro-HSC phenotype (Runx1+CD31+CD41+) were found to be in cell clusters in forming vessel of confluence and in nascent dorsal aortae lining. Pro-HSC are not directly transplantable [Cumano et al., 1996., 2001; Godin et al., 1993; 1995; Batta et al., 2016; Matsuoka et al., 2001; Nishikawa et al., 1998]. Therefore, cells and tissues containing prospective pro-HSC were initially matured using several in-vitro culture systems. According to our results, E8 mouse embryo pro-HSC are only preserved in explant cultures, but not in co-aggregate cultures with stroma cells. After culture, cells were transplanted into sub-lethally irradiated recipients. Six weeks after transplantation 19 out of 82 transplanted recipients had donor derived blood cells' chimerism at the level of 0.1-0.3%. Forty six percent of these grafts were derived from rostral part of the embryo tissues (head, heart, upper somites). Only one out of 82 recipients had donor cells contribution above 1% (1.2 %). This recipient was engrafted with cells derived from the E8 mouse embryo head and heart region. Recipients having blood chimerism at the range of 0.1-0.3% had mainly lymphoid donor derived cells in their peripheral blood. The only recipient showing the high donor cells contribution (1.2%) had contribution mainly to myeloid lineage. Recorded low levels of blood chimersims are in line with those reported by Rybtsov et al. (2014) for early E9 mouse embryos. Donor derived cells formed clearly distinguishable populations on cytometry plots. This population of cells were absent from control engraftment experiments with carrier cells only. Previously, lymphoid potential was detected in paraaortic spnanchnopleura (P-Sp) of E8.5-9 mouse embryos, but not in E8 mouse embryos (0-5 somites, pre-circulation) and later in yolk sac [Cumano et al., 1996; Nishikawa et al., 1998; Fraser et al., 2002; Yokota et al., 2006]. However, prior works used different criteria to establish recipient reconstitution. Therefore, it is possible that recipients repopulated with E8 derived cells at the level of 0.1% were not considered as repopulated and hence, presence of lymphoid lineage precursors was overlooked in early somitic mouse embryos. The only recipient showing substantial myeloid cells contribution (73% Mac1+Gr1+ cells of donor derived cells) received engrafted cells from an older (6-13 sp) embryo and therefore potentially has yolk sac derived myeloid cells. Yolk sac cell contribution to myeloid lineage, specifically to the brain microglia was reported in prior works [Samokhvalov et al., 2007]. Our data show that early E8 AGM cells do not expand in in vitro conditions. While in AGM, cells from E9 mouse embryo expand in culture [Rybtsov et al., 2014]. We have analysed Runx1 expression pattern and dorsal aorta morphology at the time when E9 HSC precursors acquire ability to expand in in vitro culture. Runx1 expression becomes clearly polarised at the time point (22-26 sp), when paired dorsal aortae fusion is initiated. We envision that intimate connection between DA fusion events and induction of pro-HSC maturation exists. According to prior reports, Bmp, Shh and VEGF signalling regulate DA fusion [Garriock et al., 2010]. Thereofore, to enhance in vitro HSC maturation system, DA fusion triggers (for example, Bmp4) might be added to culture. Since, pro-HSC maturation methods established to date are not efficient to expand and differentiate E8 pro-HSC into potent HSC, another approach had to be implemented to study HSC ontogeny. The second approach we utilized was to trace the origin of HSC in chicken embryo, starting from the very beginning of cell fate specification, i.e. from gastrulation stages. Chick embryo haematopoiesis is similar in both human and mouse: precursors of HSC arise in the embryo proper in AGM, and IAC are formed in DA ventral aspect [Dieterlen-Lièvre, 1975; Dieterlen-Lièvre and Martin, 1981; Dieterlen-Lièvre and Jaffredo, 2009; Jaffredo et al., 2000; Le Douarin and Dieterlen-Lièvre, 2013]. In contrast to mammals, chick embryo develops ex vivo, making direct labelling and cell tracing possible. We aimed to identify cells giving rise to regions of DA that produce IAC. Therefore, segments of primitive streak (PS) were labelled with lipophilic dyes or by substituting segments of host PS with PS sections derived from transgenic (GFP+) stage matched chicken embryos. Our results show that in an 18-25h chicken embryo (Hamburger and Hamilton developmental stage 4-6, HH4-6) cells giving rise to DA ingress through the wide region of PS (35-60% of its length) [Hamburger and Hamilton, 1951]. We identified that the section of DA producing HSC is formed by cells ingressing through PS in region of 40-55% of its length at 18-25h of chick embryo development. Regardless of the embryo development stage (HH4-6), in chimeras grafted at 40-55% of PS length, GFP+ cells contributed to DA and to the IAC. Within GFP+ labelled areas, we observed clusters consisting entirely of GFP+ and clusters having a mixture of GFP+ and GFP- cells. Entirely GFP+ clusters were found in the stretch of DA that had the entire aortic endothelial lining labelled. Clusters formed on the mosaic (GFP+/GFP-) aortic endothelium also had mosaic nature. According to our data, multiple descendants of PS contribute to the same stretch of dorsal aorta. This explains mosaicity of dorsal aorta lining and IAC labelling. Since we encountered clusters with mixture of GFP+ and GFP- cells, we conclude that IAC are not clonal formations. Mosaicity of IAC also does not exclude a scenario when cells migrate in and out of a cluster. Further tracing experiments are required to establish HSC nature of cells within a cluster.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: stem cells, cytokines. Includes bibliographical references (p. 79-83).

published_or_final_version / Medicine / Master / Doctor of Medicine

Thesis (MAppSc (Medical Laboratory Science)) --University of South Australia, 1993

Thesis (M.D.)--University of Adelaide, 1985. / Includes bibliographical references (leaves 1-14 of section Reference).

Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine; and, Institute of Medical and Veterinary Science, Division of Haematology, 2005. / Title from title page of source document; viewed 19 July 2005. Bibliography: p. 157-184 of source document. Also available in print.