The analysis of cellular mechanisms underlying haematopoietic stem cell (HSC) development using a novel in vitro system

The entire variety of red and white blood cells in mammals is generated by a small number of cells in the bone marrow. In this project we will investigate how blood stem cells are generated in the mammalian embryo. Blood stem cells develop in the area called the AGM region which encompasses a large vessel (the dorsal aorta) and embryonic gonads and kidneys. Two main questions will be asked here: i) what are the cells in the embryo which develop into blood stem cells? and ii) what are the helper cells which induce or facilitate this process? Until recently, owing to lack of an adequate experimental model it has not been possible to address these issues. We have developed an experimental system which allows us to culture and manipulate immature cells from the mouse AGM region and generate blood stem cells. This system will be used to test the ability of different cell types to develop into blood stem cells or to support stem cell formation. To this end different cell types from the embryo will be stained with specific reagents (antibodies) and isolated by a machine called fluorescence-activated cell sorter (FACS). However, certain important cell types cannot be isolated using antibodies. Therefore we will use genetically modified transgenic mice generated in the laboratory. In two such transgenic mouse models AML1 and HoxB4 genes known to be associated with stem cell development are labelled with green and yellow fluorescent reporter molecules. These fluorescent tags will allow us to isolate AML1 and HoxB4 cells by FACS and analyse their ability to develop in stem cells in the culture system. A complementary and important aspect of this project is to investigate the possibility of using our culture system for the generation of blood stem cells from embryonic stem cells (known as ES cells). ES cells can be used to produce entire adult mice. They are thus capable of generating all cell types in the mouse including blood stem cells. In traditional culture conditions previously used by researchers ES cells could only produce mature blood cells but not blood stem cells. Using our novel system by co-culturing ES cells and AGM cells we will be able to determine whether ES cells can generate blood stem cells under the influence of AGM cells. Here again we will isolate cell populations from cultured ES cells by FACS and test them in this system. This project will provide better understanding not only of biology of blood stem cells but also of other stem cells. It will also have important practical implications for generation and propagation of blood stem cells in culture.

The aorta-gonad-mesonephros (AGM) region is an important source of haematopoietic stem cell (HSC) activity in the developing embryo. However, the analysis of HSC development is significantly hampered by low HSC frequency, the lack of unique HSC markers as well as the difficulty of accessing HSCs in the embryo. Currently only long-term reconstitution assay (transplanting cells into irradiated recipients) enables the detection of HSCs. We aim here to identify embryonic ancestors of HSCs and accessory cells that support HSC development, using a novel in vitro system recently developed in the laboratory. This system enables robust induction of HSC development from cells isolated from the aorta-gonad-mesonephros (AGM) region and functional and phenotypic analysis of individual cell populations in the AGM region. Various cell populations isolated from the AGM region using flow cytometry will be co-cultured and thus tested for their capacity to develop into HSCs or to support HSC initiation/ expansion by the long-term repopulation assay. Flow cytometrical cell sorting will be carried out using antibodies against cell surface determinants defining endothelial, haematopoietic and mesenchymal cell compartments. Furthermore, embryonic cell populations expressing Aml1/runx1 and HoxB4 transcription factors which are implicated in HSC development will also be analysed in this system. For this purpose AML1-eGFP and HoxB4-eYFP reporter mice generated in the laboratory will be used. Embryonic stem (ES) cells are capable of generating all cell types in the mouse including HSCs. However, their haematopoietic potential in vitro is restricted to more mature myeloid and lymphoid progenitor cells. Recent progress towards generation of HSCs from ES cells has been made by overexpression of HoxB4. However, developing means for non-invasive derivation of HSCs from genetically unmodified ES cells remains an issue of significant importance. Therefore, we will test if co-culture with E11.5 AGM cells will result in maturation of HSCs from ES cells. In vitro differentiation of ES cells occurs through several stages, including formation of haemangioblasts. We expect that only at a certain stage of in vitro differentiation ES cells may respond appropriately to inducing stimuli by developing into HSCs. It may also be that some subpopulations of differentiating ES cells inhibit HSC formation. Therefore, various cell populations from different developmental stages will be isolated by flow cytometry and extensively tested in the co-culture system followed by transplantation into adult irradiated recipients. It is believed that stem cells in different tissues share some basic characteristics defining their ability to maintain sustainable generation of mature cell types. This project will contribute to understanding fundamental principles of stem cell biology and provide a basis for further analysis of genetic regulation of stem cells. Furthermore, as expected in the proposal there are good reasons to believe that the strategy proposed here will enable generation and propagation of HSCs in vitro.