Identification of functional domains in the AGM region linked to the hierarchical organisation of the developing haematopoietic stem cell lineage

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At the foundation of the blood system lie blood stem cells called haematopoietic stem cells (HSCs) which give rise to all types of blood cells. In spite of active production of mature blood cells, HSCs are not exhausted during the lifespan, since every time they divide they produce at least one copy of themselves. HSCs are the best studied stem cell type which serves a model for analysis of other stem cell types. The importance of these potent 'immortal' cells in the organism attracts considerable attention both from scientific community and general public. Despite significant progress in this field the exact origin and mechanisms whereby HSCs emerge during embryo development remain poorly understood. The aorta-gonad-mesonephros (AGM) region is an important organ in the developing embryo in which HSCs first appear. We have recently developed a powerful technique which allows us to reproduce massive generation of HSCs in cultured developing AGM regions (approximately 150-fold increase in HSCs was achieved during a four day culture period, Taoudi et al., Cell Stem Cell, 2008). Development of HSCs is a multi-step process dependent on interaction with surrounding cells. This culture system for the first time allowed us to investigate the role of individual cell populations in HSC development. Here we propose to deconstruct the AGM region into 'building blocks' suitable for manipulation and analysis. We will identify those which have functional capacity to promote development of embryonic precursors (called here pre-HSCs) into definitive HSCs. Our preliminary experiments show that during development, pre-HSCs go through distinct stages of maturation. To effectively pursue this goal we will not only purify different cell types from the AGM region but also derive a library of immortal cell lines of different types using a special transgenic mouse strain. Such cell lines will then be used as renewable standard material in our experiments. We have already identified one cell line which is capable of promoting development of one type pf pre-HSCs into definitive HSCs. Thus, important rationale for this project is that distinct stages of HSC development are associated with different microenvironments within the AGM region. Using the above strategy, we will be able to map certain stages of HSC development to specific morphological domains within the AGM region. In addition, based on the knowledge obtained, we aim to re-design the AGM region using only essential 'building blocks' identified in the analysis described above. By this we will engineer a well characterised functional AGM culture system accessible for further in-depth analysis.