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

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.

HSCs are generated during embryonic development but the mechanisms defining their specification and expansion remain obscure. The AGM region plays an important role in HSC development. A novel in vitro AGM reaggregation system which enables effective initiation and dramatic expansion of HSCs is in the core of this proposal. This system for the first time allowed us to address the question of which embryonic precursors develop into definitive HSCs. Development of HSCs in the AGM region is a stroma-dependent process; but the identity of essential stromal (accessory) elements remains unknown. We will identify those which have functional capacity to promote development of embryonic precursors (pre-HSCs) into definitive HSCs. We will first identify pre-HSCs by intrauterine injection of cells isolated from SCL-GFP mice using high precision ultrasound injection device. Our preliminary experiments show that pre-HSCs go through distinct stages of maturation and therefore can functionally depend on various domains within the AGM region. The AGM region is a complex heterogeneous organ and effective analysis of mechanisms of stromal/ HSC interactions requires focussing on essential components. To this end we will simplify the system by deconstructing the AGM region into homogenous stromal populations which can be functionally validated using our re-aggregation culture protocol. To achieve this, we will use (when possible) primary cell populations and also generate a library of stromal cell lines using novel highly efficient inducible transgenic 'immortomice'. Use of cell lines will be an important source of homogenous experimental material. Based on the above analyses we will re-design the minimal functional HSC niche. Such well characterised minimal system will in future be a valuable tool for precise in-depth system analysis of molecular mechanisms directing specification and expansion of HSCs in the AGM niche.

This project is of potential commercial value and I expect the University of Edinburgh will be able to commercialise results of this project and gain financial benefits from this work. Results of this project may provide appropriate material for graphical presentation to the general public. This culture system is expected to be of interest to the medical profession. Further development and better understanding of a similar human system may facilitate development of novel clinically relevant protocols towards generation of transplantable haematopoietic stem cells.