Towards the in vitro production of haematopoietic stem cells: lessons from the early human embryo
Easterbrook, Jennifer Elizabeth
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The production of fully functional haematopoietic stem cells (HSCs) for clinical transplantation is a highly sought after goal in the field of regenerative medicine. Given their capacity for extensive self-renewal and differentiation into any cell type, human pluripotent stem cells (hPSCs) provide a potentially limitless source of haematopoietic cells in vitro for clinical application. However, to date, fully functional HSCs have not been produced from hPSCs without the overexpression of transcription factors. In this study I first investigated the production of HSCs and haematopoietic progenitor cells (HPCs) in an established clinical-grade haematopoietic differentiation protocol. I demonstrated the efficient and reproducible production of HPCs but showed that the strategy did not produce fully functional HSCs that could repopulate the haematopoietic system of immune-deficient mice. Modification of the protocol by manipulation of the hedgehog signalling pathway and co-aggregation with OP9 stromal cells did not provide any significant enhancement of HPC production. To gain the required knowledge with which to improve our current protocol, I therefore switched my focus towards studying the development of HSCs in the early human embryo. It has been shown that HSCs first emerge from the ventral wall of the dorsal aorta in the aorta-gonad-mesonephros (AGM) region of the human embryo but the precise location and the mechanisms underpinning this process remain unknown. In this study, I established a culture system to map the spatio-temporal distribution of HSCs and to investigate the presence of HSC precursors. I showed that embryonic HSCs emerge predominantly around and above the vitelline artery entry point in the dorsal aorta and can be maintained in our explant culture system. I then performed RNA-sequencing of cells derived from AGM sub-regions, and this identified molecular signatures which could potentially underlie the ventral polarity of HSC emergence in the AGM. To elucidate the role of the stromal compartment in this unique haematopoietic niche, I derived stromal cell lines from the human AGM region and showed they were capable of supporting haematopoiesis in vitro. This work has provided some important insights into the mechanisms regulating HSC development in the human AGM region and identified interesting candidate molecules for future testing in differentiation protocols. This knowledge brings us a step closer to the successful in vitro production of HSCs for clinical use.