Final Report Summary - DENOVOHSC (Blood regeneration: de novo development of human hematopoietic stem cells) HSC transplantations represent a major curative therapy for patients with blood-related disorders and leukemia, and the need for such treatments is constantly growing. Donor samples are limiting in UK and European cord blood and bone marrow donor registries, and these insufficiencies of HSCs for clinical therapies poses an economic burden on the medical and health care systems. Thus, our objective in this ERC DE NOVO HSC project was to test out new approaches to HSC generation and growth. ERC AdG 341096 set out to de novo generate potent and robust stem cells outside the body, and use them to regenerate the entire adult blood system upon in vivo transplantation. To achieve this goal we identified and characterized the genetic program of the first hematopoietic stem cells (HSC) as they are made during embryonic development. We defined new molecules playing a role in this process and used this information to engineer novel ex vivo cultures for their growth and transdifferentiation from embryonic vascular endothelial cells. Our studies have been realized through novel reagent and mouse model creation (multi-color HSC reporter and conditional-deletion mice), directed differentiation of mouse/human reporter and mutant ESC/iPSC lines, vital imaging during embryonic development, complex cell culturing and genetic manipulation of candidate HSC regulatory genes identified through state-of-the-art single cell transcriptomic, mass spectrometry and single cell functional methodologies. Our results have brought us to a new understanding of how HSCs are generated. Rather than a simple binary formula, HSC generation requires numerous intrinsic and extrinsic factors. Much like casino slot machines, the odds of hitting the winning combination to generate an HSC is dependent on the abundance, combinations and synchrony of these numerous factors at the single cell level. We found the physiologic odds to become an HSC is about 1 in 1000 and is dependent on a highly specialized embryonic environment and cell-cell interactions that are short-lived. Our important findings now allow the field of HSC programming to go beyond the notion of transcription factor-based reprogramming of cells to HSC fate. Through the generous support of the ERC we have uncovered the novel dynamic molecular complexity necessary for production of HSCs. These discoveries are now guiding more realistic and efficient attempts to the ex vivo generation of HSCs for biomedical advancements. With this knowledge, new translational strategies for the generation of patient-specific HSCs will in the longer term relieve the economic burden of costly transplantation and immuno-therapies and provide a better quality of life for those afflicted with hematologic disorders.
