Single cell hematopoietic fate acquisition during embryonic development: To be a stem cell or not?

Rare stem cells in the adult bone marrow are responsible for the healthy life-long production of all circulating blood cells. These stem cells are used in curative clinical transplantations for blood-related diseases and for cancer therapies. Although blood banks for such stem cells exist in the UK and worldwide, there are insufficient supplies of stem cells (high quality and large numbers of cells) for transplantations and importantly, the donor stem cells need to closely match the patient's cells to avoid graft rejection.

For years, researchers have attempted to increase the number of blood stem cells in culture systems. However, blood stem cells obtained from adult bone marrow or umbilical cord blood do not retain their stem cell properties when cultured. Instead they mature into red blood cells, macrophages and immune cell types which are short-lived. Rather than furthering culture approaches to expand blood stem cells, a new reprogramming approach has been taken to generate new stem cells from skin cells and embryonic cells. To date, these reprogramming approaches have had very limited success.

In my lab, discoveries in the mouse revealed that all blood stem cells are made early in the embryo from specialized (endothelial) cells lining the blood vessels. Importantly, blood stem cells are made only once during development. Our knowledge of blood stem cell development and the molecular program directing cells to become blood stem cells is fundamental to approaches aiming to generate patient-specific stem cells and for alleviating insufficient supplies of these cells for transplantation.

This study aims to demonstrate the complete genetic program of blood stem cells as they are generated within the mouse embryonic aorta. The challenge is to identify the true blood stem cells, of which there are only 1-2 in the embryo amongst about 700 other types of blood cells. These other blood cells are very similar to blood stem cells but cannot repopulate the blood system following transplantation and thus are not curative. Because there is no current technology that allows the isolation of pure blood stem cells from the other blood cell types, we will isolate single cells from the mouse embryonic aorta using unique fluorescent reporters and test their function through transplantation. We will perform state-of-the-art single cell RNA sequencing experiments and use a new and highly innovative DNA marking technique to identify for the first time the complete genetic program that codes these unique functionally potent blood stem cells. This important information will further our understanding of how blood stem cells are made and advance strategies for generating robust patient specific stem cells for treatment of leukemia and hematologic disorders.

Adult bone marrow hematopoietic stem cells (HSC) are responsible for the healthy life-long production of all blood cells. These stem cells are used in curative clinical transplantations for blood-related diseases and cancer therapies. Currently, there are insufficient supplies of high quality stem cells for transplantations, and demands for HSCs are expected to rise as the UK population ages. Importantly, the donor stem cells must closely match the patient's cells to avoid graft rejection. Our knowledge of HSC development and the molecular program directing cells to become HSCs is fundamental to approaches aiming to generate patient-specific stem cells.

This study will examine the generation of HSCs in the mouse embryo and identify the genetic program that directs the acquisition of hematopoietic stem cell fate and function. The challenge is to identify the single functional HSCs. There are only 1-2 in the mouse embryo amongst about 700 other hematopoietic cells. While the profile of HSCs and the other hematopoietic cells are very similar, only hematopoietic stem cells can repopulate the blood system following transplantation. Currently there is no current technology that allows the isolation of pure HSCs from the other blood cell types. We will take a novel approach to isolate single cells from the mouse embryonic aorta using unique fluorescent reporters and test their function through transplantation. We will perform state-of-the-art single cell RNA sequencing experiments and use a new and highly innovative DNA marking technique to identify for the first time the complete genetic program that codes these unique functionally potent stem cells. This important information will further our understanding of how HSCs are made and advance strategies for generating robust patient-specific hematopoietic stem cells to meet the growing need for treatments of leukemia and hematologic disorders

This research will have considerable impact in several areas.

Medical, economic, societal
The incidence of blood disorders and leukemia in the UK and Europe is increasing as the population ages. These patient groups pose an economic burden on the medical and health care systems. HSC transplantations represent a major curative therapy for such patients and the most bone marrow HSC transplantations worldwide are performed in Europe. Yet despite efforts of EUROCORD, EHT, EHA and the European Network of cord blood and bone marrow donor registries and banks, donor samples are limiting and transplantations not wholly successful due to immune rejection of donor cells, resulting in insufficient supplies of HSCs for clinical therapies and also for fundamental research. The result of this BBSRC project will have great impact on how we solve this lack of HSCs through providing an understanding of how transcriptome dynamics influence HSC generation and what genetic programme makes a hematopoietic progenitor cell different from a fully functional clinically-relevant hematopoietic stem cell. With this knowledge, new translational strategies can be advanced for the generation of human (patient-specific) HSCs. The economic burden of costly transplantation and immuno-therapies following transplantation are expected to be relieved through less expensive patient-specific cell generation and transplantation.

Scientifically
This work will provide novel conceptual insights into the way embryonic endothelial cells take on a hematopoietic stem cell fate rather than a hematopoietic progenitor cell fate. Insights from the transcriptomic analyses are likely to provide evidence for new principles regarding the unstable gene expression programme and the dynamics of expression of pivotal transcription factors and master regulators.

Technologically
The work contributes to the validation of the innovative DCM-ID methylation marking technique. By identifying for the first time the active transcriptome in functional hematopoietic stem cells, this methodology will be advantageous for other stem cell systems for transcriptome identification.

Collaboratively
We have three important collaborators in this study. Links, technical exchange and intellectual sharing will take place between my lab, that of L Forrester (U of Edinburgh), B Gottgens (U of Cambridge) and J Gribnau (Erasmus MC).

Training and teaching
At the end of this project we will have trained one postdoctoral fellow and one research assistant and at least 3 MSc and 3 Honours students.
Through the MSc, ECAT and postgraduate training programmes at the U of Edinburgh, I teach in diverse courses and am an organizing for monthly lectures in Tissue Repair and Hematology.