The most powerful cell in the blood differentiation hierarchy is the haematopoietic stem cell (HSC). It is the only cell capable of building an entire haematopoietic system from scratch, i.e. long-term (LT) repopulating a multilineage blood system within a lethally irradiated recipient. Though primitive blood is generated at early embryonic stages, it is only from embryonic day (E)10.5 of mouse development (4-5 weeks in human gestation) onwards that the definitive adult haematopoietic system is established with the formation of LT-HSCs.
These first HSCs emerge in small ventral intra-aortic haematopoietic clusters (IAHCs) in the aorta-gonad-mesonephros (AGM) region. Like with many systems, a tight regulation of gene expression by master transcription factors (TF) is what ultimately drives cell fate change. Gata2 is one such TF known to play a crucial role in mouse definitive haematopoiesis. Through the work of de Pater et al. (2013) (Chapter 1) it became clear that Gata2 is both required for the generation of the very first HSCs and also, unlike other key TFs like Runx1, the survival of HSCs. The earliest HSCs are generated through an endothelial-to-haematopoietic transition (EHT), a process where flat arterial haemogenic endothelial cells (HECs) round up into IAHCs, containing HSCs.
Solaimani Kartalaei et al. (2015) (Chapter 4) discovered that Gpr56 is one of the most highly upregulated genes during EHT (in mouse) and that it is essential for HSC generation (in zebrafish). Gpr56 is also a target of the key haematopoietic ‘heptad’ TFs in both mouse and human blood progenitors. To examine another signalling pathway required in the embryo for HSC generation, we used a BMP responsive element (BRE)-GFP transgenic mouse to show that all AGM HSCs are BMP-activated (Crisan et al., 2015) (Chapter 6). At slightly later stages, in the foetal liver and bone marrow, HSC heterogeneity starts to appear with the presence of genetically distinct BMP- and non-BMP activated HSCs.
Given the importance of the ‘heptad’ TFs in establishing HSC identity, we next developed a novel Gata2-Venus (G2V) reporter mouse to isolate and examine the dynamics and function of live Gata2-expressing and non-expressing cells (Kaimakis et al., 2016) (Chapter 2). Gata2 is expressed in all HSCs, however haematopoietic progenitors can be generated either Gata2-dependently or -independently, the latter being less potent and genetically distinct from the first. With Eich et al. (2018) (Chapter 3) we went further and deciphered the role of Gata2 in haematopoietic fate establishment. By using time-lapse imaging of E10.5 live aortic sections of G2V embryos, we discovered rapid pulsatile level changes in Gata2 expression, specifically in those single cells undergoing EHT. This finding indicated the highly unstable genetic state of individual cells undergoing fate change. Furthermore, aortic cells haploinsufficient for Gata2 show many fewer pulsatile and EHT events, emphasizing the importance of Gata2 levels in this process.
Having defined the specific (medium) levels of Gata2(Venus) protein in the population containing the first HSCs, we used our G2V model to isolate a pure population of HSCs as they are generated for the first time in development (Vink et al., 2020) (Chapter 5). Through iterations of index-sorting, single-cell transcriptomics and functional analyses we were able to greatly enrich for HSCs (~70x compared to Eich et al. (2018)). Based on refined CD31, cKit and CD27 expression and specific physical parameters we isolated the first cells with an HSC identity and identified their precise gene expression profile. Immunohistochemistry localized these HSCs to only the smallest IAHCs. The combined effort of my research and novel tools generated to facilitate the creation of this body of work/portfolio have now led us for the first time to study the one or two cells per embryo that harbour HSC identity at the developmental time when they are beginning to be made. Now that we have cracked the genetic code of the true HSC, the field is one step closer to translating these findings and applying them to producing human clinically relevant HSCs, which will ultimately improve transplantation therapies and benefit the fight against leukaemia
