Blood production is coordinated by a functionally heterogeneous pool of multipotent haematopoietic stem cells (HSCs), downstream of which lineage-restricted progenitors are generated. The advent of single cell technologies has changed our view of the haematopoiesis to a dynamic continuum. Understanding the early differentiation trajectories of HSCs, and how environment and molecular factors can modify them, is vital in furthering our insight into human haematopoiesis in health and disease. Here I combined index sorting, single cell functional assays in vitro, RNA-sequencing (RNAseq) and in vivo assays to i) study lineage commitment heterogeneity within the HSC compartment of cord blood (CB) and foetal liver (FL) ii) to explore the role of inflammatory signals in HSC differentiation, using an in vitro model of human early HSC differentiation I developed.

Using in vitro functional assays, I uncovered that at single cell resolution, the CB HSC/Multipotent progenitor (MPP) compartment is polarised based on lineage output. I established novel prospective purification strategies, that maximise enrichment of cells with myeloid (My)-erythroid (Ery) (CD34lo CLEC9Ahi; Subset1) or My-lymphoid (Ly) (CD34hi CLEC9Alo; Subset2) potential in vitro. In vivo, I used an optimised NSG xenograft model for detection of erythroid potential, to show that Subset2 cells were restricted to My-Ly differentiation and displayed infrequent long-term repopulation capacity. In conclusion, I demonstrated that the first lineage restriction step in human haematopoiesis occurs within the human HSC/MPP pool and generates My-Ly committed cells with no erythroid differentiation capacity.

Using similar methodologies as above, I report 2 main findings in the human FL HSC/MPP compartment: i) there is a decrease in multipotency and Ery potential with gestational age but an increase in Ly potential and ii) there is an increased percentage of cells in G0 of the cell cycle with gestational age, indicating a progressive shift to quiescence.

Finally, I developed an in vitro model of early haematopoiesis by culturing long-term (LT-) HSCs for 5-days then performing single cell RNAseq and single cell functional assays. In this model most lineage types were produced: My, Ly, Ery, megakaryocyte (Meg) and mast cells (MC). Studying the differentiation trajectories observed in this model, I identified IL1RL1, the gene encoding the IL-33 receptor, ST2, as a potential modulator of the Ery, Meg and MC branch. When exposed to IL-33, LT-HSCs showed increased differentiation towards the Meg, MC (in vitro) and Ery lineages (in vitro and in vivo) but maintained long-term engraftment potential. This demonstrates a novel role of the pro-inflammatory cytokine IL-33 as a regulator of early LT-HSC differentiation.