Using single-cell technologies to functionally define fetal haematopoietic stem and progenitor cells

Using single cell RNA sequencing, cellular barcoding, and transplantation experiments, my study functionally benchmarks the cell types present in the fetal liver and has demonstrated the existence of functional MPP2 to 4 within the embryo. Although strikingly, FL MPP3 has significantly greater contribution to the lymphoid lineage than its adult counterpart, acting as lineage balance MPP. Our study has further revealed a large pool of functional LTHSCs were immunophenotypically hidden in a STHSC immunophenotype. While additionally, CD45 surface marker can be used to set apart all long-term reconstituting activity in the foetus. Interestingly our data indicates that the majority of a recently discovered developmentally restricted HSC population is present within our FL CD45+ STHSC compartment. Suggesting a previous unrecognised critical role for this population during development. Overall design: Single cell FL LSK subtypes (MPP2-4/ LT/ ST) were sorted and underwent library prep and RNA-sequencing to determine the molecular differences between the populations. Long-term and short-term transplantations of bulk sorted LSK subtype were performed to assess functional abilities of these populations. While lentiviral barcoding and short-term transplantation was also utilised on each population to determine their functional potential at the clonal level. Lastly, via flow cytometry and further by transplantation, a Flt3-Cre YFP reporter mouse line was utilised to investigate the presence of developmentally restricted HSCs (drHSCs) within the FL immunophenotypical STHSC compartment.