Popova, Semiramis; (2021) Human Haematopoietic Stem Cell Heterogeneity in Postnatal Haematopoiesis and Ontogeny. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Haematopoietic stem cell (HSC) transplants are upheld as one of the most successful therapies in regenerative medicine. While improved purification and functionality assays have advanced understanding of steady-state haematopoiesis and the human bona fide HSC, evidence suggests significant heterogeneity exists within the HSC compartment in post-natal and pre-natal haematopoiesis. In post-natal haematopoiesis, both CD34+ and CD34- cells possess robust in vivo repopulating potential. CD34- repopulating cells, however, exhibit distinct repopulation kinetics, capacity to produce functional CD34+ repopulating cells, and accordingly have been speculated to reside at the apex of the human haematopoietic hierarchy. But a low repopulating cell frequency has hindered efforts to study these HSCs. We thus aim to improve purification of CD34- HSCs and further expand the knowledge of this immature stem pool. We successfully identified an additional positive selection marker, CD117 (c-Kit). Through limiting dilution analysis and serial transplantations of enriched CD34- repopulating cells in enhanced NSG mouse models, we observed repopulation and lineage commitment kinetics. To investigate the molecular mechanisms, we conducted single-cell RNAseq. With these new data we have asserted the importance of human CD34- HSCs their enormous therapeutic potential. In human foetal haematopoiesis, it is unknown whether CD34- repopulating cells emerge during ontogeny and play a role in foetal haematopoiesis. While reports for HSC-purifying markers have been produced, much of these studies have been restricted to foetal liver, a single gestational stage, and the CD34+ population. In humans, little is known about how the HSC cell surface marker phenotype adapts to the dynamic niches in the liver during expansion, homing to the bone marrow, and bone marrow colonisation. To address this, we optimised a multi-parameter flow cytometry panel and used it to investigate the expression of a number of reported HSC cell surface markers across first and second trimester liver and bone marrow. Through a combination of high-dimensional data analysis and mathematical modelling we have produced an antigen-based map of foetal haematopoietic stem cell and progenitor dynamics.

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