Deciphering blood cell development one cell at a time

  • Muhammad Fadlullah Wilmot

Student thesis: Phd

Abstract

In vitro generation and expansion of haematopoietic stem cells (HSCs) holds great promise for treating genetic and blood malignancy ailments. Such technology would provide an unlimited source of cells for bone marrow or blood transplantation. Developing this in vitro approach requires first understanding how blood cells emerge in vivo. During embryonic development, haematopoietic stem and progenitor cells (HSPCs) arise from a specialised subset of endothelium cells called haemogenic endothelium (HE). The limited number of HE cells accessible, coupled with the absence of definitive markers for these cells, have hampered efforts to understand the molecular profile of HE during embryonic development. In this work, full-length single-cell RNA-sequencing (scRNA-seq) was performed on HE isolated from Runx1b and Gfi1/1b transgenic mouse reporters to unravel the underlying events leading to HSPCs emergence. In addition, cells present in the dorsal aorta microenvironment, representing the niche environment promoting HSCs emergence were also profiled by scRNA-seq and spatial transcriptomics. scRNA-seq analysis revealed that full-length scRNA-seq approach maximised the data recovery and yield in profiling HE. A robust HE expression profile was observed at embryonic day (E) E10.5 than E11.5. Further analysis of E10.5 HE uncovered a HE differentiation continuum characterised by cell cycle entry and specifically marked by Angiotensin-I converting enzyme (ACE) expression. Functional experiments confirmed the utility of ACE as a HE enrichment marker. Smooth muscle and PDGFRa mesenchymal cells were identified in the niche and shown to promote haematopoiesis. Finally, a scRNA-seq atlas of intra-embryonic and extra-embryonic HE that revealed distinct types of HE based on anatomical location. Intra-embryonic HE can be prospectively and independently isolated by the cell surface markers LYVE1, CD24 and MCAM. Co-culture experiments revealed the apparent bias of the different types of HE towards generating lymphoid-myeloid or erythro-myeloid progenitors. Collectively, these findings highlight the diversity of HE, and corresponding cell fates, and provide critical information to optimise in vitro generation of HSPCs protocols.
Date of Award1 Aug 2022
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorMagnus Rattray (Supervisor) & Georges Lacaud (Supervisor)

Keywords

  • haemogenic endothelium
  • haematopoietic stem cell
  • scRNA-seq
  • development

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