Delineating Megakaryocyte Lineage Commitment with Isoform Resolved Single Cell Transcriptomics

Scoones, Anita (2023) Delineating Megakaryocyte Lineage Commitment with Isoform Resolved Single Cell Transcriptomics. Doctoral thesis, University of East Anglia.

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Revised models of megakaryocyte (Mk) commitment from haematopoietic stem cells (HSCs) are emerging, supported by increasing evidence that heterogeneity in the HSC pool enables rapid platelet replenishment through a direct commitment to the Mk lineage (Haas et al. , 2015; Roch, Trachsel and Lutolf, 2015; Grover et al. , 2016) . Platelet-biassed differentiation potential in a subset of HSCs corresponding to high expression of von Willebrand Factor (vWF) may support this direct commitment (Sanjuan-Pla et al. , 2013; Shin et al. , 2014) , but the cellular and molecular transitions underpinning this process, and how they vary with age and under stress, remain to be elucidated.

In this thesis, single-cell transcriptomics was used to explore the continuum of differentiation between HSC and Mk, in both steady state and in response to stresses including platelet depletion and ageing. Full-length scRNA-seq of mouse bone marrow-derived Lin − c-Kit + Cd150 + (LK CD150+) cells was employed, hypothesising that this gating strategy would enable unbiased capture of HSCs, Mk progenitors as well as any intermediate cell types and states. The use of full-length scRNA-seq, generated from both short- and long-read sequencing platforms, enabled the analysis of both gene and isoform expression during megakaryopoiesis. To explore the plasticity of the system under stress, this thesis outlines the cellular and transcriptomic changes along this trajectory in response to acute platelet depletion as well as normal ageing.

scRNA-seq data from a total of 2,016 LK Cd150+ cells was generated, and pseudotime analysis used to confirm that this population captures the entire trajectory of early Mk and erythroid differentiation. Upon acute platelet depletion, global and cell-type specific changes in gene expression were observed, including an increased proportion of HSCs exiting quiescence, and the expansion of Mk progenitor populations with distinct expression profiles marked by upregulation of markers involved in Mk maturation compared to steady state control. Taken together, these data suggest an ‘emergency’ response is triggered to counteract the threat of acute thrombocytopenia at multiple levels of megakaryopoiesis, involving the activation of stem cells and the generation of novel intermediate cell types.

Differential expression programmes were identified between young and old Mk progenitors for genes related to proliferation, plasma membrane and inflammation as well as altered frequencies of HSC and progenitor populations, consistent with previous reports that show an expansion of HSCs with age.

This thesis also explores differential isoform expression arising from alternative splicing, revealing heterogeneity across HSCs in key genes for HSC and Mk function, highlighting the importance of isoform-level interrogations of single cells in megakaryopoiesis. Overall, this work adds to the understanding of the mechanisms which enable the first steps with which HSCs commit to the Mk lineage, representing an important resource for further insights into ageing, stress and plasticity in haematopoiesis.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 21 May 2024 08:22
Last Modified: 21 May 2024 08:22


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