Dissecting cellular crosstalk and genetic perturbations in haematopoietic regulation

Haematopoietic stem and progenitor cells (HSPCs) proliferate and differentiate to produce a large number of mature blood and immune cells on a daily basis while maintaining themselves as the reservoir for lifelong haematopoiesis. Haematopoiesis is dynamically regulated by activating lineage-specific and differentiation stage-specific gene programmes that determine the behaviour of HSPCs in response to physiological and pathological signals. Development is one of the most highly dynamic processes, physiologically programmed to establish mature haematopoietic homeostasis through signal interactions between HSPCs and their actively remodelling microenvironment. In contrast, genetic mutations can pathologically reprogramme key regulatory pathways and alter HSPC behaviours including differentiation fate decisions. Understanding the regulation (and therefore dysregulation) of the haematopoietic system under these conditions has significant potential to improve the clinical management of developmental and malignant blood disorders such as leukaemia. Nevertheless, the cellular and molecular mechanisms of normal and perturbed haematopoiesis remain incompletely understood. The first aim of this work is to understand the molecular and intercellular networks that regulate the development of murine bone marrow and how these regulatory networks evolve with age. To delineate the potential age-specific interactions between diverse blood and stromal cell types, comprehensive single-cell transcriptional maps of 49,009 HSPCs and 62,377 stromal cells have been generated from foetal and postnatal mouse bone marrow. In Chapter 3, I introduce these blood and stromal landscapes and give an overview of the diverse cell types captured within the developing bone marrow. Taking advantage of these developmental blood and stromal landscapes, in Chapter 4, I perform cell-cell communication analyses and characterise molecular and intercellular programmes that are differentially active in different developmental stages. My analyses show a transition from foetal chondrogenic to postnatal osteogenic and to adult homeostatic stromal environment. With these distinct stromal programmes sending age-specific signalling cues to HSPCs, I report the potential roles of the chemokine-Cxcr3 axis in foetal HSPC migration and TGF-β signalling in the postnatal establishment of haematopoietic stem cell quiescence. Mutant mouse models are a useful tool to study how genetic mutations disrupt the intricacy of physiological haematopoietic regulations and lead the system to a preleukaemic state. In Chapter 5, I characterise haematopoietic perturbations in eight different preleukaemic mouse models by further interrogating single-cell transcriptomic profiling of 269,048 mutant and wild-type HSPCs. Through development of a novel computational pipeline for integrated analysis of single-cell perturbation datasets, I present mutation-specific preleukaemic effects on cell abundance, cellular lineage fate, cellular metabolism and gene expression. An ultimate goal of developing disease models is to improve patient care and outcomes by understanding the disease mechanisms. Therefore, in Chapter 6, I explore the translational potential of molecular signatures derived from my single-cell analyses of preleukaemic mouse models. By developing a scoring system based on preleukaemic transcriptional signatures and applying it to multiple cohorts of acute myeloid leukaemia patients, I establish Stem11, an 11-gene signature that provides prognostic information independent of standard clinical risk factors, potentially enabling more precise risk stratification for leukaemia patients. Altogether, this work presents a comprehensive single-cell analysis of developmental and perturbed haematopoiesis, providing deep insights into the cellular and molecular mechanisms that govern haematopoietic regulation in health and disease. These findings have not only advanced our fundamental understanding of haematopoietic biology, but will also help improve the treatment strategies for leukaemia patients in the future.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Isobe, Tomoya

Advisor dc:contributor.advisor

• Gottgens, Berthold

• haematopoiesis
• single-cell genomics

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/4de0189a-c0a1-4163-8961-137b779ee762/download
• http://purl.org/NET/rdflicense/allrightsreserved

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.119482

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/386126