Heterogeneity within the ageing human haemopoietic compartment and predisposition to cancer

Strategic priority area:Stem Cell Science
Keywords:Single cell approaches, ageing, myeloproliferative disease

Several recent studies have described the presence of driver mutations of cancer in blood and skin tissues of healthy aged individuals, who surprisingly show no signs of disease1,2. It's unclear why healthy individuals can carry mutations that would in other circumstances cause disease. This project aims to identify cell intrinsic mechanisms that promote or suppress disease, by comparing haemopoietic stem cells (HSCs) bearing the same mutational burden from older individuals, with and without disease. Identifying disease promoting and suppressing signatures will lead to new therapeutic interventions and patient stratification. Human blood is continuously regenerated from a limited number of HSCs. With age, individual HSCs acquire leukaemia- associated mutations (e.g. DNMT3A, TET2, JAK2 or SF3B1), sometimes in the absence of clinical disease - "pre-leukaemia". Individuals with mutated HSCs will have wildtype HSCs present (proving "normal control"), giving us the opportunity to investigate the impact of these mutations within individual patients, thus avoiding patient to patient heterogeneity. To capture the intra-patient heterogeneity of HSCs will necessitate analysis of single HSCs or clones arising from single HSCs. Single cell genome and transcriptome analysis (G&T) was implemented by Dr Chandra during his time in Cambridge and is established in his lab in Edinburgh. We will also compare the impact of leukaemia associated mutations on HSCs in individuals with and without clinical disease.
Aim 1 (20:80% Glasgow:Edinburgh): What are the transcriptional consequences of leukaemia associated mutations (intra-patient comparison)? At the transcriptional level, we propose to compare mutation bearing and non-bearing HSC clones from healthy aged individuals (>70 years) and those with myeloproliferative neoplasms (MPN). We will first sequence whole blood from healthy and MPN cohorts to describe and match their mutational spectra. Since the prevalence of clonal haemopoiesis with leukaemic mutations is ~10% in the elderly, we will screen 50-100 healthy and 25 MPN patients. This will allow characterization by targeted genome sequencing of mutations of individual HSC-in vitro-derived clones, whilst simultaneously establishing the full transcriptome. Since limited material is expected, single cell approaches will be deployed to isolate DNA and RNA from the same clones. All genome-wide methods and data analysis tools are established in the Chandra lab (Edinburgh). Access to patient cohorts and ethics are in place in Glasgow.
Aim 2 (Glasgow): What are the functional consequences of leukaemia associated mutations? To assess the functional consequences underlying different mutations in healthy and disease contexts, we will use the same clones as in Aim 1 to assess HSC properties through in vitro kinetic and differentiation studies. We will follow clones derived from single HSCs short-term in vitro to determine their proliferation potential and range of progeny produced by using well-defined antibody panels for different haematological cell types. In addition, we will use serial replating assays to determine long-term "stemness "of HSCs in vitro. These assays are well established in the Kirschner and Copland lab (Glasgow) and are part of routine experiments carried out in haematology.
Training outcome:
The student will be trained in state-of-the-art experimental and computational genomics (Edinburgh) and haematological in vitro assays, human stem cell isolation and culture (Glasgow). The student will be thoroughly trained in experimental design, statistical analysis of data where applicable, data presentation in form of oral and poster presentations and is expected to participate in international conferences to discuss the work. Team work and interdisciplinary thinking will be encouraged throughout the project.