Heterogeneity within the ageing human haemopoietic compartment and predisposition to cancer

关键字：单细胞方法，衰老，骨髓增生性疾病最近的几项研究已经描述了健康老年人血液和皮肤组织中癌症驱动突变的存在，令人惊讶的是，这些老年人没有疾病的迹象。目前还不清楚为什么健康的人会携带在其他情况下会导致疾病的突变。该项目旨在通过比较患有和未患疾病的老年人携带相同突变负担的造血干细胞（hsc），确定促进或抑制疾病的细胞内在机制。识别疾病促进和抑制特征将导致新的治疗干预和患者分层。人类血液是由数量有限的造血干细胞不断再生的。随着年龄的增长，个体造血干细胞获得白血病相关突变（例如DNMT3A， TET2， JAK2或SF3B1），有时在没有临床疾病的情况下-“白血病前期”。具有突变hsc的个体将存在野生型hsc（证明“正常对照”），使我们有机会研究这些突变在个体患者中的影响，从而避免患者之间的异质性。为了捕获患者体内造血干细胞的异质性，需要分析单个造血干细胞或由单个造血干细胞产生的克隆。单细胞基因组和转录组分析（G&T）是钱德拉博士在剑桥大学期间实施的，并在他位于爱丁堡的实验室中建立起来。我们还将比较白血病相关突变对有和无临床疾病个体造血干细胞的影响。目标1（20:80格拉斯哥：爱丁堡）：白血病相关突变的转录后果是什么（患者内部比较）？在转录水平上，我们建议比较来自健康老年人（bb0 ~ 70岁）和骨髓增生性肿瘤（MPN）患者的携带突变和不携带突变的HSC克隆。我们将首先对健康人群和MPN人群的全血进行测序，以描述和匹配他们的突变谱。由于克隆造血伴白血病突变的患病率在老年人中约为10%，我们将筛选50-100名健康患者和25名MPN患者。这将允许对单个hsc体外衍生克隆的突变进行靶向基因组测序，同时建立完整的转录组。由于预计材料有限，将采用单细胞方法从同一克隆中分离DNA和RNA。所有全基因组方法和数据分析工具都是在钱德拉实验室（爱丁堡）建立的。在格拉斯哥，获得患者队列和道德规范是到位的。目标2（格拉斯哥）：白血病相关突变的功能后果是什么？为了评估健康和疾病背景下不同突变的功能后果，我们将使用与目标1中相同的克隆，通过体外动力学和分化研究来评估HSC特性。我们将对单个造血干细胞衍生的克隆进行短期体外随访，通过使用针对不同血液细胞类型的定义明确的抗体小组来确定其增殖潜力和后代的范围。此外，我们将使用连续重复试验来确定体外造血干细胞的长期“干性”。这些检测方法在科施纳和科普兰实验室（格拉斯哥）得到了很好的建立，并且是血液学常规实验的一部分。培训结果：学生将接受最先进的实验和计算基因组学（爱丁堡）和血液学体外分析、人类干细胞分离和培养（格拉斯哥）方面的培训。学生将接受实验设计、数据统计分析（如适用）、口头和海报形式的数据展示等方面的全面培训，并有望参加国际会议讨论工作。整个项目将鼓励团队合作和跨学科思维。

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