Identification and characterisation of the molecular components associated with the human erythroid island niche in normal and abnormal erythropoiesis

红细胞(RBC)是生命所必需的，因为它们将氧气输送到身体的所有组织，并以每秒超过200万的速度产生。红细胞缺乏和危及生命的贫血是由遗传疾病、慢性感染、炎症和暴露于辐射以及癌症治疗药物引起的。贫血的治疗是通过输注从健康捐赠者那里收集的红细胞来治疗的，但这只在短期内有效，而且在需要重复输血的患者中会出现重大问题。用于治疗贫血的药物数量有限，包括促红细胞生成素刺激剂，其作用是促进红细胞生成，但大多数药物并不针对这种疾病的根本原因。该项目旨在更好地了解红细胞的发育和成熟，从而改进体外生产红细胞的策略，并对先天性贫血进行更有针对性的药物治疗。大量的红细胞疾病，从相对良性的血型变异到严重的贫血病例，都与红系转录因子KLF1的突变有关。KLF1调控与红细胞结构和功能相关的基因表达。最近的研究表明，KLF1也在红细胞发育和成熟的红细胞岛(EI)生态位相关的巨噬细胞中发挥作用。在骨髓和脾的深处，人类的EI生态位是无法进行研究的，因此我们使用遗传程序诱导的多能干细胞来源的巨噬细胞(IPSC-DM)建立了EI生态位的体外模型。KLF1在IPSC-DM中的激活增强了其支持红细胞增殖和成熟的能力，我们的研究表明，其作用机制既涉及细胞间接触的因素，也涉及细胞分泌的因素。这项建议的第一个目的是评估候选EI生态位相关因子对红系细胞增殖和成熟的影响。从我们现有的KLF1靶基因数据集，我们将测试分泌型和膜相关因子使用重组蛋白和合成单生物素化多肽促进红细胞体外生产和成熟的能力。这将导致改进从无限来源(如IPSCs)生产红细胞的方案，在这些方案中，现有方案无法生产完全成熟的去核细胞。由于输血是治疗RBC疾病的第一线，这种替代来源将克服与供者来源的输血相关的问题，例如供应限制和输血传播感染。我们的第二个目标是评估KLF1突变如何影响红细胞岛生态位，并确定在遗传缺陷的生态位内异常表达的因素。我们将使用来自携带KLF1-E325K突变的先天性贫血(CDA)患者的IPSCs，我们将产生携带突变蛋白的可诱导形式的IPSCs。这些IPSCs将被分化为EI样巨噬细胞，然后我们将测试它们支持红细胞增殖和成熟的能力。我们将发现与对照IPSC-DM相比，KLF1-E325K“病态”IPSC-DM中异常表达的因子。混合共培养将被用来确定E325K突变的内在和外在效应，我们将通过RNA测序、蛋白质组学分析和染色质免疫沉淀来鉴定KLF1-E325K的巨噬细胞特异性靶点。这些研究将确定新的药物靶点，从而开发出治疗先天性贫血以及因感染、炎症和接触抗癌药物而引起的贫血的新疗法。新药的作用将使用我们的新型体外培养系统进行测试。

Red blood cells (RBCs) are essential for life as they carry oxygen to all tissues of the body and are produced at a rate of over two million per second. RBC deficiency and life-threatening anaemia are caused by genetic disorders, chronic infection, inflammation and exposure to radiation and drugs for cancer treatment. Anaemias are treated by transfusion of RBCs collected from healthy donors but this is only effective in the short term and significant problems arise in patients who require repeated transfusions. The limited number of drugs that are used to treat anaemia, including erythropoietin stimulating agents, act by enhancing RBC production but the majority are not directed to the underlying cause of the disorder. This project aims to gain a better understanding of RBC development and maturation that could lead to improved strategies for producing RBCs in vitro and more targeted drug treatment for congenital anaemia. A significant number of RBC disorders, from relatively benign blood group variants to severe cases of anaemia, have been associated with mutations in the erythroid transcription factor, KLF1. KLF1 regulates the expression of genes associated with the structure and function of RBCs. Recent studies have shown that KLF1 also plays a role in macrophages associated with the erythroid island (EI) niche where RBCs develop and mature. Deep within the bone marrow and spleen, the human EI niche is inaccessible for study so we developed in vitro model of the EI niche using genetically programmed induced pluripotent stem cell-derived macrophages (iPSC-DMs). Activation of KLF1 in iPSC-DMs enhanced their ability to support RBC proliferation and maturation and we showed that the mechanism of action involves both factors involved in cell-cell contact and factors that are secreted. The first aim of this proposal is to assess the effect of candidate EI niche-associated factors on erythroid cell proliferation and maturation. From our existing dataset of KLF1 target genes, we will test the secreted and membrane-associated factors for their ability to enhance the in vitro production and maturation of RBCs using recombinant proteins and synthetic mono-biotinylated peptides. This will lead to improved protocols for the production of RBCs from limitless sources such as iPSCs where current protocols fail to produce fully mature, enucleated cells. As blood transfusion is the first line of treatment for RBC disorders this alternative source will overcome problems associated with donor-derived transfusion such as but limitations in supply and transfusion-transmitted infection.Our second aim is to assess how mutation in KLF1 affects the erythroid island niche and to identify factors that are aberrantly expressed within the genetically defective niche. We will use iPSCs derived from congenital anaemia (CDA) patients carrying the KLF1-E325K mutation and we will generate iPSCs carrying an inducible form of the mutant protein. These iPSCs will be differentiated into EI-like macrophages and we will then test their ability to support the proliferation and maturation of RBCs. We will discover factors that are aberrantly expressed in KLF1-E325K "diseased" iPSC-DMs compared to control iPSC-DMs. Mixed co-cultures will be used to define the intrinsic and extrinsic effects of the E325K mutation and we will identify macrophage-specific targets of KLF1-E325K by RNA sequencing, proteomic analyses and chromatin immunoprecipitation. These studies will identify novel drug targets that would lead to the development of new treatments for congenital anaemia as well as those caused by infection, inflammation and exposure to anti-cancer drugs. The action of novel drugs will be tested using our novel in vitro culture system.