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

正常和异常红细胞生成中与人红细胞岛生态位相关的分子成分的鉴定和表征

• 批准号: MR/T013923/1
• 负责人: Lesley Forrester
• 金额: $ 90.47万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FT013923%2F1
• 关键词: Identification; characterisation; molecular; components; associated

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.

红细胞（RBC）是生命所必需的，因为它们将氧气运送到身体的所有组织，并以每秒超过200万的速度产生。红细胞缺乏症和危及生命的贫血症是由遗传疾病、慢性感染、炎症和暴露于辐射和癌症治疗药物引起的。贫血症通过输注从健康供体收集的红细胞来治疗，但这仅在短期内有效，并且在需要重复输血的患者中出现重大问题。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，通过增强RBC产生起作用，但大多数药物并不针对该疾病的根本原因。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，并为先天性贫血提供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，都与红细胞转录因子KLF 1的突变有关。KLF 1调节与RBC结构和功能相关的基因的表达。最近的研究表明，KLF 1还在与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。在骨髓和脾脏深处，人类EI生态位无法用于研究，因此我们使用遗传程序化诱导多能干细胞衍生的巨噬细胞（iPSC-DM）开发了EI生态位的体外模型。iPSC-DM中KLF 1的激活增强了其支持RBC增殖和成熟的能力，我们表明作用机制涉及细胞-细胞接触中涉及的因子和分泌的因子。该建议的第一个目的是评估候选EI利基相关因子对红系细胞增殖和成熟的影响。根据我们现有的KLF 1靶基因数据集，我们将使用重组蛋白和合成的单生物素化肽测试分泌和膜相关因子增强RBC体外生产和成熟的能力。这将导致从无限来源（例如iPSC）生产RBC的改进方案，而当前的方案无法生产完全成熟的无核细胞。由于输血是治疗红细胞疾病的第一线，这种替代来源将克服与供体来源的输血，如，但在供应和输血传播的infection.Our的第二个目的是评估KLF 1的突变如何影响红细胞系岛的生态位，并确定基因缺陷的生态位内异常表达的因素。我们将使用来自携带KLF 1-E325 K突变的先天性贫血（CDA）患者的iPSC，并且我们将产生携带突变蛋白的可诱导形式的iPSC。这些iPSC将分化为EI样巨噬细胞，然后我们将测试它们支持RBC增殖和成熟的能力。我们将发现与对照iPSC-DM相比，在KLF 1-E325 K“患病”iPSC-DM中异常表达的因子。混合共培养将用于定义E325 K突变的内在和外在影响，我们将通过RNA测序、蛋白质组学分析和染色质免疫沉淀来识别KLF 1-E325 K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由感染、炎症和暴露于抗癌药物引起的贫血的新疗法。将使用我们的新型体外培养系统测试新药的作用。

项目成果

Modelling the Erythroblastic Island Niche of Dyserythropoietic Anaemia Type IV patients using Induced Pluripotent Stem Cells

使用诱导多能干细胞对 IV 型红细胞生成不良性贫血患者的成红细胞岛生态位进行建模

• DOI: 10.1101/2023.02.02.526657
• 发表时间: 2023
• 作者: May A

Identification of KLF1-E325K as a loss-of-function mutation in iPSC-derived macrophages

将 KLF1-E325K 鉴定为 iPSC 衍生巨噬细胞中的功能丧失突变

• 发表时间: 2022
• 作者: May,A

Macrophages - Celebrating 140 Years of Discovery

巨噬细胞 - 庆祝发现 140 周年

• DOI: 10.5772/intechopen.104514
• 发表时间: 2022
• 作者: Sushama Jose S

KLF1-related proteins in Erythroblast Island macrophages - a proteomic study

成红细胞岛巨噬细胞中的 KLF1 相关蛋白 - 蛋白质组学研究

• 发表时间: 2021
• 作者: Ventura T

Resident Macrophages and Their Potential in Cardiac Tissue Engineering.

• DOI: 10.1089/ten.teb.2021.0036
• 发表时间: 2022-06
• 期刊: TISSUE ENGINEERING PART B-REVIEWS
• 影响因子: 6.4
• 作者: Suku, Meenakshi;Forrester, Lesley;Biggs, Manus;Monaghan, Michael G.
• 通讯作者: Monaghan, Michael G.