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.