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

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.

The transcription factor KLF1 is essential for the development and maturation of red blood cells (RBCs). A significant number of RBC disorders, including severe cases of anaemia, have been associated with KLF1 mutations. Research to date has focused on the intrinsic role of KFL1 in RBCs but KLF1 is also expressed and plays an extrinsic role in macrophages associated with the erythroid island (EI) niche. We developed a novel in vitro model of the human EI niche using induced pluripotent stem cell derived macrophages and have generated a database of EI-associated KLF1 target genes that encode factors that can promote RBC maturation. We now plan to test secreted and membrane-associated EI factors for their ability to enhance the maturation of RBCs using recombinant proteins and synthetic biotinylated peptides captured in on streptavidin-coated plates. Patients heterozygous for the dominant negative KLF1-E325K mutation present with circulating nucleated cells and a profound anaemia. To determine how KLF1 deficiency in the EI niche contributes to their pathology we will differentiate macrophages from iPSCs carrying a tamoxifen inducible iE325K-ERT2 and iPSCs derived from CDA patients. We will test their ability to support the proliferation and maturation of RBCs and identify factors that are aberrantly expressed within the genetically defective niche using RNA sequencing, comparative proteomics and chromatin immunoprecipitation. Chimeric co-cultures will be used to define the intrinsic and extrinsic effects of the E325K mutation and we will define the erythroid- and macrophage-specific targets of KLF1-E325K. Knock-in and knock-out iPSCs will be generated using CRISPR/CAS9 technology to validate the function of these targets. The characterisation of factors associated with the EI niche will impact on the treatment of anaemia, by identifying novel drug targets and designing optimised protocols for the production of therapeutic RBCs from limitless sources such as iPSCs.

This research has the potential for impact on the UK economy through commercial licensing of factors that could be used to treat patients with anaemia, improve the production of red blood cells in vitro and generate macrophages with specific functional properties for use in tissue repair. In the longer term, there could be a societal impact by improving the lives of patients and therapeutic practice by identifying novel treatments for anaemia and the supply of infection-free, immunologically compatible therapeutic blood cells.

The primary beneficiaries of this research in the shorter term will be academics working in the area of red blood cell development and maturation as our strategy provides a novel in vitro system for red blood cell production. Academics working in the area of macrophage biology will benefit as our work has developed and tested a strategy to genetically manipulate human macrophages and modulate their phenotype. Other beneficiaries of our work would include commercial companies through licensed laboratory procedures which could realise new therapies for anaemia and the production of therapeutic blood cell types from human pluripotent stem cells (hPSCs). We have established a collaboration with an industrial partner, Plasticell and were awarded funding from Innovate UK funding (2017-2019). The aim of that project is to use their unique Combicult system to simultaneous screen of large numbers of extrinsic culture conditions in the differentiation of blood cell progenitors. We can envisage that the identification of novel factors involved in the later stages of the differentiation process will improve red blood cell production. In the mid to long term, health services, health care professionals and patients worldwide could benefit from novel therapeutic strategies for anaemia and a consistent supply of infection-free cells and patient-specific red blood cells and macrophages that would be immunologically compatible.

Red blood cell (RBC) transfusion is used to treat haematological disorders as well as the effects of cytotoxic cancer treatment but is currently reliant on a consistent supply of high quality donor cells. Producing therapeutic red blood cell types from an alternative cell source such as immortalized progenitor cell lines or pluripotent stem cells (hPSCs) could provide a consistent supply of infection-free and patient-specific cells. One of the most likely impacts will be through the licensing of laboratory procedures to commercial companies within 3-5 years. Currently hPSC-derived RBCs are immature and this has been a major hurdle in the Novosang consortium's plan to take a hESC-derived RBCs to a first-in-man clinical trial.The ability to produce mature red blood cells in the lab would allow other experimentation of these cells which has been limited due to the lack of numbers such as proteomic analysis and it provides a model system to study complex signaling networks within the erythroid island niche that, to date, has relied on transgenic animal models that are costly and ethically challenged.

The ability to generate genetically-manipulated macrophages from human induced pluripotent stem cells (iPSCs) opens up an enormous opportunity to study macrophage biology and could be used in cell therapy to improve the lives of patients with a wide range of conditions including fibrosis of liver and lung. Our colleague Professor Stuart Forbes has commenced a clinical trial of patient monocyte-derived macrophages for liver fibrosis. iPSC-derived macrophages could provide an off-the-shelf therapy and macrophages that have been modulated to have a specific reparative phenotype might have more powerful properties.