3109 – INVESTIGATING A KLF1-E325K MUTATION FOUND IN PATIENTS WITH TYPE IV CONGENITAL DYSERYTHROPOIETIC ANEMIA IN THE MACROPHAGE COMPARTMENT OF THE ERYTHROBLASTIC ISLAND NICHE

Abstract

The E325K mutation in the transcription factor KLF1 causes morphological defects and gene dysregulation in red blood cells (RBCs), resulting in the disease congenital dyserythropoietic anemia type (CDA) IV. RBCs develop in erythroblastic islands (EI), structures consisting of a central macrophage surrounded by developing erythroblasts. The macrophage compartment of the EI niche has been implicated in several RBC disorders, but it is not known whether it is affected by the KLF1-E325K mutation. To address this question, we developed an in vitro model of the human EI niche using macrophages derived from induced pluripotent stem cells (iPSCs) [1]. We generated macrophages from iPSCs derived from patients carrying the KLF1-E325K mutation, these had a comparable morphology and cell surface marker expression to iPSC-derived macrophages from control, wild type (WT) iPSC lines. To assess their ability to support the differentiation and maturation of RBCs macrophages were co-cultured with CD34+ haematopoietic progenitors under erythroid culture conditions. Suspension cells were assessed for the number of mature, enucleated RBCs present using flow cytometry and image analysis. We compared the phenotype of erythroid cells that were differentiated in the presence of WT and KLF1-E325K iPSC-derived macrophages. Results of our first pilot experiments indicated that fewer enucleated cells were present in cultures with KLF1-E325K macrophages compared to WT suggesting that the mutation has a detrimental effect on the supportive role of EI macrophages. If this effect is replicated in our planned experimental assays, it would suggest that the EI macrophage might represent a possible novel target for the treatment of RBC disorders.