Dr. Korea Research Institute of Bioscience and Biotechnology (KRIBB), Republic of Korea
Abstract: Red blood cell (RBC) generation from human pluripotent stem cells (hPSCs) offers potential for innovative cell therapy in regenerative medicine as well as developmental studies. Ex vivo erythropoiesis from PSCs is currently limited by the low efficiency of functional RBCs with β-globin expression in culture systems. During induction of β-globin expression, the absence of a physiological microenvironment, such as a bone marrow niche, may impair cell maturation and lineage specification. Here, we suggest two important systems: one for RBC culture protocol and another for gene editing for Gene A knock-out. First, we describe a simple and reproducible culture system that can be used to generate erythroblasts with β-globin expression preparing a two-dimensional defined culture with ferric citrate treatment based on definitive hemogenic endothelium. Upon maturation, the erythroblasts cultured in the presence of ferric citrate showed high transcriptional levels of β-globin and enrichment of genes associated with heme synthesis and cell cycle regulation, indicating functionality. Second, Terminal erythropoiesis involves a complex differentiation process, encompassing erythropoietin (EPO) signal transduction and intracellular gene regulation. We identify gene A as a novel EPO-responsive Ca2+ channel that negatively KLF1 transcription. Using hPSCs-derived CD71+ erythroblasts, we demonstrate the down-regulation of Gene A and subsequent Ca2+ homeostasis during erythropoiesis in stage-specific manner. This gene A inactivation positively manipulated erythroid-specific genes by promoting KLF1 gene expression and erythroid maturation including increased globin expression and enucleation. Overall, when treated with Fe and modified gene A, erythroblasts exhibited advanced maturity and high β-globin transcription. These findings can aid in developing a stable protocol for generating clinically applicable RBCs.
Funding Source: Korean Cell-Based Artificial Blood Project funded (RS-2023-KH141086) the KRIBB Research Initiative Program Grants (KGM4562532) the National Research Council of Science & Technology grant by the Korea government (GTL24021-100)
