Red blood cell differentiation using canine-induced pluripotent stem cells

Abstract Background Red blood cell (RBC) transfusions are essential for treating various medical conditions, but global demand is difficult to meet due to a dwindling donor pool and compatibility issues. Pluripotent stem cells (PSCs) offer a promising alternative of blood dependent on volunteer donors for RBC production, and dogs serve as an excellent model for translational research due to their physiological and genetic similarities to humans. Methods Canine induced pluripotent stem cells (ciPSCs) were differentiated toward hematopoietic and erythroid lineages. Differentiated cells were evaluated for hematopoietic marker expression, hemoglobinization, colony-forming capacity, enucleation, and hemoglobin gene expression. Glycophorin A (GYPA)-enhanced green fluorescent protein (EGFP) reporter ciPSC lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9–mediated genome editing to visualize GYPA expression during differentiation. Results This study introduces a protocol for RBC differentiation using ciPSCs. We achieved generation of hemoglobinized RBCs, progressing through polychromatic and orthochromatic erythroblast–like stages. CiPSC–derived hematopoietic cells/RBCs were confirmed to have immature characteristics as determined by limited colony-forming capacities, low enucleation, and embryonic and fetal hemoglobin gene expression. Additionally, we created GYPA-EGFP reporter ciPSC lines using CRISPR-Cas9–mediated genome editing, enabling real-time visualization of GYPA expression. This innovation confirmed GYPA as a viable surface marker for ciPSC-derived RBCs. Conclusion Our findings mark an initial step toward establishing a canine PSC–based erythroid differentiation system, providing a foundation for future improvements and exploration of applications for canine PSC–derived RBCs.