Studies of Human Erythropoiesis Using classic 2D liquid culture and 3D scaffolds

Different bioengineering techniques have been developed to enable the production of erythrocytes in vitro. However, extrapolating the 3D structure and function of erythroblastic islands remains challenging. In our research, we employ a comprehensive strategy emphasizing physiological red blood cell (RBC) differentiation using a bespoke cocktail of cytokines and nutrients, including transferrin. The system was cultured for up to three weeks with hematopoietic stem and progenitor cells (HSPCs) in three-dimensional (3D) scaffolds engineered to mimic the physical and chemical properties of human bone marrow. Within the scaffold, HSPCs undergo erythroblastic maturation within the first 2 weeks of culture. After 3 weeks, fully mature CD36? CD71? CD235a? erythrocytes can be observed, accompanied by increased hemoglobin synthesis. In the 3D culture, diverse stages of erythroblast maturation are observed, driven by the activation of autophagy, which facilitates organelle clearance and membrane remodeling. This process leads to reduced surface integrin expression and significantly enhances RBC enucleation. Our findings underscore the importance of a 3D environment in supporting RBC maturation. Overall design: Bulk RNA-seq profiling of 2D and 3D cultures during erythroid differentiation. Differentiation is carried out in vitro.