Low initial cell density promotes the differentiation and maturation of human pluripotent stem cells into erythrocytes

Human pluripotent stem cell (hPSC)-derived red blood cells (RBCs) possess great potential for compensating for shortages in transfusion medicine. For better RBC generation from hPSCs, we compared the cell seeding density in the embryoid body formation-based hPSC induction protocol. In the selection of low- and high-density inoculation conditions, we found that low-density culture performed better in the final RBC product with more cell output and increased average cellular hemoglobin content. An elaborate study using flow cytometry demonstrated that low inoculation density promoted endothelial-to-hematopoietic transition, followed by improved hematopoietic progenitor formation and erythrocyte generation. The improved transformation from glycolysis to mitochondrial oxidation and reduced apoptosis might be responsible for this effect. Hints from RNA sequencing suggested that molecules involved in microenvironment interaction and metabolic regulation might respond for the different developmental potential. One of the possible mediators between outer message and intracellular response could be the nutrition sensors FOXO, PRKAA1 (AMPK) and MTOR genes. It is possible that low inoculation density triggered metabolic regulation signals, promoted mitochondrial oxidation, and resulted in enhanced cell amplification and hematopoietic differentiation. The low cell culture density will improve RBC generation from human PSCs. Overall design: To investigate the impact of human pluripotent stem cell seeding density on erythroid differentiation and metabolism of these cells, we seeded human embryonic stem cell H1 by 6×105/well and 2×105/well for each well of a 6-well-plate, induced these cells towards erythroid cells by step-wise induction. Comparative gene expression profiling analysis of RNA-seq data for 2×105 seeding group and its control 6×105 seeding group. Cells derived from different seeding density (6×105/well and 2×105/well) hESCs were harvested by erythroid induction day 6 and day 15. We then performed gene expression profiling alalysis using data obtained from RNA-seq of 2 different seeding desnity at two time points.