Sequential Cellular Niche Plays a Crucial Role in the Generation of Enucleated Erythrocytes from Human Pluripotent Stem Cells

In vitro generation of human red blood cells (RBC) from human pluripotent stem cells (hPSCs) provides an alternative cell source for blood transfusion medicine to overcome blood supply shortages. However , the efficacy in terms of erythroblasts enucleation remains a great challenge. In this study, we develop a scalable differentiation method to generate enucleated mature erythrocytes from hPSCs through a novel monolayer differentiation procedure with sequential niche cues.

The hematopoietic differentiation was initiated with induction of mesodermal development which was tracked by using a brachyury-GFP reporter system in hPSCs. We used a concerted activation of Activin/Nodal, BMP4, Wnt, VEGF, and bFGF signaling pathways to effectively promote a sequential development of early mesodermal progenitors (brachyury+KDR- cells), cardiovascular-hematopoietic committed mesodermal progenitors (brachyury+KDR+ cells), hematopoietic-endothelial progenitors (KDR+CD34+CD31+ cells), and hemogenic endothelial (HE) cells (CD34+CD31+CD144+CD73-CD188- cells). At day 6, the grape-like CD34+CD43+CD90+CD235a+CD41a+CD38-CD45- erythroid/myeloid progenitors(EMPs) began to appear and gradually bud off from the HE cells. Addition of EPO promoted erythroid cells differentiation, resulting in gradual loss of the expression of CD34, CD90 and CD41, and acquiring expression of CD235a and CD71, markers for erythrocytes. However, those erythrocytes had limited potential to develop into enucleated erythrocytes (<5%), and expressed mostly γ hemoglobin and low level of β hemoglobin.

To investigate the cellular niche effect on erythroid cells differentiation of hPSCs, we cultured HE cells with OP9 stromal cells or endothelial cells transduced with the adenoviral E4ORF1 gene (E4ECs). Co-culture of HE cells with either endothelial cells or OP9 cells gave rise to significantly increased number of hematopoietic cells. To further determine the cellular niche effect on erythrocyte maturation, we cultured CD235a+CD71+ cells on OP9 cells or E4ECs. Subculture of endothelial-primed CD235a+CD71+ erythrocytes on OP9 cells gave rise to 80% enucleated erythrocytes with a high level of β hemoglobin expression, whereas subculture of OP9-primed CD235a+CD71+ erythrocytes on OP9 cells give rise to only 10% enucleated erythrocytes. It was interesting that endothelial-primed CD235a+CD71+ erythrocytes were unable to generate enucleated erythrocytes on OP9-DL1 cells that ectopically express Notch ligand Delta-like 1, suggesting that Notch signaling impaired erythroid maturation. Notably, prolonged culture of HE cells on endothelial cells reduced enucleated erythrocytes to 40% in the following erythrocytes maturation stage.

In conclusion, we establishe a novel method for efficient production of enucleated erythrocytes from hPSCs. We demonstrate for the first time that sequential niche cues effectively promote erythroblasts enucleation and γ globin to β globin switch. Our method offers a novel opportunity to study erythroid development, regulation of globin switching, drug testing, and modeling of RBC diseases in vitro .