Clonal Analysis of Progressive Maturation of Erythroid Cells from Human Embryonic Stem Cell-Derived Definitive Hematopoiesis.
Authors: Ma Feng , Ebihara Yasuhiro , Umeda Katsutsugu , Sakai Hiromi , Hanada Sachiyo , Zhang Hong , Tsuchida Eishun , Nakahata Tatsutoshi , Nakauchi Hiromitsu , Tsuji Kohichiro 
Published: April 2020

Abstract
A critical issue for utilization of human embryonic stem cells (hESCs) in regenerative medicine is whether they can derive terminally mature progenies with normal function. We recently developed an efficient co-culture system of hESCs with murine fetal liver-derived stromal cells (mFLSCs), which enabled us to produce a large quantity of hESC-derived erythroid progenitor cells. By combining the hESC/mFLSC co-culture and a clonal culture, we analyzed the development of hESC-derived erythroid cells at a clone level. From day 10 of the co-culture, hESCs generated both erythroid colony- and erythroid burst-forming cells (E-CFCs and E-BFCs, respectively), and their numbers rapidly increased and reached a peak at day 14. By a clone-culture, we analyzed the erythroid cells in individually colonies and bursts by immunostaining. All erythroid cells in the colonies and bursts expressed alpha and gama globins, while beta and epsilon globins were expressed in a portion of these erythroid cells, but all the colonies and bursts contained a substantial number of beta and epsilon globin-expressing cells. We then examined the globin expression in individual bursts at day 12, 14, 16 and 18 of the co-culture. All erythroid cells in the bursts contained alpha and gama globins while beta globin-expressing erythroid cells gradually increased up to 100% until day 18, along with a decrease of epsilon globin-expressing erythroid cells in individual bursts. We then examined the globin expression of erythroid cells through day 12 to 18 in clonal culture of E-BFC at day 14 of hESC/mFLSC co-culture. Resultantly, beta globin-positive cells gradually increased up to 100% at day 18 of clonal culture, while epsilon globin-positive cells decreased. We equally divided erythroid cells in single bursts at day 12 of clonal culture; half was processed to the analysis of globin expression, and the other half was re-cultured for additional 6 days, and then their globin expression was examined. We again observed the time-dependent increase of beta globin and decrease of epsilon globin in hESC-derived erythroid cells at a clone level. On the other hand, hESC-derived erythroid cells could undergo encleation and their differentiation, when defined by co-expression of Glycophorin A, could be confirmed by up-regulation of CD71 in association with down-regulation of CD81 by flow cytometry, showing a similar pattern to human cord blood-derived erythroid cells. Furthermore, these hESC-derived erythroid cells could function as oxygen carrier, and had a sufficient glucose-6-phosphate dehydrogenase activity. The present study provides an experimental model for investigating early development of human erythropoiesis at a clone level. It enables us to conduct subtle and detailed experiment on hemoglobin switching, and the pathogenesis and discovery of drugs for hereditary diseases in human erythrocyte development. More importantly, the time-dependent maturation of hESC-derived erythroid cells indicates that hESCs can be a novel source for therapeutic transfusion.