The Characterization And In Vitro Enucleation Of CD71^high Population From Mouse Embryonic Stem Cells

Based on the previous data from our lab, we examined genes which were differentially expressed in primitive erythrocytes (EryPs) and definitive erythrocytes (EryDs), and found that the CD71high population derived from differentiated embryonic stem cells had a similar gene expression pattern with EryPs derived from E8.5 mouse yolk sac. In addition, this system could be used to screen genes which might play roles in primitive erythropoiesis. Knock-down of Eafl, a gene with a role in mouse hematopoiesis had not been reported, reduced the formation of CD71high cells dramatically. And the real-time PCR demonstrated a decreased expression level of embryonic globin (βH-,εy-globin) but no change of genes involved in megakaryopoiesis. However the miR144/451, which was reported to protect erythrocytes from oxygen stress, has no obvious effects on the derivation of CD71high population. This indicats that miR144/451 play a distinct role in EryPs and EryDs. Upon aggregation into embryoid bodies (EBs), CD71high population could further mature and even went through enucleation in vitro, rate of 2%. As it was reported that the macrophages were needed in the enucleation of EryPs in vivo, we thus induced formation of macrophages from bone marrow cells, with the purity up to 98%. We next co-cultures the CD71high population with these BM-derived-macrophages, and we observed a higher enucleation rate around 10%.Taken together, CD71high population isolated from mESCs-EB system represent a counterpart of EryPs derived from E8.5 mouse yolk sac, thus we here proposed a useful platform to obtain EryPs to investigate the molecular mechanisms that regulates the formation and maturation of primitive erythrocytes.