摘要The fetal liver(FL)is the key erythropoietic organ during fetal development,but knowl-edge on human FL erythropoiesis is very limited.In this study,we sorted primary erythroblasts from FL cells and performed RNA sequencing(RNA-seq)analyses.We found that temporal gene expression patterns reflected changes in function during primary human FL terminal erythropoiesis.Notably,the expression of genes enriched in proteolysis and autophagy was up-regulated in orthochromatic erythroblasts(OrthoEs),suggesting the involvement of these pathways in enucle-ation.We also performed RNA-seq of in vitro cultured erythroblasts derived from FL CD34+cells.Comparison of transcriptomes between the primary and cultured erythroblasts revealed significant differences,indicating impacts of the culture system on gene expression.Notably,the expression of lipid metabolism-related genes was increased in cultured erythroblasts.We further immortalized ery-throid cell lines from FL and cord blood(CB)CD34+cells(FL-iEry and CB-iEry,respectively).FL-iEry and CB-iEry were immortalized at the proerythroblast stage and can be induced to differ-entiate into OrthoEs,but their enucleation ability was very low.Comparison of the transcriptomes between OrthoEs with and without enucleation capability revealed the down-regulation of path-ways involved in chromatin organization and mitophagy in OrthoEs without enucleation capacity,indicating that defects in chromatin organization and mitophagy contribute to the inability of OrthoEs to enucleate.Additionally,the expression of HBE1,HBZ,and HBG2 was up-regulated in FL-iEry compared with CB-iEry,and this up-regulation was accompanied by down-regulated expression of BCL11A and up-regulated expression of LIN28B and IGF2BP1.Our study provides new insights into human FL erythropoiesis and rich resources for future studies.