Key Points Purification and quantification of human erythroid progenitors provides a powerful means for studying normal and disordered erythropoiesis. Transcriptome data provides a resource for the mechanistic understanding of the generation of erythroid progenitors from hematopoietic stem cells (HSCs).

The ten-eleven translocation (TET) family of proteins plays important roles in a wide range of biological processes by oxidizing 5-methylcytosine (5mC) to 5-hydroxy-methylcytosine. However, their function in erythropoiesis has remained unclear. We show here that TET2 and TET3 but not TET1 are expressed in human erythroid cells, and we explore the role of these proteins in erythropoiesis. Knockdown experiments revealed that TET2 and TET3 have different functions. Suppression of TET3 expression in human CD34 cells markedly impaired terminal erythroid differentiation, as reflected by increased apoptosis, the generation of bi/multinucleated polychromatic/orthochromatic erythroblasts, and impaired enucleation, although without effect on erythroid progenitors. In marked contrast, TET2 knockdown led to hyper-proliferation and impaired differentiation of erythroid progenitors. Surprisingly, knockdown of neither TET2 nor TET3 affected global levels of 5mC. Thus, our findings have identified distinct roles for TET2 and TET3 in human erythropoiesis, and provide new insights into their role in regulating human erythroid differentiation at distinct stages of development. Moreover, because knockdown of TET2 recapitulates certain features of erythroid development defects characteristic of myelodysplastic syndromes (MDSs), and the TET2 gene mutation is one of the most common mutations in MDS, our findings may be relevant for improved understanding of dyserythropoiesis of MDS.

Studies of human erythropoiesis have relied, for the most part, on the in vitro differentiation of hematopoietic stem and progenitor cells (HSPC) from different sources. Here, we report that despite the common core erythroid program that exists between cord blood (CB)- and peripheral blood (PB)-HSPC induced toward erythroid differentiation in vitro, significant functional differences exist. We undertook a comparative analysis of human erythropoiesis using these two different sources of HSPC. Upon in vitro erythroid differentiation, CB-derived cells proliferated 4-fold more than PB-derived cells. However, CB-derived cells exhibited a delayed kinetics of differentiation, resulting in an increased number of progenitors, notably colony-forming unit (CFU-E). The phenotypes of early erythroid differentiation stages also differed between the two sources with a significantly higher percentage of IL3R GPA CD34 CD36 cells generated from PB- than CB-HSPCs. This subset was found to generate both burst-forming unit (BFU-E) and CFU-E colonies in colony-forming assays. To further understand the differences between CB- and PB-HSPC, cells at eight stages of erythroid differentiation were sorted from each of the two sources and their transcriptional profiles were compared. We document differences at the CD34, BFU-E, poly- and orthochromatic stages. Genes exhibiting the most significant differences in expression between HSPC sources clustered into cell cycle- and autophagy-related pathways. Altogether, our studies provide a qualitative and quantitative comparative analysis of human erythropoiesis, highlighting the impact of the developmental origin of HSPCs on erythroid differentiation.

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