The maturation of human erythroblasts from adult CD

The maturation of human erythroblasts from adult CD34+ hematopoietic stem/progenitor 17 alpha hydroxylase (HSPCs) can be assessed by serially tracing the expression of erythroid lineage markers. These markers for adult-type erythroblasts\' developmental process are widely used to recognize hPSC-derived erythroblasts, along with analysis of their functional properties. However, there is no detailed information about how erythroid cells originate from hPSC-derived progenitors and which phenotypes they share during the early developmental stages, hindering the elucidation of step-by-step mechanisms that control human early erythropoiesis from hPSCs. Phenotypically, adult-type hematopoietic stem cell (HSC)-derived erythroblasts mature by losing CD34 and then gaining glycophorin A (GPA; also known as CD235a) on their surface. CD36 co-expresses with GPA when cells are committed to erythroid lineage. Expression of CD36 is then gradually downregulated during the terminal maturation stage on enucleated red blood cells (RBCs) (Okumura et al., 1992; Neildez-Nguyen et al., 2002; Fajtova et al., 2013; Li et al., 2014). However, in this study we demonstrated that the expression process of CD34, GPA, and CD36 on hPSC/AGM-S3 co-culture-derived early erythroblasts was distinct. These hPSC-derived early erythroblasts were generated from endothelium precursors, underwent maturation by losing mesodermal and endothelial properties, and gradually gained definitive erythropoietic potential. Our results suggest that a unique pathway for early definitive erythropoiesis from hPSCs is phenotypically different from that of adult HSPC-derived definitive erythropoiesis.
Results
Discussion Human early erythropoiesis has been primarily studied using adult models with definitive hematopoiesis. Through subtle investigations, several laboratories reported that the regulatory pathway of definitive erythropoiesis development from adult HSPCs can be serially tracked by the expression of erythroid lineage surface markers, particularly CD34, GPA, CD71, and CD36 (Freyssinier et al., 1999; Gregory and Eaves, 1978). However, this may not be applicable to definitive erythropoiesis of hPSCs (Giarratana et al., 2011; Lu et al., 2008). In the present study, we investigated hPSC-derived early definitive erythroblasts during their development defined by expression of GPA, CD34, and CD36 (Figure 7). A unique cell population of GPA+CD34lowCD36− is firstly generated by day 5 of H1/AGM-S3 co-culture. Then, by losing CD34 expression, they developed and matured sequentially to G+34−36−, G+34−36low/+ and then G+34−36− erythroblasts with a gradual upregulation of β-globin. It is reported that before CD34+cells occur, GPA is firstly expressed on KDR+CD34−CD144− (abbreviated as G+K+34−144−) mesodermal cells (Sturgeon et al., 2014; Vodyanik et al., 2006), which could generate exclusively primitive hematopoiesis. However, the GPA+CD34low36− cells generated in H1/AGM-S3 co-culture are different from those early mesodermal cells. Since CD34 is known as an important marker of endothelial/hematopoietic cells, according to our data GPA+CD34low36− cells in day-7 co-culture are endowed with endothelial features while a small portion of them have already shared definitive erythroid potential. More than one-quarter of them express CD71 and even 1.7% ± 0.2% have already expressed adult-type β-globin. Consequently, the GPA+CD34low36− (G+34low36−) cells in day-10 co-culture showed an enhanced generation of erythroblasts (hemoglobin-positive cells >60%). When purified by cell sorting, these day-10 co-culture-derived GPA+CD34low cells can further proliferate and generate definitive erythroblasts with very high levels of β-globin (>95%), proving their definitive erythroid potential. Accumulated data in our laboratory suggest that, by co-culture with AGM-S3, the hPSC-derived early erythroblasts may originate from definitive endothelium precursors that differ from adult-type HSC-derived erythroblasts. Human adult-type HSPCs enriched in hCB or peripheral blood (hPB) are phenotypically CD34+CD45+, while committed erythroid cells expressing GPA appear only after CD34 disappears. There is no co-expression of GPA and CD34 stage throughout the developmental process of adult-type HSC-derived erythroblasts. Clearly, the G+34low36− early precursors derived from our H1/AGM-S3 co-culture are CD45 negative, showing their lack of HSPC or myeloid source.