Regulation Of Erythroid Differentiation From Human Pluripotent Stem Cells By TGF-β Signaling

Aim:Erythropoiesis is a complex process of producing red blood cells which is stimulated by low levels of oxygen in circulation. It usually occurs within the red bone marrow in the adult body, but in the early embryos, erythropoiesis takes place in the mesodermal cells of the yolk sac. With the development of embryo and age, erythropoiesis moves to the spleen and liver, and then transferred to the bone marrow. There are some previous studies reported that the human erythroid differentiation from hematopoietic stem cells isolated from umbilical cord blood, bone marrow and peripheral blood. However, the mechanism of erythropoiesis in the early development stage is not yet clear and needs to be further investigated. The present study is aimed to establish a chemically defined, feeder-free, serum-free erythroid differentiation system for human pluripotent stem cells to explore the role of TGF-β signaling pathway during the process of erythroid differentiation.Methods:In order to define the effects of different growth factors on the direct hematopoietic differentiation of human pluripotent stem cells, we need to establish a feeder-free, serum-free hematopoietic differentiation system. In addition, the feeder-free, serum-free hematopoietic differentiation system also provides a good model for the study of extracellular matrix microenvironment of human hematopoietic differentiation. First, hPSCs were cultured as the traditional human embryonic stem cell culture methods, namely human pluripotent stem cells were cultured on the Mitomycin C-treated mice embryonic fibroblast feeder cell layer and the medium was refreshed daily. The pluripotent stem cells were then dissociated into single cell for hanging-drop embryoid body formation. After 2 days, the EBs were formed by hanging-drop method and collected to induce hematopoietic differentiation in liquid culture. After 8 days of differentiation, the EBs were dissociated to single cells and labeled with monoclonal antibody specific against CD34+to perform FACS assay. And then the hematopoietic stem cells were sorted out by magnetic bead sorting (MACS). Finally, high-purified CD34+hematopoietic stem/progenitor cells were induced differentiation into erythroid cells. During the process of hematopoietic differentiation, the cells were collected from the culture and the flow cytometric analysis, Colony-forming assay, Wright-Geimsa staining, laser scanning confocal microscope were performed to inspect and identify cell differentiation state at different time points; the gene expression of hemoglobins were detected by RT-PCR and real-time quantitative PCR; the spectrophotometer was used to detect G6PD activity. In order to investigate the mechanisms of TGF-β signaling pathway in the regulation of erythroid differentiation and the interaction between the extracellular matrix and differentiated cells in the process of erythroid differentiation, BrdU assay and TNEL assay were used to detect the cell cycle and apoptosis of hematopoietic progenitor cells.Results:It is well known that a single human pluripotent cell is difficult to survive in the culture system in vitro. Moreover, cells from single -human pluripotent cell are not easy to form embryoid bodies. We developed a method of hanging-drop EB formation by adding Y27632, in which the defined numbers of single human pluripotent stem cell aggregated together to form EBs. The efficiency of EB formation by hanging-drop was above 90%. The EBs were induced differentiation into hematopoietic stem/progenitor cells by combined different kinds of cytokines, and the result of flow cytometric analysis showed that the average percentage of CD34+hematopoietic stem/progenitor cells was 15%.The CD34+hematopoietic stem/progenitor cells purified by MACS were induced erythroid differentiation in the feeder-free, serum-free condition. At first, the pluripotent stem cell derived CD34+hematopoietic stem/progenitor cells attached the plate in the extracellular matrix-coated culture plates to survive. After two days, the loosely-attached cells were observed and with the increase of the round loosely-attached cells, the round cells gradually formed cell colonies. After 6 days of culture, there were lots of suspension cells in the well and most of them are CD71high cells. The result of flow cytometric analysis showed that the percentage of CD71high cells was 80±5%. With prolonged culture, the size of cells became smaller, the nucleus gradually condensed and the hemoglobin expression gradually increase.Giemsa staining showed that the cells were erythroid cells and enucleated CD235a+/CD71+ cells were observed under confocal microscope microscopic after 10-15 days of culture. Meanwhile, we detected the effect of different kinds of hematopoietic growth factors on the lineage differentiation of CD34+hematopoietic progenitor, and found that EPO plays a key role in the process of erythroid differentiation. The EPO significantly increased the number of CD71+/CD235a+cells and also the expression of hemoglobins. While TGF-β significantly reduced the number of suspension cells, as well as the number of BFU-E in the presence of EPO and other hematopoietic growth factors, however, the percentage of CD71+ cells did not change.Conclusions:We established a feeder-free and serum-free erythroid differentiation system, in which hPSCs derived hematopoietic stem/progenitor cells differentiated into erythroid cells. The inhibition of TGF-P synergized with EPO signaling to promote erythroid progenitor proliferation. Our studies showed that TGF-β induced cell apoptosis and suppressed cell proliferation in the early stage of hematopoietic stem/progenitor cells, while accelerated maturation of erythroid progenitor cells.