The Study Of Membrane Proteins Of Human Pluripotent Stem Cells-derived Erythroid Cells

Background:Human embryonic stem cells(hESCs)and human induced pluripotent stem cells(hiPSCs),also known as human pluripotent stem cells(hPSCs),harbor the abilities of self-renew,unlimited proliferation and differentiation to all types of cells.Clinical transfusion therapy is usually applied on current cell treatments,but shortage and infectious diseases of blood have brought huge challenges to clinical treatments.Therefore,it is an important research content of stem cell research whether we can successfully harvest a large amplification of hematopoietic stem cells and functional blood cells derived from hPSCs in vitro.hPSCs have not only provided an abundant source of cells for regenerative medicine,but also been devoted to establishing relevant disease model and finally realizing the Precision Medicine and Individual Therapy.In recent years,some laboratories have developed methods that efficiently generate functional erythrocytes derived from human pluripotent stem cells in vitro.The membrane proteins of erythrocytes,as functional proteins,play an important role in maintaining the morphology and features of erythrocytes.Therefore,it is essential to conduct the research about the developmental pattern of membrane proteins of hPSCs-derived erythroid cells.Previously we have developed a method that efficiently produce hematopoietic progenitors from hESCs by co-culturing with murine fetal liver-derived stromal cells and generating a large number of hESCs-derived erythroid cells on the condition of the colony culture,and thus we can have some studies on their functions.But there are few reports about the expression pattern of the membrane proteins of hPSCs-derived erythroid cells.Objective:This study is aimed at building a system of efficiently inducing the differentiation of hPSCs to erythroid cells in vitro.We investigate the similarities and differences of certain membrane proteins and phenotypic molecular between erythroid cells derived from hESCs and hCB-MNCs-CD34’ cells according to morphology and function.Our research is expected to provide some experiment basis for establishing the models of erythrocyte hemolytic disease.Methods:hPSCs were co-cultured with mice AGM-S3 stromal cells(mAGM-S3)irradiated by 13.3 Gray X-array with hematopoietic differentiation medium.Then substantial "cobblestone"(CS)-like hematopoietic stem/progenitor cells were harvested at day 12.Meanwhile,the CD34+ cells of umbilical cord blood mononuclear cells were sorted.Above two kinds of cells were cultured in semi-solid medium for 10-14 days so as to generate lots of erythroid colonies,which was followed by conducting a series of tests.This topic regards glycophorin A(GPA)positive cells as the object and uses the results of membrane proteins of hCB-MNCs-CD34+ cells-derived erythroid cells as reference.Then May-Grunwald-Giemsa(MGG)staining is used to compare the similarities and differences between the two sources of erythroid cells;Flow cytometry and quantitative real-time polymerase chain reaction(qRT-PCR)were applied on detecting the expression patterns of the membrane proteins of erythroid cells derived from different hematopoietic stem cells.Moreover,immunofluorescence staining was performed to explore the location and expression of intracellular and membrane proteins,especially Band3,to judge the maturity of the erythroid cells.Results:1.We can efficiently produce a higher proportion of hematopoietic progenitors and GPA positive cells derived from hESCs cocultured with murine AGM-S3 stromal cells.Above cells were cultured with semisolid medium in vitro,which eventually generate lots of erythroid colonies,including BFU-E,Mix and CFU-E as well as myeloid colonies.2.GPA+ cells appeared at day 12 in co-culture are still proerythroblast while the two kinds of stem cells-derived BFU-E are the heterogeneous population of erythroid cells in different stages mainly containing polychromatic erythroblast and orthochromatic erythroblast at the colony culture stage.3.Erythroid cells at day 10 derived from hPSCs scarcely express related genes,but increasingly express some genes encoding membrane proteins than that of the control group at the subsequent colony culture stage.Noticeably,the trend of downregulation of ITGA4 is not as obvious as that of the control group.4.The gene expression of membrane protein of hPSCs-derived Mix cells is generally lower than that of BFU-E cells.5.BFU-E cells derived from hESCs express high proportion of Band3 both in gene levels and protein levels.Conclusion:1.large quantities of hematopoietic progenitors derived from hESCs cocultured with murine AGM-S3 stromal cells,which posesses features of definitive hematopoiesis.2.Phenotypic molecules of erythroid cells from coculture of hESCs with AGM-S3 are still remain immature,but membrane proteins of eiythroid cells were constantly mature,when compared of hCB-MNCs-CD34+ cells-derived erythroid cells,which means a progressive mature process.3.The expression of Band3 of erythroid cells derived from hPSCs was paralleled with β-globin,which proves that under the condition of this experiment,it is possible to induce the hESCs to erythroid cells of adult characteristics progressively.And the comprehensive observation of β-globin and Band 3 can evaluate the maturity of erythroid cells derived from hESCs.4.The expression pattern of membrane proteins of erythroid cells is similar to the definitive hematopoiesis,but there are also have some differences between them,which contribute to the observation of the development process of erythroid cells in our experiment system.