Establishment Of Three Dimensional Culture Conditions For Erythrocytes From Human Embryonic Stem Cell-derived Definitive Hematopoiesis In Vitro

ObjectiveA significant issue for clinical utilization of human ES cells(hESCs)is whether they can generate terminally mature progenies with normal functions.Our team has already developed a method for efficient production of hESCs-derived hematopoietic progenitors by coculture with murine aorta-gonad-mesonephron(AGM)stromal cells.But multiplication of erythrocytes in vitro with current standard methods is limited and mostly insufficient for clinical applications of these cells.Therefore,engineering biomaterial-based bone marrow analogs is a promising approach for culturing erythrocytes.In our current study,the three-dimensional(3D)materials constructed by microspheres and hydrogels were used to simulate hematopoietic microenvironment in vitro,the cells harvested from different 3D culture systems were analyzed by MGG staining,flow cytometry and immunostaining,and a 3D culture method of erythroid cells in vitro was established.Methods1.Materials selectionKonjac dextran microspheres,collagen microspheres,methylcellulose,hyaluronic acid hydrogel,collagen hydrogel,hyaluronic acid scaffold,collagen scaffold,polypeptide hydrogel,fibrin hydrogel.2.Cell cultureHuman ES cells line(Hl)was used in this study.HPCs were obtained by coculture with AGM stromal cells.Human ES cells were maintained in an undifferentiated state and passaged weekly on matrigel.HPCs were sorted for the cell surface marker CD34 with the help of magnetic activated cell sorting according to the manufacturer’s instructions.The purity of the cells was assessed by flow cytometry and cells were only used if more than 90%were CD34-positive.The isolated HPCs were maintained in SFEM-2 medium with SCF(50ng/mL),FLT-3(50ng/mL)and TPO(50ng/mL)for three to five days until the CD34+CD45＋cell population ratio reached their peak.Then seeded the cells into different 3D materials and cultured in SFEM-2 medium with VEGF(20ng/mL),SCF(100ng/mL),IL-3(5ng/mL),IL-6(50ng/mL),FLT-3(5ng/mL),TPO(50ng/mL)and EPO(4IU/mL)for 5 days(SFEM-2 with 7 factors),SFEM-2 medium with SCF(1OOng/mL),IL-3(5ng/mL),EPO(4IU/mL)and dexamethasone(10-6M)for another 7 days.Cells were cultured under standard conditions at 37 ℃,5%C02 in a humidified atmosphere.All 3D materials were compared with suspension culture.We compared the proliferation of erythroid cells in different materials by flow cytometry,immunohistochemical staining and MGG staining to select the most suitable materials for 3D culture.ResultsHPCs were seeded into different 3D culture systems respectively,after 7 or 14 days of culture,we found that the quantity of erythroid progenitors from hESCs-derived hematopoietic progenitors in collagen 3D scaffold are higher than that in traditional 2D culture system and other 3D materials,and the ratios of erythroid progenitors in collagen scaffolds were similar to 2D culture,those cells also function normally equal to that in 2D culture system.Conclusion1.Three-dimensional culture systems based on collagen microspheres and konjac dextran microspheres was not conducive to the multiplication of erythrocytes.After 14 days of erythroid induction,the cell number and erythrocyte maturity were lower than that in liquid culture.2.The amplification efficiency of erythrocytes in collagen scaffold was significantly highly than that in liquid culture and other 3D hydrogel or scaffold.And there was no significant difference in the maturity of erythrocytes obtained from collagen scaffold and liquid culture.