Establish A Method Of Generating Erythroid Cells From Cord Blood And HPSCs Derived Hematopoietic Stem/Progenitor Cells In Hyaluronic Acid-Based Hydrogel

ObjectiveA three-dimensional culture system was established to culture erythroid cells derived from human umbilical cord blood CD34+ cells or hematopoietic stem/progenitor cells(HSPCs)generated from hESCs/mAGM-3 cocultured in vitro.We compared the maturity of erythroid cells cultured by traditional suspension culture methods and three-dimensional culture methods.Methods?.Materials selectionHuman umbilical cord blood CD34+ cells or HSPCs from hESCs/mAGM-S3 cocultured for 12 days were seeded into BeaverNano hydrogels,alginate hydrogels,collagen hydrogels and hyaluronic acid hydrogels respectively,and all hydrogels were compared with suspension culture.We compared the proliferation of erythroid cells in different materials by flow cytometry to select the most suitable materials for three-dimensional culture.?.Cell culture1.Cord BloodHuman umbilical cord blood CD34+ cells were grown in suspension culture and hyaluronic acid hydrogels.After ten days of culture,the cells were harvested to test their function by flow cytometry and immunofluorescence staining.Through these tests,we mainly compared the function of erythroid cells cultured by three-dimensional culture or suspension culture.2.hESCsHSPCs from day 12 of hESCs/mAGM-S3 were proliferated in suspension culture and hyaluronic acid hydrogel culture.After ten days of culture,these cells were harvested to test their function by flow cytometry.Through these tests,we compared the function of erythroid cells derived from hESCs,which were cultured by three-dimensional culture or suspension culture.Results?.Materials selectionHuman cord blood CD34+ cells were cultured in the BeaverNano hydrogel and suspension culture for ten days.The results showed that the proportion of GPA+ cells were 86.6%and GPA+CD71+ cells were 81.1%in the BeaverNano hydrogel,while the proportion of GPA+ cells and GPA+CD71+ cells reached 54.5%and 49.1%,respectively,in the suspension culture.However,eosinophils planted into BeaverNano hydrogels might also express 76.3%of GPA+ cells and 38.9%CD71+ cells.hESCs derived HSPCs were cultured in suspension culture and the GPA+ cells and GPA+CD36+ cells reached 15.7%and 6.06%respectively,while the cells planted into the alginate hydrogels expressed 3.17%of GPA+ cells and 0.89%of GPA+CD36+ cells.hESCs derived HSPCs were cultured in suspension culture and the GPA+ cells and GPA+CD36+ cells reached 35.7%and 27.7%,respectively,while cells planted into the collagen hydrogels expressed 11.1%of GPA+ cells and 5.19%of GPA+CD36+ cells.hESCs derived HSPCs were cultured in suspension culture and the GPA+ cells and GPA+CD36+ cells reached 15.7%and 6.06%,respectively,while the cells planted into the hyaluronic acid hydrogels expressed 19.6%of GPA+ cells and 6.52%of GPA+CD36+ cells.?.Cell culture1.Cord BloodUnder different culture conditions,the total numbers of erythroid cells exhibited no difference,but the numbers of GPA+ cells,GPA+CD36+ cells and GPA+CD71+ cells in hyaluronic acid group were 3.7 times,3.9 times and 5.1 times higher,respectively,compared to the control group.Immunofluorescence staining confirmed that the cells cultured in HA hydrogels were indeed erythroid cells which had some functions of mature erythroid cells.2.hESCsPrior to the experiment,flow cytometry showed that the GPA+ cells population from mAGM-S3/hESCs on D12 was 36.8%.The amount of cells was 0.6×106.However,after ten days of culture,the number of erythroid cells in the control group was 3.9×105,and in the hyaluronic acid group,it was 4.2×105.Although there was no difference in the total number of erythroid cells derived from hESCs between the control group and the hyaluronic acid group,the number of GPA+ cells from hyaluronic acid group was 1.59-fold higher than the control group.The GPA+CD36+ cells population,the GPA+CD36-cells population,the GPA+ CD71+ cells population and the GPA+CD71-cells population was 2.68-,1.54-,2.68-,1.44-fold higher than the control group respectively.Conclusion1.BeaverNano hydrogels,alginate hydrogels,collagen hydrogel is not suitable for the growth of erythroid cells,however,hyaluronic acid hydrogel favors erythroid cell growth.2.Hyaluronic acid hydrogel can promote the differentiation of hematopoietic stem/progenitor cells derived from hESCs or cord blood into erythroid cells.3.Flow cytometry results indicated that hyaluronic acid hydrogel can selectively promote the differentiation of hematopoietic stem/progenitor cells derived from hESCs into naive erythroid cells.