Quality effect of co-culturing of yolk sac cells with M2-10B4 line on their differentiation in the presence and absence of erythropoietin

ABSTRACT

The embryonic yolk sac cells have two unique characteristics high proliferative capacity and lack of MHC associated antigen. According to the positive effects of co-culturing system on the differentiation of stem cells, in this study we evaluate the effect of M2-10B4 stromal cell line and erythropoietin [EPO] on the differentiation of embryonic yolk sac cells to erythroid cells. The yolk sacs were dissected from 10-day mice and their cells were separated using syring needles and enzyme digestions [0.1% collagenase/20 fetal calf serum [FBS] or 0.25% trypsin -0.02% EDTA at 37°C]. The M2-10B4 stromal cells were cultured in the DMEM medium and mitotically inactivated using mitomycin C. These cells were co-cultured with yolk stem cells in the medium containing stem cell factor [50 ng/ml]; EPO [IU/ml] and the colony assay of these cells [5xl0[4] cells/ml] have been done in the presence of interleukin-3 [40 ng/ml], stem cell factor [50 ng/ml] and EPO [lU/ml] in semisolid medium. After 7 days, benzidine staining on the colonies was carried out and positive benzidine colonies were considered as erythroid colonies. The colony assay showed that in the presence of EPO the growth of erythroid colonies were better than the other group and they had more benzidine colonies and cells. By using the stromal feeder layer such as bone marrow stromal cells and erythropoietin the differentiation and proliferation of YSC was improved; however, further studies are needed