Combination Of Cytokines Enhances The Differentiation Of Murine Embryonic Stem Cells Into Hematopoietic Stem/Progenitor Cells

Blood transfusion and hematopoietic stem cell transplantation are the main treatment for hematologic diseases and immunodeficient diseases. There is a range of diverse sources of hematopoietic stem cells while the purification of hematopoietic stem cells is actually complicated and the content of hematopoietic stem cells is scarce. It is a huge challenge to proliferate hematopoietic stem cells in vitro. Therefore, the source of hematopoietic stem cells is the main challenge for clinical transplantation and immune reconstruction therapy method. As embryonic stem cells can not only long-term culture in vitro but also hold the capacity to produce every type of cells and tissue in the body. One report showed embryonic stem cells could be differentiated into hematopoietic stem cells and erythroid cells, myeloid and lymphoid cells. Therefore embryonic stem cells can be used as a source of hematopoietic stem cells. But the number of embryonic stem cells-derived hematopoietic stem cells is very low.The differentiation of murine embryonic stem cells into hematopoietic stem/progenitor cells is usually induced by embryoid bodies and co-culture method. We hypothesized that combination of cytokines could enhance the differentiation of murine embryonic stem cells into hematopoietic stem/progenitor cells.The mouse primary MEFs were isolated from Kunming mouse fetus at 12.5 to 16.5 day gestational ages through primary tissue digestion. The 3 passages MEFs were treated by mytomycin C to inhibit cell proliferation. The treated MEFs were used as feeder layer cells for culturing mouse embryonic stem cell E14TG2a. Taking into account the observations and drug-saving, an optimum drug level of mytomycin C to inhibit MEFs proliferation is 10 g/mL.The treated MEFs were used as feeder layer cells for culturing mouse embryonic stem cell E14TG2a. E14TG2a cells was grown to confluent clone with clear boundary in undifferentiated state. The expression of alkaline phosphatase of E14TG2a cells was positive. Detection of cell cycle of E14TG2a was achieved by flow cytometry. The normal karyotype of E14TG2a cells was detected by the chromosome G staining process. The totipotent genes OCT4 and Nanog was expressed in the E14TG2a cells. The undifferentiated state of embryonic stem was also confirmed by immunofluorescence. All the data showed that E14TG2a cells are growing well and suitable for subsequent experiments.The mice embryonic stem cell-derived hematopoietic progenitor cells, which was cultured with methylcellulose-based media, was observed. The cells of FLK1+ was significantly higher than control group at day 5. The cells of CD34+/Sca-1+ were significantly higher than control group, too. The expression of hematopoietic gene Flkl increased, and the expression of hematopoietic gene Bmp4 and CD34 were higher than control group. The expression of hematopoietic inhibition gene Smad5 was suppressed. The hematopoietic colony cells were observed during the culture.