CFU-GM Like Colonies Derived from Embryonic Stem Cells Cultured on the Bone Marrow Stromal Cells

The aim of this study was to isolate mouse embryonic stem cells from late blastocyst stage embryos and to use them as a model system for the study of hematopoietic induction outside the embryo by coculturing of embryonic stem cells with bone marrow stromal cells. Blastocyst stage embryos from pregnant NMRI mice were obtained and cultured for 1-2 days in DMEM medium. The inner cell masses formed colonies 4-6 days after embryo hatching they were collected and trypsinized. Several subcultures were prepared in the medium containing 0.1 mM 2 mercaptoethanol, 1000 U/ml leukemia inhibitory factor and 10% fetal bovine serum. The embryonic stem cells were recognized by alkaline phosphatase histochemistry. Embryonic stem cells were cultured on inactivated mouse bone marrow stromal cells for 3 weeks at 37ºC and 33ºC. The colony assay was performed on semisolid medium containing murine IL-3 and IL-6 to determine the differentiation of embryonic stem cells to hematopoietic progenitor cells in vitro. Our results showed that with the effect of bone marrow stromal cells, a highly pluripotent stem cell which is derived from blastocyst stage embryo was able to primarily differentiated into the hematopoietic progenitor cells. CFU-GM like colonies were recognized according to their morphology after culturing the embryonic stem cells on the condition medium supplemented with IL-3 and IL-6