ABSTRACT
Embryonic stem cells [ESCs] are pluripotent cells derived from the inner cell mass [ICM] of blastocysts. A feeder layer and cytokines are necessary for culture of these embryonic cells in most species. The aim of this study was application of rat mesenchymal stem cells [MSCs] as a feeder layer for the isolation and culture of mouse embryonic stem cells. Mesenchymal stem cells were isolated from rat bone marrow and cultured in DMEM [Dulbecco's modified Eagle's medium] medium supplemented with 10% FBS [fetal bovine serum]. To verify the isolated cells, they were affected by osteocyte differentiation inducer to become bone mass. After twenty-one days, the differentiation was evaluated by Alizarin red staining. Blastocysts were obtained from Balb/c pregnant mice and cultured on this MSCs feeder layer. Two days later; after hatching of blastocysts, the cells were trypsinized and the inner cell mass dissociated to the small cell clumps. These clumps were cultured on 12-well plates covered by the same MSCs without applying any cytokines or growth inducer. Two to three days after the passage, colonies appeared which were similar to embryonic stem cell colonies in morphology. These colonies were passaged two more times using the mentioned procedure and their identities were examined by morphological observation and alkalin phosphatase staining. In this study we could easily cultured MSCs using DMEM media. The mesenchimic origin of cultured cells, which showed fibroblastic morphology, was proved by differentiation to bone masses using osteocyte inducer and detection with Alizarin red. By applying DMEM media and MSCs cells, as feeder layer, we could culture ESC without any need to cytokines or growth factors. After passage to the inner cell mass colonies were formed. These colonies were formed in two more other passages. The colonies were verified with alkaline phosphatase assay. Results of this study showed mesenchymal stem cells isolated from rat bone marrow can differentiate to osteoblast line and can be used as feeder layer for isolation, culture and forming embryonic stem cells colonies. This method, by using MSCs as feeder layer and bypassing the need of cytokine and growth factors, seems to be a simple efficient method for culture and isolation of embryonic stem cells