Effects of rat mesenchymal stem cells as a feeder layer in isolation and culture of mouse embryonic stem cells

Introduction: 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 cellsin most species. The aim of this study was application of rat mesenchymal stem cells (MSCs) as afeeder layer for the isolation and culture of mouse embryonic stem cells.Materials and Methods: Mesenchymal stem cells were isolated from rat bone marrow and culturedin DMEM (Dulbecco's modified Eagle's medium) medium supplemented with 10% FBS (fetal bovineserum). To verify the isolated cells, they were affected by osteocyte differentiation inducer to becomebone 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. Twodays later; after hatching of blastocysts, the cells were trypsinized and the inner cell mass dissociatedto the small cell clumps. These clumps were cultured on 12-well plates covered by the same MSCswithout applying any cytokines or growth inducer. Two to three days after the passage, coloniesappeared which were similar to embryonic stem cell colonies in morphology. These colonies werepassaged two more times using the mentioned procedure and their identities were examined bymorphological observation and alkalin phosphatase staining.Results: In this study we could easily cultured MSCs using DMEM media. The mesenchimic originof cultured cells, which showed fibroblastic morphology, was proved by differentiation to bonemasses using osteocyte inducer and detection with Alizarin red. By applying DMEM media and MSCscells, as feeder layer, we could culture ESC without any need to cytokines or growth factors. Afterpassage to the inner cell mass colonies were formed. These colonies were formed in two more otherpassages. The colonies were verified with alkalin phosphatase assay.Conclusion: Results of this study showed mesenchymal stem cells isolated from rat bone marrowcan differentiate to osteoblaste line and can be used as feeder layer for isolation, culture and formingembryonic stem cells colonies. This method, by using MSCs as feeder layer and bypassing the need ofcytokine and growth factors, seems to be a simple efficient method for culture and isolation ofembryonic stem cells