Isolation of Mouse Ebryonic Stem Cells using Vero Cells- as a Feeder Layer- and their In Vitro Differentiation into Neuron- like Cells

Purppose: Embryonic stem (ES) ceIls are pluripotent cells conventionally isolated from the inner call mass of preimplantation embryos. ES cells can be maintained as stable, proliferative and undifferentiated cell lines if cultured in the presence of leukemia inhibitory factor (LlF) or on feeder layers (primary embryonic fibroblasts or STO fibroblasts). Feeder layers synthesize and secrete LlF into the culture medium. Since, based on several evidences, LIF is secreted by mitomycin pretreated Vero cells (a cell line isolated from the green monkey kidney epithelial cells), the main goal ofthis study was to demonstrate that ES cells can be isolated by using Vero cells as afeeder layer. The criteria used for the evaluation of undifferentiated ES cell phenotype in this study were morphology, evaluation of alkaline phosphatase activity, formation of embryoid bodies (Esb) and neural differentiation. FuIly expanded blastocysts were obtained by mating superovulated NMRI female mice and cultured individually on mitomycin C- inactivated Vero cells. After 4-5 days in the culture, The ICMs were plucked off and partially disaggregated in a 0.25% trypsin/EDTA solution. Three to four days later the compact ES cells colonies could be identified. The colonies were dissociated and reseeded on a freshly-treated Vero feeder layer. The cultures were scanned periodically and all the ES ceIl colonies were passsaged sequentially in the three days intervals. Alkaline phosphatase was detected histochemically, following the fixation ofthe colonies with 100% ethanol- using a- naphtyl phosphate as a substrate. The undifferentiated mouse ES cells derived on the Vero cells in our laboratory were induced to differentiation using retinoic acid by the 4-/4+ protocol. Several days later, some ofthe resulting cells posses a neuron-like cell morphology. The resulting ceIls had high ratio of nucleus to cytoplasm, prominent nucleuli, and a colony morphology similar to that of the murine ES ceIls. The ES cell colonies expressed the cell surface markers that characterize the undifferentiated ES cells, including alkaline phosphatase. We concluded that Vero cells as a feeder layer may present an altemative avenue in obtaining pluripotent ES celli ines. Although, the application ofVero cells in the ES cell culture still requires further investigation.