Ultrastructural characterization of potential embryonic stem cells in murine and bovine embryos

Abstract

A study was conducted to ultrastructurally characterize embryonic stem (ES) cells from an established murine ES cell line. ES cells were generally round in shape, had a prominent nucleolus and very little heterochromatin. The cytoplasm was relatively devoid of organelles but usually contained a few profiles of short endoplasmic reticulum and occasional round light staining mitochondria with distinctive tubular cristae. These data now provide an additional tool for the validation of newly isolated murine ES cells. In addition, the current study also evaluated the ultrastructure of mouse embryos cultured under the same conditions used to obtain the ES cells. These embryos were fixed either at the time of collection or on each of 5 consecutive days in culture. The ultrastructure of the embryos, primarily the ectoderm cells, was evaluated and compared to that of the established ES cells. The ultrastructure of ectoderm cells was very similar to that of the ES cells with the exception of the mitochondrial cristae. None of the cell types studied in the embryos possessed the tubular cristae observed in the ES cells. It was concluded that the optimum time for murine ES cell isolation from delayed embryos was 1-3 days following in vitro attachment. A second experiment was designed to ultrastructurally characterize the ectoderm cells in bovine embryos which were collected 7, 9 or 11 days following estrus. These ectoderm cells were also observed in cultured bovine embryos which had attached in vitro. It was found that the bovine ectoderm cells exhibited cytoplasmic differentiation during the time period evaluated and that these cells did not closely resemble either the ectoderm or ES cells of the mouse. The ectoderm cells of the attached bovine embryos appeared to be abnormal and had not yet broken through the trophoblast cell layer. Our results suggest that the attachment of bovine embryos to the feeder cells within the first few days of culture may be due to, or related to, the fact that there were irregularities detected within the ectoderm cells. Therefore, our results suggest that an alternative approach to isolating ES cells from the bovine may be indicated.