Ultrastructure of the embryonic stem cells of the 8-day pig blastocyst before and after in vitro manipulation: Development of junctional apparatus and the lethal effects of PBS mediated cell-cell dissociation

Ultrastructural examination of 8-day hatched pig blastocysts (large and sma ll), their cultured inner cell mass (ICM), and cultured epiblast tissue (em bryonic stem cells) was undertaken to assess the development of epiblast ce ll junctions and cytoskeletal elements. In small blastocysts, epiblast cell s had no desmosomes or tight junction (TJ) connections and few organized mi crofilament bundles, whereas in large blastocysts the epiblast cells were c onnected by TJ and desmosomes with associated microfilaments. ICM isolation by immunodissection damaged the endoderm. cells beneath the trophectoderm cells but did not appear to damage the epiblast cells or their associated e ndoderm cells. Epiblast cells in cultured ICMs were similar in character to those in the intact large blastocyst except that perinuclear microfilament s were observed. Isolated pig epiblasts, cultured for similar to 36 hr on S TO feeder layers, formed a monolayer whose cells were connected by TJ, adhe rens junctions and desmosomes with prominent microfilament bundles running parallel to the apical cytoplasmic membranes. Perinuclear microfilaments we re a consistent feature in the similar to 36 hr cultured epiblast cells. A feature characteristic of differentiation into notochordal cells, i.e., a s olitary cilium, was also observed in the cultured epiblast. Exposure of the cultured epiblast cells to Ca++ -Mg++-free phosphate buffered saline (PBS) for 5-10 min resulted in extensive cell blebbing and lysis. The results ma y indicate that pig epiblast cells could be more easily dissociated from ea rly blastocysts (similar to 400 mum in diameter) if immunodissection damage to the ICM can be avoided. It may be difficult, however, to establish them as embryonic stem cell lines because the cultured pig epiblast cells were easily lysed by standard cell-cell dissociation methods. Anat Rec 264:101-1 13, 2001. (C) 2001 Wiley-Liss, Inc.