Parthenogenetic embryonic stem cells are connected by functional intercellular bridges

We previously reported that parthenogenetic stem cells display abnormal centrosome and spindle formation that results in severe chromosome missegregation, with a high incidence of hypoploid karyotypes. Unexpectedly this is not accompanied by a correspondingly high rate of apoptosis and, by contrast, parthenogenetic cells share the pluripotency, self-renewal and in vitro differentiation properties of their bi-parental counterparts. We hypothesize that this is possible through a series of adaptive mechanisms that include the presence of intercellular bridges similar to those that connect germ cells during spermatogenesis. This would provide a way for mutual exchange of missing cell products, thus alleviating the unbalanced chromosome distribution that would otherwise hamper normal cell functions. The presence of intercellular bridges was investigated in pig parthenogenetic embryonic stem cells (PESCs) by transmission electron microscopy (TEM). Cultured cells were fixed in 2% glutaraldehyde and post-fixed in 1% osmic acid. After standard dehydration in ethanol series, samples were embedded in an Epon-Araldite 812 mixture and sectioned with a Reichert Ultracut S ultratome (Leica). Thin sections were stained and observed with a Jeol 1010 electron microscope. Pig PESCs were also subjected to scanning electron microscopy (SEM). To this purpose they were fixed and dehydrated as described above, covered with a 9 nm gold film by flash evaporation of carbon in an Emitech K 250 sputter coater (Emitech) and examined with a SEM-FEG Philips XL-30 microscope. To demonstrate functional trafficking activity through intercellular canals, fluroscent 10-kDa dextran was injected into the cytoplasm of a single cell with FemtoJet Microinjector (Eppendorf). Movement of the molecule from the injected cell to others was observed with a Nikon Eclipse TE200 microscope. Ultra-structural analysis of PESCs demonstrated the existence of intercellular bridges that ensured cytoplasmic continuity among cells. These canals appeared variable in size and were characterized by the presence of stabilizing actin patches. Furthermore, extensive movement of 10-kDa dextran among cells demonstrated functional intercellular trafficking through these communication canals suggesting their use for transfer of mRNAs, proteins and ribosomes among cells. Our results demonstrate that PESCs present a wide network of functional intercellular bridges that may constitute an adaptive mechanism to support normal cell functions. This process is commonly observed in transformed cells and give further support to the recent hypothesis that suggests the existence of common features and links between oncogenesis and self-renewal in pluripotent cell lines.