5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts

Phenotype definition is controlled by epigenetic regulations that allow cells to acquire their differentiated state. The process is reversible and attractive for therapeutic intervention and for the reactivation of hypermethylated pluripotency genes that facilitate transition to a higher plasticity state. We report the results obtained in human fibroblasts exposed to the epigenetic modifier 5-azacytidine (5-aza-CR), which increases adult cell plasticity and facilitates phenotype change. Although many aspects controlling its demethylating action have been widely investigated, the mechanisms underlying 5-aza-CR effects on cell plasticity are still poorly understood. Our experiments confirm decreased global methylation, but also demonstrate an increase of both Formylcytosine (5fC) and 5-Carboxylcytosine (5caC), indicating 5-aza-CR ability to activate a direct and active demethylating effect, possibly mediated via TET2 protein increased transcription. This was accompanied by transient upregulation of pluripotency markers and incremented histone expression, paralleled by changes in histone acetylating enzymes. Furthermore, adult fibroblasts reshaped into undifferentiated progenitor-like phenotype, with a sparse and open chromatin structure. Our findings indicate that 5-aza-CR induced somatic cell transition to a higher plasticity state is activated by multiple regulations that accompany the demethylating effect exerted by the modifier

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

Parthenogenetic cell lines : an unstable equilibrium between pluripotency and malignant transformation

Human parthenogenetic embryos have been recently proposed as an alternative, less controversial source of embryonic stem cells. However many aspects related to the biology of parthenogenetic cell lines are not fully understood and still need to be elucidated. These cells have great potentials; they possess most of the main features of bi-parental stem cells, show the typical morphology and express most of the pluripotency markers distinctive of ESC. They also have high telomerase activity, that disappears upon differentiation, and display great plasticity. When cultured in appropriate conditions, they are able to give rise to high specification tissues and to differentiate into mature cell types of the neural and hematopoietic lineages. However, their injection in immunodeficient mice has been reported to result in tumour formations. Aberrant levels of molecules related to spindle formation, cell cycle check points and chromosome segregation have also been detected in these cells, that are characterized by the presence of an abnormal number of centrioles and massive autophagy. All these observations indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferation versus differentiation in parthenogenetic stem cells. In this manuscript we summarize data related to these aberrant controls and describe experimental evidence indicating their uniparental origin as one of the possible cause. Finally we propose their use as an intriguing experimental tool where the pathways controlling potency, self renewal and cell plasticity are deeply interconnected with cell transformation, in an unstable, although highly controlled, equilibrium between pluripotency and malignacy

Functional amyloids in insect immune response.

The innate immune system of insects consists of humoural and cellular responses that provide protection against invading pathogens and parasites. Defence reactions against these latter include encapsulation by immune cells and targeted melanin deposition, which is usually restricted to the surface of the foreign invader, to prevent systemic damage. Here we show that a protein produced by haemocytes of Heliothis virescens (Lepidoptera, Noctuidae) larvae, belonging to XendoU family, generates amyloid fibrils, which accumulate in large cisternae of the rough endoplasmic reticulum and are released upon immune challenge, to form a layer coating non-self objects entering the haemocoel. This amyloid layer acts as a molecular scaffold that promotes localised melanin synthesis and the adhesion of immune cells around the non-self intruder during encapsulation response. Our results demonstrate a new functional role for these protein aggregates that are commonly associated with severe human diseases. We predict that insects will offer new powerful experimental systems for studying inducible amyloidogenesis, which will likely provide fresh perspectives for its prevention