Search

Phenotype definition is driven by epigenetic mechanisms as well as directly influenced by the cell microenvironment and by biophysical signals deriving from the extracellular matrix. The possibility to interact with the epigenetic signature of an adult mature cell, reversing its differentiated state and inducing a short transient high plasticity window, was previously demonstrated. In parallel, in vitro studies have shown that 3D culture systems, mimicking cell native tissue, exert significant effects on cell behavior and functions. Here we report the production of "PTFE micro-bioreactors" for long-term culture of epigenetically derived high plasticity cells. The system promotes 3D cell rearrangement, global DNA demethylation and elevated transcription of pluripotency markers, that is dependent on WW domain containing transcription regulator 1 (TAZ) nuclear accumulation and SMAD family member 2 (SMAD2) co-shuttling. Our findings demonstrate that the use of 3D culture strategies greatly improves the induction and maintenance of a high plasticity state.

Converting adult cells from one cell type to another is a particularly interesting idea for regenerative medicine. Terminally differentiated cells can be induced to de-differentiate in vitro to become multipotent progenitors. In mammals these changes do not occur naturally, however exposing differentiated adult cells to synthetic molecules capable of selectively reverting cells from their lineage commitment to a more plastic state makes it possible to re-address their fate. Only scattered information are available on the morphological changes and ultrastructural remodeling taking place when cells convert into a different and specific type. To better clarify these aspects, we derived human granulosa cell (GC) primary cultures and analyzed the morphological changes taking place in response to the exposure to the epigenetic modifier 5-azacytidine (5-aza-CR) and to the treatment with VEGF, as a stimulus for inducing differentiation into muscle cells. Ultrastructural modifications and molecular marker expression were analyzed at different intervals during the treatments. Our results indicate that the temporary up regulation of pluripotency markers is accompanied by the loss of GC-specific ultrastructural features, mainly through autophagocitosis, and is associated with a temporary chromatin decondensation. After exposure to VEGF the induction of muscle specific genes was combined with the appearance of multinucleated cells with a considerable quantity of non-spatially organized filaments. The detailed analysis of the morphological changes occurring in cells undergoing lineage re-addressing allows a better understanding of these process and may prove useful for refining the use of somatic cells in regenerative medicine and tissue replacement therapies.

Parthenogenetic cells, obtained from in vitro activated mammalian oocytes, display multipolar spindles, chromosome malsegregation and a high incidence of aneuploidy, probably due to the lack of paternal contribution. Despite this, parthenogenetic cells do not show high rates of apoptosis and are able to proliferate in a way comparable to their biparental counterpart. We hypothesize that a series of adaptive mechanisms are present in parthenogenetic cells, allowing a continuous proliferation and ordinate cell differentiation both in vitro and in vivo. Here we identify the presence of intercellular bridges that contribute to the establishment of a wide communication network among human parthenogenetic cells, providing a mutual exchange of missing products. Silencing of two molecules essential for intercellular bridge formation and maintenance demonstrates the key function played by these cytoplasmic passageways that ensure normal cell functions and survival, alleviating the unbalance in cellular component composition.

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. (C) 2011 Elsevier Ltd. All rights reserved.

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 hypothesise 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 (PESC) 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 PESC 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 an SEM-FEG Philips XL-30 microscope. To demonstrate functional trafficking activity through intercellular canals, fluorescent 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 PESC demonstrated the existence of intercellular bridges that ensured cytoplasmic continuity among cells. These canals appeared variable in size and were characterised by the presence of stabilising 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 mRNA, proteins and ribosomes among cells. Our results demonstrate that PESC 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 gives further support to the recent hypothesis that suggests the existence of common features and links between oncogenesis and self-renewal in pluripotent cell lines. Supported by AIRC IG 10376. PG was supported by INGM.