Differential response to hepatic differentiation stimuli of amniotic epithelial cells isolated from four regions of the amniotic membrane

Human Amniotic Epithelial Cells (hAEC) isolated from term placenta are a promising source for regenerative medicine. However, it has long been debated whether the hAEC population consists of heterogeneous or homogeneous cells. In a previous study, we investigated the characteristics of hAEC isolated from four different regions of the amniotic membrane finding significant heterogeneity. The aim of this study was to evaluate the hepatic differentiation capability of hAEC isolated from these four regions. Human term placentae were collected after caesarean section and hAEC were isolated from four regions of the amniotic membrane (R1-R4, according to their relative distance from the umbilical cord) and treated in hepatic differentiation conditions for 14 days. hAEC-derived hepatocyte-like cells showed marked differences in the expression of hepatic markers: R4 showed higher levels of Albumin and Hepatocyte Nuclear Factor (HNF) 4α whereas R1 expressed higher Cytochrome P450 enzymes, both at the gene and protein level. These preliminary results suggest that hAEC isolated from R1 and R4 of the amniotic membrane are more prone to hepatic differentiation. Therefore, the use of hAEC from a specific region of the amniotic membrane should be taken into consideration as it could have an impact on the outcome of therapeutic applications

Ultrastructural Patterns of Cell Damage and Death Following Gamma Radiation Exposure of Murine Erythroleukemia Cells

Radiation causes damage to cell surface membranes, cytoplasmic organelles, and the nuclear process of DNA synthesis and repair, and this eventually results in different modes of cell death. In this study we examined murine erythroleukemia (MEL) cells, exposed to 15 and 60 Gy of 10 MeV photonic energy, and left in culture for up to 96 hours. Electron microscopical analysis was performed on conventionally embedded samples and freeze-fracture replicas, in order to detect ultrastructural patterns of cell damage and death. Of interest was the observation of chromatin condensates, nuclear membrane associations and nuclear pore redistribution during early apoptosis. Pronounced rearrangements of transmembrane particles during late stages of cellular necrosis were also found. The morphological damage induced by both doses of radiation as a function of time after exposure was only quantitatively but not qualitatively different

Ionizing Radiation-Induced Apoptosis and DNA Repair in Murine Erythroleukemia Cells

A morphological study of DNA repair and apoptotic patterns in relationship with cell cycle events was performed on murine erythroleukemia cells. The presence and distribution of DNA replicon sites were evaluated through the BrdU-anti BrdU immunofluorescence and immunogold techniques in light and electron microscopy. Different patterns of labelling and percentages of BrdU positive cells were observed depending on irradiation dose (up to 60 Gy) and time in post-irradiation culture (up to 24 hours). An enlargement of the S phase of the cell cycle was evidenced 18 hours post-irradiation as determined by flow cytometry analysis. The high resolution approach showed that, in spite of several morphological alterations, BrdU labelling was present even in cells displaying early and late apoptotic features

Balance between hypertrophic and hypoxic stimulus in caspase-3 activation during rat heart development

During heart development, cell hyperplasia and hypertrophy are the main mechanisms by which cardiac mass grows. Both these processes along with programmed cell death lead to complete growth and function. In addition, since the establishment of cardiac function depends on the relationship between oxygen supply and demand, we investigated some of the molecular mechanisms at the basis of rat myocardial cell response to hypoxic stress at different times of neonatal life. In particular, the role played by hypertrophic and survival factors like NF-kB and IAP-1 (Inhibiting Apoptosis Protein) and by death factors ASK-1 (Apoptosis Signal Regulating Kinase), JNK/SAPK (Jun-N-Terminal-Kinase/Stress-Activated Protein Kinase) pathways in regulating caspase-3 expression and activity has been evaluated by immunohistochemical and Western blotting analyses, respectively. Level of phosphorylation of IkBalpha and IAP-1 expression were substantial in 8-day-old hypoxic hearts, suggesting the persistence of NF-kB driven hypertrophic signal along with a rescue attempt against hypoxic stress. In contrast, ASK-1 mediated JNK/SAPK activation, regulating Bcl(2) levels, allows Bax homodimerization and caspase-3 activation in the same experimental conditions. Thus, a regulation carried out by NF-kB and JNK/SAPK pathways on caspase-3 activation at day 8 of neonatal life can be suggested as the main factor for the heart 'adaptive' response to hypoxia

HIF-1alpha cytoplasmic accumulation is associated with cell death in old rat cerebral cortex exposed to intermittent hypoxia

Intermittent hypoxia, followed by reoxygenation, determines the production of reactive oxygen species (ROS), which may lead to accelerated aging and to the appearance of age-related diseases. The rise in ROS levels might constitute a stress-stimulus activating specific redox-sensitive signalling pathways, so inducing either damaging or protective functions. Here, we report that in old rat cerebral cortex exposed to hypoxia, the accumulation in the cytoplasm of hypoxic inducible factor 1alpha (HIF-1alpha)--the master regulator of oxygen homeostasis--concomitant with p66(Shc) activation and reduced IkBalpha phosphorylation is associated with tissue apoptosis or necrosis. In young cerebral cortex, we hypothesize that the hypoxic damage may be reversible, based on our demonstration of elevated HIF-1alpha levels, combined with a low level of IkBalpha phosphorylation, a decrease in IAP-1 and a lack of major change in Bcl2 family proteins. These observations are associated with a low level of cell death induced by hypoxia, suggesting that HIF-1alpha activation in cortical neurons may produce rescue proteins in response to intermittent hypoxia

AGE-RELATED EVENTS IN ACTIVE T LYMPHOCYTE SUBPOPULATION. A MORPHOLOGICAL STUDY

The incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of active T lymphocytes from healthy aged donors was evaluated after in vitro PHA stimulation by means of light microscopy, electron microscopy and flow cytometry. The percentage of BrdU-labelled cells differed markedly between aged and young donors after 48 h of PHA stimulation, due to an enlarged early S phase compartment. In contrast, after 72 h the percentage of positive cells was quite similar in both age groups and no significant differences in the distribution within S phase nor changes in the patterns of ultrastructural localization of DNA replicon sites were observed. Our study provides evidence that an altered synchronization and a substantial delay in the in vitro cell proliferation occur in this peculiar T subpopulation as a consequence of the ageing process