Differentiation of neuron-like cells in cultured rat optic nerves: a neuron or common neuron-glia progenitor?

The optic nerve consists of axons, glia, and undifferentiated cells; neuronal cell bodies are absent. To study the developmental potential of glia and precursor cells in vitro, we devised an original, long-term culture system of optic nerve explants, called minisegments, of newborn rats; at this stage the nerves are composed of naked axons, astrocytes, and undifferentiated cells. After about 4-5 weeks in culture, neuron-like cells appeared, which showed morphological, fine structural, and immunocytochemical properties ascribed to neurons. These neuron-like cells may be derived from a common neuron-glia progenitor or from a small population of precursors, which never produce glia in situ.     

Is sodium current present in human sinoatrial node cells?

Pacemaker activity of the sinoatrial node has been studied extensively in various animal species, but is virtually unexplored in man. As such, it is unknown whether the fast sodium current (I_Na) plays a role in the pacemaker activity of the human sinoatrial node. Recently, we had the unique opportunity to perform patch-clamp experiments on single pacemaker cells isolated from a human sinoatrial node. In 2 out of the 3 cells measured, we observed large inward currents with characteristics of I_Na. Although we were unable to analyze the current in detail, our findings provide strong evidence that I_Na is present in human sinoatrial node pacemaker cells, and that this I_Na is functionally available at potentials negative to -60 mV.     

CaV1.3 as pacemaker channels in adrenal chromaffin cells: specific role on exo- and endocytosis?

Voltage-gated L-type calcium channels (LTCCs) are expressed in adrenal chromaffin cells. Besides shaping the action potential (AP), LTCCs are involved in the excitation-secretion coupling controlling catecholamine release and in Ca (2+) -dependent vesicle retrieval. Of the two LTCCs expressed in chromaffin cells (CaV1.2 and CaV1.3), CaV1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using CaV1 .3 (-/-) KO mice and the AP-clamp it has been possible to resolve the time course of CaV1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells CaV1.3 is coupled to fast-inactivating BK channels within membrane nanodomains and controls AP repolarization. The ability to carry subthreshold Ca (2+) currents and activate BK channels confers to CaV1.3 the unique feature of driving Ca (2+) loading during long interspike intervals and, possibly, to control the Ca (2+) -dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling.     

Defining the role of 17β-estradiol in human endometrial stem cells differentiation into neuron-like cells

Human endometrial stem cells (hEnSCs) that can be differentiated into various neural cell types have been regarded as a suitable cell population for neural tissue engineering and regenerative medicine. Considering different interactions between hormones, growth factors, and other factors in the neural system, several differentiation protocols have been proposed to direct hEnSCs towards specific neural cells. The 17β-estradiol plays important roles in the processes of development, maturation, and function of nervous system. In the present research, the impact of 17β-estradiol (estrogen, E2) on the neural differentiation of hEnSCs was examined for the first time, based on the expression levels of neural genes and proteins. In this regard, hEnSCs were differentiated into neuron-like cells after exposure to retinoic acid (RA), epidermal growth factor (EGF), and also fibroblast growth factor-2 (FGF2) in the absence or presence of 17β-estradiol. The majority of cells showed a multipolar morphology. In all groups, the expression levels of nestin, Tuj-1 and NF-H (neurofilament heavy polypeptide) (as neural-specific markers) increased during 14 days. According to the outcomes of immunofluorescence (IF) and real-time PCR analyses, the neuron-specific markers were more expressed in the estrogen-treated groups, in comparison with the estrogen-free ones. These findings suggest that 17β-estradiol along with other growth factors can stimulate and upregulate the expression of neural markers during the neuronal differentiation of hEnSCs. Moreover, our findings confirm that hEnSCs can be an appropriate cell source for cell therapy of neurodegenerative diseases and neural tissue engineering.     

CaV1.3 as pacemaker channels in adrenal chromaffin cells: Specific role on exo- and endocytosis?

Voltage-gated L-type calcium channels (LTCCs) are expressed in adrenal chromaffin cells. Besides shaping the action potential (AP), LTCCs are involved in the excitation-secretion coupling controlling catecholamine release and in Ca²⁺-dependent vesicle retrieval. Of the two LTCCs expressed in chromaffin cells (Ca_V1.2 and Ca_V1.3), Ca_V1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using Ca_V1 .3^-/- KO mice and the AP-clamp it has been possible to resolve the time course of Ca_V1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells Ca_V1.3 is coupled to fast-inactivating BK channels within membrane nanodomains and controls AP repolarization. The ability to carry subthreshold Ca²⁺ currents and activate BK channels confers to Ca_V1.3 the unique feature of driving Ca²⁺ loading during long interspike intervals and, possibly, to control the Ca²⁺-dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling.     

Are Cav1.3 pacemaker channels in chromaffin cells?: Possible bias from resting cell conditions and DHP blocker usage

Mouse and rat chromaffin cells (MCCs, RCCs) fire spontaneously at rest and their activity is mainly supported by the two L-type Ca(2+) channels expressed in these cells (Ca(v)1.2 and Ca(v)1.3). Using Ca(v)1.3(-/-) KO MCCs we have shown that Ca(v)1.3 possess all the prerequisites for carrying subthreshold currents that sustain low frequency cell firing near resting (0.5 to 2 Hz at -50 mV): low-threshold and steep voltage dependence of activation, slow and incomplete inactivation during pulses of several hundreds of milliseconds. Ca(v)1.2 contributes also to pacemaking MCCs and possibly even Na(+) channels may participate in the firing of a small percentage of cells. We now show that at potentials near resting (-50 mV), Ca(v)1.3 carries equal amounts of Ca(2+) current to Ca(v)1.2 but activates at 9 mV more negative potentials. MCCs express only TTX-sensitive Na(v)1 channels that activate at 24 mV more positive potentials than Ca(v)1.3 and are fully inactivating. Their blockade prevents the firing only in a small percentage of cells (13%). This suggests that the order of importance with regard to pacemaking MCCs is: Ca(v)1.3, Ca(v)1.2 and Na(v)1. The above conclusions, however, rely on the proper use of DHPs, whose blocking potency is strongly holding potential dependent. We also show that small increases of KCl concentration steadily depolarize the MCCs causing abnormally increased firing frequencies, lowered and broadened AP waveforms and an increased facility of switching "non-firing" into "firing" cells that may lead to erroneous conclusions about the role of Ca(v)1.3 and Ca(v)1.2 as pacemaker channels in MCCs.     

Epithelial cells in culture: injured or differentiated cells?

Isolation of epithelial cells for cell culture is based on destruction of epithelial integrity. The consequences are manifold: cell polarity and specific cell functions are lost; cells acquire non‐epithelial characteristics and start to proliferate. This situation may also occur in situ when parts of the epithelium are lost, either by apoptosis or necrosis by organ or tissue injury. During recovery from this injury, surviving epithelial cells proliferate and may restore epithelial integrity and finally re‐differentiate into functional epithelial cells. In vitro, this re‐differentiation is mostly not complete due to sub‐optimal culture conditions. Therefore cultured epithelial cells resemble wounded or injured epithelia rather than healthy and well differentiated epithelia. The value of an in vitro cell model is the extent to which it helps to understand the function of the cells in situ. A variety of parameters influence the state of differentiation of cultured cells in vitro. Although each of these parameters had been studied, the picture how they co‐ordinately influence the state of differentiation of epithelial cells in vitro is incomplete. Therefore we discuss the influence of the isolation method and cell culture on epithelial cells, and outline strategies to achieve highly differentiated epithelial cells for the use as an in vitro model.     

Towards synthetic cells:are cells computers making computers?

Understanding life supposes that one will,one day,reconstruct cells. A deep analysis of what life is shows that a cell is similar to a computers making computer. This asks for several orginal levels of organisation. First,the cell needs to be seen as a machine separated from the genetic program,which it runs. Over generations the machine reproduces,while the program replicates. Reproduction is a process which is able to accumulate valuable information over generations. Extracting valuable information from an ocean of noise requires an energy-dependent process which uses energy to prevent degradation of functional entities. Analysis of bacterial genomes shows that the core set of genes which persist in most genomes code for the functions needed to perform this process of ratchet-like information accumulation. It also suggests that a mineral,polyphosphates,could be a ubiquitous (and stable) energy source essential for the process.     

Does the circadian pacemaker act through cyclic AMP to drive the melatonin rhythm in chick pineal cells?

Cyclic AMP is a key regulator of melatonin production in the chick pineal gland. Agents that raise cyclic AMP levels (such as forskolin), or cyclic AMP analogues (such as 8-bromocyclic AMP), increase melatonin synthesis and release, whereas agents that lower cyclic AMP levels (including light) decrease melatonin synthesis and release. A circadian oscillator in these cells also raises and lowers melatonin output. We have been investigating the relationships between cyclic AMP and the circadian pacemaker in the regulation of melatonin production. In the chick pineal (unlike certain neuronal systems), the weight of the evidence indicates that cyclic AMP is not on an entrainment pathway to the circadian pacemaker. Instead, cyclic AMP appears to act downstream from the pacemaker. The pacemaker might itself act directly through cyclic AMP, regulating melatonin content by raising and lowering cyclic AMP levels. If this were the case, and if the effects of cyclic AMP levels on melatonin output are saturable (as they must be), then, in the face of such saturating levels of cyclic AMP, the pacemaker should no longer raise or lower melatonin output. To test this prediction, maximally effective concentrations of forskolin and 8-bromocyclic AMP were determined. Both agents markedly increased melatonin output. After 36 hr, cells were refractory to additional stimulation of melatonin output by addition of both agents together, or by higher concentrations of forskolin (although cyclic AMP levels could still be raised further). Nonetheless, the circadian pacemaker continued to raise and lower melatonin output: The rhythm persisted in the face of saturating levels of cyclic AMP. It is therefore suggested that the circadian pacemaker in chick pineal cells acts with, not through, cyclic AMP to regulate melatonin synthesis. Cyclic AMP and the pacemaker act synergistically to regulate serotonin N-acetyltransferase activity and the melatonin rhythm, with cyclic AMP mediating acute effects and amplitude regulation.     

Endothelial progenitor cells: identity defined?

In the past decade, researchers have gained important insights on the role of bone marrow (BM)-derived cells in adult neovascularization. A subset of BM-derived cells, called endothelial progenitor cells (EPCs), has been of particular interest, as these cells were suggested to home to sites of neovascularization and neoendothelialization and differentiate into endothelial cells (ECs) in situ , a process referred to as postnatal vasculogenesis. Therefore, EPCs were proposed as a potential regenerative tool for treating human vascular disease and a possible target to restrict vessel growth in tumour pathology. However, conflicting results have been reported in the field, and the identification, characterization, and exact role of EPCs in vascular biology is still a subject of much discussion. The focus of this review is on the controversial issues in the field of EPCs which are related to the lack of a unique EPC marker, identification challenges related to the paucity of EPCs in the circulation, and the important phenotypical and functional overlap between EPCs, haematopoietic cells and mature ECs. We also discuss our recent findings on the origin of endothelial outgrowth cells (EOCs), showing that this in vitro defined EC population does not originate from circulating CD133⁺ cells or CD45⁺ haematopoietic cells.     

Kidney proximal tubule cells: epithelial cells without EGTA-extractable annexins?

The expression and the subcellular localizations of annexins I, II, IV, VI, and XIII in renal epithelial cells were investigated, using immunological techniques with specific monoclonal antibodies. Upon performing Western blotting experiments, no annexins VI and XIII were detected in kidney, whereas annexins I, II, and IV were. Immunofluorescence labelling procedure performed on thin frozen renal sections showed the presence of these three annexins along the plasma membrane of the collecting duct cells with a restricted expression of annexin I at principal cells. Annexin I was also found present in some glomerular cells. None of these annexins, however, were detected in the proximal tubular cells upon performing immunofluorescence labelling and electrophoretic analysis on an EGTA (ethylenebis(oxyethylenenitrilo)tetraacetic acid)-extractable annexin fraction prepared from freshly isolated cells. This is the first time a mammalian epithelial cell has been found to express non-typical annexin (at least partly solubilized with EGTA). However, when these cells were grown in primary culture, they were found to express annexins I, II, IV, and V. As well as being located along the basolateral membrane, annexins I and II are also present on vesicles, which suggests that these annexins may be involved in vesicular traffic under cell culture conditions.     

Delayed presentation of pacemaker Reel Syndrome and lead damage: Coincidence or consequence?

Reel syndrome is a pacemaker lead early dislodgment, characterized by reeling-in of the lead(s) without being damaged. We herein present a case of an 86-year-old woman, with medical history of single chamber pacemaker implantation two years ago, admitted in cardiology department with complete AV block. Chest-Xray revealed ventricular lead coiling around and behind the pacemaker device. Urgent extraction of the previous pacemaker was performed; however, the lead damage made its repositioning unfeasible. Successful implantation of single-chamber pacemaker has been made. This clinical case highlights the importance of adequate follow-up to timely identify lead dislodgement, avoid lethal complications and lead fracture.     

LOH-proficient embryonic stem cells: a model of cancer progenitor cells?

Cancers are thought to originate in stem cells through the accumulation of multiple mutations. Some of these mutations result in a loss of heterozygosity (LOH). A recent report demonstrates that exposure of mouse embryonic stem cells to nontoxic amounts of mutagens triggers a marked increase in the frequency of LOH. Thus, mutagen induction of LOH in embryonic stem cells suggests a new pathway to account for the multiple homozygous mutations in human tumors. This induction could mimic early mutagenic events that generate cancers in human tissue stem cells.     

Radiomic models for lymph node metastasis prediction in cervical cancer: can we think beyond sentinel lymph node?

Liu and colleagues performed a retrospective study to validate a computed tomography (CT) scan-based radiomic model to detect lymph node metastasis in cervical cancer. The proposed model incorporating the arterial and venous phase CT-scan features represented a non-invasive method exhibiting high sensitivity in the prediction of lymph node metastasis. It is well established that lymph node metastasis is one of the most significant prognostic factors in cervical cancer. For this reason, management of cervical cancer is strictly related to lymph node status, with international guidelines recommending definitive chemo-radiation in case of metastatic lymph node. More and more evidence supports the use of sentinel lymph node in early-stage cervical cancer but its frozen section analysis may result in false negative results; in locally-advanced stages staging para-aortic lymphadenectomy is proposed by many Authors to tailor chemoradiotherapy treatment, with potential intra-and post-operative related complications. The use of a validated radiomic model able to predict lymph node metastases in radiologically normal lymph nodes may represent an essential tool to possibly spare lympadenectomy related morbidity.