Placental stem cell differentiation into islets of Langerhans-like glucagon-secreting cells.

BACKGROUND AND PURPOSE: For the past few years, in an attempt to find new sources of cells that may be used in cell therapy, numerous researchers have highlighted the particular properties of mesenchymal stem cells. Mesenchymal stem cells can be isolated from adult tissues such as the bone marrow or adipose tissue, but also from other organs such as the human placenta. Our study focuses adult stem cells isolated from the chorionic villi in an attempt to differentiate them into islets of Langerhans in order to study their differentiation potential, as a future background for cell therapy. EXPERIMENTAL DESIGN: Full-term placentas were prelevated from volunteer women that have just delivered a normal pregnancy. After a mechanical fragmentation of the placenta, the chorion fragments are transferred in a dish with dispase before the enzyme is inactivated using fetal calf serum. The cell suspension is filtered in order to obtain a single-cell suspension. After the adherence of the first cells, the proliferation rate increased progressively and cell morphology is kept the same for several passages. In order to correctly differentiate placental stem cells into glucagon-secreting cells, we used a culture method on a scaffold with sequential exposure to different growth factors. The underlying substrate used contained type IV collagen, chytosan, Matrigel and laminin. Molecular biology techniques were carried out to investigate the gene expression of the stem cells. RESULTS: Our results show that exendin-4 is able to induce the differentiation of placental stem cells into glucagon-secreting cells. We also notice the absence of the insulin gene, a conclusion that may be explained by the fact that our phenotype is a partial one, incomplete, closer to islet cell progenitors than to insulin-producing progenitors. CONCLUSIONS: The identification of the placenta as a valid source for stem cells has important practical advantages because it is easily accessible, it raises no ethical issues and cells are easily to isolate in a large enough number to use. The future knowledge and manipulation of the signaling pathways that determines the dramatic phenotype shift may provide the basis for efficient cell differentiation, with great impact on regenerative medicine and tissue engineering.

Water transport in human red cells: effects of 'non-inhibitory' sulfhydryl reagents.

The water diffusional permeability of human red blood cells following exposure to various sulfhydryl group (SH) reagents have been studied using a nuclear magnetic resonance technique. Exposure of red blood cells up to 12 mM N-ethylmaleimide (NEM) or 10 mM 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNE) alone does not affect water diffusion. In contrast, when DTNB treatment follows a preincubation of the cells with NEM, a small (18% at 37 degrees C) but significant inhibition of water permeability occurs. The NEM and DTNB treatment of the cells caused no change of the cell shape and volume or of the cell water volume. Consequently, the inhibition observed after NEM and DTNB treatment has a real significance.

Effects of temperature on water diffusion in human erythrocytes and ghosts--nuclear magnetic resonance studies.

The temperature-dependence of water diffusion across human erythrocyte membrane was studied on isolated erythrocytes and resealed ghosts by a doping nuclear magnetic resonance technique. The conclusions are the following: (1) The storage of suspended erythrocytes at 2 degrees C up to 24 h or at 37 degrees C for 30 min did not change the water exchange time significantly, even if Mn2+ was present in the medium. This indicates that no significant penetration of Mn2+ is taking place under such conditions. (2) In case of cells previously incubated at 37 degrees C for longer than 30 min with concentrations of p-chloromercuribenzene sulfonate (PCMBS) greater than 0.5 mM, the water-exchange time gradually decreased if the cells were stored in the presence of Mn2+ for more than 10 min at 37 degrees C. (3) When the Arrhenius plot of the water-exchange time was calculated on the basis of measurements performed in such a way as to avoid a prolonged exposure of erythrocytes to Mn2+ no discontinuity occurred, regardless of the treatment with PCMBS. (4) No significant differences between erythrocytes and resealed ghosts regarding their permeability and the activation energy of water diffusion (Ea,d) were noticed. The mean value of Ea,d obtained on erythrocytes from 35 donors was 24.5 kJ/mol. (5) The value of Ea,d increased after treatment with PCMBS, in parallel with the percentage inhibition of water diffusion. A mean value of 41.3 kJ/mol was obtained for Ea,d of erythrocytes incubated with 1 mM PCMBS for 60 min at 37 degrees C and 28.3 kJ/mol for ghosts incubated with 0.1 mM PCMBS for 15 min, the values of inhibition being 46% and 21% respectively.

Comparison of human and rat liver microsomes by spin label and biochemical analyses.

Detailed lipid analyses of human and rat liver microsomes revealed interesting differences. It was found that human liver microsomes contain twice as much lipid as those from the rat. This increased lipid content is not associated with an increase in content of a particular lipid class; human liver microsomes contain higher amounts of each of the lipid classes. Human and rat liver microsomes differ especially in the essential fatty acid composition of total lipids and phospholipids: human liver microsomes contain more linoleic acid and less arachidonic acid than those of the rat. Such a pattern of distribution of fatty acids is similar to that previously reported for human liver mitochondria and has not been reported for other species. Although the previously reported for human liver mitochondria and has not been reported for other species. Although the unsaturation of lipids is lower in human than in rat liver microsomes, spin label studies revealed a higher fluidity in human membranes. It is suggested that this might arise from a lesser immobilization of lipids by proteins in human liver subcellular membranes.

The effect of the saturation and isomerization of dietary fatty acids on the osmotic fragility and water diffusional permeability of rat erythrocytes.

Weanling rats were fed semi-purified diets containing 15% by weight of either corn oil, a high oleic acid safflower oil, lard or hydrogenated soybean oil. Significant changes in the fatty acid composition of erythrocytes were induced by these dietary fats. The compositional changes did not effect water diffusional permeability, but did affect their osmotic fragility. An increased fragility appeared to be associated with an increased octadecenoate content of the membranes.

Effects on water diffusion of inhibitors affecting various transport processes in human red blood cells.

The water permeability of human red blood cells has been monitored by nuclear magnetic resonance (NMR) following exposure to inhibitors of various transport processes across their membranes. No significant inhibition of water diffusion could be detected after the treatment of red blood cells with the anion exchange transport inhibitor dihydro-4,4'-diisothiocyano-stilbene-2,2'-disulfonate (H2DIDS) or the glucose transport inhibitors diallyl-diethyl-stilbestrol (DADES), cytochalasin B, or 30 mM iodoacetamide. It is for the first time that the effects of glucose transport inhibitors has been studied in detail by the NMR approach. A special case proved to be phloretin, an inhibitor of anion, nonelectrolyte and glucose permeability. A small but statistically significant inhibition of water permeability (around 12% at 20 degrees C) was induced by exposure to 2 mM phloretin (for 60 min at 37 degrees C); after a pretreatment of cells with 12 mM N-ethylmaleimide (NEM), for 60 min at 37 degrees C, the degree of inhibition induced by phloretin increased (becoming 17% at 20 degrees C). None of the inhibitors prevented or potentiated the strong inhibitory effect on water diffusion of a mercurial, p-chloromercuribenzene sulfonate (PCMBS). No increase in the activation energy of water diffusion occurred by treatment with the reagents used (exception the effect of PCMBS). The present results clarify some conflicting reports concerning the effects on water permeability of inhibitors of various transport processes in red blood cells and indicate that in addition to the drastic inhibition induced by mercurials other reagents may also have inhibitory effects.

Water permeability in human erythrocytes: identification of membrane proteins involved in water transport.

The water permeability of human erythrocytes has been monitored by nuclear magnetic resonance (NMR) before and after treatment of the cells with various sulfhydryl reagents. Preincubation of the cells with N-ethylmaleimide (NEM), a non-inhibitory sulfhydryl reagent, results in a faster and more sensitive inhibition of water exchange by mercurials. The inhibition of water exchange by p-chloromercuribenzene sulfonate (PCMBS) was maximal at a binding of approximately 10 nmol PCMBS per mg protein when non-specific sulfhydryl groups are blocked by NEM. Inhibition by PCMBS has been correlated with the binding of 203Hg to erythrocyte membrane proteins. A significant binding of label to band 3 and the polypeptides in band 4.5 occurs, with approximately 1 mol of mercurial bound per mol of protein. Inhibition of water transport by sulfhydryl reagents does not induce major morphological changes in the cells as assessed by freeze-fracture and scanning electron microscopy.

Water exchange through erythrocyte membranes: p-chloromercuribenzene sulfonate inhibition of water diffusion in ghosts studied by a nuclear magnetic resonance technique.

A comparison of water diffusion in human erythrocytes and ghosts revealed a longer relaxation time in ghosts, corresponding to a decreased exchange rate. However, the diffusional permeability of ghosts was not significantly different from that of erythrocytes. The changes in water diffusion following exposure to p-chloromercuribenzene sulfonate (PCMBS) have been studied on ghosts suspended in isotonic solutions. It was found that a significant inhibitory effect of PCMBS on water diffusion occurred only after several minutes of incubation at 37 degrees C. No inhibition was noticed after short incubation at 0 degree C, as previously used in some labelling experiments. This indicates the location in the membrane interior of the SH groups involved in water diffusion across human erythrocyte membranes. The nuclear magnetic resonance (n.m.r.) method appears as a useful tool for studying changes in water diffusion in erythrocyte ghosts with the aim of locating the water channel.

Amino acid composition of band 3 protein from red blood cells of normal and epileptic children.

Amino acid analyses of the band 3 protein purified from erythrocyte membranes of control and epileptic children showed that no major structural abnormalities of this protein could be linked with the red blood cell membrane alterations previously described in child epilepsy and, consequently, the molecular basis of these alterations should be looked for elsewhere.

The basal permeability to water of human red blood cells evaluated by a nuclear magnetic resonance technique.

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes by a doping nuclear magnetic resonance technique. In order to estimate the basal permeability the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzene sulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 0.7 X 10(-3) cm s-1 at 10 degrees C, 1.2 X 10(-3) cm s-1 at 15 degrees C, 1.4 X 10(-3) cm s-1 at 20 degrees C, 1.8 X 10(-3) cm s-1 at 25 degrees C, 2.1 X 10(-3) cm s-1 at 30 degrees C and 3.5 X 10(-3) cm s-1 at 37 degrees C. The mean value of the activation energy of water diffusion (Ea,d) was 25 kJ/mol for control and 43.7 kJ/mol for PCMBS--inhibited erythrocytes. The values of P and Ea,d obtained after induction of maximal inhibition of water diffusion by PCMBS can be taken as references for the basal permeability to water of the human red blood cell membrane.

Comparison of liposome entrapment parameters by optical and atomic absorption spectrophotometry.

Methods for the complete characterization of liposomes prepared by ether-injection are described in detail. The validity of atomic absorption spectrophotometry for measuring markers of trapped volume was checked by comparative determinations of markers with established optical spectrophotometrical methods. The favorable results using atomic absorption spectrophotometry to quantitate the marker Mn2+ are of particular relevance as manganese ion is also the paramagnetic probe in n.m.r. measurements of water permeability of liposomes; our results indicate that in such measurements no other marker need be incorporated.

On measuring the diffusional water permeability of human red blood cells and ghosts by nuclear magnetic resonance.

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes and ghosts by a doping nuclear magnetic resonance technique. In contrast to all previous investigations, systematic measurements were performed on blood samples obtained from a large group of donors. The mean values of P ranged from 2.2 X 10(-3) cm.s-1 at 5 degrees C to 8.1 X 10(-3) cm.s-1 at 42 degrees C. The reasons for some of the discrepancies in the permeability coefficients reported by various authors were found. In order to estimate the basal permeability, the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzenesulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 1.3 X 10(-3) cm.s-1 at 20 degrees C, 1.6 X 10(-3) cm.s-1 at 25 degrees C, 1.9 X 10(-3) cm.s-1 at 30 degrees C and 3.2 X 10(-3) cm.s-1 at 37 degrees C. The results reported here represent the largest series of determinations of water diffusional permeability of human red blood cells (without or with exposure to mercurials) available in the literature, and consequently the best estimates of the characteristics of this transport process. The values of P can be taken as references for the studies of water permeability in various cells or in pathological conditions.

Isolation and characterization of chorionic mesenchyal stem cells from the placenta.

The aim of the study was the isolation and characterization of mesenchymal stem cells from the placental chorion from a genotypical and phenotypical point of view. The placentas included in the study were derived from term pregnancies with a normal evolution. Along with the placentas, umbilical cord blood, maternal and newborn peripheral blood samples were taken. The isolation and culture of chorionic and, incidentally, trophoblastic cells was followed by the determination of markers of the former cells. They expressed proteins and genes characteristic of stem cells. Immunofluorescence and evaluation of gene expression evidenced the pluripotential properties of these cells and also their higher position on the differentiation pathway. HLA expression provides information that might help explain the immunological mechanisms of tolerance between the maternal organism and fetal structures.

Pancreatic exocrine adult cells and placental stem cells co-culture. Working together is always the best way to go.

BACKGROUND AND AIM: The progress made in the last few years have managed to come up withy the possibility of using different stem cell types in an endeavor to correct the alterations that appear in different degenerative diseases. The pancreas, an organ with extremely low regenerative capacity, both for the endocrine and for the exocrine component, is an organ perfect for cell therapy in the hope of restoring its function and cure diabetes mellitus or chronic pancreatitis. One main issue in the stem cell transplantation problem is represented by the influence of the cellular niche, formed by completely differentiated cells, on the phenotype and function of the transplanted cells. In this study, we challenge current knowledge in the field by evaluating the influence of exocrine pancreatic cells on placental stem-like cells using the co-culture technique. METHODS: In our experiments, we used two different protocols in which adult pancreatic cells were cultured together with mesenchymal stem cells isolated from human placenta. In the case of the first protocol, we seeded pancreatic cells on a pre-adhered single-cell layer of mesenchymal stem cells and in the second one, the seeding of two cell populations in suspension was done at the same time, after passage. During the experiment, we evaluated the alteration of the morphology of the placental cells using and inverted phase microscope and reverse transcriptase-PCR. RESULTS AND CONCLUSIONS: Based on morphology, in both cases the interaction between epithelial pancreatic cells and placental ones have determined a change in phenotype from mesenchymal to epithelial-like. Taking into consideration the gene expression, placental stem cells have maintained pluripotency gene expression throughout the study. They also expressed pancreatic amylase. These experiments bring out the plasticity of placental stem cells, the cell microenvironment with a decisive part in phenotype and the level of gene expression. The results obtained in vitro can bring a new picture on the effects of the pancreatic stem cell niche.

Effect of chlorpromazine on proteins in human erythrocyte membranes as inferred from spin labeling and biochemical analyses.

ESR spectra of erythrocyte membranes labeled with a maleimide spin label (MSL) show two types of label environment: a weakly immobilized component and a strongly immobilized component. Chlorpromazine (CPZ) markedly altered the spectra: at pH 8.0, 3 mM CPZ reduced the amplitude of the spectrum by 40%, and the weakly immobilized component was almost completely removed. In order to clarify the mechanisms of these spectral changes the protein release from erythrocyte membranes induced by CPZ has been followed. CPZ had a weak solubilizing effect on erythrocyte membranes: less than 1% of the membrane protein was released, mainly Band 6. By comparison with the protein release induced by low-salt treatment it was found that the "detergent-like" property of CPZ cannot explain the alterations in the ESR spectra. The nature of the spectral changes induced by CPZ was different from that of changes induced by lowering the pH to 4.5; correlated with other data this shows that changes in organization or conformation of membrane protein cannot explain the CPZ-induced alterations in the ESR spectra. These spectral changes appeared to be due to the reduction by CPZ of the nitroxide free radical. This was documented by the marked reduction of spin concentration of the labeled ghosts in the presence of CPZ resulting in a decrease in amplitude of the ESR spectrum of MSL-labeled erythrocyte ghosts induced by CPZ. The reduction by CPZ of the nitroxide free radical was compared with that induced by ascorbate. It was found that CPZ preferentially reduces the mobile component of the ESR spectrum of MSL-labeled ghosts. The action of CPZ in reducing free radicals may have consequences for patients receiving long-term treatment with phenothiazine derivatives.

Water exchange through erythrocyte membranes. V. Incubation with papain prevents the P-chloromercuri-benzensulfonate inhibition of water diffusion studied by a nuclear magnetic resonance technique.

The changes in water diffusion across human erythrocyte membrane following exposure to proteolytic enzymes and to p-chloromercuribenzene sulfonate (PCMBS) have been studied on isolated erythrocytes suspended in isotonic solutions. Trypsin digested glycophorin without significantly changing the pattern of other polypeptides in erythrocyte membrane. On the contrary, with chymotrypsin or papain an extensive digestion of band 3 protein occurred. No changes in water diffusion were noticed after exposure of erythrocytes to trypsin, chymotrypsin or papain. Neither trypsin nor chymotrypsin treatment prevented the inhibition of water diffusion induced by PCMBS. In contrast, exposure of erythrocytes to papain did hamper the inhibitory effect of subsequent incubation with PCMBS. Taking into account the degradation of band 3 protein by papain it appears that the binding site for PCMBS playing a role in the inhibition of water diffusion is located in this protein.

p-(Chloromercuri)benzenesulfonate binding by membrane proteins and the inhibition of water transport in human erythrocytes.

The binding of [203Hg]-p-(chloromercuri)benzenesulfonate to the membrane proteins of human erythrocytes and erythrocyte ghosts was examined under conditions where binding to the bulk of membrane sulfhydryl groups was blocked by N-ethylmaleimide. Binding was essentially complete within 90 min when approximately 40 nmol was bound per milligram of membrane protein. This binding was correlated with the inhibition of water transport measured by an NMR technique. Maximal inhibition was observed with the binding of approximately 10 nmol of p-(chloromercuri)benzenesulfonate/mg of membrane protein. Under these conditions, both band 3 and band 4.5 bound 1 mol of inhibitor/mol of protein. In contrast to previous experiments, these results indicate that band 4.5 proteins as well as band 3 have to be considered as playing a role in water transport.

Water exchange through erythrocyte membranes: nuclear magnetic resonance studies on resealed ghosts compared to human erythrocytes.

The water diffusion across human erythrocyte membrane has been studied on intact cells and resealed ghosts by a doping NMR technique. Although the water exchange time of ghosts was longer than that of erythrocytes, no significant differences in their diffusional permeability were noticed for temperatures in the range 2-43 degrees C. Contrary to what was previously noticed in erythrocytes, no significant increase in the water exchange time of ghosts in the acid range of pH occurred.

Water exchange through erythrocyte membranes: nuclear magnetic resonance studies on the effects of inhibitors and of chemical modifications of human membranes.

The changes in water diffusion across human erythrocyte membranes following exposure to various inhibitors and proteolytic enzymes have been studied on isolated erythrocytes suspended in isotonic buffered solutions. An important issue was to investigate whether the sulfhydryl reacting reagents that have been applied in osmotic experiments showed similar effects on diffusional permeability. It was found that mercurials, including mersalyl, were the only sulfhydryl reacting reagents that were efficient inhibitors. Under optimal conditions a similar degree of inhibition (around 45%) was found with all mercury-containing sulfhydryl reagents. Other reagents, including the sulfhydryl reagent DTNB, phloretin, or H2DIDS, the specific inhibitor of the anion transport system in erythrocyte membrane, did not appear to inhibit significantly the diffusional permeability. No changes in water diffusion were noticed after exposure to erythrocytes to trypsin and chymotrypsin. A new kind of experiments was that in which the effects of exposure of erythrocytes to two or more agents were studied. It was found that none of the chemical manipulations of membranes that did not affect water diffusion hampered the inhibitory action of mercurials. These findings show that the SH groups involved in water diffusion across erythrocyte membrane do not react with any of the other SH reagents aside from mercurials and that the molecular mechanism of water transport is not affected by chymotryptic cleavage of band 3 protein into the 60 and 35 kD fragments. The NMR method appears as a useful tool for studying changes in water diffusion in erythrocyte membranes following various chemical manipulations of the membranes with the aim of locating the water channel.