Isolation of rat embryonic stem-like cells: a tool for stem cell research and drug discovery.

The establishment of rat embryonic stem cells constitutes a precious tool since rat has been extensively used in biomedical research, in particular for the generation of human neurodisease animal models. Up to now only a few studies have described the isolation of rat embryonic stem-like cells. One out of 9 isolated rat embryonic stem-like cell lines (B1-RESC) obtained from a 4.5-day post-coitum blastocyst were extensively characterized and kept in culture for up to 80 passages on feeders with LIF. The stable growth of these cells and the expression of pluripotent markers were confirmed up to a high number of passages in culture, also in the absence of feeders and LIF. B1-RESC expresses the three germ layers markers both in vitro, within differentiating embryoid bodies, and in vivo through teratoma formation. Collectively, the B1-RESC line with a stable near-diploid karyotype can be used as a highly sensitive tool for testing anti-proliferative molecules.

Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils.

Cytosolic free calcium ([Ca2+]i) and fusion of secondary granules with the phagosomal membrane (phagosome-lysosome fusion, P-L fusion) were assessed in single adherent human neutrophils during phagocytosis of C3bi-opsonized yeast particles. Neutrophils were loaded with the fluorescent dye fura2/AM and [Ca2+]i was assessed by dual excitation microfluorimetry. Discharge of lactoferrin, a secondary granule marker into the phagosome was verified by immunostaining using standard epifluorescence, confocal laser scanning and electron microscopy. In Ca2(+)-containing medium, upon contact with a yeast particle, a rapid rise in [Ca2+]i was observed, followed by one or more Ca2+ peaks (maximal value 1,586 nM and median duration 145 s): P-L fusion was detected in 80% of the cells after 5-10 min. In Ca2(+)-free medium the amplitude, frequency and duration of the [Ca2+]i transients were decreased (maximal value 368 nM, mostly one single Ca2+ peak and median duration 75 s): P-L fusion was decreased to 52%. Increasing the cytosolic Ca2+ buffering capacity by loading the cells with MAPT/AM led to a dose-dependent inhibition both of [Ca2+]i elevations and P-L fusion. Under conditions where basal [Ca2+]i was reduced to less than 20 nM and intracellular Ca2+ stores were depleted, P-L fusion was drastically inhibited while the cells ingested yeast particles normally. P-L fusion could be restored in Ca2(+)-buffered cells containing ingested particles by elevating [Ca2+]i with the Ca2(+)-ionophore ionomycin. The present findings directly indicate that although the ingestion step of phagocytosis is a Ca2(+)-independent event, [Ca2+]i transients triggered upon contact with opsonized particles are necessary to control the subsequent fusion of secondary granules with the phagosomal membrane.

Multiple elevations of cytosolic-free Ca2+ in human neutrophils: initiation by adherence receptors of the integrin family.

Multiple spontaneous transient elevations of cytosolic-free calcium ([Ca2+]i) are observed in single human neutrophils during adherence. The interrelation between adherence and spontaneous [Ca2+]i transients was analyzed by simultaneous monitoring of [Ca2+]i and cell morphology. Fluorescent images of fura 2-loaded neutrophils attached to albumin-coated glass were recorded with a high sensitivity CCD camera while [Ca2+]i was assessed with a dual excitation microfluorimetry. The majority of the initially round cells studied showed changes in shape which started either before or at the same time as the onset of the [Ca2+]i transients. These data suggested that a rise in [Ca2+]i is not a prerequisite for shape change. This conclusion was confirmed by observation of movement and spreading in cells whose [Ca2+]i transients were abolished by chelation of extracellular Ca2+. Instead, our data suggest that spreading or adhesion itself initiates the [Ca2+]i activity. In keeping with this hypothesis, cytochalasin B, which prevents both cell movement and adhesion, completely inhibited generation of [Ca2+]i transients. To determine if the movement alone or adhesion alone is responsible for [Ca2+]i activity, we treated cells with antibodies against the beta chain (CD18, beta 2) or the alpha subunit (CD11b, alpha m) of the dominant leukocyte integrin (CR3). Antibody-treated cells showed normal extension of pseudopods but impaired ability to adhere. Inhibition of adhesion in this way inhibited [Ca2+]i activity. Taken together these results suggest that following sequence of events after contact of neutrophils with surfaces: (a) cell movement and shape change lead to enhanced contact of integrins with the surface; and (b) integrins-mediated adhesion generates multiple [Ca2+]i transients. The [Ca2+]i transients may then control exocytic events associated with movement and may provide a link between adherence and activation or priming of neutrophils to other stimuli.

Embryonic and adult stem cell-derived cardiomyocytes: lessons from in vitro models.

For years, research has focused on how to treat heart failure by sustaining the overloaded remaining cardiomyocytes. Recently, the concept of cell replacement therapy as a treatment of heart diseases has opened a new area of investigation. In vitro-generated cardiomyocytes could be injected into the heart to rescue the function of a damaged myocardium. Embryonic and/or adult stem cells could provide cardiac cells for this purpose. Knowledge of fundamental cardiac differentiation mechanisms unraveled by studies on animal models has been improved using in vitro models of cardiogenesis such as mouse embryonal carcinoma cells, mouse embryonic stem cells and, recently, human embryonic stem cells. On the other hand, studies suggesting the existence of cardiac stem cells and the potential of adult stem cells from bone marrow or skeletal muscle to differentiate toward unexpected phenotypes raise hope and questions about their potential use for cardiac cell therapy. In this review, we compare the specificities of embryonic vs adult stem cell populations regarding their cardiac differentiation potential, and we give an overview of what in vitro models have taught us about cardiogenesis.

Stem cell science: current ethical and policy issues.

There is increasing support for embryonic stem (ES) cell research on both sides of the Atlantic. In the United States, the outcome is more funding from non-federal sources, despite the current administration's opposing views. In Europe, a similar pragmatic turn is in the making, but the future is still uncertain. Acceptance of ES cells is mitigated by the uncritical belief that their use is ethically more suspect than is the case for adult cells.

Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (mean survival: 34.2% +/- 9.0% of albumin, P less than 0.0001) despite similar efficient ingestion of extracellular bacteria. Enhancement of killing was observed when surfaces were coated with purified constituents of extracellular matrix, i.e., fibronectin, fibrinogen, laminin, vitronectin, or type IV collagen. In addition to matrix proteins, the tetrapeptide RGDS (the sequence recognized by integrins) crosslinked to surface bound albumin was also active (survival: 74.5% +/- 5.5% of albumin, P less than 0.02), and fibronectin-increased killing was inhibited by soluble RGDS. Chemiluminescence measurements and experiments with CGD neutrophils revealed that both oxygen-dependent and -independent bactericidal mechanisms are involved. In conclusion, matrix proteins enhance intracellular bactericidal activity of adherent neutrophils, presumably by integrin recognition of RGDS-containing ligands. These results indicate a role for extracellular matrix proteins in the enhancement of the host defense against pyogenic infections.

Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium.

The role of changes in cytosolic free calcium concentration ([Ca2+]i) in the assembly and disassembly of actin during adhesion and phagocytosis was evaluated. Rhodamine-phalloidin staining combined with quantitative fluorescence and confocal laser scanning microscopy was used to measure local F-actin changes in single adherent human neutrophils phagocytosing yeast particles on different surfaces and under different calcium conditions. Cells were suspended in a) calcium-containing medium (CCM) or b) calcium-free medium (CFM) or c) were first depleted of calcium (i.e., MAPT/AM-loaded in CFM) and then suspended in CFM (MAPT). In parallel, local [Ca2+]i changes were monitored using a fura-2 ratio imaging system. In CCM or CFM, attachment to the substrate and formation of pseudopods around a yeast particle generated, within a few seconds, rises in [Ca2+]i, both around the phagosome and in the cell body. During continued phagocytosis, [Ca2+]i was more elevated around the phagosome compared to the rest of the cell. No [Ca2+]i fluctuations were observed in MAPT cells. Adhesion and phagocytosis led to a several-fold increase in F-actin. The increase was transient in cells in CCM and CFM, but remained high in Ca-depleted neutrophils. A distinct ring of F-actin was formed around a phagosome with a yeast particle. Twenty min after ingestion the amount of this actin decreased more than 50% in CCM and CFM cells but increased by 40 to 100% in MAPT cells. The accumulation of F-actin in MAPT cells was reduced to resting levels by adding Ca2+ and ionomycin after ingestion. This treatment reestablished the periphagosomal [Ca2+]i rises, as observed in CCM cells. In conclusion, the present study shows that the actin polymerization, occurring in human neutrophils during adhesion and phagocytosis, is not influenced by changes in [Ca2+]i, whereas the subsequent depolymerization is. The accumulation of actin filaments around the phagosome in calcium-depleted cells could be involved in the inhibition of phagolysosome fusion seen in the absence of [Ca2+]i changes (Jaconi et al., J. Cell Biol. 110, 1555-1564 (1990)). This suggests that the actin network, controlled by [Ca2+]i, regulates the movement of granules during phagocytosis.

A spliced variant of AE1 gene encodes a truncated form of Band 3 in heart: the predominant anion exchanger in ventricular myocytes.

The anion exchangers (AE) are encoded by a multigenic family that comprises at least three genes, AE1, AE2 and AE3, and numerous splicoforms. Besides regulating intracellular pH (pHi) via the Cl-/HCO3- exchange, the AEs exert various cellular functions including generation of a senescent antigen, anchorage of the cytoskeleton to the membrane and regulation of metabolism. Most cells express several AE isoforms. Despite the key role of this family of proteins, little is known about the function of specific AE isoforms in any tissue, including the heart. We therefore chose isolated cardiac cells, in which a tight control of pHi is mandatory for the excitation-contraction coupling process, to thoroughly investigate the expression of the AE genes at both the mRNA and protein levels. RT-PCR revealed the presence of AE1, AE2 and AE3 mRNAs in both neonatal and adult rat cardiomyocytes. AE1 is expressed both as the erythroid form (Band 3 or eAE1) and a novel alternate transcript (nAE1), which was more specifically characterized using a PCR mapping strategy. Two variants of AE2 (AE2a and AE2c) were found at the mRNA level. Cardiac as well as brain AE3 mRNAs were expressed in both neonatal and adult rat cardiomyocytes. Several AE protein isoforms were found, including a truncated form of AE1 and two AE3s, but there was no evidence of AE2 protein in adult rat cardiomyocytes. In cardiomyocytes transfected with an AE3 oligodeoxynucleotide antisense, AE3 immunoreactivity was dramatically decreased but the activity of the Cl-/HCO3- exchange was unchanged. In contrast, intracellular microinjection of blocking anti-AE1 antibodies inhibited the AE activity. Altogether, our findings suggest that a specific and novel AE1 splicoform (nAE1) mediates the cardiac Cl-/HCO3- exchange. The multiple gene and protein expression within the same cell type suggest numerous functions for this protein family.

Proton channels, plasma membrane potential, and respiratory burst in human neutrophils.

When confronted with invading microorganisms, neutrophils undergo a number of nearly synchronous reactions including the generation of microbicidal reactive oxygen intermediates by the NADPH oxidase. These reactions are accompanied by a slow depolarization, from resting values of-60 mV to levels probably exceeding 0 mV. The depolarization is transient, indicating that a compensatory charge transport mechanism is activated. Activation of the oxidase system causes a massive burst of metabolic acid generation that would, if uncompensated, lower the intracellular pH of neutrophils by over 5 units, to lethal levels (pH = 2). Neutrophils must therefore possess particularly effective regulatory systems to avoid excessive cytosolic acidification. The recently described H+ conductance of neutrophils may counteract both the acidification and the depolarization. Activation of the H+ conductance occurs at depolarizing voltages and is promoted by cytosolic acidification, a combination that takes place during the respiratory burst. The NADPH oxidase of neutrophils is thus associated to an unusual, particularly efficient mechanism of H+ export and charge compensation. The sequential activation of these two systems causes neutrophils to depolarize through the activation of an electron transport chain, and to repolarize through the activation of a H+ conductance.

Short-term and long-term effects of melatonin on GnRH-stimulated gonadotropin secretion in pituitaries of sexually maturing rats.

Melatonin administration has been shown to delay sexual maturation in male rats, through an action which involves decreased binding of gonadotropin-releasing hormone (GnRH) in the pituitary and lower pituitary gonadotropin contents. It has been suggested that melatonin must act at a level higher than the pituitary to provoke these effects, but a direct action of melatonin on the pituitary has not been excluded. Using a cell culture system, the gonadotroph responsiveness to GnRH was studied. Pituitaries were obtained either from rats chronically treated with melatonin and showing delayed sexual maturation, or from control rats. In vitro luteinizing hormone and follicle-stimulating hormone response to GnRH was significantly lower when pituitaries were obtained from melatonin-treated rats. However, this diminished response was directly proportional to the amount of gonadotropin contents in cells, so that relative responsiveness, calculated as the amount of gonadotropins released in relation to the gonadotropin content was similar in cells from control and melatonin-treated rats. It is concluded that the effect of melatonin on the pituitary of male rats results from a decrease of gonadotroph growth or gonadotropin synthesis, as a consequence of a change located at the hypothalamic rather than at the pituitary level. This conclusion is further supported by results showing that melatonin added directly in culture medium prior to GnRH does not modify the pituitary responsiveness to GnRH.

The regulation of store-dependent Ca2+ influx in HL-60 granulocytes involves GTP-sensitive elements.

In granulocytes, emptying of intracellular Ca2+ stores activates Ca2+ influx across the plasma membrane. To study the putative role of GTP-binding proteins in this process, we have introduced non-hydrolyzable guanosine phosphate analogues into the cytosol of non-permeabilized HL-60 granulocytes using an endocytosis-hypoosmotic shock procedure. At the cytosolic concentrations obtained (100-500 microM), neither guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) nor guanosine 5'-3-O-(thio)diphosphate (GDP beta S) affected basal [Ca2+]i. Ca2+ release in response to the receptor agonist fMet-Leu-Phe, the Ca(2+)-ATPase inhibitor thapsigargin, or the Ca2+ ionophore ionomycin was also unaffected by GTP gamma S or GDP beta S. In contrast, the activation of the Ca2+ influx pathway by fMet-Leu-Phe or by thapsigargin was blocked by GTP gamma S but not by GDP beta S. The GTP gamma S effect was mimicked by NaF. The GTP gamma S and NaF effects were independent of protein kinase C activation and actin polymerization. Our results demonstrate that a GTP-sensitive element is involved in the signaling between intracellular Ca2+ stores and plasma membrane Ca2+ channels. The identical effects of GTP gamma S and NaF suggest that the GTP-sensitive element is a heterotrimeric G-protein.

Cytosolic free Ca2+ signals in single adherent human neutrophils: generation and functional role.

To study the role of cytosolic free calcium, [Ca2+]i, in cell activation, in particular during adhesion and movement on a surface in response to chemotactic peptide stimulation and during phagocytosis, we monitored [Ca2+]i in single human neutrophils. The neutrophils were loaded with fura-2 and allowed to adhere to albumin-coated glass coverslips. [Ca2+]i was monitored with a dual excitation microfluorimeter. Half of the cells showed spontaneous [Ca2+]i transients that lasted up to 15 min with an amplitude averaging 77 +/- 10 nM above basal levels (mean basal value of 110 +/- 20 nM) and a mean duration of 28 +/- 5 s. These repetitive [Ca2+]i elevations depended on the continuous presence of extracellular Ca2+ and could be dissociated from those triggered by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). Cell morphology was monitored in parallel by recording fluorescent images with a high sensitivity charge coupled device (CCD) camera. The majority of the cells studied showed visible changes in shape which started either before or at the same time as the onset of the [Ca2+]i transients. Removal of extracellular Ca2+ abolished [Ca2+]i transients without impairing cell movement and spreading. Blockade of adherence and cell movement with cytochalasin B markedly inhibited [Ca2+]i transients. Monoclonal antibodies directed against the leucocyte integrin CR3 (CD11b/CD18 alpha m beta 2) blocked adherence, spreading and most of the [Ca2+]i activity. Total [Ca2+]i activity was assessed during phagocytosis of C3bi-opsonized yeast particles and correlated with fusion of secondary granules with the phagosomal membrane (P-L fusion).(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous and chemoattractant-induced oscillations of cytosolic free calcium in single adherent human neutrophils.

Studies with fluorescent Ca2+ indicators in large populations of neutrophils in suspension reveal a stable base line followed by a rapid agonist-induced elevation of cytosolic free calcium, [Ca2+]i, concomitant with other parameters of cellular activation. To study the role of adhesion in cell activation, we monitored [Ca2+]i in single neutrophils adhered to albumin-coated or fibronectin-coated glass coverslips before and after stimulation with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Human neutrophils loaded with 2 microM fura 2/AM were allowed to adhere to coverslips for 15-20 min at 37 degrees C. [Ca2+]i was monitored with a dual excitation microfluorimeter with a time resolution of 200 ms. Statistical analysis was performed using an algorithm allowing to detect significant [Ca2+]i peaks. 54% of the cells showed spontaneous [Ca2+]i oscillations. The amplitude of these [Ca2+]i peaks averaged 77 +/- 10 nM above basal levels (mean value of 110 +/- 20 nM), and their mean duration was 28 +/- 5 s; periods of [Ca2+]i bursts could last up to 15 min. In "silent" cells exhibiting a stable [Ca2+]i base line without spontaneous oscillations, low concentrations of fMLP (10(-10)-10(-9) M) could induce sustained [Ca2+]i oscillations. By contrast, higher agonist concentrations (10(-6) M) induced a single [Ca2+]i transient followed by a stable base line. 47% of the cells showing spontaneous [Ca2+]i oscillations did not respond to fMLP. Spontaneous [Ca2+]i oscillations depended on the continuous presence of extracellular Ca2+. Therefore: (i) spontaneous oscillations of [Ca2+]i occur in neutrophils adherent to various substrata; (ii) these oscillations do not preclude and can be dissociated from the response to fMLP; (iii) neutrophil functions might be controlled by [Ca2+]i oscillations rather than by sustained alterations of [Ca2+]i.

Intracellular pattern of cytosolic Ca2+ changes during adhesion and multiple phagocytosis in human neutrophils. Dynamics of intracellular Ca2+ stores.

The subcellular pattern of cytosolic free Ca2+ ([Ca2+]i) changes in human polymorphonuclear neutrophils (PMNs) was studied using imaging of fura-2 fluorescence (time resolution 12.5 ratios/s) to determine whether PMNs could obtain directional information from the [Ca2+]i signal. [Ca2+]i changes were observed during initial adherence, the subsequent chemotactic movement, and the phagocytosis of opsonized yeast particles. Initial adherence was followed by a rapid increase in [Ca2+]i (from 90 +/- 10 to 290 +/- 40 nmol/L in 6.5 +/- 2.5 seconds; +/- SEM, n = 10), apparently homogeneously distributed over the entire cytoplasm, which preceded the spreading of the PMNs. [Ca2+]i increases after the contact of the PMNs with yeast particles were of lower mean amplitude; [Ca2+]i increased simultaneously throughout the cytosol. In the absence of extracellular Ca2+, multiple phagocytotic events could proceed normally without a mandatory [Ca2+]i transient. In PMNs polarized on phagocytosis, gradients in [Ca2+]i could be observed. [Ca2+]i was more elevated in the periphagosomal area than in the remaining parts. Taken together, these data show that [Ca2+]i waves do not provide the neutrophil with directional information during chemotaxis and phagocytosis. Sustained small inhomogeneity of [Ca2+]i levels are consistent with a proposed redistribution of releasable Ca2+ stores on phagocytosis.