Single cell-derived clones from human adipose stem cells present different immunomodulatory properties.

Human adipose mesenchymal stem cells are a heterogeneous population, where cell cultures derived from single-cell-expanded clones present varying degrees of differential plasticity. This work focuses on the immunomodulatory/anti-inflammatory properties of these cells. To this end, five single-cell clones were isolated (generally called 1.X and 3.X) from two volunteers. Regarding the expression level of the lineage-characteristic surface antigens, clones 1·10 and 1·22 expressed the lowest amounts, while clones 3·10 and 3·5 expressed more CD105 than the rest and clone 1·7 expressed higher amounts of CD73 and CD44. Regarding cytokine secretion, all clones were capable of spontaneously releasing high levels of interleukin (IL)-6 and low to moderate levels of IL-8. These differences can be explained in part by the distinct methylation profile exhibited by the clones. Furthermore, and after lipopolysaccharide stimulation, clone 3.X produced the highest amounts of proinflammatory cytokines such as IL-1ß, while clones 1·10 and 1·22 highly expressed IL-4 and IL-5. In co-culture experiments, clones 1.X are, together, more potent inhibitors than clones 3.X for proliferation of total, CD3(+) T, CD4(+) T and CD8(+) T lymphocytes and natural killer (NK) cells. The results of this work indicate that the adipose stem cell population is heterogeneous in cytokine production profile, and that isolation, characterization and selection of the appropriate cell clone is a more exact method for the possible treatment of different patients or pathologies.

Therapeutic potential of stem cells in diabetes.

Stem cells possess the ability to self-renew by symmetric divisions and, under certain circumstances, differentiate to a committed lineage by asymmetric cell divisions. Depending on the origin, stem cells are classified as either embryonic or adult. Embryonic stem cells are obtained from the inner cell mass of the blastocyst, a structure that appears during embryonic development at day 6 in humans and day 3.5 in mice. Adult stem cells are present within tissues of adult organisms and are responsible for cell turnover or repopulation of tissues under normal or exceptional circumstances. Taken together, stem cells might represent a potential source of tissues for cell therapy protocols, and diabetes is a candidate disease that may benefit from cell replacement protocols. The pathology of type 1 diabetes is caused by the autoimmune destruction or malfunction of pancreatic beta cells, and consequently, a lack of insulin. The absence of insulin is life-threatening, thus requiring diabetic patients to take daily hormone injections from exogenous sources; however, insulin injections do not adequately mimic beta cell function. This results in the development of diabetic complications such as neuropathy, nephropathy, retinopathy and diverse cardiovascular disorders. This chapter intends to summarize the possibilities opened by embryonic and adult stem cells in regenerative medicine for the cure of diabetes.

Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.

Embryonic stem (ES) cells display the ability to differentiate in vitro into a variety of cell lineages. Using a cell-trapping system, we have obtained an insulin-secreting cell clone from undifferentiated ES cells. The construction used allows the expression of a neomycin selection system under the control of the regulatory regions of the human insulin gene. The chimeric gene also contained a hygromycin resistance gene (pGK-hygro) to select transfected cells. A resulting clone (IB/3x-99) containing 16.5 ng/microg protein of total insulin displays regulated hormone secretion in vitro in the presence of various secretagogues. Clusters obtained from this clone were implanted (1 x 10(6) cells) in the spleen of streptozotocin-induced diabetic animals. Transplanted animals correct hyperglycemia within 1 week and restore body weight in 4 weeks. Whereas an intraperitoneal glucose tolerance test showed a slower recovery in transplanted versus control mice, blood glucose normalization after a challenge meal was similar. This approach opens new possibilities for tissue transplantation in the treatment of type 1 and type 2 diabetes and offers an alternative to gene therapy.

Palmitate and oleate induce the immediate-early response genes c-fos and nur-77 in the pancreatic beta-cell line INS-1.

To better understand the link between fatty acid signaling and the pleiotropic effects of fatty acids in the pancreatic beta-cell, we investigated whether fatty acids regulate immediate-early response genes (IEGs) coding for transcription factors implicated in cell proliferation, differentiation, and apoptosis. Palmitate and oleate, but not long-chain polyunsaturated fatty acids, caused a pronounced accumulation of c-fos and nur-77 mRNAs in beta-cells (INS cells) to an extent similar to that produced by the protein kinase C (PKC) activator phorbol myristate acetate (PMA). The effect was dose dependent and occurred at concentrations between 0.1 and 0.5 mmol/l in the presence of 0.5% albumin. The action of the fatty acid occurred at the transcriptional level, and the mRNA accumulation displayed a bell-shaped kinetics with a maximal effect at 1 h. 2-Bromopalmitate was ineffective, indicating that fatty acids must be metabolized to cause their effect. Neither fatty acid was able to induce c-fos and nur-77 in PKC-downregulated cells or cells incubated in the presence of the Ca2+ channel blocker nifedipine or the Ca2+ chelator EGTA, suggesting involvement of the PKC and Ca2+ signaling pathways. Palmitate and oleate also increased c-fos protein expression and DNA binding activity of the transcription factor AP-1. Oleate, but not palmitate, increased [3H]thymidine incorporation in INS cells. Finally, both palmitate and oleate caused c-fos and nur-77 mRNA accumulation in isolated rat islets. It is suggested that IEG induction by the most abundant circulating fatty acids plays a role in the adaptive process of the beta-cell to hyperlipidemia. These results have implications for our understanding of obesity-associated diabetes and the link between fatty acids and tumorigenesis.

From stem cells to insulin-producing cells: towards a bioartificial endocrine pancreas.

The total absence or low production of insulin by beta-cells avoids a proper control of glycemia forcing diabetic people to daily insulin injection for survival. Islet transplantation represents a hallmark in the cure of diabetes and has been successfully applied to more than 400 patients, resulting in insulin independency for periods longer than 4 years. However, transplantation trials for diabetes have to face the scarcity of islets from cadaveric donors. Therefore, the finding of renewable sources of cells could circumvent this problem. In this respect, embryonic or adult stem cells are representing an interesting alternative. Stem cells display robust proliferation and the plasticity to differentiate to other cell types, including insulin-containing cells. The current therapeutical use in the future of bioengineered insulin-secreting cells derived from stem cells needs at present to fulfill several criteria. These criteria concern to the type of stem cell to be used as starting biomaterial (embryonic or adult), the in vitro differentiation protocol applied, the functional phenotype reached for the final cell product and the transplantation associated problems (likely immune rejection and tumor formation). This review will try to focus on these different aspects in order to emphasize in the key points to consider for designing unified strategies for diabetes cell therapy.

The protein phosphatase inhibitor calyculin-A affects catecholamine secretion and granular distribution in cultured adrenomedullary chromaffin cells.

Calyculin-A, a potent inhibitor of types 1 and 2A protein phosphatases, increases basal catecholamine secretion in cultured chromaffin cells with a maximum effect observed at 100 nM. This effect was increased by forskolin and the calmodulin antagonist W7, but was modified neither by phorbol esters nor the protein kinase inhibitor, H7. The effect of the toxin, calyculin-A, on basal secretion was completely prevented by the protein kinase inhibitor K252a. In digitonin-permeabilized cells calyculin-A induced an increase in basal release, but, in contrast, it partially reduced calcium-induced secretion. Analysis of total proteins revealed that calyculin-A treatment of the cells increased the level of phosphorylation of different protein bands. Examination of the Triton X-100-insoluble fraction revealed a clear increase in the phosphorylation level of various proteins, including vimentin. Calyculin-A provoked a rapid morphological change in chromaffin cells in the same range of concentration (50-300 nM). Cells became rounder and were partially detached from the substratum forming clusters, this effect was also blocked by K252a. Transmission electron microscopy of calyculin-A-treated cells showed an increase in the proportion of chromaffin granules located closer to the membrane. These results suggest that calyculin-A induces changes both in the catecholamine secretory response and in the cytoskeletal elements of chromaffin cells by protein phosphorylation.

A peptide that mimics the carboxy-terminal domain of SNAP-25 blocks Ca(2+)-dependent exocytosis in chromaffin cells.

SNAP-25, a synaptosomal associated membrane protein of 25 kDa, participates in the presynaptic process of vesicle-plasma membrane fusion that results in neurotransmitter release at central nervous system synapses. SNAP-25 occurs in neuroendocrine cells and, in analogy to its role in neurons, has been implicated in catecholamine secretion, yet the nature of the underlying mechanism remains obscure. Here we use an anti-SNAP-25 monoclonal antibody to show that SNAP-25 is localized at the cytosolic surface of the plasma membrane of chromaffin cells. This antibody inhibited the Ca(2+)-evoked catecholamine release from digitonin-permeabilized chromaffin cells in a time- and dose-dependent manner. Remarkably, a 20-mer synthetic peptide representing the sequence of the C-terminal domain of SNAP-25 blocked Ca(2+)-dependent catecholamine release with an IC50 = 20 microM. The inhibitory activity of the peptide was sequence-specific as evidenced by the inertness of a control peptide with the same amino acid composition but random order. The C-terminal segment of SNAP-25, therefore, plays a key role in regulating Ca(2+)-dependent exocytosis, presumably mediated via interactions with other protein components of the fusion complex.

[3H]AHN 086 acylates peripheral benzodiazepine receptors in the rat pineal gland.

AHN 086, an isothiocyanato derivative of Ro 5-4864 (4'-chlorodiazepam), inhibits radioligand binding to peripheral benzodiazepine receptors with characteristics of an irreversible (acylating) ligand. We now report that [3H]AHN 086 labels a approximately 30 kDa protein in the rat pineal gland determined by both SDS-polyacrylamide gel electrophoresis and gel filtration high-performance liquid chromatography of digitonin-solubilized membranes. Specific incorporation of [3H]AHN 086 into this protein was inhibited by preincubating membranes with excess AHN 086. Moreover, significant specific binding of [3H]AHN 086 was not observed in either bovine pineal gland (which does not possess high-affinity binding sites for Ro 5-4864) or ovalbumin. These findings suggest that the approximately 30 kDa protein labeled by [3H]AHN 086 in rat pineal gland is associated with peripheral benzodiazepine receptors in this tissue.

Secretagogue-induced [Ca2+]i changes in single rat pancreatic islets and correlation with simultaneously measured insulin release.

The effects of secretagogues (glucose, tolbutamide and phorbol esters) on simultaneously measured intracellular free calcium concentration ([Ca2+]i) and insulin release were studied in rat pancreatic islets of Langerhans. Stimulatory concentrations (11mM) of glucose caused a transient [Ca2+]i. In contrast with mouse islets, rat islets scarcely showed glucose-induced [Ca2+]i oscillations. Digital image analysis showed that [Ca2+]i changes occurred synchronously across the whole islets. As expected simultaneously measured insulin release was biphasic with a clear second phase. This clearly indicated that in rat islets there is a lack of correlation between [Ca2+]i and insulin release. This was further explored using agents which separately promoted the first (tolbutamide, 200 microns and second (phorbol-12-myristate-13-acetate; PMA; 5nM) phases of insulin release. Tolbutamide induced a transient increase in [Ca2+]i. These results suggest that in rat islets the first phase of insulin release is calcium dependent, whereas the second phase is related to the activation of protein kinase C (PKC). However, the glucose-induced second phase of insulin release did not coincide with an increase in membrane-associated PKC activity. Other messengers may contribute to this late phase of insulin release.

Inhibition of nicotinic receptor-mediated responses in bovine chromaffin cells by diltiazem.

1. The effects of diltiazem on various functional parameters were studied in bovine cultured adrenal chromaffin cells stimulated with the nicotinic receptor agonist dimethylphenylpiperazinium (DMPP) or with depolarizing Krebs-HEPES solutions containing high K+ concentrations. 2. The release of [3H]-noradrenaline induced by DMPP (100 microM for 5 min) was gradually and fully inhibited by increasing concentrations of diltiazem (IC50 = 1.3 microM). In contrast, the highest concentration of diltiazem used (10 microM) inhibited the response to high K+ (59 mM for 5 min) by only 25%. 3. 45Ca2+ uptake into cells stimulated with DMPP (100 microM for 1 min) was also blocked by diltiazem in a concentration-dependent manner (IC50 = 0.4 microM). Again, diltiazem blocked the K(+)-evoked 45Ca2+ uptake (70 mM K+ for 1 min) only by 20%. In contrast, the N-P-Q-type Ca2+ channel blocker omega-conotoxin MVIIC depressed the K+ signal by 70%. In the presence of this toxin, diltiazem exhibited an additional small inhibitory effect, indicating that the compound was acting on L-type Ca2+ channels. 4. Whole-cell Ba2+ currents through Ca2+ channels in voltage-clamped chromaffin cells were inhibited by 3-10 microM diltiazem by 20-25%. The inhibition was readily reversed upon washout of the drug. 5. The whole-cell currents elicited by 100 microM DMPP (IDMPP) were inhibited in a concentration-dependent and reversible manner by diltiazem. Maximal effects were found at 10 microM, which reduced the peak IDMPP by 70%. The area of each curve represented by total current (QDMPP) was reduced more than the peak current. At 10 microM, the inhibition amounted to 80%; the IC50 for QDMPP inhibition was 0.73 microM, a figure close to the IC50 for 45Ca2+ uptake (0.4 microM) and [3H]-noradrenaline release (1.3 microM). The blocking effects of diltiazem developed very quickly and did not exhibit use-dependence; thus the drug blocked the channel in its closed state. The blocking effects of 1 microM diltiazem on IDMPP were similar at different holding potentials (inhibition by around 30% at -100, -80 or -50 mV). Diltiazem did not affect the current flow through voltage-dependent Na+ channels. 6. These data are compatible with the idea that diltiazem has little effect on Ca2+ entry through voltage-dependent Ca2+ channels in bovine chromaffin cells. Neither, does diltiazem affect INa. Rather, diltiazem acts directly on the neuronal nicotinic receptor ion channel and blocks ion fluxes, cell depolarization and the subsequent Ca2+ entry and catecholamine release. This novel effect of diltiazem might have clinical relevance since it might reduce the sympathoadrenal drive to the heart and blood vessels, thus contributing to the well established antihypertensive and cardioprotective effects of the drug.

Separate [3H]-nitrendipine binding sites in mitochondria and plasma membranes of bovine adrenal medulla.

1. Two binding sites for the 1,4-dihydropyridine (DHP) derivative [3H]-nitrendipine have been found in the bovine adrenal medulla. The high-affinity site (Kd = 0.48 nM and Bmax = 128 fmol mg-1 protein) was specifically located in purified plasma membranes. The low-affinity site (Kd = 252 nM and Bmax = 169 pmol mg-1 protein) was located only in mitochondria. Chromaffin granule membranes lacked specific binding sites for [3H]-nitrendipine. 2. Kinetic analysis of the rates of association and dissociation of [3H]-nitrendipine, saturation isotherms and displacement experiments with unlabelled nitrendipine and PN200-110 revealed single, homogeneous populations of high- and low-affinity sites in plasma and mitochondrial membranes, respectively. 3. The high affinity site was sensitive to Ca2+ deprivation and heating; it was practically unaffected by changes in ionic strength of the medium and its optimal pH was slightly alkaline. This site exhibited a strong DHP stereoselectivity; diltiazem increased and verapamil decreased the affinity of [3H]-nitrendipine. 4. In contrast, binding of [3H]-nitrendipine to the low affinity site was more heat resistant and less affected by Ca2+ removal. Its optimal pH was slightly acid and the increase in ionic strength enhanced the number of available sites. The site had no DHP stereoselectivity. Verapamil decreased the dissociation constant of [3H]-nitrendipine acting in a non-competitive manner; diltiazem did not affect equilibrium binding parameters of [3H]-nitrendipine. 5. These results suggest that both biding sites reflect different receptor entities. The high-affinity binding site corresponds to the dihydropyridine receptor associated with the L-type calcium channel. The function of the mitochondrial, low-affinity binding site is, at present, unknown.

Ruthenium red inhibits selectively chromaffin cell calcium channels.

The effect of Ruthenium red (RR) on ionic currents and catecholamine secretion was studied in chromaffin cells. This polycation inhibited 59 mM potassium-stimulated 45Ca2+ uptake in a concentration-dependent manner (IC50 = 5 +/- 0.2 microM). This effect was more evident at extracellular calcium concentrations over 1 mM and was not abolished by neuraminidase pretreatment. RR also inhibited potassium-stimulated catecholamine secretion (IC50 = 6 +/- 0.9 microM). These results were corroborated by patch-clamp in whole-cell recordings. RR inhibited chromaffin cell calcium currents (IC50 = 7 microM) without affecting significantly either sodium or potassium currents. Radioligand binding studies in adrenomedullary plasma membranes showed that RR inhibited [125I]omega-conotoxin GVIA binding but it had no effect on specific binding of [3H]nitrendipine. The effect of the RR on calcium currents was additive with the inhibitory effect observed with 10 microM nitrendipine. The residual dihydropyridine-resistant calcium current was inhibited with a potency similar to that determined under control conditions in the absence of nitrendipine. These results demonstrate that RR selectively inhibits calcium channels; however, this polycation was not selective for a particular calcium channel subtype.

The low-affinity dihydropyridine receptor and Na+/Ca2+ exchanger are associated in adrenal medullary mitochondria.

The effect of Ca2+ channel-acting drugs on bovine adrenal mitochondria Ca2+ movements was investigated. Mitochondrial Ca2+ uptake is performed by an energy-driven Ca2+ uniporter with a Km of 20.9 +/- 3.2 microM and Vmax of 148.1 +/- 7.2 nmol 45Ca2+ min-1 mg-1. Ca2+ release is performed through an Na+/Ca2+ antiporter with a Km for Na+ of 4.2 +/- 0.5 mM, a Vmax of 7.5 +/- 0.4 nmol 45Ca2+ min-1 mg-1, and a Hill coefficient of 1.4 +/- 0.2 Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger was inhibited by several dihydropyridines (nitrendipine, felodipine, nimodipine, (+)isradipine) and by the benzothiazepine diltiazem with similar potencies. In contrast, neither CGP 28392, Bay-K-8644, amlodipine, nor verapamil had any effect on Ca2+ efflux. Nitrendipine at 20 microM modified neither the Km nor the Hill coefficient for Na+, whereas the Vmax was reduced to 2.9 nmol 45Ca2+ min-1 mg-1, thus demonstrating noncompetitive modulation of the Na+/Ca2+ exchanger. None of the Ca2+ channel-acting drugs assayed at 100 microM affected Ca2+ influx through the uniporter. Ca2+ channel blockers inhibited the Na+/Ca2+ antiporter and displaced the specific binding of [3H]nitrendipine to intact mitochondria with Ki values similar to the IC50s obtained for the inhibition of the Ca2+ efflux. Ca2+ channel-acting drugs that did not inhibit the Na+/Ca2+ exchanger (amlodipine, CGP 28392, Bay-K-9644, and verapamil, at concentrations of 100 microM or higher) had no effect on [3H]nitrendipine binding. These results suggest that the adrenomedullary mitochondrial dihydropyridine receptor is associated with the Na+/Ca2+ exchanger.

Calyculin A blocks bovine chromaffin cell calcium channels independently of phosphatase inhibition.

The specific phosphatase inhibitor, Calyculin-A (CL-A), decreases high-K stimulated catecholamine secretion in bovine chromaffin cells. This effect can be split into two components: one needs long exposures to the drug to be elicited, and is sensitive to the protein kinase-inhibitor K252a; the other is observed after short incubations of CL-A, and is insensitive to K252a. Here we report that the latter component is due to an external block, by CL-A, of chromaffin cell calcium channels in a voltage-dependent, reversible and phosphorylation-independent manner.

Bio-engineering inslulin-secreting cells from embryonic stem cells: a review of progress.

According to the Edmonton protocol, human islet transplantation can result in insulin independency for periods longer than 3 years. However, this therapy for type 1 diabetes is limited by the scarcity of cadaveric donors. Owing to the ability of embryonic stem cells to expand in vitro and differentiate into a variety of cell types, research has focused on ways to manipulate these cells to overcome this problem. It has been demonstrated that mouse embryonic stem cells can differentiate into insulin-containing cells, restoring normoglycaemia in diabetic mice. To this end, mouse embryonic stem cells were transfected with a DNA construct that provides resistance to neomycin under the control of the regulatory regions of the human insulin gene. However, this protocol has a very low efficiency, needing improvements for this technology to be transferred to human stem cells. Optimum protocols will be instrumental in the production of an unlimited source of cells that synthesise, store and release insulin in a physiological manner. The review focuses on the alternative source of tissue offered by embryonic stem cells for regenerative medicine in diabetes and some key points that should be considered in order for a definitive protocol for in vitro differentiation to be established.

Nutrient toxicity in pancreatic beta-cell dysfunction.

Nutrients, such as glucose and fatty acids, have a dual effect on pancreatic beta-cell function. Acute administration of high glucose concentrations to pancreatic beta-cells stimulates insulin secretion. In addition, short term exposure of this cell type to dietary fatty acids potentiates glucose-induced insulin release. On the other hand, long-term exposure to these nutrients causes impaired insulin secretion, characterized by elevated exocytosis at low concentrations of glucose and no response when glucose increases in the extracellular medium. In addition, other phenotypic changes are observed in these conditions. One major step in linking these phenotypic changes to the diabetic pathology has been the recognition of both glucose and fatty acids as key modulators of beta-cell gene expression. This could explain the adaptative response of the cell to sustained nutrient concentration. Once this phase is exhausted, the beta-cell becomes progressively unresponsive to glucose and this alteration is accompanied by the irreversible induction of apoptotic programs. The aim of this review is to present actual data concerning the development of the toxicity to the main nutrients glucose and fatty acids in the pancreatic beta-cell and to find a possible link to the development of type 2 diabetes.

Insulin-producing cells derived from stem cells: recent progress and future directions.

Type 1 diabetes is characterized by the selective destruction of pancreatic beta-cells caused by an autoimmune attack. Type 2 diabetes is a more complex pathology which, in addition to beta-cell loss caused by apoptotic programs, includes beta-cell dedifferentiation and peripheric insulin resistance. beta-Cells are responsible for insulin production, storage and secretion in accordance to the demanding concentrations of glucose and fatty acids. The absence of insulin results in death and therefore diabetic patients require daily injections of the hormone for survival. However, they cannot avoid the appearance of secondary complications affecting the peripheral nerves as well as the eyes, kidneys and cardiovascular system. These afflictions are caused by the fact that external insulin injection does not mimic the tight control that pancreatic-derived insulin secretion exerts on the body's glycemia. Restoration of damaged beta-cells by transplantation from exogenous sources or by endocrine pancreas regeneration would be ideal therapeutic options. In this context, stem cells of both embryonic and adult origin (including beta-cell/islet progenitors) offer some interesting alternatives, taking into account the recent data indicating that these cells could be the building blocks from which insulin secreting cells could be generated in vitro under appropriate culture conditions. Although in many cases insulin-producing cells derived from stem cells have been shown to reverse experimentally induced diabetes in animal models, several concerns need to be solved before finding a definite medical application. These refer mainly to the obtainment of a cell population as similar as possible to pancreatic beta-cells, and to the problems related with the immune compatibility and tumor formation. This review will summarize the different approaches that have been used to obtain insulin-producing cells from embryonic and adult stem cells, and the main problems that hamper the clinical applications of this technology.

Submicrogram quantities of unstained proteins are visualized on polyvinylidene difluoride membranes by transillumination.

Unstained proteins on polyvinylidene difluoride membranes which have been dried and saturated with 20% methanol appear as clear areas when placed on a white light box. Sensitivity is comparable to that obtained with Coomassie Blue R250. This procedure greatly reduces the potential for unintended chemical modification during staining procedures, provides a nondestructive means of determining the major features of a protein pattern on a blot and makes it possible to initiate sequencing within minutes after blotting is complete.

Immunohistochemical demonstration of syntaxin and SNAP-25 in chromaffin cells of the frog adrenal gland.

The release of catecholamines from chromaffin cells involves specific proteins such as synaptobrevin present in the secretory vesicles as well as syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25), both present in the plasma membrane. We have found syntaxin and SNAP-25 in chromaffin cells of the frog adrenal gland by immunohistochemistry. This result suggests that the secretion of catecholamines from chromaffin cells involves these proteins in the frog.