Derivation of naive human embryonic stem cells.

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

Anchored phosphatases modulate glucose homeostasis.

Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic ß-cells involves ion channels and mobilization of Ca(2+) and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-ß-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca(2+) currents, and attenuates cytoplasmic accumulation of Ca(2+) and cAMP in ß-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.

ß1 integrin is a crucial regulator of pancreatic ß-cell expansion.

Development of the endocrine compartment of the pancreas, as represented by the islets of Langerhans, occurs through a series of highly regulated events encompassing branching of the pancreatic epithelium, delamination and differentiation of islet progenitors from ductal domains, followed by expansion and three-dimensional organization into islet clusters. Cellular interactions with the extracellular matrix (ECM) mediated by receptors of the integrin family are postulated to regulate key functions in these processes. Yet, specific events regulated by these receptors in the developing pancreas remain unknown. Here, we show that ablation of the ß1 integrin gene in developing pancreatic ß-cells reduces their ability to expand during embryonic life, during the first week of postnatal life, and thereafter. Mice lacking ß1 integrin in insulin-producing cells exhibit a dramatic reduction of the number of ß-cells to only ∼18% of wild-type levels. Despite the significant reduction in ß-cell mass, these mutant mice are not diabetic. A thorough phenotypic analysis of ß-cells lacking ß1 integrin revealed a normal expression repertoire of ß-cell markers, normal architectural organization within islet clusters, and a normal ultrastructure. Global gene expression analysis revealed that ablation of this ECM receptor in ß-cells inhibits the expression of genes regulating cell cycle progression. Collectively, our results demonstrate that ß1 integrin receptors function as crucial positive regulators of ß-cell expansion.

Treatment with testosterone or estradiol in melatonin treated females and males MRL/MpJ-Faslpr mice induces negative effects in developing systemic lupus erythematosus.

MRL/MpJ-Fas(lpr) mice is widely accepted as a valuable model of systemic lupus erythematosus. As described in a previous work, the incidence of lupus in this strain is determined by sex hormones, i.e., estrogens and androgens. Moreover, we reported that the immunomodulatory action of melatonin in these mice was gender-dependent probably through modulation and inhibition of sex hormones. Herein, we performed an experiment using hormone therapy, by treating female MRL-lpr mice with testosterone and males with estradiol and with melatonin. A decrease in total serum immunoglobulin (Ig)G and IgM immunoglobulin titers, anti-double-stranded DNA, and anti-CII autoantibodies in female mice treated with both melatonin and testosterone was revealed, along with an increase in pro-inflammatory cytokines [interleukin (IL)-2, IL-6, interferon-gamma, tumor necrosis factor-alpha, and IL-1beta), nitrite/nitrate and a decrease in anti-inflammatory cytokines (IL-10). Melatonin and estradiol treatment exhibited a similar effect in male mice. Autoantibody titer elevation and pro-inflammatory versus anti-inflammatory cytokine prevalence degraded all immunological parameters. Similar results were obtained when spleen and lymph node lymphocytes were cultured. Again, melatonin and testosterone treatment stimulated pro-inflammatory and reduced anti-inflammatory cytokines produced by lymphocytes in females. The effect was similar in males treated with melatonin and estradiol. In summary, we observed that although melatonin alone prevents lupus development in females, adding testosterone, increased pro-inflammatory cytokine pattern. In contrary, estradiol-treated males did not show any decrease in pro-inflammatory cytokines but showed an increase in regard to melatonin controls. These findings confirm that melatonin action in MRL/MpJ-Fas(lpr) mice could be gender-dependent through modulation of sex hormones.

αE-Catenin Is a Positive Regulator of Pancreatic Islet Cell Lineage Differentiation.

The development and function of epithelia depend on the establishment and maintenance of cell-cell adhesion and intercellular junctions, which operate as mechanosensor hubs for the transduction of biochemical signals regulating cell proliferation, differentiation, survival, and regeneration. Here, we show that αE-catenin, a key component of adherens junctions, functions as a positive regulator of pancreatic islet cell lineage differentiation by repressing the sonic hedgehog pathway (SHH). Thus, deletion of αE-catenin in multipotent pancreatic progenitors resulted in (1) loss of adherens junctions, (2) constitutive activation of SHH, (3) decrease in islet cell lineage differentiation, and (4) accumulation of immature Sox9+ progenitors. Pharmacological blockade of SHH signaling in pancreatic organ cultures and in vivo rescued this defect, allowing αE-catenin-null Sox9+ pancreatic progenitors to differentiate into endocrine cells. The results uncover crucial functions of αE-catenin in pancreatic islet development and harbor significant implications for the design of ß cell replacement and regeneration therapies in diabetes.

Sex-dependent effect of melatonin on systemic erythematosus lupus developed in Mrl/Mpj-Faslpr mice: it ameliorates the disease course in females, whereas it exacerbates it in males.

In this study, the effect of chronic administration of melatonin on MRL/MpJ-Fas(lpr) mice has been studied. These mice spontaneously develop an autoimmune disease that has many features resembling human systemic lupus erythematosus. In fact, histological studies showed that all female mice and most male mice exhibited glomerular abnormalities, arteritic lesions, and cellular interstitial inflammatory infiltrate ranging from mild to severe patterns. Treatment with melatonin improved the histological pattern in females and worsened it in males. Moreover, female mice treated with melatonin showed a diminution of titers of total serum IgG, IgM, and anti-double-stranded DNA and anti-CII autoantibodies; a decrease in proinflammatory cytokines (IL-2, IL-6, interferon-gamma, TNF-alpha, and IL-1beta), an increase in antiinflammatory cytokines (IL-10), and a decrease in nitrite/nitrate. In male mice, treatment with melatonin exhibited the opposite effect, worsening all the immunological parameters with an elevation of titers of autoantibodies and a prevalence of proinflammatory vs. antiinflammatory cytokines. Similar results were obtained when lymphocytes from spleen and lymph nodes were cultured. Again, melatonin treatment in females decreased proinflammatory cytokines and increased antiinflammatory cytokines produced by lymphocytes; in males, the effect was the opposite. These findings suggest that melatonin action in MRL/MpJ-Fas(lpr) mice is gender dependent, probably through modulation and inhibition of sex hormones.

Macrophage/epithelium cross-talk regulates cell cycle progression and migration in pancreatic progenitors.

Macrophages populate the mesenchymal compartment of all organs during embryogenesis and have been shown to support tissue organogenesis and regeneration by regulating remodeling of the extracellular microenvironment. Whether this mesenchymal component can also dictate select developmental decisions in epithelia is unknown. Here, using the embryonic pancreatic epithelium as model system, we show that macrophages drive the epithelium to execute two developmentally important choices, i.e. the exit from cell cycle and the acquisition of a migratory phenotype. We demonstrate that these developmental decisions are effectively imparted by macrophages activated toward an M2 fetal-like functional state, and involve modulation of the adhesion receptor NCAM and an uncommon "paired-less" isoform of the transcription factor PAX6 in the epithelium. Over-expression of this PAX6 variant in pancreatic epithelia controls both cell motility and cell cycle progression in a gene-dosage dependent fashion. Importantly, induction of these phenotypes in embryonic pancreatic transplants by M2 macrophages in vivo is associated with an increased frequency of endocrine-committed cells emerging from ductal progenitor pools. These results identify M2 macrophages as key effectors capable of coordinating epithelial cell cycle withdrawal and cell migration, two events critical to pancreatic progenitors' delamination and progression toward their differentiated fates.

Evidence for melatonin synthesis in the rat brain during development.

Melatonin production is not restricted to the pineal gland. Several extrapineal sources of this indole such as retina, Harderian gland, and immune system are well documented. Melatonin of pineal origin is not present in the rat at early stages of development. To assess the potential capacity of local melatonin synthesis by the immature brain and to gain insight into the relationship between melatonin production by the brain (without the pineal gland) and pineal gland during rat development, the melatonin content as well as the expression and activity of the melatonin-synthesizing enzymes, N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were studied at fetal and postnatal stages. Moreover, melatonin-membrane receptor (MT(1)) expression was also analyzed. Both, the expression and activity of NAT and HIOMT were found in the brain with significant day/night differences in enzymes activities. Additionally, melatonin content was detected in all stages showing day/night differences depending on the stage of development. The brain nocturnal melatonin content was higher than diurnal content on postnatal day 16 and in adult rats which is in accordance with the pineal melatonin synthesis. To investigate the origin of this brain melatonin, pinealectomized rats were used and we found that the developing brain produced its own melatonin. Also, MT(1) expression was detected in brain during development. These results demonstrate that, when the pineal is not yet producing melatonin, there is melatonin synthesis by the brain that could be used as protection from free radical damage and/or could exert some actions through MT(1) receptors.

Melatonin synthesis and melatonin-membrane receptor (MT1) expression during rat thymus development: role of the pineal gland.

To gain insight into the relationship between thymus and pineal gland during rat development, the melatonin content as well as the activity and expression of the two key enzymes for melatonin biosynthesis, i.e. N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were studied in the thymus at fetal and postnatal stages. Moreover, melatonin-membrane receptor (MT1) expression was also analyzed. We found both the expression and activity of thymic NAT and HIOMT at 18 days of fetal life. Additionally, there is production of melatonin in the thymus as well as MT1 expression at this fetal age. These results show values higher in day-time than at night-time. The pineal gland begins to produce significant levels of melatonin around postnatal day 16, and this synthesis shows a circadian rhythm with high values during the dark period; therefore the nocturnal serum melatonin may inhibit thymic melatonin production. To document this, we report an increased melatonin content of the thymus in pinealectomized rats compared with sham-pinealectomized. In conclusion, these results show, for the first time, the presence of the biosynthetic machinery of melatonin and melatonin production in developing rat thymus and that the pineal gland may regulate this process.

Dual effect of melatonin as proinflammatory and antioxidant in collagen-induced arthritis in rats.

The aim of this study was to determine the effects of melatonin on proinflammatory status of rats with collagen-induced arthritis (CIA). CIA was induced in male Wistar rats with an emulsion of type II collagen in Freund's Incomplete Adjuvant (C-II/FIA). For 14 days, control and pinealectomized rats received a subcutaneous injection of 100 microL melatonin (30 microg) or vehicle (saline on 1% ethanol). Levels of cytokines interleukin (IL)-1beta and IL-6 were determined in the serum, peripheral blood mononuclear cells, and joints. Levels of anti-type II collagen antibody, nitrite/nitrate, and lipid peroxidation (LPO) were determined in the serum, joints, and brain. Treatment with melatonin significantly increased the levels of IL-1beta, IL-6, nitrite/nitrate and LPO in joints. However, melatonin significantly reduced the levels of nitrite/nitrate and LPO in serum and brain. Moreover, CIA in pinealectomized rats presented significantly reduced levels of IL-1beta and IL-6, titers of anti-type II collagen antibodies, levels of nitrite/nitrate, and LPO in joints but elevated levels in serum and brain. Melatonin has been described as a proinflammatory and antioxidant agent. In a process of inflammation as CIA, melatonin acts with a markedly proinflammatory effect at local and peripheral levels maintaining its antioxidant effect only at peripheral level.