Diferenciación de las células troncales embrionarias humanas hacia un linaje beta-pancreático: estudios enfocados a procesos de maduración

Las células troncales embrionarias humana (CTEh) tienen la capacidad de diferenciar hacia todos los tejidos que componen el cuerpo adulto, incluido las células ß del páncreas endocrino. No obstante, un protocolo para la diferenciación de las CTEh hacia células ß-pancreáticas maduras y funcionales que sea eficiente y reproducible no ha sido todavía generado. El objetivo general de este trabajo de tesis ha sido la optimización de un protocolo de diferenciación de las CTEh hacia un fenotipo ß-pancreático que reproduzca las etapas del desarrollo embrionario del páncreas y el estudio del papel del RSV en la etapa de maduración para la obtención de una nueva y más eficaz estrategia de diferenciación. Para ello primero se ha estudiado el efecto del RSV en una línea celular de insulinoma (INS-1E) describiendo la vía de SIRT1/Ucp2 como posible mecanismo de acción. Sucesivamente, basándonos en las vías de señalización y los factores de crecimiento involucrados en el desarrollo embrionario del páncreas se ha diseñado un protocolo de diferenciación capaz de dirigir las CTEh hacia células ß-pancreáticas. Para comprobar la eficacia del protocolo de diferenciación las células han sido recogidas a diferentes etapas del protocolo y han sido caracterizadas mediante Q-PCR para la expresión de factores de transcripción que son claves en el desarrollo de las células ß. Al final del protocolo de diferenciación las células expresan marcadores de páncreas endocrino y células ß maduras como PDX1, GLUT2, GK, NKX2.2, INS, GCG, entre otros. Resultados similares se han obtenidos aplicando el protocolo de diferenciación a otro tipo de células troncales pluripotentes, la línea celular pluripotente inducida (hiPS) MSUH001. También se ha demostrado que el tratamiento con RSV durante la etapa final del protocolo de diferenciación mejora el proceso de maduración de los precursores ß-pancreáticos permitiendo duplicar el número de células productoras de insulina obtenidas. Las células tratadas con RSV presentan un incremento en la expresión de PDX1, un mayor contenido de insulina y son capaces de normalizar transitoriamente la glucemia in vivo tras el trasplante en ratones diabéticos. Finalmente se ha propuesto la activación de las vías de señalización responsables de la activación de la transcripción de PDX1 como mecanismo de acción mediante el cual el RSV mejora el proceso de maduración de las células en proceso de diferenciación. En conclusión en este trabajo de tesis se describe un protocolo de diferenciación de múltiples etapas capaz de diferenciar las células troncales pluripotentes hacia células ß-pancreáticas de forma eficiente y reproducible demostrando la implicación del RSV como factor clave en los procesos de maduración.Peer Reviewe

Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair

Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors, enhances scar formation via cell autonomous production of collagen. In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair

The Use of New Parameters to Optimize the Composting Process of Different Organic Wastes

The correct development of the composting process is essential to obtain a product of high value from organic wastes. Nowadays, some composting mixture parameters (i.e., air-filled porosity, moisture and the C/N ratio) are used to optimize the composting process, but their suitability is still debated because the literature reports contrasting results. This paper aimed to find other parameters that control the correct development of composting. The relationship between these and the compost quality was then verified. Twelve different composting mixtures were prepared using different organic wastes and bulking agents and were aerobically treated in a 300 L composter. The physico-chemical and chemical parameters of initial mixtures were analyzed, with particular regard to the total and water-extractable forms of organic C and N and their ratios and correlated with the temperature measured during composting. A positive correlation between temperature parameters during the active phase and soluble forms of N in the initial mixtures was found. A high total organic C to soluble N ratio in the composting mixtures was correlated with the low quality of the compost produced. Based on the results, a minimum content of WEN (water-extractable N) (0.4% w/w) or a TOC/WEN (total organic C/WEN) ratio in the range of 40–80 was recommended to ensure the correct development of the process and to produce compost of high quality

Functional Vascular Smooth Muscle-like Cells Derived from Adult Mouse Uterine Mesothelial Cells

In mammalian visceral organs, vascular smooth muscle cells (VSMCs) originate from an epithelial-to-mesenchymal transition (EMT) of embryonic mesothelial cells (MCs). The ability of adult MCs to recapitulate EMT and to acquire smooth muscle (SM) markers upon provasculogenic culture suggested they might retain embryonic vasculogenic differentiation potential. However, it remains unknown whether adult MCs-derived SM-like cells may acquire specific vascular SM lineage markers and the functionality of differentiated contractile VSMCs. Here, we describe how a gentle trypsinization of adult mouse uterine cords could selectively detach their outermost uterine mesothelial layer cells. As other MCs; uterine MCs (UtMCs) uniformly expressed the epithelial markers β-catenin, ZO-1, E-cadherin, CD54, CD29, and CK18. When cultured in a modified SM differentiation media (SMDM) UtMCs initiated a loss of epithelial characteristics and gained markers expression of EMT (Twist, Snail, and Slug), stem and progenitor (Nanog, Sox2, C-kit, Gata-4, Isl-1, and nestin), SM (α-SMA, calponin, caldesmon, SM22α, desmin, SM-MHC, and smoothelin-B) and cardiac (BMP2, BMP4, ACTC1, sACTN, cTnI, cTnT, ANF, Cx43, and MLC2a). UtMCs repeatedly subcultured in SMDM acquired differentiated VSM-like characteristics and expressed smoothelin-B in the typical stress-fiber pattern expression of contractile VSMCs. Relevantly, UtMCs-derived VSM-like cells could generate >mechanical force> to compact collagen lattices and displayed in diverse degree voltage (K+) and receptor (endothelin-1, oxytocin, norepinephrine, carbachol and vasopressin)-induced [Ca2+]i rises and contraction. Thus, we show for the first time that UtMCs could recapitulate in vitro differentiative events of early cardiovascular differentiation and transdifferentiate in cells exhibiting molecular and functional characteristics of VSMCs. © 2013 Lachaud et al.Authors are supported by the Fundación Progreso y Salud, Consejería de Salud, Junta de Andalucía (Grant PI-0022/2008); Consejería de Innovación Ciencia y Empresa, Junta de Andalucía (Grant CTS-6505; INP-2011-1615-900000); FEDER co-funded grants from Instituto de Salud Carlos III (Red TerCel-Grant RD06/0010/0025; PI10/00964) and the Ministry of Health and Consumer Affairs (Advanced Therapies Program Grant TRA-120). CIBERDEM is an initiative of the Instituto de Salud Carlos III.Peer Reviewe

Production of CO₂ Hydrates in Aqueous Mixtures Having (NH₄)₂SO₄ at Different Concentrations; Definition of Consequences on the Process Evolution, Quantification of CO₂ Captured and Validation of Hydrates Production as Technique for Ammonium Removal from Waste Water

Carbon dioxide hydrates were formed in fresh water and in aqueous mixtures containing ammonium sulfate, at concentrations equal to 1.9, 6.3, and 9.5 wt%. The moles of hydrates formed were compared, to define the inhibiting strength of the electrolyte solution and the dependence of inhibition from concentration. The addition of salt strongly inhibited the process and the number of hydrates produced passed from 0.204–0.256 moles, obtained in fresh water, to 0.108–0.198 moles, obtained at the lowest concentration tested. The further addition of salt still lowered the production of the hydrates; at the highest concentration tested, only 0.092–0.177 moles were obtained. The pressure-temperature evolutions of the hydrates were then discussed and compared with the ideal process and with the experimental results obtained in demineralised water. Finally, further samples of CO2 hydrates, produced in the presence of 9.5 wt% salt in the aqueous phase (corresponding to 1.5 wt% NH4+), were recovered and dissociated in a separated environment. The liquid phase, resulting from their dissociation, was subjected to spectrophotometric analyses. Its NH4+ content was measured and compared with the initial concentration in water. Therefore, it was possible to quantify the capability of the system to remove the (NH4)2SO4 from the water (involved in hydrate formation) and to concentrate it in the remaining liquid phase. Considering the portion of water involved in hydrates formation, the concentration of ammonium passed from 1.5 wt% to 0.38–0.449 wt%

The Opportunity of Valorizing Agricultural Waste, Through Its Conversion into Biostimulants, Biofertilizers, and Biopolymers

The problems arising from the limited availability of natural resources and the impact of certain anthropogenic activities on the environment must be addressed as soon as possible. To meet this challenge, it is necessary, among other things, to reconsider and redesign agricultural systems to find more sustainable and environmentally friendly solutions, paying specific attention to waste from agriculture. Indeed, the transition to a more sustainable and circular economy should also involve the effective valorization of agricultural waste, which should be seen as an excellent opportunity to obtain valuable materials. For the reasons mentioned above, this review reports and discusses updated studies dealing with the valorization of agricultural waste, through its conversion into materials to be applied to crops and soil. In particular, this review highlights the opportunity to obtain plant biostimulants, biofertilizers, and biopolymers from agricultural waste. This approach can decrease the impact of waste on the environment, allow the replacement and reduction in the use of synthetic compounds in agriculture, and facilitate the transition to a sustainable circular economy

Long-Term Effects of Amendment with Olive Mill Wastewater on Soil Chemical Properties, Microbial Community, and Olive Tree Vegetative and Productive Activities

The long-term effects of the olive mill wastewater (OMWW) spreading on soil chemical properties, microbial community, and olive tree parameters have been far poorly investigated. Therefore, this study aims to evaluate the effect of OMWW application on an olive orchard, and samples were collected at two different depths (0–20 cm and 20–40 cm) and 14 days, one year and two years from the end of the OMWW spreading on soil chemical characteristics and soil microbial structures. Variations of soil chemical parameters (pH, salinity, available P, and water-extractable organic C) were observed particularly at 14 days after spreading at both depths. All these parameters reached similar values to the soil after two years, except for available phosphorus. Firmicutes, Proteobacteria, and Actinobacteria are the most abundant phyla: only Firmicutes were negatively affected by the OMWW spreading after 14 days, suggesting that Gram-positive bacteria were probably negatively influenced by the addition of OMWW. The abundance of bacterial taxa in the soil is restored along time, except for the decrease of Firmicutes. This evidence suggests that the OMWW spreading in the long term does not affect the endemic soil bacterial community of the olive grove, as well as leaf net photosynthesis, the olive tree vegetative activity, yield, and fruits characteristics

Zebularine regulates early stages of mESC differentiation: effect on cardiac commitment

Lineage commitment during embryonic stem cell (ESC) differentiation is controlled not only by a gamut of transcription factors but also by epigenetic events, mainly histone deacetylation and promoter DNA methylation. The DNA demethylation agent 50-aza-20-deoxycytidine (AzadC) has been widely described as an effective promoter of cardiomyogenic differentiation in various stem cell types. However, its toxicity and instability complicate its use. Therefore, the purpose of this study was to examine the effects of zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-1), a stable and non-toxic DNA cytosine methylation inhibitor, on mouse ESC (mESC) differentiation. Herein, we report that treating embryoid bodies, generated from mESCs, with 30 lM zebularine for 7 days led to greater cell differentiation and induced the expression of several cardiac-specific markers that were detected using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, immunostaining and flow cytometry. Zebularine enhanced the expression of cardiac markers and the appearance of beating cells that responded to cardiac drugs, including ion channel blockers (diltiazem) and β-adrenergic stimulators (isoproterenol). Gene promoter methylation status was assessed using methylation-specific PCR (MSP) and validated by bisulfite sequencing analysis. Global gene expression profiling using microarrays showed that zebularine-differentiated cells are distinct from control ESCs. Pathway analysis revealed an enhancement of cellular processes such as embryonic development, cardiovascular system development and function. In addition, the whole-cell proteins exhibited different profiles as analyzed by two-dimensional differential-in-gel-electrophoresis. Our results indicate that zebularine regulates mesodermal differentiation of mESCs, controls promoter methylation of crucial cardiac genes and may help to improve cardiomyogenic differentiation. © 2013 Macmillan Publishers Limited All rights reserved.We were supported by Fundación Progreso y Salud, Consejería de Salud, Junta de Andalucía (Grant PI-0022/2008); Consejería de Innovación Ciencia y Empresa, Junta de Andalucía (Grant CTS-6505; INP-2011-1615-900000); FEDER co-funded grants from Instituto de Salud Carlos III (RD06/0010/0025; PI10/ 00964) and the Ministry of Health and Consumer Affairs (Advanced Therapies Program Grant TRA-120). CIBERDEM is an initiative of the Instituto de Salud Carlos III. AH was awarded a ‘Ramo´n Areces’ postgraduate scholarship.We were supported by Fundación Progreso y Salud, Consejería de Salud, Junta de Andalucía (Grant PI-0022/2008); Consejería de Innovación Ciencia y Empresa, Junta de Andalucía (Grant CTS-6505; INP-2011-1615-900000); FEDER co-funded grants from Instituto de Salud Carlos III (RD06/0010/0025; PI10/ 00964) and the Ministry of Health and Consumer Affairs (Advanced Therapies Program Grant TRA-120). CIBERDEM is an initiative of the Instituto de Salud Carlos III. AH was awarded a ‘Ramón Areces’ postgraduate scholarship.Peer Reviewe

Thyroglossal duct carcinoma: Report of a case

Summary. Aim: Thyroglossal duct carcinoma (TDCa) is a rare malignant tumor arising within a thyroglossal duct remnant (TDR) or a thyroglossal duct cyst (TDC). Controversies exist regarding its origin: whether it represents a metastatic lesion of a primary thyroid cancer or a â\u80\u9cde novoâ\u80\u9d origin. Patients and methods: Man with a visible and palpable subhyoid mass. Preoperative ultrasound scan and US-guided fine-needle aspiration cyology revealed suspicious papillary carcinoma in the TDC and thyroid nodules. Surgery consisted in removal of the hyoid bone and total thyroidectomy. Results: Histopathologic examination revealed papillary TDCa and a focus of papillary var. follicular microcarcinoma in the thyroid gland. Conclusions: Ultrasound scan with fine-needle aspiration cytology is beneficial in the preoperative diagnosis of carcinoma in TDC. Surgery is an adequate treatment for TDCa and the prognosis for TDCa is excellent. Adjuvant radioactive iodine and post-operative L-thiroxine suppressive therapy are appropriate in these cases. Our experience confirms the controversies about the origin of TDCa