En este artículo se analiza cómo los acuerdos comerciales plurilaterales pueden ser una vía eficaz para que los países que así lo deseen profundicen en la adopción de normas para un funcionamiento más competitivo de los mercados internacionales. Analiza la relación entre las normas multilaterales y los acuerdos bilaterales y plurilaterales, el marco legal aplicable para alcanzar dichos acuerdos, así como algunos ejemplos de acuerdos plurilaterales. La regulación de la economía digital es especialmente interesante para iniciativas en formato plurilateral.
The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE63710 ; A better understanding of the molecular mechanisms governing stem cell self-renewal will foster the use of different types of stem cells in disease modeling and cell therapy strategies. Immortalization, understood as the capacity for indefinite expansion, is needed for the generation of any cell line. In the case of v-myc immortalized multipotent human Neural Stem Cells (hNSCs), we hypothesized that v-myc immortalization could induce a more dedifferentiated state in v-myc hNSC lines. To test this, we investigated the expression of surface, biochemical and genetic markers of stemness and pluripotency in v-myc immortalized and control hNSCs (primary precursors, that is, neurospheres) and compared these two cell types to human Embryonic Stem Cells (hESCs) and fibroblasts. Using a Hierarchical Clustering method and a Principal Component Analysis (PCA), the v-myc hNSCs associated with their counterparts hNSCs (in the absence of v-myc) and displayed a differential expression pattern when compared to hESCs. Moreover, the expression analysis of pluripotency markers suggested no evidence supporting a reprogramming-like process despite the increment in telomerase expression. In conclusion, v-myc expression in hNSC lines ensures self-renewal through the activation of some genes involved in the maintenance of stem cell properties in multipotent cells but does not alter the expression of key pluripotency-associated genes ; This work was supported by grants from (to AMS): Spanish Ministry of Economy and Competitiveness (PLE2009–0101, SAF2010–17167), Comunidad Autónoma Madrid (S2011—BMD— 2336), Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0013) and European Union (Excell, NMP4—SL—2008–214706); (to PM): Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0006; FIS P110/0449), ERANEt ISCIII—Fondos FEDER (PI12/ 03112) and the Spanish Association of Cancer Research (CIMEN2011). MJPB was funded by MINECO (PLE2009–0101) and Instituto Salud Carlos III (RETICS TerCel, RD06/0019/0023). This work was also supported by an institutional grant from Fundación Ramón Areces to the Center of Molecular Biology Severo Ochoa. PM lab is supported by: Instituto de Salud Carlos III (ISCIII; E-Rare-2 Call PI12/03112), Ministerio de Economía y Competitividad (MINECO; SAF2013-43065), Generalitat de Catalunya (SGR330) and Obra Social La Caixa-Fundaciò Josep Carreras
During development, neurons can be generated directly from a multipotent progenitor or indirectly through an intermediate progenitor (IP). This last mode of division amplifies the progeny of neurons. The mechanisms governing the generation and behavior of IPs are not well understood. In this work, we demonstrate that the lengthening of the cell cycle enhances the generation of neurons in a human neural progenitor cell system in vitro and also the generation and expansion of IPs. These IPs are insulinoma-associated 1 (Insm1)+/BTG family member 2 (Btg2)−, which suggests an increase in a self-amplifying IP population. Later the cultures express neurogenin 2 (Ngn2) and become neurogenic. The signaling responsible for this cell cycle modulation is investigated. It is found that the release of calcium from the endoplasmic reticulum to the cytosol in response to B cell lymphoma-extra large overexpression or ATP addition lengths the cell cycle and increases the number of IPs and, in turn, the final neuron outcome. Moreover, data suggest that the p53–p21 pathway is responsible for the changes in cell cycle. In agreement with this, increased p53 levels are necessary for a calciuminduced increase in neurons. Our findings contribute to understand how calcium signaling can modulate cell cycle length during neurogenesis. ; European Union Grant NMP-SL-2008-214706 EXCELL; Spanish Ministry of Science and Technology Grant SAF2004-03405; Spanish Ministry of Science and Innovation (BIO2007-66807, PLE2009-0101, SAF2010-17167); Carlos III Institute of Health Grant RETICS TerCel RD06/0010/0009; La Caixa Foundation Grant BM05-22-0); Foundation Ramon Areces ; Peer Reviewed
Brain organoids are considered to be a highly promising in vitro model for the study of the human brain and, despite their various shortcomings, have already been used widely in neurobiological studies. Especially for drug screening applications, a highly reproducible protocol with simple tissue culture steps and consistent output, is required. Here we present an engineering approach that addresses several existing shortcomings of brain organoids. By culturing brain organoids with a polycaprolactone scaffold, we were able to modify their shape into a flat morphology. Engineered flat brain organoids (efBOs) possess advantageous diffusion conditions and thus their tissue is better supplied with oxygen and nutrients, preventing the formation of a necrotic tissue core. Moreover, the efBO protocol is highly simplified and allows to customize the organoid size directly from the start. By seeding cells onto 12 by 12 mm scaffolds, the brain organoid size can be significantly increased. In addition, we were able to observe folding reminiscent of gyrification around day 20, which was self-generated by the tissue. To our knowledge, this is the first study that reports intrinsically caused gyrification of neuronal tissue in vitro. We consider our efBO protocol as a next step towards the generation of a stable and reliable human brain model for drug screening applications and spatial patterning experiments. ; European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. 722779 and was conducted within the 'Training 4 Cell Regenerative Medicine' (T4CRM) network. Furthermore, work at the CBMSO was supported by grants (to AMS) SAF-2017-83241-R, RETICS TerCel RD16/0011/0032, and ASCTN-Training (No. 813851)
A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH+ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experiments ; This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (formerly Science and Innovation; PLE2009-0101, SAF2010-17167), Comunidad Autónoma Madrid (S2011-BMD-2336), Instituto Salud Carlos III (RETICS TerCel, RD06/0010/0009) and European Union (Excell, NMP4-SL-2008-214706). This work was also supported by an institutional grant from Foundation Ramón Areces to the Center of Molecular Biology Severo Ochoa
Calcium (Ca2+) influx into mitochondria occurs through a Ca2+-selective uniporter channel, which regulates essential cellular processes in eukaryotic organisms. Previous evolutionary analyses of its pore-forming subunits MCU and EMRE, and gatekeeper MICU1, pinpointed an evolutionary paradox: the presence of MCU homologs in fungal species devoid of any other uniporter components and of mt-Ca2+ uptake. Here, we trace the mt-Ca2+ uniporter evolution across 1,156 fully-sequenced eukaryotes and show that animal and fungal MCUs represent two distinct paralogous subfamilies originating from an ancestral duplication. Accordingly, we find EMRE orthologs outside Holoza and uncover the existence of an animal-like uniporter within chytrid fungi, which enables mt-Ca2+ uptake when reconstituted in vivo in the yeast Saccharomyces cerevisiae. Our study represents the most comprehensive phylogenomic analysis of the mt-Ca2+ uptake system and demonstrates that MCU, EMRE, and MICU formed the core of the ancestral opisthokont uniporter, with major implications for comparative structural and functional studies. ; T.G. group acknowledges support from the Spanish Ministry of Economy, Industry, and Competitiveness (MEIC) for the EMBL partnership, and grants "Centro de Excelencia Severo Ochoa 2013-2017" SEV-2012-0208 and BFU2015-67107 co-founded by European Regional Development Fund (ERDF); from the CERCA Programme/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR857; and grants from the European Union's Horizon 2020 research and innovation programme under the grant agreement ERC-2016-724173. T.G. also receives support from an INB Grant (PT17/0009/0023–ISCIII-SGEFI/ERDF). F.P. group was supported by the Munich Center for Systems Neurology (SyNergy EXC 2145/ID 390857198) and ExNet-0041-Phase2-3 ("SyNergy-HMGU") through the Initiative and Network Fund of the Helmholtz Association to F.P.; The Bert L & N Kuggie Vallee Foundation (to F.P. and J.W.); the Juniorverbund in der Systemmedizin "mitOmics" (FKZ 01ZX1405B to V.G.). A.A.P. was supported by a postdoctoral research fellowship from EMBO (118-2017) while writing this article. A.C.S. was partially supported by the Aging and Metabolic Programming project (AMPro).
Human brain tissue models such as cerebral organoids are essential tools for developmental and biomedical research. Current methods to generate cerebral organoids often utilize Matrigel as an external scaffold to provide structure and biologically relevant signals. Matrigel however is a nonspecific hydrogel of mouse tumor origin and does not represent the complexity of the brain protein environment. In this study, we investigated the application of a decellularized adult porcine brain extracellular matrix (B-ECM) which could be processed into a hydrogel (B-ECM hydrogel) to be used as a scaffold for human embryonic stem cell (hESC)-derived brain organoids. We decellularized pig brains with a novel detergent- and enzyme-based method and analyzed the biomaterial properties, including protein composition and content, DNA content, mechanical characteristics, surface structure, and antigen presence. Then, we compared the growth of human brain organoid models with the B-ECM hydrogel or Matrigel controls in vitro. We found that the native brain source material was successfully decellularized with little remaining DNA content, while Mass Spectrometry (MS) showed the loss of several brain-specific proteins, while mainly different collagen types remained in the B-ECM. Rheological results revealed stable hydrogel formation, starting from B-ECM hydrogel concentrations of 5 mg/mL. hESCs cultured in B-ECM hydrogels showed gene expression and differentiation outcomes similar to those grown in Matrigel. These results indicate that B-ECM hydrogels can be used as an alternative scaffold for human cerebral organoid formation, and may be further optimized for improved organoid growth by further improving protein retention other than collagen after decellularization. ; European Union's Horizon 2020 Research and Innovation Program under the Marie Sklodowska-Curie Grant (No. 722779) awarded to RS; AMS group (CBMSO-UAM) grant (No. MINECO SAF-2017-83241-R) awarded tp AMS, ISC-III RETICS TerCel (RD16/0011/0032) and ASCTN597 Training (No. 813851) awarded to AMS, and NIH grants ((P41EB027062 and R01NS092847)
Background Human adult adipose-derived stem cells (hADSCs) have become the most promising cell source for regenerative medicine. However the prolonged ex vivo expansion periods required to obtain the necessary therapeutic dose promotes progressive senescence, with the concomitant reduction of their therapeutic potential. Aim and scope A better understanding of the determinants of hADSC senescence is needed to improve biosafety while preserving therapeutic efficiency. Here, we investigated the association between deregulation of the imprinted DLK1-DIO3 region and replicative senescence in hADSC cultures. Methods We compared hADSC cultures at short (P S ) and prolonged (P L ) passages, both in standard and low [O 2 ] (21 and 3%, respectively), in relation to replicative senescence. hADSCs were evaluated for expression alterations in the DLK1-DIO3 region on chromosome 14q32, and particularly in its main miRNA cluster. Results Comparison of hADSCs cultured at P L or P S surprisingly showed a quite significant fraction (69%) of upregulated miRNAs in P L cultures mapping to the imprinted 14q32 locus, the largest miRNA cluster described in the genome. In agreement, expression of the lncRNA MEG3 (Maternally Expressed 3; Meg3/Gtl2), cultured at 21 and 3% [O 2 ], was also significantly higher in P L than in P S passages. During hADSC replicative senescence the AcK16H4 activating mark was found to be significantly associated with the deregulation of the entire DLK1-DIO3 locus, with a secondary regulatory role for the methylation of DMR regions. Conclusion A direct relationship between DLK1-DIO3 deregulation and replicative senescence of hADSCs is reported, involving upregulation of a very significant fraction of its largest miRNA cluster (14q32.31), paralleled by the progressive overexpression of the lncRNA MEG3, which plays a central role in the regulation of Dlk1/Dio3 activation status in mice. ; This work was supported by grants to AB from the Spanish Ministry of Economy, Industry (SAF2015-70882-R; AEI/FEDER, UE), Comunidad Autónoma de Madrid (S2010/BMD-2420), Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0018) and the European Commission (FP7-HEALTH- 2009/CARE-MI). AMS was supported by grants from the MINECO (SAF2010–17167) and Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0013), and MFF and RGU by grants from the Plan Nacional de I+D+I 2013-2016/FEDER (PI15/ 00892), the Asturias Regional Government (GRUPIN14-052), the IUOPA (Obra Social Cajastur) and the Fundación Científica de la AECC. SGL held a predoctoral fellowship from the Spanish Programa de Formación del Profesorado Universitario
This work was supported by grants to AB from the Spanish Ministry of Economy, Industry (SAF2015-70882-R; AEI/FEDER, UE), Comunidad Auto´noma de Madrid (S2010/BMD-2420), Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0018) and the European Commission (FP7-HEALTH2009/CARE-MI). AMS was supported by grants from the MINECO (SAF2010–17167) and Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0013), and MFF and RGU by grants from the Plan Nacional de I+D+I 2013-2016/FEDER (PI15/ 00892), the Asturias Regional Government (GRUPIN14-052), the IUOPA (Obra Social Cajastur) and the Fundacio´n Cientı´fica de la AECC. SGL held a predoctoral fellowship from the Spanish Programa de Formacio´n del Profesorado Universitario.
Compartmentalized microfluidic platforms are an invaluable tool in neuroscience research. However, harnessing the full potential of this technology remains hindered by the lack of a simple fabrication approach for the creation of intricate device architectures with high-aspect ratio features. Here, a hybrid additive manufacturing approach is presented for the fabrication of open-well compartmentalized neural devices that provides larger freedom of device design, removes the need for manual postprocessing, and allows an increase in the biocompatibility of the system. Suitability of the method for multimaterial integration allows to tailor the device architecture for the long-term maintenance of healthy human stem-cell derived neurons and astrocytes, spanning at least 40 days. Leveraging fast-prototyping capabilities at both micro and macroscale, a proof-of-principle human in vitro model of the nigrostriatal pathway is created. By presenting a route for novel materials and unique architectures in microfluidic systems, the method provides new possibilities in biological research beyond neuroscience applications. ; European Union Horizon 2020 Programme (H2020-MSCA-ITN-2016) under the Marie Skłodowska-Curie Innovative Training Network and Grant AgreementNo.722779. Lund University Bioimaging Centre (LBIC), Lund University, is gratefully acknowledged for providing experimental resources for SEM
