Laminin-Inspired Cell-Instructive Microenvironments for Neural Stem Cells

Laminin is a heterotrimeric glycoprotein with a key role in the formation and maintenance of the basement membrane architecture and properties, as well as on the modulation of several biological functions, including cell adhesion, migration, differentiation and matrix-mediated signaling. In the central nervous system (CNS), laminin is differentially expressed during development and homeostasis, with an impact on the modulation of cell function and fate. Within neurogenic niches, laminin is one of the most important and well described extracellular matrix (ECM) proteins. Specifically, efforts have been made to understand laminin assembly, domain architecture, and interaction of its different bioactive domains with cell surface receptors, soluble signaling molecules, and ECM proteins, to gain insight into the role of this ECM protein and its receptors on the modulation of neurogenesis, both in homeostasis and during repair. This is also expected to provide a rational basis for the design of biomaterial-based matrices mirroring the biological properties of the basement membrane of neural stem cell niches, for application in neural tissue repair and cell transplantation. This review provides a general overview of laminin structure and domain architecture, as well as the main biological functions mediated by this heterotrimeric glycoprotein. The expression and distribution of laminin in the CNS and, more specifically, its role within adult neural stem cell niches is summarized. Additionally, a detailed overview on the use of full-length laminin and laminin derived peptide/recombinant laminin fragments for the development of hydrogels for mimicking the neurogenic niche microenvironment is given. Finally, the main challenges associated with the development of laminin-inspired hydrogels and the hurdles to overcome for these to progress from bench to bedside are discussed.This work was funded by projects NORTE-01-0145-FEDER-000008 and NORTE-01-0145-FEDER000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and FEDER (Fundo Europeu de Desenvolvimento Regional) funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, by Portuguese funds through FCT/MCTES in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), and by Santa Casa da Misericordia de Lisboa through project COMBINE (Prémio Neurociências Melo e Castro 1068-2015). D.B. was supported by FCT PhD Programs (PD/BD/105953/2014) and Programa Operacional Potencial Humano (POCH), in the scope of the BiotechHealth Program (Doctoral Program on Cellular and Molecular Biotechnology Applied to Health Sciences), Programa FLAD Healthcare 2020, and the project PARES (Prémio Albino Aroso)

Ontogeny, phylogeny and functional morphology of the hominoid shoulder girdle

The shoulder is of particular relevance for resolving issues of locomotor ancestry since, as a group, living hominoids can be defined by the set of functional similarities that they share at this anatomical area (such as a scapula located on the back of the ribcage, and a shoulder joint adapted to allow extensive abduction). However, there is ongoing debate over which selective pressures are responsible for these shared morphologies. The current study addresses the question of whether the similarities in this anatomical structure in hominoids are a product of common ancestry (homology) or rather the product of parallelism (homoplasy) from an ontogenetic and phylogenetic perspectives. To this end, 30 measurements were collected on the clavicle, scapula and humerus of six hominoid species (Homo sapiens, Pan troglodytes, Pan paniscus, Gorilla gorilla, Pongo pygmaeus and Hylobates lar) and one macaque species (Macaca fascicularis); information on the dental development of each individual specimen was collected for the purpose of creating an ontogenetic sample for each species; all measurements were collected on surface scans of individual bones and analysed in a 3D environment (Geomagic Suite 12.1 and Amira 3.1), and all statistical analyses (ontogenetic, phylogenetic as well as within- and between-species differences) were conducted using R version 2.12.2 (R Core Team 2011). Overall my results provide a more detailed understanding of ontogenetic change in shoulder morphology across hominoid species, and demonstrate (1) a relative lack of phenotypic plasticity in other key traits (such as the proximal curvature of the clavicle and glenoid-axillary angle of the scapula), and (2) high levels of plasticity in key diagnostic traits of hominoid shoulder morphology in humeral torsion, the distal curvature of the clavicle, and the orientation of the scapular spine and glenoid fossa (all correlated with each other). However these seem to operate within phylogenetic constraints and to be modulated by the underlying anatomy of the thorax and shoulder girdle. Overall my results support the notion of an arboreal origin to the ape lineages and parallel evolution of quadrupedalism in the great apes

Biomimetic synthetic self-assembled hydrogels for cell transplantation

The development of three-dimensional matrices capable of recapitulating the main features of native extracellular matrix and contribute for the establishment of a favorable microenvironment for cell behavior and fate is expected to circumvent some of the main limitations of cell-based therapies. In this context, self-assembly has emerged as a promising strategy to engineer cell-compatible hydrogels. A wide number of synthetically-derived biopolymers, such as proteins, peptides and DNA/RNA, with intrinsic ability to self-assemble into well-defined nanofibrous structures, are being explored. The resulting hydrogels, in addition to closely resembling the architecture of native cellular microenvironments, present a versatile and dynamic behavior that allows them to be designed to undergo sol-to-gel transition in response to exogenous stimulus. This review presents an overview on the state-of-the-art of the different strategies being explored for the development of injectable synthetic self-assembled hydrogels for cell transplantation and/or recruitment of endogenous cells, with an emphasis on their biological performance, both in vitro and in vivo. Systems based on peptides are the most widely explored and have already generated promising results in pre-clinical in vivo studies involving different repair/regenerative scenarios, including cartilage, bone, nerve and heart. On the other hand, systems based on DNA and hybrid hydrogels are now emerging for application in the biomedical field with high potential. Finally, the main challenges hampering the translation of these systems to the clinic and the issues that need to be addressed for these to progress from bench-to-bedside are discussed.The authors would like to acknowledge the FEDER funds through the Programa Operacional Factores de Competitividade – COMPETE and the Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia (HMSPICT/0020/2010, PTDC/SAU-BMA/118869/2010 and PEst/SAU/LA0002/2013) that supported this work. D Barros is supported by FCT (PD/BD/105953/2014) and I.F. Amaral by QREN through program ON.2, in the framework of "Project on Biomedical Engineering for Regenerative Therapies and Cancer” (NORTE-07-0124-FEDER-000005)

Engineering Hydrogels with Affinity-Bound Laminin as 3D Neural Stem Cell Culture Systems

Laminin incorporation into biological or synthetic hydrogels has been explored to recapitulate the dynamic nature and biological complexity of neural stem cell (NSC) niches. However, the strategies currently explored for laminin immobilization within three-dimensional (3D) matrices do not address a critical aspect influencing cell-matrix interactions, which is the control over laminin conformation and orientation upon immobilization. This is a key feature for the preservation of the protein bioactivity. In this work, we explored an affinity-based approach to mediate the site-selective immobilization of laminin into a degradable synthetic hydrogel. Specifically, a four-arm maleimide terminated poly(ethylene glycol) (PEG-4MAL) macromer was functionalized with a mono-PEGylated recombinant human N-terminal agrin (NtA) domain, to promote high affinity binding of laminin. Different NtA concentrations (10, 50 and 100 M) were used to investigate the impact of NtA density on laminin incorporation, hydrogel biophysical properties, and biological outcome. Laminin was efficiently incorporated for all the conditions tested (laminin incorporation > 95%), and the developed hydrogels revealed mechanical properties (average storage modulus (G’) ranging from 187 to 256 Pa) within the values preferred for NSC proliferation and neurite branching and extension. Affinity-bound laminin PEG-4MAL hydrogels better preserve laminin bioactivity, compared to unmodified hydrogels and hydrogels containing physically entrapped laminin, being this effect dependent on NtA concentration. This was evidenced by the 10 M NtA-functionalized PEG-4MAL gels incorporating laminin that support enhanced human NSC proliferation and neurite extension, compared to the latter. Overall, this work highlights the potential of the proposed engineered matrices to be used as defined 3D platforms for the establishment of artificial NSC niches and as extracellular matrix-mimetic microenvironments to support human NSC transplantation.Confocal microscopy was conducted at the Bioimaging i3S Scientific Platform, member of the PPBI (PPBI-POCI-01-0145-FEDER-022122), with the assistance of Maria Lázaro. This work was funded by projects NORTE-01-0145-FEDER-000008 and NORTE-01-0145-FEDER000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020; by Portuguese funds through FCT/MCTES in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER007274), and by Santa Casa da Misericordia de Lisboa through project COMBINE (Prémio Neurociências Melo e Castro 1068-2015). DB was supported by FCT PhD Programs (PD/BD/105953/2014) and Programa Operacional Potencial Humano (POCH), in the scope of the BiotechHealth Program (Doctoral Program on Cellular and Molecular Biotechnology Applied to Health Sciences), Programa FLAD Healthcare 2020 and the project PARES (Prémio Albino Aroso). Eduardo Conde-Sousa was supported by a post-doctoral grant of the project PPBI-POCI-01-0145-FEDER-022122, in the scope of FCT National Roadmap of Research Infrastructures. AJG acknowledges support from the National Institutes of Health (R01 AR062368)

Impact of transient groundwater storage on the discharge of Himalayan rivers

International audienceIn the course of the transfer of precipitation into rivers, water is temporarily stored in reservoirs with different residence times such as soils, groundwater, snow and glaciers. In the central Himalaya, the water budget is thought to be primarily controlled by monsoon rainfall, snow and glacier melt, and secondarily by evapotranspiration. An additional contribution from deep groundwater has been deduced from the chemistry of Himalayan rivers, but its importance in the annual water budget remains to be evaluated. Here we analyse records of daily precipitation and discharge within twelve catchments in Nepal over about 30 years. We observe annual hysteresis loops--that is, a time lag between precipitation and discharge--in both glaciated and unglaciated catchments and independent of the geological setting. We infer that water is stored temporarily in a reservoir with characteristic response time of about 45 days, suggesting a diffusivity typical of fractured basement aquifers. We estimate this transient storage capacity at about 28km3 for the three main Nepal catchments; snow and glacier melt contribute around 14km3yr-1, about 10% of the annual river discharge. We conclude that groundwater storage in a fractured basement influences significantly the Himalayan river discharge cycle

Analytical tools used to distinguish chemical profiles of plants widely consumed as infusions and dietary supplements: artichoke, milk thistle and borututu

Artichoke, borututu and milk thistle are three medicinal plants widely consumed as infusions or included in dietary supplements (e.g., pills and syrups). Despite their high consumption, studies on nutritional value and primary metabolites are scarce, being only reported the composition in secondary metabolites such as phenolic compounds. Therefore, their nutritional value was assessed and analytical tools (liquid and gas chromatography coupled to different detectors) were used to distinguish the chemical profiles namely in hydrophilic (sugars and organic acids) and lipophilic (fatty acids and tocopherols) compounds. Chromatographic techniques are important analytical tools used in the identification and quantification of several molecules, also being a standard requirement to distinguish different profiles. Borututu gave the highest energetic value with the highest content of carbohydrates and fat, sucrose and total sugars, shikimic and citric acids, α-, β-, δ- and total tocopherols. Artichoke had the highest ash and protein contents, oxalic acid, SFA (mainly palmitic acid acid), and γ-tocopherol, as also the best n6/n3 ratio. Milk thistle showed the highest levels of fructose and glucose, quinic acid and total organic acids, PUFA, mainly linoleic acid, and the best PUFA/SFA ratio. The hydrophilic compounds identified in the studied plants, mostly sugars, are the responsible for the energetic contribution of their widely consumed infusions. Otherwise, the bioactivity of lipophilic compounds namely, unsaturated fatty acids and tocopherols, is lost in those preparations but can be recovered in dietary supplements based on the plants. As far as we know this is the first report on detailed composition of molecules with nutritional features.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support to the research centre CIMO (PEst-OE/AGR/UI0690/2011)

Fibrin functionalization with synthetic adhesive ligands interacting with a6ß1 integrin receptor enhance neurite outgrowth of embryonic stem cell-derived neural stem/progenitors

To enhance fibrin hydrogel affinity towards pluripotent stem cell-derived neural stem/progenitor cells (NSPCs) and its capacity to support NSPC migration and neurite extension, we explored the tethering of synthetic peptides engaging integrin a6ß1, a cell receptor enriched in NSPCs. Six a6ß1 integrin ligands were tested for their ability to support integrin a6ß1-mediated adhesion of embryonic stem cell-derived NSPCs (ES-NSPs) and sustain ES-NSPC viability, migration, and neuronal differentiation. Due to their better performance, peptides T1, HYD1, and A5G81 were immobilized into fibrin and functionalized gels characterized in terms of peptide binding efficiency, structure and viscoelastic properties. Tethering of T1 or HYD1 successfully enhanced cell outgrowth from ES-NSPC neurospheres (up to 2.4-fold increase), which exhibited a biphasic response to peptide concentration. Inhibition assays evidenced the involvement of a6ß1 and a3ß1 integrins in mediating radial outgrowth on T1-/HYD1-functionalized gels. Fibrin functionalization also promoted neurite extension of single ES-NSPCs in fibrin, without affecting cell proliferation and neuronal differentiation. Finally, HYD1-functionalized gels were found to provide a permissive environment for axonal regeneration, leading up to a 2.0-fold increase in neurite extension from rat dorsal root ganglia explants as compared to unmodified fibrin, and to significant improved locomotor function after spinal cord injury (complete transection), along with a trend toward a higher area positive for growth associated protein 43 (marker for axonal growth cone formation). Our results suggest that conjugation of a6ß1 integrin-binding motifs is of interest to increase the biofunctionality of hydrogels used in 3D platforms for ES-NSPC culture and potentially, in matrix-assisted ES-NSPC transplantation. Statement of Significance Impact statement: The transplantation of NSPCs derived from pluripotent stem cells holds much promise for the treatment of central nervous system disorders. Moreover, the combinatorial use of biodegradable hydrogels with NSPCs was shown to contribute to the establishment of a more permissive environment for survival and integration of transplanted cells. In this study, fibrin hydrogels functionalized with a synthetic peptide engaging integrin a6ß1 (HYD1) were shown to promote neurite extension of ES-NSPCs, which is fundamental for the formation of functional neuronal relay circuits after NSPC transplantation. Notably, HYD1-functionalized fibrin per se led to enhanced axonal growth ex vivo and to an improvement in locomotor function after implantation in a rat model of spinal cord injury. Conjugation of a6ß1 integrin-binding motifs may therefore be of interest to confer bioactivity to NSPC hydrogel vehicles.The authors would like to acknowledge Prof. Domingos Henrique (Instituto de Medicina Molecular, Lisbon) for providing theES 46C cell line. This work was supported by FEDER funds throughthe Programa Operacional Factores de Competitividade – COMPETE(FCOMP-01-0124-FEDER-021125) and by National Funds throughFCT – Fundação para a Ciência e a Tecnologia (PTDC/SAU-BMA/118869/2010). A.R.B. and D.B. are supported by FCT (SFRH/BD/86200/2012; PD/BD/105953/2014)

Synthetic matrix enhances transplanted satellite cell engraftment in dystrophic and aged skeletal muscle with comorbid trauma

Muscle satellite cells (MuSCs) play a central role in muscle regeneration, but their quantity and function decline with comorbidity of trauma, aging, and muscle diseases. Although transplantation of MuSCs in traumatically injured muscle in the comorbid context of aging or pathology is a strategy to boost muscle regeneration, an effective cell delivery strategy in these contexts has not been developed. We engineered a synthetic hydrogel-based matrix with optimal mechanical, cell-adhesive, and protease-degradable properties that promotes MuSC survival, proliferation, and differentiation. Furthermore, we establish a biomaterial-mediated cell delivery strategy for treating muscle trauma, where intramuscular injections may not be applicable. Delivery of MuSCs in the engineered matrix significantly improved in vivo cell survival, proliferation, and engraftment in nonirradiated and immunocompetent muscles of aged and dystrophic mice compared to collagen gels and cell-only controls. This platform may be suitable for treating craniofacial and limb muscle trauma, as well as postoperative wounds of elderly and dystrophic patients.Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH under award numbers R21AR072287 (to Y.C.J.) and R01AR062368 (to A.J.G.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This work was also funded by the Parker H. Petit Institute for Bioengineering and Bioscience Seed Grant Program (to A.J.G. and Y.C.J.)

Synthetic matrix enhances transplanted satellite cell engraftment in dystrophic and aged skeletal muscle with comorbid trauma

Muscle satellite cells (MuSCs) play a central role in muscle regeneration, but their quantity and function decline with comorbidity of trauma, aging, and muscle diseases. Although transplantation of MuSCs in traumatically injured muscle in the comorbid context of aging or pathology is a strategy to boost muscle regeneration, an effective cell delivery strategy in these contexts has not been developed. We engineered a synthetic hydrogel-based matrix with optimal mechanical, cell-adhesive, and protease-degradable properties that promotes MuSC survival, proliferation, and differentiation. Furthermore, we establish a biomaterial-mediated cell delivery strategy for treating muscle trauma, where intramuscular injections may not be applicable. Delivery of MuSCs in the engineered matrix significantly improved in vivo cell survival, proliferation, and engraftment in nonirradiated and immunocompetent muscles of aged and dystrophic mice compared to collagen gels and cell-only controls. This platform may be suitable for treating craniofacial and limb muscle trauma, as well as postoperative wounds of elderly and dystrophic patients.Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH under award numbers R21AR072287 (to Y.C.J.) and R01AR062368 (to A.J.G.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This work was also funded by the Parker H. Petit Institute for Bioengineering and Bioscience Seed Grant Program (to A.J.G. and Y.C.J.)

Sensitivity of the Himalayan orography representation in simulation of winter precipitation using Regional Climate Model (RegCM) nested in a GCM

This document is the Accepted Manuscript version of the following article: Tiwari, P.R., Kar, S.C., Mohanty, U.C., Climate Dynamics (2017). The final publication is available at Springer via https://link.springer.com/article/10.1007%2Fs00382-017-3567-3. The Accepted Manuscript is under embargo. Embargo end date: 24 February 2018.The role of the Himalayan orography representationin a Regional Climate Model (RegCM4) nested inNCMRWF global spectral model is examined in simulatingthe winter circulation and associated precipitation over theNorthwest India (NWI; 23°–37.5°N and 69°–85°E) region.For this purpose, nine different set of orography representationsfor nine distinct precipitation years (three years eachfor wet, normal and dry) have been considered by increasing(decreasing) 5, 10, 15, and 20% from the mean height(CNTRL) of the Himalaya in RegCM4 model. Validationwith various observations revealed a good improvementin reproducing the precipitation intensity and distributionwith increased model height compared to the resultsobtained from CNTRL and reduced orography experiments.Further it has been found that, increase in heightby 10% (P10) increases seasonal precipitation about 20%,while decrease in height by 10% (M10) results around 28%reduction in seasonal precipitation as compared to CNTRLexperiment over NWI region. This improvement in precipitationsimulation comes due to better representation ofvertical pressure velocity and moisture transport as thesefactors play an important role in wintertime precipitationprocesses over NWI region. Furthermore, a comparison of model-simulated precipitation with observed precipitationat 17 station locations has been also carried out. Overall,the results suggest that when the orographic increment of10% (P10) is applied on RegCM4 model, it has better skillin simulating the precipitation over the NWI region andthis model is a useful tool for further regional downscalingstudies.Peer reviewe