RESUMEN
BACKGROUND: Glioblastomas stem-like cells (GSCs) by invading the brain parenchyma, remains after resection and radiotherapy and the tumoral microenvironment become stiffer. GSC invasion is reported as stiffness sensitive and associated with altered N-glycosylation pattern. Glycocalyx thickness modulates integrins mechanosensing, but details remain elusive and glycosylation enzymes involved are unknown. Here, we studied the association between matrix stiffness modulation, GSC migration and MGAT5 induced N-glycosylation in fibrillar 3D context. METHOD: To mimic the extracellular matrix fibrillar microenvironments, we designed 3D-ex-polyacrylonitrile nanofibers scaffolds (NFS) with adjustable stiffnesses by loading multiwall carbon nanotubes (MWCNT). GSCs neurosphere were plated on NFSs, allowing GSCs migration and MGAT5 was deleted using CRISPR-Cas9. RESULTS: We found that migration of GSCs was maximum at 166 kPa. Migration rate was correlated with cell shape, expression and maturation of focal adhesion (FA), Epithelial to Mesenchymal Transition (EMT) proteins and (ß1,6) branched N-glycan binding, galectin-3. Mutation of MGAT5 in GSC inhibited N-glycans (ß1-6) branching, suppressed the stiffness dependence of migration on 166 kPa NFS as well as the associated FA and EMT protein expression. CONCLUSION: MGAT5 catalysing multibranched N-glycans is a critical regulators of stiffness induced invasion and GSCs mechanotransduction, underpinning MGAT5 as a serious target to treat cancer.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Células Madre Neoplásicas/metabolismo , Neoplasias Encefálicas/patología , Movimiento Celular/fisiología , Glioblastoma/patología , Humanos , Células Madre Neoplásicas/patología , FenotipoRESUMEN
Herein, we report the bioactivity of monodispersed nanosized reduced graphene oxide (RGO) enfolded gold nanoparticles (AuNPs) engineered polycaprolactone (PCL) based electrospun composite scaffolds. The 2D patterns of PCL based nanofibers prepared by the homogenous distribution of RGO-AuNPs exhibited unique topological and biological features such as mechanical properties, porous structure, large surface area, high electrical conductivity, biodegradability, and resemble the natural extracellular matrix (ECM) that supports the adhesion, growth, proliferation, and differentiation of stem cells. The prepared composite nanofibers based scaffolds containing RGO-AuNPs accelerated neuronal cell functions and confirmed that the optimized concentration showed cytocompatibility to PC12 and S42 cells. The 0.0005 wt% loading of RGO-AuNPs on PCL has a huge impact on neurite growth which leads to an almost one-fold increase in neurite length growth. The present study provides a new strategic design of highly efficient scaffolds that have a significant direct impact on cell activity and could be a potential bioimplant for peripheral nerve repair.
Asunto(s)
Nanopartículas del Metal , Nanofibras , Proliferación Celular , Oro , Regeneración Nerviosa , Nervios Periféricos , Poliésteres , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Damaged neural tissue is regenerated by neural stem cells (NSCs), which represent a rare and difficult-to-culture cell population. Therefore, alternative sources of stem cells are being tested to replace a shortage of NSCs. Here we show that mouse adipose tissue-derived mesenchymal stem cells (MSCs) can be effectively differentiated into cells expressing neuronal cell markers. The differentiation protocol, simulating the inflammatory site of neural injury, involved brain tissue extract, fibroblast growth factor, epidermal growth factor, supernatant from activated splenocytes and electrical stimulation under physiological conditions. MSCs differentiated using this protocol displayed neuronal cell morphology and expressed genes for neuronal cell markers, such as neurofilament light (Nf-L), medium (Nf-M) and heavy (Nf-H) polypeptides, synaptophysin (SYP), neural cell adhesion molecule (NCAM), glutamic acid decarboxylase (GAD), neuron-specific nuclear protein (NeuN), ßIII-tubulin (Tubb3) and microtubule-associated protein 2 (Mtap2), which are absent (Nf-L, Nf-H, SYP, GAD, NeuN and Mtap2) or only slightly expressed (NCAM, Tubb3 and Nf-M) in undifferentiated cells. The differentiation was further enhanced when the cells were cultured on nanofibre scaffolds. The neural differentiation of MSCs, which was detected at the level of gene expression, was confirmed by positive immunostaining for Nf-L protein. The results thus show that the simulation of conditions in an injured neural tissue and inflammatory environment, supplemented with electrical stimulation under physiological conditions and cultivation of cells on a three-dimensional (3D) nanofibre scaffold, form an effective protocol for the differentiation of MSCs into cells with neuronal markers. Copyright © 2015 John Wiley & Sons, Ltd.
Asunto(s)
Antígenos de Diferenciación/biosíntesis , Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Animales , Inflamación/metabolismo , Inflamación/patología , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos BALB C , Tejido Nervioso/patología , Células-Madre Neurales/patologíaRESUMEN
We optimised the working parameters of an innovative air spinning device to produce nanofibrous polymer scaffolds for tissue engineering applications. Scanning electron microscopy was performed on the fibre scaffolds which were then used to identify various scaffold morphologies based on the ratio of surface occupied by the polymer fibres on that covered by the entire polymer scaffold assembly. Scaffolds were then produced with the spinning experimental parameters, resulting in 90% of fibres in the overall polymer construct, and were subsequently used to perform a multiple linear regression analysis to highlight the relationship between nanofibre diameter and the air spinning parameters. Polymer solution concentration was deemed as the most significant parameter to control fibre diameter during the spinning process, despite interactions between experimental parameters. Based on these findings, viscosity measurements were performed to clarify the effect of the polymer solution property on scaffold morphology.