Centro per le Nano-Scienze della Vita

Patient-derived induced pluripotent stem cells (iPSCs) provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS) and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUS(R514S) and FUS(R521C) patient fibroblasts, whereas in the case of the severe FUS(P525L) mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis.

Acellular soft hydrogels are not ideal for hard tissue engineering given their poor mechanical stability, however, in combination with cellular components offer significant promise for tissue regeneration. Indeed, nanocomposite bioinks provide an attractive platform to deliver human bone marrow stromal cells (HBMSCs) in three dimensions producing cell-laden constructs that aim to facilitate bone repair and functionality. Here we present the in vitro, ex vivo and in vivo investigation of bioprinted HBMSCs encapsulated in a nanoclay-based bioink to produce viable and functional three-dimensional constructs. HBMSC-laden constructs remained viable over 21 d in vitro and immediately functional when conditioned with osteogenic media. 3D scaffolds seeded with human umbilical vein endothelial cells (HUVECs) and loaded with vascular endothelial growth factor (VEGF) implanted ex vivo into a chick chorioallantoic membrane (CAM) model showed integration and vascularisation after 7 d of incubation. In a pre-clinical in vivo application of a nanoclay-based bioink to regenerate skeletal tissue, we demonstrated bone morphogenetic protein-2 (BMP-2) absorbed scaffolds produced extensive mineralisation after 4 weeks (p < 0.0001) compared to the drug-free and alginate controls. In addition, HBMSC-laden 3D printed scaffolds were found to significantly (p < 0.0001) support bone tissue formation in vivo compared to acellular and cast scaffolds. These studies illustrate the potential of nanoclay-based bioink, to produce viable and functional constructs for clinically relevant skeletal tissue regeneration.

BACKGROUND: The brain barriers establish compartments in the central nervous system (CNS) that significantly differ in their communication with the peripheral immune system. In this function they strictly control T-cell entry into the CNS. T cells can reach the CNS by either crossing the endothelial blood-brain barrier (BBB) or the epithelial blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP). OBJECTIVE: T-cell subsets across the BBB versus the BCSFB. METHODS: T helper cell subsets (Th1, Th1*, Th2, Th17) across the BBB and BCSFB under inflammatory and non-inflammatory conditions in vitro. RESULTS: While under non-inflammatory conditions Th1* and Th1 cells preferentially crossed the BBB, under inflammatory conditions the migration rate of all Th subsets across the BBB was comparable. The migration of all Th subsets across the BCSFB from the same donor was 10- to 20-fold lower when compared to their migration across the BBB. Interestingly, Th17 cells preferentially crossed the BCSFB under both, non-inflamed and inflamed conditions. Barrier-crossing experienced Th cells sorted from CSF of MS patients showed migratory characteristics indistinguishable from those of circulating Th cells of healthy donors. All Th cell subsets could additionally cross the BCSFB from the CSF to ChP stroma side. T-cell migration across the BCSFB involved epithelial ICAM-1 irrespective of the direction of migration. CONCLUSIONS: Our observations underscore that different Th subsets may use different anatomical routes to enter the CNS during immune surveillance versus neuroinflammation with the BCSFB establishing a tighter barrier for T-cell entry into the CNS compared to the BBB. In addition, CNS-entry experienced Th cell subsets isolated from the CSF of MS patients do not show an increased ability to cross the brain barriers when compared to circulating Th cell subsets from healthy donors underscoring the active role of the brain barriers in controlling T-cell entry into the CNS. Also we identify ICAM-1 to mediate T cell migration across the BCSFB.

Hepatitis B virus (HBV) contributes to hepatocellular carcinoma (HCC) development through direct and indirect mechanisms. HBV-DNA integration into the host genome occurs at early steps of clonal tumor expansion and induces both genomic instability and direct insertional mutagenesis of diverse cancer-related genes. Prolonged expression of the viral regulatory protein HBx and the large envelope protein deregulate the cellular transcription program and proliferation control and sensitize liver cells to carcinogenic factors. Epigenetic changes targeting the expression of tumor suppressor genes occur early in the development of HCC. A major role is played by HBx that is recruited on cellular chromatin and modulates chromatin dynamics at specific gene loci. Compared with tumors associated with other risk factors, HBV-related tumors have a higher rate of chromosomal alterations and p53 inactivation by mutations, overexpress fetal liver/hepatic progenitor cells genes, and show a specific activation of the AKT pathway. The wnt/β-catenin pathway is also often activated, but HBV-related tumors display a low rate of activating β-catenin mutations. All available evidence strongly supports the notion that chronic HBV infection triggers both common and etiology-specific oncogenic pathways, thus playing a direct role beyond stimulation of host immune responses and chronic necroinflammatory liver disease.

The FUS gene has been linked to amyotrophic lateral sclerosis (ALS). FUS is a ubiquitous RNA-binding protein, and the mechanisms leading to selective motoneuron loss downstream of ALS-linked mutations are largely unknown. We report the transcriptome analysis of human purified motoneurons, obtained from FUS wild-type or mutant isogenic induced pluripotent stem cells (iPSCs). Gene ontology analysis of differentially expressed genes identified significant enrichment of pathways previously associated to sporadic ALS and other neurological diseases. Several microRNAs (miRNAs) were also deregulated in FUS mutant motoneurons, including miR-375, involved in motoneuron survival. We report that relevant targets of miR-375, including the neural RNA-binding protein ELAVL4 and apoptotic factors, are aberrantly increased in FUS mutant motoneurons. Characterization of transcriptome changes in the cell type primarily affected by the disease contributes to the definition of the pathogenic mechanisms of FUS-linked ALS.

In liver cirrhosis (LC), impaired intestinal functions lead to dysbiosis and possible bacterial translocation (BT). Bacteria or their byproducts within the bloodstream can thus play a role in systemic inflammation and hepatic encephalopathy (HE). We combined 16S sequencing, NMR metabolomics and network analysis to describe the interrelationships of members of the microbiota in LC biopsies, faeces, peripheral/portal blood and faecal metabolites with clinical parameters. LC faeces and biopsies showed marked dysbiosis with a heightened proportion of Enterobacteriaceae. Our approach showed impaired faecal bacterial metabolism of short-chain fatty acids (SCFAs) and carbon/methane sources in LC, along with an enhanced stress-related response. Sixteen species, mainly belonging to the Proteobacteria phylum, were shared between LC peripheral and portal blood and were functionally linked to iron metabolism. Faecal Enterobacteriaceae and trimethylamine were positively correlated with blood proinflammatory cytokines, while Ruminococcaceae and SCFAs played a protective role. Within the peripheral blood and faeces, certain species (Stenotrophomonas pavanii, Methylobacterium extorquens) and metabolites (methanol, threonine) were positively related to HE. Cirrhotic patients thus harbour a 'functional dysbiosis' in the faeces and peripheral/portal blood, with specific keystone species and metabolites related to clinical markers of systemic inflammation and HE.

The HBV covalently closed circular DNA (cccDNA) is organized as a mini-chromosome in the nuclei of infected hepatocytes by histone and non-histone proteins. Transcription from the cccDNA of the RNA replicative intermediate termed pre-genome (pgRNA), is the critical step for genome amplification and ultimately determines the rate of HBV replication. Multiple evidences suggest that cccDNA epigenetic modifications, such as histone modifications and DNA methylation, participate in regulating the transcriptional activity of the HBV cccDNA. Inflammatory cytokines (TNFα, LTβ) and the pleiotropic cytokine interleukin-6 (IL6) inhibit hepatitis B virus (HBV) replication and transcription. Here we show, in HepG2 cells transfected with linear HBV monomers and HBV-infected NTCP-HepG2 cells, that IL6 treatment leads to a reduction of cccDNA-bound histone acetylation paralleled by a rapid decrease in 3.5kb/pgRNA and subgenomic HBV RNAs transcription without affecting cccDNA chromatinization or cccDNA levels. IL6 repressive effect on HBV replication is mediated by a loss of HNF1α and HNF4α binding to the cccDNA and a redistribution of STAT3 binding from the cccDNA to IL6 cellular target genes.

Three-dimensional (3D) printing of biological material, or 3D bioprinting, is a rapidly expanding field with interesting applications in tissue engineering and regenerative medicine. Bioprinters use cells and biocompatible materials as an ink (bioink) to build 3D structures representative of organs and tissues, in a controlled manner and with micrometric resolution. Human embryonic (hESCs) and induced (hiPSCs) pluripotent stem cells are ideally able to provide all cell types found in the human body. A limited, but growing, number of recent reports suggest that cells derived by differentiation of hESCs and hiPSCs can be used as building blocks in bioprinted human 3D models, reproducing the cellular variety and cytoarchitecture of real tissues. In this review we will illustrate these examples, which include hepatic, cardiac, vascular, corneal and cartilage tissues, and discuss challenges and opportunities of bioprinting more demanding cell types, such as neurons, obtained from human pluripotent stem cells.

Amyotrophic lateral sclerosis (ALS) has been genetically linked to mutations in RNA-binding proteins (RBPs), including FUS. Here, we report the RNA interactome of wild-type and mutant FUS in human motor neurons (MNs). This analysis identified a number of RNA targets. Whereas the wild-type protein preferentially binds introns, the ALS mutation causes a shift toward 3' UTRs. Neural ELAV-like RBPs are among mutant FUS targets. As a result, ELAVL4 protein levels are increased in mutant MNs. ELAVL4 and mutant FUS interact and co-localize in cytoplasmic speckles with altered biomechanical properties. Upon oxidative stress, ELAVL4 and mutant FUS are engaged in stress granules. In the spinal cord of FUS ALS patients, ELAVL4 represents a neural-specific component of FUS-positive cytoplasmic aggregates, whereas in sporadic patients it co-localizes with phosphorylated TDP-43-positive inclusions. We propose that pathological mutations in FUS trigger an aberrant crosstalk with ELAVL4 with implications for ALS.

Glioblastoma multiforme (GB) is the most malignant primary brain tumor in humans, with an overall survival of approximatively 15 months. The molecular heterogeneity of GB, as well as its rapid progression, invasiveness and the occurrence of drug-resistant cancer stem cells, limits the efficacy of the current treatments. In order to develop an innovative therapeutic strategy, it is mandatory to identify and characterize new molecular players responsible for the GB malignant phenotype. In this study, the RNA-binding ubiquitin ligase MEX3A was selected from a gene expression analysis performed on publicly available datasets, to assess its biological and still-unknown activity in GB tumorigenesis. We find that MEX3A is strongly up-regulated in GB specimens, and this correlates with very low protein levels of RIG-I, a tumor suppressor involved in differentiation, apoptosis and innate immune response. We demonstrate that MEX3A binds RIG-I and induces its ubiquitylation and proteasome-dependent degradation. Further, the genetic depletion of MEX3A leads to an increase of RIG-I protein levels and results in the suppression of GB cell growth. Our findings unveil a novel molecular mechanism involved in GB tumorigenesis and suggest MEX3A and RIG-I as promising therapeutic targets in GB.

Bioprinting techniques use bioinks made of biocompatible non-living materials and cells to build 3D constructs in a controlled manner and with micrometric resolution. 3D bioprinted structures representative of several human tissues have been recently produced using cells derived by differentiation of induced pluripotent stem cells (iPSCs). Human iPSCs can be differentiated in a wide range of neurons and glia, providing an ideal tool for modeling the human nervous system. Here we report a neural construct generated by 3D bioprinting of cortical neurons and glial precursors derived from human iPSCs. We show that the extrusion-based printing process does not impair cell viability in the short and long term. Bioprinted cells can be further differentiated within the construct and properly express neuronal and astrocytic markers. Functional analysis of 3D bioprinted cells highlights an early stage of maturation and the establishment of early network activity behaviors. This work lays the basis for generating more complex and faithful 3D models of the human nervous systems by bioprinting neural cells derived from iPSCs.

FUsed in Sarcoma (FUS) is a multifunctional RNA binding protein (RBP). FUS mutations lead to its cytoplasmic mislocalization and cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we use mouse and human models with endogenous ALS-associated mutations to study the early consequences of increased cytoplasmic FUS. We show that in axons, mutant FUS condensates sequester and promote the phase separation of fragile X mental retardation protein (FMRP), another RBP associated with neurodegeneration. This leads to repression of translation in mouse and human FUS-ALS motor neurons and is corroborated in vitro, where FUS and FMRP copartition and repress translation. Last, we show that translation of FMRP-bound RNAs is reduced in vivo in FUS-ALS motor neurons. Our results unravel new pathomechanisms of FUS-ALS and identify a novel paradigm by which mutations in one RBP favor the formation of condensates sequestering other RBPs, affecting crucial biological functions, such as protein translation.

The uniform sampling of convex polytopes is an interesting computational problem with many applications in inference from linear constraints, but the performances of sampling algorithms can be affected by ill-conditioning. This is the case of inferring the feasible steady states in models of metabolic networks, since they can show heterogeneous time scales. In this work we focus on rounding procedures based on building an ellipsoid that closely matches the sampling space, that can be used to define an efficient hit-and-run (HR) Markov Chain Monte Carlo. In this way the uniformity of the sampling of the convex space of interest is rigorously guaranteed, at odds with non markovian methods. We analyze and compare three rounding methods in order to sample the feasible steady states of metabolic networks of three models of growing size up to genomic scale. The first is based on principal component analysis (PCA), the second on linear programming (LP) and finally we employ the Lovazs ellipsoid method (LEM). Our results show that a rounding procedure dramatically improves the performances of the HR in these inference problems and suggest that a combination of LEM or LP with a subsequent PCA perform the best. We finally compare the distributions of the HR with that of two heuristics based on the Artificially Centered hit-and-run (ACHR), gpSampler and optGpSampler. They show a good agreement with the results of the HR for the small network, while on genome scale models present inconsistencies.

α-Mangostin, one of the major xanthones isolated from pericarp of mangosteen ( Garcinia mangostana Linn), exhibits a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial as well as anticancer, both in in vitro and in vivo studies. In the present study, α-mangostin' anti-cancer and anti-parasitic properties were tested in vitro against three human cell lines, including squamous carcinoma (SCC-15) and glioblastoma multiforme (U-118 MG), compared to normal skin fibroblasts (BJ), and in vivo against Caenorhabditis elegans . The drug showed cytotoxic activity, manifested by decrease of cell viability, inhibition of proliferation, induction of apoptosis and reduction of adhesion at concentrations lower than 10 μM (the IC 50 values were 6.43, 9.59 and 8.97 μM for SCC-15, U-118 MG and BJ, respectively). The toxicity, causing cell membrane disruption and mitochondria impairment, was selective against squamous carcinoma with regard to normal cells. Moreover, for the first time anti-nematode activity of α-mangostin toward C. elegans was described (the LC 50 = 3.8 ± 0.5 μM), with similar effect exerted by mebendazole, a well-known anthelmintic drug.

Human pluripotent stem cells (PSCs) are widely used for in vitro disease modeling. One of the challenges in the field is represented by the ability of converting human PSCs into specific disease-relevant cell types. The nervous system is composed of a wide variety of neuronal types with selective vulnerability in neurodegenerative diseases. This is particularly relevant for motor neuron diseases, in which different motor neurons populations show a different susceptibility to degeneration. Here we developed a fast and efficient method to convert human induced Pluripotent Stem Cells into cranial motor neurons of the branchiomotor and visceral motor subtype. These populations represent the motor neuron subgroup that is primarily affected by a severe form of amyotrophic lateral sclerosis with bulbar onset and worst prognosis. This goal was achieved by stable integration of an inducible vector, based on the piggyBac transposon, allowing controlled activation of Ngn2, Isl1 and Phox2a (NIP). The NIP module effectively produced electrophysiologically active cranial motor neurons. Our method can be easily extended to PSCs carrying disease-associated mutations, thus providing a useful tool to shed light on the cellular and molecular bases of selective motor neuron vulnerability in pathological conditions.

Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disease characterized by progressive loss of motoneurons, muscle atrophy and paralysis. Recent evidence suggests that ALS should be considered as a multi-systemic disease, in which several cell types contribute to motoneuron degeneration. In this view, mutations in ALS linked genes in other neural and non-neural cell types may exert non-cell autonomous effects on motoneuron survival and function. Induced Pluripotent Stem Cells (iPSCs) have been recently derived from several patients with ALS mutations and it has been shown that they can generate motoneurons in vitro, providing a valuable tool to study ALS. However, the potential of iPSCs could be further valorized by generating other cell types that may be relevant to the pathology. In this paper, by taking advantage of a novel inducible system for MyoD expression, we show that both control iPSCs and iPSCs carrying mutations in ALS genes can generate skeletal muscle cells. We provide evidence that both control and mutant iPSC-derived myotubes are functionally active. This in vitro system will be instrumental to dissect the molecular and cellular pathways impairing the complex motoneuron microenvironment in ALS.

C. elegans neuronal system constitutes the ideal framework for studying simple, yet realistic, neuronal activity, since the whole nervous system is fully characterized with respect to the exact number of neurons and the neuronal connections. Most recent efforts are devoted to investigate and clarify the signal processing and functional connectivity, which are at the basis of sensing mechanisms, signal transmission, and motor control. In this framework, a refined modelof whole neuron dynamics constitutes a key ingredient to describe the electrophysiological processes, both at thecellular and at the network scale. In this work, we present Hodgkin-Huxley-based models of ion channels dynamics black, built on data available both from C. elegans and from other organisms, expressing homologous channels. We combine these channel models to simulate the electrical activity oftwo among the most studied neurons in C. elegans, which display prototypical dynamics of neuronal activation, the chemosensory AWCON and the motor neuron RMD. Our model properly describes the regenerative responses of the two cells. We analyze in detail the role of ion currents, both in wild type and in in silico knockout neurons. Moreover, we specifically investigate the behavior of RMD, identifying a heterogeneous dynamical response which includes bistable regimes and sustained oscillations. We are able to assess the critical role of T-type calcium currents, carried by CCA-1 channels, and leakage currents in the regulation of RMD response. Overall, our results provide new insights in the activity of key C. elegans neurons. The developed mathematical framework constitute a basis for single-cell and neuronal networks analyses, opening new scenarios in the in silico modeling of C. elegans neuronal system.

Targeting pharmaceuticals through the endothelial barrier is crucial for drug delivery. In this context, cavitation-assisted permeation shows promise for effective and reversible opening of intercellular junctions. A vessel-on-a-chip is exploited to investigate and quantify the effect of ultrasound-excited microbubbles-stable cavitation-on endothelial integrity. In the vessel-on-a-chip, the endothelial cells form a complete lumen under physiological shear stress, resulting in intercellular junctions that exhibit barrier functionality. Immunofluorescence microscopy is exploited to monitor vascular integrity following vascular endothelial cadherin staining. It is shown that microbubbles amplify the ultrasound effect, leading to the formation of interendothelial gaps that cause barrier permeabilization. The total gap area significantly increases with pressure amplitude compared to the control. Gap opening is fully reversible with gap area distribution returning to the control levels 45 min after insonication. The proposed integrated platform allows for precise and repeatable in vitro measurements of cavitation-enhanced endothelium permeability and shows potential for validating irradiation protocols for in vivo applications.

The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2 is the activation, stabilisation and spindle localisation of the Aurora-A kinase. High levels of TPX2 are reported in tumours and the effects of its overexpression have been investigated in cancer cell lines, while little is known in non-transformed cells. Here we studied TPX2 overexpression in hTERT RPE-1 cells, using either the full length TPX2 or a truncated form unable to bind Aurora-A, to identify effects that are dependent-or independent-on its interaction with the kinase. We observe significant defects in mitotic spindle assembly and progression through mitosis that are more severe when overexpressed TPX2 is able to interact with Aurora-A. Furthermore, we describe a peculiar, and Aurora-A-interaction-independent, phenotype in telophase cells, with aberrantly stable microtubules interfering with nuclear reconstitution and the assembly of a continuous lamin B1 network, resulting in daughter cells displaying doughnut-shaped nuclei. Our results using non-transformed cells thus reveal a previously uncharacterised consequence of abnormally high TPX2 levels on the correct microtubule cytoskeleton remodelling and G1 nuclei reformation, at the mitosis-to-interphase transition.

In this work we present an integrated biosensor that enables FTIR (Fourier Transform-Infrared) detection of analytes contained in diluted solutions. The fabricated nanosensor allows for the detection of proteins through the identification of the fine structure of their amide I and II bands, up to the nanomolar concentration range. We exploited two distinct effects to enhance the sensitivity: (i) the concentration effect due to the presence of the superhydrophobic surface that conveys molecules dispersed in solution directly inside the focus of a FTIR spectromicroscope; (ii) the plasmonic resonance of the nanoantenna array that provides electromagnetic field enhancement in the amide I and II spectral region (1500-1700 cm(-1)). We demonstrate the detection of ferritin in the nanomolar concentration range, a blood protein that is usually available in small amounts in typical blood samples.