Generation of a stably transfected mouse embryonic stem cell line for inducible differentiation to excitatory neurons.

In vitro differentiation of stem cells into various cell lineages is valuable in developmental studies and an important source of cells for modelling physiology and pathology, particularly for complex tissues such as the brain. Conventional protocols for in vitro neuronal differentiation often suffer from complicated procedures, high variability and low reproducibility. Over the last decade, the identification of cell fate-determining transcription factors has provided new tools for cellular studies in neuroscience and enabled rapid differentiation driven by ectopic transcription factor expression. As a proneural transcription factor, Neurogenin 2 (Ngn2) expression alone is sufficient to trigger rapid and robust neurogenesis from pluripotent cells. Here, we established a stable cell line, by piggyBac (PB) transposition, that conditionally expresses Ngn2 for generation of excitatory neurons from mouse embryonic stem cells (ESCs) using an all-in-one PB construct. Our results indicate that Ngn2-induced excitatory neurons have mature and functional characteristics consistent with previous studies using conventional differentiation methods. This approach provides an all-in-one PB construct for rapid and high copy number gene delivery of dox-inducible transcription factors to induce differentiation. This approach is a valuable in vitro cell model for disease modeling, drug screening and cell therapy.

Strategic Replication of the Hepatic Zonation In Vitro Employing a Biomimetic Approach.

The varied functions of the liver are dependent on the metabolic heterogeneity exhibited by the hepatocytes within the liver lobule spanning the porto-central axis. This complex phenomenon plays an important role in maintaining the physiological homeostasis of the liver. Standard in vitro culture models fail to mimic this spatial heterogeneity of hepatocytes, assuming a homogeneous population of cells, which leads to inaccurate translation of results. Here, we demonstrate the development of an in vitro model of hepatic zonation by mimicking the microarchitecture of the liver using a 3D printed mini bioreactor and decellularized liver matrix to provide the native microenvironmental cues. There was a differential expression of hypoxic and metabolic markers across the developed mini bioreactor, showing the establishment of gradients of oxygen, Wnt/ß-catenin pathway, and other metabolic pathways. The model also showed the establishment of zone-dependent toxicity on treatment with acetaminophen. The developed model would thus be a promising avenue in the field of tissue engineering for understanding the liver physiology and pathophysiology and for drug screening to evaluate the potential of new pharmaceutical interventions.

Efficient Generation of Stable Cell Lines with Inducible Neuronal Transgene Expression Using the piggyBac Transposon System.

The piggyBac transposon system has been adapted to be a highly efficient genome engineering tool for transgenesis of eukaryotic cells and organisms. As with other methods of transgenesis, incorporation of an inducible promoter, such as a tetracycline-responsive element, enables inducible transgene expression. Here, we describe an efficient method of using the piggyBac system to create stably transfected mammalian cell lines, including inducible transgene expression. Gibson assembly is used to construct the required vectors as it enables multiple DNA fragments to be seamlessly assembled in a single isothermal reaction. We demonstrate an application of this approach to generate a stably transfected pluripotent stem cell line that can be induced to express a transcription factor transgene and rapidly differentiate into neurons in a single step.

Targeting the AAVS1 Site by CRISPR/Cas9 with an Inducible Transgene Cassette for the Neuronal Differentiation of Human Pluripotent Stem Cells.

CRISPR/Cas9 system is a powerful genome-editing technology for studying genetics and cell biology. Safe harbor sites are ideal genomic locations for transgene integration with minimal interference in cellular functions. Gene targeting of the AAVS1 locus enables stable transgene expression without phenotypic effects in host cells. Here, we describe the strategy for targeting the AAVS1 site with an inducible Neurogenin-2 (Ngn2) donor template by CRISPR/Cas9 in hiPSCs, which facilitates generation of an inducible cell line that can rapidly and homogenously differentiate into excitatory neurons.

Galactose Tethered Decellularized Liver Matrix: Toward a Biomimetic and Biofunctional Matrix for Liver Tissue Engineering.

The major challenge in liver tissue engineering is the replication of the microenvironment and microarchitecture of the liver tissue at the nanoscale. Decellularized liver matrix (DLM) provides an ideal material for scaffold preparation, as it retains the relevant structural and biochemical composition. However, the loss of bioactive factors during decellularization needs to be taken into account when using DLM and should be supplemented accordingly for an expected outcome. This study reports on the modification of DLM by the addition of galactose residues using a two-step thiol-ene-mediated photoclick chemistry for the coupling of galactose moieties to the DLM. Modification with galactose enhanced the function of hepatocytes and provides many advantages over currently used DLM and DLM-based materials. The galactose modified DLM enhanced the initial HepG2 cell adhesion to the substrate with changes in dynamics over time such as spheroid formation and further migration on the matrix. Our observation is that the galactose ligand decoration can also enhance the liver-specific metabolism of HepG2 compared to unmodified DLM. Galactosylated DLM also showed a better establishment of cellular polarity which also contributes to the function of HepG2 cells. Together our results demonstrate the advantages of adding galactose residues to currently available biomaterials, which makes this approach an attractive method for ECM-based liver tissue engineering.

Comparative analysis of extracellular vesicles isolated from human mesenchymal stem cells by different isolation methods and visualisation of their uptake.

Various types of cells secrete extracellular vesicle (EVs) which contain proteins, lipids and nucleic acids and play important roles in inter-cellular signalling and pathological processes to impact the recipient cells. EVs have demonstrated their potential as biomarkers for disease and as therapeutic agents in regenerative medicine. In recent times, EVs derived from mesenchymal stem cells (MSCs), which are widely used as a promising medicinal product in many clinical applications, are being tested in many preclinical trials. However, the lack of standardization of MSC-derived EV isolation and analysis methods, restricts the utility of MSC-derived EVs in the clinical setting. Here, we focused on optimising the isolation method for EVs derived from MSCs. Four samples of EVs were isolated from human adipose derived MSC culture medium by differential ultracentrifugation with three different ultracentrifuge durations to investigate the influence of ultracentrifuge time on quality and quantity of MSC-derived EVs. Additionally, we used a commercial kit to extract EVs from MSC cultured medium and compared it with the ultracentrifugation method. The EV samples were then characterised for particle concentration, protein concentration, size distribution and the presence of known EV protein markers, by western blot and flow cytometry. A comparison of these results for the five samples demonstrated that 1 h of differential ultracentrifugation was optimal to isolate high quality and quantity of MSC-derived EVs from MSC cultured medium. Additionally, fluorescence imaging of the freshly isolated vs frozen EVs showed that freshly isolated EVs are taken up by cells more efficiently than frozen EVs. These finding establish a simple and reliable method of EV isolation from MSCs.

Influence of Liver Extracellular Matrix in Predicting Drug-Induced Liver Injury: An Alternate Paradigm.

In vitro drug-induced liver injury (DILI) models are promising tools for drug development to predict adverse events during clinical usage. However, the currently available DILI models are not specific or not able to predict the injury accurately. This is believed to be mainly because of failure to conserve the hepatocyte phenotype, lack of longevity, and difficulty in maintaining the tissue-specific microenvironment. In this study, we have assessed the potential of decellularized liver extracellular matrix (DLM) in retaining the hepatic cellular phenotype and functionality in the presence of a tissue-specific microenvironment along with its role in influencing the effect of the drug on hepatic cells. We show that DLM helps maintain the phenotype of the hepatic cell line HepG2, a well-known cell line for secretion of human proteins that is easily available. Also, the DLM enhanced the expression of a metabolic marker carbamoyl phosphate synthetase I (CPS1), a regulator of urea cycle, and bile salt export pump (BSEP), a marker of hepatocyte polarity. We further validated the DLM for its influence on the sensitivity of cells toward different classes of drugs. Interestingly, the coculture model, in the presence of endothelial cells and stellate cells, exhibited a higher sensitivity for both acetaminophen and trovafloxacin, a toxic compound that does not show any toxicity on preclinical screening. Thus, our results demonstrate for the first time that a multicellular combination along with DLM can be a potential and reliable DILI model to screen multiple drugs.

Forward Programming of Pluripotent Stem Cells to Neurons.

Pluripotent stem cells (PSCs) are powerful tools for studying developmental biology and neuronal diseases. Conventional differentiation protocols require several intermediate states and different culture conditions, inefficiently generating mixed subtypes of neuronal cells with immature characteristics. Direct programming of PSCs by forced expression of neuronal transcription factors has shown rapid cell fate determination with high purity as it can bypass sequential developmental steps that traditional culture requires. In this review, we focus on neuronal differentiation from PSCs to specific subtypes by various transcription factors. Furthermore, the potential applications and limitations of this novel technology are discussed.

Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia.

With an increasing focus on the large-scale expansion of mesenchymal stem cells (MSCs) required for clinical applications for the treatment of joint and bone diseases such as osteoarthritis, the optimisation of conditions for in vitro MSC expansion requires careful consideration to maintain native MSC characteristics. Physiological parameters such as oxygen concentration, media constituents, and passage numbers influence the properties of MSCs and may have major impact on their therapeutic potential. Cells grown under hypoxic conditions have been widely documented in clinical use. Culturing MSCs on large scale requires bioreactor culture; however, it is challenging to maintain low oxygen and other physiological parameters over several passages in large bioreactor vessels. The necessity to scale up the production of cells in vitro under normoxia may affect important attributes of MSCs. For these reasons, our study investigated the effects of normoxic and hypoxic culture condition on early- and late-passage adipose-derived MSCs. We examined effect of each condition on the expression of key stem cell marker genes POU5F1, NANOG, and KLF4, as well as differentiation genes RUNX2, COL1A1, SOX9, COL2A1, and PPARG. We found that expression levels of stem cell marker genes and osteogenic and chondrogenic genes were higher in normoxia compared to hypoxia. Furthermore, expression of these genes reduced with passage number, with the exception of PPARG, an adipose differentiation marker, possibly due to the adipose origin of the MSCs. We confirmed by flow cytometry the presence of cell surface markers CD105, CD73, and CD90 and lack of expression of CD45, CD34, CD14, and CD19 across all conditions. Furthermore, in vitro differentiation confirmed that both early- and late-passage adipose-derived MSCs grown in hypoxia or normoxia could differentiate into chondrogenic and osteogenic cell types. Our results demonstrate that the minimal standard criteria to define MSCs as suitable for laboratory-based and preclinical studies can be maintained in early- or late-passage MSCs cultured in hypoxia or normoxia. Therefore, any of these culture conditions could be used when scaling up MSCs in bioreactors for allogeneic clinical applications or tissue engineering for the treatment of joint and bone diseases such as osteoarthritis.

Mesenchymal Stem Cell-Derived Extracellular Vesicles and Their Therapeutic Potential.

Extracellular vesicles (EVs) are cell-derived membrane-bound nanoparticles, which act as shuttles, delivering a range of biomolecules to diverse target cells. They play an important role in maintenance of biophysiological homeostasis and cellular, physiological, and pathological processes. EVs have significant diagnostic and therapeutic potentials and have been studied both in vitro and in vivo in many fields. Mesenchymal stem cells (MSCs) are multipotent cells with many therapeutic applications and have also gained much attention as prolific producers of EVs. MSC-derived EVs are being explored as a therapeutic alternative to MSCs since they may have similar therapeutic effects but are cell-free. They have applications in regenerative medicine and tissue engineering and, most importantly, confer several advantages over cells such as lower immunogenicity, capacity to cross biological barriers, and less safety concerns. In this review, we introduce the biogenesis of EVs, including exosomes and microvesicles. We then turn more specifically to investigations of MSC-derived EVs. We highlight the great therapeutic potential of MSC-derived EVs and applications in regenerative medicine and tissue engineering.

3D hepatic mimics - the need for a multicentric approach.

The liver is a center of metabolic activity, including the metabolism of drugs, and consequently is prone to drug-induced liver injury. Failure to detect hepatotoxicity of drugs during their development will lead to the withdrawal of the drugs during clinical trials. To avoid such clinical and economic consequences, in vitro liver models that can precisely predict the toxicity of a drug during the pre-clinical phase is necessary. This review describes the different technologies that are used to develop in vitro liver models and the different approaches aimed at mimicking different functional aspects of the liver at the fundamental level. This involves mimicking of the functional and structural units like the sinusoid, the bile canalicular system, and the acinus.

Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7.

P2X7 receptors are important in the regulation of inflammatory responses and immune responses to intracellular pathogens such as Mycobacterium tuberculosis and Toxoplasma gondii. Enhancement of P2X7 receptor responses may be useful in pathogen clearance particularly in individuals with defective microbial killing mechanisms. Ginsenosides from Panax ginseng have been discovered to act as positive allosteric modulators of P2X7. Here we describe a novel modulator binding site identified by computational docking located in the central vestibule of P2X7 involving S60, D318, and L320 in the lower body ß-sheets lining the lateral portals. Potentiation of ATP-mediated responses by ginsenosides CK and Rd caused enhanced ionic currents, Ca2+ influx and YOPRO-1 uptake in stably transfected HEK-293 cells (HEK-hP2X7) plus enhanced cell death responses. Potentiation of ATP responses by CK and Rd was markedly reduced by mutations S59A, S60A, D318L and L320A supporting the proposed allosteric modulator binding site. Furthermore, mutation of the conserved residues S60 and D318 led to alterations in P2X7 response and a higher sensitivity to ATP in the absence of modulators suggesting residues in the connecting rods play an important role in regulating P2X7 gating. Identification of this novel binding site location in the central vestibule may also be relevant for structurally similar channels.

Ginsenosides Act As Positive Modulators of P2X4 Receptors.

We investigated the selectivity of protopanaxadiol ginsenosides from Panax ginseng acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by ∼twofold, similar to potentiation by the known positive modulator ivermectin. Investigations into the role of P2X4 in mediating a cytotoxic effect showed that only P2X7 expression in HEK-293 cells induces cell death in response to high concentrations of ATP, and that ginsenosides can enhance this process. Generation of a P2X7-deficient clone of BV-2 microglial cells using CRISPR/Cas9 gene editing enabled an investigation of endogenous P2X4 in a microglial cell line. Compared with parental BV-2 cells, P2X7-deficient BV-2 cells showed minor potentiation of ATP responses by ginsenosides, and insensitivity to ATP- or ATP+ ginsenoside-induced cell death, indicating a primary role for P2X7 receptors in both of these effects. Computational docking to a homology model of human P2X4, based on the open state of zfP2X4, yielded evidence of a putative ginsenoside binding site in P2X4 in the central vestibule region of the large ectodomain.

Abundance of ClC-1 chloride channel in human skeletal muscle: fiber type specific differences and effect of training.

Cl- channel protein 1 (ClC-1) may be important for excitability and contractility in skeletal muscle, but ClC-1 abundance has not been examined in human muscle. The aim of the present study was to examine ClC-1 abundance in human skeletal muscle, including fiber type specific differences and the effect of exercise training. A commercially available antibody was tested with positive and negative control tissue, and it recognized specifically ClC-1 in the range from 100 to 150 kDa. Abundance of ClC-1 was 38% higher ( P < 0.01) in fast twitch Type IIa muscle fibers than in slow twitch Type I. Muscle ClC-1 abundance did not change with 4 wk of training consisting of 30 min cycling at 85% of maximal heart rate (HRmax) and 3 × 30-s all out sprints or during a 7-wk training period with 10-12 × 30 s uphill cycling and 4-5 × ~4 min cycling at 90%-95% of HRmax. ClC-1 abundance correlated negatively ( P < 0.01) with maximal oxygen consumption ( r = -0.552) and incremental exercise performance ( r = -0.546). In addition, trained cyclists had lower ( P < 0.01) ClC-1 abundance than lesser trained individuals. The present observations indicate that a low abundance of muscle ClC-1 may be beneficial for exercise performance, but the role of abundance and regulation of ClC-1 in skeletal muscle of humans with respect to exercise performance and trainability need to be elucidated. NEW & NOTEWORTHY Abundance of the Cl- channel protein 1 (ClC-1) chloride channel may be important for excitability and contractility in human skeletal muscle and may therefore have implications for fatigue development. In this study, we confirmed ClC-1 specificity for a commercially available antibody, and this study is first to our knowledge to determine ClC-1 protein abundance in human muscle by Western blotting. We observed that abundance of ClC-1 was higher in fast compared with slow twitch fibers and lower in trained individuals than in recreationally active.

Design of ultra-swollen lipidic mesophases for the crystallization of membrane proteins with large extracellular domains.

In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3-5 nm) of the mesophase. Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs. We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid (Ia3d), double-diamond (Pn3m), and double-primitive (Im3m) space groups, with water channels five times larger than traditional lipidic mesophases, and showing re-entrant behavior upon increasing hydration, of sequences Ia3d→Pn3m→Ia3d and Pn3m→Im3m→Pn3m, unknown in lipid self-assembly. We use these mesophases to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein, and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology.

String method solution of the gating pathways for a pentameric ligand-gated ion channel.

Pentameric ligand-gated ion channels control synaptic neurotransmission by converting chemical signals into electrical signals. Agonist binding leads to rapid signal transduction via an allosteric mechanism, where global protein conformational changes open a pore across the nerve cell membrane. We use all-atom molecular dynamics with a swarm-based string method to solve for the minimum free-energy gating pathways of the proton-activated bacterial GLIC channel. We describe stable wetted/open and dewetted/closed states, and uncover conformational changes in the agonist-binding extracellular domain, ion-conducting transmembrane domain, and gating interface that control communication between these domains. Transition analysis is used to compute free-energy surfaces that suggest allosteric pathways; stabilization with pH; and intermediates, including states that facilitate channel closing in the presence of an agonist. We describe a switching mechanism that senses proton binding by marked reorganization of subunit interface, altering the packing of ß-sheets to induce changes that lead to asynchronous pore-lining M2 helix movements. These results provide molecular details of GLIC gating and insight into the allosteric mechanisms for the superfamily of pentameric ligand-gated channels.

Assembly, trafficking and function of α1ß2γ2 GABAA receptors are regulated by N-terminal regions, in a subunit-specific manner.

GABAA receptors are pentameric ligand-gated ion channels that mediate inhibitory fast synaptic transmission in the central nervous system. Consistent with recent pentameric ligand-gated ion channels structures, sequence analysis predicts an α-helix near the N-terminus of each GABAA receptor subunit. Preceding each α-helix are 8-36 additional residues, which we term the N-terminal extension. In homomeric GABAC receptors and nicotinic acetylcholine receptors, the N-terminal α-helix is functionally essential. Here, we determined the role of the N-terminal extension and putative α-helix in heteromeric α1ß2γ2 GABAA receptors. This role was most prominent in the α1 subunit, with deletion of the N-terminal extension or further deletion of the putative α-helix both dramatically reduced the number of functional receptors at the cell surface. Conversely, deletion of the ß2 or γ2 N-terminal extension had little effect on the number of functional cell surface receptors. Additional deletion of the putative α-helix in the ß2 or γ2 subunits did, however, decrease both functional cell surface receptors and incorporation of the γ2 subunit into mature receptors. In the ß2 subunit only, α-helix deletions affected GABA sensitivity and desensitization. Our findings demonstrate that N-terminal extensions and α-helices make key subunit-specific contributions to assembly, consistent with both regions being involved in inter-subunit interactions. N-terminal α-helices and preceding sequences of eukaryotic pentameric ligand-gated ion channels are absent in prokaryotic homologues, suggesting they may not be functionally essential. Here, we show that in heteropentameric α1ß2γ2 GABAA receptors, the role of these segments is highly subunit dependent. The extension preceding the α-helix in the α subunit is crucial for assembly and trafficking, but is of little importance in ß and γ subunits. Indeed, robust receptor levels remain when the extension and α-helix are removed in ß or γ subunits.

Molecular basis for convergent evolution of glutamate recognition by pentameric ligand-gated ion channels.

Glutamate is an indispensable neurotransmitter, triggering postsynaptic signals upon recognition by postsynaptic receptors. We questioned the phylogenetic position and the molecular details of when and where glutamate recognition arose in the glutamate-gated chloride channels. Experiments revealed that glutamate recognition requires an arginine residue in the base of the binding site, which originated at least three distinct times according to phylogenetic analysis. Most remarkably, the arginine emerged on the principal face of the binding site in the Lophotrochozoan lineage, but 65 amino acids upstream, on the complementary face, in the Ecdysozoan lineage. This combined experimental and computational approach throws new light on the evolution of synaptic signalling.

Comparative pharmacology of flatworm and roundworm glutamate-gated chloride channels: Implications for potential anthelmintics.

Pharmacological targeting of glutamate-gated chloride channels (GluCls) is a potent anthelmintic strategy, evidenced by macrocyclic lactones that eliminate numerous roundworm infections by activating roundworm GluCls. Given the recent identification of flatworm GluCls and the urgent need for drugs against schistosomiasis, flatworm GluCls should be evaluated as potential anthelmintic targets. This study sought to identify agonists or modulators of one such GluCl, SmGluCl-2 from the parasitic flatworm Schistosoma mansoni. The effects of nine glutamate-like compounds and three monoterpenoid ion channel modulators were measured by electrophysiology at SmGluCl-2 recombinantly expressed in Xenopus laevis oocytes. For comparison with an established anthelmintic target, experiments were also performed on the AVR-14B GluCl from the parasitic roundworm Haemonchus contortus. l-Glutamate was the most potent agonist at both GluCls, but l-2-aminoadipate, d-glutamate and d-2-aminoadipate activated SmGluCl-2 (EC50 1.0 ± 0.1 mM, 2.4 ± 0.4 mM, 3.6 ± 0.7 mM, respectively) more potently than AVR-14B. Quisqualate activated only SmGluCl-2 whereas l-aspartate activated only AVR-14B GluCls. Regarding the monoterpenoids, both GluCls were inhibited by propofol, thymol and menthol, SmGluCl-2 most potently by thymol (IC50 484 ± 85 µM) and least potently by menthol (IC50 > 3 mM). Computational docking suggested that agonist and inhibitor potency is attributable to particular interactions with extracellular or membrane-spanning amino acid residues. These results reveal that flatworm GluCls are pharmacologically susceptible to numerous agonists and modulators and indicate that changes to the glutamate γ-carboxyl or to the propofol 6-isopropyl group can alter the differential pharmacology at flatworm and roundworm GluCls. This should inform the development of more potent compounds and in turn lead to novel anthelmintics.

Role of the ρ1 GABA(C) receptor N-terminus in assembly, trafficking and function.

The GABAC receptor and closely related GABAA receptor are members of the pentameric ligand-gated ion channels (pLGICs) superfamily and mediate inhibitory fast synaptic transmission in the nervous system. Each pLGIC subunit comprises an N-terminal extracellular agonist-binding domain followed by a channel domain and a variable intracellular domain. Available structural information shows that the core of the agonist-binding domain is a ß sandwich of ten ß-strands, which form the agonist-binding pocket at the subunit interface. This ß-sandwich is preceded by an N-terminal α-helix in eukaryotic structures but not in prokaryotic structures. The N-terminal α-helix has been shown to be functionally essential in α7 nicotinic acetylcholine receptors. Sequence analysis of GABAC and GABAA receptors predicts an α-helix in a similar position but preceded by 8 to 46 additional residues, of unknown function, which we term the N-terminal extension. To test the functional role of both the N-terminal extension and the putative N-terminal α-helix in the ρ1 GABAC receptor, we created a series of deletions from the N-terminus. The N-terminal extension was not functionally essential, but its removal did reduce both cell surface expression and cooperativity of agonist-gated channel function. Further deletion of the putative N-terminal α-helix abolished receptor function by preventing cell-surface expression. Our results further demonstrate the essential role of the N-terminal α-helix in the assembly and trafficking of eukaryotic pLGICs. They also provide evidence that the N-terminal extension, although not essential, contributes to receptor assembly, trafficking and conformational changes associated with ligand gating.