The concept of tumor stem cells (TSCs) provides a new paradigm for understanding tumor biology, although it remains unclear whether TSCs will prove to be a more robust model than traditional cancer cell lines. We demonstrate marked phenotypic and genotypic differences between primary human tumor-derived TSCs and their matched glioma cell lines. Unlike the matched, traditionally grown tumor cell lines, TSCs derived directly from primary glioblastomas harbor extensive similarities to normal neural stem cells and recapitulate the genotype, gene expression patterns, and in vivo biology of human glioblastomas. These findings suggest that TSCs may be a more reliable model than many commonly utilized cancer cell lines for understanding the biology of primary human tumors.
Accumulating evidence shows that several cell types have the capacity to secrete membrane proteins by incorporating them into exosomes, which are small lipid vesicles derived from the intralumenal membranes of multivesicular bodies (MVBs) of the endocytic pathway. Exosomes are expelled in the extracellular space upon fusion of the MVB with the plasma membrane. Exosomal release is a way of secreting membrane proteins meant to be discarded, or to be passed on to other cells. Here, we demonstrate, using primary cortical cultures, that neurones and astrocytes can secrete exosomes. We find that exosomes released by cortical neurones contain the L1 cell adhesion molecule, the GPI-anchored prion protein, and the GluR2/3 but not the NR1 subunits of glutamate receptors. We also show that exosomal release is regulated by depolarisation. Our observation suggests that exosomes may have a regulatory function at synapses and could also allow intercellular exchange of membrane proteins within the brain.
Although understanding cells' responses to mechanical stimuli is seen as increasingly important for understanding cell biology, how to best measure, interpret, and model cells' mechanical properties remains unclear. We determine the frequency-dependent shear modulus of cultured mammalian cells by using four different methods, both unique and well established. This approach clarifies the effects of cytoskeletal heterogeneity, ATP-dependent processes, and cell regional variations on the interpretation of such measurements. Our results clearly indicate two qualitatively similar, but distinct, mechanical responses, corresponding to the cortical and intracellular networks, each having an unusual, weak power-law form at low frequency. The two frequency-dependent responses we observe are remarkably similar to those reported for a variety of cultured mammalian cells measured with different techniques, suggesting it is a useful consensus description. Finally, we discuss possible physical explanations for the observed mechanical response.
Abstract The ability to control cell proliferation/differentiation, using material surface, is a main goal in tissue engineering. The objective of this study was to evaluate the attachment, proliferation and differentiation to the osteoblastic phenotype of human marrow stromal cells (MSC) when seeded on poly- ε -caprolactone (PCL) thin films before and after irradiation with 10 keV He+ . The polymeric surface was characterized as surface chemical structure and composition, roughness and morphology on the micro- and nano-scale, wettability and surface free energy parameters. MSC were obtained from patients undergoing routine hip replacement surgery, expanded in vitro and cultured on untreated PCL and He+ irradiated PCL films for up to 4–5 weeks in osteogenic medium. He+ -irradiation led to slight smoothening of the surface and different nanoscale surface chemical structure, while surface free energy resulted unchanged in comparison to untreated PCL. The results from biological testing demonstrated that early attachment and further proliferation, as well as osteoblastic markers, were higher for MSC on He+ -irradiated PCL. In conclusion, the change of PCL surface properties induced by ion beam irradiation is confirmed to enhance the adhesion of MSC and support their differentiation.
Infections with hepatitis C virus (HCV) are marked by frequent viral persistence, chronic liver disease, and extraordinary viral genetic diversity. Although much has been learned about HCV since its discovery, progress has been slowed by a lack of permissive cell culture systems supporting its replication. Productive infections have been achieved recently with genotype 2a virus, but cirrhosis and liver cancer are typically associated with genotype 1 HCV, which is more prevalent and relatively resistant to IFN therapy. We describe production of infectious genotype la HCV in cells transfected with synthetic RNA derived from a prototype virus (H77-S). Viral proteins accumulated more slowly in H77-S transfected cells than in cells transfected with genotype 2a (JFH-1) RNA, but substantially more H77-S RNA was secreted into supernatant fluids. Most secreted RNA was noninfectious, banding in isopycnic gradients at a density of 1.04-1.07 gm/cm³, but infectivity was associated with H77-S particles possessing a density of 1.13-1.14 gm/cm³. The specific infectivity of H77-S particles (5.4 x 10⁴ RNA copies per focus-forming unit) was significantly lower than JFH-1 virus (1.4 x 10² RNA copies per focus-forming unit). Infection with either virus was blocked by CD81 antibody. Sera from genotype la-infected individuals neutralized H77-S virus, but had little activity against genotype 2a virus, suggesting that these genotypes represent different serotypes. The ability of this genotype la virus to infect cultured cells will substantially benefit antiviral and vaccine discovery programs.
Ubiquitously expressed volume‐regulated anion channels (VRACs) are chloride channels which are permeable to a variety of small organic anions, including the excitatory amino acids (EAAs) glutamate and aspartate. Broad spectrum anion channel blockers strongly reduce EAA release in cerebral ischaemia and other pathological states associated with prominent astrocytic swelling. However, it is uncertain whether VRAC serves as a major pathway for EAA release from swollen cells. In the present study, we measured swelling‐activated release of EAAs as d‐[3H]aspartate efflux, and VRAC‐mediated Cl− currents by whole‐cell patch clamp in cultured rat astrocytes. We compared the pharmacological profiles of the swelling‐activated EAA release pathway and Cl− currents. The expression of candidate Cl− channels was confirmed by RT‐PCR. The maxi Cl− channel (p‐VDAC) blocker Gd3+, the ClC‐2 inhibitor Cd2+, and the MDR‐1 blocker verapamil did not affect EAA release or VRAC currents. An antagonist of calcium‐sensitive Cl− channels (CaCC), niflumic acid, had little effect on EAA release and only partially inhibited swelling‐activated Cl− currents. The phorbol ester PDBu, which blocks ClC‐3‐mediated Cl− currents, had no effect on VRAC currents and up‐regulated EAA release. In contrast, DCPIB, which selectively inhibits VRACs, potently suppressed both EAA release and VRAC currents. Two other relatively selective VRAC inhibitors, tamoxifen and phloretin, also blocked the VRAC currents and strongly reduced EAA release. Taken together, our data suggest that (i) astrocytic volume‐dependent EAA release is largely mediated by the VRAC, and (ii) the ClC‐2, ClC‐3, ClC‐4, ClC‐5, VDAC, CaCC, MDR‐1 and CFTR gene products do not contribute to EAA permeability.
The extracellular signal-regulated kinases (ERK) signalling cascade is a key pathway that mediates the NMDA receptor (NMDAR)-dependent neuronal plasticity and survival. However, it is not clear yet how NMDARs regulate ERK activity. Stimulation of the NMDARs induces a complex modification of ERK that includes both ERK activation and inactivation and depends on particular experimental conditions. Here we show that there exists a differential restriction in the regulation of ERK activity that depends on the pool of NMDAR that was activated. The synaptic pool of NMDARs activates ERK whereas the extrasynaptic pool does not; on the contrary, it triggers a signalling pathway that results in the inactivation of ERK. As a result, simultaneous activation of both extrasynaptic and synaptic NMDAR using bath application of NMDA or glutamate (a typical protocol explored in the majority of studies) produced ERK activation that depended on the concentration of agonists and was always significantly weaker than those mediated by synaptic NMDARs. Since the activation of the extrasynaptic NMDA is attributed mainly to global release of glutamate occurring at pathological conditions including hypoxic/ischaemic insults, traumas and epileptic brain damage, the reported differential regulation of ERK cascade by NMDARs provides a unique mechanism for an early identification of the physiological and/or pathophysiological consequences of NMDAR activation. The negative regulation of the ERK activity might be one of the first signalling events determining brain injury and constitutes a putative target of new pharmacological applications.
A cultural algorithm with a differential evolution population is proposed in this paper. This cultural algorithm uses different knowledge sources to influence the variation operator of the differential evolution algorithm, in order to reduce the number of fitness function evaluations required to obtain competitive results. Comparisons are provided with respect to three techniques that are representative of the state-of-the-art in the area. The results obtained by our algorithm are similar (in quality) to those obtained by the other approaches with respect to which it was compared. However, our approach requires a lower number of fitness function evaluations than the others.
RNA interference has re-energized the field of functional genomics by enabling genome-scale loss-of-function screens in cultured cells. Looking back on the lessons that have been learned from the first wave of technology developments and applications in this exciting field, we provide both a user's guide for newcomers to the field and a detailed examination of some more complex issues, particularly concerning optimization and quality control, for more advanced users. From a discussion of cell lines, screening paradigms, reagent types and read-out methodologies, we explore in particular the complexities of designing optimal controls and normalization strategies for these challenging but extremely powerful studies.
Abstract Methods for handling burn wounds have changed in recent decades and increasingly aggressive surgical approach with early tangential excision and wound closure is being applied. Split-thickness skin (STSG) autografts are the “gold standard” for burn wound closure and remain the mainstay of treatment to provide permanent wound coverage and achieve healing. In some massively burned patients, however, the burns are so extensive that donor site availability is limited. Fortunately, considerable progress has been made in the culture of human keratinocytes and it is now possible to obtain large amounts of cultured epithelium from a small skin biopsy within 3–4 weeks. Questions related to optimal cell type for culture, culture techniques, transplantation of confluent sheets or non-confluent cells, immediate and late final take, carrier and transfer modality, as well as final outcome, ability to generate an epithelium after transplantation, and scar quality are still not fully answered. Progress accomplished since Reinwald and Green first described their keratinocyte culture technique is reviewed.