Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Exploring the cell interactome: deciphering relative impacts of cell-cell communication in cell co-culture using a novel microfluidic device.

The human body is made up of approximately 40 trillion cells in close contact, with the cellular density of individual tissues varying from 1 million to 1 billion cells per cubic centimetre. Interactions between different cell types (termed heterotypic) are thus common in vivo. Communication between cells can take the form of direct cell-cell contact mediated by plasma membrane proteins or through paracrine signalling mediated through the release, diffusion, and receipt of soluble factors. There is currently no systematic method to investigate the relative contributions of these mechanisms to cell behaviour. In this paper, we detail the conception, development and validation of a microfluidic device that allows cell-cell contact and paracrine signalling in defined areas and over a variety of biologically relevant length scales, referred to as the interactome-device or 'I-device'. Importantly, by intrinsic device design features, cells in different regions in the device are exposed to four different interaction types, including a) no heterotypic cell interaction, b) only paracrine signalling, c) only cell-cell direct contact, or d) both forms of interaction (paracrine and cell-cell direct contact) together. The device design was validated by both mathematical modelling and experiments. Perfused stem cell culture over the medium term and the formation of direct contact between cells in the culture chambers was confirmed. The I-device offers significant flexibility, being able to be applied to any combination of adherent cells to determine the relative contributions of different communication mechanisms to cellular outcomes.

S-Trap Eliminates Cell Culture Media Polymeric Surfactants for Effective Proteomic Analysis of Mammalian Cell Bioreactor Supernatants.

Proteomic analysis of bioreactor supernatants can inform on cellular metabolic status, viability, and productivity, as well as product quality, which can in turn help optimize bioreactor operation. Incubating mammalian cells in bioreactors requires the addition of polymeric surfactants such as Pluronic F68, which reduce the sheer stress caused by agitation. However, these surfactants are incompatible with mass spectrometry proteomics and must be eliminated during sample preparation. Here, we compared four different sample preparation methods to eliminate polymeric surfactants from filtered bioreactor supernatant samples: organic solvent precipitation; filter-assisted sample preparation (FASP); S-Trap; and single-pot, solid-phase, sample preparation (SP3). We found that SP3 and S-Trap substantially reduced or eliminated the polymer(s), but S-Trap provided the most robust cleanup and highest quality data. Additionally, we observed that SP3 sample preparation of our samples and in other published data sets was associated with partial alkylation of cysteines, which could impact the confidence and robustness of protein identification and quantification. Finally, we observed that several commercial mammalian cell culture media and media supplements also contained polymers with similar mass spectrometry profiles, and we suggest that proteomic analyses in these media will also benefit from the use of S-Trap sample preparation.

Encoding Stem-Cell-Secreted Extracellular Matrix Protein Capture in Two and Three Dimensions Using Protein Binding Peptides.

Capturing cell-secreted extracellular matrix (ECM) proteins through cooperative binding with high specificity and affinity is an important function of native tissue matrices during both tissue homeostasis and repair. However, while synthetic hydrogels, such as those based on poly(ethylene glycol) (PEG), are often proposed as ideal materials to deliver human mesenchymal stem cells (hMSCs) to sites of injury to enable tissue repair, they do not have this capability-a capability that would enable cells to actively remodel their local extracellular microenvironment and potentially provide the required feedback control for more effective tissue genesis. In this work, we detail a methodology that engenders poly(ethylene glycol) (PEG)-based two-dimensional substrates and three-dimensional porous hydrogels with the ability to capture desired extracellular matrix (ECM) proteins with high specificity. This "encoded" ECM protein capture is achieved by decorating the PEG-based materials with protein binding peptides (PBPs) synthesized to be specific in their binding of fibronectin, laminin, and collagen I, which are not only the most omnipresent ECM proteins in human tissues but, as we confirmed, are also secreted to differing extents by hMSCs under in vitro maintenance conditions. By encapsulating hMSCs into these PBP-functionalized hydrogels, and culturing them in protein-free maintenance media, we demonstrate that these PBPs not only actively recruit targeted ECM proteins as they are secreted from hMSCs but also retain them to much higher levels compared to nonfunctionalized gels. This novel approach thus enables the fabrication of encoded surfaces and hydrogels that capture cell-secreted proteins, with high specificity and affinity, in a programmable manner, ready for applications in many bioengineering applications, including bioactive surface coatings, bioassays, stem cell culture, tissue engineering, and regenerative medicine.

Intact mirror mechanisms for automatic facial emotions in children and adolescents with autism spectrum disorder.

It has been suggested that an early deficit in the human mirror neuron system (MNS) is an important feature of autism. Recent findings related to simple hand and finger movements do not support a general dysfunction of the MNS in autism. Studies investigating facial actions (e.g., emotional expressions) have been more consistent, however, mostly relied on passive observation tasks. We used a new variant of a compatibility task for the assessment of automatic facial mimicry responses that allowed for simultaneous control of attention to facial stimuli. We used facial electromyography in 18 children and adolescents with Autism spectrum disorder (ASD) and 18 typically developing controls (TDCs). We observed a robust compatibility effect in ASD, that is, the execution of a facial expression was facilitated if a congruent facial expression was observed. Time course analysis of RT distributions and comparison to a classic compatibility task (symbolic Simon task) revealed that the facial compatibility effect appeared early and increased with time, suggesting fast and sustained activation of motor codes during observation of facial expressions. We observed a negative correlation of the compatibility effect with age across participants and in ASD, and a positive correlation between self-rated empathy and congruency for smiling faces in TDC but not in ASD. This pattern of results suggests that basic motor mimicry is intact in ASD, but is not associated with complex social cognitive abilities such as emotion understanding and empathy. Autism Res 2017, 10: 298-310. © 2016 International Society for Autism Research, Wiley Periodicals, Inc.

Intact Rapid Facial Mimicry as well as Generally Reduced Mimic Responses in Stable Schizophrenia Patients.

Spontaneous emotional expressions (rapid facial mimicry) perform both emotional and social functions. In the current study, we sought to test whether there were deficits in automatic mimic responses to emotional facial expressions in patients (15 of them) with stable schizophrenia compared to 15 controls. In a perception-action interference paradigm (the Simon task; first experiment), and in the context of a dual-task paradigm (second experiment), the task-relevant stimulus feature was the gender of a face, which, however, displayed a smiling or frowning expression (task-irrelevant stimulus feature). We measured the electromyographical activity in the corrugator supercilii and zygomaticus major muscle regions in response to either compatible or incompatible stimuli (i.e., when the required response did or did not correspond to the depicted facial expression). The compatibility effect based on interactions between the implicit processing of a task-irrelevant emotional facial expression and the conscious production of an emotional facial expression did not differ between the groups. In stable patients (in spite of a reduced mimic reaction), we observed an intact capacity to respond spontaneously to facial emotional stimuli.

Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis.

Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1.

Concise review: tailoring bioengineered scaffolds for stem cell applications in tissue engineering and regenerative medicine.

The potential for the clinical application of stem cells in tissue regeneration is clearly significant. However, this potential has remained largely unrealized owing to the persistent challenges in reproducibly, with tight quality criteria, and expanding and controlling the fate of stem cells in vitro and in vivo. Tissue engineering approaches that rely on reformatting traditional Food and Drug Administration-approved biomedical polymers from fixation devices to porous scaffolds have been shown to lack the complexity required for in vitro stem cell culture models or translation to in vivo applications with high efficacy. This realization has spurred the development of advanced mimetic biomaterials and scaffolds to increasingly enhance our ability to control the cellular microenvironment and, consequently, stem cell fate. New insights into the biology of stem cells are expected to eventuate from these advances in material science, in particular, from synthetic hydrogels that display physicochemical properties reminiscent of the natural cell microenvironment and that can be engineered to display or encode essential biological cues. Merging these advanced biomaterials with high-throughput methods to systematically, and in an unbiased manner, probe the role of scaffold biophysical and biochemical elements on stem cell fate will permit the identification of novel key stem cell behavioral effectors, allow improved in vitro replication of requisite in vivo niche functions, and, ultimately, have a profound impact on our understanding of stem cell biology and unlock their clinical potential in tissue engineering and regenerative medicine.

Exploring cross-task compatibility in perceiving and producing facial expressions using electromyography.

Using a dual-task methodology we examined the interaction of perceiving and producing facial expressions. In one task, participants were asked to produce a smile or a frown (Task 2) in response to a tone stimulus. This auditory-facial task was embedded in a dual-task context, where the other task (Task 1) required a manual response to visual face stimuli (visual-manual task). These face stimuli showed facial expressions that were either compatible or incompatible to the to-be-produced facial expression. Both reaction times and error rates (measured by facial electromyography) revealed a robust stimulus-response compatibility effect across tasks, suggesting that perceived social actions automatically activate corresponding actions even if perceived and produced actions belong to different tasks. The dual-task nature of this compatibility effect further testifies that encoding of facial expressions is highly automatic.

Interference in simultaneously perceiving and producing facial expressions--evidence from electromyography.

The goal of the current studies was to examine perception-action interactions in a socially relevant domain. Social interactions are based on a mutual understanding of the emotions and actions of others. We assume that the perception of emotional actions also stimulates a parallel action preparation in the perceiver, underlining the common coding theory. We report two experiments aimed to examine whether the perception of socially relevant facial actions (e.g., happy vs. angry facial expressions) interact with the execution of such actions. More specifically, we use a stimulus-response compatibility paradigm, in which subjects responded to the gender of a face by either smiling or frowning while ignoring the fact that the presented face is also randomly either smiling or frowning. We measured reaction time (RT) as onset latency on the two large muscle groups used for smiling (zygomaticus major) and frowning (corrugator supercilii) using electromyography. Experiment 1 showed that on compatible trials, in which perceived facial expression and actually produced facial expression matched, RTs were shorter than on incompatible trials. Experiment 2 used pre-instructed (i.e., blocked) responses and replicated the compatibility effect, suggesting that the effect is functionally located not in response selection but in response initiation or execution. We discuss these results in relation to cognitive mechanisms of common coding of perception and action and to the human mirror neuron system.