Optimization of Oligomer Stamping Technique for Normally Closed Elastomeric Valves on Glass Substrate.

Microscale elastomeric valves are an integral part of many lab-on-chip applications. Normally closed valves require lower actuation pressures to form tight seals, making them ideal for portable devices. However, fabrication of normally closed valves is typically more difficult because the valve structure must be selectively bonded to its substrate. In this work, an oligomer stamping technique for selective bonding of normally closed valves is optimized for bonding of PDMS devices on glass substrates. Contact angle and blister bursting testing measurements are used to quantitatively characterize the oligomer stamping process for the first time, and recommendations are made for plasma treatment conditions, microstamping technique, and valve construction. Glass-PDMS devices are ideal for lab-on-chip systems that integrate electrodes on the rigid glass substrate. Here, integrated electrodes are used to assess valve performance, demonstrating electrical isolation in excess of 8 MΩ over the biologically relevant frequency range in the closed state. Further, electrical measurement is used to demonstrate that the valve design can operate under a pulsed actuation scheme, sealing to withstand fluid pressures in excess of 200 mbar.

Cellular signaling pathways as plastic, proto-cognitive systems: Implications for biomedicine.

Many aspects of health and disease are modeled using the abstraction of a "pathway"-a set of protein or other subcellular activities with specified functional linkages between them. This metaphor is a paradigmatic case of a deterministic, mechanistic framework that focuses biomedical intervention strategies on altering the members of this network or the up-/down-regulation links between them-rewiring the molecular hardware. However, protein pathways and transcriptional networks exhibit interesting and unexpected capabilities such as trainability (memory) and information processing in a context-sensitive manner. Specifically, they may be amenable to manipulation via their history of stimuli (equivalent to experiences in behavioral science). If true, this would enable a new class of biomedical interventions that target aspects of the dynamic physiological "software" implemented by pathways and gene-regulatory networks. Here, we briefly review clinical and laboratory data that show how high-level cognitive inputs and mechanistic pathway modulation interact to determine outcomes in vivo. Further, we propose an expanded view of pathways from the perspective of basal cognition and argue that a broader understanding of pathways and how they process contextual information across scales will catalyze progress in many areas of physiology and neurobiology. We argue that this fuller understanding of the functionality and tractability of pathways must go beyond a focus on the mechanistic details of protein and drug structure to encompass their physiological history as well as their embedding within higher levels of organization in the organism, with numerous implications for data science addressing health and disease. Exploiting tools and concepts from behavioral and cognitive sciences to explore a proto-cognitive metaphor for the pathways underlying health and disease is more than a philosophical stance on biochemical processes; at stake is a new roadmap for overcoming the limitations of today's pharmacological strategies and for inferring future therapeutic interventions for a wide range of disease states.

Ion Channel Drugs Suppress Cancer Phenotype in NG108-15 and U87 Cells: Toward Novel Electroceuticals for Glioblastoma.

Glioblastoma is a lethal brain cancer that commonly recurs after tumor resection and chemotherapy treatment. Depolarized resting membrane potentials and an acidic intertumoral extracellular pH have been associated with a proliferative state and drug resistance, suggesting that forced hyperpolarization and disruption of proton pumps in the plasma membrane could be a successful strategy for targeting glioblastoma overgrowth. We screened 47 compounds and compound combinations, most of which were ion-modulating, at different concentrations in the NG108-15 rodent neuroblastoma/glioma cell line. A subset of these were tested in the U87 human glioblastoma cell line. A FUCCI cell cycle reporter was stably integrated into both cell lines to monitor proliferation and cell cycle response. Immunocytochemistry, electrophysiology, and a panel of physiological dyes reporting voltage, calcium, and pH were used to characterize responses. The most effective treatments on proliferation in U87 cells were combinations of NS1643 and pantoprazole; retigabine and pantoprazole; and pantoprazole or NS1643 with temozolomide. Marker analysis and physiological dye signatures suggest that exposure to bioelectric drugs significantly reduces proliferation, makes the cells senescent, and promotes differentiation. These results, along with the observed low toxicity in human neurons, show the high efficacy of electroceuticals utilizing combinations of repurposed FDA approved drugs.

A flow through device for simultaneous dielectrophoretic cell trapping and AC electroporation.

Isolation of cells and their transfection in a controlled manner is an integral step in cell biotechnology. Electric field approaches such as dielectrophoresis (DEP) offers a more viable method for targeted immobilization of cells without any labels. For transfection of cells to incorporate exogenous materials, electrical methods such as electroporation, are preferred over chemical and viral delivery methods since they minimally affect cell viability and can target many types. However prior approaches to both methods required multiple excitation sources, an AC source for DEP-based trapping and another DC source for electroporation. In this paper, we present a first of its kind flow through lab-on-chip platform using a single AC excitation source for combined trapping using negative dielectrophoresis (nDEP) and AC electroporation. Use of AC fields for electroporation eliminates the unwanted side effects of electrolysis or joule heating at electrodes compared to DC electroporation. Adjusting the flow rate and the electrical parameters of the incident AC field precisely controls the operation (trap, trap with electroporation and release). The platform has been validated through trapping and simultaneous transfection of HEK-293 embryonic kidney cells with a plasmid vector containing a fluorescent protein tag. Numerical scaling analysis is provided that indicates promise for individual cell trapping and electroporation using low voltage AC fields.

Bioinspired Three-Dimensional Human Neuromuscular Junction Development in Suspended Hydrogel Arrays.

The physical connection between motoneurons and skeletal muscle targets is responsible for the creation of neuromuscular junctions (NMJs), which allow electrical signals to be translated to mechanical work. NMJ pathology contributes to the spectrum of neuromuscular, motoneuron, and dystrophic disease. Improving in vitro tools that allow for recapitulation of the physiology of the neuromuscular connection will enable researchers to better understand the development and maturation of NMJs, and will help to decipher mechanisms leading to NMJ degeneration. In this work, we first describe robust differentiation of bungarotoxin-positive human myotubes, as well as a reproducible method for encapsulating and aligning human myoblasts in three-dimensional (3D) suspended culture using bioprinted silk fibroin cantilevers as cell culture supports. Further analysis with coculture of motoneuron-like cells demonstrates feasibility of fully human coculture using two-dimensional and 2.5-dimensional culture methods, with appropriate differentiation of both cell types. Using these coculture differentiation conditions with motoneuron-like cells added to monocultures of 3D suspended human myotubes, we then demonstrate synaptic colocalization in coculture as well as acetylcholine and glutamic acid stimulation of human myocytes. This method represents a unique platform to coculture suspended human myoblast-seeded 3D hydrogels with integrated motoneuron-like cells derived from human induced neural stem cells. The platform described is fully customizable using 3D freeform printing into standard laboratory tissue culture materials, and allows for human myoblast alignment in 3D with precise motoneuron integration into preformed myotubes. The coculture method will ideally be useful in observation and analysis of neurite outgrowth and myogenic differentiation in 3D with quantification of several parameters of muscle innervation and function.

The body electric 2.0: recent advances in developmental bioelectricity for regenerative and synthetic bioengineering.

Breakthroughs in biomedicine and synthetic bioengineering require predictive, rational control over anatomical structure and function. Recent successes in manipulating cellular and molecular hardware have not been matched by progress in understanding the patterning software implemented during embryogenesis and regeneration. A fundamental capability gap is driving desired changes in growth and form to address birth defects and traumatic injury. Here we review new tools, results, and conceptual advances in an exciting emerging field: endogenous non-neural bioelectric signaling, which enables cellular collectives to make global decisions and implement large-scale pattern homeostasis. Spatially distributed electric circuits regulate gene expression, organ morphogenesis, and body-wide axial patterning. Developmental bioelectricity facilitates the interface to organ-level modular control points that direct patterning in vivo. Cracking the bioelectric code will enable transformative progress in bioengineering and regenerative medicine.

Gap junctional signaling in pattern regulation: Physiological network connectivity instructs growth and form.

Gap junctions (GJs) are aqueous channels that allow cells to communicate via physiological signals directly. The role of gap junctional connectivity in determining single-cell functions has long been recognized. However, GJs have another important role: the regulation of large-scale anatomical pattern. GJs are not only versatile computational elements that allow cells to control which small molecule signals they receive and emit, but also establish connectivity patterns within large groups of cells. By dynamically regulating the topology of bioelectric networks in vivo, GJs underlie the ability of many tissues to implement complex morphogenesis. Here, a review of recent data on patterning roles of GJs in growth of the zebrafish fin, the establishment of left-right patterning, the developmental dysregulation known as cancer, and the control of large-scale head-tail polarity, and head shape in planarian regeneration has been reported. A perspective in which GJs are not only molecular features functioning in single cells, but also enable global neural-like dynamics in non-neural somatic tissues has been proposed. This view suggests a rich program of future work which capitalizes on the rapid advances in the biophysics of GJs to exploit GJ-mediated global dynamics for applications in birth defects, regenerative medicine, and morphogenetic bioengineering. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 643-673, 2017.

Hypoxia Inducible Factor 1 Alpha Is Expressed in Germ Cells throughout the Murine Life Cycle.

Pluripotent stem cells of the early embryo, and germ line cells, are essential to ensure uncompromised development to adulthood as well as species propagation, respectively. Recently, the transcription factor hypoxia inducible factor 1 alpha (Hif1α) has been shown to have important roles in embryonic stem cells; in particular, regulation of conversion to glycolytic metabolism and, as we have shown, maintenance of functional levels of telomerase. In the present study, we sought to assess whether Hif1α was also expressed in the primitive cells of the murine embryo. We observed expression of Hif1α in pre-implantation embryos, specifically the 2-cell stage, morula, and blastocyst. Robust Hif1α expression was also observed in male and female primordial germ cells. We subsequently assessed whether Hif1α was expressed in adult male and female germ cells. In the testis, Hif1α was robustly expressed in spermatogonial cells, in both juvenile (6-week old) and adult (3-month old) males. In the ovaries, Hif1α was expressed in mature oocytes from adult females, as assessed both in situ and in individual oocytes flushed from super-ovulated females. Analysis of Hif1α transcript levels indicates a mechanism of regulation during early development that involves stockpiling of Hif1α protein in mature oocytes, presumably to provide protection from hypoxic stress until the gene is re-activated at the blastocyst stage. Together, these observations show that Hif1α is expressed throughout the life-cycle, including both the male and female germ line, and point to an important role for Hif1α in early progenitor cells.

Stem cells, telomerase regulation and the hypoxic state.

The cellular response to a hypoxic environment is regulated by hypoxia inducible factors. Hypoxia inducible factor 1 alpha (Hif1alpha) in particular, is tightly regulated by the hypoxic environment in most cells, and plays an important role in regulating the stress response of cells to hypoxia. Interestingly, substantial observations are now emerging that point to an important role for Hif1alpha in stem cells, including embryonic stem cells, neuronal stem cells and hematopoietic stem cells. Notably, Hif1alpha has been shown to enhance self renewal of stem cells, mediate a shift to glycolytic metabolism, and promote telomerase expression.

Human adipose stem cell and ASC-derived cardiac progenitor cellular therapy improves outcomes in a murine model of myocardial infarction.

BACKGROUND: Adipose tissue is an abundant and potent source of adult stem cells for transplant therapy. In this study, we present our findings on the potential application of adipose-derived stem cells (ASCs) as well as induced cardiac-like progenitors (iCPs) derived from ASCs for the treatment of myocardial infarction. METHODS AND RESULTS: Human bone marrow (BM)-derived stem cells, ASCs, and iCPs generated from ASCs using three defined cardiac lineage transcription factors were assessed in an immune-compromised mouse myocardial infarction model. Analysis of iCP prior to transplant confirmed changes in gene and protein expression consistent with a cardiac phenotype. Endpoint analysis was performed 1 month posttransplant. Significantly increased endpoint fractional shortening, as well as reduction in the infarct area at risk, was observed in recipients of iCPs as compared to the other recipient cohorts. Both recipients of iCPs and ASCs presented higher myocardial capillary densities than either recipients of BM-derived stem cells or the control cohort. Furthermore, mice receiving iCPs had a significantly higher cardiac retention of transplanted cells than all other groups. CONCLUSION: Overall, iCPs generated from ASCs outperform BM-derived stem cells and ASCs in facilitating recovery from induced myocardial infarction in mice.

Effective Targeted Gene Knockdown in Mammalian Cells Using the piggyBac Transposase-based Delivery System.

Nonviral gene delivery systems are rapidly becoming a desirable and applicable method to overexpress genes in various types of cells. We have recently developed a piggyBac transposase-based, helper-independent and self-inactivating delivery system (pmGENIE-3) capable of high-efficiency transfection of mammalian cells including human cells. In the following study, we have assessed the potential of this delivery system to drive the expression of short hairpin RNAs to knock down genes in human cells. Two independent pmGENIE-3 vectors were developed to specifically target knockdown of an endogenous gene, telomerase reverse transcriptase (TERT), in telomerase-positive human immortalized cell lines. As compared with a transposase-deficient vector, pmGENIE-3 showed significantly improved short-term transfection efficiency (~4-fold enhancement, 48 hours posttransfection) and long-term integration efficiency (~5-fold enhancement) following antibiotic selection. We detected a significant reduction of both TERT expression and telomerase activity in both HEK293 and MCF-7 breast carcinoma cells transfected with two pmGENIE-3 construct targeting distinct regions of TERT. Importantly, this knockdown of expression was sufficient to abrogate telomerase function since telomeres were significantly shortened (3-4 Kb, P < 0.001) in both TERT-targeted cell lines following antibiotic selection of stable integrants. Together, these data show the capacity of the piggyBac nonviral delivery system to stably knockdown gene expression in mammalian cells and indicate the potential to develop novel tumor-targeting therapies.Molecular Therapy-Nucleic Acids (2013) 2, e137; doi:10.1038/mtna.2013.61; published online 3 December 2013.