Showing papers in "Cellular and Molecular Bioengineering in 2014"

Generation of Multi-Scale Vascular Network System within 3D Hydrogel using 3D Bio-Printing Technology.

Abstract: Although 3D bio-printing technology has great potential in creating complex tissues with multiple cell types and matrices, maintaining the viability of thick tissue construct for tissue growth and maturation after the printing is challenging due to lack of vascular perfusion. Perfused capillary network can be a solution for this issue; however, construction of a complete capillary network at single cell level using the existing technology is nearly impossible due to limitations in time and spatial resolution of the dispensing technology. To address the vascularization issue, we developed a 3D printing method to construct larger (lumen size of ~1 mm) fluidic vascular channels and to create adjacent capillary network through a natural maturation process, thus providing a feasible solution to connect the capillary network to the large perfused vascular channels. In our model, microvascular bed was formed in between two large fluidic vessels, and then connected to the vessels by angiogenic sprouting from the large channel edge. Our bio-printing technology has a great potential in engineering vascularized thick tissues and vascular niches, as the vascular channels are simultaneously created while cells and matrices are printed around the channels in desired 3D patterns.

Nuclear Deformability Constitutes a Rate-Limiting Step During Cell Migration in 3-D Environments

Abstract: Cell motility plays a critical role in many physiological and pathological settings, ranging from wound healing to cancer metastasis. While cell migration on 2-dimensional (2-D) substrates has been studied for decades, the physical challenges cells face when moving in 3-D environments are only now emerging. In particular, the cell nucleus, which occupies a large fraction of the cell volume and is normally substantially stiffer than the surrounding cytoplasm, may impose a major obstacle when cells encounter narrow constrictions in the interstitial space, the extracellular matrix, or small capillaries. Using novel microfluidic devices that allow observation of cells moving through precisely defined geometries at high spatial and temporal resolution, we determined nuclear deformability as a critical factor in the cells’ ability to pass through constrictions smaller than the size of the nucleus. Furthermore, we found that cells with reduced levels of the nuclear envelope proteins lamins A/C, which are the main determinants of nuclear stiffness, passed significantly faster through narrow constrictions during active migration and passive perfusion. Given recent reports that many human cancers have altered lamin expression, our findings suggest a novel biophysical mechanism by which changes in nuclear structure and composition may promote cancer cell invasion and metastasis.

Developing Defined and Scalable 3D Culture Systems for Culturing Human Pluripotent Stem Cells at High Densities

Abstract: Human pluripotent stem cells (hPSCs)—including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs)—are very promising candidates for cell therapies, tissue engineering, high throughput pharmacology screens, and toxicity testing. These applications require large numbers of high quality cells; however, scalable production of human pluripotent stem cells and their derivatives at a high density and under well-defined conditions has been a challenge. We recently reported a simple, efficient, fully defined, scalable, and good manufacturing practice (GMP) compatible 3D culture system based on a thermoreversible hydrogel for hPSC expansion and differentiation. Here, we describe additional design rationale and characterization of this system. For instance, we have determined that culturing hPSCs as a suspension in a liquid medium can exhibit lower volumetric yields due to cell agglomeration and possible shear force-induced cell loss. By contrast, using hydrogels as 3D scaffolds for culturing hPSCs reduces aggregation and may insulate from shear forces. Additionally, hydrogel-based 3D culture systems can support efficient hPSC expansion and differentiation at a high density if compatible with hPSC biology. Finally, there are considerable opportunities for future development to further enhance hydrogel-based 3D culture systems for producing hPSCs and their progeny.

Epigenetic Mechanism in Regulation of Endothelial Function by Disturbed Flow: Induction of DNA Hypermethylation by DNMT1

Abstract: There is increasing evidence that epigenetic mechanisms such as changes in DNA methylation and histone modification play an important role in regulating cellular functions in physiological and pathophysiological states. We investigated the effects of hemodynamic force disturbance, one of the risk factors for atherogenesis, on DNA methylation in HUVECs and rat carotid arteries. Our results demonstrated that athero-prone oscillatory shear flow (OS) without a clear direction induces DNA hypermethylation in comparison to the athero-protective pulsatile shear flow (PS) with a definite direction. Furthermore, OS increases the expression and nuclear translocation of DNA methyltransferase 1 (DNMT1), which is a major maintenance DNA methyltransferase that adds methyl groups to hemi-methylated DNA to repress gene expression. Pharmacological inhibition of DNMT1 by 5-Aza-2'-deoxycytidine abolished the OS-induced DNA hypermethylation. In vivo experiments also showed increases of DNMT1 expression and DNA methylation in the partially-ligated rat carotid arteries where the shear flow is disturbed. These in vitro and in vivo findings have provided novel evidence of the differential regulation of DNA methylation by different hemodynamic forces acting on vascular endothelium and identified DNMT1 as a key protein that governs the epigenetic changes in response to the pathophysiological stimuli due to disturbed flow.

Optimization of Aqueous Biphasic Tumor Spheroid Microtechnology for Anti-Cancer Drug Testing in 3D Culture.

Abstract: Tumor spheroids are three-dimensional clusters of cancer cells that exhibit characteristics of poorly perfused tumors and hence present a relevant model for testing the efficacy of anti-cancer compounds. The use of spheroids for drug screening is hindered by technological complexities for high throughput generation of consistent size spheroids individually addressable by drug compounds. Here we present and optimize a simple spheroid technology based on the use of an aqueous two-phase system. Cancer cells confined in a drop of the denser aqueous dextran phase are robotically dispensed into a microwell containing the immersion aqueous polyethylene glycol phase. Cells remain within the drop and form a viable spheroid, without a need for any external stimuli. The size of resulting spheroids is sensitive to volume variations of dispensed drops from the air displacement pipetting head of a commercial liquid handling robot. Therefore, we parametrically optimize the process of dispensing of dextran phase drops. For a given cell density, this optimization reproducibly generates consistent size spheroids in standard 96-well plates. In addition, we evaluate the use of a commercial biochemical assay to examine cellular viability of cancer cell spheroids. Spheroids show a dose-dependent response to cisplatin similar to a monolayer culture. However unlike their two-dimensional counterpart, spheroids exhibit resistance to paclitaxel treatment. This technology, which uses only commercially-available reagents and equipment, can potentially expedite anti-cancer drug discovery. Although the use of robotics makes the ATPS spheroid technology particularly useful for drug screening applications, this approach is compatible with simpler liquid handling techniques such as manual micropipetting and offers a straightforward method of 3D cell culture in research laboratories.

Multiscale Particle-Based Modeling of Flowing Platelets in Blood Plasma Using Dissipative Particle Dynamics and Coarse Grained Molecular Dynamics

Abstract: We developed a multiscale particle-based model of platelets, to study the transport dynamics of shear stresses between the surrounding fluid and the platelet membrane. This model facilitates a more accurate prediction of the activation potential of platelets by viscous shear stresses—one of the major mechanisms leading to thrombus formation in cardiovascular diseases and in prosthetic cardiovascular devices. The interface of the model couples coarse-grained molecular dynamics (CGMD) with dissipative particle dynamics (DPD). The CGMD handles individual platelets while the DPD models the macroscopic transport of blood plasma in vessels. A hybrid force field is formulated for establishing a functional interface between the platelet membrane and the surrounding fluid, in which the microstructural changes of platelets may respond to the extracellular viscous shear stresses transferred to them. The interaction between the two systems preserves dynamic properties of the flowing platelets, such as the flipping motion. Using this multiscale particle-based approach, we have further studied the effects of the platelet elastic modulus by comparing the action of the flow-induced shear stresses on rigid and deformable platelet models. The results indicate that neglecting the platelet deformability may overestimate the stress on the platelet membrane, which in turn may lead to erroneous predictions of the platelet activation under viscous shear flow conditions. This particle-based fluid–structure interaction multiscale model offers for the first time a computationally feasible approach for simulating deformable platelets interacting with viscous blood flow, aimed at predicting flow induced platelet activation by using a highly resolved mapping of the stress distribution on the platelet membrane under dynamic flow conditions.

Activation of a Bacterial Mechanosensitive Channel in Mammalian Cells by Cytoskeletal Stress

Abstract: Cells can sense a myriad of mechanical stimuli. Mechanosensitive channel of large conductance (MscL) found in bacteria is a well-characterized mechanosensitive channel that rapidly responds to an increase in turgor pressure. Functional expression of MscL in mammalian cells has recently been demonstrated, revealing that molecular delivery or transport can be achieved by charge-induced activation of MscL. Despite a well-accepted mechanism for MscL activation by membrane tension in bacteria, it is not clear whether and how MscL can be opened by other modes of force transduction in mammalian cells. In this work, we used a variety of techniques to characterize the gating of MscL expressed in mammalian cells, using both wild type and a G22S mutant which activates at a lower threshold. In particular, employing a recently developed technique, acoustic tweezing cytometry (ATC), we show that ultrasound actuation of integrin-bound microbubbles can lead to MscL opening and that ATC induced MscL activation was dependent on the functional linkage of the microbubbles with an intact actin cytoskeleton. Our results indicate that localized mechanical stress can mediate opening of MscL that requires force transduction through the actin cytoskeleton, revealing a new mode of MscL activation that may prove to be a useful tool for mechanobiology and drug delivery research.

Substrates with Engineered Step Changes in Rigidity Induce Traction Force Polarity and Durotaxis

Abstract: Rigidity sensing plays a fundamental role in multiple cell functions ranging from migration, to proliferation and differentiation (Engler et al., Cell 126:677–689, 2006; Lo et al., Biophys. J. 79:144–152, 2000; Wells, Hepatology 47:1394–1400, 2008; Zoldan et al., Biomaterials 32:9612–9621, 2011). During migration, single cells have been reported to preferentially move toward more rigid regions of a substrate in a process termed durotaxis. Durotaxis could contribute to cell migration in wound healing and gastrulation, where local gradients in tissue rigidity have been described. Despite the potential importance of this phenomenon to physiology and disease, it remains unclear how rigidity guides these behaviors and the underlying cellular and molecular mechanisms. To investigate the functional role of subcellular distribution and dynamics of cellular traction forces during durotaxis, we developed a unique microfabrication strategy to generate elastomeric micropost arrays patterned with regions exhibiting two different rigidities juxtaposed next to each other. After initial cell attachment on the rigidity boundary of the micropost array, NIH 3T3 fibroblasts were observed to preferentially migrate toward the rigid region of the micropost array, indicative of durotaxis. Additionally, cells bridging two rigidities across the rigidity boundary on the micropost array developed stronger traction forces on the more rigid side of the substrate indistinguishable from forces generated by cells exclusively seeded on rigid regions of the micropost array. Together, our results highlighted the utility of step-rigidity micropost arrays to investigate the functional role of traction forces in rigidity sensing and durotaxis, suggesting that cells could sense substrate rigidity locally to induce an asymmetrical intracellular traction force distribution to contribute to durotaxis.

In Vitro Microvessel Growth and Remodeling within a Three-dimensional Microfluidic Environment

Abstract: This paper presents in vitro microvascular network formation within 3D gel scaffolds made from different concentrations of type-I collagen, fibrin, or a mixture of collagen and fibrin, using a simple microfluidic platform. Initially, microvascular network formation of human umbilical vein endothelial cells was examined using live time-lapse confocal microscopy every 90 min from 3 h to 12 h after seeding within three different concentrations of collagen gel scaffolds. Among the three conditions of collagen gel scaffolds (2.0 mg/ml, 2.5 mg/ml, and 3.0 mg/ml), the number of skeleton within collagen gel scaffolds was consistently the highest (3.0 mg/ml), followed by those of collagen gel scaffolds (2.5 mg/ml and 2.0 mg/ml). Results demonstrated that concentration of collagen gel scaffolds, which influences matrix stiffness and ligand density, may affect microvascular network formation during the early stages of vasculogenesis. In addition, the maturation of microvascular networks in monoculture under different gel compositions within gel scaffolds (2.5 mg/ml) was examined for 7 d using live confocal microscopy. It was confirmed that pure fibrin gel scaffolds are preferable to collagen gel or collagen/fibrin combinations, significantly reducing matrix retractions during maturation of microvascular networks for 7 d. Finally, early steps in the maturation process of microvascular networks for 14 d were characterized by demonstrating sequential steps of branching, expanding, remodeling, pruning, and clear delineation of lumens within fibrin gel scaffolds. Our findings demonstrate an in vitro model for generating mature microvascular networks within 3D microfluidic fibrin gel scaffolds (2.5 mg/ml), and furthermore suggest the importance of gel concentration and composition in promoting the maturation of microvascular networks.

Porous Membranes Promote Endothelial Differentiation of Adipose-Derived Stem Cells and Perivascular Interactions

Abstract: Efforts to create physiologically relevant microenvironments for cell differentiation have led to the creation of three-dimensional (3D) support matrices with varying physical and chemical properties. In an effort to simplify the complexity of these matrices, while maintaining their unique permeable nature, we investigated the culture and differentiation of adipose-derived stem cells (ADSCs) on porous membranes. Membranes offer many of the benefits of a 3D matrix, but simplify cell seeding, imaging, analysis and post-differentiation recovery. After inducing the differentiation of ADSCs into endothelial cells (ECs), the cells cultured on porous substrates produce more branch points and greater tube length in angiogenesis assays compared to the cells cultured on non-porous controls. While we confirm that ADSCs can be induced with vascular endothelial growth factor to express endothelial adhesion molecule CD31 (PECAM-1), only when co-cultured across a membrane with human umbilical vein endothelial cells (HUVECs), do a subset of ADSCs show appropriate CD31 distribution along cell boundaries. Others have recently described that mesenchymal stem cells derive from perivascular cells including pericytes, which are known to wrap circumferentially around microvessels. We used ultrathin porous membranes to permit limited physical interactions between polarized HUVECs and ADSCs. In this arrangement, we found that the majority of ADSCs aligned perpendicular to the polarized HUVECs even though contact between the cell types was limited by pores less than 500 nm in diameter. Together, this ADSC pericyte behavior in combination with the ability to differentiate into ECs shows the potential versatility of ADSCs in the engineering of vascular networks.

Microscale Bioadhesive Hydrogel Arrays for Cell Engineering Applications

Abstract: Bioengineered hydrogels have been explored in cell and tissue engineering applications to support cell growth and modulate its behavior. A rationally designed scaffold should allow for encapsulated cells to survive, adhere, proliferate, remodel the niche, and can be used for controlled delivery of biomolecules. Here we report a microarray of composite bioadhesive microgels with modular dimensions, tunable mechanical properties and bulk modified adhesive biomolecule composition. Composite bioadhesive microgels of maleimide functionalized polyethylene glycol (PEG-MAL) with interpenetrating network (IPN) of gelatin ionically cross-linked with silicate nanoparticles were engineered by integrating microfabrication with Michael-type addition chemistry and ionic gelation. By encapsulating clinically relevant anchorage-dependent cervical cancer cells and suspension leukemia cells as cell culture models in these composite microgels, we demonstrate enhanced cell spreading, survival, and metabolic activity compared to control gels. The composite bioadhesive hydrogels represent a platform that could be used to study independent effect of stiffness and adhesive ligand density on cell survival and function. We envision that such microarrays of cell adhesive microenvironments, which do not require harsh chemical and UV crosslinking conditions, will provide a more efficacious cell culture platform that can be used to study cell behavior and survival, function as building blocks to fabricate 3D tissue structures, cell delivery systems, and high throughput drug screening devices.

Nuclear stiffening inhibits migration of invasive melanoma cells

Abstract: During metastasis, melanoma cells must be sufficiently deformable to squeeze through extracellular barriers with small pore sizes. We visualize and quantify deformability of single cells using micropipette aspiration and examine the migration potential of a population of melanoma cells using a flow migration apparatus. We artificially stiffen the nucleus with recombinant overexpression of Δ50 lamin A, which is found in patients with Hutchison Gilford progeria syndrome and in aged individuals. Melanoma cells, both WM35 and Lu1205, both show reduced nuclear deformability and reduced cell invasion with the expression of Δ50 lamin A. These studies suggest that cellular aging including expression of Δ50 lamin A and nuclear stiffening may reduce the potential for metastatic cancer migration. Thus, the pathway of cancer metastasis may be kept in check by mechanical factors in addition to known chemical pathway regulation.

Design and Synthesis of an Artificial Pulmonary Pleura for High Throughput Studies in Acellular Human Lungs

Abstract: Whole organ decellularization of complex organs, such as lungs, presents a unique opportunity for use of acellular scaffolds for ex vivo tissue engineering or for studying cell–extracellular matrix interactions ex vivo. A growing body of literature investigating decellularizing and recellularizing rodent lungs has provided important proof of concept models and rodent lungs are readily available for high throughput studies. In contrast, comparable progress in large animal and human lungs has been impeded owing to more limited availability and difficulties in handling larger tissue. While the use of smaller segments of acellular large animal or human lungs would maximize usage from a single lung, excision of small acellular segments compromises the integrity of the pleural layer, leaving the terminal ends of blood vessels and airways exposed. We have developed a novel pleural coating using non-toxic ionically crosslinked alginate or photocrosslinked methacrylated alginate which can be applied to excised acellular lung segments, permits inflation of small segments, and significantly enhances retention of cells inoculated through cannulated airways or blood vessels. Further, photocrosslinking methacrylated alginate, using eosin Y and triethanolamine at 530 nm wavelength, results in a mechanically stable pleural coating that permits effective cyclic 3-dimensional stretch, i.e., mechanical ventilation, of individual segments.

Fluid Mechanical Forces and Endothelial Mitochondria: A Bioengineering Perspective.

Abstract: Endothelial cell dysfunction is the hallmark of every cardiovascular disease/condition, including atherosclerosis and ischemia/reperfusion injury. Fluid shear stress acting on the vascular endothelium is known to regulate cell homeostasis. Altered hemodynamics is thought to play a causative role in endothelial dysfunction. The dysfunction is associated with/preceded by mitochondrial oxidative stress. Studies by our group and others have shown that the form and/or function of the mitochondrial network are affected when endothelial cells are exposed to shear stress in the absence or presence of additional physicochemical stimuli. The present review will summarize the current knowledge on the interconnections among intracellular Ca2+ - nitric oxide - mitochondrial reactive oxygen species, mitochondrial fusion/fission, autophagy/mitophagy, and cell apoptosis vs. survival. More specifically, it will list the evidence on potential regulation of the above intracellular species and processes by the fluid shear stress acting on the endothelium under either physiological flow conditions or during reperfusion (following a period of ischemia). Understanding how the local hemodynamics affects mitochondrial physiology and the cell redox state may lead to development of novel therapeutic strategies for prevention or treatment of the endothelial dysfunction and, hence, of cardiovascular disease.

Effect of Static Pre-stretch Induced Surface Anisotropy on Orientation of Mesenchymal Stem Cells

Abstract: Mechanical cues in the cellular environment play important roles in guiding various cell behaviors, such as cell alignment, migration, and differentiation. Previous studies investigated mechanical stretch guided cell alignment predominantly with cyclic stretching whereby an external force is applied to stretch the substrate dynamically (i.e., cyclically) while the cells are attached onto the substrate. In contrast, we created a static pre-stretched anisotropic surface in which the cells were seeded subsequent to stretching the substrate. We hypothesized that the cell senses the physical environment through a more active mechanism, namely, even without external forces the cell can actively apply traction and sense an increased stiffness in the stretched direction and align in that direction. To test our hypothesis, we quantified the extent of pre-stretch induced anisotropy by employing the theory of small deformation superimposed on large and predicted the effective stiffness in the stretch direction as well as its perpendicular direction. We showed mesenchymal stem cells (MSC) aligned in the pre-stretched direction, and the cell alignment and morphology were dependent on the pre-stretch magnitude. In addition, the pre-stretched surface demonstrated an ability to promote early myoblast differentiation of the MSC. This study is the first report on MSC alignment on a statically pre-stretched surface. The cell orientation induced by the pre-stretch induced anisotropy could provide insight into tissue engineering applications involving cells that aligned in vivo in the absence of dynamic mechanical stimuli.

Depolarization of Cellular Resting Membrane Potential Promotes Neonatal Cardiomyocyte Proliferation In Vitro

Abstract: Cardiomyocytes (CMs) undergo a rapid transition from hyperplastic to hypertrophic growth soon after birth, which is a major challenge to the development of engineered cardiac tissue for pediatric patients. Resting membrane potential (Vmem) has been shown to play an important role in cell differentiation and proliferation during development. We hypothesized that depolarization of neonatal CMs would stimulate or maintain CM proliferation in vitro. To test our hypothesis, we isolated postnatal day 3 neonatal rat CMs and subjected them to sustained depolarization via the addition of potassium gluconate or Ouabain to the culture medium. Cell density and CM percentage measurements demonstrated an increase in mitotic CMs along with a ~twofold increase in CM numbers with depolarization. In addition, depolarization led to an increase in cells in G2 and S phase, indicating increased proliferation, as measured by flow cytometry. Surprisingly depolarization of Vmem with either treatment led to inhibition of proliferation in cardiac fibroblasts. This effect is abrogated when the study was carried out on postnatal day 7 neonatal CMs, which are less proliferative, indicating that the likely mechanism of depolarization is the maintenance of the proliferating CM population. In summary, our findings suggest that depolarization maintains postnatal CM proliferation and may be a novel approach to encourage growth of engineered tissue and cardiac regeneration in pediatric patients.

Characterization of mechanical and regenerative properties of human, adipose stromal cells.

Abstract: The stromal vascular fraction (SVF) of human adipose tissue is a heterogeneous population, with component cell types that may or may not contribute to its regenerative potential. Recent findings indicate that single-cell mechanical biomarkers are characteristic of cell type and can be used comparably to gene and protein expressions to identify cell populations. In this study, we characterized mechanical properties and differentiation potential of cell types present in the SVF. Fluorescence-activated cell sorting was used to isolate four distinct populations based on surface markers: endothelial cells (ECs), adipose-derived stem cells (ASCs), pre-adipocytes, and smooth muscle cells (SMCs). Atomic force microscopy was used to mechanically characterize sorted cell populations and unsorted SVF. Differentiation capabilities of sorted and unsorted populations were evaluated by quantifying lipid production and calcified matrix deposition. Cells populating the SVF exhibited a range of mechanical properties, with ECs, ASCs, pre-adipocytes, and unsorted SVF cells being significantly more compliant than SMCs. Lineage-specific metabolite production was most robust in SVF cells, followed by ASCs, with the other cell types showing little or no potential, suggesting the unsorted populations may benefit from a paracrine response that is lacking once the cells are sorted into more uniform cell populations.

Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment

Abstract: Cell–matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions in vitro can be difficult because the extracellular matrix (ECM) proteins that adsorb to in vitro cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to in vivo. Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell–matrix adhesions and cell behavior in 2D and 3D microenvironments.

Shear Stress-Induced NO Production is Dependent on ATP Autocrine Signaling and Capacitative Calcium Entry

Abstract: Flow-induced production of nitric oxide (NO) by endothelial cells plays a fundamental role in vascular homeostasis. However, the mechanisms by which shear stress activates NO production remain unclear due in part to limitations in measuring NO, especially under flow conditions. Shear stress elicits the release of ATP, but the relative contribution of autocrine stimulation by ATP to flow-induced NO production has not been established. Furthermore, the importance of calcium in shear stress-induced NO production remains controversial, and in particular the role of capacitive calcium entry (CCE) has yet to be determined. We have utilized our unique NO measurement device to investigate the role of ATP autocrine signaling and CCE in shear stress-induced NO production. We found that endogenously released ATP and downstream activation of purinergic receptors and CCE plays a significant role in shear stress-induced NO production. ATP-induced eNOS phophorylation under static conditions is also dependent on CCE. Inhibition of protein kinase C significantly inhibited eNOS phosphorylation and the calcium response. To our knowledge, we are the first to report on the role of CCE in the mechanism of acute shear stress-induced NO response. In addition, our work highlights the importance of ATP autocrine signaling in shear stress-induced NO production.

Nanofibrous Electrospun Polymers for Reprogramming Human Cells

Abstract: Forced expression of transcription factors epigenetically reprograms somatic cells harvested from routine skin biopsies into induced pluripotent stem cells (iPSCs). Human iPSCs are key resources for drug discovery, regenerative medicine and tissue engineering. Here we developed a materials approach to explore how culture substrates could impact factor-mediated reprogramming of human fibroblasts. A materials library consisting of nanofibrous substrates with randomly oriented and aligned structures was prepared by electrospinning four polymers [polylactic acid (PLA), polycaprolactone (PCL), thermoplastic polyurethane (TPU) and polypropylene carbonate (PPC)] into nanofiber orientations. Adsorbing protein to each substrate permitted robust attachment of fibroblasts to all substrates. Fibroblasts on aligned substrates had elongated nuclei, but after reprogramming factor expression, nuclei became more circular. Reprogramming factors could override the nuclear shape constraints imposed by nanofibrous substrates, and the majority of substrates supported full reprogramming. Early culture on PCL and TPU substrates promoted reprogramming, and TGF-β repressed substrate effects. Partial least squares modeling of the biochemical and biophysical cues within our reprogramming system identified TGF-β and polymer identity as important cues governing cellular reprogramming responses. We believe that our approach of using a nanofibrous materials library can be used to dissect molecular mechanisms of reprogramming and generate novel substrates that enhance epigenetic reprogramming.

Measurement science in the circulatory system

Abstract: The dynamics of the cellular and molecular constituents of the circulatory system are regulated by the biophysical properties of the heart, vasculature and blood cells and proteins. In this review, we discuss measurement techniques that have been developed to characterize the physical and mechanical parameters of the circulatory system across length scales ranging from the tissue scale (cm) to the molecular scale (nm) and time scales of years to milliseconds. We compare the utility of measurement techniques as a function of spatial resolution and penetration depth from both a diagnostic and research perspective. Together, this review provides an overview of the utility of measurement science techniques to study the spatial systems of the circulatory system in health and disease.

Effects of migrating cell-induced matrix reorganization on 3D cancer cell migration

Abstract: The migration of cells is fundamental to a number of physiological/pathological processes, ranging from embryonic development, tissue regeneration to cancer metastasis. Current research on cell migration is largely based on simplified in vitro models that assume a homogeneous microenvironment and overlook the modification of extracellular matrix (ECM) by the cells. To address this shortcoming, we developed a nested three-dimensional (3D) collagen hydrogel model mimicking the connective tissue confronted by highly malignant breast cancer cells, MDA-MB-231. Strikingly, our findings revealed two distinct cell migration patterns: a rapid and directionally persistent collective migration of the leader cells and a more randomized migration in the regions that have previously been significantly modified by cells. The cell-induced modifications, which typically include clustering and alignment of fibers, effectively segmented the matrix into smaller sub-regions. Our results suggest that in an elastic 3D matrix, the presence of adjacent cells that have modified the matrix may in fact become physical hurdle to a migrating cell. Furthermore, our study emphasizes the need for a micromechanical understanding in the context of cancer invasion that allows for cell-induced modification of ECM and a heterogeneous cell migration.

Network Modeling Approach to Predict Myofibroblast Differentiation

Abstract: Fibrotic disease is a major cause of morbidity and mortality and is characterized by the transition of resident fibroblast cells into active myofibroblasts, identified by their expression of alpha smooth muscle actin. Myofibroblast differentiation is regulated by growth factor signaling and mechanical signals transduced through integrins, which converge at focal adhesion proteins (Src and FAK) and MAPK signaling, but lead to divergent outcomes. While details are known about individual pathways, little is known about their interactions. To this end, an ODE-based model of this cell signaling network was developed in parallel with in vitro experiments to analyze potential mechanisms of crosstalk and regulation of αSMA production. We found that cells lacking Src or FAK produce significantly less or more αSMA than wild type cells, respectively. Transforming growth factor beta 1 and fibroblast growth factor signal through ERK and MAPK p38 with different dynamic profiles to increase or decrease αSMA expression, respectively. Our model effectively recreated αSMA expression levels across a set of 22 experimental conditions and matched some features of transient phosphorylation of ERK and p38. These results support a potential mechanism for regulation of fibroblast differentiation: αSMA production is promoted by active p38 and Src and opposed by ERK.

Simulation Study of Interactions Between Kinesin's Neck Linker and Motor Domain

Abstract: The docking movement of kinesin’s neck linker to the motor domain is the force generation step of kinesin. This docking movement is achieved by various non-bonding interactions between neck linker and motor domain. However, the relative strength and the structural basis for these interactions are still unclear. To elucidate these problems, we unbind the docked neck linker from motor domain using steered molecular dynamics and mutation simulations. We find that the surprisingly high strength of a structure called the “ asparagine latch” arises from exquisite cooperation between hydrogen bonds and hydrophobic interactions. A β-sheet called the “ cover-neck bundle” behaves like an elastic beam in its unbinding process. The hydrophobic interaction between ILE327 (amino acid sequence in 2KIN) and the hydrophobic pocket on the motor domain is strong and provides a fixed support for elastic bending of the cover-neck bundle.

Quantitative Evaluation and Optimization of Co-drugging to Improve Anti-HIV Latency Therapy

Abstract: Human immunodeficiency virus 1 (HIV) latency remains a significant obstacle to curing infected patients. One promising therapeutic strategy is to purge the latent cellular reservoir by activating latent HIV with latency-reversing agents (LRAs). In some cases, co-drugging with multiple LRAs is necessary to activate latent infections, but few studies have established quantitative criteria for determining when co-drugging is required. Here we systematically quantified drug interactions between histone deacetylase inhibitors and transcriptional activators of HIV and found that the need for co-drugging is determined by the proximity of latent infections to the chromatin-regulated viral gene activation threshold at the viral promoter. Our results suggest two classes of latent viral integrations: those far from the activation threshold that benefit from co-drugging, and those close to the threshold that are efficiently activated by a single drug. Using a primary T cell model of latency, we further demonstrated that the requirement for co-drugging was donor dependent, suggesting that the host may set the level of repression of latent infections. Finally, we showed that single drug or co-drugging doses could be optimized, via repeat stimulations, to minimize unwanted side effects while maintaining robust viral activation. Our results motivate further study of patient-specific latency-reversing strategies.

Efficiency of Protease-Activatable Virus Nanonodes Tuned Through Incorporation of Wild-Type Capsid Subunits

Abstract: Virus nanonodes, a tunable multi-input protease-responsive gene delivery platform, was recently built by exploiting the self-assembly property of adeno-associated virus capsids. Upon detection of specific inputs (e.g., matrix metalloproteinases—MMPs), the engineered viruses output gene delivery to targeted cells. The first generation protease-activatable viruses (PAVs) displayed the desired protease-activated cellular receptor binding and transduction behaviors. However, the less than wild type (WT) level of gene delivery achieved by the prototype viruses has left room for improvement. In this report, we have devised a method to tackle this efficiency problem. Specifically, by controlling the ratio of WT to protease-activatable subunits in the assembled 60-mer virus capsid, we can easily increase the level of overall transduction achieved by the PAVs. Since a number of MMPs are overexpressed in a vast range of human pathologies, including cancer and cardiovascular disease, the protease-sensing viruses may find broad clinical use in future gene therapy applications.

Role of Three-Dimensional Matrix Stiffness in Regulating the Response of Human Neural Cells to Toxins

Abstract: Recent studies have highlighted the strong influence of microenvironmental stiffness on various cell fates including proliferation, migration, and differentiation in both two- and three-dimensional culture conditions. Our study attempts to understand the role of 3D microenvironmental stiffness on cell cytotoxic response using neural glioblastoma U-87 cells encapsulated in alginate hydrogel matrices of varying stiffness as the model system. Interestingly, we observed that cells encapsulated in soft alginate matrices were more sensitive to the toxic chemicals relative to cells encapsulated in stiffer matrices. The observed dependence of toxic responses on matrix stiffness was also seen for other neural cell lines including U-251 glioblastoma and IMR-32 neuroblastoma cells. Given the established involvement of Rho GTPases in mediating mechanosensitive cell responses, we also performed the toxicity assays using U-87 cells expressing constitutively active mutants of the Rho GTPase RhoA. Under these conditions, the toxic responses were similar for U-87 cells cultured in both soft and stiff matrices. These results suggest a strong role of mechanotransductive signaling on the response of neural cells to toxins. We believe that determining the role of substrate stiffness on the toxic response of neural cells will be crucial to the development of optimal in vitro cell-based screens of neurotoxicity.

The Number of Lines a Cell Contacts and Cell Contractility Drive the Efficiency of Contact Guidance

Abstract: Cell migration is an important biological function that impacts many physiological and pathological processes. Often migration is directed along aligned fibers of collagen at different densities, a process called contact guidance. However, cells adhere to other components in the extracellular matrix, possibly affecting migrational behavior. Additionally, changes in intracellular contractility are well known to affect random migration, but its effect on contact guidance is less known. This study examines differences in directed migration in response to variations in the spacing of collagen, non-specific background adhesion strength and myosin II-mediated contractility. Collagen was microcontact printed onto glass substrates and timelapse live-cell microscopy was used to measure migration characteristics. Increasing the number of lines a cell contacts or decreasing contraction led to decreases in directionality, but speed changes were context dependent. This suggests that while cell migration speed is a biphasic function of contractility, directionality appears to be a monotonic, increasing function of contractility. Thus, increasing the number of lines a cell contacts or decreasing contractility degrades the contact guidance fidelity.

Target-Bound Generated Pharmacophore Model to Improve the Pharmacophore-Based Virtual Screening: Identification of G-Protein Coupled Human CCR2 Receptors Inhibitors as Anti-Inflammatory Drugs

Abstract: Pharmacophore-based virtual screening is being widely used to discover new drug candidates. Building a pharmacophore model based on a known inhibitor that is unbound to the target could be misleading and result in mining for the wrong hits. Results presented herein confirm that pharmacophore models based on unbound and bound ligand confirmations produce significantly, structurally different compound libraries and, consequently, change the outcome of the virtual screening. To further verify our findings, molecular dynamics and extensive post-dynamic analysis are performed for the best retrieved hits from each approach; these are the unbound and bound ligand pharmacophore-generated libraries. In this report, the proposed target-bound pharmacophore model is used to discover potential G-protein coupled CCR2 receptor inhibitors as potential anti-inflammatory drugs. Herein, various molecular modeling approaches are adopted including homology modeling, molecular docking, lipid bilayer molecular dynamics simulations and per-residue interaction energy decomposition analysis. The current study highlights some critical aspects in the pharmacophore-based virtual screening as a powerful tool in the drug discovery and development machinery.

Hydraulic Pressure During Fluid Flow Regulates Purinergic Signaling and Cytoskeleton Organization of Osteoblasts

Abstract: During physiological activities, osteoblasts experience a variety of mechanical forces that stimulate anabolic responses at the cellular level necessary for the formation of new bone. Previous studies have primarily investigated the osteoblastic response to individual forms of mechanical stimuli. However in this study, we evaluated the response of osteoblasts to two simultaneous, but independently controlled stimuli; fluid flow-induced shear stress (FSS) and static or cyclic hydrostatic pressure (SHP or CHP, respectively). MC3T3-E1 osteoblasts-like cells were subjected to 12 dynes/cm2 FSS along with SHP or CHP of varying magnitudes to determine if pressure enhances the anabolic response of osteoblasts during FSS. For both SHP and CHP, the magnitude of hydraulic pressure that induced the greatest release of ATP during FSS was 15 mmHg. Increasing the hydraulic pressure to 50 mmHg or 100 mmHg during FSS attenuated the ATP release compared to 15 mmHg during FSS. Decreasing the magnitude of pressure during FSS to atmospheric pressure reduced ATP release to that of basal ATP release from static cells and inhibited actin reorganization into stress fibers that normally occurred during FSS with 15 mmHg of pressure. In contrast, translocation of nuclear factor kappa B (NFκB) to the nucleus was independent of the magnitude of hydraulic pressure and was found to be mediated through the activation of phospholipase-C (PLC), but not src kinase. In conclusion, hydraulic pressure during FSS was found to regulate purinergic signaling and actin cytoskeleton reorganization in the osteoblasts in a biphasic manner, while FSS alone appeared to stimulate NFκB translocation. Understanding the effects of hydraulic pressure on the anabolic responses of osteoblasts during FSS may provide much needed insights into the physiologic effects of coupled mechanical stimuli on osteogenesis.