Overview of Micro- and Nano-Technology Tools for Stem Cell Applications: Micropatterned and Microelectronic Devices
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paper_title: Revisiting lab-on-a-chip technology for drug discovery

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Manz and colleagues discuss recent progress in the development of microfluidic techniques (lab-on-a-chip technology) and their applications in drug discovery. Highlights include high-throughput droplet technology and applications such as 'organs on a chip', which could help reduce reliance on animal testing.
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paper_title: Making a New Technology Work: The Standardization and Regulation of Microarrays

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The translation of laboratory innovations into clinical tools is dependent upon the development of regulatory arrangements designed to ensure that the new technology will be used reliably and consistently. A case study of a key post-genomic technology, gene chips or microarrays, exemplifies this claim. The number of microarray publications and patents has increased exponentially during the last decade and diagnostic microarray tests already are making their way into the clinic. Yet starting in the mid-1990s, scientific journals were overrun with criticism concerning the ambiguities involved in interpreting most of the assumptions of a microarray experiment. Questions concerning platform comparability and statistical calculations were and continue to be raised, in spite of the emergence by 2001 of an initial set of standards concerning several components of a microarray experiment. This article probes the history and ongoing efforts aimed at turning microarray experimentation into a viable, meaningful, and consensual technology by focusing on two related elements:1) The history of the development of the Microarray Gene Expression Data Society (MGED), a remarkable bottom-up initiative that brings together different kinds of specialists from academic, commercial, and hybrid settings to produce, maintain, and update microarray standards; and 2) The unusual mix of skills and expertise involved in the development and use of microarrays. The production, accumulation, storage, and mining of microarray data remain multi-skilled endeavors bridging together different types of scientists who embody a diversity of scientific traditions. Beyond standardization, the interfacing of these different skills has become a key issue for further development of the field.
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paper_title: Microfluidic Large-Scale Integration: The Evolution of Design Rules for Biological Automation

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Microfluidic large-scale integration (mLSI) refers to the development of microfluidic chips with thousands of integrated micromechanical valves and control components. This technology is utilized in many areas of biology and chemistry and is a candidate to replace today’s conventional automation paradigm, which consists of fluid-handling robots. We review the basic development of mLSI and then discuss design principles of mLSI to assess the capabilities and limitations of the current state of the art and to facilitate the application of mLSI to areas of biology. Many design and practical issues, including economies of scale, parallelization strategies, multiplexing, and multistep biochemical processing, are discussed. Several microfluidic components used as building blocks to create effective, complex, and highly integrated microfluidic networks are also highlighted.
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paper_title: Arrayed cellular microenvironments for identifying culture and differentiation conditions for stem, primary and rare cell populations

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Arrayed cellular microenvironments for identifying culture and differentiation conditions for stem, primary and rare cell populations
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paper_title: Clinical Utility of Microarrays: Current Status, Existing Challenges and Future Outlook

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Microarray-based clinical tests have become powerful tools in the diagnosis and treatment of diseases. In contrast to traditional DNA-based tests that largely focus on single genes associated with rare conditions, microarray-based tests are ideal for the study of diseases with underlying complex genetic causes. Several microarray based tests have been translated into clinical practice such as MammaPrint and AmpliChip CYP450. Additional cancer-related microarray-based tests are either in the process of FDA review or under active development, including Tissue of Tumor Origin and AmpliChip p53. All diagnostic microarray testing is ordered by physicians and tested by a Clinical Laboratories Improvement Amendment-certified (CLIA) reference laboratory. Recently, companies offering consumer based microarray testing have emerged. Individuals can order tests online and service providers deliver the results directly to the clients via a password-protected secure website. Navigenics, 23andMe and deCODE Genetics represent pioneering companies in this field. Although the progress of these microarray-based tests is extremely encouraging with the potential to revolutionize the recognition and treatment of common diseases, these tests are still in their infancy and face technical, clinical and marketing challenges. In this article, we review microarray-based tests which are currently approved or under review by the FDA, as well as the consumer-based testing. We also provide a summary of the challenges and strategic solutions in the development and clinical use of the microarray-based tests. Finally, we present a brief outlook for the future of microarray-based clinical applications.
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paper_title: From 3D cell culture to organs-on-chips.

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3D cell-culture models have recently garnered great attention because they often promote levels of cell differentiation and tissue organization not possible in conventional 2D culture systems. We review new advances in 3D culture that leverage microfabrication technologies from the microchip industry and microfluidics approaches to create cell-culture microenvironments that both support tissue differentiation and recapitulate the tissue-tissue interfaces, spatiotemporal chemical gradients, and mechanical microenvironments of living organs. These 'organs-on-chips' permit the study of human physiology in an organ-specific context, enable development of novel in vitro disease models, and could potentially serve as replacements for animals used in drug development and toxin testing.
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paper_title: Stem cell biology and drug discovery

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There are many reasons to be interested in stem cells, one of the most prominent being their potential use in finding better drugs to treat human disease. This article focuses on how this may be implemented. Recent advances in the production of reprogrammed adult cells and their regulated differentiation to disease-relevant cells are presented, and diseases that have been modeled using these methods are discussed. Remaining difficulties are highlighted, as are new therapeutic insights that have emerged.
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paper_title: Transmembrane crosstalk between the extracellular matrix--cytoskeleton crosstalk.

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Integrin-mediated cell adhesions provide dynamic, bidirectional links between the extracellular matrix and the cytoskeleton. Besides having central roles in cell migration and morphogenesis, focal adhesions and related structures convey information across the cell membrane, to regulate extracellular-matrix assembly, cell proliferation, differentiation, and death. This review describes integrin functions, mechanosensors, molecular switches and signal-transduction pathways activated and integrated by adhesion, with a unifying theme being the importance of local physical forces.
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paper_title: Human Pluripotent Stem Cells: Applications and Challenges in Neurological Diseases

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The ability to generate human pluripotent stem cells (hPSCs) holds great promise for the understanding and the treatment of human neurological diseases in modern medicine. The hPSCs are considered for their in vitro use as research tools to provide relevant cellular model for human diseases, drug discovery and toxicity assays and for their in vivo use in regenerative medicine applications. In this review, we highlight recent progress, promises and challenges of hPSC applications in human neurological disease modelling and therapies.
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paper_title: High-throughput cellular microarray platforms: applications in drug discovery, toxicology and stem cell research

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Cellular microarrays are powerful experimental tools for high-throughput screening of large numbers of test samples. Miniaturization increases assay throughput while reducing reagent consumption and the number of cells required, making these systems attractive for a wide range of assays in drug discovery, toxicology, stem cell research and potentially therapy. Here, we provide an overview of the emerging technologies that can be used to generate cellular microarrays, and we highlight recent significant advances in the field. This emerging and multidisciplinary approach offers new opportunities for the design and control of stem cells in tissue engineering and cellular therapies and promises to expedite drug discovery in the biotechnology and pharmaceutical industries.
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paper_title: Reverse transfected cell microarrays in infectious disease research.

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Several human pathogenic viruses encode large genomes with often more than 100 genes. Viral pathogenicity is determined by carefully orchestrated co-operative activities of several different viral genes which trigger the phenotypic functions of the infected cells. Systematic analyses of these complex interactions require high-throughput transfection technology. Here we have provided a laboratory manual for the reverse transfected cell microarray (RTCM; alternative name: cell chip) as a high-throughput transfection procedure, which has been successfully applied for the systematic analyses of single and combination effects of genes encoded by the human herpesvirus-8 on the NF-kappaB signal transduction pathway. In order to quantitatively determine the effects of viral genes in transfected cells, protocols for the use of GFP as an indicator gene and for indirect immunofluorescence staining of cellular target proteins have been included. RTCM provides a useful methodological approach to investigate systematically combination effects of viral genes on cellular functions.
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paper_title: An extracellular matrix microarray for probing cellular differentiation

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We present an extracellular matrix (ECM) microarray platform for the culture of patterned cells atop combinatorial matrix mixtures. This platform enables the study of differentiation in response to a multitude of microenvironments in parallel. The fabrication process required only access to a standard robotic DNA spotter, off-the-shelf materials and 1,000 times less protein than conventional means of investigating cell-ECM interactions. To demonstrate its utility, we applied this platform to study the effects of 32 different combinations of five extracellular matrix molecules (collagen I, collagen III, collagen IV, laminin and fibronectin) on cellular differentiation in two contexts: maintenance of primary rat hepatocyte phenotype indicated by intracellular albumin staining and differentiation of mouse embryonic stem (ES) cells toward an early hepatic fate, indicated by expression of a beta-galactosidase reporter fused to the fetal liver-specific gene, Ankrd17 (also known as gtar). Using this technique, we identified combinations of ECM that synergistically impacted both hepatocyte function and ES cell differentiation. This versatile technique can be easily adapted to other applications, as it is amenable to studying almost any insoluble microenvironmental cue in a combinatorial fashion and is compatible with several cell types.
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paper_title: Variable behavior and complications of autologous bone marrow mesenchymal stem cells transplanted in experimental autoimmune encephalomyelitis

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Autologous bone marrow stromal cells (BMSCs) offer significant practical advantages for potential clinical applications in multiple sclerosis (MS). Based on recent experimental data, a number of clinical trials have been designed for the intravenous (IV) and/or intrathecal (ITH) administration of BMSCs in MS patients. Delivery of BMSCs in the cerebrospinal fluid via intracerebroventricular (ICV) transplantation is a useful tool to identify mechanisms underlying the migration and function of these cells. In the current study, BMSCs were ICV administered in severe and mild EAE, as well as naive animals; neural precursor cells (NPCs) served as cellular controls. Our data indicated that ICV-transplanted BMSCs significantly ameliorated mild though not severe EAE. Moreover, BMSCs exerted significant anti-inflammatory effect on spinal cord with concomitant reduced axonopathy only in the mild EAE model. BMSCs migrated into the brain parenchyma and, depending on their cellular density, within brain parenchyma formed cellular masses characterized by focal inflammation, demyelination, axonal loss and increased collagen-fibronectin deposition. These masses were present in 64% of ICV BMASC-transplanted severe EAE animals whereas neither BMSCs transplanted in mild EAE cases nor the NPCs exhibited similar behavior. BMSCs possibly exerted their fibrogenic effect via both paracrine and autocrine manner, at least partly due to up-regulation of connective tissue growth factor (CTGF) under the trigger of TGFb1. Our findings are of substantial relevance for clinical trials in MS, particularly regarding the possibility that ICV transplanted BMSCs entering the inflamed central nervous system may exhibit - under conditions - a local pathology of yet unknown consequences.
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paper_title: Single-cell profiling of developing and mature retinal neurons.

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Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue(1-7). In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions(8). This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells(9). The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself(10). The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development. As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population(11). Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina(2,4,5,12). In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells(8). With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.
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paper_title: Telomere length changes after umbilical cord blood transplant

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BACKGROUND: The establishment of donor-derived hematopoiesis in the recipients of hematopoietic stem cell (HSC) transplants involves extensive proliferation and differentiation of HSCs. Data from long-term survivors of HSC transplants suggest that these transplanted HSCs may experience a debilitating replicative senescence. A significant posttransplant shortening of peripheral blood mononuclear cell (PBMNC) telomeres has been observed in both marrow transplant and peripheral blood progenitor cell transplant recipients. Similar studies have not been performed for umbilical cord blood (UCB) HSC transplants, which might be expected to exhibit increased posttransplant replicative potential due to their inherently greater telomere length. ::: ::: ::: ::: STUDY DESIGN AND METHODS: Blood was obtained from donor-recipient pairs of allogeneic PBHSC transplant and UCB HSC transplant, both before transplant and at follow-up treatments (minimum 1 year after transplant) after engraftment. Telomere restriction fragment length (TRFL) analysis was performed on the blood samples. The mean TRFL and posttransplant changes in the mean TRFL were analyzed. ::: ::: ::: ::: RESULTS: Measurements of telomere lengths in the PBMNCs of transplant patients revealed a significant net decrease in telomere length in all transplant recipients compared with their respective donors. Our results also revealed that the PBMNCs of umbilical cord stem cell transplant patients retain a significantly longer posttransplant telomere length. ::: ::: ::: ::: CONCLUSION: The significantly longer telomeres observed in the allogeneic UCB HSC transplant recipients compared to the allogeneic PBHSC transplant recipients in our study may be indicative of a replicative advantage inherent in the use of UCB HSC for transplant.
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paper_title: Comparison of reverse transcription–quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis

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Abstract Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription–quantitative polymerase chain reaction (RT–qPCR) is the “gold standard” for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT–qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis.
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paper_title: Microarray analysis of normal and dystrophic skeletal muscle

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The development and increasingly common use of DNA microarrays for comprehensive RNA expression analysis has had a substantial impact on the study of molecular pathology. DNA microarrays are orderly, high-density arrangements of nucleic acid spots that can be used as substrates for global gene expression analysis. Prior to their development, technical limitations necessitated that the molecular mechanisms underlying biological processes be broken down into their component parts and each gene or protein studied individually. This approach, focused as it is on a single aspect of a scientific phenomenon, does not allow appreciation or understanding of the fact that biological pathways do not exist in isolation, but are influenced by numerous factors. Enormous technological advances have been made over the past decade and now high-density DNA microarrays can provide rapid measurement of thousands of distinct transcripts simultaneously. These experiments raise the exciting opportunity to examine biological pathways in all their complexity and to compare the hypotheses deduced from the study of histological pathology with the findings of molecular pathology. This review focuses on how microarray technology has been used to interrogate muscular gene expression and, in particular, on how data generated from differential expression analysis of dystrophic and normal skeletal muscle has contributed to understanding the molecular pathophysiological pathways of muscular dystrophy.
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paper_title: Lifetime probabilities of hematopoietic stem cell transplantation in the U.S.

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Healthcare policies regarding hematopoietic stem cell transplantation (HSCT) must address the need for the procedure as well as the availability of stem cell sources: bone marrow, peripheral blood, or umbilical cord blood (UCB). However, data with respect to the lifetime probability of undergoing HSCT are lacking. This study was undertaken to estimate the latter probability in the United States (U.S.), depending on age, sex, and race. We used data from the Center for International Blood and Marrow Transplant Research, the U.S. Surveillance, Epidemiology and End Results Program, and the U.S. Census Bureau and calculated probabilities as cumulative incidences. Several scenarios were considered: assuming current indications for autologous and allogeneic HSCT, assuming universal donor availability, and assuming broadening of HSCT use in hematologic malignancies. Incidences of diseases treated with HSCT and of HSCTs performed increase with age, rising strongly after age 40. Among individuals older than 40, incidences are higher for men than for women. The lifetime probabilities of undergoing HSCT range from 0.23% to 0.98% under the various scenarios. We conclude that, given current indications, the lifetime probability of undergoing autologous or allogeneic HSCT is much higher than previously reported by others and could rise even higher with increases in donor availability and HSCT applicability.
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paper_title: Integrin-ECM interactions regulate the changes in cell shape driving the morphogenesis of the Drosophila wing epithelium.

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During development, morphogenesis involves migration and changes in the shape of epithelial sheets, both of which require coordination of cell adhesion. Thus, while modulation of integrin-mediated adhesion to the ECM regulates epithelial motility, cell-cell adhesion via cadherins controls the remodelling of epithelial sheets. We have used the Drosophila wing epithelium to demonstrate that cell-ECM interactions mediated by integrins also regulate the changes in cell shape that underly epithelial morphogenesis. We show that integrins control the transitions from columnar to cuboidal cell shape underlying wing formation, and we demonstrate that eliminating the ECM has the same effect on cell shape as inhibiting integrin function. Furthermore, lack of integrin activity also induces detachment of the basal lamina and failure to assemble the basal matrix. Hence, we propose that integrins control epithelial cell shape by mediating adherence of these cells to the ECM. Finally, we show that the ECM has an instructive rather than a structural role, because inhibition of Raf reverses the cell shape changes caused by perturbing integrins.
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paper_title: A planar interdigitated ring electrode array via dielectrophoresis for uniform patterning of cells.

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Uniform patterning of cells is highly desirable for most cellular studies involving cell-cell interactions but is often difficult in an in vitro environment. This paper presents the development of a collagen-coated planar interdigitated ring electrode (PIRE) array utilizing positive dielectrophoresis to pattern cells uniformly. Key features of the PIRE design include: (1) maximizing length along the edges where the localized maximum in the electric field exists; (2) making the inner gap slightly smaller than the outer gap in causing the electric field strength near the center of a PIRE being generally stronger than that near the outer edge of the same PIRE. Results of human hepatocellular carcinoma cells, HepG2, adhered on a 6x6 PIRE array show that cells patterned within minutes with good uniformity (48+/-6 cells per PIRE). Cell viability test revealed healthy patterned cells after 24h that were still confined to the collagen-coated PIREs. Furthermore, quantification of fluorescence intensity of living cells shows an acceptable reproducibility of cell viability among PIREs (mean normalized intensity per PIRE was 1+/-0.138). The results suggest that the PIRE array would benefit applications that desire uniform cellular patterning, and improve both response and reproducibility of cell-based biosensors.
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paper_title: Stem cell-based treatments for Type 1 diabetes mellitus: bone marrow, embryonic, hepatic, pancreatic and induced pluripotent stem cells

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Type 1 diabetes mellitus--characterized by the permanent destruction of insulin-secreting β-cells--is responsive to cell-based treatments that replace lost β-cell populations. The current gold standard of pancreas transplantation provides only temporary independence from exogenous insulin and is fraught with complications, including increased mortality. Stem cells offer a number of theoretical advantages over current therapies. Our review will focus on the development of treatments involving tissue stem cells from bone marrow, liver and pancreatic cells, as well as the potential use of embryonic and induced pluripotent stem cells for Type 1 diabetes therapy. While the body of research involving stem cells is at once promising and inconsistent, bone marrow-derived mesenchymal stem cell transplantation seems to offer the most compelling evidence of efficacy. These cells have been demonstrated to increase endogenous insulin production, while partially mitigating the autoimmune destruction of newly formed β-cells. However, recently successful experiments involving induced pluripotent stem cells could quickly move them into the foreground of therapeutic research. We address the limitations encountered by present research and look toward the future of stem cell treatments for Type 1 diabetes.
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paper_title: Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors

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Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.
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paper_title: Using a single fluorescent reporter gene to infer half-life of extrinsic noise and other parameters of gene expression.

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Fluorescent and luminescent proteins are often used as reporters of transcriptional activity. Given the prevalence of noise in biochemical systems, the time-series data arising from these is of significant interest in efforts to calibrate stochastic models of gene expression and obtain information about sources of nongenetic variability. We present a statistical inference framework that can be used to estimate kinetic parameters of gene expression, as well as the strength and half-life of extrinsic noise from single fluorescent-reporter-gene time-series data. The method takes into account stochastic variability in a fluorescent signal resulting from intrinsic noise of gene expression, kinetics of fluorescent protein maturation, and extrinsic noise, which is assumed to arise at transcriptional level. We use the linear noise approximation and derive an explicit formula for the likelihood of observed fluorescent data. The method is embedded in a Bayesian paradigm, so that certain parameters can be informed from other experiments allowing portability of results across different studies. Inference is performed using Markov chain Monte Carlo. Fluorescent reporters are primary tools to observe dynamics of gene expression and the correct interpretation of fluorescent data is crucial to investigating these fundamental processes of cellular life. As both magnitude and frequency of the noise may have a dramatic effect on the cell fitness, the quantification of stochastic fluctuation is essential to the understanding of how genes are regulated. Our method provides a framework that addresses this important question.
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paper_title: Neuronal Classification and Marker Gene Identification via Single-Cell Expression Profiling of Brainstem Vestibular Neurons Subserving Cerebellar Learning

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Identification of marker genes expressed in specific cell types is essential for the genetic dissection of neural circuits. Here we report a new strategy for classifying heterogeneous populations of neurons into functionally distinct types and for identifying associated marker genes. Quantitative single-cell expression profiling of genes related to neurotransmitters and ion channels enables functional classification of neurons; transcript profiles for marker gene candidates identify molecular handles for manipulating each cell type. We apply this strategy to the mouse medial vestibular nucleus (MVN), which comprises several types of neurons subserving cerebellar-dependent learning in the vestibulo-ocular reflex. Ion channel gene expression differed both qualitatively and quantitatively across cell types and could distinguish subtle differences in intrinsic electrophysiology. Single-cell transcript profiling of MVN neurons established six functionally distinct cell types and associated marker genes. This strategy is applicable throughout the nervous system and could facilitate the use of molecular genetic tools to examine the behavioral roles of distinct neuronal populations.
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paper_title: Transistor Probes Local Potassium Conductances in the Adhesion Region of Cultured Rat Hippocampal Neurons

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Adhesion interactions of neurons in a tissue may affect the ion conductance of the plasma membrane, inducing selective localization and modulation of channels. We studied the adhesion region of cultured neurons from rat hippocampus as a defined model where such effects could be observed electrophysiologically, taking advantage of extracellular recording by a transistor integrated in the substrate. We observed the K(+) current through the region of soma adhesion under voltage-clamp and compared it with the current through the whole cell. We found that the specific A-type conductance was depleted, even completely, in the region of adhesion, whereas the specific K-type conductance was enhanced up to a factor of 12. The electrophysiological approach opens a new way to investigate targeting of ion channels in the cell membrane as a function of adhesion processes.
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paper_title: Fluorescent proteins as a toolkit for in vivo imaging.

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Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, and its mutant variants, are the only fully genetically encoded fluorescent probes available and they have proved to be excellent tools for labeling living specimens. Since 1999, numerous GFP homologues have been discovered in Anthozoa, Hydrozoa and Copepoda species, demonstrating the broad evolutionary and spectral diversity of this protein family. Mutagenic studies gave rise to diversified and optimized variants of fluorescent proteins, which have never been encountered in nature. This article gives an overview of the GFP-like proteins developed to date and their most common applications to study living specimens using fluorescence microscopy.
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paper_title: The modulation of myogenic cells differentiation using a semiconductor-muscle junction.

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Abstract The present study is aimed to design a prototype of hybrid silicon-muscle cell junction, analog to an artificial neuromuscular junction prototype and relevant to the development of advanced neuro-prostheses and bionic systems. The device achieves focal Electric Capacitive Stimulation (ECS) by coupling of single cells and semiconductors, without electrochemical reaction with the substrate. A voltage change applied to a stimulation spot beneath an electrogenic cell leads to a capacitive current (charge accumulation) that opens voltage-gated ion channels in the membrane and generates an action potential. The myo-electronic junction was employed to chronically stimulate muscle cells via ECS and to induce cytosolic calcium transients in myotubes, fibers isolated from mouse FDB (fast [Ca 2+ ] i transients) and surprisingly also in undifferentiated myoblasts (slow [Ca 2+ ] i waves). The hybrid junction elicited, via chronic ECS, a differential reprogramming of single muscle cells by inducing early muscle contraction maturation and plasticity effects, such as NFAT-C3 nuclear translocation. In addition, in the presence of agrin, chronic ECS induced a modulation of AchR clustering which simulates in vitro synaptogenesis. This methodology can coordinate the myogenic differentiation, thus offering direct but non-invasive single cell/wiring, providing a platform for regenerative medicine strategies.
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paper_title: CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification

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Summary High-throughput sequencing has allowed for unprecedented detail in gene expression analyses, yet its efficient application to single cells is challenged by the small starting amounts of RNA. We have developed CEL-Seq, a method for overcoming this limitation by barcoding and pooling samples before linearly amplifying mRNA with the use of one round of in vitro transcription. We show that CEL-Seq gives more reproducible, linear, and sensitive results than a PCR-based amplification method. We demonstrate the power of this method by studying early C. elegans embryonic development at single-cell resolution. Differential distribution of transcripts between sister cells is seen as early as the two-cell stage embryo, and zygotic expression in the somatic cell lineages is enriched for transcription factors. The robust transcriptome quantifications enabled by CEL-Seq will be useful for transcriptomic analyses of complex tissues containing populations of diverse cell types.
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paper_title: Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines

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Background: To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery. Methodology/Principal Findings: We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with nonepithelial cancer (n=20) or healthy subjects (n=25). Microfluidic-based single cell transcriptional profiling of 87 cancerassociated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy. Conclusions/Significance: For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFs1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of ‘liquid biopsies’ to better model drug discovery.
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paper_title: Mesenchymal stem cells and progenitor cells in connective tissue engineering and regenerative medicine: is there a future for transplantation?

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PURPOSE ::: Transplantation surgery suffers from a shortage of donor organs worldwide. Cell injection and tissue engineering (TE), thus emerge as alternative therapy options. The purpose of this article is to review the progress of TE technology, focusing on mesenchymal stem cells (MSC) as a cell source for artificial functional tissue. ::: ::: ::: RESULTS ::: MSC from many different sources can be minimally invasively harvested: peripheral blood, fat tissue, bone marrow, amniotic fluid, cord blood. In comparison to embryonic stem cells (ESC), there are no ethical concerns; MSC can be extracted from autologous or allogenic tissue and cause an immune modulatory effect by suppressing the graft-versus-host reaction (GvHD). Furthermore, MSC do not develop into teratomas when transplanted, a consequence observed with ESC and iPS cells. ::: ::: ::: CONCLUSION ::: MSC as multipotent cells are capable of differentiating into mesodermal and non-mesodermal lineages. However, further studies must be performed to elucidate the differentiation capacity of MSC from different sources, and to understand the involved pathways and processes. Already, MSC have been successfully applied in clinical trials, e.g., to heal large bone defects, cartilage lesions, spinal cord injuries, cardiovascular diseases, hematological pathologies, osteogenesis imperfecta, and GvHD. A detailed understanding of the behavior and homing of MSC is desirable to enlarge the clinical application spectrum of MSC towards the in vitro generation of functional tissue for implantation, for example, resilient cartilage, contractile myocardial replacement tissue, and bioartificial heart valves.
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paper_title: Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts

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Direct reprogramming of somatic cells provides an opportunity to generate patient- or disease-specific pluripotent stem cells. Such induced pluripotent stem (iPS) cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. Mouse iPS cells are indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras. Reactivation of the c-Myc retrovirus, however, increases tumorigenicity in the chimeras and progeny mice, hindering clinical applications. Here we describe a modified protocol for the generation of iPS cells that does not require the Myc retrovirus. With this protocol, we obtained significantly fewer non-iPS background cells, and the iPS cells generated were consistently of high quality. Mice derived from Myc(-) iPS cells did not develop tumors during the study period. The protocol also enabled efficient isolation of iPS cells without drug selection. Furthermore, we generated human iPS cells from adult dermal fibroblasts without MYC.
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paper_title: Mesenchymal stem cells: therapeutic outlook for stroke.

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Adult bone marrow-derived mesenchymal stem cells (MSCs) display a spectrum of functional properties. Transplantation of these cells improves clinical outcome in models of cerebral ischemia and spinal cord injury via mechanisms that may include replacement of damaged cells, neuroprotective effects, induction of axonal sprouting, and neovascularization. Therapeutic effects have been reported in animal models of stroke after intravenous delivery of MSCs, including those derived from adult human bone marrow. Initial clinical studies on intravenously delivered MSCs have now been completed in human subjects with stroke. Here, we review the reparative and protective properties of transplanted MSCs in stroke models, describe initial human studies on intravenous MSC delivery in stroke, and provide a perspective on prospects for future progress with MSCs.
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paper_title: High-throughput analysis of signals regulating stem cell fate and function.

paper_content:

Stem cells exhibit promise in numerous areas of regenerative medicine. Their fate and function are governed by a combination of intrinsic determinants and signals from the local microenvironment, or niche. An understanding of the mechanisms underlying both embryonic and adult stem cell functions has been greatly enhanced by the recent development of several high-throughput technologies: microfabricated platforms, including cellular microarrays, to investigate the combinatorial effects of microenvironmental stimuli and large-scale screens utilizing small molecules and short interfering RNAs to identify crucial genetic and signaling elements. Furthermore, the integration of these systems with other versatile platforms, such as microfluidics and lentiviral microarrays, will continue to enable the detailed elucidation of stem cell processes, and thus, greatly contribute to the development of stem cell based therapies.
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paper_title: Fluorescent Proteins as Biomarkers and Biosensors: Throwing Color Lights on Molecular and Cellular Processes

paper_content:

Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.
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paper_title: Adult Bone Marrow: Which Stem Cells for Cellular Therapy Protocols in Neurodegenerative Disorders?

paper_content:

The generation of neuronal cells from stem cells obtained from adult bone marrow is of significant clinical interest in order to design new cell therapy protocols for several neurological disorders. The recent identification in adult bone marrow of stem cells derived from the neural crests (NCSCs) might explain the neuronal phenotypic plasticity shown by bone marrow cells. However, little information is available about the nature of these cells compared to mesenchymal stem cells (MSCs). In this paper, we will review all information available concerning NCSC from adult tissues and their possible use in regenerative medicine. Moreover, as multiple recent studies showed the beneficial effect of bone marrow stromal cells in neurodegenerative diseases, we will discuss which stem cells isolated from adult bone marrow should be more suitable for cell replacement therapy.
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paper_title: Concise review: Adipose-derived stem cells as a novel tool for future regenerative medicine.

paper_content:

The potential use of stem cell-based therapies for the repair and regeneration of various tissues and organs offers a paradigm shift that may provide alternative therapeutic solutions for a number of diseases. The use of either embryonic stem cells (ESCs) or induced pluripotent stem cells in clinical situations is limited due to cell regulations and to technical and ethical considerations involved in the genetic manipulation of human ESCs, even though these cells are, theoretically, highly beneficial. Mesenchymal stem cells seem to be an ideal population of stem cells for practical regenerative medicine, because they are not subjected to the same restrictions. In particular, large number of adipose-derived stem cells (ASCs) can be easily harvested from adipose tissue. Furthermore, recent basic research and preclinical studies have revealed that the use of ASCs in regenerative medicine is not limited to mesodermal tissue but extends to both ectodermal and endodermal tissues and organs, although ASCs originate from mesodermal lineages. Based on this background knowledge, the primary purpose of this concise review is to summarize and describe the underlying biology of ASCs and their proliferation and differentiation capacities, together with current preclinical and clinical data from a variety of medical fields regarding the use of ASCs in regenerative medicine. In addition, future directions for ASCs in terms of cell-based therapies and regenerative medicine are discussed.
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paper_title: New Array Approaches to Explore Single Cells Genomes

paper_content:

Microarray analysis enables the genome wide detection of copy number variations and the investigation of chromosomal instability. Whereas array techniques have been well established for the analysis of unamplified DNA derived from many cells, it has been more challenging to enable the accurate analysis of single cell genomes. In this review, we provide an overview of single cell DNA amplification techniques, the different array approaches and discuss their potential applications to study human embryos.
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paper_title: Methods for Synchronizing Cells at Specific Stages of the Cell Cycle

paper_content:

Exponentially growing cells are asynchronous with respect to the cell cycle stage. Detection of cell cycle-related events is improved by enriching the culture for cells at the stage during which the particular event occurs. Methods for synchronizing cells are provided here, including those based on morphological features of the cell (mitotic shake-off), cellular metabolism (thymidine inhibition, isoleucine depravation), and chemical inhibitors of cell progression in G1 (lovastatin), S (aphidicolin, mimosine), and G2/M (nocodazole). Applications of these methods and the advantages and disadvantages of each are described.
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paper_title: Cells on chips

paper_content:

Microsystems create new opportunities for the spatial and temporal control of cell growth and stimuli by combining surfaces that mimic complex biochemistries and geometries of the extracellular matrix with microfluidic channels that regulate transport of fluids and soluble factors. Further integration with bioanalytic microsystems results in multifunctional platforms for basic biological insights into cells and tissues, as well as for cell-based sensors with biochemical, biomedical and environmental functions. Highly integrated microdevices show great promise for basic biomedical and pharmaceutical research, and robust and portable point-of-care devices could be used in clinical settings, in both the developed and the developing world.
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paper_title: Gene expression analysis on a single cell level in Purkinje cells of Huntington's disease transgenic mice

paper_content:

Ataxia is a clinical feature of most polyglutamine disorders. Cerebellar neurodegeneration of Purkinje cells (PCs) in Huntington's Disease (HD) brain was described in the 1980s. PC death in the R6/2 transgenic model for HD was published by Turmaine et al. [27]. So far, PCs have not been examined on a single cell level. In order to begin to understand PC dysfunction and degeneration in HD we performed a gene expression study on laser-dissected PC based on a DNA microarray screening and quantitative real time PCR (Q-PCR). We demonstrate downregulation of the retinoid acid receptor-related orphan receptor alpha (ROR alpha) mRNA and ROR alpha-mediated mRNAs, also seen by immunofluorescent staining. As ROR alpha and ROR alpha-dependent transcriptional dysregulation is not only found in the R6/2 model for HD but also in a model for spinocerebellar ataxia type 1 (SCA1) (Serra et al. [24]) the data suggest common pathogenic mechanisms for both polyglutamine diseases. (c) 2012 Published by Elsevier Ireland Ltd.
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paper_title: Fluorescence microscopy today

paper_content:

Fluorescence microscopy has undergone a renaissance in the last decade. The introduction of green fluorescent protein (GFP) and two-photon microscopy has allowed systematic imaging studies of protein localization in living cells and of the structure and function of living tissues. The impact of these and other new imaging methods in biophysics, neuroscience, and developmental and cell biology has been remarkable. Further advances in fluorophore design, molecular biological tools and nonlinear and hyper-resolution microscopies are poised to profoundly transform many fields of biological research.
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paper_title: Dynamic culture of droplet-confined cell arrays.

paper_content:

Responding to the need of creating an accurate and controlled microenvironment surrounding the cell while meeting the requirements for biological processes or pharmacological screening tests, we aimed at designing and developing a microscaled culture system suitable for analyzing the synergic effects of extracellular matrix proteins and soluble environments on cell phenotype in a high-throughput fashion. We produced cell arrays deposing micrometer-scale protein islands on hydrogels using a robotic DNA microarrayer, constrained the culture media in a droplet-like volume and developed a suitable perfusion system. The droplet-confined cell arrays were used either with conventional culture methods (batch operating system) or with automated stable and constant perfusion (steady-state operating system). Mathematical modeling assisted the experimental design and assessed efficient mass transport and proper fluidodynamic regimes. Cells cultured on arrayed islands (500 mum diameter) maintained the correct phenotype both after static and perfused conditions, confirmed by immunostaining and gene expression analyses through total RNA extraction. The mathematical model, validated using a particle tracking experiment, predicted the constant value of velocities over the cell arrays (less than 10% variation) ensuring the same mass transport regime. BrdU analysis on an average of 96 cell spots for each experimental condition showed uniform expression inside each cell island and low variability in the data (average of 13%). Perfused arrays showed longer doubling times when compared with static cultures. In addition, perfused cultures showed a reduced variability in the collected data, allowing to detect statistically significant differences in cell behavior depending on the spotted ECM protein.
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paper_title: Derivation, propagation and differentiation of human embryonic stem cells.

paper_content:

Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug discovery, drug testing and repair of damaged or diseased tissues via transplantation.
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paper_title: An extracellular matrix microarray for probing cellular differentiation

paper_content:

We present an extracellular matrix (ECM) microarray platform for the culture of patterned cells atop combinatorial matrix mixtures. This platform enables the study of differentiation in response to a multitude of microenvironments in parallel. The fabrication process required only access to a standard robotic DNA spotter, off-the-shelf materials and 1,000 times less protein than conventional means of investigating cell-ECM interactions. To demonstrate its utility, we applied this platform to study the effects of 32 different combinations of five extracellular matrix molecules (collagen I, collagen III, collagen IV, laminin and fibronectin) on cellular differentiation in two contexts: maintenance of primary rat hepatocyte phenotype indicated by intracellular albumin staining and differentiation of mouse embryonic stem (ES) cells toward an early hepatic fate, indicated by expression of a beta-galactosidase reporter fused to the fetal liver-specific gene, Ankrd17 (also known as gtar). Using this technique, we identified combinations of ECM that synergistically impacted both hepatocyte function and ES cell differentiation. This versatile technique can be easily adapted to other applications, as it is amenable to studying almost any insoluble microenvironmental cue in a combinatorial fashion and is compatible with several cell types.
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paper_title: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

paper_content:

RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene1,2,3,4. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs5,6,7,8,9. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.
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paper_title: A resource for large-scale RNA-interference-based screens in mammals

paper_content:

Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool1,2,3,4,5,6,7,8,9,10. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA ‘bar codes’, and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery.
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paper_title: Human Cell Chips: Adapting DNA Microarray Spotting Technology to Cell-Based Imaging Assays

paper_content:

Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR), subcellular localization (nuclear translocation of NFkappaB) and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases) in response to treatment by several chemical effectors (anisomycin, TNFalpha, and interferon), and we demonstrate scalability by printing a chip with approximately 4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability.
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paper_title: Overview of Electrochemical DNA Biosensors: New Approaches to Detect the Expression of Life

paper_content:

DNA microarrays are an important tool with a variety of applications in gene expression studies, genotyping, pharmacogenomics, pathogen classification, drug discovery, sequencing and molecular diagnostics. They are having a strong impact in medical diagnostics for cancer, toxicology and infectious disease applications. A series of papers have been published describing DNA biochips as alternative to conventional microarray platforms to facilitate and ameliorate the signal readout. In this review, we will consider the different methods proposed for biochip construction, focusing on electrochemical detection of DNA. We also introduce a novel single-stranded DNA platform performing high-throughput SNP detection and gene expression profiling.
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paper_title: High-Throughput Selection of Effective RNAi Probes for Gene Silencing

paper_content:

RNA interference (RNAi) is a process of sequence-specific posttranscriptional gene silencing mediated by double-stranded RNA. RNAi has recently emerged as a powerful genetic tool to analyze gene function in mammalian cells. The power of this method is limited however, by the uncertainty in predicting the efficacy of small interfering RNAs (siRNAs) in silencing a gene. This has imposed serious limitations not only for small-scale but also for high-throughput RNAi screening initiatives in mammalian systems. We have developed a reliable and quantitative approach for the rapid and efficient identification of the most effective siRNA against any gene. The efficacy of siRNA sequences is monitored by their ability to reduce the expression of cognate target-reporter fusions with easily quantified readouts. Finally, using micro array-based cell transfections, we demonstrate an unlimited potential of this approach in high-throughput screens for identifying effective siRNA probes for silencing genes in mammalian systems. This approach is likely to have implications in the use of RNAi as a reverse genetic tool for analyzing mammalian gene function on a genome-wide scale.
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paper_title: Role of YAP/TAZ in mechanotransduction

paper_content:

Cells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behaviour, including growth, differentiation and cancer malignant progression, but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPase activity and tension of the actomyosin cytoskeleton, but is independent of the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints in dictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.
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paper_title: Microarray Transfection Analysis of Transcriptional Regulation by cAMP-dependent Protein Kinase

paper_content:

A wide variety of bioinformatic tools have been described to characterize potential transcriptional regulatory mechanisms based on genomic sequence analysis and microarray hybridization studies. However, these regulatory mechanisms are still experimentally verified using transient transfection methods. Current transfection methods are limited both by their large scale and by the low level of efficiency for certain cell types. Our goals were to develop a microarray-based transfection method that could be optimized for different cell types and that would be useful in reporter assays of transcriptional regulation. Here we describe a novel transfection method, termed STEP (surface transfection and expression protocol), which employs microarray-based DNA transfection of adherent cells in the functional analysis of transcriptional regulation. In STEP, recombinant proteins with biological activities designed to enhance transfection are complexed with expression vector DNAs prior to spotting on microscope slides. The recombinant proteins used in STEP complexes can be varied to increase the efficiency for different cell types. We demonstrate that STEP efficiently transfects both supercoiled plasmids and PCR-generated linear expression cassettes. A co-transfection assay using effector expression vectors encoding the cAMP-dependent protein kinase (PKA), as well as reporter vectors containing PKA-regulated promoters, showed that STEP transfection allows detection and quantitation of transcriptional regulation by this protein kinase. Because bioinformatic studies often result in the identification of many putative regulatory elements and signaling pathways, this approach should be of utility in high-throughput functional genomic studies of transcriptional regulation.
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paper_title: Arrayed cellular microenvironments for identifying culture and differentiation conditions for stem, primary and rare cell populations

paper_content:

Arrayed cellular microenvironments for identifying culture and differentiation conditions for stem, primary and rare cell populations
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paper_title: Microarrays of cells expressing defined cDNAs

paper_content:

Genome and expressed sequence tag projects are rapidly cataloguing and cloning the genes of higher organisms, including humans. An emerging challenge is to rapidly uncover the functions of genes and to identify gene products with desired properties. We have developed a microarray-driven gene expression system for the functional analysis of many gene products in parallel. Mammalian cells are cultured on a glass slide printed in defined locations with different DNAs. Cells growing on the printed areas take up the DNA, creating spots of localized transfection within a lawn of non-transfected cells. By printing sets of complementary DNAs cloned in expression vectors, we make microarrays whose features are clusters of live cells that express a defined cDNA at each location. Here we demonstrate two uses for our approach: as an alternative to protein microarrays for the identification of drug targets, and as an expression cloning system for the discovery of gene products that alter cellular physiology. By screening transfected cell microarrays expressing 192 different cDNAs, we identified proteins involved in tyrosine kinase signalling, apoptosis and cell adhesion, and with distinct subcellular distributions.
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paper_title: Sequencing technologies — the next generation

paper_content:

Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing (NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest.
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paper_title: The Extracellular Matrix: Not Just Pretty Fibrils

paper_content:

The extracellular matrix (ECM) and ECM proteins are important in phenomena as diverse as developmental patterning, stem cell niches, cancer, and genetic diseases. The ECM has many effects beyond providing structural support. ECM proteins typically include multiple, independently folded domains whose sequences and arrangement are highly conserved. Some of these domains bind adhesion receptors such as integrins that mediate cell-matrix adhesion and also transduce signals into cells. However, ECM proteins also bind soluble growth factors and regulate their distribution, activation, and presentation to cells. As organized, solid-phase ligands, ECM proteins can integrate complex, multivalent signals to cells in a spatially patterned and regulated fashion. These properties need to be incorporated into considerations of the functions of the ECM.
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paper_title: Strategies for Engineering the Adhesive Microenvironment

paper_content:

Cells exist within a complex tissue microenvironment, which includes soluble factors, extracellular matrix molecules, and neighboring cells. In the breast, the adhesive microenvironment plays a crucial role in driving both normal mammary gland development as well tumor initiation and progression. Researchers are designing increasingly more complex ways to mimic the in vivo microenvironment in an in vitro setting, so that cells in culture may serve as model systems for tissue structures. Here, we explore the use of microfabrication technologies to engineer the adhesive microenvironment of cells in culture. These new tools permit the culture of cells on well-defined surface chemistries, patterning of cells into defined geometries either alone or in coculture scenarios, and measurement of forces associated with cell-ECM interactions. When applied to questions in mammary gland development and neoplasia, these new tools will enable a better understanding of how adhesive, structural, and mechanical cues regulate mammary epithelial biology.
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paper_title: Soft substrates drive optimal differentiation of human healthy and dystrophic myotubes.

paper_content:

The in vitro development of human myotubes carrying genetic diseases, such as Duchenne Muscular Dystrophy, will open new perspectives in the identification of innovative therapeutic strategies. Through the proper design of the substrate, we guided the differentiation of human healthy and dystrophic myoblasts into myotubes exhibiting marked functional differentiation and highly defined sarcomeric organization. A thin film of photo cross-linkable elastic poly-acrylamide hydrogel with physiological-like and tunable mechanical properties (elastic moduli, E: 12, 15, 18 and 21 kPa) was used as substrate. The functionalization of its surface by micro-patterning in parallel lanes (75 microm wide, 100 microm spaced) of three adhesion proteins (laminin, fibronectin and matrigel) was meant to maximize human myoblasts fusion. Myotubes formed onto the hydrogel showed a remarkable sarcomere formation, with the highest percentage (60.0% +/- 3.8) of myotubes exhibiting sarcomeric organization, of myosin heavy chain II and alpha-actinin, after 7 days of culture onto an elastic (15 kPa) hydrogel and a matrigel patterning. In addition, healthy myotubes cultured in these conditions showed a significant membrane-localized dystrophin expression. In this study, the culture substrate has been adapted to human myoblasts differentiation, through an easy and rapid methodology, and has led to the development of in vitro human functional skeletal muscle myotubes useful for clinical purposes and in vitro physiological study, where to carry out a broad range of studies on human muscle physiopathology.
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paper_title: Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity

paper_content:

Abstract ::: We previously identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells, including midbrain tyrosine hydroxylase-positive (TH+) dopaminergic neurons, from mouse embryonic stem cells. We report here that SDIA induces efficient neural differentiation also in primate embryonic stem cells. Induced neurons contain TH+ neurons at a frequency of 35% and produce a significant amount of dopamine. Interestingly, differentiation of TH+ neurons from undifferentiated embryonic cells occurs much faster in vitro (10 days) than it does in the embryo (≈5 weeks). In addition, 8% of the colonies contain large patches of Pax6+-pigmented epithelium of the retina. The SDIA method provides an unlimited source of primate cells for the study of pathogenesis, drug development, and transplantation in degenerative diseases such as Parkinson's disease and retinitis pigmentosa.
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paper_title: Microarrays of small molecules embedded in biodegradable polymers for use in mammalian cell-based screens.

paper_content:

We developed a microarray-based system for screening small molecules in mammalian cells. This system is compatible with image-based screens and requires fewer than 100 cells per compound. Each compound is impregnated in a 200-microm-diameter disc composed of biodegradable poly-(D),(L)-lactide/glycolide copolymer. Cells are seeded on top of these discs, and compounds slowly diffuse out, affecting proximal cells. In contrast with microtiter-based screening, this system does not involve the use of wells or walls between each compound-treated group of cells. We demonstrate detection of the effects of a single compound in a large microarray, that diverse compounds can be released in this format, and that extended release over several days is feasible. We performed a small synthetic lethal screen and identified a compound (macbecin II) that has reduced activity in cells with RNA interference-mediated decrease in the expression of tuberous sclerosis 2. Thus, we have developed a microarray-based screening system for testing the effects of small molecules on mammalian cells by using an imaging-based readout. This method will be useful to those performing small-molecule screens to discover new chemical tools and potential therapeutic agents.
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paper_title: Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells

paper_content:

Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells
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paper_title: The Sequence of the Human Genome

paper_content:

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies—a whole-genome assembly and a regional chromosome assembly—were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional ∼12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.
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paper_title: Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT

paper_content:

To increase the coverage of secreted protein prediction, we describe a combination strategy. Instead of using a single method, we combine Hidden Markov Model (HMM)-based methods CJ-SPHMM and TMHMM with PSORT in secreted protein prediction. CJ-SPHMM is an HMM-based signal peptide prediction method, while TMHMM is an HMM-based transmembrane (TM) protein prediction algorithm. With CJ-SPHMM and TMHMM, proteins with predicted signal peptide and without predicted TM regions are taken as putative secreted proteins. This HMM-based approach predicts secreted protein with Ac (Accuracy) at 0.82 and Cc (Correlation coefficient) at 0.75, which are similar to PSORT with Ac at 0.82 and Cc at 0.76. When we further complement the HMM-based method, i.e., CJ-SPHMM + TMHMM with PSORT in secreted protein prediction, the Ac value is increased to 0.86 and the Cc value is increased to 0.81. Taking this combination strategy to search putative secreted proteins from the International Protein Index (IPI) maintained at the European Bioinformatics Institute (EBI), we constructed a putative human secretome with 5235 proteins. The prediction system described here can also be applied to predicting secreted proteins from other vertebrate proteomes.
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paper_title: Cell microarrays and RNA interference chip away at gene function

paper_content:

The recent development of cell microarrays offers the potential to accelerate high-throughput functional genetic studies. The widespread use of RNA interference (RNAi) has prompted several groups to fabricate RNAi cell microarrays that make possible discrete, in-parallel transfection with thousands of RNAi reagents on a microarray slide. Though still a budding technology, RNAi cell microarrays promise to increase the efficiency, economy and ease of genome-wide RNAi screens in metazoan cells.
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paper_title: Automatic Identification of Subcellular Phenotypes on Human Cell Arrays

paper_content:

Light microscopic analysis of cell morphology provides a high-content readout of cell function and protein localization. Cell arrays and microwell transfection assays on cultured cells have made cell phenotype analysis accessible to high-throughput experiments. Both the localization of each protein in the proteome and the effect of RNAi knock-down of individual genes on cell morphology can be assayed by manual inspection of microscopic images. However, the use of morphological readouts for functional genomics requires fast and automatic identification of complex cellular phenotypes. Here, we present a fully automated platform for high-throughput cell phenotype screening combining human live cell arrays, screening microscopy, and machine-learning-based classification methods. Efficiency of this platform is demonstrated by classification of eleven subcellular patterns marked by GFP-tagged proteins. Our classification method can be adapted to virtually any microscopic assay based on cell morphology, opening a wide range of applications including large-scale RNAi screening in human cells.
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paper_title: A large-scale RNAi screen in human cells identifies new components of the p53 pathway

paper_content:

RNA interference (RNAi) is a powerful new tool with which to perform loss-of-function genetic screens in lower organisms and can greatly facilitate the identification of components of cellular signalling pathways1,2,3. In mammalian cells, such screens have been hampered by a lack of suitable tools that can be used on a large scale. We and others have recently developed expression vectors to direct the synthesis of short hairpin RNAs (shRNAs) that act as short interfering RNA (siRNA)-like molecules to stably suppress gene expression4,5. Here we report the construction of a set of retroviral vectors encoding 23,742 distinct shRNAs, which target 7,914 different human genes for suppression. We use this RNAi library in human cells to identify one known and five new modulators of p53-dependent proliferation arrest. Suppression of these genes confers resistance to both p53-dependent and p19ARF-dependent proliferation arrest, and abolishes a DNA-damage-induced G1 cell-cycle arrest. Furthermore, we describe siRNA bar-code screens to rapidly identify individual siRNA vectors associated with a specific phenotype. These new tools will greatly facilitate large-scale loss-of-function genetic screens in mammalian cells.
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paper_title: Transfection microarray of human mesenchymal stem cells and on-chip siRNA gene knockdown.

paper_content:

The transfection efficiency of primary cells is the bottleneck for their use with miniaturized formats for gene validation assays. We have found that when formulations containing various reporter plasmids were microarrayed on glass slides (chips), hMSCs cultivated on the chip incorporated and expressed the microarrayed plasmid DNAs with high efficiency and virtually total spatial resolution. Fibronectin, as the key formulation component, was found to significantly increase the on-chip transfection efficiency in hMSCs as well as many other cells. Further, we have conclusively proven that when siRNA was co-arrayed with the target plasmid DNA, a concentration-dependent gene knockdown was observed. Thus, massively miniaturized RNAi gene knockdown experiments can now be performed in primary cells, previously unusable with transfection microarrays (TMA).
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paper_title: Cell microarray for screening feeder cells for differentiation of embryonic stem cells

paper_content:

Microarrays are currently recognized as one of major tools in the assessment of gene expression via cDNA or RNA analysis and are now accepted as a powerful experimental tool for high-throughput screening of a large number of samples, such as cDNA and siRNAs. In this study, we examined the potential of the microarray methodology for high-throughput screening of candidate cells as feeder cells which effectively differentiate embryonic stem (ES) cells to the specific lineage. Cell arrays were prepared by applying three kinds of cells, PA6, human umbilical vein endothelial, and COS-1 cells, to circular spots, 2 mm in diameter, on a glass plate, followed by the application of mouse ES cells to the cell microarray. After 8 d in culture, TuJ1 (neuron-specific class III β-tubulin) immunocytochemical staining clearly demonstrated that only PA6 cell spots had the capability to induce ES cells to neuronal differentiation. Although this is a model experiment, these findings clearly indicate that the cell microarray will become a powerful tool for high-throughput screening large numbers of candidate feeder cells for specific differentiation.
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paper_title: Induction of the Differentiation of Lentoids from Primate Embryonic Stem Cells

paper_content:

PURPOSE ::: To produce lens cells from primate embryonic stem (ES) cells in a reproducible, controlled manner. ::: ::: ::: METHODS ::: Cynomologus monkey ES cells were induced to differentiate by stromal cell-derived inducing activity (SDIA). The lentoids produced by this treatment were processed for immunohistochemical and immunoblotting analysis. The effect of varying the concentration of fibroblast growth factor (FGF)-2 and the density of the ES colonies plated during the differentiation process were also examined. ::: ::: ::: RESULTS ::: After a 2- to 3-week induction period, lentoids were produced by a subpopulation of ES colonies. Western blot analysis and immunohistochemistry revealed that these lentoids expressed alphaA-crystallin and Pax6. The number of lentoids resulting from treatment increased with increasing FGF-2 concentration and plated colony density. ::: ::: ::: CONCLUSIONS ::: The differentiation of primate ES cells into lentoids can be achieved by treatment with SIDA. ES cells can be used to facilitate a greater understanding of the mechanisms functioning in differentiation in vivo and in vitro.
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paper_title: An approach to genomewide screens of expressed small interfering RNAs in mammalian cells

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To facilitate the construction of large genomewide libraries of small interfering RNAs (siRNAs), we have developed a dual promoter system (pDual) in which a synthetic DNA encoding a gene-specific siRNA sequence is inserted between two different opposing polymerase III promoters, the mouse U6 and human H1 promoters. Upon transfection into mammalian cells, the sense and antisense strands of the duplex are transcribed by these two opposing promoters from the same template, resulting in a siRNA duplex with a uridine overhang on each 3′ terminus. A single-step PCR protocol has been developed by using this dual promoter system that allows the production of siRNA expression cassettes in a high-throughput manner. We have shown that siRNAs transcribed by either the dual promoter vector or siRNA expression cassettes can induce strong and gene-specific suppression of both endogenous genes and ectopically expressed genes in mammalian cells. Furthermore, we have constructed an arrayed siRNA expression cassette library that targets >8,000 genes with two siRNA sequences per gene. A high-throughput screen of this library has revealed both known and unique genes involved in the NF-κB signaling pathway.
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paper_title: Differentiation of Monkey Embryonic Stem Cells into Neural Lineages1

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Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (a-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (;4%) or choline acetyltransferase (;13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.
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paper_title: Induction of Midbrain Dopaminergic Neurons from ES Cells by Stromal Cell–Derived Inducing Activity

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Summary Precise specification of a particular neuronal character-istic, such as neurotransmitter choice, is crucial when We have identified a stromal cell–derived inducing ac- induced neurons are to be used for therapeutic applica- tivity (SDIA) that promotes neural differentiation of tionsorbasicneuroscienceresearch.Itisthereforepref- mouse ES cells. SDIA accumulates on the surface of erable to avoid RA treatment unless RA induces the PA6stromalcellsandinducesefficientneuronaldiffer- particular type of neurons of one’s interest. entiation of cocultured ES cells in serum-free condi- In this report, we introduce an efficient system for in tions without use of either retinoic acid or embryoid vitro neural differentiation of mouse ES cells in a serum- bodies. BMP4, which acts as an antineuralizing mor- free condition that requires neither EBs nor RA treat- phogen in Xenopus , suppresses SDIA-induced neu- ment. We also discuss the possibilities for therapeutic ralization and promotes epidermal differentiation. A
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paper_title: An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division

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RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules1,2,3,4. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions5,6,7,8. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing9,10,11. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs12 from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.
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paper_title: Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells.

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Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes.
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paper_title: The emergence and diffusion of DNA microarray technology

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The network model of innovation widely adopted among researchers in the economics of science and technology posits relatively porous boundaries between firms and academic research programs and a bi-directional flow of inventions, personnel, and tacit knowledge between sites of university and industry innovation. Moreover, the model suggests that these bi-directional flows should be considered as mutual stimulation of research and invention in both industry and academe, operating as a positive feedback loop. One side of this bi-directional flow – namely; the flow of inventions into industry through the licensing of university-based technologies – has been well studied; but the reverse phenomenon of the stimulation of university research through the absorption of new directions emanating from industry has yet to be investigated in much detail. We discuss the role of federal funding of academic research in the microarray field, and the multiple pathways through which federally supported development of commercial microarray technologies have transformed core academic research fields.Results and conclusionOur study confirms the picture put forward by several scholars that the open character of networked economies is what makes them truly innovative. In an open system innovations emerge from the network. The emergence and diffusion of microarray technologies we have traced here provides an excellent example of an open system of innovation in action. Whether they originated in a startup company environment that operated like a think-tank, such as Affymax, the research labs of a large firm, such as Agilent, or within a research university, the inventors we have followed drew heavily on knowledge resources from all parts of the network in bringing microarray platforms to light.Federal funding for high-tech startups and new industrial development was important at several phases in the early history of microarrays, and federal funding of academic researchers using microarrays was fundamental to transforming the research agendas of several fields within academe. The typical story told about the role of federal funding emphasizes the spillovers from federally funded academic research to industry. Our study shows that the knowledge spillovers worked both ways, with federal funding of non-university research providing the impetus for reshaping the research agendas of several academic fields.
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paper_title: Hepatic maturation in differentiating embryonic stem cells in vitro

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We investigated the potential of mouse embryonic stem (ES) cells to differentiate into hepatocytes in vitro. Differentiating ES cells expressed endodermal-specific genes, such as α-fetoprotein, transthyretin, α 1-anti-trypsin and albumin, when cultured without additional growth factors and late differential markers of hepatic development, such as tyrosine aminotransferase (TAT) and glucose-6-phosphatase (G6P), when cultured in the presence of growth factors critical for late embryonic liver development. Further, induction of TAT and G6P expression was induced regardless of expression of the functional SEK1 gene, which is thought to provide a survival signal for hepatocytes during an early stage of liver morphogenesis. The data indicate that the in vitro ES differentiation system has a potential to generate mature hepatocytes. The system has also been found useful in analyzing the role of growth factors and intracellular signaling molecules in hepatic development.
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paper_title: Multiplex GPCR Assay in Reverse Transfection Cell Microarrays

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G protein-coupled receptors (GPCRs) are a superfamily of proteins that include some of the most important drug targets in the pharmaceutical industry. Despite the success of this group of drugs, there remains a need to identify GPCR-targeted drugs with greater selectivity, to develop screening assays for validated targets, and to identify ligands for orphan receptors. To address these challenges, the authors have created a multiplexed GPCR assay that measures greater than 3000 receptor: ligand interactions in a single microplate. The multiplexed assay is generated by combining reverse transfection in a 96-well plate format with a calcium flux readout. This assay quantitatively measures receptor activation and inhibition and permits the determination of compound potency and selectivity for entire families of GPCRs in parallel. To expand the number of GPCR targets that may be screened in this system, receptors are cotransfected with plasmids encoding a promiscuous G protein, permitting the analysis of recep...
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paper_title: Electroactive Self-Assembled Monolayers that Permit Orthogonal Control over the Adhesion of Cells to Patterned Substrates†

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This article describes an electroactive substrate that displays two independent dynamic functions for controlling the adhesion of cells. The approach is based on self-assembled monolayers on gold that are patterned into regions presenting the Arg-Gly-Asp peptide cell adhesion ligand. The patterned regions differ in the electrochemical properties of the linkers that tether the peptides to the monolayer. In this work, three distinct chemistries are employed that provide for release of the ligand on application of a negative potential, release of the ligand on application of a positive potential, and no change in response to a potential. Cells were allowed to attach to a monolayer patterned into circular regions comprising the three chemistries. Treatment with electric potentials of 650 or -650 mV resulted in the selective release of adherent cells only from regions that display the relevant electroactive groups. This example establishes the preparation of dynamic substrates with multiple functions and will be important to preparing model cultures derived from multiple cell types, with control over the temporal interactions of each cell population.
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paper_title: Satellite cells delivered by micro-patterned scaffolds: a new strategy for cell transplantation in muscle diseases.

paper_content:

Myoblast transplantation is a potentially useful therapeutic tool in muscle diseases, but the lack of an efficient delivery system has hampered its application. Here we have combined cell biology and polymer processing to create an appropriate microenvironment for in vivo transplantation of murine satellite cells (mSCs). Cells were prepared from single muscle fibers derived from C57BL/6-Tgn enhanced green fluorescent protein (GFP) transgenic mice. mSCs were expanded and seeded within micro-patterned polyglycolic acid 3-dimensional scaffolds fabricated using soft lithography and thermal membrane lamination. Myogenicity was then evaluated in vitro using immunostaining, flow cytometry, and reverse transcription polymerase chain reaction analyses. Scaffolds containing mSCs were implanted in pre-damaged tibialis anterior muscles of GFP-negative syngenic mice. Cells detached from culture dishes were directly injected into contra-lateral limbs as controls. In both cases, delivered cells participated in muscle re...
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paper_title: Fiber Types in Mammalian Skeletal Muscles

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Mammalian skeletal muscle comprises different fiber types, whose identity is first established during embryonic development by intrinsic myogenic control mechanisms and is later modulated by neural and hormonal factors. The relative proportion of the different fiber types varies strikingly between species, and in humans shows significant variability between individuals. Myosin heavy chain isoforms, whose complete inventory and expression pattern are now available, provide a useful marker for fiber types, both for the four major forms present in trunk and limb muscles and the minor forms present in head and neck muscles. However, muscle fiber diversity involves all functional muscle cell compartments, including membrane excitation, excitation-contraction coupling, contractile machinery, cytoskeleton scaffold, and energy supply systems. Variations within each compartment are limited by the need of matching fiber type properties between different compartments. Nerve activity is a major control mechanism of the fiber type profile, and multiple signaling pathways are implicated in activity-dependent changes of muscle fibers. The characterization of these pathways is raising increasing interest in clinical medicine, given the potentially beneficial effects of muscle fiber type switching in the prevention and treatment of metabolic diseases.
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paper_title: Affinity capture of proteins from solution and their dissociation by contact printing

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Biological experiments at the solid/liquid interface, in general, require surfaces with a thin layer of purified molecules, which often represent precious material. Here, we have devised a method to extract proteins with high selectivity from crude biological sample solutions and place them on a surface in a functional, arbitrary pattern. This method, called affinity-contact printing (αCP), uses a structured elastomer derivatized with ligands against the target molecules. After the target molecules have been captured, they are printed from the elastomer onto a variety of surfaces. The ligand remains on the stamp for reuse. In contrast with conventional affinity chromatography, here dissociation and release of captured molecules to the substrate are achieved mechanically. We demonstrate this technique by extracting the cell adhesion molecule neuron-glia cell adhesion molecule (NgCAM) from tissue homogenates and cell culture lysates and patterning affinity-purified NgCAM on polystyrene to stimulate the attachment of neuronal cells and guide axon outgrowth.
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paper_title: Selective adhesion of hepatocytes on patterned surfaces.

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Successful development of cell-biased bioartificial liver devices necessitates the establishment of techniques and designs for long-term, stable hepatocellular function and efficient transport of nutrients and wastes within the device. Given the relatively large cell mass that one must consider, one possible solution involves the use of micropatterning technology to sandwich hepatocytes aligned in rows between two micropatterned surfaces. Rows of cells would alternate with hepatocyte-free areas, creating efficient transport channels for fluid flow and nutrient exchange. Ultimately, this type of device could also be used as a three-dimensional construct for investigating a variety of cell-surface, cell-extracellular matrix, and cell-cell interactions. To achieve this goal, one must develop techniques for selectively adhering hepatocytes to solid substrates. In this study, reproducible, selective adhesion of hepatocytes on a glass substrate with large regions of adhesive (AS) and nonadhesive (NAS) surfaces was obtained. The AS had hydrophilic characteristics, enhancing deposition of collagen molecules from an aqueous solution, and subsequent hepatocyte adhesion, whereas the NAS had hydrophobic properties and remained collagen-free and hepatocyte-free. In addition, a reproducible processing technique for obtaining patterns of hepatocytes was developed and optimized, using a surface with a single AS band as a first approximation to a micropatterned device. This was achieved by spincoating an aqueous collagen type I solution (0.1 mg/mL) on a banded surface at 500 rpm for 25 seconds. The morphology and long-term function of the hepatocytes attached to AS in nonbanded and banded surface configurations was assessed by mimicking sandwich culture and was shown to be similar to stable, differentiated sandwich cultures. Mathematical modeling was used to determine critical design criteria for the hypothetical micropatterned device. The oxygen distribution and viscous pressure drop were modeled along a typical microchannel and limited to in vivo values. An optimal channel length of 0.6 cm and a flow rate of 2.0 x 10(-6) mL/s were obtained for a channel of 100 microns in width and 10 microns in height. These values were reasonable in terms of practical implementation.
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paper_title: Soft substrates drive optimal differentiation of human healthy and dystrophic myotubes.

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The in vitro development of human myotubes carrying genetic diseases, such as Duchenne Muscular Dystrophy, will open new perspectives in the identification of innovative therapeutic strategies. Through the proper design of the substrate, we guided the differentiation of human healthy and dystrophic myoblasts into myotubes exhibiting marked functional differentiation and highly defined sarcomeric organization. A thin film of photo cross-linkable elastic poly-acrylamide hydrogel with physiological-like and tunable mechanical properties (elastic moduli, E: 12, 15, 18 and 21 kPa) was used as substrate. The functionalization of its surface by micro-patterning in parallel lanes (75 microm wide, 100 microm spaced) of three adhesion proteins (laminin, fibronectin and matrigel) was meant to maximize human myoblasts fusion. Myotubes formed onto the hydrogel showed a remarkable sarcomere formation, with the highest percentage (60.0% +/- 3.8) of myotubes exhibiting sarcomeric organization, of myosin heavy chain II and alpha-actinin, after 7 days of culture onto an elastic (15 kPa) hydrogel and a matrigel patterning. In addition, healthy myotubes cultured in these conditions showed a significant membrane-localized dystrophin expression. In this study, the culture substrate has been adapted to human myoblasts differentiation, through an easy and rapid methodology, and has led to the development of in vitro human functional skeletal muscle myotubes useful for clinical purposes and in vitro physiological study, where to carry out a broad range of studies on human muscle physiopathology.
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paper_title: Muscle Differentiation and Myotubes Alignment Is Influenced by Micropatterned Surfaces and Exogenous Electrical Stimulation

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An in vitro muscle-like structure with parallel-oriented contractile myotubes is needed as a model of muscle tissue regeneration. For this purpose, it is necessary to reproduce a controllable microscale environment mimicking the in vivo cues. In this work we focused on the application of topological and electrical stimuli on muscle precursor cell (MPC) culture to influence MPC orientation and induce myotube alignment. The two stimulations were tested both independently and together. A structural and topological template was achieved using micropatterned poly-(L-lactic acid) membranes. Electrical stimulation, consisting of square pulses of 70 mV/cm amplitude each 30 s, was applied to the MPC culture. The effect of different pulse durations on cultures was evaluated by galvanotaxis analysis. The highest cell displacement rate toward the cathode was observed for 3 ms pulse stimulation, which was then applied in combination with topological stimuli. Topological and electrical stimuli had an additive effect in...
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paper_title: Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

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Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a “cuboidal” epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-β-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.
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paper_title: Production of arrays of cardiac and skeletal muscle myofibers by micropatterning techniques on a soft substrate

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Micropatterning and microfabrication techniques have been widely used to pattern cells on surfaces and to have a deeper insight into many processes in cell biology such as cell adhesion and interactions with the surrounding environment. The aim of this study was the development of an easy and versatile technique for the in vitro production of arrays of functional cardiac and skeletal muscle myofibers using micropatterning techniques on soft substrates. Cardiomyocytes were used for the production of oriented cardiac myofibers whereas mouse muscle satellite cells for that of differentiated parallel myotubes. We performed micro-contact printing of extracellular matrix proteins on soft polyacrylamide-based hydrogels photopolymerized onto functionalized glass slides. Our methods proved to be simple, repeatable and effective in obtaining an extremely selective adhesion of both cardiomyocytes and satellite cells onto patterned soft hydrogel surfaces. Cardiomyocytes resulted in aligned cardiac myofibers able to exhibit a synchronous contractile activity after 2 days of culture. We demonstrated for the first time that murine satellite cells, cultured on a soft hydrogel substrate, fuse and form aligned myotubes after 7 days of culture. Immunofluorescence analyses confirmed correct expression of cell phenotype, differentiation markers and sarcomeric organization. These results were obtained in myotubes derived from satellite cells from both wild type and MDX mice which are research models for the study of muscle dystrophy. These arrays of both cardiac and skeletal muscle myofibers could be used as in vitro models for pharmacological screening tests or biological studies at the single fiber level.
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paper_title: Cell-cell signaling by direct contact increases cell proliferation via a PI3K-dependent signal

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We report a novel mechanism of cellular growth control. Increasing the density of endothelial or smooth muscle cells in culture increased cell-cell contact and decreased cell spreading, leading to growth arrest. Using a new method to independently control cell-cell contact and cell spreading, we found that introducing cell-cell contact positively regulates proliferation, but that contact-mediated proliferation can be masked by changes in cell spreading: Round cells with many contacts proliferated less than spread cells with none. Physically blocking cell-cell contact or inhibiting PI3K signaling abrogated cell-cell induced proliferation, but inhibiting diffusible paracrine signaling did not. Thus, direct cell-cell contact induces proliferation in these cells.
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paper_title: Protein and cell micropatterning and its integration with micro/nanoparticles assembly.

paper_content:

Micropatterning of proteins and cells has become very popular over the past decade due to its importance in the development of biosensors, microarrays, tissue engineering and cellular studies. This article reviews the techniques developed for protein and cell micropatterning and its biomedical applications. The prospect of integrating micro and nanoparticles with protein and cell micropatterning is discussed. The micro/nanoparticles are assembled into patterns and form the substrate for proteins and cell attachment. The assembled particles create a micro or nanotopography, depending on the size of the particles employed. The nonplanar structure can increase the surface area for biomolecules attachment and therefore enhance the sensitivity for detection in biosensors. Furthermore, a nanostructured substrate can influence the conformation and functionality of protein attached to it, while cellular response in terms of morphology, adhesion, proliferation, differentiation, etc. can be affected by a surface expressing micro or nanoscale structures. Proteins and cells tend to lose their normal functions upon attachment to substrate. By recognizing the types of topography that are favourable for preserving proteins and cell behaviour, and integrating it with micropattering will lead to the development of functional protein and cell patterns.
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paper_title: Geometric control of human stem cell morphology and differentiation

paper_content:

During tissue morphogenesis, stem cells and progenitor cells migrate, proliferate, and differentiate, with striking changes in cell shape, size, and acting mechanical stresses. The local cellular function depends on the spatial distribution of cytokines as well as local mechanical microenvironments in which the cells reside. In this study, we controlled the organization of human adipose derived stem cells using micro-patterning technologies, to investigate the influence of multi-cellular form on spatial distribution of cellular function at an early stage of cell differentiation. The underlying role of cytoskeletal tension was probed through drug treatment. Our results show that the cultivation of stem cells on geometric patterns resulted in pattern- and position-specific cell morphology, proliferation and differentiation. The highest cell proliferation occurred in the regions with large, spreading cells (such as the outer edge of a ring and the short edges of rectangles). In contrast, stem cell differentiation co-localized with the regions containing small, elongated cells (such as the inner edge of a ring and the regions next to the short edges of rectangles). The application of drugs that inhibit the formation of actomyosin resulted in the lack of geometrically specific differentiation patterns. This study confirms the role of substrate geometry on stem cell differentiation, through associated physical forces, and provides a simple and controllable system for studying biophysical regulation of cell function.
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paper_title: Biological role of connexin intercellular channels and hemichannels.

paper_content:

Gap junctions (GJ) and hemichannels (HC) formed from the protein subunits called connexins are transmembrane conduits for the exchange of small molecules and ions. Connexins and another group of HC-forming proteins, pannexins comprise the two families of transmembrane proteins ubiquitously distributed in vertebrates. Most cell types express more than one connexin or pannexin. While connexin expression and channel activity may vary as a function of physiological and pathological states of the cell and tissue, only a few studies suggest the involvement of pannexin HC in acquired pathological conditions. Importantly, genetic mutations in connexin appear to interfere with GJ and HC function which results in several diseases. Thus connexins could serve as potential drug target for therapeutic intervention. Growing evidence suggests that diseases resulting from HC dysfunction might open a new direction for development of specific HC reagents. This review provides a comprehensive overview of the current studies of GJ and HC formed by connexins and pannexins in various tissue and organ systems including heart, central nervous system, kidney, mammary glands, ovary, testis, lens, retina, inner ear, bone, cartilage, lung and liver. In addition, present knowledge of the role of GJ and HC in cell cycle progression, carcinogenesis and stem cell development is also discussed.
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paper_title: Micropatterned Surfaces for Control of Cell Shape, Position, and Function

paper_content:

The control of cell position and function is a fundamental focus in the development of applications ranging from cellular biosensors to tissue engineering. Using microcontact printing of self-assembled monolayers (SAMs) of alkanethiolates on gold, we manufactured substrates that contained micrometer-scale islands of extracellular matrix (ECM) separated by nonadhesive regions such that the pattern of islands determined the distribution and position of bovine and human endothelial cells. In addition, the size and geometry of the islands were shown to control cell shape. Traditional approaches to modulate cell shape, either by attaching suspended cells to microbeads of different sizes or by plating cells on substrates coated with different densities of ECM, suggested that cell shape may play an important role in control of apoptosis as well as growth. Data are presented which show how micropatterned substrates were used to definitively test this hypothesis. Progressively restricting bovine and human endothelial cell extension by culturing cells on smaller and smaller micropatterned adhesive islands regulated a transition from growth to apoptosis on a single continuum of cell spreading, thus confirming the central role of cell shape in cell function. The micropatterning technology is therefore essential not only for construction of biosurface devices but also for the investigation of the fundamental biology of cell-ECM interactions.
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paper_title: Degradation of Micropatterned Surfaces by Cell-Dependent and -Independent Processes†

paper_content:

This paper describes a study to determine the role of active cellular processes in the initial patterning and eventual degradation of different micropatterned substrates. We compared the effects of serum and cell type on the ability of cells to crawl onto the nonadhesive regions of a variety of patterned substrates. Cells initially patterned in the presence of serum onto substrates manufactured using agarose, pluronics, hexa(ethylene glycol), or polyacrylamide as the nonadhesive. While polyacrylamide remained inert and patterned cells for at least 28 days, agarose and pluronics degraded by gradual desorption of the nonadhesive from the surface independently of the presence of cells. Hexa(ethylene glycol) degraded by a time-dependent mechanism that could be accelerated by cell-dependent oxidative processes. In contrast to the other substrates studied, bovine serum albumin (BSA) patterned cells only under serum-free conditions. The serum did not displace BSA from the surface but instead activated cell-secreted proteases that led to degradation of the substrate. These findings illustrate the importance of specific cellular and noncellular processes in the failure of different nonadhesive chemistries commonly used to pattern cells.
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paper_title: Effect of cell – cell interactions in preservation of cellular phenotype : cocultivation of hepatocytes and nonparenchymal cells

paper_content:

Heterotypic cell interaction between parenchymal cells and nonparenchymal neighbors has been reported to modulate cell growth, migration, and/or differentiation. In both the developing and adult liver, cell–cell interactions are imperative for coordinated organ function. In vitro, cocultivation of hepatocytes and nonparenchymal cells has been used to preserve and modulate the hepatocyte phenotype. We summarize previous studies in this area as well as recent advances in microfabrication that have allowed for more precise control over cell–cell interactions through ‘cellular patterning’ or ‘micropatterning’. Although the precise mechanisms by which nonparenchymal cells modulate the hepatocyte phenotype remain unelucidated, some new insights on the modes of cell signaling, the extent of cell–cell interaction, and the ratio of cell populations are noted. Proposed clinical applications of hepatocyte cocultures, typically extracorporeal bioartificial liver support systems, are reviewed in the context of these n...
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paper_title: Haptotactic islands: a method of confining single cells to study individual cell reactions and clone formation.

paper_content:

Abstract A method is described for confining individual cells in culture to restricted areas so that they can be repeatedly identified and followed over long periods. The technique is also of value in studying clone formation and in time-lapse cinematography.
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paper_title: Micropatterning Topology on Soft Substrates Affects Myoblast Proliferation and Differentiation

paper_content:

Micropatterning techniques and substrate engineering are becoming useful tools to investigate several aspects of cell–cell interaction biology. In this work, we rationally study how different micropatterning geometries can affect myoblast behavior in the early stage of in vitro myogenesis. Soft hydrogels with physiological elastic modulus (E = 15 kPa) were micropatterned in parallel lanes (100, 300, and 500 μm width) resulting in different local and global myoblast densities. Proliferation and differentiation into multinucleated myotubes were evaluated for murine and human myoblasts. Wider lanes showed a decrease in murine myoblast proliferation: (69 ± 8)% in 100 μm wide lanes compared to (39 ± 7)% in 500 μm lanes. Conversely, fusion index increased in wider lanes: from (46 ± 7)% to (66 ± 7)% for murine myoblasts, and from (15 ± 3)% to (36 ± 2)% for human primary myoblasts, using a patterning width of 100 and 500 μm, respectively. These results are consistent with both computational modeling data and cond...
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paper_title: Tissue geometry patterns epithelial-mesenchymal transition via intercellular mechanotransduction

paper_content:

Epithelial-mesenchymal transition (EMT) is a phenotypic change in which epithelial cells detach from their neighbors and become motile. Whereas soluble signals such as growth factors and cytokines are responsible for stimulating EMT, here we show that gradients of mechanical stress define the spatial locations at which EMT occurs. When treated with transforming growth factor (TGF)-beta, cells at the corners and edges of square mammary epithelial sheets expressed EMT markers, whereas those in the center did not. Changing the shape of the epithelial sheet altered the spatial pattern of EMT. Traction force microscopy and finite element modeling demonstrated that EMT-permissive regions experienced the highest mechanical stress. Myocardin-related transcription factor (MRTF)-A was localized to the nuclei of cells located in high-stress regions, and inhibiting cytoskeletal tension or MRTF-A expression abrogated the spatial patterning of EMT. These data suggest a causal role for tissue geometry and endogenous mechanical stresses in the spatial patterning of EMT.
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paper_title: In situ collagen assembly for integrating microfabricated three-dimensional cell-seeded matrices

paper_content:

The contractile forces of cells can cause extracellular matrices to detach from their surroundings, which is problematic for biological studies and tissue engineering. Now, multiple phases of cell-seeded hydrogels can be integrated using a collagen-fibre-mediated method, resulting in the construction of well-defined and stable patterns of three-dimensional matrices.
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paper_title: Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis

paper_content:

Cell migration on 2D surfaces is governed by a balance between counteracting tractile and adhesion forces. Although biochemical factors such as adhesion receptor and ligand concentration and binding, signaling through cell adhesion complexes, and cytoskeletal structure assembly/disassembly have been studied in detail in a 2D context, the critical biochemical and biophysical parameters that affect cell migration in 3D matrices have not been quantitatively investigated. We demonstrate that, in addition to adhesion and tractile forces, matrix stiffness is a key factor that influences cell movement in 3D. Cell migration assays in which Matrigel density, fibronectin concentration, and β1 integrin binding are systematically varied show that at a specific Matrigel density the migration speed of DU-145 human prostate carcinoma cells is a balance between tractile and adhesion forces. However, when biochemical parameters such as matrix ligand and cell integrin receptor levels are held constant, maximal cell movement shifts to matrices exhibiting lesser stiffness. This behavior contradicts current 2D models but is predicted by a recent force-based computational model of cell movement in a 3D matrix. As expected, this 3D motility through an extracellular environment of pore size much smaller than cellular dimensions does depend on proteolytic activity as broad-spectrum matrix metalloproteinase (MMP) inhibitors limit the migration of DU-145 cells and also HT-1080 fibrosarcoma cells. Our experimental findings here represent, to our knowledge, discovery of a previously undescribed set of balances of cell and matrix properties that govern the ability of tumor cells to migration in 3D environments.
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paper_title: Satellite cells delivered by micro-patterned scaffolds: a new strategy for cell transplantation in muscle diseases.

paper_content:

Myoblast transplantation is a potentially useful therapeutic tool in muscle diseases, but the lack of an efficient delivery system has hampered its application. Here we have combined cell biology and polymer processing to create an appropriate microenvironment for in vivo transplantation of murine satellite cells (mSCs). Cells were prepared from single muscle fibers derived from C57BL/6-Tgn enhanced green fluorescent protein (GFP) transgenic mice. mSCs were expanded and seeded within micro-patterned polyglycolic acid 3-dimensional scaffolds fabricated using soft lithography and thermal membrane lamination. Myogenicity was then evaluated in vitro using immunostaining, flow cytometry, and reverse transcription polymerase chain reaction analyses. Scaffolds containing mSCs were implanted in pre-damaged tibialis anterior muscles of GFP-negative syngenic mice. Cells detached from culture dishes were directly injected into contra-lateral limbs as controls. In both cases, delivered cells participated in muscle re...
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paper_title: Fabrication of PLGA scaffolds using soft lithography and microsyringe deposition

paper_content:

Construction of biodegradable, three-dimensional scaffolds for tissue engineering has been previously described using a variety of molding and rapid prototyping techniques. In this study, we report and compare two methods for fabricating poly(Image -lactide-co-glycolide) (PLGA) scaffolds with feature sizes of approximately 10–30 μm. The first technique, the pressure assisted microsyringe, is based on the use of a microsyringe that utilizes a computer-controlled, three-axis micropositioner, which allows the control of motor speeds and position. A PLGA solution is deposited from the needle of a syringe by the application of a constant pressure of 20–300 mm Hg, resulting in a controlled polymer deposition. The second technique is based on ‘soft lithographic’ approaches that utilize a poly(dimethylsiloxane) mold. Three variations of the second technique are presented: polymer casting, microfluidic perfusion, and spin coating. Polymer concentration, solvent composition, and mold dimensions influenced the resulting scaffolds as evaluated by light and electron microscopy. As a proof-of-concept for scaffold utility in tissue engineering applications, multilayer structures were formed by thermal lamination, and scaffolds were rendered porous by particulate leaching. These simple methods for forming PLGA scaffolds with microscale features may serve as useful tools to explore structure/function relationships in tissue engineering.
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paper_title: Adult cell therapy for brain neuronal damages and the role of tissue engineering.

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No long term effective treatments are currently available for brain neurological disorders such as stroke/cerebral ischemia, traumatic brain injury and neurodegenerative disorders. Cell therapy is a promising strategy, although alternatives to embryonic/foetal cells are required to overcome ethical, tissue availability and graft rejection concerns. Adult cells may be easily isolated from the patient body, therefore permitting autologous grafts to be performed. Here, we describe the use of adult neural stem cells, adrenal chromaffin cells and retinal pigment epithelium cells for brain therapy, with a special emphasis on mesenchymal stromal cells. However, major problems like cell survival, control of differentiation and engraftment remain and may be overcome using a tissue engineering strategy, which provides a 3D support to grafted cells improving their survival. New developments, such as the biomimetic approach which combines the use of scaffolds with extracellular matrix molecules, may improve the control of cell proliferation, survival, migration, differentiation and engraftment in vivo. Therefore, we later discuss scaffold properties required for brain cell therapy as well as new tissue engineering advances that may be implemented in combination with adult cells for brain therapy. Finally, we describe an approach developed in our laboratory to repair/protect lesioned tissues: the pharmacologically active microcarriers.
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paper_title: Temperature measurements in microfluidic systems: Heat dissipation of negative dielectrophoresis barriers

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The manipulation of living biological cells in microfluidic channels by a combination of negative dielectrophoretic barriers and pressure-driven flows is widely employed in lab-on-a-chip systems. However, electric fields in conducting media induce Joule heating. This study investigates if the local temperatures reached under typical experimental conditions in miniaturized systems cause a potential risk for hyperthermic stress or cell damage. Two methods of optical in situ temperature detection have been tested and compared: (i) the exposure of the thermo-dependent fluorescent dye Rhodamine B to heat sources situated in microfluidic channels, and (ii) the use of thermoprecipitating N-alkyl-substituted acrylamide polymers as temperature threshold probes. Two-dimensional images of temperature distributions in the vicinity of active negative dielectrophoresis (nDEP)-barriers have been obtained and local temperature variations of more than 20 degrees C have been observed at the electrode edges. Heat propagation via both buffer and channel walls lead to significant temperature increases within a perimeter of 100 microm and more. These data indicate that power dissipation has to be taken into account when experiments at physiological temperatures are planned.
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paper_title: Reconstruction and functional analysis of altered molecular pathways in human atherosclerotic arteries

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BackgroundAtherosclerosis affects aorta, coronary, carotid, and iliac arteries most frequently than any other body vessel. There may be common molecular pathways sustaining this process. Plaque presence and diffusion is revealed by circulating factors that can mediate systemic reaction leading to plaque rupture and thrombosis.ResultsWe used DNA microarrays and meta-analysis to study how the presence of calcified plaque modifies human coronary and carotid gene expression. We identified a series of potential human atherogenic genes that are integrated in functional networks involved in atherosclerosis. Caveolae and JAK/STAT pathways, and S100A9/S100A8 interacting proteins are certainly involved in the development of vascular disease. We found that the system of caveolae is directly connected with genes that respond to hormone receptors, and indirectly with the apoptosis pathway.Cytokines, chemokines and growth factors released in the blood flux were investigated in parallel. High levels of RANTES, IL-1ra, MIP-1alpha, MIP-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-17, PDGF-BB, VEGF and IFN-gamma were found in plasma of atherosclerotic patients and might also be integrated in the molecular networks underlying atherosclerotic modifications of these vessels.ConclusionThe pattern of cytokine and S100A9/S100A8 up-regulation characterizes atherosclerosis as a proinflammatory disorder. Activation of the JAK/STAT pathway is confirmed by the up-regulation of IL-6, STAT1, ISGF3G and IL10RA genes in coronary and carotid plaques. The functional network constructed in our research is an evidence of the central role of STAT protein and the caveolae system to contribute to preserve the plaque. Moreover, Cav-1 is involved in SMC differentiation and dyslipidemia confirming the importance of lipid homeostasis in the atherosclerotic phenotype.
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paper_title: From 3D cell culture to organs-on-chips.

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3D cell-culture models have recently garnered great attention because they often promote levels of cell differentiation and tissue organization not possible in conventional 2D culture systems. We review new advances in 3D culture that leverage microfabrication technologies from the microchip industry and microfluidics approaches to create cell-culture microenvironments that both support tissue differentiation and recapitulate the tissue-tissue interfaces, spatiotemporal chemical gradients, and mechanical microenvironments of living organs. These 'organs-on-chips' permit the study of human physiology in an organ-specific context, enable development of novel in vitro disease models, and could potentially serve as replacements for animals used in drug development and toxin testing.
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paper_title: Multiphase electropatterning of cells and biomaterials{

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Tissues formed by cells encapsulated in hydrogels have uses in biotechnology, cell-based assays, and tissue engineering. We have previously presented a 3D micropatterning technique that rapidly localizes live cells within hydrogels using dielectrophoretic (DEP) forces, and have demonstrated the ability to modulate tissue function through the control of microscale cell architecture. A limitation of this method is the requirement that a single biomaterial must simultaneously harbor biological properties that support cell survival and function and material properties that permit efficient dielectrophoretic patterning. Here, we resolve this issue by forming multiphase tissues consisting of microscale tissue sub-units in a 'local phase' biomaterial, which, in turn, are organized by DEP forces in a separate, mechanically supportive 'bulk phase' material. We first define the effects of medium conductivity on the speed and quality of DEP cell patterning. As a case study, we then produce multiphase tissues with microscale architecture that combine high local hydrogel conductivity for enhanced survival of sensitive liver progenitor cells with low bulk conductivity required for efficient DEP micropatterning. This approach enables an expanded range of studies examining the influence of 3D cellular architecture on diverse cell types, and in the future may improve the biological function of inhomogeneous tissues assembled from a variety of modular tissue sub-units.
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paper_title: Cardiac regeneration: stem cells and beyond.

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After myocardial infarction, the lost healthy myocardium is replaced by non-contractile scar tissue which may lead to the development of heart failure and death. There is no curative therapy for the irreversible myocardial cell loss. This review will give an overview of the current options to restore the contractile force of the heart: the different stem cell sources as therapeutic agents in cardiac repair as well as more novel approaches like the activation of endogenous cell populations, the use of paracrine factors and engineered heart tissue.
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paper_title: Enhancement of Viability of Muscle Precursor Cells on 3D Scaffold in a Perfusion Bioreactor

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The aim of this study was to develop a methodology for the in vitro expansion of skeletal-muscle precursor cells (SMPC) in a three-dimensional (3D) environment in order to fabricate a cellularized artificial graft characterized by high density of viable cells and uniform cell distribution over the entire 3D domain. Cell seeding and culture within 3D porous scaffolds by conventional static techniques can lead to a uniform cell distribution only on the scaffold surface, whereas dynamic culture systems have the potential of allowing a uniform growth of SMPCs within the entire scaffold structure. In this work, we designed and developed a perfusion bioreactor able to ensure long-term culture conditions and uniform flow of medium through 3D collagen sponges. A mathematical model to assist the design of the experimental setup and of the operative conditions was developed. The effects of dynamic vs static culture in terms of cell viability and spatial distribution within 3D collagen scaffolds were evaluated at 1, 4 and 7 days and for different flow rates of 1, 2, 3.5 and 4.5 ml/min using C2C12 muscle cell line and SMPCs derived from satellite cells. C2C12 cells, after 7 days of culture in our bioreactor, perfused applying a 3.5 ml/min flow rate, showed a higher viability resulting in a three-fold increase when compared with the same parameter evaluated for cultures kept under static conditions. In addition, dynamic culture resulted in a more uniform 3D cell distribution. The 3.5 ml/min flow rate in the bioreactor was also applied to satellite cell-derived SMPCs cultured on 3D collagen scaffolds. The dynamic culture conditions improved cell viability leading to higher cell density and uniform distribution throughout the entire 3D collagen sponge for both C2C12 and satellite cells.
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paper_title: Microfluidic fabrication of microengineered hydrogels and their application in tissue engineering

paper_content:

Microfluidic technologies are emerging as an enabling tool for various applications in tissue engineering and cell biology. One emerging use of microfluidic systems is the generation of shape-controlled hydrogels (i.e., microfibers, microparticles, and hydrogel building blocks) for various biological applications. Furthermore, the microfluidic fabrication of cell-laden hydrogels is of great benefit for creating artificial scaffolds. In this paper, we review the current development of microfluidic-based fabrication techniques for the creation of fibers, particles, and cell-laden hydrogels. We also highlight their emerging applications in tissue engineering and regenerative medicine.
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paper_title: Fabrication of microfluidic hydrogels using molded gelatin as a sacrificial element {

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This paper describes a general procedure for the formation of hydrogels that contain microfluidic networks. In this procedure, micromolded meshes of gelatin served as sacrificial materials. Encapsulation of gelatin meshes in a hydrogel and subsequent melting and flushing of the gelatin left behind interconnected channels in the hydrogel. The channels were as narrow as approximately 6 microm, and faithfully replicated the features in the original gelatin mesh. Fifty micrometre wide microfluidic networks in collagen and fibrin readily enabled delivery of macromolecules and particles into the channels and transport of macromolecules from channels into the bulk of the gels. Microfluidic gels were also suitable as scaffolds for cell culture, and could be seeded by human microvascular endothelial cells to form rudimentary endothelial networks for potential use in tissue engineering.
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paper_title: Development of a tissue-engineered vascular graft combining a biodegradable scaffold, muscle-derived stem cells and a rotational vacuum seeding technique.

paper_content:

Abstract There is a clinical need for a tissue-engineered vascular graft (TEVG), and combining stem cells with biodegradable tubular scaffolds appears to be a promising approach. The goal of this study was to characterize the incorporation of muscle-derived stem cells (MDSCs) within tubular poly(ester urethane) urea (PEUU) scaffolds in vitro to understand their interaction, and to evaluate the mechanical properties of the constructs for vascular applications. Porous PEUU scaffolds were seeded with MDSCs using our recently described rotational vacuum seeding device, and cultured inside a spinner flask for 3 or 7 days. Cell viability, number, distribution and phenotype were assessed along with the suture retention strength and uniaxial mechanical behavior of the TEVGs. The seeding device allowed rapid even distribution of cells within the scaffolds. After 3 days, the constructs appeared completely populated with cells that were spread within the polymer. Cells underwent a population doubling of 2.1-fold, with a population doubling time of 35 h. Stem cell antigen-1 (Sca-1) expression by the cells remained high after 7 days in culture (77±20% vs. 66±6% at day 0) while CD34 expression was reduced (19±12% vs. 61±10% at day 0) and myosin heavy chain expression was scarce (not quantified). The estimated burst strength of the TEVG constructs was 2127±900 mmHg and suture retention strength was 1.3±0.3 N. We conclude from this study that MDSCs can be rapidly seeded within porous biodegradable tubular scaffolds while maintaining cell viability and high proliferation rates and without losing stem cell phenotype for up to 7 days of in-vitro culture. The successful integration of these steps is thought necessary to provide rapid availability of TEVGs, which is essential for clinical translation.
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paper_title: Matrix Elasticity Directs Stem Cell Lineage Specification

paper_content:

Microenvironments appear important in stem cell lineage specification but can be difficult to adequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs) are shown here to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. Soft matrices that mimic brain are neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. During the initial week in culture, reprogramming of these lineages is possible with addition of soluble induction factors, but after several weeks in culture, the cells commit to the lineage specified by matrix elasticity, consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibition of nonmuscle myosin II blocks all elasticity-directed lineage specification-without strongly perturbing many other aspects of cell function and shape. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells.
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paper_title: Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells.

paper_content:

This work investigates an application of the alginate encapsulation technology to the differentiation of embryonic stem (ES) cells into insulin-producing cells. It shows that the ES cells can efficiently be encapsulated within the alginate beads, retaining a high level of cell viability. The alginate encapsulation achieves approximately 10-fold increase in the cell density in the culture, in comparison to the two-dimensional conditions, opening a potential benefit of the technology in large-scale cell culture applications. Manipulations of encapsulation conditions, particularly of the initial alginate concentration, allow the control over both the diffusion of molecules into the alginate matrix (e.g. differentiation factors) as well as control over the matrix porosity/flexibility to permit the proliferation and growth of encapsulated ES aggregates within the bead. Post-differentiation analysis confirms the presence of insulin-positive cells, as judged from immunostaining, insulin ELISA and RT-PCR analysis. The functionality of the encapsulated and differentiated cells was confirmed by their insulin production capability, whereby on glucose challenge the insulin production by the cells differentiated within alginate beads was found to be statistically significantly higher than for the cells from conventional two-dimensional differentiation system.
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paper_title: Vascularized organoid engineered by modular assembly enables blood perfusion

paper_content:

Tissue engineering is one approach to address the donor-organ shortage, but to attain clinically significant viable cell densities in thick tissues, laboratory-constructed tissues must have an internal vascular supply. We have adopted a biomimetic approach and assembled microscale modular components, consisting of submillimeter-sized collagen gel rods seeded with endothelial cells (ECs) into a (micro)vascularized tissue; in some prototypes the gel contained HepG2 cells to illustrate the possibilities. The EC-covered modules then were assembled into a larger tube and perfused with medium or whole blood. The interstitial spaces among the modules formed interconnected channels that enabled this perfusion. Viable cell densities were high, within an order of magnitude of cell densities within tissues, and the percolating nature of the flow through the construct was evident in microcomputed tomography and Doppler ultrasound measurements. Most importantly, the ECs retained their nonthrombogenic phenotype and delayed clotting times and inhibited the loss of platelets associated with perfusion of whole blood through the construct. Unlike the conventional scaffold and cell-seeding paradigm of other tissue-engineering approaches, this modular construct has the potential to be scalable, uniform, and perfusable with whole blood, circumventing the limitations of other approaches.
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paper_title: Micromolding of shape-controlled, harvestable cell-laden hydrogels

paper_content:

Encapsulation of mammalian cells within hydrogels has great utility for a variety of applications ranging from tissue engineering to cell-based assays. In this work, we present a technique to encapsulate live cells in three-dimensional (3D) microscale hydrogels (microgels) of controlled shapes and sizes in the form of harvestable free standing units. Cells were suspended in methacrylated hyaluronic acid (MeHA) or poly(ethylene glycol) diacrylate (PEGDA) hydrogel precursor solution containing photoinitiator, micromolded using a hydrophilic poly(dimethylsiloxane) (PDMS) stamp, and crosslinked using ultraviolet (UV) radiation. By controlling the features on the PDMS stamp, the size and shape of the molded hydrogels were controlled. Cells within microgels were well distributed and remained viable. These shape-specific microgels could be easily retrieved, cultured and potentially assembled to generate structures with controlled spatial distribution of multiple cell types. Further development of this technique may lead to applications in 3D co-cultures for tissue/organ regeneration and cell-based assays in which it is important to mimic the architectural intricacies of physiological cell–cell interactions. r 2006 Elsevier Ltd. All rights reserved.
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paper_title: A planar interdigitated ring electrode array via dielectrophoresis for uniform patterning of cells.

paper_content:

Uniform patterning of cells is highly desirable for most cellular studies involving cell-cell interactions but is often difficult in an in vitro environment. This paper presents the development of a collagen-coated planar interdigitated ring electrode (PIRE) array utilizing positive dielectrophoresis to pattern cells uniformly. Key features of the PIRE design include: (1) maximizing length along the edges where the localized maximum in the electric field exists; (2) making the inner gap slightly smaller than the outer gap in causing the electric field strength near the center of a PIRE being generally stronger than that near the outer edge of the same PIRE. Results of human hepatocellular carcinoma cells, HepG2, adhered on a 6x6 PIRE array show that cells patterned within minutes with good uniformity (48+/-6 cells per PIRE). Cell viability test revealed healthy patterned cells after 24h that were still confined to the collagen-coated PIREs. Furthermore, quantification of fluorescence intensity of living cells shows an acceptable reproducibility of cell viability among PIREs (mean normalized intensity per PIRE was 1+/-0.138). The results suggest that the PIRE array would benefit applications that desire uniform cellular patterning, and improve both response and reproducibility of cell-based biosensors.
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paper_title: Stimulation of Ca2+ signals in neurons by electrically coupled electrolyte-oxide-semiconductor capacitors

paper_content:

Electrolyte-oxide-semiconductor capacitors (EOSCs) are a class of microtransducers for extracellular electrical stimulation that have been successfully employed to activate voltage-dependent sodium channels at the neuronal soma to generate action potentials in vitro. In the present work, we report on their use to control Ca²+ signalling in cultured mammalian cells, including neurons. Evidence is provided that EOSC stimulation with voltage waveforms in the microsecond or nanosecond range activates two distinct Ca²+ pathways, either by triggering Ca²+ entry through the plasma membrane or its release from intracellular stores. Ca²+ signals were activated in non-neuronal and neuronal cell lines, CHO-K1 and SH-SY5Y. On this basis, stimulation was tailored to rat and bovine neurons to mimic physiological somatic Ca²+ transients evoked by glutamate. Being minimally invasive and easy to use, the new method represents a versatile complement to standard electrophysiology and imaging techniques for the investigation of Ca²+ signalling in dissociated primary neurons and cell lines.
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paper_title: Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray

paper_content:

Single-cell experiments represent the next frontier for biochemical and gene expression research. Although bulk-scale methods averaging populations of cells have been traditionally used to investigate cellular behavior, they mask individual cell features and can lead to misleading or insufficient biological results. We report on a single-cell electroporation microarray enabling the transfection of pre-selected individual cells at different sites within the same culture (space-resolved), at arbitrarily chosen time points and even sequentially to the same cells (time-resolved). Delivery of impermeant molecules by single-cell electroporation was first proven to be finely tunable by acting on the electroporation protocol and then optimized for transfection of nucleic acids into Chinese Hamster Ovary (CHO-K1) cells. We focused on DNA oligonucleotides (ODNs), short interfering RNAs (siRNAs), and DNA plasmid vectors, thus providing a versatile and easy-to-use platform for time-resolved gene expression experiments in single mammalian cells.
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paper_title: A three-dimensional multi-electrode array for multi-site stimulation and recording in acute brain slices

paper_content:

Several multi-electrode array devices integrating planar metal electrodes were designed in the past 30 years for extracellular stimulation and recording from cultured neuronal cells and organotypic brain slices. However, these devices are not well suited for recordings from acute brain slice preparations due to a dead cell layer at the tissue slice border that appears during the cutting procedure. To overcome this problem, we propose the use of protruding 3D electrodes, i.e. tip-shaped electrodes, allowing tissue penetration in order to get closer to living neurons in the tissue slice. In this paper, we describe the design and fabrication of planar and 3D protruding multi-electrode arrays. The electrical differences between planar and 3D protruding electrode configuration were simulated and verified experimentally. Finally, a comparison between the planar and 3D protruding electrode configuration was realized by stimulation and recording from acute rat hippocampus slices. The results show that larger signal amplitudes in the millivolt range can be obtained with the 3D electrode devices. Spikes corresponding to single cell activity could be monitored in the hippocampus CA3 and CA1 region using 3D electrodes.
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paper_title: Electrical characterization of human mesenchymal stem cell growth on microelectrode

paper_content:

To support the development of stem cell therapies with in vitro assays, non-destructive methods are required for the quality control of stem cell culture. In this article, it was investigated whether an electrode based chip with electrical impedance spectroscopy can be used to characterize the growth of human mesenchymal stem cells on electrodes without any chemical marker. From finite element method simulations, the electrical characteristics of cell layer under weak alternating electric fields were investigated with respect to the different cell/cell or cell/substrate gap, and modelled as an equivalent circuit. The impedance spectra were measured during the long-term cultivation of human mesenchymal stem cells on platinum electrodes. By fitting analyses the equivalent circuit to measured spectra, an extra cellular resistance reflecting the cell growth was extrapolated.
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paper_title: Adult neural progenitor cells reactivate superbursting in mature neural networks

paper_content:

Behavioral recovery in animal models of human CNS syndromes suggests that transplanted stem cell derivatives can augment damaged neural networks but the mechanisms behind potentiated recovery remain elusive. Here we use microelectrode array (MEA) technology to document neural activity and network integration as rat primary neurons and rat hippocampal neural progenitor cells (NPCs) differentiate and mature. The natural transition from neuroblast to functional excitatory neuron consists of intermediate phases of differentiation characterized by coupled activity. High-frequency network-wide bursting or "superbursting" is a hallmark of early plasticity that is ultimately refined into mature stable neural network activity. Microelectrode array (MEA)-plated neurons transition through this stage of coupled superbursting before establishing mature neuronal phenotypes in vitro. When plated alone, adult rat hippocampal NPC-derived neurons fail to establish the synchronized bursting activity that neurons in primary and embryonic stem cell-derived cultures readily form. However, adult rat hippocampal NPCs evoke re-emergent superbursting in electrophysiologically mature rat primary neural cultures. Developmental superbursting is thought to accompany transient states of heightened plasticity both in culture preparations and across brain regions. Future work exploring whether NPCs can re-stimulate developmental states in injury models would be an interesting test of their regenerative potential.
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paper_title: Adult mesenchymal stem cells and cell-based tissue engineering

paper_content:

The identification of multipotential mesenchymal stem cells (MSCs) derived from adult human tissues, including bone marrow stroma and a number of connective tissues, has provided exciting prospects for cell-based tissue engineering and regeneration. This review focuses on the biology of MSCs, including their differentiation potentials in vitro and in vivo, and the application of MSCs in tissue engineering. Our current understanding of MSCs lags behind that of other stem cell types, such as hematopoietic stem cells. Future research should aim to define the cellular and molecular fingerprints of MSCs and elucidate their endogenous role(s) in normal and abnormal tissue functions.
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paper_title: Human Cell-Based Micro Electrode Array Platform for Studying Neurotoxicity

paper_content:

At present, most of the neurotoxicological analyses are based on in vitro and in vivo models utilizing animal cells or animal models. In addition, the used in vitro models are mostly based on molecular biological end-point analyses. Thus, for neurotoxicological screening, human cell-based analysis platforms in which the functional neuronal networks responses for various neurotoxicants can be also detected real-time are highly needed. Microelectrode array (MEA) is a method which enables the measurement of functional activity of neuronal cell networks in vitro for long periods of time. Here, we utilize MEA to study the neurotoxicity of methyl mercury chloride (MeHgCl, concentrations 0.5-500 nM) to human embryonic stem cell (hESC)-derived neuronal cell networks exhibiting spontaneous electrical activity. The neuronal cell cultures were matured on MEAs into networks expressing spontaneous spike train-like activity before exposing the cells to MeHgCl for 72 hours. MEA measurements were performed acutely and 24, 48, and 72 hours after the onset of the exposure. Finally, exposed cells were analyzed with traditional molecular biological methods for cell proliferation, cell survival, and gene and protein expression. Our results show that 500 nM MeHgCl decreases the electrical signaling and alters the pharmacologic response of hESC-derived neuronal networks in delayed manner whereas effects can not be detected with qRT-PCR, immunostainings, or proliferation measurements. Thus, we conclude that human cell-based MEA-platform is a sensitive online method for neurotoxicological screening.
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paper_title: Prediction of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiomyocytes.

paper_content:

Recent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. Block of the human Ether-a-go go Related Gene (hERG) ion channel in particular is associated with life-threatening arrhythmias, such as Torsade de Pointes (TdP). Here we investigated human cardiomyocytes derived from pluripotent (embryonic) stem cells (hESC) as a renewable, scalable, and reproducible system on which to base cardiac safety pharmacology assays. Analyses of extracellular field potentials in hESC-derived cardiomyocytes (hESC-CM) and generation of derivative field potential duration (FPD) values showed dose-dependent responses for 12 cardiac and noncardiac drugs. Serum levels in patients of drugs with known effects on QT interval overlapped with prolonged FPD values derived from hESC-CM, as predicted. We thus propose hESC-CM FPD prolongation as a safety criterion for preclinical evaluation of new drugs in development. This is the first study in which dose responses of such a wide range of compounds on hESC-CM have been generated and shown to be predictive of clinical effects. We propose that assays based on hESC-CM could complement or potentially replace some of the preclinical cardiac toxicity screening tests currently used for lead optimization and further development of new drugs.
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paper_title: Learning in human neural networks on microelectrode arrays

paper_content:

This paper describes experiments involving the growth of human neural networks of stem cells on a MEA (microelectrode array) support. The microelectrode arrays (MEAs) are constituted by a glass support in which a set of tungsten electrodes are inserted. The artificial neural network (ANN) paradigm was used by stimulating the neurons in parallel with digital patterns distributed on eight channels, then by analyzing a parallel multichannel output. In particular, the microelectrodes were connected following two different architectures, one inspired by the Kohonen’s SOM, the other by the Hopfield network. The output signals have been analyzed in order to evaluate the possibility of organized reactions by the natural neurons. The results show that the network of human neurons reacts selectively to the subministered digital signals, i.e., it produces similar output signals referred to identical or similar patterns, and clearly differentiates the outputs coming from different stimulations. Analyses performed with a special artificial neural network called ITSOM show the possibility to codify the neural responses to different patterns, thus to interpret the signals coming from the network of biological neurons, assigning a code to each output. It is straightforward to verify that identical codes are generated by the neural reactions to similar patterns. Further experiments are to be designed that improve the hybrid neural networks’ capabilities and to test the possibility of utilizing the organized answers of the neurons in several ways. © 2006 Elsevier Ireland Ltd. All rights reserved.
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paper_title: Real-Time Monitoring of Neural Differentiation of Human Mesenchymal Stem Cells by Electric Cell-Substrate Impedance Sensing

paper_content:

Stem cells are useful for cell replacement therapy. Stem cell differentiation must be monitored thoroughly and precisely prior to transplantation. In this study we evaluated the usefulness of electric cell-substrate impedance sensing (ECIS) for in vitro real-time monitoring of neural differentiation of human mesenchymal stem cells (hMSCs). We cultured hMSCs in neural differentiation media (NDM) for 6 days and examined the time-course of impedance changes with an ECIS array. We also monitored the expression of markers for neural differentiation, total cell count, and cell cycle profiles. Cellular expression of neuron and oligodendrocyte markers increased. The resistance value of cells cultured in NDM was automatically measured in real-time and found to increase much more slowly over time compared to cells cultured in non-differentiation media. The relatively slow resistance changes observed in differentiating MSCs were determined to be due to their lower growth capacity achieved by induction of cell cycle arrest in G0/G1. Overall results suggest that the relatively slow change in resistance values measured by ECIS method can be used as a parameter for slowly growing neural-differentiating cells. However, to enhance the competence of ECIS for in vitro real-time monitoring of neural differentiation of MSCs, more elaborate studies are needed.
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paper_title: Paracrine signalling events in embryonic stem cell renewal mediated by affinity targeted nanoparticles

paper_content:

Abstract Stem cell growth and differentiation is controlled by intrinsic and extrinsic factors. The latter includes growth factors, which are conventionally supplied in vitro in media exchanged daily. Here, we illustrate the use of affinity targeted biodegradable nanoparticles to mediate paracrine stimulation as an alternative approach to sustain the growth and pluripotency of mouse embryonic stem cells. Leukaemia Inhibitory Factor (LIF) was encapsulated in biodegradable nanoparticles and targeted to the cell surface using an antibody to the oligosaccharide antigen SSEA-1. Sustained release of LIF from nanoparticles composed of a solid Poly(lactide-co-glycolic acid) polyester or a hydrogel-based liposomal system, we term Nanolipogel, replenished once after each cell passage, proved as effective as daily replenishment with soluble LIF for maintenance of pluripotency after 5 passages using 10 4 -fold less LIF. Our study constitutes an alternative paradigm for stem cell culture, providing dynamic microenvironmental control of extrinsic bioactive factors benefiting stem cell manufacturing.
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paper_title: Real-time label-free monitoring of adipose-derived stem cell differentiation with electric cell-substrate impedance sensing

paper_content:

†Real-time monitoring of stem cells (SCs) differentiation will be critical to scale-up SC technologies, while label-free techniques will be desirable to quality-control SCs without precluding their therapeutic potential. We cultured adipose-derived stem cells (ADSCs) on top of multielectrode arrays and measured variations in the complex impedance Z* throughout induction of ADSCs toward osteoblasts and adipocytes. Z* was measured up to 17 d, every 180 s, over a 62.5–64kHz frequency range with an ECIS Zθ instrument. We found that osteogenesis and adipogenesis were characterized by distinct Z* time-courses. Significant differences were found (P = 0.007) as soon as 12 h post induction. An increase in the barrier resistance (Rb) up to 1.7 ohm·cm 2 was associated with early osteo-induction, whereas Rb peaked at 0.63 ohm·cm 2 for adipo-induced cells before falling to zero at t = 129 h. Dissimilarities in Z* throughout early induction (<24 h) were essentially attributed to variations in the cell-substrate parameter α .F our days after induction, cell membrane capacitance (Cm) of osteoinduced cells (Cm = 1.72 ± 0.10 μF/cm 2 ) was significantly different from that of adipo-induced cells (Cm = 2.25 ± 0.27 μF/cm 2 ), indicating that Cm could be used as an early marker of differentiation. Finally, we demonstrated long-term monitoring and measured a shift in the complex plane in the middle frequency range (1 kHz to 8 kHz) between early (t = 100 h) and late induction (t = 380 h). This study demonstrated that the osteoblast and adipocyte lineages have distinct dielectric properties and that such differences can be used to perform real-time label-free quantitative monitoring of adult stem cell differentiation with impedance sensing.
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paper_title: The human adipose tissue is a source of multipotent stem cells.

paper_content:

Multipotent stem cells constitute an unlimited source of differentiated cells that could be used in pharmacological studies and in medicine. Recently, several publications have reported that adipose tissue contains a population of cells able to differentiate into different cell types including adipocytes, osteoblasts, myoblasts, and chondroblasts. More recently, stem cells with a multi-lineage potential at the single cell level have been isolated from human adipose tissue. These cells, called human Multipotent Adipose-Derived Stem (hMADS) cells, have been established in culture and interestingly, maintain their characteristics with long-term passaging. The adipocyte differentiation of hMADS cells has been thoroughly studied and differentiated cells exhibit the unique feature of human adipocytes. Finally, potential applications of stem cells isolated from adipose tissue in medicine will be discussed.
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paper_title: An integrated semiconductor device enabling non-optical genome sequencing

paper_content:

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.
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paper_title: Field-effect devices for detecting cellular signals.

paper_content:

The integration of living cells together with silicon field-effect devices challenges a new generation of biosensors and bioelectronic devices. Cells are representing highly organised complex systems, optimised by millions of years of evolution and offering a broad spectrum of bioanalytical receptor "tools" such as enzymes, nucleic acids proteins, etc. Their combination with semiconductor-based electronic chips allows the construction of functional hybrid systems with unique functional and electronic properties for both fundamental studies and biosensoric applications. This review article summarises recent advances and trends in research and development of cell/transistor hybrids (cell-based field-effect transistors) as well as light-addressable potentiometric sensors.
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paper_title: A comparative study on fabrication techniques for on-chip microelectrodes

paper_content:

This paper presents an experimental study on different microelectrode fabrication techniques, with particular focus on the robustness of the surface insulation towards typical working conditions required in lab-on-a-chip applications. Pt microelectrodes with diameters of 50 μm, 100 μm and 200 μm are patterned on a Si substrate with SiO2 film. Sputtered SiO2, low-pressure chemical vapor deposition (LPCVD) low-temperature oxide (LTO), Parylene C, SU-8, and dry-film were deposited and patterned on top of the chips as the passivation layer. This paper provides the detailed fabrication processes, the adhesion enhancement strategies, and the major advantages and disadvantages of each fabrication technique. Firstly, the quality and adhesion strength of the passivations were investigated by means of hydrolysis tests, in which sputtered SiO2 and dry-film resist showed serious delamination issues and LTO showed minor defects. Secondly, the reliability of the microelectrodes was tested by impedance measurements after overnight ethanol incubation and self-assembled monolayer (SAM) formation. Thirty chips, representing a total of 300 electrodes, were measured, and statistical analyses of the results were conducted for each passivation technique. All of the electrodes passivated with these five techniques showed consistent impedance values after ethanol incubation. On the other hand, only LTO, Parylene C, and SU-8 ensured uniform electrical behavior after SAM formation. Having used both hydrolysis and impedance tests to verify the superior quality of the Parylene-based passivation, electrochemical experiments were performed to study the long-term stability of the passivation layer. Finally, the electrodes were incubated with electroactive alkanethiols functionalized with ferrocene. Square-wave voltammetry measurements demonstrated reproducible results on electrochemical label detection, which confirms the suitability of the Parylene passivation for charge-transfer-based measurements.
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paper_title: Development of an Ion-Sensitive Solid-State Device for Neurophysiological Measurements

paper_content:

The development of an ion-sensitive solid-state device is described. The device combines the principles of an MOS transistor and a glass electrode and can be used for measurements of ion activities in electrochemical and biological environments. Some preliminary results are given.
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paper_title: Macroporous nanowire nanoelectronic scaffolds for synthetic tissues

paper_content:

The development of three-dimensional (3D) synthetic biomaterials as structural and bioactive scaffolds is central to fields ranging from cellular biophysics to regenerative medicine. As of yet, these scaffolds cannot electrically probe the physicochemical and biological microenvironments throughout their 3D and macroporous interior, although this capability could have a marked impact in both electronics and biomaterials. Here, we address this challenge using macroporous, flexible and free-standing nanowire nanoelectronic scaffolds (nanoES), and their hybrids with synthetic or natural biomaterials. 3D macroporous nanoES mimic the structure of natural tissue scaffolds, and they were formed by self-organization of coplanar reticular networks with built-in strain and by manipulation of 2D mesh matrices. NanoES exhibited robust electronic properties and have been used alone or combined with other biomaterials as biocompatible extracellular scaffolds for 3D culture of neurons, cardiomyocytes and smooth muscle cells. Furthermore, we show the integrated sensory capability of the nanoES by real-time monitoring of the local electrical activity within 3D nanoES/cardiomyocyte constructs, the response of 3D-nanoES-based neural and cardiac tissue models to drugs, and distinct pH changes inside and outside tubular vascular smooth muscle constructs.
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paper_title: Integrating bio-sensing functions on CMOS chips

paper_content:

The paper discusses the recent achievements in the development of chips with integrated sensing of biomolecules. In particular, it focuses on integrated sensing electrodes on silicon and presents innovative solutions for the enhanced robustness of the electrodes towards cleaning processes and electrolytes. In this study, a microfabrication technology for 3D-integrated disposable chip layers that enables the reusability of the overall system for many times is presented.
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paper_title: Neuron–transistor coupling: interpretation of individual extracellular recorded signals

paper_content:

The electrical coupling of randomly migrating neurons from rat explant brain-stem slice cultures to the gates of non-metallized field-effect transistors (FETs) has been investigated. The objective of our work is the precise interpretation of extracellular recorded signal shapes in comparison to the usual patch-clamp protocols to evaluate the possible use of the extracellular recording technique in electrophysiology. The neurons from our explant cultures exhibited strong voltage-gated potassium currents through the plasma membrane. With an improved noise level of the FET set-up, it was possible to record individual extracellular responses without any signal averaging. Cells were attached by patch-clamp pipettes in voltage-clamp mode and stimulated by voltage step pulses. The point contact model, which is the basic model used to describe electrical contact between cell and transistor, has been implemented in the electrical simulation program PSpice. Voltage and current recordings and compensation values from the patch-clamp measurement have been used as input data for the simulation circuit. Extracellular responses were identified as composed of capacitive current and active potassium current inputs into the adhesion region between the cell and transistor gate. We evaluated the extracellular signal shapes by comparing the capacitive and the slower potassium signal amplitudes. Differences in amplitudes were found, which were interpreted in previous work as enhanced conductance of the attached membrane compared to the average value of the cellular membrane. Our results suggest rather that additional effects like electrodiffusion, ion sensitivity of the sensors or more detailed electronic models for the small cleft between the cell and transistor should be included in the coupling model.
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paper_title: Polarization-Controlled Differentiation of Human Neural Stem Cells Using Synergistic Cues from the Patterns of Carbon Nanotube Monolayer Coating

paper_content:

We report a method for selective growth and structural-polarization-controlled neuronal differentiation of human neural stem cells (hNSCs) into neurons using carbon nanotube network patterns. The CNT patterns provide synergistic cues for the differentiation of hNSCs in physiological solution and an optimal nanotopography at the same time with good biocompatibility. We demonstrated a polarization-controlled neuronal differentiation at the level of individual NSCs. This result should provide a stable and versatile platform for controlling the hNSC growth because CNT patterns are known to be stable in time unlike commonly used organic molecular patterns.
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paper_title: 3D integration technology for lab-on-a-chip applications

paper_content:

A review is presented of advances and challenges in fully integrated systems for personalised medicine applications. One key issue for the commercialisation of such systems is the disposability of the assay-substrate at a low cost. This work adds a new dimension to the integrated circuits technology for lab-on-a-chip systems by employing 3D integration for improved performance and functionality. It is proposed that a disposable biosensing layer can be aligned and temporarily attached to the 3D CMOS stack by the vertical interconnections, and can be replaced after each measurement.
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paper_title: Carbon Nanotube Monolayer Cues for Osteogenesis of Mesenchymal Stem Cells

paper_content:

Recent advances in nanotechnology present synthetic bio-inspired materials to create new controllable microenvironments for stem cell growth, which have allowed directed differentiation into specific lineages.[1,2] Carbon nanotubes (CNTs), one of the most extensively studied nanomaterials, can provide a favorable extracellular environment for intimate cell adhesion due to their similar dimension to collagen. It has been shown that CNTs support the attachment and growth of adult stem cells[3-6] and progenitor cells including osteoblasts and myoblasts.[7,8] In addition, surface-functionalized CNTs provide new opportunities in controlling cell growth. Surface functionalization improves the attachment of biomolecules, such as proteins, DNA, and aptamers, to CNTs.[9] Zanello et al. cultured osteoblasts on CNTs with various functional groups and showed reduced cell growth on positively charged CNTs.[10] Recent reports have shown that human mesenchymal stem cells (hMSCs) formed focal adhesions and grew well on single-walled CNTs (swCNTs).[5,6] However, the effect of naive swCNT substrates on the differentiation of stem cells has not been reported before. Herein, we report the osteogenic differentiation of hMSCs induced by swCNT monolayer cues without any chemical treatments. Interestingly, the surface treatment of swCNTs via oxygen plasma showed synergistic effects on the differentiation as well as the adhesion of hMSCs. The stress due to the enhanced cell spreading on swCNT layers was proposed as a possible explanation for the enhanced osteogenesis of hMSCs on the swCNT monolayers. Previous reports showed that the stress to stretch stem cells on microscale molecular patterns generated the tension on actin filaments, which eventually enhanced the osteogenesis.[11,12] Since our method relies on monolayer coating of swCNTs, it can be applied to a wide range of substrates including conventional scaffolds without any complicated fabrication processes.
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paper_title: Neurochip based on light-addressable potentiometric sensor with wavelet transform de-noising

paper_content:

Neurochip based on light-addressable potentiometric sensor (LAPS), whose sensing elements are excitable cells, can monitor electrophysiological properties of cultured neuron networks with cellular signals well analyzed. Here we report a kind of neurochip with rat pheochromocytoma (PC12) cells hybrid with LAPS and a method of de-noising signals based on wavelet transform. Cells were cultured on LAPS for several days to form networks, and we then used LAPS system to detect the extracellular potentials with signals de-noised according to decomposition in the time-frequency space. The signal was decomposed into various scales, and coefficients were processed based on the properties of each layer. At last, signal was reconstructed based on the new coefficients. The results show that after de-noising, baseline drift is removed and signal-to-noise ratio is increased. It suggests that the neurochip of PC12 cells coupled to LAPS is stable and suitable for long-term and non-invasive measurement of cell electrophysiological properties with wavelet transform, taking advantage of its time-frequency localization analysis to reduce noise.
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paper_title: Cell-based biosensors based on light-addressable potentiometric sensors for single cell monitoring

paper_content:

Abstract Cell-based biosensors incorporate cells as sensing elements that convert changes in immediate environment to signals for processing. This paper reports an investigation on light-addressable potentiometric sensor (LAPS) to be used as a possible cell-base biosensor that will enable us to monitor extracellular action potential of single living cell under stimulant. In order to modify chip surface and immobilize cells, we coat a layer of poly- l -ornithine and laminin on surface of LAPS chip on which rat cortical cells are grown well. When 10 μg/ml acetylcholine solution is administrated, the light pointer is focused on a single neuronal cell and the extracellular action potential of the targeted cell is recorded with cell-based biosensor based on LAPS. The results demonstrate that this kind of biosensor has potential to monitor electrophysiology of living cell non-invasive for a long term, and to evaluate drugs primarily.
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paper_title: Cytosensor techniques for examining signal transduction of neurohormones

paper_content:

This review describes the principles of microphysiometry and how they can be applied, using the Cytosensor, to the investigation of the signal transduction mechanisms activated by both G-protein an...
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paper_title: Detection of heavy metal toxicity using cardiac cell-based biosensor

paper_content:

Abstract Biosensors incorporating mammalian cells have a distinct advantage of responding in a manner which offers insight into the physiological effect of an analyte. To investigate the potential applications of cell-based biosensors on heavy metal toxicity detection, a novel biosensor for monitoring electrophysiological activity was developed by light-addressable potentiometric sensor (LAPS). Extracellular field potentials of spontaneously beating cardiomyocytes could be recorded by LAPS in the range of 20 μV to nearly 40 μV with frequency of 0.5–3 Hz. After exposed to different heavy metal ions (Hg 2+ , Pb 2+ , Cd 2+ , Fe 3+ , Cu 2+ , Zn 2+ ; in concentration of 10 μM), cardiomyocytes demonstrated characteristic changes in terms of beating frequency, amplitude and duration under the different toxic effects of ions in less than 15 min. This study suggests that, with the physiological monitoring, it is possible to use the cardiac cell-based biosensor to study acute and eventually chronic toxicities induced by heavy metal ions in a long-term and no-invasive way.
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paper_title: Light-addressable potentiometric sensor for biochemical systems.

paper_content:

Numerous biochemical reactions can be measured potentiometrically through changes in pH, redox potential, or transmembrane potential. An alternating photocurrent through an electrolyte-insulator-semiconductor interface provides a highly sensitive means to measure such potential changes. A spatially selectable photoresponse permits the determination of a multiplicity of chemical events with a single semiconductor device.
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paper_title: In vitro assessing the risk of drug-induced cardiotoxicity by embryonic stem cell-based biosensor

paper_content:

Abstract Drug-induced prolongation of ventricular repolarization with arrhythmia is a major concern in clinic safety pharmacology, and has been a common reason for the withdrawal of several promising drugs from the market. Therefore, novel techniques should be developed to evaluate cardiotoxicity of new drugs in preclinical research. A cardiomyocyte based biosensor was developed using the light addressable potentiometric sensor (LAPS). Mouse embryonic stem cells cultured on the surface of LAPS were induced to differentiate into synchronized spontaneity beating cardiomyocytes. Changes of extracellular potentials and cell shapes with their mechanical beatings could induce modulation of photocurrents in the LAPS system, and finally change the output of the sensor. With the characteristics of light addressability, LAPS can record cell clusters at any desired position. The sensor can be used to record the prolongation of ventricular action potentials with the cardiotoxicity induced by drugs such as amiodarone, levofloxacin, sparfloxacin, and noradrenaline. The quick and on time characteristics of the sensor were promising to establish a high-throughput platform for pharmacological toxicity investigation.
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paper_title: Embryonic Stem Cells Biosensor and Its Application in Drug Analysis and Toxin Detection

paper_content:

To investigate the use of stem cells as biosensor elements, a novel cell-based light-addressable potentiometric sensor (LAPS) was developed for monitoring cellular beating. Mouse embryonic stem cells were induced to differentiate into cardiomyocytes in vitro. Extracellular field potentials of spontaneously beating cardiomyocytes induced from stem cells were recorded by LAPS in the potential and frequency ranges of 25-45 muV and 0.5-3 Hz, respectively. Due to its capability of monitoring important physiological parameters such as potential and frequency in vitro, the sensor can be used in drug analysis and toxin detection in a long-term and noninvasive way. The pharmacological and toxicological researches make it possible to use stem cells-based biosensor for biomedical assays.
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paper_title: Applying silicon micromachining to cellular metabolism: measuring the rate of acidification induced in the extracellular environment

paper_content:

Describes how a microphysiometer works and shows how silicon micromachining can be used to provide a multichannel capability. The authors consider extracellular acidification, metabolic rate detection, LAPS pH sensors, wells for the capture of nonadherent cells, and a multichannel flow-through microphysiometer chip. >
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paper_title: Development of a Surface Plasmon Resonance Biosensor for Real-Time Detection of Osteogenic Differentiation in Live Mesenchymal Stem Cells

paper_content:

Surface plasmon resonance (SPR) biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs). OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis.
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paper_title: Osteogenic differentiation of human mesenchymal stem cells on poly(ethylene glycol)-variant biomaterials

paper_content:

This study evaluated the osteogenic differentiation of human mesenchymal stem cells (MSCs), on tyrosine-derived polycarbonates copolymerized with poly(ethylene glycol) (PEG) to determine their potential as a scaffold for bone tissue engineering applications. The addition of PEG in the backbone of polycarbonates has been shown to alter mechanical properties, degradation rates, degree of protein adsorption, and subsequent cell adhesion and motility in mature cell phenotypes. Its effect on MSC behavior is unknown. MSC morphology, motility, proliferation, and osteogenic differentiation were evaluated on polycarbonates containing 0-5% PEG over a 14 day culture. MSCs on polycarbonates containing 0% or 3% PEG content upregulated the expression of osteogenic markers as demonstrated by alkaline phosphatase activity and osteocalcin expression although at different stages in the 14 day culture. Cells on polycarbonates containing no PEG were characterized as having early onset of cell spreading and osteogenic differentiation. Cells on 3% PEG surfaces were delayed in cell spreading and osteogenic differentiation, but had the highest motility as compared with cells on substrates containing no PEG and substrates containing 5% PEG at early time points. Throughout the culture, cells on polycarbonates containing 5% PEG had the lowest levels of osteogenic markers, displayed poor cell-substrate adhesion, and established cell-cell aggregates. Thus, designing substrates with minute variations in PEG may serve as a tool to guide MSC adhesion and motility accompanying osteogenic differentiation, and may be beneficial for abundant bone tissue formation in vivo.
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paper_title: Cell shape and spreading of stromal (mesenchymal) stem cells cultured on fibronectin coated gold and hydroxyapatite surfaces.

paper_content:

In order to identify the cellular mechanisms leading to the biocompatibility of hydroxyapatite implants, we studied the interaction of human bone marrow derived stromal (mesenchymal) stem cells (hMSCs) with fibronectin-coated gold (Au) and hydroxyapatite (HA) surfaces. The adsorption of fibronectin was monitored by Quartz Crystal Microbalance with Dissipation (QCM-D) at two different concentrations, 20 μg/ml and 200 μg/ml, and the fibronectin adsorption experiments were complemented with antibody measurements. The QCM-D results show that the surface mass uptake is largest on the Au surfaces, while the number of polyclonal and monoclonal antibodies directed against the cell-binding domain (CB-domain) on the fibronectin (Fn) is significantly larger on the (HA) surfaces. Moreover, a higher number of antibodies bound to the fibronectin coatings formed from the highest bulk fibronection concentration. In subsequent cell studies with hMSC's we studied the cell spreading, cytoskeletal organization and cell morphology on the respective surfaces. When the cells were adsorbed on the uncoated substrates, a diffuse cell actin cytoskeleton was revealed, and the cells had a highly elongated shape. On the fibronectin coated surfaces the cells adapted to a more polygonal shape with a well-defined actin cytoskeleton, while a larger cell area and roundness values were observed for cells cultured on the coated surfaces. Among the coated surfaces a slightly larger cell area and roundness values was observed on HA as compared to Au. Moreover, the results revealed that the morphology of cells cultured on fibronectin coated HA surfaces were less irregular. In summary we find that fibronectin adsorbs in a more activated state on the HA surfaces, resulting in a slightly different cellular response as compared to the fibronectin coated Au surfaces.
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paper_title: Surface plasmon resonance imaging for medical and biosensing

paper_content:

A novel surface plasmon resonance (SPR) microscope/imager which allows for both angular and wavelength scanning has been constructed. Images of mesenchymal stem cells were obtained using this versatile instrument and found to be superior in clarity to those captured by the angular scanning dependent SPR microscopy/imaging techniques.
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paper_title: Revisiting lab-on-a-chip technology for drug discovery

paper_content:

Manz and colleagues discuss recent progress in the development of microfluidic techniques (lab-on-a-chip technology) and their applications in drug discovery. Highlights include high-throughput droplet technology and applications such as 'organs on a chip', which could help reduce reliance on animal testing.
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paper_title: High-throughput microfluidic single-cell RT-qPCR

paper_content:

A long-sought milestone in microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells. Here we present a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run. Our device executes all steps of single-cell processing, including cell capture, cell lysis, reverse transcription, and quanti- tative PCR. In addition to higher throughput and reduced cost, we show that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specifi- city. We apply this technology to 3,300 single-cell measurements of (i) miRNA expression in K562 cells, (ii) coregulation of a miRNA and one of its target transcripts during differentiation in embryonic stem cells, and (iii) single nucleotide variant detection in primary lobular breast cancer cells. The core functionality established here provides the foundation from which a variety of on-chip single-cell transcription analyses will be developed.
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paper_title: Microfluidic device generating stable concentration gradients for long term cell culture: application to Wnt3a regulation of β-catenin signaling

paper_content:

In developing tissues, proteins and signaling molecules present themselves in the form of concentration gradients, which determine the fate specification and behavior of the sensing cells. To mimic these conditions in vitro, we developed a microfluidic device designed to generate stable concentration gradients at low hydrodynamic shear and allowing long term culture of adhering cells. The gradient forms in a culture space between two parallel laminar flow streams of culture medium at two different concentrations of a given morphogen. The exact algorithm for defining the concentration gradients was established with the aid of mathematical modeling of flow and mass transport. Wnt3a regulation of β-catenin signaling was chosen as a case study. The highly conserved Wnt-activated β-catenin pathway plays major roles in embryonic development, stem cell proliferation and differentiation. Wnt3a stimulates the activity of β-catenin pathway, leading to translocation of β-catenin to the nucleus where it activates a series of target genes. We cultured A375 cells stably expressing a Wnt/β-catenin reporter driving the expression of Venus, pBARVS, inside the microfluidic device. The extent to which the β-catenin pathway was activated in response to a gradient of Wnt3a was assessed in real time using the BARVS reporter gene. On a single cell level, the β-catenin signaling was proportionate to the concentration gradient of Wnt3a; we thus propose that the modulation of Wnt3a gradients in real time can provide new insights into the dynamics of β-catenin pathway, under conditions that replicate some aspects of the actual cell-tissue milieu. Our device thus offers a highly controllable platform for exploring the effects of concentration gradients on cultured cells.
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paper_title: A microfluidic processor for gene expression profiling of single human embryonic stem cells.

paper_content:

The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These 'cell population averaging' data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.
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paper_title: On-chip differentiation of human mesenchymal stem cells into adipocytes

paper_content:

A microfluidic chip was designed to produce with four different cell densities after seeding has been fabricated by soft lithography. It consists of eight parallel chambers with a single inlet and a single outlet. Each chamber was divided into four compartments, separated by a row of pillars. Human mesenchymal stem cells (hMSCs) were first grown on-chip using a normal culture medium for one day. Then, the differentiation of hMSCs into adipocytes was successfully induced by the perfusion of a differentiation medium for 14days. The results showed that the differentiation rate of hMSCs in the chip critically depends on the initial cell density. Typically, with the increase of the cell density from 800 to 5000cells/cm2, the differentiation rate increases from 21% to 41%.
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paper_title: microRNAs in cancer management.

paper_content:

Since the identification of microRNAs (miRNAs) in 1993, and the subsequent discovery of their highly conserved nature in 2000, the amount of research into their function--particularly how they contribute to malignancy--has greatly increased. This class of small RNA molecules control gene expression and provide a previously unknown control mechanism for protein synthesis. As such, it is unsurprising that miRNAs are now known to play an essential part in malignancy, functioning as tumour suppressors and oncogenes. This Review summarises the present understanding of how miRNAs operate at the molecular level; how their dysregulation is a crucial part of tumour formation, maintenance, and metastasis; how they can be used as biomarkers for disease type and grade; and how miRNA-based treatments could be used for diverse types of malignancies.
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paper_title: Model of an Interdigitated Microsensor to Detect and Quantify Cells Flowing in a Test Chamber

paper_content:

A finite elements model of an interdigitated microsensor has been used to investigate the sensitivity of the sensor to the detection and the quantification of cells flowing in a test chamber. In particular the sensitivity of the sensor towards the geometry of sensors and the presence of a cell flowing through the channels was evaluated; several sensors topologies were considered in order to define proper guide-lines for the design of the microsensor.
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paper_title: Detecting particles flowing through interdigitated 3D microelectrodes

paper_content:

Counting cells in a large microchannel remains challenging and is particularly critical for in vitro assays, such as cell adhesion assays. This paper addresses this issue, by presenting the development of interdigitated three-dimensional electrodes, which are fabricated around passivated pillarshaped silicon microstructures, to detect particles in a flow. The arrays of micropillars occupy the entire channel height and detect the passage of the particle through their gaps by monitoring changes in the electrical resistance. Impedance measurements were employed in order to characterize the electrical equivalent model of the system and to detect the passage of particles in real-time. Three different geometrical micropillar configurations were evaluated and numerical simulations that supported the experimental activity were used to characterize the sensitive volume in the channel. Moreover, the signal-to-noise-ratio related to the passage of a single particle through an array was plotted as a function of the dimension and number of micropillars.
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paper_title: Automated multiparametric platform for high-content and high-Throughput Analytical screening on living cells

paper_content:

Addressing the increasing biomedical and pharmacological interest in multiparametric screening assays, a concept has been developed, which integrates multiparametric, bioelectric, and biochemical sensors for the analytical monitoring of intra- and extra cellular parameters and an automated imaging microscope for high-content screening into a single embedded platform. Utilizing a topology of distributed intelligences and hardware-based synchronization, the platform concept allows precisely timed and synchronized operation of all integrated platform components. The concept is highly modular, and its design-inherent versatility allows a multitude of platform configurations suiting widely differing user requirements. They include the future integration of probe-manipulation systems such as climate control, fluidic systems, and automated probe placement. Hardware-level synchronization is achieved with a newly developed digital signal processor based "Integration Control Unit," which runs a real-time environment and provides standard electrical interfaces to connect to other platform components. The platform can be operated "online" by user-interactive control or precisely timed and completely automated within high-throughput applications by executing experiment protocols that have been composed in advance. Possible applications of the integrated platform employ a parallel optical and sensory monitoring of extra- and intracellular parameters, yielding detailed insight into cellular functions and intercellular interrelations. Thus, the proposed automated platform may develop into an enabling technology for future screening assays, especially in the field of pharmacological drug screening. Note to Practitioners-One trend in screening applications is to measure an increasing number of cellular parameters in parallel (high-content screening) to obtain a comprehensive view on the investigated cellular process. This motivated us to develop a platform, which provides simultaneous acquisition of multisensor and fluorescence microscopic data. Since both techniques are well established in numerous screening applications, we hope that this new combination stimulates the development of new assays (e.g., in pharmacological drug screening, clinical diagnostics, or environmental monitoring). Since prototypes for high-content screening are available, we are interested in cooperation with scientists working on assay development.
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paper_title: Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays

paper_content:

We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 × 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37°C. The observed doubling time was 1.4 ± 0.1 days with a peak cell density of ∼2.5*105 cells/cm2. Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology. © 2004 Wiley Periodicals, Inc.
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paper_title: Functional cellular assays with multiparametric silicon sensor chips

paper_content:

Multiparametric silicon sensor chips mounted into biocompatible cell culture units have been used for investigations on cellular microphysiological patterns. Potentiometric, amperometric and impedimetric microsensors are combined on a common cell culture surface on the chip with an area of ∼29 mm2. Extracellular acidification rates (with pH-sensitive field effect transistors, ISFETs), cellular oxygen consumption rates (with amperometric electrode structures) and cell morphological alterations (with impedimetric electrode structures, IDES) are monitored on single chips simultaneously for up to several days. The corresponding test device accommodates six of such sensor chips in parallel, provides electronic circuitry and maintains the required cell culture conditions (temperature, fluid perfusion system). Sensor data are transformed into quantitative information about microphysiologic conditions. The outcome of this transformation as well as reliability and sensitivity in detection of drug effects is discussed. This is the first report on multiparametric cell based assays with data obtained solely with integrated sensors on silicon chips. Those assays are required in different fields of application such as pharmaceutical drug screening, tumor chemosensitivity tests and environmental monitoring.
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paper_title: Patch clamping by numbers.

paper_content:

Abstract Many ion channels are recognized as amenable targets for a range of disease states and conditions. However, the process of discovering drugs is highly influenced by the chemical doability, the biological confidence in rationale of the approach and the ‘screenability’. To date, the absence of informative high throughput technologies for ion channel screening has resulted in ion channels remaining a largely unexplored class of drug targets. This, however, is about to change – a large increase in the number of data points per day should be achieved by the introduction of automated ‘high throughput’ patch clamp machines.
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paper_title: Heterogeneity of Embryonic and Adult Stem Cells

paper_content:

New studies suggest that stem cells of embryonic, neural, and hematopoietic origin are heterogeneous, with cells moving between two or more metastable states. These cell states show a bias in their differentiation potential and correlate with specific patterns of transcription factor expression and chromatin modifications.
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paper_title: Microelectronic sensor system for microphysiological application on living cells

paper_content:

Abstract Living cells can be considered as complex biochemical plants. Biochemical and biophysical processes enable a cell to maintain itself, to grow, to reproduce and to communicate with the environment. Getting more information about the multifunctional cellular processing of input- and output-signals in different cellular plants is essential for basic research as well as for various fields of biomedical applications. For in-vitro investigations on living cells, the cellular environment differs from the native environment found in vivo. As a first approach for on-line monitoring of cellular reactions under well controlled experimental conditions we have developed the so called Cell Monitoring System (CMS ® ). It allows parallel and non-invasive measurement of different parameters from cellular systems by the use of microsensors. Microelectronic sensors are the adequate choice for the non-invasive measurement of environmental—as well as in- and output—parameters of cells. In this paper we present a measurement system with pH-sensitive ISFETs (ionsensitive fieldeffect transistors) for the measurement of extracellular pH-related signals on cells and tissues.
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paper_title: Microfabricated Platform for Studying Stem Cell Fates

paper_content:

Platforms that allow parallel, quantitative analysis of single cells will be integral to realizing the potential of postgenomic biology. In stem cell biology, the study of clonal stem cells in multiwell formats is currently both inefficient and time-consuming. Thus, to investigate low-frequency events of interest, large sample sizes must be interrogated. We report a simple, versatile, and efficient micropatterned arraying system conducive to the culture and dynamic monitoring of stem cell proliferation. This platform enables: 1) parallel, automated, long-term (∼days to weeks), live-cell microscopy of single cells in culture; 2) tracking of individual cell fates over time (proliferation, apoptosis); and 3) correlation of differentiated progeny with founder clones. To achieve these goals, we used microfabrication techniques to create an array of ∼10,000 microwells on a glass coverslip. The dimensions of the wells are tunable, ranging from 20 to >500 μm in diameter and 10–500 μm in height. The microarray can be coated with adhesive proteins and is integrated into a culture chamber that permits rapid (∼min), addressable monitoring of each well using a standard programmable microscope stage. All cells share the same media (including paracrine survival signals), as opposed to cells in multiwell formats. The incorporation of a coverslip as a substrate also renders the platform compatible with conventional, high-magnification light and fluorescent microscopy. We validated this approach by analyzing the proliferation dynamics of a heterogeneous adult rat neural stem cell population. Using this platform, one can further interrogate the response of distinct stem cell subpopulations to microenvironmental cues (mitogens, cell–cell interactions, and cell–extracellular matrix interactions) that govern their behavior. In the future, the platform may also be adapted for the study of other cell types by tailoring the surface coatings, microwell dimensions, and culture environment, thereby enabling parallel investigation of many distinct cellular responses. © 2004 Wiley Periodicals, Inc.
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paper_title: From Understanding Cellular Function to Novel Drug Discovery: The Role of Planar Patch-Clamp Array Chip Technology

paper_content:

All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions - including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells.
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paper_title: An automatic and quantitative on-chip cell migration assay using self-assembled monolayers combined with real-time cellular impedance sensing.

paper_content:

Cell migration is crucial in many physiological and pathological processes including embryonic development, immune response and cancer metastasis. Traditional methods for cell migration detection such as wound healing assay usually involve physical scraping of a cell monolayer followed by an optical observation of cell movement. However, these methods require hand-operation with low repeatability. Moreover, it's a qualitative observation not a quantitative measurement, which is hard to scale up to a high-throughput manner. In this article, a novel and reliable on-chip cell migration detection method integrating surface chemical modification of gold electrodes using self-assembled monolayers (SAMs) and real-time cellular impedance sensing is presented. The SAMs are used to inhibit cell adherence forming an area devoid of cells, which could effectively mimic wounds in a cell monolayer. After a DC electrical signal was applied, the SAMs were desorbed from the electrodes and cells started to migrate. The process of cell migration was monitored by real-time impedance sensing. This demonstrates the first occurrence of integrating cellular impedance sensing and wound-forming with SAMs, which makes cell migration assay being real-time, quantitative and fully automatic. We believe this method could be used for high-throughput anti-migratory drug screening and drug discovery.
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paper_title: Monitoring impedance changes associated with motility and mitosis of a single cell

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We present a device enabling impedance measurements that probe the motility and mitosis of a single adherent cell in a controlled way. The micrometre-sized electrodes are designed for adhesion of an isolated cell and enhanced sensitivity to cell motion. The electrode surface is switched electro-chemically to favour cell adhesion, and single cells are attracted to the electrode using positive dielectrophoresis. Periods of linear variation in impedance with time correspond to the motility of a single cell adherent to the surface estimated at 0.6 μm h−1. In the course of our study we observed the impedance changes associated with mitosis of a single cell. Electrical measurements, carried out concomitantly with optical observations, revealed three phases, prophase, metaphase and anaphase in the time variation of the impedance during cell division. Maximal impedance was observed at metaphase with a 20% increase of the impedance. We argue that at mitosis, the changes detected were due to the charge density distribution at the cell surface. Our data demonstrate subtle electrical changes associated with cell motility and for the first time with division at the single-cell level. We speculate that this could open up new avenues for characterizing healthy and pathological cells.
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paper_title: Neurochip based on light-addressable potentiometric sensor with wavelet transform de-noising

paper_content:

Neurochip based on light-addressable potentiometric sensor (LAPS), whose sensing elements are excitable cells, can monitor electrophysiological properties of cultured neuron networks with cellular signals well analyzed. Here we report a kind of neurochip with rat pheochromocytoma (PC12) cells hybrid with LAPS and a method of de-noising signals based on wavelet transform. Cells were cultured on LAPS for several days to form networks, and we then used LAPS system to detect the extracellular potentials with signals de-noised according to decomposition in the time-frequency space. The signal was decomposed into various scales, and coefficients were processed based on the properties of each layer. At last, signal was reconstructed based on the new coefficients. The results show that after de-noising, baseline drift is removed and signal-to-noise ratio is increased. It suggests that the neurochip of PC12 cells coupled to LAPS is stable and suitable for long-term and non-invasive measurement of cell electrophysiological properties with wavelet transform, taking advantage of its time-frequency localization analysis to reduce noise.
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paper_title: Cell-based biosensors based on light-addressable potentiometric sensors for single cell monitoring

paper_content:

Abstract Cell-based biosensors incorporate cells as sensing elements that convert changes in immediate environment to signals for processing. This paper reports an investigation on light-addressable potentiometric sensor (LAPS) to be used as a possible cell-base biosensor that will enable us to monitor extracellular action potential of single living cell under stimulant. In order to modify chip surface and immobilize cells, we coat a layer of poly- l -ornithine and laminin on surface of LAPS chip on which rat cortical cells are grown well. When 10 μg/ml acetylcholine solution is administrated, the light pointer is focused on a single neuronal cell and the extracellular action potential of the targeted cell is recorded with cell-based biosensor based on LAPS. The results demonstrate that this kind of biosensor has potential to monitor electrophysiology of living cell non-invasive for a long term, and to evaluate drugs primarily.
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paper_title: A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

paper_content:

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells. ::: ::: Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density. ::: ::: The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.
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paper_title: On-chip epithelial barrier function assays using electrical impedance spectroscopy.

paper_content:

A bio-impedance chip has been developed for real-time monitoring of the kinetics of epithelial cell monolayers in vitro. The human bronchial epithelial cell line (16-HBE 14o-) was cultured in Transwells creating a sustainable and interactive model of the airway epithelium. Conducting polymer polypyrrole (PPy) doped with polystyrene sulfonate (PSS) was electrochemically deposited onto the surface of gold-plated electrodes to reduce the influence of the electrical double layer on the impedance measurements. Finite element and equivalent circuit models were used to model and determine the electrical properties of the epithelial cell monolayer from the impedance spectra. Electrically tight, confluent monolayers of 16 HBE 14o- cells were treated with increasing concentrations of either Triton X-100 to solubilize cell membranes or ethylene glycol-bis(2-aminoethyl-ether)-N,N,N'N'-tetraacetic acid (EGTA) to disrupt cell-cell adhesion. Experimental impedance data showed that disruption of epithelial barrier function in response to Triton X-100 and EGTA can be successfully measured by the bio-impedance chip. The results were consistent with the conventional hand-held trans-epithelial electrical resistance measurements. Immunofluorescent staining of the ZO-1 tight junction protein in the untreated and treated 16HBEs was performed to verify the disruption of the tight junctions by EGTA.
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paper_title: Label-free electrical discrimination of cells at normal, apoptotic and necrotic status with a microfluidic device.

paper_content:

As a label-free alternative of conventional flow cytometry, chip-based impedance measurement for single cell analysis has attracted increasing attentions in recent years. In this paper, we designed a T-shape microchannel and fabricated a pair of gold electrodes located horizontally on each side of the microchannel using a transfer printing method. Instant electric signals of flowing-through single cells were then detected by connecting the electrodes to a Keithley resistance and capacitance measurement system. Experimental results based on the simultaneous measurement of resistance and capacitance demonstrated that HL-60 and SMMC-7721 cells could be differentiated effectively. Moreover, SMMC-7721 cells at normal, apoptotic and necrotic status can also be discriminated in the flow. We discussed the possible mechanism for the discrimination of cell size and cell status by electrical analysis, and it is believed that the improvement of detection with our design results from more uniform distribution of the electric field. This microfluidic design may potentially become a promising approach for the label-free cell sorting and screening.
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paper_title: Cell shape and spreading of stromal (mesenchymal) stem cells cultured on fibronectin coated gold and hydroxyapatite surfaces.

paper_content:

In order to identify the cellular mechanisms leading to the biocompatibility of hydroxyapatite implants, we studied the interaction of human bone marrow derived stromal (mesenchymal) stem cells (hMSCs) with fibronectin-coated gold (Au) and hydroxyapatite (HA) surfaces. The adsorption of fibronectin was monitored by Quartz Crystal Microbalance with Dissipation (QCM-D) at two different concentrations, 20 μg/ml and 200 μg/ml, and the fibronectin adsorption experiments were complemented with antibody measurements. The QCM-D results show that the surface mass uptake is largest on the Au surfaces, while the number of polyclonal and monoclonal antibodies directed against the cell-binding domain (CB-domain) on the fibronectin (Fn) is significantly larger on the (HA) surfaces. Moreover, a higher number of antibodies bound to the fibronectin coatings formed from the highest bulk fibronection concentration. In subsequent cell studies with hMSC's we studied the cell spreading, cytoskeletal organization and cell morphology on the respective surfaces. When the cells were adsorbed on the uncoated substrates, a diffuse cell actin cytoskeleton was revealed, and the cells had a highly elongated shape. On the fibronectin coated surfaces the cells adapted to a more polygonal shape with a well-defined actin cytoskeleton, while a larger cell area and roundness values were observed for cells cultured on the coated surfaces. Among the coated surfaces a slightly larger cell area and roundness values was observed on HA as compared to Au. Moreover, the results revealed that the morphology of cells cultured on fibronectin coated HA surfaces were less irregular. In summary we find that fibronectin adsorbs in a more activated state on the HA surfaces, resulting in a slightly different cellular response as compared to the fibronectin coated Au surfaces.
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paper_title: Capacitive microsystems for biological sensing

paper_content:

Abstract The growing interest in personalized medicine leads to the need for fast, cheap and portable devices that reveal the genetic profile easily and accurately. To this direction, several ideas to avoid the classical methods of diagnosis and treatment through miniaturized and label-free systems have emerged. Capacitive biosensors address these requirements and thus have the perspective to be used in advanced diagnostic devices that promise early detection of potential fatal conditions. The operation principles, as well as the design and fabrication of several capacitive microsystems for the detection of biomolecular interactions are presented in this review. These systems are micro-membranes based on surface stress changes, interdigitated micro-electrodes and electrode–solution interfaces. Their applications extend to DNA hybridization, protein–ligand binding, antigen–antibody binding, etc. Finally, the limitations and prospects of capacitive microsystems in biological applications are discussed.
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paper_title: Microfluidic impedance‐based flow cytometry

paper_content:

Microfabricated flow cytometers can detect, count, and analyze cells or particles using microfluidics and electronics to give impedance-based characterization. Such systems are being developed to provide simple, low-cost, label-free, and portable solutions for cell analysis. Recent work using microfabricated systems has demonstrated the capability to analyze micro-organisms, erythrocytes, leukocytes, and animal and human cell lines. Multifrequency impedance measurements can give multiparametric, high-content data that can be used to distinguish cell types. New combinations of microfluidic sample handling design and microscale flow phenomena have been used to focus and position cells within the channel for improved sensitivity. Robust designs will enable focusing at high flowrates while reducing requirements for control over multiple sample and sheath flows. Although microfluidic impedance-based flow cytometers have not yet or may never reach the extremely high throughput of conventional flow cytometers, the advantages of portability, simplicity, and ability to analyze single cells in small populations are, nevertheless, where chip-based cytometry can make a large impact. © 2010 International Society for Advancement of Cytometry
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Title: Overview of Micro- and Nano-Technology Tools for Stem Cell Applications: Micropatterned and Microelectronic Devices

Section 1: Introduction
Description 1: Introduce the exponential growth and advances in biosensor science, specifically focusing on silicon micromachining, genomics, and cell culture technology in the context of biochips, DNA microarrays, and cell-based chips.

Section 2: Sensing and Transducer Element: The Cell; Variables and Constants
Description 2: Discuss the dynamic nature of cell interactions, the importance of the cell microenvironment, and the technological advancements required to mimic in vivo conditions.

Section 3: Genechip
Description 3: Provide an overview of the development and applications of cell microarrays, emphasizing their role in high throughput screening, gene function discovery, and stem cell differentiation.

Section 4: Micropattering: Microengineering Meets Cell Biology
Description 4: Explain the use of microfabrication techniques to create well-defined surfaces and patterns for cell culture, with a focus on how these techniques can mimic natural cell niches and influence stem cell behavior.

Section 5: 3D Cell Culture and Tissue Organization
Description 5: Explore recent advances in 3D cell culture models that better simulate in vivo environments and their application in tissue differentiation and stem cell research.

Section 6: Integration of Microelectronics and Cells
Description 6: Detail the integration of cells with microelectronic devices, highlighting various types of sensors such as MEA, EICS, FET, LAPS, SPR, and QCM, and their applications in studying stem cell functions and drug discovery.

Section 7: Microelectrodes Array: MEA
Description 7: Discuss the structure, benefits, and applications of MEAs in cell pattering, drug screening, and neuronal and cardiac cell studies.

Section 8: Electric Cell-Substrate Impedance Sensor: EICS
Description 8: Examine the use of EICS sensors to study the bioelectrical properties of cells and monitor stem cell differentiation, adhesion, and response to stimuli.

Section 9: Field-Effect Transistor: FET
Description 9: Describe the development, structure, and applications of FETs in measuring extracellular potentials, pH levels, and cell signaling in stem cell research.

Section 10: Light Addressable Potentiometric Sensor: LAPS
Description 10: Present the working principle and applications of LAPS in monitoring cell metabolism, extracellular potentials, and stem cell differentiation.

Section 11: Surface Plasmon Resonance Chip (SPR) and Quartz Crystal Microbalance Chip (QCM)
Description 11: Discuss the use of SPR and QCM techniques in analyzing cell attachment, proliferation, and cell-substrate interaction, focusing on stem cell studies.

Section 12: Integration of Different Sensors and Microfluidic Approaches: Future Perspectives on Single Cell Analysis
Description 12: Explore the integration of multiple sensors and microfluidic platforms for comprehensive cell analysis, emphasizing potential advancements in single-cell analysis and drug discovery.

Section 13: Conclusions/Outlook
Description 13: Summarize the potential of micro- and nano-technology tools in revolutionizing stem cell research and personalized medicine, highlighting future challenges and opportunities in the field.