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Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic form of breast cancer that lacks the estrogen, progesterone and HER2 receptors and is resistant to targeted and hormone therapies. TNBCs express high levels of the transmembrane glycoprotein, complement C1r/C1s, Uegf, Bmp1 (CUB)-domain containing protein 1 (CDCP1), which has been correlated with the aggressiveness and poor prognosis of multiple carcinomas. Full-length CDCP1 (flCDCP1) can be proteolytically cleaved, resulting in a cleaved membrane-bound isoform (cCDCP1). CDCP1 is phosphorylated by Src family kinases in its full-length and cleaved states, which is important for its pro-metastatic signaling. We observed that cCDCP1, compared with flCDCP1, induced a dramatic increase in phosphorylation of the migration-associated proteins: PKCδ, ERK1/2 and p38 mitogen-activated protein kinase in HEK 293T. In addition, only cCDCP1 induced migration of HEK 293T cells and rescued migration of the TNBC cell lines expressing short hairpin RNA against CDCP1. Importantly, we found that only cCDCP1 is capable of dimerization, which can be blocked by expression of the extracellular portion of cCDCP1 (ECC), indicating that dimerization occurs through CDCP1's ectodomain. We found that ECC inhibited phosphorylation of PKCδ and migration of TNBC cells in two-dimensional culture. Furthermore, ECC decreased cell invasiveness, inhibited proliferation and stimulated apoptosis of TNBC cells in three-dimensional culture, indicating that the cCDCP1 dimer is an important contributor to TNBC aggressiveness. These studies have important implications for the development of a therapeutic to block CDCP1 activity and TNBC metastasis.

Undifferentiated neural stem and progenitor cells (NSPCs) encounter extracellular signals that bind plasma membrane proteins and influence differentiation. Membrane proteins are regulated by N-linked glycosylation, making it possible that glycosylation plays a critical role in cell differentiation. We assessed enzymes that control N-glycosylation in NSPCs and found that loss of the enzyme responsible for generating β1,6-branched N-glycans, N-acetylglucosaminyltransferase V (MGAT5), led to specific changes in NSPC differentiation in vitro and in vivo. Mgat5 homozygous null NSPCs in culture formed more neurons and fewer astrocytes compared with wild-type controls. In the brain cerebral cortex, loss of MGAT5 caused accelerated neuronal differentiation. Rapid neuronal differentiation led to depletion of cells in the NSPC niche, resulting in a shift in cortical neuron layers in Mgat5 null mice. Glycosylation enzyme MGAT5 plays a critical and previously unrecognized role in cell differentiation and early brain development., Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Language barriers were reported to affect timely access to health care and outcome. The aim of this study was to investigate the effect of language disparity on quality benchmarks of acute ischemic stroke therapy., Methods: Consecutive patients with acute ischemic stroke at the University of California Irvine Medical Center from 2013 to 2016 were studied. Patients were categorized into 3 groups according to their preferred language: English, Spanish, and other languages. Quality benchmarks and outcomes of the 3 language groups were analyzed., Results: Of the 928 admissions, 69.7% patients recorded English as preferred language, as compared to 17.3% Spanish and 13.0% other languages. There was no significant difference in the rate of receiving intravenous thrombolysis (24.3, 22.1 and 21.0%), last-known-well to door time, door-to-imaging time, door-to-needle time, and hospital length of stay among the 3 language groups. In univariate analysis, the other languages group had lower chance of favorable outcomes than the English-speaking group (26.3% vs 40.4, p < 0.05) while the Spanish-speaking group had lower mortality rate than English-speaking group (3.1% vs 7.7%, p = 0.05). After adjusting for age and initial NIHSS scores, multivariate regression models showed no significant difference in favorable outcomes and mortality between different language groups., Conclusion: We demonstrate no significant difference in quality benchmarks and outcome of acute ischemic stroke among 3 different language groups. Our results suggest that limited English proficiency is not a significant barrier for time-sensitive stroke care at Comprehensive Stroke Center.

Human neural stem and progenitor cells (hNSPCs) have therapeutic potential to treat neural diseases and injuries since they provide neuroprotection and differentiate into astrocytes, neurons, and oligodendrocytes. However, cultures of hNSPCs are heterogeneous, containing cells linked to distinct differentiated cell fates. HNSPCs that differentiate into astrocytes are of interest for specific neurological diseases, creating a need for approaches that can detect and isolate these cells. Astrocyte-biased hNSPCs differ from other cell types in electrophysiological properties, namely membrane capacitance, and we hypothesized that this could be used to enrich these cells using dielectrophoresis (DEP). We implemented a two-step DEP sorting scheme, consisting of analysis to define the optimal sorting frequency followed by separation of cells at that frequency, to test whether astrocyte-biased cells could be separated from the other cell types present in hNSPC cultures. We developed a novel device that increased sorting reproducibility and provided both enriched and depleted cell populations in a single sort. Astrocyte-biased cells were successfully enriched from hNSPC cultures by DEP sorting, making this the first study to use electrophysiological properties for label-free enrichment of human astrocyte-biased cells. Enriched astrocyte-biased human cells enable future experiments to determine the specific properties of these important cells and test their therapeutic efficacy in animal models of neurological diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Stem cells have attracted significant attention due to their regenerative capabilities and their potential for the treatment of disease. Consequently, significant research effort has focused on the development of protein- and polypeptide-based materials as stem cell substrates and scaffolds. Here, we explore the ability of reflectin, a cephalopod structural protein, to support the growth of murine neural stem/progenitor cells (mNSPCs). We observe that the binding, growth, and differentiation of mNSPCs on reflectin films is comparable to that on more established protein-based materials. Moreover, we find that heparin selectively inhibits the adhesion of mNSPCs on reflectin, affording spatial control of cell growth and leading to a >30-fold change in cell density on patterned substrates. The described findings highlight the potential utility of reflectin as a stem cell culture material.

We created an integrated microfluidic cell separation system that incorporates hydrophoresis and dielectrophoresis modules to facilitate high-throughput continuous cell separation. The hydrophoresis module consists of a serpentine channel with ridges and trenches to generate a diverging fluid flow that focuses cells into two streams along the channel edges. The dielectrophoresis module is composed of a chevron-shaped electrode array. Separation in the dielectrophoresis module is driven by inherent cell electrophysiological properties and does not require cell-type-specific labels. The chevron shape of the electrode array couples with fluid flow in the channel to enable continuous sorting of cells to increase throughput. We tested the new system with mouse neural stem cells since their electrophysiological properties reflect their differentiation capacity (e.g., whether they will differentiate into astrocytes or neurons). The goal of our experiments was to enrich astrocyte-biased cells. Sorting parameters were optimized for each batch of neural stem cells to ensure effective and consistent separations. The continuous sorting design of the device significantly improved sorting throughput and reproducibility. Sorting yielded two cell fractions, and we found that astrocyte-biased cells were enriched in one fraction and depleted from the other. This is an advantage of the new continuous sorting device over traditional dielectrophoresis-based sorting platforms that target a subset of cells for enrichment but do not provide a corresponding depleted population. The new microfluidic dielectrophoresis cell separation system improves label-free cell sorting by increasing throughput and delivering enriched and depleted cell subpopulations in a single sort., (© 2019 Author(s).)

Piezo channels transduce mechanical stimuli into electrical and chemical signals to powerfully influence development, tissue homeostasis, and regeneration. Studies on Piezo1 have largely focused on transduction of "outside-in" mechanical forces, and its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that cellular traction forces generate spatially-restricted Piezo1-mediated Ca 2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched near force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca 2+ flickers allow spatial segregation of mechanotransduction events, and that mobility allows Piezo1 channels to explore a large number of mechanical microdomains and thus respond to a greater diversity of mechanical cues., Competing Interests: Competing interestsThe authors declare no competing interests.

Neural stem and progenitor cells (NSPCs) are an extremely important group of cells that form the central nervous system during development and have the potential to repair damage in conditions such as stroke impairment, spinal cord injury and Parkinson's disease degradation. Current schemes for separation of NSPCs are inadequate due to the complexity and diversity of cells in the population and lack sufficient markers to distinguish diverse cell types. This study presents an unbiased high-resolution separation and characterization of NSPC subpopulations using direct current insulator-based dielectrophoresis (DC-iDEP). The properties of the cells were identified by the ratio of electrokinetic (EK) to dielectrophoretic (DEP) mobilities. The ratio factor of NSPCs showed more heterogeneity variance (SD = 3.4-3.9) than the controlled more homogeneous human embryonic kidney cells (SD = 1.1), supporting the presence of distinct subpopulations of cells in NSPC cultures. This measure reflected NSPC fate potential since the ratio factor distribution of more neurogenic populations of NSPCs was distinct from the distribution of astrogenic NSPC populations (confidence level >99.9%). The abundance of NSPCs captured with different ranges of ratio of EK to DEP mobilities also exhibit final fate trends consistent with established final fates of the chosen samples. DC-iDEP is a novel, label-free and non-destructive method for differentiating and characterizing, and potentially separating, neural stem cell subpopulations that differ in fate.

The tsetse fly is the insect vector for the Trypanosoma brucei parasite, the causative agent of human African trypanosomiasis. The colonization and spread of the trypanosome correlate positively with the presence of a secondary symbiotic bacterium, Sodalis glossinidius The metabolic requirements and interactions of the bacterium with its host are poorly understood, and herein we describe a metabolic model of S. glossinidius metabolism. The model enabled the design and experimental verification of a defined medium that supports S. glossinidius growth ex vivo This has been used subsequently to analyze in vitro aspects of S. glossinidius metabolism, revealing multiple unique adaptations of the symbiont to its environment. Continued dependence on a sugar, and the importance of the chitin monomer N- acetyl-d-glucosamine as a carbon and energy source, suggests adaptation to host-derived molecules. Adaptation to the amino acid-rich blood diet is revealed by a strong dependence on l-glutamate as a source of carbon and nitrogen and by the ability to rescue a predicted l-arginine auxotrophy. Finally, the selective loss of thiamine biosynthesis, a vitamin provided to the host by the primary symbiont Wigglesworthia glossinidia , reveals an intersymbiont dependence. The reductive evolution of S. glossinidius to exploit environmentally derived metabolites has resulted in multiple weaknesses in the metabolic network. These weaknesses may become targets for reagents that inhibit S. glossinidius growth and aid the reduction of trypanosomal transmission. IMPORTANCE Human African trypanosomiasis is caused by the Trypanosoma brucei parasite. The tsetse fly vector is of interest for its potential to prevent disease spread, as it is essential for T. brucei life cycle progression and transmission. The tsetse's mutualistic endosymbiont Sodalis glossinidius has a link to trypanosome establishment, providing a disease control target. Here, we describe a new, experimentally verified model of S. glossinidius metabolism. This model has enabled the development of a defined growth medium that was used successfully to test aspects of S. glossinidius metabolism. We present S. glossinidius as uniquely adapted to life in the tsetse, through its reliance on the blood diet and host-derived sugars. Additionally, S. glossinidius has adapted to the tsetse's obligate symbiont Wigglesworthia glossinidia by scavenging a vitamin it produces for the insect. This work highlights the use of metabolic modeling to design defined growth media for symbiotic bacteria and may provide novel inhibitory targets to block trypanosome transmission., (Copyright © 2019 Hall et al.)

Understanding the cellular properties controlling neural stem and progenitor cell (NSPC) fate choice will improve their therapeutic potential. The electrophysiological measure whole-cell membrane capacitance reflects fate bias in the neural lineage but the cellular properties underlying membrane capacitance are poorly understood. We tested the hypothesis that cell surface carbohydrates contribute to NSPC membrane capacitance and fate. We found NSPCs differing in fate potential express distinct patterns of glycosylation enzymes. Screening several glycosylation pathways revealed that the one forming highly branched N-glycans differs between neurogenic and astrogenic populations of cells in vitro and in vivo. Enhancing highly branched N-glycans on NSPCs significantly increases membrane capacitance and leads to the generation of more astrocytes at the expense of neurons with no effect on cell size, viability, or proliferation. These data identify the N-glycan branching pathway as a significant regulator of membrane capacitance and fate choice in the neural lineage., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose of Review: Advanced technologies can aid discoveries in stem cell science in surprising ways. The application of electrokinetic techniques, which use electric fields to interrogate or separate cells, to the study of stem cells has yielded important insights into stem cell function. These techniques probe inherent cell properties, obviating the need for cell-type specific labels., Recent Findings: Analysis of a variety of stem cell types including hematopoietic, mesenchymal and adipose-derived, neural, and pluripotent stem cells by electrokinetic techniques has revealed fate-specific signatures of cells. Distinct inherent cell properties are sufficient for their label-free enrichment without causing cell damage or toxicity., Summary: The successful application of label-free techniques to the analysis and sorting of stem cells open new avenues for exploring the basic biology of stem cells and optimizing their use in regenerative medicine applications., Competing Interests: Conflict of Interest Mohammad Aghaamoo and Tayloria Adams declare that they have no conflict of interest.

Adhesion to the microenvironment profoundly affects stem cell functions, including proliferation and differentiation, and understanding the interaction of stem cells with the microenvironment is important for controlling their behavior. In this study, we investigated the effects of the integrin binding epitopes GFOGER and IKVAV (natively present in collagen I and laminin, respectively) on human neural stem/progenitor cells (hNSPCs). To test the specificity of these epitopes, GFOGER or IKVAV were placed within the context of recombinant triple-helical collagen III engineered to be devoid of native integrin binding sites. HNSPCs adhered to collagen that presented GFOGER as the sole integrin-binding site, but not to IKVAV-containing collagen. For the GFOGER-containing collagens, antibodies against the β1 integrin subunit prevented cellular adhesion, antibodies against the α1 subunit reduced cell adhesion, and antibodies against α2 or α3 subunits had no significant effect. These results indicate that hNSPCs primarily interact with GFOGER through the α1β1 integrin heterodimer. These GFOGER-presenting collagen variants also supported differentiation of hNSPCs into neurons and astrocytes. Our findings show, for the first time, that hNSPCs can bind to the GFOGER sequence, and they provide motivation to develop hydrogels formed from recombinant collagen variants as a cell delivery scaffold. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1363-1372, 2018., (© 2018 Wiley Periodicals, Inc.)

This study aims to understand the phagocytic response of astrocytes to the injury of neurons or other astrocytes at the single cell level. Laser nanosurgery was used to damage individual cells in both primary mouse cortical astrocytes and an established astrocyte cell line. In both cases, the release of material/substances from laser-irradiated astrocytes or neurons induced a phagocytic response in near-by astrocytes. Propidium iodide stained DNA originating from irradiated cells was visible in vesicles of neighboring cells, confirming phagocytosis of material from damaged cortical cells. In the presence of an intracellular pH indicator dye, newly formed vesicles correspond to acidic pH fluorescence, thus suggesting lysosome bound degradation of cellular debris. Cells with shared membrane connections prior to laser damage had a significantly higher frequency of induced phagocytosis compared to isolated cells with no shared membrane. The increase in phagocytic response of cells with a shared membrane occurred regardless of the extent of shared membrane (a thin filopodial connection vs. a cell cluster with significant shared membrane). In addition to the presence (or lack) of a membrane connection, variation in phagocytic ability was also observed with differences in injury location within the cell and distance separating isolated astrocytes. These results demonstrate the ability of an astrocyte to respond to the damage of a single cell, be it another astrocyte, or a neuron. This single-cell level of analysis results in a better understanding of the role of astrocytes to maintain homeostasis in the CNS, particularly in the sensing and removal of debris in damaged or pathologic nervous tissue.

[NiFe] hydrogenases are electrocatalysts that oxidize H 2 at a rapid rate without the need for precious metals. All membrane-bound [NiFe] hydrogenases (MBH) possess a histidine residue that points to the electron-transfer iron sulfur cluster closest ("proximal") to the [NiFe] H 2 -binding active site. Replacement of this amino acid with alanine induces O 2 sensitivity, and this has been attributed to the role of the histidine in enabling the reversible O 2 -induced over-oxidation of the [Fe 4 S 3 Cys 2 ] proximal cluster possessed by all O 2 -tolerant MBH. We have created an Escherichia coli Hyd-1 His-to-Ala variant and report O 2 -free electrochemical measurements at high potential that indicate the histidine-mediated [Fe 4 S 3 Cys 2 ] cluster-opening/closing mechanism also underpins anaerobic reactivation. We validate these experiments by comparing them to the impact of an analogous His-to-Ala replacement in Escherichia coli Hyd-2, a [NiFe]-MBH that contains a [Fe 4 S 4 ] center., Competing Interests: The authors declare no conflict of interest.

Whole cell membrane capacitance is an electrophysiological property of the plasma membrane that serves as a biomarker for stem cell fate potential. Neural stem and progenitor cells (NSPCs) that differ in ability to form neurons or astrocytes are distinguished by membrane capacitance measured by dielectrophoresis (DEP). Differences in membrane capacitance are sufficient to enable the enrichment of neuron- or astrocyte-forming cells by DEP, showing the separation of stem cells on the basis of fate potential by membrane capacitance. NSPCs sorted by DEP need not be labeled and do not experience toxic effects from the sorting procedure. Other stem cell populations also display shifts in membrane capacitance as cells differentiate to a particular fate, clarifying the value of sorting a variety of stem cell types by capacitance. Here, we describe methods developed by our lab for separating NSPCs on the basis of capacitance using several types of DEP microfluidic devices, providing basic information on the sorting procedure as well as specific advantages and disadvantages of each device., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The redox chemistry of the electron entry/exit site in Escherichia coli hydrogenase-1 is shown to play a vital role in tuning biocatalysis. Inspired by nature, we generate a HyaA-R193L variant to disrupt a proposed Arg-His cation-π interaction in the secondary coordination sphere of the outermost, "distal", iron-sulfur cluster. This rewires the enzyme, enhancing the relative rate of H 2 production and the thermodynamic efficiency of H 2 oxidation catalysis. On the basis of Fourier transformed alternating current voltammetry measurements, we relate these changes in catalysis to a shift in the distal [Fe 4 S 4 ] 2+/1+ redox potential, a previously experimentally inaccessible parameter. Thus, metalloenzyme chemistry is shown to be tuned by the second coordination sphere of an electron transfer site distant from the catalytic center.

Multipotent human central nervous system-derived neural stem cells transplanted at doses ranging from 10,000 (low) to 500,000 (very high) cells differentiated predominantly into the oligodendroglial lineage. However, while the number of engrafted cells increased linearly in relationship to increasing dose, the proportion of oligodendrocytic cells declined. Increasing dose resulted in a plateau of engraftment, enhanced neuronal differentiation, and increased distal migration caudal to the transplantation sites. Dose had no effect on terminal sensory recovery or open-field locomotor scores. However, total human cell number and decreased oligodendroglial proportion were correlated with hindlimb girdle coupling errors. Conversely, greater oligodendroglial proportion was correlated with increased Ab step pattern, decreased swing speed, and increased paw intensity, consistent with improved recovery. These data suggest that transplant dose, and/or target niche parameters can regulate donor cell engraftment, differentiation/maturation, and lineage-specific migration profiles., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Human neural stem/progenitor cells (hNSPCs) are good candidates for treating central nervous system (CNS) trauma since they secrete beneficial trophic factors and differentiate into mature CNS cells; however, many cells die after transplantation. This cell death can be ameliorated by inclusion of a biomaterial scaffold, making identification of optimal scaffolds for hNSPCs a critical research focus. We investigated the properties of fibrin-based scaffolds and their effects on hNSPCs and found that fibrin generated from salmon fibrinogen and thrombin stimulates greater hNSPC proliferation than mammalian fibrin. Fibrin scaffolds degrade over the course of a few days in vivo, so we sought to develop a novel scaffold that would retain the beneficial properties of fibrin but degrade more slowly to provide longer support for hNSPCs. We found combination scaffolds of salmon fibrin with interpenetrating networks (IPNs) of hyaluronic acid (HA) with and without laminin polymerize more effectively than fibrin alone and generate compliant hydrogels matching the physical properties of brain tissue. Furthermore, combination scaffolds support hNSPC proliferation and differentiation while significantly attenuating the cell-mediated degradation seen with fibrin alone. HNSPCs express two fibrinogen-binding integrins, αVβ1 and α5β1, and several laminin binding integrins (α7β1, α6β1, α3β1) that can mediate interaction with the scaffold. Lastly, to test the ability of scaffolds to support vascularization, we analyzed human cord blood-derived endothelial cells alone and in co-culture with hNSPCs and found enhanced vessel formation and complexity in co-cultures within combination scaffolds. Overall, combination scaffolds of fibrin, HA, and laminin are excellent biomaterials for hNSPCs., Statement of Significance: Interest has increased recently in the development of biomaterials as neural stem cell transplantation scaffolds to treat central nervous system (CNS) injury since scaffolds improve survival and integration of transplanted cells. We report here on a novel combination scaffold composed of fibrin, hyaluronic acid, and laminin to support human neural stem/progenitor cell (hNSPC) function. This combined biomaterial scaffold has appropriate physical properties for hNSPCs and the CNS, supports hNSPC proliferation and differentiation, and attenuates rapid cell-mediated scaffold degradation. The hNSPCs and scaffold components synergistically encourage new vessel formation from human endothelial cells. This work marks the first report of a combination scaffold supporting human neural and vascular cells to encourage vasculogenesis, and sets a benchmark for biomaterials to treat CNS injury., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Naturally occurring oxygen tolerant NiFe membrane bound hydrogenases have a conserved catalytic bias towards hydrogen oxidation which limits their technological value. We present an Escherichia coli Hyd-1 amino acid exchange that apparently causes the catalytic rate of H2 production to double but does not impact the O2 tolerance.

Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH., (© 2016 Authors.)

Cephalopods possess remarkable camouflage capabilities, which are enabled by their complex skin structure and sophisticated nervous system. Such unique characteristics have in turn inspired the design of novel functional materials and devices. Within this context, recent studies have focused on investigating the self-assembly, optical, and electrical properties of reflectin, a protein that plays a key role in cephalopod structural coloration. Herein, we report the discovery that reflectin constitutes an effective material for the growth of human neural stem/progenitor cells. Our findings may hold relevance both for understanding cephalopod embryogenesis and for developing improved protein-based bioelectronic devices.

Neural stem and progenitor cell (NSPC) fate is strongly influenced by mechanotransduction as modulation of substrate stiffness affects lineage choice. Other types of mechanical stimuli, such as stretch (tensile strain), occur during CNS development and trauma, but their consequences for NSPC differentiation have not been reported. We delivered a 10% static equibiaxial stretch to NSPCs and examined effects on differentiation. We found static stretch specifically impacts NSPC differentiation into oligodendrocytes, but not neurons or astrocytes, and this effect is dependent on particular extracellular matrix (ECM)-integrin linkages. Generation of oligodendrocytes from NSPCs was reduced on laminin, an outcome likely mediated by the α6 laminin-binding integrin, whereas similar effects were not observed for NSPCs on fibronectin. Our data demonstrate a direct role for tensile strain in dictating the lineage choice of NSPCs and indicate the dependence of this phenomenon on specific substrate materials, which should be taken into account for the design of biomaterials for NSPC transplantation.

Dielectrophoresis (DEP) has proven an invaluable tool for the enrichment of populations of stem and progenitor cells owing to its ability to sort cells in a label-free manner and its biological safety. However, DEP separation devices have suffered from a low throughput preventing researchers from undertaking studies requiring large numbers of cells, such as needed for cell transplantation. We developed a microfluidic device designed for the enrichment of stem and progenitor cell populations that sorts cells at a rate of 150,000 cells/h, corresponding to an improvement in the throughput achieved with our previous device designs by over an order of magnitude. This advancement, coupled with data showing the DEP-sorted cells retain their enrichment and differentiation capacity when expanded in culture for periods of up to 2 weeks, provides sufficient throughput and cell numbers to enable a wider variety of experiments with enriched stem and progenitor cell populations. Furthermore, the sorting devices presented here provide ease of setup and operation, a simple fabrication process, and a low associated cost to use that makes them more amenable for use in common biological research laboratories. To our knowledge, this work represents the first to enrich stem cells and expand them in culture to generate transplantation-scale numbers of differentiation-competent cells using DEP.

Neural stem cells are multipotent cells with the ability to differentiate into neurons, astrocytes, and oligodendrocytes. Lineage specification is strongly sensitive to the mechanical properties of the cellular environment. However, molecular pathways transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification remain unclear. We found that the mechanically gated ion channel Piezo1 is expressed by brain-derived human neural stem/progenitor cells and is responsible for a mechanically induced ionic current. Piezo1 activity triggered by traction forces elicited influx of Ca(2+), a known modulator of differentiation, in a substrate-stiffness-dependent manner. Inhibition of channel activity by the pharmacological inhibitor GsMTx-4 or by siRNA-mediated Piezo1 knockdown suppressed neurogenesis and enhanced astrogenesis. Piezo1 knockdown also reduced the nuclear localization of the mechanoreactive transcriptional coactivator Yes-associated protein. We propose that the mechanically gated ion channel Piezo1 is an important determinant of mechanosensitive lineage choice in neural stem cells and may play similar roles in other multipotent stem cells.

We describe a method based on fluorescence-lifetime imaging microscopy (FLIM) to assess the fluidity of various membranes in neuronal cells at different stages of development [day 12 (E12) and day 16 (E16) of gestation]. For the FLIM measurements, we use the Laurdan probe which is commonly used to assess membrane water penetration in model and in biological membranes using spectral information. Using the FLIM approach, we build a fluidity scale based on calibration with model systems of different lipid compositions. In neuronal cells, we found a marked difference in fluidity between the internal membranes and the plasma membrane, being the plasma membrane the less fluid. However, we found no significant differences between the two cell groups, E12 and E16. Comparison with NIH3T3 cells shows that the plasma membranes of E12 and E16 cells are significantly more fluid than the plasma membrane of the cancer cells.

Neural stem and progenitor cells (NSPCs) are heterogeneous populations of self-renewing stem cells and more committed progenitors that differentiate into neurons, astrocytes, and oligodendrocytes. Accurately identifying and characterizing the different progenitor cells in this lineage has continued to be a challenge for the field. We found previously that populations of NSPCs with more neurogenic progenitors (NPs) can be distinguished from those with more astrogenic progenitors (APs) by their inherent biophysical properties, specifically the electrophysiological property of whole cell membrane capacitance, which we characterized with dielectrophoresis (DEP). Here, we hypothesize that inherent electrophysiological properties are sufficient to define NPs and APs and test this by determining whether isolation of cells solely by these properties specifically separates NPs and APs. We found NPs and APs are enriched in distinct fractions after separation by electrophysiological properties using DEP. A single round of DEP isolation provided greater NP enrichment than sorting with PSA-NCAM, which is considered an NP marker. Additionally, cell surface N-linked glycosylation was found to significantly affect cell fate-specific electrophysiological properties, providing a molecular basis for the cell membrane characteristics. Inherent plasma membrane biophysical properties are thus sufficient to define progenitor cells of differing fate potential in the neural lineage, can be used to specifically isolate these cells, and are linked to patterns of glycosylation on the cell surface., (© AlphaMed Press.)

The integration of microscale engineering, microfluidics, and AC electrokinetics such as dielectrophoresis has generated novel microsystems that enable quantitative analysis of cellular phenotype, function, and physiology. These systems are increasingly being used to assess diverse cell types, such as stem cells, so it becomes critical to thoroughly evaluate whether the systems themselves impact cell function. For example, engineered microsystems have been utilized to investigate neural stem/progenitor cells (NSPCs), which are of interest due to their potential to treat CNS disease and injury. Analysis by dielectrophoresis (DEP) microsystems determined that unlabeled NSPCs with distinct fate potential have previously unrecognized distinguishing electrophysiological characteristics, suggesting that NSPCs could be isolated by DEP microsystems without the use of cell type specific labels. To gauge the potential impact of DEP sorting on NSPCs, we investigated whether electric field exposure of varying times affected survival, proliferation, or fate potential of NSPCs in suspension. We found short-term DEP exposure (1 min or less) had no effect on NSPC survival, proliferation, or fate potential revealed by differentiation. Moreover, NSPC proliferation (measured by DNA synthesis and cell cycle kinetics) and fate potential were not altered by any length of DEP exposure (up to 30 min). However, lengthy exposure (>5 min) to frequencies near the crossover frequency (50-100 kHz) led to decreased survival of NSPCs (maximum ∼30% cell loss after 30 min). Based on experimental observations and mathematical simulations of cells in suspension, we find that frequencies near the crossover frequency generate an induced transmembrane potential that results in cell swelling and rupture. This is in contrast to the case for adherent cells since negative DEP frequencies lower than the crossover frequency generate the highest induced transmembrane potential and damage for these cells. We clarify contrasting effects of DEP on adherent and suspended cells, which are related to the cell position within the electric field and the strength of the electric field at specific distances from the electrodes. Modeling of electrode configurations predicts optimal designs to induce cell movement by DEP while limiting the induced transmembrane potential. We find DEP electric fields are not harmful to stem cells in suspension at short exposure times, thus providing a basis for developing DEP-based applications for stem cells.

We present an automated dielectrophoretic assisted cell sorting (DACS) device for dielectric characterization and isolation of neural cells. Dielectrophoretic (DEP) principles are often used to develop cell sorting techniques. Here we report the first statistically significant neuronal sorting using DACS to enrich neurons from a heterogeneous population of mouse derived neural stem/progenitor cells (NSPCs) and neurons. We also study the dielectric dispersions within a heterogeneous cell population using a Monte-Carlo (MC) simulation. This simulation model explains the trapping behavior of populations as a function of frequency and predicts sorting efficiencies. The platform consists of a DEP electrode array with three multiplexed trapping regions that can be independently activated at different frequencies. A novel microfluidic manifold enables cell sorting by trapping and collecting cells at discrete frequency bands rather than single frequencies. The device is used to first determine the percentage of cells trapped at these frequency bands. With this characterization and the MC simulation we choose the optimal parameters for neuronal sorting. Cell sorting experiments presented achieve a 1.4-fold neuronal enrichment as predicted by our model.

The neural degeneration caused by spinal cord injury leaves a cavity at the injury site that greatly inhibits repair. One approach to promoting repair is to fill the cavity with a scaffold to limit further damage and encourage regrowth. Injectable materials are advantageous scaffolds because they can be placed as a liquid in the lesion site then form a solid in vivo that precisely matches the contours of the lesion. Fibrin is one type of injectable scaffold, but risk of infection from blood borne pathogens has limited its use. We investigated the potential utility of salmon fibrin as an injectable scaffold to treat spinal cord injury since it lacks mammalian infectious agents and encourages greater neuronal extension in vitro than mammalian fibrin or Matrigel®, another injectable material. Female rats received a T9 dorsal hemisection injury and were treated with either salmon or human fibrin at the time of injury while a third group served as untreated controls. Locomotor function was assessed using the BBB scale, bladder function was analyzed by measuring residual urine, and sensory responses were tested by mechanical stimulation (von Frey hairs). Histological analyses quantified the glial scar, lesion volume, and serotonergic fiber density. Rats that received salmon fibrin exhibited significantly improved recovery of both locomotor and bladder function and a greater density of serotonergic innervation caudal to the lesion site without exacerbation of pain. Rats treated with salmon fibrin also exhibited less autophagia than those treated with human fibrin, potentially pointing to amelioration of sensory dysfunction. Glial scar formation and lesion size did not differ significantly among groups. The pattern and timing of salmon fibrin's effects suggest that it acts on neuronal populations but not by stimulating long tract regeneration. Salmon fibrin clearly has properties distinct from those of mammalian fibrin and is a beneficial injectable scaffold for treatment of spinal cord injury., (Copyright © 2012 Elsevier Inc. All rights reserved.)

This study was undertaken as part of the NIH "Facilities of Research-Spinal Cord Injury" project to support independent replication of published studies. Here, we repeated a study reporting that a combinatorial treatment with transplants of Schwann cells, systemic delivery of Rolipram to enhance cyclic AMP levels, and intra-spinal injections of dibutyryl cyclic AMP enhanced locomotor recovery in rats after contusion injuries at the thoracic level. We compared the following experimental groups: 1) rats that received Schwann cell transplants, systemic Rolipram, and injections of db-cyclic AMP (the combined treatment group that showed the greatest improvement in function); 2) rats that received Schwann cell transplants only and implantation of empty pumps as control; 3) rats that received Rolipram only and implantation of empty pumps as control, and 4) control rats that received no treatment other than the injection of DMEM into the spinal cord and implantation of empty pumps. The principal findings reported in Pearse et al. were not replicated in that the combined treatment group did not exhibit greater recovery on any of the measures, although the group that received Schwann cells only did exhibit enhanced recovery on several of the outcome measures. The failure of the combined treatment may be due in part to less successful engraftment of Schwann cells in our study vs. Pearse et al. Issues relating to failures to replicate, especially when effect size is small, are discussed., (Copyright © 2010. Published by Elsevier Inc.)

In the stem cell field there is a lack of non invasive and fast methods to identify stem cell's metabolic state, differentiation state and cell-lineage commitment. Here we describe a label-free method that uses NADH as an intrinsic biomarker and the Phasor approach to Fluorescence Lifetime microscopy to measure the metabolic fingerprint of cells. We show that different metabolic states are related to different cell differentiation stages and to stem cell bias to neuronal and glial fate, prior the expression of lineage markers. Our data demonstrate that the NADH FLIM signature distinguishes non-invasively neurons from undifferentiated neural progenitor and stem cells (NPSCs) at two different developmental stages (E12 and E16). NPSCs follow a metabolic trajectory from a glycolytic phenotype to an oxidative phosphorylation phenotype through different stages of differentiation. NSPCs are characterized by high free/bound NADH ratio, while differentiated neurons are characterized by low free/bound NADH ratio. We demonstrate that the metabolic signature of NPSCs correlates with their differentiation potential, showing that neuronal progenitors and glial progenitors have a different free/bound NADH ratio. Reducing conditions in NPSCs correlates with their neurogenic potential, while oxidative conditions correlate with glial potential. For the first time we show that FLIM NADH metabolic fingerprint provides a novel, and quantitative measure of stem cell potential and a label-free and non-invasive means to identify neuron- or glial- biased progenitors.

Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%-50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3' untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation.

Background: Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers., Methodology/principal Findings: We used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates., Conclusions/significance: We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.

Modeling the in vivo microenvironment typically involves placing cells in a three-dimensional (3D) extracellular matrix (ECM) in physiologically relevant context with respect to other cells. The mechanical and chemical features of 3D microenvironments play important roles in tissue engineering, tumor growth and metastasis, and in defining stem cell niches, and it is increasingly recognized that cells behave much differently when surrounded by a 3D ECM than when anchored to a 2D substrate. To create microenvironments that more closely mimic in vivo settings, here we describe a novel microfluidic device that allows multiple discrete constructs of 3D cell-laden hydrogels to be patterned in a sequence of simple steps. The microfluidic platform allows for real-time imaging of the interactions between multiple cell types exposed to both autocrine and paracrine signaling molecules, all within a 3D ECM environment. Detailed modeling determined that surface tension, hydrophobic interactions, and spatial geometry were important factors in containing the gels within distinct separate channels during the filling process. This allowed us to pattern multiple gel types side-by-side and pattern 3D gels spatially with tight dimensional control. Cells embedded in gels could be patterned by culturing MDA-MB-231 metastatic breast cancer cells and RAW 264.1 macrophage cells within distinct collagen type I and Matrigel ECM environments, respectively. Over a 7 day culture experiment, RAW cells invaded into neighboring gels containing MDA-MB-231 cells, but not into gels lacking cells. These studies demonstrate the versatility and potential of this new microfluidic platform to engineer 3D microscale architectures to investigate cell-cell and cell-matrix interactions.

Polymeric scaffolds formed from synthetic or natural materials have many applications in tissue engineering and medicine, and multiple material properties need to be optimized for specific applications. Recent studies have emphasized the importance of the scaffolds' mechanical properties to support specific cellular responses in addition to considerations of biochemical interactions, material transport, immunogenicity, and other factors that determine biocompatibility. Fibrin gels formed from purified fibrinogen and thrombin, the final two reactants in the blood coagulation cascade, have long been shown to be effective in wound healing and supporting the growth of cells in vitro and in vivo. Fibrin, even without additional growth factors or other components has potential for use in neuronal wound healing in part because of its mechanical compliance that supports the growth of neurons without activation of glial proliferation. This review summarizes issues related to the use of fibrin gels in neuronal cell contexts, with an emphasis on issues of immunogenicity, and considers the potential advantages and disadvantages of fibrin prepared from non-mammalian sources.

This paper presents a novel design and separation strategy for lateral flow-through separation of cells/particles in microfluidics by dual frequency coupled dielectrophoresis (DEP) forces enabled by vertical interdigitated electrodes embedded in the channel sidewalls. Unlike field-flow-fractionation-DEP separations in microfluidics, which utilize planar electrodes on the microchannel floor to generate a DEP force to balance the gravitational force and separate objects at different height locations, lateral separation is enabled by sidewall interdigitated electrodes that are used to generate non-uniform electric fields and balanced DEP forces along the width of the microchannel. In the current design, two separate AC electric fields are applied to two sets of independent interdigitated electrode arrays fabricated in the sidewalls of the microchannel to generate differential DEP forces that act on the cells/particles flowing through. Individual particles (cells or beads) will experience DEP forces differently due to the difference in their dielectric properties. The balance of the differential DEP forces from the electrode arrays will position dissimilar particles at distinct equilibrium planes across the width of the channel. When coupled with fluid flow, this results in lateral separation along the width of the microchannel and the separated particles can thus be automatically directed into branched channel outlets leading to different reservoirs for downstream processing. In this paper, we present the design and analysis of lateral separation enabled by dual frequency coupled DEP, and cell/bead and cell/cell separations are demonstrated with this lateral separation strategy. With vertical interdigitated electrodes on the sidewall, the height of the microchannel can be increased without losing the electric field strength in contrast to other multiple frequency DEP devices with planar electrodes. As a result, populations of cells can be separated simultaneously instead of one by one to enable high-throughput sorting microfluidic devices.

The relatively new field of stem cell biology is hampered by a lack of sufficient means to accurately determine the phenotype of cells. Cell-type-specific markers, such as cell surface proteins used for flow cytometry or fluorescence-activated cell sorting, are limited and often recognize multiple members of a stem cell lineage. We sought to develop a complementary approach that would be less dependent on the identification of particular markers for the subpopulations of cells and would instead measure their overall character. We tested whether a microfluidic system using dielectrophoresis (DEP), which induces a frequency-dependent dipole in cells, would be useful for characterizing stem cells and their differentiated progeny. We found that populations of mouse neural stem/precursor cells (NSPCs), differentiated neurons, and differentiated astrocytes had different dielectric properties revealed by DEP. By isolating NSPCs from developmental ages at which they are more likely to generate neurons, or astrocytes, we were able to show that a shift in dielectric property reflecting their fate bias precedes detectable marker expression in these cells and identifies specific progenitor populations. In addition, experimental data and mathematical modeling suggest that DEP curve parameters can indicate cell heterogeneity in mixed cultures. These findings provide evidence for a whole cell property that reflects stem cell fate bias and establish DEP as a tool with unique capabilities for interrogating, characterizing, and sorting stem cells.

The earliest step in creating the cerebral cortex is the specification of neuroepithelium to a cortical fate. Using mouse genetic mosaics and timed inactivations, we demonstrated that Lhx2 acts as a classic selector gene and essential intrinsic determinant of cortical identity. Lhx2 selector activity is restricted to an early critical period when stem cells comprise the cortical neuroepithelium, where it acts cell-autonomously to specify cortical identity and suppress alternative fates in a spatially dependent manner. Laterally, Lhx2 null cells adopt antihem identity, whereas medially they become cortical hem cells, which can induce and organize ectopic hippocampal fields. In addition to providing functional evidence for Lhx2 selector activity, these findings show that the cortical hem is a hippocampal organizer.

The homeostasis of both cornea and hair follicles depends on a constant supply of progeny cells produced by populations of keratin (K) 14-expressing stem cells localized in specific niches. To investigate the potential role of Co-factors of LIM domains (Clims) in epithelial tissues, we generated transgenic mice expressing a dominant-negative Clim molecule (DN-Clim) under the control of the K14 promoter. As expected, the K14 promoter directed high level expression of the transgene to the basal cells of cornea and epidermis, as well as the outer root sheath of hair follicles. In corneal epithelium, the transgene expression causes decreased expression of adhesion molecule BP180 and defective hemidesmosomes, leading to detachment of corneal epithelium from the underlying stroma, which in turn causes blisters, wounds and an inflammatory response. After a period of epithelial thinning, the corneal epithelium undergoes differentiation to an epidermis-like structure. The K14-DN-Clim mice also develop progressive hair loss due to dysfunctional hair follicles that fail to generate hair shafts. The number of hair follicle stem cells is decreased by at least 60% in K14-DN-Clim mice, indicating that Clims are required for hair follicle stem cell maintenance. In addition, Clim2 interacts with Lhx2 in vivo, suggesting that Clim2 is an essential co-factor for the LIM homeodomain factor Lhx2, which was previously shown to play a role in hair follicle stem cell maintenance. Together, these data indicate that Clim proteins play important roles in the homeostasis of corneal epithelium and hair follicles.

A novel dielectrophoresis switching with vertical electrodes in the sidewall of microchannels for multiplexed switching of objects has been designed, fabricated and tested. With appropriate electrode design, lateral DEP force can be generated so that one can dynamically position particulates along the width of the channel. A set of interdigitated electrodes in the sidewall of the microchannels is used for the generation of non-uniform electrical fields to generate negative DEP forces that repel beads/cells from the sidewalls. A countering DEP force is generated from another set of electrodes patterned on the opposing sidewall. These lateral negative DEP forces can be adjusted by the voltage and frequency applied. By manipulating the coupled DEP forces, the particles flowing through the microchannel can be positioned at different equilibrium points along the width direction and continue to flow into different outlet channels. Experimental results for switching biological cells and polystyrene microbeads to multiple outlets (up to 5) have been achieved. This novel particle switching technique can be integrated with other particle detection components to enable microfluidic flow cytometry systems.

Three-dimensional fibrin matrices have been used as cellular substrates in vitro and as bridging materials for central nervous system repair. Cells can be embedded within fibrin gels since the polymerization process is non-toxic, making fibrin an attractive scaffold for transplanted cells. Most studies have utilized fibrin prepared from human or bovine blood proteins. However, fish fibrin may be well suited for neuronal growth since fish undergo remarkable central nervous system regeneration and molecules implicated in this process are present in fibrin. We assessed the growth of mammalian central nervous system neurons in bovine, human, and salmon fibrin and found that salmon fibrin gels encouraged the greatest degree of neurite (dendrite and axon) growth and were the most resistant to degradation by cellular proteases. The neurite growth-promoting effect was not due to the thrombin used to polymerize the gels nor to any copurifying plasminogen. Copurified fibronectin partially accounted for the effect on neurites, and blockade of fibrinogen/fibrin-binding integrins markedly decreased neurite growth. Anion exchange chromatography revealed different elution profiles for salmon and mammalian fibrinogens. These data demonstrate that salmon fibrin encourages the growth of neurites from mammalian neurons and suggest that salmon fibrin may be a beneficial scaffold for neuronal regrowth after CNS injury.

Deciphering the factors that regulate human neural stem cells will greatly aid in their use as models of development and as therapeutic agents. The extracellular matrix (ECM) is a component of stem cell niches in vivo and regulates multiple functions in diverse cell types, yet little is known about its effects on human neural stem/precursor cells (NSPCs). We therefore plated human NSPCs on four different substrates (poly-L-ornithine, fibronectin, laminin, and matrigel) and compared their responses with those of mouse NSPCs. Compared with the other substrates, laminin matrices enhanced NSPC migration, expansion, differentiation into neurons and astrocytes, and elongation of neurites from NSPC-derived neurons. Laminin had a similar spectrum of effects on both human and mouse cells, highlighting the evolutionary conservation of NSPC regulation by this component of the ECM. Flow cytometry revealed that human NSPCs express on their cell surfaces the laminin-binding integrins alpha3, alpha6, alpha7, beta1, and beta4, and function-blocking antibodies to the alpha6 subunit confirmed a role for integrins in laminin-dependent migration of human NSPCs. These results define laminin and its integrin receptors as key regulators of human NSPCs.

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.

The morphology and cytoskeletal structure of fibroblasts, endothelial cells, and neutrophils are documented for cells cultured on surfaces with stiffness ranging from 2 to 55,000 Pa that have been laminated with fibronectin or collagen as adhesive ligand. When grown in sparse culture with no cell-cell contacts, fibroblasts and endothelial cells show an abrupt change in spread area that occurs at a stiffness range around 3,000 Pa. No actin stress fibers are seen in fibroblasts on soft surfaces, and the appearance of stress fibers is abrupt and complete at a stiffness range coincident with that at which they spread. Upregulation of alpha5 integrin also occurs in the same stiffness range, but exogenous expression of alpha5 integrin is not sufficient to cause cell spreading on soft surfaces. Neutrophils, in contrast, show no dependence of either resting shape or ability to spread after activation when cultured on surfaces as soft as 2 Pa compared to glass. The shape and cytoskeletal differences evident in single cells on soft compared to hard substrates are eliminated when fibroblasts or endothelial cells make cell-cell contact. These results support the hypothesis that mechanical factors impact different cell types in fundamentally different ways, and can trigger specific changes similar to those stimulated by soluble ligands., (Copyright 2004 Wiley-Liss, Inc.)

Determining whether a tumor exhibits microsatellite instability (MSI) is useful in identifying patients with hereditary non-polyposis colorectal cancer and sporadic gastrointestinal cancers with defective DNA mismatch repair (MMR). The assessment of MSI status aids in establishing a clinical prognosis and may be predictive of tumor response to chemotherapy. A reference panel of five markers was suggested for MSI analysis by a National Cancer Institute (NCI) workshop in 1997 that has helped to standardize testing. But this panel of markers has limitations resulting from the inclusion of dinucleotide markers, which are less sensitive and specific for detection of tumors with MMR deficiencies compared to other types of markers that are currently available. This study demonstrates that mononucleotides are the most sensitive and specific markers for detection of tumors with defects in MMR and identifies an optimal panel of markers for detection of MSI-H tumors. A set of 266 mono-, di-, tetra- and penta-nucleotide repeat microsatellite markers were used to screen for MSI in colorectal tumors. The best markers for detection of MSI-H tumors were selected for a MSI Multiplex System, which included five mononucleotide markers: BAT-25, BAT-26, NR-21, NR-24 and MONO-27. In addition, two pentanucleotide markers were added to identify sample mix-ups and/or contamination. We classified 153 colorectal tumors using the new MSI Multiplex System and compared the results to those obtained with a panel of 10 microsatellite markers combined with immunohistochemical (IHC) analysis. We observed 99% concordance between the two methods with nearly 100% accuracy in detection of MSI-H tumors. Approximately 5% of the MSI-H tumors had normal levels of four MMR proteins and as a result would have been misclassified based solely on IHC analysis, emphasizing the importance of performing MSI testing. The new MSI Multiplex System offers several distinct advantages over other methods of MSI testing in that it is both extremely sensitive and specific and amenable to high-throughput analysis. The MSI Multiplex System meets the new recommendations proposed at the recent 2002 NCI workshop on HNPCC and MSI testing and overcomes problems inherent to the original five-marker panel. The use of a single multiplex fluorescent MSI assay reduces the time and costs involved in MSI testing with increased reliability and accuracy and thus should facilitate widespread screening for microsatellite instability in tumors of patients with gastrointestinal cancers.

A clamp-and-ratchet microstructure based on poly crystalline silicon (polysilicon) microelectromechanical systems (MEMS) technology has been designed to exert mechanical tension along radial glial processes between groups of neural stem cells to study the effect of tension on cerebral cortex neurogenesis. FEA analysis shows that the design should not fail under expected loading conditions. Preliminary studies show that embryonic brain tissue survives under tension for at least six days. Neurospheres have been successfully cultured on Poly(dimethylsiloxane) (PDMS) for eight days and exhibit radial extensions which appear to support neuronal migration. Stretching the PDMS using the clamp and ratchet will produce tension in these radial extensions which may modulate neuronal migration, a key process in cerebral cortex development.

The mechanical properties of substrates underlying cells can have profound effects on cell structure and function. To examine the effect of substrate deformability on neuronal cell growth, protein-laminated polyacrylamide gels were prepared with differing amounts of bisacrylamide to generate substrates of varying deformability with elastic moduli ranging from 500 to 5500 dyne/cm. Mouse spinal cord primary neuronal cells were plated on the gels and allowed to grow and extend neurites for several weeks in culture. While neurons grew well on the gels, glia, which are normally co-cultured with the neurons, did not survive on these deformable substrates even though the chemical environment was permissive for their growth. Substrate flexibility also had a significant effect on neurite branching. Neurons grown on softer substrates formed more than three times as many branches as those grown on stiffer gels. These results show that mechanical properties of the substrate specifically direct the formation of neurite branches, which are critical for appropriate synaptic connections during development and regeneration.

The Arp2/3 complex and filamin A (FLNa) branch actin filaments. To define the role of these actin-binding proteins in cellular actin architecture, we compared the morphology of FLNa-deficient human melanoma (M2) cells and three stable derivatives of these cells expressing normal FLNa concentrations. All the cell lines contain similar amounts of the Arp2/3 complex. Serum addition causes serum-starved M2 cells to extend flat protrusions transiently; thereafter, the protrusions turn into spherical blebs and the cells do not crawl. The short-lived lamellae of M2 cells contain a dense mat of long actin filaments in contrast to a more three-dimensional orthogonal network of shorter actin filaments in lamellae of identically treated FLNa-expressing cells capable of translational locomotion. FLNa-specific antibodies localize throughout the leading lamellae of these cells at junctions between orthogonally intersecting actin filaments. Arp2/3 complex-specific antibodies stain diffusely and label a few, although not the same, actin filament overlap sites as FLNa antibody. We conclude that FLNa is essential in cells that express it for stabilizing orthogonal actin networks suitable for locomotion. Contrary to some proposals, Arp2/3 complex-mediated branching of actin alone is insufficient for establishing an orthogonal actin organization or maintaining mechanical stability at the leading edge.

The recent demonstration that the fast axonal transport motors kinesin and dynein participate in axonal transport of neurofilaments--known to undergo slow transport--supports and extends recent studies indicating that some neurofilaments exhibit alternating bursts of fast axonal transport interspersed with periods of non-motility. In addition, these findings unify both certain aspects of axonal transport and neurofilament biology. We discuss these data herein in the context of both older and more recent studies of neurofilament dynamics.

LIS1, a microtubule-associated protein, is required for neuronal migration, but the precise mechanism of LIS1 function is unknown. We identified a LIS1 interacting protein encoded by a mouse homolog of NUDE, a nuclear distribution gene in A. nidulans and a multicopy suppressor of the LIS1 homolog, NUDF. mNudE is located in the centrosome or microtubule organizing center (MTOC), and interacts with six different centrosomal proteins. Overexpression of mNudE dissociates gamma-tubulin from the centrosome and disrupts microtubule organization. Missense mutations that disrupt LIS1 function block LIS1-mNudE binding. Moreover, misexpression of the LIS1 binding domain of mNudE in Xenopus embryos disrupts the architecture and lamination of the CNS. Thus, LIS1-mNudE interactions may regulate neuronal migration through dynamic reorganization of the MTOC.