Neurite Branching on Deformable Substrates

Neuroreport. Dec, 2002  |  Pubmed ID: 12499839

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.

Development of a MEMS Microsystem to Study the Effect of Mechanical Tension on Cerebral Cortex Neurogenesis

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2004  |  Pubmed ID: 17270809

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.

Effects of Substrate Stiffness on Cell Morphology, Cytoskeletal Structure, and Adhesion

Cell Motility and the Cytoskeleton. Jan, 2005  |  Pubmed ID: 15573414

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.

Human Neural Stem Cell Growth and Differentiation in a Gradient-generating Microfluidic Device

Lab on a Chip. Apr, 2005  |  Pubmed ID: 15791337

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.

Regulation of Human Neural Precursor Cells by Laminin and Integrins

Journal of Neuroscience Research. Apr, 2006  |  Pubmed ID: 16477652

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.

Enhanced Neurite Growth from Mammalian Neurons in Three-dimensional Salmon Fibrin Gels

Biomaterials. Apr, 2007  |  Pubmed ID: 17258313

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.

The Value of D-dimer in the Diagnosis of Acute Aortic Dissection

International Journal of Cardiology. Jun, 2007  |  Pubmed ID: 17275937

Dielectrophoresis Switching with Vertical Sidewall Electrodes for Microfluidic Flow Cytometry

Lab on a Chip. Sep, 2007  |  Pubmed ID: 17713608

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.

Co-factors of LIM Domains (Clims/Ldb/Nli) Regulate Corneal Homeostasis and Maintenance of Hair Follicle Stem Cells

Developmental Biology. Dec, 2007  |  Pubmed ID: 17991461

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.

Unique Dielectric Properties Distinguish Stem Cells and Their Differentiated Progeny

Stem Cells (Dayton, Ohio). Mar, 2008  |  Pubmed ID: 18096719

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.

Lhx2 Selector Activity Specifies Cortical Identity and Suppresses Hippocampal Organizer Fate

Science (New York, N.Y.). Jan, 2008  |  Pubmed ID: 18202285

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.

Dual Frequency Dielectrophoresis with Interdigitated Sidewall Electrodes for Microfluidic Flow-through Separation of Beads and Cells

Electrophoresis. Mar, 2009  |  Pubmed ID: 19197906

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.

Soft Materials to Treat Central Nervous System Injuries: Evaluation of the Suitability of Non-mammalian Fibrin Gels

Biochimica Et Biophysica Acta. May, 2009  |  Pubmed ID: 19344675

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.

Engineering Microscale Cellular Niches for Three-dimensional Multicellular Co-cultures

Lab on a Chip. Jun, 2009  |  Pubmed ID: 19495458

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.

Systematic Review of the Management of Incontinence and Promotion of Continence in Older People in Care Homes: Descriptive Studies with Urinary Incontinence As Primary Focus

Journal of Advanced Nursing. Feb, 2011  |  Pubmed ID: 21105895

This is a review of descriptive studies with incontinence as the primary focus in older people in care homes.

Complex and Dynamic Landscape of RNA Polyadenylation Revealed by PAS-Seq

RNA (New York, N.Y.). Apr, 2011  |  Pubmed ID: 21343387

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.

Biophysical Characteristics Reveal Neural Stem Cell Differentiation Potential

PloS One. 2011  |  Pubmed ID: 21980464

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.

Systematic Review of Descriptive Studies That Investigated Associated Factors with the Management of Incontinence in Older People in Care Homes

International Journal of Older People Nursing. Dec, 2011  |  Pubmed ID: 22182302

roe b, flanagan l, jack b, shaw c, williams k, chung a & barrett j (2011) Systematic review of descriptive studies that investigated associated factors with the management of incontinence in older people in care homes. International Journal of Older People Nursing doi: 10.1111/j.1748-3743.2011.00300.x Background.  Incontinence is prevalent among older populations and residents in care homes. Aim and objectives.  This paper is a review of descriptive studies that investigated associated factors related to managing urinary incontinence in older people in care homes. A systematic literature review was undertaken. MEDLINE and CINHAL were searched from 1996-2010 using the Cochrane Incontinence Review Group search strings for urinary and faecal incontinence including all research designs. Results.  Sixteen studies were identified that reported on associated factors related to comorbidities, management preferences, policies, staff views and knowledge or methodological studies. Non-invasive methods involving toileting and use of pads were common management approaches. No studies aimed at maintaining continence were identified. Conclusions.  Factors associated with incontinence need to also be considered when planning and managing care for individuals, and developing and designing systems of care within care homes. Further study in care home populations to change or inform practice and provide effective care is warranted. Preventive studies that maintain continence are required. Implications for practice.  Older people and their families should be involved with decisions regarding their preferred care, goals, management and outcomes for managing incontinence, promoting or maintaining continence.

A Re-assessment of a Combinatorial Treatment Involving Schwann Cell Transplants and Elevation of Cyclic AMP on Recovery of Motor Function Following Thoracic Spinal Cord Injury in Rats

Experimental Neurology. Feb, 2012  |  Pubmed ID: 21195070

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.