Real time imaging of human progenitor neurogenesis.

Human neural progenitors are increasingly being employed in drug screens and emerging cell therapies targeted towards neurological disorders where neurogenesis is thought to play a key role including developmental disorders, Alzheimer's disease, and depression. Key to the success of these applications is understanding the mechanisms by which neurons arise. Our understanding of development can provide some guidance but since little is known about the specifics of human neural development and the requirement that cultures be expanded in vitro prior to use, it is unclear whether neural progenitors obey the same developmental mechanisms that exist in vivo. In previous studies we have shown that progenitors derived from fetal cortex can be cultured for many weeks in vitro as undifferentiated neurospheres and then induced to undergo neurogenesis by removing mitogens and exposing them to supportive substrates. Here we use live time lapse imaging and immunocytochemical analysis to show that neural progenitors use developmental mechanisms to generate neurons. Cells with morphologies and marker profiles consistent with radial glia and recently described outer radial glia divide asymmetrically and symmetrically to generate multipolar intermediate progenitors, a portion of which express ASCL1. These multipolar intermediate progenitors subsequently divide symmetrically to produce CTIP2(+) neurons. This 3-cell neurogenic scheme echoes observations in rodents in vivo and in human fetal slice cultures in vitro, providing evidence that hNPCs represent a renewable and robust in vitro assay system to explore mechanisms of human neurogenesis without the continual need for fresh primary human fetal tissue. Knowledge provided by this and future explorations of human neural progenitor neurogenesis will help maximize the safety and efficacy of new stem cell therapies by providing an understanding of how to generate physiologically-relevant cell types that maintain their identities when placed in diagnostic or transplantation environments.

A neuron-benign microfluidic gradient generator for studying the response of mammalian neurons towards axon guidance factors.

Investigation of biochemical cues in isolation or in combinations in cell culture systems is crucial for unraveling the mechanisms that govern neural development and repair. The most widely used experimental paradigms that elicit axon guidance in vitro utilize as the source of the gradient a pulsatile pipette, transfected cells, or a loaded gel, producing time-varying gradients of poor reproducibility which are not well suited for studying slow-growing mammalian cells. Although microfluidic device design have allowed for generating stable, complex gradients of diffusible molecules, the flow-induced shear forces in a microchannel has made it impossible to maintain viable mammalian neuronal cultures for sufficiently long times. In this paper, we describe axonal responses of mouse cortical neurons in a "neuron-benign" gradient-generator device based on an open chamber that can establish highly stable gradients of diffusible molecules for at least 6 h with negligible shear stress, and also allows the neurons to thrive for at least 2 weeks. Except for the period when the gradient is on, the cells in the gradient are under the same conditions as the cells on the control surfaces, which ensure a consistent set of micro-environmental variables. The gradient stability and uniformity over the cell culture surface achieved by the device, together with our software platform for acquiring, post-processing and quantitatively analyzing the large number of images allowed us to extract valuable information even from small datasets. We report a directed response of primary mammalian neurons (from E14 embryonic mice cortex) to a diffusible gradient of netrin in vitro. We infer from our studies that a large majority (∼73%) of the neurons that extend axons during the gradient application grow towards the netrin source, and our data analysis also indicates that netrin acts as a growth factor for this same population of neurons.

A new method for studying gradient-induced neutrophil desensitization based on an open microfluidic chamber.

During inflammation neutrophils rapidly migrate to the site of tissue damage or infection by following complex gradients of bacterial peptides and host-derived chemokines. The efficiency and speed of neutrophil migration is critically dependent upon the ability of neutrophils to sense new gradients and utilize only those that provide the most direct path to the damaged or infected site. Receptor desensitization plays an important role in migration efficiency and is most commonly studied using bath application of chemotactic factor solutions instead of presenting cells with gradients analogous to those they would experience in vivo. Here we describe a new method for examining gradient-induced neutrophil desensitization using a previously-developed open-chamber microfluidic gradient generator.

Biomolecular gradients in cell culture systems.

Biomolecule gradients have been shown to play roles in a wide range of biological processes including development, inflammation, wound healing, and cancer metastasis. Elucidation of these phenomena requires the ability to expose cells to biomolecule gradients that are quantifiable, controllable, and mimic those that are present in vivo. Here we review the major biological phenomena in which biomolecule gradients are employed, traditional in vitro gradient-generating methods developed over the past 50 years, and new microfluidic devices for generating gradients. Microfluidic gradient generators offer greater levels of precision, quantitation, and spatiotemporal gradient control than traditional methods, and may greatly enhance our understanding of many biological phenomena. For each method, we outline the salient features, capabilities, and applications.

Measurement of cell migration in response to an evolving radial chemokine gradient triggered by a microvalve.

We describe a novel chemotaxis assay based on the microvalve-actuated release of a chemoattractant from a cell-free microchamber into a cell-containing microchamber. The microvalve chemotaxis device (microVCD) was placed on the stage of a conventional inverted microscope to obtain time-lapse micrographs of neutrophils migrating in a radially-symmetric evolving gradient of the chemotactic factor CXCL8/Interleukin-8. A fluorescent tracer was added to the CXCL8 solution to visualize the evolution of the gradient profile, so that at each time point the cell positions could be assigned CXCL8 concentration values. Tracking of individual neutrophils for 90 minutes showed that (a) the neutrophil migratory response is, on average, radially directed towards the CXCL8 source; (b) significant non-radial displacements occur frequently; and (c) there is considerable heterogeneity in the migration speeds and directions amongst the neutrophil population. A custom-made imaging analysis tool was used to extract measurements of migratory behavior such as speed, velocity along the gradient's radial axis, and the cosine of the turning angle as a function of CXCL8 concentration. The microVCD can be easily adapted to study the migratory behavior of cultured cells other than neutrophils.

Polyhydroxyalkanoate copolymers from forest biomass.

The potential for the use of woody biomass in poly-beta-hydroxyalkanoate (PHA) biosynthesis is reviewed. Based on previously cited work indicating incorporation of xylose or levulinic acid (LA) into PHAs by several bacterial strains, we have initiated a study for exploring bioconversion of forest resources to technically relevant copolymers. Initially, PHA was synthesized in shake-flask cultures of Burkholderia cepacia grown on 2.2% (w/v) xylose, periodically amended with varying concentrations of levulinic acid [0.07-0.67% (w/v)]. Yields of poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) [P(3HB-co-3HV)] from 1.3 to 4.2 g/l were obtained and could be modulated to contain from 1.0 to 61 mol% 3-hydroxyvalerate (3HV), as determined by 1H and 13C NMR analyses. No evidence for either the 3HB or 4HV monomers was found. Characterization of these P(3HB-co-3HV) samples, which ranged in molecular mass (viscometric, Mv) from 511-919 kDa, by differential scanning calorimetry and thermogravimetric analyses (TGA) provided data which were in agreement for previously reported P(3HB-co-3HV) copolymers. For these samples, it was noted that melting temperature (Tm) and glass transition temperature (Tg) decreased as a function of 3HVcontent, with Tm demonstrating a pseudoeutectic profile as a function of mol% 3HV content. In order to extend these findings to the use of hemicellulosic process streams as an inexpensive carbon source, a detoxification procedure involving sequential overliming and activated charcoal treatments was developed. Two such detoxified process hydrolysates (NREL CF: aspen and CESF: maple) were each fermented with appropriate LA supplementation. For the NREL CF hydrolysate-based cultures amended with 0.25-0.5% LA, P(3HB-co-3HV) yields, PHA contents (PHA as percent of dry biomass), and mol% 3HV compositions of 2.0 g/l, 40% (w/w), and 16-52 mol% were obtained, respectively. Similarly, the CESF hydrolysate-based shake-flask cultures yielded 1.6 g/l PHA, 39% (w/w) PHA contents, and 4-67 mol% 3HV compositions. These data are comparable to copolymer yields and cellular contents reported for hexose plus levulinic acid-based shake-flask cultures, as reported using Alcaligenes eutrophus and Pseudomonas putida. However, our findings presage a conceivable alternative, forestry-based biorefinery approach for the production of value-added biodegradable PHA polymers. Specifically, this review describes the current and potential utilization of lignocellulosic process streams as platform precursors to PHA polymers including hemicellulosic hydrolysates, residual cellulose-derived levulinic acid, tall oil fatty acids (Kraft pulping residual), and lignin-derived aromatics.

Automated identification of axonal growth cones in time-lapse image sequences.

The isolation and purification of axon guidance molecules has enabled in vitro studies of the effects of axon guidance molecule gradients on numerous neuronal cell types. In a typical experiment, cultured neurons are exposed to a chemotactic gradient and their growth is recorded by manual identification of the axon tip position from two or more micrographs. Detailed and statistically valid quantification of axon growth requires evaluation of a large number of neurons at closely spaced time points (e.g. using a time-lapse microscopy setup). However, manual tracing becomes increasingly impractical for recording axon growth as the number of time points and/or neurons increases. We present a software tool that automatically identifies and records the axon tip position in each phase-contrast image of a time-lapse series with minimal user involvement. The software outputs several quantitative measures of axon growth, and allows users to develop custom measurements. For, example analysis of growth velocity for a dissociated E13 mouse cortical neuron revealed frequent extension and retraction events with an average growth velocity of 0.05 +/- 0.14 microm/min. Comparison of software-identified axon tip positions with manually identified axon tip positions shows that the software's performance is indistinguishable from that of skilled human users.

Production and characterization of poly-beta-hydroxyalkanoate copolymers from Burkholderia cepacia utilizing xylose and levulinic acid.

Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (P(3HB-co-3HV)) copolymers were prepared via shake-flask fermentations of Burkholderia cepacia (formerly Pseudomonas cepacia) containing 2.2% (w/v) xylose and concentrations of levulinic acid ranging from 0.07% to 0.67% (w/v). Periodic harvest of shake-flask cultures from 48 to 92 h post-inoculation yielded 4.4-5.3 g/L of dry cell biomass, containing 42-56% (w/w) P(3HB-co-3HV), with optimal product yield occurring between 66 and 74 h. Growth and PHA accumulation enhancement were observed with concentrations of levulinic acid from 0.07 to 0.52% (w/v), producing dry cell biomass and P(3HB-co-3HV) yields of 9.5 and 4.2 g/L, respectively, at the 0.52% (w/v) concentration of levulinic acid. Representative samples were subjected to compositional analysis by 300 MHz 1H and 150 MHz 13C NMR, indicating that these random copolymers contained between 0.8 and 61 mol % 3-hydroxyvalerate (3HV). Solvent-cast film samples were characterized by differential scanning calorimetry, which demonstrated melting temperatures (Tm) to decrease in a pseudoeutectic fashion from 174.3 degrees C (0.8 mol % 3HV) to a minimum of 154.2 degrees C (25 mol % 3HV) and the glass transition temperatures (Tg) to decrease linearly from 2.1 to -11.9 degrees C as a function of increasing mol % 3HV. Thermogravimetric analysis of the copolymer series showed the temperature for onset of thermal decomposition (T(decomp)) to vary as a function of mol % 3HV from 273.4 to 225.5 degrees C. Intrinsic viscosities (eta) varied from 3.2 to 5.4 dL/g, as determined by dilute solution viscometry. Viscosity average molecular weights (Mv) of the copolymers were determined to range from 469 to 919 kDa, indicating that these P(3HB-co-3HV) copolymers are of sufficient molecular mass for commercial application.