Lelkes Lab Abstracts:

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• Honesto Poblete

Title: In Vivo Model of Engineered, Vascularized Murine Fetal Lung Constructs
Abstract: The repair of dysfunctional lungs with engineered functional lung tissue might be beneficial for treating many pediatric and adult pulmonary disease states. We recently assembled in vitro, mixed populations of isolated murine fetal pulmonary cells into 3-D tissue-like constructs which morphologically resembled alveolar forming units (AFUs) lined with type II pneumocytes. We hypothesized that an in vivo model might be more suitable for creating fetal pulmonary cell constructs with invading blood vessels providing the essential nutrients and the capillary-alveolar interface necessary for gas exchange. Mixed populations of pulmonary cells isolated from d17 mouse fetuses were admixed with liquid Matrigel™ (murine basement membrane preparation) in the absence or presence of basic fibroblast growth factor (bFGF), injected into the subcutaneous layer of the anterior abdominal wall of C57BL/6 mice and allowed to solidify. The plugs were harvested at 1, 2 and 3 week intervals. Vascularization of the plugs was ascertained by tail vein injection with FITC-dextran at harvest and fluorescent microscopy of harvested plugs. Histological sections of the plugs were stained with H&E, (Griffonia simplifolica isotype I 4B lectin (a murine endothelial marker) and antibodies against vimentin, cytokeratin and surfactant protein C. All harvested plugs contained AFUs some of which exhibited branching morphogenesis. Patent blood vessels invaded the Matrigel™ plugs in the absence of bFGF. bFGF significantly enhanced neovascularization with evidence for close apposition of capillaries and AFUs suggesting the possibility for vascular-AFU interactions. Taken together, we present here a first in vivo model of engineered lung constructs with bFGF-enhanced vascularization.
 

• Donnie Simmons

Depressed immune function is a well documented effect of spaceflight. Prior studies, both in-flight, and in ground-based microgravity analogs, such as Rotating Wall Vessel (RWV) bioreactors, have demonstrated that T lymphocytes do not proliferate or express activation markers in response to mitogenic stimuli in microgravity culture. The purpose of this study was to investigate the kinetics of RWV-induced T lymphocyte inhibition by monitoring the cells’ ability to become activated at 1-g following culture in the RWV. Murine splenocytes (containing 20-% T lymphocytes) were stimulated with concanavalin A (Con A) and cultured for up to 24-hrs in the RWV before being allowed to “recover” for up to 72-hrs at 1-g in the continued presence of Con A. Following recovery the cells were assayed for proliferation and expression of phenotype and activation markers. Our results indicate that T cells were unaffected by up to 8-hrs of culture in the RWV. However, after 12- and 16-hrs of culture in the RWV, the cells’ ability to proliferate was reduced by 33- and 50-% respectively, and by 24-hrs, exposure to simulated microgravity completely suppressed the cells’ ability to proliferate. This effect appears to be specifically due to inhibition of the proliferative mechanism, as decreased proliferation did not correlate with a loss of responding cells or induction of upstream events, such as expression of activation markers, Taken together these results delineate a window of time between 8- and 24-hrs during which simulated microgravity specifically affects the cells’ ability to proliferate in response to mitogen.

• Prakash Manley

NOVEL IMAGING SYSTEM TO MONITOR AGGREGATION KINETICS AND TRAJECTORIES OF CELLS SUSPENDED IN RWV BIOREACTORS

Rotating Wall Vessel Bioreactors (RWVs) constitute a dynamic suspension culture venue uniquely suited for tissue engineering applications. Quantitative real-time assessment of the kinetics and extent of cell-cell aggregation in RWVs, not available to date, can yield mechanistic information about the crucial initial steps leading from individual cells to tissues. We designed a novel imaging system, in which fluorescently labeled cells/particles, suspended in a High Aspect Ratio Vessel (HARV), are illuminated laterally by an excitation laser-beam. Fluorescence emission is recorded using a high-resolution CCD video camera, mounted in front of the rotating bioreactor. The camera is fitted with powerful macro lenses that can resolve particles of ~ 2 μm in diameter from a 5 cm working distance. The system was validated using calibrated fluorescent microspheres. The experimentally determined sizes distributions of 20, 45 and 90 μm microspheres rotating in a HARV match supplier specifications. Importantly, our system can distinguish aggregated 20 μm particles from single larger (45 and 90 μm) spheres and allows for plotting local trajectories of individual microspheres. Experiments are under way to measure the aggregation kinetics of fluorescently labeled cells. Our system provides a novel method for analyzing the mechanism of 3-D tissue-like assembly of diverse cell types, such as tumor cells, into spheroids in real-time. As a long-term goal, this imaging system will be used to design intelligent feedback algorithms which allow continuous monitoring and control of the environment required to maintain cell aggregates and nascent tissues in continual free fall in RWV Bioreactors.

• Jennifer Frank

Global Analysis  Pheochromocytoma Cell (PC12) Phosphoproteins by Two-Dimensional SDS-PAGE Electrophoresis Method

PC12 pheochromocytoma cells, originally developed from tumors of rat adrenal medullary chromaffin cells, have been widely used as a cellular model to investigate neuronal differentiation induced by neurotrophins, such as nerve growth factor (NGF). To study short-term effects on protein phosphorylation, total proteins from NGF (50 ng/ml, 15 min) – treated PC12 cells were first isoelectrofocused on pH 3-10 strips on the Biorad Protean IEF apparatus followed by separation on 4-20% gradient polyacrylamide gel using the Biorad Criterion TM system. Phosphoproteins were analyzed by Pro-Q Diamond Phosphostain and normalized to total protein as assessed by staining with Sypro Ruby.  Separation of PC12 proteins by 2D SDS PAGE revealed several hundred protein spots with molecular weight (Mw) in the range of 10 – 200 kD and isoelectric points (pI) from 4 to 10 pH, as assessed by comparison with protein markers of known molecular weight and pIs. Preliminary analysis indicates that reproducibly NGF-induced the specific phosphorylation of several dozen phosphoproteins, most which were in the molecular weight range between 70 and 100 kDa and pI’s of 4.5 -8 p.  We hypothesize that NGF-inducible phosphoproteins as detected by 2D SDS-PAGE followed by phosphoprotein-specific staining represent important cellular targets involved in NGF-signaling and/or NGF-induced PC12 differentiation. To test this hypothesis, identification of selected NGF-induced phosphoproteins will be performed by mass spectroscopy.  Tentative identification of phosphoproteins such as Tyrosine-protein phosphatase, protein phosphatase 2A,  non-receptor type 5, and phospholipase A2, were identified using Tag-Indent of EXPASY Proteomics server.