Exploiting decellularized cochleae as scaffolds for inner ear tissue engineering.

BACKGROUND: Use of decellularized tissues has become popular in tissue engineering applications as the natural extracellular matrix can provide necessary physical cues that help induce the restoration and development of functional tissues. In relation to cochlear tissue engineering, the question of whether decellularized cochlear tissue can act as a scaffold and support the incorporation of exogenous cells has not been addressed. Investigators have explored the composition of the cochlear extracellular matrix and developed multiple strategies for decellularizing a variety of different tissues; however, no one has investigated whether decellularized cochlear tissue can support implantation of exogenous cells. METHODS: As a proof-of-concept study, human Wharton's jelly cells were perfused into decellularized cochleae isolated from C57BL/6 mice to determine if human Wharton's jelly cells could implant into decellularized cochlear tissue. Decellularization was verified through scanning electron microscopy. Cocheae were stained with DAPI and immunostained with Myosin VIIa to identify cells. Perfused cochleae were imaged using confocal microscopy. RESULTS: Features of the organ of Corti were clearly identified in the native cochleae when imaged with scanning electron microscopy and confocal microscopy. Acellular structures were identified in decellularized cochleae; however, no cellular structures or lipid membranes were present within the decellularized cochleae when imaged via scanning electron microscopy. Confocal microscopy revealed positive identification and adherence of cells in decellularized cochleae after perfusion with human Wharton's jelly cells. Some cells positively expressed Myosin VIIa after perfusion. CONCLUSIONS: Human Wharton's jelly cells are capable of successfully implanting into decellularized cochlear extracellular matrix. The identification of Myosin VIIa expression in human Wharton's jelly cells after implantation into the decellularized cochlear extracellular matrix suggest that components of the cochlear extracellular matrix may be involved in differentiation.

Endoplasmic Reticulum-resident Heat Shock Protein 90 (HSP90) Isoform Glucose-regulated Protein 94 (GRP94) Regulates Cell Polarity and Cancer Cell Migration by Affecting Intracellular Transport.

Heat shock protein 90 (HSP90) is a molecular chaperone that is up-regulated in cancer and is required for the folding of numerous signaling proteins. Consequently, HSP90 represents an ideal target for the development of new anti-cancer agents. The human HSP90 isoform, glucose-regulated protein 94 (GRP94), resides in the endoplasmic reticulum and regulates secretory pathways, integrins, and Toll-like receptors, which contribute to regulating immunity and metastasis. However, the cellular function of GRP94 remains underinvestigated. We report that GRP94 knockdown cells are defective in intracellular transport and, consequently, negatively impact the trafficking of F-actin toward the cellular cortex, integrin α2 and integrin αL toward the cell membrane and filopodia, and secretory vesicles containing the HSP90α-AHA1-survivin complex toward the leading edge. As a result, GRP94 knockdown cells form a multipolar spindle instead of bipolar morphology and consequently manifest a defect in cell migration and adhesion.

Development of a novel cell-based assay to monitor the transactivation activity of the HSV-1 protein ICP0.

The herpes simplex virus type 1 (HSV-1) immediate-early phosphoprotein infected cell protein 0 (ICP0) is a potent transcriptional activator of viral genes and is required for efficient viral replication and reactivation from latency. However, it is largely unknown what role specific cellular factors play in the transactivator function of ICP0. With the long-term goal of identifying these factors, we developed a cell-based assay in a 96-well format to measure this activity of ICP0. We designed a system using a set of HSV-1 GFP reporter viruses in which the expression of GFP is potently induced by ICP0 in cell culture. The initial feasibility of this system was confirmed over a 24-h period by fluorescence microscopy. We adapted this assay to a 96-well plate format, quantifying GFP expression with a fluorescence scanner. Our results indicate that the cell-based assay we developed is a valid and effective method for examining the transactivating activity of ICP0. This assay can be used to identify cellular factors that regulate the transactivating activity of ICP0.

Fluorescent Photo-conversion: A second chance to label unique cells.

Not all cells behave uniformly after treatment in tissue engineering studies. In fact, some treated cells display no signs of treatment or show unique characteristics not consistent with other treated cells. What if the "unique" cells could be isolated from a treated population, and further studied? Photo-convertible reporter proteins, such as Dendra2, allow for the ability to selectively identify unique cells with a secondary label within a primary labeled treated population. In the current study, select cells were identified and labeled through photo-conversion of Dendra2-transfected human Wharton's Jelly cells (hWJCs) for the first time. Robust photo-conversion of green-to-red fluorescence was achieved consistently in arbitrarily selected cells, allowing for precise cell identification of select hWJCs. The current study demonstrates a method that offers investigators the opportunity to selectively label and identify unique cells within a treated population for further study or isolation from the treatment population. Photo-convertible reporter proteins, such as Dendra2, offer the ability over non-photo-convertible reporter proteins, such as green fluorescent protein, to analyze unique individual cells within a treated population, which allows investigators to gain more meaningful information on how a treatment affects all cells within a target population.

Physical characterization and in vitro biological impact of highly aggregated antibodies separated into size-enriched populations by fluorescence-activated cell sorting.

An IgG2 monoclonal antibody (mAb) solution was subjected to stirring, generating high concentrations of nanometer and subvisible particles, which were then successfully size-enriched into different size bins by low-speed centrifugation or a combination of gravitational sedimentation and fluorescence-activated cell sorting (FACS). The size-fractionated mAb particles were assessed for their ability to elicit the release of cytokines from a population of donor-derived human peripheral blood mononuclear cells (PBMC) at two phases of the immune response. Fractions enriched in nanometer-sized particles showed a lower response than those enriched in micron-sized particles in this assay. Particles of 5-10 µm in size displayed elevated cytokine release profiles compared with other size ranges. Stir-stressed mAb particles had amorphous morphology, contained protein with partially altered secondary structure, elevated surface hydrophobicity (compared with controls), and trace levels of elemental fluorine. FACS size-enriched the mAb particle samples, yet did not notably alter the overall morphology or composition of particles as measured by microflow imaging, transmission electron microscopy, and scanning electron microscopy-energy dispersive X-ray spectroscopy. The utility and limitations of FACS for size separation of mAb particles and potential of in vitro PBMC studies to rank-order the immunogenic potential of various types of mAb particles are discussed.

Nonviral Reprogramming of Human Wharton's Jelly Cells Reveals Differences Between ATOH1 Homologues.

The transcription factor atonal homolog 1 (ATOH1) has multiple homologues that are functionally conserved across species and is responsible for the generation of sensory hair cells. To evaluate potential functional differences between homologues, human and mouse ATOH1 (HATH1 and MATH-1, respectively) were nonvirally delivered to human Wharton's jelly cells (hWJCs) for the first time. Delivery of HATH1 to hWJCs demonstrated superior expression of inner ear hair cell markers and characteristics than delivery of MATH-1. Inhibition of HES1 and HES5 signaling further increased the atonal effect. Transfection of hWJCs with HATH1 DNA, HES1 siRNA, and HES5 siRNA displayed positive identification of key hair cell and support cell markers found in the cochlea, as well as a variety of cell shapes, sizes, and features not native to hair cells, suggesting the need for further examination of other cell types induced by HATH1 expression. In the first side-by-side evaluation of HATH1 and MATH-1 in human cells, substantial differences were observed, suggesting that the two atonal homologues may not be interchangeable in human cells, and artificial expression of HATH1 in hWJCs requires further study. In the future, this line of research may lead to engineered systems that would allow for evaluation of drug ototoxicity or potentially even direct therapeutic use.

Hsp90 C-terminal inhibitors exhibit antimigratory activity by disrupting the Hsp90α/Aha1 complex in PC3-MM2 cells.

Human Hsp90 isoforms are molecular chaperones that are often up-regulated in malignances and represent a primary target for Hsp90 inhibitors undergoing clinical evaluation. Hsp90α is a stress-inducible isoform of Hsp90 that plays a significant role in apoptosis and metastasis. Though Hsp90α is secreted into the extracellular space under metastatic conditions, its role in cancer biology is poorly understood. We report that Hsp90α associates with the Aha1 co-chaperone and found this complex to localize in secretory vesicles and at the leading edge of migrating cells. Knockdown of Hsp90α resulted in a defect in cell migration. The functional role of Hsp90α/Aha1 was studied by treating the cells with various novobiocin-based Hsp90 C-terminal inhibitors. These inhibitors disrupted the Hsp90α/Aha1 complex, caused a cytoplasmic redistribution of Hsp90α and Aha1, and decreased cell migration. Structure-function studies determined that disruption of Hsp90α/Aha1 association and inhibition of cell migration correlated with the presence of a benzamide side chain, since an acetamide substituted analog was less effective. Our results show that disruption of Hsp90α/Aha1 interactions with novobiocin-based Hsp90 C-terminal inhibitors may limit the metastatic potential of tumors.

Improving viability and transfection efficiency with human umbilical cord wharton's jelly cells through use of a ROCK inhibitor.

Differentiating stem cells using gene delivery is a key strategy in tissue engineering and regenerative medicine applications. Nonviral gene delivery bypasses several safety concerns associated with viral gene delivery; however, leading nonviral techniques, such as electroporation, subject cells to high stress and can result in poor cell viabilities. Inhibition of Rho-associated coiled-coil kinase (ROCK) has been shown to mitigate apoptotic mechanisms associated with detachment and freezing of induced pluripotent stem cells and embryonic stem cells; however, inhibiting ROCK in mesenchymal stromal cells (MSCs) for improving gene delivery applications has not been reported previously. In this study, we hypothesized that ROCK Inhibitor (RI) would improve cell viability and gene expression in primary human umbilical cord mesenchymal stromal cells (hUCMSCs) when transfected via Nucleofection™. As hypothesized, the pre-treatment and post-treatment of hUCMSCs transfected via nucleofection with Y-27632-RI significantly improved survival rates of hUCMSCs and gene expression as measured by green fluorescent protein intensity. This study provides the first comparative look at the effect of Y-27632-RI on hUCMSCs that underwent transfection via nucleofection and shows that using Y-27632-RI in concert with nucleofection could greatly enhance the utility of differentiating and reprogramming hUCMSCs for tissue engineering applications.

Development of a Grp94 inhibitor.

Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/ß (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/ß client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein.

An automated image-based method for rapid analysis of Chlamydia infection as a tool for screening antichlamydial agents.

A major limitation in the identification of novel antichlamydial compounds is the paucity of effective methods for large-scale compound screening. The immunofluorescence assay is the preferred approach for accurate quantification of the intracellular growth of Chlamydia. In this study, an immunofluorescence image-based method (termed image-based automated chlamydial identification and enumeration [iBAChIE]) was customized for fully automated quantification of Chlamydia infection using the freely available open-source image analysis software program CellProfiler and the complementary data exploration software program CellProfiler Analyst. The method yielded enumeration of different species and strains of Chlamydia highly comparably to the conventional manual methods while drastically reducing the analysis time. The inhibitory capability of established antichlamydial activity was also evaluated. Overall, these data support that iBAChIE is a highly effective tool for automated quantification of Chlamydia infection and assessment of antichlamydial activities of molecules. Furthermore, iBAChIE is expected to be amenable to high-throughput screening studies for inhibitory compounds and fluorescently labeled molecules to study host-pathogen interactions.

Using spectroscopic and microscopic methods to probe the structural stability of human adenovirus type 4.

Adenovirus serotype 4 (Ad4) is a major cause of Ad-associated human diseases. Ad4 is also considered to be a potential delivery vector for gene therapy. In this study, multiple spectroscopic techniques together with transmission electron microscopy (TEM) were employed to probe viral stability and to improve pharmaceutical formulations of Ad4-based vaccines and DNA carriers. Perturbations of secondary, tertiary and quaternary structure of Ad4 proteins induced by elevated temperatures over a wide pH range (3-8) were analyzed using circular dichroism, UV absorption and intrinsic and extrinsic fluorescence spectroscopy as well as static and dynamic light scattering. The spectroscopic results obtained indicate a decrease in Ad4 stability as pH increases from 4 to 8, similar to the behavior reported previously for Ad2 and Ad5, although the Ad4 virion appears to possess slightly more tolerance to thermal stress. An empirical phase diagram (EPD) approach was used to summarize the data in the form of a colored map. In addition, the different physical states of Ad4 identified by the EPD were confirmed by TEM images. The results obtained in this study reveal both structural similarities among three commonly employed Ad subtypes (2, 4 and 5) as well as unique properties of Ad4.

Clearance and phosphorylation of alpha-synuclein are inhibited in methionine sulfoxide reductase a null yeast cells.

Aggregated alpha-synuclein and the point mutations Ala30Pro and Ala53Thr of alpha-synuclein are associated with Parkinson's disease. The physiological roles of alpha-synuclein and methionine oxidation of the alpha-synuclein protein structure and function are not fully understood. Methionine sulfoxide reductase A (MsrA) reduces methionine sulfoxide residues and functions as an antioxidant. To monitor the effect of methionine oxidation to alpha-synuclein on basic cellular processes, alpha-synucleins were expressed in msrA null mutant and wild-type yeast cells. Protein degradation was inhibited in the alpha-synuclein-expressing msrA null mutant cells compared to alpha-synuclein-expressing wild-type cells. Increased inhibition of degradation and elevated accumulations of fibrillated proteins were observed in SynA30P-expressing msrA null mutant cells. Additionally, methionine oxidation inhibited alpha-synuclein phosphorylation in yeast cells and in vitro by casein kinase 2. Thus, a compromised MsrA function combined with alpha-synuclein overexpression may promote processes leading to synucleinopathies.