P. Scott Pine

The Determination of Stem Cell Fate by 3D Scaffold Structures through the Control of Cell Shape

Stem cell response to a library of scaffolds with varied 3D structures was investigated. Microarray screening revealed that each type of scaffold structure induced a unique gene expression signature in primary human bone marrow stromal cells (hBMSCs). Hierarchical cluster analysis showed that treatments sorted by scaffold structure and not by polymer chemistry suggesting that scaffold structure was more influential than scaffold composition. Further, the effects of scaffold structure on hBMSC function were mediated by cell shape. Of all the scaffolds tested, only scaffolds with a nanofibrous morphology were able to drive the hBMSCs down an osteogenic lineage in the absence of osteogenic supplements. Nanofiber scaffolds forced the hBMSCs to assume an elongated, highly branched morphology. This same morphology was seen in osteogenic controls where hBMSCs were cultured on flat polymer films in the presence of osteogenic supplements (OS). In contrast, hBMSCs cultured on flat polymer films in the absence of OS assumed a more rounded and less-branched morphology. These results indicate that cells are more sensitive to scaffold structure than previously appreciated and suggest that scaffold efficacy can be optimized by tailoring the scaffold structure to force cells into morphologies that direct them to differentiate down the desired lineage.

Using RNA sample titrations to assess microarray platform performance and normalization techniques

We have assessed the utility of RNA titration samples for evaluating microarray platform performance and the impact of different normalization methods on the results obtained. As part of the MicroArray Quality Control project, we investigated the performance of five commercial microarray platforms using two independent RNA samples and two titration mixtures of these samples. Focusing on 12,091 genes common across all platforms, we determined the ability of each platform to detect the correct titration response across the samples. Global deviations from the response predicted by the titration ratios were observed. These differences could be explained by variations in relative amounts of messenger RNA as a fraction of total RNA between the two independent samples. Overall, both the qualitative and quantitative correspondence across platforms was high. In summary, titration samples may be regarded as a valuable tool, not only for assessing microarray platform performance and different analysis methods, but also for determining some underlying biological features of the samples.

Laser scanning and confocal microscopy of daunorubicin, doxorubicin, and rhodamine 123 in multidrug-resistant cells.

The multidrug-resistant gene (MDR1) encodes an energy-dependent drug efflux pump (P-glycoprotein) for many anti-cancer drugs. We have studied the intracellular distribution of rhodamine 123 (R123), daunorubicin (DN), and doxorubicin (DOX) in cells expressing a human MDR1 gene. The distribution of these fluorescent drugs was measured by laser scanning microscopy and confocal microscopy. We devised a new method for analysis of fluorescence line scan data to determine the intracellular distribution of fluorescent probes. This method and confocal microscopy showed that R123, DN, and DOX are localized to both plasma membrane and intracellular compartments in multidrug-resistant cells. When the cells are treated with verapamil, an inhibitor of the multidrug transporter, the amount of DOX, DN, and R123 associated with the cell rises. After inhibition, the relative distribution of DOX and DN between the cell surface and intracellular structures does not change dramatically. However, R123 tends to relocalize to intracellular sites from predominantly plasma membrane sites, indicating that this dye behaves differently than the anti-cancer drugs. These results show the subcellular distributions of R123, DN, and DOX in plasma membrane, cytoplasm, and intracellular membrane systems, but do not allow definitive distinctions among existing models of how P-glycoprotein affects the distribution of drugs.

Improvement in the Reproducibility and Accuracy of DNA Microarray Quantification by Optimizing Hybridization Conditions

BackgroundDNA microarrays, which have been increasingly used to monitor mRNA transcripts at a global level, can provide detailed insight into cellular processes involved in response to drugs and toxins. This is leading to new understandings of signaling networks that operate in the cell, and the molecular basis of diseases. Custom printed oligonucleotide arrays have proven to be an effective way to facilitate the applications of DNA microarray technology. A successful microarray experiment, however, involves many steps: well-designed oligonucleotide probes, printing, RNA extraction and labeling, hybridization, and imaging. Optimization is essential to generate reliable microarray data.ResultsHybridization and washing steps are crucial for a successful microarray experiment. By following the hybridization and washing conditions recommended by an oligonucleotide provider, it was found that the expression ratios were compressed greater than expected and data analysis revealed a high degree of non-specific binding. A series of experiments was conducted using rat mixed tissue RNA reference material (MTRRM) and other RNA samples to optimize the hybridization and washing conditions. The optimized hybridization and washing conditions greatly reduced the non-specific binding and improved the accuracy of spot intensity measurements.ConclusionThe results from the optimized hybridization and washing conditions greatly improved the reproducibility and accuracy of expression ratios. These experiments also suggested the importance of probe designs using better bioinformatics approaches and the need for common reference RNA samples for platform performance evaluation in order to fulfill the potential of DNA microarray technology.

Prevention of binding of rgp120 by anti-HIV active tannins.

Several tannins with anti-HIV activity have been described previously (Nonaka et al., J Nat Prod 53: 587-595, 1990). We have shown that the tannins chebulinic acid and punicalin were able to block the binding of HIV rgp120 to CD4. These compounds were not toxic to stimulated human peripheral blood lymphocytes at concentrations ten times above their maximal effective concentration.

Use of a mixed tissue RNA design for performance assessments on multiple microarray formats

The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes). The design provides four known ratio measurements of >200 reference probes, which were chosen for their tissue-selectivity, dynamic range coverage and alignment to the same exemplar transcript sequence across all three platforms. The data generated from testing three biological replicates of the reagent at eight laboratories on three array formats provides a benchmark set for both laboratory and data processing performance assessments. Close agreement with target ratios adjusted for sample complexity was achieved on all platforms and low variance was observed among platforms, replicates and sites. The mixed tissue design produces a reagent with known gene expression changes within a complex sample and can serve as a paradigm for performance standards for microarrays that target other species.

Workgroup report : Review of genomics data based on experience with mock submissions-view of the CDER pharmacology toxicology nonclinical pharmacogenomics subcommittee

Over the past few years, both the U.S. Food and Drug Administration (FDA) and the pharmaceutical industry have recognized the potential importance of pharmacogenomics and toxicogenomics to drug development. To resolve the uncertainties surrounding the use of microarray technology and the presentation of genomics data for regulatory purposes, several pharmaceutical companies and genomics technology providers have provided the FDA with reports of genomics studies that included supporting toxicology data (e.g., serum chemistry, histopathology). These studies were not associated with any active drug application and were exploratory or hypothesis generating in nature. For training purposes, these reports were reviewed by the Nonclinical Pharmacogenomics Subcommittee consisting of the Center for Drug Evaluation and Research pharmacology and toxicology researchers and reviewers. In this article, we describe some of these submissions and report on our assessment of data content, format, and quality control metrics that were useful for evaluating these nonclinical genomics submissions, specifically in relation to the proposed MIAME/MINTox (minimum information about a microarray experiment/minimum information needed for a toxicology experiment) recommendations. These genomics submissions allowed both researchers and regulators to gain experience in the process of reviewing and analyzing toxicogenomics data. The experience will allow development of recommendations for the submission and review of these data as the state of the science evolves.

Effect of combination of suboptimal concentrations of p-glycoprotein blockers on the proliferation of MDR1 gene expressing cells

Pharmacologically active in vivo doses of P‐glycoprotein (Pgp) blockers, specifically verapamil, Cremophor EL and PSC833 cause toxicity in addition to that from the concomitantly used cancer chemotherapeutic drugs. It was shown before that these blockers cause different types of toxicities in vivo. We found that these 3 chemically distinct Pgp blockers exert different biophysical effects on the membranes of L1210 MDR cells. They also affect the general metabolism of these cells differently, but all block affinity labeling of Pgp. We could also show that the combination of suboptimal doses of these blockers can restore the uptake of the Pgp substrate rhodamine 123 into L1210MDR, 3T3MDR and KB‐VI cells and can reduce the survival rate of these cells when treated in combination with daunorubicin. Our results suggest that the combination of suboptimal doses of these Pgp blockers may be advantageous in clinical practice.

Inhibition of the Binding of HIV rgp120 to CD4 by Dyes

We have found that several dye compounds have the ability to inhibit the binding of HIV rgpl 20 to the Leu3a epitope of CD4 on PBL. One of these compounds, selected for further testing as the best candidate, can inhibit the growth of HIV In vitro. The tested dyes have varying degrees of specificity and efficacy in inhibiting the binding of rgpl 20. These results may point towards compounds that can be useful therapeutics against HIV.

Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films

Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-β and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation.