Tissue Engineering to Repair Diaphragmatic Defect in a Rat Model.

Tissue engineering is an emerging strategy for repairing damaged tissues or organs. The current study explored using decellularized rat diaphragm scaffolds combined with human amniotic fluid-derived multipotent stromal cells (hAFMSC) to provide a scaffold, stem cell construct that would allow structural barrier function during tissue ingrowth/regeneration. We created an innovative cell infusion system that allowed hAFMSC to embed into scaffolds and then implanted the composite tissues into rats with surgically created left-sided diaphragmatic defects. Control rats received decellularized diaphragm scaffolds alone. We found that the composite tissues that combined hAFMSCs demonstrated improved physiological function as well as the muscular-tendon structure, compared with the native contralateral hemidiaphragm of the same rat. Our results indicate that the decellularized diaphragm scaffolds are a potential support material for diaphragmatic hernia repair and the composite grafts with hAFMSC are able to accelerate the functional recovery of diaphragmatic hernia.

A tanscriptomics study to elucidate the toxicological mechanism of methylmercury chloride in a human stem cell based in vitro test.

Traditional approaches in evaluating the hazard of drug candidates on the developing offspring are often time-consuming and cost-intensive. Moreover, variations in the toxicological response of different animal species to the tested substance cause severe problems when extrapolating safety dosages for humans. Therefore, more predictive and relevant toxicological systems based on human cell models are required. In the presented study the environmental toxicant methylmercury chloride (MeHgCl), known to cause structural developmental abnormalities in the brain, was used as reference compound to develop a concept contributing to a mechanistic understanding of the toxicity of an investigated substance. Despite the fact, that there are significant data available from animal studies and from poisonings in Japan and Iraq, uncertainties on the mechanism of MeHgCl during human development are still remaining and qualify the substance for further analysis. Transcriptomics analysis in combination with a human cell based in vitro model has been used in order to elucidate the toxicity of MeHgCl at molecular level. Differentiating neural precursor cells that have been exposed continuously to non- and low-cytotoxic concentrations of MeHgCl were investigated. Quantitative change in the mRNA expression profiles of selected genes demonstrated the sensitivity of the cell model and its qualification for a transcriptomics study screening changes in the expression profile of the complete human genome of MeHgCl-treated human neural cells. Potential biomarkers were identified and these candidate marker genes as well as their involvement in a possible toxic mechanism of MeHgCl during the human neurulation process are hereby introduced. The study confirmed the hypothesis that a cellular model based on a human stem cell line can be applied for elucidating unknown mode of actions of developmental toxicants.

MicroRNA profiling as a tool for pathway analysis in a human in vitro model for neural development.

MicroRNAs (miRNA) are a recently recognised class of small, non-coding RNAs involved in the post-transcriptional regulation of gene expression and with crucial implication for mammalian development. In particular, they play key roles in neuronal development, from early neurogenesis to neuronal differentiation and synaptic development, and also in in vitro systems. The detection of embryotoxic hazards in the preclinical phase is still a challenge, often due to species-species variations. In this study we analysed whether miRNA expression profiles in a human pluripotent cell model can be a helpful tool for a more mechanistic approach to pharmacology and toxicology. Differentiating human pluripotent cells were repeatedly treated with non-cytotoxic doses of methylmercury chloride (MeHgCl), a well known brain developmental toxicant. The expression of proteins, mRNA and miRNAs were used to monitor successful neural differentiation. Significant changes in the expression of 12 miRNAs were detected. By using available bioinformatics tools, we obtained validated and predicted targets for the identified miRNAs, on which we performed functional clustering analysis. Through this approach, we identified several terms and functional clusters associated with neural development, together with indicators of general toxic effect, such as apoptosis or stress response-related genes. Interestingly, our results also suggest a previously undiscovered association between MeHgCl and the ubiquitin-proteasome protein degradation pathway. Although further investigations are needed, our results suggest that miRNA expression analysis is a powerful tool in pathway-oriented toxicity and could improve early-phase hazard assessments.

Calcein assay: a high-throughput method to assess P-gp inhibition.

Transporter mediated drug-drug interactions (tDDI) mediated by ABCB1 have been shown to be clinically relevant. Hence, the assessment of the ABCB1 tDDI potential early in the drug development process has gained interest. We have evaluated the Calcein assay as a means of assessing the ABCB1 tDDI that is amenable to high throughout and compared it with the monolayer efflux assay. We found the Calcein assay, when performed in K562MDR cells using the protocol originally published more sensitive than digoxin transport inhibition in MDCKII-MDR1 cells. Application of the Calcein assay to cell lines containing different amounts of ABCB1, yielded IC(50) values that varied 10-100-fold. The differences observed for IC(50) values for the same compounds were in the following rank order: IC(50, MDCKII-MDR1) >IC(50, K562MDR)>IC(50, hCMEC/D3). Higher IC(50) values were obtained in cells with higher ABCB1 expression. The Calcein assay is a high-throughput alternative to digoxin transport inhibition as it appears to have a comparable selectivity but higher sensitivity than previously published digoxin transport inhibition in MDCKII-MDR1 cells. In addition, it can be performed in a barrier-specific manner highlighting the dependence of ABCB1 IC(50) values on different ABCB1 expression levels.