Phenotypic assessment of liver-derived cell cultures during in vitro expansion.

Background: Liver cells represent an attractive source of cells for autologous regenerative medicine. The present study assesses the liver cells' stability during in vitro expansion, as a prerequisite for therapeutic use. Results: The human liver cell cultures in this study were propagated efficiently in vitro for at least 12 passages. No significant changes in morphology, intracellular ultrastructures and characteristic markers expression were found during in vitro expansion of cells from all analyzed donors. However, expanded cells derived from male donors of >60 years old, lost the Y chromosome. Conclusion: Liver-derived cell cultures adopt a proliferative, stable mesenchymal phenotype, through an epithelial to mesenchymal transition process. The molecular and phenotypic changes of the cells during propagation are uniform, despite the heterogeneity of the different donors. Loss of Y chromosome occurs after cells' propagation in elder male donors.

Adenovirus-Mediated FasL Minigene Transfer Endows Transduced Cells with Killer Potential.

Fas ligand (First apoptosis signal ligand, FasL, also known as CD95L) is the common executioner of apoptosis within the tumor necrosis factor (TNF) superfamily. We aimed to induce functional FasL expression in transduced cells using an adenovirus vector, which has the advantage of strong and transient induction of the gene included in the adenoviral genome. Here, we report that the adenovirus carrying a truncated FasL gene, named FasL minigene, encoding the full-length FasL protein (Ad-gFasL) is more efficient than the adenovirus carrying FasL cDNA (Ad-cFasL) in the induction of FasL expression in transduced cells. FasL minigene (2887 bp) lacking the second intron and a part of the 3'-UTR was created to reduce the gene length due to the size limitation of the adenoviral genome. The results show that, in transduced hepatocytes, strong expression of mRNA FasL appeared after 10 h for Ad-gFasL, while for Ad-cFasL, a faint expression appeared after 16 h. For Ad-gFasL, the protein expression was noticed starting with 0.5 transfection units (TU)/cell, while for Ad-cFasL, it could not be revealed. FasL-expressing endothelial cells induced apoptosis of A20 cells in co-culture experiments. FasL-expressing cells may be exploitable in various autoimmune diseases such as graft-versus-host disease, chronic colitis, and type I diabetes.

Plasma Small Extracellular Vesicles Derived miR-21-5p and miR-92a-3p as Potential Biomarkers for Hepatocellular Carcinoma Screening.

INTRODUCTION: Liquid biopsy using circulating microvesicles and exosomes is emerging as a new diagnostic tool that could improve hepatocellular carcinoma (HCC) early diagnosis and screening protocols. Our study aimed to investigate the utility of plasma exosomal miR-21-5p and miR-92-3p for HCC diagnosis during screening protocols. METHODS: The study group included 106 subjects: 48 patients diagnosed with HCC during screening, who underwent a potentially curative treatment (surgical resection or liver transplantation), 38 patients with liver cirrhosis (LC) on the waiting list for liver transplantation, and 20 healthy volunteers. The exosomes were isolated by precipitation with a reagent based on polyethylene glycol and were characterized based on morphological aspects (i.e., diameter); molecular weight; CD63, CD9, and CD81 protein markers; and exosomal miR-21-5p and miR-92a-3p expression levels. RESULTS: We first demonstrate that the exosome population isolated with the commercially available Total Exosome Isolation kit respects the same size ranging, morphological, and protein expression aspects compared to the traditional ultracentrifugation technique. The analysis of the expression profile indicates that miR-21-5p was upregulated (p = 0.017), and miR-92a-3p was downregulated (p = 0.0005) in plasma-derived exosomes from HCC subjects, independently from the patient's characteristics. AUROC for HCC diagnosis based on AFP (alpha-fetoprotein) was 0.72. By integrating AFP and the relative expression of exosomal miR-21-5p and miR-92a-3p in a logistic regression equation for HCC diagnosis, the combined AUROC of the new exosomal miR HCC score was 0.85-significantly better than serum AFP alone (p = 0.0007). CONCLUSION: Together with serum AFP, plasma exosomal miR-21-5p and miR-92a-3p could be used as potential biomarkers for HCC diagnosis in patients with LC subjected to screening and surveillance.

Detection of human papilloma viruses using nanostructurated silicon support in microarray technology.

In a typical microarray experiment, DNA is arrayed on a solid substrate as spots, the array being probed with a sample or a capture molecule of interest and the interaction monitored through different detection methods. The present study evaluates the possibility to use micro-array technology to genotype samples with Human Papilloma Viruses (HPV). The performance of DNA microarrays strongly depend on their surface properties. The efficiency of DNA immobilization in terms of sensitivity and specificity is one of the most important step in obtaining a microarray chip for diagnosis of HPV family viruses. Here we report the preparation and evaluation of nano-porous silicon surfaces for HPV detection based on DNA micro-array technique. Two different surfaces based on similar porous structure chemically modified in order to efficiently immobilize ss-DNA specific for HPV viruses were investigate.