Modified mesenchymal stromal cells by in vitro transcribed mRNA: a therapeutic strategy for hepatocellular carcinoma.

BACKGROUND: Mesenchymal stromal cells (MSCs) tropism for tumours allows their use as carriers of antitumoural factors and in vitro transcribed mRNA (IVT mRNA) is a promising tool for effective transient expression without insertional mutagenesis risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with antitumor properties by stimulating the specific immune response. The aim of this work was to generate modified MSCs by IVT mRNA transfection to overexpress GM-CSF and determine their therapeutic effect alone or in combination with doxorubicin (Dox) in a murine model of hepatocellular carcinoma (HCC). METHODS: DsRed or GM-CSF IVT mRNAs were generated from a cDNA template designed with specific primers followed by reverse transcription. Lipofectamine was used to transfect MSCs with DsRed (MSC/DsRed) or GM-CSF IVT mRNA (MSC/GM-CSF). Gene expression and cell surface markers were determined by flow cytometry. GM-CSF secretion was determined by ELISA. For in vitro experiments, the J774 macrophage line and bone marrow monocytes from mice were used to test GM-CSF function. An HCC model was developed by subcutaneous inoculation (s.c.) of Hepa129 cells into C3H/HeN mice. After s.c. injection of MSC/GM-CSF, Dox, or their combination, tumour size and mouse survival were evaluated. Tumour samples were collected for mRNA analysis and flow cytometry. RESULTS: DsRed expression by MSCs was observed from 2 h to 15 days after IVT mRNA transfection. Tumour growth remained unaltered after the administration of DsRed-expressing MSCs in a murine model of HCC and MSCs expressing GM-CSF maintained their phenotypic characteristic and migration capability. GM-CSF secreted by modified MSCs induced the differentiation of murine monocytes to dendritic cells and promoted a proinflammatory phenotype in the J774 macrophage cell line. In vivo, MSC/GM-CSF in combination with Dox strongly reduced HCC tumour growth in C3H/HeN mice and extended mouse survival in comparison with individual treatments. In addition, the tumours in the MSC/GM-CSF + Dox treated group exhibited elevated expression of proinflammatory genes and increased infiltration of CD8 + T cells and macrophages. CONCLUSIONS: Our results showed that IVT mRNA transfection is a suitable strategy for obtaining modified MSCs for therapeutic purposes. MSC/GM-CSF in combination with low doses of Dox led to a synergistic effect by increasing the proinflammatory tumour microenvironment, enhancing the antitumoural response in HCC.

Hepatic SPARC Expression Is Associated with Inflammasome Activation during the Progression of Non-Alcoholic Fatty Liver Disease in Both Mice and Morbidly Obese Patients.

The severity of non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to steatohepatitis, and it is not yet clearly understood which patients will progress to liver fibrosis or cirrhosis. SPARC (Secreted Protein Acidic and Rich in Cysteine) has been involved in NAFLD pathogenesis in mice and humans. The aim of this study was to investigate the role of SPARC in inflammasome activation, and to evaluate the relationship between the hepatic expression of inflammasome genes and the biochemical and histological characteristics of NAFLD in obese patients. In vitro studies were conducted in a macrophage cell line and primary hepatocyte cultures to assess the effect of SPARC on inflammasome. A NAFLD model was established in SPARC knockout (SPARC-/-) and SPARC+/+ mice to explore inflammasome activation. A hepatic RNAseq database from NAFLD patients was analyzed to identify genes associated with SPARC expression. The results were validated in a prospective cohort of 59 morbidly obese patients with NAFLD undergoing bariatric surgery. Our results reveal that SPARC alone or in combination with saturated fatty acids promoted IL-1ß expression in cell cultures. SPARC-/- mice had reduced hepatic inflammasome activation during the progression of NAFLD. NAFLD patients showed increased expression of SPARC, NLRP3, CASP1, and IL-1ß. Gene ontology analysis revealed that genes positively correlated with SPARC are linked to inflammasome-related pathways during the progression of the disease, enabling the differentiation of patients between steatosis and steatohepatitis. In conclusion, SPARC may play a role in hepatic inflammasome activation in NAFLD.

Chromatographic Scalable Method to Isolate Engineered Extracellular Vesicles Derived from Mesenchymal Stem Cells for the Treatment of Liver Fibrosis in Mice.

New therapeutic options for liver cirrhosis are needed. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have emerged as a promising tool for delivering therapeutic factors in regenerative medicine. Our aim is to establish a new therapeutic tool that employs EVs derived from MSCs to deliver therapeutic factors for liver fibrosis. EVs were isolated from supernatants of adipose tissue MSCs, induced-pluripotent-stem-cell-derived MSCs, and umbilical cord perivascular cells (HUCPVC-EVs) by ion exchange chromatography (IEC). To produce engineered EVs, HUCPVCs were transduced with adenoviruses that code for insulin-like growth factor 1 (AdhIGF-I-HUCPVC-EVs) or green fluorescent protein. EVs were characterized by electron microscopy, flow cytometry, ELISA, and proteomic analysis. We evaluated EVs' antifibrotic effect in thioacetamide-induced liver fibrosis in mice and on hepatic stellate cells in vitro. We found that IEC-isolated HUCPVC-EVs have an analogous phenotype and antifibrotic activity to those isolated by ultracentrifugation. EVs derived from the three MSCs sources showed a similar phenotype and antifibrotic potential. EVs derived from AdhIGF-I-HUCPVC carried IGF-1 and showed a higher therapeutic effect in vitro and in vivo. Remarkably, proteomic analysis revealed that HUCPVC-EVs carry key proteins involved in their antifibrotic process. This scalable MSC-derived EV manufacturing strategy is a promising therapeutic tool for liver fibrosis.

Evaluation of cancer stem cells markers expression in HCC trough real-time polymerase chain reaction.

Quantitative real-time polymerase chain reaction (qRT-PCR) flexibility, robustness and reproducibility have rapidly extended the scope of the method. Cancer stem cells are gaining increasing importance since their role in cancer initiation, treatment resistance and recurrence give rise to a wide range of potential diagnostic and therapeutic applications. The expression of several characteristic markers is proven a reliable method to assess stem-like-phenotype of cancer cells. Here, we provided a thorough protocol for the study of cancer stem cells in hepatocellular carcinoma mouse models and cell cultures using qRT-PCR.

Design of nalidixic acid­vanadium complex loaded into chitosan hybrid nanoparticles as smart strategy to inhibit bacterial growth and quorum sensing.

The discovery of new alternatives for the treatment of infectious diseases has become the focus of burgeoning global interest. The complexation of the wide-spectrum antibiotic nalidixic acid (NA) with oxidovanadium(IV) ion and its incorporation into hybrid nanoparticulate systems were explored. The V-NA complex proved to be a stronger antimicrobial agent against E. coli, B. cereus, S. aureus and P. aeruginosa than NA, based on inhibition experiments. Myristyl myristate nanostructured lipid carriers (NLCs) and polymeric nanoparticles of Eudragit NE30D (EuNPs) were hybridized with chitosan (chi) to increase their stability and mucoadhesivity. They showed V-NA encapsulation of 97.8 ± 0.5% and 96.1 ± 0.1% respectively. TEM and DLS characterization ascertained the presence of spherical positive charged NPs ranging from 170 to 330 nm. Controlled release of V-NA from NPs was observed with 30-40% release in 3 days. A considerable potentiation of V-NA antimicrobial activity from 5 to 10 times was elucidated against P. aeruginosa with MIC values of 59.3 and 129.9 µM for NLC/chi and EuNPs/chi respectively, in comparison with 625 µM of the free complex. Hybrid NPs were able to interfere with the quorum sensing of the reporter Chromobacterium violaceum. Cytotoxicity on mouse fibroblast L929 cells was evaluated in the range of 29.7-519 µM by MTT assay showing that, NLC/chi particles supported cell growth in the range of at 29.7-60 µM while Eu/chi do not exert cytotoxicity between 29.7 and 120 µM. These results suggest that nanoparticles are suitable systems for drug delivery applications.