Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue.

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.

1,4-Dihydropyridine as a Promising Scaffold for Novel Antimicrobials Against Helicobacter pylori.

The increasing occurrence of multidrug-resistant strains of the gastric carcinogenic bacterium Helicobacter pylori threatens the efficacy of current eradication therapies. In a previous work, we found that several 1,4-dihydropyridine (DHP)-based antihypertensive drugs exhibited strong bactericidal activities against H. pylori by targeting the essential response regulator HsrA. To further evaluate the potential of 1,4-DHP as a scaffold for novel antimicrobials against H. pylori, we determined the antibacterial effects of 12 novel DHP derivatives that have previously failed to effectively block L- and T-type calcium channels. Six of these molecules exhibited potent antimicrobial activities (MIC ≤ 8 mg/L) against three different antibiotic-resistant strains of H. pylori, while at least one compound resulted as effective as metronidazole. Such antimicrobial actions appeared to be specific against Epsilonproteobacteria, since no deleterious effects were appreciated on Escherichia coli and Staphylococcus epidermidis. The new bactericidal DHP derivatives targeted the H. pylori regulator HsrA and inhibited its DNA binding activity according to both in vitro and in vivo analyses. Molecular docking predicted a potential druggable binding pocket in HsrA, which could open the door to structure-based design of novel anti-H. pylori drugs.