Human Fibroblast Sheet Promotes Human Pancreatic Islet Survival and Function In Vitro

In previous work, we engineered functional cell sheets using bone marrow-derived mesenchymal stem cells (BM-MSCs) to promote islet graft survival. In the present study, we hypothesized that a cell sheet using dermal fibroblasts could be an alternative to MSCs, and then we aimed to evaluate the effects of this cell sheet on the functional viability of human islets. Fibroblast sheets were fabricated using temperature-responsive culture dishes. Human islets were seeded onto fibroblast sheets. The efficacy of the fibroblast sheets was evaluated by dividing islets into three groups: the islets-alone group, the coculture with fibroblasts group, and the islet culture on fibroblast sheet group. The ultrastructure of the islets cultured on each fibroblast sheet was examined by electron microscopy. The fibroblast sheet expression of fibronectin (as a component of the extracellular matrix) was quantified by Western blotting. After 3 days of culture, islet viabilities were 70.2 ± 9.8%, 87.4 ± 5.8%, and 88.6 ± 4.5%, and survival rates were 60.3 ± 6.8%, 65.3 ± 3.0%, and 75.8 ± 5.6%, respectively. Insulin secretions in response to high-glucose stimulation were 5.1 ± 1.6, 9.4 ± 3.8, and 23.5 ± 12.4 μIU/islet, and interleukin-6 (IL-6) secretions were 3.0 ± 0.7, 5.1 ± 1.2, and 7.3 ± 1.0 ng/day, respectively. Islets were found to incorporate into the fibroblast sheets while maintaining a three-dimensional structure and well-preserved extracellular matrix. The fibroblast sheets exhibited a higher expression of fibronectin compared to fibroblasts alone. In conclusion, human dermal fibroblast sheets fabricated by tissue-engineering techniques could provide an optimal substrate for human islets, as a source of cytokines and extracellular matrix

A novel multilocus variable-number tandem repeat analysis for Bordetella parapertussis.

Purpose. Human-adapted Bordetella parapertussis is one of the causative agents of whooping cough; however, there are currently no genotyping systems with high discriminatory power for this bacterial pathogen. We therefore aimed to develop a multilocus variable-number tandem repeat analysis (MLVA) for human-adapted B. parapertussis.Methodology. Four highly polymorphic variable number tandem repeat (VNTR) loci in the B. parapertussis genome were selected and amplified by multiplex PCR. MLVA was performed based on the number of tandem repeats at VNTR loci. The discriminatory power of MLVA was evaluated with three laboratory reference strains and 50 human isolates of B. parapertussis.Results. Multiplex PCR-based MLVA characterized 53 B. parapertussis reference strains and isolates into 25 MLVA types and the Simpson diversity index was 0.91 (95 % confidence interval, 0.86-0.97). The three reference strains exhibited different MLVA types. Thirty-one Japanese isolates, ten French isolates and three Taiwanese isolates belonged to fourteen, nine and three MLVA types, respectively. In contrast, all five Australian isolates belonged to the same type. Two Japanese isolates collected from patients with known epidemiological links had the same type.Conclusion. Our novel MLVA method has high discriminatory power for genotyping human B. parapertussis. Regarding this organism, this genotyping system is a promising tool for epidemiological surveillance and investigating outbreaks