A Sendai Virus-Based Cytoplasmic RNA Vector as a Novel Platform for Long-Term Expression of MicroRNAs.

Cytoplasmic RNA virus-derived vectors have emerged as attractive vehicles for microRNA (miRNA) delivery as they possess no potential risk of chromosomal insertion. However, their relatively short-term expression limits their use in biological applications that require long-term miRNA manipulation, such as somatic cell reprogramming. Here, we show that a cytoplasmic RNA virus vector based on a replication-defective and persistent Sendai virus (SeVdp) serves as an effective platform for long-term production of miRNAs capable of inducing sequence-specific target suppression. The SeVdp vector was able to simultaneously deliver embryonic stem cell-enriched miRNAs, as well as multiple transcription factors, into fibroblasts, resulting in effective reprogramming into induced pluripotent stem cells. Furthermore, we report that the murine miR-367 hairpin produced elevated levels of mature miRNA when it was incorporated into the SeVdp vector and served as an effective backbone for production of artificial miRNAs. These SeVdp vector-derived artificial miRNAs efficiently inhibited expression of target genes. Our findings provide novel insights into a powerful tool for long-term and targeted gene silencing in areas such as regenerative medicine, gene therapy, and cell therapy.

Sensitive and long-term monitoring of intracellular microRNAs using a non-integrating cytoplasmic RNA vector.

MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression at the post-transcriptional level. Different types of cells express unique sets of miRNAs that can be exploited as potential molecular markers to identify specific cell types. Among the variety of miRNA detection methods, a fluorescence-based imaging system that utilises a fluorescent-reporter gene regulated by a target miRNA offers a major advantage for long-term tracking of the miRNA in living cells. In this study, we developed a novel fluorescence-based miRNA-monitoring system using a non-integrating cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because SeVdp vectors robustly and stably express transgenes, this system enabled sensitive monitoring of miRNAs by fluorescence microscopy. By applying this system for cellular reprogramming, we found that miR-124, but not miR-9, was significantly upregulated during direct neuronal conversion. Additionally, we were able to isolate integration-free human induced pluripotent stem cells by long-term tracking of let-7 expression. Notably, this system was easily expandable to allow detection of multiple miRNAs separately and simultaneously. Our findings provide insight into a powerful tool for evaluating miRNA expression during the cellular reprogramming process and for isolating reprogrammed cells potentially useful for medical applications.

An antithrombogenic citric acid-crosslinked gelatin with endothelialization activity.

A novel citric acid-crosslinked gelatin matrix with endothelialization activity and anti-adhesive properties for platelets is prepared. The matrix is characterized using an endothelial cell culture and an antithrombogenic activity test. The number of endothelial cells cultured on the surface of the trisuccinimidyl citrate (TSC)-crosslinked gelatin increases as the concentration of TSC increases to 20 mM, and then decreases with further increases in TSC concentration. Compared with glutaraldehyde-crosslinked gelatin, platelet number and fibrin network formation on the TSC-crosslinked gelatin are minimal at high TSC concentration. The biocompatibility of the matrix is evaluated by bioluminescence imaging. This indicates that the inflammation reaction of the TSC-crosslinked gelatin is lower than that of glutaraldehyde-crosslinked gelatin. Physicochemical analysis of TSC-crosslinked gelatin with different TSC concentrations shows that the high concentration of the cell adhesion sequence, arginine-glycine-aspartic acid, contributes to the promotion of endothelial cell adhesion and subsequent endothelial cell growth. Analysis of the carboxyl groups in the TSC-crosslinked gelatin showed that the antithrombogenic activity is due to the increased negative charge derived from the hydrolyzed active ester groups of TSC. These findings show that TSC-crosslinked gelatin has the potential for use in biomedical devices in contact with blood, such as stents, artificial blood vessels, and artificial heart valves.

In vitro osteogenic differentiation of HOS cells on two types of collagen gels.

HOS cell is a model strain of human osteoblasts derived from human osteosarcoma. We cultured the HOS cells on both the conventional collagen gel (neutral gel), and the gamma-crosslinked collagen gel without collagen fibrils (acidic gel). The shape of HOS cells on the neutral gel was similar to that on the culture dish. However, HOS cells on acidic gel had an elongated shape and attached each other to form a mesh-like pattern. The cells attached to the surface of both gels but scarcely penetrated their depths. We measured the biochemical markers for osteogenic differentiation in the HOS cells cultured on both the neutral gel and the acidic gel. The expressions of alkaline phosphatase and osteocalcin were detected in the HOS cells on both types of collagen gel. Deposition of the calcium also occurred on both gels although it was higher in the neutral gel than the acidic one. These results indicate the importance of collagen for the differentiation of HOS cells, but it is not dependent on the molecular structure (fibril formation) of collagen.