Tonsil-derived stem cells as a new source of adult stem cells.

Located near the oropharynx, the tonsils are the primary mucosal immune organ. Tonsil tissue is a promising alternative source for the high-yield isolation of adult stem cells, and recent studies have reported the identification and isolation of tonsil-derived stem cells (T-SCs) from waste surgical tissue following tonsillectomies in relatively young donors (i.e., under 10 years old). As such, T-SCs offer several advantages, including superior proliferation and a shorter doubling time compared to bone marrow-derived mesenchymal stem cells (MSCs). T-SCs also exhibit multi-lineage differentiation, including mesodermal, endodermal (e.g., hepatocytes and parathyroid-like cells), and even ectodermal cells (e.g., Schwann cells). To this end, numbers of researchers have evaluated the practical use of T-SCs as an alternative source of autologous or allogenic MSCs. In this review, we summarize the details of T-SC isolation and identification and provide an overview of their application in cell therapy and regenerative medicine.

Enzymatic Synthesis of Self-assembled Dicer Substrate RNA Nanostructures for Programmable Gene Silencing.

Enzymatic synthesis of RNA nanostructures is achieved by isothermal rolling circle transcription (RCT). Each arm of RNA nanostructures provides a functional role of Dicer substrate RNA inducing sequence specific RNA interference (RNAi). Three different RNAi sequences (GFP, RFP, and BFP) are incorporated within the three-arm junction RNA nanostructures (Y-RNA). The template and helper DNA strands are designed for the large-scale in vitro synthesis of RNA strands to prepare self-assembled Y-RNA. Interestingly, Dicer processing of Y-RNA is highly influenced by its physical structure and different gene silencing activity is achieved depending on its arm length and overhang. In addition, enzymatic synthesis allows the preparation of various Y-RNA structures using a single DNA template offering on demand regulation of multiple target genes.

Self-assembled mirror DNA nanostructures for tumor-specific delivery of anticancer drugs.

Nanoparticle delivery systems have been extensively investigated for targeted delivery of anticancer drugs over the past decades. However, it is still a great challenge to overcome the drawbacks of conventional nanoparticle systems such as liposomes and micelles. Various novel nanomaterials consist of natural polymers are proposed to enhance the therapeutic efficacy of anticancer drugs. Among them, deoxyribonucleic acid (DNA) has received much attention as an emerging material for preparation of self-assembled nanostructures with precise control of size and shape for tailored uses. In this study, self-assembled mirror DNA tetrahedron nanostructures is developed for tumor-specific delivery of anticancer drugs. l-DNA, a mirror form of natural d-DNA, is utilized for resolving a poor serum stability of natural d-DNA. The mirror DNA nanostructures show identical thermodynamic properties to that of natural d-DNA, while possessing far enhanced serum stability. This unique characteristic results in a significant effect on the pharmacokinetics and biodistribution of DNA nanostructures. It is demonstrated that the mirror DNA nanostructures can deliver anticancer drugs selectively to tumors with enhanced cellular and tissue penetration. Furthermore, the mirror DNA nanostructures show greater anticancer effects as compared to that of conventional PEGylated liposomes. Our new approach provides an alternative strategy for tumor-specific delivery of anticancer drugs and highlights the promising potential of the mirror DNA nanostructures as a novel drug delivery platform.

Bioreducible-Cationic Poly(amido amine)s for Enhanced Gene Delivery and Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

The development of efficient and safe gene delivery carriers has been a major challenge in the clinical application of non-viral gene therapy. Herein, we report novel bioreducible poly(amido amine)s for the efficient delivery of genetic material such as plasmid DNA. A library of 34 different bioreducible polymer compounds was synthesized and screened to find lead materials for in vitro gene transfection. Our lead material (CBA-106) allows effortless polyplex formation with genetic materials by electrostatic interactions at the weight ratio of 1:5 (DNA/polymer). Polyplexes were further characterized by DLS and AFM analysis. Enhanced serum stability and bioreducibility under physiological conditions were confirmed, in addition to low cellular cytotoxicity. When compared with a commercially available gene delivery carrier (Lipofectamine 2000), CBA-1 06 shows comparable or even surpassing gene transfection efficiency. Furthermore, BMP-2 plasmids were efficiently delivered to tonsil-derived mesenchymal stem cells (TMSCs) for osteogenic commitment in vitro and in vivo. Taken together, our results clearly demonstrate the potential of novel bioreducible polymeric systems for gene delivery applications. We suggest that our system can provide a valuable platform for the broad application of gene regulation in cell therapy and regenerative medicine.

Viral Effects of a dsRNA Mycovirus (PoV-ASI2792) on the Vegetative Growth of the Edible Mushroom Pleurotus ostreatus.

A double-stranded RNA (dsRNA) mycovirus was detected in malformed fruiting bodies of Pleurotus ostreatus strain ASI2792, one of bottle cultivated commercial strains of the edible oyster mushroom. The partial RNA-dependent RNA polymerase (RdRp) gene of the P. ostreatus ASI2792 mycovirus (PoV-ASI2792) was cloned, and a cDNA sequences alignment revealed that the sequence was identical to the RdRp gene of a known PoSV found in the P. ostreatus strain. To investigate the symptoms of PoV-ASI2792 infection by comparing the isogenic virus-free P. ostreatus strains with a virus-infected strain, isogenic virus-cured P. ostreatus strains were obtained by the mycelial fragmentation method for virus curing. The absence of virus was verified with gel electrophoresis after dsRNA-specific virus purification and Northern blot analysis using a partial RdRp cDNA of PoV-ASI2792. The growth rate and mycelial dry weight of virus-infected P. ostreatus strain with PoV-ASI2792 mycovirus were compared to those of three virus-free isogenic strains on 10 different media. The virus-cured strains showed distinctly higher mycelial growth rates and dry weights on all kinds of experimental culture media, with at least a 2.2-fold higher mycelial growth rate on mushroom complete media (MCM) and Hamada media, and a 2.7-fold higher mycelial dry weight on MCM and yeastmalt-glucose agar media than those of the virus-infected strain. These results suggest that the infection of PoV mycovirus has a deleterious effect on the vegetative growth of P. ostreatus.

DhITACT: DNA Hydrogel Formation by Isothermal Amplification of Complementary Target in Fluidic Channels.

DNA hydrogel formation by isothermal amplification of complementary targets in microfluidic channels (DhITACT) is a new platform for rapid and accurate detection of infectious pathogens. DNA hydrogel is formed in situ within microfluidic channels by the isothermal rolling circle amplification process upon the selective binding of target strands from the biological fluid. Once the volume of DNA hydrogel sufficiently enlarges, it can selectively block the matching channels with target pathogens.

Induced myogenic commitment of human chondrocytes via non-viral delivery of minicircle DNA.

Lineage conversion from one somatic cell type to another is an attractive approach for deriving specific therapeutic cell generation. In order to bypass inducing pluripotent stage, transdifferentiation/direct conversion technologies have been recently developed. We report the development of a direct conversion methodology in which cells are transdifferentiated through a plastic intermediate state induced by exposure to non-integrative minicircle DNA (MCDNA)-based reprogramming factors, followed by differentiation into myoblasts. In order to increase the MCDNA delivery efficiency, reprogramming factors were delivered into the chondrocytes via electroporation followed by poly (ß-amino esters) (PBAE) transfection. We used this approach to convert human chondrocytes to myoblast, and with treatment of SB-431542, an inhibitor of the activin receptor-like kinase receptors, to enhance myogenic commitment. Differentiated cells exhibited expression of myogenic markers such as MyoD and Myog. This methodology for direct lineage conversion from chondrocytes to myoblast represents a novel non-viral Method to convert hard-to-transfect cells to other lineage.

Efficient delivery of siRNAs by a photothermal approach using plant flavonoid-inspired gold nanoshells.

Polymer gold nanoshells (PGNs) are prepared by a novel plant-inspired flavonoid surface modification method. The PGNs show dramatic photothermal properties, which can facilitate the endosomal escape and delivery of siRNA into the cytoplasm of cells. Efficient gene silencing has been achieved using siRNA immobilized PGNs, suggesting the potential applications of in vitro gene regulation by an external NIR stimulus.

Self-assembled DNA nanostructures prepared by rolling circle amplification for the delivery of siRNA conjugates.

Inspired by the isothermal enzymatic process of rolling circle amplification (RCA) of DNA strands, we have developed a system to achieve more than a 200-fold increase in the synthesis of DNA nanostructures using a single-stranded circular DNA template. The amplified DNA nanostructures have shown efficient delivery of folic acid (FA) conjugated siRNAs into KB cells with a dose dependent gene silencing.

Selective semihydrogenation of alkynes on shape-controlled palladium nanocrystals.

A systematic study on the selective semihydrogenation of alkynes to alkenes on shape-controlled palladium (Pd) nanocrystals was performed. Pd nanocrystals with a cubic shape and thus exposed {100} facets were synthesized in an aqueous solution through the reduction of Na2PdCl4 with L-ascorbic acid in the presence of bromide ions. The Pd nanocubes were tested as catalysts for the semihydrogenation of various alkynes such as 5-decyne, 2-butyne-1,4-diol, and phenylacetylene. For all substrates, the Pd nanocubes exhibited higher alkene selectivity (>90 %) than a commercial Pd/C catalyst (75-90 %), which was attributed to a large adsorption energy of the carbon-carbon triple bond on the {100} facets of the Pd nanocubes. Our approach based on the shape control of Pd nanocrystals offers a simple and effective route to the development of a highly selective catalyst for alkyne semihydrogenation.