Animal Cells and Systems 

Abstract: Generation of induced pluripotent stem cells (iPSCs) by defined factors (OCT4, SOX2, C-MYC, and KLF4) from various human primary cells has been reported. Human fibroblasts have been widely used as a cellular source in reprogramming studies over recent decades. The original method of iPSC generation uses retro- or lentivirus vectors that require integration of viral DNA into the target cells. The integration of exogenous genes encoding transcription factors (OCT4, SOX2, C-MYC, and KLF4) can be detected in iPSCs, raising concern about the risk of mutagenesis and tumor formation. Therefore, stem cell therapy would ideally require generation of integration-free iPSCs using non-integration gene delivery system such as Sendai virus, recombinant proteins, synthetic mRNA, and episomal vectors. Several groups have reported that episomal vectors are capable of reprogramming human fibroblasts into iPSCs. Although vector concentration and cell density are important in the episomal vector reprogramming method, optimization of this method for human fibroblasts has not been reported. In this study, we determined optimal conditions for generating integration-free iPSCs from human fibroblasts through the use of different concentrations of episomal vectors (OCT4/p53, SOX2/KLF4, L-MYC/LIN28A) and different plating cell density. We found that optimized vector concentration and cell density accelerate reprogramming and improve iPSC generation. Our study provides a detailed stepwise protocol for improved generation of integration-free iPSCs from human fibroblasts by transfection with episomal vectors.

Abstract: Programmed cell death, or apoptosis, is one of the most studied areas of modern biology. Apoptosis is a genetically regulated process, which plays an essential role in the development and homeostasis of higher organisms. Mitochondria, known to play a central role in regulating cellular metabolism, was found to be critical for regulating apoptosis induced under both physiological and pathological conditions. Mitochondria are a major source of reactive oxygen species (ROS) but they can also serve as its target during the apoptosis process. Release of apoptogenic factors from mitochondria, the best known of which is cytochrome c, leads to assembly of a large apoptosis‐inducing complex called the apoptosome. Cysteine proteases (called caspases) are recruited to this complex and, following their activation by protectlytic cleavage, activate other caspases, which in turn target for specific cleavage a large number of cellular proteins. The redox regulation of apoptosis during and after cytochrome c release is a...

Abstract: Neural stem cells (NSCs) are capable of self-renewal and can differentiate into neurons and two types of glial cells. NSCs have great potential for basic studies of neurogenesis and neurodegenerative diseases, and for therapeutic transplantation applications. NSCs can be derived from the fetal or adult brain. In vitro expansion of NSCs may help to elucidate their properties involved in neurogenesis and is crucial to obtain on-demand the large numbers of these cells needed for clinical transplantation trials. Laminin is an extracellular matrix molecule commonly used to culture NSCs as an attached monolayer. However, laminin is costly if NSCs need to be cultured in large quantities. Matrigel is a soluble basement membrane biomaterial, which has been widely used for feeder-free cultures of human embryonic stem cells. In the present study, we evaluated the ability of Matrigel to support the attachment and long-term proliferation of NSCs. We investigated the growth and adherence of NSCs derived from th...

Abstract: We analyzed and compared profiles of flavonols extracted from leaves and exocarps of the grape Kyoho by TLC, HPLC and UV spectrophotometry. In the exocarps, quercetin 3‐O‐glucoside was the main compound while isorhamnetin 3‐O‐glycoside (I) was present in minor amounts. In leaves, on the other hand, quercetin 3‐O‐glucoside and quercetin 3‐O‐glucoside‐7‐O‐glucronide were the major compounds while isorhamnetin 3‐O‐glycoside (II) and kaempferol 3, 7‐O‐diglycoside were present in minor amounts.