Serum-free culture of rhesus monkey embryonic stem cells

Previous studies have shown that the maintenance and proliferation of undifferentiated rhesus monkey embryonic stem (rES) cells requires medium supplemented with fetal bovine serum (FBS). Due to the uncharacterized composition and variation in serum nature, the present study aimed to replace the serum-containing medium with a serum-free medium in the rES cell culture. The results showed that after the initial 48-h culture in the routinely used serum-containing medium, rES cells can grow and proliferate for a prolonged period in the serum-free medium composed of DMEM supplemented with a cocktail of BSA, IGF-1, TGF-alpha, bFGF, aFGF, estradiol, and progesterone. rES cells cultured in the serum-free medium maintained high level of alkaline phosphatase activity and OCT4 level. There was no indication of differentiation as judged by the marker gene expression of all three embryonic germ layers and trophoblast. In addition, serum-free culture would not affect the passage capacity and differentiation potential of rES cells. This work will facilitate the future study of induced differentiation of rES cells and other applications.

Feeder layer- and serum-free culture of rhesus monkey embryonic stem cells

The common culture system of rhesus monkey embryonic stem (rES) cells depends largely on feeder cells and serum, which limits the research and application of rES cells. This study reports a feeder layer-free and serum-free system for culture of rES cells.

Hepatocyte growth factor enhances the generation of high-purity oligodendrocytes from human embryonic stem cells

Generation of homogeneous oligodendrocytes as donor cells is essential for human embryonic stem cell (hESC)-based cell therapy for demylinating diseases. Herein we present a novel method for efficiently obtaining mature oligodendrocytes from hESCs with high purity (79.7 +/- 6.9%), using hepatocyte growth factor (HGF) and G5 supplement(containing insulin, transferrin, selenite, biotin, hydrocortisone, basic fibroblast growth factor and epidermal growth factor) in a four-step method. We induced hESCs into neural progenitors (NP) with HGF (5 ng/ml) and G5 (1 x) supplemented medium in an adherent differentiation system. The purified NPs were amplified in suspension as neurospheres for 1 month, and terminal oligodendrocyte differentiation was then induced by G5 supplement withdrawal and HGF treatment (20 ng/ml). The cells generated displayed typical morphologies of mature oligodendrocytes and expressed oligodendrocyte markers O4 and myelin basic protein (MBP). Our result revealed that HGF significantly enhanced the proliferation of hESC-derived NPs and promoted the differentiation as well as the maturation of oligodendrocytes from NPs. Further studies suggest that HGF/c-Met signaling pathway might play an important role in oligodendrocyte differentiation in our system. Our studies provide a means for generating the clinically relevant cell type and a platform for deciphering the molecular mechanisms that control oligodendrocyte differentiation. (C) 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.

Statistical correlation between protein secondary structure and messenger RNA stem-loop structure

Anew integrated sequence-structure database, called IADE (Integrated ASTRAL-DSSP-EMBL), incorporating matching mRNA sequence, amino acid sequence, and protein secondary structural data, is constructed. It includes 648 protein domains. Based on the IADE database, we studied the relation between RNA stem-loop frequencies and protein secondary structure. It was found that the alpha-helices and beta-strands on proteins tend to be preferably "coded" by mRNA stem region, while the coils on proteins tend to be preferably "coded" by mRNA loop region. These tendencies are more obvious if we observe the structural words (SWs). An SW is defined by a four-amino-acid-fragment that shows the pronounced secondary structural (alpha-helix or beta-strand) propensity. It is demonstrated that the deduced correlation between protein and mRNA structure can hardly be explained as the stochastic fluctuation effect. (C) 2003 Wiley Periodicals, Inc.

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

Two new benzochromone glycosides from the stem of Berchemia racemosa

Two new benzochromone glycosides, rubrofusarin 6-O-alpha-L-rhamnosyl- (1 -> 6)-O-beta-D-glucopyranoside (1) and demethylflavasperone 10-O-beta-D-glucopyranoside (2), have been isolated from the stem of Berchemia racemosa Sieb. et Zucc. (Rhamnaceae). Their structures were elucidated on the basis of spectroscopic evidence.