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.

Molecular evolution of connective tissue growth factor in Cyprinidae (Teleostei : Cypriniformes)

Connective tissue growth factor (CTGF) plays an important role in regulation of cell growth, differentiation, apoptosis and individual development in animals. The study of sequences variation and molecular evolution of CTGF gene across various species of the cyprinid could be helpful for understanding of speciation and gene divergence in this kind of fish. In this study, 19 novel sequences of CTGF gene were obtained from the representative species of the family Cyprinidae using PCR amplification, cloning and sequencing. Phylogenetic relationships of Cyprinidae were reconstructed by neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian method. Oryzias latipes from the family Cyprinodontidae was assigned to be the outgroup taxon. Leuciscini and Barbini were clustered into the monophyletic lineages, respectively, with the high nodal supports. The estimation of the ratio of non-synonymous to synonymous substitution (dN/dS) for the various branches indicated that there stood the different evolution rates between the Leuciscini and the Barbini. With the ratio of dN/dS of the Leuciscini being lower than that of the Barbini, species within the Barbini were demonstrated to be subjected to the relatively less selection pressure and under the relaxable evolution background. A 6 by indel (insertion/deletion) was found at the 5' end of CTGF gene of Cyprinidae, and this 6 by deletion only appeared in the Leuciscini, which is a typical characteristic of the Leuciscini and provides evidence for the monophylogeny of the Leuciscini. For the amino acid sequences of CTGF protein, the most variations and indels were distributed in the signal region and IGFBP region of this protein, implying that these variations were correlated with the regulation of the CTGF gene expression and protein activity. (c) 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Destabilization of basic fibroblast growth factor by oligodeoxynucleotides

The effects of oligodeoxynucleotide (ODN) on the conformation of basic fibroblast growth factor (bFGF) were studied by spectral method. The results showed that ODN destabilized the protein.

Insulin-like growth factor-binding protein-3 plays an important role in regulating pharyngeal skeleton and inner ear formation and differentiation

Insulin-like growth factor-binding protein (IGFBP)-3 is the major insulin-like growth factor (IGF) carrier protein in the bloodstream. IGFBP-3 prolongs the half-life of circulating IGFs and prevents their potential hypo-glycemic effect. IGFBP-3 is also expressed in many peripheral tissues in fetal and adult stages. In vitro, IGFBP-3 can inhibit or potentiate IGF actions and even possesses IGF-independent activities, suggesting that local IGFBP-3 may also have paracrine/autocrine function(s). The in vivo function of IGFBP-3, however, is unclear. In this study, we elucidate the developmental role of IGFBP-3 using the zebrafish model. IGFBP-3 mRNA expression is first detected in the migrating cranial neural crest cells and subsequently in pharyngeal arches in zebrafish embryos. IGFBP-3 mRNA is also persistently expressed in the developing inner ears. To determine the role of IGFBP-3 in these tissues, we ablated the IGFBP-3 gene product using morpholino-modified antisense oligonucleotides (MOs). The IGFBP-3 knocked down embryos had delayed pharyngeal skeleton morphogenesis and greatly reduced pharyngeal cartilage differentiation. Knockdown of IGFBP-3 also significantly decreased inner ear size and disrupted hair cell differentiation and semicircular canal formation. Furthermore, reintroduction of a MO-resistant form of IGFBP-3 "rescued" the MO-induced defects. These findings suggest that IGFBP-3 plays an important role in regulating pharyngeal cartilage and inner car development and growth in zebrafish.

Expression of human epidermal growth factor gene in cyanobacteria

The human epidermal growth factor (hEGF) is a small single-chain polypeptide of 53 amino acid residues. It can stimulate the proliferation of many cell types, mainly those of epidermal and epithelial tissues both in vivo and in vitro. A vector pRL-hEGF was constructed using plasmids pRL-489 and pUC-hEGF. The synthetic hEGF gene was recombined into the downstream of strong promoter psbA in plasmids pRL-489. Then, the vector was introduced into Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120 by triparental conjugative transfer. The transformation was confirmed by PCR amplification. The pRL-hEGF is thought to be retained as a plasmid form in the transgenic Anabaena sp. PCC 7120, since it can be recovered. However, it has been integrated into the chromosome of Synechococcus sp. PCC 7002 as there is no duplication origin in the pRL-hEGF in this cyanobacterium. and plasmid cannot be isolated from the Synechococcus sp. PCC 7002 either. The radioimmunoassay (RIA) proved that the hEGF gene has been expressed as the protein existed in these two strains of transgenic cyanobacteria, and the hEGF protein in Anabaena sp. PCC 7002 could be secreted into the medium.

A role for LH in the regulation of expression of mRNAs encoding components of the insulin-like growth factor (IGF) system in the ovine corpus luteum

P.M. Hastie and W. Haresign (2006). A role for LH in the regulation of expression of mRNAs encoding components of the insulin-like growth factor (IGF) system in the ovine corpus luteum. Animal Reproduction Science, 96(1-2), 196-209. Sponsorship: DEFRA RAE2008

The rpf gene of Micrococcus luteus encodes an essential secreted growth factor

Galina V. Mukamolova, Obolbek A. Turapov, Konstantin Kazarian, Miroslav Telkov, Arseny S. Kaprelyants, Douglas B. Kell and Michael Young (2002). The rpf gene of Micrococcus luteus encodes an essential secreted growth factor. Molecular Microbiology, 46 (3), 611-621. Sponsorship: BBSRC / Russian Foundation for Basic Research (grant 00-04-48691)/ WHO Global Programme for Vaccines and Immunization / Wellcome Trust RAE2008

Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in vitro oocyte maturation, and embryo development in mice

Insulin-like growth factor-I (IGF-I) is involved in the regulation of ovarian follicular development and has been shown to potentiate the FSH responsiveness of granulosa cells from preantral follicles. The aim of the present study was to investigate the effect of IGF-I during preantral follicular culture on steroidogenesis, subsequent oocyte maturation, fertilization, and embryo development in mice. Preantral follicles were isolated mechanically and cultured for 12 days in a simplified culture medium supplemented with 1% fetal calf serum, recombinant human FSH, transferrin, and selenium. In these conditions, follicles were able to grow and produce oocytes that could be matured and fertilized. The first experiment analyzed the effect of different concentrations of IGF-I (0, 10, 50, or 100 ng/ml) added to the culture medium on the follicular survival, steroidogenesis, and the oocyte maturation process. The presence of IGF-I during follicular growth increased the secretion of estradiol but had no effect on the subsequent oocyte survival and maturation rates. In the second experiment, IGF-I (0 or 50 ng/ml) was added to the culture medium during follicular growth, oocyte maturation, or both, and subsequent oocyte fertilization and embryo development rates were evaluated. Oocyte fertilization rates were comparable in the presence or absence of IGF-I. However, the blastocyst development rate was enhanced after follicular culture in the presence of IGF-I. Moreover, the total cell number of the blastocysts observed after differential labeling staining was also higher when follicles were cultured or matured in the presence of IGF-I.

Growth factor erv1-like modulates Drp1 to preserve mitochondrial dynamics and function in mouse embryonic stem cells.

The relationship of mitochondrial dynamics and function to pluripotency are rather poorly understood aspects of stem cell biology. Here we show that growth factor erv1-like (Gfer) is involved in preserving mouse embryonic stem cell (ESC) mitochondrial morphology and function. Knockdown (KD) of Gfer in ESCs leads to decreased pluripotency marker expression, embryoid body (EB) formation, cell survival, and loss of mitochondrial function. Mitochondria in Gfer-KD ESCs undergo excessive fragmentation and mitophagy, whereas those in ESCs overexpressing Gfer appear elongated. Levels of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) are highly elevated in Gfer-KD ESCs and decreased in Gfer-overexpressing cells. Treatment with a specific inhibitor of Drp1 rescues mitochondrial function and apoptosis, whereas expression of Drp1-dominant negative resulted in the restoration of pluripotency marker expression in Gfer-KD ESCs. Altogether, our data reveal a novel prosurvival role for Gfer in maintaining mitochondrial fission-fusion dynamics in pluripotent ESCs.

The influence of transforming growth factor-beta1 gene polymorphisms on the severity of gingival overgrowth associated with concomitant use of cyclosporin A and a calcium channel blocker

The purpose of this study was to determine whether the prevalence and severity of gingival overgrowth in renal transplant recipients concomitantly treated with cyclosporin and a calcium channel blocker was associated with functional polymorphisms within the signal sequence of the transforming growth factor-(TGF)beta1 gene.