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.

Auxetic nuclei in embryonic stem cells exiting pluripotency.

Embryonic stem cells (ESCs) self-renew in a state of naïve pluripotency in which they are competent to generate all somatic cells. It has been hypothesized that, before irreversibly committing, ESCs pass through at least one metastable transition state. This transition would represent a gateway for differentiation and reprogramming of somatic cells. Here, we show that during the transition, the nuclei of ESCs are auxetic: they exhibit a cross-sectional expansion when stretched and a cross-sectional contraction when compressed, and their stiffness increases under compression. We also show that the auxetic phenotype of transition ESC nuclei is driven at least in part by global chromatin decondensation. Through the regulation of molecular turnover in the differentiating nucleus by external forces, auxeticity could be a key element in mechanotransduction. Our findings highlight the importance of nuclear structure in the regulation of differentiation and reprogramming.

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.

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.

miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro

The mammalian nervous system exerts essential control on many physiological processes in the organism and is itself controlled extensively by a variety of genetic regulatory mechanisms. microRNA (miR), an abundant class of small non-coding RNA, are emerging as important post-transcriptional regulators of gene expression in the brain. Increasing evidence indicates that miR regulate both the development and function of the nervous system. Moreover, deficiency in miR function has also been implicated in a number of neurological disorders. Expression profile analysis of miR is necessary to understand their complex role in the regulation of gene expression during the development and differentiation of cells. Here we present a comparative study of miR expression profiles in neuroblastoma, in cortical development, and in neuronal differentiation of embryonic stem (ES) cells. By microarray profiling in combination with real time PCR we show that miR-7 and miR-214 are modulated in neuronal differentiation (as compared to miR-1, -16 and -133a), and control neurite outgrowth in vitro. These findings provide an important step toward further elucidation of miR function and miR-related gene regulatory networks in the mammalian central nervous system. (C) 2010 Elsevier Inc. All rights reserved.

Sca-1+ progenitors derived from embryonic stem cells differentiate into endothelial cells capable of vascular repair after arterial injury

Embryonic stem cells possess the ability to differentiate into endothelium. The ability to produce large volumes of endothelium from embryonic stem cells could provide a potential therapeutic modality for vascular injury. We describe an approach that selects endothelial cells using magnetic beads that may be used therapeutically to treat arterial injury.

From embryonic stem cells to sensory hair cells : a programming approach

Tese de doutoramento, Ciências Biomédicas (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Medicina, 2014

Enhancing and diminishing gene function in human embryonic stem cells

It is widely recognized that gain- and loss-of-function approaches are essential for understanding the functions of specific genes, and such approaches would be particularly valuable in studies involving human embryonic stem (hES) cells. We describe a simple and efficient approach using lipofection to transfect hES cells, which enabled us to generate hES cell lines expressing naturally fluorescent green or red proteins without affecting cell pluripotency. We used these cell lines to establish a means of diminishing gene function using small interfering (si)RNAs, which were effective at knocking down gene expression in hES cells. We then demonstrated that stable expression of siRNA could knock down the expression of endogenous genes. Application of these gain- and loss-of-function approaches should have widespread use, not only in revealing the developmental roles of specific human genes, but also for their utility in modulating differentiation.

Establishment of a Brazilian Line of Human Embryonic Stem Cells in Defined Medium: Implications for Cell Therapy in an Ethnically Diverse Population

Pluripotent human embryonic stem (hES) cells are an important experimental tool for basic and applied research, and a potential source of different tissues for transplantation. However, one important challenge for the clinical use of these cells is the issue of immunocompatibility, which may be dealt with by the establishment of hES cell banks to attend different populations. Here we describe the derivation and characterization of a line of hES cells from the Brazilian population, named BR-I, in commercial defined medium. In contrast to the other hES cell lines established in defined medium, BR-I maintained a stable normal karyotype as determined by genomic array analysis after 6 months in continuous culture (passage 29). To our knowledge, this is the first reported line of hES cells derived in South America. We have determined its genomic ancestry and compared the HLA-profile of BR-I and another 22 hES cell lines established elsewhere with those of the Brazilian population, finding they would match only 0.011% of those individuals. Our results highlight the challenges involved in hES cell banking for populations with a high degree of ethnic admixture.

Phosphoproteomics Profiling Suggests a Role for Nuclear beta IPKC in Transcription Processes of Undifferentiated Murine Embryonic Stem Cells

Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal and differentiation However, the function of specific PKC Isoenzymes have yet to be determined Of the PKCs expressed in undifferentiated ESCs, beta IPKC was the only isoenzyme abundantly expressed in the nuclei To investigate the role of beta IPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one beta IPKC-specific inhibitor peptide We identified 13 nuclear proteins that are direct or indirect beta IPKC substrates in undifferentiated ESCs These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation Inhibiting beta IPKC had no effect on DNA synthesis in undifferentiated ESCs However, upon differentiation many cells seized to express beta IPKC and beta IPKC was frequently found in the cytoplasm Taken together, our results suggest that beta IPKC takes part in the processes that maintain ESCs in their undifferentiated state