Characterization of trimethylpsoralen as a mutagen for mouse embryonic stem cells

Given a large number of genes with unknown functions in model organisms, collections of mutants are valuable resources for studying gene function. For the mouse, embryonic stem cell technology offers the possibility to manipulate the genome and select for mutations in vitro. Mutant mice can then be generated from clones of interest to study the phenotype of these animals. We manipulate the genome of mouse embryonic stem (ES) cells chemically using the mutagen trimethylpsoralen (TMP). TMP predominantly causes deletions in the genome of Caenorhabditis elegans and Escherichia coli, but has not been established as a mutagen in mammalian systems yet. We have characterized TMP as a mutagen for mouse ES cells regarding death rates, mutation frequencies, and mutation spectrum. Allowing for 12.5% of cell survival, the mutation frequency at the mouse hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus was 3.5 x 10(-5) on average. The characterization of a non-redundant set of 17 Hprt-deficient ES clones revealed that only 12% of clones contained genomic deletions and almost 50% were point mutations. Base substitutions were mostly transversions and all affected AT base pairs. We conclude that the mutation spectrum of TMP in mouse ES cells is different from that observed in C. elegans and E. coli.     

Microarray analysis of LTR retrotransposon silencing identifies Hdac1 as a regulator of retrotransposon expression in mouse embryonic stem cells

Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells.     

ARHGAP21 as a master regulator of multiple cellular processes

The cellular cytoskeleton is involved with multiple biological processes and is tightly regulated by multiple proteins and effectors. Among these, the RhoGTPases family is one of the most important players. RhoGTPAses are, in turn, regulated by many other elements. In the past decade, one of those regulators, the RhoGAP Rho GTPase Activating Protein 21 (ARHGAP21), has been overlooked, despite being implied as having an important role on many of those processes. In this paper, we aimed to review the available literature regarding ARHGAP21 to highlight its importance and the mechanisms of action that have been found so far for this still unknown protein involved with cell adhesion, migration, Golgi regulation, cell trafficking, and even insulin secretion.     

Derivation of mouse embryonic stem cells

Here we describe a simple and efficient protocol for derivation of germline chimera-competent mouse embryonic stem cells (mESCs) from embryonic day 3.5 (E3.5) blastocysts. The protocol involves the use of early-passage mouse embryonic fibroblast feeders (MEF) and the alternation of fetal bovine serum- and serum replacement (SR)-containing media. As compared to other available protocols for mESCs derivation, our protocol differs in the combination of commercial availability of all reagents, technical simplicity and high efficiency. mESC lines are derived with approximately 50% efficiency (50 independent mESC lines derived from 96 blastocysts). We believe that this protocol could be a good starting point for (i) setting up the derivation of mESC lines in a laboratory and (ii) incorporating further steps to improve efficiency or adapt the protocol to other applications. The whole process (from blastocyst extraction to the freezing of mESC line) usually takes between 15 and 20 d.     

Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells

Telomeres are essential for protecting the ends of chromosomes and preventing chromosome fusion. Telomere loss has been proposed to play an important role in the chromosomal rearrangements associated with tumorigenesis. To determine the relationship between telomere loss and chromosome instability in mammalian cells, we investigated the events resulting from the introduction of a double-strand break near a telomere with I-SceI endonuclease in mouse embryonic stem cells. The inactivation of a selectable marker gene adjacent to a telomere as a result of the I-SceI-induced double-strand break involved either the addition of a telomere at the site of the break or the formation of inverted repeats and large tandem duplications on the end of the chromosome. Nucleotide sequence analysis demonstrated large deletions and little or no complementarity at the recombination sites involved in the formation of the inverted repeats. The formation of inverted repeats was followed by a period of chromosome instability, characterized by amplification of the subtelomeric region, translocation of chromosomal fragments onto the end of the chromosome, and the formation of dicentric chromosomes. Despite this heterogeneity, the rearranged chromosomes eventually acquired telomeres and were stable in most of the cells in the population at the time of analysis. Our observations are consistent with a model in which broken chromosomes that do not regain a telomere undergo sister chromatid fusion involving nonhomologous end joining. Sister chromatid fusion is followed by chromosome instability resulting from breakage-fusion-bridge cycles involving the sister chromatids and rearrangements with other chromosomes. This process results in highly rearranged chromosomes that eventually become stable through the addition of a telomere onto the broken end. We have observed similar events after spontaneous telomere loss in a human tumor cell line, suggesting that chromosome instability resulting from telomere loss plays a role in chromosomal rearrangements associated with tumor cell progression.     

Parthenotes as a source of embryonic stem cells

Abstract.  The derivation and study of human embryonic stem cell lines, despite their potential therapeutic usefulness, raise considerable ethical, religious, legal and political concerns because it inevitably leads to the destruction of viable embryos. In an attempt to bridge the division between ethical questions and potential scientific and medical benefits, considerable efforts have been devoted to the search for alternative sources of pluripotent cell lines. In this review we discuss the use of artificial parthenogenesis as a way to create entities, called parthenotes, that may represent an alternative ethical source for pluripotent cell lines. We describe the biological differences between parthenotes and embryos, in order to provide a rationale for the discussion on whether their use can be acceptable as a source of stem cells. We present data derived from animal models on the extent parthenogenetic stem cells are similar to biparental cell lines and discuss these aspects in the context of their extension to the human species. Finally, we present experiments recently carried out in our laboratory that allowed us to generate human parthenotes through artificial activation of human oocytes and to use them as a source for the derivation of parthenogenetic pluripotent cell lines.     

Enhanced gene trapping in mouse embryonic stem cells

Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.     

Citrinin induces apoptosis in mouse embryonic stem cells

The mycotoxin citrinin (CTN) is a natural contaminant in foodstuffs and animal feeds, and exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis. However, its precise regulatory mechanisms of action, particularly in stem cells and embryos, are currently unclear. Recent studies show that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Experiments with the embryonic stem cell line, ESC-B5, disclose that CTN induces apoptosis via several mechanisms, including ROS generation, increased cytoplasmic free calcium levels, intracellular nitric oxide production, enhanced Bax/Bcl-2 ratio, loss of mitochondrial membrane potential, cytochrome c release, activation of caspase-9 and caspase-3, and p21-activated protein kinase 2 and c-Jun N-terminal protein kinase activation. Additional studies show that CTN promotes cell death via inactivation of the HSP90/multi-chaperone complex and subsequent degradation of Ras and Raf-1, further inhibiting anti-apoptotic processes such as the Ras-->ERK signal transduction pathway. On the basis of these findings, we propose a model for CTN-induced cell injury signalling cascades in embryonic stem cells and blastocysts.     

Bi-allelic gene targeting in mouse embryonic stem cells

The EUCOMM and KOMP programs have generated targeted conditional alleles in mouse embryonic stem cells for nearly 10,000 genes. The availability of these stem cell resources will greatly accelerate the functional analysis of genes in mice and in cultured cells. We present a method for conditional ablation of genes in ES cells using vectors and targeted clones from the EUCOMM and KOMP conditional resources. Inducible homozygous cells described here provide a precisely controlled experimental system to study gene function in a model cell.     

E-cadherin as a novel surface marker of spermatogonial stem cells

Spermatogenesis is a fundamental biological process that ensures the transition of a gene from one ganeration to another via male gametes. This process relies on a rare population of testicular cells called spermatogonial stem cells (SSCs), which self-renew throughout adult male life and differentiate into mature gametes. Despite the longstanding study of SSCs, their biological properties remain largely unknown, which is partly due to the very limited availability of these cells. Here, we show that cell adhesion protein E-cadherin is a highly specific surface marker of mouse SSCs that can be successfully used to enrich them.     

Generation of insulin-expressing cells from mouse embryonic stem cells

The therapeutic potential of transplantation of insulin-secreting pancreatic beta-cells has stimulated interest in using pluripotent embryonic stem (ES) cells as a starting material from which to generate insulin secreting cells in vitro. Mature beta-cells are endodermal in origin so most reported differentiation protocols rely on the identification of endoderm-specific markers. However, endoderm development is an early event in embryogenesis that produces cells destined for the gut and associated organs in the embryo, and for the development of extra-embryonic structures such as the yolk sac. We have demonstrated that mouse ES cells readily differentiate into extra-embryonic endoderm in vitro, and that these cell populations express the insulin gene and other functional elements associated with beta-cells. We suggest that the insulin-expressing cells generated in this and other studies are not authentic pancreatic beta-cells, but may be of extra-embryonic endodermal origin.     

Expression and knockdown of cellular prion protein (PrPC) in differentiating mouse embryonic stem cells

The mammalian cellular prion protein (PrP(C)) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrP(Sc)), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about pathogenic PrP conversion and its role in TSEs, the normal function of PrP(C) is poorly understood. Given the abundant expression of PrP(C) in the developing mammalian CNS and the spatial association with differentiated stages of neurogenesis, recently it has been proposed that PrP(C) participates in neural cell differentiation. In the present study, we investigated the role of PrP(C) in neural development during early embryogenesis. In bovine fetuses, PrP(C) was differentially expressed in the neuroepithelium, showing higher levels at the intermediate and marginal layers where more differentiated states of neurogenesis were located. We utilized differentiating mouse embryonic stem (ES) cells to test whether PrP(C) contributed to the process of neural differentiation during early embryogenesis. PrP(C) showed increasing levels of expression starting on Day 9 until Day 18 of ES cell differentiation. PrP(C) expression was negatively correlated with pluripotency marker Oct-4 confirming that ES cells had indeed differentiated. Induction of ES cells differentiation by retinoic acid (RA) resulted in up-regulation of PrP(C) at Day 20 and nestin at Day 12. PrP(C) expression was knocked down in PrP-targeted siRNA ES cells between Days 12 and 20. PrP(C) knockdown in ES cells resulted in nestin reduction at Days 16 and 20. Analysis of bovine fetuses suggests the participation of PrP(C) in neural cell differentiation during early embryogenesis. The positive association between PrP(C) and nestin expression provide evidence for the contribution of PrP(C) to ES cell differentiation into neural progenitor cells.     

Semliki Forest virus capsid protein acts as a pleiotropic regulator of host cellular protein synthesis

The Semliki Forest virus capsid (C) protein was introduced into various target cells by electroporation-, liposome-, and erythrocyte-ghost-mediated delivery. Data are presented which show that the incorporated C protein is biologically active and, at low concentrations (10(3) to 10(4) molecules per cell), markedly induces host cellular protein synthesis (average value, up to 90%). On the other hand, high concentrations (10(5) to 10(6) molecules per cell) led to a significant inhibition (average value, up to 60%). The cellular response to C protein was found to be identical in P3X63Ag8 suspension cells, CV-1 cells, and GpBind4 cells. Following electroporation-mediated delivery of C-protein molecules, both induction and repression of cellular protein synthesis were immediate, whereas with liposome-mediated delivery these events were delayed by about 1 h. Maximum stimulation and repression occurred between 0 and 1 h after delivery of C protein and decreased thereafter to reach control values at about 4 h. The analysis of the proteins synthesized suggests that low amounts of microinjected C protein are responsible for the induction of classes with specific Mrs, whereas high amounts lead to an inhibition of overall protein synthesis.     

Mouse embryonic stem cell-derived feeder cells support the growth of their own mouse embryonic stem cells

Feeder cells are usually used in culturing embryonic stem cells (ESCs) to maintain their undifferentiated and pluripotent status. To test whether mouse embryonic stem cells (mESCs) may be a source of feeder cells to support their own growth, 48 fibroblast-like cell lines were isolated from the same mouse embryoid bodies (mEBs) at three phases (10th day, 15th day, 20th day), and five of them, mostly derived from 15th day mEBs, were capable of maintaining mESCs in an undifferentiated and pluripotent state over 10 passages, even up to passage 20. mESCs cultured on the feeder system derived from these five cell lines expressed alkaline phosphatase and specific mESCs markers, including SSEA-1, Oct-4, Nanog, and formed mEBs in vitro and teratomas in vivo. These results suggest that mEB-derived fibroblasts (mEB-dFs) could serve as feeder cells that could sustain the undifferentiated growth and pluripotency of their own mESCs in culture. This study not only provides a novel feeder system for mESCs culture, avoiding a lot of disadvantages of commonly used mouse embryonic fibroblasts as feeder cells, but also indicates that fibroblast-like cells derived from mESCs take on different functions. Investigating the molecular mechanisms of these different functional fibroblast-like cells to act on mESCs will contribute to the understanding of the mechanisms of mESCs self-renewal.