Mouse ES Cell Line Research Articles

MPP8 Governs the Activity of the LIF/STAT3 Pathway and Plays a Crucial Role in the Differentiation of Mouse Embryonic Stem Cells.

Mouse embryonic stem cells (mESCs) possess the remarkable characteristics of unlimited self-renewal and pluripotency, which render them highly valuable for both fundamental research and clinical applications. A comprehensive understanding of the molecular mechanisms underlying mESC function is of the utmost importance. The Human Silence Hub (HUSH) complex, comprising FAM208A, MPP8, and periphilin, constitutes an epigenetic silencing complex involved in suppressing retroviruses and transposons during early embryonic development. However, its precise role in regulating mESC pluripotency and differentiation remains elusive. In this study, we generated homogenous miniIAA7-tagged Mpp8 mouse ES cell lines. Upon induction of MPP8 protein degradation, we observed the impaired proliferation and reduced colony formation ability of mESCs. Furthermore, this study unveils the involvement of MPP8 in regulating the activity of the LIF/STAT3 signaling pathway and Nanog expression in mESCs. Finally, we provide compelling evidence that degradation of the MPP8 protein impairs the differentiation of mESC.

On the accuracy of the epigenetic copy machine: comprehensive specificity analysis of the DNMT1 DNA methyltransferase.

The specificity of DNMT1 for hemimethylated DNA is a central feature for the inheritance of DNA methylation. We investigated this property in competitive methylation kinetics using hemimethylated (HM), hemihydroxymethylated (OH) and unmethylated (UM) substrates with single CpG sites in a randomized sequence context. DNMT1 shows a strong flanking sequence dependent HM/UM specificity of 80-fold on average, which is slightly enhanced on long hemimethylated DNA substrates. To explain this strong effect of a single methyl group, we propose a novel model in which the presence of the 5mC methyl group changes the conformation of the DNMT1-DNA complex into an active conformation by steric repulsion. The HM/OH preference is flanking sequence dependent and on average only 13-fold, indicating that passive DNA demethylation by 5hmC generation is not efficient in many flanking contexts. The CXXC domain of DNMT1 has a moderate flanking sequence dependent contribution to HM/UM specificity during DNA association to DNMT1, but not if DNMT1 methylates long DNA molecules in processive methylation mode. Comparison of genomic methylation patterns from mouse ES cell lines with various deletions of DNMTs and TETs with our data revealed that the UM specificity profile is most related to cellular methylation patterns, indicating that de novo methylation activity of DNMT1 shapes the DNA methylome in these cells.

Overcoming off-targets: assessing Western blot signals for Bcnt/Cfdp1, a tentative component of the chromatin remodeling complex

The Bucentaur (BCNT) protein family is characterized by a conserved amino acid sequence at the C-terminus (BCNT-C domain) and plays an essential role in gene expression and chromosomal maintenance in yeast and Drosophila. The mammalian Bucentaur/Craniofacial developmental protein 1 (Bcnt/Cfdp1) is also a tentative component of the SNF2-related CBP activator protein (Srcap) chromatin remodeling complex, but little is known about its properties, partly because few antibodies are available to examine the endogenous protein. In this paper, we assigned the Western blot signal against the mouse Bcnt/Cfdp1 as a doublet of approximately 45 kDa using anti-Bcnt/Cfdp1 antibodies, which were generated against either of two unrelated immunogens, BCNT-C domain or mouse N-terminal peptide, and in addition, the Cfdp1 knockdown mouse ES cell line and bovine tissue were used as potential negative controls. Moreover, LC-MS/MS analysis of the corresponding doublet to the Flag-tagged mouse Bcnt/Cfdp1 that was constitutively expressed in a HEK293 cell exhibited that the upper band was much more phosphorylated than the lower band with preferential Ser phosphorylation in the WESF motif of BCNT-C domain. Western blot analysis with these evaluated antibodies indicated a preferential expression of Bcnt/Cfdp1 in the early stages of brain development of mouse and rat, which is consistent with a data file of the expression of Bcnt/Cfdp1 mRNA.

Genome-wide tracking of dCas9-methyltransferase footprints

In normal mammalian development cytosine methylation is essential and is directed to specific regions of the genome. Despite notable advances through mapping its genome-wide distribution, studying the direct contribution of DNA methylation to gene and genome regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of the CRISPR–Cas9 system with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9 (dCas9) methyltransferase fusion protein is challenging within a highly methylated genome. Here, we report the generation and use of an engineered, methylation depleted but maintenance competent mouse ES cell line and find surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Subsequent experiments in human somatic cells refine these observations and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations.

Utilising the resources of the International Knockout Mouse Consortium: the Australian experience

Mouse models play a key role in the understanding gene function, human development and disease. In 2007, the Australian Government provided funding to establish the Monash University embryonic stem cell-to-mouse (ES2M) facility. This was part of the broader Australian Phenomics Network, a national infrastructure initiative aimed at maximising access to global resources for understanding gene function in the mouse. The remit of the ES2M facility is to provide subsidised access for Australian biomedical researchers to the ES cell resources available from the International Knockout Mouse Consortium (IKMC). The stated aim of the IKMC is to generate a genetically modified mouse ES cell line for all of the ~23,000 genes in the mouse genome. The principal function of the Monash University ES2M service is to import genetically modified ES cells into Australia and to convert them into live mice with the potential to study human disease. Through advantages of economy of scale and established relationships with ES cell repositories worldwide, we have created over 110 germline mouse strains sourced from all of the major ES providers worldwide. We comment on our experience in generating these mouse lines; providing a snapshot of a "clients" perspective of using the IKMC resource and one which we hope will serve as a guide to other institutions or organisations contemplating establishing a similar centralised service.

Expression of Histocompatibility 2 Blastocyst (H2-Bl) in Embryonic Stem Cells Inhibits CD8+ T-Cell Activation but is not Sufficient to Facilitate Graft Tolerance

Universal embryonic stem cell donor lines would greatly facilitate stem cell based regenerative medicine and permit facile testing of human pluripotent stem cell derived grafts in xenogeneic settings. Because HLA-G overcomes immune cell mediated attack of foetal tissues during pregnancy and inhibits T-cell responses and dendritic cell antigen maturation in vitro and in vivo, it is an attractive candidate molecule for achieving this goal. Here we investigated whether enforced expression of either the soluble or the membrane bound form of the HLA-G mouse homologue, H2- Bl, in human and mouse ES cell lines would allow engraftment in immunocompetent mice. Despite evidence for robust expression of soluble or membrane bound H2-Bl molecules and effective inhibition of CD8+ T-cell proliferation by all H2-Bl engineered ES cell lines, all failed to generate teratomas in immunocompetent mice, despite doing so in NODSCID mice. We conclude that expression of H2-Bl in human and mouse embryonic stem cells alone is insufficient to overcome xenogeneic rejection.

Generation of induced pluripotent stem cells from virus-free in vivo reprogramming of BALB/c mouse liver cells

The in vivo cell reprogramming of terminally differentiated somatic cells to a pluripotent state by the ectopic expression of defined transcription factors has been previously shown in the BALB/c mouse liver upon plasmid DNA injection with no teratoma formation in the host tissue. Here, we hypothesized that the reprogrammed cells could be extracted from the tissue and cultured in vitro. We called these cells in vivo induced pluripotent stem (i2PS) cells because they showed pluripotent characteristics equivalent to a standard mouse ES cell line (E14TG2A). The pluripotent character of i2PS cells was determined by a battery of morphological, molecular and functional assays, including their contribution to adult tissues of chimeric mice upon blastocyst injection. These observations further confirm that terminally differentiated somatic cells in wild type, adult animals can be reprogrammed in vivo using virus-free methodologies. The reprogrammed cells can generate in vitro stem cell colonies that exhibit pluripotency similar to ES cells with numerous implications for the application of in vivo reprogramming for tissue regenerative purposes.

Targeting human embryonic stem cells with quantum dot-conjugated phages

Targeting embryonic stem cells (ESCs) is important for ESC labeling, drug delivery and cell fate control. In this study, we identified twenty-two phage clones that bind specifically to the hESC cell line X-01, which was derived from human blastocysts of Chinese origin. One phage (H178), which displays the sequence VGGEAWSSPTDL, showed higher binding affinity to hESCs than to a monkey ES cell line (RS366.4) and two mouse ES cell lines (R1 and E14). Using quantum dots (QDs) conjugated to the H178 phage, we demonstrate that the phage can specifically bind to hESCs in vitro. Our results suggest a possible interaction between the selected peptide and the stem cell extracellular matrix (ECM). The selection method described here allows rapid and efficient screening of unique phage clones and targeting cells. The phages displaying peptides identified by this study have potential applications for cargo delivery and receptor studies.

MacroH2A1 histone variant represses rDNA transcription

The regulation of ribosomal DNA transcription is an important step for the control of cell growth. Epigenetic marks such as DNA methylation and posttranslational modifications of canonical histones have been involved in this regulation, but much less is known about the role of histone variants. In this work, we show that the histone variant macroH2A1 is present on the promoter of methylated rDNA genes. The inhibition of the expression of macroH2A1 in human HeLa and HepG2 cells and in a mouse ES cell line resulted in an up to 5-fold increase of pre-rRNA levels. This increased accumulation of pre-rRNA is accompanied by an increase of the loading of RNA polymerase I and UBF on the rDNA without any changes in the number of active rDNA genes. The inhibition of RNA polymerase I transcription by actinomycin D or by knocking down nucleolin, induces the recruitment of macroH2A1 on the rDNA and the relocalization of macroH2A1 in the nucleolus. Interestingly, the inhibition of rDNA transcription induced by nucleolin depletion is alleviated by the inactivation of macroH2A1. These results demonstrate that macroH2A1 is a new factor involved in the regulation of rDNA transcription.

IGF promotes cardiac lineage induction by selective expansion of cardiogenic mesoderm in vitro

Purpose: A thorough understanding of the developmental processes that direct embryonic stem cells (ESCs) towards a cardiac fate is essential for translational application of ESCs in cardiac disease modeling and therapy. Methods: This study set out to identify, by high throughput screening, key signaling pathways that regulate early cardiac lineage formation. Results: We screened a panel of 42 cytokines/growth factors for their ability to induce greater Nkx2.5+ cardiac progenitor cell (CPC) formation during ES cell in vitro differentiation using an Nkx2.5 cardiac enhancer-eGFP transgenic mouse ES cell line. Consistent with previous reports, the treatment of differentiating ESCs with Wnt3a and FGF-2 resulted in increased proportion of Nkx2.5-eGFP+ cells (Wnt3a; 5.3±0.05 and FGF-2; 4.0-fold ± 1.3 over untreated baseline, respectively). Surprisingly, treatment with insulin-like growth factor (IGF) family of ligands (e.g. IGF-1, IGF-2, insulin) resulted in expansion of Nkx2.5+ CPCs by 7.4-fold ± 0.79 (IGF-1; 2.71%±0.27 vs. 0.37%±0.03); 6.3-fold ± 0.46 (IGF-2; 2.96%±0.25 vs. 0.47%±0.07) and 6.3-fold ± 0.35 (insulin; 2.85%±0.01 vs. 0.45%±0.03), respectively. To examine the mechanism of IGF-mediated cardiac lineage expansion, we quantified the abundance of endoderm, ectoderm, and mesoderm populations after IGF treatment and found that IGF selectively increased the number of mesendodermal cells (Brachyury+ cell population: IGF-I treated; 14.3%±1.51, IGF-II treated; 19.3%±0.95, and insulin treated 17.3%±1.51 compared with negative control; 3.95%±0.56), but not of ectodermal (Nestin+) populations. This was corroborated by analysis of lineage marker expression using quantitative PCR. Mechanistically, IGF treatment selectively enhanced the proliferation of Brachyury+ cells (Brachyury+/Ki-67+ cells = 46.2%±7.27, 46.0%±12.7, and 50.3%±8.58 after IGF-I, IGF-II, or insulin supplementation vs. 26.0%±8.34 in untreated control). Furthermore, IGFs appear to directly signal to mesodermal cells through phosphorylation of the Akt/mTOR pathway. Conclusions: Our data uncover a novel role for IGF family ligands to regulate cardiac lineage induction through its ability to expand the mesodermal cell population. These findings contribute to our understanding of early developmental events during germ layer induction and provide new approaches to enhance in vitro cardiogenesis for regenerative medicine applications.

Directed neuronal differentiation of mouse embryonic and induced pluripotent stem cells and their gene expression profiles

Embryonic stem cells (ESCs) may be useful as a therapeutic source of cells for the production of healthy tissue; however, they are associated with certain challenges including immunorejection as well as ethical issues. Induced pluripotent stem cells (iPSCs) are a promising substitute since a patient's own adult cells would serve as tissue precursors. Ethical concerns prevent a full evaluation of the developmental potency of human ESCs and iPSCs, therefore, mouse iPSC models are required for protocol development and safety assessments. We used a modified culturing protocol to differentiate pluripotent cells from a mouse iPS cell line and two mouse ES cell lines into neurons. Our results indicated that all three pluripotent stem cell lines underwent nearly the same differentiation process when induced to form neurons in vitro. Genomic expression microarray profiling and single-cell RT-qPCR were used to analyze the neural lineage differentiation process, and more than one thousand differentially expressed genes involved in multiple molecular processes relevant to neural development were identified.

Feeder Independent Culture of Mouse Embryonic Stem Cells

Embryonic stem cell classically cultured on feeder layer with FBS contained ES medium. Feeder-free mouse ES cell culture systems are essential to avoid the possible contamination of nonES cells. First we determined the difference between ES cell and MEF by Oct4 population. We demonstrate to culture and to induce differentiation on feeder free condition using a commercially available mouse ES cell lines.

Lack of huntingtin promotes neural stem cells differentiation into glial cells while neurons expressing huntingtin with expanded polyglutamine tracts undergo cell death

Huntington's disease (HD) is a neurodegenerative disorder that affects muscle coordination and diminishes cognitive abilities. The genetic basis of the disease is an expansion of CAG repeats in the Huntingtin (Htt) gene. Here we aimed to generate a series of mouse neural stem (NS) cell lines that carried varying numbers of CAG repeats in the mouse Htt gene (Hdh CAG knock-in NS cells) or that had Hdh null alleles (Hdh knock-out NS cells). Towards this end, Hdh CAG knock-in mouse ES cell lines that carried an Htt gene with 20, 50, 111, or 140 CAG repeats or that were Htt null were neuralized and converted into self-renewing NS cells. The resulting NS cell lines were immunopositive for the neural stem cell markers NESTIN, SOX2, and BLBP and had similar proliferative rates and cell cycle distributions. After 14days in vitro, wild-type NS cells gave rise to cultures composed of 70% MAP2+ neurons and 30% GFAP+ astrocytes. In contrast, NS cells with expanded CAG repeats underwent neuronal cell death, with only 38%±15% of the MAP2+ cells remaining at the end of the differentiation period. Cell death was verified by increased caspase 3/7 activity on day 14 of the neuronal differentiation protocol. Interestingly, Hdh knock-out NS cells treated using the same neuronal differentiation protocol showed a dramatic increase in the number of GFAP+ cells on day 14 (61%±20% versus 24%±10% in controls), and a massive decrease of MAP2+ neurons (30%±11% versus 64%±17% in controls). Both Hdh CAG knock-in NS cells and Hdh knock-out NS cells showed reduced levels of Bdnf mRNA during neuronal differentiation, in agreement with data obtained previously in HD mouse models and in post-mortem brain samples from HD patients. We concluded that Hdh CAG knock-in and Hdh knock-out NS cells have potential as tools for investigating the roles of normal and mutant HTT in differentiated neurons and glial cells of the brain.

A Novel and Stable Mouse Artificial Chromosome Vector

Human chromosome fragments (hCFs) and human artificial chromosomes (HACs) can be transferred into mouse ES cells to produce trans-chromosomic (Tc) mice. Although hCFs and HACs containing large genomic DNAs can be autonomously maintained in Tc mice, their retention rate is variable in mouse ES cell lines and Tc mouse tissues, possibly because of centromere differences between the species. To improve the retention rate of artificial chromosomes in mouse cells, we constructed novel mouse artificial chromosome (MAC) vectors by truncating a natural mouse chromosome at a site adjacent to the centromeric region. We obtained cell clones containing the MAC vectors that were stably maintained in mouse ES cells and various tissues in Tc mice. The MACs possess acceptor sites into which a desired gene or genes can be inserted. Thus, Tc mice harboring the MAC vectors may be valuable tools for functional analyses of desired genes, producing humanized model mice, and synthetic biology.

Generation of mouse ES cell lines engineered for the forced induction of transcription factors

Here we report the generation and characterization of 84 mouse ES cell lines with doxycycline-controllable transcription factors (TFs) which, together with the previous 53 lines, cover 7–10% of all TFs encoded in the mouse genome. Global gene expression profiles of all 137 lines after the induction of TFs for 48 hrs can associate each TF with the direction of ES cell differentiation, regulatory pathways, and mouse phenotypes. These cell lines and microarray data provide building blocks for a variety of future biomedical research applications as a community resource.

DREME: motif discovery in transcription factor ChIP-seq data

Motivation: Transcription factor (TF) ChIP-seq datasets have particular characteristics that provide unique challenges and opportunities for motif discovery. Most existing motif discovery algorithms do not scale well to such large datasets, or fail to report many motifs associated with cofactors of the ChIP-ed TF.Results: We present DREME, a motif discovery algorithm specifically designed to find the short, core DNA-binding motifs of eukaryotic TFs, and optimized to analyze very large ChIP-seq datasets in minutes. Using DREME, we discover the binding motifs of the the ChIP-ed TF and many cofactors in mouse ES cell (mESC), mouse erythrocyte and human cell line ChIP-seq datasets. For example, in mESC ChIP-seq data for the TF Esrrb, we discover the binding motifs for eight cofactor TFs important in the maintenance of pluripotency. Several other commonly used algorithms find at most two cofactor motifs in this same dataset. DREME can also perform discriminative motif discovery, and we use this feature to provide evidence that Sox2 and Oct4 do not bind in mES cells as an obligate heterodimer. DREME is much faster than many commonly used algorithms, scales linearly in dataset size, finds multiple, non-redundant motifs and reports a reliable measure of statistical significance for each motif found. DREME is available as part of the MEME Suite of motif-based sequence analysis tools (http://meme.nbcr.net).Contact: t.bailey@uq.edu.auSupplementary information: Supplementary data are available at Bioinformatics online.

Human and mouse natural killer cell cytotoxicity towards embryonic stem cells (169.41)

Abstract Embryonic stem cells (ESC) have tremendous potential for the treatment of numerous diseases but little is known regarding the immune response towards them. Human ESC (hESC) express very low levels of MHC class I, rendering them resistant to T cell-mediated rejection but making them likely targets of natural killer (NK) cells. As clinical use of MHC-matched ESC is unlikely, we examined the susceptibility of ESC to NK cell mediated killing. We tested the ability of activated human NK cells from several different donors to lyse the H9 hES cell line in vitro. Activated peripheral blood NK cells achieved significant H9 killing in a 4 hour chromium release assay. Similar experiments using mouse ES cell lines (JM8A3.N1, B6 background) with syngeneic (B6) or allogeneic (BALB/c) activated NK effector cells showed significant mouse ESC killing, regardless of NK cell source. This could be explained by very low MHC class I expression on the ESC targets leading to NK cell-mediated lysis due to lack of MHC class I, rather than allogeneic recognition. Experiments determining mouse ESC in vivo clearance by NK cells are under way. Together these data underscore the susceptibility of human and mouse ESC to NK cell-mediated lysis. This suggests that therapeutic applications of ESC are susceptible to host NK-cell mediated resistance.

An Inducible Expression System of the Calcium-Activated Potassium Channel 4 to Study the Differential Impact on Embryonic Stem Cells

Rationale. The family of calcium-activated potassium channels consists of four members with varying biological functions and conductances. Besides membrane potential modulation, SK channels have been found to be involved in cardiac pacemaker cell development from ES cells and morphological shaping of neural stem cells. Objective. Distinct SK channel subtype expression in ES cells might elucidate their precise impact during cardiac development. We chose SK channel subtype 4 as a potential candidate influencing embryonic stem cell differentiation. Methods. We generated a doxycycline inducible mouse ES cell line via targeted homologous recombination of a cassette expressing a bicistronic construct encoding SK4 and a fluorophore from the murine HPRT locus. Conclusion. We characterized the mouse ES cell line iSK4-AcGFP. The cassette is readily expressed under the control of doxycycline, and the overexpression of SK4 led to an increase in cardiac and pacemaker cell differentiation thereby serving as a unique tool to characterize the cell biological variances due to specific SK channel overexpression.

The Efficacy of Human Placenta as a Source of the Universal Feeder in Human and Mouse Pluripotent Stem Cell Culture

The use of a mouse embryonic fibroblast (MEF) feeder for culture of embryonic stem cells (ESCs) is a widely accepted method, regardless of the ESCs' origin and type. In this study, we performed the undifferentiated propagation of human ES cell lines (hESCs, H1, and HSF6) and mouse ES cell lines (mESCs, D3, and CE3), which were previously maintained on an MEF feeder, using human placenta-derived fibroblast-like cell (HPC) feeders originated from chorionic villi of women who had undergone therapeutic abortion due to known maternal disease that is aggravated by pregnancy. Moreover, we tried to introduce the HPC feeder for the establishment of inducible pluripotent stem cells (iPSCs) from human placental mesenchymal stem cells (MSCs). On the HPC feeder we were able to propagate ESCs and iPSCs colonies as an undifferentiated state up to the 50th passage and 20th passage, respectively. Maintenance of undifferentiated ESCs was identified by the expression of ALP, SSEA-1, SSEA-4, TRA-81, TRA-60, Oct-4, Nanog, or Rex-1. Also, addition of leukemia inhibitory factor was not required for undifferentiated propagation of mESCs on the HPC feeder. The efficiency and expression of three germ layer markers of embryoid bodies (EBs) from ESCs were satisfactory in both the MEF and HPC group. EBs formed from iPSCs were scant, and differentiation to the three germ layers was identifiable by reverst transcription-polymerase chain reactio (RT-PCR) only in the HPC group. In conclusion, the HPC feeder can efficiently support the undifferentiated propagation of hESCs, mESCs, and iPSCs, suggesting that human placenta may be a useful source of universal feeder cells for hESC, mESC, and iPSC culture.

Abstract 3876: Cell cycle checkpoint in vivo in developing mouse embryo liver cells

Abstract Embryonic stem (ES) cells have a distinct cell cycle distribution and cyclins/CDKs expression. ES cells are characterized by a short G1 phase and a high proportion of cell in S and G2 phase, maintain consistent level of cyclins expression seems not to be phase dependent. However, most cell cycle studies are on mouse or human ES cell line, whether that it is really reflective in vivo is not well defined. By extracting fresh liver cells from mouse embryo, we have investigated cell cycle distribution, correspondent cyclins and CDK expression in vivo in mouse embryo from embryonic days 10.5(E10.5, liver start to develop at E9.5) to post-natal 21 days(P21). We found that most of the cells (62%) were in S and G2 phase, 38% of cells in G1 at E11.5. Upon embryo development, the proportion of cells in G1 increased, although there were still large proportion of cells in S and G2 phase (55%) at P0. The short G1 phase in early embryonic day suggests that cell phase distribution may be related with embryo cells’ pluripotency in vivo. Distinct high levels of cyclin D1, E1, A, B1 and CDK1(cdc2) expressed at E10.5, which seemed to be pluripotency related but not cell phase dependent. The cyclins and CDK level started to drop from E11.5 to post-natal (P0) suggesting that cyclins/CDKs expression reduced and became regulated with differentiation. In ES cell line, where G1 population increased with differentiation, G1 DNA-damage checkpoint was still absent. However, in response to ionizing irradiation, P0 mouse liver cells showed a short G2 population increase followed by G1 cell phase arrest indicating that at P0 or even earlier stage, G1 DNA-damage checkpoint had been established in vivo. Thus, cell cycle and cell cycle checkpoint in vivo in mouse embryo liver cells displayed both similar and differential pattern compared to that in mES cell line in vitro. Underlying mechanism such as DNA-damage checkpoint patterns and its regulation will be further discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3876.