Epigenetic status of human embryonic stem cells

We examined the allele-specific expression of six imprinted genes and the methylation profiles of three imprinting control regions to assess the epigenetic status of human embryonic stem cells. We identified generally monoallelic gene expression and normal methylation patterns. During prolonged passage, one cell line became biallelic with respect to H19, but without loss of the gametic methylation imprint. These data argue for a substantial degree of epigenetic stability in human embryonic stem cells.     

Mouse mutants from chemically mutagenized embryonic stem cells

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.     

Global hypomethylation of the genome in XX embryonic stem cells

Embryonic stem (ES) cells are important tools in the study of gene function and may also become important in cell therapy applications. Establishment of stable XX ES cell lines from mouse blastocysts is relatively problematic owing to frequent loss of one of the two X chromosomes. Here we show that DNA methylation is globally reduced in XX ES cell lines and that this is attributable to the presence of two active X chromosomes. Hypomethylation affects both repetitive and unique sequences, the latter including differentially methylated regions that regulate expression of parentally imprinted genes. Methylation of differentially methylated regions can be restored coincident with elimination of an X chromosome in early-passage parthenogenetic ES cells, suggesting that selection against loss of methylation may provide the basis for X-chromosome instability. Finally, we show that hypomethylation is associated with reduced levels of the de novo DNA methyltransferases Dnmt3a and Dnmt3b and that ectopic expression of these factors restores global methylation levels.     

Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells

The ability to generate mutations is a prerequisite to functional genetic analysis. Despite a long history of using mice as a model system for genetic analysis, the scientific community has not generated a comprehensive collection of multiple alleles for most mouse genes. The chemical mutagen of choice for mouse has been N-ethyl-N-nitrosourea (ENU), an alkylating agent that mainly causes base substitutions in DNA, and therefore allows for recovery of complete and partial loss-, as well as gain-, of-function alleles . Specific locus tests designed to detect recessive mutations showed that ENU is the most efficient mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes. In fact, several genome-wide and region-specific screens based on phenotypes have been carried out. The anticipation of the completion of the human and mouse genome projects, however, now emphasizes genotype-driven genetics--from sequence to mutants. To take advantage of the mutagenicity of ENU and its ability to create allelic series of mutations, we have developed a complementary approach to generating mutations using mouse embryonic stem (ES) cells. We show that a high mutation frequency can be achieved and that modulating DNA-repair activities can enhance this frequency. The treated cells retain germline competency, thereby rendering this approach applicable for efficient generation of an allelic series of mutations pivotal to a fine-tuned dissection of biological pathways.     

Efficient Cre-loxP-induced mitotic recombination in mouse embryonic stem cells.

FLP/FRT-induced mitotic recombination provides a powerful method for creating genetic mosaics in Drosophila and for discerning the function of recessive genes in a heterozygous individual. Here we show that mitotic recombination can be reproducibly induced in mouse embryonic stem (ES) cells, by Cre/loxP technology, at frequencies ranging from 4.2 x 10(-5) (Snrpn) to 7.0 x 10(-3) (D7Mit178) for single allelic loxP sites, and to 5.0 x 10(-2) (D7Mit178) for multiple allelic lox sites, after transient Cre expression. Notably, much of the recombination occurs in G2 and is followed by X segregation, where the recombinant chromatids segregate away from each other during mitosis. It is X segregation that is useful for genetic mosaic analysis because it produces clones of homozygous mutant daughter cells from heterozygous mothers. Our studies confirm the predictions made from studies in Drosophila that suggest that X segregation will not be limited to organisms with strong mitotic pairing, because the forces (sister-chromatid cohesion) responsible for X segregation are an elemental feature of mitosis in all eukaryotes. Our studies also show that genetic mosaic analysis in mice is feasible, at least for certain chromosomal regions.     

Selection for transgene homozygosity in embryonic stem cells results in extensive loss of heterozygosity

Embryonic stem cells offer unprecedented opportunities for random or targeted genome alterations in the mouse. We present here an efficient strategy to create chromosome-specific loss of heterozygosity in embryonic stem cells. The combination of this method with genome-wide mutagenesis in ES cells (using chemical mutagens or gene-trap vectors) opens up the possibility for in vitro or in vivo functional screening of recessive mutations in the mouse.     

Ras-MAPK signaling promotes trophectoderm formation from embryonic stem cells and mouse embryos

In blastocyst chimeras, embryonic stem (ES) cells contribute to embryonic tissues but not extraembryonic trophectoderm. Conditional activation of HRas1(Q61L) in ES cells in vitro induces the trophectoderm marker Cdx2 and enables derivation of trophoblast stem (TS) cell lines that, when injected into blastocysts, chimerize placental tissues. Erk2, the downstream effector of Ras-mitogen-activated protein kinase (MAPK) signaling, is asymmetrically expressed in the apical membranes of the 8-cell-stage embryo just before morula compaction. Inhibition of MAPK signaling in cultured mouse embryos compromises Cdx2 expression, delays blastocyst development and reduces trophectoderm outgrowth from embryo explants. These data show that ectopic Ras activation can divert ES cells toward extraembryonic trophoblastic fates and implicate Ras-MAPK signaling in promoting trophectoderm formation from mouse embryos.     

DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal

The molecular controls that govern the differentiation of embryonic stem (ES) cells remain poorly understood. DGCR8 is an RNA-binding protein that assists the RNase III enzyme Drosha in the processing of microRNAs (miRNAs), a subclass of small RNAs. Here we study the role of miRNAs in ES cell differentiation by generating a Dgcr8 knockout model. Analysis of mouse knockout ES cells shows that DGCR8 is essential for biogenesis of miRNAs. On the induction of differentiation, DGCR8-deficient ES cells do not fully downregulate pluripotency markers and retain the ability to produce ES cell colonies; however, they do express some markers of differentiation. This phenotype differs from that reported for Dicer1 knockout cells, suggesting that Dicer has miRNA-independent roles in ES cell function. Our findings indicate that miRNAs function in the silencing of ES cell self-renewal that normally occurs with the induction of differentiation.     

Bmi1 is expressed in vivo in intestinal stem cells

Bmi1 plays an essential part in the self-renewal of hematopoietic and neural stem cells. To investigate its role in other adult stem cell populations, we generated a mouse expressing a tamoxifen-inducible Cre from the Bmi1 locus. We found that Bmi1 is expressed in discrete cells located near the bottom of crypts in the small intestine, predominantly four cells above the base of the crypt (+4 position). Over time, these cells proliferate, expand, self-renew and give rise to all the differentiated cell lineages of the small intestine epithelium. The induction of a stable form of beta-catenin in these cells was sufficient to rapidly generate adenomas. Moreover, ablation of Bmi1(+) cells using a Rosa26 conditional allele, expressing diphtheria toxin, led to crypt loss. These experiments identify Bmi1 as an intestinal stem cell marker in vivo. Unexpectedly, the distribution of Bmi1-expressing stem cells along the length of the small intestine suggested that mammals use more than one molecularly distinguishable adult stem cell subpopulation to maintain organ homeostasis.     

PTEN-deficient intestinal stem cells initiate intestinal polyposis

Intestinal polyposis, a precancerous neoplasia, results primarily from an abnormal increase in the number of crypts, which contain intestinal stem cells (ISCs). In mice, widespread deletion of the tumor suppressor Phosphatase and tensin homolog (PTEN) generates hamartomatous intestinal polyps with epithelial and stromal involvement. Using this model, we have established the relationship between stem cells and polyp and tumor formation. PTEN helps govern the proliferation rate and number of ISCs and loss of PTEN results in an excess of ISCs. In PTEN-deficient mice, excess ISCs initiate de novo crypt formation and crypt fission, recapitulating crypt production in fetal and neonatal intestine. The PTEN-Akt pathway probably governs stem cell activation by helping control nuclear localization of the Wnt pathway effector beta-catenin. Akt phosphorylates beta-catenin at Ser552, resulting in a nuclear-localized form in ISCs. Our observations show that intestinal polyposis is initiated by PTEN-deficient ISCs that undergo excessive proliferation driven by Akt activation and nuclear localization of beta-catenin.