Summary Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and non-tumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429 and miR-183-96-182 were down-regulated in human BCSCs, normal human and murine mammary stem/progenitor cells and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. MiR-200c inhibited the clonogenicity of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated down-regulation of three microRNA clusters and the similar functional regulation of clonogenicity by miR-200c provide a molecular link that connects breast cancer stem cells with normal stem cells.

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes, ID1, BCL2L1 and HM13, expressed in human ES cells, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.

Salmonella typhimurium is a facultative intracellular pathogen capable of sving within host phagocytic cells. Salmonela strains carrying phoP mutations are avirulent, unable to survive in macrophages, and extremely sensitive to peptides having antimicrobial activity such as the hostderived defensins. We present here the DNA sequence of the phoP gene and show that the deduced amino acid sequence of phoP has extensive homology with the Escherichia coli transcriptional regulators PhoB and OmpR, which control the expression of loci in response to different environmental stimuli. The psiD locus, which is regulated by phosphate availability, was found to be under the control ofthephoP gene product.Sequences homologous to phoP were found in several Gramnegative species and in the yeast Saccharomyces cerevisiae.Facultative intracellular pathogens are organisms that can survive and replicate in phagocytic cells. Because of this property, they usually cause long and debilitating diseases and in many cases fatal infections if untreated. Facultative intracellular pathogens, which include the protozoa Trypanosoma cruzi and Leishmania and bacterial species such as Mycobacterium tuberculosis, Mycobacterium leprae, Listeria monocytogenes, Brucella abortus, Legionella pneumophila, and Salmonella typhimurium, utilize different strategies to survive within phagocytic cells (1). These strategies, which include inhibiting the fusion of lysosomes with the phagocytic vacuole and escape from or survival within the phagolysosome, have molecular mechanisms that remain largely unknown. S. typhimurium causes a typhoid-like syndrome in mice and is frequently used as a model system for human typhoid fever, a worldwide problem with over 30 million cases annually (2). Our laboratory has been studying the molecular mechanisms by which S. typhimurium is able to survive and replicate in murine macrophages (3,4 Table 1. Strain JM103 (10) was used as a host for bacteriophages M13mp18 and M13mp19 (11). The construction of plasmids pEG5381 and pEG5433 (Fig. lA) and the isolation and mapping of transposon insertions in these plasmids (Fig. 1B) will be described elsewhere. Media composition (7, 12) and general bacterial genetic techniques (13) have been described. 5-Bromo-4-chloro-3-indolyl (3-D-galactoside (X-Gal; Boehringer Mannheim) was added to agar plates to a final concentration of 60 gg/ml. M13 phage was manipulated as described (10). The nonspecific acid phosphatase staining protocol has been described (5). ,3-Galactosidase assays were carried out as described (12).Restriction endonucleases and phage T4 ligase were purchased from Amersham, Bethesda Research Laboratories, Boehringer Mannheim, New England BioLabs, or P-L Biochemicals and were used according to the suppliers' specifications. Large-scale isolation ofplasmid DNA was done by the procedure described by Kupersztoch and Helinski (14). For nucleotide sequencing, DNA restriction fiagments were purified from acrylamide gels and cloned in the phages M13mp18 and M13mp19. Clones were...

To investigate the role of DNA methylation during human development, we developed Methyl-seq, a method that assays DNA methylation at more than 90,000 regions throughout the genome. Performing Methyl-seq on human embryonic stem cells (hESCs), their derivatives, and human tissues allowed us to identify several trends during hESC and in vivo liver differentiation. First, differentiation results in DNA methylation changes at a minimal number of assayed regions, both in vitro and in vivo (2%-11%). Second, in vitro hESC differentiation is characterized by both de novo methylation and demethylation, whereas in vivo fetal liver development is characterized predominantly by demethylation. Third, hESC differentiation is uniquely characterized by methylation changes specifically at H3K27me3-occupied regions, bivalent domains, and low density CpG promoters (LCPs), suggesting that these regions are more likely to be involved in transcriptional regulation during hESC differentiation. Although both H3K27me3-occupied domains and LCPs are also regions of high variability in DNA methylation state during human liver development, these regions become highly unmethylated, which is a distinct trend from that observed in hESCs. Taken together, our results indicate that hESC differentiation has a unique DNA methylation signature that may not be indicative of in vivo differentiation.