NucTools: analysis of chromatin feature occupancy profiles from high-throughput sequencing data

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

Embryonic stem (ES) cells are derived from the epiblast of the inner cell mass (ICM) of a blastocyst. These are pluripotent cells and can give rise to all derivatives of the three primary germ layers ectoderm, endoderm, and mesoderm. ES cells require specific signals for specific lineage of differentiation if injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma (Wu et al., 2007). The techniques for culturing mouse embryonic stem (ES) cells from the inner cell mass of the preimplantation blastocyst were first done in 1981 (Martin, 1981), and versions of these standard procedures are used today in laboratories throughout the world. The first successful derivation of human ES (hES) cells was reported by Thomson et al. (Thomson et al., 1998). They isolated and plated the cells onto mitotically inactivated MEF (mouse embryonic fibroblast) cells. In 2000, Reubinoff et al. confirmed that hES cells could be efficiently derived from surplus embryos and possess the differentiation potential under in vitro conditions. Since then, there has been rapid progress made and numerous studies have described the derivation of new hES cell lines including methods of growing both undifferentiated hES cells and their differentiated progeny. In last 6 years, there has been exponential increase in methods to improve culture conditions, differentiation patterns to produce human cells for transplantation and drug testing (Trounson, 2006, Gepstein, 2002) and genetic manipulation (Draper et al., 2004, Zwaka and Thomson, 2003). Generating cultures of mouse or human ES cells that remain in a proliferating and undifferentiated state is multistep process. Typically, the inner cell mass of a preimplantation blastocyst is removed from the trophectoderm that surrounds it and cultured in the small plastic culture dishes containing growth medium supplemented with fetal calf serum. The culture dishes are sometimes coated with a layer of nondividing cells, which are often MEF cells that have been chemically inactivated so they will not divide. Mouse ES cells can be grown in vitro without feeder layers if the cytokine leukemia inhibitory factor (LIF) is added to the culture medium, but human ES cells do not respond to LIF. The process of generating an embryonic stem cell line is somewhat difficult, so lines are not produced every time from the preimplantation-stage embryo maintained in a culture dish. However, if the plated cells survive, divide and multiply enough to crowd the dish, they are removed, and plated into several fresh culture dishes. The process of re-plating or

Human and mouse embryonic stem (ES) cells have the capacity to differentiate into derivatives of all three germ layers, suggesting novel therapies for degenerative, metabolic, and traumatic disorders. ES-based regenerative medicine will be further advanced by the development of reliable methods for transgene introduction and expression. Here, we show infection of human and mouse embryonic stem (ES) cells with two of the most popular vectors in gene transfer, adenovirus type 5 (Ad5) and adeno-associated virus (AAV; serotypes 2, 4, and 5). All vectors express the nuclear-localized marker gene beta-galactosidase expressed from the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Both Ad5 and AAV2 infected human and mouse ES cells and gave rise to beta-galactosidase expression. AAV4 and 5 did not yield detectable levels of beta-galactosidase expression. Quantitative PCR analysis of virally infected human and mouse ES cells revealed that only Ad5 and AAV2 are capable of transducing both cell-types. No viral DNA was detected in cells infected with either AAV4 or AAV5. Infection and subsequent differentiation of mouse and human ES cells with Ad5 showed that beta-galactosidase-expressing cells were restricted to cells in the interior of the embryoid body mass. No beta-galactosidase expression was observed in AAV-infected cells following differentiation. There was no difference in morphology or differentiation patterns between infected and noninfected differentiating mouse and human ES cells. Differentiation of hES cells prior to infection led to transduction of neuronally differentiated cells with good efficiency using all vectors. These data show that Ad5- and AAV2-based vectors are capable of infecting both human and mouse ES cells, in both their undifferentiated and differentiated states, whereas AAV4 and AAV5 can infect human and mouse ES cells only following differentiation.

IntroductionEmbryonic stem cells (ESCs) provide an attractive cell source for basic research and disease treatment. Currently, the common culture system for mouse ESC requires mouse embryonic fibroblast (MEF) as a feeder layer supplemented with leukemia inhibitory factor (LIF). The drawbacks associated with MEF and the cost of LIF have motivated exploration of new feeder cell types to maintain self-renewal of mouse ESCs without the need of exogenous LIF. However, why these feeder cells could maintain ESCs at the undifferentiated state independent of exogenous LIF is unclear.MethodsWe derived mouse ESC lines using human foreskin fibroblast (HFF) in the absence of exogenous LIF. We also examined the dependence of HFF on the JAK-Stat3 pathway to maintain ESC identities and explored the potential molecular basis for HFF to support self-renewal of ESCs.ResultsHFF supported mouse ESC self-renewal superiorly to MEFs. Using the HFF system, multiple lines of mouse ESCs were successfully derived without addition of exogenous LIF and any small molecular inhibitors. These ESCs had capacities to self-renew for a long period of time and to differentiate into various cell types of the three germ layers both in vitro and in vivo. Moreover, the ESCs participated in embryonic development and contributed to germ cell lineages in the chimeric mouse. At a molecular level, HFF was dependent on the JAK-Stat3 pathway to maintain ESC self-renewal. The high level of interleukin-6 (IL-6) produced by HFF might be responsible for the exogenous LIF-independent effect.ConclusionThis study describes an efficient, convenient and economic system to establish and maintain mouse ESC lines, and provides insights into the functional difference in the support of ESC culture between MEF and HFF.

It is important to estimate the influence of space radiation on human body during a longer stay in space, including missions to International Space Station (ISS), the moon, or Mars.We use mouse embryonic stem (ES) cells, which have normal karyotypes to estimate the effects of space radiation.For this purpose, mouse ES cells have been launched to the Japanese experiment module called 'KIBO' in ISS and stored in a freezer (MELFI) at -95°C until the return to the Earth (return in five times in 3 years).After returning to the Earth, the DNA damages of space-radiated ES cells will be

Gene silencing by RNA interference (RNAi) using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool for silencing specific genes in various organisms. As compared with transgene-based RNAi using shRNA expression vectors, chemically synthesized siRNAs have an advantage in that they do not modify genome organization. This nature is ideal as a differentiation method for embryonic stem (ES) cells. The objective of this work was to develop an efficient method to repress a specific gene expression in mouse and monkey ES cells using chemically synthesized siRNA, and to investigate whether this transient gene silencing can be used as a new differentiation method of ES cells. First, we tried to knock down the expression of enhanced green fluorescent protein (EGFP) gene in GFP-expressing mouse ES cells. We found that lipofection was effective to deliver siRNA into mouse ES cells. Suppression of EGFP expression was observed by fluorescence microscopy after 24 h of transfection and the silencing continued at least for 5 days. FACS analysis revealed that 86% of cells showed suppression of EGFP at 48 h after transfection. Then we tried to suppress endogenous gene Oct4 that plays an important role in the maintenance of pluripotency and the lineage commitment in mouse ES cells. We performed RT-PCR analysis and western blotting to assay for Oct4 mRNA and protein at 24, 48, 72, and 96 h after transfection. Hand1 and Cdx2, transcription factors implicated in trophoblast differentiation, were also analyzed. RT-PCR analysis showed a reduced level of Oct4 mRNA at 24–96 h. Reduction of Oct4 protein was confirmed by western blotting and the reduced level was still maintained at 96 h after transfection. RT-PCR analysis also showed up-regulation of Hand1 and Cdx2 concomitant with the suppression of Oct4. Furthermore, ES cells changed their morphology into a large and flattened shape that is characteristic of trophoblast cells. These results suggested that the transient suppression of Oct4 induced differentiation of mouse ES cells to trophectoderm cells as expected. Therefore our data imply that the chemically synthesized siRNA can be used to differentiate ES cells. Next, we tried to suppress EGFP and Oct4 in monkey ES cells. In monkey ES cells, we found that the Sendai virus (hemagglutinating virus of Japan, HVJ) envelope was suitable to deliver siRNA into cells. With this method, we detected efficient silencing of EGFP and Oct4 by fluorescence microscopy, RT-PCR analysis, and western blotting. In the case of monkey ES cells, however, no morphological change was observed by Oct4 suppression at 96 h after transfection. These results suggest possible diversity between murine and primate ES cells in the differentiation process. This work was supported by grants from the Ministry of Education, Science, Sports, and Culture (14580798, 13358014 and 14380382).

In eukaryotes, chromatin provides a way to compact the genetic material into the confined space of a nucleus. It is also a means to store the same genetic information in different chromatin states. Alteration of these states is enabled by chromatin modifying and remodeling machineries – enzymes that utilize a diverse range of structural changes to chromatin. Despite their apparent importance in gene regulation, it is unclear how they facilitate the transition between chromatin states. Within two distinct projects, we aimed to (1) decipher how chromatin modifying complexes, namely the Polycomb group proteins, are targeted to chromatin and (2) how chromatin remodelers, specifically the ISWI remodeling complexes, change chromatin structure. Polycomb group proteins assemble as chromatin-modifying complexes that maintain the memory of the silent transcriptional state, in part through methylation of lysine 27 on histone H3. Despite their established importance during development, it is largely unclear how these complexes are recruited to specific target genes and how they impair transcription. In flies, Polycomb is recruited by Polycomb response elements that are abundant in various DNA-binding factor motifs. However, the contribution of individual motifs is not yet resolved. In mammals, equivalents of Polycomb response elements are not yet characterized. Here, we aimed to dissect Polycomb-mediated silencing in the mouse genome by identifying DNA determinants of Polycomb recruitment and investigating the role of Polycomb recruitment in transcriptional silencing. More specifically, we developed an assay to test many DNA sequences with various sequence properties for their ability to drive PRC2 recruitment in mouse embryonic stem cells. The assay enabled integration of hundreds of sequences into a defined genomic location in parallel. We found that high density of unmethylated CG motifs within a synthetic backbone sequence is sufficient to recruit PRC2. Furthermore, to link PRC2 recruitment with transcriptional repression, we used CRISPR/Cas9 technology to delete the core PRC2 (Eed) component and monitored the transcriptional response by RNA-seq. Upon depletion of global H3K27me3 levels, we observed no significant changes in gene expression in mouse embryonic stem cells but global deregulation of PRC2 targets during differentiation into neuronal progenitors. These results indicate that recruitment of PRC2 and subsequent H3K27 methylation is important for cell-fate transition, but not required for gene repression in mouse embryonic stem cells. For the second project, we were interested in chromatin remodelers (ISWI) and their role in regulating chromatin structure. Chromatin remodelers are known to use the energy of ATP hydrolysis to evict, slide and reposition nucleosomes, yet we do not fully understand how nucleosome positioning and occupancy affects transcription factor binding. To this aim, we deleted Snf2h, the ATPase subunit of the ISWI chromatin remodeling family, in mouse embryonic stem cells. The Snf2h knockout mouse embryonic stem cells are viable with unchanged expression of pluripotency markers, which is exciting as this is the first viable knockout of an ATPase remodeler subunit. To determine global changes upon deletion of Snf2h, we monitored nucleosome positioning, chromatin accessibility and transcriptional response in Snf2h knockout cells using MNase, ATAC and RNA sequencing, respectively. Extensive data analysis revealed global changes in nucleosome positioning proximal to transcription start sites and transcription factor motifs. Analyzing nucleosome positioning and chromatin accessibility data, we identified transcription factors that require Snf2h to bind their target sites, such as CTCF. It seems that in the absence of Snf2h, nucleosomes cannot be evicted from CTCF motifs, which in turn results in loss of CTCF binding. Taken together, these results indicate that ISWI complexes enable transcription factor binding, at both promoters and distal regulatory regions, by sliding of motif-bound nucleosomes.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Derivation, propagation and differentiation of human embryonic stem cells

Sox family transcription factors play essential roles in cell differentiation, development, and sex determination. Sox2 was previously thought to be the sole Sox protein expressed in mouse embryonic stem (ES) cells. Sox2 associates with Oct3/4 to maintain self-renewal of ES cells. In the current study, digital differential display identified transcripts for an additional Sox family member, Sox15, enriched in mouse ES cells. Reverse transcription-PCR confirmed that Sox15 expression is highest in undifferentiated ES cells and repressed upon differentiation. Sox15 is expressed at low levels in several tissues, including testis and muscle. In vitro studies showed that Sox15, like Sox2, associated with Oct3/4 on DNA sequences containing the octamer motif and Sox-binding site. Gel mobility shift assays and SELEX analyses showed that Sox15 binds similar DNA sequences as Sox2 but with weaker affinity. In contrast to the early embryonic lethality observed in Sox2-null mice, Sox15-null ES cells and mice were grossly normal. DNA microarray analyses revealed that Otx2, Ctgf, Ebaf, and Hrc are dysregulated in Sox15-null ES cells, however. Chromatin immunoprecipitation showed that Sox15, but not Sox2, bound to a Sox consensus binding site within the Hrc gene. Taken together, these data demonstrate differential roles for Sox15 and Sox2 in transcriptional control in mouse ES cells.

Self-renewal is a complex biological process necessary for maintaining the pluripotency of embryonic stem cells (ESCs). Recent studies have used global proteomic techniques to identify proteins that associate with the master regulators Oct4, Nanog and Sox2 in ESCs or in ESCs during the early stages of differentiation. Through an unbiased proteomic screen, Banf1 was identified as a Sox2-associated protein. Banf1 has been shown to be essential for worm and fly development but, until now, its role in mammalian development and ESCs has not been explored. In this study, we examined the effect of knocking down Banf1 on ESCs. We demonstrate that the knockdown of Banf1 promotes the differentiation of mouse ESCs and decreases the survival of both mouse and human ESCs. For mouse ESCs, we demonstrate that knocking down Banf1 promotes their differentiation into cells that exhibit markers primarily associated with mesoderm and trophectoderm. Interestingly, knockdown of Banf1 disrupts the survival of human ESCs without significantly reducing the expression levels of the master regulators Sox2, Oct4 and Nanog or inducing the expression of markers of differentiation. Furthermore, we determined that the knockdown of Banf1 alters the cell cycle distribution of both human and mouse ESCs by causing an uncharacteristic increase in the proportion of cells in the G2-M phase of the cell cycle.

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

An improved cryopreservation method for a mouse embryonic stem cell line

The Homeoprotein Nanog Is Required for Maintenance of Pluripotency in Mouse Epiblast and ES Cells

Classic derivation of mouse embryonic stem (ES) cells from blastocysts is inefficient, strain-dependent, and requires expert skills. Over recent years, several major improvements have greatly increased the success rate for deriving mouse ES cell lines. The first improvement was the establishment of a user-friendly and reproducible medium-alternating protocol that allows isolation of ES cells from C57BL/6 transgenic mice with efficiencies of up to 75%. A recent report describes the use of this protocol in combination with leukemia inhibitory factor and pluripotin treatment, which made it possible to obtain ES cells from F1 strains with high efficiency. We report modifications of these protocols for user-friendly and reproducible derivation of mouse ES cells with efficiencies of up to 100%. Our protocol involves a long initial incubation of primary outgrowths from blastocysts with pluripotin, which results in the formation of large spherical outgrowths. These outgrowths are morphologically distinct from classical inner cell mass (ICM) outgrowths and can be easily picked and trypsinized. Pluripotin was omitted after the first trypsinization because we found that it blocks attachment of ES cells to the feeder layer and its removal facilitated formation of ES cell colonies. The newly established ES cells exhibited normal karyotypes and generated chimeras. In summary, our user-friendly modified protocol allows formation of large spherical ICM outgrowths in a robust and reliable manner. These outgrowths gave rise to ES cell lines with success rates of up to 100%.