Inhibition of HDAC activity directly reprograms murine embryonic stem cells to trophoblast stem cells

Embryonic stem cells (ESCs) can differentiate into all cell types of the embryonic germ layers. ESCs can also generate totipotent 2C-like cells and trophectodermal cells. However, these latter transitions occur at low frequency due to epigenetic barriers, the nature of which is not fully understood. Here, we show that treating mouse ESCs with sodium butyrate (NaB) increases the population of 2C-like cells and enables direct reprogramming of ESCs into trophoblast stem cells (TSCs) without a transition through a 2C-like state. Mechanistically, NaB inhibits histone deacetylase activities in the LSD1-HDAC1/2 corepressor complex. This increases acetylation levels in the regulatory regions of both 2C- and TSC-specific genes, promoting their expression. In addition, NaB-treated cells acquire the capacity to generate blastocyst-like structures that can develop beyond the implantation stage in vitro and form deciduae in vivo. These results identify how epigenetics restrict the totipotent and trophectoderm fate in mouse ESCs.</p

Development of Space-Time-Controlled Multi-Stage Pulsed Magnetic Field Forming and Manufacturing Technology at the WHMFC*

In November 2011, the Project of Basic Research of Forming by Space-Time-Controlled Multi-Stage Pulsed Magnetic Field (Stic-Must-PMF) was supported by the National Basic Research Program of China (973 Project, 2011.11-2016.08). It is aimed at achieving breakthroughs in manufacturing technology to solve current problems in forming largescale and complex sheet and tube parts and components, imposed by the limitations of existing equipment and materials forming properties. The objective of our research group focuses on the design principles and structural layout optimization of Stic-Must-PMF facility. And this paper will report the development of Stic-Must-PMF forming and manufacturing technology at the Wuhan National High Magnetic Field Center (WHMFC) including numerical modeling, experimental setup and experimental studies

OP9-Lhx2 stromal cells facilitate derivation of hematopoietic progenitors both in vitro and in vivo

AbstractGenerating engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is an ideal approach for obtaining induced HSCs for cell therapy. However, the path from PSCs to robustly induced HSCs (iHSCs) in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study, we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2) accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly, the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore, the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo

Tet and TDG Mediate DNA Demethylation Essential for Mesenchymal-to-Epithelial Transition in Somatic Cell Reprogramming

SummaryTet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion

Vitamin C Enhances the Generation of Mouse and Human Induced Pluripotent Stem Cells

SummarySomatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However, the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound, vitamin C (Vc), enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence, a recently identified roadblock for reprogramming. In addition, Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process

WNT signalling control by KDM5C during development affects cognition

Although KDM5C is one of the most frequently mutated genes in X-linked intellectual disability, the exact mechanisms that lead to cognitive impairment remain unknown. Here we use human patient-derived induced pluripotent stem cells and Kdm5c knockout mice to conduct cellular, transcriptomic, chromatin and behavioural studies. KDM5C is identified as a safeguard to ensure that neurodevelopment occurs at an appropriate timescale, the disruption of which leads to intellectual disability. Specifically, there is a developmental window during which KDM5C directly controls WNT output to regulate the timely transition of primary to intermediate progenitor cells and consequently neurogenesis. Treatment with WNT signalling modulators at specific times reveal that only a transient alteration of the canonical WNT signalling pathway is sufficient to rescue the transcriptomic and chromatin landscapes in patient-derived cells and to induce these changes in wild-type cells. Notably, WNT inhibition during this developmental period also rescues behavioural changes of Kdm5c knockout mice. Conversely, a single injection of WNT3A into the brains of wild-type embryonic mice cause anxiety and memory alterations. Our work identifies KDM5C as a crucial sentinel for neurodevelopment and sheds new light on KDM5C mutation-associated intellectual disability. The results also increase our general understanding of memory and anxiety formation, with the identification of WNT functioning in a transient nature to affect long-lasting cognitive function

Lithium, an anti-psychotic drug, greatly enhances the generation of induced pluripotent stem cells

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. The low efficiency of reprogramming and genomic integration of oncogenes and viral vectors limited the potential application of iPSCs. Here we report that Lithium (Li), a drug used to treat mood disorders, greatly enhances iPSC generation from both mouse embryonic fibroblast and human umbilical vein endothelial cells. Li facilitates iPSC generation with one (Oct4) or two factors (OS or OK). The effect of Li on promoting reprogramming only partially depends on its major target GSK3β. Unlike other GSK3β inhibitors, Li not only increases the expression of Nanog, but also enhances the transcriptional activity of Nanog. We also found that Li exerts its effect by promoting epigenetic modifications via downregulation of LSD1, a H3K4-specific histone demethylase. Knocking down LSD1 partially mimics Li's effect in enhancing reprogramming. Our results not only provide a straightforward method to improve the iPSC generation efficiency, but also identified a histone demethylase as a critical modulator for somatic cell reprogramming

Critical current density: Measurements vs. reality

Different experimental techniques are employed to evaluate the critical current density (Jc), namely transport current measurements and two different magnetisation measurements forming quasi-equilibrium and dynamic critical states. Our technique-dependent results for superconducting YBa 2Cu3O7 (YBCO) film and MgB2 bulk samples show an extremely high sensitivity of Jc and associated interpretations, such as irreversibility fields and Kramer plots, which lose meaning without a universal approach. We propose such approach for YBCO films based on their unique pinning features. This approach allows us to accurately recalculate the magnetic-field-dependent Jc obtained by any technique into the Jc behaviour, which would have been measured by any other method without performing the corresponding experiments. We also discovered low-frequency-dependent phenomena, governing flux dynamics, but contradicting the considered ones in the literature. The understanding of these phenomena, relevant to applications with moving superconductors, can clarify their dramatic impact on the electric-field criterion through flux diffusivity and corresponding measurements. © Copyright EPLA, 2013

Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms

Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substraterecognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals crucial and distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis

Identification of Novel SNPs by Next-Generation Sequencing of the Genomic Region Containing the APC Gene in Colorectal Cancer Patients in China

We described an approach of identifying single nucleotide polymorphisms (SNPs) in complete genomic regions of key genes including promoters, exons, introns, and downstream sequences by combining long-range polymerase chain reaction (PCR) or NimbleGen sequence capture with next-generation sequencing. Using the adenomatous polyposis coli (APC) gene as an example, we identified 210 highly reliable SNPs by next-generation sequencing analysis program MAQ and Samtools, of which 69 were novel ones, in the 123-kb APC genomic region in 27 pair of colorectal cancers and normal adjacent tissues. We confirmed all of the eight randomly selected high-quality SNPs by allele-specific PCR, suggesting that our false discovery rate is negligible. We identified 11 SNPs in the exonic region, including one novel SNP that was not previously reported. Although 10 of them are synonymous, they were predicted to affect splicing by creating or removing exonic splicing enhancers or exonic splicing silencers. We also identified seven SNPs in the upstream region of the APC gene, three of which were only identified in the cancer tissues. Six of these upstream SNPs were predicted to affect transcription factor binding. We also observed that long-range PCR was better in capturing GC-rich regions than the NimbleGen sequence capture technique.MOST, Chin