Rolling back human pluripotent stem cells to an eight-cell embryo-like stage.

After fertilization, the quiescent zygote experiences a burst of genome activation that initiates a short-lived totipotent state. Understanding the process of totipotency in human cells would have broad applications. However, in contrast to in mice1,2, demonstration of the time of zygotic genome activation or the eight-cell (8C) stage in in vitro cultured human cells has not yet been reported, and the study of embryos is limited by ethical and practical considerations. Here we describe a transgene-free, rapid and controllable method for producing 8C-like cells (8CLCs) from human pluripotent stem cells. Single-cell analysis identified key molecular events and gene networks associated with this conversion. Loss-of-function experiments identified fundamental roles for DPPA3, a master regulator of DNA methylation in oocytes3, and TPRX1, a eutherian totipotent cell homeobox (ETCHbox) family transcription factor that is absent in mice4. DPPA3 induces DNA demethylation throughout the 8CLC conversion process, whereas TPRX1 is a key executor of 8CLC gene networks. We further demonstrate that 8CLCs can produce embryonic and extraembryonic lineages in vitro or in vivo in the form of blastoids5 and complex teratomas. Our approach provides a resource to uncover the molecular process of early human embryogenesis.

The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity.

The RNA modification N6-methyladenosine (m6A) has critical roles in many biological processes1,2. However, the function of m6A in the early phase of mammalian development remains poorly understood. Here we show that the m6A reader YT521-B homology-domain-containing protein 1 (YTHDC1) is required for the maintenance of mouse embryonic stem (ES) cells in an m6A-dependent manner, and that its deletion initiates cellular reprogramming to a 2C-like state. Mechanistically, YTHDC1 binds to the transcripts of retrotransposons (such as intracisternal A particles, ERVK and LINE1) in mouse ES cells and its depletion results in the reactivation of these silenced retrotransposons, accompanied by a global decrease in SETDB1-mediated trimethylation at lysine 9 of histone H3 (H3K9me3). We further demonstrate that YTHDC1 and its target m6A RNAs act upstream of SETDB1 to repress retrotransposons and Dux, the master inducer of the two-cell stage (2C)-like program. This study reveals an essential role for m6A RNA and YTHDC1 in chromatin modification and retrotransposon repression.

Dux activates metabolism-lactylation-MET network during early iPSC reprogramming with Brg1 as the histone lactylation reader.

The process of induced pluripotent stem cells (iPSCs) reprogramming involves several crucial events, including the mesenchymal-epithelial transition (MET), activation of pluripotent genes, metabolic reprogramming, and epigenetic rewiring. Although these events intricately interact and influence each other, the specific element that regulates the reprogramming network remains unclear. Dux, a factor known to promote totipotency during the transition from embryonic stem cells (ESC) to 2C-like ESC (2CLC), has not been extensively studied in the context of iPSC reprogramming. In this study, we demonstrate that the modification of H3K18la induced by Dux overexpression controls the metabolism-H3K18la-MET network, enhancing the efficiency of iPSC reprogramming through a metabolic switch and the recruitment of p300 via its C-terminal domain. Furthermore, our proteomic analysis of H3K18la immunoprecipitation experiment uncovers the specific recruitment of Brg1 during reprogramming, with both H3K18la and Brg1 being enriched on the promoters of genes associated with pluripotency and epithelial junction. In summary, our study has demonstrated the significant role of Dux-induced H3K18la in the early reprogramming process, highlighting its function as a potent trigger. Additionally, our research has revealed, for the first time, the binding of Brg1 to H3K18la, indicating its role as a reader of histone lactylation.

Chromatin-Remodeling Components of the BAF Complex Facilitate Reprogramming.

Reprogramming of somatic cells achieved by combination of the four transcription factors Oct4, Sox2, Klf4, and c-Myc has very low efficiency. To increase the reprogramming efficiency and better understand the process, we sought to identify factors that mediate reprogramming with higher efficiency. We established an assay to screen nuclear fractions from extracts of pluripotent mouse cells based on Oct4 reactivation. Using proteomics, we identified components of the ATP-dependent BAF chromatin-remodeling complex, which significantly increases reprogramming efficiency when used together with the four factors. The reprogrammed cells could transmit to the germline and exhibited pluripotency. Reprogramming remained highly efficient when c-Myc was not present but BAF components were overexpressed. BAF complex components mediate this effect by facilitating enhanced Oct4 binding to target promoters during reprogramming. Thus, somatic cell reprogramming using chromatin-remodeling molecules represents an efficient method of generating reprogrammed cells.

Epiblast stem cell subpopulations represent mouse embryos of distinct pregastrulation stages.

Embryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast.

YAP establishes epiblast responsiveness to inductive signals for germ cell fate.

The germ cell lineage in mammals is induced by the stimulation of pluripotent epiblast cells by signaling molecules. Previous studies have suggested that the germ cell differentiation competence or responsiveness of epiblast cells to signaling molecules is established and maintained in epiblast cells of a specific differentiation state. However, the molecular mechanism underlying this process has not been well defined. Here, using the differentiation model of mouse epiblast stem cells (EpiSCs), we have shown that two defined EpiSC lines have robust germ cell differentiation competence. However, another defined EpiSC line has no competence. By evaluating the molecular basis of EpiSCs with distinct germ cell differentiation competence, we identified YAP, an intracellular mediator of the Hippo signaling pathway, as crucial for the establishment of germ cell induction. Strikingly, deletion of YAP severely affected responsiveness to inductive stimuli, leading to a defect in WNT target activation and germ cell differentiation. In conclusion, we propose that the Hippo/YAP signaling pathway creates a potential for germ cell fate induction via mesodermal WNT signaling in pluripotent epiblast cells.

Biomimetic Transparent Layered Tough Aerogels for Thermal Superinsulation and Triboelectric Nanogenerator.

Transparent aerogels are ideal candidates for thermally insulating windows, solar thermal receivers, electronics, etc. However, they are usually prepared via energy-consuming supercritical drying and show brittleness and low tensile strength, significantly restricting their practical applications. It remains a great challenge to prepare transparent aerogels with high tensile strength and toughness. Herein, biomimetic transparent tough cellulose nanofiber-based nanocomposite aerogels with a layered nanofibrous structure are achieved by vacuum-assisted self-assembly combined with ambient pressure drying. The nacre-like layered homogeneous nanoporous structures can reduce light scattering and effectively transfer stress and prevent stress concentration under external forces. The aerogels exhibit an attractive combination of excellent transparency and hydrophobicity, high compressive and tensile strengths, high toughness, excellent machinability, thermal superinsulation, and wide working temperature range (-196 to 230 °C). It is demonstrated that they can be used for superinsulating windows of buildings and high-efficient thermal management for electronics and human bodies. In addition, a prototype of transparent flexible aerogel-based triboelectric nanogenerator is developed. This work provides a promising pathway toward transparent tough porous materials for energy saving/harvesting, thermal management, electronics, sensors, etc.

Adenosylhomocysteinase plays multiple roles in maintaining the identity and pluripotency of mouse embryonic stem cells†.

Adenosylhomocysteinase (AHCY), a key enzyme in the methionine cycle, is essential for the development of embryos and the maintenance of mouse embryonic stem cells (mESCs). However, the precise underlying mechanism of Ahcy in regulating pluripotency remains unclear. As the only enzyme that can hydrolyze S-adenosylhomocysteine in mammals, AHCY plays a critical role in the metabolic homeostasis, epigenetic remodeling, and transcriptional regulation. Here, we identified Ahcy as a direct target of OCT4 and unveiled that AHCY regulates the self-renewal and differentiation potency of mESCs through multiple mechanisms. Our study demonstrated that AHCY is required for the metabolic homeostasis of mESCs. We revealed the dual role of Ahcy in both transcriptional activation and inhibition, which is accomplished via the maintenance of H3K4me3 and H3K27me3, respectively. We found that Ahcy is required for H3K4me3-dependent transcriptional activation in mESCs. We also demonstrated that AHCY interacts with polycomb repressive complex 2 (PRC2), thereby maintaining the pluripotency of mESCs by sustaining the H3K27me3-regulated transcriptional repression of related genes. These results reveal a previously unrecognized OCT4-AHCY-PRC2 axis in the regulation of mESCs' pluripotency and provide insights into the interplay between transcriptional factors, cellular metabolism, chromatin dynamics and pluripotency regulation.

Attributing Atmospheric Phosphorus in the Himalayas: Biomass Burning vs Mineral Dust.

Atmospheric phosphorus is a vital nutrient for ecosystems whose sources and fate are still debated in the fragile Himalayan region, hindering our comprehension of its local ecological impact. This study provides novel insights into atmospheric phosphorus based on the study of total suspended particulate matter at the Qomolangma station. Contrary to the prevailing assumptions, we show that biomass burning (BB), not mineral dust, dominates total dissolved phosphorus (TDP, bioavailable) deposition in this arid region, especially during spring. While total phosphorus is mainly derived from dust (77% annually), TDP is largely affected by the transport of regional biomass-burning plumes from South Asia. During BB pollution episodes, TDP causing springtime TDP fluxes alone accounts for 43% of the annual budget. This suggests that BB outweighs dust in supplying bioavailable phosphorus, a critical nutrient, required to sustain Himalayas' ecological functions. Overall, this first-hand field evidence refines the regional and global phosphorus budget by demonstrating that BB emission, while still unrecognized, is a significant source of P, even in the remote mountains of the Himalayas. It also reveals the heterogeneity of atmospheric phosphorus deposition in that region, which will help predict changes in the impacted ecosystems as the deposition patterns vary.

Oct4-induced pluripotency in adult neural stem cells.

The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.

Progress on the relationship between tumor suppressor PDCD4 and diseases based on the analysis of structural characteristics.

The tumor suppressor programmed cell death 4 (PDCD4) is downregulated in various tumor tissues indicating poor prognosis. PDCD4 is the first protein found to resist tumor transformation, invasion, and metastasis by inhibiting translation. The functions of PDCD4 dependent on its structures are affected by extracellular signals. It regulates tumor-related proteins through a variety of mechanisms, especially involved in two major signaling pathways, PI3K-Akt-mTOR and MAPK. By analyzing the relationship between the structures, functions and diseases of PDCD4, this review summarizes the roles of PDCD4 in several physiological processes and diseases such as apoptosis, autophagy, tumor, and inflammation in recent years, thereby providing insights for the study of the signaling pathways of PDCD4 and related proteins and the treatment of diseases targeting them.

Permissive epigenomes endow reprogramming competence to transcriptional regulators.

Identifying molecular and cellular processes that regulate reprogramming competence of transcription factors broadens our understanding of reprogramming mechanisms. In the present study, by a chemical screen targeting major epigenetic pathways in human reprogramming, we discovered that inhibiting specific epigenetic roadblocks including disruptor of telomeric silencing 1-like (DOT1L)-mediated H3K79/K27 methylation, but also other epigenetic pathways, catalyzed by lysine-specific histone demethylase 1A, DNA methyltransferases and histone deacetylases, allows induced pluripotent stem cell generation with almost all OCT factors. We found that simultaneous inhibition of these pathways not only dramatically enhances reprogramming competence of most OCT factors, but in fact enables dismantling of species-dependent reprogramming competence of OCT6, NR5A1, NR5A2, TET1 and GATA3. Harnessing these induced permissive epigenetic states, we performed an additional screen with 98 candidate genes. Thereby, we identified 25 transcriptional regulators (OTX2, SIX3, and so on) that can functionally replace OCT4 in inducing pluripotency. Our findings provide a conceptual framework for understanding how transcription factors elicit reprogramming in dependency of the donor cell epigenome that differs across species.

Residual pluripotency is required for inductive germ cell segregation.

Fine-tuned dissolution of pluripotency is critical for proper cell differentiation. Here we show that the mesodermal transcription factor, T, globally affects the properties of pluripotency through binding to Oct4 and to the loci of other pluripotency regulators. Strikingly, lower T levels coordinately affect naïve pluripotency, thereby directly activating the germ cell differentiation program, in contrast to the induction of germ cell fate of primed models. Contrary to the effect of lower T levels, higher T levels more severely affect the pluripotency state, concomitantly enhancing the somatic differentiation program and repressing the germ cell differentiation program. Consistent with such in vitro findings, nascent germ cells in vivo are detected in the region of lower T levels at the posterior primitive streak. Furthermore, T and core pluripotency regulators co-localize at the loci of multiple germ cell determinants responsible for germ cell development. In conclusion, our findings indicate that residual pluripotency establishes the earliest and fundamental regulatory mechanism for inductive germline segregation from somatic lineages.

Wildfire-Derived Nitrogen Aerosols Threaten the Fragile Ecosystem in Himalayas and Tibetan Plateau.

Himalayas and Tibetan Plateau (HTP) is important for global biodiversity and regional sustainable development. While numerous studies have revealed that the ecosystem in this unique and pristine region is changing, their exact causes are still poorly understood. Here, we present a year-round (23 March 2017 to 19 March 2018) ground- and satellite-based atmospheric observation at the Qomolangma monitoring station (QOMS, 4276 m a.s.l.). Based on a comprehensive chemical and stable isotope (15N) analysis of nitrogen compounds and satellite observations, we provide unequivocal evidence that wildfire emissions in South Asia can come across the Himalayas and threaten the HTP's ecosystem. Such wildfire episodes, mostly occurring in spring (March-April), not only substantially enhanced the aerosol nitrogen concentration but also altered its composition (i.e., rendering it more bioavailable). We estimated a nitrogen deposition flux at QOMS of ∼10 kg N ha-1 yr-1, which is approximately twice the lower value of the critical load range reported for the Alpine ecosystem. Such adverse impact is particularly concerning, given the anticipated increase of wildfire activities in the future under climate change.

Genome-wide analysis of cell-Free DNA methylation profiling with MeDIP-seq identified potential biomarkers for colorectal cancer.

BACKGROUND: Colorectal cancer is the most common malignancy and the third leading cause of cancer-related death worldwide. This study aimed to identify potential diagnostic biomarkers for colorectal cancer by genome-wide plasma cell-free DNA (cfDNA) methylation analysis. METHODS: Peripheral blood from colorectal cancer patients and healthy controls was collected for cfDNA extraction. Genome-wide cfDNA methylation profiling, especially differential methylation profiling between colorectal cancer patients and healthy controls, was performed by methylated DNA immunoprecipitation coupled with high-throughput sequencing (MeDIP-seq). Logistic regression models were established, and the accuracy of this diagnostic model for colorectal cancer was verified using tissue-sourced data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) due to the lack of cfDNA methylation data in public datasets. RESULTS: Compared with the control group, 939 differentially methylated regions (DMRs) located in promoter regions were found in colorectal cancer patients; 16 of these DMRs were hypermethylated, and the remaining 923 were hypomethylated. In addition, these hypermethylated genes, mainly PRDM14, RALYL, ELMOD1, and TMEM132E, were validated and confirmed in colorectal cancer by using publicly available DNA methylation data. CONCLUSIONS: MeDIP-seq can be used as an optimal approach for analyzing cfDNA methylomes, and 12 probes of four differentially methylated genes identified by MeDIP-seq (PRDM14, RALYL, ELMOD1, and TMEM132E) could serve as potential biomarkers for clinical application in patients with colorectal cancer.

Histone demethylase KDM6A promotes somatic cell reprogramming by epigenetically regulating the PTEN and IL-6 signal pathways.

Aberrant epigenetic reprogramming is one of the major barriers for somatic cell reprogramming. Although our previous study has indicated that H3K27me3 demethylase KDM6A can improve the nuclear reprogramming efficiency, the mechanism remains unclear. In this study, we demonstrate that the overexpression of Kdm6a may improve induced pluripotent stem cell (iPSC) reprogramming efficiency in a demethylase enzymatic activity-dependent manner. KDM6A erased H3K27me3 on pluripotency- and metabolism-related genes, and consequently facilitated changing the gene expression profile and metabolic pattern to an intermediate state. Furthermore, KDM6A may promote IL-6 expression, and the secreted IL-6 may further improve iPSC reprogramming efficiency. In addition, KDM6A may promote PTEN expression to decrease p-AKT and p-mTOR levels, which in turn facilitates reprogramming. Overall, our results reveal that KDM6A may promote iPSC reprogramming efficiency by accelerating changes in the gene expression profile and the metabolic pattern in a demethylation-activity-dependent manner. These results may provide an insight into the relationship between epigenomics, transcriptomics, metabolomics, and reprogramming.

Formation Mechanisms and Source Apportionments of Airborne Nitrate Aerosols at a Himalayan-Tibetan Plateau Site: Insights from Nitrogen and Oxygen Isotopic Compositions.

Formation pathways and sources of atmosphere nitrate (NO3-) have attracted much attention as NO3- had detrimental effects on Earth's ecosystem and climate change. Here, we measured nitrogen (δ15N-NO3-) and oxygen (δ18O-NO3- and Δ17O-NO3-) isotope compositions in nitrate aerosols at the Qomolangma station (QOMS) over the Himalayan-Tibetan Plateau (HTP) to quantify the formation mechanisms and emission sources of nitrate at the background site. At QOMS, the enhanced NO3- concentrations were observed in the springtime. The average δ15N-NO3-, δ18O-NO3-, and Δ17O-NO3- values were 0.4 ± 4.9, 64.7 ± 11.5 and 27.6 ± 6.9‰, respectively. Seasonal variations of isotope ratios at QOMS can be explained by the different emissions and formation pathways to nitrate. The average fractions of NO2 + OH and N2O5 + H2O to nitrate production were estimated to be 43 and 52%, respectively, when the NO3 + hydrocarbon (HC)/dimethyl sulfide (DMS) (NO3 + HC/DMS) pathway was assumed to be 5%. Using stable isotope analysis in the R (SIAR) model, the relative contributions of biomass burning (BB), biogenic soil emission, traffic, and coal combustion to nitrate were estimated to be 28, 25, 24, and 23%, respectively, on yearly basis. By FLEXible PARTicle (FLEXPART) dispersion model, we highlighted that NOx from BB emission over South Asia that had undergone N2O5 + H2O processes enhanced the nitrate concentrations in the springtime over the HTP region.

hnRNP-K Targets Open Chromatin in Mouse Embryonic Stem Cells in Concert with Multiple Regulators.

The transcription factor Oct4 plays a key regulatory role in the induction and maintenance of cellular pluripotency. In this article, we show that ubiquitous and multifunctional poly(C) DNA/RNA-binding protein hnRNP-K occupies Oct4 (Pou5f1) enhancers in embryonic stem cells (ESCs) but is dispensable for the initiation, maintenance, and downregulation of Oct4 gene expression. Nevertheless, hnRNP-K has an essential cell-autonomous function in ESCs to maintain their proliferation and viability. To better understand mechanisms of hnRNP-K action in ESCs, we have performed ChIP-seq analysis of genome-wide binding of hnRNP-K and identified several thousands of hnRNP-K target sites that are frequently co-occupied by pluripotency-related and common factors (Oct4, TATA-box binding protein, Sox2, Nanog, Otx2, etc.), as well as active histone marks. Furthermore, hnRNP-K localizes exclusively within open chromatin, implying its role in the onset and/or maintenance of this chromatin state. Stem Cells 2019;37:1018-1029.

Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos.

Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are 'trapped' inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative applications, as donated or discarded embryos are more accessible than unfertilized MII oocytes.

Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile.

Primordial germ cells (PGCs) develop only into sperm and oocytes in vivo. The molecular mechanisms underlying human PGC specification are poorly understood due to inaccessibility of cell materials and lack of in vitro models for tracking the earliest stages of germ cell development. Here, we describe a defined and stepwise differentiation system for inducing pre-migratory PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs). In response to cytokines, PSCs differentiate first into a heterogeneous mesoderm-like cell population and then into PGCLCs, which exhibit minimal PRDM14 expression. PGC specification in humans is similar to the murine process, with the sequential activation of mesodermal and PGC genes, and the suppression of neural induction and of de novo DNA methylation, suggesting that human PGC formation is induced via epigenesis, the process of germ cell specification via inductive signals from surrounding somatic cells. This study demonstrates that PGC commitment in humans shares key features with that of the mouse, but also highlights key differences, including transcriptional regulation during the early stage of human PGC development (3-6 weeks). A more comprehensive understanding of human germ cell development may lead to methodology for successfully generating PSC-derived gametes for reproductive medicine.