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Strong epigenetic pan-cancer biomarkers are required to meet several current, urgent clinical needs and to further improve the present chemotherapeutic standard. We have concentrated on the investigation of epigenetic alteration of the hTERT gene, which is frequently epigenetically dysregulated in a number of cancers in specific developmental stages. Distinct DNA methylation profiles were identified in our data on early urothelial cancer. An efficient EpihTERT assay could be developed utilizing suitable combinations with sequence-dependent thermodynamic parameters to distinguish between differentially methylated states. We infer from this data set, the epigenetic context, and the related literature that a CpG-rich, 2800 bp region, a prominent CpG island, surrounding the transcription start of the hTERT gene is the crucial epigenetic zone for the development of a potent biomarker. In order to accurately describe this region, we have named it "Acheron" (Ἀχέρων). In Greek mythology, this is the river of woe and misery and the path to the underworld. Exploitation of the DNA methylation profiles focused on this region, e.g., idiolocal normalized Methylation Specific PCR (IDLN-MSP), opens up a wide range of new possibilities for diagnosis, determination of prognosis, follow-up, and detection of residual disease. It may also have broad implications for the choice of chemotherapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer treatment could be revolutionized by using particular CAR-T-cell therapies for all solid tumors. Finding appropriate CAR-T-cell antigens for every tumor entity would be necessary for this though. Our findings provide new insight into the possibility of employing CAR-T-cell therapy to treat nearly all cancers, as genome-wide screening following consistent occurring DNA hypomethylations may uncover novel antigens for every cancer entity. Based on the impressive success of Car-T-cell therapy in the treatment of hematological malignancies, a broad application for solid tumors also appears promising. However, some important hurdles need to be overcome. One of these is certainly the identification of specific target antigens on cancer cells. Hypomethylation is a characteristic epigenetic aberration in many tumor entities. Genome-wide screenings for consistent DNA hypomethylations in tumors enable the identification of aberrantly upregulated transcripts, which might result in cell surface proteins. Thus, this approach provides a new perspective for the discovery of potential new Car-T-cell target antigens for almost every tumor entity. First, we focus on this approach as a possible treatment for prostate cancer. [ABSTRACT FROM AUTHOR]

Background: Recombinant human bone morphogenetic protein 2 (rhBMP-2) and human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied for research and translational bone regeneration purposes. rhBMP-2 induces bone formation in vivo, and hBM-MSCs are its target, bone-forming cells. In this article, we studied how rhBMP-2 drives the multilineage differentiation of hBM-MSCs both in vivo and in vitro. Methods: rhBMP-2 and hBM-MSCs were tested in an in vivo subcutaneous implantation model to assess their ability to form mature bone and undergo multilineage differentiation. Then, the hBM-MSCs were treated in vitro with rhBMP-2 for short-term or long-term cell-culture periods, alone or in combination with osteogenic, adipogenic or chondrogenic media, aiming to determine the role of rhBMP-2 in these differentiation processes. Results: The data indicate that hBM-MSCs respond to rhBMP-2 in the short term but fail to differentiate in long-term culture conditions; these cells overexpress the rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, in combination with other differentiation signals, rhBMP-2 acts as a potentiator of multilineage differentiation, not only of osteogenesis but also of adipogenesis and chondrogenesis, both in vitro and in vivo. Conclusions: Altogether, our data indicate that rhBMP-2 alone is unable to induce in vitro osteogenic terminal differentiation of hBM-MSCs, but synergizes with other signals to potentiate multiple differentiation phenotypes. Therefore, rhBMP-2 triggers on hBM-MSCs different specific phenotype differentiation depending on the signalling environment. [ABSTRACT FROM AUTHOR]

Background: Frailty is a geriatric syndrome associated with negative health outcomes that represents a dynamic condition with a potential of reversibility after physical exercise interventions. Typically, inflammatory and senescence markers are increased in frail individuals. However, the impact that physical exercise exerts on inflammatory and senescence biomarkers remains unknown. We assessed the effect of physical intervention in old individuals and mice and determined the expression of inflammatory and senescence markers. Methods: Twelve elderly individuals were enrolled from a primary care setting to a 3-month intervention. Frailty was measured by SPPB and the expression of biomarkers by cytokine array and RT-qPCR. In addition, 12 aged C57BL/6 mice completed an intervention, and inflammation and senescence markers were studied. Results: The physical intervention improved the SPPB score, reducing frail and pre-frail individuals. This was correlated with a reduction in several pro-inflammatory biomarkers such as IL-6, CXCL-1, CXCL-10, IL-1β, IL-7, GM-CSF as well as p16INK4a and p21CIP1 senescence markers. Otherwise, the levels of anti-inflammatory biomarker IL-4 were significantly increased. Moreover, the physical intervention in mice also improved their functional capacity and restored the expression of inflammatory (Il-1β, Cxcl-10, Il-6, and Cxcl-1) and senescence (p21Cip1) markers. Additionally, PLSDA and ROC curve analysis revealed CXCL-10 and IL-1β to be the biomarkers of functional improvement in both cohorts. Conclusions: Our results showed that a physical intervention improves physical frailty, and reverses inflammation and senescence biomarkers comprising CXCL-10 and IL-1β. [ABSTRACT FROM AUTHOR]

Introduction: Bipolar disorder (BD) is a chronically progressive mental condition, associated with a reduced quality of life and greater disability. Patient admissions are preventable events with a considerable impact on global functioning and social adjustment. While machine learning (ML) approaches have proven prediction ability in other diseases, little is known about their utility to predict patient admissions in this pathology. Aim: To develop prediction models for hospital admission/readmission within 5 years of diagnosis in patients with BD using ML techniques. Methods: The study utilized data from patients diagnosed with BD in a major healthcare organization in Colombia. Candidate predictors were selected from Electronic Health Records (EHRs) and included sociodemographic and clinical variables. ML algorithms, including Decision Trees, Random Forests, Logistic Regressions, and Support Vector Machines, were used to predict patient admission or readmission. Survival models, including a penalized Cox Model and Random Survival Forest, were used to predict time to admission and first readmission. Model performance was evaluated using accuracy, precision, recall, F1 score, area under the receiver operating characteristic curve (AUC) and concordance index. Results: The admission dataset included 2,726 BD patients, with 354 admissions, while the readmission dataset included 352 patients, with almost half being readmitted. The best-performing model for predicting admission was the Random Forest, with an accuracy score of 0.951 and an AUC of 0.98. The variables with the greatest predictive power in the Recursive Feature Elimination (RFE) importance analysis were the number of psychiatric emergency visits, the number of outpatient follow-up appointments and age. Survival models showed similar results, with the Random Survival Forest performing best, achieving an AUC of 0.95. However, the prediction models for patient readmission had poorer performance, with the Random Forest model being again the best performer but with an AUC below 0.70. Conclusion: ML models, particularly the Random Forest model, outperformed traditional statistical techniques for admission prediction. However, readmission prediction models had poorer performance. This study demonstrates the potential of ML techniques in improving prediction accuracy for BD patient admissions. [ABSTRACT FROM AUTHOR]

Cell-free extrachromosomal circular DNA (cf-eccDNA) has been proposed as a promising early biomarker for disease diagnosis, progression and drug response. Its established biomarker features are changes in the number and length distribution of cf-eccDNA. Another novel promising biomarker is a set of eccDNA excised from a panel of genes specific to a condition compared to a control. Deficiencies in two endonucleases that specifically target DNA, Dnase1 and Dnase1l3, are associated with systemic lupus erythematosus (SLE). To study the genic eccDNA profiles in the case of their deficiencies, we mapped sequenced eccDNA data from plasma, liver and buffy coat from Dnase1 and Dnase1l3 knockouts (KOs), and wild type controls in mouse. Next, we performed an eccDNA differential analysis between KO and control groups using our DifCir algorithm. We found a specific genic cf-eccDNA fingerprint of the Dnase1l3 group compared to the wild type controls involving 131 genes; 26% of them were associated with human chromosomal fragile sites (CFSs) and with a statistically significant enrichment of CFS-associated genes. We found six genes in common with the genic cf-eccDNA profile of SLE patients with DNASE1L3 deficiency, namely Rorb, Mvb12b, Osbpl10, Fto, Tnik and Arhgap10; all of them were specific and present in all human plasma samples, and none of them were associated with CFSs. A not so distinctive genic cf-eccDNA difference involving only seven genes was observed in the case of the Dnase1 group compared to the wild type. In tissue—liver and buffy coat—we did not detect the same genic eccDNA difference observed in the plasma samples. These results point to a specific role of a set of genic eccDNA in plasma from DNase KOs, as well as a relation with CFS genes, confirming the promise of the genic cf-eccDNA in studying diseases and the need for further research on the relationship between eccDNA and CFSs. [ABSTRACT FROM AUTHOR]

Background: Numerous genes, including SOD1, mutated in familial and sporadic amyotrophic lateral sclerosis (f/sALS) share a role in DNA damage and repair, emphasizing genome disintegration in ALS. One possible outcome of chromosomal instability and repair processes is extrachromosomal circular DNA (eccDNA) formation. Therefore, eccDNA might accumulate in f/sALS with yet unknown function. Methods: We combined rolling circle amplification with linear DNA digestion to purify eccDNA from the cervical spinal cord of 9 co-isogenic symptomatic hSOD1G93A mutants and 10 controls, followed by deep short-read sequencing. We mapped the eccDNAs and performed differential analysis based on the split read signal of the eccDNAs, referred as DifCir, between the ALS and control specimens, to find differentially produced per gene circles (DPpGC) in the two groups. Compared were eccDNA abundances, length distributions and genic profiles. We further assessed proteome alterations in ALS by mass spectrometry, and matched the DPpGCs with differentially expressed proteins (DEPs) in ALS. Additionally, we aligned the ALS-specific DPpGCs to ALS risk gene databases. Results: We found a six-fold enrichment in the number of unique eccDNAs in the genotoxic ALS-model relative to controls. We uncovered a distinct genic circulome profile characterized by 225 up-DPpGCs, i.e., genes that produced more eccDNAs from distinct gene sequences in ALS than under control conditions. The inter-sample recurrence rate was at least 89% for the top 6 up-DPpGCs. ALS proteome analyses revealed 42 corresponding DEPs, of which 19 underlying genes were itemized for an ALS risk in GWAS databases. The up-DPpGCs and their DEP tandems mainly impart neuron-specific functions, and gene set enrichment analyses indicated an overrepresentation of the adenylate cyclase modulating G protein pathway. Conclusions: We prove, for the first time, a significant enrichment of eccDNA in the ALS-affected spinal cord. Our triple circulome, proteome and genome approach provide indication for a potential importance of certain eccDNAs in ALS neurodegeneration and a yet unconsidered role as ALS biomarkers. The related functional pathways might open up new targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Background: Native bone marrow (BM) mesenchymal stem/stromal cells (BM-MSCs) participate in generating and shaping the skeleton and BM throughout the lifespan. Moreover, BM-MSCs regulate hematopoiesis by contributing to the hematopoietic stem cell niche in providing critical cytokines, chemokines and extracellular matrix components. However, BM-MSCs contain a heterogeneous cell population that remains ill-defined. Although studies on the taxonomy of native BM-MSCs in mice have just started to emerge, the taxonomy of native human BM-MSCs remains unelucidated. Methods: By using single-cell RNA sequencing (scRNA-seq), we aimed to define a proper taxonomy for native human BM non-hematopoietic subsets including endothelial cells (ECs) and mural cells (MCs) but with a focal point on MSCs. To this end, transcriptomic scRNA-seq data were generated from 5 distinct BM donors and were analyzed together with other transcriptomic data and with computational biology analyses at different levels to identify, characterize and classify distinct native cell subsets with relevant biomarkers. Results: We could ascribe novel specific biomarkers to ECs, MCs and MSCs. Unlike ECs and MCs, MSCs exhibited an adipogenic transcriptomic pattern while co-expressing genes related to hematopoiesis support and multilineage commitment potential. Furthermore, by a comparative analysis of scRNA-seq of BM cells from humans and mice, we identified core genes conserved in both species. Notably, we identified MARCKS, CXCL12, PDGFRA, and LEPR together with adipogenic factors as archetypal biomarkers of native MSCs within BM. In addition, our data suggest some complex gene nodes regulating critical biological functions of native BM-MSCs together with a preferential commitment toward an adipocyte lineage. Conclusions: Overall, our taxonomy for native BM non-hematopoietic compartment provides an explicit depiction of gene expression in human ECs, MCs and MSCs at single-cell resolution. This analysis helps enhance our understanding of the phenotype and the complexity of biological functions of native human BM-MSCs. [ABSTRACT FROM AUTHOR]

Cell-free (cf) extrachromosomal circular DNA (eccDNA) has a potential clinical application as a biomarker. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a complex immunological pathogenesis, associated with autoantibody synthesis. A previous study found that SLE patients with deoxyribonuclease 1-like 3 (DNASE1L3) deficiency exhibit changes in the frequency of short and long eccDNA in plasma compared to controls. Here, using the DifCir method for differential analysis of short-read sequenced purified eccDNA data based on the split-read signal of the eccDNA on circulomics data, we show that SLE patients with DNASE1L3 deficiency have a distinctive profile of eccDNA excised by gene regions compared to controls. Moreover, this profile is specific; cf-eccDNA from the top 93 genes is detected in all SLE with DNASE1L3 deficiency samples, and none in the control plasma. The top protein coding gene producing eccDNA-carrying gene fragments is the transcription factor BARX2, which is involved in skeletal muscle morphogenesis and connective tissue development. The top gene ontology terms are 'positive regulation of torc1 signaling' and 'chondrocyte development'. The top Harmonizome terms are 'lymphopenia', 'metabolic syndrome x', 'asthma', 'cardiovascular system disease', 'leukemia', and 'immune system disease'. Here, we show that gene associations of cf-eccDNA can serve as a biomarker in the autoimmune rheumatic diseases. [ABSTRACT FROM AUTHOR]

To bring new extrachromosomal circular DNA (eccDNA) enrichment technologies closer to the clinic, specifically for screening, early diagnosis, and monitoring of diseases or lifestyle conditions, it is paramount to identify the differential pattern of the genic eccDNA signal between two states. Current studies using short-read sequenced purified eccDNA data are based on absolute numbers of unique eccDNAs per sample or per gene, length distributions, or standard methods for RNA-seq differential analysis. Previous analyses of RNA-seq data found significant transcriptomics difference between sedentary and active life style skeletal muscle (SkM) in young people but very few in old. The first attempt using circulomics data from SkM and blood of aged lifelong sedentary and physically active males found no difference at eccDNA level. To improve the capability of finding differences between circulomics data groups, we designed a computational method to identify Differentially Produced per Gene Circles (DPpGCs) from short-read sequenced purified eccDNA data based on the circular junction, split-read signal, of the eccDNA, and implemented it into a software tool DifCir in Matlab. We employed DifCir to find to the distinctive features of the influence of the physical activity or inactivity in the aged SkM that would have remained undetected by transcriptomics methods. We mapped the data from tissue from SkM and blood from two groups of aged lifelong sedentary and physically active males using Circle_finder and subsequent merging and filtering, to find the number and length distribution of the unique eccDNA. Next, we used DifCir to find up-DPpGCs in the SkM of the sedentary and active groups. We assessed the functional enrichment of the DPpGCs using Disease Gene Network and Gene Set Enrichment Analysis. To find genes that produce eccDNA in a group without comparison with another group, we introduced a method to find Common PpGCs (CPpGCs) and used it to find CPpGCs in the SkM of the sedentary and active group. Finally, we found the eccDNA that carries whole genes. We discovered that the eccDNA in the SkM of the sedentary group is not statistically different from that of physically active aged men in terms of number and length distribution of eccDNA. In contrast, with DifCir we found distinctive gene-associated eccDNA fingerprints. We identified statistically significant up-DPpGCs in the two groups, with the top up-DPpGCs shed by the genes AGBL4, RNF213, DNAH7, MED13, and WWTR1 in the sedentary group, and ZBTB7C, TBCD, ITPR2, and DDX11-AS1 in the active group. The up-DPpGCs in both groups carry mostly gene fragments rather than whole genes. Though the subtle transcriptomics difference, we found RYR1 to be both transcriptionally up-regulated and up-DPpGCs gene in sedentary SkM. DifCir emphasizes the high sensitivity of the circulome compared to the transcriptome to detect the molecular fingerprints of exercise in aged SkM. It allows efficient identification of gene hotspots that excise more eccDNA in a health state or disease compared to a control condition. [ABSTRACT FROM AUTHOR]

With approximately 1.4 million men annually diagnosed with prostate cancer (PCa) worldwide, PCa remains a dreaded threat to life and source of devastating morbidity. In recent decades, a significant decrease in age-specific PCa mortality has been achieved by increasing prostate-specific antigen (PSA) screening and improving treatments. Nevertheless, upcoming, augmented recommendations against PSA screening underline an escalating disproportion between the benefit and harm of current diagnosis/prognosis and application of radical treatment standards. Undoubtedly, new potent diagnostic and prognostic tools are urgently needed to alleviate this tensed situation. They should allow a more reliable early assessment of the upcoming threat, in order to enable applying timely adjusted and personalized therapy and monitoring. Here, we present a basic study on an epigenetic screening approach by Methylated DNA Immunoprecipitation (MeDIP). We identified genes associated with hypomethylated CpG islands in three PCa sample cohorts. By adjusting our computational biology analyses to focus on single CpG-enriched 60-nucleotide-long DNA probes, we revealed numerous consistently differential methylated DNA segments in PCa. They were associated among other genes with NOTCH3, CDK2AP1, KLK4, and ADAM15. These can be used for early discrimination, and might contribute to a new epigenetic tumor classification system of PCa. Our analysis shows that we can dissect short, differential methylated CpG-rich DNA fragments and combinations of them that are consistently present in all tumors. We name them tumor cell-specific differential methylated CpG dinucleotide signatures (TUMS). [ABSTRACT FROM AUTHOR]

The transplantation of pluripotent stem cell (PSC)-derived liver organoids has been studied to solve the current donor shortage. However, the differentiation of unintended cell populations, difficulty in generating multi-lineage organoids, and tumorigenicity of PSC-derived organoids are challenges. However, direct conversion technology has allowed for the generation lineage-restricted induced stem cells from somatic cells bypassing the pluripotent state, thereby eliminating tumorigenic risks. Here, liver assembloids (iHEAs) were generated by integrating induced endothelial cells (iECs) into the liver organoids (iHLOs) generated with induced hepatic stem cells (iHepSCs). Liver assembloids showed enhanced functional maturity compared to iHLOs in vitro and improved therapeutic effects on cholestatic liver fibrosis animals in vivo. Mechanistically, FN1 expressed from iECs led to the upregulation of Itgα5/β1 and Hnf4α in iHEAs and were correlated to the decreased expression of genes related to hepatic stellate cell activation such as Lox and Spp1 in the cholestatic liver fibrosis animals. In conclusion, our study demonstrates the possibility of generating transplantable iHEAs with directly converted cells, and our results evidence that integrating iECs allows iHEAs to have enhanced hepatic maturation compared to iHLOs. [ABSTRACT FROM AUTHOR]

Background: Previously, we collected age-stratified incidence data of 404 epidemiological datasets of 10 neurodegenerative diseases (NDs), namely Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), Fronto Temporal Dementia (FTD), Dementia with Lewy Bodies (DLB), Parkinsonism (PDM), Parkinson's disease with Dementia (PDD), Creutzfeldt–Jakob disease (CJD), and Multiple Sclerosis (MS). We tested whether each ND follows a multistep model, found the number of steps necessary for the onset of each ND, found the number of common steps with other NDs and the number of specific steps of each ND, and built a parsimony tree of the genealogy of the NDs. The tree disclosed three groups of NDs: the stem NDs with less than 3 steps; the trunk NDs with 5–7 steps; and the crown NDs with more than 7 steps. Methods: We made a multidimensional reduction of the previously collected age-stratified incidence epidemiological data of the 10 NDs. We studied the general range of incidence of the 10 NDs using the age- and sex-stratified incidence data. First, we calculated the log of the incidence versus the log of the age for each ND. Next, we calculated the age intervals of the spread of the incidence of each ND. We calculated the regression of the steps obtained with the multistep model versus the age of incidence of the NDs. Results: We found that the number of steps of the NDs is inversely correlated with the age of incidence of the NDs, and calculated the number of years required for a single step for each ND. Based on these results, we extended the genealogy tree model of the NDs to account for the time needed for a ND step to occur. Conclusion: The extended genealogy tree disclosed three groups of NDs according to the estimated time needed for a step to occur: the stem ND, HD, with 32.5 years/step, the trunk NDs ALS, FTD, PD and CJD, with 6.7–13.7 years/step; and the crown NDs PDM, PDD, AD and DLB, with 2.3–3.8 years/step. Thus, the NDs cluster into three groups according to both the number of steps and the number of years for a step to occur. [ABSTRACT FROM AUTHOR]

Background Feature selection is a relevant step in the analysis of single-cell RNA sequencing datasets. Most of the current feature selection methods are based on general univariate descriptors of the data such as the dispersion or the percentage of zeros. Despite the use of correction methods, the generality of these feature selection methods biases the genes selected towards highly expressed genes, instead of the genes defining the cell populations of the dataset. Results Triku is a feature selection method that favors genes defining the main cell populations. It does so by selecting genes expressed by groups of cells that are close in the k -nearest neighbor graph. The expression of these genes is higher than the expected expression if the k -cells were chosen at random. Triku efficiently recovers cell populations present in artificial and biological benchmarking datasets, based on adjusted Rand index, normalized mutual information, supervised classification, and silhouette coefficient measurements. Additionally, gene sets selected by triku are more likely to be related to relevant Gene Ontology terms and contain fewer ribosomal and mitochondrial genes. Conclusion Triku is developed in Python 3 and is available at https://github.com/alexmascension/triku. [ABSTRACT FROM AUTHOR]

(E) Quantification of mRNA levels of p21cip, Bax and SerpinB5 in cells transfected with ASOs for PR-LncRNA1 and 10 and compared to cells with a control ASO (F) Expression of PR-LncRNA 1,5 and 10 in indicated conventional cell lines (U87-MG, U251, U373 and A172) and glioma stem cells (GNS166, GNS179 and GB1) (G) Quantification of primary oncospheres formed in ASO-transfected cells after 10 days in culture (n = 3). (H) Quantification of number of secondary oncospheres generated from disaggregating primary oncospheres in ASO-transfected and control cells. [Extracted from the article]

Background: Osteogenesis imperfecta (OI) is a rare genetic disease characterized by bone fragility, with a wide range in the severity of clinical manifestations. The majority of cases are due to mutations in the COL1A1 or COL1A2 genes, which encode type I collagen. Mesenchymal stem cells (MSCs), as the progenitors of the osteoblasts, the main type I collagen secreting cell type in the bone, have been proposed and tested as an innovative therapy for OI with promising but transient outcomes. Methods: To overcome the short‐term effect of MSCs therapy, we performed a phase I clinical trial based on reiterative infusions of histocompatible MSCs, administered in a 2.5‐year period, in two pediatric patients affected by severe and moderate OI. The aim of this study was to assess the safety and effectiveness of this cell therapy in nonimmunosuppressed OI patients. The host response to MSCs was studied by analyzing the sera from OI patients, collected before, during, and after the cell therapy. Results: We first demonstrated that the sequential administration of MSCs was safe and improved the bone parameters and quality of life of OI patients along the cell treatment plus 2‐year follow‐up period. Moreover, the study of the mechanism of action indicated that MSCs therapy elicited a pro‐osteogenic paracrine response in patients, especially noticeable in the patient affected by severe OI. Conclusions: Our results demonstrate the feasibility and potential of reiterative MSCs infusion for two pediatric OI and highlight the paracrine response shown by patients as a consequence of MSCs treatment. [ABSTRACT FROM AUTHOR]

Urothelial carcinoma (UC) is a common disease causing significant morbidity and mortality as well as considerable costs for health systems. Extensive aberrant methylation of DNA is broadly documented in early UC, contributing to genetic instability, altered gene expression and tumor progression. However the triggers initiating aberrant methylation are unknown. Recently we discovered that several genes encoding key enzymes of methyl group and polyamine metabolism, including Ornithine Decarboxylase 1 (ODC1), are affected by DNA methylation in early stage UC. In this study, we investigated the hypothesis that these epigenetic alterations act in a feed-forward fashion to promote aberrant DNA methylation in UC. We demonstrate that siRNA-mediated knockdown of ODC1 expression elicits genome-wide LINE-1 demethylation, induction of LINE-1 transcripts and double-strand DNA breaks and decreases viability in primary cultured uroepithelial cells. Similarly, following siRNA-mediated knockdown of ODC1, UC cells undergo double-strand DNA breaks and apoptosis. Collectively, our findings provide evidence that ODC1 gene hypermethylation could be a starting point for the onset of genome-wide epigenetic aberrations in urothelial carcinogenesis. Furthermore, LINE-1 induction enabled by ODC1 interference provides a new experimental model to study mechanisms and consequences of LINE-1 activation in the etiology and progression of UC as well as presumably other cancers. [ABSTRACT FROM AUTHOR]

Background: Vaccination induces survival of otherwise lethal blood-stage infections of the experimental malaria Plasmodium chabaudi. Blood-stage malaria induces extramedullary erythropoiesis in the liver. This study investigates how vaccination affects the course of malaria-induced expression of erythrocytic genes in the liver. Methods: Female Balb/c mice were vaccinated at week 3 and week 1 before challenging with 106P. chabaudi-parasitized erythrocytes. The non-infectious vaccine consisted of erythrocyte ghosts isolated from P. chabaudi-infected erythrocytes. Gene expression microarrays and quantitative real-time PCR were used to compare mRNA expression of different erythrocytic genes in the liver of vaccination-protected and non-protected mice during infections on days 0, 1, 4, 8, and 11 p.i. Results: Global transcriptomics analyses reveal vaccination-induced modifications of malaria-induced increases in hepatic gene expression on days 4 and 11 p.i. On these days, vaccination also alters hepatic expression of the erythropoiesis-involved genes Ermap, Kel, Rhd, Rhag, Slc4a1, Gypa, Add2, Ank1, Epb4.1, Epb4.2, Epb4.9, Spta1, Sptb, Tmod1, Ahsp, Acyp1, Gata1, Gfi1b, Tal1, Klf1, Epor, and Cldn13. In vaccination-protected mice, expression of these genes, except Epb4.1, is significantly higher on day 4 p.i. than in un-protected non-vaccinated mice, reaches maximal expression at peak parasitaemia on day 8 p.i., and is slowed down or even decreased towards the end of crisis phase on day 11 p.i.. After day 1 p.i., Epor expression takes about the same course as that of the other erythroid genes. Hepatic expression of Epo, however, is delayed in both vaccinated and non-vaccinated mice for the first 4 days p.i. and is maximal at significantly higher levels in vaccinated mice on day 8 p.i., before declining towards the end of crisis phase on day 11 p.i. Conclusion: The present data indicate that vaccination accelerates malaria-induced erythroblastosis in the liver for 1–2 days. This may contribute to earlier replenishment of peripheral red blood cells by liver-derived reticulocytes, which may favour final survival of otherwise lethal blood-stage malaria, since reticulocytes are not preferred as host cells by P. chabaudi. [ABSTRACT FROM AUTHOR]

Direct conversion from fibroblasts to generate hepatocyte like-cells (iHeps) bypassing the pluripotent state has been described in previous reports as an attractive method acquiring hepatocytes for cell-based therapy. The limited proliferation of iHeps, however, has hampered it uses in cell-based therapy. Since hepatic stem cells (HepSCs) possess self-renewal and bipotency with the capacity to differentiate into both hepatocytes and cholangiocytes, they have therapeutic potential for treating liver disease. Here, we investigated the therapeutic effects of induced HepSCs (iHepSCs) on a carbon tetrachloride (CCl4)-induced liver fibrosis model. We demonstrate that Oct4 and Hnf4a are sufficient to convert fibroblasts into expandable iHepSCs. Hepatocyte-like cells derived from iHepSCs (iHepSC-HEPs) exhibit the typical morphology of hepatocytes and hepatic functions, including glycogen storage, low-density lipoprotein (LDL) uptake, Indocyanine green (ICG) detoxification, drug metabolism, urea production, and albumin secretion. iHepSCs-derived cholangiocyte-like cells (iHepSC-CLCs) expressed cholangiocyte-specific markers and formed cysts and tubule-like structures with apical-basal polarity and secretory function in three-dimensional culture condition. Furthermore, iHepSCs showed anti-inflammatory and anti-fibrotic effects in CCl4-induced liver fibrosis. This study demonstrates that Oct4 and Hnf4α-induced HepSCs show typical hepatic and biliary functionality in vitro. It also presents the therapeutic effect of iHepSCs in liver fibrosis. Therefore, directly converting iHepSCs from somatic cells may facilitate the development of patient-specific cell-based therapy for chronic liver damage. [ABSTRACT FROM AUTHOR]

GABA-B receptors are important for Schwann cell (SC) commitment to a non-myelinating phenotype during development. However, the P0-GABA-B1fl/fl conditional knockout mice, lacking the GABA-B1 receptor specifically in SCs, also presented axon modifications, suggesting SC non-autonomous effects through the neuronal compartment. In this in vitro study, we evaluated whether the specific deletion of the GABA-B1 receptor in SCs may induce autonomous or non-autonomous cross-changes in sensory dorsal root ganglia (DRG) neurons. To this end, we performed an in vitro biomolecular and transcriptomic analysis of SC and DRG neuron primary cultures from P0-GABA-B1fl/fl mice. We found that cells from conditional P0-GABA-B1fl/fl mice exhibited proliferative, migratory and myelinating alterations. Moreover, we found transcriptomic changes in novel molecules that are involved in peripheral neuron-SC interaction. [ABSTRACT FROM AUTHOR]

Phenotypic transformation of liver sinusoidal endothelial cells is one of the most important stages of liver metastasis progression. The miRNA effects on liver sinusoidal endothelial cells during liver metastasis have not yet been studied. Herein, whole genome analysis of miRNA expression in these cells during colorectal liver metastasis revealed repressed expression of microRNA‐20a. Importantly, downregulation of miR‐20a occurs in parallel with upregulation of its known protein targets. To restore normal miR‐20a levels in liver sinusoidal endothelial cells, we developed chondroitin sulfate‐sorbitan ester nanoparticles conjugated with miR‐20a in a delivery system that specifically targets liver sinusoidal endothelial cells. The restoration of normal mir‐20a levels in these cells induced downregulation of the expression of its protein targets, and this also resulted in a reduction of in vitro LSEC migration and a reduction of in vivo activation and tumor‐infiltrating capacity and ability of the tumor decreased by ∼80% in a murine liver metastasis model. [ABSTRACT FROM AUTHOR]

Background: DNA methylation at promoters is largely correlated with inhibition of gene expression. However, the role of DNA methylation at enhancers is not fully understood, although a crosstalk with chromatin marks is expected. Actually, there exist contradictory reports about positive and negative correlations between DNA methylation and H3K4me1, a chromatin hallmark of enhancers. Results: We investigated the relationship between DNA methylation and active chromatin marks through genomewide correlations, and found anti-correlation between H3K4me1 and H3K4me3 enrichment at low and intermediate DNA methylation loci. We hypothesized "seesaw" dynamics between H3K4me1 and H3K4me3 in the low and intermediate DNA methylation range, in which DNA methylation discriminates between enhancers and promoters, marked by H3K4me1 and H3K4me3, respectively. Low methylated regions are H3K4me3 enriched, while those with intermediate DNA methylation levels are progressively H3K4me1 enriched. Additionally, the enrichment of H3K27ac, distinguishing active from primed enhancers, follows a plateau in the lower range of the intermediate DNA methylation level, corresponding to active enhancers, and decreases linearly in the higher range of the intermediate DNA methylation. Thus, the decrease of the DNA methylation switches smoothly the state of the enhancers from a primed to an active state. We summarize these observations into a rule of thumb of one-out-of-three methylation marks: "In each genomic region only one out of these three methylation marks {DNA methylation, H3K4me1, H3K4me3} is high. If it is the DNA methylation, the region is inactive. If it is H3K4me1, the region is an enhancer, and if it is H3K4me3, the region is a promoter". To test our model, we used available genome-wide datasets of H3K4 methyltransferases knockouts. Our analysis suggests that CXXC proteins, as readers of non-methylated CpGs would regulate the "seesaw" mechanism that focuses H3K4me3 to unmethylated sites, while being repulsed from H3K4me1 decorated enhancers and CpG island shores. Conclusions: Our results show that DNA methylation discriminates promoters from enhancers through H3K4me1- H3K4me3 seesaw mechanism, and suggest its possible function in the inheritance of chromatin marks after cell division. Our analyses suggest aberrant formation of promoter-like regions and ectopic transcription of hypomethylated regions of DNA. Such mechanism process can have important implications in biological process in where it has been reported abnormal DNA methylation status such as cancer and aging. [ABSTRACT FROM AUTHOR]

Background: Superenhancers are crucial structural genomic elements determining cell fate, and they are also involved in the determination of several diseases, such as cancer or neurodegeneration. Although there are pipelines which use independent pieces of software to predict the presence of superenhancers from genome-wide chromatin marks or DNA-interaction protein binding sites, there is not yet an integrated software tool that processes automatically algebra combinations of raw data sequencing into a comprehensive final annotated report of predicted superenhancers. Results: We have developed NaviSE, a user-friendly streamlined tool which performs a fully-automated parallel processing of genome-wide epigenomics data from sequencing files into a final report, built with a comprehensive set of annotated files that are navigated through a graphic user interface dynamically generated by NaviSE. NaviSE also implements an 'epigenomics signal algebra' that allows the combination of multiple activation and repression epigenomics signals. NaviSE provides an interactive chromosomal landscaping of the locations of superenhancers, which can be navigated to obtain annotated information about superenhancer signal profile, associated genes, gene ontology enrichment analysis, motifs of transcription factor binding sites enriched in superenhancers, graphs of the metrics evaluating the superenhancers quality, protein-protein interaction networks and enriched metabolic pathways among other features. We have parallelised the most time-consuming tasks achieving a reduction up to 30% for a 15 CPUs machine. We have optimized the default parameters of NaviSE to facilitate its use. NaviSE allows different entry levels of data processing, from sra-fastq files to bed files; and unifies the processing of multiple replicates. NaviSE outperforms the more time-consuming processes required in a non-integrated pipeline. Alongside its high performance, NaviSE is able to provide biological insights, predicting cell type specific markers, such as SOX2 and ZIC3 in embryonic stem cells, CDK5R1 and REST in neurons and CD86 and TLR2 in monocytes. Conclusions: NaviSE is a user-friendly streamlined solution for superenhancer analysis, annotation and navigation, requiring only basic computer and next generation sequencing knowledge. NaviSE binaries and documentation are available at: https://sourceforge.net/projects/navise-superenhancer/. [ABSTRACT FROM AUTHOR]

Motivation: Bisulfite sequencing (BSseq) processing is among the most cumbersome next generation sequencing (NGS) applications. Though some BSseq processing tools are available, they are scattered, require puzzling parameters and are running-time and memory-usage demanding. Results: We developed P3BSseq, a parallel processing pipeline for fast, accurate and automatic analysis of BSseq reads that trims, aligns, annotates, records the intermediate results, performs bisulfite conversion quality assessment, generates BED methylome and report files following the NIH standards. P3BSseq outperforms the known BSseq mappers regarding running time, computer hardware requirements (processing power and memory use) and is optimized to process the upcoming, extended BSseq reads. We optimized the P3BSseq parameters for directional and non-directional libraries, and for single-end and paired-end reads of Whole Genome and Reduced Representation BSseq. P3BSseq is a user-friendly streamlined solution for BSseq upstream analysis, requiring only basic computer and NGS knowledge. [ABSTRACT FROM AUTHOR]

In the last decades extracellular vesicles (EVs) have emerged as key players for intercellular communication. In the case of inflammation, several studies have reported that EV levels are increased in circulation during inflammatory episodes. Based on this, we investigated whether aging results in elevated EV number, as a basal proinflammatory status termed "inflammaging" has been described in aged individuals. Moreover, we also hypothesized that frailty and dependence conditions of the elderly could affect EV concentration in plasma. Results showed that inflammaging, frailty or dependence status do not result in EV increase, at least in the total number of EVs in circulation. These results open a new perspective for investigating the role of EVs in human aging and in the inflammaging process. [ABSTRACT FROM AUTHOR]

Summary It has previously been reported that mouse epiblast stem cell (EpiSC) lines comprise heterogeneous cell populations that are functionally equivalent to cells of either early- or late-stage postimplantation development. So far, the establishment of the embryonic stem cell (ESC) pluripotency gene regulatory network through the widely known chemical inhibition of MEK and GSK3beta has been impractical in late-stage EpiSCs. Here, we show that chemical inhibition of casein kinase 1alpha (CK1alpha) induces the conversion of recalcitrant late-stage EpiSCs into ESC pluripotency. CK1alpha inhibition directly results in the simultaneous activation of the WNT signaling pathway, together with inhibition of the TGFbeta/SMAD2 signaling pathway, mediating the rewiring of the gene regulatory network in favor of an ESC-like state. Our findings uncover a molecular mechanism that links CK1alpha to ESC pluripotency through the direct modulation of WNT and TGFbeta signaling. [ABSTRACT FROM AUTHOR]

The generation of patient-specific oligodendrocyte progenitor cells (OPCs) holds great potential as an expandable cell source for cell replacement therapy as well as drug screening in spinal cord injury or demyelinating diseases. Here, we demonstrate that induced OPCs (iOPCs) can be directly derived from adult mouse fibroblasts by Oct4-mediated direct reprogramming, using anchorageindependent growth to ensure high purity. Homogeneous iOPCs exhibit typical small-bipolar morphology, maintain their selfrenewal capacity and OPC marker expression for more than 31 passages, share high similarity in the global gene expression profile to wild-type OPCs, and give rise to mature oligodendrocytes and astrocytes in vitro and in vivo. Notably, transplanted iOPCs contribute to functional recovery in a spinal cord injury (SCI) model without tumor formation. This study provides a simple strategy to generate functional self-renewing iOPCs and yields insights for the in-depth study of demyelination and regenerative medicine. [ABSTRACT FROM AUTHOR]

Background: Understanding the mechanisms by which hundreds of diverse cell types develop from a single mammalian zygote has been a central challenge of developmental biology. Conrad H. Waddington, in his metaphoric "epigenetic landscape" visualized the early embryogenesis as a hierarchy of lineage bifurcations. In each bifurcation, a single progenitor cell type produces two different cell lineages. The tristable dynamical systems are used to model the lineage bifurcations. It is also shown that a genetic circuit consisting of two auto-activating transcription factors (TFs) with cross inhibitions can form a tristable dynamical system. Results: We used gene expression profiles of pre-implantation mouse embryos at the single cell resolution to visualize the Waddington landscape of the early embryogenesis. For each lineage bifurcation we identified two clusters of TFs -- rather than two single TFs as previously proposed -- that had opposite expression patterns between the pair of bifurcated cell types. The regulatory circuitry among each pair of TF clusters resembled a genetic circuit of a pair of single TFs; it consisted of positive feedbacks among the TFs of the same cluster, and negative interactions among the members of the opposite clusters. Our analyses indicated that the tristable dynamical system of the two-cluster regulatory circuitry is more robust than the genetic circuit of two single TFs. Conclusions: We propose that a modular hierarchy of regulatory circuits, each consisting of two mutually inhibiting and auto-activating TF clusters, can form hierarchical lineage bifurcations with improved safeguarding of critical early embryogenesis against biological perturbations. Furthermore, our computationally fast framework for modeling and visualizing the epigenetic landscape can be used to obtain insights from experimental data of development at the single cell resolution. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Until recently, acquired resistance to cytostatics had mostly been attributed to biochemical mechanisms such as decreased intake and/or increased efflux of therapeutics, enhanced DNA repair, and altered activity or deregulation of target proteins. Although these mechanisms have been widely investigated, little is known about membrane barriers responsible for the chemical imperviousness of cell compartments and cellular segregation in cytostatic-treated tumors. In highly heterogeneous cross-resistant and radiorefractory cell populations selected by exposure to anticancer agents, we found a number of atypical recurrent cell types in (1) tumor cell cultures of different embryonic origins, (2) mouse xenografts, and (3) paraffin sections from patient tumors. Alongside morphologic peculiarities, these populations presented cancer stem cell markers, aberrant signaling pathways, and a set of deregulated miRNAs known to confer both stem-cell phenotypes and highly aggressive tumor behavior. The first type, named spiral cells, is marked by a spiral arrangement of nuclei. The second type, monastery cells, is characterized by prominent walls inside which daughter cells can be seen maturing amid a rich mitochondrial environment. The third type, called pregnant cells, is a giant cell with a syncytium-like morphology, a main nucleus, and many endoreplicative functional progeny cells. A rare fourth cell type identified in leukemia was christened shepherd cells, as it was always associated with clusters of smaller cells. Furthermore, a portion of resistant tumor cells displayed nuclear encapsulation via mitochondrial aggregation in the nuclear perimeter in response to cytostatic insults, probably conferring imperviousness to drugs and long periods of dormancy until nuclear eclosion takes place. This phenomenon was correlated with an increase in both intracellular and intercellular mitochondrial traffic as well as with the uptake of free extracellular mitochondria. All these cellular disorders could, in fact, be found in untreated tumor cells but were more pronounced in resistant entities, suggesting a natural mechanism of cell survival triggered by chemical injury, or a primitive strategy to ensure stemming, self-renewal, and differentiation under adverse conditions, a fact that may play a significant role in chemotherapy outcomes. [ABSTRACT FROM AUTHOR]

Summary The differentiation capability of induced pluripotent stem cells (iPSCs) toward certain cell types for disease modeling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from fetal neural stem cells (fNSCs), dermal fibroblasts, or cord blood CD34 + hematopoietic progenitor cells. Neural progenitor cells (NPCs) and neurons could be generated at similar efficiencies from all iPSCs. Transcriptomics analysis of the whole genome and of neural genes revealed a separation of neuroectoderm-derived iPSC-NPCs from mesoderm-derived iPSC-NPCs. Furthermore, we found genes that were similarly expressed in fNSCs and neuroectoderm, but not in mesoderm-derived iPSC-NPCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival of neuroectoderm-derived cells in vivo. These results indicate distinct origin-dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Urinary DNA is increasingly gaining importance in diagnosis of urological malignancies. Especially cell-free DNA originating from apoptotic and necrotic cells of the early tumor could become a key target for early stage tumor diagnosis. Aberrant DNA methylation forms tumor cell characteristic epigenetic profiles which are covalently established before any tumor related aberration at transcriptional or protein level has occurred. In addition, these epigenetic signatures are alterably adapted to and accompanying the individual stages of multistep, progressive tumorigenesis. Hence, they seem very promising for diagnosis as well as for monitoring the patient's follow-up care and even for decisions regarding personalized therapeutic options. The essential prerequisite at this approach will be a reliable methodological handling of the biological material of interest. In this study we present detailed analyses of LINE-1 DNA methylation profiles and demonstrate the sensitive detection of LINE-1 DNA methylation differences as well as between cancer patients and healthy individuals, between urinary cellular and cell-free DNA. In addition, we show methylome differences between both DNA fractions from a healthy individual and bladder cancer patients. In conclusion, we demonstrate here the unrestricted amenability of urinary cell-free DNA for both, a detailed characterization of a distinct DNA methylation alteration and its sensitive detection and a comprehensive global, array-based screening for DNA methylation differences. [ABSTRACT FROM AUTHOR]

Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU-domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for longterm periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feederfree culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate. [ABSTRACT FROM AUTHOR]

Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24–36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1) and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle. [ABSTRACT FROM AUTHOR]

It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterized the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells. [ABSTRACT FROM AUTHOR]

It has recently been established that synthesis of double-stranded cDNA can be done from a single cell for use in DNA sequencing. Global gene expression can be quantified from the number of reads mapping to each gene, and mutations and mRNA splicing variants determined from the sequence reads. Here we demonstrate that this method of transcriptomic analysis can be done using the extremely low levels of mRNA in a single nucleus, isolated from a mouse neural progenitor cell line and from dissected hippocampal tissue. This method is characterized by excellent coverage and technical reproducibility. On average, more than 16,000 of the 24,057 mouse protein-coding genes were detected from single nuclei, and the amount of gene-expression variation was similar when measured between single nuclei and single cells. Several major advantages of the method exist: first, nuclei, compared with whole cells, have the advantage of being easily isolated from complex tissues and organs, such as those in the CNS. Second, the method can be widely applied to eukaryotic species, including those of different kingdoms. The method also provides insight into regulatory mechanisms specific to the nucleus. Finally, the method enables dissection of regulatory events at the single-cell level; pooling of 10 nuclei or 10 cells obscures some of the variability measured in transcript levels, implying that single nuclei and cells will be extremely useful in revealing the physiological state and interconnectedness of gene regulation in a manner that avoids the masking inherent to conventional transcriptomics using bulk cells or tissues. [ABSTRACT FROM AUTHOR]