Itay Maza

Derivation of novel human ground state naive pluripotent stem cells

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.

Deterministic direct reprogramming of somatic cells to pluripotency

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation

mRNA modification regulates pluripotency When stem cells progress from an embryonic pluripotent state toward a particular lineage, molecular switches dismantle the transcription factor network that keeps the cell pluripotent. Geula et al. now show that N6-methyladenosine (m6A), a messenger RNA (mRNA) modification present on transcripts of pluripotency factors, drives this transition. Methylation destabilized mRNA transcripts and limited their translation efficiency, which promoted the timely decay of naïve pluripotency. This m6A methylation was also critical for mammalian development. Science, this issue p. 1002 A messenger RNA epigenetic modification regulates stem cell progression from the pluripotent to the differentiated state. Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N6-methyladenosine (m6A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m6A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m6A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.

The H3K27 demethylase Utx regulates somatic and germ cell epigenetic reprogramming

Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by ectopic expression of different transcription factors, classically Oct4 (also known as Pou5f1), Sox2, Klf4 and Myc (abbreviated as OSKM). This process is accompanied by genome-wide epigenetic changes, but how these chromatin modifications are biochemically determined requires further investigation. Here we show in mice and humans that the histone H3 methylated Lys 27 (H3K27) demethylase Utx (also known as Kdm6a) regulates the efficient induction, rather than maintenance, of pluripotency. Murine embryonic stem cells lacking Utx can execute lineage commitment and contribute to adult chimaeric animals; however, somatic cells lacking Utx fail to robustly reprogram back to the ground state of pluripotency. Utx directly partners with OSK reprogramming factors and uses its histone demethylase catalytic activity to facilitate iPSC formation. Genomic analysis indicates that Utx depletion results in aberrant dynamics of H3K27me3 repressive chromatin demethylation in somatic cells undergoing reprogramming. The latter directly hampers the derepression of potent pluripotency promoting gene modules (including Sall1, Sall4 and Utf1), which can cooperatively substitute for exogenous OSK supplementation in iPSC formation. Remarkably, Utx safeguards the timely execution of H3K27me3 demethylation observed in embryonic day 10.5–11 primordial germ cells (PGCs), and Utx-deficient PGCs show cell-autonomous aberrant epigenetic reprogramming dynamics during their embryonic maturation in vivo. Subsequently, this disrupts PGC development by embryonic day 12.5, and leads to diminished germline transmission in mouse chimaeras generated from Utx-knockout pluripotent cells. Thus, we identify Utx as a novel mediator with distinct functions during the re-establishment of pluripotency and germ cell development. Furthermore, our findings highlight the principle that molecular regulators mediating loss of repressive chromatin during in vivo germ cell reprogramming can be co-opted during in vitro reprogramming towards ground state pluripotency.

Evolutionary analysis across mammals reveals distinct classes of long non-coding RNAs

BackgroundRecent advances in transcriptome sequencing have enabled the discovery of thousands of long non-coding RNAs (lncRNAs) across many species. Though several lncRNAs have been shown to play important roles in diverse biological processes, the functions and mechanisms of most lncRNAs remain unknown. Two significant obstacles lie between transcriptome sequencing and functional characterization of lncRNAs: identifying truly non-coding genes from de novo reconstructed transcriptomes, and prioritizing the hundreds of resulting putative lncRNAs for downstream experimental interrogation.ResultsWe present slncky, a lncRNA discovery tool that produces a high-quality set of lncRNAs from RNA-sequencing data and further uses evolutionary constraint to prioritize lncRNAs that are likely to be functionally important. Our automated filtering pipeline is comparable to manual curation efforts and more sensitive than previously published computational approaches. Furthermore, we developed a sensitive alignment pipeline for aligning lncRNA loci and propose new evolutionary metrics relevant for analyzing sequence and transcript evolution. Our analysis reveals that evolutionary selection acts in several distinct patterns, and uncovers two notable classes of intergenic lncRNAs: one showing strong purifying selection on RNA sequence and another where constraint is restricted to the regulation but not the sequence of the transcript.ConclusionOur results highlight that lncRNAs are not a homogenous class of molecules but rather a mixture of multiple functional classes with distinct biological mechanism and/or roles. Our novel comparative methods for lncRNAs reveals 233 constrained lncRNAs out of tens of thousands of currently annotated transcripts, which we make available through the slncky Evolution Browser.

Prediction of disease complication occurrence in Crohn's disease using phenotype and genotype parameters at diagnosis.

BACKGROUND AND AIMS Complications associated with Crohns disease (CD) are common and influence treatment decisions and outcomes. Appropriate early treatment may offer a therapeutic advantage to patients. The aim of our study was to indentify predictive factors for occurrence of complications at the time of CD diagnosis. METHODS The study population consisted of 269 CD patients treated during a ten year period. Risk factors compared between complicated and non-complicated disease included phenotypical characteristics, disease classification and the presence of NOD2/CARD15 mutations and single nucleotide polymorphisms in selected autophagy and phagosome genes. RESULTS Complete data was obtained for 146 patients with an average follow up of 12years. Sixty five patients (44%) developed a complication during follow up. The only independent risk factors associated with developing a complication were smoking and male gender. There was no association between developing complications and the presence of selected SNPs (P=0.07 for Tyrosine residue on both alleles in NCF4 SNP rs4821544 and P=0.06 for a Guanine residue on both alleles in ATG16L SNP rs2241880). Multivariate analysis using a backwards logistic regression model left only male gender as an independent statistically significant association with complicated disease (OR 2.6017, 95% CI: 1.17 to 5.75). The median time to developing a complication was 4years, and the most common complication was the need for surgical intervention (54%). CONCLUSIONS In the present study, a risk factor for developing CD complication was male gender. Further studies are warranted to assess additional risk factors and how such findings should affect therapy.

Color-Coded Duplex Sonography Compared to Multidetector Computed Tomography for the Diagnosis of Crohn Disease Relapse and Complications

The purpose of this study was to evaluate the accuracy of color‐coded duplex sonography for the diagnosis of Crohn disease relapse and complications compared to multidetector computed tomography (CT).

The association of Haptoglobin polymorphism with Crohn's disease in Israel

OBJECTIVES Haptoglobin is a α(2)-sialoglycoprotein with hemoglobin binding capacity. Functional differences between the Hp phenotypes with the Hp 1-1 protein being a superior anti-inflammatory to the Hp 2-2 protein have been identified. The aim of our study was to investigate the possible role of Hp polymorphism in the susceptibility to Crohns disease and its clinical course. METHODS Hp phenotypes were determined for 382 Israeli CD patients and 3243 healthy controls. Phenotypic data for all Crohns disease patients were carefully characterized. Analysis was preformed to evaluate the association between Hp polymorphism and Crohns disease. RESULTS The frequency of Haptoglobin 1-1 was lower in Crohns disease patients than in healthy individuals (6.28% vs. 9.28%, P=0.057). There was no association between Haptoglobin phenotypes and disease location, behavior or extra-intestinal manifestations. No association was found between the Haptoglobin polymorphism and the frequency of the three Crohns disease associated NOD2 mutations examined. CONCLUSIONS We found a borderline significant decrease of the Haptoglobin 1-1 phenotype in Israeli Crohns disease patients compared to healthy controls. Our findings may support the importance of inflammation in Crohns disease pathogenesis and the protective function of Haptoglobin 1-1 in the susceptibility for Crohns disease.

Rectal bleeding as a presenting symptom of AL amyloidosis and multiple myeloma.

Amyloidosis of the gastrointestinal tract is a rare disease that presents with common, nonspecific signs and symptoms. It may affect any part of the gastrointestinal tract from mouth to anus. The clinical and endoscopic features are diverse and may mimic other diseases, such as inflammatory bowel disease, malignancy, ischemic colitis and, at times, collagenous colitis. We describe an uncommon case of rectal bleeding and anemia with polypoid lesions and ulcerations in the colon, as the presenting symptom of AL amyloidosis and light chain multiple myeloma.

High-Resolution Dissection of Conducive Reprogramming Trajectory to Ground State Pluripotency

The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) with four transcription factors Oct4, Sox2, Klf4 and cMyc (abbreviated as OSKM)1 has provoked interest to define the molecular characteristics of this process2-7. Despite important progress, the dynamics of epigenetic reprogramming at high resolution in correctly reprogrammed iPSCs and throughout the entire process remain largely undefined. This gap in understanding results from the inefficiency of conventional reprogramming methods coupled with the difficulty of prospectively isolating the rare cells that eventually correctly reprogram into iPSCs. Here we characterize cell fate conversion from fibroblast to iPSC using a highly efficient deterministic murine reprogramming system engineered through optimized inhibition of Gatad2a-Mbd3/NuRD repressive sub-complex. This comprehensive characterization provides single-day resolution of dynamic changes in levels of gene expression, chromatin modifications, TF binding, DNA accessibility and DNA methylation. The integrative analysis identified two transcriptional modules that dominate successful reprogramming. One consists of genes whose transcription is regulated by on/off epigenetic switching of modifications in their promoters (abbreviated as ESPGs), and the second consists of genes with promoters in a constitutively active chromatin state, but a dynamic expression pattern (abbreviated as CAPGs). ESPGs are mainly regulated by OSK, rather than Myc, and are enriched for cell fate determinants and pluripotency factors. CAPGs are predominantly regulated by Myc, and are enriched for cell biosynthetic regulatory functions. We used the ESPG module to study the identity and temporal occurrence of activating and repressing epigenetic switching during reprogramming. Removal of repressive chromatin modifications precedes chromatin opening and binding of RNA polymerase II at enhancers and promoters, and the opposite dynamics occur during repression of enhancers and promoters. Genome wide DNA methylation analysis demonstrated that de novo DNA methylation is not required for highly efficient conducive iPSC reprogramming, and identified a group of super-enhancers targeted by OSK, whose early demethylation marks commitment to a successful reprogramming trajectory also in inefficient conventional reprogramming systems. CAPGs are distinctively regulated by multiple synergystic ways: 1) Myc activity, delivered either endogenously or exogenously, dominates CAPG expression changes and is indispensable for induction of pluripotency in somatic cells; 2) A change in tRNA codon usage which is specific to CAPGs, but not ESPGs, and favors their translation. In summary, our unbiased high-resolution mapping of epigenetic changes on somatic cells that are committed to undergo successful reprogramming reveals interleaved epigenetic and biosynthetic reconfigurations that rapidly commission and propel conducive reprogramming toward naïve pluripotency.