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Identifying the relationships between chromosome structures, nuclear bodies, chromatin states and gene expression is an overarching goal of nuclear-organization studies.sup.1-4. Because individual cells appear to be highly variable at all these levels.sup.5, it is essential to map different modalities in the same cells. Here we report the imaging of 3,660 chromosomal loci in single mouse embryonic stem (ES) cells using DNA seqFISH+, along with 17 chromatin marks and subnuclear structures by sequential immunofluorescence and the expression profile of 70 RNAs. Many loci were invariably associated with immunofluorescence marks in single mouse ES cells. These loci form 'fixed points' in the nuclear organizations of single cells and often appear on the surfaces of nuclear bodies and zones defined by combinatorial chromatin marks. Furthermore, highly expressed genes appear to be pre-positioned to active nuclear zones, independent of bursting dynamics in single cells. Our analysis also uncovered several distinct mouse ES cell subpopulations with characteristic combinatorial chromatin states. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3 trimethylation at lysine 27 (H3K27me3) and macroH2A1 (mH2A1), are heritable over at least 3-4 generations, whereas other marks fluctuate on a faster time scale. This seqFISH+-based spatial multimodal approach can be used to explore nuclear organization and cell states in diverse biological systems. Multiplexed imaging of 3,660 chromosomal loci in individual mouse embryonic stem cells by DNA seqFISH+ with immunofluorescence of 17 chromatin marks and subnuclear structures reveals invariant organization of loci within individual cells, and heterogeneous and long-lived distinct combinatorial chromatin states in cellular subpopulations., Author(s): Yodai Takei [sup.1] , Jina Yun [sup.1] , Shiwei Zheng [sup.2] [sup.4] , Noah Ollikainen [sup.1] , Nico Pierson [sup.1] , Jonathan White [sup.1] , Sheel Shah [sup.1] , [...]

Haematopoietic stem cells self-renew and differentiate into all blood lineages throughout life, and can repair damaged blood systems upon transplantation. Asymmetric cell division has previously been suspected to be a regulator of haematopoietic-stem-cell fate, but its existence has not directly been shown.sup.1. In asymmetric cell division, asymmetric fates of future daughter cells are prospectively determined by a mechanism that is linked to mitosis. This can be mediated by asymmetric inheritance of cell-extrinsic niche signals by, for example, orienting the divisional plane, or by the asymmetric inheritance of cell-intrinsic fate determinants. Observations of asymmetric inheritance or of asymmetric daughter-cell fates alone are not sufficient to demonstrate asymmetric cell division.sup.2. In both cases, sister-cell fates could be controlled by mechanisms that are independent of division. Here we demonstrate that the cellular degradative machinery--including lysosomes, autophagosomes, mitophagosomes and the protein NUMB--can be asymmetrically inherited into haematopoietic-stem-cell daughter cells. This asymmetric inheritance predicts the asymmetric future metabolic and translational activation and fates of haematopoietic-stem-cell daughter cells and their offspring. Therefore, our studies provide evidence for the existence of asymmetric cell division in haematopoietic stem cells. The cellular degradative machinery can be asymmetrically inherited upon haematopoietic-stem-cell division, which predicts the future metabolic and translational activation of their daughter cells., Author(s): Dirk Loeffler [sup.1] [sup.2] , Arne Wehling [sup.1] , Florin Schneiter [sup.1] , Yang Zhang [sup.1] , Niklas Müller-Bötticher [sup.1] , Philipp S. Hoppe [sup.1] [sup.2] , Oliver Hilsenbeck [...]

The emergence of multiple axes is an essential element in the establishment of the mammalian body plan. This process takes place shortly after implantation of the embryo within the uterus and relies on the activity of gene regulatory networks that coordinate transcription in space and time. Whereas genetic approaches have revealed important aspects of these processes.sup.1, a mechanistic understanding is hampered by the poor experimental accessibility of early post-implantation stages. Here we show that small aggregates of mouse embryonic stem cells (ESCs), when stimulated to undergo gastrulation-like events and elongation in vitro, can organize a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimic embryonic spatial and temporal gene expression. The establishment of the three major body axes in these 'gastruloids'.sup.2,3 suggests that the mechanisms involved are interdependent. Specifically, gastruloids display the hallmarks of axial gene regulatory systems as exemplified by the implementation of collinear Hox transcriptional patterns along an extending antero-posterior axis. These results reveal an unanticipated self-organizing capacity of aggregated ESCs and suggest that gastruloids could be used as a complementary system to study early developmental events in the mammalian embryo.Cultures grown from small aggregates of mouse embryonic stem cells can be induced to organize spatial and temporal patterns of gene expression that parallel those of the early embryo, offering a potentially useful system for studying early development., Author(s): Leonardo Beccari [sup.1] , Naomi Moris [sup.2] , Mehmet Girgin [sup.3] , David A. Turner [sup.2] , Peter Baillie-Johnson [sup.2] [sup.5] , Anne-Catherine Cossy [sup.4] , Matthias P. Lutolf [...]

Author(s): Joana Carrelha [1, 2]; Yiran Meng [1, 2]; Laura M. Kettyle [3, 4]; Tiago C. Luis [1, 2]; Ruggiero Norfo [1, 2]; Vernica Alcolea [1, 2]; Hanane Boukarabila [1, [...]

From antibiotics and organoids to CRISPR, improved biomedical methods and apparatus are enabling new therapies. From antibiotics and organoids to CRISPR, improved biomedical methods and apparatus are enabling new therapies., Author(s): Jennifer Cooke, Bec Crew Author Affiliations: New tools for new treatments Organoids mimic the early stages of major organs and tissues. [see PDF for image] Credit: Pamela Kairath Oliva/MCRI [...]

It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify [N.sup.6]-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for [N.sup.6]-methyIadenine. An increase of W6-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. W6-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (6 million years old) L1 elements. The deposition of [N.sup.6]-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, [N.sup.6]-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that [N.sup.6]- methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes., DNA methylation is a crucial component of epigenetic regulation that controls many important aspects of mammalian biology, such as imprinting, X chromosome inactivation and tumorigenesis (1,2). The prevailing dogma states [...]

Blood vessels define local microenvironments in the skeletal system, play crucial roles in osteogenesis and provide niches for haematopoietic stem cells (1-6). The properties of niche-forming vessels and their changes [...]

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols., Vascular-forming endothelial cells form a vast network which participates in homeostasis and metabolism regulation, delivering oxygen, nutrients and other building blocks to distinct organs. This diverse network also serves as [...]

The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina, for example, develops from neuroectoderm via the optic vesicle, the corneal [...]

Stem cells underlie tissue homeostasis, but their dynamics during ageing--and the relevance of these dynamics to organ ageing--remain unknown. Here we report that the expression of the hemidesmosome component collagen XVII (COL17A1) by epidermal stem cells fluctuates physiologically through genomic/oxidative stress-induced proteolysis, and that the resulting differential expression of COL17A1 in individual stem cells generates a driving force for cell competition. In vivo clonal analysis in mice and in vitro 3D modelling show that clones that express high levels of COL17A1, which divide symmetrically, outcompete and eliminate adjacent stressed clones that express low levels of COL17A1, which divide asymmetrically. Stem cells with higher potential or quality are thus selected for homeostasis, but their eventual loss of COL17A1 limits their competition, thereby causing ageing. The resultant hemidesmosome fragility and stem cell delamination deplete adjacent melanocytes and fibroblasts to promote skin ageing. Conversely, the forced maintenance of COL17A1 rescues skin organ ageing, thereby indicating potential angles for anti-ageing therapeutic intervention. COL17A1-driven stem cell competition and symmetric cell divisions initially govern skin homeostasis, but the same mechanisms result in skin ageing later in life., Author(s): Nan Liu [sup.1] , Hiroyuki Matsumura [sup.1] , Tomoki Kato [sup.1] , Shizuko Ichinose [sup.2] , Aki Takada [sup.1] , Takeshi Namiki [sup.3] , Kyosuke Asakawa [sup.1] , Hironobu [...]

Overflow metabolism refers to the seemingly wasteful strategy in which cells use fermentation instead of the more efficient respiration to generate energy, despite the availability of oxygen. Known as the Warburg effect in the context of cancer growth, this phenomenon occurs ubiquitously for fast-growing cells, including bacteria, fungi and mammalian cells, but its origin has remained unclear despite decades of research. Here we study metabolic overflow in Escherichia coli, and show that it is a global physiological response used to cope with changing proteomic demands of energy biogenesis and biomass synthesis under different growth conditions. A simple model of proteomic resource allocation can quantitatively account for all of the observed behaviours, and accurately predict responses to new perturbations. The key hypothesis of the model, that the proteome cost of energy biogenesis by respiration exceeds that by fermentation, is quantitatively confirmed by direct measurement of protein abundances via quantitative mass spectrometry., Under anaerobic conditions, organisms ranging from bacteria to mammalian cells excrete large quantities of fermentation products such as acetate or lactate. Notably, the excretion of these fermentation products occurs widely [...]

Haematopoietic stresses mobilize haematopoietic stem cells (HSCs) from the bone marrow to the spleen and induce extramedullary haematopoiesis (EMH). However, the cellular nature of the EMH niche is unknown. Here [...]

The gut epithelium has remarkable self-renewal capacity that under homeostatic conditions is driven by Wnt signalling in [Lgr5.sup.+] intestinal stem cells (ISCs) (1). However, the mechanisms underlying ISC regeneration after injury remain poorly understood. The Hippo signalling pathway mediates tissue growth and is important for regeneration (2,3). Here we demonstrate in mice that Yap, a downstream transcriptional effector of Hippo, is critical for recovery of intestinal epithelium after exposure to ionizing radiation. Yap transiently reprograms [Lgr5.sup.+] ISCs by suppressing Wnt signalling and excessive Paneth cell differentiation, while promoting cell survival and inducing a regenerative program that includes Egf pathway activation. Accordingly, growth of Yap-deficient organoids is rescued by the Egfr ligand epiregulin, and we find that non-cell-autonomous production of stromal epiregulin may compensate for Yap loss in vivo. Consistent with key roles for regenerative signalling in tumorigenesis, we further demonstrate that Yap inactivation abolishes adenomas in the [Apc.sup.Min] mouse model of colon cancer, and that Yap-driven expansion of [Apc.sup.-/-] organoids requires the Egfr module of the Yap regenerative program. Finally, we show that in vivo Yap is required for progression of early Apc mutant tumour-initiating cells, suppresses their differentiation into Paneth cells, and induces a regenerative program and Egfr signalling. Our studies reveal that upon tissue injury, Yap reprograms [Lgr5.sup.+] ISCs by inhibiting the Wnt homeostatic program, while inducing a regenerative program that includes activation of Egfr signalling. Moreover, our findings reveal a key role for the Yap regenerative pathway in driving cancer initiation., To examine Yap function in intestinal epithelial cells we first analysed Yap localization. This revealed cytoplasmic localization in most cells but absence in Paneth cells (Fig. 1a and Extended Data [...]

The binding motifs for many RNA-binding proteins are normally buried within structured regions; now, the N.sup.6-methyladenosine modification is shown to act as a switch to remodel these regions, expose the motif, and thereby facilitate binding of RNA-binding proteins. N.sup.6-methyladenosine influences RNA-protein interactions N.sup.6-methyladenosine (m.sup.6A) is a common internal modification found in many eukaryotic mRNAs and long non-coding RNAs. It is involved in the control of various cellular functions including circadian rhythm, meiosis and stem cell development. This study uncovers a previously unknown mechanism by which m.sup.6A regulates RNA-protein interactions. Tao Pan and colleagues show that the binding motifs for many RNA binding proteins (RBPs) are normally buried within structured regions. The m.sup.6A modification acts as a switch to reorganize these regions, inducing structural changes that expose the motif and thereby facilitate RBP binding. This finding implies that m.sup.6A functions as an RNA structure remodeller, for example, to affect mRNA maturation through interference with post-transcriptional regulator binding activities. RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs).sup.1,2,3. However, RBMs can be buried within their local RNA structures.sup.4,5,6,7, thus inhibiting RNA-protein interactions. N.sup.6-methyladenosine (m.sup.6A), the most abundant and dynamic internal modification in eukaryotic messenger RNA.sup.8,9,10,11,12,13,14,15,16,17,18,19, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs.sup.15, but how m.sup.6A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m.sup.6A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism 'the m.sup.6A-switch'. We found that m.sup.6A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing.sup.20,21,22,23,24. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m.sup.6A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m.sup.6A-switches among HNRNPC-binding sites; and global m.sup.6A reduction decreased HNRNPC binding at 2,798 high-confidence m.sup.6A-switches. We determined that these m.sup.6A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m.sup.6A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m.sup.6A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology., Author(s): Nian Liu [sup.1] , Qing Dai [sup.1] , Guanqun Zheng [sup.2] , Chuan He [sup.1] [sup.2] [sup.3] [sup.4] , Marc Parisien [sup.2] [sup.5] , Tao Pan [sup.2] [sup.3] Author [...]

Author(s): Simon J. Elssser (corresponding author) [1]; Kyung-Min Noh [2]; Nichole Diaz [2]; C. David Allis [2]; Laura A. Banaszynski (corresponding author) [3] replying to G. Wolf et al . [...]

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age (1,2). Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation (2). While many drivers of HSC ageing have been proposed (2-5), the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies., Both human and mouse HSCs accumulate γH2AX signals with age (6,7). This is taken as direct evidence of DNA damage occurring in old HSCs, since phosphorylation of histone H2AX by [...]

Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homologydirected DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases., Haematopoietic stem cell (HSC)-based gene therapy has provided therapeutic benefit in primary immunodeficiencies (1,2), thalassaemia (3) and leukodystrophies (4,5). Whereas more advanced vectors, such as lentiviral vectors (6), have shown [...]

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure (1) by providing human cardiomyocytes to support heart regeneration (2). Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment (3-7). However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intramyocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models (7),nonfatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome., Human pluripotent stem cells have indisputable cardiomyocyte-generating abilities and have been extensively investigated for repair of the injured heart (3,4,6,10). These stem cells are derived either from developing blastocytsts (human [...]

Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells (1). Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types (2-4). However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis (5), expression of hypoxia inducible factor-1a (Hif-1a) and related genes (6), and staining with surrogate hypoxic markers (for example, pimonidazole) (6-8). Here we perform direct in vivo measurements of local oxygen tension [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] of the bone marrow to be quite low (, The precise location of a haematopoietic stem cell (HSC) niche within the bone marrow (BM) remains elusive, with evidence supporting the existence of both a vascular niche (9,10) and an [...]

During both embryonic development and adult tissue regeneration, changes in chromatin structure driven by master transcription factors lead to stimulus-responsive transcriptional programs. A thorough understanding of how stem cells in the skeleton interpret mechanical stimuli and enact regeneration would shed light on how forces are transduced to the nucleus in regenerative processes. Here we develop a genetically dissectible mouse model of mandibular distraction osteogenesis--which is a process that is used in humans to correct an undersized lower jaw that involves surgically separating the jaw bone, which elicits new bone growth in the gap. We use this model to show that regions of newly formed bone are clonally derived from stem cells that reside in the skeleton. Using chromatin and transcriptional profiling, we show that these stem-cell populations gain activity within the focal adhesion kinase (FAK) signalling pathway, and that inhibiting FAK abolishes new bone formation. Mechanotransduction via FAK in skeletal stem cells during distraction activates a gene-regulatory program and retrotransposons that are normally active in primitive neural crest cells, from which skeletal stem cells arise during development. This reversion to a developmental state underlies the robust tissue growth that facilitates stem-cell-based regeneration of adult skeletal tissue.Reversion of adult skeletal stem cells to a developmental state underlies the growth of new bone during jaw regeneration, in a process that relies on mechanotransduction via the focal adhesion kinase protein., Author(s): Ryan C. Ransom [sup.1] [sup.2] , Ava C. Carter [sup.3] , Ankit Salhotra [sup.1] [sup.2] , Tripp Leavitt [sup.1] , Owen Marecic [sup.1] [sup.2] , Matthew P. Murphy [sup.1] [...]

De novo mutations in ADNP, which encodes activity-dependent neuroprotective protein (ADNP), have recently been found to underlie Helsmoortel-Van der Aa syndrome, a complex neurological developmental disorder that also affects several other organ functions.sup.1. ADNP is a putative transcription factor that is essential for embryonic development.sup.2. However, its precise roles in transcriptional regulation and development are not understood. Here we show that ADNP interacts with the chromatin remodeller CHD4 and the chromatin architectural protein HP1 to form a stable complex, which we refer to as ChAHP. Besides mediating complex assembly, ADNP recognizes DNA motifs that specify binding of ChAHP to euchromatin. Genetic ablation of ChAHP components in mouse embryonic stem cells results in spontaneous differentiation concomitant with premature activation of lineage-specific genes and in a failure to differentiate towards the neuronal lineage. Molecularly, ChAHP-mediated repression is fundamentally different from canonical HP1-mediated silencing: HP1 proteins, in conjunction with histone H3 lysine 9 trimethylation (H3K9me3), are thought to assemble broad heterochromatin domains that are refractory to transcription. ChAHP-mediated repression, however, acts in a locally restricted manner by establishing inaccessible chromatin around its DNA-binding sites and does not depend on H3K9me3-modified nucleosomes. Together, our results reveal that ADNP, via the recruitment of HP1 and CHD4, regulates the expression of genes that are crucial for maintaining distinct cellular states and assures accurate cell fate decisions upon external cues. Such a general role of ChAHP in governing cell fate plasticity may explain why ADNP mutations affect several organs and body functions and contribute to cancer progression.sup.1,3,4. Notably, we found that the integrity of the ChAHP complex is disrupted by nonsense mutations identified in patients with Helsmoortel-Van der Aa syndrome, and this could be rescued by aminoglycosides that suppress translation termination.sup.5. Therefore, patients might benefit from therapeutic agents that are being developed to promote ribosomal read-through of premature stop codons.sup.6,7.ADNP interacts with the chromatin remodeller CHD4 and the heterochromatin protein HP1 to form a complex termed ChAHP that represses gene expression independently of the histone H3K9me3 modification., Author(s): Veronika Ostapcuk [sup.1] [sup.2] , Fabio Mohn [sup.1] , Sarah H. Carl [sup.1] [sup.3] , Anja Basters [sup.1] , Daniel Hess [sup.1] , Vytautas Iesmantavicius [sup.1] , Lisa Lampersberger [...]

Author(s): Anna Alemany [1]; Maria Florescu [1]; Chlo S. Baron [1]; Josi Peterson-Maduro [1]; Alexander van Oudenaarden (corresponding author) [1] Embryonic development is a crucial period in the life of [...]

After 20 years of hope, promise and controversy, human embryonic stem cells are reshaping biological concepts and starting to move into the clinic.After 20 years of hope, promise and controversy, human embryonic stem cells are reshaping biological concepts and starting to move into the clinic., Author(s): David CyranoskiAuthor Affiliations:How human embryonic stem cells sparked a revolution Neural rosettes, derived from human embryonic stem cells, assemble into spheres in culture. [see PDF for image]Neural rosettes, derived [...]

Author(s): Li He (corresponding author) [1]; Guangwei Si [2]; Jiuhong Huang [3]; Aravinthan D. T. Samuel [2]; Norbert Perrimon (corresponding author) [1, 4] Somatic stem cells constantly adjust their self-renewal [...]

Down's syndrome results from full or partial trisomy of chromosome 21. However, the consequences of the underlying gene-dosage imbalance on adult tissues remain poorly understood. Here we show that in Ts65Dn mice, which are trisomic for 132 genes homologous to genes on human chromosome 21, triplication of Usp16 reduces the self-renewal of haematopoietic stem cells and the expansion of mammary epithelial cells, neural progenitors and fibroblasts. In addition, Usp16 is associated with decreased ubiquitination of Cdkn2a and accelerated senescence in Ts65Dn fibroblasts. Usp16 can remove ubiquitin from histone H2A on lysine 119, a critical mark for the maintenance of multiple somatic tissues. Downregulation of Usp16, either by mutation of a single normal Usp16 allele or by short interfering RNAs, largely rescues all of these defects. Furthermore, in human tissues overexpression of USP16 reduces the expansion of normal fibroblasts and postnatal neural progenitors, whereas downregulation of USP16 partially rescues the proliferation defects of Down's syndrome fibroblasts. Taken together, these results suggest that USP16 has an important role in antagonizing the self-renewal and/or senescence pathways in Down's syndrome and could serve as an attractive target to ameliorate some of the associated pathologies., Down's syndrome is a complex clinical syndrome associated with multiple pathological conditions, including deficits in motor skills and learning (1,2). It has also been associated with early onset and higher [...]

Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops (1,2). ES-cell identity is maintained by a [...]

To maintain lifelong production of blood cells, haematopoietic stem cells (HSCs) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSCs reside [...]

One goal of regenerative medicine, to use stem cells to replace cells lost by injury or disease, depends on producing an excess of the relevant cell for study or transplantation. [...]

Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts (1-3), but two critical issues related to their electrophysiological behaviour [...]

Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-P precursor protein gene (1) (APP termed [APP.sup.Dp]), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two [APP.sup.Dp] patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1-40), phospho-tau(Thr231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from [APP.sup.Dp] and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients., Alzheimer's disease is a common neurodegenerative disorder, defined post mortem by the increased presence of amyloid plaques and neurofibrillary tangles in the brain (2). Amyloid plaques are extracellular deposits consisting [...]

Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development (1,2). Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation (3,4). During differentiation a subset of these enhancers must be silenced, but the mechanisms under-lying enhancer silencing are poorly understood. Here we show that the histone demethylase lysine-specific demethylase 1 (LSD1; ref. 5), which demethylates histone H3 on Lys 4 or Lys 9 (H3K4/ K9), is essential in decommissioning enhancers during the differentiation of mouse embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes that are critical for control of the state of ESCs. However, LSD1 is not essential for the maintenance of ESC identity. Instead, ESCs lacking LSD1 activity fail to differentiate fully, and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At active enhancers, LSD1 is a component of the NuRD (nucleosome remodelling and histone deacetylase) complex, which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1-NuRD complex decommissions enhancers of the pluripotency program during differentiation, which is essential for the complete shutdown of the ESC gene expression program and the transition to new cell states., The histone H3K4/K9 demethylase LSD1 (also known as KDM1A) is one of the chromatin regulators that have been implicated in the control of early embryogenesis (6-8). Loss of LSD1 leads [...]

Fundamental questions remain unanswered about the transcriptional networks that control the identity and self-renewal of neural stem cells (NSCs), a specialized subset of astroglial cells that are endowed with stem properties and neurogenic capacity. Here we report that the zinc finger protein Ars2 (arsenite-resistance protein 2; also known as Srrt) is expressed by adult NSCs from the subventricular zone (SVZ) of mice, and that selective knockdown of Ars2in cells expressing glial fibrillary acidic protein within the adult SVZ depletes the number of NSCs and their neurogenic capacity. These phenotypes are recapitulated in the postnatal SVZ of hGFAP-cre:[Ars2.sup.fl/fl] conditional knockout mice, but are more severe. Ex vivo assays show that Ars2 is necessary and sufficient to promote NSC self-renewal, and that it does so by positively regulating the expression of Sox2. Although plant (1-3) and animal (4,5) orthologues of Ars2 are known for their conserved roles in microRNA biogenesis, we unexpectedly observed that Ars2 retains its capacity to promote self-renewal in Drosha and Dicerl knockout NSCs. Instead, chromatin immunoprecipitation revealed that Ars2 binds a specific region within the 6-kilobase NSC enhancer of SoxX2. This association is RNA-independent, and the region that is bound is required for Ars2-mediated activation of SoxX2. We used gel-shift analysis to refine the Sox2 region bound by Ars2 to a specific conserved DNA sequence. The importance of SoxX2 as a critical downstream effector is shown by its ability to restore the self-renewal and multipotency defects of Ars2 knockout NSCs. Our findings reveal Ars2 as a new transcription factor that controls the multipotent progenitor state of NSCs through direct activation of the pluripotency factor Sox2., Stem cells reside in most mammalian tissues throughout adult life, and contribute to normal homeostasis and repair after injury (6). They are defined by their capacity to both self-renew and [...]

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL., Acute lymphoblastic leukaemia (ALL) is the most common malignancy of childhood, with 85% of cases being of B-cell lineage, and 15% T-cell lineage (1). Recent studies have identified a subtype [...]

Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which are often refractory to conventional therapies (1). Many MLL-fusion partners are members of the super elongation complex (SEC), a critical regulator of transcriptional elongation, suggesting that aberrant control of this process has an important role in leukaemia induction (2,3). Here we use a global proteomic strategy to demonstrate that MLL fusions, as part of SEC (2,3) and the polymerase-associated factor complex (PAFc) (4,5), are associated with the BET family of acetyl-lysine recognizing, chromatin 'adaptor' proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. We show that a novel small molecule inhibitor of the BET family, GSK1210151A (I-BET151), has profound efficacy against human and murine MLL-fusion leukaemic cell lines, through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukaemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is, at least in part, due to the inhibition of transcription at key genes (BCL2, C-MYC and CDK6) through the displacement of BRD3/4, PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value, providing survival benefit in two distinct mouse models of murine MLL-AF9 and human MLL-AF4 leukaemia. Finally, the efficacy of I-BET151 against human leukaemia stem cells is demonstrated, providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukaemias., Dysregulation of chromatin modifiers is a recurrent and sentinel event in oncogenesis (6). Therapeutic strategies that selectively alter the recruitment and/or catalytic activity of these enzymes at chromatin therefore hold [...]

Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs (1). Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states (2). By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention., AML represents a paradigm for understanding how complex patterns of cooperating genetic and epigenetic alterations lead to tumorigenesis (3,4). Although this complexity poses a challenge for the development of targeted [...]

The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues., The genes Lgr4, Lgr5 and Lgr6 encode orphan 7-transmembrane receptors that are close relatives of the receptors for follicle stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) (1). [...]

The gene for the atypical NOTCH ligand delta-like homologue 1 (Dlk1) encodes membrane-bound and secreted isoforms that function in several developmental processes in vitro and in vivo. Dlk1, a member of a cluster of imprinted genes, is expressed from the paternally inherited chromosome12. Here we show that mice that are deficient in Dlk1 have defects in postnatal neurogenesis in the subventricular zone: a developmental continuum that results in depletion of mature neurons in the olfactory bulb. We show that DLK1 is secreted by niche astrocytes, whereas its membrane-bound isoform is present in neural stem cells (NSCs) and is required for the inductive effect of secreted DLK1 on self-renewal. Notably, we find that there is a requirement for Dlk1 to be expressed from both maternally and paternally inherited chromosomes. Selective absence of Dlk1 imprinting in both NSCs and niche astrocytes is associated with postnatal acquisition of DNA methylation at the germ-line-derived imprinting control region. The results emphasize molecular relationships between NSCs and the niche astrocyte cells of the microenvironment, identifying a signalling system encoded by a single gene that functions coordinately in both cell types. The modulation of genomic imprinting in a stem-cell environment adds a new level of epigenetic regulation to the establishment and maintenance of the niche, raising wider questions about the adaptability, function and evolution of imprinting in specific developmental contexts., The mammalian adult brain is generally postmitotic, but reservoirs of NSCs with features of astroglial cells exist in the hippocampus and subventricular zone (SVZ), supporting lifelong neurogenesis (3,4). The SVZ [...]

Schizophrenia (SCZD) is a debilitating neurological disorder with a world-wide prevalence of 1%; there is a strong genetic component, with an estimated heritability of 80-85% (1). Although post-mortem studies have revealed reduced brain volume, cell size, spine density and abnormal neural distribution in the prefrontal cortex and hippocampus of SCZD brain tissue (2) and neuropharmacological studies have implicated dopaminergic, glutamatergic and GABAergic activity in SCZD (3), the cell types affected in SCZD and the molecular mechanisms underlying the disease state remain unclear. To elucidate the cellular and molecular defects of SCZD, we directly reprogrammed fibroblasts from SCZD patients into human induced pluripotent stem cells (hiPSCs) and subsequently differentiated these disorder-specific hiPSCs into neurons (Supplementary Fig. 1). SCZD hiPSC neurons showed diminished neuronal connectivity in conjunction with decreased neurite number, PSD95-protein levels and glutamate receptor expression. Gene expression profiles of SCZD hiPSC neurons identified altered expression ofmany components of the cyclic AMP and WNT signalling pathways. Key cellular and molecular elements of the SCZD phenotype were ameliorated following treatment of SCZD hiPSC neurons with the antipsychotic loxapine. To date, hiPSC neuronal pathology has only been demonstrated in diseases characterized by both the loss of function of a single gene product and rapid disease progression in early childhood (4-6).We now report hiPSC neuronal phenotypes and gene expression changes associated with SCZD, a complex genetic psychiatric disorder., Four SCZD patients were selected: patient 1, diagnosed at 6 years of age, had childhood-onset SCZD; patients 2, 3 and 4 were from families in which all offspring and one [...]

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease (1-5), characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs) (6-8). HGPS is caused [...]

The neural fate is generally considered to be the intrinsic direction of embryonic stem (ES) cell differentiation. However, little is known about the intracellular mechanism that leads undifferentiated cells to adopt the neural fate in the absence of extrinsic inductive signals. Here we show that the zinc-finger nuclear protein Zfp521 is essential and sufficient for driving the intrinsic neural differentiation of mouse ES cells. In the absence of the neural differentiation inhibitor BMP4, strong Zfp521 expression is intrinsically induced in differentiating ES cells. Forced expression of Zfp521 enables the neural conversion of ES cells even in the presence of BMP4. Conversely, in differentiation culture, Zfp521-depleted ES cells do not undergo neural conversion but tend to halt at the epiblast state. Zfp521 directly activates early neural genes by working with the co-activator p300. Thus, the transition of ES cell differentiation from the epiblast state into neuroectodermal progenitors specifically depends on the cell-intrinsic expression and activator function of Zfp521., Previous studies, particularly in amphibians, have demonstrated that the initial step of vertebrate neural differentiation from uncommitted embryonic ectodermal cells occurs via a cell-intrinsic mechanism and requires only that inhibitory [...]

Polycomb group proteins maintain the gene-expression pattern of different cells that is set during early development by regulating chromatin structure. In mammals, two main Polycomb group complexes exist--Polycomb repressive complex 1 (PRC1) and 2 (PRC2). PRC1 compacts chromatin and catalyses the monoubiquitylation of histone H2A. PRC2 also contributes to chromatin compaction, and catalyses the methylation of histone H3 at lysine 27. PRC2 is involved in various biological processes, including differentiation, maintaining cell identity and proliferation, and stem-cell plasticity. Recent studies of PRC2 have expanded our perspectives on its function and regulation, and uncovered a role for non-coding RNA in the recruitment of PRC2 to target genes., The term Polycomb (Pc) initially referred to a Drosophila mutant that displayed improper body segmentation (1). It was suggested that Polycomb encodes a negative regulator of the homoerotic genes that [...]

The capacity to fine-tune cellular bioenergetics with the demands of stem-cell maintenance and regeneration is central to normal development and ageing, and to organismal survival during periods of acute stress. How energy metabolism and stem-cell homeostatic processes are coordinated is not well understood. Lkb1 acts as an evolutionarily conserved regulator of cellular energy metabolism in eukaryotic cells and functions as the major upstream kinase to phosphorylate AMP-activated protein kinase (AMPK) and 12 other AMPK-related kinases (1-3). Whether Lkb1 regulates stem-cell maintenance remains unknown. Here we show that Lkb1 has an essential role in haematopoietic stem cell (HSC) homeostasis. We demonstrate that ablation of Lkb1 in adult mice results in severe pancytopenia and subsequent lethality. Loss of Lkb1 leads to impaired survival and escape from quiescence of HSCs, resulting in exhaustion of the HSC pool and a marked reduction of HSC repopulating potential in vivo. Lkb1 deletion has an impact on cell proliferation in HSCs, but not on more committed compartments, pointing to context-specific functions for Lkb1 in haematopoiesis. The adverse impact of Lkb1 deletion on haematopoiesis was predominantly cell-autonomous and mTOR complex 1 (mTORC1)-independent, and involves multiple mechanisms converging on mitochondrial apoptosis and possibly downregulation of PGC-1 coactivators and their transcriptional network, which have critical roles in mitochondrial biogenesis and function. Thus, Lkb1 serves as an essential regulator of HSCs and haematopoiesis, and more generally, points to the critical importance of coupling energy metabolism and stem-cell homeostasis., HSCs function to replenish the blood system and maintain haematopoietic homeostasis in response to either physiological or imposed stress demands (4-6). To explore the role of Lkb1 in various aspects [...]

Little is known about metabolic regulation in stem cells and how this modulates tissue regeneration or tumour suppression. We studied the Lkb1 tumour suppressor and its substrate AMP-activated protein kinase (AMPK), kinases that coordinate metabolism with cell growth. Deletion of the Lkb1 (also called Stldl) gene in mice caused increased haematopoietic stem cell (HSC) division, rapid HSC depletion and pancytopenia. HSCs depended more acutely on Lkb1 for cell-cycle regulation and survival than many other haematopoietic cells. HSC depletion did not depend on mTOR activation or oxidative stress. Lkb1-deficient HSCs, but not myeloid progenitors, had reduced mitochondrial membrane potential and ATP levels. HSCs deficient for two catalytic α-subunits of AMPK (AMPK-deficient HSCs) showed similar changes in mitochondrial function but remained able to reconstitute irradiated mice. Lkb1-deficient HSCs, but not AMPK-deficient HSCs, exhibited defects in centrosomes and mitotic spindles in culture, and became aneuploid. Lkb1 is therefore required for HSC maintenance through AMPK-dependent and AMPK-independent mechanisms, revealing differences in metabolic and cell-cycle regulation between HSCs and some other haematopoietic progenitors., Lkb1 coordinates cell growth with energy metabolism. Energy stress prompts Lkb1 to activate catabolic processes and mitochondrial biogenesis and to inactivate anabolic processes including mammalian target of rapamycin (mTOR)-mediated protein [...]

The stable states of differentiated cells are now known to be controlled by dynamic mechanisms that can easily be perturbed. An adult cell can therefore be reprogrammed, altering its pattern of gene expression, and hence its fate, to that typical of another cell type. This has been shown by three distinct experimental approaches to nuclear reprogramming: nuclear transfer, cell fusion and transcription-factor transduction. Using these approaches, nuclei from 'terminally differentiated' somatic cells can be induced to express genes that are typical of embryonic stem cells, which can differentiate to form all of the cell types in the body. This remarkable discovery of cellular plasticity has important medical applications., In the early embryo of vertebrates, totipotent cells have the potential to differentiate and give rise to cells that function in specific tissues, ultimately forming an entire organism, including the [...]

During early mammalian development, as the pluripotent cells that give rise to all of the tissues of the body proliferate and expand in number, they pass through transition states marked by a stepwise restriction in developmental potential and by changes in the expression of key regulatory genes. Recent findings show that cultured stem-cell lines derived from different stages of mouse development can mimic these transition states. They further reveal that there is a high degree of heterogeneity and plasticity in pluripotent populations in vitro and that these properties are modulated by extrinsic signalling. Understanding the extrinsic control of plasticity will guide efforts to use human pluripotent stem cells in research and therapy., Pluripotent stem cells have two remarkable properties: immortality, or the capacity for indefinite self-renewal; and pluripotency, the ability to give rise to all the tissues of the adult body. The [...]

The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS--mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems (1,2). The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype., Approximately 90% of LEOPARD syndrome cases, and 45% of Noonan syndrome cases, are caused by missense mutations in the PTPN11 gene which encodes the protein tyrosine phosphatase SHP2. PTPN11 is [...]

Age-dependent changes in their environment can impair stem cells' function. The finding that T cells infiltrate the brains of aged mice and cause dysfunction of neural stem cells reveals a potential therapeutic target. Immune cells enter ageing mouse brains and hinder nerve-cell production., Author(s): Allison M. Bond, Hongjun Song Author Affiliations: Infiltration of old brains by T cells causes dysfunction of neural stem cells In a healthy adult, tissue-specific stem cells replenish damaged [...]

A glue ingredient was the secret to getting the mouse cells to multiply outside the body. A glue ingredient was the secret to getting the mouse cells to multiply outside the body., Author(s): David Cyranoski Author Affiliations: Blood stem cells produced in vast quantities in the lab Scientists have produced vast quantities of blood stem cells (pictured) in the lab. [see PDF [...]

Influenza A virus, being responsible for seasonal epidemics and reoccurring pandemics, represents a worldwide threat to public health (1). High mutation rates facilitate the generation of viral escape mutants, rendering vaccines and drugs directed against virus-encoded targets potentially ineffective (2). In contrast, targeting host cell determinants temporarily dispensable for the host but crucial for virus replication could prevent viral escape. Here we report the discovery of 287 human host cell genes influencing influenza A virus replication in a genome-wide RNA interference (RNAi) screen. Using an independent assay we confirmed 168 hits (59%) inhibiting either the endemic H1N1 (119 hits) or the current pandemic swine-origin (121 hits) influenza A virus strains, with an overlap of 60%. Notably, a subset of these common hits was also essential for replication of a highly pathogenic avian H5N1 strain. In-depth analyses of several factors provided insights into their infection stage relevance. Notably, SON DNA binding protein (SON) (3) was found to be important for normal trafficking of influenza virions to late endosomes early in infection. We also show that a small molecule inhibitor of CDC-like kinase 1 (CLK1) (4) reduces influenza virus replication by more than two orders of magnitude, an effect connected with impaired splicing of the viral M2 messenger RNA. Furthermore, influenza-virus-infected [p27.sup.-/-] (cyclin-dependent kinase inhibitor 1B; Cdkn1b) mice accumulated significantly lower viral titres in the lung, providing in vivo evidence for the importance of this gene. Thus, our results highlight the potency of genome-wide RNAi screening for the dissection of virus-host interactions and the identification of drug targets for a broad range of influenza viruses., During the course of infection, the influenza virus encounters numerous bottlenecks, constituted by host cell functions essential or inhibitory for viral propagation (5). Comprehensive knowledge of such critical host cell [...]

When embryonic stem cells (ESCs) differentiate, they must both silence the ESC self-renewal program and activate new tissue-specific programs. In the absence of DGCR8 ([Dgcr8.sup.-/-]), a protein required for microRNA (miRNA) biogenesis, mouse ESCs are unable to silence self-renewal. Here we show that the introduction of let-7 miRNAs--a family of miRNAs highly expressed in somatic cells--can suppress self-renewal in [Dgcr8.sup.-/-] but not wild-type ESCs. Introduction of ESC cell cycle regulating (ESCC) miRNAs into the [Dgcr8.sup.-/-] ESCs blocks the capacity of let-7 to suppress self-renewal. Profiling and bioinformatic analyses show that let-7 inhibits whereas ESCC miRNAs indirectly activate numerous self-renewal genes. Furthermore, inhibition of the let-7 family promotes de-differentiation of somatic cells to induced pluripotent stem cells. Together, these findings show how the ESCC and let-7 miRNAs act through common pathways to alternatively stabilize the self-renewing versus differentiated cell fates., Mammalian development follows a carefully orchestrated unfolding of cell fate transitions leading to a complex set of highly specialized cell types. These cell fate transitions involve the silencing of previously [...]