Characterization of the Nonendocrine Cell Populations in Human Embryonic Stem Cell-Derived (hESC) Islet-Like Clusters Posttransplantation.

Islet-like clusters derived from human embryonic stem cells (hESC) hold the potential to cure type 1 diabetes mellitus. Differentiation protocols of islet-like clusters lead to the generation of minor fractions of nonendocrine cells, which are mainly from endodermal and mesodermal lineages, and the risk of implanting these is unclear. In the present study, the histogenesis and the tumorigenicity of nonendocrine cells were investigated in vivo. Immunodeficient mice were implanted under the kidney capsule with islet-like clusters which were derived from differentiation of cells batches with either an intermediate or poor cell purity and followed for 8 or 26 weeks. Using immunohistochemistry and other techniques, it was found that the intermediate differentiated cell implants had limited numbers of small duct-like cysts and nonpancreatic tissue resembling gastrointestinal and retinal pigmented epithelium. In contrast, highly proliferative cystic teratomas were found at a high incidence at the implant site after 8 weeks, only in the animals implanted with the poorly differentiated cells. These findings indicate that the risk for teratoma formation and the amount of nonpancreatic tissue can be minimized by careful in-process characterization of the cells and thus highlights the importance of high purity at transplantation and a thorough ex-vivo characterization during cell product development.

Maker Buoy Variants for Water Level Monitoring and Tracking Drifting Objects in Remote Areas of Greenland.

Meltwater runoff from the Greenland Ice Sheet changes water levels in glacial lakes and can lead to glacial lake outburst flooding (GLOF) events that threaten lives and property. Icebergs produced at Greenland's marine terminating glaciers drift into Baffin Bay and the North Atlantic, where they can threaten shipping and offshore installations. Thus, monitoring glacial lake water levels and the drift of icebergs can enhance safety and aid in the scientific studies of glacial hydrology and iceberg-ocean interactions. The Maker Buoy was originally designed as a low-cost and open source sensor to monitor surface ocean currents. The open source framework, low-cost components, rugged construction and affordable satellite data transmission capabilities make it easy to customize for environmental monitoring in remote areas and under harsh conditions. Here, we present two such Maker Buoy variants that were developed to monitor water level in an ice-infested glacial lake in southern Greenland and to track drifting icebergs and moorings in the Vaigat Strait (Northwest Greenland). We describe the construction of each design variant, methods to access data in the field without an internet connection, and deployments in Greenland in summer 2019. The successful deployments of each Maker Buoy variant suggest that they may also be useful in operational iceberg management strategies and in GLOF monitoring programs.

Predominant archaea in marine sediments degrade detrital proteins.

Half of the microbial cells in the Earth's oceans are found in sediments. Many of these cells are members of the Archaea, single-celled prokaryotes in a domain of life separate from Bacteria and Eukaryota. However, most of these archaea lack cultured representatives, leaving their physiologies and placement on the tree of life uncertain. Here we show that the uncultured miscellaneous crenarchaeotal group (MCG) and marine benthic group-D (MBG-D) are among the most numerous archaea in the marine sub-sea floor. Single-cell genomic sequencing of one cell of MCG and three cells of MBG-D indicated that they form new branches basal to the archaeal phyla Thaumarchaeota and Aigarchaeota, for MCG, and the order Thermoplasmatales, for MBG-D. All four cells encoded extracellular protein-degrading enzymes such as gingipain and clostripain that are known to be effective in environments chemically similar to marine sediments. Furthermore, we found these two types of peptidase to be abundant and active in marine sediments, indicating that uncultured archaea may have a previously undiscovered role in protein remineralization in anoxic marine sediments.

Surrogate markers of visceral fat and response to anti-depressive treatment in patients with major depressive disorder: a prospective exploratory analysis.

BACKGROUND: Body mass index (BMI) and body weight have been shown to be associated to treatment outcome in patients with major depressive disorder, but this relationship is not clear. Visceral fat might be an underlying mechanism explaining this relationship. AIMS: The aim of this study was to prospectively investigate whether visceral fat, as measured by hip-to-waist ratio and waist circumference, affects treatment outcome in patients with major depressive disorder in patients attending a hospital psychiatric care unit in Denmark. METHODS: The study was conducted as an observational prospective study including 33 patients with major depressive disorder. Assessments were made at enrolment and after 8 weeks. Primary variables were hip-to-waist ratio and waist circumference. Outcome were remission or response of depressive symptoms measured with the Hamilton Depression Rating Scale (HAM-D17) interviews and HAM-D6 self-rating questionnaires. RESULTS: No differences were found in outcome between groups of patients with high vs low visceral fat in this population. CONCLUSIONS: The lack of association was evident for all surrogate markers of visceral fat, and suggests that visceral fat has no impact on outcomes of depressive symptoms. However, study limitations might have contributed to this lack of association, especially sample size and considerable variations on multiple parameters including treatment received during the 8 weeks of follow-up.

Adverse life events as risk factors for behavioural and emotional problems in a 7-year follow-up of a population-based child cohort.

BACKGROUND AND AIM: The aim of the study was to identify risk factors for significant changes in emotional and behavioural problem load in a community-based cohort of Danish children aged 9-16 years, the risk factors being seven parental and two child-related adverse life events. METHODS: Data on emotional and behavioural problems was obtained from parents filling in the Child Behavior Checklist (CBCL) when the child was 8-9 and again when 15 years old. Data on risk factors was drawn from Danish registers. Analysis used was logistic regression for crude and adjusted change. RESULTS: Parental divorce significantly raised the odds ratio of an increase in emotional and behavioural problems; furthermore, the risk of deterioration in problem behaviour rose significantly with increasing number of adverse life events. By dividing the children into four groups based on the pathway in problem load (increasers, decreasers, high persisters and low persisters), we found that children with a consistently high level of behavioural problems also had the highest number of adverse life events compared with any other group. CONCLUSIONS: Family break-up was found to be a significant risk factor. This supports findings in previous studies. The fact that no other risk factor proved to be of significance might be due to lack of power in the study. Children experiencing high levels of adverse life events are at high risk of chronic problem behaviour. Thus these risk factors should be assessed in daily clinical practice.

Indoor radon survey in Greenland and dose assessment.

Indoor radon and its decay products are the primary sources of the population's exposure to background ionizing radiation. Radon decay products are one of the leading causes of lung cancer, with a higher lung cancer risk for smokers due to the synergistic effects of radon decay products and cigarette smoking. A total of 459 year-long radon measurements in 257 detached and semi-detached residential homes in southwest and south Greenland were carried out, and a dose assessment for adults was performed. The annual arithmetic and geometric means of indoor radon concentrations was 10.5 ± 0.2 Bq m-3 and 8.0 ± 2.3 Bq m-3 in Nuuk, 139.0 ± 1.0 Bq m-3 and 97.3 ± 2.1 Bq m-3 in Narsaq, and 42.1 ± 0.7 Bq m-3 and 22.0 ± 3.1 Bq m-3 in Qaqortoq. Arithmetic and geometric mean radon concentration of 79.0 Bq m-3 and 50.3 Bq m-3 were estimated for adult, person-weighted living in south Greenland. The total number of detached and semi-detached residential homes where indoor radon is exceeding 100 Bq m-3, 200 Bq m-3, and 300 Bq m-3 is 37 homes (15.0%), 13 homes (5.2%), and 8 homes (3.2%), respectively. A positive correlation between indoor air radon concentrations and underlying geology was observed. The indoor radon contribution to the annual inhalation effective dose to an average adult was 0.5 mSv in Nuuk, 6.5 mSv in Narsaq, 2.0 mSv in Qaqortoq, and 4.0 mSv for south Greenland adult person weighted. The estimated annual average effective dose to adults in Narsaq is higher than the world's average annual effective dose of 1.3 mSv due to inhalation of indoor radon. Cost-efficient mitigation methods exist to reduce radon in existing buildings, and to prevent radon entry into new buildings.

Abundance of microbial genes associated with nitrogen cycling as indices of biogeochemical process rates across a vegetation gradient in Alaska.

Nitrification and denitrification processes are crucial to plant nutrient availability, eutrophication and greenhouse gas production both locally and globally. Unravelling the major environmental predictors for nitrification and denitrification is thus pivotal in order to understand and model environmental nitrogen (N) cycling. Here, we sampled five plant community types characteristic of interior Alaska, including black spruce, bog birch, tussock grass and two fens. We assessed abundance of functional genes affiliated with nitrification (bacterial and archaeal amoA) and denitrification (nirK/S and nosZ) using qPCR, soil characteristics, potential nitrification and denitrification rates (PNR and PDR) and gross mineralization rates. The main chemical and biological predictors for PNR and PDR were assigned through path analysis. The potential N cycling rates varied dramatically between sites, from some of the highest (in fens) to some of the lowest (in black spruce) measured globally. Based on path analysis, functional gene abundances were the most important variables to predict potential rates. PNR was best explained by bacterial amoA gene abundance followed by ammonium content, whereas PDR was best explained directly by nosZ gene abundance and indirectly by nirK/S gene abundance and nitrate. Hence, functional gene abundance is a valuable index that integrates recent environmental history and recent process activity, and therefore is a good predictor of potential rates. The results of this study contribute to our understanding of the relative importance of different biological and chemical factors in driving the potential for nitrification and denitrification across terrestrial ecosystems.

Retinoic acid synthesis promotes development of neural progenitors from mouse embryonic stem cells by suppressing endogenous, Wnt-dependent nodal signaling.

Embryonic stem (ES) cells differentiate spontaneously toward a neuroectodermal fate in serum-free, adherent monocultures. Here, we show that this spontaneous neural fate requires retinoic acid (RA) synthesis. We monitor ES cells containing reporter genes for markers of the early neural plate as well as the primitive streak and its progeny to determine the cell fates induced when RA signaling is perturbed. We demonstrate that the spontaneous neural commitment of mouse ES cells requires endogenous RA production from vitamin A (vitA) in the medium. Formation of neural progenitors is inhibited by removing vitA from the medium, by inhibiting the enzymes that catalyze the synthesis of RA, or by inhibiting RA receptors. We show that subnanomolar concentrations of RA restore neuroectodermal differentiation when RA synthesis is blocked. We demonstrate that a neural to mesodermal fate change occurring when RA signaling is inhibited is dependent on Nodal-, Wnt-, and fibroblast growth factor-signaling. We show that Nodal suppresses neural development in a Wnt-dependent manner and that Wnt-mediated inhibition of neural development is reversed by inhibition of Nodal signaling. Together, our results show that neural induction in ES cells requires RA at subnanomolar levels to suppress Nodal signaling and suggest that the mechanism by which Wnt signaling suppresses neural development is through facilitation of Nodal signaling.

Novel method to immobilize phosphate in lakes using sediment microbial fuel cells.

Phosphate pollution in lakes poses an intractable remediation challenge. Accumulated stocks of phosphorus in sediments cause high concentrations in the overlying water despite elimination of external sources. We propose to use sediment microbial fuel cells (SMFCs) for lake remediation by sediment phosphorus immobilization. The hypothesis is that SMFCs can increase sediment redox potential at the top layer, and that such changes will allow the sediment to retain phosphorus as immobile species. This study placed an emphasis on scalability, practicality, and use of low-cost materials. Stainless steel net was selected as electrode material, and modifications were tested: (i) chronoamperometric operation with anode poised at +399 mV (versus standard hydrogen potential); (ii) injection of graphite slurry; and (iii) coating with nickel-carbon matrix. Stainless steel electrodes were implemented in laboratory microcosms (1.3 L) and at field scale in a eutrophic freshwater lake. All tests were carried out in untreated sediment and water from Lake Søllerød, Denmark. Phosphate immobilization was shown at lab scale, with 85% decrease in overlying water using steel electrodes. At field scale maximum phosphate decrease of 94% was achieved in the water body above a 16 m2 stainless steel SMFC electrode. Results are promising and warrant further study, including remediation trials at full scale. Added benefits include degradation of sediment organic matter and pollutants, inhibition of methane and sulfide emission and production of electricity.

Length effects in pseudo-word spelling: stronger in dyslexic than in non-dyslexic students.

It is often discussed whether dyslexics show a deviant pattern of reading and spelling development when compared to typically developing students, or whether they follow the same pattern as other students, only at markedly slower rate. The present cross-sectional study investigated phonological encoding skills in dyslexic Danish students. We compared dyslexic and non-dyslexic students from grades 3, 5, 7, and 9 and examined whether effects of item length were stronger in the dyslexic groups. Mixed between-within subjects analyses of variance revealed significant interactions between dyslexia status and item length as the dyslexics at all grade levels were more affected by item length than their non-dyslexic peers. A marked developmental delay was apparent as the dyslexic group from grade 9 performed on approximately the same level as the non-dyslexic group from grade 3. Although the overall difference between these two groups was not significant, a significant interaction between dyslexia status and item length remained because the grade 9 dyslexics were more affected by item length than the younger non-dyslexic students. This difference in error profiles suggests a difference in the developmental patterns of dyslexic vs. non-dyslexic students.

Abrupt shift in the observed runoff from the southwestern Greenland ice sheet.

The recent decades of accelerating mass loss of the Greenland ice sheet have arisen from an increase in both surface meltwater runoff and ice flow discharge from tidewater glaciers. Despite the role of the Greenland ice sheet as the dominant individual cryospheric contributor to sea level rise in recent decades, no observational record of its mass loss spans the 30-year period needed to assess its climatological state. We present for the first time a 40-year (1975-2014) time series of observed meltwater discharge from a >6500-km2 catchment of the southwestern Greenland ice sheet. We find that an abrupt 80% increase in runoff occurring between the 1976-2002 and 2003-2014 periods is due to a shift in atmospheric circulation, with meridional exchange events occurring more frequently over Greenland, establishing the first observation-based connection between ice sheet runoff and climate change.

Single-Cell Genome and Group-Specific dsrAB Sequencing Implicate Marine Members of the Class Dehalococcoidia (Phylum Chloroflexi) in Sulfur Cycling.

UNLABELLED: The marine subsurface sediment biosphere is widely inhabited by bacteria affiliated with the class Dehalococcoidia (DEH), phylum Chloroflexi, and yet little is known regarding their metabolisms. In this report, genomic content from a single DEH cell (DEH-C11) with a 16S rRNA gene that was affiliated with a diverse cluster of 16S rRNA gene sequences prevalent in marine sediments was obtained from sediments of Aarhus Bay, Denmark. The distinctive gene content of this cell suggests metabolic characteristics that differ from those of known DEH and Chloroflexi The presence of genes encoding dissimilatory sulfite reductase (Dsr) suggests that DEH could respire oxidized sulfur compounds, although Chloroflexi have never been implicated in this mode of sulfur cycling. Using long-range PCR assays targeting DEH dsr loci, dsrAB genes were amplified and sequenced from various marine sediments. Many of the amplified dsrAB sequences were affiliated with the DEH Dsr clade, which we propose equates to a family-level clade. This provides supporting evidence for the potential for sulfite reduction by diverse DEH species. DEH-C11 also harbored genes encoding reductases for arsenate, dimethyl sulfoxide, and halogenated organics. The reductive dehalogenase homolog (RdhA) forms a monophyletic clade along with RdhA sequences from various DEH-derived contigs retrieved from available metagenomes. Multiple facts indicate that this RdhA may not be a terminal reductase. The presence of other genes indicated that nutrients and energy may be derived from the oxidation of substituted homocyclic and heterocyclic aromatic compounds. Together, these results suggest that marine DEH play a previously unrecognized role in sulfur cycling and reveal the potential for expanded catabolic and respiratory functions among subsurface DEH. IMPORTANCE: Sediments underlying our oceans are inhabited by microorganisms in cell numbers similar to those estimated to inhabit the oceans. Microorganisms in sediments consist of various diverse and uncharacterized groups that contribute substantially to global biogeochemical cycles. Since most subsurface microorganisms continue to evade cultivation, possibly due to very slow growth, we obtained and analyzed genomic information from a representative of one of the most widespread and abundant, yet uncharacterized bacterial groups of the marine subsurface. We describe several key features that may contribute to their widespread distribution, such as respiratory flexibility and the potential to use oxidized sulfur compounds, which are abundant in marine environments, as electron acceptors. Together, these data provide important information that can be used to assist in designing enrichment strategies or other postgenomic studies, while also improving our understanding of the diversity and distribution of dsrAB genes, which are widely used functional marker genes for sulfur-cycling microbes.

Improvements for comparative analysis of changes in diversity of microbial communities using internal standards in PCR-DGGE.

The use of internal standards both during DNA extraction and PCR-DGGE procedure gives the opportunity to analyse the relative abundance of individual species back to the original sample, thereby facilitating relative comparative analysis of diversity. Internal standards were used throughout the DNA extraction and PCR-DGGE to compensate for experimental variability. Such variability causes decreased reproducibility among replicate samples as well as compromise comparisons between samples, since experimental errors cannot be differentiated from actual changes in the community abundance and structure. The use of internal standards during DNA extraction and PCR-DGGE is suitable for ecological and ecotoxicological experiments with microbial communities, where relative changes in the community abundance and structure are studied. We have developed a protocol Internal Standards in Molecular Analysis of Diversity (ISMAD) that is simple to use, inexpensive, rapid to perform and it does not require additional samples to be processed. The internal standard for DNA extraction (ExtrIS) is a fluorescent 510-basepair PCR product which is added to the samples prior to DNA extraction, recovered together with the extracted DNA from the samples and analysed with fluorescence spectrophotometry. The use of ExtrIS during isolation of sample DNA significantly reduced variation among replicate samples. The PCR internal standard (PCR(IS)) originates from the Drosophila melanogaster genome and is a 140-basepair long PCR product, which is amplified by non-competitive primers in the same PCR reaction tubes as the target DNA and analysed together with the target PCR product on the same DGGE gel. The use of PCR(IS) during PCR significantly reduced variation among replicate samples both when assessing total PCR product and when comparing bands representing species on a DGGE gel. The entire ISMAD protocol was shown to accurately describe changes in relative abundance in an environmental sample using PCR-DGGE. It should, however, be mentioned that despite the use of ISMAD some inherent biases still exist in DNA extraction and PCR-DGGE and these should be taken into consideration when interpreting the diversity in a sample based on a DGGE gel.

Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm.

Human embryonic stem cells have the ability to generate all cell types in the body and can potentially provide an unlimited source of cells for cell replacement therapy to treat degenerative diseases such as diabetes. Current differentiation protocols of human embryonic stem cells towards insulin producing beta cells focus on soluble molecules whereas the impact of cell-matrix interactions has been mainly unattended. In this study almost 500 different extracellular matrix protein combinations were screened to systemically identify extracellular matrix proteins that influence differentiation of human embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed by time lapse studies compared to cells on the other tested substrates. Global gene expression analysis showed that cells differentiated on collagen I were largely similar to cells on fibronectin after completed differentiation. Collectively, the data suggest that collagen I induces a more rapid and consistent differentiation of stem cells to definitive endoderm. The results shed light on the importance of extracellular matrix proteins for differentiation and also points to a cost effective and easy method to improve differentiation.

Ammonia-oxidizing Bacteria of the Nitrosospira cluster 1 dominate over ammonia-oxidizing Archaea in oligotrophic surface sediments near the South Atlantic Gyre.

Sediments across the Namibian continental margin feature a strong microbial activity gradient at their surface. This is reflected in ammonium concentrations of < 10 µM in oligotrophic abyssal plain sediments near the South Atlantic Gyre compared with ammonium concentrations of > 700 µM in upwelling areas near the coast. Here we address changes in apparent abundance and structure of ammonia-oxidizing archaeal and bacterial communities (AOA and AOB) along a transect of seven sediment stations across the Namibian shelf by analysing their respective ammonia monooxygenase genes (amoA). The relative abundance of archaeal and bacterial amoA (g(-1) DNA) decreased with increasing ammonium concentrations, and bacterial amoA frequently outnumbered archaeal amoA at the sediment-water interface [0-1 cm below seafloor (cmbsf)]. In contrast, AOA were apparently as abundant as AOB or dominated in several deeper (> 10 cmbsf), anoxic sediment layers. Phylogenetic analyses showed a change within the AOA community along the transect, from two clusters without cultured representatives at the gyre to Nitrososphaera and Nitrosopumilus clusters in the upwelling region. AOB almost exclusively belonged to the Nitrosospira cluster 1. Our results suggest that this predominantly marine AOB lineage without cultured representatives can thrive at low ammonium concentrations and is active in the marine nitrogen cycle.

Effects of zinc pyrithione and copper pyrithione on microbial community function and structure in sediments.

The effects of the new antifouling biocides, zinc pyrithione (ZPT) and copper pyrithione (CPT), on microbial communities in estuarine sediments were studied in microcosms. As functional endpoints, fluxes of nutrients (NO3-, NH4+, HPO4(2-), Si(OH)4) and protein synthesis ([14C]leucine incorporation) were used, whereas molecular fingerprinting methods (polymerase chain reaction/denaturing gradient gel electrophoresis) were used to describe the bacterial community structure. The lowest-observed-effect concentration (LOEC) for ZPT was 0.001 nmol/g dry sediment for the phosphate flux and total DNA content, whereas the LOEC for CPT was 0.1 nmol/g dry sediment for the nitrate flux and total DNA content. Nitrate fluxes increased significantly following additions of both ZPT and CPT, whereas ammonium fluxes decreased significantly after ZPT addition, suggesting changes in the nitrification and denitrification processes. The total DNA content decreased significantly following addition of both ZPT and CPT, but at the highest addition of ZPT (10 nmol ZPT/g dry sediment), an increase in total DNA content was found. Increased protein synthesis and bacterial diversity were also observed at this concentration of ZPT, suggesting growth of tolerant opportunistic species.

Genome sequencing of a single cell of the widely distributed marine subsurface Dehalococcoidia, phylum Chloroflexi.

Bacteria of the class Dehalococcoidia (DEH), phylum Chloroflexi, are widely distributed in the marine subsurface, yet metabolic properties of the many uncultivated lineages are completely unknown. This study therefore analysed genomic content from a single DEH cell designated 'DEH-J10' obtained from the sediments of Aarhus Bay, Denmark. Real-time PCR showed the DEH-J10 phylotype was abundant in upper sediments but was absent below 160 cm below sea floor. A 1.44 Mbp assembly was obtained and was estimated to represent up to 60.8% of the full genome. The predicted genome is much larger than genomes of cultivated DEH and appears to confer metabolic versatility. Numerous genes encoding enzymes of core and auxiliary beta-oxidation pathways were identified, suggesting that this organism is capable of oxidising various fatty acids and/or structurally related substrates. Additional substrate versatility was indicated by genes, which may enable the bacterium to oxidise aromatic compounds. Genes encoding enzymes of the reductive acetyl-CoA pathway were identified, which may also enable the fixation of CO2 or oxidation of organics completely to CO2. Genes encoding a putative dimethylsulphoxide reductase were the only evidence for a respiratory terminal reductase. No evidence for reductive dehalogenase genes was found. Genetic evidence also suggests that the organism could synthesise ATP by converting acetyl-CoA to acetate by substrate-level phosphorylation. Other encoded enzymes putatively conferring marine adaptations such as salt tolerance and organo-sulphate sulfohydrolysis were identified. Together, these analyses provide the first insights into the potential metabolic traits that may enable members of the DEH to occupy an ecological niche in marine sediments.

Influence of oxic/anoxic fluctuations on ammonia oxidizers and nitrification potential in a wet tropical soil.

Ammonia oxidation is a key process in the global nitrogen cycle. However, in tropical soils, little is known about ammonia-oxidizing microorganisms and how characteristically variable oxygen regimes affect their activity. We investigated the influence of brief anaerobic periods on ammonia oxidation along an elevation, moisture, and oxygen availability gradient in wet tropical soils. Soils from three forest types were incubated for up to 36 weeks in lab microcosms under three regimes: (1) static aerobic; (2) static anaerobic; and (3) fluctuating (aerobic/anaerobic). Nitrification potential was measured in field-fresh soils and incubated soils. The native ammonia-oxidizing community was also characterized, based on diversity assessments (clone libraries) and quantification of the ammonia monooxygenase α-subunit (amoA) gene. These relatively low pH soils appear to be dominated by ammonia-oxidizing archaea (AOA), and AOA communities in the three soil types differed significantly in their ability to oxidize ammonia. Soils from an intermediate elevation, and those incubated with fluctuating redox conditions, tended to have the highest nitrification potential following an influx of oxygen, although all soils retained the capacity to nitrify even after long anoxic periods. Together, these results suggest that wet tropical soil AOA are tolerant of extended periods of anoxia.

Engineering artificial signaling centers to polarize embryoid body differentiation.

Embryonic stem (ES) cells differentiating as aggregates self-organize dependent on Wnt signaling that is initially localized to discrete sites in the aggregate. As differentiation proceeds, Wnt signaling expands to most of the aggregates, thus resulting in widespread differentiation of mesendodermal progenitors. This process resembles primitive streak formation, but the lack of organized positional information makes the differentiating aggregates develop in a disorganized fashion. Here, we report that exogenous, cellular signaling sources can control the site where differentiation initiates in ES cell aggregates. Fibroblasts engineered to express cadherins are assembled with ES cells to form composite aggregates where the fibroblasts are positioned as a discrete pole. When engineered to express secreted Wnt agonists or antagonists, this pole functions to localize signaling in a way that polarizes the differentiating aggregates. The use of cell adhesion molecules to control morphology of developing stem cell aggregates should be widely applicable in tissue engineering.

Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice.

Extracellular signals in development, physiology, homeostasis and disease often act by regulating transcription. Herein we describe a general method and specific resources for determining where and when such signaling occurs in live animals and for systematically comparing the timing and extent of different signals in different cellular contexts. We used recombinase-mediated cassette exchange (RMCE) to test the effect of successively deleting conserved genomic regions of the ubiquitously active Rosa26 promoter and substituting the deleted regions for regulatory sequences that respond to diverse extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can be adapted to any pathway that acts via DNA elements.