Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications. © 2013 Goh et al

Multi annual comparisons of summer and under ice phytoplankton communities of a mountain lake

Little is known on the dynamics of under-ice phytoplankton communities. We investigated phytoplankton communities in the upper (0-20 m) and lower (30-35 m) layer of oligotrophic Lake Tovel, Brenta Dolomites (Italy) over six years during summer and under ice. Winter conditions were different from one year to another with respect to ice thickness and snow cover. Proxies for light transmission (Secchi disk transparency, light attenuation) were similar between seasons, even though the incident solar radiation was lower in winter. Algal richness and chlorophyll-a were not different between seasons while biomass was higher during summer. In four of the six years, Bacillariophyta dominated during summer and Miozoa (class Dinophyceae) under ice while in two years Bacillariophyta also dominated under ice. Generally, a shift to larger size classes from summer to under ice was observed for Bacillariophyta, Chlorophyta, and Ochrophyta (class Chrysophyceae) while Dinophyceae showed the opposite pattern. No strong links between phytoplankton community composition and abiotic factors (under-ice convective mixing, snow on ice, under-ice light) were found. We suggest that inter-species relationships and more precise indicators of under-ice light should be considered to better understand under-ice processes

Mapping Differentiation under Mixed Culture Conditions Reveals a Tunable Continuum of T Cell Fates

Cell differentiation is typically directed by external signals that drive opposing regulatory pathways. Studying differentiation under polarizing conditions, with only one input signal provided, is limited in its ability to resolve the logic of interactions between opposing pathways. Dissection of this logic can be facilitated by mapping the system's response to mixtures of input signals, which are expected to occur in vivo, where cells are simultaneously exposed to various signals with potentially opposing effects. Here, we systematically map the response of naïve T cells to mixtures of signals driving differentiation into the Th1 and Th2 lineages. We characterize cell state at the single cell level by measuring levels of the two lineage-specific transcription factors (T-bet and GATA3) and two lineage characteristic cytokines (IFN-γ and IL-4) that are driven by these transcription regulators. We find a continuum of mixed phenotypes in which individual cells co-express the two lineage-specific master regulators at levels that gradually depend on levels of the two input signals. Using mathematical modeling we show that such tunable mixed phenotype arises if autoregulatory positive feedback loops in the gene network regulating this process are gradual and dominant over cross-pathway inhibition. We also find that expression of the lineage-specific cytokines follows two independent stochastic processes that are biased by expression levels of the master regulators. Thus, cytokine expression is highly heterogeneous under mixed conditions, with subpopulations of cells expressing only IFN-γ, only IL-4, both cytokines, or neither. The fraction of cells in each of these subpopulations changes gradually with input conditions, reproducing the continuous internal state at the cell population level. These results suggest a differentiation scheme in which cells reflect uncertainty through a continuously tuneable mixed phenotype combined with a biased stochastic decision rather than a binary phenotype with a deterministic decision

Perceptions of employability among London's low-paid: 'self-determination' or ethnicity?

We investigate how ethnicity, gender and other characteristics affect low-paid workers’ perceptions of their employability in London’s labour market, examining ‘self-determination’, ethnic and dual labour market theories. We find that perceptions vary considerably, both between genders and ethnicities and in the extent to which they are ‘justified’ by human capital attributes. Optimism varies between genders and ethnic groups but individuals’ perceptions vary to an even greater extent within genders and ethnic groups. Hence, individual-level ‘self-determination’ explanations of these perceptions appear to have greatest explanatory power though ethnic theories also have utility

A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis

Extracellular matrix interactions have essential roles in normal physiology and many pathological processes. Although the importance of extracellular matrix interactions in metastasis is well documented, systematic approaches to identify their roles in distinct stages of tumorigenesis have not been described. Here we report a novel-screening platform capable of measuring phenotypic responses to combinations of extracellular matrix molecules. Using a genetic mouse model of lung adenocarcinoma, we measure the extracellular matrix-dependent adhesion of tumour-derived cells. Hierarchical clustering of the adhesion profiles differentiates metastatic cell lines from primary tumour lines. Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8 or laminin. We show that these molecules correlate with human disease and that their interactions are mediated in part by α3β1 integrin. Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified extracellular matrix and integrin interactions that could serve as therapeutic targets.National Institutes of Health (U.S.) (Grant K99-CA151968)National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service AwardStand Up To Cancer (SU2C/AACR)David H. Koch Institute for Integrative Cancer Research at MIT (CTC Project)Harvard Stem Cell Institute (SG-0046-08-00)National Cancer Center (Postdoctoral Fellowship)National Cancer Institute (U.S.) (U54CA126515)National Cancer Institute (U.S.) (U54CA112967)Howard Hughes Medical InstituteMassachusetts Institute of Technology. Ludwig Center for Molecular Oncolog

Mechanisms of transmurally varying myocyte electromechanics in an integrated computational model

The mechanical properties of myocardium vary across the transmural aspect of the left ventricular wall. Some of these functional heterogeneities may be related to differences in excitation–contraction coupling characteristics that have been observed in cells isolated from the epicardial, mid-myocardial and endocardial regions of the left ventricle of many species, including canine. Integrative models of coupled myocyte electromechanics are reviewed and used here to investigate sources of heterogeneous electromechanical behaviour in these cells. The simulations (i) illustrate a previously unrecognized role of the transient outward potassium current in mechanical function and (ii) suggest that there may also exist additional heterogeneities affecting crossbridge cycling rates in cells from different transmural regions

Amplified surface temperature response of cold, deep lakes to inter-annual air temperature variability

Summer lake surface water temperatures (LSWTs) have previously been shown to respond more rapidly to climatic warming compared to local summer surface air temperatures (SATs). In a global- scale analysis, we explore the factors underpinning the observation of an amplified response of summer LSWT to SAT variability using 20 years of satellite-derived temperatures from 144 lakes. We demonstrate that the degree of amplification in inter-annual summer LSWT is variable, and is greater for cold lakes (e.g. high latitude and high altitude), which are characterised by a short warming season, and deep lakes, that exhibit long correlation timescales of temperature anomalies due to increased thermal inertia. Such lakes are more likely to display responses in excess of local inter-annual summer SAT variability. Climatic modification of LSWT has numerous consequences for water quality and lake ecosystems, so quantifying this amplified response at a global scale is important

High throughput methods applied in biomaterial development and discovery

The high throughput discovery of new materials can be achieved by rapidly screening many different materials synthesised by a combinatorial approach to identify the optimal material that fulfils a particular biomedical application. Here we review the literature in this area and conclude that for polymers, this process is best achieved in a microarray format, which enable thousands of cell-material interactions to be monitored on a single chip. Polymer microarrays can be formed by printing pre-synthesised polymers or by printing monomers onto the chip where on-slide polymerisation is initiated. The surface properties of the material can be analysed and correlated to the biological performance using high throughput surface analysis, including time-of-flight secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA) measurements. This approach enables the surface properties responsible for the success of a material to be understood, which in turn provides the foundations of future material design. The high throughput discovery of materials using polymer microarrays has been explored for many cell-based applications including the isolation of specific cells from heterogeneous populations, the attachment and differentiation of stem cells and the controlled transfection of cells. Further development of polymerisation techniques and high throughput biological assays amenable to the polymer microarray format will broaden the combinatorial space and biological phenomenon that polymer microarrays can explore, and increase their efficacy. This will, in turn, result in the discovery of optimised polymeric materials for many biomaterial applications

Warming of Central European lakes and their response to the 1980s climate regime shift

Lake surface water temperatures (LSWTs) are sensitive to atmospheric warming and have previously been shown to respond to regional changes in the climate. Using a combination of in situ and simulated surface temperatures from 20 Central European lakes, with data spanning between 50 and ∼100 years, we investigate the long-term increase in annually averaged LSWT. We demonstrate that Central European lakes are warming most in spring and experience a seasonal variation in LSWT trends. We calculate significant LSWT warming during the past few decades and illustrate, using a sequential t test analysis of regime shifts, a substantial increase in annually averaged LSWT during the late 1980s, in response to an abrupt shift in the climate. Surface air temperature measurements from 122 meteorological stations situated throughout Central Europe demonstrate similar increases at this time. Climatic modification of LSWT has numerous consequences for water quality and lake ecosystems. Quantifying the response of LSWT increase to large-scale and abrupt climatic shifts is essential to understand how lakes will respond in the future

Single Cell Deposition and Patterning with a Robotic System

Integrating single-cell manipulation techniques in traditional and emerging biological culture systems is challenging. Microfabricated devices for single cell studies in particular often require cells to be spatially positioned at specific culture sites on the device surface. This paper presents a robotic micromanipulation system for pick-and-place positioning of single cells. By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location. A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells. Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate. In contrast, the whole-cell aspiration method required 30 seconds per cell and achieved a success rate of 80%. The broad applicability of this robotic manipulation technique is demonstrated using multiple cell types on traditional substrates and on open-top microfabricated devices, without requiring modifications to device designs. Furthermore, we used this serial deposition process in conjunction with an established parallel cell manipulation technique to improve the efficiency of single cell capture from ∼80% to 100%. Using a robotic micromanipulation system to position single cells on a substrate is demonstrated as an effective stand-alone or bolstering technology for single-cell studies, eliminating some of the drawbacks associated with standard single-cell handling and manipulation techniques