Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation

Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs) in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG). After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin

In Situ Spatiotemporal Mapping of Flow Fields around Seeded Stem Cells at the Subcellular Length Scale

A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV) for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD) predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms

Complementarity, completeness and quality of long-term faunal archives in an Asian biodiversity hotspot

Long-term baselines on biodiversity change through time are crucial to inform conservation decision-making in biodiversity hotspots, but environmental archives remain unavailable for many regions. Extensive palaeontological, zooarchaeological and historical records and indigenous knowledge about past environmental conditions exist for China, a megadiverse country experiencing large-scale biodiversity loss, but their potential to understand past human-caused faunal turnover is not fully assessed. We investigate a series of complementary environmental archives to evaluate the quality of the Holocene–historical faunal record of Hainan Island, China's southernmost province, for establishing new baselines on postglacial mammalian diversity and extinction dynamics. Synthesis of multiple archives provides an integrated model of long-term biodiversity change, revealing that Hainan has experienced protracted and ongoing human-caused depletion of its mammal fauna from prehistory to the present, and that past baselines can inform practical conservation management. However, China's Holocene–historical archives exhibit substantial incompleteness and bias at regional and country-wide scales, with limited taxonomic representation especially for small-bodied species, and poor sampling of high-elevation landscapes facing current-day climate change risks. Establishing a clearer understanding of the quality of environmental archives in threatened ecoregions, and their ability to provide a meaningful understanding of the past, is needed to identify future conservation-relevant historical research priorities

Stakeholder perceptions of job stress in an industrialized country: implications for policy and practice

We used a secondary, qualitative analysis of stakeholder perceptions of work stress in Australia to characterize the context for policy and practice intervention. Themes included: Individual versus contextual descriptions of stress; perceived ‘gender’ differences in manifesting and reporting of stress; the work/home interface; and perceived sectoral and occupational differences in compensation claim rates. We found that people often still perceive stress as an individual rather than organizational problem and view work stress as a stereotypically feminine weakness that affects only certain people. Organizations downplay and overlook risks, increasing worker reluctance to report stressors, creating barriers to job stress interventions. Our study may be relevant to other industrial countries where researchers currently study job stress interventions to improve their effectiveness. Comprehensive approaches can increase knowledge and decrease stigma about job stress and mental illness, and target both work- and non-work-related influences on mental health

Controlled embryoid body formation via surface modification and avidin–biotin cross-linking

Cell–cell interaction is an integral part of embryoid body (EB) formation controlling 3D aggregation. Manipulation of embryonic stem (ES) cell interactions could provide control over EB formation. Studies have shown a direct relationship between EB formation and ES cell differentiation. We have previously described a cell surface modification and cross-linking method for influencing cell–cell interaction and formation of multicellular constructs. Here we show further characterisation of this engineered aggregation. We demonstrate that engineering accelerates ES cell aggregation, forming larger, denser and more stable EBs than control samples, with no significant decrease in constituent ES cell viability. However, extended culture ≥5 days reveals significant core necrosis creating a layered EB structure. Accelerated aggregation through engineering circumvents this problem as EB formation time is reduced. We conclude that the proposed engineering method influences initial ES cell-ES cell interactions and EB formation. This methodology could be employed to further our understanding of intrinsic EB properties and their effect on ES cell differentiation