The genetic basis of inter-individual variation in recovery from traumatic brain injury.

Traumatic brain injury (TBI) is one of the leading causes of death among young people, and is increasingly prevalent in the aging population. Survivors of TBI face a spectrum of outcomes from short-term non-incapacitating injuries to long-lasting serious and deteriorating sequelae. TBI is a highly complex condition to treat; many variables can account for the observed heterogeneity in patient outcome. The limited success of neuroprotection strategies in the clinic has led to a new emphasis on neurorestorative approaches. In TBI, it is well recognized clinically that patients with similar lesions, age, and health status often display differences in recovery of function after injury. Despite this heterogeneity of outcomes in TBI, restorative treatment has remained generic. There is now a new emphasis on developing a personalized medicine approach in TBI, and this will require an improved understanding of how genetics impacts on long-term outcomes. Studies in animal model systems indicate clearly that the genetic background plays a role in determining the extent of recovery following an insult. A candidate gene approach in human studies has led to the identification of factors that can influence recovery. Here we review studies of the genetic basis for individual differences in functional recovery in the CNS in animals and man. The application of in vitro modeling with human cells and organoid cultures, along with whole-organism studies, will help to identify genes and networks that account for individual variation in recovery from brain injury, and will point the way towards the development of new therapeutic approaches

Cancer Stem Cells: Notes for Authors.

Stem Cell Reports frequently receives manuscripts dealing with the topic of cancer stem cells. Many of the submissions on this topic have major shortcomings in their content or limits to the conclusions that can be drawn from the results presented. The purpose of this Commentary is to highlight some of the underlying issues so that authors can enhance the strength of their research contributions

Comprehensive characterization of molecular interactions based on nanomechanics

Molecular interaction is a key concept in our understanding of the biological mechanisms of life. Two physical properties change when one molecular partner binds to another. Firstly, the masses combine and secondly, the structure of at least one binding partner is altered, mechanically transducing the binding into subsequent biological reactions. Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro. This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology. To prove the concept we measured lipid vesicle (approximately 748*10(6) Da) adsorption on the sensor interface followed by subsequent binding of the bee venom peptide melittin (2840 Da) to the vesicles. The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions

Biomedical and societal impacts of in vitro embryo models of mammalian development.

In recent years, a diverse array of in vitro cell-derived models of mammalian development have been described that hold immense potential for exploring fundamental questions in developmental biology, particularly in the case of the human embryo where ethical and technical limitations restrict research. These models open up new avenues toward biomedical advances in in vitro fertilization, clinical research, and drug screening with potential to impact wider society across many diverse fields. These technologies raise challenging questions with profound ethical, regulatory, and social implications that deserve due consideration. Here, we discuss the potential impacts of embryo-like models, and their biomedical potential and current limitations

Human Induced Pluripotent Stem Cells on Autologous Feeders

BACKGROUND: For therapeutic usage of induced Pluripotent Stem (iPS) cells, to accomplish xeno-free culture is critical. Previous reports have shown that human embryonic stem (ES) cells can be maintained in feeder-free condition. However, absence of feeder cells can be a hostile environment for pluripotent cells and often results in karyotype abnormalities. Instead of animal feeders, human fibroblasts can be used as feeder cells of human ES cells. However, one still has to be concerned about the existence of unidentified pathogens, such as viruses and prions in these non-autologous feeders. METHODOLOGY/PRINCIPAL FINDINGS: This report demonstrates that human induced Pluripotent Stem (iPS) cells can be established and maintained on isogenic parental feeder cells. We tested four independent human skin fibroblasts for the potential to maintain self-renewal of iPS cells. All the fibroblasts tested, as well as their conditioned medium, were capable of maintaining the undifferentiated state and normal karyotypes of iPS cells. Furthermore, human iPS cells can be generated on isogenic parental fibroblasts as feeders. These iPS cells carried on proliferation over 19 passages with undifferentiated morphologies. They expressed undifferentiated pluripotent cell markers, and could differentiate into all three germ layers via embryoid body and teratoma formation. CONCLUSIONS/SIGNIFICANCE: These results suggest that autologous fibroblasts can be not only a source for iPS cells but also be feeder layers. Our results provide a possibility to solve the dilemma by using isogenic fibroblasts as feeder layers of iPS cells. This is an important step toward the establishment of clinical grade iPS cells

Toward Guidelines for Research on Human Embryo Models Formed from Stem Cells.

Over the past few years, a number of research groups have reported striking progress on the generation of in vitro models from mouse and human stem cells that replicate aspects of early embryonic development. Not only do these models reproduce some key cell fate decisions but, especially in the mouse system, they also mimic the spatiotemporal arrangements of embryonic and extraembryonic tissues that are required for developmental patterning and implantation in the uterus. If such models could be developed for the early human embryo, they would have great potential benefits for understanding early human development, for biomedical science, and for reducing the use of animals and human embryos in research. However, guidelines for the ethical conduct of this line of work are at present not well defined. In this Forum article, we discuss some key aspects of this emerging area of research and provide some recommendations for its ethical oversight

Use of the Nanobridge system for the Rapid Production of Pluipotent Stem Cells and Neural Progenitor Cells

The novel Nanobridge system allows the formation of cellular aggregates of pluripotent stem cells, which can then be grown in suspensions cultures allowing accurate control of the environment in which the cells are growing. The Nanobridge system utilizes a thermo-responsive poly N-isopropyl acrylamide (PNIPAM) polymer decorated with extracellular matrix (ECM) protein fragments (fibronectin or vitronectin) to bind to and bridge between adjacent cells and form cell aggregates at 370 C. A temperature shift from 370 C to 320 C causes the PNIPAM to become water soluble weakening the bonding between adjacent PNIPAM chains and allowing the aggregates to be broken down to smaller aggregates by increased shear forces. By returning the temperature to 370 C and increasing the culture volume with additional medium, the increased number of smaller aggregates are able to grow to a larger diameter. Repeating this cycle allows for the rapid expansion in cell numbers. In addition, the ability to vary the concentrations and ratios of the two components in the Nanobridge system, when coupled with the temperature shift procedure during passaging, allows for tight control over the aggregate diameters at all stages of the expansion process. In this paper, two examples of using the Nanobridge system to culture stem cells will be described: firstly using the system for the rapid expansion of human embryonic stem cells whilst maintaining high viability and pluripotency, and secondly; using the system to develop a process to form neural cell aggregates and maintain and expand cells at a stem/progenitor (NPC) stage, obviating the need for the current cumbersome manual methods to produce larger numbers of NPCs. In the first example, embryonic stem cells (hESC) WA09 were cultured in spinner flasks with the Nanobridge system. At the end of the growth phase, aggregates of 348 micron average diameter were reduced to an average diameter of 139microns after sub-passaging. When this cycle was repeated five times, there was a 500 fold increase in the number of cells produced, with a viability at the end of the process of 90% while maintaining key pluripotent markers NANOG, OCT3/4, SOX2, and DNMT3B. Characterization of the hESC aggregates was performed using the IN Cell Analyser 2200, which demonstrated that there was uniform cell viability and pluripotency marker distribution throughout the aggregates, ie there was no evidence of any diffusional limitations or necrotic regions within the aggregates. At the end of the expansion process it was shown that the cells were able to differentiate into all three germ layers, and that the cells could be converted, to cells types such as cardiomyocytes. The results demonstrate that the Nanobridge system is a simple and scalable method of producing large numbers of PSCs without the need for enzymes during passaging. For the production of the neural progenitors (NPCs), hESC (WA09) cells were formed and cultured as Nanobridge aggregates with diameters of 200-300 mm. Differentiation was initiated by culturing the aggregates in mTESR medium with 5uM SB431542 and 100 nM LDN for 5 days. At day 5, the medium was changed to neural basal medium (NBM) supplemented with EGF and FGF2 for the next 5 days of culture. Cultures were maintained in NBM from day 10 onwards. Passaging was performed at day 5 and day 10 and thereafter on a weekly basis for 4 weeks. Temperature shift and mechanical shear were utilized to breakup aggregates and Nanobridge components and medium were replaced during passaging. Cells demonstrated upregulation and subsequent maintenance of neural-associated markers (PAX6, SOX1, and NCAM) in aggregate culture. Passaging resulted in an overall seven fold increase in the number of cells expressing the neural-associated markers. Furthermore, neural progenitor cell aggregates exhibited the capacity to differentiate towards a more mature phenotype as demonstrated by the outgrowth of neurites. This demonstrated that the Nanobridge system has the potential to facilitate the scale-up of NPC production in bioreactors for applications in regenerative medicine and pharmacological testing

Prepatterning in the Stem Cell Compartment

The mechanism by which an apparently uniform population of cells can generate a heterogeneous population of differentiated derivatives is a fundamental aspect of pluripotent and multipotent stem cell behaviour. One possibility is that the environment and the differentiation cues to which the cells are exposed are not uniform. An alternative, but not mutually exclusive possibility is that the observed heterogeneity arises from the stem cells themselves through the existence of different interconvertible substates that pre-exist before the cells commit to differentiate. We have tested this hypothesis in the case of apparently homogeneous pluripotent human embryonal carcinoma (EC) stem cells, which do not follow a uniform pattern of differentiation when exposed to retinoic acid. Instead, they produce differentiated progeny that include both neuronal and non-neural phenotypes. Our results suggest that pluripotent NTERA2 stem cells oscillate between functionally distinct substates that are primed to select distinct lineages when differentiation is induced

Fibronectin-conjugated thermoresponsive nanobridges generate three dimensional human pluripotent stem cell cultures for differentiation towards the neural lineages.

Production of 3-dimensional neural progenitor cultures from human pluripotent stem cells offers the potential to generate large numbers of cells. We utilised our nanobridge system to generate 3D hPSC aggregates for differentiation towards the neural lineage, and investigate the ability to passage aggregates while maintaining cells at a stem/progenitor stage. Over 38 days, aggregate cultures exhibited upregulation and maintenance of neural-associated markers and demonstrated up to 10 fold increase in cell number. Aggregates undergoing neural induction in the presence or absence of nanobridges demonstrated no differences in marker expression, proliferation or viability. However, aggregates formed without nanobridges were statistically significantly fewer and smaller by passage 3. Organoids, cultured from aggregates, and treated with retinoic acid or rock inhibitor demonstrated terminal differentiation as assessed by immunohistochemistry. These data demonstrate that nanobridge 3D hPSC can differentiate to neural stem/progenitor cells, and be maintained at this stage through serial passaging and expansion

Metal-organic framework based mixed matrix membranes: a solution for highly efficient CO2 capture?

The field of metal-organic framework based mixed matrix membranes (M(4)s) is critically reviewed, with special emphasis on their application in CO2 capture during energy generation. After introducing the most relevant parameters affecting membrane performance, we define targets in terms of selectivity and productivity based on existing literature on process design for pre- and post-combustion CO2 capture. Subsequently, the state of the art in M(4)s is reviewed against these targets. Because final application of these membranes will only be possible if thin separation layers can be produced, the latest advances in the manufacture of M-4 hollow fibers are discussed. Finally, the recent efforts in understanding the separation performance of these complex composite materials and future research directions are outlined.European Commission FP7 608490 ERC 33574