From 2D to 3D: novel nanostructured scaffolds to investigate signalling in reconstructed neuronal networks

To recreate in vitro 3D neuronal circuits will ultimately increase the relevance of results from cultured to whole-brain networks and will promote enabling technologies for neuro-engineering applications. Here we fabricate novel elastomeric scaffolds able to instruct 3D growth of living primary neurons. Such systems allow investigating the emerging activity, in terms of calcium signals, of small clusters of neurons as a function of the interplay between the 2D or 3D architectures and network dynamics. We report the ability of 3D geometry to improve functional organization and synchronization in small neuronal assemblies. We propose a mathematical modelling of network dynamics that supports such a result. Entrapping carbon nanotubes in the scaffolds remarkably boosted synaptic activity, thus allowing for the first time to exploit nanomaterial/cell interfacing in 3D growth support. Our 3D system represents a simple and reliable construct, able to improve the complexity of current tissue culture models

Cell tracking in cardiac repair: what to image and how to image

Stem cell therapies hold the great promise and interest for cardiac regeneration among scientists, clinicians and patients. However, advancement and distillation of a standard treatment regimen are not yet finalised. Into this breach step recent developments in the imaging biosciences. Thus far, these technical and protocol refinements have played a critical role not only in the evaluation of the recovery of cardiac function but also in providing important insights into the mechanism of action of stem cells. Molecular imaging, in its many forms, has rapidly become a necessary tool for the validation and optimisation of stem cell engrafting strategies in preclinical studies. These include a suite of radionuclide, magnetic resonance and optical imaging strategies to evaluate non-invasively the fate of transplanted cells. In this review, we highlight the state-of-the-art of the various imaging techniques for cardiac stem cell presenting the strengths and limitations of each approach, with a particular focus on clinical applicability

A Biological Global Positioning System: Considerations for Tracking Stem Cell Behaviors in the Whole Body

Many recent research studies have proposed stem cell therapy as a treatment for cancer, spinal cord injuries, brain damage, cardiovascular disease, and other conditions. Some of these experimental therapies have been tested in small animals and, in rare cases, in humans. Medical researchers anticipate extensive clinical applications of stem cell therapy in the future. The lack of basic knowledge concerning basic stem cell biology-survival, migration, differentiation, integration in a real time manner when transplanted into damaged CNS remains an absolute bottleneck for attempt to design stem cell therapies for CNS diseases. A major challenge to the development of clinical applied stem cell therapy in medical practice remains the lack of efficient stem cell tracking methods. As a result, the fate of the vast majority of stem cells transplanted in the human central nervous system (CNS), particularly in the detrimental effects, remains unknown. The paucity of knowledge concerning basic stem cell biology—survival, migration, differentiation, integration in real-time when transplanted into damaged CNS remains a bottleneck in the attempt to design stem cell therapies for CNS diseases. Even though excellent histological techniques remain as the gold standard, no good in vivo techniques are currently available to assess the transplanted graft for migration, differentiation, or survival. To address these issues, herein we propose strategies to investigate the lineage fate determination of derived human embryonic stem cells (hESC) transplanted in vivo into the CNS. Here, we describe a comprehensive biological Global Positioning System (bGPS) to track transplanted stem cells. But, first, we review, four currently used standard methods for tracking stem cells in vivo: magnetic resonance imaging (MRI), bioluminescence imaging (BLI), positron emission tomography (PET) imaging and fluorescence imaging (FLI) with quantum dots. We summarize these modalities and propose criteria that can be employed to rank the practical usefulness for specific applications. Based on the results of this review, we argue that additional qualities are still needed to advance these modalities toward clinical applications. We then discuss an ideal procedure for labeling and tracking stem cells in vivo, finally, we present a novel imaging system based on our experiments

A Research Agenda for Helminth Diseases of Humans: Social Ecology, Environmental Determinants, and Health Systems

In this paper, the Disease Reference Group on Helminth Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR), with the mandate to review helminthiases research and identify research priorities and gaps, focuses on the environmental, social, behavioural, and political determinants of human helminth infections and outlines a research and development agenda for the socioeconomic and health systems research required for the development of sustainable control programmes. Using Stockols' social-ecological approach, we describe the role of various social (poverty, policy, stigma, culture, and migration) and environmental determinants (the home environment, water resources development, and climate change) in the perpetuation of helminthic diseases, as well as their impact as contextual factors on health promotion interventions through both the regular and community-based health systems. We examine these interactions in regard to community participation, intersectoral collaboration, gender, and possibilities for upscaling helminthic disease control and elimination programmes within the context of integrated and interdisciplinary approaches. The research agenda summarises major gaps that need to be addressed

RNA Interference in Schistosoma mansoni Schistosomula: Selectivity, Sensitivity and Operation for Larger-Scale Screening

RNA interference (RNAi) is a technique to selectively suppress mRNA of individual genes and, consequently, their cognate proteins. RNAi using double-stranded (ds) RNA has been used to interrogate the function of mainly single genes in the flatworm, Schistosoma mansoni, one of a number of schistosome species causing schistosomiasis. In consideration of large-scale screens to identify candidate drug targets, we examined the selectivity and sensitivity (the degree of suppression) of RNAi for 11 genes produced in different tissues of the parasite: the gut, tegument (surface) and otherwise. We used the schistosomulum stage prepared from infective cercariae larvae which are accessible in large numbers and adaptable to automated screening platforms. We found that RNAi suppresses transcripts selectively, however, the sensitivity of suppression varies (40%–>75%). No obvious changes in the parasite occurred post-RNAi, including after targeting the mRNA of genes that had been computationally predicted to be essential for survival. Additionally, we defined operational parameters to facilitate large-scale RNAi, including choice of culture medium, transfection strategy to deliver dsRNA, dose- and time-dependency, and dosing limits. Finally, using fluorescent probes, we show that the developing gut allows rapid entrance of dsRNA into the parasite to initiate RNAi

Effects of gamma irradiation on the biomechanical properties of peroneus tendons

Christopher M Aguila,1 Gaëtan J-R Delcroix,2–5 David N Kaimrajh,6 Edward L Milne,6 H Thomas Temple,5,7 Loren L Latta2,6 1Department of Biological Sciences, Florida International University, Miami, FL, USA; 2Department of Orthopaedics, Miller School of Medicine, University of Miami, Miami, FL, USA; 3Research Service & Geriatric Research, Education, and Clinical Center, Bruce W. Carter Veterans Affairs Medical Center, Miami, FL, USA; 4Interdisciplinary Stem Cell Institute, University of Miami, Miami, FL, USA; 5Vivex Biomedical Inc., Marietta, GA, USA; 6Max Biedermann Institute for Biomechanics, Miami Beach, FL, USA; 7Translational Research and Economic Development, Nova Southeastern University, Fort-Lauderdale, FL, USA Purpose: This study was designed to investigate the biomechanical properties of nonirradiated (NI) and irradiated (IR) peroneus tendons to determine if they would be suitable allografts, in regards to biomechanical properties, for anterior cruciate ligament reconstruction after a dose of 1.5–2.5 Mrad.Methods: Seven pairs of peroneus longus (PL) and ten pairs of peroneus brevis (PB) tendons were procured from human cadavers. The diameter of each allograft was measured. The left side of each allograft was IR at 1.5–2.5 Mrad, whereas the right side was kept aseptic and NI. The allografts were thawed, kept wet with saline, and attached in a single-strand fashion to custom freeze grips using liquid nitrogen. A preload of 10 N was then applied and, after it had reached steady state, the allografts were pulled at 4 cm/sec. The parameters recorded were the displacement and force.Results: The elongation at the peak load was 10.3±2.3 mm for the PB NI side and 13.5±3.3 mm for the PB IR side. The elongation at the peak load was 17.4±5.3 mm for the PL NI side and 16.3±2.0 mm for the PL IR side. For PL, the ultimate load was 2,091.6±148.7 N for NI and 2,122.8±380.0 N for IR. The ultimate load for the PB tendons was 1,485.7±209.3 N for NI and 1,318.4±296.9 N for the IR group. The ultimate stress calculations for PL were 90.3±11.3 MPa for NI and 94.8±21.0 MPa for IR. For the PB, the ultimate stress was 82.4±19.0 MPa for NI and 72.5±16.6 MPa for the IR group. The structural stiffness was 216.1±59.0 N/mm for the NI PL and 195.7±51.4 N/mm for the IR side. None of these measures were significantly different between the NI and IR groups. The structural stiffness was 232.1±45.7 N/mm for the NI PB and 161.9±74.0 N/mm for the IR side, and this was the only statistically significant difference found in this study (P=0.034).Conclusion: Our statistical comparisons found no significant differences in terms of elongation, ultimate load, or ultimate stress between IR and NI PB and PL tendons. Only the PB structural stiffness was affected by irradiation. Thus, sterilizing allografts at 1.5–2.5 Mrad of gamma irradiation does not cause major alterations in the tendons’ biomechanical properties while still providing a suitable amount of sterilization for anterior cruciate ligament reconstruction. Keywords: ACL reconstruction, allograft, sterilization, tissue bankin

TAp63γ and ΔNp63β Promote Osteoblastic Differentiation of Human Mesenchymal Stem Cells: Regulation by Vitamin D3 Metabolites

The transcription factor p63 is required for skeletal formation, and is important for the regulation of 1α,25(OH)2D3 receptor (VDR) in human mesenchymal stem cells (hMSC). Herein we report that TAp63γ and ΔNp63β appear to be an integral part of the osteoblastic differentiation of hMSC and are differentially regulated by the vitamin D3 metabolites 1α,25(OH)2D3 and 24R,25(OH)2D3. We compared the endogenous expression of p63 isoforms (TA- and ΔNp63) and splice variants (p63α, -β, -γ), in naive hMSC and during osteoblastic differentiation of hMSC. TAp63α and -β were the predominant p63 variants in naive, proliferating hMSC. In contrast, under osteoblastic differentiation conditions, expression of p63 changed from the TAp63α and -β to the TAp63γ and ΔNp63β variants. Transient overexpression of the p63 variants demonstrated that TAp63β, ΔNp63β, and ΔNp63γ increased alkaline phosphatase activity and ΔNp63α and -γ increased the expression of mRNA for osteocalcin and osterix. Our results support the hypothesis that TAp63α and -β promote a naive state in hMSC. Moreover, TAp63γ is increased during and promotes early osteoblastic differentiation through the expression of pro-osteogenic genes; VDR, Osterix, Runx2 and Osteopontin. ΔNp63β also appears to support osteogenic maturation through increased alkaline phosphatase activity. Treatment with 1α,25(OH)2D3 increased the expression of mRNA for ΔNp63, while addition of 24R,25(OH)2D3 increased the expression of TA- and ΔNp63γ variants. These novel findings demonstrate for the first time that p63 variants are differentially expressed in naive hMSC (TAp63α,β), are important during the osteoblastic differentiation of hMSC (TAp63γ and ΔNp63β), and are differentially regulated by the vitamin D3 metabolites, 1α,25(OH)2D3 and 24R,25(OH)2D3. The molecular nuances and mechanisms of osteoblastic differentiation presented here will hopefully improve our understanding of bone development, complications in bone repair (mal- and non-union fractures), osteoporosis and possibly lead to new modalities of treatment