Aryl Hydrocarbon Receptor (AhR)-Mediated Signaling in iPSC-Derived Human Motor Neurons.

Exposure to environmental pollutants and endogenous metabolites that induce aryl hydrocarbon receptor (AhR) expression has been suggested to affect cognitive development and, particularly in boys, also motor function. As current knowledge is based on epidemiological and animal studies, in vitro models are needed to better understand the effects of these compounds in the human nervous system at the molecular level. Here, we investigated expression of AhR pathway components and how they are regulated by AhR ligands in human motor neurons. Motor neurons generated from human induced pluripotent stem cells (hiPSCs) were characterized at the molecular level and by electrophysiology. mRNA levels of AhR target genes, CYP1A1 and CYP1B1 (cytochromes P450 1A1/1B1), and AhR signaling components were monitored in hiPSCs and in differentiated neurons following treatment with AhR ligands, 2,3,7,8,-tetrachlodibenzo-p-dioxin (TCDD), L-kynurenine (L-Kyn), and kynurenic acid (KA), by RT-qPCR. Changes in AhR cellular localization and CYP1A1 activity in neurons treated with AhR ligands were also assessed. The neurons we generated express motor neuron-specific markers and are functional. Transcript levels of CYP1B1, AhR nuclear translocators (ARNT1 and ARNT2) and the AhR repressor (AhRR) change with neuronal differentiation, being significantly higher in neurons than hiPSCs. In contrast, CYP1A1 and AhR transcript levels are slightly lower in neurons than in hiPSCs. The response to TCDD treatment differs in hiPSCs and neurons, with only the latter showing significant CYP1A1 up-regulation. In contrast, TCDD slightly up-regulates CYP1B1 mRNA in hiPSCs, but downregulates it in neurons. Comparison of the effects of different AhR ligands on AhR and some of its target genes in neurons shows that L-Kyn and KA, but not TCDD, regulate AhR expression and differently affect CYP1A1 and CYP1B1 expression. Finally, although TCDD does not significantly affect AhR transcript levels, it induces AhR protein translocation to the nucleus and increases CYP1A1 activity. This is in contrast to L-Kyn and KA, which either do not affect or reduce, respectively, CYP1A1 activity. Expression of components of the AhR signaling pathway are regulated with neuronal differentiation and are differently affected by TCDD, suggesting that pluripotent stem cells might be less sensitive to this toxin than neurons. Crucially, AhR signaling is affected differently by TCDD and other AhR ligands in human motor neurons, suggesting that they can provide a valuable tool for assessing the impact of environmental pollutants.

Modelling human CNS injury with human neural stem cells in 2- and 3-Dimensional cultures.

The adult human central nervous system (CNS) has very limited regenerative capability, and injury at the cellular and molecular level cannot be studied in vivo. Modelling neural damage in human systems is crucial to identifying species-specific responses to injury and potentially neurotoxic compounds leading to development of more effective neuroprotective agents. Hence we developed human neural stem cell (hNSC) 3-dimensional (3D) cultures and tested their potential for modelling neural insults, including hypoxic-ischaemic and Ca2+-dependent injury. Standard 3D conditions for rodent cells support neuroblastoma lines used as human CNS models, but not hNSCs, but in all cases changes in culture architecture alter gene expression. Importantly, response to damage differs in 2D and 3D cultures and this is not due to reduced drug accessibility. Together, this study highlights the impact of culture cytoarchitecture on hNSC phenotype and damage response, indicating that 3D models may be better predictors of in vivo response to damage and compound toxicity.

Loss of Tiparp Results in Aberrant Layering of the Cerebral Cortex.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP) is an enzyme that adds a single ADP-ribose moiety to itself or other proteins. Tiparp is highly expressed in the brain; however, its function in this organ is unknown. Here, we used Tiparp-/- mice to determine Tiparp's role in the development of the prefrontal cortex. Loss of Tiparp resulted in an aberrant organization of the mouse cortex, where the upper layers presented increased cell density in the knock-out mice compared with wild type. Tiparp loss predominantly affected the correct distribution and number of GABAergic neurons. Furthermore, neural progenitor cell proliferation was significantly reduced. Neural stem cells (NSCs) derived from Tiparp-/- mice showed a slower rate of migration. Cytoskeletal components, such as α-tubulin are key regulators of neuronal differentiation and cortical development. α-tubulin mono-ADP ribosylation (MAR) levels were reduced in Tiparp-/- cells, suggesting that Tiparp plays a role in the MAR of α-tubulin. Despite the mild phenotype presented by Tiparp-/- mice, our findings reveal an important function for Tiparp and MAR in the correct development of the cortex. Unravelling Tiparp's role in the cortex, could pave the way to a better understanding of a wide spectrum of neurological diseases which are known to have increased expression of TIPARP.

Toward modeling the human nervous system in a dish: recent progress and outstanding challenges.

Studying the cellular and molecular bases governing development, and normal and abnormal functions of the human CNS is hampered by its complexity and the very limited possibility of experimentally manipulating it in vivo. Development of 3D, tissue-like culture systems offers much promise for boosting our understanding of human neural development, birth defects, neurodegenerative diseases and neural injury, and for providing platforms that will more accurately predict efficacy of putative therapeutic compounds and assess responses to potentially neurotoxic agents. Although novel technological developments and a more interdisciplinary approach to modeling the human CNS are accelerating the pace of discovery, increasing the complexity of in vitro systems increases the ordeals to be overcome to establish highly reproducible models amenable to quantitative analysis.

Biocompatibility of nanostructured boron doped diamond for the attachment and proliferation of human neural stem cells.

OBJECTIVE: We quantitatively investigate the biocompatibility of chemical vapour deposited (CVD) nanocrystalline diamond (NCD) after the inclusion of boron, with and without nanostructuring. The nanostructuring method involves a novel approach of growing NCD over carbon nanotubes (CNTs) that act as a 3D scaffold. This nanostructuring of BNCD leads to a material with increased capacitance, and this along with wide electrochemical window makes BNCD an ideal material for neural interface applications, and thus it is essential that their biocompatibility is investigated. APPROACH: Biocompatibility was assessed by observing the interaction of human neural stem cells (hNSCs) with a variety of NCD substrates including un-doped ones, and NCD doped with boron, which are both planar, and nanostructured. hNSCs were chosen due to their sensitivity, and various methods including cell population and confluency were used to quantify biocompatibility. MAIN RESULTS: Boron inclusion into NCD film was shown to have no observable effect on hNSC attachment, proliferation and viability. Furthermore, the biocompatibility of nanostructured boron-doped NCD is increased upon nanostructuring, potentially due to the increased surface area. SIGNIFICANCE: Diamond is an attractive material for supporting the attachment and development of cells as it can show exceptional biocompatibility. When boron is used as a dopant within diamond it becomes a p-type semiconductor, and at high concentrations the diamond becomes quasi-metallic, offering the prospect of a direct electrical device-cell interfacing system.

Chondrogenic differentiation of adipose tissue-derived stem cells within nanocaged POSS-PCU scaffolds: a new tool for nanomedicine.

Scaffold cellularization for cartilage engineering can aid implant properties, their retention and minimize repeated intervention, particularly in paediatric reconstructive craniofacial surgery. We developed novel bionanoscaffolds using paediatric adipose tissue-derived stem cells (hADSCs), an accessible autologous cell source, and POSS-PCU. Little is known about cellular responses to this nanomaterial, though it was used in human. We assessed: 1) POSS-PCU cellularization and bioaffinity to hADSCs; 2) hADSC chondrogenic differentiation ability in POSS-PCU; 3) whether bionanoscaffolds became encased within a vascular network and/or vascularised. POSS-PCU supported ADSC survival and proliferation and their migration and differentiation into cartilage within the nanoscaffold. Furthermore, after CAM-grafting, bionanoscaffolds were rapidly surrounded by blood vessels without any apparent negative reaction and erythrocytes of host origin were detected inside the scaffold, suggesting invasion from some capillaries. Altogether, this study demonstrates that POSS-PCU displays excellent bioactivity and hADSC/POSS-PCU bionanoscaffolds offer much promise for autologous cell-based tissue engineering for clinical applications. FROM THE CLINICAL EDITOR: In this study, human adipose tissue derived stem cells were used in combination with POSS-PCU nanoscaffolds to generate cartilage tissue demonstrating excellent bioactivity for autologous cell-based tissue engineering for clinical applications.

Cord blood Lin(-)CD45(-) embryonic-like stem cells are a heterogeneous population that lack self-renewal capacity.

Human umbilical cord blood (hUCB) has been proposed to contain not only haematopoietic stem cells, but also a rare pluripotent embryonic-like stem cell (ELSc) population that is negative for hematopoietic markers (Lin(-)CD45(-)) and expresses markers typical of pluripotent cells. The aim of this work was to isolate, characterise and expand this ELSc fraction from hUCB, as it may provide a valuable cell source for regenerative medicine applications. We found that we could indeed isolate a Lin(-)CD45(-) population of small cells (3-10 µm diameter) with a high nucleus to cytoplasm ratio that expressed the stem cell markers CD34 and CXCR4. However, in contrast to some previous reports, this fraction was not positive for CD133. Furthermore, although these cells expressed transcripts typical of pluripotent cells, such as SOX2, OCT3/4, and NANOG, they were not able to proliferate in any of the culture media known to support stem cell growth that we tested. Further analysis of the Lin(-)CD45(-) population by flow cytometry showed the presence of a Lin(-)CD45(-)Nestin(+) population that were also positive for CD34 (20%) but negative for CXCR4. These data suggest that the Lin(-)CD45(-) stem cell fraction present in the cord blood represents a small heterogeneous population with phenotypic characteristics of stem cells, including a Lin(-)CD45(-)Nestin(+) population not previously described. This study also suggests that heterogeneity within the Lin(-)CD45(-) cell fraction is the likely explanation for differences in the hUCB cell populations described by different groups that were isolated using different methods. These populations have been widely called "embryonic-like stem cell" on the basis of their phenotypical similarity to embryonic stem cells. However, the fact they do not seem to be able to self-renew casts some doubt on their identity, and warns against defining them as "embryonic-like stem cell" at this stage.

Selective sterilization of Vibro parahaemolyticus from a bacterial mixture by low-amperage electric current.

The objective of this study was to investigate the possibility of using low-amperage electrical treatment (LAET) as a selective bacteriocide. Mixtures containing Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus were treated with different electric current intensities and for different times. The results showed that at 263 mA, treating bacteria for 100 ms eliminated all V. parahaemolyticus colonies. Although LAET reduced the populations of the three microorganisms, V. parahaemolyticus was more injured by LAET than S. aureus and E. coli when treated at the same processing conditions.