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Graphene oxide shows great promise as a material for biomedical applications, e.g., as a multi-drug delivery platform. With this in view, reports of studies on the interaction between nanosized graphene oxide flakes and biological cells are beginning to emerge. However, the number of studies remains limited, and most used labeled graphene oxide samples to track the material upon endocytosis. Unfortunately, the labeling process alters the surface functionality of the graphene oxide, and this additional functionalization has been shown to alter the cellular response. Hence, in this work we used label-free graphene oxide. We carefully tracked the uptake of three different nanoscale graphene oxide flake size distributions using scanning/transmission electron microscopy. Uptake was investigated in undifferentiated human monocyte cells (THP-1) and differentiated macrophage cells. The data show clear size dependence for uptake, such that larger graphene oxide flakes (and clusters) are more easily taken up by the cells compared to smaller flakes. Moreover, uptake is shown to occur very rapidly, within two min of incubation with THP-1 cells. The data highlights a crucial need for cellular incubation studies with nanoparticles, to be conducted for short incubation periods as certain dependencies (e.g., size and concentration) are lost with longer incubation periods.

We present artificially motorized sperm cells-a novel type of hybrid micromotor, where customized microhelices serve as motors for transporting sperm cells with motion deficiencies to help them carry out their natural function. Our results indicate that metal-coated polymer microhelices are suitable for this task due to potent, controllable, and nonharmful 3D motion behavior. We manage to capture, transport, and release single immotile live sperm cells in fluidic channels that allow mimicking physiological conditions. Important steps toward fertilization are addressed by employing proper means of sperm selection and oocyte culturing. Despite the fact that there still remain some challenges on the way to achieve successful fertilization with artificially motorized sperms, we believe that the potential of this novel approach toward assisted reproduction can be already put into perspective with the present work.

The mammalian neocortex shows a conserved six-layered structure that differs between species in the total number of cortical neurons produced owing to differences in the relative abundance of distinct progenitor populations. Recent studies have identified a new class of proliferative neurogenic cells in the outer subventricular zone (OSVZ) in gyrencephalic species such as primates and ferrets. Lissencephalic brains of mice possess fewer OSVZ-like progenitor cells and these do not constitute a distinct layer. Most in vitro and in vivo studies have shown that oxygen regulates the maintenance, proliferation and differentiation of neural progenitor cells. Here we dissect the effects of fetal brain oxygen tension on neural progenitor cell activity using a novel mouse model that allows oxygen tension to be controlled within the hypoxic microenvironment in the neurogenic niche of the fetal brain in vivo. Indeed, maternal oxygen treatment of 10%, 21% and 75% atmospheric oxygen tension for 48 h translates into robust changes in fetal brain oxygenation. Increased oxygen tension in fetal mouse forebrain in vivo leads to a marked expansion of a distinct proliferative cell population, basal to the SVZ. These cells constitute a novel neurogenic cell layer, similar to the OSVZ, and contribute to corticogenesis by heading for deeper cortical layers as a part of the cortical plate.

Artificial microvasculature, particularly as part of the blood-brain barrier, has a high benefit for pharmacological drug discovery and uptake regulation. We demonstrate the fabrication of tubular structures with patterns of holes, which are capable of mimicking microvasculatures. By using photolithography, the dimensions of the cylindrical scaffolds can be precisely tuned as well as the alignment and size of holes. Overlapping holes can be tailored to create diverse three-dimensional configurations, for example, periodic nanoscaled apertures. The porous tubes, which can be made from diverse materials for differential functionalization, are biocompatible and can be modified to be biodegradable in the culture medium. As a proof of concept, endothelial cells (ECs) as well as astrocytes were cultured on these scaffolds. They form monolayers along the scaffolds, are guided by the array of holes and express tight junctions. Nanoscaled filaments of cells on these scaffolds were visualized by scanning electron microscopy (SEM). This work provides the basic concept mainly for an in vitro model of microvasculature which could also be possibly implanted in vivo due to its biodegradability.

Self-folding films are a unique kind of thin film. They are able to deform in response to a change in environmental conditions or internal stress and form complex 3D structures. They are very promising candidates for the design of bioscaffolds, which resemble different kinds of biological tissues. In this paper, a very simple and cheap approach for the fabrication of fully biodegradable and biocompatible self-rolled tubes is reported. The tubes' folding can be triggered by temperature. A bilayer approach is used, where one component is active and another one is passive. The passive one can be any biocompatible, biodegradable, hydrophobic polymer. Gelatin is used as an active component: it allows the design of (i) self-folding polymer films, which fold at room temperature (22 °C) and irreversibly unfold at 37 °C, and (ii) films, which are unfolded at room temperature (22 °C), but irreversibly fold at 37 °C. The possibilities of encapsulation of neural stem cells are also demonstrated using self-folded tubes.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder and is characterized by a continuous and selective loss of dopaminergic neurons in the midbrain with a subsequent reduction of the neurotransmitter dopamine in the striatum. Strategies to overcome limitations of conventional symptomatic treatment have employed cell-based strategies including transplantation of developing neural tissue or neural stem cells (NSCs) into the degenerated host brain. Still there is a tug of war for determining the ideal cell source for transplantation strategies. ES cells have the widest and most blatant potential to become the winner because they promise to be made in high quantities and to hold large amounts of the desired cell type. Adult and fetal neural stem cells have the capacity to self-renew and they are able to differentiate into all major cell-types of the brain without bearing tumorigenic potential. They can be isolated and expanded in vitro for a long time retaining the potential to differentiate into important neural cell types including dopaminergic neurons. Another source for cell-replacement are bone marrow stromal cells (MSCs). These cells can be converted into a cell type with all major features of NSCs. Efforts are made to improve these cell sources for transplantation or finding new cell sources like induced pluripotent stem cells (iPS). However, novel grounds are broken: bridging transplantations might improve the clinical outcome by restoring the nigro-striatal pathway and recruitment of endogenous stem cells by pharmacological manipulations uses the inherent regenerative potential of the diseased brain. This review discusses recent data on stem cell technology with respect to cell replacement strategies in PD as well as endogenous dopaminergic regeneration.

Novel approaches to develop naturally-induced drug delivery in tumor environments in a deterministic and controlled manner have become of growing interest in recent years. Different polymeric-based microstructures and other biocompatible substances have been studied taking advantage of lactic acidosis phenomena in tumor cells, which decrease the tumor extracellular pH down to 6.8. Micromotors have recently demonstrated a high performance in living systems, revealing autonomous movement in the acidic environment of the stomach or moving inside living cells by using acoustic waves, opening the doors for implementation of such smart microengines into living entities. The need to develop biocompatible motors which are driven by natural fuel sources inherently created in biological systems has thus become of crucial importance. As a proof of principle, we here demonstrate calcium carbonate Janus particles moving in extremely light acidic environments (pH 6.5), whose motion is induced in conditioned acidic medium generated by HeLa cells in situ. Our system not only obviates the need for an external fuel, but also presents a selective activation of the micromotors which promotes their motion and consequent dissolution in presence of a quickly propagating cell source (i.e. tumor cells), therefore inspiring new micromotor configurations for potential drug delivery systems.

Smart biomimetics, a unique class of devices combining the mechanical adaptivity of soft actuators with the imperceptibility of microelectronics, is introduced. Due to their inherent ability to self-assemble, biomimetic microelectronics can firmly yet gently attach to an inorganic or biological tissue enabling enclosure of, for example, nervous fibers, or guide the growth of neuronal cells during regeneration.

We employ glass microtube structures fabricated by rolled-up nanotechnology to infer the influence of scaffold dimensionality and cell confinement on neural stem cell (NSC) migration. Thereby, we observe a pronounced morphology change that marks a reversible mesenchymal to amoeboid migration mode transition. Space restrictions preset by the diameter of nanomembrane topography modify the cell shape toward characteristics found in living tissue. We demonstrate the importance of substrate dimensionality for the migration mode of NSCs and thereby define rolled-up nanomembranes as the ultimate tool for single-cell migration studies.

Leucine-rich repeat kinase 2 (Lrrk2) is a Parkinson’s disease (PD)-related protein involved in sporadic and familial cases of the disease. Lrrk2 is a kinase and may be involved in intracellular signaling pathways. We established a neural stem cell (NSC)-based model system for Lrrk2-associated PD. We investigated its physiological and pathophysiological expression in human and mouse brain and NSCs using different antibodies defining dopaminergic NSCs as a relevant cells population to study Lrrk2 dysfunction. Using small interfering RNA duplexes targeting Lrrk2 in fetal mouse NSCs, we observed higher Tyrosine hydroxylase ( Th ) mRNA levels. Conversely, when overexpressing human LRRK2 in fetal mouse NSCs, Th , mRNA levels were reduced. Interestingly, in mutant LRRK2 overexpression Map2 and Kif3A mRNA levels were reduced. In conclusion, we demonstrate that overexpressing LRRK2 in neural stem cells results in a cell culture model for LRRK2-mediated PD. Lrrk2 is conversely linked to Th expression and expression of microtubule genes, implicating a role in the pathomechanism towards dopaminergic neurodegeneration.

Reduced oxygen levels (1-5% O2, named herein 'physioxia') are beneficial for stem cell cultures leading to enhanced proliferation, better survival and higher differentiation potential, but the underlying molecular mechanisms remain elusive. A potential link between physioxia and the canonical Wnt pathway was found recently, but the differential involvement of this signalling pathway for the various stem cell properties such as proliferation, stem cell maintenance, and differentiation capacity remains enigmatic. We here demonstrate increased Wnt target gene transcription and stabilised active β-catenin upon physioxic cell culture in primary tissue-specific foetal mouse neural stem cells. Knock-out of the main oxygen sensing molecule, hypoxia-inducible factor-1α (Hif-1α), had no impact on Wnt activation assuming that physioxia induces the Wnt pathway independently of Hif-1α. To determine the physiological relevance of physioxia-induced Wnt/β-catenin signalling, we examined proliferation, cell cycle kinetics, survival and stem cell maintenance upon Wnt activation and inhibition. Whereas survival and stem cell maintenance seem to be independent of the Wnt pathway, our studies provide first evidence that Wnt/β-catenin signalling positively stimulates proliferation of physioxic cells by affecting cell cycle regulation. Together, our results provide mechanistic insight into oxygen-mediated regulation of the self-renewal activity of neural stem cells.

Stem cells have one major advantage over primary cells for regenerative therapies in neurodegenerative diseases. They are able to self-renew making sufficient quantities of cells available for transplantation. Embryonic stem cells and fetal neural progenitor cells (NPCs) have been transplanted into models for PD with functional recovery of motor deficits. However, their precise characteristics are still unknown and ideal conditions for their long-term expansion and differentiation into dopamine neurons remain to be explored. Mouse fetal NPCs are commonly grown as characteristic neurospheres, but they also proliferate under monolayer culture conditions. We investigated the proliferative behavior and dopaminergic differentiation capacity of fetal mouse midbrain NPCs derived from E10 to E14 embryos expanded either as neurosphere or monolayer culture. We found similar proliferation capacities in NPCs of all embryonic stages. Neuronal differentiation capacity is higher in neurosphere cultures compared to monolayer NPCs and persists in long-term cultures. We did not find dopaminergic differentiation in long-term expanded mouse NPC types, which is in contrast to rat and human fetal midbrain NPCs. Mouse NPCs generate dopaminergic neurons until up to three weeks in vitro but they do not incorporate BrdU. Quantitative analysis showed that they were not just primary neurons from the isolation process but formed to a great extent in vitro during differentiation suggesting that they are formed by promotion of post-mitotic neuroblasts. A detailed transcription profile reveals de-specification processes during in vitro cultivation, which matches their NPC behavior. We provide the constitutive work for studies using fetal midbrain NPCs of mouse including transplantation studies and transgenic models.

The “low-energy checkpoint” SNF1-related protein kinases, which are conserved in all eukaryotes, play an important role in cellular metabolic adaptation to differences in energy and oxygen availability. Although the signaling pathways involved in such metabolic adaptations are well understood in yeast and mammals, they have been poorly understood in plants. A recent study revealed that calcineurin B–like interacting protein kinase 15 (CIPK15) acted as a global regulator of such adaptations, linking the response to O 2 deficiency with the response to carbohydrate starvation in rice ( Oryza sativa ). Knockout mutants of Nipponbare rice CIPK15 failed to initiate transcription of the glycolytic enzymes α-amylase 3 and alcohol dehydrogenase 2, which mediate fermentative metabolism for adenosine triphosphate generation under anaerobic conditions. Targeted manipulation of OsCIPK15 might facilitate rice cultivation and ensure agricultural productivity in regions subject to flooding. Here, we highlight the importance of the energy- and oxygen-sensing pathway indicated by its conservation among different eukaryotic kingdoms.

Interleukin-1 (IL-1) plays a pivotal role in terminal dopaminergic differentiation of midbrain-derived neural precursor cells already committed to the mesencephalic dopaminergic phenotype (named mdNPCs for mesencephalic dopaminergic neural precursor cells). Here we characterized the molecular events in long-term expanded rat nuclear receptor related-1− (Nurr1−) mdNPCs in response to IL-1β during their terminal dopaminergic specification. We showed that IL-1β induced a rapid induction of mRNA of dopaminergic key fate-determining transcription factors, such as Nurr1 and Pitx3, and a subsequent increase of tyrosine hydroxylase protein as an early marker for dopaminergic neurons in vitro. These effects of IL-1β were specific for mdNPCs and were not observed in striatal neural precursor cells (NPCs). Surprisingly, IL-1β did not activate the NF-κB pathway or the transcription factor activating protein 1 (AP-1), but inhibition of nuclear translocation of NF-κB by SN50 facilitated IL-1β-induced Nurr1 expression and dopaminergic differentiation of mdNPCs. Incubation of mdNPCs with IL-1β led to a rapid phosphorylation of ERK1/2 and p38 mitogen-activated protein (MAP) kinases within 1 to 3 hours, whereas Jun kinase was not phosphorylated in response to IL-1β. Consistently, inhibition of the ERK1/2 pathway or p38 MAP kinase blocked Nurr1 upregulation and further dopaminergic specification of mdNPCs, but not differentiation into MAP2ab+ neurons. IL-1 receptor antagonist did not block early dopaminergic differentiation events, suggesting that the effects of IL-1β are not mediated through activation of IL-1 receptor type I. Our results indicate that induction of terminal dopaminergic specification of Nurr1− mdNPCs by IL-1β depends on activation of the ERK1/2 and p38 MAP kinase pathway. Disclosure of potential conflicts of interest is found at the end of this article.

Despite a comprehensive mapping of the Parkinson's disease (PD)-related mRNA and protein leucine-rich repeat kinase 2 (LRRK2) in the mammalian brain, its physiological function in healthy individuals remains enigmatic. Based on its structural features and kinase properties, LRRK2 may interact with other proteins involved in signalling pathways. Here, we show a widespread LRRK2 mRNA and/or protein expression in expanded or differentiated human mesencephalic neural progenitor cells (hmNPCs) and in post-mortem substantia nigra PD patients. Using small interfering RNA duplexes targeting LRRK2 in hmNPCs following their differentiation into glia and neurons, we observed a reduced number of dopaminergic neurons due to apoptosis in LRRK2 knockdown samples. LRRK2-deficient hmNPCs exhibited elevated cell cycle- and cell death-related markers. In conclusion, a reduction of LRRK2 expression in hmNPCs severely impaired dopaminergic differentiation and/or survival of dopaminergic neurons most likely via preserving or reactivating the cell cycle.