Transcription factor combinations that define human astrocyte identity encode significant variation of maturity and function.

Increasing evidence indicates that cellular identity can be reduced to the distinct gene regulatory networks controlled by transcription factors (TFs). However, redundancy exists in these states as different combinations of TFs can induce broadly similar cell types. We previously demonstrated that by overcoming gene silencing, it is possible to deterministically reprogram human pluripotent stem cells directly into cell types of various lineages. In the present study we leverage the consistency and precision of our approach to explore four different TF combinations encoding astrocyte identity, based on previously published reports. Analysis of the resulting induced astrocytes (iAs) demonstrated that all four cassettes generate cells with the typical morphology of in vitro astrocytes, which expressed astrocyte-specific markers. The transcriptional profiles of all four iAs clustered tightly together and displayed similarities with mature human astrocytes, although maturity levels differed between cells. Importantly, we found that the TF cassettes induced iAs with distinct differences with regards to their cytokine response and calcium signaling. In vivo transplantation of selected iAs into immunocompromised rat brains demonstrated long term stability and integration. In conclusion, all four TF combinations were able to induce stable astrocyte-like cells that were morphologically similar but showed subtle differences with respect to their transcriptome. These subtle differences translated into distinct differences with regards to cell function, that could be related to maturation state and/or regional identity of the resulting cells. This insight opens an opportunity to precision-engineer cells to meet functional requirements, for example, in the context of therapeutic cell transplantation.

Comparing Transcriptome Profiles of Neurons Interfacing Adjacent Cells and Nanopatterned Substrates Reveals Fundamental Neuronal Interactions.

Developing neuronal axons are directed by chemical and physical signals toward a myriad of target cells. According to current dogma, the resulting network architecture is critically shaped by electrical interconnections, the synapses; however, key mechanisms translating neuronal interactions into neuronal growth behavior during network formation are still unresolved. To elucidate these mechanisms, we examined neurons interfacing nanopatterned substrates and compared them to natural interneuron interactions. We grew similar neuronal populations under three connectivity conditions, (1) the neurons are isolated, (2) the neurons are interconnected, and (3) the neurons are connected only to artificial substrates, then quantitatively compared both the cell morphologies and the transcriptome-expression profiles. Our analysis shows that whereas axon-guidance signaling pathways in isolated neurons are predominant, in isolated neurons interfacing nanotopography, these pathways are downregulated, similar to the interconnected neurons. Moreover, in nanotopography, interfacing neuron genes related to synaptogenesis and synaptic regulation are highly expressed, that is, again resembling the behavior of interconnected neurons. These molecular findings demonstrate that interactions with nanotopographies, although not leading to electrical coupling, play a comparable functional role in two major routes, neuronal guidance and network formation, with high relevance to the design of regenerative interfaces.

Interactions of Neurons with Physical Environments.

Nerve growth strongly relies on multiple chemical and physical signals throughout development and regeneration. Currently, a cure for injured neuronal tissue is an unmet need. Recent advances in fabrication technologies and materials led to the development of synthetic interfaces for neurons. Such engineered platforms that come in 2D and 3D forms can mimic the native extracellular environment and create a deeper understanding of neuronal growth mechanisms, and ultimately advance the development of potential therapies for neuronal regeneration. This progress report aims to present a comprehensive discussion of this field, focusing on physical feature design and fabrication with additional information about considerations of chemical modifications. We review studies of platforms generated with a range of topographies, from micro-scale features down to topographical elements at the nanoscale that demonstrate effective interactions with neuronal cells. Fabrication methods are discussed as well as their biological outcomes. This report highlights the interplay between neuronal systems and the important roles played by topography on neuronal differentiation, outgrowth, and development. The influence of substrate structures on different neuronal cells and parameters including cell fate, outgrowth, intracellular remodeling, gene expression and activity is discussed. Matching these effects to specific needs may lead to the emergence of clinical solutions for patients suffering from neuronal injuries or brain-machine interface (BMI) applications.

Iron oxide nanoparticles for neuronal cell applications: uptake study and magnetic manipulations.

BACKGROUND: The ability to direct and manipulate neuronal cells has important potential in therapeutics and neural network studies. An emerging approach for remotely guiding cells is by incorporating magnetic nanoparticles (MNPs) into cells and transferring the cells into magnetic sensitive units. Recent developments offer exciting possibilities of magnetic manipulations of MNPs-loaded cells by external magnetic fields. In the present study, we evaluated and characterized uptake properties for optimal loading of cells by MNPs. We examined the interactions between MNPs of different cores and coatings, with primary neurons and neuron-like cells. RESULTS: We found that uncoated-maghemite iron oxide nanoparticles maximally interact and penetrate into cells with no cytotoxic effect. We observed that the cellular uptake of the MNPs depends on the time of incubation and the concentration of nanoparticles in the medium. The morphology patterns of the neuronal cells were not affected by MNPs uptake and neurons remained electrically active. We theoretically modeled magnetic fluxes and demonstrated experimentally the response of MNP-loaded cells to the magnetic fields affecting cell motility. Furthermore, we successfully directed neurite growth orientation along regeneration. CONCLUSIONS: Applying mechanical forces via magnetic mediators is a useful approach for biomedical applications. We have examined several types of MNPs and studied the uptake behavior optimized for magnetic neuronal manipulations.

Gold Nanoparticle-Decorated Scaffolds Promote Neuronal Differentiation and Maturation.

Engineered 3D neuronal networks are considered a promising approach for repairing the damaged spinal cord. However, the lack of a technological platform encouraging axonal elongation over branching may jeopardize the success of such treatment. To address this issue we have decorated gold nanoparticles on the surface of electrospun nanofiber scaffolds, characterized the composite material, and investigated their effect on the differentiation, maturation, and morphogenesis of primary neurons and on an immature neuronal cell line. We have shown that the nanocomposite scaffolds have encouraged a longer outgrowth of the neurites, as judged by the total length of the branching trees and the length and total distance of neurites. Moreover, neurons grown on the nanocomposite scaffolds had less neurites originating out of the soma and lower number of branches. Taken together, these results indicate that neurons cultivated on the gold nanoparticle scaffolds prefer axonal elongation over forming complex branching trees. We envision that such cellular constructs may be useful in the future as implantable cellular devices for repairing damaged neuronal tissues, such as the spinal cord.

In-vitro Optimization of Nanoparticle-Cell Labeling Protocols for In-vivo Cell Tracking Applications.

Recent advances in theranostic nanomedicine can promote stem cell and immune cell-based therapy. Gold nanoparticles (GNPs) have been shown to be promising agents for in-vivo cell-tracking in cell-based therapy applications. Yet a crucial challenge is to develop a reliable protocol for cell upload with, on the one hand, sufficient nanoparticles to achieve maximum visibility of cells, while on the other hand, assuring minimal effect of particles on cell function and viability. Previous studies have demonstrated that the physicochemical parameters of GNPs have a critical impact on their efficient uptake by cells. In the current study we have examined possible variations in GNP uptake, resulting from different incubation period and concentrations in different cell-lines. We have found that GNPs effectively labeled three different cell-lines - stem, immune and cancer cells, with minimal impairment to cell viability and functionality. We further found that uptake efficiency of GNPs into cells stabilized after a short period of time, while GNP concentration had a significant impact on cellular uptake, revealing cell-dependent differences. Our results suggest that while heeding the slight variations within cell lines, modifying the loading time and concentration of GNPs, can promote cell visibility in various nanoparticle-dependent in-vivo cell tracking and imaging applications.

The effect of nanoparticle size on the probability to cross the blood-brain barrier: an in-vitro endothelial cell model.

BACKGROUND: During the last decade nanoparticles have gained attention as promising drug delivery agents that can transport through the blood brain barrier. Recently, several studies have demonstrated that specifically targeted nanoparticles which carry a large payload of therapeutic agents can effectively enhance therapeutic agent delivery to the brain. However, it is difficult to draw definite design principles across these studies, owing to the differences in material, size, shape and targeting agents of the nanoparticles. Therefore, the main objective of this study is to develop general design principles that link the size of the nanoparticle with the probability to cross the blood brain barrier. Specifically, we investigate the effect of the nanoparticle size on the probability of barbiturate coated GNPs to cross the blood brain barrier by using bEnd.3 brain endothelial cells as an in vitro blood brain barrier model. RESULTS: The results show that GNPs of size 70 nm are optimal for the maximum amount of gold within the brain cells, and that 20 nm GNPs are the optimal size for maximum free surface area. CONCLUSIONS: These findings can help understand the effect of particle size on the ability to cross the blood brain barrier through the endothelial cell model, and design nanoparticles for brain imaging/therapy contrast agents.

Neuronal growth on L- and D-cysteine self-assembled monolayers reveals neuronal chiral sensitivity.

Studying the interaction between neuronal cells and chiral molecules is fundamental for the design of novel biomaterials and drugs. Chirality influences all biological processes that involve intermolecular interaction. One common method used to study cellular interactions with different enantiomeric targets is the use of chiral surfaces. Based on previous studies that demonstrated the importance of cysteine in the nervous system, we studied the effect of L- and D-cysteine on single neuronal growth. L-Cysteine, which normally functions as a neuromodulator or a neuroprotective antioxidant, causes damage at elevated levels, which may occur post trauma. In this study, we grew adult neurons in culture enriched with L- and D-cysteine as free compounds or as self-assembled monolayers of chiral surfaces and examined the effect on the neuronal morphology and adhesion. Notably, we have found that exposure to the L-cysteine enantiomer inhibited, and even prevented, neuronal attachment more severely than exposure to the D-cysteine enantiomer. Atop the L-cysteine surfaces, neuronal growth was reduced and degenerated. Since the cysteine molecules were attached to the surface via the thiol groups, the neuronal membrane was exposed to the molecular chiral site. Thus, our results have demonstrated high neuronal chiral sensitivity, revealing chiral surfaces as indirect regulators of neuronal cells and providing a reference for studying chiral drugs.

Interactions of neurons with topographic nano cues affect branching morphology mimicking neuron-neuron interactions.

We study the effect of topographic nano-cues on neuronal growth-morphology using invertebrate neurons in culture. We use photolithography to fabricate substrates with repeatable line-pattern ridges of nano-scale heights of 10-150 nm. We plate leech neurons atop the patterned-substrates and compare their growth pattern to neurons plated atop non-patterned substrates. The model system allows us the analysis of single neurite-single ridge interactions. The use of high resolution electron microscopy reveals small filopodia processes that attach to the line-pattern ridges. These fine processes, that cannot be detected in light microscopy, add anchoring sites onto the side of the ridges, thus additional physical support. These interactions of the neuronal process dominantly affect the neuronal growth direction. We analyze the response of the entire neuronal branching tree to the patterned substrates and find significant effect on the growth patterns compared to non-patterned substrates. Moreover, interactions with the nano-cues trigger a growth strategy similarly to interactions with other neuronal cells, as reflected in their morphometric parameters. The number of branches and the number of neurites originating from the soma decrease following the interaction demonstrating a tendency to a more simplified neuronal branching tree. The effect of the nano-cues on the neuronal function deserves further investigation and will strengthen our understanding of the interplay between function and form.

Topographic cues of nano-scale height direct neuronal growth pattern.

We study the role of nano-scale cues in controlling neuronal growth. We use photolithography to fabricate substrates with repeatable line-pattern ridges of nano-scale heights. We find that neuronal processes, which are of micron size, have strong interactions with ridges even as low as 10 nm. The interaction between the neuronal process and the ridge leads to a deflection of growth direction and a preferred alignment with the ridges. The interaction strength clearly depends on the ridges' height. For 25 nm ridges approximately half of the neuronal processes are modified, while at 100 nm the majority of neurites change their original growth direction post interaction. In addition, the effect on growth correlates with the incoming angle between the neuronal process and the ridge. We underline the adhesion as a key mechanism in directing neuronal growth. Our study highlights the sensitivity of growing neurites to nano-scale cues thus opens a new avenue of research for pre-designed neuronal growth and circuitry.

Conditional inactivation of the NBS1 gene in the mouse central nervous system leads to neurodegeneration and disorganization of the visual system.

Nijmegen breakage syndrome (NBS) is a genomic instability disease caused by hypomorphic mutations in the NBS1 gene encoding the Nbs1 (nibrin) protein. Nbs1 is a component of the Mre11/Rad50/Nbs1 (MRN) complex that acts as a sensor of double strand breaks (DSBs) in the DNA and is critical for proper activation of the broad cellular response to DSBs. Conditional disruption of the murine ortholog of the human NBS1, Nbs1, in the CNS of mice was previously reported to cause microcephaly, severe cerebellar atrophy and ataxia. Here we report that conditional targeted disruption of the murine NBS1 gene in the CNS results in mal-development, degeneration, disorganization and dysfunction of the murine visual system, especially in the optic nerve. Nbs1 deletion resulted in reduced diameters of Nbs1-CNS-Delta eye and optic nerve. MRI analysis revealed defective white matter development and organization. Nbs1 inactivation altered the morphology and organization of the glial cells. Interestingly, at the age of two-month-old the levels of the axonal guidance molecule semaphorin-3A and its receptor neuropilin-1 were up-regulated in the retina of the mutant mice, a typical injury response. Electroretinogram analysis revealed marked reduction in a- and b-waves, indicative of decreased retinal function. Our study points to a novel role for Nbs1 in the development, organization and function of the visual system.