Jelena Ban

Comparison of the Force Exerted by Hippocampal and DRG Growth Cones

Mechanical properties such as force generation are fundamental for neuronal motility, development and regeneration. We used optical tweezers to compare the force exerted by growth cones (GCs) of neurons from the Peripheral Nervous System (PNS), such as Dorsal Root Ganglia (DRG) neurons, and from the Central Nervous System (CNS) such as hippocampal neurons. Developing GCs from dissociated DRG and hippocampal neurons were obtained from P1-P2 and P10-P12 rats. Comparing their morphology, we observed that the area of GCs of hippocampal neurons was 8-10 µm2 and did not vary between P1-P2 and P10-P12 rats, but GCs of DRG neurons were larger and their area increased from P1-P2 to P10-P12 by 2-4 times. The force exerted by DRG filopodia was in the order of 1-2 pN and never exceeded 5 pN, while hippocampal filopodia exerted a larger force, often in the order of 5 pN. Hippocampal and DRG lamellipodia exerted lateral forces up to 20 pN, but lamellipodia of DRG neurons could exert a vertical force larger than that of hippocampal neurons. Force-velocity relationships (Fv) in both types of neurons had the same qualitative behaviour, consistent with a common autocatalytic model of force generation. These results indicate that molecular mechanisms of force generation of GC from CNS and PNS neurons are similar but the amplitude of generated force is influenced by their cytoskeletal properties.

Acceleration of neuronal precursors differentiation induced by substrate nanotopography

Embryonic stem (ES) cell differentiation in specific cell lineages is a major issue in cell biology particularly in regenerative medicine. Differentiation is usually achieved by using biochemical factors and it is not clear whether mechanical properties of the substrate over which cells are grown can affect proliferation and differentiation. Therefore, we produced patterns in polydimethylsiloxane (PDMS) consisting of groove and pillar arrays of sub‐micrometric lateral resolution as substrates for cell cultures. We analyzed the effect of different nanostructures on differentiation of ES‐derived neuronal precursors into neuronal lineage without adding biochemical factors. Neuronal precursors adhered on PDMS more effectively than on glass coverslips. We demonstrated that neuronal yield was enhanced by increasing pillars height from 35 to 400 nm. On higher pillar neuronal differentiation reaches ∼80% 96 h after plating and the largest differentiation enhancement of pillars over flat PDMS was observed during the first 6 h of culture. We conclude that PDMS nanopillars accelerate and increase neuronal differentiation. Biotechnol. Bioeng. 2011;108: 2736–2746.

Embryonic Stem Cell‐Derived Neurons Form Functional Networks In Vitro

Embryonic stem (ES) cells provide a flexible and unlimited source for a variety of neuronal types. Because mature neurons establish neuronal networks very easily, we tested whether ES‐derived neurons are capable of generating functional networks and whether these networks, generated in vitro, are capable of processing information. Single‐cell electrophysiology with pharmacological antagonists demonstrated the presence of both excitatory and inhibitory synaptic connections. Extracellular recording with planar multielectrode arrays showed that spontaneous bursts of electrical activity are present in ES‐derived networks with properties remarkably similar to those of hippocampal neurons. When stimulated with extracellular electrodes, ES‐derived neurons fired action potentials, and the evoked electrical activity spread throughout the culture. A statistical analysis indicated that ES‐derived networks discriminated between stimuli of different intensity at a single trial level, a key feature for an efficient information processing. Thus, ES‐derived neurons provide a novel in vitro strategy to create functional networks with defined computational properties.

Nanomechanics controls neuronal precursors adhesion and differentiation

The ability to control the differentiation of stem cells into specific neuronal types has a tremendous potential for the treatment of neurodegenerative diseases. In vitro neuronal differentiation can be guided by the interplay of biochemical and biophysical cues. Different strategies to increase the differentiation yield have been proposed, focusing everything on substrate topography, or, alternatively on substrate stiffness. Both strategies demonstrated an improvement of the cellular response. However it was often impossible to separate the topographical and the mechanical contributions. Here we investigate the role of the mechanical properties of nanostructured substrates, aiming at understanding the ultimate parameters which govern the stem cell differentiation. To this purpose a set of different substrates with controlled stiffness and with or without nanopatterning are used for stem cell differentiation. Our results show that the neuronal differentiation yield depends mainly on the substrate mechanical properties while the geometry plays a minor role. In particular nanostructured and flat polydimethylsiloxane (PDMS) substrates with comparable stiffness show the same neuronal yield. The improvement in the differentiation yield obtained through surface nanopatterning in the submicrometer scale could be explained as a consequence of a substrate softening effect. Finally we investigate by single cell force spectroscopy the neuronal precursor adhesion on the substrate immediately after seeding, as a possible critical step governing the neuronal differentiation efficiency. We observed that neuronal precursor adhesion depends on substrate stiffness but not on surface structure, and in particular it is higher on softer substrates. Our results suggest that cell–substrate adhesion forces and mechanical response are the key parameters to be considered for substrate design in neuronal regenerative medicine. Biotechnol. Bioeng. 2013; 110: 2301–2310.

Less than 5 Netrin-1 molecules initiate attraction but 200 Sema3A molecules are necessary for repulsion

Guidance molecules, such as Sema3A or Netrin-1, induce growth cone (GC) repulsion or attraction. In order to determine the speed of action and efficiency of these guidance cues we developed an experimental procedure to deliver controlled amounts of these molecules. Lipid vesicles encapsulating 10–104 molecules of Sema3A or Netrin-1 were manipulated with high spatial and temporal resolution by optical tweezers and their photolysis triggered by laser pulses. Guidance molecules released from the vesicles diffused and reached the GC membrane in a few seconds. Following their arrival, GCs retracted or grew in 20–120 s. By determining the number of guidance molecules trapped inside vesicles and estimating the fraction of guidance molecules reaching the GC, we show that the arrival of less than 5 Netrin-1 molecules on the GC membrane is sufficient to induce growth. In contrast, the arrival of about 200 Sema3A molecules is necessary to induce filopodia repulsion.

The role of dimensionality in neuronal network dynamics

Recent results from network theory show that complexity affects several dynamical properties of networks that favor synchronization. Here we show that synchronization in 2D and 3D neuronal networks is significantly different. Using dissociated hippocampal neurons we compared properties of cultures grown on a flat 2D substrates with those formed on 3D graphene foam scaffolds. Both 2D and 3D cultures had comparable glia to neuron ratio and the percentage of GABAergic inhibitory neurons. 3D cultures because of their dimension have many connections among distant neurons leading to small-world networks and their characteristic dynamics. After one week, calcium imaging revealed moderately synchronous activity in 2D networks, but the degree of synchrony of 3D networks was higher and had two regimes: a highly synchronized (HS) and a moderately synchronized (MS) regime. The HS regime was never observed in 2D networks. During the MS regime, neuronal assemblies in synchrony changed with time as observed in mammalian brains. After two weeks, the degree of synchrony in 3D networks decreased, as observed in vivo. These results show that dimensionality determines properties of neuronal networks and that several features of brain dynamics are a consequence of its 3D topology.

Fragmentation as a mechanism for growth cone pruning and degeneration.

During early development of the central nervous system, there is an excessive outgrowth of neuronal projections, which later need to be refined to achieve precise connectivity. Axon pruning and degeneration are strategies used to remove exuberant neurites and connections in the immature nervous system to ensure the proper formation of functional circuitry. To observe morphological changes and physical mechanisms underlying this process, early differentiating embryonic stem cell-derived neurons were used combining video imaging of live growth cones (GCs) with confocal laser scanning microscopy and atomic force microscopy, both on fixed and living neurons. Using this method, we could highlight the presence of submicrometric fragments in still and in some of the retracting GCs. The observed fragmentation is not an artifact of atomic force microscopy scanning or fixation, or the result of apoptosis. Therefore, the morphology of GCs depends on their overall motility, and fragmentation seems to be the fate of GCs that have not found a correct destination.

An improved method for growing neurons: Comparison with standard protocols

BACKGROUND Since different culturing parameters - such as media composition or cell density - lead to different experimental results, it is important to define the protocol used for neuronal cultures. The vital role of astrocytes in maintaining homeostasis of neurons - both in vivo and in vitro - is well established: the majority of improved culturing conditions for primary dissociated neuronal cultures rely on astrocytes. NEW METHOD Our culturing protocol is based on a novel serum-free preparation of astrocyte - conditioned medium (ACM). We compared the proposed ACM culturing method with other two commonly used methods Neurobasal/B27- and FBS- based media. We performed morphometric characterization by immunocytochemistry and functional analysis by calcium imaging for all three culture methods at 1, 7, 14 and 60days in vitro (DIV). RESULTS ACM-based cultures gave the best results for all tested criteria, i.e. growth cones size and shape, neuronal outgrowth and branching, network activity and synchronization, maturation and long-term survival. The differences were more pronounced when compared with FBS-based medium. Neurobasal/B27 cultures were comparable to ACM for young cultures (DIV1), but not for culturing times longer than DIV7. COMPARISON WITH EXISTING METHOD(S) ACM-based cultures showed more robust neuronal outgrowth at DIV1. At DIV7 and 60, the activity of neuronal network grown in ACM had a more vigorous spontaneous electrical activity and a higher degree of synchronization. CONCLUSIONS We propose our ACM-based culture protocol as an improved and more suitable method for both short- and long-term neuronal cultures.

Can hippocampal neurites and growth cones climb over obstacles

Guidance molecules, such as Sema3A or Netrin-1, can induce growth cone (GC) repulsion or attraction in the presence of a flat surface, but very little is known of the action of guidance molecules in the presence of obstacles. Therefore we combined chemical and mechanical cues by applying a steady Netrin-1 stream to the GCs of dissociated hippocampal neurons plated on polydimethylsiloxane (PDMS) surfaces patterned with lines 2 µm wide, with 4 µm period and with a height varying from 100 to 600 nm. GC turning experiments performed 24 hours after plating showed that filopodia crawl over these lines within minutes. These filopodia do not show staining for the adhesion marker Paxillin. GCs and neurites crawl over lines 100 nm high, but less frequently and on a longer time scale over lines higher than 300 nm; neurites never crawl over lines 600 nm high. When neurons are grown for 3 days over patterned surfaces, also neurites can cross lines 300 nm and 600 nm high, grow parallel to and on top of these lines and express Paxillin. Axons - selectively stained with SMI 312 – do not differ from dendrites in their ability to cross these lines. Our results show that highly motile structures such as filopodia climb over high obstacle in response to chemical cues, but larger neuronal structures are less prompt and require hours or days to climb similar obstacles.

Improved neuron culture using scaffolds made of three-dimensional PDMS micro-lattices

Tissue engineering strives to create functional components of organs with different cell types in vitro. One of the challenges is to fabricate scaffolds for three-dimensional (3D) cell culture under physiological conditions. Of particular interest is the investigation of the morphology and function of the central nervous system cultured using such scaffolds. Here, we used an elastomer-polydimethylsiloxane (PDMS)-to produce lattice-type scaffolds from a photolithography-defined template. The photomask with antidot arrays was spin-coated by a thick layer of resist, and was downward mounted on a rotating stage at an angle of 45°. After the exposure was repeated three or more times, maintaining the same exposure plan but rotated by the same angle, a photoresist was developed to produce a 3D porous template. Afterwards, a pre-polymer mixture of PDMS was poured in and cured, followed by a resist etch, resulting in lattice-type PDMS features. Before cell culture, the PDMS lattices were surface functionalized. A culture test was conducted using NIH-3T3 cells and primary hippocampal cells from rats, showing homogenous cell infiltration and 3D attachment. As expected, a much higher cell number was found in the 3D PDMS lattices compared to the 2D culture. We also found a higher neuron-to-astrocyte ratio and a higher degree of cell ramification in the 3D culture compared to the 2D culture due to the change of scaffold topography and the elastic properties of the PDMS micro-lattices. Our results demonstrate that the 3D PDMS micro-lattices improve the survival and growth of cells, as well as the network formation of neurons. We believe that such an enabling technology is useful for research and clinical applications, including disease modeling, regenerative medicine, and drug discovery/drug cytotoxicity studies.