Chemical surface modification of poly-ε-caprolactone improves Schwann cell proliferation for peripheral nerve repair.

Poly-ε-caprolactone (PCL) is a biodegradable and biocompatible polymer used in tissue engineering for various clinical applications. Schwann cells (SCs) play an important role in nerve regeneration and repair. SCs attach and proliferate on PCL films but cellular responses are weak due to the hydrophobicity and neutrality of PCL. In this study, PCL films were hydrolysed and aminolysed to modify the surface with different functional groups and improve hydrophilicity. Hydrolysed films showed a significant increase in hydrophilicity while maintaining surface topography. A significant decrease in mechanical properties was also observed in the case of aminolysis. In vitro tests with Schwann cells (SCs) were performed to assess film biocompatibility. A short-time experiment showed improved cell attachment on modified films, in particular when amino groups were present on the material surface. Cell proliferation significantly increased when both treatments were performed, indicating that surface treatments are necessary for SC response. It was also demonstrated that cell morphology was influenced by physico-chemical surface properties. PCL can be used to make artificial conduits and chemical modification of the inner lumen improves biocompatibility.

Generation of Schwann cell-derived multipotent neurospheres isolated from intact sciatic nerve

Schwann cells (SCs) are the supporting cells of the peripheral nervous system and originate from the neural crest. They play a unique role in the regeneration of injured peripheral nerves and have themselves a highly unstable phenotype as demonstrated by their unexpectedly broad differentiation potential. Thus, SCs can be considered as dormant, multipotent neural crest-derived progenitors or stem cells. Upon injury they de-differentiate via cellular reprogramming, re-enter the cell cycle and participate in the regeneration of the nerve. Here we describe a protocol for efficient generation of neurospheres from intact adult rat and murine sciatic nerve without the need of experimental in vivo pre-degeneration of the nerve prior to Schwann cell isolation. After isolation and removal of the connective tissue, the nerves are initially plated on poly-D-lysine coated cell culture plates followed by migration of the cells up to 80% confluence and a subsequent switch to serum-free medium leading to formation of multipotent neurospheres. In this context, migration of SCs from the isolated nerve, followed by serum-free cultivation of isolated SCs as neurospheres mimics the injury and reprograms fully differentiated SCs into a multipotent, neural crest-derived stem cell phenotype. This protocol allows reproducible generation of multipotent Schwann cell-derived neurospheres from sciatic nerve through cellular reprogramming by culture, potentially marking a starting point for future detailed investigations of the de-differentiation process.

Mesenchymal stem cells engrafted in a fibrin scaffold stimulate Schwann cell reactivity and axonal regeneration following sciatic nerve tubulization

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A mouse model of Schwartz-Jampel syndrome reveals myelinating Schwann cell dysfunction with persistent axonal depolarization in vitro and distal peripheral nerve hyperexcitability when perlecan is lacking

Congenital peripheral nerve hyperexcitability (PNH) is usually associated with impaired function of voltage-gated K(+) channels (VGKCs) in neuromyotonia and demyelination in peripheral neuropathies. Schwartz-Jampel syndrome (SJS) is a form of PNH that is due to hypomorphic mutations of perlecan, the major proteoglycan of basement membranes. Schwann cell basement membrane and its cell receptors are critical for the myelination and organization of the nodes of Ranvier. We therefore studied a mouse model of SJS to determine whether a role for perlecan in these functions could account for PNH when perlecan is lacking. We revealed a role for perlecan in the longitudinal elongation and organization of myelinating Schwann cells because perlecan-deficient mice had shorter internodes, more numerous Schmidt-Lanterman incisures, and increased amounts of internodal fast VGKCs. Perlecan-deficient mice did not display demyelination events along the nerve trunk but developed dysmyelination of the preterminal segment associated with denervation processes at the neuromuscular junction. Investigating the excitability properties of the peripheral nerve suggested a persistent axonal depolarization during nerve firing in vitro, most likely due to defective K(+) homeostasis, and excluded the nerve trunk as the original site for PNH. Altogether, our data shed light on perlecan function by revealing critical roles in Schwann cell physiology and suggest that PNH in SJS originates distally from synergistic actions of peripheral nerve and neuromuscular junction changes.

Cell death in the Schwann cell lineage and its regulation by neuregulin.

The development of Schwann cells, the myelin-forming glial cells of the vertebrate peripheral nervous system, involves a neonatal phase of proliferation in which cells migrate along and segregate newly formed axons. Withdrawal from the cell cycle, around postnatal days 2-4 in rodents, initiates terminal differentiation to the myelinating state. During this time, Schwann cell number is subject to stringent regulation such that within the first postnatal week, axons and myelinating Schwann cells attain the one-to-one relationship characteristic of the mature nerve. The mechanisms that underly this developmental control remain largely undefined. In this report, we examine the role of apoptosis in the determination of postnatal Schwann cell number. We find that Schwann cells isolated from postnatal day 3 rat sciatic nerve undergo apoptosis in vitro upon serum withdrawal and that Schwann cell death can be prevented by beta forms of neuregulin (NRG-beta) but not by fibroblast growth factor 2 or platelet-derived growth factors AA and BB. This NRG-beta-mediated Schwann cell survival is apparently transduced through an ErbB2/ErbB3 receptor heterodimer. We also provide evidence that postnatal Schwann cells undergo developmentally regulated apoptosis in vivo. Together with other recent findings, these results suggest that Schwann cell apoptosis may play an important role in peripheral nerve development and that Schwann cell survival may be regulated by access to axonally derived NRG.

Intracellular reactive oxidative stress, cell proliferation and apoptosis of Schwann cells on carbon nanofibrous substrates

Despite considerable research to develop carbon based materials for biomedical applications, the toxicity of carbon remains a major concern. In order to address this issue as well as to investigate the cell fate processes of neural cells from the perspective of neural tissue engineering applications, the in vitro cytocompatibility of polyacrylonitrile (PAN) derived continuous carbon nanofibers and PAN derived carbon thin films were investigated both quantitatively and qualitatively using in vitro biochemical assays followed by extensive flow cytometry analysis. The experimental results of Schwann cell fate, i.e. cell proliferation, cell metabolic activity and cell apoptosis on amorphous carbon substrates are discussed in reference to the time dependent evolution of intracellular oxidative stress. Apart from providing evidence that an electrospun carbon nanofibrous substrate can physically guide the cultured Schwann cells, this study suggested that continuous carbon nanofibers and amorphous carbon films are not cytotoxic in vitro and do not significantly induce apoptosis of Schwann cells, but in fact even facilitate their proliferation and growth.

Immobilization of cell-binding peptides on poly-ε-caprolactone film surface to biomimic the peripheral nervous system.

Cell-material interactions are crucial for cell adhesion and proliferation on biomaterial surfaces. Immobilization of biomolecules leads to the formation of biomimetic substrates, improving cell response. We introduced RGD (Arg-Gly-Asp) sequences on poly-ε-caprolactone (PCL) film surfaces using thiol chemistry to enhance Schwann cell (SC) response. XPS elemental analysis indicated an estimate of 2-3% peptide functionalization on the PCL surface, comparable with carbodiimide chemistry. Contact angle was not remarkably reduced; hence, cell response was only affected by chemical cues on the film surface. Adhesion and proliferation of Schwann cells were enhanced after PCL modification. Particularly, RGD immobilization increased cell attachment up to 40% after 6 h of culture. It was demonstrated that SC morphology changed from round to very elongated shape when surface modification was carried out, with an increase in the length of cellular processes up to 50% after 5 days of culture. Finally RGD immobilization triggered the formation of focal adhesion related to higher cell spreading. In summary, this study provides a method for immobilization of biomolecules on PCL films to be used in peripheral nerve repair, as demonstrated by the enhanced response of Schwann cells.

Schwann cells can be reprogrammed to multipotency by culture

Adult neural crest related-stem cells persist in adulthood, making them an ideal and easily accessible source of multipotent cells for potential clinical use. Recently, we reported the presence of neural crest-related stem cells within adult palatal ridges, thus raising the question of their localization in their endogenous niche. Using immunocytochemistry, reverse transcription-polymerase chain reaction, and correlative fluorescence and transmission electron microscopy, we identified myelinating Schwann cells within palatal ridges as a putative neural crest stem cell source. Palatal Schwann cells expressed nestin, p75(NTR), and S100. Correlative fluorescence and transmission electron microscopy revealed the exclusive nestin expression within myelinating Schwann cells. Palatal neural crest stem cells and nestin-positive Schwann cells isolated from adult sciatic nerves were able to grow under serum-free conditions as neurospheres in presence of FGF-2 and EGF. Spheres of palatal and sciatic origin showed overlapping expression pattern of neural crest stem cell and Schwann cell markers. Expression of the pluripotency factors Sox2, Klf4, c-Myc, Oct4, the NF-κB subunits p65, p50, and the NF-κB-inhibitor IκB-β were up-regulated in conventionally cultivated sciatic nerve Schwann cells and in neurosphere cultures. Finally, neurospheres of palatal and sciatic origin were able to differentiate into ectodermal, mesodermal, and endodermal cell types emphasizing their multipotency. Taken together, we show that nestin-positive myelinating Schwann cells can be reprogrammed into multipotent adult neural crest stem cells under appropriate culture conditions.

Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues.

The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. β-Galactosidase (β-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5–15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.

Granular cell tumour (Abrikossoff's tumour): Case series

This case series describes three unreported cases of an uncommon benign neoplasm named Abrikossoff's tumour or granular cell tumour (GCT). This mesenchymal neoplasm apparently arises from neural or Schwann cell origin with benign and malignant forms. All cases presented here were unique nodules on oral mucosa, coming out from the connective tissue, and occurring in women with age ranging from 30 to 42 years. The histological aspects of the GCT showed large granular cells arranged in groups and nests of connective tissue separating pseudoepitheliomatous overlying surface. This feature can possibly be misunderstood with squamous cell carcinoma. The differentiation between malignant and benign GCT is evaluated by the presence of metastases which is considered the only reliable criterion for malignancy. © 2005 Elsevier Ltd. All rights reserved.

The Ets domain transcription factor Erm distinguishes rat satellite glia from Schwann cells and is regulated in satellite cells by neuregulin signaling

Distinct glial cell types of the vertebrate peripheral nervous system (PNS) are derived from the neural crest. Here we show that the expression of the Ets domain transcription factor Erm distinguishes satellite glia from Schwann cells beginning early in rat PNS development. In developing dorsal root ganglia (DRG), Erm is present both in presumptive satellite glia and in neurons. In contrast, Erm is not detectable at any developmental stage in Schwann cells in peripheral nerves. In addition, Erm is downregulated in DRG-derived glia adopting Schwann cell traits in culture. Thus, Erm is the first described transcription factor expressed in satellite glia but not in Schwann cells. In culture, the Neuregulin1 (NRG1) isoform GGF2 maintains Erm expression in presumptive satellite cells and reinduces Erm expression in DRG-derived glia but not in Schwann cells from sciatic nerve. These data demonstrate that there are intrinsic differences between these glial subtypes in their response to NRG1 signaling. In neural crest cultures, Erm-positive progenitor cells give rise to two distinct glial subtypes: Erm-positive, Oct-6-negative satellite glia in response to GGF2, and Erm-negative, Oct-6-positive Schwann cells in the presence of serum and the adenylate cyclase activator forskolin. Thus, Erm-positive neural crest-derived progenitor cells and presumptive satellite glia are able to acquire Schwann cell features. Given the in vivo expression of Erm in peripheral ganglia, we suggest that ganglionic Erm-positive cells may be precursors of Schwann cells.

Plasticité présynaptique et gliale à long-terme en réponse à un changement chronique de l’activité synaptique,à la jonction neuromusculaire d’amphibien

La plasticité synaptique est une importante propriété du système nerveux, impliquée dans l’intégration de l’information. Cette plasticité a généralement été décrite par des changements aux niveaux pré et postsynaptiques. Notamment, l’efficacité présynaptique, soit la probabilité de libération de neurotransmetteurs associée au contenu quantique d’une synapse, peut être augmentée ou diminuée selon l’activité antérieure de la synapse. Malgré cette caractérisation, les mécanismes à l’origine de la détermination de l’efficacité présynaptique demeurent obscurs. Également, la plasticité synaptique reste encore mal définie au niveau glial, limitant, de ce fait, notre compréhension de l’intégration de l’information. Pourtant, la dernière décennie a mené à une redéfinition du rôle des cellules gliales. Autrefois reléguées à un rôle de support passif aux neurones, elles sont désormais reconnues comme étant impliquées dans la régulation de la neurotransmission. Notamment, à la jonction neuromusculaire (JNM), les cellules de Schwann périsynaptiques (CSPs) sont reconnues pour moduler l’efficacité présynaptique et les phénomènes de plasticité. Un tel rôle actif dans la modulation de la neurotransmission implique cependant que les CSPs soient en mesure de s’adapter aux besoins changeants des JNMs auxquelles elles sont associées. La plasticité synaptique devrait donc sous-tendre une forme de plasticité gliale. Nous savons, en effet, que la JNM est capable de modifications tant morphologiques que physiologiques en réponse à des altérations de l'activité synaptique. Par exemple, la stimulation chronique des terminaisons nerveuses entraîne une diminution persistante de l’efficacité présynaptique et une augmentation de la résistance à la dépression. À l’opposé, le blocage chronique des récepteurs nicotiniques entraîne une augmentation prolongée de l’efficacité présynaptique. Aussi, compte tenu que les CSPs détectent et répondent à la neurotransmission et qu’elles réagissent à certains stimuli environnementaux par des changements morphologiques, physiologiques et d’expression génique, nous proposons que le changement d'efficacité présynaptique imposé à la synapse, soit par une stimulation nerveuse chronique ou par blocage chronique des récepteurs nicotiniques, résulte en une adaptation des propriétés des CSPs. Cette thèse propose donc d’étudier, en parallèle, la plasticité présynaptique et gliale à long-terme, en réponse à un changement chronique de l’activité synaptique, à la JNM d’amphibien. Nos résultats démontrent les adaptations présynaptiques de l’efficacité présynaptique, des phénomènes de plasticité à court-terme, du contenu mitochondrial et de la signalisation calcique. De même, ils révèlent différentes adaptations gliales, notamment au niveau de la sensibilité des CSPs aux neurotransmetteurs et des propriétés de leur réponse calcique. Les adaptations présynaptiques et gliales sont discutées, en parallèle, en termes de mécanismes et de fonctions possibles dans la régulation de la neurotransmission. Nos travaux confirment donc la coïncidence de la plasticité présynaptique et gliale et, en ce sens, soulèvent l’importance des adaptations gliales pour le maintien de la fonction synaptique.

Engineering a multimodal nerve conduit for repair of injured peripheral nerve

Injury to nerve tissue in the peripheral nervous system (PNS) results in long-term impairment of limb function, dysaesthesia and pain, often with associated psychological effects. Whilst minor injuries can be left to regenerate without intervention and short gaps up to 2 cm can be sutured, larger or more severe injuries commonly require autogenous nerve grafts harvested from elsewhere in the body (usually sensory nerves). Functional recovery is often suboptimal and associated with loss of sensation from the tissue innervated by the harvested nerve. The challenges that persist with nerve repair have resulted in development of nerve guides or conduits from non-neural biological tissues and various polymers to improve the prognosis for the repair of damaged nerves in the PNS. This study describes the design and fabrication of a multimodal controlled pore size nerve regeneration conduit using polylactic acid (PLA) and (PLA):poly(lactic-co-glycolic) acid (PLGA) fibers within a neurotrophin-enriched alginate hydrogel. The nerve repair conduit design consists of two types of PLGA fibers selected specifically for promotion of axonal outgrowth and Schwann cell growth (75:25 for axons; 85:15 for Schwann cells). These aligned fibers are contained within the lumen of a knitted PLA sheath coated with electrospun PLA nanofibers to control pore size. The PLGA guidance fibers within the nerve repair conduit lumen are supported within an alginate hydrogel impregnated with neurotrophic factors (NT-3 or BDNF with LIF, SMDF and MGF-1) to provide neuroprotection, stimulation of axonal growth and Schwann cell migration. The conduit was used to promote repair of transected sciatic nerve in rats over a period of 4 weeks. Over this period, it was observed that over-grooming and self-mutilation (autotomy) of the limb implanted with the conduit was significantly reduced in rats implanted with the full-configuration conduit compared to rats implanted with conduits containing only an alginate hydrogel. This indicates return of some feeling to the limb via the fully-configured conduit. Immunohistochemical analysis of the implanted conduits removed from the rats after the four-week implantation period confirmed the presence of myelinated axons within the conduit and distal to the site of implantation, further supporting that the conduit promoted nerve repair over this period of time. This study describes the design considerations and fabrication of a novel multicomponent, multimodal bio-engineered synthetic conduit for peripheral nerve repair.

Telomere dynamics and homeostasis in a transmissible cancer

Devil Facial Tumour Disease (DFTD) is a unique clonal cancer that threatens the world's largest carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii) with extinction. This transmissible cancer is passed between individual devils by cell implantation during social interactions. The tumour arose in a Schwann cell of a single devil over 15 years ago and since then has expanded clonally, without showing signs of replicative senescence; in stark contrast to a somatic cell that displays a finite capacity for replication, known as the “Hayflick limit”.