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1.
PLoS One ; 12(7): e0180427, 2017.
Article in English | MEDLINE | ID: mdl-28672008

ABSTRACT

Impairment of spiral ganglion neurons (SGNs) of the auditory nerve is a major cause for hearing loss occurring independently or in addition to sensory hair cell damage. Unfortunately, mammalian SGNs lack the potential for autonomous regeneration. Stem cell based therapy is a promising approach for auditory nerve regeneration, but proper integration of exogenous cells into the auditory circuit remains a fundamental challenge. Here, we present novel nanofibrous scaffolds designed to guide the integration of human stem cell-derived neurons in the internal auditory meatus (IAM), the foramen allowing passage of the spiral ganglion to the auditory brainstem. Human embryonic stem cells (hESC) were differentiated into neural precursor cells (NPCs) and seeded onto aligned nanofiber mats. The NPCs terminally differentiated into glutamatergic neurons with high efficiency, and neurite projections aligned with nanofibers in vitro. Scaffolds were assembled by seeding GFP-labeled NPCs on nanofibers integrated in a polymer sheath. Biocompatibility and functionality of the NPC-seeded scaffolds were evaluated in vivo in deafened guinea pigs (Cavia porcellus). To this end, we established an ouabain-based deafening procedure that depleted an average 72% of SGNs from apex to base of the cochleae and caused profound hearing loss. Further, we developed a surgical procedure to implant seeded scaffolds directly into the guinea pig IAM. No evidence of an inflammatory response was observed, but post-surgery tissue repair appeared to be facilitated by infiltrating Schwann cells. While NPC survival was found to be poor, both subjects implanted with NPC-seeded and cell-free control scaffolds showed partial recovery of electrically-evoked auditory brainstem thresholds. Thus, while future studies must address cell survival, nanofibrous scaffolds pose a promising strategy for auditory nerve regeneration.


Subject(s)
Cochlear Nerve/physiology , Embryonic Stem Cells/cytology , Nanofibers , Nerve Regeneration/physiology , Neurons/cytology , Tissue Engineering , Animals , Biocompatible Materials , Brain Stem/physiology , Cell Differentiation , Cell Transplantation , Deafness/therapy , Female , Green Fluorescent Proteins/genetics , Guinea Pigs , Humans , Male
2.
Int J Mol Sci ; 16(6): 13885-907, 2015 Jun 17.
Article in English | MEDLINE | ID: mdl-26090715

ABSTRACT

Multiple sclerosis (MS) is the most common multifocal inflammatory demyelinating disease of the central nervous system (CNS). Due to the progressive neurodegenerative nature of MS, developing treatments that exhibit direct neuroprotective effects are needed. Tecfidera™ (BG-12) is an oral formulation of the fumaric acid esters (FAE), containing the active metabolite dimethyl fumarate (DMF). Although BG-12 showed remarkable efficacy in lowering relapse rates in clinical trials, its mechanism of action in MS is not yet well understood. In this study, we reported the potential neuroprotective effects of dimethyl fumarate (DMF) on mouse and rat neural stem/progenitor cells (NPCs) and neurons. We found that DMF increased the frequency of the multipotent neurospheres and the survival of NPCs following oxidative stress with hydrogen peroxide (H2O2) treatment. In addition, utilizing the reactive oxygen species (ROS) assay, we showed that DMF reduced ROS production induced by H2O2. DMF also decreased oxidative stress-induced apoptosis. Using motor neuron survival assay, DMF significantly promoted survival of motor neurons under oxidative stress. We further analyzed the expression of oxidative stress-induced genes in the NPC cultures and showed that DMF increased the expression of transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) at both levels of RNA and protein. Furthermore, we demonstrated the involvement of Nrf2-ERK1/2 MAPK pathway in DMF-mediated neuroprotection. Finally, we utilized SuperArray gene screen technology to identify additional anti-oxidative stress genes (Gstp1, Sod2, Nqo1, Srxn1, Fth1). Our data suggests that analysis of anti-oxidative stress mechanisms may yield further insights into new targets for treatment of multiple sclerosis (MS).


Subject(s)
Dimethyl Fumarate/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , NF-E2-Related Factor 2/metabolism , Neural Stem Cells/drug effects , Neurons/drug effects , Neuroprotective Agents/pharmacology , Oxidative Stress/drug effects , Animals , Apoptosis/drug effects , Blotting, Western , Cells, Cultured , Chick Embryo , Female , Hydrogen Peroxide/pharmacology , Immunosuppressive Agents/pharmacology , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , NF-E2-Related Factor 2/genetics , Neural Stem Cells/metabolism , Neural Stem Cells/pathology , Neurons/metabolism , Neurons/pathology , Oxidants/pharmacology , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain Reaction
3.
Nat Protoc ; 8(4): 771-82, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23589937

ABSTRACT

Current methods for studying oligodendrocyte myelination using primary neurons are limited by the time, cost and reproducibility of myelination in vitro. Nanofibers with diameters of >0.4 µm fabricated from electrospinning of liquid polystyrene are suitable scaffolds for concentric membrane wrapping by oligodendrocytes. With the advent of aligned electrospinning technology, nanofibers can be rapidly fabricated, standardized, and configured into various densities and patterns as desired. Notably, the minimally permissive culture environment of fibers provides investigators with an opportunity to explore the autonomous oligodendrocyte cellular processes underlying differentiation and myelination. The simplicity of the system is conducive to monitoring oligodendrocyte proliferation, migration, differentiation and membrane wrapping in the absence of neuronal signals. Here we describe protocols for the fabrication and preparation of nanofibers aligned on glass coverslips for the study of membrane wrapping by rodent oligodendrocytes. The entire protocol can be completed within 2 weeks.


Subject(s)
Cell Culture Techniques , Myelin Sheath/metabolism , Nanofibers/chemistry , Animals , Cell Differentiation , Cell Movement , Cell Proliferation , Cells, Cultured , Mice , Models, Biological , Myelin Sheath/physiology , Nanofibers/ultrastructure , Oligodendroglia/cytology , Oligodendroglia/ultrastructure , Rats , Reproducibility of Results , Surface Properties , Tissue Scaffolds
4.
Nat Methods ; 9(9): 917-22, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22796663

ABSTRACT

Current methods for studying central nervous system myelination necessitate permissive axonal substrates conducive to myelin wrapping by oligodendrocytes. We have developed a neuron-free culture system in which electron-spun nanofibers of varying sizes substitute for axons as a substrate for oligodendrocyte myelination, thereby allowing manipulation of the biophysical elements of axonal-oligodendroglial interactions. To investigate axonal regulation of myelination, this system effectively uncouples the role of molecular (inductive) cues from that of biophysical properties of the axon. We use this method to uncover the causation and sufficiency of fiber diameter in the initiation of concentric wrapping by rat oligodendrocytes. We also show that oligodendrocyte precursor cells display sensitivity to the biophysical properties of fiber diameter and initiate membrane ensheathment before differentiation. The use of nanofiber scaffolds will enable screening for potential therapeutic agents that promote oligodendrocyte differentiation and myelination and will also provide valuable insight into the processes involved in remyelination.


Subject(s)
Cell Culture Techniques/methods , Myelin Sheath/physiology , Nanofibers/chemistry , Nanotechnology/methods , Oligodendroglia/cytology , Animals , Cell Proliferation , Female , Male , Microscopy, Electron, Scanning , Polylysine/chemistry , Rats , Rats, Sprague-Dawley
5.
Mater Sci Eng C Mater Biol Appl ; 32(7): 1779-1784, 2012 Oct 01.
Article in English | MEDLINE | ID: mdl-34062655

ABSTRACT

Electrospun polymer nanofibers show promise as components of scaffolds for tissue engineering because of their ability to orient regenerating cells. Our research focuses on aligned electrospun fiber scaffolds for nerve regeneration. Critical to this are highly aligned fibers, which are frequently difficult to manufacture reproducibly. Here we show that three variables: the distance between the spinneret tip and collector, the addition of DMF to the solvent, and placement of an aluminum sheet on the spinneret together greatly improve the alignment of electrospun poly-L-lactide (PLLA) nanofibers. We identified the most important variable as tip-to-collector distance. Nanofiber alignment was maximal at 30cm compared to shorter distances. DMF:chloroform (1:9) improved nanofiber uniformity and was integral to maintaining a uniform stream over the 30cm tip-to-collector distance. Other ratios caused splattering of the solution or flattening or beading of the fibers and non-uniform fiber diameter. The aluminum sheet helped to stabilize the electric field and improve fiber alignment provided that it was placed at 1cm behind the tip, while other distances destabilized the stream and worsened alignment. This study demonstrates that control of these variables produces dramatic improvement in reproducibly obtaining high alignment and uniform morphology of electrospun PLLA nanofibers.

6.
J Vis Exp ; (48)2011 Feb 15.
Article in English | MEDLINE | ID: mdl-21372783

ABSTRACT

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.


Subject(s)
Cytological Techniques/methods , Motor Neurons/cytology , Nanofibers/chemistry , Nanotechnology/methods , Polyesters/chemistry , Sensory Receptor Cells/cytology , Animals , Cells, Cultured , Culture Media , Embryo, Mammalian/cytology , Female , Pregnancy , Rats
7.
J Vis Exp ; (47)2011 Jan 21.
Article in English | MEDLINE | ID: mdl-21304466

ABSTRACT

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.


Subject(s)
Electrochemical Techniques/methods , Nanofibers/chemistry , Polymers/chemistry , Electrochemical Techniques/instrumentation , Nanotechnology/instrumentation , Nanotechnology/methods , Solutions/chemistry
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