Elsevier

Neuroscience

Volume 277, 26 September 2014, Pages 690-699
Neuroscience

Development of a cell-based treatment for long-term neurotrophin expression and spiral ganglion neuron survival

https://doi.org/10.1016/j.neuroscience.2014.07.044Get rights and content

Highlights

  • Primary fibroblasts can be nucleofected to express neurotrophins.

  • Nucleofection was superior to lipofection in terms of longevity of expression.

  • Nucleofected fibroblasts expressed BDNF for at least six months.

  • Nucleofected BDNF-fibroblasts support spiral ganglion neuron survival in vitro.

Abstract

Spiral ganglion neurons (SGNs), the target cells of the cochlear implant, undergo gradual degeneration following loss of the sensory epithelium in deafness. The preservation of a viable population of SGNs in deafness can be achieved in animal models with exogenous application of neurotrophins such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3. For translation into clinical application, a suitable delivery strategy that provides ongoing neurotrophic support and promotes long-term SGN survival is required. Cell-based neurotrophin treatment has the potential to meet the specific requirements for clinical application, and we have previously reported that Schwann cells genetically modified to express BDNF can support SGN survival in deafness for 4 weeks. This study aimed to investigate various parameters important for the development of a long-term cell-based neurotrophin treatment to support SGN survival. Specifically, we investigated different (i) cell types, (ii) gene transfer methods and (iii) neurotrophins, in order to determine which variables may provide long-term neurotrophin expression and which, therefore, may be the most effective for supporting long-term SGN survival in vivo. We found that fibroblasts that were nucleofected to express BDNF provided the most sustained neurotrophin expression, with ongoing BDNF expression for at least 30 weeks. In addition, the secreted neurotrophin was biologically active and elicited survival effects on SGNs in vitro. Nucleofected fibroblasts may therefore represent a method for safe, long-term delivery of neurotrophins to the deafened cochlea to support SGN survival in deafness.

Introduction

Sensorineural hearing loss (SNHL), the most common form of deafness, is typically caused by the loss of cochlear hair cells. The only therapeutic treatment for patients with severe-profound SNHL is a cochlear implant – a neural prosthesis that electrically stimulates the residual spiral ganglion neuron (SGN) population to provide the rate and pitch cues necessary for speech perception. In the normal cochlea, the hair cells and supporting cells of the organ of Corti support the survival of SGNs through endogenous neurotrophin secretion (Fritzsch et al., 2004, Stankovic et al., 2004, Green et al., 2012, Zilberstein et al., 2012), and therefore damage to the organ of Corti and loss of this neurotrophin support, as occurs in SNHL, leads to the loss of SGNs. Since SGNs are the target cells for the cochlear implant, the loss of a significant population of SGNs may compromise the function of the device (Pfingst and Sutton, 1983, Shepherd and Javel, 1997, Hardie and Shepherd, 1999). Furthermore, future developments in device and software design may also benefit from an enhanced SGN population (Wise and Gillespie, 2012).

Exogenous delivery of neurotrophins such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) can support SGN survival in models of deafness (Ernfors et al., 1996, Miller et al., 1997, Gillespie et al., 2003, Gillespie et al., 2004, Yamagata et al., 2004, Richardson et al., 2005, Shepherd et al., 2005). However, the cessation of exogenous neurotrophin treatment can result in a loss of these survival effects (Gillespie et al., 2003, Shepherd et al., 2005). While others have reported continued auditory neuron survival for 2 weeks post-treatment (Agterberg et al., 2009), long-term outcomes remain unknown. Chronic electrical stimulation via a cochlear implant extends neurotrophin-based survival effects past the end of neurotrophin treatment; however, to maximize SGN rescue, long-term neurotrophin delivery is desirable (Shepherd et al., 2005, Shepherd et al., 2008).

Current methods of neurotrophin delivery into the cochlea, such as osmotic pumps, are not considered suitable for clinical application (Pettingill et al., 2007). Alternative pump-based delivery systems must be re-filled at regular intervals, necessitating multiple surgical procedures. This poses a small but significant risk of infection, which could result in labyrinthitis and meningitis (Wei et al., 2008). Furthermore, neurotrophins have a short half-life (Lindholm et al., 1988, Matsuoka et al., 1991, Poduslo and Curran, 1996, Kishino et al., 2001), meaning that the use of long-term pump delivery systems with high volume capacities may be complicated by the unknown bioactivity of neurotrophins maintained at body temperature for long periods.

Cell-based therapies, in which cells secreting a therapeutic substance are implanted into a patient, are an alternative mechanism for continuous delivery of neurotrophins into the cochlea (for review see Zanin et al., 2012). Cell-based therapies may utilize cells which naturally secrete therapeutic agents (Wise et al., 2011), or may be combined with gene transfer techniques to genetically modify cells to secrete the desired therapeutic agent(s) (Pettingill et al., 2008, Pettingill et al., 2011). Cell-based therapies provide an avenue for delivering neurotrophins at physiological levels and in a consistent manner, and also overcome issues of infection (Shepherd, 2011) and longevity of survival effects (Gillespie et al., 2003, Shepherd et al., 2005) associated with other experimental delivery methods. In addition, cell-based therapies have the potential for long-term neurotrophin expression (Winn et al., 1996). For these reasons, cell-based therapies are considered clinically viable, and have already been implemented for therapeutic drug delivery in clinical trials for various neurodegenerative conditions (for review see Zanin et al., 2012).

Previously, we successfully genetically modified Schwann cells using lipofection to express BDNF or NT3 and demonstrated that these cells could support SGN survival in vitro (Pettingill et al., 2008). Furthermore, we reported that the implantation of encapsulated, BDNF-secreting Schwann cells into the deaf guinea pig cochlea successfully supported SGN survival over 2- and 4-week implantation periods (Pettingill et al., 2011). While promising, longer term studies, using cells with a greater duration of neurotrophin secretion, are required in order to best assess the potential of this therapy for ongoing SGN survival (Pettingill et al., 2011).

There are numerous experimental parameters that may play an important role in achieving longer term neurotrophin expression from a cell-based treatment. In the current study, we investigated different (i) cell types, (ii) gene transfer techniques and (iii) neurotrophins, with the aim of developing a population of cells that reliably secreted neurotrophin for periods of time significantly greater than 4 weeks.

Section snippets

Experimental procedures

We utilized an array of test conditions in order to develop cells with long-term neurotrophin expression. Specifically, Schwann cells and fibroblasts were genetically modified using lipofection or nucleofection to express BDNF or NT3. Schwann cells were also genetically modified using lentiviral vectors to express these neurotrophins. The resultant neurotrophin-expressing cells were compared in terms of transfection efficiency, and duration and quantity of neurotrophin expression. The

Results

In this study we compared various approaches for cell transfection with the aim of developing a protocol that would generate cells with long-term neurotrophin secretion at levels sufficient to support SGN survival in vitro. Specifically, we genetically modified Schwann cells and fibroblasts to express EGFP, BDNF or NT3 using lipofection and nucleofection, and also genetically modified Schwann cells to express these genes using lentiviral vectors. We assessed the outcomes by quantifying the

Discussion

This study was undertaken to produce a population of primary cells that secrete neurotrophin for extended periods of time at concentrations that support SGN survival. We genetically modified Schwann cells and fibroblasts using lipofection and nucleofection, and also genetically modified Schwann cells using lentiviral vectors, to express two genes known to be important in the auditory system – BDNF and NT3. From the parameters tested we demonstrated that nucleofected BDNF-expressing fibroblasts

Conclusions

We utilized different methods of gene transfer to genetically modify Schwann cells or fibroblasts to secrete BDNF or NT3. We established that fibroblasts that were nucleofected to express BDNF provided the greatest duration of neurotrophin secretion of at least 30 weeks, and that these BDNF-expressing fibroblasts were effective in supporting SGN survival in an in vitro model of deafness, which indicates that therapeutic levels of neurotrophins can be delivered using cell-based methods. Since a

Author contributions

MPZ, RKS and LNG designed the experiments; MH and ARH produced the lentiviral Schwann cells used for the experiments; MPZ and LNG performed the experiments and wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgments

We thank Rebecca Argent for technical assistance, and Professor Johnson Mak for access to the AMAXA Nucleofector II. This work was supported by the National Health and Medical Research Council of Australia (APP526901) and the Garnett Passe and Rodney Williams Memorial Foundation. The Bionics Institute acknowledges the support it receives from the Victorian Government through its Operational Infrastructure Support Program. The funding bodies had no role in study design, data collection and

References (56)

  • J.F. Poduslo et al.

    Permeability at the blood–brain and blood–nerve barriers of the neurotrophic factors: NGF, CNTF, NT-3, BDNF

    Brain Res Mol Brain Res

    (1996)
  • R.T. Richardson et al.

    A single dose of neurotrophin-3 to the cochlea surrounds spiral ganglion neurons and provides trophic support

    Hear Res

    (2005)
  • R.K. Shepherd et al.

    Neurotrophins and electrical stimulation for protection and repair of spiral ganglion neurons following sensorineural hearing loss

    Hear Res

    (2008)
  • R.K. Shepherd et al.

    Electrical stimulation of the auditory nerve. I. Correlation of physiological responses with cochlear status

    Hear Res

    (1997)
  • M. Skrzyszowska et al.

    Development of porcine transgenic nuclear-transferred embryos derived from fibroblast cells transfected by the novel technique of nucleofection or standard lipofection

    Theriogenology

    (2008)
  • A. Warnecke et al.

    Stable release of BDNF from the fibroblast cell line NIH3T3 grown on silicone elastomers enhances survival of spiral ganglion cells in vitro and in vivo

    Hear Res

    (2012)
  • S.R. Winn et al.

    Polymer-encapsulated genetically modified cells continue to secrete human nerve growth factor for over one year in rat ventricles: behavioral and anatomical consequences

    Exp Neurol

    (1996)
  • A.K. Wise et al.

    Combining cell-based therapies and neural prostheses to promote neural survival

    Neurotherapeutics

    (2011)
  • M.P. Zanin et al.

    The development of encapsulated cell technologies as therapies for neurological and sensory diseases

    J Control Release

    (2012)
  • M.J. Agterberg et al.

    Enhanced survival of spiral ganglion cells after cessation of treatment with brain-derived neurotrophic factor in deafened guinea pigs

    J Assoc Res Otolaryngol

    (2009)
  • R. Badakov et al.

    Efficient transfection of primary zebrafish fibroblasts by nucleofection

    Cytotechnology

    (2006)
  • T. Brigadski et al.

    Differential vesicular targeting and time course of synaptic secretion of the mammalian neurotrophins

    J Neurosci

    (2005)
  • J.P. Brockes et al.

    A surface antigenic marker for rat Schwann cells

    Nature

    (1977)
  • D.F. Emerich et al.

    NT-501: an ophthalmic implant of polymer-encapsulated ciliary neurotrophic factor-producing cells

    Curr Opin Mol Ther

    (2008)
  • P. Ernfors et al.

    Protection of auditory neurons from aminoglycoside toxicity by neurotrophin-3

    Nat Med

    (1996)
  • L.N. Gillespie et al.

    LIF is more potent than BDNF in promoting neurite outgrowth of mammalian auditory neurons in vitro

    Neuroreport

    (2001)
  • L.N. Gillespie et al.

    BDNF-induced survival of auditory neurons in vivo: cessation of treatment leads to an accelerated loss of survival effects

    J Neurosci Res

    (2003)
  • L.N. Gillespie et al.

    Delayed neurotrophin treatment supports auditory neuron survival in deaf guinea pigs

    Neuroreport

    (2004)
  • Cited by (11)

    • Bridging the electrode–neuron gap: finite element modeling of in vitro neurotrophin gradients to optimize neuroelectronic interfaces in the inner ear

      2022, Acta Biomaterialia
      Citation Excerpt :

      Over the course of the past 20–30 years, it has been established that BDNF mediates survival and differentiation activities of SGNs by binding and activating tyrosine kinase receptor kinase B (TrkB), a member of the larger family of Trk receptors [20]. Numerous studies have reported that BDNF can palliate SGN degeneration in ototoxically deafened animals, a widely accepted model for retrograde trans-synaptic SGN degeneration secondary to hair cell destruction [13,14,57,58]. Additionally, it has been confirmed that there is a positive correlation between SGN counts and CI performance [59].

    • Recent advances in the implant-based drug delivery in otorhinolaryngology

      2020, Acta Biomaterialia
      Citation Excerpt :

      Like NIH3T3 cells, primary fibroblasts have also been successfully explored. One noteworthy study focused on the optimization of important parameters of a cell-mediated neurotrophin-delivering implant, i.e., cell type, gene transfer method, and neurotrophin type [24]. Their study concluded that nucleofecting fibroblasts to express BDNF yields the most sustained neurotrophin expression.

    • Scaffolds for auditory nerve regeneration

      2019, Handbook of Tissue Engineering Scaffolds: Volume Two
    • Cell-based neurotrophin treatment supports long-term auditory neuron survival in the deaf guinea pig

      2015, Journal of Controlled Release
      Citation Excerpt :

      Specifically, we combined cell- and gene therapies with alginate encapsulation technology to produce encapsulated BDNF-expressing fibroblasts, and assessed the survival-promoting effects on ANs in the deaf guinea pig following implantation for periods of up to six months, and with and without chronic electrical stimulation. Fibroblasts that were nucleofected to express BDNF were used for this study based upon findings from in vitro experiments [27], which demonstrated that these parameters were most efficacious in terms of gene transfer, and produced cells which had the greatest duration of neurotrophin expression. Specifically, fibroblasts were isolated from rat sciatic nerve explants using a method similar to that used to isolate Schwann cells [21,28].

    View all citing articles on Scopus

    Current address: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis, TN, USA.

    Current address: Laboratory for Transplantation and Regenerative Medicine, Department of Obstetrics and Gynecology, Sahlgrenska Academy, University of Gothenburg, Sweden.

    View full text