Abstract
Stem-cell-based repair of auditory neurons may represent an attractive therapeutic option to restore sensorineural hearing loss. Hair-follicle-bulge-derived stem cells (HFBSCs) are promising candidates for this type of therapy, because they (1) have migratory properties, enabling migration after transplantation, (2) can differentiate into sensory neurons and glial cells, and (3) can easily be harvested in relatively high numbers. However, HFBSCs have never been used for this purpose. We hypothesized that HFBSCs can be used for cell-based repair of the auditory nerve and we have examined their migration and incorporation into cochlear modiolus explants and their subsequent differentiation. Modiolus explants obtained from adult wild-type mice were cultured in the presence of EF1α-copGFP-transduced HFBSCs, constitutively expressing copepod green fluorescent protein (copGFP). Also, modiolus explants without hair cells were co-cultured with DCX-copGFP-transduced HFBSCs, which demonstrate copGFP upon doublecortin expression during neuronal differentiation. Velocity of HFBSC migration towards modiolus explants was calculated, and after two weeks, co-cultures were fixed and processed for immunohistochemical staining. EF1α-copGFP HFBSC migration velocity was fast: 80.5 ± 6.1 μm/h. After arrival in the explant, the cells formed a fascicular pattern and changed their phenotype into an ATOH1-positive neuronal cell type. DCX-copGFP HFBSCs became green-fluorescent after integration into the explants, confirming neuronal differentiation of the cells. These results show that HFBSC-derived neuronal progenitors are migratory and can integrate into cochlear modiolus explants, while adapting their phenotype depending on this micro-environment. Thus, HFBSCs show potential to be employed in cell-based therapies for auditory nerve repair.
Highlights
basic growth medium (BGM) consists of DMEM/Ham’s F-12 1:1 (Biochrom AG, Berlin, Germany), 1% GlutaMaxTM (100x; Gibco, Bleiswijk, the Netherlands) and 1% antibiotic/antimycotic solution supplemented with 10% fetal bovine serum (FBS; Gibco), 2% B-271 supplement without vitamin A (50x; Gibco), 1% N-2 MAX media supplement (100x; R&D SystemsTM, Minneapolis, MN, USA), recombinant human basic fibroblast growth factor (20 ng/ml; R&D Systems), and recombinant human epidermal growth factor (20 ng/ml; R&D Systems)
After obtaining a fascicular pattern of elongation factor 1α (EF1α)-copepod green fluorescent protein (copGFP) Hair-follicle-bulge-derived stem cells (HFBSCs) in quarter-turn explants of adult mice (Fig 4A), we investigated the role of the neuronal migration protein DCX in the process of pattern formation and seeded DCX-copGFP HFBSCs onto quarter-turn explants
The results show that our population of HFBSCs meets three important requirements to enable their contribution to successful inner ear regeneration: they are migratory, they integrate into cochlear tissue, and have the capability to adapt a neuronal phenotype depending on the microenvironment
Summary
The degree of neuron loss is of significance for hearing-impaired patients using a cochlear implant (CI), because this device directly stimulates auditory neurons. Stem cells may serve different purposes: their descendants can replace spiral ganglion cells and/or glial cells, but regeneration may be stimulated by stem-cell-driven paracrine secretion of cytokines and growth factors [3]. This could be of interest in clinical applications, because it can be surmised that in deaf patients stem cells will lack trophic support from hair cells and perhaps from non-sensory cells
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