In vitro Methods to Cultivate Spiral Ganglion Cells, and Purification of Cellular Subtypes for Induced Neuronal Reprogramming

Abstract

Hearing loss can develop as a consequence of primary auditory neuron degeneration. These neurons are present within the spiral ganglion of the inner ear and co-exist with glial cells that assist in neuronal maintenance and function. There are limited interventions for individuals with hearing impairment, hence novel biological solutions must be explored. Regenerative strategies can benefit from in vitro methods to examine the long-term culture of purified cell populations. The culturing of neuronal, glial, and non-neuronal, non-glial cell types in both neonatal and adult mice is presented along with the whole-organ explant culture of the spiral ganglion. High yields of spiral ganglion glial and non-glial cells were cultured from both neonatal and adult mice. Dissociated spiral ganglion cells from Sox2-EGFP mice were sorted based on EGFP expression using fluorescence activated cell sorting. The EGFP+ fraction included purified glial populations, whereas the EGFP- fraction contained non-glial cells. Purified glial cells could be reprogrammed into induced neurons displaying neuronal markers and morphology at a higher efficiency than non-glial cells. Previous studies have only allowed for the short-term culturing of spiral ganglion cell populations and have placed emphasis on neonatal cells. There has also been a lack of methods able to cultivate pure cell populations. Here, the coupling of transgenic mouse lines, fluorescence activated cell sorting and advanced culture conditions allow cultivation and characterization of neuronal, glial and non-neuronal, non-glial cells from the spiral ganglion. These techniques are used to demonstrate that different spiral ganglion cell subtypes (glial vs. non-glial) display different competencies for direct neuronal reprogramming.

Lin28 reprograms inner ear glia to a neuronal fate

“…49 Proneural bHLH transcription factors are upregulated in reactive glia after damage to adult auditory neurons, 31 and forced overexpression of Ascl1 or Neurod1 in non-neural inner ear cells in vitro can generate neuron-like cells. 50,51 We show here that Lin28 initiates proliferation of Pl p1-positive cochlear glial cells isolated and grown as neurospheres in vitro and promotes neurogenesis by activating proneural bHLH transcription factors through inhibition of Let-7. We further show that transient Lin28 overexpression reprograms inner ear glial cells to neurons in an in vivo model of auditory neuropathy.…”

“…Prolonged expression of Lin28 delays cell cycle exit and maintains an undifferentiated state in inner ear sensory cells 49 . Proneural bHLH transcription factors are upregulated in reactive glia after damage to adult auditory neurons, 31 and forced overexpression of Ascl1 or Neurod1 in non‐neural inner ear cells in vitro can generate neuron‐like cells 50,51 …”

Atrial Natriuretic Peptide Improves Neurite Outgrowth from Spiral Ganglion Neurons In Vitro through a cGMP-Dependent Manner

“…Cultures. The cochlea dissection procedures were performed as described in previous studies with slight modifications [56][57][58][59]. The cochleae from P3 rats were immersed in icecold HBSS; then, the cochlear capsule was opened by fine forceps and the membranous labyrinth was removed from the modiolus under a dissecting microscope.…”

Cochlear Sox2+ Glial Cells Are Potent Progenitors for Spiral Ganglion Neuron Reprogramming Induced by Small Molecules

“…The Plp1 + cochlear glial cells serve as potent progenitors for neurons, astrocytes and oligodendrocytes, but not hair cells in vitro ( McLean et al, 2016 ). Cochlear glial cells isolated from Sox2-eGFP reporter mice also displayed potent neurogenic potential in vitro ( Lang et al, 2015 ; Meas et al, 2018a ). However, a direct comparison of glial subpopulations is lacking and the iNs did not appear to mature as SGNs.…”