Clin Exp Otorhinolaryngol.  2018 Dec;11(4):224-232. 10.21053/ceo.2018.00878.

The Presence of Neural Stem Cells and Changes in Stem Cell-Like Activity With Age in Mouse Spiral Ganglion Cells In Vivo and In Vitro

• Moon BS
• Ammothumkandy A
• Zhang N
• Peng L
• Ibrayeva A
• Bay M
• Pratap A
• Park HJ
• Bonaguidi
• Lu W

• KMID: 2439848
• DOI: http://doi.org/10.21053/ceo.2018.00878

Abstract

OBJECTIVES
Spiral ganglion neurons (SGNs) include potential endogenous progenitor populations for the regeneration of the peripheral auditory system. However, whether these populations are present in adult mice is largely unknown. We examined the presence and characteristics of SGN-neural stem cells (NSCs) in mice as a function of age.
METHODS
The expression of Nestin and Ki67 was examined in sequentially dissected cochlear modiolar tissues from mice of different ages (from postnatal day to 24 weeks) and the sphere-forming populations from the SGNs were isolated and differentiated into different cell types.
RESULTS
There were significant decreases in Nestin and Ki67 double-positive mitotic progenitor cells in vivo with increasing mouse age. The SGNs formed spheres exhibiting self-renewing activity and multipotent capacity, which were seen in NSCs and were capable of differentiating into neuron and glial cell types. The SGN spheres derived from mice at an early age (postnatal day or 2 weeks) contained more mitotic stem cells than those from mice at a late age.
CONCLUSION
Our findings showed the presence of self-renewing and proliferative subtypes of SGN-NSCs which might serve as a promising source for the regeneration of auditory neurons even in adult mice.

Keyword

Spiral Ganglion; Neural Stem Cells; Deafness; Hearing Loss; Cochlea

MeSH Terms

Adult
Animals
Cochlea
Deafness
Hearing Loss
Humans
In Vitro Techniques*
Mice*
Nestin
Neural Stem Cells*
Neuroglia
Neurons
Regeneration
Spiral Ganglion*
Stem Cells

Figure

• 

  Fig. 1. Surgical dissection of the cochlea and sections for spiral ganglion neurons in postnatal mice as a function of age. (A) The bony shell of the cochlea (red box) was removed and then spiral cochlear tissue was removed, resulting in the modiolus containing spiral ganglion neurons. (B) Fixed coronal sections of the spiral ganglion neurons (yellow boxes) stained with antibodies against Nestin (green) and Ki67 (red). White arrows indicate Nestin and Ki67 double positive cells. (C) Quantification of the data shown in (B) (postnatal day 1 [P1], n=4; 2 weeks, n=3; 4 weeks, n=3; 12 weeks, n=3; and 24 weeks, n=3). All individual quantification data underlying (C) can be found in Supplementary Table 1. Values are presented as mean±standard deviation. Analysis of variance tests were performed to calculate significance (***P<0.0001). DAPI, 4´,6-diamidino-2-phenylindole.

• 

  Fig. 2. Spiral ganglion cells exhibit self-renewing and proliferative activity. (A) Spiral ganglions were separated from mice of age postnatal day 1 (P1), 2 weeks, and 4 weeks, and sphere formation was induced for 7 days in vitro. (B) Quantification of the relative sizes and numbers of primary or secondary spheres in (A). The number of P1 spiral ganglion neuron-derived neural stem cell spheres formed during 1 day culture was normalized to 1. Values are presented as mean±standard deviation. Analysis of variance tests were performed to calculate significance (*P<0.01, **P<0.001, ***P<0.0001). (C) P1 spiral ganglion cells dissociated from the spheres (upper) were grown and were subjected to immunofluorescence labeling using antibodies specific for Sox2 (green), Ki67 (green), and Nestin (red). (D) Quantification of anti-Sox2-positive (P1: +bFGF, n=3; –bFGF, n=5; 2 weeks: +bFGF, n=3; –bFGF, n=4), or anti-Nestin and anti-Ki67 double-positive cells (P1: +bFGF, n=6; –bFGF, n=5; 2 weeks: +bFGF, n=4; –bFGF, n=3) in (C). The t-tests were performed to calculate significance (*P<0.05, **P<0.005, ***P<0.0005). bFGF, basic fibroblast growth factor; DAPI, 4´,6-diamidino-2-phenylindole; Un, undifferentiated; 2DIV, 2 days differentiation in vitro.

• 

  Fig. 3. The spiral ganglion cells exhibited a multipotent capacity. (A) Spiral ganglions were grown and their differentiation capabilities monitored by changes in morphology. (B) Quantitative polymerase chain reaction analysis of the indicated mRNA sequences in the postnatal day 1 (P1) mice spiral ganglions (n=4). Values are presented as mean±standard deviation. (C) Immunostaining with Tuj1, MAP2, and GFAP (all red) antibodies in P1 and 2-week-old mice under undifferentiation and differentiation conditions. (D) Quantification of Tuj1-positive (P1: +bFGF, n=3; –bFGF, n=5; 2 weeks: +bFGF, n=3; –bFGF, n=4) and GFAP-positive cells (P1: +bFGF, n=5; –bFGF, n=5; 2 weeks: +bFGF, n=4; –bFGF, n=4) in (C). The t-tests were conducted to calculate the statistical significance (*P<0.05, **P<0.005, ***P<0.0005). Un, undifferentiated; 2DIV, 2 days differentiation in vitro; bFGF, basic fibroblast growth factor; MAP2, mature microtubule-associated protein 2; DAPI, 4´,6-diamidino-2-phenylindole.

• 

  Fig. 4. Spiral ganglion cells consist of heterogeneous cell populations, of which spiral ganglion neuron-derived neural stem cell spheres (SGNNSCs) could be a regenerative source for damaged cell repair. (A) Fixed coronal sections of the cochlea stained with antibodies against GFAP (green), Tuj1 (red), and MAP2 (green). (B) Quantification of Tuj1-positive (postnatal day 1 [P1], n=4; 2 weeks, n=4; 4 weeks, n=3; 12 weeks, n=4; 24 weeks, n=4), MAP2-positive (P1, n=4; 2 weeks, n=4; 4 weeks, n=4; 12 weeks, n=4; 24 weeks, n=4), and GFAP-positive (P1, n=4; 2 weeks, n=4; 4 weeks, n=3; 12 weeks, n=4; 24 weeks, n=4), in (A). (C) Schematic showing the existence of regenerative and self-renewing SGN-NSCs. DAPI, 4´,6-diamidino-2-phenylindole; MAP2, mature microtubule-associated protein 2.

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