Clin Exp Otorhinolaryngol.  2015 Jun;8(2):83-91. 10.3342/ceo.2015.8.2.83.

Neural-Induced Human Mesenchymal Stem Cells Promote Cochlear Cell Regeneration in Deaf Guinea Pigs

• Jang S
• Cho HH
• Kim SH
• Lee KH
• Jun JY
• Park JS
• Jeong HS
• Cho YB

• KMID: 2360776
• DOI: http://doi.org/10.3342/ceo.2015.8.2.83

Abstract

OBJECTIVES
In mammals, cochlear hair cell loss is irreversible and may result in a permanent sensorineural hearing loss. Secondary to this hair cell loss, a progressive loss of spiral ganglion neurons (SGNs) is presented. In this study, we have investigated the effects of neural-induced human mesenchymal stem cells (NI-hMSCs) from human bone marrow on sensory neuronal regeneration from neomycin treated deafened guinea pig cochleae.
METHODS
HMSCs were isolated from the bone marrow which was obtained from the mastoid process during mastoidectomy for ear surgery. Following neural induction with basic fibroblast growth factor and forskolin, we studied the several neural marker and performed electrophysiological analysis. NI-hMSCs were transplanted into the neomycin treated deafened guinea pig cochlea. Engraftment of NI-hMSCs was evaluated immunohistologically at 8 weeks after transplantation.
RESULTS
Following neural differentiation, hMSCs expressed high levels of neural markers, ionic channel markers, which are important in neural function, and tetrodotoxin-sensitive voltage-dependent sodium currents. After transplantation into the scala tympani of damaged cochlea, NI-hMSCs-injected animals exhibited a significant increase in the number of SGNs compared to Hanks balanced salt solution-injected animals. Transplanted NI-hMSCs were found within the perilymphatic space, the organ of Corti, along the cochlear nerve fibers, and in the spiral ganglion. Furthermore, the grafted NI-hMSCs migrated into the spiral ganglion where they expressed the neuron-specific marker, NeuN.
CONCLUSION
The results show the potential of NI-hMSCs to give rise to replace the lost cochlear cells in hearing loss mammals.

Keyword

Hair Cells, Auditory; Mesenchymal Stem Cells; Hearing Loss; Cell Differentiation; Transplantation

MeSH Terms

Animals
Bone Marrow
Cell Differentiation
Cochlea
Cochlear Nerve
Colforsin
Ear
Fibroblast Growth Factor 2
Guinea Pigs*
Hair
Hearing Loss
Hearing Loss, Sensorineural
Humans
Ion Channels
Mammals
Mastoid
Mesenchymal Stromal Cells*
Neomycin
Neurons
Organ of Corti
Regeneration*
Scala Tympani
Sensory Receptor Cells
Sodium
Spiral Ganglion
Transplantation
Transplants
Colforsin
Fibroblast Growth Factor 2
Ion Channels
Neomycin
Sodium

Figure

• 

  Fig. 1 Characterization of bone marrow-derived human mesenchymal stem cells (hMSCs). Primary hMSCs expressed MSC specific markers including CD13 (+), CD44 (+), CD90 (+), and CD166 (+), but did not express hematopoietic stem cell markers including CD14 (-), CD34 (-), and CD45 (-).

• 

  Fig. 2 In vitro differentiation of human mesenchymal stem cells (hMSCs). hMSCs were induced to differentiate into neural cells in the presence of basic fibroblast growth factor and forskolin for two weeks. (A) Immunocytochemistry revealed that the expressions of neurofilament-L (NF-L), neurofilament-M (NF-M), neurofilament-H (NF-H), microtubule-associated protein (MAP2), neuronal nuclei (NeuN), β-tubulin III (Tuj1), and glial fibrillary acidic protein (GFAP) in neural-induced hMSCs (NI-hMSCs) were increased than those in primary hMSCs. (B) Immunocytochemical data depicted the high ratio of NI-hMSCs expressing above neural markers. The number of positive cells was counted and the ratio to the number of nuclei was analyzed for each antigen (n=8, *P<0.05 compared with primary hMSCs).

• 

  Fig. 3 Electrophysiological features of the neural-induced human mesenchymal stem cells (NI-hMSCs). (A) hMSCs demonstrated neuronal characteristics after neural differentiation under voltage-clamp recording. The holding potential was -80 mV and depolarizing steps were applied from -80 mV to +40 mV in 10 mV increments. Large voltage-dependent sodium currents were activated evidently from a depolarizing step of -30 mV and blocked reversibly by tetrodotoxin (TTX) 100 nM. In addition, the hMSCs grown with basic fibroblast growth factor (bFGF) and forskolin showed sustained outward potassium currents as well. Peak current-voltage relationship was plotted against the voltages and demonstrated the voltage-dependence of potassium currents (IK) and sodium currents (INa) (n=16). (B) Under current clamp condition, the resting membrane potential of NI-hMSCs was recorded more negatively than that of control hMSCs grown without bFGF and forskolin (n=16, **P<0.01 compare with primary hMSCs). (C) Expression of molecular markers for ion channel subunits was increased after neural differentiation in hMSCs. The mRNA expression of human large-conductance, voltage- and calcium-dependent K+ channel marker, MaxiK; voltage-dependent K+ channel marker, Kv1.4, Kv4.2, and Kv4.3; either-à-go-go K+ channel marker, Eag1 and Eag2; tetrodotoxin-sensitive Na+ channel marker, NE-Na; voltage-dependent L-type Ca2+ channel, alpha 1C subunit marker, CACNA1C; and voltage-dependent T-type Ca2+ channel, alpha 1G subunit marker, CACNA1G were increased in NI-hMSCs, however, that of TTX-insensitive sodium channel marker, SCN5A were not detected. Reverse transcription-polymerase chain reaction assay was repeated five times independently from different cells. The representative data are shown. (D) The intensity of each gene was normalized to GAPDH and these results were repeated at least three times (*P<0.05 compared with primary hMSCs).

• 

  Fig. 4 Hematoxylin and eosin staining at eight weeks after neural-induced human mesenchymal stem cells (NI-hMSCs) transplantation. (A, B) All turns of cochlea were studied and basal one was magnified as a box, individually. (A) In a Hanks balanced salt solution (HBSS) alone-injected animal, severe loss of sensory hair cells in the organ of Corti and the degeneration of spiral ganglion neurons were observed. (B) However, cochlea of a NI-hMSCs-injected guinea pig demonstrated relatively preserved organ of Corti and spiral ganglion. (C) Quantification of spiral ganglion cell counts demonstrated that cochleae of NI-hMSCs-injected animals exhibited a significant increase in the number of cell body compared to HBSS alone-injected animals (n=8, **P<0.01). Normal animals (n=10) were used as a control. OC, organ of Corti; SG, spiral ganglion; SM, scala media; ST, scala tympani; SV, scala vestibule; IHC, inner hair cell; OHC, outer hair cell. Scale bars indicate 50 µm.

• 

  Fig. 5 Localization and in vivo differentiation of transplanted human mesenchymal stem cells (hMSCs). (A-D) Immunohistochemical study was performed eight weeks after neural-induced hMSCs (NI-hMSCs) transplantation into 10% neomycin-treated guinea pig inner ear. (A) Transplanted hMSCs stained with human nuclei (hNuclei; red) were found along the cochlear nerve fibers close to the organ of Corti. (B) Human nuclei expressing transplant derived-hMSCs (red) were located in the inner hair cell layer (arrow) and supporting cell layer (arrowhead). (C) NI-hMSCs (green) migrated into the damaged spiral ganglion were stained with microtubule-associated protein (MAP2) (red), and merged as yellow. These results depict that NI-hMSCs have the capacity not only to survive and localize in the inner ear, but also to regenerate or replace the damaged cochlear cell types. (D) Quantification of spiral ganglion neuron (MAP2+) and transplant-derived cell (hNuclei+) counts indicated that transplanted hMSCs were transdifferentiated into mature neurons in the spiral ganglion. CN, cochlear nerve fibers; OC, organ of Corti; SG, spiral ganglion; SL, spiral limbus; SM, scala media; ST, scala tympani; SV, scala vestibule.

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