Postnatal Changes of Neural Stem Cells in the Mammalian Auditory Cortex.

Our previous study reported neural stem cells (NSCs) in the auditory cortex (AC) of postnatal day 3 (P3) mice in vitro. It is unclear whether AC-NSCs exist in vivo. This study aims to determine the presence and changes of AC-NSCs during postnatal development and maturation both in vitro and in vivo. P3, postnatal day 14 (P14), 2-month-old (2M), and 4-month-old (4M) mouse brain tissues were fixed and cryosectioned for NSC marker immunostaining. In vitro, P3, P14, and 2M AC tissues were dissected and cultured in suspension to study NSCs. NSC proliferation was examined by EdU incorporation and cell doubling time assays in vitro. The results show that Nestin and Sox2 double expressing NSCs were observed in the AC area from P3 to 4M in vivo, in which the number of NSCs remarkably reduced with age. In vitro, the neurosphere forming capability, cell proliferation, and percentage of Nestin and Sox2 double expressing NSCs significantly diminished with age. These results suggest that AC-NSCs exist in the mouse AC area both in vitro and in vivo, and the percentage of AC-NSCs decreases during postnatal development and maturation. The results may provide important cues for the future research of the central auditory system.

Generation of Cochlear Hair Cells from Sox2 Positive Supporting Cells via DNA Demethylation.

Regeneration of auditory hair cells in adult mammals is challenging. It is also difficult to track the sources of regenerated hair cells, especially in vivo. Previous paper found newly generated hair cells in deafened mouse by injecting a DNA methyltransferase inhibitor 5-azacytidine into the inner ear. This paper aims to investigate the cell sources of new hair cells. Transgenic mice with enhanced green fluorescent protein (EGFP) expression controlled by the Sox2 gene were used in the study. A combination of kanamycin and furosemide was applied to deafen adult mice, which received 4 mM 5-azacytidine injection into the inner ear three days later. Mice were followed for 3, 5, 7 and 14 days after surgery to track hair cell regeneration. Immunostaining of Myosin VIIa and EGFP signals were used to track the fate of Sox2-expressing supporting cells. The results show that (i) expression of EGFP in the transgenic mice colocalized the supporting cells in the organ of Corti, and (ii) the cell source of regenerated hair cells following 5-azacytidine treatment may be supporting cells during 5-7 days post 5-azacytidine injection. In conclusion, 5-azacytidine may promote the conversion of supporting cells to hair cells in chemically deafened adult mice.

Angiotensin II Facilitates Matrix Metalloproteinase-9-Mediated Myosin Light Chain Kinase Degradation in Pressure Overload-Induced Cardiac Hypertrophy.

BACKGROUND/AIMS: Angiotensin II (Ang II) has been shown to promote cardiac remodeling during the process of hypertrophy. Myosin light chain kinase (MLCK), a specific kinase for the phosphorylation of myosin light chain 2 (MLC2), plays an important role in regulating cardiac muscle contraction and hypertrophy. However, whether Ang II could facilitate cardiac hypertrophy by altering the expression of MLCK remains unclear. This study aimed to investigate this effect and the underlying mechanisms. METHODS: Cardiac hypertrophy was induced via pressure overload in rats, which were then evaluated via histological and biochemical measurements and echocardiography. Angiotensin-converting enzyme inhibitor (ACEI) was used to inhibit Ang II. Neonatal rat cardiomyocytes were stimulated with Ang II to induce hypertrophy and were treated with a matrix metalloproteinase 9 (MMP9) inhibitor. Myocyte hypertrophy was evaluated using immunofluorescence and qRT-PCR. Degradation of recombinant human MLCK by recombinant human MMP9 was tested using a cleavage assay. The expression levels of MLCK, MLC2, phospho-myosin light chain 2 (p-MLC2), myosin phosphatase 2 (MYPT2), and calmodulin (CaM) were measured using western blotting. RESULTS: ACEI improved cardiac function and remodeling and increased the levels of MLCK and p-MLC2 as well as reduced the expression of MMP9 in pressure overload-induced cardiac hypertrophy. Moreover, the MMP9 inhibitor alleviated myocyte hypertrophy and upregulated the levels of MLCK and p-MLC2 in Ang II-induced cardiomyocyte hypertrophy. Recombinant human MLCK was concentration- and time-dependently degraded by recombinant human MMP9 in vitro, and this process was prevented by the MMP9 inhibitor. CONCLUSION: Our results suggest that Ang II is involved in the degradation of MLCK in pressure overload-induced cardiac hypertrophy and that this process was mediated by MMP9.

Hearing Recovery Induced by DNA Demethylation in a Chemically Deafened Adult Mouse Model.

Functional hair cell regeneration in the adult mammalian inner ear remains challenging. This study aimed to study the function of new hair cells induced by a DNA demethylating agent 5-azacytidine. Adult mice were deafened chemically, followed by injection of 5-azacytidine or vehicle into the inner ear. Functionality of regenerated hair cells was evaluated by expression of hair cell proteins, auditory brainstem response (ABR), and distortion-product otoacoustic emission (DPOAE) tests for 6 weeks. In the vehicle-treated group, no cells expressed the hair cell-specific protein myosin VIIa in the cochlea, whereas numerous myosin VIIa-expressing cells were found in the 5-azacytidine-treated cochlea, suggesting the regeneration of auditory hair cells. Moreover, regenerated hair cells were co-labeled with functional proteins espin and prestin. Expression of ribbon synapse proteins suggested synapse formation between new hair cells and neurons. In hearing tests, progressive improvements in ABR [5-30 dB sound pressure level (SPL)] and DPOAE (5-20 dB) thresholds were observed in 5-azacytidine-treated mice. In vehicle-treated mice, there were <5 dB threshold changes in hearing tests. This study demonstrated the ability of 5-azacytidine to promote the functional regeneration of auditory hair cells in a mature mouse model via DNA demethylation, which may provide insights into hearing regeneration using an epigenetic approach.

Tripartite motif 27 promotes cardiac hypertrophy via PTEN/Akt/mTOR signal pathways.

Tripartite motif-containing 27 (Trim27) is highly expressed in tumor cells and regulates natural immunity and apoptosis. However, the effects of Trim27 in cardiac hypertrophy are not fully elucidated. In this study, we tried to explore the potential role of Trim27 in pressure overload-induced cardiac hypertrophy and the underlying mechanism. The results indicated that compared to sham operation (Sham) group, transverse aortic constriction (TAC) group showed significantly up-regulated Trim27 protein expression (P < 0.05). The neonatal rat cardiomyocytes (NRCMs) were isolated and stimulated with PBS, angiotensin (AngII) and phenylephrine (PE). NRCMs were collected to detect the protein expression of Trim27. The results were consistent with the results in vivo. Compared to PBS treatment, the expression of Trim27 protein in NRCMs was significantly increased after PE or AngII stimulation (P < 0.05, respectively). Knockout of Trim27 can reduce the size of cardiomyocytes and reduce the proteins expression of ANP, BNP, and ß-MHC, improve cardiac function, and reverse myocardial hypertrophy (P < 0.05). Trim27 may be involved in regulating the development of cardiac hypertrophy. Further results showed that Trim27 can increase the protein expression of phosphorylation of Akt, GSK3ß, mTOR, and P70s6k by interacting with PTEN (phosphatase tensin homolog). These findings revealed that Trim27 can promote cardiac hypertrophy by activating PTEN/Akt/GSK3ß/mTOR signaling pathway.

Insights into the molecular mechanisms regulating mammalian hair cell regeneration.

PURPOSE OF REVIEW: To give an overview of recent advances in mammalian auditory hair cell regeneration. RECENT FINDINGS: Supporting cells act as progenitors to regenerate hair cells in the prehearing mammalian cochlea but not in the mature cochlea. To overcome this developmental obstacle, manipulation of multiple genes and intracellular pathways has been investigated, which has obtained promising data. This review focuses on recent advances in auditory hair cell regeneration, including synergic gene regulation associated with Atoh1 and Notch signaling, epigenetics, and functional recovery of regenerated hair cells. Co-manipulation of genes critical for hair cell development and cell cycle re-entry, including Atoh1, Isl1, Pou4f3, Gata3, Gfi1, P27kip1, RB, Myc, and Notch-signaling genes, has generated hair cell-like cells in the adult cochlea both in vitro and in vivo. The epigenetic mechanism has been studied in hair cell development and regeneration. Regeneration of hair cell function has a very limited progress, which lacks in-vitro and in-vivo electrophysiology data. SUMMARY: Regeneration of adult auditory hair cells remains a major challenge. Manipulation of multiple genes and pathways together with epigenetic regulation might potentially regenerate functional hair cells in the adult mammalian cochlea.

Generation of a Spiral Ganglion Neuron Degeneration Mouse Model.

Spiral ganglion neurons (SGNs) can be injured by a wide variety of insults. However, there still is a lack of degeneration models to specifically damage the SGNs without disturbing other types of cells in the inner ear. This study aims to generate an SGN-specific damage model using the Cre-LoxP transgenic mouse strains. The Cre-inducible diphtheria toxin receptor (iDTR+/+ ) knock-in mouse strain was crossed with a mouse strain with Cre activity specific to neurons (Nefl CreER/CreER ). Expression of the Cre-recombinase activity was evaluated using the reporter mouse strain Ai9 at pre-hearing, hearing onset, and post-hearing stages. Accordingly, heterozygous Nefl CreER/+;iDTR+/- mice were treated with tamoxifen on postnatal days 1-5 (P1-5), followed by diphtheria toxin (DT) or vehicle injection on P7, P14, and P21 to evaluate the SGN loss. Robust tamoxifen-induced Cre-mediated Ai9 tdTomato fluorescence was observed in the SGN area of heterozygous Nefl CreER/+;Ai9+/- mice treated with tamoxifen, whereas vehicle-treated heterozygote mice did not show tdTomato fluorescence. Compared to vehicle-treated Nefl CreER/+;iDTR+/- mice, DT-treated Nefl CreER/+;iDTR+/- mice showed significant auditory brainstem response (ABR) threshold shifts and SGN cell loss. Hair cell count and functional study did not show significant changes. These results demonstrate that the Nefl CreER/CreER mouse strain exhibits inducible SGN-specific Cre activity in the inner ear, which may serve as a valuable SGN damage model for regeneration research of the inner ear.

Generation of new hair cells by DNA methyltransferase (Dnmt) inhibitor 5-azacytidine in a chemically-deafened mouse model.

Regeneration of mature mammalian inner ear hair cells remains to be a challenge. This study aims to evaluate the ability of DNA methyltransferase (Dnmt) inhibitor 5-azacytidine (5-aza) to generate outer hair cells (OHCs) in a chemically-deafened adult mouse model. 5-aza was administrated into the mouse inner ear via the round window. Immunofluorescence was used to examine the expression of hair cell specific proteins following 5-aza treatment. The results showed that in the chemically-deafened mouse cochlea, new OHCs were found post 5-aza treatment, whereas OHCs were completely lost in saline-treated mice. New hair cells expressed multiple hair cell markers included Myosin VIIa, Pou4f3 and Myosin VI. Newly-generated hair cells presented in three cochlear turns and were able to survive for at least six weeks. The effects of new hair cells generation by 5-aza were concentration dependent. Quantitative PCR study indicates that 5-aza may function through Dnmt1 inhibition. The results of this report suggest that the Dnmt inhibitor 5-aza may promote hair cell regeneration in a chemically-deafened mouse model.

Identification of Neural Stem Cells from Postnatal Mouse Auditory Cortex In Vitro.

Auditory signals are processed in multiple central nervous system structures, including the auditory cortex (AC). Development of stem cell biology provides the opportunity to identify neural stem cells (NSCs) in the central nervous system. However, it is unclear whether NSCs exist in the AC. The aim of this study is to determine the existence of NSCs in the postnatal mouse AC. To accomplish this aim, postnatal mouse AC tissues were dissected and dissociated into singular cells and small cell clumps, which were suspended in the culture medium to observe neurosphere formation. The spheres were examined by quantitative real-time polymerase chain reaction and immunofluorescence to determine expression of NSC genes and proteins. In addition, AC-spheres were cultured in the presence or absence of astrocyte-conditioned medium (ACM) to study neural differentiation. The results show that AC-derived cells were able to proliferate to form neurospheres, which expressed multiple NSC genes and proteins, including SOX2 and NESTIN. AC-derived NSCs (AC-NSCs) differentiated into cells expressing neuronal and glial cell markers. However, the neuronal generation rate is low in the culture medium containing nerve growth factor, ∼8%. To stimulate neuronal generation, AC-NSCs were cultured in the culture medium containing ACM. In the presence of ACM, ∼29% AC-NSCs differentiated into cells expressing neuronal marker class III ß-tubulin (TUJ1). It was observed that the length of neurites of AC-NSC-derived neurons in the ACM group was significantly longer than that of the control group. In addition, synaptic protein immunostaining showed significantly higher expression of synaptic proteins in the ACM group. These results suggest that ACM is able to stimulate neuronal differentiation, extension of neurites, and expression of synaptic proteins. Identifying AC-NSCs and determining effects of ACM on NSC differentiation will be important for the auditory research and other neural systems.

Aligned contiguous microfiber platform enhances neural differentiation of embryonic stem cells.

A microfiber platform that is able to enhance neuronal differentiation and guide aligned neurite outgrowths is essential to the repair of nerve damage. To achieve this aim, we utilized biocompatible and biodegradable poly lactic-co-glycolic acid (PLGA) to design a novel Aligned Contiguous Microfiber Platform (ACMFP) as substrates for the neuronal induction of mouse embryonic stem (ES) cells. To generate the ACMFP, a modified micro-fluid chip system was established to control microfiber parameters including fiber diameter, alignment, and the distance between fibers. Further, Pluronic-F127 was applied to the ACMFP system to maintain a stable and highly aligned fiber platform for at least 12 days. We found that the ACMFP can enhance the neuronal differentiation of mouse ES cells. The ACMFP system showed significantly better neurite outgrowth alignment guidance compared to the control substrate. The effects of alignment guidance were inversely proportionate to the diameter of the fiber, with the optimal diameter size of 60 µm. This study demonstrates a novel ACMFP system that can be used as a biomaterial substrate for neurite outgrowth alignment guidance, which may provide a new model for the development of a multidisciplinary treatment option for nerve injuries.

Embryonic Stem Cell-Derived Peripheral Auditory Neurons Form Neural Connections with Mouse Central Auditory Neurons In Vitro via the α2δ1 Receptor.

Integration of stem cell-derived neurons into the central nervous system (CNS) remains a challenge. A co-culture system is developed to understand whether mouse embryonic stem cell (ESC)-derived spiral ganglion neuron (SGN)-like cells (ESNs) synapse with native mouse cochlear nucleus (CN) neurons. A Cre system is used to generate Cop-GFP ESCs, which are induced into ESNs expressing features similar to auditory SGNs. Neural connections are observed between ESNs and CN neurons 4-6 days after co-culturing, which is stimulated by thrombospondin-1 (TSP1). Antagonist and loss-of-function small hairpin RNA studies indicate that the α2δ1 receptor is critical for TSP1-induced synaptogenesis effects. Newly generated synapse-like structures express pre- and post-synaptic proteins. Synaptic vesicle recycling, pair recording, and blocker electrophysiology suggest functional synaptic vesicles, transsynaptic activities, and formation of glutamatergic synapses. These results demonstrate the synaptogenesis capability of ESNs, which is important for pluripotent ESC-derived neurons to form functional synaptic connections to CNS neurons.

Effect of the Shensong Yangxin Capsule on Energy Metabolism in Angiotensin II-Induced Cardiac Hypertrophy.

BACKGROUND: Shensong Yangxin Capsule (SSYX), traditional Chinese medicine, has been used to treat arrhythmias, angina, cardiac remodeling, cardiac fibrosis, and so on, but its effect on cardiac energy metabolism is still not clear. The objective of this study was to investigate the effects of SSYX on myocardium energy metabolism in angiotensin (Ang) II-induced cardiac hypertrophy. METHODS: We used 2 µl (10-6 mol/L) AngII to treat neonatal rat cardiomyocytes (NRCMs) for 48 h. Myocardial α-actinin staining showed that the myocardial cell volume increased. Expression of the cardiac hypertrophic marker-brain natriuretic peptide (BNP) messenger RNA (mRNA) also increased by real-time polymerase chain reaction (PCR). Therefore, it can be assumed that the model of hypertrophic cardiomyocytes was successfully constructed. Then, NRCMs were treated with 1 µl of different concentrations of SSYX (0.25, 0.5, and 1.0 µg/ml) for another 24 h. To explore the time-depend effect of SSYX on energy metabolism, 0.5 µg/ml SSYX was added into cells for 0, 6, 12, 24, and 48 h. Mitochondria was assessed by MitoTracker staining and confocal microscopy. mRNA and protein expression of mitochondrial biogenesis-related genes - Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), energy balance key factor - adenosine monophosphate-activated protein kinase (AMPK), fatty acids oxidation factor - carnitine palmitoyltransferase-1 (CPT-1), and glucose oxidation factor - glucose transporter- 4 (GLUT-4) were measured by PCR and Western blotting analysis. RESULTS: With the increase in the concentration of SSYX (from 0.25 to 1.0 µg/ml), an increased mitochondrial density in AngII-induced cardiomyocytes was found compared to that of those treated with AngII only (0.25 µg/ml, 18.3300 ± 0.8895 vs. 24.4900 ± 0.9041, t = 10.240, P < 0.0001; 0.5 µg/ml, 18.3300 ± 0.8895 vs. 25.9800 ± 0.8187, t = 12.710, P < 0.0001; and 1.0 µg/ml, 18.3300 ± 0.8895 vs. 24.2900 ± 1.3120, t = 9.902, P < 0.0001; n = 5 per dosage group). SSYX also increased the mRNA and protein expression of PGC-1α (0.25 µg/ml, 0.8892 ± 0.0848 vs. 1.0970 ± 0.0994, t = 4.319, P = 0.0013; 0.5 µg/ml, 0.8892 ± 0.0848 vs. 1.2330 ± 0.0564, t = 7.150, P < 0.0001; and 1.0 µg/ml, 0.8892 ± 0.0848 vs. 1.1640 ± 0.0755, t = 5.720, P < 0.0001; n = 5 per dosage group), AMPK (0.25 µg/ml, 0.8872 ± 0.0779 vs. 1.1500 ± 0.0507, t = 7.239, P < 0.0001; 0.5 µg/ml, 0.8872 ± 0.0779 vs. 1.2280 ± 0.0623, t = 9.379, P < 0.0001; and 1.0 µg/ml, 0.8872 ± 0.0779 vs. 1.3020 ± 0.0450, t = 11.400, P < 0.0001; n = 5 per dosage group), CPT-1 (1.0 µg/ml, 0.7348 ± 0.0594 vs. 0.9880 ± 0.0851, t = 4.994, P = 0.0007, n = 5), and GLUT-4 (0.5 µg/ml, 1.5640 ± 0.0599 vs. 1.7720 ± 0.0660, t = 3.783, P = 0.0117; 1.0 µg/ml, 1.5640 ± 0.0599 vs. 2.0490 ± 0.1280, t = 8.808, P < 0.0001; n = 5 per dosage group). The effect became more obvious with the increasing concentration of SSYX. When 0.5 µg/ml SSYX was added into cells for 0, 6, 12, 24, and 48 h, the expression of AMPK (6 h, 14.6100 ± 0.6205 vs. 16.5200 ± 0.7450, t = 3.456, P = 0.0250; 12 h, 14.6100 ± 0.6205 vs. 18.3200 ± 0.9965, t = 6.720, P < 0.0001; 24 h, 14.6100 ± 0.6205 vs. 21.8800 ± 0.8208, t = 13.160, P < 0.0001; and 48 h, 14.6100 ± 0.6205 vs. 23.7400 ± 1.0970, t = 16.530, P < 0.0001; n = 5 per dosage group), PGC-1α (12 h, 11.4700 ± 0.7252 vs. 16.9000 ± 1.0150, t = 7.910, P < 0.0001; 24 h, 11.4700 ± 0.7252 vs. 20.8800 ± 1.2340, t = 13.710, P < 0.0001; and 48 h, 11.4700 ± 0.7252 vs. 22.0300 ± 1.4180, t = 15.390; n = 5 per dosage group), CPT-1 (24 h, 15.1600 ± 1.0960 vs. 18.5800 ± 0.9049, t = 6.048, P < 0.0001, n = 5), and GLUT-4 (6 h, 10.2100 ± 0.9485 vs. 12.9700 ± 0.8221, t = 4.763, P = 0.0012; 12 h, 10.2100 ± 0.9485 vs. 16.9100 ± 0.8481, t = 11.590, P < 0.0001; 24 h, 10.2100 ± 0.9485 vs. 19.0900 ± 0.9797, t = 15.360, P < 0.0001; and 48 h, 10.2100 ± 0.9485 vs. 14.1900 ± 0.9611, t = 6.877, P < 0.0001; n = 5 per dosage group) mRNA and protein increased gradually with the prolongation of drug action time. CONCLUSIONS: SSYX could increase myocardial energy metabolism in AngII-induced cardiac hypertrophy. Therefore, SSYX might be considered to be an alternative therapeutic remedy for myocardial hypertrophy.

A cell therapy approach to substitute neural elements in the inner ear.

Three different donor tissues were tested for their capacity to survive, integrate and differentiate in the adult inner ear. Surviving embryonic dorsal root ganglion cells were found within the spiral ganglion neuron region and along the auditory nerve fibers. In the presence of exogenous nerve growth factor (NGF), the dorsal root ganglion cells formed extensive growth of neurites that seemed to contact the host neurons. Adult neural stem cells survived relative poorly in the inner ear whereas embryonic stem cells showed a somewhat greater capacity for survival and integration. Overall, the survival rate of implanted tissue was quite low in the cochlea. It is concluded that an inner ear cell therapy approach based on the implantation of exogenous cells will require that important survival factors are identified and supplied. In addition, it is possible that the physical properties of the cochlea, e.g., fluid-filled compartments and very limited space for cell proliferation, are unfavorable, at least in the normal cochlea.

Stimulation of synapse formation between stem cell-derived neurons and native brainstem auditory neurons.

Integration of stem cell-derived cells into native cellular environment remains a challenge in the field. This study developed novel methods to co-culture neural stem cell-derived spiral ganglion-like neurons (ScNs) and mouse auditory cochlear nucleus (CN) neurons to understand whether ScNs of the peripheral nervous system (PNS) synapse with CN neurons of the central nervous system (CNS). ScNs were obtained from neural stem cells that were derived from transgenic mouse pre-labeled with enhanced green fluorescent protein (EGFP), whereas CN neurons were from postnatal mouse primary cultures. ScNs and CN neurons were co-cultured  for 4-6 days in the absence or presence of astrocyte-conditioned medium (ACM). Class III ß-tubulin (TUJ1)-expressing connections were found between ScNs and CN neurons. Expression of the synaptic vesicle marker SV2 was significantly increased along connections between ScNs and CN neurons in the presence of ACM. Immunodepletion and knockout studies indicated that thrombospodin-1 played an important role in ACM-exerted synaptogenic effects. Newly-generated synapse-like structures expressed glutamatergic marker VGluT1, pre- and post-synaptic proteins. Synaptic vesicle recycling studies suggested functional synaptic vesicle retrieval. These results reveal that stem cell-derived PNS neurons are able to form functional connections with native CNS neurons, which is critical for stem cell-based neural pathway regeneration.

Expression of Oligodendrocyte Marker during Peripheral-Central Transitional Zone Formation of the Postnatal Mouse Cochlear Nerve.

Objective To better understand oligodendrocyte protein expression along the mouse cochlear nerve in postnatal mice. Study Design In vivo murine study. Setting Research laboratory. Subjects and Methods Swiss Webster mice used at multiple postnatal days (0, 1, 3, 5, 7, 8, 10, 14, 30, and 60). There were 5 replicates at each postnatal day. Cryosection was done to produce sections that included the cochlear nucleus, cochlear nerve, and cochlea in a single sample. Differential interference contrast (DIC) microscopy and immunofluorescence with antibodies specific to the oligodendrocyte protein Olig2 were used to study the cochlear nerve of Swiss Webster mice at postnatal days. Results The myelination of central nervous system projections initiates in close proximity to the peripheral nervous system-central nervous system transitional zone (PCTZ), and oligodendrocytes in neonatal mice are seen with immunohistochemistry peripheral to the DIC-PCTZ interface. Conclusions As the PCTZ migrates from the brain to the cochlea, oligodendrocytes are a part of peripheral extension of central nervous system tissue along the cochlear nerve. Expression of oligodendrocyte marker Oligo2 was observed peripherally to the formation of PCTZ, as determined by DIC microscopy.

Cell replacement therapy in the inner ear.

The mammalian inner ear is vulnerable to genetic disorders and aging, as well as to injuries caused by overstimulation, ototoxic drugs, and viral infections. Due to the poor regeneration of the sensory epithelium and the spiral ganglion neurons in the adult mammalian inner ear, cell replacement therapy strategies have been proposed to compensate for degeneration and loss of sensory and neuronal cells. Transplantation of stem cells and embryonic neurons into the inner ear has revealed that exogenous cells can survive, migrate, differentiate, and extend neuritic projections in the auditory system of adult mammals. These results suggest that cell replacement therapy could provide an effective future treatment alternative for hearing loss and other inner ear disorders.

Epigenetic DNA Demethylation Causes Inner Ear Stem Cell Differentiation into Hair Cell-Like Cells.

The DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-aza) causes genomic demethylation to regulate gene expression. However, it remains unclear whether 5-aza affects gene expression and cell fate determination of stem cells. In this study, 5-aza was applied to mouse utricle sensory epithelia-derived progenitor cells (MUCs) to investigate whether 5-aza stimulated MUCs to become sensory hair cells. After treatment, MUCs increased expression of hair cell genes and proteins. The DNA methylation level (indicated by percentage of 5-methylcytosine) showed a 28.57% decrease after treatment, which causes significantly repressed DNMT1 protein expression and DNMT activity. Additionally, FM1-43 permeation assays indicated that the permeability of 5-aza-treated MUCs was similar to that of sensory hair cells, which may result from mechanotransduction channels. This study not only demonstrates a possible epigenetic approach to induce tissue specific stem/progenitor cells to become sensory hair cell-like cells, but also provides a cell model to epigenetically modulate stem cell fate determination.

Differentiation of Spiral Ganglion-Derived Neural Stem Cells into Functional Synaptogenetic Neurons.

Spiral ganglion neurons (SGNs) are usually damaged in sensorineural hearing loss. SGN-derived neural stem cells (NSCs) have been identified and proposed to differentiate into neurons to replace damaged SGNs. However, it remains obscure whether SGN-NSC-derived neurons (ScNs) are electrophysiologically functional and possess the capability to form neural connections. Here, we found that SGN-derived cells demonstrated NSC characteristics and differentiated into SGN-like glutamatergic neurons. Neurotrophins significantly increased neuronal differentiation and neurite length of ScNs. Patch clamp recording revealed that ScNs possessed SGN-like NaV and HCN channels, suggesting electrophysiological function. FM1-43 staining and synaptic protein immunofluorescence showed ScNs possess the ability to form neural connections. Astrocyte-conditioned medium was able to stimulate ScNs to express synaptic proteins. These data suggested that neurotrophins are able to stimulate postnatal SGN-NSCs to differentiate into functional glutamatergic ScNs with the capability to form synaptic connections in vitro.

Neural cograft stimulates the survival and differentiation of embryonic stem cells in the adult mammalian auditory system.

Mouse embryonic stem (ES) cells were transplanted into the cochlea of adult guinea pigs in order to explore their survival, differentiation, and possible integration with the host tissue. With the purpose of investigating the possible effect of manipulating the local embryonic microenvironment, ES cells were transplanted into the cochlea with or without an embryonic neuronal cograft consisting of dorsal root ganglion (DRG) tissue. To detect the survival and differentiation of ES cells, cells expressing green fluorescent protein (GFP) were used in combination with immunohistochemical detection of a neuronal marker, neural class III beta-tubulin (TUJ1 antibody). At 4 weeks following transplantation implanted ES cells were found close both to the sensory epithelium, and the spiral ganglion neurons (SGNs) with their peripheral dendritic processes projecting to the organ of Corti. There was a significant difference in the number of surviving TUJ1 (+) ES cells between the DRG cograft group and the non-cograft group (P < 0.01, ANOVA). Neurite-like projections were also identified between TUJ1-positive ES cells and the peripheral dendritic processes from SGNs. The results suggest that an embryonic neuronal microenvironment may be one of the key factors in the survival and differentiation of ES cells in the adult auditory system.