Hyong-Ho Cho

Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin.

BackgroundAdult mesenchymal stem cells (MSCs) derived from adipose tissue have the capacity to differentiate into mesenchymal as well as endodermal and ectodermal cell lineage in vitro. We characterized the multipotent ability of human adipose tissue-derived stem cells (hADSCs) as MSCs and investigated the neural differentiation potential of these cells.ResultsHuman ADSCs from earlobe fat maintained self-renewing capacity and differentiated into adipocytes, osteoblasts, or chondrocytes under specific culture conditions. Following neural induction with bFGF and forskolin, hADSCs were differentiated into various types of neural cells including neurons and glia in vitro. In neural differentiated-hADSCs (NI-hADSCs), the immunoreactivities for neural stem cell marker (nestin), neuronal markers (Tuj1, MAP2, NFL, NFM, NFH, NSE, and NeuN), astrocyte marker (GFAP), and oligodendrocyte marker (CNPase) were significantly increased than in the primary hADSCs. RT-PCR analysis demonstrated that the mRNA levels encoding for ABCG2, nestin, Tuj1, MAP2, NFL, NFM, NSE, GAP43, SNAP25, GFAP, and CNPase were also highly increased in NI-hADSCs. Moreover, NI-hADSCs acquired neuron-like functions characterized by the display of voltage-dependent tetrodotoxin (TTX)-sensitive sodium currents, outward potassium currents, and prominent negative resting membrane potentials under whole-cell patch clamp recordings. Further examination by RT-PCR showed that NI-hADSCs expressed high level of ionic channel genes for sodium (SCN5A), potassium (MaxiK, Kv4.2, and EAG2), and calcium channels (CACNA1C and CACNA1G), which were expressed constitutively in the primary hADSCs. In addition, we demonstrated that Kv4.3 and Eag1, potassium channel genes, and NE-Na, a TTX-sensitive sodium channel gene, were highly induced following neural differentiation.ConclusionsThese combined results indicate that hADSCs have the same self-renewing capacity and multipotency as stem cells, and can be differentiated into functional neurons using bFGF and forskolin.

Effect of neural-induced mesenchymal stem cells and platelet-rich plasma on facial nerve regeneration in an acute nerve injury model.

The purpose of this study was to investigate the effects of platelet‐rich plasma (PRP) and neural‐induced human mesenchymal stem cells (nMSCs) on axonal regeneration from a facial nerve axotomy injury in a guinea pig model.

Transplantation of Neural Differentiated Human Mesenchymal Stem Cells into the Cochlea of an Auditory-neuropathy Guinea Pig Model

The aim of this study was to determine the effects of transplanted neural differentiated human mesenchymal stem cells (hMSCs) in a guinea pig model of auditory neuropathy. In this study, hMSCs were pretreated with a neural-induction protocol and transplanted into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. A control model was made by injection of Hanks balanced salt solution alone into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. We established the auditory neuropathy guinea pig model using 1 mM ouabain application to the round window niche. After application of ouabain to the round window niche, degeneration of most spiral ganglion neurons (SGNs) without the loss of hair cells within the organ of Corti and increasing the auditory brain responses (ABR) threshold were found. After transplantation of neural differentiated hMSCs, the number of SGNs was increased, and some of the SGNs expressed immunoreactivity with human nuclear antibody under confocal laser scanning microscopy. ABR results showed mild hearing recovery after transplantation. Based on an auditory neuropathy animal model, these findings suggest that it may be possible to replace degenerated SGNs by grafting stem cells into the scala tympani.

Otologic complications caused by hearing aid mold impression material.

We report two extremely rare cases of otologic complications caused by hearing aid mold impression material. The symptoms of patients with retained impression material are characteristic of the length of time the impression material is retained. In case 1 had a chronic discharge and granulation tissue of the middle ear, while case 2 presented with acute pain and dizziness. The management for retained impression material may require surgical interventions, which can be safely accomplished by standard otologic techniques.

Non-canonical Wnt mediated neurogenic differentiation of human bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BM-MSCs), which are characterized by multipotency and self-renewal, are responsible for tissue regeneration and repair. We have previously reported in adipose tissue-derived MSCs that only Wnt5a is enhanced at neurogenic differentiation, and the mechanism of differentiation is dependent on the Wnt5a/JNK pathway; however, the role of Wnt/MAPK pathway is yet to be investigated in neurogenic differentiation in BM-MSCs. We compared the transcriptional expression of Wnt in neurogenic induced-hBM-MSCs (NI-hBM-MSCs) with that in primary hBM-MSCs, using RT-PCR, qPCR, and western blotting. Although the expression of Wnt1 and Wnt2 was unchanged, the expression of Wnt4, Wnt5a, and Wnt11 increased after neurogenic differentiation. In addition, only the expression of frizzled class receptor (Fzd) 3 gene was increased, but not of most of the Fzds and Wnt ligands in NI-hBM-MSCs. Interestingly, Wnt4, Wnt5a, and Wnt11 gene expressions significantly increased in NI-hBM-MSCs by qPCR. In addition, the protein expression level of Wnt4 and Wnt5a, but not Wnt3, increased after neurogenic induction. Furthermore, the expressions of phosphorylated-GSK-3β, ERK1/2, and PKC decreased; however, JNK was activated after neurogenic differentiation. Thus, non-canonical Wnts, i.e., Wnt4, Wnt5a, and Wnt11, regulate neurogenic differentiation through Fzd3 activation and the increase in downstream targets of JNK, which is one of the non-canonical pathways, in hBM-MSCs.

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

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.

Transplantation of human adipose tissue-derived stem cells for repair of injured spiral ganglion neurons in deaf guinea pigs

Excessive noise, ototoxic drugs, infections, autoimmune diseases, and aging can cause loss of spiral ganglion neurons, leading to permanent sensorineural hearing loss in mammals. Stem cells have been confirmed to be able to differentiate into spiral ganglion neurons. Little has been reported on adipose tissue-derived stem cells (ADSCs) for repair of injured spiral ganglion neurons. In this study, we hypothesized that transplantation of neural induced-human ADSCs (NI-hADSCs) can repair the injured spiral ganglion neurons in guinea pigs with neomycin-induced sensorineural hearing loss. NI-hADSCs were induced with culture medium containing basic fibroblast growth factor and forskolin and then injected to the injured cochleae. Guinea pigs that received injection of Hanks′ balanced salt solution into the cochleae were used as controls. Hematoxylin-eosin staining showed that at 8 weeks after cell transplantation, the number of surviving spiral ganglion neurons in the cell transplantation group was significantly increased than that in the control group. Also at 8 weeks after cell transplantation, immunohistochemical staining showed that a greater number of NI-hADSCs in the spiral ganglions were detected in the cell transplantation group than in the control group, and these NI-hADSCs expressed neuronal markers neurofilament protein and microtubule-associated protein 2. Within 8 weeks after cell transplantation, the guinea pigs in the cell transplantation group had a gradually decreased auditory brainstem response threshold, while those in the control group had almost no response to 80 dB of clicks or pure tone burst. These findings suggest that a large amount of NI-hADSCs migrated to the spiral ganglions, survived for a period of time, repaired the injured spiral ganglion cells, and thereby contributed to the recovery of sensorineural hearing loss in guinea pigs.

Efficacy of Concomitant Intratympanic Steroid Injection for Sudden Deafness According to Initial Hearing Loss.

Objectives/Hypothesis: We identified the efficacy and appropriate target group for intratympanic steroid injections (ITS) in patients with idiopathic sudden sensorineural hearing loss (ISSNHL). Study Design: Retrospective case-control study. Methods: Fifty-five ears treated with concomitant ITS and systemic steroids and 165 ears without ITS treatment were identified. Each case had three controls matched according to the age, sex, and the presence of vertigo. Patients were divided into subgroups according to pure tone audiometric configuration and the levels of initial hearing loss. Hearing results and frequency-related hearing gain were investigated. Results: Additional ITS was effective for patients with an ascending type audiogram or patients with Grade 3 (60–90 dB) initial hearing loss. Concomitant ITS, however, resulted in a negative effect on hearing in patients with Grades 1 and 2 (<60 dB). ITS resulted in significant (p < 0.05) hearing improvement at 250 Hz in the Grade 3 (60–90 dB) and Grade 4 (>90 dB) ITS subgroups. Conclusions: The efficacy of additional ITS for ISSNHL was different among subgroups with various levels of initial hearing loss or audiogram configuration.

Donor-Site Morbidity Following Minimally Invasive Costal Cartilage Harvest Technique

Objectives Autologous costal cartilage is a promising alternative for mastoid obliteration. However, donor-site morbidities of the chest wall limit the use of this graft. To address this issue, we have developed a minimally-invasive technique of harvesting costal cartilage and report donor site morbidity associated with the procedure. Methods Donor site morbidities were evaluated for 151 patients who underwent costal cartilage harvest, canal wall down mastoidectomy, and mastoid obliteration. Pain and cosmetic concern were evaluated via visual analogue scale (VAS). Scars were evaluated via the modified Vancouver Scar Scale (VSS) and the Patient and Observer Scar Assessment Scale (POSAS). Postoperative complications were assessed during the follow-up period. Results The mean duration of noticeable pain was 5.3 days post operation. The mean VAS score for pain was 3.0 of 10 on the first day after the operation and gradually declined. At the 6 months post operation, the mean VAS cosmetic score at the costal cartilage harvest site was 0.6 of 10. The mean VSS score was 9.5 out of 10 total, and the mean POSAS score was 23.27 out of 110 total. Conclusion The minimally-invasive chopped costal cartilage harvest technique resulted in acceptable pain, cosmetic concern, and postoperative complications for most patients. There were no major postoperative complications. Costal cartilage is an acceptable donor for mastoid obliteration in canal wall down mastoidectomy, especially in the context of the extremely low donor site morbidity of the minimally-invasive technique presented in the study.

Atoh1 as a Coordinator of Sensory Hair Cell Development and Regeneration in the Cochlea

Cochlear sensory hair cells (HCs) are crucial for hearing as mechanoreceptors of the auditory systems. Clarification of transcriptional regulation for the cochlear sensory HC development is crucial for the improvement of cell replacement therapies for hearing loss. Transcription factor Atoh1 is the key player during HC development and regeneration. In this review, we will focus on Atoh1 and its related signaling pathways (Notch, fibroblast growth factor, and Wnt/β-catenin signaling) involved in the development of cochlear sensory HCs. We will also discuss the potential applicability of these signals for the induction of HC regeneration.