Lili Ren

The differentiation of mesenchymal stem cells into inner ear hair cell-like cells in vitro

Abstract Conclusion. Bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into hair cells, and this method of culturing MSCs provides a useful tool for studies on mammalian cochlear hair cell regeneration. Objective: To investigate a method to induce bone marrow MSCs to differentiate into inner ear hair cells. Methods: Rat bone marrow MSCs were isolated from healthy rats and cultured in vitro. To make sure that the cultured cells were bone marrow MSCs, the expression of MSC markers such as SH2, CD31, CD34, and CD44 genes on the cultured cells was assessed by RT-PCR. Adipogenic cells and osteogenic cells were induced by the differentiation of the cultured cells, respectively, suggesting that the cultured cells have the characteristic of pluripotent differentiation. Then they were induced to differentiate into neural stem cells and hair cell progenitor cells. Immunohistochemistry experiments were carried out to detect the expression of molecular markers. Scanning electron microscope samples were prepared for observation of the morphology of the cells. Results: Rat bone marrow MSCs were successfully isolated, purified, cultured, and identified in vitro. They were also successfully induced to differentiate into neural progenitor cells and then hair cell-like cells that expressed myosin VIIa.

The Morphology and Electrophysiology of the Cochlea of the Miniature Pig

To report the cochlear morphology and electrophysiology of Chinese experimental miniature pigs. Twenty Chinese experimental miniature pigs were used in this study. Auditory brainstem responses (ABR), cochlear endolymphatic potentials (EP), and the potassium concentrations of cochlear endolymph were recorded. Hair cell morphology was examined using electron microscopy. The capsule of cochlea of the miniature pig has three and one‐half turns which contains a 39‐mm long membranous labyrinth. The organ of Corti in the labyrinth encompasses three rows of outer hair cells and one row of inner hair cells. The stereocilia of the hair cells in the apical turn of the cochlea were significantly longer than those in the basal turn. The vestibular apparatus consists of three semicircular canals and the otolith organs. The average threshold of the ABR was 35–45 dB SPL (n = 20) from 4 to 32 kHz. There was no significant difference in the threshold or latency of the ABR between 1‐day‐old and 30‐day‐old miniature pigs. The average EP value was 77.3 ± 14 mV (n = 9) and the average potassium concentration was 147.1 ± 13 mM (n = 5) recorded from the second turn of the cochlea. These studies on the cochlear morphology and electrophysiology of the miniature pigs help to establish the Chinese experimental miniature pig as an animal model for future studies in otology and audiology. Anat Rec, 298:494–500, 2015.

Migration and differentiation of mouse embryonic stem cells transplanted into mature cochlea of rats with aminoglycoside-induced hearing loss

Abstract Conclusion: Mouse embryonic stem cells (ESCs) transplanted into the scala tympani are able to migrate in the cochlea of rats deafened with aminoglycoside and partly restore the structure of sensory epithelia of the inner ear. Objectives: To explore the migration and differentiation of enhanced green fluorescence protein (EGFP)-expressing ESCs by transplanting them into the scala tympani of rats with amikacin sulfate-induced hearing loss. Methods: Adult Sprague-Dawley (SD) rats were deafened with amikacin sulfate. Mouse ESCs expressing EGFP (EGFP-ESCs) were transplanted into the scala tympani. The migration and differentiation were observed at different time points. Results: EGFP-ESCs transplanted into normal cochlea did not migrate, but those in the amikacin-damaged cochlea could survive and migrate into the scala media and the vestibular cisterna. For the first time, we observed that the EGFP-ESCs migrated into the scala media, took the place of the organ of Corti, and formed a structure just like the cochlear tunnel. Some grafted stem cells even expressed myosin VIIa, the molecular marker of hair cells. Some nerve fibers reached to the bottom of the hair cell-like cells. The ESCs migrated into the vestibule and restored the sensory epithelia of the ampullary crest. The number of the transplanted ESCs reduced over the 6 week period of the study.

The diagnosis and surgical treatment of occult otogenic CSF leakage

Abstract Conclusions: The diagnosis of occult otogenic cerebrospinal fluid (CSF) leakage is challenging and it can easily be misdiagnosed. Some characteristics of clinical presentation can supply important clues and confirmed diagnosis should be obtained according to these clues and suitable imaging studies before meningitis develops. Different surgical techniques should be adopted to treat the CSF leakage according to different leakage etiologies, and good results can be obtained. Objective: The aim of the study was to evaluate the diagnosis and surgical treatment of occult otogenic CSF leakage, including the characteristics of clinical presentation, imaging studies, and operation methods in order to decrease the rate of misdiagnosis and obtain a good curative effect. Methods: We performed a retrospective review of 11 cases of CSF leakage that were all misdiagnosed and accompanied by meningitis, operated in our department from 2007 to 2012 after a mean follow-up of 3 years. In this context, the characteristics of clinical presentation, imaging studies, and management of CSF leakage were studied. Results: The CSF leakage had arisen traumatically (n = 9) or congenitally (n = 2). The medical history and special clinical presentation such as repeated otorrhea or rhinorrhea, fever, headache, and unilateral deafness can supply important diagnostic clues. Imaging studies including high-resolution noncontrast CT (HRCT), CT cisternography, and magnetic resonance imaging (MRI) are very important diagnostic methods. The surgical repairs were performed via a transmastoid approach (n = 8), packing the vestibule (n = 1) or a translabyrithine approach (n = 2). Recurrent leakage did not occur.

The morphological and functional development of the stria vascularis in miniature pigs

The purpose of this study was to examine the morphological and functional development of the lateral wall of the scala media of the cochlea in miniature pigs; light and transmission electron microscopy and electrophysiology were used for this purpose. We showed that the lateral wall of the scala media of the cochlea appears at embryonic Day 21 (E21) when the cochlear duct begins to form. From E28 to E49, the lateral wall can be distinguished according to its position along the cochlea. At E56, cells in the lateral wall begin to differentiate into three different types. At E70, three cell types, marginal, intermediate and basal, can be clearly distinguished. At E91, the stria vascularis is adult-like and the organ of Corti is also morphologically mature. The average endocochlear potential measured from the second turn of the cochlea (at E98, postnatal Day 1 (P1), P13 and P30) was 71.4 ± 2.5 (n = 7), 78.8 ± 1.5 (n = 10), 77.3 ± 2.3 (n = 10) and 78.0 ± 2.1 mV (n = 10), respectively. Our results suggest that in miniature pigs the stria vascularis develops during the embryonic period, concurrent with maturation of the organ of Corti. The magnitude of the endocochlear potential reached its mature level when the stria vascularis was morphologically adult-like at E98. These findings provide a morphological and functional basis for future animal studies using the miniature pig model concerning the pathogenesis of various inner-ear diseases.The purpose of this study was to examine the morphological and functional development of the lateral wall of the scala media of the cochlea in miniature pigs; light and transmission electron microscopy and electrophysiology were used for this purpose. We showed that the lateral wall of the scala media of the cochlea appears at embryonic Day 21 (E21) when the cochlear duct begins to form. From E28 to E49, the lateral wall can be distinguished according to its position along the cochlea. At E56, cells in the lateral wall begin to differentiate into three different types. At E70, three cell types, marginal, intermediate and basal, can be clearly distinguished. At E91, the stria vascularis is adult-like and the organ of Corti is also morphologically mature. The average endocochlear potential measured from the second turn of the cochlea (at E98, postnatal Day 1 (P1), P13 and P30) was 71.4±2.5 (n=7), 78.8±1.5 (n=10), 77.3±2.3 (n=10) and 78.0±2.1 mV (n=10), respectively. Our results suggest that in miniature pigs the stria vascularis develops during the embryonic period, concurrent with maturation of the organ of Corti. The magnitude of the endocochlear potential reached its mature level when the stria vascularis was morphologically adult-like at E98. These findings provide a morphological and functional basis for future animal studies using the miniature pig model concerning the pathogenesis of various inner-ear diseases.

The temporal bone microdissection of miniature pigs as a useful large animal model for otologic research

Abstract Conclusion: Compared with traditional animal models, the miniature pig may be a better model for biomedical research because its morphology has many similarities with that of humans. Objective: To investigate the suitability of the miniature pig as an animal model for otological research as regards morphology. Methods: Microdissection of the temporal bone of 10 miniature pigs was performed and recorded on photographs. Results: The morphology and measurements of the external, middle, and inner ear, and the lateral recess of the miniature pigs were completed by microdissection. The temporal bone structures, including the external, middle, inner ear, and the lateral recess, were similar in the miniature pig and humans.

Establishing the standard method of cochlear implant in Rongchang pig

Abstract Conclusions: In this investigation, a large mammal, Rongchang pigs were used to successfully establish a research platform for cochlear implant study on the routine use of it in clinic. Objective: The aim of this study was to establish a standard method of cochlear implant in a large mammal—pig. Methods: Rongchang pigs were selected, then divided into two groups: normal-hearing group (Mitf +/+) and mutation group with hearing loss (Mitf −/−). Cochlear implants were used and ABR and EABR were recorded. The implanted electrodes were observed by X-ray and HE stains. Results: The success with cochlear implant and the best electrode position could be defined in all animals, the coiling of the cochlea reached 1.5–1.75 turns. Immediately after the operation of cochlear implants, the ABR threshold of the operated ear (right) could not be derived for each frequency at 120 dB SPL. Moreover, 7 days after surgery, the low-frequency ABR threshold of the operated ear (right) could be derived partly at 100 dB SPL, but the high-frequency ABR threshold could not be derived at 120 dB SPL. Immediately or 1 week after cochlear implants, the EABR threshold was 90 CL in the Mitf +/+ group. This was obviously lower than the 190 CL in the Mitf −/− group.

Transcript variants and expression profiles analysis of Mitf gene in minipigs

Object To identify transcript variants and expression patterns of porcine Mitf. Materials and methods A pairwise BLAST search at NCBI database was performed to deduce the structure of porcine Mitf gene. Subsequently, 5′ RACE and fluorescent quantitative RT-PCR were used to analyze the expression pattern of porcine Mitf in different tissues. Results Four transcript variants of porcine Mitf, MITF-A, MITF-H, MITF-M and MITF-SUS were identified, all sharing high homology with those in humans, except Mitf-SUS. Conclusion The sequence of porcine Mitf appear highly homologous to human MITF. However, only 4 transcript variants of porcine Mitf were identified in these minipigs, less than the 9 transcript variants in human MITF.