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This study investigated the potential of bone-marrow stromal cell transplantation for cell replacement therapy in the cochlea. Bone-marrow stromal cells labeled with enhanced green fluorescent protein were injected into the perilymphatic space of normal cochleae in mice. Histological analysis 2 weeks after transplantation demonstrated that transplanted cells settled within the cochlear tissues, especially in the spiral ligament and the spiral limbus, although most transplants were located in the perilymphatic space. Some of the transplanted cells expressed the cochlear gap-junction protein connexin 26. These findings indicate the potential of bone-marrow stromal cells for delivering therapeutic molecules and for the restoration of cochlear cells, particularly in the spiral ligament and the spiral limbus.
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... Bone marrow MSCs were cultured according to the standard protocol . Briefly, femur and tibia were flushed with DMEM supplemented with 10% fetal bovine serum (FBS), 4mM L-glutamine, 1mM sodium pyruvate, 100 units/ml penicillin, and 100μg/ml streptomycin. ...
... In the present study, we demonstrated an improved and cytokine-free method for the regeneration of hepatic-like cells from BMMSCs by NaBu pre-treatment in the 3D collagen scaffold. In this study, rat bone marrow was used for the isolation of MSCs in accordance with a previously published protocol . Immunocytochemistry and flow cytometry analysis showed that bone marrow cells expressed MSC-specific surface markers CD29, CD90, vimentin, and CD44, whereas hematopoietic marker CD45 was not detected. ...
Stem cell-based therapy is considered an attractive tool to overcome the burden of liver diseases. However, efficient hepatic differentiation is still a big challenge for the research community. In this study, we explored a novel method for differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into hepatic-like cells using 3D culture conditions and histone deacetylase inhibitor, sodium butyrate (NaBu). MSCs were characterized by the presence of cell surface markers via immunocytochemistry, flow cytometry, and by their potential for osteogenic, adipogenic, and chondrogenic differentiation. MSCs were treated with 1mM NaBu in 2D and 3D environments for 21 days. The hepatic differentiation was confirmed by qPCR and immunostaining. According to qPCR data, the 3D culture of NaBu-treated MSCs has shown significant upregulation of hepatic gene, CK-18 (P < 0.01), and hepatic proteins, AFP (P < 0.01) and ALB (P < 0.01). In addition, immunocytochemistry analysis showed significant increase (P < 0.05) in the acetylation of histones (H3 and H4) in NaBu-pretreated cells. It can be concluded from the study that NaBu-treated MSCs in 3D culture conditions can induce hepatic differentiation without the use of additional cytokines and growth factors. The method shown in this study represents an improved protocol for hepatic differentiation and could contribute to improvement in future cell-based therapeutics.
... Adult stem cells also have the potential to deliver gene and therapeutic molecules to other parts of the inner ear. For example, Connexin 26, a protein present in cochlear gap junctions and supporting cells was expressed when bone marrow stromal cells were transplanted into the perilymphatic space of the mouse cochlea . Bone marrow mesenchymal stem cells also have the potential to differentiate into auditory neurons in vitro and in vivo [106,107], demonstrating that a wide variety of inner ear cell types can be generated from stem cells. ...
... These cells are not concerned with any ethical issues and can enter clinical trials involving autologous transplantation therapies and used in bioengineered products. In treating inner ear disorders, bone marrow-derived stem cells have shown the most favorable results [105,106,110,111]. ...
Sensory neural hearing loss and vestibular dysfunction have become the most common forms of sensory defects, affecting millions of people worldwide. Developing effective therapies to restore hearing loss is challenging, owing to the limited regenerative capacity of the inner ear hair cells. With recent advances in understanding the developmental biology of mammalian and non-mammalian hair cells a variety of strategies have emerged to restore lost hair cells are being developed. Two predominant strategies have developed to restore hair cells: transfer of genes responsible for hair cell genesis and replacement of missing cells via transfer of stem cells. In this review article, we evaluate the use of several genes involved in hair cell regeneration, the advantages and disadvantages of the different viral vectors employed in inner ear gene delivery and the insights gained from the use of embryonic, adult and induced pluripotent stem cells in generating inner ear hair cells. Understanding the role of genes, vectors and stem cells in therapeutic strategies led us to explore potential solutions to overcome the limitations associated with their use in hair cell regeneration.
... There are few previous reports of experiments on the transplanting MSCs into the cochlea . These studies used the primary undifferentiated MSCs and showed that primary MSCs can survive in normal or damaged inner ear. ...
... However, the distribution of transplant-derived MSCs was different from the experiments. In detail, engrafted MSCs were localized mostly in the perilymphatic space of cochleae in normal mice . However, in the animal of damaged cochlea, stem cells were found mainly in the modiolus and lateral wall [25,26]. ...
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.
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.
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.
The results show the potential of NI-hMSCs to give rise to replace the lost cochlear cells in hearing loss mammals.
... We review previous reports and discuss obstacles to overcome for successful functional recovery. Several kinds of pluripotent stem cells have been delivered into the cochlea for the regeneration of SGNs, including NSCs [10,13,46], ESCs [4,5,11,12,29,34,36,38], bone marrow stem cells (BMSCs) [26,28,40], and iPSCs . Tamura et al., evaluated the ability of NSCs to achieve neural differentiation in the modiolus of the cochlea and demonstrated that some grafted NSCs expressed β-III tubulin, a neuronal marker, although the majority of them differentiated into glial cells . ...
... BMSCs, which can be readily obtained from an individual's own bone marrow, are also good candidates as transplants, because recent studies have shown that BMSCs can produce not only osteoblasts, chondrocytes, adipocytes, or myoblasts, but also neurons [15,16]. The survival of autologous BMSCs grafted in the cochlea was proven [26,28,40]. The enhanced survival of BMSCs was confirmed in deafened cochleae . ...
... The major findings from these studies are summarized in Table 1, and the reader is referred back to Figure 1 for anatomical details. Several stem cell types have now been delivered into the mammalian cochlea for the replacement of auditory neurons, including bone marrow stem cells [72,81], neural stem cells [73,74,76,77], and embryonic stem cells [71, 74, 75, 78 -80]. Although transplantation methodology varies among publications, all authors report the survival of exogenous stem cells in the inner ear for periods of between 3 and 13 weeks. ...
Sensory hair cells in the mammalian cochlea are sensitive to many insults including loud noise, ototoxic drugs, and ageing. Damage to these hair cells results in deafness and sets in place a number of irreversible changes that eventually result in the progressive degeneration of auditory neurons, the target cells of the cochlear implant. Techniques designed to preserve the density and integrity of auditory neurons in the deafened cochlea are envisaged to provide improved outcomes for cochlear implant recipients. This review examines the potential of embryonic stem cells to generate new neurons for the deafened mammalian cochlea, including the directed differentiation of stem cells toward a sensory neural lineage and the engraftment of exogenous stem cells into the deafened auditory system. Although still in its infancy the aim of this therapy is to restore a critical number of auditory neurons, thereby improving the benefits derived from a cochlear implant.Disclosure of potential conflicts of interest is found at the end of this article.
... In another similar experiment, some of the implanted cells expressed conexin-26 protein, which belongs to the intercellular junction of support cells and fibroblasts of Corti cells and essential for the maintenance of endocochlear potential. Such finding indicates a possible use of transplant of bone marrow SC to restore intercellular cells in the cochlear conjunctive tissues (19). ...
... In this study, the cochlear bony walls of living mice were exposed through the removal of the bulla. Mice are known to survive after the removal of the bulla, even when followed by cell transplantation (21,22). Furthermore, we showed that repeated visualization of the cochlea in living mice 3 or 7 days after the first visualization was possible by this method without inducing endolymphatic hydrops, otitis media, or labyrinthitis. ...
Cochlear pathology can be evaluated in living animals using optical coherence tomography (OCT).
The current imaging methods available for the detailed analysis of cochlear pathology in a clinical setting provide only limited information. Thus, a cochlear imaging modality with high definition is needed for improving the diagnosis of cochlear pathology. OCT has been used in other fields for obtaining high-resolution subsurface images, and its use could potentially be extended to the analysis of cochlear pathogenesis.
Slc26a4 mice, which generate endolymphatic hydrops, and their littermates were used in this study. Auditory function was monitored by the auditory brainstem responses (ABR). After the mice were placed under general anesthesia, OCT images of the cochlea were captured. The cochlea was subsequently dissected out and histologically evaluated. Three or 7 days later, the wild-type mice cochleae were visualized again.
In ABR assessments, Slc26a4 mice showed severe hearing loss, while no significant hearing loss was found in Slc26a4 or Slc26a4 mice. OCT demonstrated normal morphology in the cochlea of both Slc26a4 and Slc26a4 mice, including the location of Reissner's membrane. Meanwhile, in Slc26a4 mice, obvious dislocation of Reissner's membrane was observed, indicating severe endolymphatic hydrops. These findings in the OCT images were consistent with the histologic results for the cochlear morphology, as observed with hematoxylin and eosin staining. Three or 7 days later, wild-type cochleae were successfully visualized using OCT, and no otitis media or labyrinthitis was observed.
OCT can be applied in the detection of endolymphatic hydrops in living mice, indicating the potential of OCT for cochlear imaging analyses for clinical use in the near future.
... Furthermore, BMSCs have been shown to be useful for the treatment of inner ear inflammatory damage. Sharif et al.  showed that BMSCs carrying the enhanced green fluorescent protein (EGFP) gene transplanted into the inner ear of healthy C57BL/6 mice migrated to the cochlea. Despite the distribution of transplanted cells in the perilymphatic space, fluorescence was also detected in the spiral ligament and spiral limb, which was consistent with the fluorescence distribution observed in our study. ...
Bone marrow mesenchymal stem cells (BMSCs) expressing recombinant IL-4 have the potential to remediate inflammatory diseases. We thus investigated whether BMSCs expressing exogenous IL-4 could alleviate autoimmune sensorineural hearing loss. BMSCs isolated from guinea pigs were transfected with recombinant lentivirus expressing IL-4. A total of 33 animals were divided into three groups. Group A received scala tympani injection of IL-4-expressing BMSCs, and Group B received control vector-expressing BMSCs, and Group C received phosphate-buffered saline. The distribution of implanted BMSCs in the inner ears was assessed by immunohistochemistry and fluorescence microscopy. Auditory brain-stem response (ABR) was monitored to evaluate the auditory changes. Following BMSCs transplantation, the threshold levels of ABR wave III decreased in Groups A and B and significant differences were observed between these two groups
. Transplanted BMSCs distributed in the scala tympani and scala vestibuli. In some ears with hearing loss, there was a decrease in the number of spiral ganglion cells and varying degrees of endolymphatic hydrops or floccule. Following transplantation, the lentivirus-infected BMSCs migrated to the inner ear and produced IL-4. Our results demonstrate that, upon transplantation, BMSCs and BMSCs expressing recombinant IL-4 have the ability to remediate the inflammatory injury in autoimmune inner ear diseases.
... These cells can enter clinical trials involving autologous transplantation therapies and be used in bioengineered products. In treating inner ear disorders, bone marrowderived stem cells have shown the most favorable results (69,70,74,75). ...
... AS cells also have the potential to deliver gene and therapeutic molecules to other parts of the inner ear. For example, a protein present in cochlear gap junctions and supporting cells, Connexin 26, was expressed when bone marrow stromal cells were transplanted into the perilymphatic space of the mouse cochlea . AS cells isolated from olfactory neuroepithelium expressed hair cell markers and bear a phenotypic resemblance to hair cells when cocultured with cochlear cell . ...
... GFP transgenic mouse BMSCs transplantation into the perilymphatic space of normal cochleae in mice displayed that transplanted cells could settle within the cochlear tissues, especially in the SLs and the spiral limbus, although most transplants were located in the perilymphatic space. Some of the transplanted cells expressed the cochlear gap-junction protein connexin 26, indicating their potential for restoration of cochlear cells . Tan et al. in autoimmune deafened adult Guinea pig by use of IL-4-expressing BMSCs into scala tympani, via lateral wall, reported homing and survival capability of cells to the deafened cochlea, and transdifferentiation of them to any cochlea cell types . ...
Otorhinolaryngology enrolls head and neck surgery in various tissues such as ear, nose, and throat (ENT) that govern different activities such as hearing, breathing, smelling, production of vocal sounds, the balance, deglutition, facial animation, air filtration and humidification, and articulation during speech, while absence of these functions can lead to high morbidity and even mortality. Conventional therapies for head and neck damaged tissues include grafts, transplants, and artificial materials, but grafts have limited availability and cause morbidity in the donor site. To improve these limitations, regenerative medicine, as a novel and rapidly growing field, has opened a new therapeutic window in otorhinolaryngology by using cell transplantation to target the healing and replacement of injured tissues. There is a high risk of rejection and tumor formation for transplantation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs); mesenchymal stem cells (MSCs) lack these drawbacks. They have easy expansion and antiapoptotic properties with a wide range of healing and aesthetic functions that make them a novel candidate in otorhinolaryngology for craniofacial defects and diseases and hold immense promise for bone tissue healing; even the tissue sources and types of MSCs, the method of cell introduction and their preparation quality can influence the final outcome in the injured tissue. In this review, we demonstrated the anti-inflammatory and immunomodulatory properties of MSCs, from different sources, to be safely used for cell-based therapies in otorhinolaryngology, while their achievements and challenges have been described too.
... Bone marrow stem cells (BMSCs) were isolated from GFP expressing transgenic mice (3 months old) as described previously (Sharif et al., 2007). BMSCs were grown until second passage, after which they were used for experiments. ...
Stem cell capability enhanced with cytokine administration is a promising treatment for myocardial infarction. Bone marrow stem cells (BMSCs) were isolated from C57BL/6 mice (8–12 weeks old) expressing GFP and characterized with c-kit and CD34. Infarcted heart tissue fragments were placed into dishes with BMSCs and medium supplemented with G-CSF, SCF, IGF-1 or combinations thereof were given to the BMSC-infarcted myocardium in vitro model. The IGF-1–G-CSF group showed significantly higher migration (67.7% ± 2.6) of c-kit+ BMSCs towards the ischemic tissue and expressed MEF-2 (43.7% ± 1.7). Of the single treatment groups, the G-CSF group demonstrated significantly higher migration of c-kit+ BMSCs (60.5 ± 2.7) with MEF-2 expression (38.7 ± 1.4). IGF-1 complements G-CSF and was relatively more significant in its effects on BMSC migration and cardiac lineage commitment towards ischemic heart tissue.
... Another possibility is that TTFderived iPS cells have a higher risk for tumorigenesis formation. The number of cells that can be transplanted into cochleae is limited because of their tiny size, and than MEF-derived iPS cells, which is consistent with previous observations of transplantation into nonobese the numbers of settled transplant-derived cells in cochleae are reportedly low (4,20,34). Therefore, the diabetic/severe combined immune deficiency (NOD/ SCID) mouse brains (19). ...
This study evaluated the tumorigenesis risk of induced pluripotent stem (iPS) cells after transplantation into the cochlea. One mouse embryonic stem (ES) cell line and three mouse iPS cell lines, one derived from adult mouse tail-tip fibroblasts (TTFs) and two from mouse embryonic fibroblasts (MEFs), were neurally induced by stromal cell-inducing activity. Before transplantation, the efficiency of neural induction and the proportion of residual undifferentiated cells were evaluated using immunocytochemistry, and no significant differences were observed in the ratios of colonies expressing βIII tubulin, nestin, or octamer (Oct)3/4. Four weeks after transplantation into the cochleae of neonatal mice, the number of surviving transplants of TTF-derived iPS cells generated by retroviral infection was significantly higher than those of MEF-derived iPS cells generated by plasmid transfection. Teratoma formation was identified in one of five cochleae transplanted with TTF-derived iPS cells. However, no significant differences were found in the cell-proliferation activity or the extent of differentiation into mature neurons among the cell lines. These findings emphasize the necessity of selecting appropriate iPS cell lines and developing methods to eliminate undifferentiated cells after neural induction, in order to establish safe iPS cell-based therapy for the inner ear.
... Bone marrow-derived mesenchymal stem cells can be differentiated into hepatic, renal, and cardiac lineages (Mohsin et al., 2011;Yeagy and Cherqui, 2011;Choudhery et al., 2012) due to their immense plasticity and easy availability. Potential of transplantation of BMSCs has been demonstrated in the repair of various disorders such as hearing (Sharif et al., 2007), myocardial infarction (Khan et al., 2009), renal failure (Morigi et al., 2008), stroke (Hayase et al., 2009), neuronal defects (Keilhoff et al., 2006), and bone defects (Granero-Molto et al., 2009). Therefore, BMSCs represent an attractive autologous source of cells for cell therapy in liver diseases (Bird et al., 2013). ...
Hepatic oval cells are likely to be activated during advanced stage of liver fibrosis to reconstruct damaged hepatic tissue. However, their scarcity, difficulties in isolation, and in vitro expansion hampered their transplantation in fibrotic liver. This study was aimed to investigate the repair potential of in vitro differentiated hepatic oval-like cells in CCl4 -induced liver fibrosis. BMSCs and oval cells were isolated and characterized from C57BL/6 GFP(+) mice. BMSCs were differentiated into oval cells by preconditioning with HGF, EGF, SCF, and LIF and analyzed for the oval cells-specific genes. Efficiency of oval cells to reduce hepatocyte injury was studied by determining cell viability, release of LDH, and biochemical tests in a co-culture system. Further, in vivo repair potential of differentiated oval cells was determined in CCl4 -induced fibrotic model by gene expression analysis, biochemical tests, mason trichrome, and Sirius red staining. Differentiated oval cells expressed hepatic oval cells-specific markers AFP, ALB, CK8, CK18, CK19. These differentiated cells when co-cultured with injured hepatocytes showed significant hepato-protection as measured by reduction in apoptosis, LDH release, and improvement in liver functions. Transplantation of differentiated oval cells like cells in fibrotic livers exhibited enhanced homing, reduced liver fibrosis, and improved liver functions by augmenting hepatic microenvironment by improved liver functions. This preconditioning strategy to differentiate BMSCs into oval cell leads to improved survival and homing of transplanted cells. In addition, reduction in fibrosis and functional improvement in mice with CCl4 -induced liver fibrosis was achieved.
... The extent of integration of mesenchymal stem cells into host tissues after cochlear transplantation has differed between studies. Although Sharif et al.  described integration of bone marrow-derived stromal cells into mouse cochlear tissues following transplantation via a lateral wall cochleostomy, Matsuoka et al.  reported no integration of mesenchymal stem cells following transplantation via a lateral wall cochleostomy but extensive integration after injection directly into cochlear tissues. Direct transplantation of stem cells into cochlea tissues has not yet been examined in mice because of technical difficulties relating to the size of the murine cochlea. ...
Transplantation of exogenous stem cells has been proposed as a treatment to prevent or reverse sensorineural hearing loss. Here, we investigate the effects of transplantation of adult human olfactory mucosa-derived stem cells on auditory function in A/J mice, a strain exhibiting early-onset progressive sensorineural hearing loss. Recent evidence indicates that these stem cells exhibit multipotency in transplantation settings and may represent a subtype of mesenchymal stem cell. Olfactory stem cells were injected into the cochleae of A/J mice via a lateral wall cochleostomy during the time period in which hearing loss first becomes apparent. Changes in auditory function were assessed 1 month after transplantation and compared against animals that received sham injections. Hearing threshold levels in stem cell-transplanted mice were found to be significantly lower than those of sham-injected mice (p < .05) for both click and pure tone stimuli. Transplanted cells survived within the perilymphatic compartments but did not integrate into cochlear tissues. These results indicate that transplantation of adult human olfactory mucosa-derived stem cells can help preserve auditory function during early-onset progressive sensorineural hearing loss.
... In addition, the transplanted BMSCs migrate to different parts of the cochlea. Consistent findings have been reported regarding the transplantation of the BMSCs 13 . The transplanted BMSCs migrated widely into the spiral ligament, which was used to regenerate the specific fibrocytes in the spiral ligament. ...
In this study, we attempted to differentiated human bone marrow-derived mesenchymal stem cells (hBMSCs) to auditory hair cells using growth factors.
Retinoic acid (RA), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) were added to hBMSCs cell culture medium. The cells were evaluated morphologically and the expression of SOX2, POU4F3, MYO7A, and Calretinin at mRNA level and ATOH1 mRNA and protein expression.
After treatment with the growth factors, the morphology of the cells did not change, but evaluation of gene expression at the mRNA level increased the expression of the ATOH1, SOX2, and POU4F3 markers. Growth factors increased the expression of ATOH1 at the protein level. The expression of calretinin showed decreased and MYO7A no significant change in expression.
hBMSCs have the potential to differentiate to hair cell-like using the RA, bFGF, and EGF.
... Multi differentiation potential of MSCs and their capability to migrate into acute injury location make these cells suitable candidate for gene and cell therapy  . MSCs have been demonstrated to be helpful in treating inner ear inflammatory damage because they exhibit multidirectional differentiation potential, immunosuppressive function and low immunogenicity 75 . Among MSCs, BMSCs have been widely studied and are comparably more practical 58 . ...
In this review, we compared the potential of mesenchymal stem cells derived from bone marrow, adipose tissue and umbilical cord as suitable sources for regeneration of inner ear hair cells and auditory neurons. Our intensive literature search indicates that stem cells in some of adult mammalian tissues, such as bone marrow, can generate new cells under physiological and pathological conditions. Among various types of stem cells, bone marrow-derived mesenchymal stem cells are one of the most promising candidates for cell replacement therapy. Mesenchymal stem cells have been reported to invade the damaged area, contribute to the structural reorganization of the damaged cochlea and improve incomplete hearing recovery. We suggest that bone marrow-derived mesenchymal stem cells would be more beneficial than other mesenchymal stem cells.
... The impact of cochlear matrix on the phenotype of genetically modified stem cells was evaluated by injecting human MSC that had been pre-treated with transfection of hath1 and hes1/5 RNAi (Sharif et al., 2007;Parker and Cotanche, 2004) in decellularized cochleae. The pre-treatment of MSC led to a decreased number of cells engrafting within the cochlear scaffold when compared to the delivery of native untreated MSC (Fig. 4 vs. ...
Transplantation of mesenchymal stromal cells (MSC) presents a promising approach not only for the replacement of lost or degenerated cells in diseased organs but also for local drug delivery. It can potentially be used to enhance the safety and efficacy of inner ear surgeries such as cochlear implantation. Options for enhancing the effects of MSC therapy include modulating cell behaviour with customized bio-matrixes or modulating their behaviour by ex vivo transfection of the cells with a variety of genes. In this study, we demonstrate that MSC delivered to the inner ear of guinea pigs or to decellularized cochleae preferentially bind to areas of high heparin concentration. This presents an opportunity for modulating cell behaviour ex vivo. We evaluated the effect of carboxymethylglucose sulfate (Cacicol®), a heparan sulfate analogue on spiral ganglion cells and MSC and demonstrated support of neuronal survival and support of stem cell proliferation.
... In another study, bone-marrow stromal cells labeled with enhanced green fluorescent protein injected into the perilymphatic space of normal cochleae in mice. After 2 weeks it has been demonstrated that, transplanted cells settled within the cochlear tissues, especially in the spiral ligament and the spiral limbus, but most transplants were located at perilymphatic space itself . ...
Genetical, ageing, excessive noise and certain antibiotics are account for majority of permanent hearing loss in humans. Hearing loss is caused by dysfunction of the sensory epithelium (the organ of Corti) within the inner ear (cochlea). It is associated with irreversible loss of sensory hair cells and spiral ganglion neurons. Inner ear hair cells are specialized mechanoreceptors converts mechanical stimuli into neural information for transmission to the brain. Several areas of research have addressed the treatment of hearing loss. There are evidences for regeneration of sensory hair cells in non-mammalian species; however, studies in mammals have failed to find evidence of cochlear hair cells regeneration. Stem cell regenerative medicine is the current focus on cure for deafness while through regeneration of specific cell types from different sources of stem cells. This review deled present stem cell regenerative therapy promising outcome on clinical application.
... Mesenchymal stem cells (MSCs) from bone marrow have gained significance because of their immense plasticity and easy availability . Recent studies conducted by our group and others have demonstrated the therapeutic potency of MSCs for numerous disorders such as hearing (Sharif et al., 2007), myocardial infarction (Khan et al., 2009a), renal failure (Morigi et al., 2008), stroke (Hayase et al., 2009), neuronal defects (Keilhoff et al., 2006) and bone defects (Granero-Moltó et al., 2009). Liver diseases are also possible targets for stem cell therapy with the aim to reduce fibrosis induced cirrhosis and liver failure (Wynn, 2008). ...
Liver failure represents a serious challenge for cell based therapies. Mesenchymal stem cells (MSCs) possess potential for regeneration of fibrotic liver; however, there is a dire need to improve their hepatic differentiation. This study examines a pretreatment strategy to augment the differentiation potential of MSCs towards hepatic lineage. MSCs were isolated from C57BL/6 wild type mice and were characterized by flow cytometry for CD44 (92.4%), CD90 (96.6%), CD105 (94.7%), CD45 (0.8%) and CD34 (1.4%) markers. To improve the differentiation potential of MSCs towards hepatic lineage, cells were pretreated with injured liver tissue in an in-vitro model, which resulted in high expression of albumin, cytokeratin 8, 18, TAT and HNF1α as compared to untreated MSCs. The efficacy of pretreated MSCs was evaluated by preparing in-vivo mouse model with liver fibrosis by intraperitoneal administration of CCl(4). Pretreated MSCs were transplanted in the left lateral lobe of mice with liver fibrosis and showed enhanced localization and differentiation abilities after 1 month. The expression for cytokeratin 8, 18, albumin and Bcl-xl was up-regulated and that of HGF, Bax and Caspase- 3 was down-regulated in animals transplanted with pretreated MSCs. Sirus red staining also confirmed a significant reduction in the fibrotic area in liver tissue transplanted with pretreated MSCs as compared to untreated MSCs and was concomitant with improved serum levels of bilirubin and alkaline phosphatase (ALP). Therefore, it was concluded that pretreatment with injured liver tissue augment homing and hepatic differentiation abilities of MSCs and provides an improved procedure for the treatment of liver fibrosis.
... Mesenchymal stem cells (MSCs) possess multilineage differentiation potential [13,14] and provide a good source for cell-based therapies [15,16]. Nevertheless, effect of diabetes on the function of MSCs has not been previously documented and is an area of great interest with implications for the repair of diabetic heart failure. ...
Bone marrow-derived mesenchymal stem cells (MSCs) possess multilineage differentiation potential and can be used for the treatment of diabetic heart failure. However, hyperglycemia can affect the function of MSCs adversely and merits the requirement for a strategy to correct this anomaly. MSCs were isolated from the tibias and femurs of C57BL/6 wild-type mice at 60 days after induction of diabetes by streptozotocin. MSCs were characterized by flow cytometry for CD44 (97.7%), CD90 (95.4%), and CD105 (92.3%) markers and were preconditioned with insulin-like growth factor-1 (IGF-1) (50 ng/mL) and fibroblast growth factor-2 (FGF-2) (50 ng/mL) in combination for 1 h in serum-free Iscove's modified Dulbecco's medium. This was followed by hypoxic and high glucose insults to mimic diabetic heart microenvironment and to study the effect of preconditioning. Diabetic MSCs after treatment showed upregulation of IGF-1, FGF-2, Akt, GATA-4, and Nkx 2.5 and downregulation of p16(INK4a), p66(shc), p53, Bax, and Bak. Under hypoxic stress, preconditioned diabetic MSCs showed high superoxide dismutase activity (52.3%) compared with untreated cells (36.9%). This was concomitant with low numbers of annexin-V-positive cells, high in vitro tube-forming ability, and high chemotactic mobility to stromal cell-derived factor-1α after preconditioning in diabetic MSCs. Upregulation of Ang-I and VEGF and downregulation of p16(INK4a) were also observed in preconditioned cells under conditions of high glucose insult. Therefore, preconditioning with IGF-1 and FGF-2 in combination represents a novel strategy to augment MSC function affected by diabetes and holds significance for future strategies to treat diabetic heart failure.
... Mesenchymal stem cells (MSCs) have been shown to differentiate into cells of many organs [1,2] including cardiomyocytes [3,4] and can therefore effectively repair infarcted myocardium resulting in an improved cardiac function [5,6]. Since the regenerative potential of the body declines with age, stem cells like other cells of the body are also prone to the adverse effects of aging. ...
Myocardial infarction is one of the leading causes of mortality in aged people. Whether age of donors of mesenchymal stem cells (MSCs) affects its ability to repair the senescent heart tissue is unknown. In the present study, MSCs from young (2 months) and aged (18 months) green fluorescent protein expressing C57BL/6 mice were characterized with p16(INK4a) and β-gal associated senescence. Myocardial infarction was produced in 18-month-old wild-type C57BL/6 mice transplanted with MSCs from young and aged animals in the border of the infarct region. Expression of p16(INK4a) in MSCs from aged animals was significantly higher (21.5%± 1.2, P < 0.05) as compared to those from young animals (9.2%± 2.8). A decline in the tube-forming ability on Matrigel was also observed in aged MSCs as well as down-regulation of insulin-like growth factor-1, fibroblast growth factor (FGF-2), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) compared to young cells. Mice transplanted with young MSCs exhibited significant improvement in their left ventricle (LV) systolic and diastolic function as demonstrated by dp/dt(max) , dp/dt(min) , P(max) . Reduction in the LV fibrotic area was concomitant with neovascularization as demonstrated by CD31 and smooth muscle actin (SMA) expression. Real-time RT-PCR analysis for VEGF, stromal cell derived factor (SDF-1α) and GATA binding factor 4 (GATA-4) genes further confirmed the effect of age on MSC differentiation towards cardiac lineages and enhanced angiogenesis. These studies lead to the conclusion that repair potential of MSCs is dependent on the age of donors and the repair of senescent infarcted myocardium requires young healthy MSCs.
... In another study, bone-marrow stromal cells labeled with enhanced green fluorescent protein injected into the perilymphatic space of normal cochleae in mice. After 2 weeks it has been demonstrated that, transplanted cells settled within the cochlear tissues, especially in the spiral ligament and the spiral limbus, but most transplants were located at perilymphatic space itself . ...
Genetical, ageing, excessive noise and certain antibiotics are account for majority of permanent hearing loss in humans. Hearing loss is caused by dysfunction of the sensory epithelium (the organ of Corti) within the inner ear (cochlea). It is associated with irreversible loss of sensory hair cells and spiral ganglion neurons.Inner ear hair cells are specialized mechanoreceptors converts mechanical stimuli into neural information for transmission to the brain. Several areas of research have addressed the treatment of hearing loss. There are evidences for regeneration of sensory hair cells in non-mammalian species; however, studies in mammals have failed to find evidence of cochlear hair cells regeneration.Stem cell regenerative medicine is the current focus on cure for deafness while through regeneration of specific cell types from different sources of stem cells.This review deled present stem cell regenerative therapy promising outcome on clinical application.
... Bone marrow MSCs were isolated from the femurs and tibias of rat according to the standard protocol . The isolated MSCs were cultured in DMEM (Gibco) containing 10% fetal bovine serum (FBS) (Sigma), 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 4 mM L-glutamine, at 37 °C in humidified incubator with 5% CO 2 . ...
Mesenchymal stem cells (MSCs) have multi-lineage differentiation potential which make them an excellent source for cell-based therapies. Histone modification is one of the major epigenetic regulations that play central role in stem cell differentiation. Keeping in view their ability to maintain gene expression essential for successful differentiation, it was interesting to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, in the hepatic differentiation of MSCs within the 3D scaffold. MSCs were treated with the optimized concentration of VPA in the 3D collagen scaffold. Analyses of hepatic differentiation potential of treated MSCs were performed by qPCR, immunostaining and periodic acid Schiff assay. Our results demonstrate that MSCs differentiate into hepatic-like cells when treated with 5 mM VPA for 24 h. The VPA-treated MSCs have shown significant upregulation in the expression of hepatic genes, CK-18 (P < 0.05), TAT (P < 0.01), and AFP (P < 0.001), and hepatic proteins, AFP (P < 0.05) and ALB (P < 0.01). In addition, acetylation of histones (H3 and H4) was significantly increased (P < 0.001) in VPA-pretreated cells. Further analysis showed that VPA treatment significantly enhanced (P < 0.01) glycogen storage, an important functional aspect of hepatic cells. The present study revealed the effectiveness of VPA in hepatic differentiation within the 3D collagen scaffold. These hepatic-like cells may have an extended clinical applicability in future for successful liver regeneration.
... 6,7) Adult stem cells also have the potential to deliver gene and therapeutic molecules to other parts of the inner ear. 8) Stem cells can be categorized as fetal stem cells, embryonic stem cells, adult stem cells, induced pluripotent stem cells and perinatal stem cells. 9) Adult stem cells play a key role in tissue regeneration and have multipotent cells present in most adult tissues. ...
Background and Objectives Ototoxic sensorineural hearing loss causes permanent hearing loss in most cases. Recently there have been many reports describing cell base therapy with stem cells that has some effect on hearing recovery. We evaluated the efficacy of clinical grade, pre-made, human bone marrow derived mesenchymal stem cells (BM-MSCs) in ototoxic deaf animal model.Materials and Method BM-MSCs were cultured in a clinical grade laboratory. The animals were divided into 2 groups as follows: a saline injected control group and a stem cell injected group (MSC-group). Cultured MSCs were transplanted into the brachial vein of the deaf mice model. We recorded auditory brainstem response (ABR) and conducted immunohistochemistry at 1, 3, and 5 weeks.Results After the transplantation of MSC, a significant improvement in the hearing threshold of ABR was observed in the MSC transplanted group. Five weeks after transplantation of MSCs, hair cell regeneration was confirmed from the basal to the apex of the cochlea in fluorescent dyed image under the microscope compared to the control group.Conclusion BM-MSCs were effective in an acute ototoxic deaf animal model. These results show that stem cell transplantation mediate inner ear regeneration.
... Stem cell is thought to be an undifferentiated master cell of the body, having phenomenal aptitude to differentiate into many different specialized cell types of the body (Krause et al., 2002;Vieyra et al., 2005;Sharif et al., 2007). The major role of stem cell is its ability to differentiate into a wide-range of cell types to renew the injured or harmed tissue, and upgrade its performance in a living body (Najmaldin et al., 2013). ...
Wharton's jelly derived mesenchymal stem cells (WJ-MSCs) have remarkable potential in regenerative medicine and cellular therapy. Cell therapy is based on transplantation of living cells to repair damaged tissues. The viability of the transplanted cells leads to the successful stem cell therapy. However, the therapeutic efficiency is greatly limited by apoptosis due to existing oxidative, ischemic and hypoxic conditions in the tissue. The high glucose concentration especially in diabetic patients may also diminish the effectiveness of stem cell therapy. The poor survival of WJ-MSCs compromised the therapeutic efficiency of stem cells. Therefore, it is necessary to identify the factors behind this drawback of stem cell therapy. The present study is aimed to investigate the effect of high glucose and hydrogen peroxide (H2O2) on WJ-MSCs. For this purpose these cells were treated with various concentration of glucose (5.5 and 25mM) and H2O2 (100, 200 and 300µM) for 3 hours. Cell viability, proliferation and cytotoxicity were evaluated by crystal violet assay, MTT and LDH release respectively. Estimation of glucose utilization of the cells was performed by glucose utilization assay. The oxidative stress is assessed by measuring the activity of reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT). It was concluded that high glucose and 300µl H2O2 reduces the WJ-MSCs survival and proliferation.
... Restoration of neoangiogenesis within a diabetic heart remains a desirable treatment modality and may augment the depleted diabetic heart function. Mesenchymal stem cells (MSCs) exhibit multilineage differentiation potential678 and have been widely used in cell based therapies for the alleviation of a number of impairments including cardiovascular disorders [9,10]. Even though MSCs represent a feasible choice for myocardial repair, recent evidence implicates that the capability of MSCs to repair damaged tissues declines with age and disease11121314. ...
Mesenchymal stem cells (MSCs) have the potential for treatment of diabetic cardiomyopathy; however, the repair capability of MSCs declines with age and disease. MSCs from diabetic animals exhibit impaired survival, proliferation, and differentiation and therefore require a strategy to improve their function. The aim of the study was to develop a preconditioning strategy to augment the ability of MSCs from diabetes patients to repair the diabetic heart.
Diabetes was induced in C57BL/6 mice (6 to 8 weeks) with streptozotocin injections (55 mg/kg) for 5 consecutive days. MSCs isolated from diabetic animals were preconditioned with medium from cardiomyocytes exposed to oxidative stress and high glucose (HG/H-CCM).
Gene expression of VEGF, ANG-1, GATA-4, NKx2.5 MEF2c, PCNA, and eNOS was upregulated after preconditioning with HG/H-CCM, as evidenced by reverse transcriptase/polymerase chain reaction (RT-PCR). Concurrently, increased AKT phosphorylation, proliferation, angiogenic ability, and reduced levels of apoptosis were observed in HG/H-CCM-preconditioned diabetic MSCs compared with nontreated controls. HG/H-CCM-preconditioned diabetic-mouse-derived MSCs (dmMSCs) were transplanted in diabetic animals and demonstrated increased homing concomitant with augmented heart function. Gene expression of angiogenic and cardiac markers was significantly upregulated in conjunction with paracrine factors (IGF-1, HGF, SDF-1, FGF-2) and, in addition, reduced fibrosis, apoptosis, and increased angiogenesis was observed in diabetic hearts 4 weeks after transplantation of preconditioned dmMSCs compared with hearts with nontreated diabetic MSCs.
Preconditioning with HG/H-CCM enhances survival, proliferation, and the angiogenic ability of dmMSCs, augmenting their ability to improve function in a diabetic heart.
According to 2010 estimates from The National Institute on Deafness and other Communication Disorders, approximately 17% (36 million) American adults have reported some degree of hearing loss. Currently, the only clinical treatment available for those with severe-to-profound hearing loss is a cochlear implant, which is designed to electrically stimulate the auditory nerve in the absence of hair cells. Whilst the cochlear implant has been revolutionary in terms of providing hearing to the severe-to-profoundly deaf, there are variations in cochlear implant performance which may be related to the degree of degeneration of auditory neurons following hearing loss. Hence, numerous experimental studies have focused on enhancing the efficacy of cochlear implants by using neurotrophins to preserve the auditory neurons, and more recently, attempting to replace these dying cells with new neurons derived from stem cells. As a result, several groups are now investigating the potential for both embryonic and adult stem cells to replace the degenerating sensory elements in the deaf cochlea. Recent advances in our knowledge of stem cells and the development of induced pluripotency by Takahashi and Yamanaka in 2006, have opened a new realm of science focused on the use of induced pluripotent stem (iPS) cells for therapeutic purposes. This review will provide a broad overview of the potential benefits and challenges of using iPS cells in combination with a cochlear implant for the treatment of hearing loss, including differentiation of iPS cells into an auditory neural lineage and clinically relevant transplantation approaches.
To investigate the potential of neurally induced bone marrow stromal cells (BMSCs) as transplants for replacement of spiral ganglion neurons.
BMSCs were harvested from the femurs and tibias of adult guinea pigs. BMSCs were cultured with neural induction media and formed spheres. The capacity of BMSC-derived spheres for neural differentiation was examined by immunocytochemistry in vitro. BMSC-derived spheres were injected into the modiolus of the intact cochleae or those locally damaged by ouabain, followed by histological and functional analyses.
In vitro analysis revealed a high capacity of BMSC-derived spheres for neural differentiation. After transplantation into the cochlear modiolus, the survival and neural differentiation of BMSC-derived spheres was observed in both the intact and damaged cochleae. In intact cochleae, transplants settled in various portions of the cochlea, including the cochlear modiolus, whereas in damaged cochleae, transplants were predominantly observed in the internal auditory meatus. Transplantation of BMSC-derived spheres resulted in no functional recovery of the cochlea or protection of host spiral ganglion neurons.
The present findings indicate that BMSC-derived spheres can be a source for replacement of spiral ganglion neurons, although further manipulations are required for functional recovery.
Strategies to restore sensorineural hearing loss focus on the replacement of lost hair cells, the specialized mechanoreceptors in the organ of Corti that convert the mechanical energy of sound into electrical energy. Hair cells in mammalian systems do not have the capacity to regenerate, but two exciting lines of research hold promise in restoring inner ear function. Here we review basic principles of gene therapy and discuss its application in the inner ear. We survey the various viral vectors and routes of delivery into the inner ear. Applications of gene therapy in the inner include hair cell protection in the face of chemical or noise-induced ototoxicity, spiral ganglion cell survival following hair cell death or injury, and hair cell regeneration. More recently, the viability of gene therapy in human inner ear tissue has been reported. Transplantation of progenitor cells that can differentiate into functioning hair cells with the appropriate connections to their corresponding spiral ganglion cells is yet another strategy to restore sensorineural hearing loss. Neonatal or embryonic stem cells, adult mouse inner ear stem cells, and stem cells from the central nervous system have been shown to differentiate into cells containing hair cell markers and proteins. Prospects for stem cell therapy in the inner ear, and its limitations, will also be examined.
Hearing impairment is one of the most common sensory disorders in human. The major causes of sensorineural hearing loss are aging, noise, genetics, ototoxicity, and autoimmune. A better understanding of sensorineural hearing loss is required to understand the mechanisms by which specific causes lead to hearing impairment. The study of sensorineural hearing impairment in humans is limited by the inability to follow inner ear development and elucidate the true mechanism of hearing loss in human. Animal experiment is an essential part of research and testing in the life sciences. Because of the complicated structures of the inner ear, every morphological study is made to be appropriate for such a special tissue. Here I will outline the several morphological methods of the rodent inner ear covering such basic things including fixation, and sectioning.
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.
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.
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.
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.
Sensorineural hearing loss (SNHL) is one of the most common disabilities in our society. Experimentally, many candidates for use as therapeutic molecules have been discovered. However, a considerable obstacle to clinical application is the lack of an effective method for drug delivery to the cochlea. In order to overcome this obstacle, there needs to be development of a local cochlear drug delivery system. Advances in pharmacological technology have provided various drug delivery systems that use biomaterials, and which can be utilized for local drug delivery to the cochlea. Indeed, recent studies have demonstrated the potential of synthetic and natural biomaterials for local drug delivery to the cochlea, indicating that the clinical application of such local drug delivery systems could be used in the near future for therapeutic treatments. Recent progress in cell therapy research also offers a novel drug delivery method for the cochlea. In addition, transplantation of stem cells into the cochlea has been demonstrated to provide protective effects for the auditory function. Transplantation of genetically engineered cells has also resulted in the sustained delivery of aimed therapeutic molecules within the inner ear. Although problems involving clinical application still need to be resolved, these drug delivery systems for the inner ear may hold the future therapeutic options for treatment of SNHL.
A number of somatic stem cells have been used in inner ear research. In this chapter, two stem cells, mesenchymal and hematopoietic stem cells, are the focus, because these stem cells have already utilized in clinical settings. Of mesenchymal stem cells, the potential of bone marrow- and adipose tissue-derived stem cells for the treatment of the inner ear is reviewed. As for hematopoietic stem cells, their contribution to the maintenance of inner ear cell circumstances is introduced.
BACKGROUND: The sensorineural deafness occurs as a result of loss of inner ear hair cells in the cochlea or of their primary afferent the spiral ganglion neurons. Stem cells to restore hearing following inner ear cell death has become a focus in recent years. OBJECTIVE: To summarize research progress in stem cells differentiating into inner ear cells in vitro and in vivo and to review the achievement in stem cells replacing inner ear cells in treating sensorineural deafness. METHODS: With "inner ear, stem cells" as key words, a computer-based online search of Pubmed and CNKI was performed for articles published from January 2000 to August 2009. RESULTS AND CONCLUSION: A total of 170 articles were collected, and experimental studies and review articles on stem cells in sensorineural deafness were included, while repetitive articles were excluded. Finally, 32 articles were summarized and analyzed. Different types of stem cells have the capacity to differentiate into inner ear cells. They can differentiate into neural cell types. Stem cells can live and migrate, differentiating into cell types of the sites of injury. It provides a therapy strategy to restore hearing following sensorineural deafness by he capacity of stem cells differentiating into inner ear cells. However, it remains further investigation how to function following cell differentiation and how to form the appropriate neural pathways by stem cell transplantation in sensorineural deafness.
As the regeneration capacity of hair cells is limited, inner ear stem cell therapies hold promise. Effects of mouse induced pluripotent stem cells (IPSCs) on Wistar albino rats (WARs) with hearing impairment were investigated.
Materials and methods:
Thirty-five adult WARs with normal hearing were divided into 4 groups. Excluding the study group (n = 15), the other three groups served as control groups for ototoxicity and IPSC injection models. IPSC injections were performed via cochleostomy after a retroauricular approach. Auditory functions were evaluated with auditory brainstem responses (ABRs) before and after the injections. After a final hearing assessment the WARs were sacrificed and cochleae were extracted to see the biologic behavior of IPSCs in the inner ear by light microscopy and immunohistochemistry.
There were no significant differences in the click-ABR thresholds in the study group after IPSC transplantation. The mean hearing threshold in the study group after ototoxic agent injection was 53.2 dB (10-90 dB). There was no significant difference between groups (P > 0.05) and no differentiated stem cells were observed immunohistochemically.
Transplanted IPSCs did not show a therapeutic effect in this trial. We discuss potential pitfalls and factors affecting the therapeutic effect.
The olfactory mucosa contains cells that enable it to generate new neurons and other supporting cells throughout life, allowing it to replace cells of the mucosa that have been damaged by exposure to various insults. In this article, we discuss the different types of stem cell found within the olfactory mucosa and their properties. In particular, the mesenchymal-like cells found within the lamina propria will be reviewed in detail. In addition, we discuss potential applications of olfactory-derived stem cells toward hearing regeneration secondary to either inner hair cell loss or primary or secondary auditory nerve degeneration.
Mammalian cochleae have limited capacity for regeneration, which is one of the major difficulties in the treatment of sensorineural hearing loss. In the current study, we examined the potential of bone marrow‐derived stromal cells (BMSCs) for functional restoration of mouse cochleae through regeneration or maintenance of cochlear fibrocytes in the spiral ligament. We used a mouse model of degeneration of cochlear fibrocytes in the spiral ligament (SL) using local application of 3‐nitropropionic acid (3‐NP), in which disruption of the gap junction network in the SL resulted in reduction of the endocochlear potential (EP). Mouse BMSCs were infused into the posterior semicircular canal 7 days after 3‐NP application. Transplanted BMSCs were frequently observed in the cochlear fluid space 4 weeks after transplantation, although a few transplants had migrated into the cochlear tissues including the spiral ligament. BMSC‐treated cochleae exhibited higher cell densities in the spiral ligament and greater EP levels than the control ones. Immunohistochemistry further demonstrated the restoration of functional proteins in the spiral ligament. Significant recovery in thresholds of auditory brainstem responses (ABRs) following BMSC transplantation was found only at 40 kHz in a mild degeneration model. Our cumulative findings indicated that BMSCs accelerated regeneration or maintenance of fibrocytes in damaged spiral ligaments, leading to partial functional restoration of the mouse cochleae.
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In the auditory system, the primary sensory neurons, spiral ganglion neurons (SGNs), transmit complex acoustic information from hair cells to the second-order sensory neurons in the cochlear nucleus for sound processing, thus building the initial bridge between the physical world of sound and the perception of that sound. Cochlear SGN loss causes irreversible hearing impairment because this type of neural cell cannot regenerate. A better understanding of the molecular mechanisms of formation, structure, degeneration, and protection of SGNs will help to design potential therapeutic strategies for preservation and replacement of them in the cochlear implant recipient. In this review, we described and summarized the following about SGNs: (1) their cell biology and their peripheral and central connections, (2) mechanisms of their neuronal damage and their protection, and (3) the neural and synaptic mechanism of auditory neuropathy and current options for hearing rehabilitation from auditory neuropathy. The updates of the research progress and the significant issues on these topics were discussed.
Most cases of sensorineural deafness are caused by degeneration of hair cells. Although stem/progenitor cell therapy is becoming a promising treatment strategy in a variety of organ systems, cell engraftment in the adult mammalian cochlea has not yet been demonstrated. In this study, we generated human otic progenitor cells (hOPCs) from induced pluripotent stem cells (iPSCs) in vitro and identified these cells by the expression of known otic markers. We showed successful cell transplantation of iPSC-derived-hOPCs in an in vivo adult guinea pig model of ototoxicity. The delivered hOPCs migrated throughout the cochlea, engrafted in non-sensory regions, and survived up to 4 weeks post-transplantation. Some of the engrafted hOPCs responded to environmental cues within the cochlear sensory epithelium and displayed molecular features of early sensory differentiation. We confirmed these results with hair cell progenitors derived from Atoh1-GFP mice as donor cells. These mouse otic progenitors transplanted using the same in vivo delivery system migrated into damaged cochlear sensory epithelium and adopted a partial sensory cell fate. This is the first report of the survival and differentiation of hOPCs in ototoxic-injured mature cochlear epithelium, and it should stimulate further research into cell-based therapies for treatment of deafness.
The aim of this study was to investigate the potential of using bone marrow mesenchymal stem cells (BMSCs) for treatment of inflammation and autoimmune sensorineural hearing loss.
Fifty-five immunized guinea pigs were divided into five groups. Group A received BMSCs expressing IL-4, group B received BMSCs expressing an empty carrier vector, group C received recombinant lentivirus expressing IL-4, group D received recombinant lentivirus expressing an empty carrier vector, and group E received phosphate-buffered saline. Auditory function was monitored using brain stem responses (ABRs) to evaluate the auditory changes. The distribution of implanted BMSCs in the inner ear was estimated using fluorescence microscopy. The distribution and expression of IL-4 gene products in the inner ear were detected via immunohistochemistry.
After transplantation, the ABR III wave threshold decreased significantly in BMSCs expressing exogenous IL-4 group (group A), BMSCs expressing empty carrier vector group (group B), and recombinant lentivirus expressing IL-4 group (group C) (P < 0.001), which means the auditory functions of the experimental guinea pigs were improved. Further statistical analysis revealed that BMSCs expressing exogenous IL-4 group (group A) and BMSCs expressing empty carrier vector group (group B) were able to improve the auditory function more obviously (P < 0.05). Lentivirus-infected BMSCs were able to migrate to the inner ear. Fluorescence-positive BMSCs were scattered in the scala tympani and vestibule.
These results demonstrated that BMSCs expressing exogenous IL-4 successfully migrated into the inner ear in an in vitro study. BMSCs expressing exogenous IL-4 and BMSCs can be used to treat inflammatory injury in autoimmune inner ear diseases.
The high degree of bone marrow cell (BMC) plasticity has prompted us to test its restoration possibility in inner ear repair. Our aim was to determine the potential of these cells to transdifferentiate into specialized cochlea cell types after acoustic injury and BMC mobilization. Lethally irradiated mice were transplanted with BMCs from green fluorescent protein (GFP) transgenic mice and subjected to acoustic deafening 3 months later. In a separate experiment, stem cell factor and granulocyte colony-stimulating factor were administered to test the effect of BMC mobilization on bone marrow-derived cell (BMDC) transdifferentiation. All mice showed almost complete chimerism 3 months after bone marrow transplantation. Upon acoustic trauma, robust BMDC migration was observed in the deafened cochlea. GFP+ cell migration was most prominent during the first week after acoustic deafening, and these cells accumulated significantly at the spiral ligament, perilymphatic compartment walls, and limbus regions. Most of the BMDCs expressed CD45 and CD68 and were identified as macrophages. Upregulation of stromal-derived factor 1 (SDF-1) was also observed in the spiral ligament during the first week after acoustic deafening. Cytokine treatment resulted in increased BMC mobilization in the systemic circulation. However, the presence of any stem cell progenitors or the differentiation of BMDCs into any cell types expressing cochlea sensory, supporting, fibrocytic, or neuronal markers were not detected in the deafened cochlea. In conclusion, we have demonstrated the homing capability of BMDCs to the deafened cochlea, and these cells displayed mature hematopoietic properties without spontaneous transdifferentiation to any cochlea cell types after acoustic trauma or bone marrow mobilization.
Ototoxicity is a major dose-limiting side effect of cisplatin (DDP) administration due to its propensity to induce destruction of hair cells and neurons in the auditory system. Previous studies demonstrated that TrkC-expressing spiral ganglion neurons (SGN) are protected from the cytotoxic effects of DDP by localized delivery of the trophic factor neurotrophin-3 (NT-3). Successful in vivo implementation of such a therapy requires the development of an efficient gene delivery vehicle for expression of NT-3 within the cochlea. To this end, we constructed a herpes simplex virus (HSV) amplicon vector that expressed a c-Myc-tagged NT-3 chimera (HSVnt-3myc). Helper virus-free vector stocks were initially evaluated in vitro for their capacity to direct expression of NT-3 mRNA and protein. Transduction of cultured murine cochlear explants with HSVnt-3myc resulted in production of NT-3 mRNA and protein up to 3 ng/ml as measured over a 48-h period in culture supernatants. To determine whether NT-3 overexpression could abrogate DDP toxicity, cochlear explants were transduced with HSVnt-3myc or a murine intestinal alkaline phosphatase-expressing control vector, HSVmiap, and then exposed to cisplatin. HSVnt-3myc-transduced cochlear explants harbored significantly greater numbers of surviving SGNs than those infected with control virus. These data demonstrate that amplicon-mediated NT-3 transduction can attenuate the ototoxic action of DDP on organotypic culture. The potency of NT-3 in protecting spiral ganglion neurons from degeneration suggests that in vivo neurotrophin-based gene therapy may be useful for the prevention and/or treatment of hearing disorders.
The use of adenoviral vectors has recently provided a novel strategy for direct gene transfer into the cochlea. In this study, we assessed the utility of an adenoviral vector expressing glial-cell-derived neurotrophic factor (GDNF) in ischemia-reperfusion injury of the gerbil cochlea. The vector was injected through the round window 4 days before ischemic insult. The distribution of a reporter transgene was confirmed throughout the cochlea from the basal to the apical turn and Western blot analysis indicated significant upregulation of GDNF protein 11 days following virus inoculation. Hearing ability was assessed by sequentially recording compound action potentials (CAP), and the degree of hair cell loss in the organ of Corti was evaluated in specimens stained with rhodamine-phalloidin and Hoechst 33342. On the seventh day of ischemia, the CAP threshold shift and inner hair cell loss were remarkably suppressed in the Ad-GDNF group compared with the control group. These results suggest that adenovirus-mediated overexpression of GDNF is useful for protection against hair cell damage, which otherwise eventually occurs after transient ischemia of the cochlea.
Hearing impairment, which is the most prevalent sensory deficit of human beings, needs a breakthrough in therapeutic technologies. One technology is the usage of a vector system to reach the inner ear, and another is by a therapeutic molecule. Here we developed a novel gene therapy strategy by combining hepatocyte growth factor (HGF) with hemagglutinating virus of Japan envelope (HVJ-E) vector. When HVJ-E containing human HGF gene was injected intrathecally into the cerebrospinal fluid via cisterna magna of rats, the vector reached the inner ear region, and human HGF gene expression was detected in the spiral ganglion cells (SGCs) of the inner ear. Expression of endogenous rat HGF and its receptor, c-Met, was also induced in SGCs by human HGF. Kanamycin treatment results in hearing impairment by inducing degeneration of hair cells (HCs) and apoptosis of SGCs in rats. By HGF gene transfer before kanamycin treatment, both loss of HCs and apoptosis of SGCs were prevented. Furthermore, hearing function, evaluated by auditory brainstem response, was maintained at a normal level. When HGF gene transfer was performed 2 wk after kanamycin treatment, hearing impairment was significantly recovered. These results indicate a novel and effective therapeutic strategy against sensorineural hearing impairment.
In the mammalian auditory system, sensory cell loss resulting from aging, ototoxic drugs, infections, overstimulation and other causes is irreversible and leads to permanent sensorineural hearing loss. To restore hearing, it is necessary to generate new functional hair cells. One potential way to regenerate hair cells is to induce a phenotypic transdifferentiation of nonsensory cells that remain in the deaf cochlea. Here we report that Atoh1, a gene also known as Math1 encoding a basic helix-loop-helix transcription factor and key regulator of hair cell development, induces regeneration of hair cells and substantially improves hearing thresholds in the mature deaf inner ear after delivery to nonsensory cells through adenovectors. This is the first demonstration of cellular and functional repair in the organ of Corti of a mature deaf mammal. The data suggest a new therapeutic approach based on expressing crucial developmental genes for cellular and functional restoration in the damaged auditory epithelium and other sensory systems.
Sensorineural hearing loss is a common disability, but treatment options are currently limited to cochlear implants and hearing aids. Studies are therefore being conducted to provide alternative means of biological therapy, including gene therapy. Safe and effective methods of gene delivery to the cochlea need to be developed to facilitate the clinical application of these therapeutic treatments for hearing loss. In this study, we examined the potential of cell-gene therapy with nonviral vectors for delivery of therapeutic molecules into the cochlea. NIH3T3 cells were transfected with the brain-derived neurotrophic factor (Bdnf) gene using lipofection and then transplanted into the mouse inner ear. Immunohistochemistry and Western blotting demonstrated the survival of grafted cells in the cochlea for up to 4 weeks after transplantation. No significant hearing loss was induced by the transplantation procedure. A Bdnf-specific enzyme-linked immunosorbent assay revealed a significant increase in Bdnf production in the inner ear following transplantation of engineered cells. These findings indicate that cell-gene delivery with nonviral vectors may be applicable for the local, sustained delivery of therapeutic molecules into the cochlea.
The aim of this experimental study was to examine the potential of local recombinant human insulin-like growth factor-1 (rhIGF-1) application through a biodegradable hydrogel for the treatment of cochleae.
A hydrogel immersed with rhIGF-1 was placed on the round window membrane of Sprague-Dawley rats while a hydrogel immersed with physiological saline was applied to control animals. On day 3 after drug application, the animals were exposed to white noise at 120 dB sound pressure level (SPL) for 2 hours. Cochlear function was monitored using measurements of auditory brain stem responses (ABRs) at frequencies of 8, 16, and 32 kHz. The temporal bones were collected 7 or 30 days after noise exposure and the loss of hair cells was quantitatively analyzed.
Local rhIGF-1 treatment significantly reduced the elevation of ABR thresholds on days 7 and 30 after noise exposure. Histologic analysis revealed that local rhIGF-1 treatment significantly prohibited the loss of outer hair cells.
These findings demonstrate that local IGF-1 application through the biodegradable hydrogel has the potential for protection of cochleae from noise trauma.
The green fluorescent protein (GFP) is responsible for the green bioluminescence of the jellyfish Aequorea victoria. Many classes of GFP mutants exist that display modified fluorescence spectra and an increased extinction coefficient. We produced transgenic mouse lines with an 'enhanced' GFP (EGFP) cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer. All of the tissues from these transgenic lines, with the exception of erythrocytes and hair, were green under excitation light. The fluorescent nature of the cells from these transgenic mouse lines would facilitate their use in many kinds of cell transplantation experiments.
Aminoglycosides are commonly used antimicrobial drugs that often have ototoxic side effects. The ototoxicity often involves permanent loss of cochlear hair cells (HCs). Neurotrophic factors have been shown to protect a variety of tissues, including HCs, from toxic trauma. To determine if glial cell line-derived neurotrophic factor (GDNF) can protect cochlear HCs from trauma, we inoculated an adenoviral vector encoding the human GDNF gene into guinea pig cochleae via the round window membrane 4 days prior to injection of aminoglycosides. Control groups showed little or no negative influence of the viral inoculation on cochlear structure and function. In contrast, ears that were inoculated with the GDNF vector had better hearing and fewer missing HCs after exposure to the ototoxins, as compared with controls. Our results demonstrate the feasibility of gene therapy for cochlear application and suggest that virus-mediated overexpression of GDNF may be developed as a valuable prevention against trauma-induced HC death.
The immunohistochemical localization of connexin 26 (a gap junction protein) and Na,K-ATPase in the mouse cochlear lateral wall was studied at different ages between 0 and 30 days after birth (DAB). Connexin 26-like immunoreactivity was sparsely distributed among the connective tissue cells just lateral to the future marginal cells of the stria vascularis on 0 DAB. In the mice of 3-6 DAB, connexin 26 was observed in the strial basal cell area, and was increased in its distribution density on 10 DAB. Connexin 26 was sparsely distributed among the fibrocytes in the spiral ligament and the suprastrial zone on 10 DAB, and its distribution density increased rapidly in the mouse on 12 DAB. The immunohistochemical distribution reached the adult pattern in the cochlear lateral wall on 15 DAB. Weak Na, K-ATPase-like immunoreactivity was observed in the epithelial cells, corresponding to the future strial marginal cells, on 0 DAB. Its staining intensity was enhanced with the increase of age, and reached the adult pattern on 10 DAB. In contrast, Na,K-ATPase-like immunoreactivity in the type II fibrocytes and suprastrial fibrocytes was first detected on 12 DAB, and reached the mature level on 15 DAB. It is well known that the endolymphatic potential (EP) reaches the adult level 2 weeks after birth. The expression patterns of connexin 26 and Na,K-ATPase in the fibrocytes of the spiral ligament and the suprastrial zone coincided with the rapid growth and maturation of EP. These findings may suggest a role for the gap junctional communications and Na,K-ATPase activity of the fibrocytes within the cochlear lateral wall in the generation and maturation of EP.
Loss of sensory hair cells in the inner ear is a major cause of permanent hearing loss, since regeneration of hair cells rarely occurs in mammals. The aim of this study was to examine the potential of neural stem cell transplantation to restore inner ear hair cells in mice. Fetal neural stem cells were transplanted into the mouse inner ear after drug-induced injury. Histological analysis demonstrates that the majority of grafted cells differentiated into glial or neural cells in the inner ear. Strikingly, however, we show that grafted cells integrate in vestibular sensory epithelia and express specific markers for hair cells. This finding suggests that transplanted neural stem cells have the potential to differentiate and restore inner ear hair cells.
Noise exposure damages the stria and spiral ligament and may contribute to noise-induced threshold shift by altering the endocochlear potential (EP). The aim of this study was to correlate lateral wall histopathology with changes in EP and ABR thresholds. CBA/CaJ mice were exposed to octave band (8-16 kHz) noise for 2 h at intensities ranging from 94 to 116 dB SPL and evaluated 0 h to 8 weeks postexposure. EP in control mice averaged 86 and 101 mV in apical and basal turns, respectively. The 94 dB exposures caused a 40 dB temporary threshold shift (TTS), and there was with no corresponding change in EP. The 112 and 116 dB exposures caused >60 dB threshold shifts at 24 h, and EP was transiently decreased, e.g., to 21 and 27 mV in apical and basal turns after 116 dB. By 1 week postexposure, EP returned to control values in all exposure groups, although those exposed to 112 or 116 dB showed large permanent threshold shifts (PTS). Cochleas were plastic-embedded and serial-sectioned for light microscopic and ultrastructural analysis. Acute changes included degeneration of type II fibrocytes of the spiral ligament and strial edema. The strial swelling peaked at 24 h when significant EP recovery, had taken place, suggesting that these changes reflect compensatory volume changes. In the chronic state, massive loss of type II fibrocytes and degeneration of strial intermediate and marginal cells was observed with drastic reduction in membrane surface area. The results suggest that EP shifts do not occur with TTS and also do not add significantly to PTS in the steady state. However, EP loss could contribute to acute threshold shifts that resolve to a PTS. EP recovery despite significant strial degeneration may be partly due to decreased transduction current caused by hair cell damage.
This study aimed to evaluate the potential of bone marrow stromal cells for treatment of inner ear diseases. Autologous marrow cells labeled with Dil were implanted into the inner ear of five gentamicin-treated chinchillas. Histological analysis 3 weeks later revealed robust survival of grafted marrow cells in multiple regions within the cochlea. Marrow cells implanted in the basal turn of the cochlea migrated as far as the apical end or into the spiral ligament of the cochlea. Some grafted cells expressed a neuronal or glial cell marker, indicating their ability to differentiate into neuronal or glial cells. Survival, migrational mobility and differentiation of autologous marrow cells in damaged cochlea suggest their potential as transplants for treatment of various degenerative inner ear diseases.
Unilateral lesions to the anterior thalamic nuclei (ATN) and the hippocampus (H) were made in opposite hemispheres in the rat to examine whether these brain structures form part of a functional neural pathway underlying spatial learning and memory. In the first experiment, rats were tested on a spatial-visual conditional associative task in which they had to learn to approach one of two stimuli depending on the spatial context in which the stimuli were embedded. The rats were subsequently trained on delayed forced alternation, a spatial working memory task known to be sensitive to the effects of ATNxH damage. Rats with ATNxH lesions were impaired in the acquisition of both tasks in comparison with normal control animals. The findings support the idea that the anterior thalamic nuclei and the hippocampus are critical components of an anatomical system subserving spatial memory and suggest that these brain regions work in a dependent fashion during the performance of certain spatial learning tasks.
Many neurodegenerative diseases are attributed to the degeneration of neurons with subsequent functional loss. Cell transplantation is a strategy with potential for treating such diseases, and many kinds of cells are considered candidates for transplantation therapy. Bone marrow stromal cells (MSCs) have great potential as therapeutic agents, as they are easy to isolate and expand from patients without serious ethical and technical problems. The authors have found a method for the highly efficient, exclusive and specific induction of functional postmitotic neuronal cells from both rat and human MSCs. Gene transfer of Notch intracellular domain (NICD) followed by the administration of certain trophic factors induced mature neurons expressing neuronal markers, some of which showed action potentials. Induced neurons were transplanted to animal models of neurodegenerative disorders, including Parkinson's disease and ischaemic brain injury, resulting in the successful integration of transplanted cells and improvement in function of the transplanted animals. This review summarises the respective potentials, benefits and drawbacks of MSC-derived neurons, and discusses the possibility of their clinical application in neurodegenerative diseases.
Pluripotent mesenchymal stem cells (MSCs) differentiate into a variety of cells, including cardiomyocytes and vascular endothelial cells. However, little information is available about the therapeutic potency of MSC transplantation in cases of dilated cardiomyopathy (DCM), an important cause of heart failure.
We investigated whether transplanted MSCs induce myogenesis and angiogenesis and improve cardiac function in a rat model of DCM. MSCs were isolated from bone marrow aspirates of isogenic adult rats and expanded ex vivo. Cultured MSCs secreted large amounts of the angiogenic, antiapoptotic, and mitogenic factors vascular endothelial growth factor, hepatocyte growth factor, adrenomedullin, and insulin-like growth factor-1. Five weeks after immunization, MSCs or vehicle was injected into the myocardium. Some engrafted MSCs were positive for the cardiac markers desmin, cardiac troponin T, and connexin-43, whereas others formed vascular structures and were positive for von Willebrand factor or smooth muscle actin. Compared with vehicle injection, MSC transplantation significantly increased capillary density and decreased the collagen volume fraction in the myocardium, resulting in decreased left ventricular end-diastolic pressure (11+/-1 versus 16+/-1 mm Hg, P<0.05) and increased left ventricular maximum dP/dt (6767+/-323 versus 5138+/-280 mm Hg/s, P<0.05).
MSC transplantation improved cardiac function in a rat model of DCM, possibly through induction of myogenesis and angiogenesis, as well as by inhibition of myocardial fibrosis. The beneficial effects of MSCs might be mediated not only by their differentiation into cardiomyocytes and vascular cells but also by their ability to supply large amounts of angiogenic, antiapoptotic, and mitogenic factors.
This study aimed to evaluate the potential of embryonic stem cell-derived neural progenitors for use as transplants for the replacement of the auditory primary neurons, spiral ganglion neurons. Mouse embryonic stem cell-derived neural progenitors were implanted into the base of the cochlear modiolus of normal or deafened guinea pigs, which contains spiral ganglion neurons and cochlear nerve fibers. Histological analysis demonstrated the survival and neural differentiation of transplants in the cochlear modiolus and active neurite outgrowth of transplants toward host peripheral or central auditory systems. Functional assessments indicated the potential of transplanted embryonic stem cell-derived neural progenitors to elicit the functional recovery of damaged cochleae. These findings support the hypothesis that transplantation of embryonic stem cell-derived neural progenitors can contribute to the functional restoration of spiral ganglion neurons.
This study aims to investigate the therapeutic potential of adult bone marrow stromal cells (BMSCs). Exposed to a cocktail of induction medium, some BMSCs could differentiate into cell types with phenotypes of neural lineages in vitro. These cells expressed neural markers nestin, GFAP, 68-kDa neurofilament and beta-tubulin III as detected by immunohistochemistry and RT-PCR. Fluorescence-labeled cells were injected intravenously at 72 h after traumatic brain injury. Transplanted cells survived and migrated to the ipsilateral cerebral cortex at different time points after injection. They were immunopositive for neuronal marker MAP-2, oligodendrocyte marker CNPase, astrocytic maker GFAP or microglial marker OX-42 in vivo. In rats receiving BMSC transplants, there were significant improvements in motor and neurological functions when compared with the control groups. Hence, the therapeutic potential of BMSCs for traumatic brain injury is further amplified.
Noise-induced hearing loss has been associated with alterations in cochlear blood flow. Our study analyzed the expression of Vascular Endothelial Growth Factor (VEGF) and its functional receptors, Flt-1 and Flk-1, in the cochlear structures of noise-exposed and unexposed guinea pigs. VEGF is a prototypical angiogenic agent, with multiple functions on vascular biology, ranging from vascular permeability to endothelial cell migration, proliferation, differentiation, and survival. Acoustic trauma was induced by a continuous pure tone of 6 kHz, at 120 dB SPL for 30 min. Auditory function was evaluated by electrocochleographic recordings at 2-20 kHz for 7 days. Noise-induced cochlear morphological changes were studied by immunohistochemistry and scanning electron microscopy. The expression of VEGF and its receptors was examined by immunohistochemistry and western blotting analysis. The hearing threshold shift reached a level of 60 dB SPL on day 1 after trauma and underwent a partial recovery over time, reaching a value of about 20 dB SPL on day 7. Outer hair cell loss was more prominent in the area located 14-16 mm from the apex. Increased cochlear VEGF expression was observed in noise-exposed animals, in particular at the level of stria vascularis, spiral ligament, and spiral ganglion cells. No changes were observed in the expression of VEGF-receptors. Our data suggest a role for VEGF in the regulation of the vascular network in the inner ear after acoustic trauma and during auditory recovery, with potentially important clinical and therapeutic implications.