Differential distribution of stem cells in the auditory and vestibular organs of the inner ear

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.

[Stem-cell-based approaches for treating inner ear diseases]

The capacity of stem cells to regenerate lost tissue cells has gained recognition among physicians. Despite the successful use of blood stem cells for treating blood cancers, other stem cell types have not yet been widely introduced into clinical practice. Therapy options involving stem cells for inner ear diseases consequently have not been implemented. Nonetheless, several reports have recently been published describing the generation of morphologically and immunologically distinctive inner ear cell types-such as hair cells, supporting cells, and spiral ganglion neurons-from stem cells. Although promising, all of these studies still lack functional results regarding hearing restoration or vestibular function.

Recovery of visual field and acuity after removal of epiretinal and inner limiting membranes

BACKGROUND: Visual acuity serves as only a rough gauge of macular function. The aim therefore was to ascertain whether central an assessment of the central visual field afforded a closer insight into visual function after removal of epiretinal membranes and Infracyanine-Green- or Trypan-Blue-assisted peeling of the inner limiting membrane. Patients and methods: Fourty-three patients undergoing pars-plana vitrectomy for the removal of epimacular membranes and dye-assisted peeling of the inner limiting membrane using either Infracyanine Green (n = 29; group 1) or Trypan Blue (n = 14; group 2) were monitored prospectively for 12 months. Preoperatively, and 1, 6 and 12 months postoperatively, distance and reading visual acuities were evaluated; the central visual field was assessed by automated static perimetry. RESULTS: Twelve months after surgery, distance and reading visual acuities had improved in both groups, but to a significant degree only in Trypan-Blue-treated eyes. The difference between the two groups was not significant. Likewise at this juncture, the mean size of the visual-field defect remained unchanged in Trypan-Blue-treated eyes (preoperative: 4.3 (SD 2.1) dB; 12 months: 4.0 (2.1) dB (p = 0.15)), but had increased in Infracyanine-Green-treated ones (from 5.3 (3.7) dB to 8.0 (5.2) dB (p = 0.027)). CONCLUSION: Unlike visual acuity, the central visual field had deteriorated in Infracyanine-Green-treated eyes but not in Trypan-Blue-treated eyes 12 months after surgery. Hence, as a predictor of functional outcome, testing of the central visual field may be a more sensitive gauge than visual acuity. Furthermore, Infracyanine Green may have a chronic and potentially clinically relevant effect on the macula which is not reflected in the visual acuity.

Isolated development of inner (wall) caries like lesions in a bacterial-based in vitro model

The study conducted in a bacterial-based in vitro caries model aimed to determine whether typical inner secondary caries lesions can be detected at cavity walls of restorations with selected gap widths when the development of outer lesions is inhibited. Sixty bovine tooth specimens were randomly assigned to the following groups: test group 50 (TG50; gap, 50 microm), test group 100 (TG100; gap, 100 microm), test group 250 (TG250; gap, 250 microm) and a control group (CG; gap, 250 microm). The outer tooth surface of the test group specimens was covered with an acid-resistant varnish to inhibit the development of an outer caries lesion. After incubation in the caries model, the area of demineralization at the cavity wall was determined by confocal laser scanning microscopy. All test group specimens demonstrated only wall lesions. The CG specimens developed outer and wall lesions. The TG250 specimens showed significantly less wall lesion area compared to the CG (p < 0.05). In the test groups, a statistically significant increase (p < 0.05) in lesion area could be detected in enamel between TG50 and TG250 and in dentine between TG50 and TG100. In conclusion, the inner wall lesions of secondary caries can develop without the presence of outer lesions and therefore can be regarded as an entity on their own. The extent of independently developed wall lesions increased with gap width in the present setting.

Isolation of sphere-forming stem cells from the mouse inner ear

The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-forming stem cells from neonatal vestibular and cochlear sensory epithelia as well as from the spiral ganglion. We further describe procedures for sphere propagation, cell differentiation, and characterization of inner ear cell types derived from spheres. Sphere-forming stem cells from the mouse inner ear are an important tool for the development of cellular replacement strategies of damaged inner ears and are a bona fide progenitor cell source for transplantation studies.

Phosphatidylglycerophosphate synthase associates with a mitochondrial inner membrane complex and is essential for growth of Trypanosoma brucei

Maintenance of the lipid composition is important for proper function and homeostasis of the mitochondrion. In Trypanosoma brucei, the enzymes involved in the biosynthesis of the mitochondrial phospholipid, phosphatidylglycerol (PG), have not been studied experimentally. We now report the characterization of T. brucei phosphatidylglycerophosphate synthase (TbPgps), the rate-limiting enzyme in PG formation, which was identified based on its homology to other eukaryotic Pgps. Lipid quantification and metabolic labelling experiments show that TbPgps gene knock-down results in loss of PG and a reduction of another mitochondria-specific phospholipid, cardiolipin. Using immunohistochemistry and immunoblotting of digitonin-isolated mitochondria, we show that TbPgps localizes to the mitochondrion. Moreover, reduced TbPgps expression in T. brucei procyclic forms leads to alterations in mitochondrial morphology, reduction in the amounts of respiratory complexes III and IV and, ultimately, parasite death. Using native polyacrylamide gel electrophoresis we demonstrate for the first time in a eukaryotic organism that TbPgps is a component of a 720 kDa protein complex, co-migrating with T. brucei cardiolipin synthase and cytochrome c1, a protein of respiratory complex III.

Creatine supports propagation and promotes neuronal differentiation of inner ear progenitor cells

Long-term propagation of inner ear-derived progenitor/stem cells beyond the third generation and differentiation into inner ear cell types has been shown to be feasible, but challenging. We investigated whether the known neuroprotective guanidine compound creatine (Cr) promotes propagation of inner ear progenitor/stem cells as mitogen-expanded neurosphere cultures judged from the formation of spheres over passages. In addition, we studied whether Cr alone or in combination with brain-derived neurotrophic factor (BDNF) promotes neuronal differentiation of inner ear progenitors. For this purpose, early postnatal rat spiral ganglia, utricle, and organ of Corti-derived progenitors were grown as floating spheres in the absence (controls) or presence of Cr (5 mM) from passage 3 onward. Similarly, dissociated sphere-derived cultures were differentiated for 14 days in the presence or absence of Cr (5 mM) and spiral ganglia sphere-derived cultures in a combination of Cr with the neurotrophin BDNF (50 ng/ml). We found that the cumulative total number of spheres over all passages was significantly higher after Cr supplementation as compared with controls in all the three inner ear cultures. In contrast, sphere sizes were not affected by the administration of Cr. Administration of Cr during differentiation of spiral ganglia cells resulted in a significantly higher density of β-III-tubulin-positive cells compared with controls, whereas densities of myosin VIIa-positive cells in cultures of utricle and organ of Corti were not affected by the treatment. Importantly, a combination of Cr with the neurotrophin BDNF resulted in further significantly increased densities of β-III-tubulin-positive cells in cultures of spiral ganglia cells as compared with single treatments. In sum, Cr promoted continuing propagation of rat inner ear-derived progenitor cells and supported specifically in combination with BDNF the differentiation of neuronal cell types from spiral ganglion-derived spheres.