Kristen Rak

Effects of salinomycin on human bone marrow-derived mesenchymal stem cells in vitro

Various hypotheses on the origin of cancer stem cells (CSCs) exist, including that CSCs develop from transformed human bone marrow mesenchymal stem cells (hBMSC). Since the polyether antibiotic salinomycin selectively kills CSCs, the present study aims to elucidate the effects of salinomycin on normal hBMSC. The immunophenotype of hBMSC after salinomycin exposure was observed by flow cytometry. The multi-differentiation capacity of hBMSC was evaluated by Oil Red O and van Kossa staining. Cytotoxic effects of salinomycin were monitored by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay. Furthermore, spheroid formation and migration capacity were assessed. There were no differences in the immunophenotype and multi-differentiation capacity of hBMSC induced by salinomycin treatment. Cytotoxic effects were observed at concentrations of 30 μM and above. Neither the migration capability nor the ability to form spheroids was affected. Essential functional properties of hBMSC were unaffected by salinomycin. However, dose-dependent cytotoxicity effects could be observed. Overall, low dose salinomycin showed no negative effects on hBMSC. Since mesenchymal stem cells from various sources respond differently, further in vitro studies are needed to clarify the effect of salinomycin on tissue-specific stem cells.

Valproic acid blocks excitability in SMA type I mouse motor neurons.

Valproic acid (VPA), an antiepileptic drug and HDAC inhibitor, has been identified as a drug candidate for spinal muscular atrophy (SMA), a motoneuron disorder for which currently no effective therapy is available. Based on its potential to up-regulate SMN expression from the SMN2 gene in fibroblasts and lymphoblastoid cell lines from SMA patients, we analysed the effects of VPA in isolated motoneurons from Smn(-/-);SMN2 mice, a model for SMA type I. Treatment with VPA increased Smn expression but unexpectedly also led to reduced growth cone size and reduced excitability in axon terminals of mutant motoneurons. Analysis of Ca2+ currents and distribution of voltage-gated Ca2+ channels revealed an inhibitory function of VPA on voltage-gated Ca2+ channels and possibly also other ion channels that contribute to presynaptic excitability of motoneurons. Our data indicate effects of VPA which might aggravate disease-specific symptoms in SMA patients.

Polyethylene glycol-coupled IGF1 delays motor function defects in a mouse model of spinal muscular atrophy with respiratory distress type 1

Spinal muscular atrophy with respiratory distress type 1 is a neuromuscular disorder characterized by progressive weakness and atrophy of the diaphragm and skeletal muscles, leading to death in childhood. No effective treatment is available. The neuromuscular degeneration (Nmd(2J)) mouse shares a crucial mutation in the immunoglobulin mu-binding protein 2 gene (Ighmbp2) with spinal muscular atrophy with respiratory distress type 1 patients and also displays some basic features of the human disease. This model serves as a promising tool in understanding the complex mechanisms of the disease and in exploring novel treatment modalities such as insulin-like growth factor 1 (IGF1) which supports myogenic and neurogenic survival and stimulates differentiation during development. Here we investigated the treatment effects with polyethylene glycol-coupled IGF1 and its mechanisms of action in neurons and muscles. Polyethylene glycol-coupled IGF1 was applied subcutaneously every second day from post-natal Day 14 to post-natal Day 42 and the outcome was assessed by morphology, electromyography, and molecular studies. We found reduced IGF1 serum levels in Nmd(2J) mice 2 weeks after birth, which was normalized by polyethylene glycol-coupled IGF1 treatment. Nmd(2J) mice showed marked neurogenic muscle fibre atrophy in the gastrocnemius muscle and polyethylene glycol-coupled IGF1 treatment resulted in muscle fibre hypertrophy and slowed fibre degeneration along with significantly higher numbers of functionally active axonal sprouts. In the diaphragm with predominant myogenic changes a profound protection from muscle fibre degeneration was observed under treatment. No effects of polyethylene glycol-coupled IGF1 were monitored at the level of motor neuron survival. The beneficial effects of polyethylene glycol-coupled IGF1 corresponded to a marked activation of the IGF1 receptor, resulting in enhanced phosphorylation of Akt (protein kinase B) and the ribosomal protein S6 kinase in striated muscles and spinal cord from Nmd(2J) mice. Based on these findings, polyethylene glycol-coupled IGF1 may hold promise as a candidate for future treatment trials in human patients with spinal muscular atrophy with respiratory distress type 1.

Isolation and characterization of neural stem cells from the neonatal rat cochlear nucleus

Neural stem cells have been identified in multiple parts of the postnatal mammalian brain, as well as in the inner ear. No investigation of potential neural stem cells in the cochlear nucleus has yet been performed. The aim of this study was to investigate potential neural stem cells from the cochlear nucleus by neurosphere assay and in histological sections to prove their capacity for self-renewal and for differentiation into progenitor cells and cells of the neuronal lineage. For this purpose, cells of the cochlear nucleus of postnatal day 6 rats were isolated and cultured for generation of primary neurospheres. Spheres were dissociated and cells analyzed for capacity for mitosis and differentiation. Cell division was detected by cell-counting assay and BrdU incorporation. Differentiated neural progenitor cells showed distinct labeling for Nestin and for Atoh1. Positive staining of ß-III Tubulin, glial fibrillary acid protein (GFAP) and myelin basic protein (MBP) showed differentiation into neurons, astrocytes and oligodendrocytes. Furthermore, Nestin- and BrdU-labeled cells could also be detected in histological sections. In conclusion, the isolated cells from the cochlear nucleus presented all the features of neural stem cells: cell division, presence of progenitor cells and differentiation into different cells of the neuronal lineage. The existence of neural stem cells may add to the understanding of developmental features in the cochlear nucleus.

Standardized Active Middle-Ear Implant Coupling to the Short Incus Process.

Introduction Active middle-ear implants with floating-mass transducer (FMT) technology are used to treat mild-to-severe sensorineural hearing losses. The standard surgical approach for incus vibroplasty is a mastoidectomy and a posterior tympanotomy, crimping the FMT to the long incus process. An alternative fixation side with less surgical trauma might be the short incus process and incus body. The aim of this study was to develop and test a short incus process coupling device for its functional properties in temporal bone preparations and clinical practice. Materials and Methods An extended antrotomy and a posterior tympanotomy were performed in 10 fresh human temporal bones. As a control for normal middle-ear function, the tympanic membrane was stimulated acoustically, and the vibration of the stapes footplate was measured using laser Doppler vibrometry. FMT-induced vibration responses of the stapes were then measured for standard attachment at the long process and for 2 types of couplers designed for attachment at the short process of the incus (SP1 and SP2 coupler). Additionally, the functional outcome in 2 patients provided with an SP2 coupler was assessed postoperatively at 2 weeks, 3 months, and then 11 months, using pure-tone audiometry, auditory thresholds for frequency-modulated (warble) tones, vibroplasty thresholds, and speech audiometry in quiet and noise. Results For the SP2 coupler, velocity-amplitude responses in temporal-bone preparations showed generally similar mean amplitudes as compared with the standard coupling of the FMT to the long process but with clearly increased mean amplitudes between 0.7 and 1.5 kHz and with reduced interindividual variation between 0.5 and 3 kHz. The clinical data of 2 patients with mild-to-severe sensory hearing loss showed good vibroplasty thresholds and convincing results for speech audiometry in quiet (Freiburger monosyllables at 65 dB SPL, 23 ± 31% unaided versus 83 ± 4% aided) and noise (Hochmair-Schulz-Moser-test at 65 dB SPL at 10 dB SNR, 32 ± 45% unaided and 42 ± 29% aided). Conclusion The attachment of the FMT to the short incus process with the SP2 coupler leads to good mechanical and functional coupling in an experimental setup and clinical practice.

A hydrogel coating for cochlear implant arrays with encapsulated adipose-derived stem cells allows brain-derived neurotrophic factor delivery

Abstract Conclusion: Human adipose-derived stem cells (ASCs), encapsulated in a fibrin-collagen hydrogel for the coating of an electrode array, produce sufficient amounts of neurotrophic factors and may be suitable for enhancing the bioelectric interface of cochlear implants (CIs). Objectives: To evaluate different hydrogel compositions loaded with ASCs with regard to delivery of neuroactive substances and mechanical suitability for the coating of a CI electrode array. Methods: ASCs were cultivated in hydrogels consisting of collagen and fibrin in varying fractions (0:1, 1:1, 1:2, and 1:0). The cell proliferation and viability, as well as the production of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and laminin were determined. Two hydrogel compositions were used as a coating for CI electrode arrays and tested in a scala tympani model. Results: Cell proliferation was best in collagen/fibrin hydrogel compositions (1:1 and 1:2) and increasing amounts of BDNF (up to 2.59 ng/ml) and laminin (up to 320 ng/ml) were detected. GDNF production was inconsistent and markedly lower. A sufficient coating of a CI electrode carrier in terms of stability and flexibility was achieved only with mixed compositions, although hydrogels formed bulky and uneven layers on the silicone surfaces.

Long-term Stability of the Active Middle-ear Implant with Floating-mass Transducer Technology: A Single-center Study.

Objective: To examine the long-term results of an active middle-ear implant (AMEI) with floating-mass transducer (FMT) technology. Study Design: Prospective cohort study of German-speaking patients implanted with an AMEI between 2006 and 2013. Setting: Single-center study. Patients: Eighty-three patients. Intervention: AMEI with FMT technology implantation. Main Outcome Measures: Long-term outcome (27 mo; range, 12–84 mo) for FMT position in correlation with pure-tone audiometry, auditory thresholds for frequency-modulated (warble) tones, vibroplasty thresholds for pure tones, and speech audiometry in quiet and noise. Results: In 15.6% of patients, a revision surgery was necessary to improve functional performance of the AMEI, and the highest revision rate was found with FMT coupling to the round window not using couplers. A peak number of revision surgeries were observed 3 years after the initial surgery. Stable audiological results (pure-tone audiometry and speech audiometry in quiet and noise) were observed up to 84-month post-surgery. Incus vibroplasty (classic indication) showed a significantly lower functional gain compared with oval and round window vibroplasty. Vibroplasty in combined or conductive hearing loss showed no functional difference between forward and reverse stimulation of the cochlea; however, significantly lower vibroplasty thresholds were detected when using a coupler. Conclusions: The AMEI with FMT technology can be safely used in treatment of patients with mild-to-severe sensorineural, conductive, or mixed hearing loss. Optimized coupling, especially in incus vibroplasty, has to be developed to achieve enhanced audiological results.

Human mesenchymal stem cells enhance cancer cell proliferation via IL-6 secretion and activation of ERK1/2

Human mesenchymal stem cells (hMSC) are frequently used in tissue engineering. Due to their strong tumor tropism, hMSC seem to be a promising vehicle for anticancer drugs. However, interactions between hMSC and cancer are ambiguous. Particularly the cytokines and growth factors seem to play an important role in cancer progression and metastasis. The present study evaluated the effects of hMSC on head and neck squamous cell carcinoma (HNSCC) cell lines (FaDu and HLaC78) in vitro. hMSC released several cytokines and growth factors. FaDu and HLaC78 showed a significant enhancement of cell proliferation after cultivation with hMSC-conditioned medium as compared to control. This proliferation improvement was inhibited by the addition of anti-IL-6. The western blot showed an activation of Erk1/2 in FaDu and HLaC78 by hMSC-conditioned medium. HNSCC cell lines expressed EGFR. The current study confirms the importance of cytokines secreted by hMSC in cancer biology. Especially IL-6 seems to play a key role in cancer progression. Thus, the use of hMSC as a carrier for cancer therapy must be discussed critically. Future studies should evaluate the possibility of generating genetically engineered hMSC with, for example, the absence of IL-6 secretion.

Intraoperative monitoring of active middle ear implant function in patients with normal and pathologic middle ears.

Objective: To describe a technique enabling the intraoperative assessment of the mechanical coupling of active middle ear implants in patients with normal or dysfunctional middle ears. Patients: Patients with sensorineural or mixed hearing loss subjected to revision surgery of an active middle ear implant. Intervention: Recording of compound action potentials (CAPs) of the auditory nerve in response to stimulation via an active middle ear implant during revision surgery. Main Outcome Measures: CAP thresholds as a measure of mechanical coupling and system integrity. Conclusion: Determining CAP thresholds may be suitable for identifying a proper mechanical coupling of active middle ear implants in patients with a dysfunctional middle ear, even if the ossicular chain is disrupted.

Growth behavior of cochlear nucleus neuronal cells on semiconductor substrates.

Auditory brainstem implants provide sound information by direct stimulation of the cochlear nucleus to patients with dysfunctional or absent cranial nerve VIII. In contrast to patients with cochlear implants, the use of the auditory brainstem implants is less successful. This cannot be fully explained by the difference location of stimulation but a rather unspecific neuronal stimulation. The aim of this study was to further examine neuronal cells of the cochlear nucleus and to test their interactions with semiconductor substrates as a potential electrode material for improved auditory brainstem implants. The cochlear nuclei of postnatal day 7 rats were microsurgically dissected. The tissue was dissociated enzymatically and plated on coverslips as control and on the semiconductor substrates silicon or silicon nitride. After 4 days in culture the morphology and growth of dissociated cells was determined by fluorescence and scanning electron microscopy. Dissociated cells of the cochlear nucleus showed reduced cell growth on semiconductor substrates compared with controls. SEM analysis demonstrated close contact of neurons with supporting cells in culture and good adherence of neuronal growth cones on the used materials. These findings present basic knowledge for the development of neuron-electrode interfaces for future auditory brainstem implants.