446 Stem cells and development
Creatine supports propagation and promotes neuronal differentiation of inner ear progenitor cells Stefano Di Santob,*, Amir Minaa,*, Ange´lique Ducrayb, Hans R. Widmerb,w and Pascal Senna,w 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 brainderived 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 b-III-tubulin-positive cells compared with controls, whereas densities of myosin
Introduction The loss of mammalian inner ear sensory hair cells leads to a permanent functional loss of hearing and balance because there is no effective regeneration of these cells. The discovery of functional regeneration in the inner ears of nonmammalian vertebrates [1] raised hopes that regenerative principles could be transferred to the mammalian inner ear and ultimately translated into a human hearing loss therapy in the future. Currently, however, no effective cell-based therapy for hearing loss exists. Nevertheless, significant progress has been made at the preclinical level mostly with rodent animal models recently. Sphere-forming stem/progenitor cells have been discovered in the inner ears of mice that can be propagated over multiple generations and differentiated into inner ear cell types such as hair cells, supporting cells, and auditory neurons [2]. Despite most careful handling of primary tissues and stem/progenitor cells, the propagation of inner ear-derived stem cells beyond the third generation remains challenging [3]. Similarly, c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4965
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 b-III-tubulinpositive 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 c ganglion-derived spheres. NeuroReport 25:446–451 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. NeuroReport 2014, 25:446–451 Keywords: auditory neurons, cochlea, creatine, differentiation, hearing loss therapy, inner ear stem cells, propagation, spiral ganglion, utricle a Inner Ear Research Laboratory, Department of Otorhinolaryngology, Head & Neck Surgery and bDepartment of Neurosurgery, Neurocenter and Regenerative Neuroscience Cluster, University of Bern, Inselspital, Bern, Switzerland
Correspondence to Pascal Senn, MD, University Department of Otorhinolaryngology, Inselspital, CH-3010 Bern, Switzerland Tel: + 41 31 632 3347; fax: + 41 31 632 49 00; e-mail: [email protected] *Stefano Di Santo and Amir Mina contributed equally to the writing of this article. wHans R. Widmer and Pascal Senn shared senior authorship. Received 7 November 2013 accepted 25 November 2013
differentiation of meaningful quantities of inner ear cell types such as hair cells and auditory neurons from stem/ progenitor cells is not optimal at present. Hence, ways to improve propagation and to advance differentiation of inner ear cells are needed. An extensive scale of beneficial effects has been attributed to the guanidine compound creatine (Naminoiminomethyl-N-methylglycine, Cr) [4]. In addition, Cr supplementation enhanced neuronal differentiation in striatal and spinal cord cultures [5,6]. Recent evidence indicates that Cr exerts its functions not only by improving cellular energy metabolism but also by nonenergetic mechanisms including antiapoptotic, antioxidant effects, and protection from hyperosmotic shock (for a review, see Andres et al. [7]). The Cr/creatine kinase (Cr/ CK) system also plays an important role in the cellular energy metabolism in mechanotransduction and ion homeostasis in the inner ear [8]. Recently, CK was detected in hair bundles of chicken and mice [9]. Moreover, the Cr/CK system has also been found to be DOI: 10.1097/WNR.0000000000000112
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Creatine supports inner ear cell cultures Di Santo et al. 447
functionally relevant for hearing. CK knockout mice showed elevated auditory brain stem responses [9], whereas the administration of Cr was protective against noise-induced hearing loss in guinea pigs [10]. In the present study, we tested the hypothesis that supplementation of Cr in the culture media improves propagation of neurosphere cultures and neuronal differentiation of postnatal rat inner ear stem/progenitor cells.
Materials and methods
microscopy (Leica Labovert, Solms, Germany) in the culture wells before the dissociation procedure. The diameters of spheres (50 spheres per each condition) were measured systematically by acquiring digital images with calibrated image acquisition and analysis software on an inverted microscope equipped with a digital camera [Leitz Labovert Microscope (Leica, Solms, Germany) equipped with Moticam 2300 camera (Motic Deutschland GmbH, Wetzlar, Germany) and the Motic Image Plus 2.0 software (Motic Deutschland GmbH)].
Isolation of primary inner ear tissues from rat temporal bones
Differentiation of propagated rat inner ear cells
Temporal bones were isolated from the skulls of postnatal days 1–4 Wistar rats (Janvier Elevage, Saint Berthevin, France). The University of Bern Animal Care and Use Committee, Switzerland, approved all experimental protocols. In brief, rats were decapitated under inhalation anesthesia, the heads were briefly rinsed in 70% ethanol, and the temporal bones were isolated. Spiral ganglia, utricles, and organs of Corti were harvested and dissected under the stereomicroscope in Dulbecco’s modified Eagle medium (DMEM; Gibco, Carlsbad, California, USA) as described in detail by Senn et al. [3]. Tissues were transferred into a 50 ml drop of sterile, chilled PBS, and then subjected to a 5–10 min treatment with 0.125% trypsin EDTA (Gibco) at 371C in a total volume of 100 ml. The enzymatic reaction was blocked by adding 100 ml of 0.5% soybean trypsin inhibitor (Worthington, Lakewood, New Jersey, USA) and 2 mg/ml DNAse I (Worthington) in DMEM/high glucose and F12 media (mixed 1 : 1; Gibco). The cells were carefully triturated 20–50 with plastic pipette tips. Cell separation was insured by microscopic inspection. Cell cultures
The freshly dissociated cells (from a total of 16 animals) were cultured as free-floating aggregates of cells (called ‘neurospheres’) in six-well plates in 2 ml of mitogencontaining medium consisting of DMEM/high glucose and F12 media (1 : 1) supplemented with N2 (1%) and B27 (2%) (Invitrogen, Carlsbad, California, USA) supplements, EGF (20 ng/ml; Sigma, St Louis, Missouri, USA), bFGF (10 ng/ml; Sigma), IGF-1 (50 ng/ml; Sigma), and heparan sulfate (50 ng/ml; Sigma). Cultures were placed in a humidified incubator at 371C and 5% CO2. Fresh mitogen-containing media were added every other day from stock solutions. After 1 week, primary spheres (1200–2000 spheres) were passaged by dissociation with trypsin EDTA or Accumax (Innovative Cell Technologies, San Diego, California, USA), followed by careful trituration. After blocking trypsin and Accumax activity, cell suspensions were replated in mitogen-containing medium for 7 days to induce the next generation of spheres and further passaging into higher generations. Sphere formation was assessed comparatively in the absence (control) or presence of Cr (5 mM) (Creapure; SKW, Trostberg, Germany), starting from the third generation. Total sphere numbers were assessed by phase-contrast
Dissociated cell suspensions from second-generation spheres were plated onto poly-DL-lysine (1 mg/ml; Sigma)/ fibronectin (1 mg/ml; Sigma)-coated glass cover slides in 24-well plates (Falcon; BD Biosciences, Franklin Lakes, New Jersey, USA) in DMEM/high glucose and F12 medium (mixed 1 : 1) supplemented with N2 (1%) and B27 (2%) supplements (Invitrogen). A total of 40 000 cells/well (corresponding to 20 000 cells/cm2) were incubated for 14 days and then fixed for further analysis. During differentiation, 80% of the medium was replaced every other day. Differentiation was assessed comparatively across four conditions, that is, in untreated controls, presence of Cr (5 mM), presence of brain-derived neurotrophic factor (BDNF; 50 ng/ml; Promega, Madison, Wisconsin, USA) and presence of a combination of Cr (5 mM) and BDNF (50 ng/ml; Promega). Immunohistochemistry and cell counts
Cultures were fixed for 20 min using 4% paraformaldehyde in PBS, washed with PBS, and blocked for 2 h [0.1% Triton X-100 (Sigma) in PBS, 10% horse serum]. After incubation overnight at 41C with primary antibodies [mouse monoclonal antibody to neuron-specific b-IIItubulin (1 : 1000 TuJ; Promega) or rabbit polyclonal antimyosin VIIa (1 : 1000; Proteus BioSciences, Ramona, California, USA)], cells were labeled using Alexa Fluor 488 donkey anti-mouse or Alexa Fluor 594 donkey anti-rabbit secondary antibodies (1 : 250; Invitrogen). 40 ,6-Diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen) was used to visualize nuclei. The slides were analyzed using an epifluorescence microscope equipped with a digital camera and image acquisition software [Nikon Eclipse 800 with NIS-Elements BR 3.0 (Nikon,Tokyo, Japan) running on a Dell Optiplex PC ((Dell, Austin, Texas, USA)]. Six random fields (700 565 mm) were analyzed from each well. The number of stained cells was counted and related to the total (DAPI positive) cell count. A mean of 265.8±11.2 DAPI-positive cells was analyzed per culture well (mean±SEM). A total of seven cultures from three independent experiments were analyzed. Statistical analyses
A commercially available software package (GraphPad Software, La Jolla, California, USA) was used for statistical analyses. The analyses were carried out using
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448 NeuroReport 2014, Vol 25 No 7
the unpaired Student’s t-test and, for nonparametric analyses, the Mann–Whitney U-test. The Pearson’s coefficient of correlation (r) was calculated for time in culture using a linear regression model for quantification of correlations. Data are presented as mean ± SEM. Statistical significance was set at P value less than 0.05.
Results Propagation of spheres over multiple passages
Neonatal rat inner ear-derived stem/progenitor cells from the utricle and organ of Corti could be propagated up to 11 generations and up to six generations for spiral ganglionderived cells. However, correlation analyses showed that sphere numbers derived from all three inner ear tissues considerably decreased in higher generations compared with the number of primary spheres (r2 = 0.507, 0.188, and 0.546, P < 0.05, for spiral ganglia-derived, utricle-derived, and organ of Corti-derived spheres, respectively; n = 5–6 independent experiments) (Fig. 1a). Diameters of utriclederived and organ of Corti-derived spheres decreased slightly in higher generations (r2 = 0.338 and 0.312,
P < 0.05), whereas no correlation could be found for spiral ganglia-derived spheres (r2 = 0.001; Fig. 1b). Spheres at early passages presented as healthy in appearance (Fig. 1), whereas at prolonged time points, an increasing number of spheres appeared hollow or with irregular spherical shapes (data not shown). Notably, analyses for spiral gangliaderived sphere numbers and total cell numbers at passages 1 and 2 showed a significant correlation at both time points investigated (r2 = 0.647 and 0.611, respectively; P < 0.05; n = 9 and 8 independent experiments), indicating that at least in the early passages more spheres correspond to higher cell numbers. Notably, we have to point out that the outcome presented refers to passages 1 and 2 only and that there is a need for more in-depth studies here to investigate whether more spheres clearly mean more cells. Effects of creatin treatment on sphere numbers over multiple passages
Cr treatment starting at the third passage resulted in higher sphere numbers of propagated spiral gangliaderived, utricle-derived, and organ of Corti-derived cells
Fig. 1
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Correlation analyses for numbers (a, b, c) and diameters (d, e, f) of spiral ganglia-derived, utricle-derived, and organ of Corti-derived spheres, respectively, over passages. For all three inner ear tissue sources, the decrease in sphere numbers was statistically significant over passages. Diameters of utricle-derived and organ of Corti-derived spheres decreased slightly in higher generations, whereas no correlation could be found for spiral ganglia-derived spheres (n = 5–6 independent experiments for both sphere numbers and diameter analyses). Data are presented as percentage of corresponding sphere numbers at first passage, that is, primary passage (PS). Representative phase-contrast photomicrographs of a spiral ganglia-derived, utricle-derived, and organ of Corti-derived spheres at passage 2 (scale bar: 50 mm).
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Creatine supports inner ear cell cultures Di Santo et al. 449
at all time points investigated (Fig. 2). The cumulative total number of spheres combined over all investigated passages was significantly increased after Cr supplementation compared with controls by a factor of 1.40, 1.26, and 1.30 for spiral ganglion, utricle, and organ of Corti (n = 5–6 independent experiments). Sphere sizes were not affected by the administration of Cr compared with untreated controls (data not shown). Effect of creatine on differentiation of sphere-derived cells
As our earlier study showed that fresh dissociated inner ear-derived tissue indicated an overall higher cell survival Fig. 2
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compared with passaged sphere-derived cells (data not shown), we aimed to investigate whether Cr supplementation would result in improved outcome as compared with the control. Cr supplementation during differentiation of second-generation spiral ganglion-derived spheres led to a significantly higher density of b-III-tubulinpositive cells compared with untreated controls, whereas the total cell numbers were not altered by Cr treatment, resulting in a significantly higher percentage of b-IIItubulin-positive neurons (Fig. 3a and b). b-III-tubulin (Tuj1) has been used as a reliable marker to show neuronal differentiation of embryonic stem cell-derived otic cells [11] and for the assessment of neural induction of induced pluripotent stem cells [12]. Similarly, administration of BDNF (50 ng/ml) significantly augmented densities and percentage of b-III-tubulin-positive cells (139.1%±14.1 vs. 100%±7.0 and 128.5%±12.4 vs. 100%±5.5, respectively; n = 9 cultures; 4 independent experiments), whereas no effects were observed for total cell numbers (108.9%±6.8 vs. 100%±4.7) (n = 9 cultures; 4 independent experiments). Most importantly, the combined administration of Cr and BDNF further increased densities (by 1.8-fold) and percentage (by 1.7fold) of b-III-tubulin-positive cells, with no effects for total cell numbers (Fig. 3c). To investigate the effect of Cr on the neuronal differentiation of utricle-derived and organ of Corti-derived cells, we have used the specific marker for hair cells, myosin VIIa. This marker has been used to identify undamaged hair cells in the organ of Corti after a toxic insult [11] or for identification of neurite extension of pluripotent stem cell-derived neurons toward hair cells in a cochlear implant [12]. In contrast to the effects observed for spiral ganglia-derived cells, Cr treatment did not significantly affect the total cell number (data not shown), densities of myosin VIIapositive cells (data not shown), and percentage of myosin VIIa-positive cells of cultures derived from utriclederived (115.6±34.8 vs. 100±10.6; mean±SEM; n = 8 cultures; 2 independent experiments) and organ of Cortiderived (97.5±18.4 vs. 100±15.2; mean±SEM; n = 8 cultures; 2 independent experiments) spheres as compared with controls.
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Discussion
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Propagation of inner ear-derived stem cells for extended generations has been reported to be challenging [3]. Accordingly, in the present study, we observed a critical decrease in sphere cultures particularly after the third passage. On the basis of our previous findings that Cr promoted survival and/or differentiation of neuronal stem/progenitor cells, we assessed the potential of Cr supplementation in advancing propagation of inner ear-derived stem/progenitor cells [5]. Indeed, the administration of Cr significantly attenuated the decrease in sphere numbers at higher generations in all three inner ear organs, reaching roughly 30% when cumulated over all
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Graphical representation of sphere numbers derived from neonatal rat spiral ganglion, utricle, and organ of Corti over multiple passages with 5 mM creatine supplementation starting at passage 3 (filled bars) versus untreated controls (open bars). Data are expressed as percentage of corresponding control numbers at passage 3 (P3) and are given as mean ± SEM (n = 5–6, corresponding to all sphere numbers analyzed per group in independent experiments).
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NeuroReport 2014, Vol 25 No 7
Fig. 3
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Representative photomicrographs of b-III-tubulin-positive cells in differentiated rat spiral ganglia cultures grown in the absence (control) or presence of creatine (Cr) (5 mM) (scale bar: 50 mm) (a). Quantitative analyses showed that Cr treatment (5 mM, filled bars) significantly increased the densities of b-III-tubulin-positive cells (b-III-tub-cell numbers) compared with controls (open bars). Although not altering total cell numbers (DAPI cell numbers), Cr administration resulted in a significantly higher percentage of b-III-tubulin-positive cells per total cell numbers (ratio) (b). Notably, a combination of Cr (5 mM) and brain-derived neurotrophic factor (50 ng/ml) (gray bars) resulted in an even higher density and ratio of b-III-tubulin-positive cells compared with controls (open bars) (c). Values are expressed as percentage of corresponding controls and are presented as mean ± SEM versus corresponding controls (*P < 0.05; n = 7; 3 independent experiments).
passages as compared with untreated controls. Even though we did not specifically address the underlying mechanisms, the observed increase in sphere numbers likely involves augmented survival associated with energy metabolism and additional mechanisms reported previously for neuronal cells [7]. The finding of a missing effect of Cr on mean sphere sizes over passages may suggest that the proliferative capacity of the stem/ progenitor cells was not affected by the administration of Cr. This assumption is in line with the observation of
a lack of neuronal progenitor proliferation in the presence of Cr [5,6,13]. Another key finding of the present study in terms of Cr treatment consists of the significantly higher density of b-III-tubulin-positive cells but not of total cell numbers during differentiation of propagated rat spiral ganglion spheres. The observation of a significantly higher yield of b-III-tubulin-positive neurons thus advocates for a promotion of neuronal differentiation by Cr exposure. This observation is in agreement with our previous study showing enhanced differentiation of GABA-ergic neurons in striatal cultures from embryonic day 14 rats after chronic and short-term Cr treatment [5]. In similar agreement, we reported beneficial effects in fetal rat and human spinal cord cultures following Cr administration [5,6,13]. Similarly, the BDNF-induced neuronal differentiation effect on the spiral ganglionderived cells is in line with previous studies [14–16]. Notably, the neuronal differentiation-inducing actions of both Cr and BDNF were observed to be in the same range of 40%. Importantly, we found that the combination of Cr with BDNF resulted in significantly higher densities of b-III-tubulin-positive cells compared with either single treatments, indicating that likely different signaling pathways were involved in the actions of Cr and BDNF. As a number of other neurotrophic factors including leukemia inhibitory factor [17] stimulate differentiation of spiral ganglion-derived cells, it is tempting to speculate that combinations of Cr with neurotrophic factors may offer a tool for further optimization of neuronal differentiation. In contrast to the finding obtained for spiral ganglion-derived cells, the administration of Cr did not have any effects on differentiation of hair cell-like cells from utricle-derived and organ of Corti-derived spheres. The dissimilarity in findings across inner ear compartments indicate that hair cells, although they share the same neuroectodermal origin with the spiral ganglion neurons, are not targets for the neurotrophic effects elicited through Cr. Notably, the experiments were conducted using early passaged sphere-derived cells and future studies are needed to unravel whether similar effects of Cr will be achieved on survival and/or differentiation of higher passaged sphere-derived cells. Given that Cr is a natural constituent of the regular diet and is also synthesized endogenously, potential benefits of Cr administration can therefore be expected in the absence of any major side effects [7].
Conclusion Taken together, our findings show that the administration of Cr promoted propagation of rat inner ear-derived stem/ progenitor cells and supported differentiation of neuronal cell types from spiral ganglion-derived spheres. Importantly, the observation that the combination of Cr and BDNF treatment resulted in a further augmented yield of neuronal cells from spiral ganglion-derived spheres suggests that the combination of Cr with other
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Creatine supports inner ear cell cultures Di Santo et al. 451
neurotrophic factors may be a potential strategy for optimization of neuronal differentiation of spiral ganglion cultures.
7
Acknowledgements
8
The expert technical assistance for the cell culture work from Susanne Wa¨lchli is gratefully acknowledged.
9
This research was supported by a grant from the Foundation ‘Stiftung fu ¨r Ho¨rgescha¨digte Luzern’ and by an industrial grant from MED-EL GmbH, Innsbruck. Conflicts of interest
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There are no conflicts of interest. 13
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Mackenzie SM, Raible DW. Proliferative regeneration of zebrafish lateral line hair cells after different ototoxic insults. PLoS One 2012; 7:e47257. Li H, Corrales CE, Edge A, Heller S. Stem cells as therapy for hearing loss. Trends Mol Med 2004; 10:309–315. Senn P, Oshima K, Teo D, Grimm C, Heller S. Robust postmortem survival of murine vestibular and cochlear stem cells. J Assoc Res Otolaryngol 2007; 8:194–204. Tokarska-Schlattner M, Epand RF, Meiler F, Zandomeneghi G, Neumann D, Widmer HR, et al. Phosphocreatine interacts with phospholipids, affects membrane properties and exerts membrane-protective effects. PLoS One 2012; 7:e43178. Andres RH, Ducray AD, Huber AW, Perez-Bouza A, Krebs SH, Schlattner U, et al. Effects of creatine treatment on survival and differentiation of GABAergic neurons in cultured striatal tissue. J Neurochem 2005; 95:33–45. Ducray AD, Schlappi JA, Qualls R, Andres RH, Seiler RW, Schlattner U, et al. Creatine treatment promotes differentiation of GABA-ergic neuronal
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precursors in cultured fetal rat spinal cord. J Neurosci Res 2007; 85: 1863–1875. Andres RH, Ducray AD, Schlattner U, Wallimann T, Widmer HR. Functions and effects of creatine in the central nervous system. Brain Res Bull 2008; 76:329–343. Spicer SS, Schulte BA. Creatine kinase in epithelium of the inner ear. J Histochem Cytochem 1992; 40:185–192. Shin JB, Streijger F, Beynon A, Peters T, Gadzala L, McMillen D, et al. Hair bundles are specialized for ATP delivery via creatine kinase. Neuron 2007; 53:371–386. Minami SB, Yamashita D, Ogawa K, Schacht J, Miller JM. Creatine and tempol attenuate noise-induced hearing loss. Brain Res 2007; 1148:83–89. Chen W, Jongkamonwiwat N, Abbas L, Eshtan SJ, Johnson SL, Kuhn S, et al. Restoration of auditory evoked responses by human ES-cell-derived otic progenitors. Nature 2012; 490:278–282. Nishimura K, Nakagawa T, Ono K, Ogita H, Sakamoto T, Yamamoto N, et al. Transplantation of mouse induced pluripotent stem cells into the cochlea. Neuroreport 2009; 20:1250–1254. Ducray AD, Qualls R, Schlattner U, Andres RH, Dreher E, Seiler RW, et al. Creatine promotes the GABAergic phenotype in human fetal spinal cord cultures. Brain Res 2007; 1137:50–57. Marzella PL, Gillespie LN, Clark GM, Bartlett PF, Kilpatrick TJ. The neurotrophins act synergistically with LIF and members of the TGF-beta superfamily to promote the survival of spiral ganglia neurons in vitro. Hear Res 1999; 138 (1–2):73–80. Jin Y, Kondo K, Ushio M, Kaga K, Ryan AF, Yamasoba T. Developmental changes in the responsiveness of rat spiral ganglion neurons to neurotrophic factors in dissociated culture: differential responses for survival, neuritogenesis and neuronal morphology. Cell Tissue Res 2013; 351: 15–27. Zuccotti A, Kuhn S, Johnson SL, Franz C, Singer W, Hecker D, et al. Lack of brain-derived neurotrophic factor hampers inner hair cell synapse physiology, but protects against noise-induced hearing loss. J Neurosci 2012; 32:8545–8553. Oshima K, Teo DT, Senn P, Starlinger V, Heller S. LIF promotes neurogenesis and maintains neural precursors in cell populations derived from spiral ganglion stem cells. BMC Dev Biol 2007; 7:112.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Creatine supports propagation and promotes neuronal differentiation of inner ear progenitor cells Stefano Di Santob,*, Amir Minaa,*, Ange´lique Ducrayb, Hans R. Widmerb,w and Pascal Senna,w 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 brainderived 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 b-III-tubulin-positive cells compared with controls, whereas densities of myosin
Introduction The loss of mammalian inner ear sensory hair cells leads to a permanent functional loss of hearing and balance because there is no effective regeneration of these cells. The discovery of functional regeneration in the inner ears of nonmammalian vertebrates [1] raised hopes that regenerative principles could be transferred to the mammalian inner ear and ultimately translated into a human hearing loss therapy in the future. Currently, however, no effective cell-based therapy for hearing loss exists. Nevertheless, significant progress has been made at the preclinical level mostly with rodent animal models recently. Sphere-forming stem/progenitor cells have been discovered in the inner ears of mice that can be propagated over multiple generations and differentiated into inner ear cell types such as hair cells, supporting cells, and auditory neurons [2]. Despite most careful handling of primary tissues and stem/progenitor cells, the propagation of inner ear-derived stem cells beyond the third generation remains challenging [3]. Similarly, c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4965
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 b-III-tubulinpositive 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 c ganglion-derived spheres. NeuroReport 25:446–451 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. NeuroReport 2014, 25:446–451 Keywords: auditory neurons, cochlea, creatine, differentiation, hearing loss therapy, inner ear stem cells, propagation, spiral ganglion, utricle a Inner Ear Research Laboratory, Department of Otorhinolaryngology, Head & Neck Surgery and bDepartment of Neurosurgery, Neurocenter and Regenerative Neuroscience Cluster, University of Bern, Inselspital, Bern, Switzerland
Correspondence to Pascal Senn, MD, University Department of Otorhinolaryngology, Inselspital, CH-3010 Bern, Switzerland Tel: + 41 31 632 3347; fax: + 41 31 632 49 00; e-mail: [email protected] *Stefano Di Santo and Amir Mina contributed equally to the writing of this article. wHans R. Widmer and Pascal Senn shared senior authorship. Received 7 November 2013 accepted 25 November 2013
differentiation of meaningful quantities of inner ear cell types such as hair cells and auditory neurons from stem/ progenitor cells is not optimal at present. Hence, ways to improve propagation and to advance differentiation of inner ear cells are needed. An extensive scale of beneficial effects has been attributed to the guanidine compound creatine (Naminoiminomethyl-N-methylglycine, Cr) [4]. In addition, Cr supplementation enhanced neuronal differentiation in striatal and spinal cord cultures [5,6]. Recent evidence indicates that Cr exerts its functions not only by improving cellular energy metabolism but also by nonenergetic mechanisms including antiapoptotic, antioxidant effects, and protection from hyperosmotic shock (for a review, see Andres et al. [7]). The Cr/creatine kinase (Cr/ CK) system also plays an important role in the cellular energy metabolism in mechanotransduction and ion homeostasis in the inner ear [8]. Recently, CK was detected in hair bundles of chicken and mice [9]. Moreover, the Cr/CK system has also been found to be DOI: 10.1097/WNR.0000000000000112
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Creatine supports inner ear cell cultures Di Santo et al. 447
functionally relevant for hearing. CK knockout mice showed elevated auditory brain stem responses [9], whereas the administration of Cr was protective against noise-induced hearing loss in guinea pigs [10]. In the present study, we tested the hypothesis that supplementation of Cr in the culture media improves propagation of neurosphere cultures and neuronal differentiation of postnatal rat inner ear stem/progenitor cells.
Materials and methods
microscopy (Leica Labovert, Solms, Germany) in the culture wells before the dissociation procedure. The diameters of spheres (50 spheres per each condition) were measured systematically by acquiring digital images with calibrated image acquisition and analysis software on an inverted microscope equipped with a digital camera [Leitz Labovert Microscope (Leica, Solms, Germany) equipped with Moticam 2300 camera (Motic Deutschland GmbH, Wetzlar, Germany) and the Motic Image Plus 2.0 software (Motic Deutschland GmbH)].
Isolation of primary inner ear tissues from rat temporal bones
Differentiation of propagated rat inner ear cells
Temporal bones were isolated from the skulls of postnatal days 1–4 Wistar rats (Janvier Elevage, Saint Berthevin, France). The University of Bern Animal Care and Use Committee, Switzerland, approved all experimental protocols. In brief, rats were decapitated under inhalation anesthesia, the heads were briefly rinsed in 70% ethanol, and the temporal bones were isolated. Spiral ganglia, utricles, and organs of Corti were harvested and dissected under the stereomicroscope in Dulbecco’s modified Eagle medium (DMEM; Gibco, Carlsbad, California, USA) as described in detail by Senn et al. [3]. Tissues were transferred into a 50 ml drop of sterile, chilled PBS, and then subjected to a 5–10 min treatment with 0.125% trypsin EDTA (Gibco) at 371C in a total volume of 100 ml. The enzymatic reaction was blocked by adding 100 ml of 0.5% soybean trypsin inhibitor (Worthington, Lakewood, New Jersey, USA) and 2 mg/ml DNAse I (Worthington) in DMEM/high glucose and F12 media (mixed 1 : 1; Gibco). The cells were carefully triturated 20–50 with plastic pipette tips. Cell separation was insured by microscopic inspection. Cell cultures
The freshly dissociated cells (from a total of 16 animals) were cultured as free-floating aggregates of cells (called ‘neurospheres’) in six-well plates in 2 ml of mitogencontaining medium consisting of DMEM/high glucose and F12 media (1 : 1) supplemented with N2 (1%) and B27 (2%) (Invitrogen, Carlsbad, California, USA) supplements, EGF (20 ng/ml; Sigma, St Louis, Missouri, USA), bFGF (10 ng/ml; Sigma), IGF-1 (50 ng/ml; Sigma), and heparan sulfate (50 ng/ml; Sigma). Cultures were placed in a humidified incubator at 371C and 5% CO2. Fresh mitogen-containing media were added every other day from stock solutions. After 1 week, primary spheres (1200–2000 spheres) were passaged by dissociation with trypsin EDTA or Accumax (Innovative Cell Technologies, San Diego, California, USA), followed by careful trituration. After blocking trypsin and Accumax activity, cell suspensions were replated in mitogen-containing medium for 7 days to induce the next generation of spheres and further passaging into higher generations. Sphere formation was assessed comparatively in the absence (control) or presence of Cr (5 mM) (Creapure; SKW, Trostberg, Germany), starting from the third generation. Total sphere numbers were assessed by phase-contrast
Dissociated cell suspensions from second-generation spheres were plated onto poly-DL-lysine (1 mg/ml; Sigma)/ fibronectin (1 mg/ml; Sigma)-coated glass cover slides in 24-well plates (Falcon; BD Biosciences, Franklin Lakes, New Jersey, USA) in DMEM/high glucose and F12 medium (mixed 1 : 1) supplemented with N2 (1%) and B27 (2%) supplements (Invitrogen). A total of 40 000 cells/well (corresponding to 20 000 cells/cm2) were incubated for 14 days and then fixed for further analysis. During differentiation, 80% of the medium was replaced every other day. Differentiation was assessed comparatively across four conditions, that is, in untreated controls, presence of Cr (5 mM), presence of brain-derived neurotrophic factor (BDNF; 50 ng/ml; Promega, Madison, Wisconsin, USA) and presence of a combination of Cr (5 mM) and BDNF (50 ng/ml; Promega). Immunohistochemistry and cell counts
Cultures were fixed for 20 min using 4% paraformaldehyde in PBS, washed with PBS, and blocked for 2 h [0.1% Triton X-100 (Sigma) in PBS, 10% horse serum]. After incubation overnight at 41C with primary antibodies [mouse monoclonal antibody to neuron-specific b-IIItubulin (1 : 1000 TuJ; Promega) or rabbit polyclonal antimyosin VIIa (1 : 1000; Proteus BioSciences, Ramona, California, USA)], cells were labeled using Alexa Fluor 488 donkey anti-mouse or Alexa Fluor 594 donkey anti-rabbit secondary antibodies (1 : 250; Invitrogen). 40 ,6-Diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen) was used to visualize nuclei. The slides were analyzed using an epifluorescence microscope equipped with a digital camera and image acquisition software [Nikon Eclipse 800 with NIS-Elements BR 3.0 (Nikon,Tokyo, Japan) running on a Dell Optiplex PC ((Dell, Austin, Texas, USA)]. Six random fields (700 565 mm) were analyzed from each well. The number of stained cells was counted and related to the total (DAPI positive) cell count. A mean of 265.8±11.2 DAPI-positive cells was analyzed per culture well (mean±SEM). A total of seven cultures from three independent experiments were analyzed. Statistical analyses
A commercially available software package (GraphPad Software, La Jolla, California, USA) was used for statistical analyses. The analyses were carried out using
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448 NeuroReport 2014, Vol 25 No 7
the unpaired Student’s t-test and, for nonparametric analyses, the Mann–Whitney U-test. The Pearson’s coefficient of correlation (r) was calculated for time in culture using a linear regression model for quantification of correlations. Data are presented as mean ± SEM. Statistical significance was set at P value less than 0.05.
Results Propagation of spheres over multiple passages
Neonatal rat inner ear-derived stem/progenitor cells from the utricle and organ of Corti could be propagated up to 11 generations and up to six generations for spiral ganglionderived cells. However, correlation analyses showed that sphere numbers derived from all three inner ear tissues considerably decreased in higher generations compared with the number of primary spheres (r2 = 0.507, 0.188, and 0.546, P < 0.05, for spiral ganglia-derived, utricle-derived, and organ of Corti-derived spheres, respectively; n = 5–6 independent experiments) (Fig. 1a). Diameters of utriclederived and organ of Corti-derived spheres decreased slightly in higher generations (r2 = 0.338 and 0.312,
P < 0.05), whereas no correlation could be found for spiral ganglia-derived spheres (r2 = 0.001; Fig. 1b). Spheres at early passages presented as healthy in appearance (Fig. 1), whereas at prolonged time points, an increasing number of spheres appeared hollow or with irregular spherical shapes (data not shown). Notably, analyses for spiral gangliaderived sphere numbers and total cell numbers at passages 1 and 2 showed a significant correlation at both time points investigated (r2 = 0.647 and 0.611, respectively; P < 0.05; n = 9 and 8 independent experiments), indicating that at least in the early passages more spheres correspond to higher cell numbers. Notably, we have to point out that the outcome presented refers to passages 1 and 2 only and that there is a need for more in-depth studies here to investigate whether more spheres clearly mean more cells. Effects of creatin treatment on sphere numbers over multiple passages
Cr treatment starting at the third passage resulted in higher sphere numbers of propagated spiral gangliaderived, utricle-derived, and organ of Corti-derived cells
Fig. 1
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Correlation analyses for numbers (a, b, c) and diameters (d, e, f) of spiral ganglia-derived, utricle-derived, and organ of Corti-derived spheres, respectively, over passages. For all three inner ear tissue sources, the decrease in sphere numbers was statistically significant over passages. Diameters of utricle-derived and organ of Corti-derived spheres decreased slightly in higher generations, whereas no correlation could be found for spiral ganglia-derived spheres (n = 5–6 independent experiments for both sphere numbers and diameter analyses). Data are presented as percentage of corresponding sphere numbers at first passage, that is, primary passage (PS). Representative phase-contrast photomicrographs of a spiral ganglia-derived, utricle-derived, and organ of Corti-derived spheres at passage 2 (scale bar: 50 mm).
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Creatine supports inner ear cell cultures Di Santo et al. 449
at all time points investigated (Fig. 2). The cumulative total number of spheres combined over all investigated passages was significantly increased after Cr supplementation compared with controls by a factor of 1.40, 1.26, and 1.30 for spiral ganglion, utricle, and organ of Corti (n = 5–6 independent experiments). Sphere sizes were not affected by the administration of Cr compared with untreated controls (data not shown). Effect of creatine on differentiation of sphere-derived cells
As our earlier study showed that fresh dissociated inner ear-derived tissue indicated an overall higher cell survival Fig. 2
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compared with passaged sphere-derived cells (data not shown), we aimed to investigate whether Cr supplementation would result in improved outcome as compared with the control. Cr supplementation during differentiation of second-generation spiral ganglion-derived spheres led to a significantly higher density of b-III-tubulinpositive cells compared with untreated controls, whereas the total cell numbers were not altered by Cr treatment, resulting in a significantly higher percentage of b-IIItubulin-positive neurons (Fig. 3a and b). b-III-tubulin (Tuj1) has been used as a reliable marker to show neuronal differentiation of embryonic stem cell-derived otic cells [11] and for the assessment of neural induction of induced pluripotent stem cells [12]. Similarly, administration of BDNF (50 ng/ml) significantly augmented densities and percentage of b-III-tubulin-positive cells (139.1%±14.1 vs. 100%±7.0 and 128.5%±12.4 vs. 100%±5.5, respectively; n = 9 cultures; 4 independent experiments), whereas no effects were observed for total cell numbers (108.9%±6.8 vs. 100%±4.7) (n = 9 cultures; 4 independent experiments). Most importantly, the combined administration of Cr and BDNF further increased densities (by 1.8-fold) and percentage (by 1.7fold) of b-III-tubulin-positive cells, with no effects for total cell numbers (Fig. 3c). To investigate the effect of Cr on the neuronal differentiation of utricle-derived and organ of Corti-derived cells, we have used the specific marker for hair cells, myosin VIIa. This marker has been used to identify undamaged hair cells in the organ of Corti after a toxic insult [11] or for identification of neurite extension of pluripotent stem cell-derived neurons toward hair cells in a cochlear implant [12]. In contrast to the effects observed for spiral ganglia-derived cells, Cr treatment did not significantly affect the total cell number (data not shown), densities of myosin VIIapositive cells (data not shown), and percentage of myosin VIIa-positive cells of cultures derived from utriclederived (115.6±34.8 vs. 100±10.6; mean±SEM; n = 8 cultures; 2 independent experiments) and organ of Cortiderived (97.5±18.4 vs. 100±15.2; mean±SEM; n = 8 cultures; 2 independent experiments) spheres as compared with controls.
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Discussion
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Propagation of inner ear-derived stem cells for extended generations has been reported to be challenging [3]. Accordingly, in the present study, we observed a critical decrease in sphere cultures particularly after the third passage. On the basis of our previous findings that Cr promoted survival and/or differentiation of neuronal stem/progenitor cells, we assessed the potential of Cr supplementation in advancing propagation of inner ear-derived stem/progenitor cells [5]. Indeed, the administration of Cr significantly attenuated the decrease in sphere numbers at higher generations in all three inner ear organs, reaching roughly 30% when cumulated over all
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Graphical representation of sphere numbers derived from neonatal rat spiral ganglion, utricle, and organ of Corti over multiple passages with 5 mM creatine supplementation starting at passage 3 (filled bars) versus untreated controls (open bars). Data are expressed as percentage of corresponding control numbers at passage 3 (P3) and are given as mean ± SEM (n = 5–6, corresponding to all sphere numbers analyzed per group in independent experiments).
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NeuroReport 2014, Vol 25 No 7
Fig. 3
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Representative photomicrographs of b-III-tubulin-positive cells in differentiated rat spiral ganglia cultures grown in the absence (control) or presence of creatine (Cr) (5 mM) (scale bar: 50 mm) (a). Quantitative analyses showed that Cr treatment (5 mM, filled bars) significantly increased the densities of b-III-tubulin-positive cells (b-III-tub-cell numbers) compared with controls (open bars). Although not altering total cell numbers (DAPI cell numbers), Cr administration resulted in a significantly higher percentage of b-III-tubulin-positive cells per total cell numbers (ratio) (b). Notably, a combination of Cr (5 mM) and brain-derived neurotrophic factor (50 ng/ml) (gray bars) resulted in an even higher density and ratio of b-III-tubulin-positive cells compared with controls (open bars) (c). Values are expressed as percentage of corresponding controls and are presented as mean ± SEM versus corresponding controls (*P < 0.05; n = 7; 3 independent experiments).
passages as compared with untreated controls. Even though we did not specifically address the underlying mechanisms, the observed increase in sphere numbers likely involves augmented survival associated with energy metabolism and additional mechanisms reported previously for neuronal cells [7]. The finding of a missing effect of Cr on mean sphere sizes over passages may suggest that the proliferative capacity of the stem/ progenitor cells was not affected by the administration of Cr. This assumption is in line with the observation of
a lack of neuronal progenitor proliferation in the presence of Cr [5,6,13]. Another key finding of the present study in terms of Cr treatment consists of the significantly higher density of b-III-tubulin-positive cells but not of total cell numbers during differentiation of propagated rat spiral ganglion spheres. The observation of a significantly higher yield of b-III-tubulin-positive neurons thus advocates for a promotion of neuronal differentiation by Cr exposure. This observation is in agreement with our previous study showing enhanced differentiation of GABA-ergic neurons in striatal cultures from embryonic day 14 rats after chronic and short-term Cr treatment [5]. In similar agreement, we reported beneficial effects in fetal rat and human spinal cord cultures following Cr administration [5,6,13]. Similarly, the BDNF-induced neuronal differentiation effect on the spiral ganglionderived cells is in line with previous studies [14–16]. Notably, the neuronal differentiation-inducing actions of both Cr and BDNF were observed to be in the same range of 40%. Importantly, we found that the combination of Cr with BDNF resulted in significantly higher densities of b-III-tubulin-positive cells compared with either single treatments, indicating that likely different signaling pathways were involved in the actions of Cr and BDNF. As a number of other neurotrophic factors including leukemia inhibitory factor [17] stimulate differentiation of spiral ganglion-derived cells, it is tempting to speculate that combinations of Cr with neurotrophic factors may offer a tool for further optimization of neuronal differentiation. In contrast to the finding obtained for spiral ganglion-derived cells, the administration of Cr did not have any effects on differentiation of hair cell-like cells from utricle-derived and organ of Corti-derived spheres. The dissimilarity in findings across inner ear compartments indicate that hair cells, although they share the same neuroectodermal origin with the spiral ganglion neurons, are not targets for the neurotrophic effects elicited through Cr. Notably, the experiments were conducted using early passaged sphere-derived cells and future studies are needed to unravel whether similar effects of Cr will be achieved on survival and/or differentiation of higher passaged sphere-derived cells. Given that Cr is a natural constituent of the regular diet and is also synthesized endogenously, potential benefits of Cr administration can therefore be expected in the absence of any major side effects [7].
Conclusion Taken together, our findings show that the administration of Cr promoted propagation of rat inner ear-derived stem/ progenitor cells and supported differentiation of neuronal cell types from spiral ganglion-derived spheres. Importantly, the observation that the combination of Cr and BDNF treatment resulted in a further augmented yield of neuronal cells from spiral ganglion-derived spheres suggests that the combination of Cr with other
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Creatine supports inner ear cell cultures Di Santo et al. 451
neurotrophic factors may be a potential strategy for optimization of neuronal differentiation of spiral ganglion cultures.
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Acknowledgements
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The expert technical assistance for the cell culture work from Susanne Wa¨lchli is gratefully acknowledged.
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This research was supported by a grant from the Foundation ‘Stiftung fu ¨r Ho¨rgescha¨digte Luzern’ and by an industrial grant from MED-EL GmbH, Innsbruck. Conflicts of interest
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There are no conflicts of interest. 13
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