Hearing Research 316 (2014) 57e64
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Research paper
Inner ear stem cells derived feeder layer promote directional differentiation of amniotic fluid stem cells into functional neurons Ling Zong a, Kaitian Chen a, Wei Zhou a, Di Jiang a, b, Liang Sun a, c, Xuemei Zhang a, d, Hongyan Jiang a, * a
Department of Otorhinolaryngology, The First Affiliated Hospital, Sun Yat-Sen University and Institute of Otorhinolaryngology, Sun Yat-Sen University, Guangzhou 510080, PR China Department of Otorhinolaryngology, Dongguan People's Hospital, Dongguan 523059, PR China c Department of Otorhinolaryngology, Hainan General Hospital, Haikou 570311, PR China d Department of Otorhinolaryngology, The Second Hospital of Hebei Medical University, Hebei 050000, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2014 Received in revised form 15 July 2014 Accepted 29 July 2014 Available online 11 August 2014
Intact spiral ganglion neurons are required for cochlear implantation or conventional hearing amplification as an intervention for sensorineural hearing loss. Treatment strategies to replace the loss of spiral ganglion neurons are needed. Recent reports have suggested that amniotic fluid-derived stem cells are capable of differentiating into neuron-like cells in response to cytokines and are not tumorigenic. Amniotic fluid stem cells represent a potential resource for cellular therapy of neural deafness due to spiral ganglion pathology. However, the directional differentiation of amniotic fluid stem cells is undetermined in the absence of cytokines and the consequence of inner ear supporting cells from the mouse cochlea organ of Corti on the differentiation of amniotic fluid stem cells remains to be defined. In an effort to circumvent these limitations, we investigated the effect of inner ear stem cells derived feeder layer on amniotic fluid stem cells differentiation in vitro. An inner ear stem cells derived feeder layer direct contact system was established to induce differentiation of amniotic fluid stem cells. Our results showed that inner ear stem cells derived feeder layer successfully promoted directional differentiation of amniotic fluid stem cells into neurons with characteristics of functionality. Furthermore, we showed that Wnt signaling may play an essential role in triggering neurogenesis. These findings indicate the potential use of inner ear stem cells derived feeder layer as a nerve-regenerative scaffold. A reliable and effective amniotic fluid stem cell differentiation support structure provided by inner ear stem cells derived feeder layer should contribute to efforts to translate cell-based strategies to the clinic. © 2014 Elsevier B.V. All rights reserved.
1. Introduction A loss of sensory hair cells or spiral ganglion neurons from the inner ear causes sensorineural deafness. The quest to restore inner ear tissues remains a major challenge. At present, for profound hearing loss that is not improved by hearing-aid amplification, cochlear implantation surgery remains the only treatment option to restore input. However, intact spiral ganglion neurons are required for effective cochlear implantation and the clinical outcome is closely related to the residual amount of spiral ganglion neurons. Efforts to develop effective treatments for deafness associated with spiral ganglion pathology have demonstrated progress, most notably utilizing stem cell transplantation (Bas et al., 2014; Chen et al., 2012; Kondo et al., 2011; Pandit et al., 2011). * Corresponding author. Tel./fax: þ86 020 87333733. E-mail addresses: [email protected], [email protected] (H. Jiang). http://dx.doi.org/10.1016/j.heares.2014.07.012 0378-5955/© 2014 Elsevier B.V. All rights reserved.
Amniotic fluid has been reported to contain a population of pluripotent stem cells, which is known to have the capacity to differentiate into different cell types representative of three embryonic germ layers in response to appropriate chemical cues (Fauza, 2004; In et al., 2003; McLaughlin et al., 2006; Prusa and Hengstschlager, 2002; Prusa et al., 2003; Tsai et al., 2004, 2006). Several studies have shown that amniotic fluid-derived stem (AFS) cells are capable of differentiating into neuron-like cells under specific conditions in vitro (De Coppi et al., 2007; Donaldson et al., 2009; Prusa et al., 2004). Unlike embryonic stem cells (ES), these cells harbor a low risk of tumor development when implanted in vivo (De Coppi et al., 2007) and as such, represent the basis of a potential cellular therapy for neural deafness. The application of a typical combination of cytokines is known to stimulate neuronal differentiation of AFS cells (De Coppi et al., 2007; Donaldson et al., 2009; Prusa et al., 2004). Nevertheless, this directional differentiation is undetermined in the absence of cytokines. Additionally,
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following transplantation into the inner ear, the implanted cells were located along the auditory nerve fibers close to the organ of Corti (Corrales et al., 2006; Hu et al., 2005). The effect of inner ear supporting cells from the organ of Corti on AFS cell differentiation is still unknown and such limitations may impede the clinical application of AFS cell transplantation. The mechanisms involved in AFS cell neuronal differentiation are poorly understood. Wnt proteins constitute a large family of signaling molecules that have important roles in the regulation of embryonic patterning, cell proliferation and determination (Wodarz and Nusse, 1998). Adult hippocampal stem/progenitor cells express receptors and signaling components for Wnt proteins (Kleber and Sommer, 2004). Overexpression of Wnt-1 in embryonic carcinoma P19 cells promotes neuronal differentiation (Tang et al., 2002). Moreover, it has been reported that Wnt signaling inhibits the self-renewal capacity of neural precursor cells, and instructively promotes differentiation of neurons and astrocytes (Hirabayashi et al., 2004; Muroyama et al., 2004). All this evidence suggests that Wnt signaling plays an important role in triggering neurogenesis; however, the effects of Wnt signaling on the differentiation of AFS cells remains to be elucidated. In this study, we sought to investigate the consequence of inner ear stem cells derived feeder layer on AFS cell directional differentiation in the absence of cytokines in vitro and to elucidate the underlying mechanism. The establishment of an effective AFS cell differentiation relay for inner ear stem cells derived feeder layer should contribute to efforts to translate cell-based strategies to the clinic.
and Use Committee, following the Guidelines for the Care and Use of Laboratory Animals set forth by the National Institutes of Health. Isolation of inner ear stem cells from the organ of Corti of neonatal C57BL/6 mice was performed as described previously (Oshima et al., 2009). The stem cells were cultured at 37 C under 5% CO2 in DMEM/F12 supplemented with N2 supplement, B27 supplement, 20 ng/ml EGF, 20 ng/ml b-FGF and 50 ng/ml IGF-1 in the absence of serum. All growth factors and supplements were obtained from R&D Systems or Invitrogen/GIBCO/BRL. After 7 d, inner ear stem cell-derived hollow spheres were harvested, centrifuged at 2000g for 5 min and dissociated into a single cell suspension with 0.25% trypsin at 37 C for 3 min. The 2 105 single cell suspension (in DMEM/F12) was plated in 6-well culture plates containing uncoated glass cover slips for immunocytochemical analysis in each experiment. Cells were allowed to attach to the substratum for the preparation of a monolayer of feeder cells. AFS cells were then cultured at 37 C under 5% CO2 on the surface of the monolayer to provide a direct contact environment. After 6 d of culture in DMEM/F12, neurons were stained with Tuj1. 2.4. RT-PCR Total RNA was extracted from cultured cells using RNeasy kits (Qiagen, Valencia, CA). Reverse transcription was carried out with Superscript II reverse transcriptase (Invitrogen). PCR Primers were synthesized and PCR were conducted as previous reports (Donaldson et al., 2009; Zhang et al., 2011). The PCR fragments were confirmed by 3% agarose gel electrophoresis with ethidium bromide staining.
2. Materials and methods 2.5. Immunofluorescence 2.1. Amniotic fluid cell culture and isolation of AFS cells Samples of amniotic fluid were obtained from the First Affiliated Hospital of Sun Yat-Sen University following routine amniocentesis carried out on pregnant women at 16e24 weeks of gestation. This project has been reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-Sen University. Written informed consent was obtained from each woman before amniocentesis. Amniotic fluid cells were cultured (Tsai et al., 2004) and CD117-positive stem cell lines were established by magnetic cell sorting using a previously described protocol (De Coppi et al., 2007). AFS cells were labeled with green fluorescent protein (GFP) to permit subsequent detection. For GFP labeling, lentiviral vector encoding GFP (Genechem, Shanghai, China) was used to transfect the AFS cells, as per the manufacturer's protocol. Briefly, replicationincompetent GFP control lentiviral particles (1:10, catalog number: LVCON063) were incubated for 24 h with adherent cells. Cells were washed and re-seeded in feeder layer for further experiments. 2.2. Flow cytometry analysis AFS cell-specific antigen expressions (passage 3) were characterized by flow cytometry analysis. AFS cells were washed with PBS, trypsinized and stained with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies against CD73, CD133, CD44, CD90, CD34, CD117, Oct-4 and MHC-II (BD Biosciences). Positive cells were identified by comparison with cells stained with isotypic control antibodies. 2.3. Isolation of inner ear stem cells, feeder layer cell preparation, direct contact with the AFS cells Animal care and euthanization were conducted according to methods approved by the University of Sun Yat-sen Animal Care
The primary antibodies used were as follows: anti-Oct-4 (1:400; Santa Cruz, CA, USA), anti-Sox2 (1:500; Abcam, Cambridge, UK), anti-Tuj1 (1:500; Abcam), anti-P27kip1 (1:300; Abcam), anti-PSD95 (1:500; Abcam), anti-HuNu (1:100, Millipore, MA, USA), anti-GATA3 (1:100; Abcam). The secondary antibodies used were Alexa-488conjugated anti-mouse or rabbit antibody and Alexa-594conjugated anti-mouse or rabbit antibody (1:400; Molecular Probes, Oregon, USA). Nuclei were stained with DAPI. 2.6. Separate transwell culture system A separate transwell culture system was established to examine the regulatory effects of inner ear stem cells derived feeder layer on AFS cells. For immunocytochemical analysis, AFS cells were seeded in the transwell membrane insert (pore diameter, 0.4 mm, Corning) in a 6-well culture plate containing uncoated glass cover slips. The monolayer of inner ear feeder cells prepared as described above were cultured for 6 d in DMEM/F12 in the lower chamber of the coculture system. The ratio of AFS cells to feeder layer cells was controlled at 1:1. These culture conditions were designed to allow for the secretion of factors and crosstalk by both cell types without cell contact. The percentages of cells that differentiated into morphological neurons per field of vision at 20 magnification were recorded. 2.7. FM 1-43 dye loading FM1-43 dye loading was applied to measure the kinetics of synaptic vesicles exocytosis and endocytosis as previously described (Amaral et al., 2011; Ryan et al., 1993). Neurons were labeled by superfusion in a chamber with the fluorescent styryl membrane probe FM1-43 (10 mM in 45 mM KCl) for 1 min and washed with a 3 mM KCl solution for 1e5 min. Subsequently, the
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same neurons were destained in 90 mM KCl solution for 5 min. The off-rate of the dye from neuronal membranes was measured directly by recording the decrease in fluorescence intensity as a function of time during the period after staining. 2.8. Inhibition of Wnt signaling In the treatment group, mouse Dickkopf related protein 1 (Dkk1) was added at 100 ng/ml (R&D Systems) to the DMEM/F12 culture medium for 6 days. The following concentration ranges were tested to determine the optimal concentration of the reagent: Dkk1 at 50e200 ng/ml. Half of the medium was replaced on the third day of incubation. A two stages culture protocol for AFS differentiation was introduced as a standard comparison (De Coppi et al., 2007; Zsebo et al., 1990). The percentage and number of cells that differentiated into morphological neurons per field of vision at 20 magnification were recorded. TUNEL staining (KeyGen, Nanjing, China) was used to exclude the possibility of cells death caused by DKK1. Positive control of apoptosis was designed according to the procedure by the manufacturer. 2.9. Determination of neurite length and neuron survival The number of processes and neurite length was determined as previously described from digital images (7e10) randomly selected fields (20 magnification), with approximately equal cell density, for each experimental condition (Atkinson et al., 2011; Xu et al., 2012).
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3. Results 3.1. Isolation and characterization of AFS cells After 7e9 days of in vitro culture of amniotic fluid, both fibroblast-like cells and epithelioid cells appeared. For the 2 to 3 passages, most cells exhibited a fibroblast-like phenotype (Fig. 1A). The AFS cells proliferated as a monolayer, with a typical doublingtime (approximately 48 h). Occasional ES-like AFS cell colonies were observed (Fig. 1B). The AFS cells were negative for hematopoietic stem cell markers (CD34, CD133) and Class II major histocompatibility (MHC) antigens (HLA-DR) (Fig. 1C). However, they were positive for surface markers characteristic of mesenchymal stem cells, including CD73, CD90, and some were weakly positive for CD44. About 75.0 ± 4.6% of cells expressed transcription factor Oct-4, which is considered to be a marker of pluripotent stem cells (Donovan, 2001; Pesce and Scholer, 2001) and is especially associated with the maintenance of the undifferentiated state of ES (Pan et al., 2002). AFS cell expression of Oct-4 was confirmed by immunofluorescence. Besides, about 86.0 ± 2.8% of cells expressed Sox2, which is a transcription factor expressed in the neural progenitors, neural stem cells, and is involved in neural cell commitment and self-renewal (Ferri et al., 2004; Neves et al., 2007; Pevny and Nicolis, 2010). Co-expression of Oct-4 and Sox2 were identified in 72 ± 4.9% of cells (Fig. 1D). The expressions pattern varied amongst different cell lines and were confirmed by RT-PCR (supplementary information Fig. 1).
3.2. Neuronal differentiation in the inner ear stem cells derived feeder layer direct contact system
2.10. Statistical analysis Data were obtained from at least three replicate tests and then presented as means ± standard derivation. Statistical analysis was carried out by means of Student's t-tests, One-way ANOVA using SPSS 11.0. A value of P < 0.05 was considered statistically significant.
After 7 days of in vitro culture, inner ear stem cells formed hollow spheres (Fig. 2A) that were harvested and dissociated into single cell suspensions for the preparation of feeder cell monolayer (Fig. 2B), which were positive for P27kip1 (Fig. 2C), a marker of inner ear supporting cells of the sensory epithelium (Chen and Segil,
Fig. 1. (A) The morphology of amniotic fluid cells after culturing for 3 passages in vitro. (B) Embryonic stem cell-like AFS cell colonies appeared occasionally in the primary culture. (C) Expression of surface antigens was detected by flow cytometry (filled curve) in AFS cells. (D) AFS cells were positive for Oct-4 and Sox2. Scale bar: A, B ¼ 100 mm; D ¼ 50 mm.
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Fig. 2. Neuronal differentiation in the inner ear stem cells derived feeder layer. (A) Typical hollow spheres generated after 7 d of culture in vitro. (B) A monolayer of feeder cells from hollow spheres. (C) Feeder layer cells are positive for P27kip1. (D) AFS cells are cultured on top of the feeding monolayer, forming a direct contact system. (E) GFPþ AFS cells in the direct contact system, showing large numbers of neurons with strong expression of Tuj1 and long neurites at the site of a massive accumulation of cell nuclei. (F, G) Neuronal cell bodies, fibers and terminals observed in direct contact system, nuclei are stained with HuNu or DAPI. Scale bar: A ¼ 100 mm; C, E ¼ 50 mm; B, D ¼ 25 mm; F ¼ 12.5 mm.
1999). According to our previous study, the otic hollow spheres used to produce these feeder cells were not able to differentiate into neurons in the present study, probably due to their lack of differentiation capacity of stem cells (unpublished data). Subsequently, a direct contact system by culturing lentiviraltransfected GFPþ AFS cells on the feeder cell monolayer was established to visualize the AFS cells' differentiation (Fig. 2D). The morphology of AFS cells changed obviously from the third day compared with that of uninduced AFS cells. The cell body exhibited a long process extending to join and overlap cells. The GFPþ AFS cells gave rise to large numbers of neurons, not only having strong Tuj1 expression but also exhibiting typical neuronal morphological characteristics and long neurites after 6 days’ culture (Fig. 2E). The neurons appeared at the site of a massive accumulation of cell nuclei belonging to the seeded AFS cells. Furthermore, the neurons showed direct contact with the structure of the inner ear stem cells derived feeder layer. Human cells were distinguished from the
mouse cells by staining their nuclei with an antibody against the human nuclear (HuNu) antigen (Fig. 2F). Neuronal cell bodies, fibers and terminals were observed in this culture system at high magnification (Fig. 2FeG). The neurons developed normal polarity; an average of 79 ± 2.0% had two neurite outgrowths, while a small percentage (19 ± 3.2%) had multiple processes. Interestingly, 30 ± 6.0% of these neurons was positive for GATA-3 (Fig. 3), a marker of spiral ganglion neurons, indicating that AFS cells could differentiate into auditory neurons under certain condition. The Tuj1þ and GFPþ cells became elongated with neuron-like processes up to 320 mm. The AFS cells cultivated alone for 6 days exhibited specific Tuj1 expression, but no typical neuron was seen (Fig. 4A). Tuj1 expression by feeder layer cells cultured alone for 6 days was negligible (Fig. 4B). Altogether, these observations confirmed the effectiveness of the inner ear stem cells derived feeder layer on AFS cell neurogenesis.
Fig. 3. The direct contact co-culture produced neurons that were positive for GATA-3.
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Fig. 4. AFS cells (A) or feeder layer cells (B) that were cultured alone showed mere expression of Tuj1. (C) In transwell system, no typical pyramidal morphological neuron was observed. Scale bar: ¼ 50 mm.
The co-culture of AFS cells with normal neonatal cochlea were further investigated, but our results indicated that normal neonatal cochlea could promote neither disperse nor spherical AFS cells into differentiation (data not shown). Combined with the present study, intact inner ear supporting cells structures seemed not to be able to promote AFS cells into differentiation until lesion occurred. The specific differentiations of AFS cells co-culture with neonatal cochlea after lesion are under study. Further studies regarding the nature and functional characterization of the neurons in the present study are still required. 3.3. Separate transwell culture system In this system, the two cell types are physically separated to avoid direct cell-to-cell contact, while intercellular communication occurs only through substances released by either cell type into the shared culture medium. After 6 d of co-culture, the fraction of Tuj1þ expressing cells did not change obviously. Although the AFS cells exhibited specific Tuj1 expression, typical neuronal morphology was scarcely observed (0.6 ± 0.18%). The feeder layer cells exhibited minimal or scarcely detectable expression of Tuj1 (1.4 ± 0.8%). These results were distinguished from direct contact co-culture, whose percentage was 37 ± 3.0% (P < 0.01, One-way ANOVA). No clear evidence of morphological characteristics typical of neurons was observed in the AFS cells and feeder cell layer cultured separately in the transwell culture system (Fig. 4C). 3.4. Neurons derived from inner ear stem cells derived feeder layer system show characteristics of functionality Synapse formation between neurons occurred within 6 days, as indicated by punctuate staining of the postsynaptic density protein
(PSD95), which was concentrated at the synapses in mature neurons (Fig. 5A). About 90 ± 1.2% of neurons were positive for PSD95 from 700 neurons that were counted. As seen in Fig. 5B, typical synaptic structures were associated with the dendrites, spines, and cell bodies of neurons derived from AFS cells. Synaptic gaps between two neurons were identified. Additionally, AFS cells in the direct contact system were observed to undergo active synaptic vesicle recycling that was monitored by visualizing the FM1-43 dye uptake and release after potassium stimulation. Fig. 5C illustrated neurons derived from AFS cells in the direct contact system during superfusion with a depolarizing solution (45 mM KCl) containing 10 mm FM1-43. The cell body showed two neurites traversing its length. Fig. 5D shows the same cells after a second 60 s superfusion in 90 mM KCl solution, in this case, in the absence of dye. The fluorescence intensity decreased significantly during this period (n ¼ 6 independent specimens). 3.5. The feeder layer direct contact system may depends on Wnt signaling In the control group (without Dkk1), an average of 37 ± 3.0% of AFS cells differentiated into morphologically neurons with long neurite outgrowths compared to 21 ± 2.5% in DKK1-treated group (Fig. 6A). The percentage of processes derived from DKK1-treated AFS cells decreased greatly to 33.3 ± 3.8% by contrast with 60.4 ± 4.6% in the control group (P < 0.01, Fig. 6B), suggesting that AFS cells differentiation toward neurons were inhibited when the Wnt signaling was suppressed. Whereas about 18.4 ± 1.4% AFS cells were induced into morphologically neurons according to the two stage differentiation model (De Coppi et al., 2007; Zsebo et al., 1990), a number less than Dkk1 treatment group or control group (Fig. 6C, P < 0.01, One-way ANOVA). In addition to the obvious
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Fig. 5. Functional bioassay on neurons derived from AFS cells in direct contact co-culture. (A) Synapse formation was positive for Tuj1 and PSD95. (B) Enlarged picture showed typical synapse formation. (C) The neurons being superfused in a 45 mM KCl solution containing FM1-43; (D) The same field of view as in (C), after a 60s superfusion in 90 mM KCl. Note the marked decrease in fluorescence intensity. Scale bar: A, C, D ¼ 25 mm.
decrease in the number of neurites, Dkk1 treatment led to differentiated neurons with shortened outgrowths than in the direct contact system (Fig. 6D). Dkk1 significantly reduced neurite length; the average length was 51 ± 1.5 mm in the Dkk1 treatment group, compared to 94 ± 3.7 mm in the control group (P < 0.01). In addition, the absence of TUNEL expression confirmed that Dkk1 prevent differentiation mainly, instead of causing cell death (supplementary information Fig. 2). The percentage of Sox2positive cells with/without DKK1 treatment was similar (81.2 ± 3.8% vs. 82.4 ± 2.6%, P > 0.05). Taken together, these findings indicate that Wnt signaling may play an important role in neuronal differentiation of AFS cells. 4. Discussion The present study aimed to examine the possibility of utilizing inner ear stem cells derived feeder layer in a direct contact system to promote directional differentiation of AFS cells into functional neurons; in principle, an efficacious AFS cell source could translate to clinical use rapidly. First, we established an inner ear stem cells derived feeder layer direct contact system for AFS cells. In this direct contact system, AFS cells were cultured for 6 days on the surface of a feeder cell layer derived from inner ear stem cells obtained from the organ of Corti of neonatal mice. Using this approach, we found neurons developed normal polarity, most with two neurites and a small percentage with multiple neurites and long processes. These exciting results may be attributed, in part, to directional induction by feeder layer. The combination of AFS cells and inner ear stem cells derived feeder layer are suspected to induce stable neuron differentiation.
It is known that inner ear, which mediates hearing and sensory equilibrium, develops from the ectodermal placode. During inner ear development, the majorities of neural stem cells are generated and differentiate into cell types including sensory hair cells and spiral ganglion neurons. It can be speculated that the mechanism underlying this effect is based on the stimulation of the production of numerous neurons as a result of direct cell-to-cell contact between the two cell types although further research is required to confirm this hypothesis. However, in comparison with reported methods (De Coppi et al., 2007; Donaldson et al., 2009; Prusa et al., 2004), the direct contact system is a cost-effective method that allows stable induction of the neuron-oriented differentiation of AFS cells. Inner ear stem cells derived feeder layer may serve as a nerve-regenerative scaffold. Using a transwell culture system to prevent direct cell-to-cell contact, we showed that there was no obvious change in the fraction of AFS cells expressing Tuj1þ and no specific neuron-like cell morphology was observed. Thus, we are able to speculate that the process of neuron-oriented differentiation does not depend on secreted factors and molecules; while direct cell-to-cell interactions, especially mechanical signals provided by inner ear stem cells derived feeder layer, might regulate the fate of AFS cells. It is important to assess whether the morphological differentiation of neuritis in the direct contact system concurred with the acquisition of functional properties. Synaptogenesis, indicated by the concentration of PSD95 at synapses in mature neurons, was confirmed in this system. Meanwhile, by monitoring the uptake and release of the fluorescent marker dye, FM1-43, to show active synaptic vesicle recycling (Amaral et al., 2011; Ryan et al., 1993), we further demonstrated the functional trait of the cells differentiated
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Fig. 6. Wnt signaling is involved in direct contact system. Immunofluorescent staining for Tuj1 in the direct contact without Dkk1 treatment (A) and with Dkk1 treatment (B). (C) The number of processes was measured in different experimental groups. (D) The length of neurites was measured in different experimental groups compared to the two stage differentiation protocol (De Coppi et al., 2007; Zsebo et al., 1990). Scale bar: A, B ¼ 50 mm.
in our study. Thus, we consider this approach using inner ear stem cells derived feeder layer provides a microenvironment to support morphological and functional neuronal differentiation and regulate neurogenesis by directing AFS cells to adopt a neuronal fate. The Wnt/b-catenin signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations (Chai et al., 2012). It has been reported that in the neonatal cochlea, the Lgr5 marker of adult stem cells, is expressed in a subset of inner ear supporting cells and that Lgr5-expressing inner ear supporting cells are Wnt-responsive sensory precursor cells (Chai et al., 2012; Shi et al., 2012). The feeder layer derived from inner ear stem cells has the specific marker of inner ear supporting cells. We therefore investigated whether feeder layer cells from the organ of Corti of neonatal mice provided Wnt signaling to support the neuronal differentiation of AFS cells observed in the present study. Our results suggested that blocking Wnt signaling activity significantly affected the number of neurons and neurite length. The possibility that Wnt signaling is playing a role in neurite outgrowth in the present study was accord with the conclusion by Blakely et al. (2011). We speculated that inner ear stem cells derived feeder layer trigger Wnt signaling cell-surface stretch receptors and adhesion sites, resulting in signaling cascades including the activation of genes responsible for the synthesis of functional neurons. Inner ear stem cells derived feeder layer may represent a new nerve-regenerative scaffold and possess broad application prospects in the field of neuron regeneration. The hypothesis that Wnt signaling activators may improve the efficacy of AFS transplantation remains to be investigated.
The present results have raised great significance for clinical practices. It is important to note that inner ear stem cells derived feeder layer exert positive effects on AFS cells differentiation and AFS cell transplantation may be feasible. The two types of cells have
Fig. 7. Flow chart documenting the total numbers and percentages of cells with a given characteristic in each stage.
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direct cell-to-cell contact and produce certain amount of functional neurons to replace lesion spiral ganglion. This culture system was also shown to be effective and stable for the neuron-oriented differentiation in the absence of any cytokines (Fig. 7). In combination with a previous study (De Coppi et al., 2007), our study indicates that AFS cell transplantation seems to be practicable with low risk of tumor development. Understanding the functional roles of feeder cells on AFS cells would provide useful information for clinical translation. However, an in vivo study is necessary to determine the effects of differences in the inner ear environment, including factors such as pH and ion types and concentrations. In conclusion, our study shows that inner ear stem cells derived feeder layer successfully promote the directional differentiation of AFS cells into functional neurons and that Wnt signaling may play an essential role in triggering neurogenesis. Though the nature of neurons in the present study remained to be defined, the reliability of an effective AFS cell differentiation system on inner ear stem cells derived feeder layer should contribute to efforts to translate cellbased strategies to the clinic. Conflict of interests None. Acknowledgements The study was supported by grants from the National Basic Research Program of China (No. 2011CB504502), the National Natural Science fund of China (No. 81271076 and No. 81200748) and the Minster of Health of China (No. 201202005). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.heares.2014.07.012. References Amaral, E., Guatimosim, S., Guatimosim, C., 2011. Using the fluorescent styryl dye FM1-43 to visualize synaptic vesicles exocytosis and endocytosis in motor nerve terminals. Methods Mol. Biol. 689, 137e148. Atkinson, P.J., Cho, C.H., Hansen, M.R., Green, S.H., 2011. Activity of all JNK isoforms contributes to neurite growth in spiral ganglion neurons. Hear Res. 278, 77e85. Bas, E., Van De Water, T.R., Lumbreras, V., Rajguru, S., Goss, G., Hare, J.M., Goldstein, B.J., 2014. Adult human nasal mesenchymal-like stem cells restore cochlear spiral ganglion neurons after experimental lesion. Stem Cells Dev. 23, 502e514. Blakely, B.D., Bye, C.R., Fernando, C.V., Horne, M.K., Macheda, M.L., Stacker, S.A., Arenas, E., Parish, C.L., 2011. Wnt5a regulates midbrain dopaminergic axon growth and guidance. PLoS One 6, e18373. Chai, R., Kuo, B., Wang, T., Liaw, E.J., Xia, A., Jan, T.A., Liu, Z., Taketo, M.M., Oghalai, J.S., Nusse, R., Zuo, J., Cheng, A.G., 2012. Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea. Proc. Natl. Acad. Sci. U. S. A. 109, 8167e8172. Chen, P., Segil, N., 1999. p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti. Development 126, 1581e1590. Chen, W., Jongkamonwiwat, N., Abbas, L., Eshtan, S.J., Johnson, S.L., Kuhn, S., Milo, M., Thurlow, J.K., Andrews, P.W., Marcotti, W., Moore, H.D., Rivolta, M.N., 2012. Restoration of auditory evoked responses by human ES-cell-derived otic progenitors. Nature 490, 278e282. Corrales, C.E., Pan, L., Li, H., Liberman, M.C., Heller, S., Edge, A.S., 2006. Engraftment and differentiation of embryonic stem cell-derived neural progenitor cells in the cochlear nerve trunk: growth of processes into the organ of Corti. J. Neurobiol. 66, 1489e1500. De Coppi, P., Bartsch, G.J., Siddiqui, M.M., Xu, T., Santos, C.C., Perin, L., Mostoslavsky, G., Serre, A.C., Snyder, E.Y., Yoo, J.J., Furth, M.E., Soker, S., Atala, A., 2007. Isolation of amniotic stem cell lines with potential for therapy. Nat. Biotechnol. 25, 100e106.
Donaldson, A.E., Cai, J., Yang, M., Iacovitti, L., 2009. Human amniotic fluid stem cells do not differentiate into dopamine neurons in vitro or after transplantation in vivo. Stem Cells Dev. 18, 1003e1012. Donovan, P.J., 2001. High Oct-ane fuel powers the stem cell. Nat. Genet. 29, 246e247. Fauza, D., 2004. Amniotic fluid and placental stem cells. Best. Pract. Res. Clin. Obstet. Gynaecol. 18, 877e891. Ferri, A.L., Cavallaro, M., Braida, D., Di Cristofano, A., Canta, A., Vezzani, A., Ottolenghi, S., Pandolfi, P.P., Sala, M., DeBiasi, S., Nicolis, S.K., 2004. Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain. Development 131, 3805e3819. Hirabayashi, Y., Itoh, Y., Tabata, H., Nakajima, K., Akiyama, T., Masuyama, N., Gotoh, Y., 2004. The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells. Development 131, 2791e2801. Hu, Z., Wei, D., Johansson, C.B., Holmstrom, N., Duan, M., Frisen, J., Ulfendahl, M., 2005. Survival and neural differentiation of adult neural stem cells transplanted into the mature inner ear. Exp. Cell. Res. 302, 40e47. In, T.A.P., Scherjon, S.A., Kleijburg-van, D.K.C., Noort, W.A., Claas, F.H., Willemze, R., Fibbe, W.E., Kanhai, H.H., 2003. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 102, 1548e1549. Kleber, M., Sommer, L., 2004. Wnt signaling and the regulation of stem cell function. Curr. Opin. Cell. Biol. 16, 681e687. Kondo, T., Matsuoka, A.J., Shimomura, A., Koehler, K.R., Chan, R.J., Miller, J.M., Srour, E.F., Hashino, E., 2011. Wnt signaling promotes neuronal differentiation from mesenchymal stem cells through activation of Tlx3. Stem Cells 29, 836e846. McLaughlin, D., Tsirimonaki, E., Vallianatos, G., Sakellaridis, N., Chatzistamatiou, T., Stavropoulos-Gioka, C., Tsezou, A., Messinis, I., Mangoura, D., 2006. Stable expression of a neuronal dopaminergic progenitor phenotype in cell lines derived from human amniotic fluid cells. J. Neurosci. Res. 83, 1190e1200. Muroyama, Y., Kondoh, H., Takada, S., 2004. Wnt proteins promote neuronal differentiation in neural stem cell culture. Biochem Biophys. Res. Commun. 313, 915e921. Neves, J., Kamaid, A., Alsina, B., Giraldez, F., 2007. Differential expression of Sox2 and Sox3 in neuronal and sensory progenitors of the developing inner ear of the chick. J. Comp. Neurol. 503, 487e500. Oshima, K., Senn, P., Heller, S., 2009. Isolation of sphere-forming stem cells from the mouse inner ear. Methods Mol. Biol. 493, 141e162. Pan, G.J., Chang, Z.Y., Scholer, H.R., Pei, D., 2002. Stem cell pluripotency and transcription factor Oct4. Cell. Res. 12, 321e329. Pandit, S.R., Sullivan, J.M., Egger, V., Borecki, A.A., Oleskevich, S., 2011. Functional effects of adult human olfactory stem cells on early-onset sensorineural hearing loss. Stem Cells 29, 670e677. Pesce, M., Scholer, H.R., 2001. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19, 271e278. Pevny, L.H., Nicolis, S.K., 2010. Sox2 roles in neural stem cells. Int. J. Biochem Cell. Biol. 42, 421e424. Prusa, A.R., Hengstschlager, M., 2002. Amniotic fluid cells and human stem cell research: a new connection. Med. Sci. Monit. 8, RA253e7. Prusa, A.R., Marton, E., Rosner, M., Bernaschek, G., Hengstschlager, M., 2003. Oct-4expressing cells in human amniotic fluid: a new source for stem cell research? Hum. Reprod. 18, 1489e1493. Prusa, A.R., Marton, E., Rosner, M., Bettelheim, D., Lubec, G., Pollack, A., Bernaschek, G., Hengstschlager, M., 2004. Neurogenic cells in human amniotic fluid. Am. J. Obstet. Gynecol. 191, 309e314. Ryan, T.A., Reuter, H., Wendland, B., Schweizer, F.E., Tsien, R.W., Smith, S.J., 1993. The kinetics of synaptic vesicle recycling measured at single presynaptic boutons. Neuron 11, 713e724. Shi, F., Kempfle, J.S., Edge, A.S., 2012. Wnt-responsive Lgr5-expressing stem cells are hair cell progenitors in the cochlea. J. Neurosci. 32, 9639e9648. Tang, K., Yang, J., Gao, X., Wang, C., Liu, L., Kitani, H., Atsumi, T., Jing, N., 2002. Wnt-1 promotes neuronal differentiation and inhibits gliogenesis in P19 cells. Biochem Biophys. Res. Commun. 293, 167e173. Tsai, M.S., Lee, J.L., Chang, Y.J., Hwang, S.M., 2004. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum. Reprod. 19, 1450e1456. Tsai, M.S., Hwang, S.M., Tsai, Y.L., Cheng, F.C., Lee, J.L., Chang, Y.J., 2006. Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells. Biol. Reprod. 74, 545e551. Wodarz, A., Nusse, R., 1998. Mechanisms of Wnt signaling in development. Annu Rev. Cell. Dev. Biol. 14, 59e88. Xu, N., Engbers, J., Khaja, S., Xu, L., Clark, J.J., Hansen, M.R., 2012. Influence of cAMP and protein kinase A on neurite length from spiral ganglion neurons. Hear Res. 283, 33e44. Zhang, M., Ma, Q., Hu, H., Zhang, D., Li, J., Ma, G., Bhat, K., Wu, E., 2011. Stem cell factor/c-kit signaling enhances invasion of pancreatic cancer cells via HIF1alpha under normoxic condition. Cancer Lett. 303, 108e117. Zsebo, K.M., Williams, D.A., Geissler, E.N., Broudy, V.C., Martin, F.H., Atkins, H.L., Hsu, R.Y., Birkett, N.C., Okino, K.H., Murdock, D.C., et al., 1990. Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63, 213e224.
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Research paper
Inner ear stem cells derived feeder layer promote directional differentiation of amniotic fluid stem cells into functional neurons Ling Zong a, Kaitian Chen a, Wei Zhou a, Di Jiang a, b, Liang Sun a, c, Xuemei Zhang a, d, Hongyan Jiang a, * a
Department of Otorhinolaryngology, The First Affiliated Hospital, Sun Yat-Sen University and Institute of Otorhinolaryngology, Sun Yat-Sen University, Guangzhou 510080, PR China Department of Otorhinolaryngology, Dongguan People's Hospital, Dongguan 523059, PR China c Department of Otorhinolaryngology, Hainan General Hospital, Haikou 570311, PR China d Department of Otorhinolaryngology, The Second Hospital of Hebei Medical University, Hebei 050000, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2014 Received in revised form 15 July 2014 Accepted 29 July 2014 Available online 11 August 2014
Intact spiral ganglion neurons are required for cochlear implantation or conventional hearing amplification as an intervention for sensorineural hearing loss. Treatment strategies to replace the loss of spiral ganglion neurons are needed. Recent reports have suggested that amniotic fluid-derived stem cells are capable of differentiating into neuron-like cells in response to cytokines and are not tumorigenic. Amniotic fluid stem cells represent a potential resource for cellular therapy of neural deafness due to spiral ganglion pathology. However, the directional differentiation of amniotic fluid stem cells is undetermined in the absence of cytokines and the consequence of inner ear supporting cells from the mouse cochlea organ of Corti on the differentiation of amniotic fluid stem cells remains to be defined. In an effort to circumvent these limitations, we investigated the effect of inner ear stem cells derived feeder layer on amniotic fluid stem cells differentiation in vitro. An inner ear stem cells derived feeder layer direct contact system was established to induce differentiation of amniotic fluid stem cells. Our results showed that inner ear stem cells derived feeder layer successfully promoted directional differentiation of amniotic fluid stem cells into neurons with characteristics of functionality. Furthermore, we showed that Wnt signaling may play an essential role in triggering neurogenesis. These findings indicate the potential use of inner ear stem cells derived feeder layer as a nerve-regenerative scaffold. A reliable and effective amniotic fluid stem cell differentiation support structure provided by inner ear stem cells derived feeder layer should contribute to efforts to translate cell-based strategies to the clinic. © 2014 Elsevier B.V. All rights reserved.
1. Introduction A loss of sensory hair cells or spiral ganglion neurons from the inner ear causes sensorineural deafness. The quest to restore inner ear tissues remains a major challenge. At present, for profound hearing loss that is not improved by hearing-aid amplification, cochlear implantation surgery remains the only treatment option to restore input. However, intact spiral ganglion neurons are required for effective cochlear implantation and the clinical outcome is closely related to the residual amount of spiral ganglion neurons. Efforts to develop effective treatments for deafness associated with spiral ganglion pathology have demonstrated progress, most notably utilizing stem cell transplantation (Bas et al., 2014; Chen et al., 2012; Kondo et al., 2011; Pandit et al., 2011). * Corresponding author. Tel./fax: þ86 020 87333733. E-mail addresses: [email protected], [email protected] (H. Jiang). http://dx.doi.org/10.1016/j.heares.2014.07.012 0378-5955/© 2014 Elsevier B.V. All rights reserved.
Amniotic fluid has been reported to contain a population of pluripotent stem cells, which is known to have the capacity to differentiate into different cell types representative of three embryonic germ layers in response to appropriate chemical cues (Fauza, 2004; In et al., 2003; McLaughlin et al., 2006; Prusa and Hengstschlager, 2002; Prusa et al., 2003; Tsai et al., 2004, 2006). Several studies have shown that amniotic fluid-derived stem (AFS) cells are capable of differentiating into neuron-like cells under specific conditions in vitro (De Coppi et al., 2007; Donaldson et al., 2009; Prusa et al., 2004). Unlike embryonic stem cells (ES), these cells harbor a low risk of tumor development when implanted in vivo (De Coppi et al., 2007) and as such, represent the basis of a potential cellular therapy for neural deafness. The application of a typical combination of cytokines is known to stimulate neuronal differentiation of AFS cells (De Coppi et al., 2007; Donaldson et al., 2009; Prusa et al., 2004). Nevertheless, this directional differentiation is undetermined in the absence of cytokines. Additionally,
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following transplantation into the inner ear, the implanted cells were located along the auditory nerve fibers close to the organ of Corti (Corrales et al., 2006; Hu et al., 2005). The effect of inner ear supporting cells from the organ of Corti on AFS cell differentiation is still unknown and such limitations may impede the clinical application of AFS cell transplantation. The mechanisms involved in AFS cell neuronal differentiation are poorly understood. Wnt proteins constitute a large family of signaling molecules that have important roles in the regulation of embryonic patterning, cell proliferation and determination (Wodarz and Nusse, 1998). Adult hippocampal stem/progenitor cells express receptors and signaling components for Wnt proteins (Kleber and Sommer, 2004). Overexpression of Wnt-1 in embryonic carcinoma P19 cells promotes neuronal differentiation (Tang et al., 2002). Moreover, it has been reported that Wnt signaling inhibits the self-renewal capacity of neural precursor cells, and instructively promotes differentiation of neurons and astrocytes (Hirabayashi et al., 2004; Muroyama et al., 2004). All this evidence suggests that Wnt signaling plays an important role in triggering neurogenesis; however, the effects of Wnt signaling on the differentiation of AFS cells remains to be elucidated. In this study, we sought to investigate the consequence of inner ear stem cells derived feeder layer on AFS cell directional differentiation in the absence of cytokines in vitro and to elucidate the underlying mechanism. The establishment of an effective AFS cell differentiation relay for inner ear stem cells derived feeder layer should contribute to efforts to translate cell-based strategies to the clinic.
and Use Committee, following the Guidelines for the Care and Use of Laboratory Animals set forth by the National Institutes of Health. Isolation of inner ear stem cells from the organ of Corti of neonatal C57BL/6 mice was performed as described previously (Oshima et al., 2009). The stem cells were cultured at 37 C under 5% CO2 in DMEM/F12 supplemented with N2 supplement, B27 supplement, 20 ng/ml EGF, 20 ng/ml b-FGF and 50 ng/ml IGF-1 in the absence of serum. All growth factors and supplements were obtained from R&D Systems or Invitrogen/GIBCO/BRL. After 7 d, inner ear stem cell-derived hollow spheres were harvested, centrifuged at 2000g for 5 min and dissociated into a single cell suspension with 0.25% trypsin at 37 C for 3 min. The 2 105 single cell suspension (in DMEM/F12) was plated in 6-well culture plates containing uncoated glass cover slips for immunocytochemical analysis in each experiment. Cells were allowed to attach to the substratum for the preparation of a monolayer of feeder cells. AFS cells were then cultured at 37 C under 5% CO2 on the surface of the monolayer to provide a direct contact environment. After 6 d of culture in DMEM/F12, neurons were stained with Tuj1. 2.4. RT-PCR Total RNA was extracted from cultured cells using RNeasy kits (Qiagen, Valencia, CA). Reverse transcription was carried out with Superscript II reverse transcriptase (Invitrogen). PCR Primers were synthesized and PCR were conducted as previous reports (Donaldson et al., 2009; Zhang et al., 2011). The PCR fragments were confirmed by 3% agarose gel electrophoresis with ethidium bromide staining.
2. Materials and methods 2.5. Immunofluorescence 2.1. Amniotic fluid cell culture and isolation of AFS cells Samples of amniotic fluid were obtained from the First Affiliated Hospital of Sun Yat-Sen University following routine amniocentesis carried out on pregnant women at 16e24 weeks of gestation. This project has been reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-Sen University. Written informed consent was obtained from each woman before amniocentesis. Amniotic fluid cells were cultured (Tsai et al., 2004) and CD117-positive stem cell lines were established by magnetic cell sorting using a previously described protocol (De Coppi et al., 2007). AFS cells were labeled with green fluorescent protein (GFP) to permit subsequent detection. For GFP labeling, lentiviral vector encoding GFP (Genechem, Shanghai, China) was used to transfect the AFS cells, as per the manufacturer's protocol. Briefly, replicationincompetent GFP control lentiviral particles (1:10, catalog number: LVCON063) were incubated for 24 h with adherent cells. Cells were washed and re-seeded in feeder layer for further experiments. 2.2. Flow cytometry analysis AFS cell-specific antigen expressions (passage 3) were characterized by flow cytometry analysis. AFS cells were washed with PBS, trypsinized and stained with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies against CD73, CD133, CD44, CD90, CD34, CD117, Oct-4 and MHC-II (BD Biosciences). Positive cells were identified by comparison with cells stained with isotypic control antibodies. 2.3. Isolation of inner ear stem cells, feeder layer cell preparation, direct contact with the AFS cells Animal care and euthanization were conducted according to methods approved by the University of Sun Yat-sen Animal Care
The primary antibodies used were as follows: anti-Oct-4 (1:400; Santa Cruz, CA, USA), anti-Sox2 (1:500; Abcam, Cambridge, UK), anti-Tuj1 (1:500; Abcam), anti-P27kip1 (1:300; Abcam), anti-PSD95 (1:500; Abcam), anti-HuNu (1:100, Millipore, MA, USA), anti-GATA3 (1:100; Abcam). The secondary antibodies used were Alexa-488conjugated anti-mouse or rabbit antibody and Alexa-594conjugated anti-mouse or rabbit antibody (1:400; Molecular Probes, Oregon, USA). Nuclei were stained with DAPI. 2.6. Separate transwell culture system A separate transwell culture system was established to examine the regulatory effects of inner ear stem cells derived feeder layer on AFS cells. For immunocytochemical analysis, AFS cells were seeded in the transwell membrane insert (pore diameter, 0.4 mm, Corning) in a 6-well culture plate containing uncoated glass cover slips. The monolayer of inner ear feeder cells prepared as described above were cultured for 6 d in DMEM/F12 in the lower chamber of the coculture system. The ratio of AFS cells to feeder layer cells was controlled at 1:1. These culture conditions were designed to allow for the secretion of factors and crosstalk by both cell types without cell contact. The percentages of cells that differentiated into morphological neurons per field of vision at 20 magnification were recorded. 2.7. FM 1-43 dye loading FM1-43 dye loading was applied to measure the kinetics of synaptic vesicles exocytosis and endocytosis as previously described (Amaral et al., 2011; Ryan et al., 1993). Neurons were labeled by superfusion in a chamber with the fluorescent styryl membrane probe FM1-43 (10 mM in 45 mM KCl) for 1 min and washed with a 3 mM KCl solution for 1e5 min. Subsequently, the
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same neurons were destained in 90 mM KCl solution for 5 min. The off-rate of the dye from neuronal membranes was measured directly by recording the decrease in fluorescence intensity as a function of time during the period after staining. 2.8. Inhibition of Wnt signaling In the treatment group, mouse Dickkopf related protein 1 (Dkk1) was added at 100 ng/ml (R&D Systems) to the DMEM/F12 culture medium for 6 days. The following concentration ranges were tested to determine the optimal concentration of the reagent: Dkk1 at 50e200 ng/ml. Half of the medium was replaced on the third day of incubation. A two stages culture protocol for AFS differentiation was introduced as a standard comparison (De Coppi et al., 2007; Zsebo et al., 1990). The percentage and number of cells that differentiated into morphological neurons per field of vision at 20 magnification were recorded. TUNEL staining (KeyGen, Nanjing, China) was used to exclude the possibility of cells death caused by DKK1. Positive control of apoptosis was designed according to the procedure by the manufacturer. 2.9. Determination of neurite length and neuron survival The number of processes and neurite length was determined as previously described from digital images (7e10) randomly selected fields (20 magnification), with approximately equal cell density, for each experimental condition (Atkinson et al., 2011; Xu et al., 2012).
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3. Results 3.1. Isolation and characterization of AFS cells After 7e9 days of in vitro culture of amniotic fluid, both fibroblast-like cells and epithelioid cells appeared. For the 2 to 3 passages, most cells exhibited a fibroblast-like phenotype (Fig. 1A). The AFS cells proliferated as a monolayer, with a typical doublingtime (approximately 48 h). Occasional ES-like AFS cell colonies were observed (Fig. 1B). The AFS cells were negative for hematopoietic stem cell markers (CD34, CD133) and Class II major histocompatibility (MHC) antigens (HLA-DR) (Fig. 1C). However, they were positive for surface markers characteristic of mesenchymal stem cells, including CD73, CD90, and some were weakly positive for CD44. About 75.0 ± 4.6% of cells expressed transcription factor Oct-4, which is considered to be a marker of pluripotent stem cells (Donovan, 2001; Pesce and Scholer, 2001) and is especially associated with the maintenance of the undifferentiated state of ES (Pan et al., 2002). AFS cell expression of Oct-4 was confirmed by immunofluorescence. Besides, about 86.0 ± 2.8% of cells expressed Sox2, which is a transcription factor expressed in the neural progenitors, neural stem cells, and is involved in neural cell commitment and self-renewal (Ferri et al., 2004; Neves et al., 2007; Pevny and Nicolis, 2010). Co-expression of Oct-4 and Sox2 were identified in 72 ± 4.9% of cells (Fig. 1D). The expressions pattern varied amongst different cell lines and were confirmed by RT-PCR (supplementary information Fig. 1).
3.2. Neuronal differentiation in the inner ear stem cells derived feeder layer direct contact system
2.10. Statistical analysis Data were obtained from at least three replicate tests and then presented as means ± standard derivation. Statistical analysis was carried out by means of Student's t-tests, One-way ANOVA using SPSS 11.0. A value of P < 0.05 was considered statistically significant.
After 7 days of in vitro culture, inner ear stem cells formed hollow spheres (Fig. 2A) that were harvested and dissociated into single cell suspensions for the preparation of feeder cell monolayer (Fig. 2B), which were positive for P27kip1 (Fig. 2C), a marker of inner ear supporting cells of the sensory epithelium (Chen and Segil,
Fig. 1. (A) The morphology of amniotic fluid cells after culturing for 3 passages in vitro. (B) Embryonic stem cell-like AFS cell colonies appeared occasionally in the primary culture. (C) Expression of surface antigens was detected by flow cytometry (filled curve) in AFS cells. (D) AFS cells were positive for Oct-4 and Sox2. Scale bar: A, B ¼ 100 mm; D ¼ 50 mm.
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Fig. 2. Neuronal differentiation in the inner ear stem cells derived feeder layer. (A) Typical hollow spheres generated after 7 d of culture in vitro. (B) A monolayer of feeder cells from hollow spheres. (C) Feeder layer cells are positive for P27kip1. (D) AFS cells are cultured on top of the feeding monolayer, forming a direct contact system. (E) GFPþ AFS cells in the direct contact system, showing large numbers of neurons with strong expression of Tuj1 and long neurites at the site of a massive accumulation of cell nuclei. (F, G) Neuronal cell bodies, fibers and terminals observed in direct contact system, nuclei are stained with HuNu or DAPI. Scale bar: A ¼ 100 mm; C, E ¼ 50 mm; B, D ¼ 25 mm; F ¼ 12.5 mm.
1999). According to our previous study, the otic hollow spheres used to produce these feeder cells were not able to differentiate into neurons in the present study, probably due to their lack of differentiation capacity of stem cells (unpublished data). Subsequently, a direct contact system by culturing lentiviraltransfected GFPþ AFS cells on the feeder cell monolayer was established to visualize the AFS cells' differentiation (Fig. 2D). The morphology of AFS cells changed obviously from the third day compared with that of uninduced AFS cells. The cell body exhibited a long process extending to join and overlap cells. The GFPþ AFS cells gave rise to large numbers of neurons, not only having strong Tuj1 expression but also exhibiting typical neuronal morphological characteristics and long neurites after 6 days’ culture (Fig. 2E). The neurons appeared at the site of a massive accumulation of cell nuclei belonging to the seeded AFS cells. Furthermore, the neurons showed direct contact with the structure of the inner ear stem cells derived feeder layer. Human cells were distinguished from the
mouse cells by staining their nuclei with an antibody against the human nuclear (HuNu) antigen (Fig. 2F). Neuronal cell bodies, fibers and terminals were observed in this culture system at high magnification (Fig. 2FeG). The neurons developed normal polarity; an average of 79 ± 2.0% had two neurite outgrowths, while a small percentage (19 ± 3.2%) had multiple processes. Interestingly, 30 ± 6.0% of these neurons was positive for GATA-3 (Fig. 3), a marker of spiral ganglion neurons, indicating that AFS cells could differentiate into auditory neurons under certain condition. The Tuj1þ and GFPþ cells became elongated with neuron-like processes up to 320 mm. The AFS cells cultivated alone for 6 days exhibited specific Tuj1 expression, but no typical neuron was seen (Fig. 4A). Tuj1 expression by feeder layer cells cultured alone for 6 days was negligible (Fig. 4B). Altogether, these observations confirmed the effectiveness of the inner ear stem cells derived feeder layer on AFS cell neurogenesis.
Fig. 3. The direct contact co-culture produced neurons that were positive for GATA-3.
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Fig. 4. AFS cells (A) or feeder layer cells (B) that were cultured alone showed mere expression of Tuj1. (C) In transwell system, no typical pyramidal morphological neuron was observed. Scale bar: ¼ 50 mm.
The co-culture of AFS cells with normal neonatal cochlea were further investigated, but our results indicated that normal neonatal cochlea could promote neither disperse nor spherical AFS cells into differentiation (data not shown). Combined with the present study, intact inner ear supporting cells structures seemed not to be able to promote AFS cells into differentiation until lesion occurred. The specific differentiations of AFS cells co-culture with neonatal cochlea after lesion are under study. Further studies regarding the nature and functional characterization of the neurons in the present study are still required. 3.3. Separate transwell culture system In this system, the two cell types are physically separated to avoid direct cell-to-cell contact, while intercellular communication occurs only through substances released by either cell type into the shared culture medium. After 6 d of co-culture, the fraction of Tuj1þ expressing cells did not change obviously. Although the AFS cells exhibited specific Tuj1 expression, typical neuronal morphology was scarcely observed (0.6 ± 0.18%). The feeder layer cells exhibited minimal or scarcely detectable expression of Tuj1 (1.4 ± 0.8%). These results were distinguished from direct contact co-culture, whose percentage was 37 ± 3.0% (P < 0.01, One-way ANOVA). No clear evidence of morphological characteristics typical of neurons was observed in the AFS cells and feeder cell layer cultured separately in the transwell culture system (Fig. 4C). 3.4. Neurons derived from inner ear stem cells derived feeder layer system show characteristics of functionality Synapse formation between neurons occurred within 6 days, as indicated by punctuate staining of the postsynaptic density protein
(PSD95), which was concentrated at the synapses in mature neurons (Fig. 5A). About 90 ± 1.2% of neurons were positive for PSD95 from 700 neurons that were counted. As seen in Fig. 5B, typical synaptic structures were associated with the dendrites, spines, and cell bodies of neurons derived from AFS cells. Synaptic gaps between two neurons were identified. Additionally, AFS cells in the direct contact system were observed to undergo active synaptic vesicle recycling that was monitored by visualizing the FM1-43 dye uptake and release after potassium stimulation. Fig. 5C illustrated neurons derived from AFS cells in the direct contact system during superfusion with a depolarizing solution (45 mM KCl) containing 10 mm FM1-43. The cell body showed two neurites traversing its length. Fig. 5D shows the same cells after a second 60 s superfusion in 90 mM KCl solution, in this case, in the absence of dye. The fluorescence intensity decreased significantly during this period (n ¼ 6 independent specimens). 3.5. The feeder layer direct contact system may depends on Wnt signaling In the control group (without Dkk1), an average of 37 ± 3.0% of AFS cells differentiated into morphologically neurons with long neurite outgrowths compared to 21 ± 2.5% in DKK1-treated group (Fig. 6A). The percentage of processes derived from DKK1-treated AFS cells decreased greatly to 33.3 ± 3.8% by contrast with 60.4 ± 4.6% in the control group (P < 0.01, Fig. 6B), suggesting that AFS cells differentiation toward neurons were inhibited when the Wnt signaling was suppressed. Whereas about 18.4 ± 1.4% AFS cells were induced into morphologically neurons according to the two stage differentiation model (De Coppi et al., 2007; Zsebo et al., 1990), a number less than Dkk1 treatment group or control group (Fig. 6C, P < 0.01, One-way ANOVA). In addition to the obvious
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Fig. 5. Functional bioassay on neurons derived from AFS cells in direct contact co-culture. (A) Synapse formation was positive for Tuj1 and PSD95. (B) Enlarged picture showed typical synapse formation. (C) The neurons being superfused in a 45 mM KCl solution containing FM1-43; (D) The same field of view as in (C), after a 60s superfusion in 90 mM KCl. Note the marked decrease in fluorescence intensity. Scale bar: A, C, D ¼ 25 mm.
decrease in the number of neurites, Dkk1 treatment led to differentiated neurons with shortened outgrowths than in the direct contact system (Fig. 6D). Dkk1 significantly reduced neurite length; the average length was 51 ± 1.5 mm in the Dkk1 treatment group, compared to 94 ± 3.7 mm in the control group (P < 0.01). In addition, the absence of TUNEL expression confirmed that Dkk1 prevent differentiation mainly, instead of causing cell death (supplementary information Fig. 2). The percentage of Sox2positive cells with/without DKK1 treatment was similar (81.2 ± 3.8% vs. 82.4 ± 2.6%, P > 0.05). Taken together, these findings indicate that Wnt signaling may play an important role in neuronal differentiation of AFS cells. 4. Discussion The present study aimed to examine the possibility of utilizing inner ear stem cells derived feeder layer in a direct contact system to promote directional differentiation of AFS cells into functional neurons; in principle, an efficacious AFS cell source could translate to clinical use rapidly. First, we established an inner ear stem cells derived feeder layer direct contact system for AFS cells. In this direct contact system, AFS cells were cultured for 6 days on the surface of a feeder cell layer derived from inner ear stem cells obtained from the organ of Corti of neonatal mice. Using this approach, we found neurons developed normal polarity, most with two neurites and a small percentage with multiple neurites and long processes. These exciting results may be attributed, in part, to directional induction by feeder layer. The combination of AFS cells and inner ear stem cells derived feeder layer are suspected to induce stable neuron differentiation.
It is known that inner ear, which mediates hearing and sensory equilibrium, develops from the ectodermal placode. During inner ear development, the majorities of neural stem cells are generated and differentiate into cell types including sensory hair cells and spiral ganglion neurons. It can be speculated that the mechanism underlying this effect is based on the stimulation of the production of numerous neurons as a result of direct cell-to-cell contact between the two cell types although further research is required to confirm this hypothesis. However, in comparison with reported methods (De Coppi et al., 2007; Donaldson et al., 2009; Prusa et al., 2004), the direct contact system is a cost-effective method that allows stable induction of the neuron-oriented differentiation of AFS cells. Inner ear stem cells derived feeder layer may serve as a nerve-regenerative scaffold. Using a transwell culture system to prevent direct cell-to-cell contact, we showed that there was no obvious change in the fraction of AFS cells expressing Tuj1þ and no specific neuron-like cell morphology was observed. Thus, we are able to speculate that the process of neuron-oriented differentiation does not depend on secreted factors and molecules; while direct cell-to-cell interactions, especially mechanical signals provided by inner ear stem cells derived feeder layer, might regulate the fate of AFS cells. It is important to assess whether the morphological differentiation of neuritis in the direct contact system concurred with the acquisition of functional properties. Synaptogenesis, indicated by the concentration of PSD95 at synapses in mature neurons, was confirmed in this system. Meanwhile, by monitoring the uptake and release of the fluorescent marker dye, FM1-43, to show active synaptic vesicle recycling (Amaral et al., 2011; Ryan et al., 1993), we further demonstrated the functional trait of the cells differentiated
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Fig. 6. Wnt signaling is involved in direct contact system. Immunofluorescent staining for Tuj1 in the direct contact without Dkk1 treatment (A) and with Dkk1 treatment (B). (C) The number of processes was measured in different experimental groups. (D) The length of neurites was measured in different experimental groups compared to the two stage differentiation protocol (De Coppi et al., 2007; Zsebo et al., 1990). Scale bar: A, B ¼ 50 mm.
in our study. Thus, we consider this approach using inner ear stem cells derived feeder layer provides a microenvironment to support morphological and functional neuronal differentiation and regulate neurogenesis by directing AFS cells to adopt a neuronal fate. The Wnt/b-catenin signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations (Chai et al., 2012). It has been reported that in the neonatal cochlea, the Lgr5 marker of adult stem cells, is expressed in a subset of inner ear supporting cells and that Lgr5-expressing inner ear supporting cells are Wnt-responsive sensory precursor cells (Chai et al., 2012; Shi et al., 2012). The feeder layer derived from inner ear stem cells has the specific marker of inner ear supporting cells. We therefore investigated whether feeder layer cells from the organ of Corti of neonatal mice provided Wnt signaling to support the neuronal differentiation of AFS cells observed in the present study. Our results suggested that blocking Wnt signaling activity significantly affected the number of neurons and neurite length. The possibility that Wnt signaling is playing a role in neurite outgrowth in the present study was accord with the conclusion by Blakely et al. (2011). We speculated that inner ear stem cells derived feeder layer trigger Wnt signaling cell-surface stretch receptors and adhesion sites, resulting in signaling cascades including the activation of genes responsible for the synthesis of functional neurons. Inner ear stem cells derived feeder layer may represent a new nerve-regenerative scaffold and possess broad application prospects in the field of neuron regeneration. The hypothesis that Wnt signaling activators may improve the efficacy of AFS transplantation remains to be investigated.
The present results have raised great significance for clinical practices. It is important to note that inner ear stem cells derived feeder layer exert positive effects on AFS cells differentiation and AFS cell transplantation may be feasible. The two types of cells have
Fig. 7. Flow chart documenting the total numbers and percentages of cells with a given characteristic in each stage.
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direct cell-to-cell contact and produce certain amount of functional neurons to replace lesion spiral ganglion. This culture system was also shown to be effective and stable for the neuron-oriented differentiation in the absence of any cytokines (Fig. 7). In combination with a previous study (De Coppi et al., 2007), our study indicates that AFS cell transplantation seems to be practicable with low risk of tumor development. Understanding the functional roles of feeder cells on AFS cells would provide useful information for clinical translation. However, an in vivo study is necessary to determine the effects of differences in the inner ear environment, including factors such as pH and ion types and concentrations. In conclusion, our study shows that inner ear stem cells derived feeder layer successfully promote the directional differentiation of AFS cells into functional neurons and that Wnt signaling may play an essential role in triggering neurogenesis. Though the nature of neurons in the present study remained to be defined, the reliability of an effective AFS cell differentiation system on inner ear stem cells derived feeder layer should contribute to efforts to translate cellbased strategies to the clinic. Conflict of interests None. Acknowledgements The study was supported by grants from the National Basic Research Program of China (No. 2011CB504502), the National Natural Science fund of China (No. 81271076 and No. 81200748) and the Minster of Health of China (No. 201202005). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.heares.2014.07.012. References Amaral, E., Guatimosim, S., Guatimosim, C., 2011. Using the fluorescent styryl dye FM1-43 to visualize synaptic vesicles exocytosis and endocytosis in motor nerve terminals. Methods Mol. Biol. 689, 137e148. Atkinson, P.J., Cho, C.H., Hansen, M.R., Green, S.H., 2011. Activity of all JNK isoforms contributes to neurite growth in spiral ganglion neurons. Hear Res. 278, 77e85. Bas, E., Van De Water, T.R., Lumbreras, V., Rajguru, S., Goss, G., Hare, J.M., Goldstein, B.J., 2014. Adult human nasal mesenchymal-like stem cells restore cochlear spiral ganglion neurons after experimental lesion. Stem Cells Dev. 23, 502e514. Blakely, B.D., Bye, C.R., Fernando, C.V., Horne, M.K., Macheda, M.L., Stacker, S.A., Arenas, E., Parish, C.L., 2011. Wnt5a regulates midbrain dopaminergic axon growth and guidance. PLoS One 6, e18373. Chai, R., Kuo, B., Wang, T., Liaw, E.J., Xia, A., Jan, T.A., Liu, Z., Taketo, M.M., Oghalai, J.S., Nusse, R., Zuo, J., Cheng, A.G., 2012. Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea. Proc. Natl. Acad. Sci. U. S. A. 109, 8167e8172. Chen, P., Segil, N., 1999. p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti. Development 126, 1581e1590. Chen, W., Jongkamonwiwat, N., Abbas, L., Eshtan, S.J., Johnson, S.L., Kuhn, S., Milo, M., Thurlow, J.K., Andrews, P.W., Marcotti, W., Moore, H.D., Rivolta, M.N., 2012. Restoration of auditory evoked responses by human ES-cell-derived otic progenitors. Nature 490, 278e282. Corrales, C.E., Pan, L., Li, H., Liberman, M.C., Heller, S., Edge, A.S., 2006. Engraftment and differentiation of embryonic stem cell-derived neural progenitor cells in the cochlear nerve trunk: growth of processes into the organ of Corti. J. Neurobiol. 66, 1489e1500. De Coppi, P., Bartsch, G.J., Siddiqui, M.M., Xu, T., Santos, C.C., Perin, L., Mostoslavsky, G., Serre, A.C., Snyder, E.Y., Yoo, J.J., Furth, M.E., Soker, S., Atala, A., 2007. Isolation of amniotic stem cell lines with potential for therapy. Nat. Biotechnol. 25, 100e106.
Donaldson, A.E., Cai, J., Yang, M., Iacovitti, L., 2009. Human amniotic fluid stem cells do not differentiate into dopamine neurons in vitro or after transplantation in vivo. Stem Cells Dev. 18, 1003e1012. Donovan, P.J., 2001. High Oct-ane fuel powers the stem cell. Nat. Genet. 29, 246e247. Fauza, D., 2004. Amniotic fluid and placental stem cells. Best. Pract. Res. Clin. Obstet. Gynaecol. 18, 877e891. Ferri, A.L., Cavallaro, M., Braida, D., Di Cristofano, A., Canta, A., Vezzani, A., Ottolenghi, S., Pandolfi, P.P., Sala, M., DeBiasi, S., Nicolis, S.K., 2004. Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain. Development 131, 3805e3819. Hirabayashi, Y., Itoh, Y., Tabata, H., Nakajima, K., Akiyama, T., Masuyama, N., Gotoh, Y., 2004. The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells. Development 131, 2791e2801. Hu, Z., Wei, D., Johansson, C.B., Holmstrom, N., Duan, M., Frisen, J., Ulfendahl, M., 2005. Survival and neural differentiation of adult neural stem cells transplanted into the mature inner ear. Exp. Cell. Res. 302, 40e47. In, T.A.P., Scherjon, S.A., Kleijburg-van, D.K.C., Noort, W.A., Claas, F.H., Willemze, R., Fibbe, W.E., Kanhai, H.H., 2003. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 102, 1548e1549. Kleber, M., Sommer, L., 2004. Wnt signaling and the regulation of stem cell function. Curr. Opin. Cell. Biol. 16, 681e687. Kondo, T., Matsuoka, A.J., Shimomura, A., Koehler, K.R., Chan, R.J., Miller, J.M., Srour, E.F., Hashino, E., 2011. Wnt signaling promotes neuronal differentiation from mesenchymal stem cells through activation of Tlx3. Stem Cells 29, 836e846. McLaughlin, D., Tsirimonaki, E., Vallianatos, G., Sakellaridis, N., Chatzistamatiou, T., Stavropoulos-Gioka, C., Tsezou, A., Messinis, I., Mangoura, D., 2006. Stable expression of a neuronal dopaminergic progenitor phenotype in cell lines derived from human amniotic fluid cells. J. Neurosci. Res. 83, 1190e1200. Muroyama, Y., Kondoh, H., Takada, S., 2004. Wnt proteins promote neuronal differentiation in neural stem cell culture. Biochem Biophys. Res. Commun. 313, 915e921. Neves, J., Kamaid, A., Alsina, B., Giraldez, F., 2007. Differential expression of Sox2 and Sox3 in neuronal and sensory progenitors of the developing inner ear of the chick. J. Comp. Neurol. 503, 487e500. Oshima, K., Senn, P., Heller, S., 2009. Isolation of sphere-forming stem cells from the mouse inner ear. Methods Mol. Biol. 493, 141e162. Pan, G.J., Chang, Z.Y., Scholer, H.R., Pei, D., 2002. Stem cell pluripotency and transcription factor Oct4. Cell. Res. 12, 321e329. Pandit, S.R., Sullivan, J.M., Egger, V., Borecki, A.A., Oleskevich, S., 2011. Functional effects of adult human olfactory stem cells on early-onset sensorineural hearing loss. Stem Cells 29, 670e677. Pesce, M., Scholer, H.R., 2001. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19, 271e278. Pevny, L.H., Nicolis, S.K., 2010. Sox2 roles in neural stem cells. Int. J. Biochem Cell. Biol. 42, 421e424. Prusa, A.R., Hengstschlager, M., 2002. Amniotic fluid cells and human stem cell research: a new connection. Med. Sci. Monit. 8, RA253e7. Prusa, A.R., Marton, E., Rosner, M., Bernaschek, G., Hengstschlager, M., 2003. Oct-4expressing cells in human amniotic fluid: a new source for stem cell research? Hum. Reprod. 18, 1489e1493. Prusa, A.R., Marton, E., Rosner, M., Bettelheim, D., Lubec, G., Pollack, A., Bernaschek, G., Hengstschlager, M., 2004. Neurogenic cells in human amniotic fluid. Am. J. Obstet. Gynecol. 191, 309e314. Ryan, T.A., Reuter, H., Wendland, B., Schweizer, F.E., Tsien, R.W., Smith, S.J., 1993. The kinetics of synaptic vesicle recycling measured at single presynaptic boutons. Neuron 11, 713e724. Shi, F., Kempfle, J.S., Edge, A.S., 2012. Wnt-responsive Lgr5-expressing stem cells are hair cell progenitors in the cochlea. J. Neurosci. 32, 9639e9648. Tang, K., Yang, J., Gao, X., Wang, C., Liu, L., Kitani, H., Atsumi, T., Jing, N., 2002. Wnt-1 promotes neuronal differentiation and inhibits gliogenesis in P19 cells. Biochem Biophys. Res. Commun. 293, 167e173. Tsai, M.S., Lee, J.L., Chang, Y.J., Hwang, S.M., 2004. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum. Reprod. 19, 1450e1456. Tsai, M.S., Hwang, S.M., Tsai, Y.L., Cheng, F.C., Lee, J.L., Chang, Y.J., 2006. Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells. Biol. Reprod. 74, 545e551. Wodarz, A., Nusse, R., 1998. Mechanisms of Wnt signaling in development. Annu Rev. Cell. Dev. Biol. 14, 59e88. Xu, N., Engbers, J., Khaja, S., Xu, L., Clark, J.J., Hansen, M.R., 2012. Influence of cAMP and protein kinase A on neurite length from spiral ganglion neurons. Hear Res. 283, 33e44. Zhang, M., Ma, Q., Hu, H., Zhang, D., Li, J., Ma, G., Bhat, K., Wu, E., 2011. Stem cell factor/c-kit signaling enhances invasion of pancreatic cancer cells via HIF1alpha under normoxic condition. Cancer Lett. 303, 108e117. Zsebo, K.M., Williams, D.A., Geissler, E.N., Broudy, V.C., Martin, F.H., Atkins, H.L., Hsu, R.Y., Birkett, N.C., Okino, K.H., Murdock, D.C., et al., 1990. Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63, 213e224.
