lable at ScienceDirect

Hearing Research 275 (2011) 43e52

Contents lists avai

Hearing Research

journal homepage: www.elsevier .com/locate/heares

Research paper

Hypoxia enhances the stemness markers of cochlear stem/progenitor cells andexpands sphere formation through activation of hypoxia-inducible factor-1alpha

Hsin-Chien Chen a,b, Huey-Kang Sytwu c,1, Junn-Liang Chang d,e,1, Hsing-Won Wang b, Hang-Kang Chen b,Bor-Hwang Kang b,f, Dai-Wei Liu g,h, Chi-Huang Chen i, Ting-Ting Chao b, Chih-Hung Wang a,b,c,*

aGraduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, ROCbDepartment of Otolaryngology-Head and Neck Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROCc Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, ROCdDepartment of Pathology and Laboratory Medicine, Taoyuan Armed Forces General Hospital, Taoyuan, Taiwan, ROCeDepartment of Biomedical Engineering, Ming Chuan University, Taoyuan, Taiwan, ROCf Institute of Undersea and Hyperbaric Medicine, National Defense Medical Center, Taipei, Taiwan, ROCgDepartment of Radiation Oncology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan, ROChDepartment of Radiology, Tzu Chi University, Hualien, Taiwan, ROCiDepartment of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC

a r t i c l e i n f o

Article history:Received 31 March 2010Received in revised form22 November 2010Accepted 1 December 2010Available online 10 December 2010

Abbreviations: SPCs, stem/progenitor cells; DIV, dinducible factor-1alpha; Vegf, vascular endothelial grgrowth factor; b-FGF, basic fibroblast growth factoDMEM, Dulbecco’s Modified Eagle Medium; EDTA,acid; EGFP, enhanced green fluorescence protein; PBWST-1, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tfonate; FITC, fluorescein isothiocyanate; TRITC, tetramDAPI, 40 , 6-diamidino-2-phenylindole; SEM, standardReverse Transcription-Polymerase Chain Reaction;subfamily G, member 2; Gapdh, glyceraldehyde-3p27Kip1, cyclin-dependent kinase inhibitor 1B; siRNA,* Corresponding author. Department of Otolaryngo

Tri-Service General Hospital, National Defense MedicKung Rd., Neihu District, Taipei 114, Taiwan, ROC. Tel.:2 8792 7193.

E-mail address: chw@ms3.hinet.net (C.-H. Wang).1 These authors contribute equally.

0378-5955/$ e see front matter � 2010 Elsevier B.V.doi:10.1016/j.heares.2010.12.004

a b s t r a c t

Unlike neural stem cells that maintain populations in the adult brains of both rodents and humans,cochlear stem cells appear to diminish in number after birth and may become quiescent in adultmammalian cochleae. Hypoxia has been observed to promote an undifferentiated cell state in variousstem cell populations; however, little is known about such an effect on cochlear stem/progenitor cells(SPCs). The aims of this study were to assess the effect of hypoxia on cochlear SPCs and to examine theimpact of hypoxia-inducible factor-1alpha (Hif-1a) on regulating such an effect. Our data demonstratethat hypoxic culturing for 24 h significantly increased sphere formation and viability of cochlear SPCscompared with those cultured under normoxic conditions. Concurrent with these proliferation promo-tion effects are changes in the expression of multiple stemness and cell-cycle quiescent associated genetargets, including Abcg2, nestin, p27Kip1and Vegf. Knockdown of Hif-1a expression by small-interferingRNA inhibited hypoxia-induced cochlear SPC expansion and resulted in downregulation of Vegf, Abcg2,and nestin and upregulation of p27Kip1 gene expression. These results suggest that Hif-1a plays animportant role in the stimulation of the proliferation of cochlear SPCs, which confers a great benefit ofexpanding cochlear SPCs via hypoxic conditions.

� 2010 Elsevier B.V. All rights reserved.

ays in vitro; Hif-1a, hypoxia-owth factor; EGF, epidermalr; FBS, fetal bovine serum;ethylenediamine tetraaceticS, Phosphate buffered saline;etrazolio]-1,3-benzene disul-ethyl rhodamine isocyanate;error of the mean; RT-PCR,

Abcg2, ATP-binding cassette,-phosphate dehydrogenase;small-interfering RNA.logy-Head and Neck Surgery,al Center, 325, Sec. 2, Cheng-þ886 2 8792 7192; fax: þ886

All rights reserved.

1. Introduction

Degeneration of cochlear hair cells and spiral ganglion neuronsoften causes irreversible hearing impairment. The loss of regener-ative ability of hair cells in the organ of Corti and spiral ganglionscontributes to the progression of hearing loss. Cell replacementtherapies for cochlear diseases have thus attracted considerableattention. Recently, tissue-specific cochlear stem/progenitor cells(SPCs) have been identified in the inner ear and have been char-acterized in several reports as having pluripotency (Li et al., 2003;Oshima et al., 2007; Savary et al., 2007; White et al., 2006). Theo-retically, stem/progenitor cells derived from the cochlea should bethe best candidate for replacing damaged cells in the inner ear;however, these SPCs appear to diminish in number after birth andmay become quiescent in the adult mammalian organ of Corti(Oshima et al., 2007). Therefore, the development of strategies to

H.-C. Chen et al. / Hearing Research 275 (2011) 43e5244

expand a considerable number of implanted cells ex vivo or inducegreater numbers of endogenous stem cells in the inner ear isimperative, but its execution remains a challenge.

Oxygen is important for cellular energy production andmetabolism. Cellular processes affected by oxygen tension includeembryonic development (Dumoulin et al., 1999; Orsi and Leese,2001; Thompson et al., 1990), cell proliferation (Packer and Fuehr,1977; Wing et al., 1988), angiogenesis (Choi et al., 2003),apoptosis (Carmeliet et al., 1998), and differentiation (Malladi et al.,2006). The influence of hypoxia has also been observed on theproliferation and differentiation of stem cells, including neuralstem cells (Panchision, 2009), mesenchymal stem cells (Graysonet al., 2006), hematopoietic stem cells (Cipolleschi et al., 1993)and induced pluripotent stem cells (Yoshida et al., 2009). Indeed,physiological normoxia usually involves a much lower oxygenconcentration than does ambient air (Simon and Keith, 2008).Cochlear SPCs might also exist in a local hypoxic microenvironment(Lamm and Arnold, 2000); however, the effect of hypoxia oncochlear SPCs has never been investigated. In the present study, wefocused exclusively on the effects of hypoxia on cochlear SPCs andcharacterized its molecular mechanism. Our study demonstratedthat hypoxia enhanced the stemness markers and sphere expan-sion of postnatal cochlear SPCs, which would contribute toa potential new strategy of stem cell application aimed at regen-erating a damaged inner ear.

2. Materials and methods

2.1. Animals

To assess the effect of hypoxia on cochlear SPCs from differentmouse strains and to examine the impact of hypoxia-induciblefactor-1alpha (Hif-1a) on regulating such an effect, cochlear SPCswere isolated from the cochleae of postnatal day 1 (P1) pups ofCBA/CaJ mice, FVB/N-Tg (PolII-luc) transgenic mice, and FVB/NCrl-Tg (Pgk1-EGFP) 1Narl transgenic mice. The experimental protocolwas approved by the Animal Care and Use Committee at theNational Defense Medical Center, Taipei, Taiwan. In this experi-ment, FVB/N-Tg (PolII-luc) transgenicmice expressing the luciferasegene, which not only serves as an optical indicator but also allowsfor examination of the spheres in thewhole dish, were used. Briefly,FVB/N-Tg (PolII-luc) transgenic mice were generated by pronucleimicroinjection of PolII-luc transgene into FVB/N fertilized embryos.The construct (gene fragment) PolII-luc contains a 712-bp mouseRNA Polymerase II promoter (PolII) and modified firefly luciferasecDNA (Promega pGL-2). Because pgk-1 is one of the Hif-1a targetgenes, the FVB/NCrl-Tg (Pgk1-EGFP) 1Narl transgenic mice (Huanget al., 2009) obtained from the National Laboratory Animal Center(Taipei, Taiwan), which ubiquitously expressed EGFP that is drivenby the mouse Pgk1 promoter in all tissues, allowed us to examinethe impact of Hif-1a and served as another source of cochlear SPCs.

2.2. Cell culture

Cochleae of P1 pups were excised and transferred into Petridishes containing phosphate buffered saline glucose solution (BSG;116 mM NaCl, 27.2 mM Na2HPO4, 6.1 mM KH2PO4, 11.4 mMglucose). The bony capsules were removed and the membranouscochleae were retained. To prepare the dissociated cell cultures,approximately 20 membranous cochleae were pooled and theirfragments were cut into small pieces and incubated in a mixture of0.05% trypsin/0.02% (w/v) EDTA at 37 �C for 15 min, followed byrepeated pipetting. The enzymatic digest was inactivated by adding10% fetal bovine serum (FBS, Biological Industries) in a mixture ofDMEM/F12. Tissue dissociates were filtered through a 40-mm mesh

to remove cell aggregates and debris. These newly dissociatedprimary cochlear cells were plated in non-coated T25 flasks (Nunc)at 37 �C in a 5% CO2 atmosphere in serum-free DMEM/F12 sup-plemented with penicillin, 20 ng/ml epidermal growth factor (EGF,Sigma), 10 ng/ml basic fibroblast growth factor (b-FGF, Sigma), andN2 and B27 (GIBCO) supplements on the first day in vitro (DIV). Themedium was changed every 3 days. After 7 DIV, primary sphereswere observed and collected by centrifugation at 1000 rpm for5 min followed by mechanical dissociation with a Pasteur pipetteand 0.05% trypsin. The dissociated primary spheres were main-tained in ultralow attachment 6-well plates (Corning) forsecondary sphere formation. To allow for continuous expansion,half of this medium was replaced every day and cultures werepassaged every seventh day.

To test the self-renewal property of the cochlea SPCs, celldissociates from P1 mouse cochlear sensory epithelia (organ ofCorti) were specifically isolated and followed by the same cellculture protocol as mentioned above.

2.3. Immunocytochemistry

For immunocytochemical studies, secondary spheres derived fromCBA/CaJ mice were either transferred into 24-well plates with cover-slips and cultured in DMEM/F12mediumsupplementedwith 10% FBSovernight or prepared using a cytospin at 1200 rpm for 5 min. Theattached spheres were fixed in phosphate buffered saline (PBS)-buff-ered 4% paraformaldehyde and 2% sucrose, washed three times withPBS, permeabilized with 3% BSA in PBS containing 0.3% Triton X-100,and blocked with 5% normal goat serum. Coverslips were incubatedwith mouse monoclonal anti-Abcg2 antibody (1:100, Santa Cruz),mouse monoclonal anti-nestin antibody (1:500, Abcam), and rabbitpolyclonal anti-Nanog antibody (1:200, Abcam) at 4 �C overnight.After three washes with PBS, coverslips were incubated with fluo-rescein isothiocyanate (FITC)- or tetramethyl rhodamine isocyanate(TRITC)-conjugated secondary antibody (1:200, Thermo FisherScientific) to reveal thecellmarkers,andstainedwithDAPI (0.66mg/mlin PBS; Molecular Probes) for nuclei visualization and rhodamine-conjugated phalloidin (1:200, Molecular Probes) to visualize actinfilaments in cell structures. Coverslips were mounted onto slides andexamined under an epifluorescence microscope.

2.4. Cell differentiation

For cell differentiation secondary spheres were cultured underadherent conditions in 24-well plates filled with DMEM/10% FBS.The medium was replaced every second day. After 96 h differen-tiated cells were analyzed by immunocytochemistry. We usedpolyclonal antibody to myosinVIIA (1:200, Nevus), monoclonalantibody to p27Kip1 (1:100, Neo-Markers), and monoclonal anti-body to b-III tubulin (1:500, Thermo).

2.5. Bromodeoxyuridine (BrdU) incorporation

Detection of BrdU incorporation in DNA-synthesizing cells wasperformed by adding 10 mM BrdU (Sigma) to the secondary spherecultures. After a 72-h incubation period, spheres were plated ontocoverslips in a 24-well plate containing DMEM/F12 and 10% FBS.After 24 h of cell seeding, coverslips were fixed and incubated in 2NHCl for 30 min at 37 �C. Immunodetection of BrdU was performedusing a monoclonal antibody against BrdU (1:500; Sigma). Fluo-rescence TRITC-tagged secondary antibody (1:200) was employedfor visualization. For quantitativemeasurement of cell proliferation,chemiluminescent detection of BrdU using a cell proliferation ELISAkit (Roche) was performed according to the manufacturer’sinstructions. Briefly, cells were seeded in black 96-well plates

H.-C. Chen et al. / Hearing Research 275 (2011) 43e52 45

(1�104 cells/well) and underwent hypoxic incubation for 24 h priorto be maintained in normoxic culture conditions for 24 h. BrdUlabeling solution (final concentration of 10 mM) was added andincubated for 16 h. After labeling, the cells were fixed and incubatedwith anti-BrdU antibody peroxidase conjugate (anti-BrdU-POD).Excess antibodywas removed by PBS washing. Chemiluminescencewas determined by a luminometer (Fluoroskan Ascent FL, Thermo)and expressed as relative luminescence unit/second.

2.6. Hypoxia/reoxygenation experiments and counting of spheres

Hypoxic culture conditions were created by incubating newlyisolated cochlear cells or dissociated primary spheres in an N2/CO2multigas incubator (APM-50D, Astec, Japan) and were exposed tohypoxic (1%O2 and5%CO2) conditions at 37 �C for 24h. The cellswereexposed to a normoxic atmosphere (reoxygenated) upon replace-mentofmediumsaturatedwith95%air and5%CO2andmaintained ina normoxic incubator at 37 �C for the indicated time interval. Themedium was changed every 3e4 days. The number of spheres wascounted either under lightmicroscopyorepifluorescencemicroscopyusing MetaMorph image analysis software (MDS Analytical Tech-nologies, USA). Briefly, ten randomly selected high-power field(�100) images obtained from thenormoxia andhypoxia groupsweretaken and loaded into MetaMorph software. Sphere cells larger than75 mm in diameter were calculated in the present study. The resultsobtained by eye and software analysis were similar.

2.7. In vitro bioluminescence imaging

For bioluminescence imagings, 50 ml of 15 mg/ml D-luciferin(Xenogen Corp, Alameda, CA, USA) were added to each well ofultralow attachment 6-well plates containing primary spheresderived from the FVB/N-Tg (PolII-luc) transgenic mice. Biolumi-nescence signals were monitored using the IVIS Imaging System100 Series (Xenogen) and analyzed with Living Image software(Xenogen). The acquired fluorescence images were displayed usinga pseudo-color scale to represent photon signal intensity. Luciferaseactivity was verified in advance with a serial dilution of 3.2 � 105

primary cochlear cells in a 12-well plate by using the total photonflux emission (photons/second) in the regions of interest (ROI)covering the entire well. Peak luminescence was found in the initial4 min after D-luciferin was administered to the in vitro samples.

2.8. Measurement of the fluorescence intensity and viability assay

Dissociated primary spheres derived fromFVB/Tg transgenicmicewere seeded in a 96-well plate (1 � 104 cells/well) and underwenthypoxic incubation for 24 h prior to be maintained in normoxicculture conditions for 48 h. Fluorescence intensity caused by EGFPexpression on cells in hypoxic or normoxic control conditions wasdetected by a fluorometer (Fluoroskan Ascent FL, Thermo). To deter-mine cell viability, 10% WST-1 agent (Roche) was added to each welland incubated for 4 h. This colorimetric assay is based on cleavage ofthe membrane-permeable, water soluble tetrazolium monosodiumsalt (WST-1) to produce a formazan-class dye. The reaction is cata-lyzed by a mitochondrial reductase in active cells, and the amount offormazan dye can be quantified by measuring the absorbance at450 nm using a Bio-Rad ELISA reader to calculate the optical density(OD) values (A450nm � A655nm). Statistical analysis was determinedusing Student’s t-test with P values <0.05 considered significant.

2.9. RNA isolation and RT-PCR

For immediate gene expression after hypoxia, newly isolatedcochlear cells or primary spheres that underwent hypoxic culturing

for 24 h were collected for immediate mRNA extraction. For lategene expression related to hypoxia, cells first underwent hypoxicculturing andwere thenmaintained in normoxic culture conditionsfor another 7 days, followed by mRNA extraction. The total RNA ofcochlear cells or primary spheres was extracted with an RNeasyMicro Kit (Qiagen, Australia) according to the manufacturer’sinstructions. Three micrograms of total RNAwas used to synthesizecDNA with random primers (Invitrogen, USA). The cDNAs weresynthesized using a Transcriptor cDNA Synthesis Kit (Roche).Polymerase chain reaction (PCR) was conducted with a Taq DNAPolymerase Kit (Invitrogen). The sequences of all primer pairs andthe annealing temperature are as follows: Vegf, forward (50-ATGTGA CAA GCC AAG G-30), reverse (50-CGG ACC CAA AGT GCT C-30),and 60 �C; Hif-1a, forward (50-GTA AGT GGTATTATT CAG CAC G-30),reverse (50-TAA AGG AGA CAT TGC CAG G-30), and 50 �C; nestin,forward (50-GGA ACA GAG ATT GGA AGG C-30), reverse (50-TGT CACAGG AGT CTC AAG G-30), and 50 �C; Abcg2, forward (50-TCG CAGAAGGAGATGTGT-30), reverse (50-TTG TTG GAAGTC GAAGAG C-30),and 50 �C; p27Kip1, forward (50-GGA CTT GGA GAA GCA CTG-30),reverse (50-GGG AAC CGT CTG AAA CAT-30), and 50 �C; Nanog,forward (50-AAG TAC CTC AGC CTC CAG-30), reverse (50-AGA AAGTCC TCC CCG AAG-30), and 50 �C; Oct4, forward (50- GGA GAA GGTGGA ACC AAC-30), reverse (50-GTG AGT GAT CTG CTG TAG G-30), and50 �C; Musashi1, forward (50- TAC GCC AGC CGG AGT TAC A-30),reverse (50- AAG CCG TGA GAG GGA TAG CT-30), and 50 �C; Gapdh,forward (50-AAC GGG AAG CCC ATC ACC A-30), reverse (50-CAG CCTTGG CAG CAC CAG-30), and 50 �C. Amplified fragment sizes for eachgene are: Vegf (197-bp), Hif-1a (333-bp), nestin (382-bp), Abcg2(357-bp), p27Kip1 (407-bp), Nanog (413-bp), Oct4 (409-bp), Musa-shi1 (119-bp) and Gapdh (442-bp). PCR products were electro-phoresed on a 1.5% agarose gel and visualized with ethidiumbromide under ultraviolet light. Reverse transcription (RT)-PCRanalysis of at least three independent cultures was performed forall experiments.

2.10. RNA interference and transfection

Three small-interfering RNA (siRNA) duplexes designed to Hif-1a (Genbank accession no. NM_010431) were evaluated. The mostefficient duplex for silencing Hif-1a targeted nucleotides 1294 to1314 (Sense Strand: 50-CAC CAU GAU AUG UUU ACU ATT-30) wasobtained from Santa Cruz Biotechnology. A non-silencing control(50-UUC UCC GAA CGU GUC ACG UTT-30) was obtained from SantaCruz Biotechnology, and this control was used in all experiments. Inaddition, a negative control duplex (50-UUC UCC GAA CGU GUC ACGUTT-30) labeled with FITC was used to monitor transfection effi-ciency. Cells were transfected with siRNA duplexes using siRNAtransfection reagent (Santa Cruz Biotechnology, Cat. No: sc-35562),according to the manufacturer’s protocol. The primary spheres,after culture for 7 DIV, were dissociated into single cells incubatedwith Hif-1a siRNA or non-silencing control siRNA for 6 h. Aftera medium change, spheres were exposed to hypoxic or normoxicconditions for 24 h, followed by RT-PCR (2� 105 cells/well in 6-wellplates), fluorescence intensity measurement, and WST-1 assays(1 � 104 cells/well in 96-well plates).

2.11. Western blot analysis

Total cell lysates were prepared by lysing the cochlear SPCs ina sample buffer (66 mM TriseHCl, pH 7.4, 2% SDS) at 90 �C. Lysatescontaining equal amounts of protein were loaded and separated on8% SDS polyacrylamide gels. After electrophoresis, the gels weretransferred to polyvinylidene difluoride (PVDF) membranes (Mil-lipore), blocked with 5% skim milk in TBST (0.2 M Tris-base, 1.37 MNaCl, and 0.1% Tween 20), and probed with the indicated antibody

H.-C. Chen et al. / Hearing Research 275 (2011) 43e5246

at 4 �C overnight. After washing three times with TBST, themembranes were then incubated with a peroxidase-conjugatedsecondary antibody for 1 h at RT and washed another three timeswith TBST. The immunoreactive bands were stained using a light-emitting nonradioactive method (ECL; Millipore). Antibodies usedfor western blot analyses were mouse anti-Hif-1a monoclonalantibody (1:500; Santa Cruz) and rabbit anti-Actin polyclonalantibody (1:2000; Chemicon).

2.12. Statistical analysis

Statistical analysis was performed using a two-tailed Student’st-test. Results are expressed as means� standard error of the mean(SEM). Differences were considered significant at P < 0.05.

3. Results

3.1. Morphologic change and growth characteristics of spheresderived from neonatal cochleae

A hallmark of pluripotent cochlear stem cells is their ability toform clonal, free-floating colonies, called spheres (Oshima et al.,2007). Normally, small sphere populations were observed after2e3 DIV after isolation from approximately 20 cochleae of P1neonates (Fig. 1A). Hundreds of spheres with a heterogeneousmorphologic population were produced after 7 DIV (Fig. 1B).Primary spheres were collected, dissociated, and transferred intoultralow attachment 6-well plates for another 7 days. At that time,most secondary spheres presented more dominantly with a solidappearance (Fig. 1C). After propagation, most solid spheres tendedto convert their appearance into a transitional formation witha hollow appearance (Fig. 1D). These observations were compatiblewith a previous report (Diensthuber et al., 2009) in which solidspheres might have self-renewing capacity before subsequentlyconverting to hollow spheres.

3.2. Sphere cells expressed stem/progenitor cell markers

To characterize the stemness properties of sphere cells, theexpression of stem/progenitor cell markers (nestin and Abcg2) was

Fig. 1. Morphological changes in spheres derived from P1 cochleae. (A) Phase contrast imageafter 3 DIV. Small sphere populations could be observed (original magnification �40). (B) Hu�40). (C) Both hollow and solid appearances were noted in these spheres after 14 DIV (origshown (original magnification �200). DIV: days in vitro.

further established by immunocytochemistry. Nestin, an interme-diate filament protein expressed in neural stem cells (Lendahl et al.,1990), has been recognized as a cochlear stem cell marker becauseof the close relationship between cochlear and neural stem cells(Malgrange et al., 2002; Yerukhimovich et al., 2007). Abcg2,a member of the ATP-binding cassette transporters exclusivelyexpressed in the plasma membrane, is also considered a universalstem/progenitor cell marker (Savary et al., 2008). As shown inFig. 2A, we detected variable expression patterns of nestin in spherecells, indicating that they may contain distinct populations ofneural cells. Significant expression of Abcg2 was also seen in thesolid spheres (Fig. 2B).

3.3. Proliferation and self-renewal of cochlear stem/progenitor cells

To determine whether spheres were capable of proliferation,primary spheres derived from FVB/NCrl-Tg (Pgk1-EGFP) 1Narltransgenic mice expressing EGFP as marker were observed fora period of 21 DIV.Without subculture, these primary spheres werefound to be able to increase in size when maintained in non-adherent culture conditions (Fig. 3A). The EGFP expression of thesespheres inferred the existence of cell viability. The BrdU labeling inthe spheres revealed that the cochlear SPCs prepared in thisexperiment were capable of proliferation (Fig. 3B). In addition,24e29% of the cells in spheres were BrdU-positive in the presentstudy. Our data are in agreement with a previous report dealingwith cochlear SPCs from a postnatal cochlea (Savary et al., 2008),which demonstrated that 13e27% of the cells in spheres hadincorporated BrdU and approximately 55e65% of BrdU-positivecells were immunopositive for Abcg2.

To examine whether spheres following passage maintained thecapability of proliferation, primary spheres were passaged at every7 DIV and observed for a period of 21 DIV. As shown in Fig. 3C,increased sphere number could be observed following passages.Furthermore, BrdU incorporation was detected in each passagedspheres (Fig. 3C).

To further investigate whether spheres generated in this studywere capable of self-renewal, the hallmark property of SPCs, weculturedprimary spheres for 21DIVandexamined sphere formationnumber at each passage, initially from a dissociated cell suspension

of newly isolated cochlear cells derived frommice P1 neonatal cochlea tissues culturedndreds of spheres were produced after 7 DIV in ultralow plates (original magnificationinal magnification �100). (D) Representative solid, transitional and hollow spheres are

Fig. 2. Stem/progenitor cell markers revealed by immunocytochemistry. (A) Different expression patterns of nestin seen in the solid, transitional, and hollow forms of spheres.(B) Abcg2 expressed in the cytoplasmic membrane of spheres. Original magnification �200.

H.-C. Chen et al. / Hearing Research 275 (2011) 43e52 47

following single primary spheredissociation. As shown in Fig. 3D, bydissociating a single primary sphere at DIV 7 (passage 0) intoa single-cell suspension (passage 1), increased sphere number wasobtained, and this clonal propagation phenomenon could beobserved in the subsequent passage (passage 2). We also examinedthe characteristic self-renewal property in sensory epithelium(organof Corti only)-derived cochlear SPCs. After passageof primaryspheres, secondary spheres could also express the stemnessmarkerNanog as expressed and shown in primary spheres, suggesting thatan unchanged stem cell population is maintained. Furthermore,under adherent differentiating conditions, both primary spheresand secondary spheres were shown to develop antigenic propertiesof organof Corti cells (i.e., p27 andmyosinVIIA); thesemarkerswere

Fig. 3. Proliferation and self-renewal of cochlea-derived spheres. (A) Primary spheres derivwere cultured in a non-adherent condition for a period of 21 DIV. The proliferated spheres wincorporation seen in the nuclei of spheres. Scale bar ¼ 25 mm. (C) Primary spheres, when pnumber following passages. BrdU incorporation was also detected in each passaged spheresdissociated to a single cell, and cultured at a relative low density (approximately 1 cell/ml) toThe average number of sphere formations at each passage was calculated (n ¼ 6).

not detected under undifferentiating conditions (SupplementaryFig. S1). This evidence suggests that cochlear SPCs derived fromnewborn mice retain characteristic proliferation and self-renewalpotentiality, and this is basically in agreement with the previousliterature (Li et al., 2003;Malgrange et al., 2002; Oshima et al., 2007;Yerukhimovich et al., 2007).

3.4. Differentiation and the expression of specific markers fromcochlea-derived spheres

We examined whether spheres are able to form different differ-entiated cell types. Spheres derived from cochlear SPCs werecultured in adherent conditions. Using immunostaining with

ed from FVB/NCrl-Tg (Pgk1-EGFP) 1Narl transgenic mice expressing EGFP as a markerere found to increase in size in a time-dependent manner. Scale bar ¼ 500 mm (B) BrdUassaged at every 7 DIV for a period of 21 DIV, were found to increase secondary sphere. Scale bars ¼ 300 mm (Phase), 100 mm (ICC). (D) A single primary sphere was isolated,generate new spheres (passage 1), followed by another passage (passage 2) 7 DIV later.

H.-C. Chen et al. / Hearing Research 275 (2011) 43e5248

antibodies specific for the hair cell marker myosin VIIA (Sahly et al.,1997) and the supporting cell marker p27Kip1 (Chen and Segil, 1999)to characterize the differentiated cells, we detected that 11 � 3.6%cells in the islandswere positive formyosin VIIa (Fig. 4A), indicatingthat some of cochlea-derived spheres had differentiated into haircell-like cells. Double immunocytochemical staining of the cultureswith an anti-p27 antibody revealed that 13.3� 4.9% cells within theepithelial islands also became supporting cell-like cells (Fig. 4A).Interestingly, those cells that expressed significant p27 were foundto have no myosin VIIA co-expression. In contrast, cells thatexpressed significant myosin VIIA also downregulated the expres-sion of p27 (Fig. 4A). A subset of cells after in vitro differentiationpresented neurite-like extensions, which confirmed the expressionof neuron-specific b-III tubulin (Fig. 4B). These data indicated thatcochlear sphere-derived cells are capable of differentiating into haircell- and supporting cell-like cells, and neurons.

3.5. Hypoxia promotes cochlear SPC expansion

To determine the effects of hypoxia on cochlear SPCs, newlyisolated primary cochlear cells (2 � 104 cells/well in 24-well plates)were cultured in hypoxic conditions with 1% oxygen tension for24 h followed by normoxic culture conditions for another 7 days.The number of spheres was calculated. As shown in Fig. 5A and B,hypoxia yielded a significantly higher number of spheres(19.7 � 3.1) than did normoxia (10.3 � 1.5). To test whether sucha phenomenon is shared by other mouse strains, we used anothersource of cochlear SPCs derived from FVB/N-Tg (PolII-luc) trans-genic mice with a visible bioluminescence marker and found byluminescent emission analysis that the numbers of spheres werealso strikingly increased in hypoxic conditions (Fig. 5C and D).

3.6. Coordination of upregulated Abcg2 and nestin anddownregulated p27Kip1 during hypoxia

It has been shown that Abcg2 is involved in survival enhancementof hematopoietic stem cells in hypoxia (Krishnamurthy et al., 2004).Abcg2 found in primitive stem cells might play a significant role inmaintaining stemcells in anundifferentiated state (Zhou et al., 2001).Robust expression of the neural precursor cellmarker nestinhas beendemonstrated in spheres derived from the cochlea, suggestinga neural precursor in cochlear SPCs. p27Kip1 (p27), a cyclin-dependent

Fig. 4. Differentiation of multiple cell types from cochlea-derived spheres. (A) Differentiatedp27 (white arrowhead). The expression pattern of both proteins are mutually exclusive (mergextension morphology. Nuclei are visualized with blue DAPI staining and cell structure with rto colour in this figure legend, the reader is referred to the web version of this article).

kinase inhibitor, functions as an inhibitor of cell cycle progression. Inmice, p27 expression is induced in the primitive organ of CortibetweenE12andE14, the critical time forprogenitorcells of theorganof Corti to exit the cell cycle (Ruben, 1967). Differences in abundanceofp27Kip1 inmouseneuroblastoma cells havebeen shown to correlatewell with neuronal differentiation (Kranenburg et al., 1995). More-over, mitotically active cells can still be observed in the organ of Cortiin P6 animals in the absence of p27 (Chen and Segil, 1999), indicatingthat the downregulation of p27 expression may be related to themaintenanceof cell divisionand theundifferentiated stateof cochlearprogenitor cells.Wewere thus interested inevaluatingwhether thesestemness- and cell-cycle quiescent-related cell markers were regu-lated in hypoxia-induced cochlear SPC proliferation. In RT-PCR, boththe Abcg2 and the nestin gene showed higher levels of mRNAexpression immediately after hypoxia for 24 h compared with thoseafter normoxia (Fig. 6). After reoxygenation by culturing in normoxicconditions for 7 DIV, higher levels of Abcg2 and nestin expressionremained in the hypoxia group compared with control. When theexpression levels of the immediate and 7DIV groupswere compared,bothgeneswere found tobeupregulated in the7DIVgroupcomparedwith those in the immediate group. On the contrary, the expression ofp27 in newly isolated cochlear cells was significantly downregulatedafter hypoxia for 24 h in the immediate group. After 7 DIV, thishypoxia-induced downregulation effect of p27 could still be observedcompared with cells exposed to normoxic conditions. These resultsimply that newly isolated cochlear cells will enhance their stemnessafter hypoxia stimulation by upregulating expression of the stem-ness-related genes Abcg2 and nestin. Concurrent downregulation ofp27 in hypoxia also contributed to the maintenance of the undiffer-entiated state of cochlear SPCs. Furthermore, these hypoxia-inducedexpressional differences could be observed until 7 DIV after 24-hhypoxia. In addition to Abcg2 and nestin, we also examined severalstemness-relatedmarkers (Nanog,Oct4, andMusashi1) in response tohypoxia by RT-PCR analysis. The results revealed that an hypoxiaculture condition predominantly upregulated those correspondinggenes transcripts (Fig. 6B).

3.7. Augmentation of stemness-related gene expression in hypoxiais regulated by Hif-1a activation

The transcription factor Hif-1a plays an essential role in regu-lating gene expression in response to hypoxia/ischemia. Several

spheres in adherent culture show immunoreactivity to myosin VIIA (yellow arrow) ande). (B) Other cells were b-III tubulin immunoreactive (green) and presented neurite-likeed Rhodamine staining to actin. Scale bar ¼ 25 mm. (For interpretation of the references

Fig. 5. Sphere formation assays of newly isolated cochlear cells in normoxic and hypoxic conditions. (A) Newly isolated cochlear cells cultured in serum-free medium supplementedwith EGF (20 ng/ml), b-FGF (10 ng/ml), N2, and B27 were incubated at 1% oxygen (hypoxia) and 20% oxygen (normoxia) for 24 h, respectively. After 7 DIV, a representative wellviewed with phase contrast microscopy shows that the number of formed spheres increased when incubated in hypoxic versus in normoxic conditions. The counted number ofspheres was statistically higher after hypoxia than after normoxia. Scale bar ¼ 150 mm. (B) Newly isolated cochlear cells derived from FVB/N-Tg (polII-Luc) transgenic neonates andexpressing bioluminescence signals as markers were tested under the same hypoxic and normoxic conditions. After 7 DIV, a representative well viewed with the IVIS ImagingSystem showed that numbers of spheres are also increased after hypoxia versus after normoxia. Significantly stronger bioluminescence signal measurement was observed inhypoxic conditions. Data are expressed as means � standard error of the mean (SEM) of three independent experiments. DIV: days in vitro; * indicates P < 0.05.

H.-C. Chen et al. / Hearing Research 275 (2011) 43e52 49

reports have suggested a role of Hif-1a in regulating cellulardifferentiation during hypoxia (Koay and Athanasiou, 2008; Luet al., 2009; Yun et al., 2002). We thus were interested in exam-ining the impact of hypoxia on the abundance of Hif-1a and Hif-1aregulated Vegf gene expression. In addition to newly isolatedcochlear cells, the primary spheres derived from isolated cochlearcells and representing a substantially more homogeneous pop-ulation of stem/progenitors were also investigated. As shown inFig. 7A, Hif-1a and its target gene Vegf were upregulated in bothnewly isolated cochlear cells and primary spheres after 24-hhypoxia; meanwhile, the expression of the cell-cycle quiescentmarker p27 was downregulated in hypoxic conditions. Theseresults indicate that Hif-1a in cochlear SPCs can be transcriptionallyregulated under hypoxic conditions.

To further determine whether Hif-1a could influence the fate ofcochlear SPCs in response to hypoxia and its association withstemness- and cell cycle quiescent-related gene expression, weevaluated knockdown Hif-1a expression in primary spheres by

Fig. 6. Effects of hypoxia-induced expression of Abcg2, nestin, p27, Nanog, Oct4, andMusashi1 mRNA in newly isolated cochlear cells. (A) After 24-h hypoxia, the expressionof Abcg2 and nestin mRNAwas significantly upregulated while p27 was downregulatedcompared to normoxic controls at both the immediate and 7 DIV time points. (B)Enhanced expression of the stem/progenitor cell markers Nanog, Oct4, and Musashi1from newly isolated cochlear cells after hypoxia were observed. A representative ofthree independent experiments is shown. N: normoxia; H: hypoxia; Gapdh: glycer-aldhyde-3-phosphate dehydrogenase.

using siRNAs. The transfection efficiency, as determined usinga FITC-labeled non-silencing control siRNA, was 60e70% (Fig. 7B).We also determined the efficiency of Hif-1a-specific siRNA trans-fection, using a western blot analysis (Fig. 7C). The Hif-1a siRNAsignificantly inhibited Hif-1a expression (Fig. 7C). Fig. 7D showsthat hypoxia-induced upregulation of Abcg2 and nestin mRNA wasattenuated when Hif-1a expression was knocked down by siRNA,concurrent with the downregulation of Vegf expression. On thecontrary, downregulated p27 expression in hypoxic conditions wasupregulated after transient Hif-1a knockdown (Fig. 7D). Theseresults indicate that Hif-1amay directly regulate stemness- and cellcycle quiescent-related gene expression in cochlear SPCs.

3.8. Hif-1a contributes to hypoxia-promoted cell proliferation andviability of cochlear SPCs

We finally examinedwhether the promotion of cell proliferationand viability of cochlear SPCs in hypoxia stimulation was throughthe activation of Hif-1a. To this end, we isolated primary cochlearspheres from FVB/Tg transgenicmice inwhich EGFP expressionwasdriven by themouse Pgk1 promoter. Because pgk-1 is one of the Hif-1a target genes (Gustafsson et al., 2005), these primary spheresthus could be used to examine pgk-1 hypoxia response element-mediated EGFP expression levels in the presence or absence ofHif-1a under normoxic or hypoxic conditions. After knockdown ofHif-1a by siRNA, primary spheres were exposed to hypoxic ornormoxic conditions for 24 h. After another 2 DIV in normoxicconditions, measurements of EGFP fluorescence intensity (byfluorometer detection), cell proliferation (by BrdU incorporationassay), and cell viability (by WST-1 assay) were conducted. Weobserved that hypoxia significantly increased the EGFP expressionlevel, while Hif-1a siRNA significantly reversed this hypoxia-induced effect (Fig. 8A). Knockdown of Hif-1a induced paralleldecreases in proliferation of primary spheres in hypoxic conditions(Fig. 8B). Consistent with this, the promotion of primary sphere cellviability in hypoxic conditions was also reversed by Hif-1a siRNA

Fig. 7. Hif-1a expression and augmentation of stemness-related gene expression inhypoxia. (A) Hif-1a and Vegf mRNA levels were enhanced after 24-h hypoxia in newlyisolated cochlear cells with concurrent upregulated expression of the stem/progenitorcell markers Abcg2 and nestin while the cell-cycle quiescent marker p27 was down-regulated compared to the control. A similar gene expression profile was also observedin the primary spheres. (B) Photographs of cochlear SPCs transfected with a negativecontrol duplex labeled with FITC (green) revealed a transfection efficiency of 60e70%.Scale bar ¼ 100 mm. (C) Knockdown of Hif-1a gene expression by siRNA transfectionresulted in reduced amounts of Hif-1a protein in cochlear spheres under hypoxicculture conditions. (D) After transient knockdown of Hif-1a by siRNA, the expressionlevels of Abcg2 and nestin were decreased compared to those of the controls. Incontrast, p27 was upregulated in the Hif-1a knockdown group. These experimentswere repeated three times with similar results. N: normoxia; H: hypoxia; Gapdh:glyceraldehyde-3-phosphate dehydrogenase. (For interpretation of the references tocolour in this figure legend, the reader is referred to the web version of this article).

Fig. 8. Measurement of EGFP expression levels in assays of cell proliferation andviability to examine the effect of Hif-1a on cochlear SPCs in hypoxic conditions. (A) Thefluorescence intensity of cochlear spheres derived from FVB/NCrl-Tg (Pgk1-EGFP) 1Narltransgenic mice was significantly affected by downregulation of Hif-1a in hypoxicconditions. (B) BrdU incorporation assay shows that the proliferative capacity ofprimary cochlear spheres significant decreased in hypoxic conditions after Hif-1aknockdown compared with controls. (C) After hypoxia stimulation, cell viability(represented as OD values) of Hif-1a siRNA-transfected spheres was significantlydecreased compared with that of control siRNA-transfected spheres. *indicatesP < 0.05, ** indicates P < 0.01.

H.-C. Chen et al. / Hearing Research 275 (2011) 43e5250

(Fig. 8C). These results indicate that in this cochlear SPC culturesystem, Hif-1a plays a role in regulating hypoxia-induced stem cellsurvival and proliferation.

4. Discussion

The cochlea contains cells with the ability to form clonal floatingcolonies, which are termed spheres when cultured in non-adherentconditions (Diensthuber et al., 2009; Malgrange et al., 2002;Yerukhimovich et al., 2007). Spheres are also a population ofstem/progenitor cells capable of extensive self-renewal and differ-entiation into multiple lineages. Like neurospheres, which aregenerated from neural stem cells and express nestin as a marker(Bauer, 2009), spheres generated from cochlear SPCs originatingfrom neural ectoderm can express nestin as a characteristic SPCmarker. Since cochlear SPCs cannot be identified with uniquemarkers, an in vitro functional assay is used to identify the maincharacteristics of cochlear SPCs, including spheres (Fig. 1), prolif-eration (Fig. 3), and self-renewal (Fig. 3 and Supplementary Fig. S1).Although multilineage cell markers could be shown on spheresfollowing culture under differentiating conditions (Fig. 4), furtherstudies will be needed to confirm these findings as multipotencyfrom clonal-derived spheres because the cochlear SPCs currentlybeing tested were derived from several cochlear partitions.

The newly isolated cochlear cells used in this study, which wereacutely harvested from cochleae of P1 neonates, contained differentcell types originating from several cochlear tissues, including theorgan of Corti, stria vascularis, spiral ligament, and modiolus.

Although their composition is heterogeneous, dissociated cellsderived fromwhole cochlea (Yerukhimovich et al., 2007) as shownin our study or individual cochlear partition (Malgrange et al.,2002; Oshima et al., 2007; Rask-Andersen et al., 2005; Savaryet al., 2007) have all been shown to be able to generate spheres.Therefore, by investigating the effects of low-oxygen on sphereformation, we dissociated the whole cochlear tissues regardless ofindividual cochlear tissue isolation. We observed that hypoxiadrastically increased sphere formation in newly isolated cochlearcells (an increase of 76%) in both CBA/CaJ mice (Fig. 5A and B) andFVB/N-Tg (polII-Luc) transgenic mice (Fig. 5C and D). These resultscorrelated with previous observations that hypoxic culture resultsin the enhancement of the progenitor cells of several cell types,including the bone marrow cells (Cipolleschi et al., 1993, 2000),neural cells (Studer et al., 2000; Zhang et al., 2006), and limbalepithelial cells (Miyashita et al., 2007). Although the proliferationcapacity and the undifferentiated state of neural stem cells (NSCs)are known to be promoted by hypoxia (Gustafsson et al., 2005),such an effect on cochlear SPCs has never been shown. Ourdemonstration is the first to reveal that low-oxygen culturingpromotes the proliferation capacities of cochlear SPCs, which isessential in order to produce sufficient cells for future applications.

H.-C. Chen et al. / Hearing Research 275 (2011) 43e52 51

In addition to promoting proliferation capacities, hypoxia wasalso shown to contribute to the maintenance of the stemness orimmature state of cochlear SPCs by upregulating nestin, Abcg2,Nanog, Oct4, and Musashi1 gene expression compared to those innormoxia (Fig. 6). Nestin is an intermediate filament proteinexpressed by stem cells and progenitors early in development andthroughout the early postnatal period in the central and peripheralnervous systems (Breuskin et al., 2008). High expression of nestinhas been recognized as a distinct molecular marker of neural stem/progenitor cells (Felling et al., 2006; Lendahl et al.,1990;Wang et al.,2007; Yang and Levison, 2006). On the contrary, loss of nestinexpression usually suggests that phenotypic changes in differenti-ation occurred in both NSCs (Mellodew et al., 2004) and cochlearSPCs (Breuskin et al., 2008). By using nestin promoter-green fluo-rescent protein (GFP) transgenic mice to determine the presence ofSPCs in the mouse inner ear, Lopez et al. (Lopez et al., 2004)demonstrated that GFP-positive cells could be observed in the innerborder and inner phalangeal cells, Dieters cells, and cells in the greatepithelial ridge in P0 to P15 animals. However, only moderate GFPexpression was found in a few Dieters cells in P60 animals, sug-gesting adevelopmentally regulatednestin expressionpattern in themouse inner ear. In contrast, our data strongly imply that theexpression levels of both nestin and Abcg2 increase after hypoxia,sustaining the idea that amore immature or undifferentiated extentof SPCs may be induced after hypoxia stimulation. In addition,downregulated p27 in response to hypoxia also contributed to themaintenance of immature SPCs in the in vitro culture systems. It hasbeen observed that over-expression of the negative cell cycle regu-lator p27 enhances premature neuronal differentiation (Tarui et al.,2005). Thus, modulation of cell cycle regulator expression may bea prominent mechanism for the maintenance of the cochlearprecursor pool. Since those isolated cochlear cells were cultured ina serum-free medium, the presence of p27 expressional differencesmay be largely related to cell cycle progression rather than thespecified hair cell and supporting cell patterning (differentiation ofhair cells and supporting cells) at a later embryogenesis stage.

Hif-1a, a global regulator of oxygen homeostasis, is known topromote the undifferentiated neural stem cell phenotypes(Gustafsson et al., 2005) and increase proliferation of mouseembryonic stem cells (Gibbons et al., 2006). In the present study,stabilization of Hif-1a was also found in newly isolated cochlearcells and primary spheres under hypoxic conditions (Fig. 7A). siRNAexperiments revealed that Hif-1a determines the expression ofnestin, Abcg2, and the direct transcriptional target Vegf (Fig. 7D),which suggests that Hif-1a participates in the promotion ofcochlear SPC expansion and maintenance of an undifferentiatedstate. Genetic analysis of Hif function in multiple species and thedevelopmental defects in Hif-deficient embryos have revealed theimportance of oxygen availability and Hif as a key regulator ofontogeny (Simon and Keith, 2008). In another report, the protectivebenefits of hypoxic preconditioning against permanent noise-induced hearing loss (NIHL) shown in CBA mice were associatedwith significant upregulation of Hif-1a within the organ of Corti(Gagnon et al., 2007). Accordingly, these evidences implicatea great survival promotion benefit of Hif-1a in response to hypoxiastimulation, although the mechanisms regulating cochlear SPCbehavior via Hif have yet to be elucidated.

5. Conclusions

Collectively, our results suggest that hypoxia facilitated theexpansion of sphere formation and maintenance of the immaturestate of cochlear SPCs in vitro. Activation of Hif-1a is shown tocontribute to these hypoxia-induced effects. Our findings provideinsights into potential future strategies of expansion of the ex vivo

pool of cochlear SPCs for transplantation or endogenous augmen-tation of SPCs for the development of therapies for inner earregeneration.

Acknowledgements

This work was supported in part by grants from the NationalScience Council, Taiwan (NSC 98-2314-B-016-017-MY3), Tri-ServiceGeneral Hospital (TSGH C99e40 and C99-42) and National DefenseMedical Research grants, Taiwan, ROC (DOD 98-11-05, DOD 99-09-01, and DOD 99-10-05).

Appendix. Supplementary data

Supplementary data associated with this article can be found inthe on-line version, at doi:10.1016/j.heares.2010.12.004.

References

Bauer, S., 2009. Cytokine control of adult neural stem cells. Ann. N. Y. Acad. Sci. 1153,48e56.

Breuskin, I., Bodson, M., Thelen, N., Thiry, M., Nguyen, L., Belachew, S., Lefebvre, P.P.,Malgrange, B., 2008. Strategies to regenerate hair cells: identification ofprogenitors and critical genes. Hear Res. 236, 1e10.

Carmeliet, P., Dor, Y., Herbert, J.M., Fukumura, D., Brusselmans, K., Dewerchin, M.,Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C.J., Ratcliffe, P.,Moons, L., Jain, R.K., Collen, D., Keshert, E., 1998. Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394,485e490.

Chen, P., Segil, N., 1999. p27(Kip1) links cell proliferation to morphogenesis in thedeveloping organ of Corti. Development 126, 1581e1590.

Choi, K.S., Bae, M.K., Jeong, J.W., Moon, H.E., Kim, K.W., 2003. Hypoxia-inducedangiogenesis during carcinogenesis. J. Biochem. Mol. Biol. 36, 120e127.

Cipolleschi, M.G., Dello Sbarba, P., Olivotto, M., 1993. The role of hypoxia in themaintenance of hematopoietic stem cells. Blood 82, 2031e2037.

Cipolleschi, M.G., Rovida, E., Ivanovic, Z., Praloran, V., Olivotto, M., Dello Sbarba, P.,2000. The expansion of murine bone marrow cells preincubated in hypoxia asan in vitro indicator of their marrow-repopulating ability. Leukemia 14,735e739.

Diensthuber, M., Oshima, K., Heller, S., 2009. Stem/progenitor cells derived from thecochlear sensory epithelium give rise to spheres with distinct morphologiesand features. J. Assoc. Res. Otolaryngol. 10, 173e190.

Dumoulin, J.C., Meijers, C.J., Bras, M., Coonen, E., Geraedts, J.P., Evers, J.L., 1999. Effectof oxygen concentration on human in-vitro fertilization and embryo culture.Hum. Reprod. 14, 465e469.

Felling, R.J., Snyder, M.J., Romanko, M.J., Rothstein, R.P., Ziegler, A.N., Yang, Z.,Givogri, M.I., Bongarzone, E.R., Levison, S.W., 2006. Neural stem/progenitor cellsparticipate in the regenerative response to perinatal hypoxia/ischemia.J. Neurosci. 26, 4359e4369.

Gagnon, P.M., Simmons, D.D., Bao, J., Lei, D., Ortmann, A.J., Ohlemiller, K.K., 2007.Temporal and genetic influences on protection against noise-induced hearingloss by hypoxic preconditioning in mice. Hear Res. 226, 79e91.

Gibbons, J., Hewitt, E., Gardner, D.K., 2006. Effects of oxygen tension on theestablishment and lactate dehydrogenase activity of murine embryonic stemcells. Cloning Stem Cells 8, 117e122.

Grayson, W.L., Zhao, F., Izadpanah, R., Bunnell, B., Ma, T., 2006. Effects of hypoxia onhuman mesenchymal stem cell expansion and plasticity in 3D constructs. J. CellPhysiol. 207, 331e339.

Gustafsson, M.V., Zheng, X., Pereira, T., Gradin, K., Jin, S., Lundkvist, J., Ruas, J.L.,Poellinger, L., Lendahl, U., Bondesson, M., 2005. Hypoxia requires notchsignaling to maintain the undifferentiated cell state. Dev. Cell 9, 617e628.

Huang, Y.H., Chin, C.C., Ho, H.N., Chou, C.K., Shen, C.N., Kuo, H.C., Wu, T.J., Wu, Y.C.,Hung, Y.C., Chang, C.C., Ling, T.Y., 2009. Pluripotency of mouse spermatogonialstem cells maintained by IGF-1-dependent pathway. FASEB J. 23, 2076e2087.

Koay, E.J., Athanasiou, K.A., 2008. Hypoxic chondrogenic differentiation of humanembryonic stem cells enhances cartilage protein synthesis and biomechanicalfunctionality. Osteoarthr. Cartil. 16, 1450e1456.

Kranenburg, O., Scharnhorst, V., Van der Eb, A.J., Zantema, A., 1995. Inhibition ofcyclin-dependent kinase activity triggers neuronal differentiation of mouseneuroblastoma cells. J. Cell Biol. 131, 227e234.

Krishnamurthy, P., Ross, D.D., Nakanishi, T., Bailey-Dell, K., Zhou, S., Mercer, K.E.,Sarkadi, B., Sorrentino, B.P., Schuetz, J.D., 2004. The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions with heme. J. Biol.Chem. 279, 24218e24225.

Lamm, K., Arnold, W., 2000. The effect of blood flow promoting drugs on cochlearblood flow, perilymphatic pO(2) and auditory function in the normal and noise-damaged hypoxic and ischemic guinea pig inner ear. Hear Res. 141, 199e219.

Lendahl, U., Zimmerman, L.B., McKay, R.D., 1990. CNS stem cells express a new classof intermediate filament protein. Cell 60, 585e595.

H.-C. Chen et al. / Hearing Research 275 (2011) 43e5252

Li, H., Liu, H., Heller, S., 2003. Pluripotent stem cells from the adult mouse inner ear.Nat. Med. 9, 1293e1299.

Lopez, I.A., Zhao, P.M., Yamaguchi, M., de Vellis, J., Espinosa-Jeffrey, A., 2004. Stem/progenitor cells in the postnatal inner ear of the GFP-nestin transgenic mouse.Int. J. Dev. Neurosci. 22, 205e213.

Lu, H., Li, Y., Shu, M., Tang, J., Huang, Y., Zhou, Y., Liang, Y., Yan, G., 2009. Hypoxia-inducible factor-1alpha blocks differentiation of malignant gliomas. FEBS J. 276,7291e7304.

Malgrange, B., Belachew, S., Thiry, M., Nguyen, L., Rogister, B., Alvarez, M.L.,Rigo, J.M., Van De Water, T.R., Moonen, G., Lefebvre, P.P., 2002. Proliferativegeneration of mammalian auditory hair cells in culture. Mech. Dev. 112, 79e88.

Malladi, P., Xu, Y., Chiou, M., Giaccia, A.J., Longaker, M.T., 2006. Effect of reducedoxygen tension on chondrogenesis and osteogenesis in adipose-derivedmesenchymal cells. Am. J. Physiol. Cell Physiol. 290, C1139eC1146.

Mellodew, K., Suhr, R., Uwanogho, D.A., Reuter, I., Lendahl, U., Hodges, H., Price, J.,2004. Nestin expression is lost in a neural stem cell line through a mechanisminvolving the proteasome and notch signalling. Brain Res. Dev. Brain Res. 151,13e23.

Miyashita, H., Higa, K., Kato, N., Kawakita, T., Yoshida, S., Tsubota, K., Shimmura, S.,2007. Hypoxia enhances the expansion of human limbal epithelial progenitorcells in vitro. Invest. Ophthalmol. Vis. Sci. 48, 3586e3593.

Orsi, N.M., Leese, H.J., 2001. Protection against reactive oxygen species duringmouse preimplantation embryo development: role of EDTA, oxygen tension,catalase, superoxide dismutase and pyruvate. Mol. Reprod. Dev. 59, 44e53.

Oshima, K., Grimm, C.M., Corrales, C.E., Senn, P., Martinez Monedero, R., Geleoc, G.S.,Edge, A., Holt, J.R., Heller, S., 2007. Differential distribution of stem cells in theauditory and vestibular organs of the inner ear. J. Assoc. Res. Otolaryngol. 8,18e31.

Packer, L., Fuehr, K., 1977. Low oxygen concentration extends the lifespan ofcultured human diploid cells. Nature 267, 423e425.

Panchision, D.M., 2009. The role of oxygen in regulating neural stem cells indevelopment and disease. J. Cell Physiol. 220, 562e568.

Rask-Andersen, H., Bostrom, M., Gerdin, B., Kinnefors, A., Nyberg, G., Engstrand, T.,Miller, J.M., Lindholm, D., 2005. Regeneration of human auditory nerve. In vitro/in video demonstration of neural progenitor cells in adult human and guineapig spiral ganglion. Hear Res. 203, 180e191.

Ruben, R.J., 1967. Development of the inner ear of the mouse: a radioautographicstudy of terminal mitoses. Acta Otolaryngol. (Suppl. 220), 1e44.

Sahly, I., El-Amraoui, A., Abitbol, M., Petit, C., Dufier, J.L., 1997. Expression of myosinVIIA during mouse embryogenesis. Anat. Embryol. (Berl) 196, 159e170.

Savary, E., Hugnot, J.P., Chassigneux, Y., Travo, C., Duperray, C., Van De Water, T.,Zine, A., 2007. Distinct population of hair cell progenitors can be isolated fromthe postnatal mouse cochlea using side population analysis. Stem Cells 25,332e339.

Savary, E., Sabourin, J.C., Santo, J., Hugnot, J.P., Chabbert, C., Van De Water, T.,Uziel, A., Zine, A., 2008. Cochlear stem/progenitor cells from a postnatal cochlearespond to jagged1 and demonstrate that notch signaling promotes sphereformation and sensory potential. Mech. Dev. 125, 674e686.

Simon, M.C., Keith, B., 2008. The role of oxygen availability in embryonic devel-opment and stem cell function. Nat. Rev. Mol. Cell Biol. 9, 285e296.

Studer, L., Csete, M., Lee, S.H., Kabbani, N., Walikonis, J., Wold, B., McKay, R., 2000.Enhanced proliferation, survival, and dopaminergic differentiation of CNSprecursors in lowered oxygen. J. Neurosci. 20, 7377e7383.

Tarui, T., Takahashi, T., Nowakowski, R.S., Hayes, N.L., Bhide, P.G., Caviness, V.S.,2005. Overexpression of p27 Kip 1, probability of cell cycle exit, and laminardestination of neocortical neurons. Cereb. Cortex 15, 1343e1355.

Thompson, J.G., Simpson, A.C., Pugh, P.A., Donnelly, P.E., Tervit, H.R., 1990. Effect ofoxygen concentration on in-vitro development of preimplantation sheep andcattle embryos. J. Reprod. Fertil. 89, 573e578.

Wang, X.L., Yang, Y.J., Xie, M., Yu, X.H., Liu, C.T., Wang, X., 2007. Proliferation ofneural stem cells correlates with Wnt-3 protein in hypoxic-ischemic neonaterats after hyperbaric oxygen therapy. Neuroreport 18, 1753e1756.

White, P.M., Doetzlhofer, A., Lee, Y.S., Groves, A.K., Segil, N., 2006. Mammaliancochlear supporting cells can divide and trans-differentiate into hair cells.Nature 441, 984e987.

Wing, D.A., Talley, G.D., Storch, T.G., 1988. Oxygen concentration regulates EGF-induced proliferation and EGF-receptor down regulation. Biochem. Biophys.Res. Commun. 153, 952e958.

Yang, Z., Levison, S.W., 2006. Hypoxia/ischemia expands the regenerative capacityof progenitors in the perinatal subventricular zone. Neuroscience 139,555e564.

Yerukhimovich, M.V., Bai, L., Chen, D.H., Miller, R.H., Alagramam, K.N., 2007. Iden-tification and characterization of mouse cochlear stem cells. Dev. Neurosci. 29,251e260.

Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., Yamanaka, S., 2009. Hypoxiaenhances the generation of induced pluripotent stem cells. Cell Stem Cell 5,237e241.

Yun, Z., Maecker, H.L., Johnson, R.S., Giaccia, A.J., 2002. Inhibition of PPAR gamma 2gene expression by the HIF-1-regulated gene DEC1/Stra13: a mechanism forregulation of adipogenesis by hypoxia. Dev. Cell 2, 331e341.

Zhang, C.P., Zhu, L.L., Zhao, T., Zhao, H., Huang, X., Ma, X., Wang, H., Fan, M., 2006.Characteristics of neural stem cells expanded in lowered oxygen and thepotential role of hypoxia-inducible factor-1alpha. Neurosignals 15, 259e265.

Zhou, S., Schuetz, J.D., Bunting, K.D., Colapietro, A.M., Sampath, J., Morris, J.J.,Lagutina, I., Grosveld, G.C., Osawa, M., Nakauchi, H., Sorrentino, B.P., 2001. TheABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and isa molecular determinant of the side-population phenotype. Nat. Med. 7,1028e1034.