Research ArticleRegenerating Salivary Glands in the Microenvironment ofInduced Pluripotent Stem Cells

Hitomi Ono1 Aya Obana1 Yu Usami2 Manabu Sakai3 Kanji Nohara1

Hiroshi Egusa4 and Takayoshi Sakai1

1Department of Oral-Facial Disorders Osaka University Graduate School of Dentistry Suita Osaka 565-0871 Japan2Division of Orthopedic Surgery The Childrenrsquos Hospital of Philadelphia Research Institute Philadelphia PA 19104 USA3Clinical Laboratory Osaka University Dental Hospital Suita Osaka 565-0871 Japan4Division of Molecular and Regenerative Prosthodontics Graduate School of Dentistry Tohoku University SendaiMiyagi 980-8575 Japan

Correspondence should be addressed to Takayoshi Sakai sakaidentosaka-uacjp

Received 5 March 2015 Accepted 4 June 2015

Academic Editor Chung-Liang Chien

Copyright copy 2015 Hitomi Ono et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This report describes our initial attempt to regenerate salivary glands using induced pluripotent stem (iPS) cells in vivo and in vitroGlandular tissues that were similar to the adult submandibular glands (SMGs) and sublingual glands could be partially produced bythe transplantation of iPS cells into mouse salivary glands However the tumorigenicity of iPS cells has not been resolved yet It iswell known that stem cells affect their microenvironment known as a stem cell niche We focused on the niche and the interactionbetween iPS cells and salivary gland cells in our study on salivary gland regeneration Coculture of embryonic SMG cells and iPScells have better-developed epithelial structures and fewer undifferentiated specific markers than monoculture of embryonic SMGcells in vitro These results suggest that iPS cells have a potential ability to accelerate differentiation for salivary gland developmentand regeneration

1 Introduction

Salivary glands have important functions in maintaining oralhealth [1] Hypofunction of the salivary glands can causevarious life-disrupting side effects such as dental cariesswallowing difficulties loss of taste and oral candidiasisIrradiation therapy and Sjogrenrsquos syndrome can cause salivarygland hypofunction and xerostomia No satisfactory therapyhas been established to treat salivary hypofunction [1ndash3]

Induced pluripotent stem (iPS) cells can be generatedfrom fully differentiated nonpluripotent cells and possesspluripotency similar to that of embryonic stem (ES) cells [4]iPS cells could be a powerful tool in regenerative medicinebut their potential tumorigenicity is a significant challenge forclinical use [5 6]

On the other hand ES cells and other stem cells have amicroenvironment that is self-renewing and have multilin-eage developmental potential In vivo these properties are

not autonomous to stem cells and recent evidence pointsto a level of external control from the microenvironmentthat defines the stem cell niche The stem cell niche mayrepresent a significant entry point for therapeuticmodulationof stem cell behavior [7 8] Although it is known that themicroenvironment is derived from iPS cells the niche of iPScells has not been well studied [9 10]

In this study we tried to regenerate salivary glandsusing both embryonic salivary glands and iPS cells andwe identified putative functions of the iPS cell niche Manyresearchers have tried to regenerate organs such as salivaryglands kidneys and lungs [11ndash13] A recent study describeda therapy using stemprogenitor cell transplantation afterradiation to restore long-term saliva production [3] It iscurrently proposed that stemprogenitor cells reside in theducts of salivary glands [14] There is a possibility thatthe stem cell niche affects salivary gland development andregeneration These analyses may provide new concepts for

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 293570 11 pageshttpdxdoiorg1011552015293570

2 BioMed Research International

the functional regeneration of salivary glands using tissueengineering

2 Methods

21 Animals All animal experiments were performed instrict accordance with the guidelines of the Animal Care andUse Committee of the Osaka University Graduate Schoolof Dentistry Osaka Japan The protocol was approved bythe Committee on the Ethics of Animal Experiments ofOsaka University Graduate School of Dentistry (permitnumber 25-004-0) All surgeries were performed undersodium pentobarbital anesthesia and all efforts were madeto minimize suffering SMGs were dissected from embryonicand postnatal ICRmice (Japan SLC Inc Hamamatsu Japan)For regenerating cultures of salivary glands three mice inembryonic day 135 (E135) were used For the analysis ofdevelopmental process three mice in E135 to E175 andthree mice in postnatal eight weeks were used For teratomaformation assays three immunodeficient mice (CB-17 SCIDClea Japan Tokyo Japan) that are eight-week-old were usedEach experiment was repeated at least three times with datashowing a representative experiment

22 Cell Culture The mouse green fluorescent protein-(GFP-) iPS cell line (APS0006 iPS-Stm-FBgfp-99-3)derived from stomach cells was provided by the RIKEN BRCthrough the National Bio-Resource Project of the MEXTJapan The iPS cells express GFP by the CAG promoterThe cells were disseminated in a 6-well dish with gelatinsolution and cultured with ESGRO complete plus serum-freeclonal grade medium (Millipore Billerica MA USA) Thecells were washed once with D-PBS (Millipore BillericaMA USA) and dissociated with Accutase cell detachment(Millipore Billerica MA USA)

23 Teratoma Formation Assay To analyze the multipotencyof GFP-iPS cells 20 times 105 GFP-iPS cells were collected asone pellet in cold Dulbeccorsquos Modified Eaglersquos MediumF12(DMEMF12) (Invitrogen) and transplanted into the SMG ofanesthetized SCID mouse The mice were thereafter housedwith free access to water and food under specific pathogen-free conditions The teratomas were excised after 4 weeksfixed in 4 PFA embedded in paraffin and sectioned at3 120583m Teratoma formation assays were repeated at leastthree times using three mice per group with data showinga representative experiment The histology of the formedteratomas was analyzed using hematoxylin and eosin (HampE)staining

24 Regenerating Salivary Gland Culture SMGs were treatedwith trypLE (Life technologies Carlsbad CA USA) andthe dissociated cells were seeded on 20 120583L matrigel (BDBiosciences Franklin Lakes NJ USA) coated U-bottomplates (Thermo Waltham MA USA) to yield 20 times 105 cellsper well Then the cells were cultured with 250120583L serum-free DMEMF12 medium for 96 h as previously describedMonoculture of SMG cells is defined as SG and coculture of

SMGcells andGFP-iPS cells is defined as iSG 5 iSG consistsof 5 iPS cells and 95 SMG cells 20 iSG consists of 20iPS cells and 80 SMG cells

25 Immunofluorescence Paraffin-embedded tissues of ter-atomas and regenerated salivary glands were evaluated Tis-sue sections were deparaffinized and antigen retrieval wasperformed by autoclave heating (instant antigen retrieval Hbuffer 121∘C for 5min)The slides were washed in phosphate-buffered saline (PBS) The samples were first incubated withMOM Mouse Ig Blocking Reagent (Vector LaboratoriesInc Burlingame CA USA) and then with primary anti-bodies in diluent (1x PBS containing 8 protein concen-trate MOM Kit Vector Laboratories Inc) overnight at4∘C Specific antibodies used were anti-120572-Amylase (dilution1 100 Sigma-Aldrich St Louis MO USA) anti-parotidsecretory protein (PSP) (dilution 1 100 Everest BiotechOxfordshire UK) anti-E-cadherin (dilution 1 100 BD Bio-sciences Franklin Lakes NJ USA) anti-Green FluorescentProtein (GFP) (dilution 1 100 MBL Nagoya Japan) anti-SRY (sex determining region Y) box 2 (Sox2) (dilution1 100 Santa Cruz Dallas TX USA) and anti-Aquaporin5 (AQP5) (dilution 1 100 Alomone Har Hotzvim Israel)After washing with PBS the tissues were incubated with Cy2-labelled donkey anti-Goat and Cy3-labelled donkey anti-mouse andCy5-labelled donkey anti-rabbit or anti-rat IgG for2 h at room temperature (dilution 1 100 Life TechnologiesCarlsbad CAUSA) in diluent (5donkey serum containing8 protein concentrate) Immunostaining was repeated atleast three times

26 Extraction of the SMG Cells from Regenerated SalivaryGlands by Cell Sorting SG and iSG were treated with trypLE(Life technologies Carlsbad CA USA) and SMG cells ineach SG and iSG were dissociated to single cells SMGcells were separated from GFP-iPS cells using a fluorescent-activated cell sorter (FACSAria BD Biosciences FranklinLakes NJ USA) GFP-iPS cells were separated from the GFPfraction by fluorescent-activated cell sorting SMG cells fromSG and from iSG were analyzed by quantitative RT-PCR(qPCR) and by western blotting

27 Quantitative Real-Time Reverse Transcription-PolymeraseChain Reaction SMG cells were separated from iPS cells bycell sorting Total RNAwas isolated fromboth embryonic andpostnatal tissues using TRIzol Reagent (Life TechnologiesCarlsbad CA USA) according to the manufacturerrsquos instruc-tions and was treated with DNase I (Roche Applied SciencePenzberg Upper Bavaria Germany) to avoid genomic DNAcontamination For cDNA synthesis reverse transcriptionwas performed using the Prime Script RT Reagent Kit(Takara Bio Inc Otsu Shiga Japan) Quantification of PCRproducts was performed using the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad Laboratories IncHercules CA USA) with iQ SYBRGreen Supermix (Bio-RadLaboratories Inc Hercules CA USA) The amplificationprogram comprised 40 cycles of denaturation at 95∘C for5 s annealing at 55∘C for 20 s and extension at 72∘C for

BioMed Research International 3

20 s Quantitative real-time reverse transcription-polymerasechain reaction (qPCR) results for each sample were normal-ized by glyceraldehyde-3-phosphate dehydrogenase (Gapdh)The results were expressed as normalized ratios and experi-ments were repeated three times The primer sequences usedwere as follows

Sox2 51015840-GGGAAATGGGAGGGGTGCAAAAGAGG-3101584051015840-TTGCGTGAGTGTGGATGGGATTGGTG-31015840

c-Myc 51015840-CAGAGGAGGAACGAGCTGAAGCGC-3101584051015840-TTATGCACCAGAGTTTCGAAGCTGTTCG-31015840

Klf4 51015840-CACCATGGACCCGGGCGTGGCTGCCAGAAA-3101584051015840-TTAGGCTGTTCTTTTCCGGGGCCACGA-31015840

Aqp5 51015840-TGGAGCAGGCATCCTGTACT-3101584051015840-CGTGGAGGAGAAGATGCAGA-31015840

Amy 51015840-GGATGGAGAAAAGATGTCCTAC-3101584051015840-CATCACCCGTGTGAAACC-31015840

M3r 51015840-TCGGTAGAGCGGACTGGACA-3101584051015840-TCCACTGAGCAAGTCAGAAGTGAAG-31015840

Gapdh 51015840-CCATCACCATCTTCCAGGAG-3101584051015840-GCATGGACTGTGGTCATGAG-31015840

28 Statistical Analysis Statistical analysis was performed asindicated in the figure legends and text One-way analysis ofthe 119905-test was performed to determine the statistical differ-ence between the means of each treatment for experimentsin which multiple treatments were compared

3 Results

31 Teratoma Formation in SMG after Transplantation ofMouse GFP-iPS Cells The pluripotency of GFP-iPS cellswas validated by teratoma formation in the SMG of SCIDmice Four weeks after implantation histological analysisdemonstrated that the formed teratomas were derived fromall three primary germ layers Gut-like epithelium (endo-derm) adipose tissues (mesoderm) muscles (mesoderm)neural tissues (ectoderm) and epidermis (ectoderm) wereall identified histologically in the GFP-iPS-derived teratomas(Figure 1)The teratomas contained different types of salivarygland-like tissues By immunohistochemical analysis onewas similar to the SMG and the other was similar to thepattern of the SLG in adult mice (Figure 2)

32 Localization of iPS Cells in Regenerated SMGs (SGand iSG) In SMG monoculture the epithelial cells aggre-gated and formed salivary gland-like tissue (SG) after 96 h(Figure 3) Coculture of SMG cells and GFP-iPS cells (iSG)also formed many acinar-like structures similar to the SGIn immunohistochemical analysis GFP-iPS cells did notuniformly mix with SMG cells (Figure 4(a)) GFP-iPS cellsexisted around acinar-like epithelial cells (Figure 4(b))

Endo

derm

Gut-like epithelium(a)

(b)

(c)

Gut-like epithelium

Mes

oder

m

Adipose tissue Muscle

EpidermisNeural tissue

Ecto

derm

Figure 1 Histological analysis of teratoma formation after trans-plantation of GFP-iPS cells Images of sections of teratomas formedafter transplantation of GFP-iPS cells into salivary glands of SCIDmice A teratoma formed from transplantation of 50 times 105 GFP-iPS cells HampE stained sections of a teratoma showing derivativesof all three germ layers including gut-like epithelium tissues ((a)endoderm) adipose tissues andmuscles ((b)mesoderm) andneuraltissues and keratin-containing epidermal tissues ((c) ectoderm)Scale bars 50 120583m

33 Morphological Analysis of Regenerated Salivary Glands(SG 5 iSG and 20 iSG) To investigate the effect of GFP-iPS cells on SMG cells the morphology of SG and iSG wasanalyzed (Figure 5(a))The size of regenerated salivary glandswas statistically evaluated There were no significant sizedifferences between SG 5 iSG and 20 iSG (Figure 5(b))The number of acinar-like aggregations in 5 iSG and 20iSGwasmore than that in SG (Figure 5(c))The size of acinar-like structures was significantly reduced in 5 iSG and 20iSG in comparison with that of SG (Figure 5(d))

34 Expression of Differentiation Markers in RegeneratedSalivary Glands (SG and iSG) To evaluate the differentiationof SMG cells embryonic stem cell markers and salivary glandmarkers were used for qPCR and for immunohistologicalanalysis Sox2 is known as a transcription factor involved

4 BioMed Research International

AmyPSPE-cadE-cadAmyPSP

WT

HESM

GSL

GSM

GSL

G

Tera

tom

a

AmyPSPE-cadE-cadAmyPSP HE(a)

(b)

Figure 2 Histological analysis of salivary gland-like tissue in teratomas formed after transplantation of GFP-iPS cells Immunofluorescenceand HampE staining of sections showed submandibular glands (SMGs) and sublingual glands (SLGs) of adult mouse PSP (green) Amy (blue)and E-cadherin (red) (a) Scale bars 25 120583m Immunofluorescence andHampE staining of sections of GFP-iPS-grafted teratoma revealed salivarygland-like tissue (b) Scale bars 25120583m

0h 96h24h

Figure 3 Aggregation of SMG cells in SG Phase-contrast image of regenerated SMG in organ culture for 0 24 and 96 h The regeneratedSMG contained many acinar-like structures Scale bar 05mm

BioMed Research International 5

GFPPCMPCMGFP

(a)

GFP E-cad GFPE-cad

(b)

Figure 4 Aggregation of SMG cells in iSG SMG cells and GFP-iPS cells were cocultured with DMEMF12 for 96 h Localization of GFP-iPS cells in iSG iSG consists of 20 iPS cells and 80 SMG cells Phase-contrast image and immunological stained image GFP-iPS (green)and pericentriolar material (PCM) (a) Scale bar 25 120583m Immunofluorescence of paraffin-embedded tissue of regenerated salivary glandsGFP-iPS (green) and E-cadherin (red) (b) Scale bar 25120583m

in regulating the self-renewal of embryonic stem cells Thedevelopment of the SMG is an ideal model to study thedifferential stage of regenerated salivary glands Sox2 wasexpressed both in the epithelium and mesenchyme of E135mouse SMGs and in the epithelium of E155 to E175 Sox2was gradually decreased from E175 to the adult stage and wasexpressed in the nucleus of ductal epithelium but not in thecytoplasm (Figure 6(a)) The expression of Sox2 was inducedin the cytoplasm of SG but decreased in the cytoplasm of iSG(Figure 6(b))

AQP5 is known as one of the salivary gland markers forfluid secretion AQP5 was expressed at a low level in theterminal bud and in the epithelial stalk of E135 and E155SMGs AQP5 was increased in the organized proacinar cellsof SMGs in E175 to adult (Figure 7(a)) SMG cells in SGexpressed AQP5 at the same level as E135 SMG HoweverSMG cells in iSG highly expressed AQP5 at the same level asthat in E175 SMG (Figure 7(b))

To analyze specific gene expression SMG cells from SGand iSG were isolated by a cell sorter The gene expressionof markers related to embryonic stem cell maintenance(Sox2 Nanog c-Myc and Klf4) and submandibular markers(Aqp5 Amy and M3r) was investigated by qPCR The geneexpression of Sox2 c-Myc and Nanog was decreased in iSGcompared with that in SG However Klf4 was increased iniSG (Figure 8(a)ndash8(d)) Aqp5 gene expression was higher iniSG than that in SG (Figure 8(e)) The expression of Amy andM3r was not detected by qPCR (Figures 8(f) and 8(g))

4 Discussion

Salivary glands have a potential ability to recover theirfunction over time [15] Several studies showed thatstemprogenitor cells in salivary glands restored salivation[16ndash18] Various kinds of cells have been used fortransplantation in regenerative medicine Many studiesshowed that the stem cell itself differentiates in the part ofthe organ where the function was restored Recent studiesrevealed that various kinds of cytokines secreted by stemcells are related to the mechanism of regeneration Thetransplantation of bone marrow stromal cells for spinal cordinjury produced extensive outgrowth of regenerating axonsassociated with Schwann cells through the extracellularmatrices [8 19] The restoration of morphology and functionwas induced by the combination of several factors such as theparacrine effect cell transdifferentiation and vasculogenesisby hematopoietic andor mesenchymal cells derived frombone marrow [20]

Many studies have tried to use iPS cells as a source ofthe transplant for treatment [21 22] However the functionof the microenvironment that iPS cells produce has not beeninvestigated yet In this study we examined the differentiationof salivary gland cells using iPS cells in a microenvironment

At first to confirm the pluripotency of iPS cells GFP-iPScells were transplanted into SCID mice Histological analysisof the produced teratoma demonstrated all three primarygerm layers It suggested that GFP-iPS cells had pluripotency

6 BioMed Research International

SG

PCM

GFP

E-c

ad

5 iSG 20 iSG

SG 5iSG iSG

20

20

0

10

05

15

The a

rea o

f a re

gene

rate

dsa

livar

y gl

and

(mm

2) 80

0

40

20

60

SG 5iSG iSG

20

The n

umbe

r of a

n ac

inar

-like

struc

ture

(m

m2)

lowastlowast

lowast

80

0

40

20

60

The d

iam

eter

of a

n ac

inar

-like

struc

ture

(120583m

2)

lowastlowast

lowast

SG 5iSG iSG

20

(a)

(b) (c) (d)

Figure 5 Morphological analysis of regenerated SMGs (SG 5 iSG and 20 iSG) PCM and immunostained images indicated a reductionin size and an increase in the number of acinar-like structures in regenerated salivary glands (a) Size of whole regenerated salivary glands(119899 = 3) (b) Number of acinar-like structures in regenerated salivary glands (119899 = 3) (c) Size of acinar-like structures in regenerated salivaryglands (119899 = 24) (d) lowast119901 lt 005 lowastlowast119901 lt 001 versus control

for differentiation Furthermore teratomas contained twokinds of glandular tissueswhichwere similar to both the SMGand the SLG comparedwith the pattern of the salivary glandsin adult mice [23] GFP-iPS cells have a potential ability toregenerate SMG and SLG cells However only few parts ofthe tissues differentiated from salivary glands The potentialtumorigenicity of iPS cells has to be considered prior toclinical application

The embryonic SMG is a remarkably adaptable tissueand dissociated SMG cells can self-assemble in serum-free

medium [24] The cell aggregation assay is a useful methodfor studying mechanisms of tissue assembly as the structureof the regenerated glands is similar to salivary glands [25 26]Embryonic SMG cells and GFP-iPS cells were cocultured toidentify the roles of the microenvironment around the iPScells Regenerated salivary glands (SG and iSG) formedmanyacinar-like structures similar to embryonic salivary glands for96 h in vitro [27] It is very difficult for our conventional organculture without serum and other factors to grow salivarygland in vitro for longer time such as several weeks So

BioMed Research International 7

E135 E175E155 Ad

Sox2

Sox2

E-c

ad

E-ca

d

iSGSG

Sox2

Sox2

GFP

E-c

ad

(a)

(b)

Figure 6 Distribution of Sox2 in developing SMG and in regenerated SMGs (SG and iSG) Sox2 is expressed in the cytoplasm of bothepithelial cells and mesenchymal cells in E135 E155 and E175 developing SMGs and is gradually decreased in later embryonic stages Sox2is expressed in the nucleus of epithelial cells in both postnatal and adult SMGs Sox2 (cyan) and E-cadherin (red) (a) Scale bar 25 120583m Sox2expression in the cytoplasm of both epithelial cells and mesenchymal cells in iSG (20 iSG) is less than that in SG The nucleus of GFP-iPScells highly expressed Sox2 as a positive control Sox2 (cyan) GFP (green) and E-cadherin (red) (b) Scale bar 25 120583m

8 BioMed Research International

AQP5

AQP5

E-c

ad

E-ca

d

E135 E175E155 Ad

iSGSG(a)

(b)

AQP5

AQP5

GFP

E-c

ad

Figure 7 Distribution of AQP5 in developing SMG and regenerated SMGs (SG and iSG) AQP5 is gradually increased in the epithelial cellsof SMGs in E135 E155 E175 and adult AQP5 is expressed in the cell membrane of epithelial cells in iSG (20 iSG) more than that in SGAQP5 (white) and E-cadherin (red) (a) Scale bar 25 120583m Distribution on AQP5 in regenerated SMG (b) AQP5 (white) GFP (green) andE-cadherin (red) Scale bar 25 120583m

BioMed Research International 9

Sox2

SG iSG

100

0

60

40

80

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

c-Myc

100

0

60

40

80

lowastlowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

Nanog

100

0

60

40

80

lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG0

200

100

Klf4lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

AQP5

0

200

100

300

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

Amy

Not detected

Relat

ive e

xpre

ssio

n (

)

(a) (b) (c) (d)

(e) (f) (g)

SG iSG

M3R

Not detected

Relat

ive e

xpre

ssio

n (

)

Figure 8 Gene expression of salivary gland cells in regenerated SMGs between SG and iSG Gene expression of Sox2 (a) c-Myc (b) andNanog (c) was decreased in iSG and gene expression of Klf4 (d) was increased in iSG Aqp5 (e) gene expression was increased in iSG butAmy (f) andM3r (g) gene expression were not detected by qPCR similar to early embryonic SMG cells lowastlowast119901 lt 001 versus control

salivary gland cells needed to be transplanted to mouse [2829]

Because the water channel protein AQP5 is found inthe lumen of the acinar-like structures regenerated sali-vary glands may have an ability to secrete saliva [30 31]Morphological analysis of the regenerated salivary glandsindicated that the difference between SG and iSG was notstatistically significant However iSG had a larger number ofsmall acinar-like structures more than that in SG (Figure 9)Developing salivary glands in epithelial tissue increases thesurface area by branching morphogenesis and increases theability of saliva secretion [32ndash34] Coculture of embryonicSMG cells and iPS cells had more developed epithelialstructures than that in monoculture of embryonic SMG cells

Previous studies show the gene expression of stem cellmarkers (Sox2 c-Myc Nanog and Klf4) in E13 E15 and adult

(Ad) SMGs Sox2 c-Myc and Nanog decreased during SMGdevelopment but Klf4 increased [16] The submandibularmarker AQP5 belongs to a family of water channel proteinsthat allow water to pass through the plasma membrane byosmosis E14 SMG expressed AQP5 as a first indication [31]Our results showed that the gene expressions of Sox2 c-Myc and Nanog were decreased in iSG and that the geneexpression of Klf4 and Aqp5 was increased in iSG Immuno-histochemical analysis also showed that the undifferentiatedmarker of salivary gland cells decreased in iSG comparedwith that in SG It was suggested that iPS cells induce thecharacteristic differentiation resulting in the morphologicalchanges observed in salivary gland cell formation Theseresults indicate that iPS cells have a potential ability toaccelerate differentiation of salivary gland development andregeneration Regenerated salivary glands produced from

10 BioMed Research International

iPS cell

Acinar-like structure

iSGSGRegenerated salivary gland

Sox2 c-Myc NanogAqp5

Figure 9 Effect of iPS cells on aggregation of SMG cells iPS cells(green balls) reduced the size of the epithelial tissue but acinar-likestructure (orange balls) increased their number iPS cells cannotmixcompletely with SMG cells and instead surround the epitheliumof SMG Stem cell markers such as Sox2 c-Myc and Nanog aredecreased but Aqp5 is increased in iSG

E135 SMG cells were cultured for four daysAmy andM3r areusually expressed after birth Amy and M3r gene expressionalso occurred in the regenerated salivary gland cells

Various approaches would be necessary to organize thecomplex tissues such as the salivary gland kidney and lungOur finding that coculture of salivary glands cells with iPScells formed differentiated salivary glands is significant andfuture elucidation of the mechanism could lead to viableregeneration therapy of functional organs using iPS cellsOur study provides new insights for future research into theregeneration of organs such as salivary glands

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HitomiOno andAyaObana contributed equally to this work

Acknowledgments

This work was supported by JSPS KAKENHI Grant no24390452 The authors thank Dr Kenji Mishima Division ofPathology Department of Oral Diagnostic Sciences Schoolof Dentistry Showa University for helpful advice regard-ing salivary markers The authors also thank Dr HirokoOkawa Department of Fixed Prosthodontics Osaka Uni-versity Graduate School of Dentistry for excellent technicalsupport in iPS cell cultures

References

[1] S P Humphrey and R T Williamson ldquoA review of salivanormal composition flow and functionrdquo Journal of ProstheticDentistry vol 85 no 2 pp 162ndash169 2001

[2] S B Jensen A M L Pedersen A Vissink et al ldquoA systematicreview of salivary gland hypofunction and xerostomia inducedby cancer therapies management strategies and economicimpactrdquo Supportive Care in Cancer vol 18 no 8 pp 1061ndash10792010

[3] J-Y Lim J C Ra I S Shin et al ldquoSystemic transplantationof human adipose tissue-derived mesenchymal stem cells forthe regeneration of irradiation-induced salivary gland damagerdquoPLoS ONE vol 8 no 8 Article ID e71167 2013

[4] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007

[5] H Kamao M Mandai S Okamoto et al ldquoCharacterization ofhuman induced pluripotent stem cell-derived retinal pigmentepithelium cell sheets aiming for clinical applicationrdquo Stem CellReports vol 2 no 2 pp 205ndash218 2014

[6] H Egusa K Okita H Kayashima et al ldquoGingival fibroblastsas a promising source of induced pluripotent stem cellsrdquo PLoSONE vol 5 no 9 Article ID e12743 12 pages 2010

[7] S C Bendall M H Stewart P Menendez et al ldquoIGF andFGF cooperatively establish the regulatory stem cell niche ofpluripotent human cells in vitrordquo Nature vol 448 no 7157 pp1015ndash1021 2007

[8] C Ide Y Nakai N Nakano et al ldquoBone marrow stromal celltransplantation for treatment of sub-acute spinal cord injury inthe ratrdquo Brain Research vol 1332 pp 32ndash47 2010

[9] T Takebe K SekineM Enomura et al ldquoVascularized and func-tional human liver from an iPSC-derived organ bud transplantrdquoNature vol 499 no 7459 pp 481ndash484 2013

[10] M Eiraku K Watanabe M Matsuo-Takasaki et al ldquoSelf-organized formation of polarized cortical tissues fromESCs andits active manipulation by extrinsic signalsrdquo Cell Stem Cell vol3 no 5 pp 519ndash532 2008

[11] M Kawakami H Ishikawa T Tachibana A Tanaka andI Mataga ldquoFunctional transplantation of salivary gland cellsdifferentiated from mouse early ES cells in vitrordquo Human Cellvol 26 no 2 pp 80ndash90 2013

[12] K Mishima H Inoue T Nishiyama et al ldquoTransplantation ofside population cells restores the function of damaged exocrineglands through clusterinrdquo Stem Cells vol 30 no 9 pp 1925ndash1937 2012

[13] J Mima A Koshino K Oka et al ldquoRegulation of the epithelialadhesion molecule CEACAM1 is important for palate forma-tionrdquo PLoS ONE vol 8 no 4 Article ID e61653 2013

[14] V N Pateland andM PHoffman ldquoSalivary gland developmenta template for regenerationrdquo Seminars in Cell amp DevelopmentalBiology vol 25-26 pp 52ndash60 2014

[15] S B Jensen A M Pedersen J Reibel and B NauntofteldquoXerostomia and hypofunction of the salivary glands in cancertherapyrdquo Supportive Care in Cancer vol 11 no 4 pp 207ndash2252003

[16] I M A Lombaert and M P Hoffman ldquoEpithelialstemprogenitor cells in the embryonic mouse submandibularglandrdquo Frontiers of Oral Biology vol 14 pp 90ndash106 2010

[17] A Angelova Volponi M Kawasaki and P T Sharpe ldquoAdulthuman gingival epithelial cells as a source for whole-toothbioengineeringrdquo Journal of Dental Research vol 92 no 4 pp329ndash334 2013

[18] A V Vanikar H L Trivedi S C Gopal A Kumar and SD Dave ldquoPre-transplant co-infusion of donor-adipose tissuederived mesenchymal stem cells and hematopoietic stem cells

BioMed Research International 11

may help in achieving tolerance in living donor renal transplan-tationrdquo Renal Failure vol 36 no 3 pp 457ndash460 2014

[19] J P M Issa R Tiossi A S D S Mello R A Lopes M A S DiMatteo and M M Iyomasa ldquoPRP a possibility in regenerativetherapyrdquo International Journal of Morphology vol 25 no 3 pp587ndash590 2007

[20] Y Sumita Y Liu S Khalili et al ldquoBone marrow-derived cellsrescue salivary gland function in mice with head and neckirradiationrdquo The International Journal of Biochemistry amp CellBiology vol 43 no 1 pp 80ndash87 2011

[21] B Song A M Smink C V Jones et al ldquoThe directeddifferentiation of human iPS cells into kidney podocytesrdquo PLoSONE vol 7 no 9 Article ID e46453 2012

[22] H Saito M Takeuchi K Chida and A Miyajima ldquoGenerationof glucose-responsive functional islets with a three-dimensionalstructure frommouse fetal pancreatic cells and ips cells in vitrordquoPLoS ONE vol 6 no 12 Article ID e28209 2011

[23] D A Nelson C Manhardt V Kamath et al ldquoQuantitativesingle cell analysis of cell population dynamics during sub-mandibular salivary gland development and differentiationrdquoBiology Open vol 2 no 5 pp 439ndash447 2013

[24] C Wei M Larsen M P Hoffman and K M Yamada ldquoSelf-organization and branchingmorphogenesis of primary salivaryepithelial cellsrdquo Tissue Engineering vol 13 no 4 pp 721ndash7352007

[25] TMisteli ldquoThe concept of self-organization in cellular architec-turerdquo Journal of Cell Biology vol 155 no 2 pp 181ndash185 2001

[26] L S Y Nanduri I M A Lombaert M van der Zwaaget al ldquoSalisphere derived c-Kit+ cell transplantation restorestissue homeostasis in irradiated salivary glandrdquo Radiotherapy ampOncology vol 108 no 3 pp 458ndash463 2013

[27] J C-F Hsu and K M Yamada ldquoSalivary gland branchingmorphogenesismdashrecent progress and future opportunitiesrdquoInternational Journal of Oral Science vol 2 no 3 pp 117ndash1262010

[28] MHirayamaMOgawaMOshima et al ldquoFunctional lacrimalgland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2497 2013

[29] M Ogawa M Oshima A Imamura et al ldquoFunctional salivarygland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2498 2013

[30] K Ishibashi S Hara and S Kondo ldquoAquaporin water channelsin mammalsrdquo Clinical and Experimental Nephrology vol 13 no2 pp 107ndash117 2009

[31] H S Larsen M H Aure S B Peters M Larsen E BMesselt and H Kanli Galtung ldquoLocalization of AQP5 duringdevelopment of the mouse submandibular salivary glandrdquoJournal of Molecular Histology vol 42 no 1 pp 71ndash81 2011

[32] J E Fata Z Werb and M J Bissell ldquoRegulation of mammarygland branchingmorphogenesis by the extracellular matrix andits remodeling enzymesrdquo Breast Cancer Research vol 6 no 1pp 1ndash11 2004

[33] T Sakai M Larsen and K M Yamada ldquoFibronectin require-ment in branching morphogenesisrdquo Nature vol 423 no 6942pp 876ndash881 2003

[34] T Onodera T Sakai J C-F Hsu K Matsumoto J A ChioriniandKMYamada ldquoBtbd7 regulates epithelial cell dynamics andbranching morphogenesisrdquo Science vol 329 no 5991 pp 562ndash565 2010

BioMed Research International 3

20 s Quantitative real-time reverse transcription-polymerasechain reaction (qPCR) results for each sample were normal-ized by glyceraldehyde-3-phosphate dehydrogenase (Gapdh)The results were expressed as normalized ratios and experi-ments were repeated three times The primer sequences usedwere as follows

Sox2 51015840-GGGAAATGGGAGGGGTGCAAAAGAGG-3101584051015840-TTGCGTGAGTGTGGATGGGATTGGTG-31015840

c-Myc 51015840-CAGAGGAGGAACGAGCTGAAGCGC-3101584051015840-TTATGCACCAGAGTTTCGAAGCTGTTCG-31015840

Klf4 51015840-CACCATGGACCCGGGCGTGGCTGCCAGAAA-3101584051015840-TTAGGCTGTTCTTTTCCGGGGCCACGA-31015840

Aqp5 51015840-TGGAGCAGGCATCCTGTACT-3101584051015840-CGTGGAGGAGAAGATGCAGA-31015840

Amy 51015840-GGATGGAGAAAAGATGTCCTAC-3101584051015840-CATCACCCGTGTGAAACC-31015840

M3r 51015840-TCGGTAGAGCGGACTGGACA-3101584051015840-TCCACTGAGCAAGTCAGAAGTGAAG-31015840

Gapdh 51015840-CCATCACCATCTTCCAGGAG-3101584051015840-GCATGGACTGTGGTCATGAG-31015840

28 Statistical Analysis Statistical analysis was performed asindicated in the figure legends and text One-way analysis ofthe 119905-test was performed to determine the statistical differ-ence between the means of each treatment for experimentsin which multiple treatments were compared

3 Results

31 Teratoma Formation in SMG after Transplantation ofMouse GFP-iPS Cells The pluripotency of GFP-iPS cellswas validated by teratoma formation in the SMG of SCIDmice Four weeks after implantation histological analysisdemonstrated that the formed teratomas were derived fromall three primary germ layers Gut-like epithelium (endo-derm) adipose tissues (mesoderm) muscles (mesoderm)neural tissues (ectoderm) and epidermis (ectoderm) wereall identified histologically in the GFP-iPS-derived teratomas(Figure 1)The teratomas contained different types of salivarygland-like tissues By immunohistochemical analysis onewas similar to the SMG and the other was similar to thepattern of the SLG in adult mice (Figure 2)

32 Localization of iPS Cells in Regenerated SMGs (SGand iSG) In SMG monoculture the epithelial cells aggre-gated and formed salivary gland-like tissue (SG) after 96 h(Figure 3) Coculture of SMG cells and GFP-iPS cells (iSG)also formed many acinar-like structures similar to the SGIn immunohistochemical analysis GFP-iPS cells did notuniformly mix with SMG cells (Figure 4(a)) GFP-iPS cellsexisted around acinar-like epithelial cells (Figure 4(b))

Endo

derm

Gut-like epithelium(a)

(b)

(c)

Gut-like epithelium

Mes

oder

m

Adipose tissue Muscle

EpidermisNeural tissue

Ecto

derm

Figure 1 Histological analysis of teratoma formation after trans-plantation of GFP-iPS cells Images of sections of teratomas formedafter transplantation of GFP-iPS cells into salivary glands of SCIDmice A teratoma formed from transplantation of 50 times 105 GFP-iPS cells HampE stained sections of a teratoma showing derivativesof all three germ layers including gut-like epithelium tissues ((a)endoderm) adipose tissues andmuscles ((b)mesoderm) andneuraltissues and keratin-containing epidermal tissues ((c) ectoderm)Scale bars 50 120583m

33 Morphological Analysis of Regenerated Salivary Glands(SG 5 iSG and 20 iSG) To investigate the effect of GFP-iPS cells on SMG cells the morphology of SG and iSG wasanalyzed (Figure 5(a))The size of regenerated salivary glandswas statistically evaluated There were no significant sizedifferences between SG 5 iSG and 20 iSG (Figure 5(b))The number of acinar-like aggregations in 5 iSG and 20iSGwasmore than that in SG (Figure 5(c))The size of acinar-like structures was significantly reduced in 5 iSG and 20iSG in comparison with that of SG (Figure 5(d))

34 Expression of Differentiation Markers in RegeneratedSalivary Glands (SG and iSG) To evaluate the differentiationof SMG cells embryonic stem cell markers and salivary glandmarkers were used for qPCR and for immunohistologicalanalysis Sox2 is known as a transcription factor involved

4 BioMed Research International

AmyPSPE-cadE-cadAmyPSP

WT

HESM

GSL

GSM

GSL

G

Tera

tom

a

AmyPSPE-cadE-cadAmyPSP HE(a)

(b)

Figure 2 Histological analysis of salivary gland-like tissue in teratomas formed after transplantation of GFP-iPS cells Immunofluorescenceand HampE staining of sections showed submandibular glands (SMGs) and sublingual glands (SLGs) of adult mouse PSP (green) Amy (blue)and E-cadherin (red) (a) Scale bars 25 120583m Immunofluorescence andHampE staining of sections of GFP-iPS-grafted teratoma revealed salivarygland-like tissue (b) Scale bars 25120583m

0h 96h24h

Figure 3 Aggregation of SMG cells in SG Phase-contrast image of regenerated SMG in organ culture for 0 24 and 96 h The regeneratedSMG contained many acinar-like structures Scale bar 05mm

BioMed Research International 5

GFPPCMPCMGFP

(a)

GFP E-cad GFPE-cad

(b)

Figure 4 Aggregation of SMG cells in iSG SMG cells and GFP-iPS cells were cocultured with DMEMF12 for 96 h Localization of GFP-iPS cells in iSG iSG consists of 20 iPS cells and 80 SMG cells Phase-contrast image and immunological stained image GFP-iPS (green)and pericentriolar material (PCM) (a) Scale bar 25 120583m Immunofluorescence of paraffin-embedded tissue of regenerated salivary glandsGFP-iPS (green) and E-cadherin (red) (b) Scale bar 25120583m

in regulating the self-renewal of embryonic stem cells Thedevelopment of the SMG is an ideal model to study thedifferential stage of regenerated salivary glands Sox2 wasexpressed both in the epithelium and mesenchyme of E135mouse SMGs and in the epithelium of E155 to E175 Sox2was gradually decreased from E175 to the adult stage and wasexpressed in the nucleus of ductal epithelium but not in thecytoplasm (Figure 6(a)) The expression of Sox2 was inducedin the cytoplasm of SG but decreased in the cytoplasm of iSG(Figure 6(b))

AQP5 is known as one of the salivary gland markers forfluid secretion AQP5 was expressed at a low level in theterminal bud and in the epithelial stalk of E135 and E155SMGs AQP5 was increased in the organized proacinar cellsof SMGs in E175 to adult (Figure 7(a)) SMG cells in SGexpressed AQP5 at the same level as E135 SMG HoweverSMG cells in iSG highly expressed AQP5 at the same level asthat in E175 SMG (Figure 7(b))

To analyze specific gene expression SMG cells from SGand iSG were isolated by a cell sorter The gene expressionof markers related to embryonic stem cell maintenance(Sox2 Nanog c-Myc and Klf4) and submandibular markers(Aqp5 Amy and M3r) was investigated by qPCR The geneexpression of Sox2 c-Myc and Nanog was decreased in iSGcompared with that in SG However Klf4 was increased iniSG (Figure 8(a)ndash8(d)) Aqp5 gene expression was higher iniSG than that in SG (Figure 8(e)) The expression of Amy andM3r was not detected by qPCR (Figures 8(f) and 8(g))

4 Discussion

Salivary glands have a potential ability to recover theirfunction over time [15] Several studies showed thatstemprogenitor cells in salivary glands restored salivation[16ndash18] Various kinds of cells have been used fortransplantation in regenerative medicine Many studiesshowed that the stem cell itself differentiates in the part ofthe organ where the function was restored Recent studiesrevealed that various kinds of cytokines secreted by stemcells are related to the mechanism of regeneration Thetransplantation of bone marrow stromal cells for spinal cordinjury produced extensive outgrowth of regenerating axonsassociated with Schwann cells through the extracellularmatrices [8 19] The restoration of morphology and functionwas induced by the combination of several factors such as theparacrine effect cell transdifferentiation and vasculogenesisby hematopoietic andor mesenchymal cells derived frombone marrow [20]

Many studies have tried to use iPS cells as a source ofthe transplant for treatment [21 22] However the functionof the microenvironment that iPS cells produce has not beeninvestigated yet In this study we examined the differentiationof salivary gland cells using iPS cells in a microenvironment

At first to confirm the pluripotency of iPS cells GFP-iPScells were transplanted into SCID mice Histological analysisof the produced teratoma demonstrated all three primarygerm layers It suggested that GFP-iPS cells had pluripotency

6 BioMed Research International

SG

PCM

GFP

E-c

ad

5 iSG 20 iSG

SG 5iSG iSG

20

20

0

10

05

15

The a

rea o

f a re

gene

rate

dsa

livar

y gl

and

(mm

2) 80

0

40

20

60

SG 5iSG iSG

20

The n

umbe

r of a

n ac

inar

-like

struc

ture

(m

m2)

lowastlowast

lowast

80

0

40

20

60

The d

iam

eter

of a

n ac

inar

-like

struc

ture

(120583m

2)

lowastlowast

lowast

SG 5iSG iSG

20

(a)

(b) (c) (d)

Figure 5 Morphological analysis of regenerated SMGs (SG 5 iSG and 20 iSG) PCM and immunostained images indicated a reductionin size and an increase in the number of acinar-like structures in regenerated salivary glands (a) Size of whole regenerated salivary glands(119899 = 3) (b) Number of acinar-like structures in regenerated salivary glands (119899 = 3) (c) Size of acinar-like structures in regenerated salivaryglands (119899 = 24) (d) lowast119901 lt 005 lowastlowast119901 lt 001 versus control

for differentiation Furthermore teratomas contained twokinds of glandular tissueswhichwere similar to both the SMGand the SLG comparedwith the pattern of the salivary glandsin adult mice [23] GFP-iPS cells have a potential ability toregenerate SMG and SLG cells However only few parts ofthe tissues differentiated from salivary glands The potentialtumorigenicity of iPS cells has to be considered prior toclinical application

The embryonic SMG is a remarkably adaptable tissueand dissociated SMG cells can self-assemble in serum-free

medium [24] The cell aggregation assay is a useful methodfor studying mechanisms of tissue assembly as the structureof the regenerated glands is similar to salivary glands [25 26]Embryonic SMG cells and GFP-iPS cells were cocultured toidentify the roles of the microenvironment around the iPScells Regenerated salivary glands (SG and iSG) formedmanyacinar-like structures similar to embryonic salivary glands for96 h in vitro [27] It is very difficult for our conventional organculture without serum and other factors to grow salivarygland in vitro for longer time such as several weeks So

BioMed Research International 7

E135 E175E155 Ad

Sox2

Sox2

E-c

ad

E-ca

d

iSGSG

Sox2

Sox2

GFP

E-c

ad

(a)

(b)

Figure 6 Distribution of Sox2 in developing SMG and in regenerated SMGs (SG and iSG) Sox2 is expressed in the cytoplasm of bothepithelial cells and mesenchymal cells in E135 E155 and E175 developing SMGs and is gradually decreased in later embryonic stages Sox2is expressed in the nucleus of epithelial cells in both postnatal and adult SMGs Sox2 (cyan) and E-cadherin (red) (a) Scale bar 25 120583m Sox2expression in the cytoplasm of both epithelial cells and mesenchymal cells in iSG (20 iSG) is less than that in SG The nucleus of GFP-iPScells highly expressed Sox2 as a positive control Sox2 (cyan) GFP (green) and E-cadherin (red) (b) Scale bar 25 120583m

8 BioMed Research International

AQP5

AQP5

E-c

ad

E-ca

d

E135 E175E155 Ad

iSGSG(a)

(b)

AQP5

AQP5

GFP

E-c

ad

Figure 7 Distribution of AQP5 in developing SMG and regenerated SMGs (SG and iSG) AQP5 is gradually increased in the epithelial cellsof SMGs in E135 E155 E175 and adult AQP5 is expressed in the cell membrane of epithelial cells in iSG (20 iSG) more than that in SGAQP5 (white) and E-cadherin (red) (a) Scale bar 25 120583m Distribution on AQP5 in regenerated SMG (b) AQP5 (white) GFP (green) andE-cadherin (red) Scale bar 25 120583m

BioMed Research International 9

Sox2

SG iSG

100

0

60

40

80

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

c-Myc

100

0

60

40

80

lowastlowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

Nanog

100

0

60

40

80

lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG0

200

100

Klf4lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

AQP5

0

200

100

300

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

Amy

Not detected

Relat

ive e

xpre

ssio

n (

)

(a) (b) (c) (d)

(e) (f) (g)

SG iSG

M3R

Not detected

Relat

ive e

xpre

ssio

n (

)

Figure 8 Gene expression of salivary gland cells in regenerated SMGs between SG and iSG Gene expression of Sox2 (a) c-Myc (b) andNanog (c) was decreased in iSG and gene expression of Klf4 (d) was increased in iSG Aqp5 (e) gene expression was increased in iSG butAmy (f) andM3r (g) gene expression were not detected by qPCR similar to early embryonic SMG cells lowastlowast119901 lt 001 versus control

salivary gland cells needed to be transplanted to mouse [2829]

Because the water channel protein AQP5 is found inthe lumen of the acinar-like structures regenerated sali-vary glands may have an ability to secrete saliva [30 31]Morphological analysis of the regenerated salivary glandsindicated that the difference between SG and iSG was notstatistically significant However iSG had a larger number ofsmall acinar-like structures more than that in SG (Figure 9)Developing salivary glands in epithelial tissue increases thesurface area by branching morphogenesis and increases theability of saliva secretion [32ndash34] Coculture of embryonicSMG cells and iPS cells had more developed epithelialstructures than that in monoculture of embryonic SMG cells

Previous studies show the gene expression of stem cellmarkers (Sox2 c-Myc Nanog and Klf4) in E13 E15 and adult

(Ad) SMGs Sox2 c-Myc and Nanog decreased during SMGdevelopment but Klf4 increased [16] The submandibularmarker AQP5 belongs to a family of water channel proteinsthat allow water to pass through the plasma membrane byosmosis E14 SMG expressed AQP5 as a first indication [31]Our results showed that the gene expressions of Sox2 c-Myc and Nanog were decreased in iSG and that the geneexpression of Klf4 and Aqp5 was increased in iSG Immuno-histochemical analysis also showed that the undifferentiatedmarker of salivary gland cells decreased in iSG comparedwith that in SG It was suggested that iPS cells induce thecharacteristic differentiation resulting in the morphologicalchanges observed in salivary gland cell formation Theseresults indicate that iPS cells have a potential ability toaccelerate differentiation of salivary gland development andregeneration Regenerated salivary glands produced from

10 BioMed Research International

iPS cell

Acinar-like structure

iSGSGRegenerated salivary gland

Sox2 c-Myc NanogAqp5

Figure 9 Effect of iPS cells on aggregation of SMG cells iPS cells(green balls) reduced the size of the epithelial tissue but acinar-likestructure (orange balls) increased their number iPS cells cannotmixcompletely with SMG cells and instead surround the epitheliumof SMG Stem cell markers such as Sox2 c-Myc and Nanog aredecreased but Aqp5 is increased in iSG

E135 SMG cells were cultured for four daysAmy andM3r areusually expressed after birth Amy and M3r gene expressionalso occurred in the regenerated salivary gland cells

Various approaches would be necessary to organize thecomplex tissues such as the salivary gland kidney and lungOur finding that coculture of salivary glands cells with iPScells formed differentiated salivary glands is significant andfuture elucidation of the mechanism could lead to viableregeneration therapy of functional organs using iPS cellsOur study provides new insights for future research into theregeneration of organs such as salivary glands

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HitomiOno andAyaObana contributed equally to this work

Acknowledgments

This work was supported by JSPS KAKENHI Grant no24390452 The authors thank Dr Kenji Mishima Division ofPathology Department of Oral Diagnostic Sciences Schoolof Dentistry Showa University for helpful advice regard-ing salivary markers The authors also thank Dr HirokoOkawa Department of Fixed Prosthodontics Osaka Uni-versity Graduate School of Dentistry for excellent technicalsupport in iPS cell cultures

References

[1] S P Humphrey and R T Williamson ldquoA review of salivanormal composition flow and functionrdquo Journal of ProstheticDentistry vol 85 no 2 pp 162ndash169 2001

[2] S B Jensen A M L Pedersen A Vissink et al ldquoA systematicreview of salivary gland hypofunction and xerostomia inducedby cancer therapies management strategies and economicimpactrdquo Supportive Care in Cancer vol 18 no 8 pp 1061ndash10792010

[3] J-Y Lim J C Ra I S Shin et al ldquoSystemic transplantationof human adipose tissue-derived mesenchymal stem cells forthe regeneration of irradiation-induced salivary gland damagerdquoPLoS ONE vol 8 no 8 Article ID e71167 2013

[4] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007

[5] H Kamao M Mandai S Okamoto et al ldquoCharacterization ofhuman induced pluripotent stem cell-derived retinal pigmentepithelium cell sheets aiming for clinical applicationrdquo Stem CellReports vol 2 no 2 pp 205ndash218 2014

[6] H Egusa K Okita H Kayashima et al ldquoGingival fibroblastsas a promising source of induced pluripotent stem cellsrdquo PLoSONE vol 5 no 9 Article ID e12743 12 pages 2010

[7] S C Bendall M H Stewart P Menendez et al ldquoIGF andFGF cooperatively establish the regulatory stem cell niche ofpluripotent human cells in vitrordquo Nature vol 448 no 7157 pp1015ndash1021 2007

[8] C Ide Y Nakai N Nakano et al ldquoBone marrow stromal celltransplantation for treatment of sub-acute spinal cord injury inthe ratrdquo Brain Research vol 1332 pp 32ndash47 2010

[9] T Takebe K SekineM Enomura et al ldquoVascularized and func-tional human liver from an iPSC-derived organ bud transplantrdquoNature vol 499 no 7459 pp 481ndash484 2013

[10] M Eiraku K Watanabe M Matsuo-Takasaki et al ldquoSelf-organized formation of polarized cortical tissues fromESCs andits active manipulation by extrinsic signalsrdquo Cell Stem Cell vol3 no 5 pp 519ndash532 2008

[11] M Kawakami H Ishikawa T Tachibana A Tanaka andI Mataga ldquoFunctional transplantation of salivary gland cellsdifferentiated from mouse early ES cells in vitrordquo Human Cellvol 26 no 2 pp 80ndash90 2013

[12] K Mishima H Inoue T Nishiyama et al ldquoTransplantation ofside population cells restores the function of damaged exocrineglands through clusterinrdquo Stem Cells vol 30 no 9 pp 1925ndash1937 2012

[13] J Mima A Koshino K Oka et al ldquoRegulation of the epithelialadhesion molecule CEACAM1 is important for palate forma-tionrdquo PLoS ONE vol 8 no 4 Article ID e61653 2013

[14] V N Pateland andM PHoffman ldquoSalivary gland developmenta template for regenerationrdquo Seminars in Cell amp DevelopmentalBiology vol 25-26 pp 52ndash60 2014

[15] S B Jensen A M Pedersen J Reibel and B NauntofteldquoXerostomia and hypofunction of the salivary glands in cancertherapyrdquo Supportive Care in Cancer vol 11 no 4 pp 207ndash2252003

[16] I M A Lombaert and M P Hoffman ldquoEpithelialstemprogenitor cells in the embryonic mouse submandibularglandrdquo Frontiers of Oral Biology vol 14 pp 90ndash106 2010

[17] A Angelova Volponi M Kawasaki and P T Sharpe ldquoAdulthuman gingival epithelial cells as a source for whole-toothbioengineeringrdquo Journal of Dental Research vol 92 no 4 pp329ndash334 2013

[18] A V Vanikar H L Trivedi S C Gopal A Kumar and SD Dave ldquoPre-transplant co-infusion of donor-adipose tissuederived mesenchymal stem cells and hematopoietic stem cells

BioMed Research International 11

may help in achieving tolerance in living donor renal transplan-tationrdquo Renal Failure vol 36 no 3 pp 457ndash460 2014

[19] J P M Issa R Tiossi A S D S Mello R A Lopes M A S DiMatteo and M M Iyomasa ldquoPRP a possibility in regenerativetherapyrdquo International Journal of Morphology vol 25 no 3 pp587ndash590 2007

[20] Y Sumita Y Liu S Khalili et al ldquoBone marrow-derived cellsrescue salivary gland function in mice with head and neckirradiationrdquo The International Journal of Biochemistry amp CellBiology vol 43 no 1 pp 80ndash87 2011

[21] B Song A M Smink C V Jones et al ldquoThe directeddifferentiation of human iPS cells into kidney podocytesrdquo PLoSONE vol 7 no 9 Article ID e46453 2012

[22] H Saito M Takeuchi K Chida and A Miyajima ldquoGenerationof glucose-responsive functional islets with a three-dimensionalstructure frommouse fetal pancreatic cells and ips cells in vitrordquoPLoS ONE vol 6 no 12 Article ID e28209 2011

[23] D A Nelson C Manhardt V Kamath et al ldquoQuantitativesingle cell analysis of cell population dynamics during sub-mandibular salivary gland development and differentiationrdquoBiology Open vol 2 no 5 pp 439ndash447 2013

[24] C Wei M Larsen M P Hoffman and K M Yamada ldquoSelf-organization and branchingmorphogenesis of primary salivaryepithelial cellsrdquo Tissue Engineering vol 13 no 4 pp 721ndash7352007

[25] TMisteli ldquoThe concept of self-organization in cellular architec-turerdquo Journal of Cell Biology vol 155 no 2 pp 181ndash185 2001

[26] L S Y Nanduri I M A Lombaert M van der Zwaaget al ldquoSalisphere derived c-Kit+ cell transplantation restorestissue homeostasis in irradiated salivary glandrdquo Radiotherapy ampOncology vol 108 no 3 pp 458ndash463 2013

[27] J C-F Hsu and K M Yamada ldquoSalivary gland branchingmorphogenesismdashrecent progress and future opportunitiesrdquoInternational Journal of Oral Science vol 2 no 3 pp 117ndash1262010

[28] MHirayamaMOgawaMOshima et al ldquoFunctional lacrimalgland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2497 2013

[29] M Ogawa M Oshima A Imamura et al ldquoFunctional salivarygland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2498 2013

[30] K Ishibashi S Hara and S Kondo ldquoAquaporin water channelsin mammalsrdquo Clinical and Experimental Nephrology vol 13 no2 pp 107ndash117 2009

[31] H S Larsen M H Aure S B Peters M Larsen E BMesselt and H Kanli Galtung ldquoLocalization of AQP5 duringdevelopment of the mouse submandibular salivary glandrdquoJournal of Molecular Histology vol 42 no 1 pp 71ndash81 2011

[32] J E Fata Z Werb and M J Bissell ldquoRegulation of mammarygland branchingmorphogenesis by the extracellular matrix andits remodeling enzymesrdquo Breast Cancer Research vol 6 no 1pp 1ndash11 2004

[33] T Sakai M Larsen and K M Yamada ldquoFibronectin require-ment in branching morphogenesisrdquo Nature vol 423 no 6942pp 876ndash881 2003

[34] T Onodera T Sakai J C-F Hsu K Matsumoto J A ChioriniandKMYamada ldquoBtbd7 regulates epithelial cell dynamics andbranching morphogenesisrdquo Science vol 329 no 5991 pp 562ndash565 2010

4 BioMed Research International

AmyPSPE-cadE-cadAmyPSP

WT

HESM

GSL

GSM

GSL

G

Tera

tom

a

AmyPSPE-cadE-cadAmyPSP HE(a)

(b)

Figure 2 Histological analysis of salivary gland-like tissue in teratomas formed after transplantation of GFP-iPS cells Immunofluorescenceand HampE staining of sections showed submandibular glands (SMGs) and sublingual glands (SLGs) of adult mouse PSP (green) Amy (blue)and E-cadherin (red) (a) Scale bars 25 120583m Immunofluorescence andHampE staining of sections of GFP-iPS-grafted teratoma revealed salivarygland-like tissue (b) Scale bars 25120583m

0h 96h24h

Figure 3 Aggregation of SMG cells in SG Phase-contrast image of regenerated SMG in organ culture for 0 24 and 96 h The regeneratedSMG contained many acinar-like structures Scale bar 05mm

BioMed Research International 5

GFPPCMPCMGFP

(a)

GFP E-cad GFPE-cad

(b)

Figure 4 Aggregation of SMG cells in iSG SMG cells and GFP-iPS cells were cocultured with DMEMF12 for 96 h Localization of GFP-iPS cells in iSG iSG consists of 20 iPS cells and 80 SMG cells Phase-contrast image and immunological stained image GFP-iPS (green)and pericentriolar material (PCM) (a) Scale bar 25 120583m Immunofluorescence of paraffin-embedded tissue of regenerated salivary glandsGFP-iPS (green) and E-cadherin (red) (b) Scale bar 25120583m

in regulating the self-renewal of embryonic stem cells Thedevelopment of the SMG is an ideal model to study thedifferential stage of regenerated salivary glands Sox2 wasexpressed both in the epithelium and mesenchyme of E135mouse SMGs and in the epithelium of E155 to E175 Sox2was gradually decreased from E175 to the adult stage and wasexpressed in the nucleus of ductal epithelium but not in thecytoplasm (Figure 6(a)) The expression of Sox2 was inducedin the cytoplasm of SG but decreased in the cytoplasm of iSG(Figure 6(b))

AQP5 is known as one of the salivary gland markers forfluid secretion AQP5 was expressed at a low level in theterminal bud and in the epithelial stalk of E135 and E155SMGs AQP5 was increased in the organized proacinar cellsof SMGs in E175 to adult (Figure 7(a)) SMG cells in SGexpressed AQP5 at the same level as E135 SMG HoweverSMG cells in iSG highly expressed AQP5 at the same level asthat in E175 SMG (Figure 7(b))

To analyze specific gene expression SMG cells from SGand iSG were isolated by a cell sorter The gene expressionof markers related to embryonic stem cell maintenance(Sox2 Nanog c-Myc and Klf4) and submandibular markers(Aqp5 Amy and M3r) was investigated by qPCR The geneexpression of Sox2 c-Myc and Nanog was decreased in iSGcompared with that in SG However Klf4 was increased iniSG (Figure 8(a)ndash8(d)) Aqp5 gene expression was higher iniSG than that in SG (Figure 8(e)) The expression of Amy andM3r was not detected by qPCR (Figures 8(f) and 8(g))

4 Discussion

Salivary glands have a potential ability to recover theirfunction over time [15] Several studies showed thatstemprogenitor cells in salivary glands restored salivation[16ndash18] Various kinds of cells have been used fortransplantation in regenerative medicine Many studiesshowed that the stem cell itself differentiates in the part ofthe organ where the function was restored Recent studiesrevealed that various kinds of cytokines secreted by stemcells are related to the mechanism of regeneration Thetransplantation of bone marrow stromal cells for spinal cordinjury produced extensive outgrowth of regenerating axonsassociated with Schwann cells through the extracellularmatrices [8 19] The restoration of morphology and functionwas induced by the combination of several factors such as theparacrine effect cell transdifferentiation and vasculogenesisby hematopoietic andor mesenchymal cells derived frombone marrow [20]

Many studies have tried to use iPS cells as a source ofthe transplant for treatment [21 22] However the functionof the microenvironment that iPS cells produce has not beeninvestigated yet In this study we examined the differentiationof salivary gland cells using iPS cells in a microenvironment

At first to confirm the pluripotency of iPS cells GFP-iPScells were transplanted into SCID mice Histological analysisof the produced teratoma demonstrated all three primarygerm layers It suggested that GFP-iPS cells had pluripotency

6 BioMed Research International

SG

PCM

GFP

E-c

ad

5 iSG 20 iSG

SG 5iSG iSG

20

20

0

10

05

15

The a

rea o

f a re

gene

rate

dsa

livar

y gl

and

(mm

2) 80

0

40

20

60

SG 5iSG iSG

20

The n

umbe

r of a

n ac

inar

-like

struc

ture

(m

m2)

lowastlowast

lowast

80

0

40

20

60

The d

iam

eter

of a

n ac

inar

-like

struc

ture

(120583m

2)

lowastlowast

lowast

SG 5iSG iSG

20

(a)

(b) (c) (d)

Figure 5 Morphological analysis of regenerated SMGs (SG 5 iSG and 20 iSG) PCM and immunostained images indicated a reductionin size and an increase in the number of acinar-like structures in regenerated salivary glands (a) Size of whole regenerated salivary glands(119899 = 3) (b) Number of acinar-like structures in regenerated salivary glands (119899 = 3) (c) Size of acinar-like structures in regenerated salivaryglands (119899 = 24) (d) lowast119901 lt 005 lowastlowast119901 lt 001 versus control

for differentiation Furthermore teratomas contained twokinds of glandular tissueswhichwere similar to both the SMGand the SLG comparedwith the pattern of the salivary glandsin adult mice [23] GFP-iPS cells have a potential ability toregenerate SMG and SLG cells However only few parts ofthe tissues differentiated from salivary glands The potentialtumorigenicity of iPS cells has to be considered prior toclinical application

The embryonic SMG is a remarkably adaptable tissueand dissociated SMG cells can self-assemble in serum-free

medium [24] The cell aggregation assay is a useful methodfor studying mechanisms of tissue assembly as the structureof the regenerated glands is similar to salivary glands [25 26]Embryonic SMG cells and GFP-iPS cells were cocultured toidentify the roles of the microenvironment around the iPScells Regenerated salivary glands (SG and iSG) formedmanyacinar-like structures similar to embryonic salivary glands for96 h in vitro [27] It is very difficult for our conventional organculture without serum and other factors to grow salivarygland in vitro for longer time such as several weeks So

BioMed Research International 7

E135 E175E155 Ad

Sox2

Sox2

E-c

ad

E-ca

d

iSGSG

Sox2

Sox2

GFP

E-c

ad

(a)

(b)

Figure 6 Distribution of Sox2 in developing SMG and in regenerated SMGs (SG and iSG) Sox2 is expressed in the cytoplasm of bothepithelial cells and mesenchymal cells in E135 E155 and E175 developing SMGs and is gradually decreased in later embryonic stages Sox2is expressed in the nucleus of epithelial cells in both postnatal and adult SMGs Sox2 (cyan) and E-cadherin (red) (a) Scale bar 25 120583m Sox2expression in the cytoplasm of both epithelial cells and mesenchymal cells in iSG (20 iSG) is less than that in SG The nucleus of GFP-iPScells highly expressed Sox2 as a positive control Sox2 (cyan) GFP (green) and E-cadherin (red) (b) Scale bar 25 120583m

8 BioMed Research International

AQP5

AQP5

E-c

ad

E-ca

d

E135 E175E155 Ad

iSGSG(a)

(b)

AQP5

AQP5

GFP

E-c

ad

Figure 7 Distribution of AQP5 in developing SMG and regenerated SMGs (SG and iSG) AQP5 is gradually increased in the epithelial cellsof SMGs in E135 E155 E175 and adult AQP5 is expressed in the cell membrane of epithelial cells in iSG (20 iSG) more than that in SGAQP5 (white) and E-cadherin (red) (a) Scale bar 25 120583m Distribution on AQP5 in regenerated SMG (b) AQP5 (white) GFP (green) andE-cadherin (red) Scale bar 25 120583m

BioMed Research International 9

Sox2

SG iSG

100

0

60

40

80

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

c-Myc

100

0

60

40

80

lowastlowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

Nanog

100

0

60

40

80

lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG0

200

100

Klf4lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

AQP5

0

200

100

300

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

Amy

Not detected

Relat

ive e

xpre

ssio

n (

)

(a) (b) (c) (d)

(e) (f) (g)

SG iSG

M3R

Not detected

Relat

ive e

xpre

ssio

n (

)

Figure 8 Gene expression of salivary gland cells in regenerated SMGs between SG and iSG Gene expression of Sox2 (a) c-Myc (b) andNanog (c) was decreased in iSG and gene expression of Klf4 (d) was increased in iSG Aqp5 (e) gene expression was increased in iSG butAmy (f) andM3r (g) gene expression were not detected by qPCR similar to early embryonic SMG cells lowastlowast119901 lt 001 versus control

salivary gland cells needed to be transplanted to mouse [2829]

Because the water channel protein AQP5 is found inthe lumen of the acinar-like structures regenerated sali-vary glands may have an ability to secrete saliva [30 31]Morphological analysis of the regenerated salivary glandsindicated that the difference between SG and iSG was notstatistically significant However iSG had a larger number ofsmall acinar-like structures more than that in SG (Figure 9)Developing salivary glands in epithelial tissue increases thesurface area by branching morphogenesis and increases theability of saliva secretion [32ndash34] Coculture of embryonicSMG cells and iPS cells had more developed epithelialstructures than that in monoculture of embryonic SMG cells

Previous studies show the gene expression of stem cellmarkers (Sox2 c-Myc Nanog and Klf4) in E13 E15 and adult

(Ad) SMGs Sox2 c-Myc and Nanog decreased during SMGdevelopment but Klf4 increased [16] The submandibularmarker AQP5 belongs to a family of water channel proteinsthat allow water to pass through the plasma membrane byosmosis E14 SMG expressed AQP5 as a first indication [31]Our results showed that the gene expressions of Sox2 c-Myc and Nanog were decreased in iSG and that the geneexpression of Klf4 and Aqp5 was increased in iSG Immuno-histochemical analysis also showed that the undifferentiatedmarker of salivary gland cells decreased in iSG comparedwith that in SG It was suggested that iPS cells induce thecharacteristic differentiation resulting in the morphologicalchanges observed in salivary gland cell formation Theseresults indicate that iPS cells have a potential ability toaccelerate differentiation of salivary gland development andregeneration Regenerated salivary glands produced from

10 BioMed Research International

iPS cell

Acinar-like structure

iSGSGRegenerated salivary gland

Sox2 c-Myc NanogAqp5

Figure 9 Effect of iPS cells on aggregation of SMG cells iPS cells(green balls) reduced the size of the epithelial tissue but acinar-likestructure (orange balls) increased their number iPS cells cannotmixcompletely with SMG cells and instead surround the epitheliumof SMG Stem cell markers such as Sox2 c-Myc and Nanog aredecreased but Aqp5 is increased in iSG

E135 SMG cells were cultured for four daysAmy andM3r areusually expressed after birth Amy and M3r gene expressionalso occurred in the regenerated salivary gland cells

Various approaches would be necessary to organize thecomplex tissues such as the salivary gland kidney and lungOur finding that coculture of salivary glands cells with iPScells formed differentiated salivary glands is significant andfuture elucidation of the mechanism could lead to viableregeneration therapy of functional organs using iPS cellsOur study provides new insights for future research into theregeneration of organs such as salivary glands

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HitomiOno andAyaObana contributed equally to this work

Acknowledgments

This work was supported by JSPS KAKENHI Grant no24390452 The authors thank Dr Kenji Mishima Division ofPathology Department of Oral Diagnostic Sciences Schoolof Dentistry Showa University for helpful advice regard-ing salivary markers The authors also thank Dr HirokoOkawa Department of Fixed Prosthodontics Osaka Uni-versity Graduate School of Dentistry for excellent technicalsupport in iPS cell cultures

References

[1] S P Humphrey and R T Williamson ldquoA review of salivanormal composition flow and functionrdquo Journal of ProstheticDentistry vol 85 no 2 pp 162ndash169 2001

[2] S B Jensen A M L Pedersen A Vissink et al ldquoA systematicreview of salivary gland hypofunction and xerostomia inducedby cancer therapies management strategies and economicimpactrdquo Supportive Care in Cancer vol 18 no 8 pp 1061ndash10792010

[3] J-Y Lim J C Ra I S Shin et al ldquoSystemic transplantationof human adipose tissue-derived mesenchymal stem cells forthe regeneration of irradiation-induced salivary gland damagerdquoPLoS ONE vol 8 no 8 Article ID e71167 2013

[4] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007

[5] H Kamao M Mandai S Okamoto et al ldquoCharacterization ofhuman induced pluripotent stem cell-derived retinal pigmentepithelium cell sheets aiming for clinical applicationrdquo Stem CellReports vol 2 no 2 pp 205ndash218 2014

[6] H Egusa K Okita H Kayashima et al ldquoGingival fibroblastsas a promising source of induced pluripotent stem cellsrdquo PLoSONE vol 5 no 9 Article ID e12743 12 pages 2010

[7] S C Bendall M H Stewart P Menendez et al ldquoIGF andFGF cooperatively establish the regulatory stem cell niche ofpluripotent human cells in vitrordquo Nature vol 448 no 7157 pp1015ndash1021 2007

[8] C Ide Y Nakai N Nakano et al ldquoBone marrow stromal celltransplantation for treatment of sub-acute spinal cord injury inthe ratrdquo Brain Research vol 1332 pp 32ndash47 2010

[9] T Takebe K SekineM Enomura et al ldquoVascularized and func-tional human liver from an iPSC-derived organ bud transplantrdquoNature vol 499 no 7459 pp 481ndash484 2013

[10] M Eiraku K Watanabe M Matsuo-Takasaki et al ldquoSelf-organized formation of polarized cortical tissues fromESCs andits active manipulation by extrinsic signalsrdquo Cell Stem Cell vol3 no 5 pp 519ndash532 2008

[11] M Kawakami H Ishikawa T Tachibana A Tanaka andI Mataga ldquoFunctional transplantation of salivary gland cellsdifferentiated from mouse early ES cells in vitrordquo Human Cellvol 26 no 2 pp 80ndash90 2013

[12] K Mishima H Inoue T Nishiyama et al ldquoTransplantation ofside population cells restores the function of damaged exocrineglands through clusterinrdquo Stem Cells vol 30 no 9 pp 1925ndash1937 2012

[13] J Mima A Koshino K Oka et al ldquoRegulation of the epithelialadhesion molecule CEACAM1 is important for palate forma-tionrdquo PLoS ONE vol 8 no 4 Article ID e61653 2013

[14] V N Pateland andM PHoffman ldquoSalivary gland developmenta template for regenerationrdquo Seminars in Cell amp DevelopmentalBiology vol 25-26 pp 52ndash60 2014

[15] S B Jensen A M Pedersen J Reibel and B NauntofteldquoXerostomia and hypofunction of the salivary glands in cancertherapyrdquo Supportive Care in Cancer vol 11 no 4 pp 207ndash2252003

[16] I M A Lombaert and M P Hoffman ldquoEpithelialstemprogenitor cells in the embryonic mouse submandibularglandrdquo Frontiers of Oral Biology vol 14 pp 90ndash106 2010

[17] A Angelova Volponi M Kawasaki and P T Sharpe ldquoAdulthuman gingival epithelial cells as a source for whole-toothbioengineeringrdquo Journal of Dental Research vol 92 no 4 pp329ndash334 2013

[18] A V Vanikar H L Trivedi S C Gopal A Kumar and SD Dave ldquoPre-transplant co-infusion of donor-adipose tissuederived mesenchymal stem cells and hematopoietic stem cells

BioMed Research International 11

may help in achieving tolerance in living donor renal transplan-tationrdquo Renal Failure vol 36 no 3 pp 457ndash460 2014

[19] J P M Issa R Tiossi A S D S Mello R A Lopes M A S DiMatteo and M M Iyomasa ldquoPRP a possibility in regenerativetherapyrdquo International Journal of Morphology vol 25 no 3 pp587ndash590 2007

[20] Y Sumita Y Liu S Khalili et al ldquoBone marrow-derived cellsrescue salivary gland function in mice with head and neckirradiationrdquo The International Journal of Biochemistry amp CellBiology vol 43 no 1 pp 80ndash87 2011

[21] B Song A M Smink C V Jones et al ldquoThe directeddifferentiation of human iPS cells into kidney podocytesrdquo PLoSONE vol 7 no 9 Article ID e46453 2012

[22] H Saito M Takeuchi K Chida and A Miyajima ldquoGenerationof glucose-responsive functional islets with a three-dimensionalstructure frommouse fetal pancreatic cells and ips cells in vitrordquoPLoS ONE vol 6 no 12 Article ID e28209 2011

[23] D A Nelson C Manhardt V Kamath et al ldquoQuantitativesingle cell analysis of cell population dynamics during sub-mandibular salivary gland development and differentiationrdquoBiology Open vol 2 no 5 pp 439ndash447 2013

[24] C Wei M Larsen M P Hoffman and K M Yamada ldquoSelf-organization and branchingmorphogenesis of primary salivaryepithelial cellsrdquo Tissue Engineering vol 13 no 4 pp 721ndash7352007

[25] TMisteli ldquoThe concept of self-organization in cellular architec-turerdquo Journal of Cell Biology vol 155 no 2 pp 181ndash185 2001

[26] L S Y Nanduri I M A Lombaert M van der Zwaaget al ldquoSalisphere derived c-Kit+ cell transplantation restorestissue homeostasis in irradiated salivary glandrdquo Radiotherapy ampOncology vol 108 no 3 pp 458ndash463 2013

[27] J C-F Hsu and K M Yamada ldquoSalivary gland branchingmorphogenesismdashrecent progress and future opportunitiesrdquoInternational Journal of Oral Science vol 2 no 3 pp 117ndash1262010

[28] MHirayamaMOgawaMOshima et al ldquoFunctional lacrimalgland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2497 2013

[29] M Ogawa M Oshima A Imamura et al ldquoFunctional salivarygland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2498 2013

[30] K Ishibashi S Hara and S Kondo ldquoAquaporin water channelsin mammalsrdquo Clinical and Experimental Nephrology vol 13 no2 pp 107ndash117 2009

[31] H S Larsen M H Aure S B Peters M Larsen E BMesselt and H Kanli Galtung ldquoLocalization of AQP5 duringdevelopment of the mouse submandibular salivary glandrdquoJournal of Molecular Histology vol 42 no 1 pp 71ndash81 2011

[32] J E Fata Z Werb and M J Bissell ldquoRegulation of mammarygland branchingmorphogenesis by the extracellular matrix andits remodeling enzymesrdquo Breast Cancer Research vol 6 no 1pp 1ndash11 2004

[33] T Sakai M Larsen and K M Yamada ldquoFibronectin require-ment in branching morphogenesisrdquo Nature vol 423 no 6942pp 876ndash881 2003

[34] T Onodera T Sakai J C-F Hsu K Matsumoto J A ChioriniandKMYamada ldquoBtbd7 regulates epithelial cell dynamics andbranching morphogenesisrdquo Science vol 329 no 5991 pp 562ndash565 2010

BioMed Research International 5

GFPPCMPCMGFP

(a)

GFP E-cad GFPE-cad

(b)

Figure 4 Aggregation of SMG cells in iSG SMG cells and GFP-iPS cells were cocultured with DMEMF12 for 96 h Localization of GFP-iPS cells in iSG iSG consists of 20 iPS cells and 80 SMG cells Phase-contrast image and immunological stained image GFP-iPS (green)and pericentriolar material (PCM) (a) Scale bar 25 120583m Immunofluorescence of paraffin-embedded tissue of regenerated salivary glandsGFP-iPS (green) and E-cadherin (red) (b) Scale bar 25120583m

in regulating the self-renewal of embryonic stem cells Thedevelopment of the SMG is an ideal model to study thedifferential stage of regenerated salivary glands Sox2 wasexpressed both in the epithelium and mesenchyme of E135mouse SMGs and in the epithelium of E155 to E175 Sox2was gradually decreased from E175 to the adult stage and wasexpressed in the nucleus of ductal epithelium but not in thecytoplasm (Figure 6(a)) The expression of Sox2 was inducedin the cytoplasm of SG but decreased in the cytoplasm of iSG(Figure 6(b))

AQP5 is known as one of the salivary gland markers forfluid secretion AQP5 was expressed at a low level in theterminal bud and in the epithelial stalk of E135 and E155SMGs AQP5 was increased in the organized proacinar cellsof SMGs in E175 to adult (Figure 7(a)) SMG cells in SGexpressed AQP5 at the same level as E135 SMG HoweverSMG cells in iSG highly expressed AQP5 at the same level asthat in E175 SMG (Figure 7(b))

To analyze specific gene expression SMG cells from SGand iSG were isolated by a cell sorter The gene expressionof markers related to embryonic stem cell maintenance(Sox2 Nanog c-Myc and Klf4) and submandibular markers(Aqp5 Amy and M3r) was investigated by qPCR The geneexpression of Sox2 c-Myc and Nanog was decreased in iSGcompared with that in SG However Klf4 was increased iniSG (Figure 8(a)ndash8(d)) Aqp5 gene expression was higher iniSG than that in SG (Figure 8(e)) The expression of Amy andM3r was not detected by qPCR (Figures 8(f) and 8(g))

4 Discussion

Salivary glands have a potential ability to recover theirfunction over time [15] Several studies showed thatstemprogenitor cells in salivary glands restored salivation[16ndash18] Various kinds of cells have been used fortransplantation in regenerative medicine Many studiesshowed that the stem cell itself differentiates in the part ofthe organ where the function was restored Recent studiesrevealed that various kinds of cytokines secreted by stemcells are related to the mechanism of regeneration Thetransplantation of bone marrow stromal cells for spinal cordinjury produced extensive outgrowth of regenerating axonsassociated with Schwann cells through the extracellularmatrices [8 19] The restoration of morphology and functionwas induced by the combination of several factors such as theparacrine effect cell transdifferentiation and vasculogenesisby hematopoietic andor mesenchymal cells derived frombone marrow [20]

Many studies have tried to use iPS cells as a source ofthe transplant for treatment [21 22] However the functionof the microenvironment that iPS cells produce has not beeninvestigated yet In this study we examined the differentiationof salivary gland cells using iPS cells in a microenvironment

At first to confirm the pluripotency of iPS cells GFP-iPScells were transplanted into SCID mice Histological analysisof the produced teratoma demonstrated all three primarygerm layers It suggested that GFP-iPS cells had pluripotency

6 BioMed Research International

SG

PCM

GFP

E-c

ad

5 iSG 20 iSG

SG 5iSG iSG

20

20

0

10

05

15

The a

rea o

f a re

gene

rate

dsa

livar

y gl

and

(mm

2) 80

0

40

20

60

SG 5iSG iSG

20

The n

umbe

r of a

n ac

inar

-like

struc

ture

(m

m2)

lowastlowast

lowast

80

0

40

20

60

The d

iam

eter

of a

n ac

inar

-like

struc

ture

(120583m

2)

lowastlowast

lowast

SG 5iSG iSG

20

(a)

(b) (c) (d)

Figure 5 Morphological analysis of regenerated SMGs (SG 5 iSG and 20 iSG) PCM and immunostained images indicated a reductionin size and an increase in the number of acinar-like structures in regenerated salivary glands (a) Size of whole regenerated salivary glands(119899 = 3) (b) Number of acinar-like structures in regenerated salivary glands (119899 = 3) (c) Size of acinar-like structures in regenerated salivaryglands (119899 = 24) (d) lowast119901 lt 005 lowastlowast119901 lt 001 versus control

for differentiation Furthermore teratomas contained twokinds of glandular tissueswhichwere similar to both the SMGand the SLG comparedwith the pattern of the salivary glandsin adult mice [23] GFP-iPS cells have a potential ability toregenerate SMG and SLG cells However only few parts ofthe tissues differentiated from salivary glands The potentialtumorigenicity of iPS cells has to be considered prior toclinical application

The embryonic SMG is a remarkably adaptable tissueand dissociated SMG cells can self-assemble in serum-free

medium [24] The cell aggregation assay is a useful methodfor studying mechanisms of tissue assembly as the structureof the regenerated glands is similar to salivary glands [25 26]Embryonic SMG cells and GFP-iPS cells were cocultured toidentify the roles of the microenvironment around the iPScells Regenerated salivary glands (SG and iSG) formedmanyacinar-like structures similar to embryonic salivary glands for96 h in vitro [27] It is very difficult for our conventional organculture without serum and other factors to grow salivarygland in vitro for longer time such as several weeks So

BioMed Research International 7

E135 E175E155 Ad

Sox2

Sox2

E-c

ad

E-ca

d

iSGSG

Sox2

Sox2

GFP

E-c

ad

(a)

(b)

Figure 6 Distribution of Sox2 in developing SMG and in regenerated SMGs (SG and iSG) Sox2 is expressed in the cytoplasm of bothepithelial cells and mesenchymal cells in E135 E155 and E175 developing SMGs and is gradually decreased in later embryonic stages Sox2is expressed in the nucleus of epithelial cells in both postnatal and adult SMGs Sox2 (cyan) and E-cadherin (red) (a) Scale bar 25 120583m Sox2expression in the cytoplasm of both epithelial cells and mesenchymal cells in iSG (20 iSG) is less than that in SG The nucleus of GFP-iPScells highly expressed Sox2 as a positive control Sox2 (cyan) GFP (green) and E-cadherin (red) (b) Scale bar 25 120583m

8 BioMed Research International

AQP5

AQP5

E-c

ad

E-ca

d

E135 E175E155 Ad

iSGSG(a)

(b)

AQP5

AQP5

GFP

E-c

ad

Figure 7 Distribution of AQP5 in developing SMG and regenerated SMGs (SG and iSG) AQP5 is gradually increased in the epithelial cellsof SMGs in E135 E155 E175 and adult AQP5 is expressed in the cell membrane of epithelial cells in iSG (20 iSG) more than that in SGAQP5 (white) and E-cadherin (red) (a) Scale bar 25 120583m Distribution on AQP5 in regenerated SMG (b) AQP5 (white) GFP (green) andE-cadherin (red) Scale bar 25 120583m

BioMed Research International 9

Sox2

SG iSG

100

0

60

40

80

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

c-Myc

100

0

60

40

80

lowastlowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

Nanog

100

0

60

40

80

lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG0

200

100

Klf4lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

AQP5

0

200

100

300

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

Amy

Not detected

Relat

ive e

xpre

ssio

n (

)

(a) (b) (c) (d)

(e) (f) (g)

SG iSG

M3R

Not detected

Relat

ive e

xpre

ssio

n (

)

Figure 8 Gene expression of salivary gland cells in regenerated SMGs between SG and iSG Gene expression of Sox2 (a) c-Myc (b) andNanog (c) was decreased in iSG and gene expression of Klf4 (d) was increased in iSG Aqp5 (e) gene expression was increased in iSG butAmy (f) andM3r (g) gene expression were not detected by qPCR similar to early embryonic SMG cells lowastlowast119901 lt 001 versus control

salivary gland cells needed to be transplanted to mouse [2829]

Because the water channel protein AQP5 is found inthe lumen of the acinar-like structures regenerated sali-vary glands may have an ability to secrete saliva [30 31]Morphological analysis of the regenerated salivary glandsindicated that the difference between SG and iSG was notstatistically significant However iSG had a larger number ofsmall acinar-like structures more than that in SG (Figure 9)Developing salivary glands in epithelial tissue increases thesurface area by branching morphogenesis and increases theability of saliva secretion [32ndash34] Coculture of embryonicSMG cells and iPS cells had more developed epithelialstructures than that in monoculture of embryonic SMG cells

Previous studies show the gene expression of stem cellmarkers (Sox2 c-Myc Nanog and Klf4) in E13 E15 and adult

(Ad) SMGs Sox2 c-Myc and Nanog decreased during SMGdevelopment but Klf4 increased [16] The submandibularmarker AQP5 belongs to a family of water channel proteinsthat allow water to pass through the plasma membrane byosmosis E14 SMG expressed AQP5 as a first indication [31]Our results showed that the gene expressions of Sox2 c-Myc and Nanog were decreased in iSG and that the geneexpression of Klf4 and Aqp5 was increased in iSG Immuno-histochemical analysis also showed that the undifferentiatedmarker of salivary gland cells decreased in iSG comparedwith that in SG It was suggested that iPS cells induce thecharacteristic differentiation resulting in the morphologicalchanges observed in salivary gland cell formation Theseresults indicate that iPS cells have a potential ability toaccelerate differentiation of salivary gland development andregeneration Regenerated salivary glands produced from

10 BioMed Research International

iPS cell

Acinar-like structure

iSGSGRegenerated salivary gland

Sox2 c-Myc NanogAqp5

Figure 9 Effect of iPS cells on aggregation of SMG cells iPS cells(green balls) reduced the size of the epithelial tissue but acinar-likestructure (orange balls) increased their number iPS cells cannotmixcompletely with SMG cells and instead surround the epitheliumof SMG Stem cell markers such as Sox2 c-Myc and Nanog aredecreased but Aqp5 is increased in iSG

E135 SMG cells were cultured for four daysAmy andM3r areusually expressed after birth Amy and M3r gene expressionalso occurred in the regenerated salivary gland cells

Various approaches would be necessary to organize thecomplex tissues such as the salivary gland kidney and lungOur finding that coculture of salivary glands cells with iPScells formed differentiated salivary glands is significant andfuture elucidation of the mechanism could lead to viableregeneration therapy of functional organs using iPS cellsOur study provides new insights for future research into theregeneration of organs such as salivary glands

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HitomiOno andAyaObana contributed equally to this work

Acknowledgments

This work was supported by JSPS KAKENHI Grant no24390452 The authors thank Dr Kenji Mishima Division ofPathology Department of Oral Diagnostic Sciences Schoolof Dentistry Showa University for helpful advice regard-ing salivary markers The authors also thank Dr HirokoOkawa Department of Fixed Prosthodontics Osaka Uni-versity Graduate School of Dentistry for excellent technicalsupport in iPS cell cultures

References

[1] S P Humphrey and R T Williamson ldquoA review of salivanormal composition flow and functionrdquo Journal of ProstheticDentistry vol 85 no 2 pp 162ndash169 2001

[2] S B Jensen A M L Pedersen A Vissink et al ldquoA systematicreview of salivary gland hypofunction and xerostomia inducedby cancer therapies management strategies and economicimpactrdquo Supportive Care in Cancer vol 18 no 8 pp 1061ndash10792010

[3] J-Y Lim J C Ra I S Shin et al ldquoSystemic transplantationof human adipose tissue-derived mesenchymal stem cells forthe regeneration of irradiation-induced salivary gland damagerdquoPLoS ONE vol 8 no 8 Article ID e71167 2013

[4] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007

[5] H Kamao M Mandai S Okamoto et al ldquoCharacterization ofhuman induced pluripotent stem cell-derived retinal pigmentepithelium cell sheets aiming for clinical applicationrdquo Stem CellReports vol 2 no 2 pp 205ndash218 2014

[6] H Egusa K Okita H Kayashima et al ldquoGingival fibroblastsas a promising source of induced pluripotent stem cellsrdquo PLoSONE vol 5 no 9 Article ID e12743 12 pages 2010

[7] S C Bendall M H Stewart P Menendez et al ldquoIGF andFGF cooperatively establish the regulatory stem cell niche ofpluripotent human cells in vitrordquo Nature vol 448 no 7157 pp1015ndash1021 2007

[8] C Ide Y Nakai N Nakano et al ldquoBone marrow stromal celltransplantation for treatment of sub-acute spinal cord injury inthe ratrdquo Brain Research vol 1332 pp 32ndash47 2010

[9] T Takebe K SekineM Enomura et al ldquoVascularized and func-tional human liver from an iPSC-derived organ bud transplantrdquoNature vol 499 no 7459 pp 481ndash484 2013

[10] M Eiraku K Watanabe M Matsuo-Takasaki et al ldquoSelf-organized formation of polarized cortical tissues fromESCs andits active manipulation by extrinsic signalsrdquo Cell Stem Cell vol3 no 5 pp 519ndash532 2008

[11] M Kawakami H Ishikawa T Tachibana A Tanaka andI Mataga ldquoFunctional transplantation of salivary gland cellsdifferentiated from mouse early ES cells in vitrordquo Human Cellvol 26 no 2 pp 80ndash90 2013

[12] K Mishima H Inoue T Nishiyama et al ldquoTransplantation ofside population cells restores the function of damaged exocrineglands through clusterinrdquo Stem Cells vol 30 no 9 pp 1925ndash1937 2012

[13] J Mima A Koshino K Oka et al ldquoRegulation of the epithelialadhesion molecule CEACAM1 is important for palate forma-tionrdquo PLoS ONE vol 8 no 4 Article ID e61653 2013

[14] V N Pateland andM PHoffman ldquoSalivary gland developmenta template for regenerationrdquo Seminars in Cell amp DevelopmentalBiology vol 25-26 pp 52ndash60 2014

[15] S B Jensen A M Pedersen J Reibel and B NauntofteldquoXerostomia and hypofunction of the salivary glands in cancertherapyrdquo Supportive Care in Cancer vol 11 no 4 pp 207ndash2252003

[16] I M A Lombaert and M P Hoffman ldquoEpithelialstemprogenitor cells in the embryonic mouse submandibularglandrdquo Frontiers of Oral Biology vol 14 pp 90ndash106 2010

[17] A Angelova Volponi M Kawasaki and P T Sharpe ldquoAdulthuman gingival epithelial cells as a source for whole-toothbioengineeringrdquo Journal of Dental Research vol 92 no 4 pp329ndash334 2013

[18] A V Vanikar H L Trivedi S C Gopal A Kumar and SD Dave ldquoPre-transplant co-infusion of donor-adipose tissuederived mesenchymal stem cells and hematopoietic stem cells

BioMed Research International 11

may help in achieving tolerance in living donor renal transplan-tationrdquo Renal Failure vol 36 no 3 pp 457ndash460 2014

[19] J P M Issa R Tiossi A S D S Mello R A Lopes M A S DiMatteo and M M Iyomasa ldquoPRP a possibility in regenerativetherapyrdquo International Journal of Morphology vol 25 no 3 pp587ndash590 2007

[20] Y Sumita Y Liu S Khalili et al ldquoBone marrow-derived cellsrescue salivary gland function in mice with head and neckirradiationrdquo The International Journal of Biochemistry amp CellBiology vol 43 no 1 pp 80ndash87 2011

[21] B Song A M Smink C V Jones et al ldquoThe directeddifferentiation of human iPS cells into kidney podocytesrdquo PLoSONE vol 7 no 9 Article ID e46453 2012

[22] H Saito M Takeuchi K Chida and A Miyajima ldquoGenerationof glucose-responsive functional islets with a three-dimensionalstructure frommouse fetal pancreatic cells and ips cells in vitrordquoPLoS ONE vol 6 no 12 Article ID e28209 2011

[23] D A Nelson C Manhardt V Kamath et al ldquoQuantitativesingle cell analysis of cell population dynamics during sub-mandibular salivary gland development and differentiationrdquoBiology Open vol 2 no 5 pp 439ndash447 2013

[24] C Wei M Larsen M P Hoffman and K M Yamada ldquoSelf-organization and branchingmorphogenesis of primary salivaryepithelial cellsrdquo Tissue Engineering vol 13 no 4 pp 721ndash7352007

[25] TMisteli ldquoThe concept of self-organization in cellular architec-turerdquo Journal of Cell Biology vol 155 no 2 pp 181ndash185 2001

[26] L S Y Nanduri I M A Lombaert M van der Zwaaget al ldquoSalisphere derived c-Kit+ cell transplantation restorestissue homeostasis in irradiated salivary glandrdquo Radiotherapy ampOncology vol 108 no 3 pp 458ndash463 2013

[27] J C-F Hsu and K M Yamada ldquoSalivary gland branchingmorphogenesismdashrecent progress and future opportunitiesrdquoInternational Journal of Oral Science vol 2 no 3 pp 117ndash1262010

[28] MHirayamaMOgawaMOshima et al ldquoFunctional lacrimalgland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2497 2013

[29] M Ogawa M Oshima A Imamura et al ldquoFunctional salivarygland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2498 2013

[30] K Ishibashi S Hara and S Kondo ldquoAquaporin water channelsin mammalsrdquo Clinical and Experimental Nephrology vol 13 no2 pp 107ndash117 2009

[31] H S Larsen M H Aure S B Peters M Larsen E BMesselt and H Kanli Galtung ldquoLocalization of AQP5 duringdevelopment of the mouse submandibular salivary glandrdquoJournal of Molecular Histology vol 42 no 1 pp 71ndash81 2011

[32] J E Fata Z Werb and M J Bissell ldquoRegulation of mammarygland branchingmorphogenesis by the extracellular matrix andits remodeling enzymesrdquo Breast Cancer Research vol 6 no 1pp 1ndash11 2004

[33] T Sakai M Larsen and K M Yamada ldquoFibronectin require-ment in branching morphogenesisrdquo Nature vol 423 no 6942pp 876ndash881 2003

[34] T Onodera T Sakai J C-F Hsu K Matsumoto J A ChioriniandKMYamada ldquoBtbd7 regulates epithelial cell dynamics andbranching morphogenesisrdquo Science vol 329 no 5991 pp 562ndash565 2010

6 BioMed Research International

SG

PCM

GFP

E-c

ad

5 iSG 20 iSG

SG 5iSG iSG

20

20

0

10

05

15

The a

rea o

f a re

gene

rate

dsa

livar

y gl

and

(mm

2) 80

0

40

20

60

SG 5iSG iSG

20

The n

umbe

r of a

n ac

inar

-like

struc

ture

(m

m2)

lowastlowast

lowast

80

0

40

20

60

The d

iam

eter

of a

n ac

inar

-like

struc

ture

(120583m

2)

lowastlowast

lowast

SG 5iSG iSG

20

(a)

(b) (c) (d)

Figure 5 Morphological analysis of regenerated SMGs (SG 5 iSG and 20 iSG) PCM and immunostained images indicated a reductionin size and an increase in the number of acinar-like structures in regenerated salivary glands (a) Size of whole regenerated salivary glands(119899 = 3) (b) Number of acinar-like structures in regenerated salivary glands (119899 = 3) (c) Size of acinar-like structures in regenerated salivaryglands (119899 = 24) (d) lowast119901 lt 005 lowastlowast119901 lt 001 versus control

for differentiation Furthermore teratomas contained twokinds of glandular tissueswhichwere similar to both the SMGand the SLG comparedwith the pattern of the salivary glandsin adult mice [23] GFP-iPS cells have a potential ability toregenerate SMG and SLG cells However only few parts ofthe tissues differentiated from salivary glands The potentialtumorigenicity of iPS cells has to be considered prior toclinical application

The embryonic SMG is a remarkably adaptable tissueand dissociated SMG cells can self-assemble in serum-free

medium [24] The cell aggregation assay is a useful methodfor studying mechanisms of tissue assembly as the structureof the regenerated glands is similar to salivary glands [25 26]Embryonic SMG cells and GFP-iPS cells were cocultured toidentify the roles of the microenvironment around the iPScells Regenerated salivary glands (SG and iSG) formedmanyacinar-like structures similar to embryonic salivary glands for96 h in vitro [27] It is very difficult for our conventional organculture without serum and other factors to grow salivarygland in vitro for longer time such as several weeks So

BioMed Research International 7

E135 E175E155 Ad

Sox2

Sox2

E-c

ad

E-ca

d

iSGSG

Sox2

Sox2

GFP

E-c

ad

(a)

(b)

Figure 6 Distribution of Sox2 in developing SMG and in regenerated SMGs (SG and iSG) Sox2 is expressed in the cytoplasm of bothepithelial cells and mesenchymal cells in E135 E155 and E175 developing SMGs and is gradually decreased in later embryonic stages Sox2is expressed in the nucleus of epithelial cells in both postnatal and adult SMGs Sox2 (cyan) and E-cadherin (red) (a) Scale bar 25 120583m Sox2expression in the cytoplasm of both epithelial cells and mesenchymal cells in iSG (20 iSG) is less than that in SG The nucleus of GFP-iPScells highly expressed Sox2 as a positive control Sox2 (cyan) GFP (green) and E-cadherin (red) (b) Scale bar 25 120583m

8 BioMed Research International

AQP5

AQP5

E-c

ad

E-ca

d

E135 E175E155 Ad

iSGSG(a)

(b)

AQP5

AQP5

GFP

E-c

ad

Figure 7 Distribution of AQP5 in developing SMG and regenerated SMGs (SG and iSG) AQP5 is gradually increased in the epithelial cellsof SMGs in E135 E155 E175 and adult AQP5 is expressed in the cell membrane of epithelial cells in iSG (20 iSG) more than that in SGAQP5 (white) and E-cadherin (red) (a) Scale bar 25 120583m Distribution on AQP5 in regenerated SMG (b) AQP5 (white) GFP (green) andE-cadherin (red) Scale bar 25 120583m

BioMed Research International 9

Sox2

SG iSG

100

0

60

40

80

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

c-Myc

100

0

60

40

80

lowastlowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

Nanog

100

0

60

40

80

lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG0

200

100

Klf4lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

AQP5

0

200

100

300

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

Amy

Not detected

Relat

ive e

xpre

ssio

n (

)

(a) (b) (c) (d)

(e) (f) (g)

SG iSG

M3R

Not detected

Relat

ive e

xpre

ssio

n (

)

Figure 8 Gene expression of salivary gland cells in regenerated SMGs between SG and iSG Gene expression of Sox2 (a) c-Myc (b) andNanog (c) was decreased in iSG and gene expression of Klf4 (d) was increased in iSG Aqp5 (e) gene expression was increased in iSG butAmy (f) andM3r (g) gene expression were not detected by qPCR similar to early embryonic SMG cells lowastlowast119901 lt 001 versus control

salivary gland cells needed to be transplanted to mouse [2829]

Because the water channel protein AQP5 is found inthe lumen of the acinar-like structures regenerated sali-vary glands may have an ability to secrete saliva [30 31]Morphological analysis of the regenerated salivary glandsindicated that the difference between SG and iSG was notstatistically significant However iSG had a larger number ofsmall acinar-like structures more than that in SG (Figure 9)Developing salivary glands in epithelial tissue increases thesurface area by branching morphogenesis and increases theability of saliva secretion [32ndash34] Coculture of embryonicSMG cells and iPS cells had more developed epithelialstructures than that in monoculture of embryonic SMG cells

Previous studies show the gene expression of stem cellmarkers (Sox2 c-Myc Nanog and Klf4) in E13 E15 and adult

(Ad) SMGs Sox2 c-Myc and Nanog decreased during SMGdevelopment but Klf4 increased [16] The submandibularmarker AQP5 belongs to a family of water channel proteinsthat allow water to pass through the plasma membrane byosmosis E14 SMG expressed AQP5 as a first indication [31]Our results showed that the gene expressions of Sox2 c-Myc and Nanog were decreased in iSG and that the geneexpression of Klf4 and Aqp5 was increased in iSG Immuno-histochemical analysis also showed that the undifferentiatedmarker of salivary gland cells decreased in iSG comparedwith that in SG It was suggested that iPS cells induce thecharacteristic differentiation resulting in the morphologicalchanges observed in salivary gland cell formation Theseresults indicate that iPS cells have a potential ability toaccelerate differentiation of salivary gland development andregeneration Regenerated salivary glands produced from

10 BioMed Research International

iPS cell

Acinar-like structure

iSGSGRegenerated salivary gland

Sox2 c-Myc NanogAqp5

Figure 9 Effect of iPS cells on aggregation of SMG cells iPS cells(green balls) reduced the size of the epithelial tissue but acinar-likestructure (orange balls) increased their number iPS cells cannotmixcompletely with SMG cells and instead surround the epitheliumof SMG Stem cell markers such as Sox2 c-Myc and Nanog aredecreased but Aqp5 is increased in iSG

E135 SMG cells were cultured for four daysAmy andM3r areusually expressed after birth Amy and M3r gene expressionalso occurred in the regenerated salivary gland cells

Various approaches would be necessary to organize thecomplex tissues such as the salivary gland kidney and lungOur finding that coculture of salivary glands cells with iPScells formed differentiated salivary glands is significant andfuture elucidation of the mechanism could lead to viableregeneration therapy of functional organs using iPS cellsOur study provides new insights for future research into theregeneration of organs such as salivary glands

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HitomiOno andAyaObana contributed equally to this work

Acknowledgments

This work was supported by JSPS KAKENHI Grant no24390452 The authors thank Dr Kenji Mishima Division ofPathology Department of Oral Diagnostic Sciences Schoolof Dentistry Showa University for helpful advice regard-ing salivary markers The authors also thank Dr HirokoOkawa Department of Fixed Prosthodontics Osaka Uni-versity Graduate School of Dentistry for excellent technicalsupport in iPS cell cultures

References

[1] S P Humphrey and R T Williamson ldquoA review of salivanormal composition flow and functionrdquo Journal of ProstheticDentistry vol 85 no 2 pp 162ndash169 2001

[2] S B Jensen A M L Pedersen A Vissink et al ldquoA systematicreview of salivary gland hypofunction and xerostomia inducedby cancer therapies management strategies and economicimpactrdquo Supportive Care in Cancer vol 18 no 8 pp 1061ndash10792010

[3] J-Y Lim J C Ra I S Shin et al ldquoSystemic transplantationof human adipose tissue-derived mesenchymal stem cells forthe regeneration of irradiation-induced salivary gland damagerdquoPLoS ONE vol 8 no 8 Article ID e71167 2013

[4] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007

[5] H Kamao M Mandai S Okamoto et al ldquoCharacterization ofhuman induced pluripotent stem cell-derived retinal pigmentepithelium cell sheets aiming for clinical applicationrdquo Stem CellReports vol 2 no 2 pp 205ndash218 2014

[6] H Egusa K Okita H Kayashima et al ldquoGingival fibroblastsas a promising source of induced pluripotent stem cellsrdquo PLoSONE vol 5 no 9 Article ID e12743 12 pages 2010

[7] S C Bendall M H Stewart P Menendez et al ldquoIGF andFGF cooperatively establish the regulatory stem cell niche ofpluripotent human cells in vitrordquo Nature vol 448 no 7157 pp1015ndash1021 2007

[8] C Ide Y Nakai N Nakano et al ldquoBone marrow stromal celltransplantation for treatment of sub-acute spinal cord injury inthe ratrdquo Brain Research vol 1332 pp 32ndash47 2010

[9] T Takebe K SekineM Enomura et al ldquoVascularized and func-tional human liver from an iPSC-derived organ bud transplantrdquoNature vol 499 no 7459 pp 481ndash484 2013

[10] M Eiraku K Watanabe M Matsuo-Takasaki et al ldquoSelf-organized formation of polarized cortical tissues fromESCs andits active manipulation by extrinsic signalsrdquo Cell Stem Cell vol3 no 5 pp 519ndash532 2008

[11] M Kawakami H Ishikawa T Tachibana A Tanaka andI Mataga ldquoFunctional transplantation of salivary gland cellsdifferentiated from mouse early ES cells in vitrordquo Human Cellvol 26 no 2 pp 80ndash90 2013

[12] K Mishima H Inoue T Nishiyama et al ldquoTransplantation ofside population cells restores the function of damaged exocrineglands through clusterinrdquo Stem Cells vol 30 no 9 pp 1925ndash1937 2012

[13] J Mima A Koshino K Oka et al ldquoRegulation of the epithelialadhesion molecule CEACAM1 is important for palate forma-tionrdquo PLoS ONE vol 8 no 4 Article ID e61653 2013

[14] V N Pateland andM PHoffman ldquoSalivary gland developmenta template for regenerationrdquo Seminars in Cell amp DevelopmentalBiology vol 25-26 pp 52ndash60 2014

[15] S B Jensen A M Pedersen J Reibel and B NauntofteldquoXerostomia and hypofunction of the salivary glands in cancertherapyrdquo Supportive Care in Cancer vol 11 no 4 pp 207ndash2252003

[16] I M A Lombaert and M P Hoffman ldquoEpithelialstemprogenitor cells in the embryonic mouse submandibularglandrdquo Frontiers of Oral Biology vol 14 pp 90ndash106 2010

[17] A Angelova Volponi M Kawasaki and P T Sharpe ldquoAdulthuman gingival epithelial cells as a source for whole-toothbioengineeringrdquo Journal of Dental Research vol 92 no 4 pp329ndash334 2013

[18] A V Vanikar H L Trivedi S C Gopal A Kumar and SD Dave ldquoPre-transplant co-infusion of donor-adipose tissuederived mesenchymal stem cells and hematopoietic stem cells

BioMed Research International 11

may help in achieving tolerance in living donor renal transplan-tationrdquo Renal Failure vol 36 no 3 pp 457ndash460 2014

[19] J P M Issa R Tiossi A S D S Mello R A Lopes M A S DiMatteo and M M Iyomasa ldquoPRP a possibility in regenerativetherapyrdquo International Journal of Morphology vol 25 no 3 pp587ndash590 2007

[20] Y Sumita Y Liu S Khalili et al ldquoBone marrow-derived cellsrescue salivary gland function in mice with head and neckirradiationrdquo The International Journal of Biochemistry amp CellBiology vol 43 no 1 pp 80ndash87 2011

[21] B Song A M Smink C V Jones et al ldquoThe directeddifferentiation of human iPS cells into kidney podocytesrdquo PLoSONE vol 7 no 9 Article ID e46453 2012

[22] H Saito M Takeuchi K Chida and A Miyajima ldquoGenerationof glucose-responsive functional islets with a three-dimensionalstructure frommouse fetal pancreatic cells and ips cells in vitrordquoPLoS ONE vol 6 no 12 Article ID e28209 2011

[23] D A Nelson C Manhardt V Kamath et al ldquoQuantitativesingle cell analysis of cell population dynamics during sub-mandibular salivary gland development and differentiationrdquoBiology Open vol 2 no 5 pp 439ndash447 2013

[24] C Wei M Larsen M P Hoffman and K M Yamada ldquoSelf-organization and branchingmorphogenesis of primary salivaryepithelial cellsrdquo Tissue Engineering vol 13 no 4 pp 721ndash7352007

[25] TMisteli ldquoThe concept of self-organization in cellular architec-turerdquo Journal of Cell Biology vol 155 no 2 pp 181ndash185 2001

[26] L S Y Nanduri I M A Lombaert M van der Zwaaget al ldquoSalisphere derived c-Kit+ cell transplantation restorestissue homeostasis in irradiated salivary glandrdquo Radiotherapy ampOncology vol 108 no 3 pp 458ndash463 2013

[27] J C-F Hsu and K M Yamada ldquoSalivary gland branchingmorphogenesismdashrecent progress and future opportunitiesrdquoInternational Journal of Oral Science vol 2 no 3 pp 117ndash1262010

[28] MHirayamaMOgawaMOshima et al ldquoFunctional lacrimalgland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2497 2013

[29] M Ogawa M Oshima A Imamura et al ldquoFunctional salivarygland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2498 2013

[30] K Ishibashi S Hara and S Kondo ldquoAquaporin water channelsin mammalsrdquo Clinical and Experimental Nephrology vol 13 no2 pp 107ndash117 2009

[31] H S Larsen M H Aure S B Peters M Larsen E BMesselt and H Kanli Galtung ldquoLocalization of AQP5 duringdevelopment of the mouse submandibular salivary glandrdquoJournal of Molecular Histology vol 42 no 1 pp 71ndash81 2011

[32] J E Fata Z Werb and M J Bissell ldquoRegulation of mammarygland branchingmorphogenesis by the extracellular matrix andits remodeling enzymesrdquo Breast Cancer Research vol 6 no 1pp 1ndash11 2004

[33] T Sakai M Larsen and K M Yamada ldquoFibronectin require-ment in branching morphogenesisrdquo Nature vol 423 no 6942pp 876ndash881 2003

[34] T Onodera T Sakai J C-F Hsu K Matsumoto J A ChioriniandKMYamada ldquoBtbd7 regulates epithelial cell dynamics andbranching morphogenesisrdquo Science vol 329 no 5991 pp 562ndash565 2010

BioMed Research International 7

E135 E175E155 Ad

Sox2

Sox2

E-c

ad

E-ca

d

iSGSG

Sox2

Sox2

GFP

E-c

ad

(a)

(b)

Figure 6 Distribution of Sox2 in developing SMG and in regenerated SMGs (SG and iSG) Sox2 is expressed in the cytoplasm of bothepithelial cells and mesenchymal cells in E135 E155 and E175 developing SMGs and is gradually decreased in later embryonic stages Sox2is expressed in the nucleus of epithelial cells in both postnatal and adult SMGs Sox2 (cyan) and E-cadherin (red) (a) Scale bar 25 120583m Sox2expression in the cytoplasm of both epithelial cells and mesenchymal cells in iSG (20 iSG) is less than that in SG The nucleus of GFP-iPScells highly expressed Sox2 as a positive control Sox2 (cyan) GFP (green) and E-cadherin (red) (b) Scale bar 25 120583m

8 BioMed Research International

AQP5

AQP5

E-c

ad

E-ca

d

E135 E175E155 Ad

iSGSG(a)

(b)

AQP5

AQP5

GFP

E-c

ad

Figure 7 Distribution of AQP5 in developing SMG and regenerated SMGs (SG and iSG) AQP5 is gradually increased in the epithelial cellsof SMGs in E135 E155 E175 and adult AQP5 is expressed in the cell membrane of epithelial cells in iSG (20 iSG) more than that in SGAQP5 (white) and E-cadherin (red) (a) Scale bar 25 120583m Distribution on AQP5 in regenerated SMG (b) AQP5 (white) GFP (green) andE-cadherin (red) Scale bar 25 120583m

BioMed Research International 9

Sox2

SG iSG

100

0

60

40

80

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

c-Myc

100

0

60

40

80

lowastlowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

Nanog

100

0

60

40

80

lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG0

200

100

Klf4lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

AQP5

0

200

100

300

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

Amy

Not detected

Relat

ive e

xpre

ssio

n (

)

(a) (b) (c) (d)

(e) (f) (g)

SG iSG

M3R

Not detected

Relat

ive e

xpre

ssio

n (

)

Figure 8 Gene expression of salivary gland cells in regenerated SMGs between SG and iSG Gene expression of Sox2 (a) c-Myc (b) andNanog (c) was decreased in iSG and gene expression of Klf4 (d) was increased in iSG Aqp5 (e) gene expression was increased in iSG butAmy (f) andM3r (g) gene expression were not detected by qPCR similar to early embryonic SMG cells lowastlowast119901 lt 001 versus control

salivary gland cells needed to be transplanted to mouse [2829]

Because the water channel protein AQP5 is found inthe lumen of the acinar-like structures regenerated sali-vary glands may have an ability to secrete saliva [30 31]Morphological analysis of the regenerated salivary glandsindicated that the difference between SG and iSG was notstatistically significant However iSG had a larger number ofsmall acinar-like structures more than that in SG (Figure 9)Developing salivary glands in epithelial tissue increases thesurface area by branching morphogenesis and increases theability of saliva secretion [32ndash34] Coculture of embryonicSMG cells and iPS cells had more developed epithelialstructures than that in monoculture of embryonic SMG cells

Previous studies show the gene expression of stem cellmarkers (Sox2 c-Myc Nanog and Klf4) in E13 E15 and adult

(Ad) SMGs Sox2 c-Myc and Nanog decreased during SMGdevelopment but Klf4 increased [16] The submandibularmarker AQP5 belongs to a family of water channel proteinsthat allow water to pass through the plasma membrane byosmosis E14 SMG expressed AQP5 as a first indication [31]Our results showed that the gene expressions of Sox2 c-Myc and Nanog were decreased in iSG and that the geneexpression of Klf4 and Aqp5 was increased in iSG Immuno-histochemical analysis also showed that the undifferentiatedmarker of salivary gland cells decreased in iSG comparedwith that in SG It was suggested that iPS cells induce thecharacteristic differentiation resulting in the morphologicalchanges observed in salivary gland cell formation Theseresults indicate that iPS cells have a potential ability toaccelerate differentiation of salivary gland development andregeneration Regenerated salivary glands produced from

10 BioMed Research International

iPS cell

Acinar-like structure

iSGSGRegenerated salivary gland

Sox2 c-Myc NanogAqp5

Figure 9 Effect of iPS cells on aggregation of SMG cells iPS cells(green balls) reduced the size of the epithelial tissue but acinar-likestructure (orange balls) increased their number iPS cells cannotmixcompletely with SMG cells and instead surround the epitheliumof SMG Stem cell markers such as Sox2 c-Myc and Nanog aredecreased but Aqp5 is increased in iSG

E135 SMG cells were cultured for four daysAmy andM3r areusually expressed after birth Amy and M3r gene expressionalso occurred in the regenerated salivary gland cells

Various approaches would be necessary to organize thecomplex tissues such as the salivary gland kidney and lungOur finding that coculture of salivary glands cells with iPScells formed differentiated salivary glands is significant andfuture elucidation of the mechanism could lead to viableregeneration therapy of functional organs using iPS cellsOur study provides new insights for future research into theregeneration of organs such as salivary glands

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HitomiOno andAyaObana contributed equally to this work

Acknowledgments

This work was supported by JSPS KAKENHI Grant no24390452 The authors thank Dr Kenji Mishima Division ofPathology Department of Oral Diagnostic Sciences Schoolof Dentistry Showa University for helpful advice regard-ing salivary markers The authors also thank Dr HirokoOkawa Department of Fixed Prosthodontics Osaka Uni-versity Graduate School of Dentistry for excellent technicalsupport in iPS cell cultures

References

[1] S P Humphrey and R T Williamson ldquoA review of salivanormal composition flow and functionrdquo Journal of ProstheticDentistry vol 85 no 2 pp 162ndash169 2001

[2] S B Jensen A M L Pedersen A Vissink et al ldquoA systematicreview of salivary gland hypofunction and xerostomia inducedby cancer therapies management strategies and economicimpactrdquo Supportive Care in Cancer vol 18 no 8 pp 1061ndash10792010

[3] J-Y Lim J C Ra I S Shin et al ldquoSystemic transplantationof human adipose tissue-derived mesenchymal stem cells forthe regeneration of irradiation-induced salivary gland damagerdquoPLoS ONE vol 8 no 8 Article ID e71167 2013

[4] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007

[5] H Kamao M Mandai S Okamoto et al ldquoCharacterization ofhuman induced pluripotent stem cell-derived retinal pigmentepithelium cell sheets aiming for clinical applicationrdquo Stem CellReports vol 2 no 2 pp 205ndash218 2014

[6] H Egusa K Okita H Kayashima et al ldquoGingival fibroblastsas a promising source of induced pluripotent stem cellsrdquo PLoSONE vol 5 no 9 Article ID e12743 12 pages 2010

[7] S C Bendall M H Stewart P Menendez et al ldquoIGF andFGF cooperatively establish the regulatory stem cell niche ofpluripotent human cells in vitrordquo Nature vol 448 no 7157 pp1015ndash1021 2007

[8] C Ide Y Nakai N Nakano et al ldquoBone marrow stromal celltransplantation for treatment of sub-acute spinal cord injury inthe ratrdquo Brain Research vol 1332 pp 32ndash47 2010

[9] T Takebe K SekineM Enomura et al ldquoVascularized and func-tional human liver from an iPSC-derived organ bud transplantrdquoNature vol 499 no 7459 pp 481ndash484 2013

[10] M Eiraku K Watanabe M Matsuo-Takasaki et al ldquoSelf-organized formation of polarized cortical tissues fromESCs andits active manipulation by extrinsic signalsrdquo Cell Stem Cell vol3 no 5 pp 519ndash532 2008

[11] M Kawakami H Ishikawa T Tachibana A Tanaka andI Mataga ldquoFunctional transplantation of salivary gland cellsdifferentiated from mouse early ES cells in vitrordquo Human Cellvol 26 no 2 pp 80ndash90 2013

[12] K Mishima H Inoue T Nishiyama et al ldquoTransplantation ofside population cells restores the function of damaged exocrineglands through clusterinrdquo Stem Cells vol 30 no 9 pp 1925ndash1937 2012

[13] J Mima A Koshino K Oka et al ldquoRegulation of the epithelialadhesion molecule CEACAM1 is important for palate forma-tionrdquo PLoS ONE vol 8 no 4 Article ID e61653 2013

[14] V N Pateland andM PHoffman ldquoSalivary gland developmenta template for regenerationrdquo Seminars in Cell amp DevelopmentalBiology vol 25-26 pp 52ndash60 2014

[15] S B Jensen A M Pedersen J Reibel and B NauntofteldquoXerostomia and hypofunction of the salivary glands in cancertherapyrdquo Supportive Care in Cancer vol 11 no 4 pp 207ndash2252003

[16] I M A Lombaert and M P Hoffman ldquoEpithelialstemprogenitor cells in the embryonic mouse submandibularglandrdquo Frontiers of Oral Biology vol 14 pp 90ndash106 2010

[17] A Angelova Volponi M Kawasaki and P T Sharpe ldquoAdulthuman gingival epithelial cells as a source for whole-toothbioengineeringrdquo Journal of Dental Research vol 92 no 4 pp329ndash334 2013

[18] A V Vanikar H L Trivedi S C Gopal A Kumar and SD Dave ldquoPre-transplant co-infusion of donor-adipose tissuederived mesenchymal stem cells and hematopoietic stem cells

BioMed Research International 11

may help in achieving tolerance in living donor renal transplan-tationrdquo Renal Failure vol 36 no 3 pp 457ndash460 2014

[19] J P M Issa R Tiossi A S D S Mello R A Lopes M A S DiMatteo and M M Iyomasa ldquoPRP a possibility in regenerativetherapyrdquo International Journal of Morphology vol 25 no 3 pp587ndash590 2007

[20] Y Sumita Y Liu S Khalili et al ldquoBone marrow-derived cellsrescue salivary gland function in mice with head and neckirradiationrdquo The International Journal of Biochemistry amp CellBiology vol 43 no 1 pp 80ndash87 2011

[21] B Song A M Smink C V Jones et al ldquoThe directeddifferentiation of human iPS cells into kidney podocytesrdquo PLoSONE vol 7 no 9 Article ID e46453 2012

[22] H Saito M Takeuchi K Chida and A Miyajima ldquoGenerationof glucose-responsive functional islets with a three-dimensionalstructure frommouse fetal pancreatic cells and ips cells in vitrordquoPLoS ONE vol 6 no 12 Article ID e28209 2011

[23] D A Nelson C Manhardt V Kamath et al ldquoQuantitativesingle cell analysis of cell population dynamics during sub-mandibular salivary gland development and differentiationrdquoBiology Open vol 2 no 5 pp 439ndash447 2013

[24] C Wei M Larsen M P Hoffman and K M Yamada ldquoSelf-organization and branchingmorphogenesis of primary salivaryepithelial cellsrdquo Tissue Engineering vol 13 no 4 pp 721ndash7352007

[25] TMisteli ldquoThe concept of self-organization in cellular architec-turerdquo Journal of Cell Biology vol 155 no 2 pp 181ndash185 2001

[26] L S Y Nanduri I M A Lombaert M van der Zwaaget al ldquoSalisphere derived c-Kit+ cell transplantation restorestissue homeostasis in irradiated salivary glandrdquo Radiotherapy ampOncology vol 108 no 3 pp 458ndash463 2013

[27] J C-F Hsu and K M Yamada ldquoSalivary gland branchingmorphogenesismdashrecent progress and future opportunitiesrdquoInternational Journal of Oral Science vol 2 no 3 pp 117ndash1262010

[28] MHirayamaMOgawaMOshima et al ldquoFunctional lacrimalgland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2497 2013

[29] M Ogawa M Oshima A Imamura et al ldquoFunctional salivarygland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2498 2013

[30] K Ishibashi S Hara and S Kondo ldquoAquaporin water channelsin mammalsrdquo Clinical and Experimental Nephrology vol 13 no2 pp 107ndash117 2009

[31] H S Larsen M H Aure S B Peters M Larsen E BMesselt and H Kanli Galtung ldquoLocalization of AQP5 duringdevelopment of the mouse submandibular salivary glandrdquoJournal of Molecular Histology vol 42 no 1 pp 71ndash81 2011

[32] J E Fata Z Werb and M J Bissell ldquoRegulation of mammarygland branchingmorphogenesis by the extracellular matrix andits remodeling enzymesrdquo Breast Cancer Research vol 6 no 1pp 1ndash11 2004

[33] T Sakai M Larsen and K M Yamada ldquoFibronectin require-ment in branching morphogenesisrdquo Nature vol 423 no 6942pp 876ndash881 2003

[34] T Onodera T Sakai J C-F Hsu K Matsumoto J A ChioriniandKMYamada ldquoBtbd7 regulates epithelial cell dynamics andbranching morphogenesisrdquo Science vol 329 no 5991 pp 562ndash565 2010

8 BioMed Research International

AQP5

AQP5

E-c

ad

E-ca

d

E135 E175E155 Ad

iSGSG(a)

(b)

AQP5

AQP5

GFP

E-c

ad

Figure 7 Distribution of AQP5 in developing SMG and regenerated SMGs (SG and iSG) AQP5 is gradually increased in the epithelial cellsof SMGs in E135 E155 E175 and adult AQP5 is expressed in the cell membrane of epithelial cells in iSG (20 iSG) more than that in SGAQP5 (white) and E-cadherin (red) (a) Scale bar 25 120583m Distribution on AQP5 in regenerated SMG (b) AQP5 (white) GFP (green) andE-cadherin (red) Scale bar 25 120583m

BioMed Research International 9

Sox2

SG iSG

100

0

60

40

80

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

c-Myc

100

0

60

40

80

lowastlowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

Nanog

100

0

60

40

80

lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG0

200

100

Klf4lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

AQP5

0

200

100

300

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

Amy

Not detected

Relat

ive e

xpre

ssio

n (

)

(a) (b) (c) (d)

(e) (f) (g)

SG iSG

M3R

Not detected

Relat

ive e

xpre

ssio

n (

)

Figure 8 Gene expression of salivary gland cells in regenerated SMGs between SG and iSG Gene expression of Sox2 (a) c-Myc (b) andNanog (c) was decreased in iSG and gene expression of Klf4 (d) was increased in iSG Aqp5 (e) gene expression was increased in iSG butAmy (f) andM3r (g) gene expression were not detected by qPCR similar to early embryonic SMG cells lowastlowast119901 lt 001 versus control

salivary gland cells needed to be transplanted to mouse [2829]

Because the water channel protein AQP5 is found inthe lumen of the acinar-like structures regenerated sali-vary glands may have an ability to secrete saliva [30 31]Morphological analysis of the regenerated salivary glandsindicated that the difference between SG and iSG was notstatistically significant However iSG had a larger number ofsmall acinar-like structures more than that in SG (Figure 9)Developing salivary glands in epithelial tissue increases thesurface area by branching morphogenesis and increases theability of saliva secretion [32ndash34] Coculture of embryonicSMG cells and iPS cells had more developed epithelialstructures than that in monoculture of embryonic SMG cells

Previous studies show the gene expression of stem cellmarkers (Sox2 c-Myc Nanog and Klf4) in E13 E15 and adult

(Ad) SMGs Sox2 c-Myc and Nanog decreased during SMGdevelopment but Klf4 increased [16] The submandibularmarker AQP5 belongs to a family of water channel proteinsthat allow water to pass through the plasma membrane byosmosis E14 SMG expressed AQP5 as a first indication [31]Our results showed that the gene expressions of Sox2 c-Myc and Nanog were decreased in iSG and that the geneexpression of Klf4 and Aqp5 was increased in iSG Immuno-histochemical analysis also showed that the undifferentiatedmarker of salivary gland cells decreased in iSG comparedwith that in SG It was suggested that iPS cells induce thecharacteristic differentiation resulting in the morphologicalchanges observed in salivary gland cell formation Theseresults indicate that iPS cells have a potential ability toaccelerate differentiation of salivary gland development andregeneration Regenerated salivary glands produced from

10 BioMed Research International

iPS cell

Acinar-like structure

iSGSGRegenerated salivary gland

Sox2 c-Myc NanogAqp5

Figure 9 Effect of iPS cells on aggregation of SMG cells iPS cells(green balls) reduced the size of the epithelial tissue but acinar-likestructure (orange balls) increased their number iPS cells cannotmixcompletely with SMG cells and instead surround the epitheliumof SMG Stem cell markers such as Sox2 c-Myc and Nanog aredecreased but Aqp5 is increased in iSG

E135 SMG cells were cultured for four daysAmy andM3r areusually expressed after birth Amy and M3r gene expressionalso occurred in the regenerated salivary gland cells

Various approaches would be necessary to organize thecomplex tissues such as the salivary gland kidney and lungOur finding that coculture of salivary glands cells with iPScells formed differentiated salivary glands is significant andfuture elucidation of the mechanism could lead to viableregeneration therapy of functional organs using iPS cellsOur study provides new insights for future research into theregeneration of organs such as salivary glands

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HitomiOno andAyaObana contributed equally to this work

Acknowledgments

This work was supported by JSPS KAKENHI Grant no24390452 The authors thank Dr Kenji Mishima Division ofPathology Department of Oral Diagnostic Sciences Schoolof Dentistry Showa University for helpful advice regard-ing salivary markers The authors also thank Dr HirokoOkawa Department of Fixed Prosthodontics Osaka Uni-versity Graduate School of Dentistry for excellent technicalsupport in iPS cell cultures

References

[1] S P Humphrey and R T Williamson ldquoA review of salivanormal composition flow and functionrdquo Journal of ProstheticDentistry vol 85 no 2 pp 162ndash169 2001

[2] S B Jensen A M L Pedersen A Vissink et al ldquoA systematicreview of salivary gland hypofunction and xerostomia inducedby cancer therapies management strategies and economicimpactrdquo Supportive Care in Cancer vol 18 no 8 pp 1061ndash10792010

[3] J-Y Lim J C Ra I S Shin et al ldquoSystemic transplantationof human adipose tissue-derived mesenchymal stem cells forthe regeneration of irradiation-induced salivary gland damagerdquoPLoS ONE vol 8 no 8 Article ID e71167 2013

[4] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007

[5] H Kamao M Mandai S Okamoto et al ldquoCharacterization ofhuman induced pluripotent stem cell-derived retinal pigmentepithelium cell sheets aiming for clinical applicationrdquo Stem CellReports vol 2 no 2 pp 205ndash218 2014

[6] H Egusa K Okita H Kayashima et al ldquoGingival fibroblastsas a promising source of induced pluripotent stem cellsrdquo PLoSONE vol 5 no 9 Article ID e12743 12 pages 2010

[7] S C Bendall M H Stewart P Menendez et al ldquoIGF andFGF cooperatively establish the regulatory stem cell niche ofpluripotent human cells in vitrordquo Nature vol 448 no 7157 pp1015ndash1021 2007

[8] C Ide Y Nakai N Nakano et al ldquoBone marrow stromal celltransplantation for treatment of sub-acute spinal cord injury inthe ratrdquo Brain Research vol 1332 pp 32ndash47 2010

[9] T Takebe K SekineM Enomura et al ldquoVascularized and func-tional human liver from an iPSC-derived organ bud transplantrdquoNature vol 499 no 7459 pp 481ndash484 2013

[10] M Eiraku K Watanabe M Matsuo-Takasaki et al ldquoSelf-organized formation of polarized cortical tissues fromESCs andits active manipulation by extrinsic signalsrdquo Cell Stem Cell vol3 no 5 pp 519ndash532 2008

[11] M Kawakami H Ishikawa T Tachibana A Tanaka andI Mataga ldquoFunctional transplantation of salivary gland cellsdifferentiated from mouse early ES cells in vitrordquo Human Cellvol 26 no 2 pp 80ndash90 2013

[12] K Mishima H Inoue T Nishiyama et al ldquoTransplantation ofside population cells restores the function of damaged exocrineglands through clusterinrdquo Stem Cells vol 30 no 9 pp 1925ndash1937 2012

[13] J Mima A Koshino K Oka et al ldquoRegulation of the epithelialadhesion molecule CEACAM1 is important for palate forma-tionrdquo PLoS ONE vol 8 no 4 Article ID e61653 2013

[14] V N Pateland andM PHoffman ldquoSalivary gland developmenta template for regenerationrdquo Seminars in Cell amp DevelopmentalBiology vol 25-26 pp 52ndash60 2014

[15] S B Jensen A M Pedersen J Reibel and B NauntofteldquoXerostomia and hypofunction of the salivary glands in cancertherapyrdquo Supportive Care in Cancer vol 11 no 4 pp 207ndash2252003

[16] I M A Lombaert and M P Hoffman ldquoEpithelialstemprogenitor cells in the embryonic mouse submandibularglandrdquo Frontiers of Oral Biology vol 14 pp 90ndash106 2010

[17] A Angelova Volponi M Kawasaki and P T Sharpe ldquoAdulthuman gingival epithelial cells as a source for whole-toothbioengineeringrdquo Journal of Dental Research vol 92 no 4 pp329ndash334 2013

[18] A V Vanikar H L Trivedi S C Gopal A Kumar and SD Dave ldquoPre-transplant co-infusion of donor-adipose tissuederived mesenchymal stem cells and hematopoietic stem cells

BioMed Research International 11

may help in achieving tolerance in living donor renal transplan-tationrdquo Renal Failure vol 36 no 3 pp 457ndash460 2014

[19] J P M Issa R Tiossi A S D S Mello R A Lopes M A S DiMatteo and M M Iyomasa ldquoPRP a possibility in regenerativetherapyrdquo International Journal of Morphology vol 25 no 3 pp587ndash590 2007

[20] Y Sumita Y Liu S Khalili et al ldquoBone marrow-derived cellsrescue salivary gland function in mice with head and neckirradiationrdquo The International Journal of Biochemistry amp CellBiology vol 43 no 1 pp 80ndash87 2011

[21] B Song A M Smink C V Jones et al ldquoThe directeddifferentiation of human iPS cells into kidney podocytesrdquo PLoSONE vol 7 no 9 Article ID e46453 2012

[22] H Saito M Takeuchi K Chida and A Miyajima ldquoGenerationof glucose-responsive functional islets with a three-dimensionalstructure frommouse fetal pancreatic cells and ips cells in vitrordquoPLoS ONE vol 6 no 12 Article ID e28209 2011

[23] D A Nelson C Manhardt V Kamath et al ldquoQuantitativesingle cell analysis of cell population dynamics during sub-mandibular salivary gland development and differentiationrdquoBiology Open vol 2 no 5 pp 439ndash447 2013

[24] C Wei M Larsen M P Hoffman and K M Yamada ldquoSelf-organization and branchingmorphogenesis of primary salivaryepithelial cellsrdquo Tissue Engineering vol 13 no 4 pp 721ndash7352007

[25] TMisteli ldquoThe concept of self-organization in cellular architec-turerdquo Journal of Cell Biology vol 155 no 2 pp 181ndash185 2001

[26] L S Y Nanduri I M A Lombaert M van der Zwaaget al ldquoSalisphere derived c-Kit+ cell transplantation restorestissue homeostasis in irradiated salivary glandrdquo Radiotherapy ampOncology vol 108 no 3 pp 458ndash463 2013

[27] J C-F Hsu and K M Yamada ldquoSalivary gland branchingmorphogenesismdashrecent progress and future opportunitiesrdquoInternational Journal of Oral Science vol 2 no 3 pp 117ndash1262010

[28] MHirayamaMOgawaMOshima et al ldquoFunctional lacrimalgland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2497 2013

[29] M Ogawa M Oshima A Imamura et al ldquoFunctional salivarygland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2498 2013

[30] K Ishibashi S Hara and S Kondo ldquoAquaporin water channelsin mammalsrdquo Clinical and Experimental Nephrology vol 13 no2 pp 107ndash117 2009

[31] H S Larsen M H Aure S B Peters M Larsen E BMesselt and H Kanli Galtung ldquoLocalization of AQP5 duringdevelopment of the mouse submandibular salivary glandrdquoJournal of Molecular Histology vol 42 no 1 pp 71ndash81 2011

[32] J E Fata Z Werb and M J Bissell ldquoRegulation of mammarygland branchingmorphogenesis by the extracellular matrix andits remodeling enzymesrdquo Breast Cancer Research vol 6 no 1pp 1ndash11 2004

[33] T Sakai M Larsen and K M Yamada ldquoFibronectin require-ment in branching morphogenesisrdquo Nature vol 423 no 6942pp 876ndash881 2003

[34] T Onodera T Sakai J C-F Hsu K Matsumoto J A ChioriniandKMYamada ldquoBtbd7 regulates epithelial cell dynamics andbranching morphogenesisrdquo Science vol 329 no 5991 pp 562ndash565 2010

BioMed Research International 9

Sox2

SG iSG

100

0

60

40

80

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

c-Myc

100

0

60

40

80

lowastlowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

Nanog

100

0

60

40

80

lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG0

200

100

Klf4lowastlowast

Relat

ive e

xpre

ssio

n (

)

SG iSG

AQP5

0

200

100

300

Relat

ive e

xpre

ssio

n (

)

lowastlowast

SG iSG

Amy

Not detected

Relat

ive e

xpre

ssio

n (

)

(a) (b) (c) (d)

(e) (f) (g)

SG iSG

M3R

Not detected

Relat

ive e

xpre

ssio

n (

)

Figure 8 Gene expression of salivary gland cells in regenerated SMGs between SG and iSG Gene expression of Sox2 (a) c-Myc (b) andNanog (c) was decreased in iSG and gene expression of Klf4 (d) was increased in iSG Aqp5 (e) gene expression was increased in iSG butAmy (f) andM3r (g) gene expression were not detected by qPCR similar to early embryonic SMG cells lowastlowast119901 lt 001 versus control

salivary gland cells needed to be transplanted to mouse [2829]

Because the water channel protein AQP5 is found inthe lumen of the acinar-like structures regenerated sali-vary glands may have an ability to secrete saliva [30 31]Morphological analysis of the regenerated salivary glandsindicated that the difference between SG and iSG was notstatistically significant However iSG had a larger number ofsmall acinar-like structures more than that in SG (Figure 9)Developing salivary glands in epithelial tissue increases thesurface area by branching morphogenesis and increases theability of saliva secretion [32ndash34] Coculture of embryonicSMG cells and iPS cells had more developed epithelialstructures than that in monoculture of embryonic SMG cells

Previous studies show the gene expression of stem cellmarkers (Sox2 c-Myc Nanog and Klf4) in E13 E15 and adult

(Ad) SMGs Sox2 c-Myc and Nanog decreased during SMGdevelopment but Klf4 increased [16] The submandibularmarker AQP5 belongs to a family of water channel proteinsthat allow water to pass through the plasma membrane byosmosis E14 SMG expressed AQP5 as a first indication [31]Our results showed that the gene expressions of Sox2 c-Myc and Nanog were decreased in iSG and that the geneexpression of Klf4 and Aqp5 was increased in iSG Immuno-histochemical analysis also showed that the undifferentiatedmarker of salivary gland cells decreased in iSG comparedwith that in SG It was suggested that iPS cells induce thecharacteristic differentiation resulting in the morphologicalchanges observed in salivary gland cell formation Theseresults indicate that iPS cells have a potential ability toaccelerate differentiation of salivary gland development andregeneration Regenerated salivary glands produced from

10 BioMed Research International

iPS cell

Acinar-like structure

iSGSGRegenerated salivary gland

Sox2 c-Myc NanogAqp5

Figure 9 Effect of iPS cells on aggregation of SMG cells iPS cells(green balls) reduced the size of the epithelial tissue but acinar-likestructure (orange balls) increased their number iPS cells cannotmixcompletely with SMG cells and instead surround the epitheliumof SMG Stem cell markers such as Sox2 c-Myc and Nanog aredecreased but Aqp5 is increased in iSG

E135 SMG cells were cultured for four daysAmy andM3r areusually expressed after birth Amy and M3r gene expressionalso occurred in the regenerated salivary gland cells

Various approaches would be necessary to organize thecomplex tissues such as the salivary gland kidney and lungOur finding that coculture of salivary glands cells with iPScells formed differentiated salivary glands is significant andfuture elucidation of the mechanism could lead to viableregeneration therapy of functional organs using iPS cellsOur study provides new insights for future research into theregeneration of organs such as salivary glands

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HitomiOno andAyaObana contributed equally to this work

Acknowledgments

This work was supported by JSPS KAKENHI Grant no24390452 The authors thank Dr Kenji Mishima Division ofPathology Department of Oral Diagnostic Sciences Schoolof Dentistry Showa University for helpful advice regard-ing salivary markers The authors also thank Dr HirokoOkawa Department of Fixed Prosthodontics Osaka Uni-versity Graduate School of Dentistry for excellent technicalsupport in iPS cell cultures

References

[1] S P Humphrey and R T Williamson ldquoA review of salivanormal composition flow and functionrdquo Journal of ProstheticDentistry vol 85 no 2 pp 162ndash169 2001

[2] S B Jensen A M L Pedersen A Vissink et al ldquoA systematicreview of salivary gland hypofunction and xerostomia inducedby cancer therapies management strategies and economicimpactrdquo Supportive Care in Cancer vol 18 no 8 pp 1061ndash10792010

[3] J-Y Lim J C Ra I S Shin et al ldquoSystemic transplantationof human adipose tissue-derived mesenchymal stem cells forthe regeneration of irradiation-induced salivary gland damagerdquoPLoS ONE vol 8 no 8 Article ID e71167 2013

[4] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007

[5] H Kamao M Mandai S Okamoto et al ldquoCharacterization ofhuman induced pluripotent stem cell-derived retinal pigmentepithelium cell sheets aiming for clinical applicationrdquo Stem CellReports vol 2 no 2 pp 205ndash218 2014

[6] H Egusa K Okita H Kayashima et al ldquoGingival fibroblastsas a promising source of induced pluripotent stem cellsrdquo PLoSONE vol 5 no 9 Article ID e12743 12 pages 2010

[7] S C Bendall M H Stewart P Menendez et al ldquoIGF andFGF cooperatively establish the regulatory stem cell niche ofpluripotent human cells in vitrordquo Nature vol 448 no 7157 pp1015ndash1021 2007

[8] C Ide Y Nakai N Nakano et al ldquoBone marrow stromal celltransplantation for treatment of sub-acute spinal cord injury inthe ratrdquo Brain Research vol 1332 pp 32ndash47 2010

[9] T Takebe K SekineM Enomura et al ldquoVascularized and func-tional human liver from an iPSC-derived organ bud transplantrdquoNature vol 499 no 7459 pp 481ndash484 2013

[10] M Eiraku K Watanabe M Matsuo-Takasaki et al ldquoSelf-organized formation of polarized cortical tissues fromESCs andits active manipulation by extrinsic signalsrdquo Cell Stem Cell vol3 no 5 pp 519ndash532 2008

[11] M Kawakami H Ishikawa T Tachibana A Tanaka andI Mataga ldquoFunctional transplantation of salivary gland cellsdifferentiated from mouse early ES cells in vitrordquo Human Cellvol 26 no 2 pp 80ndash90 2013

[12] K Mishima H Inoue T Nishiyama et al ldquoTransplantation ofside population cells restores the function of damaged exocrineglands through clusterinrdquo Stem Cells vol 30 no 9 pp 1925ndash1937 2012

[13] J Mima A Koshino K Oka et al ldquoRegulation of the epithelialadhesion molecule CEACAM1 is important for palate forma-tionrdquo PLoS ONE vol 8 no 4 Article ID e61653 2013

[14] V N Pateland andM PHoffman ldquoSalivary gland developmenta template for regenerationrdquo Seminars in Cell amp DevelopmentalBiology vol 25-26 pp 52ndash60 2014

[15] S B Jensen A M Pedersen J Reibel and B NauntofteldquoXerostomia and hypofunction of the salivary glands in cancertherapyrdquo Supportive Care in Cancer vol 11 no 4 pp 207ndash2252003

[16] I M A Lombaert and M P Hoffman ldquoEpithelialstemprogenitor cells in the embryonic mouse submandibularglandrdquo Frontiers of Oral Biology vol 14 pp 90ndash106 2010

[17] A Angelova Volponi M Kawasaki and P T Sharpe ldquoAdulthuman gingival epithelial cells as a source for whole-toothbioengineeringrdquo Journal of Dental Research vol 92 no 4 pp329ndash334 2013

[18] A V Vanikar H L Trivedi S C Gopal A Kumar and SD Dave ldquoPre-transplant co-infusion of donor-adipose tissuederived mesenchymal stem cells and hematopoietic stem cells

BioMed Research International 11

may help in achieving tolerance in living donor renal transplan-tationrdquo Renal Failure vol 36 no 3 pp 457ndash460 2014

[19] J P M Issa R Tiossi A S D S Mello R A Lopes M A S DiMatteo and M M Iyomasa ldquoPRP a possibility in regenerativetherapyrdquo International Journal of Morphology vol 25 no 3 pp587ndash590 2007

[20] Y Sumita Y Liu S Khalili et al ldquoBone marrow-derived cellsrescue salivary gland function in mice with head and neckirradiationrdquo The International Journal of Biochemistry amp CellBiology vol 43 no 1 pp 80ndash87 2011

[21] B Song A M Smink C V Jones et al ldquoThe directeddifferentiation of human iPS cells into kidney podocytesrdquo PLoSONE vol 7 no 9 Article ID e46453 2012

[22] H Saito M Takeuchi K Chida and A Miyajima ldquoGenerationof glucose-responsive functional islets with a three-dimensionalstructure frommouse fetal pancreatic cells and ips cells in vitrordquoPLoS ONE vol 6 no 12 Article ID e28209 2011

[23] D A Nelson C Manhardt V Kamath et al ldquoQuantitativesingle cell analysis of cell population dynamics during sub-mandibular salivary gland development and differentiationrdquoBiology Open vol 2 no 5 pp 439ndash447 2013

[24] C Wei M Larsen M P Hoffman and K M Yamada ldquoSelf-organization and branchingmorphogenesis of primary salivaryepithelial cellsrdquo Tissue Engineering vol 13 no 4 pp 721ndash7352007

[25] TMisteli ldquoThe concept of self-organization in cellular architec-turerdquo Journal of Cell Biology vol 155 no 2 pp 181ndash185 2001

[26] L S Y Nanduri I M A Lombaert M van der Zwaaget al ldquoSalisphere derived c-Kit+ cell transplantation restorestissue homeostasis in irradiated salivary glandrdquo Radiotherapy ampOncology vol 108 no 3 pp 458ndash463 2013

[27] J C-F Hsu and K M Yamada ldquoSalivary gland branchingmorphogenesismdashrecent progress and future opportunitiesrdquoInternational Journal of Oral Science vol 2 no 3 pp 117ndash1262010

[28] MHirayamaMOgawaMOshima et al ldquoFunctional lacrimalgland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2497 2013

[29] M Ogawa M Oshima A Imamura et al ldquoFunctional salivarygland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2498 2013

[30] K Ishibashi S Hara and S Kondo ldquoAquaporin water channelsin mammalsrdquo Clinical and Experimental Nephrology vol 13 no2 pp 107ndash117 2009

[31] H S Larsen M H Aure S B Peters M Larsen E BMesselt and H Kanli Galtung ldquoLocalization of AQP5 duringdevelopment of the mouse submandibular salivary glandrdquoJournal of Molecular Histology vol 42 no 1 pp 71ndash81 2011

[32] J E Fata Z Werb and M J Bissell ldquoRegulation of mammarygland branchingmorphogenesis by the extracellular matrix andits remodeling enzymesrdquo Breast Cancer Research vol 6 no 1pp 1ndash11 2004

[33] T Sakai M Larsen and K M Yamada ldquoFibronectin require-ment in branching morphogenesisrdquo Nature vol 423 no 6942pp 876ndash881 2003

[34] T Onodera T Sakai J C-F Hsu K Matsumoto J A ChioriniandKMYamada ldquoBtbd7 regulates epithelial cell dynamics andbranching morphogenesisrdquo Science vol 329 no 5991 pp 562ndash565 2010

10 BioMed Research International

iPS cell

Acinar-like structure

iSGSGRegenerated salivary gland

Sox2 c-Myc NanogAqp5

Figure 9 Effect of iPS cells on aggregation of SMG cells iPS cells(green balls) reduced the size of the epithelial tissue but acinar-likestructure (orange balls) increased their number iPS cells cannotmixcompletely with SMG cells and instead surround the epitheliumof SMG Stem cell markers such as Sox2 c-Myc and Nanog aredecreased but Aqp5 is increased in iSG

E135 SMG cells were cultured for four daysAmy andM3r areusually expressed after birth Amy and M3r gene expressionalso occurred in the regenerated salivary gland cells

Various approaches would be necessary to organize thecomplex tissues such as the salivary gland kidney and lungOur finding that coculture of salivary glands cells with iPScells formed differentiated salivary glands is significant andfuture elucidation of the mechanism could lead to viableregeneration therapy of functional organs using iPS cellsOur study provides new insights for future research into theregeneration of organs such as salivary glands

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HitomiOno andAyaObana contributed equally to this work

Acknowledgments

This work was supported by JSPS KAKENHI Grant no24390452 The authors thank Dr Kenji Mishima Division ofPathology Department of Oral Diagnostic Sciences Schoolof Dentistry Showa University for helpful advice regard-ing salivary markers The authors also thank Dr HirokoOkawa Department of Fixed Prosthodontics Osaka Uni-versity Graduate School of Dentistry for excellent technicalsupport in iPS cell cultures

References

[1] S P Humphrey and R T Williamson ldquoA review of salivanormal composition flow and functionrdquo Journal of ProstheticDentistry vol 85 no 2 pp 162ndash169 2001

[2] S B Jensen A M L Pedersen A Vissink et al ldquoA systematicreview of salivary gland hypofunction and xerostomia inducedby cancer therapies management strategies and economicimpactrdquo Supportive Care in Cancer vol 18 no 8 pp 1061ndash10792010

[3] J-Y Lim J C Ra I S Shin et al ldquoSystemic transplantationof human adipose tissue-derived mesenchymal stem cells forthe regeneration of irradiation-induced salivary gland damagerdquoPLoS ONE vol 8 no 8 Article ID e71167 2013

[4] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007

[5] H Kamao M Mandai S Okamoto et al ldquoCharacterization ofhuman induced pluripotent stem cell-derived retinal pigmentepithelium cell sheets aiming for clinical applicationrdquo Stem CellReports vol 2 no 2 pp 205ndash218 2014

[6] H Egusa K Okita H Kayashima et al ldquoGingival fibroblastsas a promising source of induced pluripotent stem cellsrdquo PLoSONE vol 5 no 9 Article ID e12743 12 pages 2010

[7] S C Bendall M H Stewart P Menendez et al ldquoIGF andFGF cooperatively establish the regulatory stem cell niche ofpluripotent human cells in vitrordquo Nature vol 448 no 7157 pp1015ndash1021 2007

[8] C Ide Y Nakai N Nakano et al ldquoBone marrow stromal celltransplantation for treatment of sub-acute spinal cord injury inthe ratrdquo Brain Research vol 1332 pp 32ndash47 2010

[9] T Takebe K SekineM Enomura et al ldquoVascularized and func-tional human liver from an iPSC-derived organ bud transplantrdquoNature vol 499 no 7459 pp 481ndash484 2013

[10] M Eiraku K Watanabe M Matsuo-Takasaki et al ldquoSelf-organized formation of polarized cortical tissues fromESCs andits active manipulation by extrinsic signalsrdquo Cell Stem Cell vol3 no 5 pp 519ndash532 2008

[11] M Kawakami H Ishikawa T Tachibana A Tanaka andI Mataga ldquoFunctional transplantation of salivary gland cellsdifferentiated from mouse early ES cells in vitrordquo Human Cellvol 26 no 2 pp 80ndash90 2013

[12] K Mishima H Inoue T Nishiyama et al ldquoTransplantation ofside population cells restores the function of damaged exocrineglands through clusterinrdquo Stem Cells vol 30 no 9 pp 1925ndash1937 2012

[13] J Mima A Koshino K Oka et al ldquoRegulation of the epithelialadhesion molecule CEACAM1 is important for palate forma-tionrdquo PLoS ONE vol 8 no 4 Article ID e61653 2013

[14] V N Pateland andM PHoffman ldquoSalivary gland developmenta template for regenerationrdquo Seminars in Cell amp DevelopmentalBiology vol 25-26 pp 52ndash60 2014

[15] S B Jensen A M Pedersen J Reibel and B NauntofteldquoXerostomia and hypofunction of the salivary glands in cancertherapyrdquo Supportive Care in Cancer vol 11 no 4 pp 207ndash2252003

[16] I M A Lombaert and M P Hoffman ldquoEpithelialstemprogenitor cells in the embryonic mouse submandibularglandrdquo Frontiers of Oral Biology vol 14 pp 90ndash106 2010

[17] A Angelova Volponi M Kawasaki and P T Sharpe ldquoAdulthuman gingival epithelial cells as a source for whole-toothbioengineeringrdquo Journal of Dental Research vol 92 no 4 pp329ndash334 2013

[18] A V Vanikar H L Trivedi S C Gopal A Kumar and SD Dave ldquoPre-transplant co-infusion of donor-adipose tissuederived mesenchymal stem cells and hematopoietic stem cells

BioMed Research International 11

may help in achieving tolerance in living donor renal transplan-tationrdquo Renal Failure vol 36 no 3 pp 457ndash460 2014

[19] J P M Issa R Tiossi A S D S Mello R A Lopes M A S DiMatteo and M M Iyomasa ldquoPRP a possibility in regenerativetherapyrdquo International Journal of Morphology vol 25 no 3 pp587ndash590 2007

[20] Y Sumita Y Liu S Khalili et al ldquoBone marrow-derived cellsrescue salivary gland function in mice with head and neckirradiationrdquo The International Journal of Biochemistry amp CellBiology vol 43 no 1 pp 80ndash87 2011

[21] B Song A M Smink C V Jones et al ldquoThe directeddifferentiation of human iPS cells into kidney podocytesrdquo PLoSONE vol 7 no 9 Article ID e46453 2012

[22] H Saito M Takeuchi K Chida and A Miyajima ldquoGenerationof glucose-responsive functional islets with a three-dimensionalstructure frommouse fetal pancreatic cells and ips cells in vitrordquoPLoS ONE vol 6 no 12 Article ID e28209 2011

[23] D A Nelson C Manhardt V Kamath et al ldquoQuantitativesingle cell analysis of cell population dynamics during sub-mandibular salivary gland development and differentiationrdquoBiology Open vol 2 no 5 pp 439ndash447 2013

[24] C Wei M Larsen M P Hoffman and K M Yamada ldquoSelf-organization and branchingmorphogenesis of primary salivaryepithelial cellsrdquo Tissue Engineering vol 13 no 4 pp 721ndash7352007

[25] TMisteli ldquoThe concept of self-organization in cellular architec-turerdquo Journal of Cell Biology vol 155 no 2 pp 181ndash185 2001

[26] L S Y Nanduri I M A Lombaert M van der Zwaaget al ldquoSalisphere derived c-Kit+ cell transplantation restorestissue homeostasis in irradiated salivary glandrdquo Radiotherapy ampOncology vol 108 no 3 pp 458ndash463 2013

[27] J C-F Hsu and K M Yamada ldquoSalivary gland branchingmorphogenesismdashrecent progress and future opportunitiesrdquoInternational Journal of Oral Science vol 2 no 3 pp 117ndash1262010

[28] MHirayamaMOgawaMOshima et al ldquoFunctional lacrimalgland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2497 2013

[29] M Ogawa M Oshima A Imamura et al ldquoFunctional salivarygland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2498 2013

[30] K Ishibashi S Hara and S Kondo ldquoAquaporin water channelsin mammalsrdquo Clinical and Experimental Nephrology vol 13 no2 pp 107ndash117 2009

[31] H S Larsen M H Aure S B Peters M Larsen E BMesselt and H Kanli Galtung ldquoLocalization of AQP5 duringdevelopment of the mouse submandibular salivary glandrdquoJournal of Molecular Histology vol 42 no 1 pp 71ndash81 2011

[32] J E Fata Z Werb and M J Bissell ldquoRegulation of mammarygland branchingmorphogenesis by the extracellular matrix andits remodeling enzymesrdquo Breast Cancer Research vol 6 no 1pp 1ndash11 2004

[33] T Sakai M Larsen and K M Yamada ldquoFibronectin require-ment in branching morphogenesisrdquo Nature vol 423 no 6942pp 876ndash881 2003

[34] T Onodera T Sakai J C-F Hsu K Matsumoto J A ChioriniandKMYamada ldquoBtbd7 regulates epithelial cell dynamics andbranching morphogenesisrdquo Science vol 329 no 5991 pp 562ndash565 2010

BioMed Research International 11

may help in achieving tolerance in living donor renal transplan-tationrdquo Renal Failure vol 36 no 3 pp 457ndash460 2014

[19] J P M Issa R Tiossi A S D S Mello R A Lopes M A S DiMatteo and M M Iyomasa ldquoPRP a possibility in regenerativetherapyrdquo International Journal of Morphology vol 25 no 3 pp587ndash590 2007

[20] Y Sumita Y Liu S Khalili et al ldquoBone marrow-derived cellsrescue salivary gland function in mice with head and neckirradiationrdquo The International Journal of Biochemistry amp CellBiology vol 43 no 1 pp 80ndash87 2011

[21] B Song A M Smink C V Jones et al ldquoThe directeddifferentiation of human iPS cells into kidney podocytesrdquo PLoSONE vol 7 no 9 Article ID e46453 2012

[22] H Saito M Takeuchi K Chida and A Miyajima ldquoGenerationof glucose-responsive functional islets with a three-dimensionalstructure frommouse fetal pancreatic cells and ips cells in vitrordquoPLoS ONE vol 6 no 12 Article ID e28209 2011

[23] D A Nelson C Manhardt V Kamath et al ldquoQuantitativesingle cell analysis of cell population dynamics during sub-mandibular salivary gland development and differentiationrdquoBiology Open vol 2 no 5 pp 439ndash447 2013

[24] C Wei M Larsen M P Hoffman and K M Yamada ldquoSelf-organization and branchingmorphogenesis of primary salivaryepithelial cellsrdquo Tissue Engineering vol 13 no 4 pp 721ndash7352007

[25] TMisteli ldquoThe concept of self-organization in cellular architec-turerdquo Journal of Cell Biology vol 155 no 2 pp 181ndash185 2001

[26] L S Y Nanduri I M A Lombaert M van der Zwaaget al ldquoSalisphere derived c-Kit+ cell transplantation restorestissue homeostasis in irradiated salivary glandrdquo Radiotherapy ampOncology vol 108 no 3 pp 458ndash463 2013

[27] J C-F Hsu and K M Yamada ldquoSalivary gland branchingmorphogenesismdashrecent progress and future opportunitiesrdquoInternational Journal of Oral Science vol 2 no 3 pp 117ndash1262010

[28] MHirayamaMOgawaMOshima et al ldquoFunctional lacrimalgland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2497 2013

[29] M Ogawa M Oshima A Imamura et al ldquoFunctional salivarygland regeneration by transplantation of a bioengineered organgermrdquo Nature Communications vol 4 article 2498 2013

[30] K Ishibashi S Hara and S Kondo ldquoAquaporin water channelsin mammalsrdquo Clinical and Experimental Nephrology vol 13 no2 pp 107ndash117 2009

[31] H S Larsen M H Aure S B Peters M Larsen E BMesselt and H Kanli Galtung ldquoLocalization of AQP5 duringdevelopment of the mouse submandibular salivary glandrdquoJournal of Molecular Histology vol 42 no 1 pp 71ndash81 2011

[32] J E Fata Z Werb and M J Bissell ldquoRegulation of mammarygland branchingmorphogenesis by the extracellular matrix andits remodeling enzymesrdquo Breast Cancer Research vol 6 no 1pp 1ndash11 2004

[33] T Sakai M Larsen and K M Yamada ldquoFibronectin require-ment in branching morphogenesisrdquo Nature vol 423 no 6942pp 876ndash881 2003

[34] T Onodera T Sakai J C-F Hsu K Matsumoto J A ChioriniandKMYamada ldquoBtbd7 regulates epithelial cell dynamics andbranching morphogenesisrdquo Science vol 329 no 5991 pp 562ndash565 2010