Matrigel improves functional properties of human submandibular salivary gland cell line

Page 1

Mg

Oa

b

a

ARRAA

KSMCEH

1

otsc1liv

H

os

C

C

1d

The International Journal of Biochemistry & Cell Biology 43 (2011) 622–631

Contents lists available at ScienceDirect

The International Journal of Biochemistry& Cell Biology

journa l homepage: www.e lsev ier .com/ locate /b ioce l

atrigel improves functional properties of human submandibular salivaryland cell line

la M. Mariaa,b,1, Osama Mariaa,2, Younan Liua,3, Svetlana V. Komarovaa,4, Simon D. Trana,∗

Faculty of Dentistry, McGill University, Montreal, Quebec H3A 2B2, CanadaFaculty of Dentistry, Mansoura University, Mansoura 35516, Egypt

r t i c l e i n f o

rticle history:eceived 30 July 2010eceived in revised form 3 December 2010ccepted 3 January 2011vailable online 7 January 2011

eywords:alivary glandatrigel

ell differentiation

a b s t r a c t

Sjogren’s syndrome and radiotherapy for head and neck cancers result in irreversible damage to functionalsalivary tissue, for which no adequate treatment is available. The microenvironment for salivary glandcell cytodifferentiation is critical for the future development of salivary gland regeneration, repair andtissue engineering treatments. Results from this study indicate that human submandibular cell line (HSG)cultured on Matrigel (2 mg/ml) could be induced to differentiate into polarized secretory acinar-likecells. The HSG cells grown on Matrigel were evaluated by physiological functional assays, molecularand immunohistochemistry, immunofluorescence, and morphological assessments. The results showed(1) a decrease in cell proliferation; (2) an increase in cell apoptosis; (3) cellular polarization evidentby transepithelial electrical resistance (TER), expressions of tight junction proteins (claudin-1, -2, -3, -4,

xtracellular matrixuman cell line

occludin, JAM-A, and ZO-1) and transmission electron microscopy (TEM); (4) an increase in the productionand/or secretion of acinar cell proteins, i.e., alpha-amylase, aquaporin-5, cytokeratins, and mucin-1, thatwere not associated with increases in mRNA transcription; (5) a decrease in vimentin expression; and (6)expression of potential stem cell biomarkers CD44 and CD166. The data indicated that Matrigel provideda suitable microenvironment for morphological and functional differentiation of HSG cells into 3D acinarlike cells. This study provides an in vitro model and baseline data on future developments of new strategies

ation

for salivary gland regener

. Introduction

Salivary tissue is a densely packed epithelium composed mainlyf acinar and ductal cells (Cook et al., 1994). Acinar cells representhe major glandular cell type that are salt-secreting and the onlyite of fluid movement (Baum, 1993; Cook et al., 1994) while ductalells are absorptive and relatively water-impermeable cells (Baum,

993; Cook et al., 1994). Both cell types are organized as a mono-

ayer around an extensively branching lumen that opens directlynto the oral cavity (Cook et al., 1994). Acinar secretory cells are irre-ersibly damaged following therapeutic irradiation for head and

∗ Corresponding author at: 3640 University Street, Room M43, Montreal, Quebec3A 2B2, Canada. Tel.: +1 514 398 7203x09182; fax: +1 514 398 8900.

E-mail addresses: [email protected] (O.M. Maria),[email protected] (O. Maria), [email protected] (Y. Liu),[email protected] (S.V. Komarova), [email protected] (S.D. Tran).

1 Address: 3640 University Street, Room M33, Montreal, Quebec H3A 2B2, Canada.2 Address: 740 Dr. Penfield Avenue, Room 2301, Montreal, Quebec H3A 1A4,anada.3 Address: 3640 University Street, Room M36, Montreal, Quebec H3A 2B2, Canada.4 Address: 740 Dr. Penfield Avenue, Room 2201, Montreal, Quebec H3A 1A4,anada.

357-2725/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.biocel.2011.01.001

and replacement.© 2011 Elsevier Ltd. All rights reserved.

neck cancers or in association with the autoimmune exocrinopa-thy Sjogren’s syndrome (Fox et al., 1985; Fox and Maruyama, 1997).In the absence of adequate number of functioning acinar cells, thegeneration of salivary fluid is impossible. Patients afflicted withthis condition experience rampant dental caries, mucositis, can-didiasis, dysphagia, and considerable pain and discomfort (Baumand O’Connell, 1995; Fox et al., 1985, 1998, 2000; Kashima et al.,1965). Currently, there is no satisfactory therapy for such patients.One therapeutic strategy we have been working on is to develop atissue-engineered artificial salivary gland device (Baum and Tran,2006). In an earlier report, we found that the human submandibulargland (HSG) cell line was unsuitable as an allogeneic graft cells inour envisioned salivary gland device because these cells were inca-pable of forming a polarized epithelial layer, did not express tightjunctions (TJs), and consequently had a weak transepithelial elec-trical resistance (TER) and no control in water movement (Aframianet al., 2002). Even after stable transfection of HSG cells with cDNAsencoding claudin-1 or claudin-2, these cells were unable to form

TJs. Since that report, our group has found other sources of graftcells that could form TJs, such as the primary salivary cells obtainedfrom human submandibular glands (huSMGs) and from rhesusparotid glands (RPGs) (Tran et al., 2005, 2006). However a limitationof all these cells (HSG, huSMG, and RPG cells) was their inability to

Page 2

of Bioc

bodt

tAcOdodeda1dmtskaeea2swir2rc

tmt(Mmia

2

2

vM1pT3ctiHsmaeesD

O.M. Maria et al. / The International Journal

e grown as acinar cells. huSMGs and RPGs retained at most 5–10%f acinar cells during culture and the remaining 90–95% were of auctal phenotype. The aim of this paper was to develop a modelhat would allow salivary acinar cell differentiation.

Maintenance of an acinar phenotype in primary salivary cell cul-ures has been proved to be difficult (Redman and Quissell, 1993).

critical factor required for maintaining an acinar phenotype inulture is the presence of an extracellular matrix (Durban, 1990;liver et al., 1987). The HSG cell line is derived from intercalateduct cells (Shirasuna et al., 1981). During salivary gland devel-pment, intercalated duct cells are believed to be stem cells ofuctal, acinar and myoepithelial cell types (Batsakis, 1980; Dennyt al., 1997; Eversole, 1971; Pierce, 1974). HSG cells with variousifferentiation inducers results in generation of myoepithelial orcinar cells (Azuma et al., 1986; Hayashi et al., 1985; Sato et al.,987; Yoshida et al., 1986;). Many trials have been reported usingifferent salivary cell lines; Motegi et al. (2005) reported that treat-ent of ductal cells with 5-aza-2′-deoxycytidine could result in

he expression of the AQP5 gene, thus leading to increased waterecretion. Moreover, retinoic acid induced HSG cells to adopt aeratinocyte-like phenotype expressing well-developed cytoker-tin filaments (Azuma et al., 1988). Matrigel, a basement membranextract, (Becton Dickinson Biosciences, Bedford, MA) (Vukicevict al., 1992) is the only substrate that promotes complete differenti-tion including the expression of salivary acinar markers (Lam et al.,005). Matrigel is the trade name for a gelatinous protein mixtureecreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cellshich contains collagen IV, laminin, fibronectin, entactin, perlecan,

n addition to multiple angiogentic and growth factors essential foregulation of cell growth and differentiation (Kleinman and Martin,005; Roberts and Sporn, 1990). HSG cells grown on Matrigel wereeported to form acinar structures that expressed �-amylase andystatin proteins (Hoffman et al., 1996; Royce et al., 1993).

Our group has continued to work with the HSG cell line becausehey can be expanded rapidly in culture (as compared to pri-

ary salivary cells) and can provide a standard cell source foresting seeding efficiency on different tissue-engineered scaffoldsAframian et al., 2000). The aim of the current study was to test

atrigel as a three-dimensional (3D) salivary acinar cell cultureodel for HSG cells, and to study their characteristics/behaviors

n vitro. These acinar cells would be used in the fabrication of anrtificial salivary gland device to be tested in an animal model.

. Materials and methods

.1. HSG cell culture

HSG cells (a generous gift from Prof. M. Sato, Tokushuma Uni-ersity, Japan (Sato et al., 1987)) were maintained in Dulbecco’sodified Eagle’s Medium (DMEM)/Ham’s F-12 (1:1), containing

0% fetal bovine serum (FBS, Biofluids, Rockville, MD), 100 U/mlenicillin, 100 �g/ml streptomycin, and 10 �g/ml gentamicin (Lifeechnologies Inc., Gaithersburg, MD). The cells were maintained at7 ◦C in a humidified 5% CO2 and 95% air atmosphere incubator. HSGells were detached from confluent plates with a solution of 0.05%rypsin, 0.02% versene (Biofluids, Rockville, MD) and re-suspendedn fresh tissue culture media. For all experiments in this manuscript,SG cells were plated at a density of 5 × 104 cell/cm2. Cells were

eeded on: (a) 24-mm Transwell-Clear filters for TER measure-ents, (b) 8-well slide chambers for confocal microscopy and

poptosis analyses, and (c) 12-well/or 6-well dish for TEM, west-rn blot and quantitative RT-PCR analyses. Cells were cultured onither non-coated or Matrigel-coated surfaces (19.6 mg/ml, BD Bio-ciences, Bedford, MA). Matrigel was thawed on ice and diluted inMEM (1:6, final concentration = 2 mg/ml). The plates/slide cham-

hemistry & Cell Biology 43 (2011) 622–631 623

bers/polyester filters were coated with a thin layer of this dilutedMatrigel and incubated at 37 ◦C for 60 min before cell seeding. Forexample, one well in an 8-well slide chamber of 0.8 cm2 surface areareceived 30 �l of this diluted Matrigel mix. The Matrigel acted as arigid thin layer that allowed cells to grow on it. The concentrationof Matrigel and seeding density were optimized to ensure repro-ducible 3D formation, which occurred after 24 h (Fig. 1). Culturemedium was changed every 2 days. The morphology of the cells wasobserved using phase contrast microscopy. MDCK-II cells obtainedfrom BD Biosciences Clontech (Palo Alto, CA) were maintained inDMEM/F-12 supplemented with 10% FBS (HyClone).

2.2. Measurements of transepithelial electrical resistance (TER)

HSG cells were seeded on 24-mm Transwell-Clear polyester fil-ters that were either un-coated or Matrigel-coated and grown asmentioned above. The upper chamber containing the cells received1.5 mL of medium, and the lower chamber (no cells) received 2.6 mLof medium. TER was measured after 3, 5 and 7 days (from 6 sepa-rate determinations) using a Millicell ERS epithelial volt-ohmmeter(Millipore Corp., Allen, TX) as described by the manufacturer.MDCK-II cells cultured on filters were used as a positive controlbecause they possess high TER due to their TJs (Aframian, 2002).TER readings from Transwell chambers without cells on non-coatedand Matrigel-coated filters were subtracted from readings obtainedfrom filters seeded with HSG cells.

2.3. Evaluation of cell apoptosis

An ApopTag® peroxidase in situ apoptosis detection kit(Chemicon International, MA, USA) was used to evaluate HSGcells apoptosis activity. ApopTag® is a mixed molecular andbiological–histo-chemical system that allows for sensitive and spe-cific staining of apoptotic bodies. Apoptotic reaction in three 8-wellslide chambers of HSG cultured on either plastic or Matrigel at day 7was scored at magnification of 400×. In addition, cytospin was usedto collect all floating/disintegrating cells in the media and evaluatedtheir apoptotic activity. The slides were assessed by two observersin a blinded manner in 10 randomly chosen fields per slide. Themean of all apoptotic cells per field was calculated.

2.4. Evaluation of cell proliferation

Proliferating cell nuclear antigen (PCNA) stain was performed tomonitor HSG cells proliferation using Zymed kit (Invitrogen, Carls-bad, CA, USA). After endogenous peroxidase activity was blocked for10 min, three 8-well slide chambers of HSG cells cultured on eitherplastic or Matrigel (at day 7) were processed with routine indirectimmunoperoxidase technique. In addition, cytospin was used tocollect all floating/disintegrating cells in the media and evaluatetheir mitotic activity. Two examiners independently counted thenumber of PCNA positive cells in a blinded manner in 10 randomlychosen fields per slide (400×). The mean of all PCNA positive cellsper field was calculated.

2.5. Immunofluorescence and confocal imaging

HSG cells cultured for 5 days were fixed with 10% paraformalde-hyde for 30 min and permeabilized with methanol at −20 ◦C for5 min followed by 0.2% Triton X-100 for 10 min at room tempera-ture. We used the following primary antibodies to characterize HSG

cultured on plastic and on Matrigel: rabbit anti-claudin-1, claudin-2, claudin-3, occludin, JAM-A and mouse anti-ZO-1, claudin-4, fromZymed; mouse anti-cytokeratin (panel), vimentin, and �-smoothmuscle actin, from Invitrogen; goat anti-aquaporin-5 (AQP-5),mouse anti-mucin-1 from Santa Cruz; rabbit anti-�-amylase

Page 3

624 O.M. Maria et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 622–631

F layer.w

fraamchaaa(

n0w1

roL123sriyui

2

cwcffw(fPMTai(a

ig. 1. (A) HSG cells cultured on plastic formed isolated clusters of epithelial monoithin 24 h and monolayer [scale bar = 200 �m, 200×].

rom Sigma–Aldrich. These antibodies were reactive against theirespective proteins from human and several other species. Welso tested stem cell markers by using these antibodies: goatnti-Musashi-1 and mouse multipotent mesenchymal/stromal cellarker (MSCs) antibody panel kit from R&D Systems. This panel

ontains a group of antibodies for the positive identification ofuman MSCs: anti-Stro-1, anti-CD90, anti-CD106, anti-CD105,nti-CD146, anti-CD166, anti-CD44, plus leukocytes markers;nti-CD19 and anti-CD45. As negative controls for the primaryntibodies tested, we used goat (R&D Systems), rabbit and mouseZymed Labs) isotype antibodies.

HSG cells were incubated with a blocking solution containing 5%ormal donkey serum (Jackson ImmunoResearch Laboratories) and.5% bovine serum albumin for 1 h at room temperature. HSG cellsere incubated with their respective primary antibodies (diluted

:100 in blocking solution) overnight at 4 ◦C.Secondary antibodies were either donkey anti-mouse/anti-

abbit/or anti-goat fluorescein isothiocyanate-conjugated (FITC)r Rhodamine Red-X-conjugated (RRX) (Jackson ImmunoResearchaboratories) incubated for 1 h at room temperature (diluted:100 in blocking solution) in the dark. Finally, 4′,6-diamidino--phenylindole dihydrochloride (DAPI, Invitrogen) was added for–5 min. Fluorescence images were taken using Zeiss LSM 510 lasercanning confocal microscope (Jena, Germany). Images shown areepresentative of at least 3 separate experiments, with multiplemages taken per slide. Imaris 3D/4D image visualization and anal-sis software (Bitplane AG, Zurich, Switzerland; version 7.0.0), weresed in the creation of 3D isosurface renderings for CLDN-1 (shown

n Fig. 4).

.6. Western blot analysis

Media from HSG cultures (i.e. the conditioned media) wereollected at days 1, 3, 5 and 7. HSG cells remaining on the dishere lysed in 200 �l/well of cold RIPA buffer. Cell lysates were

ollected into microcentrifuge tubes and centrifuged at 1000 rpmor 20 min to pellet the cell debris. The supernatants were keptor further analysis. Protein concentrations of all preparationsere determined with a bichinchonic acid (BCA) protein assay kit

Pierce Biotechnology, Rockford, IL). Protein samples (30 �g each)rom the cell or the collected media were subjected to 10% SDS-AGE on mini-gels and transferred to nitrocellulose membranes.embranes were blocked for 1 h with 5% fat-free dry milk in

ris-buffered saline [0.137 M NaCl, 0.025 M Tris (hydroxymethyl)-minomethane, pH 7.4] containing 0.1% Tween-20 (TBST) andmmune-blotted overnight with rabbit anti-�-amylase antibody1:2000 dilution, Sigma–Aldrich) at 4 ◦C in TBST containing 5% BSAnd 0.02% sodium azide. Human salivary �-amylase purchased

(B) HSG cells cultured on Matrigel (2 mg/ml) formed both 3D acinar-like structures

from Sigma–Aldrich was used as a positive control for the antibody.In addition, �-tubulin (mouse monoclonal, clone DM1A, Sigma) wasused as an internal control to normalize the protein-fold changefor �-amylase from HSG grown on Matrigel or plastic. After incu-bation with the primary antibodies, membranes were washed 3times for 15 min each with TBST and incubated with Horseradishperoxidase (HRP)-conjugated anti-rabbit (1:5000 dilution, SantaCruz Biotechnology) and anti-mouse IgG (1:50,000 dilution, Amer-sham, Biosciences, Piscataway, NJ) at room temperature for 1 h.The membranes were washed 3 times for 15 min each with TBST,then treated with chemiluminescence detection reagent containing20 mM Tris buffer, pH 8.5, 250 mM Luminol and 90 mM coumaricacid (Sigma–Aldrich) and protein bands were visualized on X-rayfilms. Quantification of bands was done using NIH J Image software(NIH, Bethesda, USA). All experiments were performed in duplicateand repeated at least 3 separate times.

2.7. Transmission electron microscopy (TEM)

HSG cells cultured on un-coated or Matrigel-coated 12-wellplates for 1, 2, 3, 5 and 7 days were fixed in 2% formaldehyde and2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h, rinsedin 0.1 M cacodylate buffer, and post-fixed in 1% osmium tetroxidefor 1 h. After rinsing in cacodylate buffer, samples were dehydratedthrough an ethanol series and infiltrated and embedded in Epon812(Electron Microscopy Sciences, Fort Washington, PA). Thin (70 nm)sections were cut with a diamond knife and mounted onto coppergrids. Grids were stained with 3% uranyl acetate for 30 min and 2%lead citrate for 5 min, to be examined at different magnificationsusing a 100-CXII transmission electron microscopy (JEOL, Tokyo,Japan) at an accelerating voltage of 120 kV.

2.8. Quantitative real-time polymerase chain reaction analysis

At days 3, 5, 7, and 9, HSG cells grown on either plastic orMatrigel were washed in PBS, then, total RNA was extracted usingthe RNeasy micro kit (Qiagen Ltd, Crawly, UK) with in-columnDNase digestion. The concentration of RNA was determined usingQubit (Molecular Probes). Total RNA (2 �g per sample) was reversetranscribed using the high-capacity cDNA Archive Kit (AppliedBiosystems, Foster City, CA) in 20 �L volume. For polymerase chainreaction (PCR) amplification, 5% of the cDNA was used with real-time PCR primers and 6-carboxy-fluorescein (FAM)-labeled minor

groove binder probes (MGB). The probes and primers for claudin-1 (CLDN-1), claudin-2 (CLDN-2), �-amylase (AMY), aquaporin-5(AQP-5), cytokeratin-18 (CK-18), epidermal growth factor (EGF),and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, used asan endogenous reference) were selected from the Applied Biosys-

Page 4

of Biochemistry & Cell Biology 43 (2011) 622–631 625

tTSdfliRtg

2

safpl

3

3

eufwftatt

3

tHkeseaT4to(a

3

Mwftncmc

2 1 3

401 417

332

470

568601

0

100

200

300

400

500

600

700

Day 3 Day 5 Day 7

HSG HSG + Matrigel MDCK-II

TE

R (

Ώc

m2

SE

M)

* *

*

* **

Fig. 2. Transepithelial electrical resistance (TER) measurements at days 3, 5 and 7.HSG grown on uncoated Transwell filters exhibited almost no TERs while HSG grown

CD105, CD106, CD90, CD146, Stro-1, CD45, and CD19 (Table 1B andC). Many HSG cells were positive for vimentin (88.4 ± 0.7%) butfew for �-amylase (5 ± 1%). However on Matrigel, HSG cells (bothmonolayers and 3D structures) expressed AQP5 (on the lateral cell

90

3838

78

0

10

20

30

40

50

60

70

80

90

100

HSG HSG + Matrigel

Mitotic Nuclei Apoptotic Nuclei

Av

era

ge

% o

f N

uc

lei

SE

Ma

t d

ay

7

*

*

O.M. Maria et al. / The International Journal

ems. PCR reactions (20 �L) were performed in duplicates usingaqMan Universal Master Mix (Applied Biosystems) on a Prismequence Detection System 7500 (Applied Biosystems) with theefault settings (50 ◦C for 2 min, 95 ◦C for 10 min, 40 cycles [95 ◦Cor 15 s, 60 ◦C for 1 min]). Every culture experiment was repeated ateast 3 times. Gene expression levels were calculated by normaliz-ng the target RNA value to the value of GAPDH in the same sample.esults are expressed as fold-changes in gene expression relativeo control (HSG cultured on plastic for 9 days, expressing the lowestene levels) sample.

.9. Statistical analysis

Data are presented as means ± SEM of results from 3 or moreeparate experiments. Our data were analyzed by Student’s t-testnd one-way ANOVA where P value < 0.05 represents significant dif-erences between both groups at specified times. Cell percentagesresented in this study were determined upon examination of at

east 1000 cells per slide.

. Results

.1. Cell morphology of HSG

HSG cells cultured on plastic formed monolayers; makingpithelial isolated clusters (Fig. 1A) that proliferated continuouslyntil confluence. On Matrigel coated surfaces (2 mg/ml), HSG cellsormed both spherical 3D acinar-like structures (20% of cells plated)ithin 24 h and monolayer (Fig. 1B). Most 3D structures initially

ormed, consisted of 6–8 cells, then, the number of cells/3D struc-ure increased up to 12 cells at day 3 where total 3D structures werepproximately 40% of total cells attached. At day 5, some 3D struc-ures started to detach from the Matrigel. By days 7–9, 20–30% ofhese 3D structures were seen floating in the media.

.2. Formation of a functional epithelial barrier

Because of the crucial role of tight junctions (TJs) in the secre-ion of salt, fluids, and proteins by epithelial cells, the ability ofSG cells to form a functional epithelial barrier and express severaley TJ proteins (claudin-1, -2, -3, -4, occludin, ZO-1, JAM-A) werevaluated first, on both Matrigel and plastic. As a standard mea-ure of TJs formation, TER was assessed across HSG cells seeded onither un-coated or Matrigel-coated Transwell filters at days 3, 5nd 7 (Fig. 2). HSG grown on un-coated filters exhibited almost noERs (1–3 � cm2) when compared to MDCK cells (positive control,70–601 � cm2). However HSG cells grown on Matrigel-coated fil-ers, exhibited moderate TERs (332–417 � cm2). TER values of HSGn Matrigel were significantly different from HSG culture on plasticP < 0.01). These results indicate that Matrigel allowed HSG to formrelatively tight epithelial barrier.

.3. Apoptosis versus mitosis

The apoptotic activity increased significantly (P < 0.01) onatrigel (78%) compared to plastic (38%) (Fig. 3). Apoptotic cellsere seen mainly at the centre of intact 3D structures where the

uture lumen is supposed to form and in the cell clumps forming

he disintegrating 3D (see Section 3.6). PCNA stain revealed a sig-ificant (P < 0.01) decrease of the mitotic activity on Matrigel (38%)ompared to plastic (90%) (Fig. 3). The mitotic activity was seenainly among monolayers and fewer mitosis was evident among

ells forming 3D structures.

on Matrigel (2 mg/ml), exhibited moderate TERs, when compared to MDCK-II cells(positive control). TER values of HSG on Matrigel were significantly different fromHSG culture on uncoated filters (*P < 0.01). Results represent the average ± SEM of 6separate determinations.

3.4. Protein expression

HSG cultured on plastic did not express any of the TJ pro-teins tested (Table 1A and Fig. 4) while HSG cultured on Matrigelexpressed all of them: claudin-1, -2, -3, -4, occludin, JAM-A, ZO-1(Table 1A and Fig. 4). Confocal microscopy localized these TJ pro-teins at the apicolateral sides of the cells. The relative position of TJsto the media-facing (analogous to the “apical” or “lumenal” side invivo) and filter-facing (“basolateral”) sides of the cells is depictedin the XZ and YZ planes of Fig. 4. The correct location of claudin-1 in 3D-acinar-like structures was further confirmed by using the3D-reconstruction software (Imaris 3D/4D) (Fig. 4). These imagesindicated that HSG cells formed TJs and were correctly polarizedon Matrigel.

HSG cells on plastic were negative for AQP5 (Fig. 5), cytoker-atins, mucin-1, �-smooth muscle actin, Musashi-1, CD44, CD166,

Fig. 3. Apoptotic and mitotic activities of HSG cultured on plastic and Matrigel atday 7. The apoptotic activity (evaluated by ApopTag kit) increased significantly onMatrigel (78%) when compared to HSG on plastic (38%). The mitotic activity (evalu-ated by PCNA stain) was significantly decreased on Matrigel (38%) when comparedto HSG on plastic (90%) (*P < 0.01).

Page 5

626 O.M. Maria et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 622–631

Fig. 4. Confocal micrographs of HSG. On plastic HSG were negative to claudin-1 and other TJ proteins. However on Matrigel, HSG expressed all TJ proteins tested. The XY, XZand YZ planes demonstrate the presence of the TJ proteins: claudin-1, -2, -3, -4, occludin, JAM-A, ZO-1, respectively (shown in red). The nuclei are stained with DAPI (shownin blue). Both XZ and YZ planes show TJ proteins were localized at the apicolateral membranes of HSG cells cultured on Matrigel. The Matrigel coated surface is indicated bya white bar/line at the basal side of HSG cells. The apical/lumenal side of HSG cells is indicated by a dash-line. [Scale bar = 34 �m, 400× for CLDN-1 on plastic and CLDN-4,occludin, JAM-A, ZO-1 on Matrigel; scale bar = 40 �m, 630× for CLDN-1, -2, -3 on Matrigel]. Series of confocal images of CLDN-1s taken on Matrigel at 1 �m intervals. Theseimages were then used by the Imaris 3D/4D image software to re-create a 3D isosurface picture of the location of CLDN-1 (in red) in HSG cells (cell nuclei are represented inblue) [scale bar = 15 �m]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Page 6

O.M. Maria et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 622–631 627

Table 1Expression of different markers/proteins in HSG cells grown on plastic and on Matrigel.

Marker Claudin-1 Claudin-2 Claudin-3 Claudin-4 Occludin JAM-A ZO-1

AHSG + Matrigel 99 ± 0.3%* 98 ± 0.5%* 98 ± 1%* 98 ± 0.8%* 98 ± 0.6%* 99 ± 0.1%* 98 ± 2%*HSG 0% 0% 0% 0% 0% 0% 0%

Marker Cytokeratin Vimentin �-SMA �-Amylase AQP5 Mucin-1

BHSG + Matrigel 95 ± 0.9%* 60.5 ± 2%* 0% 98 ± 0.9%* 98 ± 0.5%* 30 ± 2.4%*HSG 0% 88.4 ± 0.7% 0% 5 ± 1% 0% 0%

Marker Msi-1 Stro-1 CD44 CD166 CD105 CD106 CD90 CD146 CD45 CD19

CHSG + Matrigel 0% 0% 95 ± 2% 50 ± 4% 0% 0% 0% 0% 0% 0%HSG 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%

Results are based on the examination of 5 slides per culture type. At least 1000 cells were examined per slide. *The expression level of each marker/protein is indicated asp ompar- nchym� Stem(

mae9

FiiCv

ercentage ± SEM. All results from HSG on Matrigel were statistically significant in c3, -4, occludin, JAM-A, and ZO-1. (B) Cytokeratin-panel (epithelial), vimentin (mese-amylase (salivary serous), AQP-5 (salivary acinar), mucin-1 (salivary mucous). (C)

mesenchymal), CD45, CD19 (leukocyte).

embrane) and �-amylase (in the cytoplasm) strongly in 98 ± 0.5%nd 98 ± 0.9% of cells, respectively (Fig. 5A and B). Vimentin, cytok-ratins (Fig. 5C and D) and mucin-1 were expressed by 60.5 ± 2%,5 ± 0.9%, and 30 ± 2.4% of the cells, respectively (Table 1B). Inter-

ig. 5. Confocal micrographs of HSG. On plastic HSG were negative for AQP-5. However onn both monolayer and 3D structures, and �-amylase (98 ± 0.9%, shown in red). On Matrign 95 ± 0.9% of cells (shown in green), and CD44 in 95 ± 2% of cells (shown in red). [ScalD44 on Matrigel; scale bar = 40 �m, 630× for �-amylase on Matrigel]. (For interpretatioersion of the article.)

ison to HSG cultured on plastic (*P < 0.01). (A) Tight junction proteins: claudin-1, -2,al, salivary ductal, immature acinar and their progenitors), �-SMA (myoepithelial),

cell markers: Musashi-1 (Neural), Stro-1, CD44, CD166, CD105, CD106, CD90, CD146

estingly, CD44 (Fig. 5E) and CD166 were expressed by HSG cells(95 ± 2% and 50 ± 4%, respectively) while the other stem cell mark-ers were absent (CD105, CD106, CD90, CD146, Stro-1, CD45, CD19and Musashi-1; Table 1C).

Matrigel HSG expressed acinar secretory proteins: AQP-5 (98 ± 0.5%, shown in red)el, vimentin was expressed in 60.5 ± 2% of cells (shown in green), cytokeratin-panele bar = 34 �m, 400× for AQP-5 on plastic and AQP-5, vimentin, cytokeratin-panel,n of the references to color in this figure legend, the reader is referred to the web

Page 7

628 O.M. Maria et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 622–631

F f HSGi roteis s used

3

�lpt(t5c

3

ocpordhtsac(et(eocTipmtom

3

1

ig. 6. Western blot analysis of �-amylase (60 kDa) secreted in the cultured media on their media. However HSG cells cultured on plastic did not secrete �-amylase pecretion at days 5 and 7, respectively. The �-tubulin protein (from cell lysates) wa

.5. Protein synthesis and secretion

At days 3, 5 and 7, HSG cells cultured on Matrigel could secrete-amylase protein into the media (supernatant samples from cell

ysates showed similar results). However, HSG cells cultured onlastic did not show either synthesis or secretion of �-amylase pro-ein until day 5 of culture. Western blot data from cultured mediaFig. 6) indicated that Matrigel increased the synthesis and secre-ion of �-amylase protein to approximately 3- and 2-fold at daysand 7, respectively. The �-tubulin protein shown in Fig. 6 (from

ell lysates) is used as an internal control.

.6. Ultrastructural changes

Ultrastructural analysis by TEM revealed that HSG cells grownn plastic resembled ductal-like cells arranged in monolayers. Itsytoplasm showed scarce and small secretory granules among thelentiful large and elongated mitochondria, small isolated islandsf glycogen, and an absence of Golgi apparatus, rough endoplasmiceticulum and microvilli (Fig. 7A). On Matrigel, many electron-ense secretory-like granules (similar to those observed in normaluman salivary glands by Takano et al. (1991) were detected inhe cytoplasm at day 3 (Fig. 7B). Features of active protein synthe-is were observed at days 3, 5 and 7 where many Golgi sacculesnd rough endoplasmic reticulum were seen everywhere in theytoplasm (Fig. 7C). On Matrigel, approximately 90% of HSG cellsfrom both monolayers and 3D acinar-like structures) producedlectron-dense secretory granules at days 5 and 7; at day 3, 60% ofhese cells showed secretory granules. TJ structures were observedfrom day 2) at the apicolateral cell membrane (Fig. 7D). How-ver, no basal lamina could be viewed under HSG cells grownn Matrigel. Membrane-bound apoptotic bodies were observed inells located at the centre of 3D acinar-like structures (Fig. 8A).his suggested the formation of a central lumen with surround-ng cells showing well-developed microvilli, as an indication ofrotein secretion (Fig. 8A). These microvilli resembled those of nor-al salivary epithelial cells. Apoptotic cells had many lysosomes in

heir cytoplasm and showed no signs of protein synthesis. More-ver, their chromatin became broken and both nuclear and cellularembranes lost their integrities (Fig. 8B).

.7. Gene expression

HSG cells cultured on plastic were found to express claudin-, -2, AQP5 and cytokeratin18 genes by real-time PCR (Fig. 9)

cells. At days 3, 5 and 7 HSG cells cultured on Matrigel secreted �-amylase proteinn until day 5. On Matrigel, HSG cells showed 3- and 2-fold increase in �-amylaseas an internal control.

but were unable to translate these into proteins (as indicated bytheir absence under immunofluorescence staining in Table 1 andFigs. 4 and 5, as well as an absence of TJ structures under TEM).This suggested that HSG cells cultured on plastic might experi-ence a protein translation defect. When HSG cells were culturedon Matrigel, the expression of genes for tight junctions proteins(claudin-1, -2), acinar-cell proteins (�-amylase, AQP5), and ductal-cell proteins (cytokeratin-18, EGF) decreased. The down-regulationof the AQP5 gene was statistically significant (P < 0.01). A similartrend was observed for day 3, 5 and 7 post-plating. These datasuggested that the protein expression pattern associated with HSGcultured on Matrigel was achieved through regulation of transla-tion rather than through the transcription of these relevant genes.

4. Discussion

The findings of this report indicate that Matrigel inducedmorphologic changes and cytodifferentiation of HSG cells. Theseincluded the formation of polarized 3D acinar-like structuresexpressing TJs with reasonable TER. The formation of TJs is an essen-tial differentiation step for functional secretory epithelial cells. Thisstudy thoroughly documented the expression of TJ proteins in HSGcells grown on Matrigel (claudin-1, -2, -3, -4, occludin, JAM-A,ZO-1). TEM showed TJ structures, well-developed Golgi appara-tus, rough endoplasmic reticulum and secretory-like granules. Thestudy further confirmed the relationships between salivary cellproliferation, apoptosis and expressions of acinar cell markers (�-amylase, AQP5, mucin-1). Interestingly, the increase/changes in theexpression of TJ, ductal and acinar proteins (on Matrigel) were notdue to increases in mRNAs transcription.

Vag et al. (2007) reported that HSG formed few acini by pro-liferation in low concentrations of basement membrane extract(BME). HSG sank into this soft gel and were less polarized whencompared to those cultured in higher concentrations of BME. Thisresulted in the formation of 2D reticular networks rather than 3Dstructures. In a higher concentration of BME, the gel was morerigid and allowed cell movement, differentiation and formation of3D structures (Vag et al., 2007). Here we used Matrigel at a lowconcentration (2 mg/ml) and were able to obtain 3D acinar struc-tures within 24 h (but not reticular networks). These 3D acinar-like

structures were comparable in size, shape and number of cellsas those found in normal human salivary acini; with the excep-tion that 3D acinar-like structures observed in this study werenot connected with ducts. Expression of acinar-specific markers,such as �-amylase and cystatin, from previous reports demon-

Page 8

O.M. Maria et al. / The International Journal of Biochemistry & Cell Biology 43 (2011) 622–631 629

Fig. 7.

Fig. 8. Transmission electron microscope (TEM) micrograph of HSG cells stainedwith lead citrate and uranyl acetate staining. (A) 3D acinar-like structure of HSGcells cultured on Matrigel (at day 3) exhibiting features of apoptosis (AP) in the cen-tre for the formation of a central lumen [4200×]. (B) Inset of the apoptotic cell at ahigher magnification showing membrane-bound apoptotic bodies (AB), few secre-

tory granules (SG) among many lysosomes (LY). Apoptotic nucleus (N) with brokenchromatin, the nuclear (arrows) and cellular membranes (arrow heads) lost theirintegrity. The other surrounding cells show well-developed microvilli (M) suggest-ing protein secretion [9900×].

strated that HSG could differentiate into acinar cells, but therewas no evaluation of the secretion of these proteins into the cul-tured media (Lam et al., 2005; Royce et al., 1993; Zheng et al.,1998). In our study, we confirmed that HSG grown on Matrigelsecreted �-amylase into the conditioned media from days 3 to 7.These results implied a consecutive (constitutive) secretion of amy-lase by HSG cells without beta-adrenergic receptor stimulation.However, future studies are essential to evaluate the mechanism

and direction of secretion involved in this model in presence ofbeta-adrenergic stimulants. Zheng et al. (1998) reported that bothTGF-� and HGF when added to laminin were able to activate the �-amylase promoter in HSG cells. In the current study, these growthfactors were already components of Matrigel. We also found that

Fig. 7. Transmission electron microscope (TEM) micrographs of HSG cells stainedwith lead citrate and uranyl acetate staining. (A) HSG cell grown on plastic exhibitedfeatures of ductal cells: scarce small secretory granules (SG) among the plentifullarge elongated mitochondria (M), small isolated island of glycogen (GL) [9900×].Panels B–D show HSG cells grown on Matrigel. (B) HSG cell (at day 7) exhibitingmultiple electron-dense secretory granules (SG) among multiple mitochondria (M)[16,500×]. (C) HSG cell (at day 3) exhibiting features of protein synthesis: Golgisaccules (G) and rough endoplasmic reticulum (rER), multiple mitochondria (M)and a secretory granule (SG) budding out of Golgi saccule, all swimming close to thenucleus (N) [16,500×]. (D) Two adjacent HSG cells (at day 3) exhibiting characteristicfeatures of salivary epithelial cells: joined by tight junction structure (TJ) at theapicolateral cell membrane, electron-dense secretory granule (SG), and microvilli(M) at the luminal cell membrane [43,000×].

Page 9

630 O.M. Maria et al. / The International Journal of Bioc

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

CLDN-1 CLDN-2 AMY AQP5 CK-18 EGF

HSG HSG +Mat

Gen

e e

xp

ressio

n (

fold

ch

an

ge)

+ S

EM

at

day 3

*

Fig. 9. Gene expression levels (at day 3) indicated in fold-change for HSG culturedon Matrigel and on plastic for: claudin-1 (CLDN-1), claudin-2 (CLDN-2), �-amylase(AMY), Aquaporin-5 (AQP-5), cytokeratin-18 (CK18) and epidermal growth factor(EGF) genes, relative to samples from cultures grown on plastic for 9 days and nor-malized to GAPDH levels using quantitative RT-PCR (at least 3 separate experimentswere performed/gene). Results are reported as means ± SEM of parallel cultures(*P < 0.01). A similar trend was observed for day 3, 5 and 7 post-plating. ThesedMto

3ee2toc

3teh1tfectbItHlwSbMrwMvi

ce2mo

ata suggest that the protein expression pattern associated with HSG cultured onatrigel was achieved through regulation of translation rather than through the

ranscription of these relevant genes (i.e. the increase in TJ and acinar proteinsbserved by confocal microscopy was not due to increase in mRNAs).

D acinar-like structures and monolayer of HSG cells on Matrigelxpressed AQP-5 (in 98 ± 0.5% of cells), a water channel protein nec-ssary for water secretion in human salivary glands (Beroukas et al.,001; Gresz et al., 2001). Moreover, these 3D structures expressedhe adhesion-related proteins CD44 and CD166 which are used asne of a panel of markers to identify mesenchymal stem/progenitorells.

Our study found that the distribution of TJ proteins in HSGD acinar-like structures and monolayers (on Matrigel) resembledhose found in normal human and rodent salivary glands (Mariat al., 2008; Peppi and Ghabriel, 2004). Although salivary gland cellsave been characterized in 3D culture previously (Hoffman et al.,996; Joraku et al., 2007; Szlávik et al., 2008; Wei et al., 2007),his is the first study to demonstrate that 3D acinar-like structuresrom HSG cells were capable of establishing a reasonable TER andxpressed TJ proteins. Aframian et al. (2002), reported that HSGells did not express TJ structures and therefore had low TER. Afterransfection with claudin-1 and claudin-2 genes, HSG expressedoth proteins at their membranes but did not acquire adequate TER.

t seems that Matrigel drives a specific mechanism through whichhe translation of specific proteins such as TJ proteins is triggered.owever, Matrigel might not be the only required factor for trans-

ation of all proteins. We report here that �-smooth muscle actinas not expressed in HSG cultured on either plastic or Matrigel.

ato et al. (1985) reported that 95% of HSG cells on plastic expressedoth keratin and vimentin. In the current study, on both plastic andatrigel, 88.4 ± 0.7% and 60.5 ± 2% of HSG cells expressed vimentin,

espectively while cytokeratin was not expressed on plastic butas expressed in 95 ± 0.9% of the cells on Matrigel. It seems thatatrigel drove HSG cells to a keratinocyte-like direction. Normally,

imentin is expressed in immature salivary acini and their progen-tors, some ductal cells and myoepithelial cells (Ogawa, 2003).

In this study, the reduction in mitotic activity (38%) of HSGultured on Matrigel was consistent with previous reports (Gresz

t al., 2001; Hoffman et al., 1998; Royce et al., 1993; Szlávik et al.,008). Royce et al. (1993) reported that Matrigel decreased HSGitotic activity to 50%. In contrast, on very low concentration

f either Matrigel or laminin, few acini differentiated from HSG

hemistry & Cell Biology 43 (2011) 622–631

cells (Hoffman et al., 1996). Moreover, Lam et al. (2005) reportedthat HSG cells cultured on either Matrigel or collagen-I had anearly increase in mitotic activity in comparison to cells culturedon uncoated substrates. In this study, apoptosis was observed incells that lost contact with the Matrigel as they became internal-ized in the 3D acinar structures leading to a central lumen formation(Boudreau et al., 1995; Dirami et al., 1995; Hoffman et al., 1996).During salivary gland development, balanced mechanisms are gov-erning the interactions between cell proliferation, apoptosis andcytodifferentiation (Jaskoll and Melnick, 1999).

Neither Matrigel nor the HSG cell line would be used in clinicalapplications as both are derived from mouse sarcoma and humanadenocarcinoma, respectively. However, together they representan excellent and easily reproducible model to study salivary aci-nar cell formation, physiology, gene expression, morphogenesis,and cytodifferentiation. In addition HSG, as a long-term dividinghuman salivary cell source, would allow long-term studies in ani-mal models. The next step would be testing HSG (as a graft cell type)grown on Matrigel (as an extracellular matrix) carried on a scaf-fold designed to be implanted in animal models (mouse, rat, rabbit,monkey, etc.) to further examine our envisioned artificial salivarygland device in vivo. In analyzing safer substitutes of Matrigel, weare also testing several 3D gel types that would be acceptable forclinical applications. Certainly, all these studies will provide newtreatments for salivary gland hypofunction.

Author disclosure statement

No competing financial interests exist.

Acknowledgements

We are most grateful to Cory Glowinski for the creation of 3Disosurface renderings for both CLDN-1 and DAPI channels. We areequally appreciative of the technical help provided by Yunlin Tai,and Jeannie Mui from the facility of electron microscopy research(FEMR) at McGill University. The authors would like to thank Dr.John Presley and Dr. Judith Lacoste (from the Cell Imaging andAnalysis Network, CIAN) for Confocal Microscopy use. We are alsograteful to Dr. Mari Kaartinen and Dr. Monzur Murshed for sharingtheir laboratory equipments. The authors would like to thank thesefunding institutions: Canada Research Chair, NSERC, Oral Healthand Bone Network, and the Egyptian Ministry of Higher Educationand Scientific Research.

References

Aframian DJ, Cukierman E, Nikolovski J, Mooney DJ, Yamada KM, Baum BJ. Thegrowth and morphological behavior of salivary epithelial cells on matrixprotein-coated biodegradable substrata. Tissue Eng 2000;6:209–16.

Aframian DJ, Tran SD, Cukierman E, Yamada KM, Baum BJ. Absence of tight junc-tion formation in an allogeneic graft cell line used for developing an engineeredartificial salivary gland. Tissue Eng 2002;8:871–8.

Azuma M, Hayashi Y, Yoshida H, Yanagawa T, Yura Y, Ueno A, et al. Emergence ofdifferentiated subclones from a human salivary adenocarcinoma cell clone inculture after treatment with sodium butyrate. Cancer Res 1986;46:770–7.

Azuma M, Kawamata H, Kasai Y, Nagamine S, Yoshida H, Yanagawa T, et al.Effects of retinoic acid on morphological features and biological markers ofa neoplastic human salivary intercalated duct cell line in culture. Cancer Res1988;48:7219–25.

Batsakis JG. Salivary gland neoplasia: an outcome of modified morphogenesis andcytodifferentiation. Oral Surg Oral Med Oral Pathol 1980;49:229–32.

Baum BJ, O’Connell BC. The impact of gene therapy on dentistry. J Am Dent Assoc1995;126:179–89.

Baum BJ. Principles of saliva secretion. Ann N Y Acad Sci 1993;694:17–23.Baum BJ, Tran SD. Synergy between genetic and tissue engineering: creating an

artificial salivary gland. Periodontology 2000 2006;41:218–23.Beroukas D, Hiscock J, Jonsson R, Waterman SA, Gordon TP. Subcellular distribu-

tion of aquaporin 5 in salivary glands in primary Sjogren’s syndrome. Lancet2001;358:1875–6.

Page 10

of Bioc

B

C

D

D

D

E

F

F

F

F

G

H

H

H

J

J

K

K

L

M

O.M. Maria et al. / The International Journal

oudreau N, Sympson CJ, Werb Z, Bissell MJ. Suppression of ICE and apoptosis inmammary epithelial cells by extracellular matrix. Science 1995;267:891–3.

ook DI, Van Lennep EW, Roberts ML, Young JA. Secretion by the major salivaryglands. In: Johnson LR, editor. Physiology of the gastrointestinal tract. New York:Raven; 1994. p. 1061–117.

enny PC, Ball WD, Redman RS. Salivary glands: a paradigm for diversity of glanddevelopment. Crit Rev Oral Biol Med 1997;8:51–75.

irami G, Ravindranath N, Kleinman HK, Dym M. Evidence that basement membraneprevents apoptosis of sertoli cells in vitro in the absence of known regulators ofsertoli cell function. Endocrinology 1995;136:4439–47.

urban EM. Mouse submandibular salivary epithelial cell growth and differentiationin long term culture: influence of the extracellular matrix. In Vitro Cell Dev Biol1990;26:33–43.

versole LR. Histogenic classification of salivary tumors. Arch Pathol1971;92:433–43.

ox PC, Brennan M, Pillemer S, Radfar L, Yamano S, Baum BJ. Sjogren’s syndrome: amodel for dental care in the 21st century. J Am Dent Assoc 1998;129:719–28.

ox PC, Van der Ven PF, Sonies BC, Weiffenbach JM, Baum BJ. Xerostomia: evaluationof a symptom with increasing significance. J Am Dent Assoc 1985;110:519–25.

ox RI, Maruyama T. Pathogenesis and treatment of Sjögren’s syndrome. Curr OpinRheumatol 1997;9:393–9.

ox RI, Michelson P, Casiano CA, Hayashi J, Stern M. Sjogren’s syndrome. Clin Der-matol 2000;18:589–600.

resz V, Kwon TH, Hurley PT, Varga G, Zelles T, Nielsen S, et al. Identification andlocalization of aquaporin water channels in human salivary glands. Am J PhysiolGastrointest Liver Physiol 2001;281:G247–54.

ayashi Y, Yanagawa T, Azuma M, Yura Y, Yoshida H, Sato M. Induction of cellswith a myoepithelial cell phenotype by treatment with dibutyryl cyclic AMP inhuman salivary adenocarcinoma cells grown in athymic nude mice. VirchowsArch Zellpathol 1985;50:1–11.

offman MP, Kibbey MC, Letterio JJ, Kleinman HK. Role of laminin-1 and TGF-b3 inacinar differentiation of a human submandibular gland cell line (HSG). J Cell Sci1996;109:2013–21.

offman MP, Nomizu M, Roque E, Lee S, Jung DW, Yamada Y, et al. Laminin-1and laminin-2 Gdomain synthetic peptides bind syndecan-1 and are involvedin acinar formation of a human submandibular gland cell line. J Biol Chem1998;273:28633–41.

askoll T, Melnick M. Submandibular gland morphogenesis: stage-specific expres-sion of TGF-alpha/EGF, IGF, TGF-beta, TNF, and IL-6 signal transduction in normalembryonic mice and the phenotypic effects of TGF-beta2, TGF-beta3, and EGF-rnull mutations. Anat Rec 1999;256:252–68.

oraku A, Sullivan CA, Yoo J, Atala A. In-vitro reconstitution of three dimensionalhuman salivary gland tissue structures. Differentiation 2007;75:318–24.

ashima HK, Kirkham JR, Andrews JR. Post-irradiation sialadenitis. A study of theclinical features, histopathologic changes and serum enzyme variations follow-ing irradiation of human salivary glands. Am J Roentgenol Radium Ther NuclMed 1965;94:271–91.

leinman HK, Martin JR. Matrigel: basement membrane matrix with biological activ-ity. Semin Cancer Biol 2005;15:378–86.

am K, Zheng L, Bewick M, Lafrenie RM. HSG cells differentiated by culture on extra-cellular matrix involves induction of s-adenosylmethione decarboxylase andornithine decarboxylase. J Cell Physiol 2005;203:353–61.

aria OM, Kim JW, Gerstenhaber JA, Baum BJ, Tran SD. Distribution of tightjunction proteins in adult human salivary glands. J Histochem Cytochem2008;56:1093–8.

hemistry & Cell Biology 43 (2011) 622–631 631

Motegi K, Azuma M, Tamatani T, Ashida Y, Sato M. Expression of aquaporin-5 in andfluid secretion from immortalized human salivary gland ductal cells by treat-ment with 5-aza-2′-deoxycytidine: a possibility for improvement of xerostomiain patients with Sjogren’s syndrome. Lab Invest 2005;85:342–53.

Ogawa Y. Immunocytochemistry of myoepithelial cells in the salivary glands. ProgHistochem Cytochem 2003;38:343–426.

Oliver C, Water JF, Tolbert CC, Kleinman HK. Growth of exocrine acinar cells on areconstituted basement membrane gel. In Vitro Cell Dev Biol 1987;23:465–73.

Peppi M, Ghabriel MN. Tissue-specific expression of the tight junction proteinsclaudins and occludin in the rat salivary glands. J Anat 2004;205:257–66.

Pierce GB. Neoplasms, differentiations and mutations. Am J Pathol 1974;77:103–18.Redman RS, Quissell DO. Isolation and maintenance of submandibular gland cells.

In: Dobrosielski-Vergona K, editor. Biology of the Salivary Glands. Boca Raton,FL: CRC Press, Inc.; 1993. p. 285–306.

Roberts AB, Sporn MB. The transforming growth factor-bs. In: Sporn MB, RobertsAB, editors. Peptide growth factors and their receptors. Part 1. Berlin: Springer-Verlag; 1990. p. 419–72 [Chapter 8].

Royce LS, Kibbey MC, Mertz P, Kleinman HK, Baum BJ. Human neoplastic sub-mandibular intercalated duct cells express an acinar phenotype when culturedon a basement membrane matrix. Differentiation 1993;52:247–55.

Sato M, Azuma M, Hayashi Y, Yoshida H, Yanagawa T, Yura Y. 5-Azacytidine inductionof stable myoepithelial and acinar cells from a human salivary intercalated ductcell clone. Cancer Res 1987;47:4453–9.

Sato M, Hayashi Y, Yanagawa T, Yoshida H, Yura Y, Azuma M, et al. Intermediate-sized filaments and specific markers in a human salivary gland adenocarcinomacell line and its nude mouse tumors. Cancer Res 1985;45:3878–90.

Shirasuna K, Sato M, Miyazaki T. A neoplastic epithelial duct cell line establishedfrom an irradiated human salivary gland. Cancer 1981;48:745–52.

Szlávik V, Vág J, Markó K, Demeter K, Madarász E, Oláh I, et al. Matrigel-inducedacinar differentiation is followed by apoptosis in HSG cells. J Cell Biochem2008;103:284–95.

Takano K, Bogert M, Malamud D, Lally E, Hand AR. Differential distribution of salivaryagglutinin and amylase in the golgi apparatus and secretory granules of thehuman salivary gland acinar cells. Anat Rec 1991;230:307–18.

Tran SD, Wang J, Bandyopadhyay BC, Redman RS, Dutra A, Pak E, et al. Primaryculture of polarized human salivary epithelial cells for use in developing anartificial salivary gland. Tissue Eng 2005;11:172–81.

Tran SD, Sugito T, Dipasquale G, Cotrim AP, Bandyopadhyay BC, Riddle K, et al. Re-engineering primary epithelial cells from rhesus monkey parotid glands for usein developing an artificial salivary gland. Tissue Eng 2006;12:2939–48.

Vag J, Byrne EM, Hughes DH, Hoffman M, Ambudkar I, Maguire P, et al. Morphologicaland functional differentiation of HSG cells: role of extracellular matrix and trpc1. J Cell Physiol 2007;212:416–23.

Vukicevic S, Kleinman HK, Luyten FP, Roberts AB, Roche NS, Reddi AH. Identifica-tion of multiple active growth factors in basement membrane Matrigel suggestscaution in interpretation of cellular activity related to extracellular matrix com-ponents. Exp Cell Res 1992;202:1–8.

Wei C, Larsen M, Hoffman MP, Yamada KM. Self-organization and branching mor-phogenesis of primary salivary epithelial cells. Tissue Eng 2007;13:721–35.

Yoshida H, Azuma M, Yanagawa T, Yura Y, Hayashi Y, Sato M. Effect of dibutyrylcyclic AMP on morphologic features and biologic markers of a human salivarygland adenocarcinoma cell line in culture. Cancer 1986;57:1011–8.

Zheng C, Hoffman MP, McMillan T, Kleinman HK, O’connell BC. Growth factor regu-lation of the amylase promoter in a differentiating salivary acinar cell line. J CellPhysiol 1998;177:628–35.