MR

MJF

*PFSF

BtdpafgpcepeacmbntmatTuogCpitaaffv

Pes(

GASTROENTEROLOGY 2008;135:1301–1310

urine Embryonic Stem Cell–Derived Pancreatic Acinar Cellsecapitulate Features of Early Pancreatic Differentiation

ERITXELL ROVIRA,* FABIEN DELASPRE,* MOHAMMAD MASSUMI,* SELMA A. SERRA,‡ MIGUEL ANGEL VALVERDE,‡

OSEP LLORETA,§,� MARLÈNE DUFRESNE,¶ BRUNO PAYRÉ,# STEPHEN F. KONIECZNY,** PIERRE SAVATIER,‡‡,§§,� �

RANCISCO X. REAL,*,� and ANOUCHKA SKOUDY*

Cancer Research Program, Institut Municipal d’Investigació Mèdica, ‡Laboratory of Molecular Physiology and Channelopathies, §Hospital del Mar, Department deatologia, �Universitat Pompeu Fabra, Barcelona, Spain; ¶INSERM U858/I2MR, Institut Fédératif de Recherche 31, Toulouse, France, Université de Toulouse 3,rance; #Centre de Microscopie Électronique Appliquée à la Biologie, Faculté de Médecine de Rangueil, Toulouse Cedex, France; **Department of Biologicalciences and the Purdue Cancer Center, Purdue University, West Lafayette, Indiana; ‡‡Inserm U846, Bron, France; §§Stem Cells and Brain Research Institute, Bron,

rance; and the � �Université de Lyon, Université Lyon 1, Lyon, France

ike

paaifmsvasftmpaadcadt(r(lzap

Cstt

BA

SIC–L

IVER

,PA

NCREA

S,A

ND

BIL

IARY

TRA

CT

ackground & Aims: Acinar cells constitute 90% ofhe pancreas epithelium, are polarized, and secreteigestive enzymes. These cells play a crucial role inancreatitis and pancreatic cancer. However, therere limited models to study normal acinar cell dif-erentiation in vitro. The aim of this work was toenerate and characterize purified populations ofancreatic acinar cells from embryonic stem (ES)ells. Methods: Reporter ES cells (Ela-pur) were gen-rated that stably expressed both �-galactosidase anduromycin resistance genes under the control of thelastase I promoter. Directed differentiation waschieved by incubation with conditioned media ofultured fetal pancreatic rudiments and adenoviral-ediated co-expression of p48/Ptf1a and Mist1, 2

asic helix-loop-helix transcription factors crucial forormal pancreatic acinar development and differen-

iation. Results: Selected cells expressed multiplearkers of acinar cells, including digestive enzymes

nd proteins of the secretory pathway, indicating ac-ivation of a coordinated differentiation program.he genes coding for digestive enzymes were not reg-lated as a single module, thus recapitulating whatccurs during in vivo pancreatic development. Theenerated cells displayed transient agonist-induceda2� mobilization and showed a typical response tohysiologic concentrations of secretagogues, includ-

ng enzyme synthesis and secretion. Importantly,hese effects did not imply the acquisition of a mixedcinar-ductal phenotype. Conclusions: These studiesllow the generation of almost pure acinar-like cellsrom ES cells, providing a normal cell-based modelor the study of the acinar differentiation program initro.

ancreatic acinar cells play a key role in digestion invertebrates. The synthesis and secretion of digestive

nzymes is finely regulated: many enzymes are synthe-ized as propeptides and stored in zymogen granules

ZG), the exocytosis of which is tightly controlled. Bind-

ng of secretagogues to the muscarinic 3 and cholecysto-inin (CCK) receptors leads to Ca2� release from thendoplasmic reticulum and triggers ZG secretion.1

Acinar cells also play an important role in exocrineancreatic diseases, including acute and chronic pancre-titis and, possibly, ductal adenocarcinoma. Upon stress,cinar cells readily undergo a phenotypic switch, result-ng in loss of differentiation and acquisition of duct-likeeatures both in vivo and in vitro.2– 6 Together with their

inimal proliferative potential, these effects hamper thetudy of acinar differentiation. Furthermore, there areery few acinar tumor cell lines and they lack differenti-ted features (ie, ZG). Because of their tumor origin, theirtudy is also of questionable physiologic relevance. There-ore, the establishment of normal-cell– derived acinar cul-ures remains a high priority to better understand and

anipulate acinar differentiation. In particular, it is im-ortant to better characterize and dissect substages ofcinar differentiation during pancreatic developmentnd regeneration to better interfere with their loss ofifferentiation properties. For instance, it generally isonsidered that genes coding for digestive enzymes arectivated simultaneously and that their expression is un-er identical regulatory control mechanisms, yet conven-ional reverse-transcription polymerase chain reactionRT-PCR) data support the notion that these genes areegulated sequentially.7 Because nonquantitative RT-PCRnon– qRT-PCR) has technical limitations, the relativeevels of expression of the genes coding for acinar en-ymes at different developmental stages remains to benalyzed. This is important because in vivo tracing ex-eriments evaluating the contribution of acinar cells to

Abbreviations used in this paper: Ad, adenovirus; Amyl, �-amylase;PA, carboxypeptidase A; CCK, cholecystokinin; ChymoB, chymotryp-inogen B; CM, conditioned medium; EB, embryoid bodies; Ela1, elas-ase 1; ES, embryonic stem; qRT-PCR, quantitative reverse-transcrip-ion polymerase chain reaction; ZG, zymogen granules.

© 2008 by the AGA Institute0016-5085/08/$34.00

doi:10.1053/j.gastro.2008.06.049

od

cIogfknrtaabdpsaeappaisouiosNdca

mcf(ipDcvpOscclisst

AracppfAsau�(f

BMrtNImt

acs3dspp(chtEt�wr3

BA

SIC–LIV

ER,

PA

NCREA

S,A

ND

BILIA

RY

TRA

CT

1302 ROVIRA ET AL GASTROENTEROLOGY Vol. 135, No. 4

ther pancreatic cell types currently are performed usingigestive enzyme gene promoters.Acinar cells originate from multipotent precursors lo-

ated in the foregut giving rise to all pancreatic cell types.n mice, exocrine pancreas specification occurs at embry-nic day 10 (E10). Cells with acinar features are distin-uished at E14, ZGs accumulate from E16 to birth, andull maturation occurs postnatally.8 –10 However, little isnown about the precise molecular events heralding aci-ar differentiation. To date, few transcription factorsegulating exocrine differentiation have been identified:he basic helix-loop-helix proteins p48/Ptf1a and Mist1,nd RBPL. Ptf1a messenger RNA (mRNA) is first detectedround E9.5 in pancreatic primordia and, in the adult,ecomes restricted to acinar cells. Ptf1a originally wasescribed as part of the heterotrimeric transcription com-lex PTF1 involved in digestive enzyme gene expres-ion.11,12 Ptf1a also is essential for pancreas formationnd for the development of all pancreatic cell lin-ages.13–15 In its absence, foregut endoderm precursorsssume an intestinal fate.14 Ptf1a also promotes ectopicancreas fates from endoderm tissue.16 –18 Mist1 is ex-ressed in a wide array of secretory tissues and, in thedult pancreas, is detected only in acinar cells.19,20 Mist1nactivation or inhibition of Mist1 function results in aevere impairment of acinar organization, including lossf gap junctions, structural alterations of secretory gran-les, and acinar-ductal metaplasia.20,21 Therefore, Mist1

s necessary for proper cell polarization and maintenancef acinar identity. RBPL is essential for the high tran-criptional activity of the PTF1 complex independently ofotch signaling.22 Interestingly, RBPL is expressed in theeveloping pancreas tips at E14.5,23 when exocrine pre-ursors expand and differentiate, confirming a role incinar differentiation during the secondary transition.24

Embryonic stem (ES) cells, derived from the inner-ass of pre-implantation embryos, can differentiate into

ells of the 3 germ layers, making them an excellent toolor differentiation studies in vitro. Via embryoid bodyEB) formation, endodermal precursors can be specifiednto endocrine and exocrine lineages in a process thatartially recapitulates early pancreatic development.25,26

espite promising advances,27 this process is very ineffi-ient. Here, we aimed to generate ES cells that had acti-ated a pancreatic acinar differentiation program to ap-ly this knowledge to study exocrine pancreatic diseases.ur strategy relied on the ability of ES cells to respond to

oluble factors involved in pancreatic embryogenesis inonjunction with expression of multiple exocrine trans-ription factors and genetic selection. This system al-owed us to isolate normal cell populations displayingmmature acinar phenotypes. These cells reproducepecific substages of acinar cell differentiation andhould be valuable for studying the exocrine differen-

iation program. X

Materials and MethodsES Cell Culture and TransfectionCGR8 ES cells were maintained as published.25

reporter gene construct containing the -500/�8EIat elastase I (Ela1) enhancer/promoter28 was gener-ted by subcloning the -500 fragment-driven puromy-in resistance complementary DNA (cDNA) into pKSlasmid. An IresLacZ-mouse phosphoglycerate-kinaseromoter-driven hygromycin resistance cassette (a giftrom E. Maandag, The Netherlands Cancer Institute,msterdam, The Netherlands) was inserted down-

tream. Cells were electroporated (260V, 500 �F) usingBio-Rad (Hercules, CA) gene pulser and selected

sing 200 �g/mL hygromycin to establish Ela-pur-resLacZ clones (Ela-pur). Pancreatic acinar tumor cellsAR42J and 266-6) as well as NIH3T3 cells, were trans-ected as controls.

Adenoviral Generation and GeneTransductionAdenoviral vectors were obtained as described.29

riefly, full-length cDNAs encoding rat Ptf1a30 or mouseist131 were inserted into the pAd-shuttle-cytomegalovi-

us vector32; recombinant adenoviruses were produced byhe Laboratory of Gene Therapy (Gene Vector Productionetwork, Nantes, France) using the Ad-Easy system.32

nfections were performed as described in the Supple-entary Materials and Methods section (see Supplemen-

ary material online at www.gastrojournal.org).

In Vitro Differentiation and SelectionProcedureTo direct differentiation, Ela-pur ES clones were

llowed to aggregate in bacterial Petri dishes (3.3 � 104

ells/mL) in medium supplemented with 3% fetal bovineerum without leukemia inhibitory factor. After 7 days,0 –50 EBs were plated in gelatin-coated, 6-well cultureishes for 7 additional days in 10% fetal bovine serum–upplemented medium (differentiation step). In some ex-eriments, conditioned medium (CM) from E16.5 fetalancreas cultures was added during this 14-day period

1:1 dilution with normal medium).25 Medium washanged every 2 days. In addition, cells were infected 36ours after EB plating with adenoviruses as described inhe Supplementary Materials and Methods section. ForS–Ela-pur selection, differentiated cultures were main-

ained in differentiation medium plus puromycin (0.8g/mL) (Calbiochem, Darmstadt, Germany). After 2eeks of selection, cells were re-infected with adenovi-

uses and cultured for an additional week (selection step,5 days).

Molecular, Ultrastructural, and FunctionalCharacterization of Selected Ela-Pur CellsCells were processed by immunocytochemistry,

-Gal staining, and electron microscopy as described

iEtt(M

d

nmtdWqueAN1cmAemucaodsc2b

ecdcptghEtp

wadp

Fpmqpt

BA

SIC–L

IVER

,PA

NCREA

S,A

ND

BIL

IARY

TRA

CT

October 2008 PANCREATIC ACINAR DIFFERENTIATION OF ES CELLS 1303

n the Supplementary Materials and Methods section.xpression analyses were performed by RT-PCR. Func-

ional assays included bromodeoxyuridine incorpora-ion, cytosolic Ca2� measurements, and �-amylaseAmyl) secretion (see the Supplementary Materials and

ethods section).

StatisticsStatistical differences were analyzed by the Stu-

ent t test.

ResultsDigestive Enzyme Gene Expression Is NotActivated as a Single Regulatory ModuleDuring Pancreatic DevelopmentNinety percent of acinar mRNA encodes a small

umber of enzymes, widely used as cytodifferentiationarkers. However, little is known about the quantita-

ive regulation of expression during mouse pancreaticevelopment after initiation of acinar differentiation.e assessed the relative levels of selected mRNAs using

uantitative RT-PCR and RNA from E12.5 pancreasntil the postnatal period. We found that acinar genexpression does not follow a single pattern (Figure 1A).fter an increase in levels of all digestive enzyme mR-As at E14.5, distinct patterns were identified (Figure

A). This diversity is better reflected by comparinghanges in mRNA levels of chymotrypsinogen B (Chy-oB), Amyl, and Ela1 with those of carboxypeptidase1 (CPA). We chose CPA as a reference because it is thearliest enzyme transcript detected during develop-ent.23 The CPA:ChymoB ratio remained essentially

nchanged from E15.5 to adulthood (Figure 1B), indi-ating similar regulation. By contrast, the CPA:Amylnd CPA:Ela1 ratios (Figure 1B) decreased markedlyver time, indicating that Ela1 and Amyl mRNAs un-ergo major up-regulation at later developmentaltages. Moreover, the increase in Ela1 expression oc-urs mainly in the adult compared with postnatal day(Figure 1). These regulatory patterns can be used to

etter characterize acinar differentiation in vitro.

Genetic Selection of ES Cells Engaging theAcinar Differentiation ProgramDifferentiating ES cells recapitulate many aspects of

mbryonic development in vivo; therefore, reporter ESlones were generated to study acinar differentiation. Un-ifferentiated ES cells were transfected with a constructonferring puromycin-resistance and �-galactosidase ex-ression under the control of the rat Ela1 -500 enhancer28;he construct also contains a phosphoglycerate-kinase-Hy-ro unit for selection of transfectants (Figure 2A). Fourygromycin-resistant clones (Ela-pur) of undifferentiatedS cells screened by PCR to verify transgene integration (see

he supplementary Materials and Methods section and sup-

lementary Figure 1; see supplementary material online at u

ww.gastrojournal.org) showed similar growth propertiess parental cells and displayed similar expression profiles onifferentiation (not shown). Because Ela1 already is ex-ressed at E12 of development (Figure 1), this system will be

igure 1. Expression of transcripts coding for digestive enzymes duringancreatic development and in adult pancreas assessed by qRT-PCR. (A)RNAs from embryonic pancreatic rudiments and adult pancreas wereuantified and transcript levels are expressed relative to those in the E12.5ancreas. (B) Ratios of mRNAs levels of ChymoB, Amyl, and Ela1 relativeo CPA mRNA levels (A) during pancreatic development.

seful to trace activation of acinar gene expression.

ceeftbpc1

twor(rwt(ttnecci(eces

trTeatlmaw(pAMstce

ewmiclCeIc

FdpoddwCFCbwsl

BA

SIC–LIV

ER,

PA

NCREA

S,A

ND

BILIA

RY

TRA

CT

1304 ROVIRA ET AL GASTROENTEROLOGY Vol. 135, No. 4

Differential Regulation of Digestive EnzymeGene Expression by Ptf1a and Mist1 inDifferentiating Ela-Pur ES Cell ClonesSoluble signals present in CM from E16.5 pan-

reas cultures, superimposed to adenoviral-mediatedxpression of Ptf1a, result in a synergistic induction ofxocrine genes in differentiating ES cells.29 We there-ore investigated the effect of the combined transduc-ion of Ptf1a and Mist1 on acinar gene expressionecause both transcription factors are co-expressed inancreatic cells starting at E10.5. EBs from the Ela-purlones were generated in the presence of CM and after

igure 2. Generation of the reporter Ela-pur ES cell clones and theifferentiation and selection protocol. (A) Structure of the Ela promoter-uror-IresLacZ- phosphoglycerate-kinase-Hygror construct. (B) Schemef the protocol and experimental conditions used. Ela-pur cells wereifferentiated in suspension as EB for 7 days, plated in cell cultureishes, and grown for 7 days. During this period, adenoviral infectionsere performed in adhered EB and cultures were supplemented withM from the culture of E16.5 fetal pancreatic rudiments, as indicated.or the sake of simplicity, this step was designated as differentiation.ells having activated an acinar differentiation program were selectedy incubation with puromycin. Two weeks later, cells were re-infectedith the same adenoviruses and cultured for an additional week. Thisecond step was designated as selection, although differentiation-re-

ated events could take place during this phase.

week were allowed to adhere for 7 days (differentia- o

ion step) (Figure 2B). In addition, EBs were infectedith adenovirus expressing either GFP cDNA (AdGFP),r rat Ptf1a (Adp48) and mouse Mist1 (AdMist1),29

esulting in 60%–70% of cells expressing transgenessupplementary Figure 2A–C; see supplementary mate-ial online at www.gastrojournal.org). Control cellsere generated by inducing spontaneous differentia-

ion without additional experimental manipulationFigure 2B). Importantly, qRT-PCR showed a differen-ial regulation of acinar genes by Ptf1a and Mist1 athis stage: Ptf1a induced a marked increase in CPA butot in Ela1 mRNA whereas Mist1 did not have anyffect on gene expression on its own. By contrast, theombination of both led to a significantly higher in-rease of CPA mRNA levels (P � .003 vs control) and,mportantly, to a 2.5-fold increase in Ela1 mRNA levelsFigure 3). Therefore, Ptf1a and Mist1 activated a genexpression pattern similar to that of early exocrineells (Figure 1). Because Ela1 mRNA was enhancedxclusively in this condition, it was chosen for furthertudies.

To select for cells having activated an acinar differen-iation program, puromycin was added and cells weree-infected with both Adp48 and AdMist1 (Figure 2B).ransient sequential gene expression obtained with ad-noviruses, rather than sustained expression, was used tovoid the strong antiproliferative effect of Ptf1a30; mul-iplicity of infection was optimized to favor cell survival,eading to 40% of the cells infected at this stage (supple-

entary Figure 2E–G; see supplementary material onlinet www.gastrojournal.org). Cells were analyzed after 3eeks of selection, when isolated colonies had expanded

n � 8) (selection step, Figure 2B). Undifferentiated Ela-ur ES cells did not survive puromycin selection (n � 3).fter inducing differentiation with CM and Ptf1a andist1 adenoviruses (referred to as Ela-purp48-Mist1 after

election), greater than 95% cells were strongly � galac-osidase positive (n � 6) (Figure 4A). In conclusion,ell-trapping results in an efficient selection of elastase-xpressing cells.

Cells Having Activated the Acinar GeneExpression Program Display a ReducedProliferative AbilityTo characterize the features of the acinar differ-

ntiation program in genetically selected cells, qRT-PCRas used (Figure 4B and C). The highest digestive enzymeRNA levels were achieved in Ela-purp48-Mist1. Interest-

ngly, only CPA and ChymoB increased up to 300-fold inomparison with spontaneously differentiated and se-ected cells (Ela-purC) (Figure 4B). The ratio of CPA/hymoB mRNA levels was 0.43, a value similar to that in

arly (E12.5–E14.5) pancreatic development (Figure 1B).mportantly, these effects were associated with an in-rease in endogenous Ptf1a and RBPL mRNAs, but not

f endogenous Mist1 (Figure 4C). As expected, high

lmwvwclpaccIied

patcn

ewp�p(drpoRuigCa(pb5sftapcltd

epw(tftmum

wimmr

FethAi

BA

SIC–L

IVER

,PA

NCREA

S,A

ND

BIL

IARY

TRA

CT

October 2008 PANCREATIC ACINAR DIFFERENTIATION OF ES CELLS 1305

evels of ectopic Mist1 mRNA were detected (supple-entary Figure 3; see supplementary material online atww.gastrojournal.org). Pdx1, a transcription factor in-

olved in both exocrine and endocrine differentiation,as expressed— but not significantly modulated—in these

onditions (Figure 4C), possibly reflecting the low Pdx1evels characteristic of acinar cells.33,34 In addition, ex-ression of selected genes whose products are involved incinar secretion was analyzed (Figure 4C). Ela-purp48-Mist1

ells expressed mRNAs coding for cholecystokinin A re-eptor (CCKAR) and muscarinic 3 receptors as well asP3R3, an important signaling mediator. Transcripts cod-ng for connexin 32, a gap junctional protein required forxocrine secretion,35 also were detected. By contrast, en-

igure 3. Expression of digestive enzyme transcripts after the differ-ntiation step by qRT-PCR. mRNA transcripts were analyzed just afterhe first step of differentiation as described in Figure 2B. After EB ad-esion, cells were infected—or not—with GFP cDNA (AdGFP), Adp48,dMist1, or with a combination of both Adp48 and AdMist1. Error bars

ndicate the standard deviations of 2 independent experiments.

ocrine marker levels were very low (Ngn3, Nkx6.1) (sup- i

lementary Figure 4; see supplementary material onlinet www.gastrojournal.org) or undetectable (insulin). Cy-okeratin 19 (CK19) and cystic fibrosis transmembraneonductance regulator (CFTR) (ductal) mRNA levels wereot modulated (Figure 4C).To assess whether physiologic cytodifferentiation was

ngaged, expression of selected corresponding proteinsas studied using immunofluorescence. Ninety-eightercent of Ela-purp48-Mist1 cells expressed E-cadherin and-catenin at cell– cell contact sites (results from 3 inde-endent experiments), showing their epithelial nature

Figure 5A and B). Cytoplasmatic Amyl and CPA wereetected in most cells (67% � 3.8% and 80% � 1.54%,espectively) (Figure 5C and D), as in AR42J cells (sup-lementary Figure 5A and B; see supplementary materialnline at www.gastrojournal.org). In agreement with theT-PCR results, nuclear Pdx1 (Figure 5E) and RBPL (Fig-re 5F) were expressed. These proteins were undetectable

n undifferentiated Ela-pur cells (Figure 5C’ and F’). Re-arding the secretory signaling pathway and machinery,CKAR (Figure 5I), VAMP8 —a SNARE protein associ-ted with ZG whose function is crucial for secretion36

Figure 5G), and syntaxin 4 —a membrane protein ofancreatic acinar cells36 (Figure 5H), displayed mem-rane (Figure 5H and I) or cytoplasmic patterns (FigureG). By contrast, Dolichos biflorus agglutinin, whichelectively recognizes ductal cells,37 was restricted to veryew cells (1.8% � 1.82%) that were �-galactosidase nega-ive (Figure 5J). Moreover, bromodeoxyuridine uptakessays showed that Ela-purp48-Mist1 cells showed reducedroliferative ability as compared with other experimentalonditions (Figure 4D). In addition, cyclin D1 mRNAevels were reduced in these cells (Figure 4E). Collectively,hese data show the coordinated activation of an acinarifferentiation program in Ela-purp48-Mist1 cells.

Ela-purp48-Mist1 Cells Contain ZG and Respondto Acinar SecretagoguesA hallmark of acinar cells is enzyme storage in

lectron-dense ZG (Figure 6A). Ela-purp48-Mist1 cells dis-layed 250- to 600-nm ZG-like vesicles (Figure 6B), whichere very scarce in spontaneously differentiated cells

Figure 6C), but present in selected CM-treated cellsransduced with GFP (Ela-purGFP), suggesting that CMavors ZG formation and contributes to improved secre-ion to secretagogues (see below). Gold immunoelectron

icroscopy showed that these vesicles contain Amyl (Fig-re 6E and supplementary Figure 6; see supplementaryaterial online at www.gastrojournal.org).To further investigate their functional properties, cells

ere loaded with the calcium-sensitive dye fura-2 and thentracellular Ca2� response to 5 �mol/L carbachol was

onitored. Figure 7A summarizes results from 4 experi-ents. Ela-purp48-Mist1 cells (Figure 7A, right) displayed a

apid and transient increase in Ca2� concentration, sim-

lar to that described for primary isolated mouse acinar

cindv(h(s

plsiupsae

cm�bt

bhsldmfat

BA

SIC–LIV

ER,

PA

NCREA

S,A

ND

BILIA

RY

TRA

CT

1306 ROVIRA ET AL GASTROENTEROLOGY Vol. 135, No. 4

ells.38 In contrast, Ela-purC cells (left) showed a smallerncrease in Ca2� concentration lacking the typical transient-ess of the normal response. Peak increases were statisticallyifferent for the 2 conditions (P � .05), whereas the plateaualues at the end of the carbachol pulse were not differentP � .05). The percentage of carbachol-responsive cells wasigher in Ela-purp48-Mist1 (35%; 97 of 280) than in Ela-purC

14%; 33 of 328). Ela-purGFP displayed an intermediate re-ponse (not shown).

In acinar cells, intracellular free Ca2� is considered therimary trigger for enzyme secretion. Therefore, we ana-

yzed the ability of cells to secrete Amyl in response toecretagogues in vitro (Figure 7B). Carbachol and CCKnduced a significant increase in total Amyl activity (Fig-re 7B, left); this effect was more pronounced in Ela-urp48-Mist1 cells (P � .05 compared with Ela-purC cells),uggesting de novo synthesis of digestive enzymes medi-ted by the secretagogues, a known effect of these mol-

cules. Both secretagogues also induced a significant in- h

rease in the extracellular Amyl activity (Figure 7B,iddle), being more pronounced in Ela-purp48-Mist1 cells (P.05). Altogether, these findings support the notion that

oth increased synthesis and secretion occur in responseo secretagogues.

DiscussionPrevious attempts to generate acinar cells in vitro,

ased on the improvement of primary culture conditions,ave been unsuccessful. In all cases, acinar-ductal metapla-ia/transdifferentiation occurs in association with a rapidoss of functional properties.39–42 Here, we describe theevelopment of immature acinar cells using the ES–EBodel, which recapitulates many cues and signals required

or the exocrine development in vivo. Our strategy takesdvantage of the genetic selection of acinar lineage commit-ed cells using an acinar-specific elastase promoter. The

Figure 4. Selection of elas-tase-producing cells. (A) Analy-sis of the reporter �-galactosi-dase activity. X-Gal staining wasperformed after selection of dif-ferentiated cells. For each con-dition, representative staining ofthe selected colonies is shown.Scale bar, 100 �m. (B) Digestiveenzyme and (C) exocrine geneexpression by q-PCR. Histo-grams show the relative expres-sion levels normalized to theloading control Hprt. Error barsindicate the standard deviationsof 4 experiments in B and of 2–3experiments in C. *P � .05 and**P � .01 compared with Ela-purC cells. (D) Bromodeoxyuri-dine (BrdU) incorporation in thedifferentiation conditions ana-lyzed by fluorescence-activatedcell sorter (n � 3). *P � .05 and**P � .01 compared with Ela-purC cells. (E) Cyclin D1 expres-sion by q-RT-PCR analyzed asin C.

igh efficiency of our strategy was confirmed by X-Gal

sco

pF

aenttraealrcrpolmowESFMeke

ssIlmabotzdamtzpCplirEu

ovdi

Ff(CatsEbsFsf

BA

SIC–L

IVER

,PA

NCREA

S,A

ND

BIL

IARY

TRA

CT

October 2008 PANCREATIC ACINAR DIFFERENTIATION OF ES CELLS 1307

taining, showing that nearly all cells expressed the reporteronstruct. Contamination by other cell types (ie, endocriner ductal) was minimal (data not shown).

The ability to generate essentially pure acinar-like cellsrovides a powerful model to study acinar differentiation.

igure 5. Immunofluorescence analysis of Ela-purp48-Mist1 cells. Con-ocal images of cells immunostained for (A) E-cadherin, (B) �-catenin,C) Amyl, (D) CPA, (E) Pdx1, (F) RBPL, (G) VAMP8, (H) syntaxin 4, and (I)CKAR. As negative controls, we show the staining without primaryntibodies and with anti-mouse (A’) and anti-rabbit (B’) secondary an-ibodies on Ela-purp48-Mist1 cells, whereas C’, D’, E’, and F’, show thetaining with the corresponding primaries antibodies on undifferentiatedla-pur ES cells. Nuclei were labeled with ToPro-3 iodide (blue). (J) Theox shows a brown cell stained for Dolichos biflorus agglutinin in doubletaining with X-Gal. Scale bars: A–I, 50 �m; J, 100 �m. Supplementaryigure 5 (see supplementary material online at www.gastrojournal.org)hows immunostaining of AR42J acinar cells with the same antibodies,or comparison.

or instance, this system has major potential to directly c

ssess the functional role of candidate genes involved inxocrine development or function: cells selected on sponta-eous differentiation indeed express Ptf1a and Mist1, al-hough at levels that are much lower than found in cellsransduced with Ptf1a and Mist1, providing evidence thategulation of transcription factor levels is a key step duringcinar differentiation. In this regard, we found that endog-nous Ptf1a also was up-regulated by co-expression of Ptf1and Mist-1, suggesting the existence of an autoregulatoryoop favoring exocrine differentiation. Indeed, such auto-egulatory circuits have been described recently during exo-rine pancreatic development. Thus, RBPL is able to auto-egulate its expression as acinar differentiation progresses,roviding a possible mechanism to ensure the maintenancef the acinar phenotype.24 Interestingly, such positive regu-

ation was not observed for Mist1 because endogenousRNA levels were not modulated (Figure 4C), probably

wing to the high levels achieved after gene transduction,hich were found to be quite similar to those observed at16.5 and in adult pancreas (Supplementary Figure 7; seeupplementary material online at www.gastrojournal.org).urthermore, we showed that the combination of Ptf1a andist1 activates acinar cell gene expression in short-term

xperiments. In this sense, it has been described that Mist1nock-out mice show a reduced amount of Amyl duringarly stages of pancreatic development.19

The differentiated cells produced using this strategyhowed a significant up-regulation of acinar gene expres-ion as shown by qRT-PCR and immunocytochemistry.n particular, several digestive enzymes were up-regu-ated, reflecting not only an increase in the Ptf1a/p48-

ediated PTF1 activity, but also the true activation of thecinar differentiation program. This notion is supportedy the accompanying increase in RBPL, a key componentf the PTF1 complex conferring its high activation po-ential. Importantly, the genes coding for digestive en-ymes are not regulated as a single module, but insteadisplay distinct regulatory patterns at different stages ofcinar cell differentiation as suggested by earlier experi-ents.7 In this sense, it was reported very recently that

he ability of RBPL to regulate the expression of theymogen genes is not universal.24 Interestingly, it wasroposed that a cell population expressing high levels ofPA corresponds to the main multipotent progenitoropulation during pancreatic development.23 These cells,

acking expression of Amyl, are incompletely character-zed. On the basis of our results showing a strong up-egulation of CPA and ChymoB expression, but not oflas1 and Amyl, we suggest that ChymoB also could besed to trace early exocrine precursors.In addition to generating a highly enriched population

f cells expressing digestive enzymes and proteins in-olved in the secretory pathway, nearly 40% of the cellsisplay functional properties of native acinar cells. It is

ntriguing that this figure is similar to the proportion of

ells that could be transduced efficiently by adenoviral

tcr

asC

Fwnbs

BA

SIC–LIV

ER,

PA

NCREA

S,A

ND

BILIA

RY

TRA

CT

1308 ROVIRA ET AL GASTROENTEROLOGY Vol. 135, No. 4

ransgene delivery during selection. Because Ela-purC

ells that differentiated spontaneously showed an aber-ant pattern of Ca2� response, similar to that described in

igure 7. Carbachol-evoked Ca2� signaling and exocytosis in Ela-purith 5 �mol/L carbachol for 2.5 minutes. At the indicated times, the sec� 97. (B) Selected cells were stimulated for 30 minutes with the indicoth the supernatant and cell lysates. Total activity corresponds to th

tandard deviation of 2 experiments performed in triplicate. �, Ela-purC; □,

cinar cells of Mist-1– deficient mice,38 it is tempting topeculate that efficient expression of Mist1 renders thea2� signaling machinery more mature in elastase-pro-

Figure 6. Immunoelectronmicroscopy analysis of Ela-purp48-Mist1 cells. Electron mi-crographs illustrating electron-dense vesicles in (A) murinepancreas (inset) and (B) Ela-purp48-Mist1 cells (arrows). (C)Histograms of data from 2 ex-periments showing the per-centage of cells displayingthese vesicles in the indicatedculture conditions. Immuno-gold labeling with anti-Amyl an-tibody in (D) murine pancreasand (E) Elas-purop48-Mist1. Scalebars: B, 1 �m; D, 0.6 �m; E,1.7 �m.

ted cells. (A) Cytosolic Ca2� signals in Fura-2–loaded cells stimulatedogue was superfused and removed. Ela-purC, n � 33; Ela-purp48-Mist1,concentrations of carbachol and CCK. Amyl activity was measured in

of secreted and intracellular Amyl activities. Error bars indicate the

selecretagatede sum

Ela-purGFP; , Ela-purp48-Mist1.

dpEdZsPea

lacdadtaaatettwnlomppcned

nbsaccKnttCgdci

c

G1

1

1

1

1

1

1

1

1

1

1

BA

SIC–L

IVER

,PA

NCREA

S,A

ND

BIL

IARY

TRA

CT

October 2008 PANCREATIC ACINAR DIFFERENTIATION OF ES CELLS 1309

ucing cells, as has been proposed in vivo. Also, theercentage of cells showing ZG-like vesicles was higher inla-purp48-Mist1 cells than in any other experimental con-ition, in agreement with the fact that the number ofGs is reduced in Mist1 knock-out mice.19 Overall, wehow that the directed-differentiated cellular response totf1a and Mist1 is similar to early stages of pancreaticxocrine development, supporting the validity of ourpproach to develop functional genomic assays.

A major contribution of this work is that the cell popu-ation generated largely consists of cells lacking a mixedcinar-ductal phenotype, in contrast to reports based on theulture of purified pancreatic acinar cells in which a de-ifferentiation process takes place. Because we have takendvantage of the fact that ES cells recapitulate embryonicevelopmental processes, we may have been able to avoidhe signals that are triggered on mature acinar cell isolationnd in vitro culture. The signaling pathways proposed to bectivated under these conditions are mediated by Notchnd epidermal growth factor transduction, which lead tohe transdifferentiation of acinar cells to a ductal lin-age.2,3,40 We have not detected clear changes in the activa-ion of the Notch signaling pathway in the culture condi-ions tested here (not shown), and the expression of Pdx1,hich is induced during the acinar-ductal switch, also wasot regulated.40 Because acinar cells express much higher

evels of digestive enzymes in the adult than during devel-pment, it is possible that only when committed cells ter-inally achieve a mature differentiation program are the

rotective mechanisms to bypass the lytic potential of theirroducts activated. This fully differentiated state may in-lude a higher potential of cell plasticity in vitro. An alter-ate explanation is that in vivo metaplasia occurs because ofxpansion of duct-like cells without dedifferentiation/trans-ifferentiation of acinar cells.37

The importance of the molecular characterization of aci-ar cell populations undergoing embryonic maturation haseen emphasized by several recent experimental modelshowing that pancreatic ductal neoplasia can be induced byctivation of oncogenes (ie, K-ras) in all cells of the pan-reas43,44 as well as in acinar cells.6,45,46 Immature acinarells are more susceptible to the transforming effects of-ras activation than mature acinar cells in vivo.45 Providingew insights into the identity of the immature acinar cellsargeted during cancer development45 is an important issueo understand the nature of pancreatic cancer precursors.onsequently, the identification of new markers distin-uishing acinar cell populations during development and inisease conditions remains an important aim. Global trans-riptome analyses of ES-derived exocrine cells should benformative and currently are underway in our laboratories.

Supplementary Data

Note: To access the supplementary material ac-

ompanying this article, visit the online version of

astroenterology at www.gastrojournal.org, and at doi:0.1053/j.gastro.2008.06.049.

References

1. Williams JA. Regulation of pancreatic acinar cell function. CurrOpin Gastroenterol 2006;22:498–504.

2. Means AL, Meszoely IM, Suzuki K, et al. Pancreatic epithelialplasticity mediated by acinar cell transdifferentiation and gener-ation of nestin-positive intermediates. Development 2005;132:3767–3776.

3. Miyamoto Y, Maitra A, Ghosh B, et al. Notch mediates TGFalpha-induced changes in epithelial differentiation during pancre-atic tumorigenesis. Cancer Cell 2003;3:565–576.

4. Sandgren EP, Luetteke NC, Palmiter RD, et al. Overexpression ofTGF alpha in transgenic mice: induction of epithelial hyperplasia,pancreatic metaplasia, and carcinoma of the breast. Cell 1990;61:1121–1135.

5. Schmid RM. Acinar-to-ductal metaplasia in pancreatic cancer de-velopment. J Clin Invest 2002;109:1403–1404.

6. Zhu L, Shi G, Schmidt CM, et al. Acinar cells contribute to themolecular heterogeneity of pancreatic intraepithelial neoplasia.Am J Pathol 2007;171:263–273.

7. Gittes GK, Rutter WJ. Onset of cell-specific gene expression in thedeveloping mouse pancreas. Proc Natl Acad Sci U S A 1992;89:1128–1132.

8. Kim SK, MacDonald RJ. Signaling and transcriptional control ofpancreatic organogenesis. Curr Opin Genet Dev 2002;12:540–547.

9. Jensen J. Gene regulatory factors in pancreatic development. DevDyn 2004;229:176–200.

0. Murtaugh LC. Pancreas and beta-cell development: from theactual to the possible. Development 2007;134:427–438.

1. Rose SD, Swift GH, Peyton MJ, et al. The role of PTF1-P48in pancreatic acinar gene expression. J Biol Chem 2001;276:44018–44026.

2. Petrucco S, Wellauer PK, Hagenbuchle O. The DNA-binding activ-ity of transcription factor PTF1 parallels the synthesis of pan-creas-specific mRNAs during mouse development. Mol Cell Biol1990;10:254–264.

3. Krapp A, Knofler M, Ledermann B, et al. The bHLH protein PTF1-p48 is essential for the formation of the exocrine and the correctspatial organization of the endocrine pancreas. Genes Dev 1998;12:3752–3763.

4. Kawaguchi Y, Cooper B, Gannon M, et al. The role of the trans-criptional regulator Ptf1a in converting intestinal to pancreaticprogenitors. Nat Genet 2002;32:128–134.

5. Lin JW, Biankin AV, Horb ME, et al. Differential requirement forptf1a in endocrine and exocrine lineages of developing zebrafishpancreas. Dev Biol 2004;270:474–486.

6. Afelik S, Chen YL, Pieler T. Combined ectopic expression of Pdx1and Ptf1a/p48 results in the stable conversion of posteriorendoderm into endocrine and exocrine pancreatic tissue. GenesDev 2006;20:1441–1446.

7. Jarikji ZH, Vanamala S, Beck CW, et al. Differential ability of Ptf1aand Ptf1a-VP16 to convert stomach, duodenum and liver to pan-creas. Dev Biol 2007;304:786–799.

8. Fukuda A, Kawaguchi Y, Furuyama K, et al. Ectopic pancreasformation in Hes1-knockout mice reveals plasticity of endoder-mal progenitors of the gut, bile duct, and pancreas. J Clin Invest2006;116:1484–1493.

9. Johnson CL, Kowalik AS, Rajakumar N, et al. Mist1 is neces-sary for the establishment of granule organization in serousexocrine cells of the gastrointestinal tract. Mech Dev 2004;

121:261–272.

2

2

2

2

2

2

2

2

2

2

3

3

3

3

3

3

3

3

3

3

4

4

4

4

4

4

4

i(a

SG4PmHdFS

GLXvo

c

BA

SIC–LIV

ER,

PA

NCREA

S,A

ND

BILIA

RY

TRA

CT

1310 ROVIRA ET AL GASTROENTEROLOGY Vol. 135, No. 4

0. Pin CL, Rukstalis JM, Johnson C, et al. The bHLH transcriptionfactor Mist1 is required to maintain exocrine pancreas cell orga-nization and acinar cell identity. J Cell Biol 2001;155:519–530.

1. Zhu L, Tran T, Rukstalis JM, et al. Inhibition of Mist1 homodimerformation induces pancreatic acinar-to-ductal metaplasia. MolCell Biol 2004;24:2673–2681.

2. Beres TM, Masui T, Swift GH, et al. PTF1 is an organ-specific andnotch-independent basic helix-loop-helix complex containing themammalian suppressor of hairless (RBP-J) or its paralogue,RBP-L. Mol Cell Biol 2006;26:117–130.

3. Zhou Q, Law AC, Rajagopal J, et al. A multipotent progenitordomain guides pancreatic organogenesis. Dev Cell 2007;13:103–114.

4. Masui T, Long Q, Beres TM, et al. Early pancreatic developmentrequires the vertebrate Suppressor of Hairless (RBPJ) in the PTF1bHLH complex. Genes Dev 2007;21:2629–2643.

5. Skoudy A, Rovira M, Savatier P, et al. Transforming growth factor(TGF)beta, fibroblast growth factor (FGF) and retinoid signallingpathways promote pancreatic exocrine gene expression inmouse embryonic stem cells. Biochem J 2004;379:749–756.

6. Wobus AM, Boheler KR. Embryonic stem cells: prospects fordevelopmental biology and cell therapy. Physiol Rev 2005;85:635–678.

7. D’Amour KA, Bang AG, Eliazer S, et al. Production of pancreatichormone-expressing endocrine cells from human embryonic stemcells. Nat Biotechnol 2006;24:1392–1401.

8. MacDonald RJ, Swift GH. Analysis of transcriptional regulatoryregions in vivo. Int J Dev Biol 1998;42:983–994.

9. Rovira M, Jane-Valbuena J, Marchand M, et al. Viral-mediatedcoexpression of Pdx1 and p48 regulates exocrine pancreaticdifferentiation in mouse ES cells. Cloning Stem Cells 2007;9:327–338.

0. Rodolosse A, Chalaux E, Adell T, et al. PTF1 alpha/p48 transcrip-tion factor couples proliferation and differentiation in the exo-crine pancreas and cell proliferation. Gastroenterology 2004;127:937–949.

1. Pin CL, Lemercier C, Konieczny SF. Cloning of the murine Mist1gene and assignment to mouse chromosome band 5G2-5G3.Cytogenet Cell Genet 1999;86:219–222.

2. He TC, Zhou S, da Costa LT, et al. A simplified system forgenerating recombinant adenoviruses. Proc Natl Acad Sci U S A1998;95:2509–2514.

3. Heller RS, Stoffers DA, Bock T, et al. Improved glucose toleranceand acinar dysmorphogenesis by targeted expression of trans-cription factor PDX-1 to the exocrine pancreas. Diabetes 2001;50:1553–1561.

4. Miyatsuka T, Kaneto H, Shiraiwa T, et al. Persistent expression ofPDX-1 in the pancreas causes acinar-to-ductal metaplasiathrough Stat3 activation. Genes Dev 2006;20:1435–1440.

5. Chanson M, Fanjul M, Bosco D, et al. Enhanced secretion ofamylase from exocrine pancreas of connexin32-deficient mice.J Cell Biol 1998;141:1267–1275.

6. Wang CC, Ng CP, Lu L, et al. A role of VAMP8/endobrevin inregulated exocytosis of pancreatic acinar cells. Dev Cell 2004;7:359–371.

7. Stanger BZ, Stiles B, Lauwers GY, et al. Pten constrains cen-troacinar cell expansion and malignant transformation in the

pancreas. Cancer Cell 2005;8:185–195.

8. Luo X, Shin DM, Wang XH, et al. Aberrant localization of intracel-lular organelles, Ca2� signaling, and exocytosis in Mist1 nullmice. J Biol Chem 2005;280:12668–12675.

9. Vila MR, Lloreta J, Real FX. Normal human pancreas culturesdisplay functional ductal characteristics. Lab Invest 1994;71:423–431.

0. Rooman I, De Medts N, Baeyens L, et al. Expression of the Notchsignaling pathway and effect on exocrine cell proliferation in adultrat pancreas. Am J Pathol 2006;169:1206–1214.

1. Delisle RC, Logsdon CD. Pancreatic acinar-cells in culture—ex-pression of acinar and ductal antigens in a growth-related man-ner. Eur J Clin Invest 1990;51:64–75.

2. Sphyris N, Logsdon CD, Harrison DJ. Improved retention of zymo-gen granules in cultured murine pancreatic acinar cells and in-duction of acinar-ductal transdifferentiation in vitro. Pancreas2005;30:148–157.

3. Hruban RH, Adsay NV, Albores-Saavedra J, et al. Pathology ofgenetically engineered mouse models of pancreatic exocrinecancer: consensus report and recommendations. Cancer Res2006;66:95–106.

4. Hingorani SR, Petricoin EF, Maitra A, et al. Preinvasive and inva-sive ductal pancreatic cancer and its early detection in themouse. Cancer Cell 2003;4:437–450.

5. Guerra C, Schuhmacher AJ, Canamero M, et al. Chronic pancre-atitis is essential for induction of pancreatic ductal adenocarci-noma by k-Ras oncogenes in adult mice. Cancer Cell 2007;11:291–302.

6. Sandgren EP, Quaife CJ, Paulovich AG, et al. Pancreatic tumorpathogenesis reflects the causative genetic lesion. Proc NatlAcad Sci U S A 1991;88:93–97.

Received December 27, 2007. Accepted June 12, 2008.Address reprint requests to: Anouchka Skoudy, PhD, Institut Munic-

pal d’Investigació Mèdica, Barcelona Biomedical Research ParkBBRP), Dr Aiguader 88, 08003 Barcelona, Spain. e-mail:skoudy@imim.es; fax: (34) 93-3160410.This study was supported by Spanish Ministry of Education and

cience Grants (SAF2001-0432 and GEN2001-4748-C05 to A.S.;EN2001-4748-C01 and SAF2004-01137 to F.X.R. and SAF2006-973 to M.A.V.), Instituto de Salud Carlos III grants (02/3053 andI05/2738 to A.S.; and Red HERACLES to M.A.V.), Catalan Govern-ent grants (SGR2005 to M.A.V.), and by a National Institutes ofealth grant (DK55489 to S.F.K.). A.S. was supported by the Institutoe Salud Carlos III; M.R., F.D. and M. M. were recipients of a Graduateellowship from the Ministry of Education and Science, Instituto dealud Carlos III, and the Catalan Government, respectively.The authors thank P. Navarro and Y. Tor for technical support; M.

arrido for the processing of samples for electron microscopy; theaboratory of Gene Therapy, Nantes, for producing adenoviral vectors;. Molero and S. Leach for valuable discussions; R. Wagener and M.on Harrach for the gift of the anti-RBPL; and E. Maandag for the giftf the pEA6 plasmid.Present address of F.X.R.: Centro Nacional de Investigaciones On-

ológicas, Melchor Fernández Almagro 3, Madrid, Spain.

F.D. and M.M. contributed equally to this work.

fftaE

fuiAnr

ecowsUns

tDfDBMCffMwTtfC(CCCCGGAC

AaaaMATTGdw

ghpMatrwbtwtWRCsatr(cBIwLi(�p

ogfipuDIda

October 2008 PANCREATIC ACINAR DIFFERENTIATION OF ES CELLS 1310.e1

Supplementary Material and Methods

Tissue IsolationMurine embryonic pancreas isolation was per-

ormed under a stereomicroscope and tissues were trans-erred to RNAlater (Qiagen, Valencia, CA) for RNA ex-raction. All animal experiments were performed inccordance with the guidelines of the Animal Researchthical Committee of our Institution.

Genomic PCR for Screening ES Stable ClonesPCR amplification on genomic DNA was per-

ormed for the detection of the elastase-puror transgenesing EcoTaq (Ecogen, Barcelona, Spain) and the follow-

ng primers: 5=-TTAACTGAGTGCCGGCCTT-3= and 5=-ACTGCAGTGTGGTATGGCTGATTATGA-3= that an-eal to the 5= region of the elastase I enhancer and the 3=egion of the puror cDNA, respectively.

Adenoviral Gene TransductionCells were washed 3 times with phosphate-buff-

red saline (PBS) and infected for 12 hours with re-ombinant adenoviruses at a multiplicity of infectionf 1:25 in serum-free medium. Virus was eliminated byashing and cells were cultured further in medium

upplemented with fetal bovine serum or with CM.nder these conditions, the viability of the cells wasot compromised as scored by trypan blue dye exclu-ion in replicated wells.

qRT-PCRRNA was prepared using the GenElute mammalian

otal RNA kit (Sigma, St. Louis, MO) and treated withNase I (Ambion, Austin, TX). Real-time RT-PCR was per-

ormed in triplicate on an ABI Prism 7900HT Sequenceetector with the following TaqMan reagent kits (Appliediosystems, Foster City, CA): Mm00481616_m1 for CPA,m02342486_mH for Amyl, Mm00481616_m1 for

hymoB, Mm00712898_m1 for Ela1, Mm00479622_m1or Ptf1a, Mm00437606_s1 for ngn3, Mm00454962_m1or nkx6.1, Mm01259683_g1 for insulin I, and

m00446968_m1 for Hprt. Expression for other markersas performed with standard techniques and using theaqMan RT reagents for retrotranscription, the quantita-

ive SYBR Green PCR kit (Applied Biosystems), and theollowing primers: CK19 (S: CCTCCCGAGATTACAAC-ACT and AS: GGCGAGCATTGTCAATCTGT), CFTR

S: ACAAAACCTGGATCCCAATG and S: GAGCTGTC-AGGAAACTGCT), Pdx1 (S: AAATCCACCAAAGCT-ACGC and AS: CGGTCAAGTTCAACATCACTGC),x32 (S: CCCACCGAGAAAACCGTCTT and AS: AGGC-CGGATGATGAGGTA), CCKAR (S: CTGTCCCTG-CTGTCAGTGA and AS: ACACGGCACTTCCGAA-ATG), M3R (S: GAAGCGGAAGCAGAAAACTTTG andS: TCCTCCTAGATGACCGTTTCGT), IP3R3 (S: CCTC-

CCCAAGGCAAACT and AS: GACCTAGCATCCTCTA- a

GGCACTAA), RBPL (S: ACTCCGGTGCCTCTCATCAGnd AS: CTACGCACACCAAGGAACGA), endogenousnd ectopic Mist1 (S: GAACACCCATGCAGGACACAGnd AS: CCTGGAAGGCATTGTTGAG), endogenousist1 (Mist1e) (S: TGAACTCCTGATCCTCCTC and AS:

GTTGAAGGCCCAGATCAC), cyclin D1 (S: CCCTCCG-ATCTTACTTCAA and AS: GGAATGGTCTCCT-CATCTT) and Hprt (S: GGCCAGACTTTGTTG-ATTTG and AS: TGCGCTCATCTTAGGCTTTGT). Theata were processed using SDS 2.1 software and resultsere normalized to Hprt mRNA levels.

ImmunocytochemistryFor the detection of adenoviral-mediated trans-

ene expression, immunostaining was performed for 1our in PBS containing 1% bovine serum albumin withrimary rabbit antibodies raised against Ptf1a1 andist1. Cells were incubated with the Envision second-

ry reagent (Dako, Glostrup, Denmark) and the reac-ions were developed using 3,3’-diaminobenzidine tet-ahydrochloride as a chromogen. This substrate alsoas used to detect the duct-specific lectin Dolichosiflorus agglutinin coupled to peroxidase (EY Labora-ories, San Mateo, CA). Immunofluorescence stainingas conducted using the Tyramide Signal Amplifica-

ion method (TSA Fluorescence Systems; Perkin Elmer,altham, MA) and rabbit antibodies against Pdx1,25,29

BPL2 (a kind gift from R. Wagener, University ofologne, Germany), Amyl (Sigma), and CPA (Biogene-

is, Sandown, NH). Alternatively, staining with mousentibodies against E-cadherin (Transduction Labora-ories, Lexington, KY), � -catenin (Transduction Labo-atories), and rabbit antibodies against syntaxin 4Synaptic Systems, Göttingen, Germany), VAMP8 (Ab-am, Cambridge, UK), and CCKAR (Euro-diagnostica,N Arnhem, The Netherlands) was revealed using anti-

gG coupled to fluorescein (Dako). Nuclear labelingas performed with ToPro-3 iodide (Molecular Probes,eiden, The Netherlands). Immunofluorescence stain-

ng and GFP expression were visualized with a LeicaHouston, TX) TCS-SP2 confocal microscope. In situ-galactosidase activity was detected using standardrocedures.

Bromodeoxyuridine IncorporationCells were incubated with 1 �mol/L bromode-

xyuridine (Sigma) for 16 hours, dissociated into sin-le-cell suspensions, and resuspended in 300 �L ofxation and permeabilization solution (Cytofix/Cyto-erm kit; BD Biosciences, San Diego, CA) for 20 min-tes at 4°C. After washing with Perm/Wash buffer, theNA was denatured with 2 mol/L HCl for 20 minutes.

mmunofluorescence was performed following a stan-ard protocol and using a mouse monoclonal antibodygainst bromodeoxyuridine (BD Biosciences) and an

nti-mouse IgG coupled to fluorescein (Dako). Nuclear

s�n(

1doblTgacSgh

wbmm3mwmoia

isFp

mmomltdaamtdPl

1

2

3

1310.e2 ROVIRA ET AL GASTROENTEROLOGY Vol. 135, No. 4

taining was performed by incubating the cells with 5 g/mL propidium iodine and 150 � g/mL RNase over-ight at 4°C. Staining was analyzed by flow cytometry

Becton Dickinson).

Electron MicroscopyCells were fixed in 2% glutaraldehyde, postfixed in

% osmium tetroxide, dehydrated in ethanol, and embed-ed in Epon 812 resin as described.39 Semithin sectionsf cell layers were examined after staining with toluidinelue. Thin sections were stained with uranyl acetate and

ead citrate and examined using a Philips (Eindhoven,he Netherlands) 301 electron microscope. For immuno-old labeling the anti-Amyl antibody, followed by a goatnti-rabbit antibody conjugated with 15-nm gold parti-les (Aurion, Wageningen, The Netherlands), were used.amples were fixed with 4% paraformaldehyde and 0.1%lutaraldehyde in PBS and embedded in Unicryl (Eppel-eim, Germany).

Cytosolic Ca2� MeasurementsIntracellular Ca2� concentration measurements

ere performed as described previously.3 Cells were incu-ated in a bathing solution (140 mmol/L NaCl, 2.5mol/L KCl, 1.2 mmol/L CaCl2, 0.5 mmol/L MgCl2, 5mol/L glucose, and 10 mmol/L HEPES, pH 7.25 and

05 mOsm) containing 5 �mol/L Fura-2 AM for 45inutes at room temperature and washed thoroughlyith fura-2–free solution before initiating the experi-ent. Video microscopic measurements of [Ca2�] were

btained using an Olympus (Center Valley, PA) IX70nverted microscope with a 40� oil-immersion objective,

Polychrome IV monochromator (Till Photonics), a dig-

tal charge-coupled device camera, and the AquaCosmosoftware program (both from Hamamatsu Photonics).luorescence ratio (340 nm/380 nm) images were com-uted every 2 seconds.

Amylase Secretion AssayCells were washed with PBS and fresh cell culture

edium without fetal bovine serum was added, supple-ented or not with cholecystokinin octapeptide (Sigma)

r with carbachol at the indicated concentrations for 30inutes at 37°C. Culture supernatants then were col-

ected and cells were lysed in Krebs–Ringer buffer con-aining 0.2% bovine serum albumin. Amyl activity wasetermined using the Infinity Amylase Liquid Stable Re-gent (Termo Electron, Woburn, MA). To normalize themount of Amyl secretion, the total protein content waseasured by the Bradford method. Amyl released into

he supernatant and Amyl content of the cell pellets wasetermined. Each experiment was performed in triplicate.ositive control experiments were performed using iso-

ated mouse pancreatic acinar cells (not shown).

References

. Adell T, Gomez-Cuadrado A, Skoudy A, et al. Role of the basichelix-loop-helix transcription factor p48 in the differentiation phe-notype of exocrine pancreas cancer cells. Cell Growth Differ 2000;11:137–147.

. Wagener R, Kobbe B, Aszodi A, et al. Characterization of themouse matrilin-4 gene: a 5’ antiparallel overlap with the geneencoding the transcription factor RBP-L. Genomics 2001;76:89–98.

. Arniges M, Vazquez E, Fernandez-Fernandez JM, et al. Swelling-activated Ca2� entry via TRPV4 channel is defective in cysticfibrosis airway epithelia. J Biol Chem 2004;279:54062–54068.

SIPt

Spccc

October 2008 PANCREATIC ACINAR DIFFERENTIATION OF ES CELLS 1310.e3

upplementary Figure 1. Integration of the Ela promoter-puror-resLacZ-pgk-Hygror construct into ES cell genomic DNA (lanes 1–4).rimers are located (bold) as indicated in Figure 2A. (�), negative con-

rol using DNA from CGR8 cells; (�), positive control.

upplementary Figure 2. Adenoviral transduction in Ela-pur ES cells.rocess with (A and E) Adp48, (B and F) AdMist1, or (C, D, F, and G)hemistry 2 days after infection (P2 and P23, respectively) using (A anonfocal microscopy. P2 and P23 refer to the number of days in cultu

Cells were infected during the (A–D) differentiation step or the (E–H) selectionAdGFP. The expression of the transgenes was analyzed by immunocyto-d E) rabbit anti-Ptf1a and (B and F) anti-Mist1 antibodies or by (C and G)

re after EB plating. Contrast phase images are provided to indicate the cell

onfluency in each field. Scale bars: 50 �m.

Sefnd

Sefnd

SnEfaScale bar: 50 �m.

1310.e4 ROVIRA ET AL GASTROENTEROLOGY Vol. 135, No. 4

upplementary Figure 3. Quantitative RT-PCR for the expression ofndogenous and ectopic Mist1 in selected Ela-pur cells in various dif-erentiation conditions. Histograms show the relative expression levelsormalized to the loading control Hprt. Error bars indicate the standardeviations of 3 experiments. *P � .01 compared with Ela-purC.

upplementary Figure 4. qRT-PCR for the expression of pancreaticndocrine transcription factors in selected Ela-pur cells in various dif-erentiation conditions. Histograms show the relative expression levelsormalized to the loading control Hprt. Error bars indicate the standard

eviations of 4 experiments.

upplementary Figure 5. Immunofluorescence of AR42J rat aci-ar tumor cells for the expression of the same markers studied onla-purp48-Mist1 cells. Confocal images of AR42J cells immunostained

or (A) Amyl, (B) CPA, (C) Pdx1, (D) RBPL, (E) VAMP8, (F) syntaxin 4,nd (G) CCKAR. Nuclei were labeled with Tropo-3 iodide (blue).

Sci

Sb

October 2008 PANCREATIC ACINAR DIFFERENTIATION OF ES CELLS 1310.e5

upplementary Figure 6. Immunogold labeling with irrelevant anti-

odies in Ela-purp48-Mist1 cells. Scale bar: 1.7 �m.

upplementary Figure 7. qRT-PCR for the expression of pancreatic exocrine markers in developing and adult pancreas as well as in Ela-purp48-Mist1

ells. mRNAs from embryonic pancreatic rudiments, adult pancreas, and Ela-purp48-Mist1 cells were quantified and transcripts are expressed relative to those

n the E12.5 pancreas.