β-catenin signalling in mesenchymal islet-derived precursor cells

L. Ikonomou, E. Geras-Raaka, B. M. Raaka, and M. C. GershengornClinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases,National Institutes of Health

AbstractObjectives—Previously, we characterized human islet-derived precursor cells (hIPCs) asmesenchymal stem cells that migrate out from islets in vitro and can differentiate into functionalislet-like structures following proliferative expansion. Here, we investigate the role of β-cateninsignalling in derivation and proliferation of hIPCs.

Materials and methods—Localization of β-catenin was performed using confocal microscopy.Expression levels of β-catenin target genes were measured by quantitative real-time polymerase chainreaction. Loss-of-function studies were performed using specific short interfering RNAs.

Results—Immunostaining of islet outgrowths revealed translocation of β-catenin from plasmamembranes in intact islets to the nucleus in cells migrating out. There were no nuclear β-catenin-positive cells in intact islets whereas between 35% and 70% of cells in established hIPC culturesexhibited nuclear β-catenin. Transcripts for β-catenin target genes were increased in hIPCs comparedto those in islets. β-Catenin translocated to the cell membrane when hIPCs formed epithelial cellclusters. In proliferating hIPCs, there was a strong correlation between markers of proliferation andnuclear β-catenin. Treatment of hIPCs with the glycogen synthase kinase-3β inhibitor (2′Z,3′E)-6-Bromoindirubin-3′-oxime increased intracellular β-catenin but reduced nuclear β-catenin, and wasassociated with reduced cell proliferation. Finally, knockdown of β-catenin decreased β-catenintarget gene expression and hIPC proliferation.

Conclusions—These results support a functional role for β-catenin during proliferation of hIPCsand suggest that activated β-catenin signalling may also be important during hIPC derivation fromislets.

IntroductionType 1 diabetes is a debilitating autoimmune disease that results in dysfunction of glucosehomeostasis (Bach 1994). A shortage of donors for islet transplantation has brought aboutefforts to isolate and expand progenitor cells from the pancreas (Nir & Dor 2005). Our grouphas characterized a possible mesenchymal-like progenitor cell derived, in vitro, from humanislets (Gershengorn et al. 2004), and a number of laboratories have reported derivation ofsimilar mesenchymal progenitor cell populations from islets (Gao et al. 2005; Lechner et al.2005; Ouziel-Yahalom et al. 2006). Although we previously suggested that these progenitorcells may arise by dedifferentiation of islet endocrine cells by epithelial-to-mesenchymaltransition (Gershengorn et al. 2004), recent work from several laboratories including ours(Atouf et al. 2007; Chase et al. 2007; Morton et al. 2007; Weinberg et al. 2007) suggests thatthese mesenchymal precursors may not be derived from β-cells. This conclusion, however, isbased on studies with mouse cultures and may not apply to human cells. Nevertheless, it is

Correspondence: Marvin C. Gershengorn, Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and KidneyDiseases, National Institutes of Health, 50 South Dr., Rm. 4134, Bethesda, MD 20892-8029, USA. Tel.: 301-451-6305; Fax:301-480-4214; E-mail: E-mail: marving@intra.niddk.nih.gov.

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Published in final edited form as:Cell Prolif. 2008 June ; 41(3): 474–491. doi:10.1111/j.1365-2184.2008.00527.x.

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perhaps more likely that these precursor cells may represent a pancreas-specific mesenchymalstem cell (MSC) population (Chase et al. 2007; Davani et al. 2007). In any case, the pathwaysthat control the transitions of these mesenchymal-like progenitors to cells with an epithelialphenotype remain largely unknown.

One pathway essential for epithelial-to-mesenchymal transition (Huber et al. 2005; Thiery &Sleeman 2006) and for MSC proliferation (Boland et al. 2004; Etheridge et al. 2004; Cho etal. 2006) in other systems is the canonical Wnt/β-catenin pathway. When Wnt signalling isabsent, β-catenin is part of the ‘destruction complex’ that is composed of scaffolding proteinssuch as adenomatous polyposis coli (APC) and axin, and serine/threonine kinases such asglycogen synthase kinase (GSK)-3β and CK1. This complex hyperphosphorylates β-cateninand marks it for proteasomal degradation (Aberle et al. 1997). Wnt signalling, which can beactivated by Wnt ligand binding to frizzled receptors and LRP5/6 co-receptors (Logan & Nusse2004), inhibits phosphorylation of β-catenin causing it to accumulate in the cytoplasm andsubsequently to translocate to the nucleus. There, β-catenin can associate with members of theT-cell factor/lymphocyte enhancer factor (TCF/LEF) family of DNA binding proteins andinitiate transcription of Wnt-responsive genes. β-catenin signalling can also be activated in aWnt-independent way by growth factor signalling via receptor tyrosine kinases (Monga etal. 2002; Muller et al. 2002) or by cell adhesion using the integrin-linked kinase (Novak etal. 1998; Oloumi et al. 2004). Because β-catenin may also participate in the formation of cell–cell (adherens) junctions by its association with type 1 cadherins, it is clear that its subcellularlocation can dramatically affect processes such as cell differentiation, proliferation andadhesion (Jamora et al. 2003; Nelson & Nusse 2004). In in vitro cell systems, several reportsshow that incubation with growth factors such as insulin-like growth factor-II, epidermalgrowth factor and hepatocyte growth factor induce β-catenin nuclear translocation (Morali etal. 2001; Muller et al. 2002; Lu et al. 2003). Furthermore, recent work shows that vascularsmooth muscle cells exhibit nuclear activation of β-catenin in both in vivo and in vitro modelsof arterial injury (Wang et al. 2002; George & Beeching 2006; Quasnichka et al. 2006).

Because human islet-derived precursor cells (hIPCs) originate during in vitro culture of isletsin a growth factor-rich environment, we hypothesized that β-catenin may play a role in thisprocess. We observed clear transition of β-catenin from surface membrane localization in intactislets to nuclear localization in proliferating mesenchymal-like hIPCs. To investigate whetherthe observed β-catenin translocation activated Wnt responses, we measured mRNA and proteinlevels for known Wnt target genes. Up-regulation of genes such as DKK1, BIRC5, CCND1and FZD7 occurred during transition from islets to hIPCs. Short interfering RNA (siRNA)-mediated knockdown of β-catenin in hIPCs resulted in reduced 5-bromo-2′-deoxyuridine(BrdU) incorporation, indicating that β-catenin signalling controls, at least partially, hIPCproliferation. When hIPCs were induced to cluster and differentiate to epithelial-like cells thatdo not proliferate, β-catenin translocated to the cell membrane. This study, along with ourrecent work (Davani et al. 2007), is consistent with the idea that proliferative hIPCs aremesenchymal-like progenitor cells that exhibit activated β-catenin signalling.

Materials and MethodsReagents

All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwiseindicated.

Cell cultureHuman islet-derived precursor cells were maintained in 150-mm tissue culture dishes (Falcon,Becton Dickinson Labware, Franklin Lakes, NJ, USA) in growth medium CMRL-1066 (Gibco,

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Grand Island, NY, USA) supplemented with 10% foetal bovine serum, Prime (Biosource,Rockville, MD, USA) and 2 mM L-glutamine, 100× (Gibco). Cells were passaged every 3–4days at 80–90% confluence. Briefly, medium was removed and 10 mL trypsinethylenediaminetetraacetic acid, 1× (Cellgro, Mediatech Inc., Herndon, VA, USA) was addedper dish. Trypsin was inactivated by addition of growth medium and cells were collected bycentrifugation (160 g, 5 min). They were then re-suspended in fresh growth medium, countedby means of a Vi-Cell XR analyser (Beckman Coulter Inc., Fullerton, CA, USA) and seededin new dishes at a density of 1.6 × 104/cm2. Passage 2 bone marrow-derived MSCs werepurchased from Cambrex Bio Science (Walkersville, MD, USA) and maintained in proprietarymesenchymal stem cell growth medium. Cells were passaged every 6 days and seeded at adensity of 6000 cells/cm2. Clusters of either hIPCs or MSCs were formed following seedingof 330 000 cells per well of a 6-well plate. Induction media were the respective basal media(CMRL-1066 for hIPCs and mesenchymal stem cell basal medium for mesenchymal cells)supplemented with 2 mM L-glutamine, ITS-A supplement (Invitrogen, Carlsbad, CA, USA) and1% (w/v) fatty acid-free bovine serum albumin (MP Biomedicals, Solon, OH, USA). Cellclusters were harvested by gentle pipetting 4 or 7 days after serum withdrawal, for furtherprocessing.

Quantitative real-time polymerase chain reactionThe RNeasy mini kit (Qiagen Sciences, Germantown, MD, USA) was used for total RNAextraction. hIPC pellets were re-suspended in lysis buffer and processed according to themanufacturer's protocol. Concentration of total RNA was measured using the ND-1000spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). cDNA was preparedusing the high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA) andquantitative real-time polymerase chain reaction (qRT-PCR) was performed in an Mx3000Pinstrument (Stratagene, La Jolla, CA, USA) using 96-well optical reaction plates and universalPCR master mix (both from Applied Biosystems). Pre-designed TaqMan gene expressionassays from Applied Biosystems were: DKK1, Hs00183740_m1; FZD7, Hs00275833_s1;BIRC5, Hs00153353_ml; MYC, Hs00153408_m1; CCND1, Hs00277039_m1; LEF1,Hs00212390_m1; WNT5A, Hs00180103_m1; FZD2, Hs00361432_s1; DKK3,Hs00247426_m1; TCF4, Hs00162613_m1; GSK3β, Hs00275656_m1; CTNNB1,Hs00170025_m1; JUP, Hs00158408_m1; CLDN3, Hs00265816_s1; CLDN4,Hs00533616_s1; CDH2, Hs00169953_m1; CDH1, Hs00170423_m1. Each PCR reactioncontained cDNA prepared from 100 ng total RNA. Cycle threshold values were normalized toeither 18S rRNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

DKK1 ELISAThe DKK1 ELISA was performed as previously described (Tian et al. 2003). Recombinanthuman DKK1, anti-DKK1 antibody and biotinylated anti-DKK1 antibody were obtained fromR&D Systems (Minneapolis, MN, USA).

ImmunocytochemistryRabbit antihuman C-peptide, antihuman somatostatin and antihuman glucagon (Linco, St.Charles, MO, USA), mouse antihuman β-catenin, mouse antihuman E-cadherin, and rabbitantihistone 3 (phospho S10) (Abcam, Cambridge, MA, USA) and mouse antihuman N-cadherin (BD Biosciences, San Jose, CA, USA) antibodies were used at 1 : 100 dilution inblocking buffer [4% donkey serum in Dulbecco's phosphate-buffered saline (DPBS)]. Rabbitantihuman Ki-67 (Abcam) was used at 1 : 500 dilution; Alexa-Fluor 488 and 546 F(ab')2secondary antibodies (Molecular Probes, Eugene, OR, USA) were added at 1 : 100 dilution.For staining of hIPC monolayers, cells were cultured on two-chamber Lab-Tek Permanoxslides (Nalge Nunc International, Rochester, NY, USA) and were fixed with 4%

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paraformaldehyde (EMS, Hatfield, PA, USA) in 1× DPBS and permeabilized with chilled 50%methanol in PBS for 15 min. Whole human pancreas was fixed in 4% paraformaldehyde,embedded in paraffin wax and 10 μm sections were prepared. Embedded sections weredeparaffinized following standard procedures. For antigen retrieval, sections were incubatedin citrate buffer (10 mM sodium citrate, pH 6.0) for 20 min at 95 °C and blocking was performedwith 4% normal donkey serum in 1× DPBS for 30 min at room temperature. Slides were thenincubated with primary antibodies for 1.5–2 h at 37 °C in a humidified chamber, washedextensively with DPBS and incubated with secondary antibodies at 37 °C for 1.5–2 h. Slideswere mounted in Prolong Gold antifade reagent (Invitrogen) containing Hoechst 33342 (10μg/mL) or 4′,6-diamidino-2-phenylindole (10 μg/mL) to visualize nuclei. Confocal imageswere captured with a Zeiss LSM 510 Meta NLO laser scanning inverted microscope. Themicroscope was equipped with Argon/2 (488 nm line), HeNe (546 nm line) (Carl ZeissMicroImaging, Thornwood, NY, USA) and Titanium/Sapphire Chameleon (Coherent, SantaClara, CA, USA) lasers. Slides were viewed with either a 25×, 0.80 ImmCorr DIC or a 40×,1.3 Oil DIC or a 63×, 1.3 Oil DIC or a 100×, 1.4 Oil DIC objectives at room temperature andimages were captured using the LSM510 META software. Confocal micrographs wereconverted to TIFF format and arranged using Photoshop 6.0 (Adobe Systems Incorporated,San Jose, CA, USA).

BIO experimentsA 10-mM solution of (2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO) in dimethyl sulfoxide waspurchased from Calbiochem (San Diego, CA, USA). hIPCs were seeded in 6-well plates and2-well Permanox chambers in growth media, at a density of 1 × 104 cells/cm2 and let to attachovernight. BIO was diluted in growth media and added to the cells. For experiments measuringBrdU incorporation, BrdU was added to the culture 24 h after BIO addition for a 24-h period.Cells were either trypsin-harvested for cell count by Vi-Cell or ethanol-fixed for cell cycleanalysis (see below) or paraformaldehyde-fixed for immunocytochemistry.

Cell cycle analysisFor cell cycle analysis, hIPCs were harvested with trypsin, collected by centrifugation at 200g for 5 min and re-suspended in PBS. Cells were again collected by centrifugation,monodispersed in 0.5 mL PBS and transferred to tubes containing 4.5 mL of cold 70% ethanol;tubes were stored at −20 °C until analysis. Before analysis, ethanol-suspended cells werecollected by centrifugation at 200 g for 5 min, rinsed with PBS and re-suspended in PBScontaining propidium iodide (50 μg/mL) and RNase A (0.2 mg/mL). A FACS Caliburcytometer operated with CellQuest software (BD Biosciences) was used for data collection.Histogram analysis and calculation of G1, S and G2/M percentages was performed with ModFitLT (Verity Software House, Topsham, ME, USA).

ImmunoblottingCell pellets were dissolved in RIPA buffer (Pierce, Rockford, IL, USA) containing proteaseinhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). After addition of β-mercaptoethanol, samples were heated at 100 °C for 5 min. Proteins were resolved by 7.5%SDS-PAGE and transferred to nitrocellulose membranes and membranes were blocked with5% non-fat dry milk. After incubation with primary mouse anti-β-catenin (cat. no. 610153, BDBiosciences) and secondary antibodies, bands were visualized with SuperSignalchemiluminescent substrate (Pierce). GAPDH served as loading control.

β-Catenin siRNAValidated Stealth RNAi DuoPak β-catenin siRNAs were purchased from Invitrogen. ForsiRNA delivery by nucleofection, the Human Dermal Fibroblast-Adult Nucleofector kit

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(Amaxa Inc., Gaithersburg, MD, USA) was used. A total of 1.0–1.5 × 106 cells per siRNAwere transfected (1.5 μg of plasmid/0.5 × 106 cells). Controls included mock-transfected cellsand cells transfected with a Stealth Negative Control Medium GC siRNA. hIPCs weretransferred to 10-cm culture dishes in growth medium. For siRNA experiments relating to β-catenin target gene expression and hIPC proliferation, cells were co-transfected with one oftwo different siRNAs directed at β-catenin mRNA or both siRNAs, or siRNA buffer and agreen fluorescent protein plasmid (1–1.5 μg of plasmid) and were seeded in 10-cm or 15-cmculture dishes. After 24 h in culture, cells were harvested, sorted by FACS and seeded in 1- or2-chamber Lab-Tek slides in growth medium. After 24 h, BrdU (Amersham Biosciences,Piscataway, NJ, USA) was added to the medium for an additional 24h incubation prior tofixation and staining as described in the ‘Immunocytochemistry’ section above. A monoclonalanti-BrdU antibody was used (Amersham Biosciences). Additional cells were harvested withtrypsin and cell pellets were stored at − 80 °C for RNA isolation, RT-PCR analysis and Westernblotting. Changes in gene expression were quantified by qRT-PCR and cycle threshold valueswere normalized to Ct = 15 of GAPDH. Fold change relative to control siRNA was calculatedbased on the 2(−ΔΔCt) method (Livak & Schmittgen 2001).

Short interfering RNA experiments were also performed using the INTERFERin deliveryreagent (Bridge Bioscience Corp., Portsmouth, NH, USA). Briefly, hIPCs were seeded in 2-chamber Lab-Tek slides or 12-well plates. Following cell attachment overnight, siRNAs werecomplexed with INTERFERin reagent according to the manufacturer's instructions and wereadded to the hIPC monolayer (40 nM final concentration). After 48 h, BrdU was added to themedium and 24 h later samples were fixed and processed as described for the nucleofectionprocedure. Because the effects of one siRNA or the other and combination of both siRNAsyielded the same effects, we combined these data.

ResultsWhen human islets are cultured in growth media, they flatten and mesenchymal-like cellsmigrate out (Gershengorn et al. 2004). The expanded population is highly proliferative andvisually homogeneous after several doublings in culture. hIPCs exhibited nuclear staining forβ-catenin along with some cytoplasmic staining (Fig. 1a). There was a dramatic shift in β-catenin localization in this population relative to intact islets (compare Fig. 1a,c). Nuclear β-catenin staining varied from 30% to about 70% in hIPCs from different donors duringlogarithmic growth (Table 1). In contrast, β-catenin exhibited surface membrane localizationin cells expressing insulin (measured as C-peptide) (β-cells), glucagon (α-cells) andsomatostatin (δ-cells) within islets (Fig. 1c). However, cells that had migrated out of the isletand assumed a mesenchymal-like phenotype were characterized by diminished membrane β-catenin and intense nuclear staining (Fig. 1b). Some cells that had migrated out of islets co-expressed C-peptide (Fig. 1b, arrows), while other cells expressing nuclear β-catenin had nodetectable C-peptide (Fig. 1b, arrowheads). We suggest that the cells that co-express C-peptideand nuclear β-catenin may represent a transient population of mesenchymal cells derived fromhormone-expressing cells similar to those that we (Morton et al. 2007) and others (Weinberget al. 2007) have observed in cultures of mouse islets.

Nuclear localization of β-catenin is a sine qua non for canonical Wnt signalling activation(Cong et al. 2003). Because a large percentage of the expanded hIPC population expressednuclear β-catenin during proliferation (Table 1), we next examined whether β-cateninsignalling was active in established hIPC cultures. A first step was to assess transcriptionalchanges of Wnt target genes. Table 2 shows that mRNA levels of Wnt target genes such asDKK1 (Niida et al. 2004; Gonzalez-Sancho et al. 2005), BIRC5 (survivin) (Zhang et al.2001), CCND1 (Shtutman et al. 1999; Tetsu & McCormick 1999), MYC (He et al. 1998),LEF1 (Filali et al. 2002) and FZD7 (Willert et al. 2002) were higher in hIPCs than in freshly

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isolated human islets. Up-regulation of these genes was observed with varying magnitudes insamples from all three donors, suggesting these changes were characteristic of the expansionof mesenchymal pancreatic precursors. Of note, the transcript levels of TCF4, the prototypicalmember of the TCF/LEF family of DNA binding proteins, as well the transcript levels ofWNT5A and FZD2, genes involved in activation of the non-canonical Wnt/Ca2+ pathway(Slusarski et al. 1997), increased in hIPCs compared to islets also (Table 2). mRNA levels ofβ-catenin and GSK-3β were modestly increased during hIPC derivation. Therefore, theobserved β-catenin translocation probably reflects release of membrane-associated β-cateninrather than increased expression of β-catenin.

Because hIPCs may arise from expansion of a mesenchymal stem cell-like population withinhuman islets (Davani et al. 2007), expression levels of Wnt-related genes in hIPCs and bonemarrow-derived MSCs were compared. The molecular signatures of Wnt-related genesexpressed in hIPCs and bone marrow-derived MSCs were similar (Table 2). This result isconsistent with our previous observations that hIPCs share many similarities with bonemarrow-derived MSCs, including the ability to differentiate into adipocytes, chondrocytes andosteocytes (Davani et al. 2007).

One of the most up-regulated Wnt target genes in hIPCs relative to islets was DKK1 (Table 2).DKK1 protein is a potent-secreted inhibitor of the canonical Wnt pathway and it has beensuggested to be part of a negative feedback loop in normal tissues (Gonzalez-Sancho et al.2005). hIPCs produced and secreted DKK1 during proliferation (Fig. 2). Interestingly, theproduction rate of DKK1 correlated with exponential growth and was greatly reduced whencells entered the plateau phase, as indicated by the plateau in its cumulative concentration.Additional experiments revealed that production of DKK1 by hIPCs was switched off whencells were transferred to differentiation media and were induced to cluster (data not shown).Similar observations on changes of DKK1 expression have recently been reported by others(Kayali et al. 2007) when they expanded pancreatic cells using our protocol. Furthermore,DKK1, contrary to DKK3, is not expressed in the adult human pancreas (Hermann et al.2007) and, based on these results, its expression appears to be closely related to themesenchymal state of the cells. Interestingly, DKK1 has been implicated in cell cycle controlof mesenchymal stem cells (Gregory et al. 2003). These results suggest that β-catenin signallingis activated during hIPC derivation and expansion, due to nuclear translocation of β-catenin inmesenchymal hIPCs versus epithelial endocrine cells.

To confirm the importance of nuclear β-catenin for hIPC population growth, we assessedexpression of proliferation markers along with β-catenin in hIPCs during exponential growth(Fig. 3). Phosphorylation of the serine 10 residue of the N-terminal tail of histone H3 occursduring cell cycle progression through mitosis (Nowak & Corces 2004). We used an anti-H3phosphoS10 antibody to identify mitotic cells and these cells showed nuclear β-cateninstaining also. In addition, cells that stained for Ki-67, a marker expressed only in cells in theactive stages of the cell cycle but not in G0 (Scholzen & Gerdes 2000), co-stained with nuclearβ-catenin.

Work from our laboratory has suggested that hIPCs exhibit remarkable plasticity and that theirdifferentiation programme may be initiated by mesenchymal-to-epithelial transition(Gershengorn et al. 2004; Gershengorn et al. 2005). As described previously, hIPCs migratetogether to form epithelial cell clusters (ECC) when monodispersed and transferred to mediumwithout serum. In contrast to its nuclear localization in proliferative hIPC monolayers, β-catenin was localized at the cell periphery in ECCs (Fig. 4a). This translocation wasaccompanied by an increase in mRNA transcript levels of β-catenin and plakoglobin, whichis the main catenin in formation of desmosomes (Fig. 4b). Transcripts for epithelial markersclaudin-3 and claudin-4 also increased. Surprisingly, transcripts for the epithelial gene E-

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cadherin decreased (Fig. 4b). Immunostaining of days 4 and 7 cell clusters confirmed that E-cadherin protein was absent (data not shown). Nevertheless, membrane localization of β-catenin (Fig. 4a,c, lower left panel) and N-cadherin (Fig. 4c, upper left panel) in ECCs implythat the latter and another adhesion molecule, or both, may sequester β-catenin at the cellsurface during the initial in vitro epithelialization of hIPCs. Taken together, these resultssuggest that β-catenin participates in the organization of ECCs and are consistent withmesenchymal-to-epithelial transition when hIPCs differentiate into epithelial-like structures.When compared to hIPC ECCs, MSC clusters displayed similar distribution of N-cadherin andβ-catenin (Fig. 4c, compare left and right panels).

We then attempted to modulate β-catenin signalling in hIPCs by changing the distribution ofβ-catenin. We treated hIPCs with BIO, a specific GSK-3β inhibitor that can mimic Wntsignalling by increasing the cellular pool of β-catenin in some systems (Meijer et al. 2003).Paradoxically, the number of hIPCs with distinct nuclear β-catenin was reduced after treatmentwith BIO (Fig. 5a,b), contrary to previous reports on BIO use (Sato et al. 2004). β-Cateninappeared to be mostly perinuclear, with staining intensity proportional to BIO concentration(Fig. 5a, compare middle and lower panels). Treatment of bone marrow-derived MSCs by thesame concentrations of BIO led to similar distribution of β-catenin (data not shown). Nuclearexclusion of β-catenin correlated with decreased proliferation (Fig. 5d, left panel). Cell cycleanalysis showed that BIO addition led to a decrease in the percentage of cells in S phase (Fig.5c). Consistent with this, BIO reduced BrdU incorporation in hIPCs (Fig. 5d, right panel).These results taken together indicate that treatment of proliferating hIPCs with BIO leads tonuclear exclusion of β-catenin, depletion of S-phase cells and over all decreased proliferation.

To confirm the functional roles of β-catenin, we studied the effect of its reduced expressionon β-catenin target genes and hIPC proliferation. We used RNA interference to specificallydown-regulate β-catenin in hIPC cultures. hIPCs were transfected with β-catenin siRNAs orwith control siRNA using either nucleofection or a transfection reagent designed to deliversiRNA oligonucleotides. Two commercial β-catenin siRNAs each led to about 10-foldreduction in β-catenin mRNA levels (Fig. 6b) after 72 h. Specific reduction of β-catenin proteinexpression by these siRNAs was confirmed by Western blot analysis (Fig. 6a). The siRNAsappeared to have similar effects decreasing both protein bands appearing in the region of the95 kDa standard, suggesting that these may represent β-catenin and a degradation or alternativesplice product; there were decreases in DKK1, BIRC5 and FZD7 mRNA levels (Fig. 6b). Theeffect of β-catenin knockdown on hIPC proliferation was assessed by BrdU incorporation (Fig.6c,d). There was a highly significant reduction (∼50%) in the fraction of BrdU-positive cellswhen β-catenin expression was reduced (Fig. 6d). These results clearly show that nuclear β-catenin plays a critical regulatory role in the expression of these genes and the control of hIPCproliferation.

DiscussionShortage of islet donors for the treatment of Type 1 diabetes has led to attempts by manylaboratories to identify and expand progenitor cell populations that have the potential todifferentiate into functional, islet-like structures. Our laboratory has previously described sucha population, mesenchymal hIPCs, which may arise by expansion of cells from the endocrinepancreas (Gershengorn et al. 2004). The present study provides a more detailedcharacterization of hIPCs in terms of β-catenin signalling. In this work, these cells were shownfor the first time to exhibit intense β-catenin nuclear staining. Perhaps more interestingly, cellswith nuclear β-catenin also appeared in islet outgrowths, that is, cells migrating out from islets.Established hIPC cultures contained high proportions of cells with nuclear β-catenin,independent of donor age or sex. The up-regulation of several β-catenin target genes, relativeto their expression levels in islets (Table 2), supports the idea of activation of β-catenin

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signalling during hIPC derivation. One of the most up-regulated genes was DKK1; DKK1protein is a potent soluble inhibitor of Wnt signalling (Glinka et al. 1998). That hIPCs exhibitnuclear β-catenin throughout exponential population growth, while continuously secretingDKK1 is consistent with DKK1 being part of a negative feedback loop (Niida et al. 2004;Gonzalez-Sancho et al. 2005). Because DKK1 is not expressed in human pancreas (Hermannet al. 2007) but is highly up-regulated during hIPC expansion (Kayali et al. 2007 and this work),it may have a role as a marker of the mesenchymal state of the cells and its high expression inhIPCs may be a regulatory part of the hIPC proliferation programme.

The concept that β-catenin signalling relates to and is indispensable for hIPC proliferation issupported by several lines of evidence. First, hIPCs co-expressed markers of cell proliferationsuch as Ki-67 and H3phosphoS10 with nuclear β-catenin. When we knocked β-catenin downby means of RNAi, we observed reduction of hIPC proliferation in growth media, as measuredby BrdU incorporation. Hence, it appears that there is a tight link between nuclear localizationof β-catenin in hIPCs and their proliferation. Less direct results suggest that activated β-cateninsignalling may also be important during hIPC derivation from islets.

Some interesting parallels exist between this work and the role of Wnt signalling in vertebratepancreas development. Recent publications suggest that β-catenin signalling has complex rolesin the different stages of pancreatic development (Dessimoz et al. 2005; Murtaugh et al.2005; Papadopoulou & Edlund 2005; Heiser et al. 2006). Wnt signalling appears to be activein pancreatic epithelial cell progenitors and to decline thereafter (Dessimoz et al. 2005;Murtaugh et al. 2005). To determine its role in endocrine pancreas development, β-catenin-mediated signalling was modulated. Canonical Wnt signalling was reduced or abolished indeveloping mice either by overexpression of the cysteine-rich domain of Frz8, which acts asa decoy Wnt receptor (Papadopoulou & Edlund 2005), or by pancreas-specific deletion of β-catenin (Dessimoz et al. 2005; Murtaugh et al. 2005; Wells et al. 2007). Although one studyhas reported a decrease in endocrine cell numbers (Dessimoz et al. 2005), others have not(Murtaugh et al. 2005; Wells et al. 2007) and architecture and function of mature islets has notappeared to be perturbed. In contrast, overexpression of stabilized (unphosphorylated) β-catenin under the control of the Pdx1 promoter (Pdx1Crelate) at E13.5 resulted in increasedsize of the exocrine pancreas without affecting islet size, the latter being attributed to resistanceof pancreatic endocrine cells to nuclear accumulation of β-catenin (Heiser et al. 2006). Asimilar phenomenon was observed when we tried to increase cellular β-catenin by inhibitingGSK-3β activity. hIPCs as well as bone marrow-derived MSCs appear to exclude increasedβ-catenin from the nucleus (Fig. 5 and data not shown). Although this contrasts with the nuclearaccumulation of β-catenin when MSCs were treated with lithium chloride, another GSK-3βinhibitor (Etheridge et al. 2004), it may reflect differences in the mode of action of lithiumchloride and BIO on MSCs. Addition of BIO led to depletion of S-phase cells and decreasedproliferation of hIPCs. On the contrary, a recent study on the effect of GSK inhibitors on ratpancreatic islets, reported an increase in the β-cell replication rate (Mussmann et al. 2007).Thus, GSK inhibition may have different effects on β-cell and mesenchymal islet precursorreplication. Another striking feature of the mature (1-year-old) animals following β-cateninoverexpression in the mouse pancreas was the presence of cells with nuclear localization ofβ-catenin within a subset of islets (Heiser et al. 2006). These cells did not express insulin orany other marker of mature β-cells. Thus, it is possible that nuclear presence of β-catenininduces islet precursor cells to revert to a mesenchymal, proliferative phenotype in vivo also.In a recent publication, addition of Wnt3a, a canonical Wnt ligand, stimulated ex vivoproliferation of human β-cells and increased expression of cyclin D2 (Rulifson et al. 2007).We have observed nuclear β-catenin in C-peptide expressing cells migrating, from cadaverichuman islets. Because these cells may not persist in culture (Morton et al. 2007; Weinberg etal. 2007), Wnt signalling may not be sufficient for β-cell expansion during in vitro culture

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(Rulifson et al. 2007), while it appears to have a significant role in the proliferation of MSC-like cells residing in the islets.

The latter idea seems particularly interesting given the similarity of hIPCs and MSCs. hIPCsadhere to plastic and proliferate in serum-containing media. Furthermore, they appear to meetmost of the criteria set to identify MSCs, because they express MSC surface markers (CD105/CD73/CD90), are CD14-, CD19- and CD34-negative and some hIPC preparations have beenshown to be able to differentiate to adipocytes, osteocytes and chondrocytes (Davani et al.2007). Bone-marrow MSCs are established in culture as the fraction of cells from bone aspiratethat adhere to plastic. Wnt signalling has been shown to be active in MSCs (Etheridge et al.2004) and it is known to block adipogenic differentiation of pre-adipocytes (Ross et al.2000) and osteogenic differentiation of adult human MSCs (Boland et al. 2004). In the latterwork, infection of MSCs with an adenovirus encoding Wnt3a, a ligand of the canonical Wntpathway, resulted in increased proliferation and substantially higher cell counts relative tomock-infected cells. Recent work from the same group has indicated that two Wnt pathway-related genes, namely FZD7 and DKK3, are part of the ‘stemness’ gene group for MSCs (Songet al. 2006). These two genes are down-regulated during adipogenesis and osteogenesis ofhMSCs, but their expression is increased when differentiated cells of these lineages revert toan undifferentiated state. Interestingly, it has been recently shown that DKK3 is expressed inhuman β-cells (Hermann et al. 2007) and by in situ hybridization that FZD7 (among genes forother frizzled receptors) is mainly localized in pancreatic islets (Heller et al. 2003). Both genesare highly up-regulated in hIPCs relative to human islets (Table 2). Thus, if hIPCs are an islet-derived stem cell population, insights from MSC biology may be useful for their furthercharacterization.

Certain analogies that exist between hIPCs and other non-transformed cells in culture may alsobe of interest. For example, it has been shown that increased expression of β-catenin and itsnuclear translocation increased proliferation and inhibited apoptosis of vascular smooth musclecells (VSMC) following carotid injury in Sprague-Dawley rats (Wang et al. 2002). Recently,Fat1 cadherin, whose expression is increased in hIPCs relative to islets (unpublishedobservation), has been shown to interact with β-catenin and modulate its proliferative effectwhile increasing migration of VSMCs (Hou et al. 2006). N-cadherin was also found to complexwith β-catenin in the membrane of quiescent VSMCs (Uglow et al. 2003). Serum stimulationof these cells led to adherens junction dismantling and nuclear translocation of β-catenin. Thus,information from VSMC biology, especially with relevance to cadherin–catenin interactions,may help us to gain insights in the regulation of proliferation of islet progenitor cells.

β-Catenin localization may be one of the cues regulating hIPC proliferation and differentiation.Indeed, the reversible translocation of β-catenin from the cell membrane to the nucleus suggeststhat β-catenin may mediate, in part, the remarkable plasticity exhibited by hIPCs. When theyare induced to differentiate by removal of growth factors from the culture media, β-catenintranslocates to the membrane. Because we hypothesize that mesenchymal-to-epithelialtransition is an essential step in the hIPC differentiation programme, enhancement of β-catenintranslocation could produce ECCs that are more stable and poised to differentiate followingtransplantation. In conclusion, in the present work, we show that human mesenchymal islet-derived precursor cells exhibit dynamic β-catenin signalling. Although derivation and invitro expansion of similar populations of proliferative precursor cells have been reported, thisis the first time, to our knowledge, that these cells have been demonstrated to exhibit activationof this major developmental pathway. Because it is known that β-catenin signalling is not activein normal human pancreas, our results may have implications for both pancreas neogenesisand development of better in vitro protocols for progenitor cell expansion and differentiation.

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AcknowledgementsThis work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive andKidney Diseases, National Institutes of Health. The authors thank Behrous Davani for helpful suggestions and criticalreading of the manuscript.

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Figure 1. Localization of β-catenin in expanded human islet-derived precursor cells (hIPCs), isletoutgrowths and mature human pancreas(a) Proliferating hIPCs were fixed 72 h after re-seeding in culture and were stained with amonoclonal antibody against β-catenin. Nuclei were stained with Hoechst 33342. Bar, 20 μm.(b) High purity islet fractions were cultured in 2-chamber Permanox slides in growth media.Islets were fixed after 48 h in culture and stained for β-catenin and C-peptide. A representativefield showing islet outgrowth is presented. Cells co-expressing C-peptide and nuclear β-catenin(arrows) or cells expressing nuclear β-catenin but no detectable C-peptide (arrowheads) canbe seen. Bar, 10 μm. (c) High magnification confocal micrographs of islet endocrine cells.Paraffin wax-embedded sections of human pancreas were deparaffinized and stained for β-catenin (green). Sections were co-stained with C-peptide, glucagon or somatostatin (red) toidentify β-, α- and δ-cells, respectively. Bar, 10 μm.

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Figure 2. DKK1 production during exponential human islet-derived precursor cell (hIPC) growthhIPCs were seeded in 6-well plates at between 0.5–1.5 × 104 cells/cm2. Cell counts wereperformed in triplicate every day and supernatants were collected for measuring secretedDKK1, by ELISA. Cumulative DKK1 concentration is presented as mean ± SD for each day(left axis, filled diamonds). Viable cell density of the three growth curves are presented as mean± SD (right axis, filled squares). The right axis is in log scale to clearly show the lag, exponentialand plateau phases of hIPC growth.

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Figure 3. Nuclear β-catenin is co-expressed with proliferation markersHuman islet-derived precursor cells (hIPC) were seeded in 2-well Permanox slides in growthmedium and were stained during exponential growth for H3phosphoS10 (red) (upper panel;bar, 20 μm) or Ki-67 (red) (lower panel; bar, 20 μm) and β-catenin (green). All of the Ki-67and H3phosphoS10-positive cells were found to express nuclear β-catenin.

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Figure 4. Cell surface translocation of β-catenin during epithelialization of human islet-derivedprecursor cells (hIPC)(a) hIPCs exhibit nuclear β-catenin staining in monolayer culture in growth medium andmembrane staining in epithelial cell clusters (ECC) at day 7. ECCs were harvested either onday 4 or day 7 after switch to differentiation medium and were paraffin wax-embedded. hIPCsfrom three different donors were used to create ECCs. In all experiments, β-catenin had asimilar expression pattern in ECCs. A representative 10-μm section is shown. Bar, 20 μm. (b)Total RNA was prepared from monolayer hIPCs in growth medium or from ECCs after 4 daysin differentiation medium (day 4 ECCs). After cDNA preparation, transcript levels for theindicated genes were determined by qRT-PCR. The fold change relative to day 0 hIPCs wascalculated using the 2(−ΔΔCt) method. (c) N-cadherin and β-catenin staining in day 4 ECCs.Paraffin wax-embedded ECCs of hIPCs or MSCs were deparaffinized and stained for N-cadherin or β-catenin (green). Nuclear counterstain is DAPI. Bar, 20 μm.

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Figure 5. Treatment of human islet-derived precursor cells (hIPCs) with BIO leads to reduced cellgrowth(a) Confocal micrographs of hIPCs treated with BIO. Cells were stained for β-catenin (green)72 h after addition of dimethyl sulfoxide (DMSO) (control, upper panel), 1 μM BIO (middlepanel) or 3 μM BIO (lower panel). Nuclear counterstain was Hoechst 33342. Bar, 20 μm. (b)Quantification of nuclear staining in a typical experiment. (c) Cell cycle analysis of cells treatedwith BIO or DMSO (control). Cells were stained with propidium iodide and analysed by meansof a BD FACS Calibur cytometer. A representative set of data from one of three independentexperiments is shown. (d) For cell number (left panel), hIPCs were seeded in 6-well plates at1 × 104 cells/cm2 (∼0.1 × 106 cells/well). BIO was added at the indicated concentrations afterovernight cell attachment. Cells were harvested 48 h after BIO addition. Six wells were usedfor cell counting and the experiment was performed twice. Results are shown as mean ± SD.Significant statistical differences between control and treated conditions were calculated byone-way ANOVA followed by Dunnett's multiple comparison test (*P < 0.01). For BrdU staining(right panel), hIPCs were seeded in 2-well Permanox chambers, BrdU was added 24 h afterBIO addition and slides were fixed after 24-h incubation. Areas with varied degrees of cellconfluence were included in the analysis. To avoid biased selection of fields, slides were viewedusing the DAPI filter (nuclear staining).

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Figure 6. Reduced expression of β-catenin inhibits human islet-derived precursor cell (hIPC)proliferation(a) Western blot of total cell extracts for β-catenin 72 h after transfection with a control siRNA,β-catenin siRNA1, β-catenin siRNA2 or pooled β-catenin siRNAs (50 : 50 ratio). The twoprotein bands near the 95 kDa standard are both reduced similarly following siRNA treatmentsand likely reflect β-catenin and a degradation or alternative splice product. The band just belowthe 37 kDa standard is GAPDH, documenting similar protein loading in the four lanes. (b)Levels of DKK1, BIRC5 and FZD7 mRNAs were measured by qRT-PCR. Results are shownas mean ± SEM. (c) Confocal micrographs of BrdU-incorporating hIPCs at 72 h aftertransfection with siRNA1 + siRNA2 (left panel) or Control siRNA (right panel). Bar, 50 μm.(d) BrdU incorporation in hIPCs is decreased following transfection with β-catenin siRNAs.A total number of 500–700 nuclei were counted for each condition in each experiment tocalculate the fraction of BrdU+ cells. Areas with varied degrees of cell confluence wereincluded in the analysis. To avoid biased selection of fields, slides were viewed using the DAPIfilter (nuclear staining). The results of four independent experiments are compiled. Results areshown as mean ± SEM. Significant statistical differences between control siRNA and β-cateninsiRNA were calculated by paired two-tailed t-test (*P < 0.0001).

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Table 1Percentage of nuclear β-catenin-positive cells in exponentially growing human islet-derived precursor cell cultures

Preparation Passage number Time after re-seeding (h) Nuclear β-catenin positivity(%)

A 15 24 68 (189/278)

A 15 72 72 (231/321)

D 3 48 35 (97/274)

E 5 24 32 (88/273)

F 13 48 51 (389/757)

F 13 72 52 (159/306)

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ed p

recu

rsor

cel

l; M

SC, m

esen

chym

al st

em c

ell;

qRT-

PCR

, qua

ntita

tive

real

-tim

e po

lym

eras

e ch

ain

reac

tion.

Cell Prolif. Author manuscript; available in PMC 2009 June 1.