Sonic Hedgehog and Other Soluble Factors from Differentiating Embryoid Bodies Inhibit Pancreas Development

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1 EMBRYONIC STEM CELLS Sonic Hedgehog and Other Soluble Factors from Differentiating Embryoid Bodies Inhibit Pancreas Development JOSUÉ K. MFOPOU, a VÉRONIQUE DE GROOTE, a XIABO XU, b HARRY HEIMBERG, b LUC BOUWENS a a Cell Differentiation Unit; b Medical Biochemistry Unit, Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium Key Words. Activin Beta cells Definitive endoderm Embryonic stem cells Hedgehog Pancreas development ABSTRACT Success of cell-replacement therapy for diabetes will largely depend on the establishment of alternative sources of pancreatic islet grafts. Embryonic stem (ES) cell differentiation toward pancreatic insulin-producing cells offers such perspectives, but there are still many challenges to overcome. Our previous studies suggested that the limited amount of insulin-positive cells derived from ES cells is related to the activation of pancreas inhibitory signals. To confirm this hypothesis, we report here that exposure of mouse embryonic pancreas explants to soluble factors from embryoid bodies (EBs) inhibits growth, morphogenesis, and endocrine and exocrine differentiation as evaluated by explant size and mrna and protein expression. Sonic Hedgehog (Shh), an established pancreas repressor both at early and late developmental stages, was produced and secreted by EBs, and participated in the inhibitory effect by inducing its target Disclosure of potential conflicts of interest is found at the end of this article. Gli1 in the explants. Inhibition of Hedgehog pathway rescued the differentiation of Insulin-positive cells in the explants. In contrast to pancreatic cells, hepatic progenitors exposed to EB-conditioned medium showed improved differentiation of albumin-positive cells. In a model system of ES cell differentiation in vitro, we found that definitive endoderm induction by serum removal or activin A treatment further increased Hedgehog production and activity in EBs. Concomitantly, downregulation of the pancreas marker Pdx1 was recorded in activin-treated EBs, a phenomenon that was prevented by antagonizing Hedgehog signaling with Hedgehog interacting protein. These data strongly suggest that Hedgehog production in EBs limits pancreatic fate acquisition and forms a major obstacle in the specification of pancreatic cells from ES-derived definitive endoderm. STEM CELLS 2007;25: INTRODUCTION During development, pancreatic cells differentiate from definitive endoderm-derived progenitor cells. The definitive endoderm portion fated to become pancreas receives signals from adjacent layers and becomes responsive to pancreas-inducing cues. In mammals, notochord, aortic endothelium, and neighboring mesenchymal cells coordinate pancreas specification within the competent endoderm. Signals from these tissues initiate a cascade of transcription factors (TFs), setting up a Pdx1 /Ptf1a pancreas precursor population that will further differentiate into exocrine, endocrine, and duct cells [1 3]. Embryonic stem (ES) cell differentiation toward endocrine insulin-producing cells constitutes a field of intensive investigation and represents a possible alternative to shortened human islets needed for transplantation in diabetes therapy. Several studies described the generation of insulin-producing cells from both mouse and human ES cells following diverse strategies including exposure to extracellular signaling molecules and overexpression of key pancreatic TFs [4 9]. Reassessment of some protocols yielded controversial data, indicating uptake of insulin from the medium or its expression by neuronal progenitors known to frequently appear in ES cell cultures [10 12]. Until very recently, no protocol allowing for straightforward in vitro differentiation of functional beta cells from ES cells existed. This contrasts with what is known for neuronal cell types [13 15] and could be linked either to the paucity of definitive endoderm population that can be obtained with standard embryoid body (EB) cultures or to the lack of a comprehensive protocol recapitulating all aspects of in vivo pancreas morphogenesis. Recent studies revealed an enhanced definitive endoderm derivation from mouse and human ES cells after serum removal and exposure to activin A [16 19], thereby validating the role of transforming growth factor (TGF)- signaling in endoderm development, as known from Xenopus experimentation [20, 21]. Although these ES-derived definitive endoderm cells gave rise to hepatocytes, pneumocytes, and intestinal cells after implantation in mice or additional differentiation in vitro, no evidence for pancreas differentiation was provided. Nevertheless, these studies have opened new perspectives in the quest for true beta cells by setting up conditions required for the endoderm progenitor pool from which the pancreas is known to develop. Sonic Hedgehog (Shh) expression is normally restricted to embryogenesis and is instrumental in patterning several tissues of the early embryo. However, a few organs such as the pancreas and pituitary require the absence of Hedgehog signals to initiate and bud out of their respective germ layers [22 25]. We recently reported that cells within EBs express numerous pancreas regulatory genes including Shh both at messenger and protein levels. When compared to their expression during em- Correspondence: Luc Bouwens, Ph.D., Cell Differentiation Unit, Diabetes Research Centre, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium. Telephone: ; Fax: ; lucbo@vub.ac.be Received November 7, 2006; accepted for publication January 22, 2007; first published online in STEM CELLS EXPRESS February 1, AlphaMed Press /2007/$30.00/0 doi: /stemcells STEM CELLS 2007;25:

2 Mfopou, De Groote, Xu et al bryonic development, the overall profile of pancreas regulators appeared incompatible with differentiation of pancreatic cells from ES cells [26]. Although very suggestive, demonstration of this hypothesis was made extremely difficult by the lack of a thorough protocol that would associate blockade of all inhibitory signals and supplementation of all defective factors in correct amounts and at needed time points. D Amour et al. [27], in a very recent study, followed a stepwise strategy including induction of definitive endoderm, posterior foregut endoderm, pancreatic epithelium, and, finally, insulin-producing cells by sequential treatment with extracellular molecules that mimic pancreas developmental signals. Improved derivation of insulinproducing cells from ES cells subjected to fetal pancreas signals was also recently shown [28, 29], but to our knowledge, no effects of signals released by differentiating ES cells on developing pancreas have been critically investigated. Using embryonic pancreas explants, we provide evidence that soluble factors released by EBs severely alter pancreas morphogenesis and differentiation in vitro and that this effect is partly mediated by Shh. Moreover, we show that serum removal with or without addition of activin A to induce definitive endoderm in ES cells further upregulates Hedgehog activity, thereby suppressing Pdx1-dependent pancreas fate acquisition in EBs. MATERIALS AND METHODS ES Cell Culture and Endoderm Induction ES cell (E14 mouse cell line) colonies were maintained undifferentiated on mitomycin C-inactivated (STO cell line) in the presence of 1,000 units/ml leukemia inhibitory factor (LIF; Sigma-Aldrich, St. Louis, The culture medium consisted of knockout-dulbecco s modified Eagle s medium (DMEM; Invitrogen, Carlsbad, CA, supplemented with penicillin-streptomycin (Sigma-Aldrich), monothioglycerol (Sigma-Aldrich), nonessential amino acids (Sigma-Aldrich), L-glutamine (Sigma-Aldrich), and 15% knockout serum replacement (KOSR; Invitrogen). For EB initiation, colonies were washed in Dulbecco s phosphate buffered saline (D-PBS), dissociated with 0.25% Trypsin-EDTA solution (Sigma-Aldrich), depleted of feeder cells and cultured in suspension (10 5 cells per milliliter) in a similar medium but with 10% fetal bovine serum (FBS; PAA, Pasching, Austria, and without LIF. Serum was lowered to 2% from day 3, and conditioned medium (EB-CM) was harvested twice between days 6 and 14, passed through a m filter (Pall Corporation, East Hills, NY, and kept at 20 C until use in pancreas explant culture. For definitive endoderm induction, EBs were initiated in Stem- Pro34 medium (Invitrogen) supplemented with 10 ng/ml of stem cell factor (SCF; Sigma-Aldrich) as previously described [17]. On day 3, control EBs initiated in serum were maintained in 10% FBS or transferred to 15% KOSR until day 10, whereas those initiated in StemPro34 were treated with 15% KOSR and 100 ng/ml activin A or B (Sigma-Aldrich). These cultures were performed in suspension or in gelatin-coated 24-well plates and media were refreshed every 2 3 days. Hedgehog inhibition was investigated by addition of monoclonal anti-shh (10 and 50 g/ml; R&D Systems, Minneapolis, Hedgehog interacting protein (HIP; 2 and 10 g/ml; Sigma-Aldrich), or cyclopamine (1 and 5 M; Biomol Research Laboratories, Plymouth Meeting, PA, with or without 20 mm forskolin (Sigma-Aldrich). Explants Isolation and Culture C57Bl6 pregnant mice were obtained from Janvier (Le Genest Saint Isle, France) and sacrificed according to the regulations of the local ethical committee. Pancreas explants were microdissected at embryonic day (E) 12.5 and embedded in collagen R (Serva, Heidelberg, Germany, matrix. Explants were cultured in DMEM with 2% FBS for 7 days either alone (control) or together with 7-day-old EBs seeded in a transwell chamber separated by a m filter (Corning, Corning, NY, corning.com). Other explants were cultured with EB-CM at various dilutions or in control medium supplemented with 2.5 g/ml of Shh-N peptide (Sigma-Aldrich). Immediately after isolation and on day 7 of culture, explants were photographed and their sizes were measured using Image J software (NIH, Bethesda, MD). Liver buds were concomitantly isolated, dissociated in enzymefree dissociation medium (Invitrogen), and seeded in gelatin-coated 24-well plates (one bud over four wells). After overnight culture in control medium (2% FBS), nonattached cells were washed off, and the remaining cultured either in control medium, EB-CM, EB-CM supplemented with recombinant HIP (5 g/ml), or control medium supplemented with recombinant Shh-N peptide (0.75 g/ml). Media were refreshed every 2 3 days until day 7. RNA Extraction and Real-Time Polymerase Chain Reaction Total RNA was extracted from different samples using a columnbased kit (Sigma-Aldrich) and 1 g (ES cells and EBs), 500 ng (cultured embryonic liver cells), or 100 ng (embryonic pancreas explants) used in reverse transcription. Real-time polymerase chain reaction (PCR) was carried out with a cdna pool corresponding to 50 ng (ES cells, EBs, cultured liver cells) or 7.5 ng (pancreas explants) RNA equivalent using SYBR green I (Sigma-Aldrich), as previously described [26]. Amplicon specificities were ensured by primer design encompassing introns of mouse mrnas, primer sequences matching up with Basic Local Alignment Search Tool (BLASTing) against the National Center for Biotechnology Information database, and recording of melting profiles during each run. The complete list of primers is available as supplemental online Table 4. Data were normalized to Gapdh and to the maximal expression level, and then to a reference sample (control pancreas explants, control liver cells, or ES cells) as applicable. Data Analysis and Statistics Explant sizes (ratio of day 7 to day 1) and relative gene expression levels (mean SEM) from at least four independent experiments (2 4 explants per experiment) are presented. Differences between treatments were tested for significance (p.05) using unpaired Student s t test or one-way analysis of variance with Bonferroni s multiple comparisons. Immunocytochemistry Pancreas explants were washed twice in D-PBS, fixed for 20 minutes in 4% formaldehyde, enclosed in a drop of 2% agarose, and processed for paraffin embedding. Sections (5 m) were incubated overnight with primary antibodies against amylase (polyclonal, 1/5,000; Sigma-Aldrich), glucagon (polyclonal, 1/3,000; Dr. C. Van Schravendijk, Vrije Universiteit Brussel, Brussels), insulin (monoclonal, 1/2,000; Sigma-Aldrich), Ngn3 (polyclonal, 1/1,000; the laboratory of Michael German, University of California, San Francisco), and Pdx1 (polyclonal, 1/1,000; Beta Cell Biology Consortium, Nashville, TN, and stained as previously described [26]. Attached liver cells were washed with PBS, fixed for 20 minutes in 4% formaldehyde, permeabilized in ice-cold methanol, and stained overnight with primary antibodies against -catenin (polyclonal, 1/100; BD Biosciences, San Diego, bdbiosciences.com), -fetoprotein (AFP; polyclonal, 1/200; Dako- Cytomation, Glostrup, Denmark, albumin (polyclonal, 1/100; Biogenesis, Munich, Germany, biogenesis.co.uk), and Foxa2 (polyclonal, 1/50; Santa Cruz Biotechnology Inc., Santa Cruz, CA, within 24- well plates. EBs cultured in suspension or attached EBs in 24-well plates were washed with PBS, fixed for 20 minutes in 4% formaldehyde, and processed for immunostaining. Antibodies tested were polyclonal anti-shh (1/100; Santa Cruz Biotechnology), polyclonal anti- Gli1 (1/100; Santa Cruz Biotechnology), and polyclonal anti-foxa2 (1/50; Santa Cruz Biotechnology). Aspecific binding was blocked

3 1158 EBs Factors Inhibit Pancreas Development before exposure to primary antibodies by incubation in relevant nonimmune serum (goat, rat, or donkey). For both liver cells and EBs, tetramethylrhodamine isothiocyanate, fluorescein isothiocyanate, or biotinylated secondary antibodies were used and processed as previously described [26] with 4,6-diamidino-2-phenylindole or hematoxylin-eosin used for counterstaining. Dot and Western Blotting We evaluated the presence of Shh in fresh media, conditioned media, and sera used at different stages of cultures by dot blot analysis. One microliter of the corresponding medium was mixed with sampling buffer, spotted on a nitrocellulose membrane, and allowed to dry. The membrane was further treated in a similar way as for Western blotting. For protein isolation, ES cells and EBs were washed in D-PBS and lysed in RIPA buffer supplemented with protease inhibitors. After 1-hour incubation at 4 C, samples were sonicated 3 20 seconds and centrifuged for 10 minutes (4 C; 14,000 rpm). Protein concentration in the supernatant was evaluated with a BCA kit (Bio-Rad, Hercules, CA, and 50 g were loaded on 10% polyacrylamide gel. Blotted nitrocellulose membranes were incubated overnight with a primary anti-shh antibody (goat polyclonal, 1/200; Santa Cruz Biotechnology) allowing for detection of both full-length and processed N-terminal Shh (48 and 19 kda, respectively). Signals were developed after binding of the secondary anti-goat horseradish peroxidase-labeled antibody (1/ 500; Santa Cruz Biotechnology) using ECL technology (Amersham Biosciences, Buckinghamshire, U.K., and archived on Kodak films (Kodak, Rochester, NY, kodak.com). Enzyme-Linked Immunosorbent Assay for Shh Concentrations in Conditioned Media Conditioned media or protein extracts were harvested from EBs at different time points and stored ( 20 C or 80 C) until use. Indirect enzyme-linked immunosorbent assays (ELISAs) were performed from 150 l of medium in a 96-well flat-bottom ELISA plate initially coated overnight with monoclonal anti-shh capture antibody (100 l, 4 g/ml; R&D Systems) and blocked for 1 hour with 300 l of blocking buffer (0.05% sodium azide, 1% BSA, and 5% sucrose in PBS). After three washes with 0.05% Tween 20 in PBS (PBS-T; Sigma-Aldrich), the biotinylated detection antibody (100 l, 62.5 ng/ml; R&D Systems) was added for 1 hour at 37 C, followed by three washes in PBS-T, and binding of horseradish peroxidase conjugate (ABC kit, DakoCytomation). The chromogenic substrates (H 2 O 2 and tetramethylbenzidine; R&D Systems) were used for color development, and the plates were read at OD450 and OD540 using a Benchmark microplate reader (Bio-Rad). Each analysis was carried out in duplicate from at least three independent samples. Shh concentrations were estimated from standard curves generated from serial dilutions of recombinant Shh-N peptide (Sigma-Aldrich) after subtraction of background blank values. RESULTS Conditioned Medium from Embryoid Bodies Alters the Growth and Morphology of Embryonic Pancreas Explants To detect potential inhibitors of pancreas development that could be produced by differentiating ES cells, we cultured E12.5 embryonic pancreas explants in the presence of conditioned medium from EBs or we adopted a coculture system using transwells, thereby keeping EBs physically separated from pancreas explants by a m filter. After 7 days of culture in control medium, pancreatic explants showed progressive growth and presented a dense and lobulated appearance in bright-field microscopy (Fig. 1A). In contrast, coculture with EBs or culture in EB-CM inhibited explant growth, and many cystic or ductlike structures of various sizes were apparent by day 4 5, Figure 1. Morphology and growth of embryonic day-12.5 mouse pancreatic explants exposed to EB-secreted factors. (A): Control pancreas cultured for 7 days, showing a dense lobular structure. (B): Pancreas appearance after 7-day coculture with EBs. (C): Pancreas cultured for 7 days in conditioned medium from EBs. Note the reduced density by loss of bulk pancreatic tissue, as well as numerous enlarged cystic structures. Scale bar 500 m. (D): High magnification of the duct-like structure indicated in (C). Scale bar 100 m. (E): The explant size significantly increases between culture days 1 and 7 in control condition, but not in coculture, nor in EB-CM. Abbreviations: CM, conditioned medium; EB, embryoid body; NS, nonsignificant., p.001. reducing the lobulation and density of the explants (Fig. 1B 1D). These morphological changes recalled the phenotype of transgenic mice expressing Shh or Indian Hedgehog (Ihh) in the pancreas [30], and prompted us to test the effect of recombinant Shh-N peptide. As for EB-CM, similar alterations in the explants were observed when control medium was supplemented with 2.5 g/ml recombinant Shh-N peptide for 7 days (data not shown). To quantify the effect on growth, explant size was measured, indicating an increase between days 1 and 7 in control medium (twofold; p.001). Coculture with EBs or culture in EB-CM significantly inhibited pancreatic growth (Fig. 1E). Conditioned Medium from Embryoid Bodies Alters Both Endocrine and Exocrine Gene Expression in the Explants We assessed the expression patterns of major pancreatic TFs and cell differentiation markers. Preliminary data suggested analogous profiles in coculture as in EB-CM. For ease of experimentation, we focused on EB-CM rather than coculture. TFs expressed in pancreas progenitor cells (Pdx1, Ngn3, Islet1) seemed to increase on treatment with EB-CM, but the differences did not reach statistical significance. On the contrary, explant culture in EB-CM significantly reduced expression of the TFs Ptf1a and Mist1, which are typically expressed in terminally differentiated exocrine pancreas (Fig. 2A). Expression of Insulin (endocrine marker) decreased by fourfold, and that of Amylase (exocrine marker) decreased more than 50-fold in EB-CM (Fig. 2A). Several of these mrna findings were confirmed by immunostaining of explants, where a severe reduction in Insulin- and Amylase-positive cells was seen after culture in EB-CM, in contrast to control explants, which displayed well-organized Insulin-positive islets and Amylase-positive acini (Fig. 2B 2E). In addition, Pdx1 and Ngn3 signals were observed in more cells and at higher staining intensity in treated explants, mainly in cells lining the enlarged cystic or duct-like structures (Fig. 2F 2I). Surprisingly, we observed a fivefold increase in Glucagon expression, together with higher levels of TFs required for alpha cell differentiation (Arx and Brn4; supplemental online Fig. 3). These observations indicate

4 Mfopou, De Groote, Xu et al Figure 2. Gene expression in embryonic day-12.5 mouse pancreas explants exposed to an embryoid body (EB) environment for 7 days. (A): Real-time polymerase chain reaction (PCR) analysis of pancreas transcription factor (TF) and marker expression in explants cultured in EB-conditioned medium (CM). Exposure to EB-CM does not significantly alter the profile of TFs common to pancreas progenitors (Pdx1, Islet1), nor that of Ngn3, which is restricted to endocrine progenitors. These genes show a tendency for upregulation. However, markers of terminal differentiation are critically affected, with roughly a 50-fold decrease in exocrine genes (Ptf1a, Mist1, Amylase) and fourfold decrease in the beta cell marker Insulin. Immunoreactivity for insulin (B, C), amylase (D, E), Pdx1 (F, G), and Ngn3 (H, I) in control pancreas explants (B, D, F, H) and in pancreas explants cultured in EB-CM (C, E, G, I). Note the loss of Insulin- and Amylase-positive cells induced by EB-CM, as well as the sustained expression of Pdx1 and Ngn3 by duct cells, all concordant with PCR data. Scale bars 250 (C, E) and 100 m (G, I). Abbreviation: NS, nonsignificant., p.05;, p.01;, p.001. that factors secreted from EBs inhibit the differentiation of pancreas progenitor cells to endocrine and exocrine acinar cells, thereby relatively increasing the pool of Pdx1- and Ngn3- positive precursor cells. Hedgehog Signals Are Implicated in the Observed Pancreas Inhibition by EBs-Secreted Factors The alteration in expression of pancreatic TFs and markers observed in EB-CM could be reproduced by Shh-N supplementation to control medium, except for Pdx1, Islet1, and Ngn3 expression, the latter being reduced (Fig. 3A). Considering the similar effects of EB-CM and Shh-N peptide supplementation Figure 3. Hedgehog involvement in the inhibition of explants differentiation. (A): Real-time polymerase chain reaction (PCR) analysis of the expression of pancreas transcription factors (TFs) and markers in explants cultured in the presence of Shh-N peptide (2.5 g/ml). Culture in control medium supplemented with Shh-N results in a similar expression profile of terminal differentiation markers (Ptf1a, Mist1, Amylase, Insulin) as with EB-CM. In contrast to EB-CM, Shh-N induces Ngn3 downregulation. (B): Dot blot detection of Shh in serum-free media from ES cells and EBs. Note the strong signal at days 7 and 28, which suggests EBs as the source of Shh. (C): Western blot with anti-shh (sc1194) identifies the 48-kDa full-length and the 19-kDa processed fragment in 8-, 10- and 14-day-old EB extracts. (D): Realtime PCR indicating a 17-fold induction of the ultimate Hedgehog target Gli1 in pancreas explants exposed to EB-CM. This expression level is higher than that induced by Shh-N peptide at 2.5 g/ml (fivefold). Dilution of EB-CM to 10% allows for lower Hedgehog target activation (fourfold), which could be inhibited by supplementation of HIP at 5 g/ml. (E): Enzyme-linked immunosorbent assay for Shh in fresh and conditioned media from EBs cultured in varying concentrations of serum, and in protein extracts from ES cells and EBs cultured in 10% serum or in serum-free medium. EBs are the source of Shh detected in conditioned media, and serum removal increases Shh production and release. (F): Addition of HIP to 10% EB-CM dilution led to increased occurrence of duct-associated and small clusters of Insulin-positive beta cells. (G): The percentage of Insulin-positive cells after HIP supplementation to 10% EB-CM increased by fourfold up to control level (minimum 6,000 cells counted in each condition). Scale bar 250 m (F). Abbreviations: BSA, bovine serum albumin; CM, conditioned medium; EB, embryoid body; ES, embryonic stem; HIP, Hedgehog interacting protein; Shh, Sonic Hedgehog; STO, feeders cell line., p.05;, p.01;, p.001. on the expression of Ptf1a, Mist1, Amylase, and Insulin, secretion of Hedgehog proteins by EBs may be at least partially responsible for the observed inhibition of pancreas explant growth and differentiation. We therefore screened for the presence of Shh in EB-CM. To ascertain its expression and secretion

5 1160 EBs Factors Inhibit Pancreas Development by EBs, we performed dot blot for Shh on serum-free EB-CM. This analysis revealed a drastic increase in secreted Shh from day 7 and its maintenance until as late as day 28 (Fig. 3B), which is concordant with the mrna profile that we have previously observed [26]. Western blot analysis of EBs protein extracts confirmed these data and also denoted full-length Shh processing to the N-terminal 19-kDa active fragment (Fig. 3C). To validate the functionality of EB-derived Shh protein, we further analyzed the expression of Hedgehog targets in pancreatic explants. Indeed, Ptc1 and Gli1 transcripts were significantly induced in explants on exposure to EB-CM (4- and 17-fold, respectively). Their induction by EB-CM was stronger than recorded after Shh-N peptide supplementation (three- and fivefold respectively; Fig. 3D and data not shown), suggesting that conditioned medium contained higher concentrations of Shh-N. To clarify this issue, we performed an indirect ELISA for Shh on EB-CM. To our surprise, the concentrations measured were far lower ( pg/ml) compared to 2.5 g/ml recombinant Shh-N peptide used in culture. Lowering serum concentration to 2% (for EB-CM harvesting) or culturing in serum-free medium (for endoderm induction) appeared to significantly increase Shh release by 2-fold and 13-fold respectively. In serum-free condition, Shh amounted for up to 2 ng/mg of EBs protein at day 14, representing a sixfold increase over control (Fig. 3E). We used several EB-CM dilutions (2%, 5%, 10%, and 25%) and noticed a dose-dependent effect on expression of Hedgehog targets and pancreas markers in the explants (data not shown). In 2% EB-CM dilution, nearly all explants differentiated normally. Exposure to 10% EB-CM dilution still inhibited the differentiation and was associated with activation of the Hedgehog target Gli1 (Fig. 3D). Hedgehog Blockade in Conditioned Medium Rescues Beta Cell Differentiation Considering the secretion of Shh by EBs and its inhibitory effects on pancreas development in vitro, we attempted to block Hedgehog signaling in EB-CM by use of its recombinant antagonist HIP. HIP was supplemented at a concentration of 5 g/ml to EB-CM during the 7 days of culture. We focused our analysis on differentiation of insulin-producing cells because these represent the most likely application of this study. Our data indicated no rescue of beta cell differentiation in explants after HIP supplementation to undiluted EB-CM where antagonism with the pathway was not optimal (data not shown). However, addition of HIP to 10% EB-CM dilution reduced Gli1 expression to control levels (Fig. 3D), indicating inhibition of the Hedgehog pathway. Concomitantly, antagonizing Hedgehog activity allowed for the appearance of duct-lining Insulin-positive cells as well as numerous small foci of Insulin-positive cells by day 7 (Fig. 3F). The percentage of Insulin-positive cells increased fourfold (8.79%; p.0001) on HIP supplementation, thereby returning to normal values (Fig. 3G). Nevertheless, the normal morphology of the pancreas was not restored, and still more ductal structures and fewer acini were observed. In view of these results, we assume that Hedgehog is not the unique factor responsible for pancreas inhibition by this medium, but that it plays a major role in beta cell differentiation. Differentiation of Hepatic Progenitors Is Promoted by EB-Derived Factors During development, a bipotential endoderm progenitor in the ventral endoderm follows either a pancreatic or hepatic fate depending on extracellular cues. Shh induced by factors from the neighboring cardiac mesoderm controls this fate selection [22]. Thus, in contrast to pancreas that requires Shh repression to develop, the liver benefits from Shh signaling. We demonstrated a deleterious effect of EB-CM containing active Shh-N peptide on pancreatic explant growth and differentiation, and we expected this medium not to counter liver differentiation. After culture in EB-CM, cells from dissociated E12.5 mouse liver buds formed discrete colonies intermingled with spindle-shaped cells, a feature quite different from that in control medium, where cells grew as large monolayers. -catenin staining could easily reveal this cellular organization, and suggested activation of canonical Wnt pathway (enhanced -catenin stabilization) after culture in EB-CM (Fig. 4A). Many cells showed immunoreactivity for AFP and albumin, but the occurrence of positive cells and the signal intensity were higher after culture in EB-CM (Fig. 4B, 4C). This improved expression of liver markers in EB-CM was concordant with mrna expression analysis. Indeed, more than 15-fold Gli1 activation in liver cells was recorded, together with a 2.5-fold increase in both Afp and Alb gene expression (p.001, Fig. 4D). On the contrary, expression of the general hepatic TF Foxa2 was lower in EB-CM (data not shown), a pattern that was confirmed by immunocytochemistry, in which treated hepatocytes displayed larger nuclei with lower signal (Fig. 4E). Gli1 activation induced by Shh-N peptide in liver cells was 65% lower than in cells exposed to EB-CM, and no increase in Afp or Alb was noticed in that condition (Fig. 4D). On the other hand, antagonizing Hedgehog signaling reduced Afp expression to control levels, but had no effect on Alb (data not shown), suggesting a differential response of hepatic progenitors and differentiated hepatocytes to Hedgehog signaling. These observations indicate that EB-CM promoted differentiation of hepatic precursors cells and that altered pancreatic differentiation is a specific response in the context of Hedgehog signaling. Definitive Endoderm Induction in EBs Increases Hedgehog Production Definitive endoderm differentiation was recently induced in mouse and in human ES cells by serum removal and activin A supplementation. We wondered whether these treatments affect Hedgehog signaling that was previously shown to be induced on EB formation [26]. We therefore screened several Hedgehog and pancreatic genes to estimate the potential of ES-derived definitive endoderm cells to acquire a pancreatic fate within EBs (Fig. 5A). When exposed to activin A or B (100 ng/ml), EBs showed higher activation of the corresponding intracellular Smad components and endoderm-related genes on days 3, 6, and 10, confirming that endoderm was induced (Fig. 5B, supplemental online Fig. 1, and data not shown). Gene expression analysis revealed higher levels for Shh and its targets (Ptc1, Gli1, Hhip) in serum-deprived as well as in activin A-treated EBs at days 6 and 10 (Fig. 5C). The expression of Gli2, which is not directly regulated by Hedgehog signaling in contrast to Ptc1 and Gli1, was not significantly altered by these treatments (data not shown). Shh, Gli1 and Foxa2 expression were further analyzed by immunostaining. Activin-treated EBs showed a more widespread Shh immunoreactivity in contrast to control EBs, in which mainly the outer visceral endoderm layer stained positive. Concomitantly, higher reactivity for Hedgehog targets Gli1 and Foxa2 was noticed in differentiated cells on endoderm induction (Fig. 5D 5F). Pdx1 Expression in Activin A-Treated EBs Is Secondarily Repressed, and This Effect Can Be Reversed on Hedgehog Antagonism Taking into account the findings of greater Hedgehog activation on definitive endoderm induction and its deleterious effects on pancreas initiation and maintenance as known in vivo, we re-

6 Mfopou, De Groote, Xu et al Figure 4. In contrast to its effect in the pancreas, in the liver, EB-CM does not block but rather improves cell differentiation. (A): Immunoreactivity for -catenin. Note the formation of discrete colonies and the diffuse staining suggestive of -catenin stabilization in treated cells, contrasting with large monolayers and essentially membrane localization of -catenin in control. (B): Immunofluorescence showing increased AFP expression in liver cells cultured for 7 days in EB-CM. (C): Increased albumin expression is also observed in hepatocytes colonies in EB-CM as compared to monolayers in control. Width of magnified images is 100 m. (D): Realtime polymerase chain reaction indicates that expression of Hedgehog targets (Ptc1, Gli1) is induced in liver cells by EB-CM (2- and 17-fold, respectively) more than by Shh-N peptide (1.2- and 6-fold, respectively). Expression of Afp and Alb are also increased, but not after Shh-N peptide treatment. (E): Immunoreactivity for FOXA2. Note smaller size of nuclei with higher intensity in control as compared to EB-CM. Scale bars 100 (A, B, E) and 200 m (C). Abbreviations: AFP, -fetoprotein; CM, conditioned medium; DAPI, 4,6-diamidino-2-phenylindole; EB, embryoid body; Shh, Sonic Hedgehog. a, significant versus control;, p.05;, p.001. solved to assess the short-term profile of few pancreas markers in activin A-induced EBs. Serum removal and treatment with activin A were associated with reduced Pdx1 expression and further decrease between days 6 and 10, which was concomitant to the sharp increase in Shh release ( 50-fold) and activation of its targets (Figs. 5C, 6A, 6B). Compared to control (10% FBS) and contrary to Pdx1, activin A treatment increased Ptf1a expression on day 10 by threefold (p.05; Fig. 6A), indicating the independence of Ptf1a from Hedgehog activity. Because activin B is required for Shh repression and pancreatic initiation in the gut endoderm, we also tested its effects on EBs. When activin B was used in substitution to activin A, the expression of endoderm genes, Hedgehog ligands, and targets were approximately 30% lower, and Pdx1 expression did not show a significantly different pattern (supplemental online Fig. 1 and data not shown). To ascertain the repressive function of increased Hedgehog signaling on pancreas-related gene expression in activin-induced EBs, we made use of several Hedgehog antagonists including monoclonal anti-shh antibody (10 and 50 g/ml), cyclopamine (1 and 5 ), forskolin (20 mm), and recombinant HIP (2 and 10 g/ml). Inhibition of the pathway was assessed by expression analysis of Ptc1, Gli1, and Hhip. Several molecules inhibited Hedgehog targets expression by 20% 50% when compared to activin A (supplemental online Fig. 2); however, only very high concentrations of HIP (10 g/ml) gave a more than 50% inhibition that was associated with an increase in Pdx1 and Insulin1 expression (5-fold and 1.8-fold, respectively; Fig. 6C, 6D), hence confirming that Hedgehog signaling in differentiating ES cells is a restrictive factor for Pdx1 expression, pancreas fate acquisition and beta cell differentiation. DISCUSSION Current protocols aimed at differentiating endocrine beta cells from ES cells strive to recapitulate stepwise the normal features of embryonic pancreas development [31 33]. Since the first proof of principle of beta cell commitment from ES cells, it has not been possible to go any further than definitive endoderm; but a very recent and elegant study by D Amour et al., which sequentially integrates several signal transductions implicated in pancreas development, allowed for differentiation of C-peptidepositive insulin-producing cells from human ES cells on definitive endoderm induction [4, 16, 17, 27]. A major problem concerning pancreas differentiation from ES cells is that several signaling pathways that are known to inhibit in vivo pancreas initiation and maintenance are activated on differentiation, including Hedgehog [26]. Considering that no study reports on a possible role of EB-derived molecules on pancreas differentiation from ES-cells and taking advantage of the well-described growth and differentiation of embryonic pancreas explants in vitro, we aimed at testing our hypothesis [26] by exposing E12.5 mouse pancreas explants to EBs environment ex vivo. Our experimental model denotes that EB-derived soluble factors alter ex vivo pancreas growth and differentiation. The cystic morphology of treated explants recalls pancreas remnants of transgenic mice that express Shh or Ihh in the pancreas, as well as the effect of activin on developing glandular organs, and of Ptf1a inactivation in the pancreas [30, 34 37]. This similarity was underscored by identification of Shh-N active fragments in EBs extracts on one hand, and by strong activation of Hedgehog targets in explants exposed to EB-CM on the other hand. Gene expression analysis in explants confirmed these observations, with almost a total loss of exocrine genes Amylase, Ptf1a, and

7 1162 EBs Factors Inhibit Pancreas Development Figure 5. Definitive endoderm induction from embryonic stem cells and Hedgehog pathway activation. (A): The bipotential ventral endoderm progenitor can adopt a pancreatic or hepatic fate depending on repression or expression of Shh as dictated by the cardiac mesenchyme. Adapted from [52]. (B): Treatment of EBs with activin A in serum-free condition upregulated Smad2/3 mrna expression at days 6 and 10. (C): Expression of Hedgehog ligand (Shh) and targets (Ptc1, Gli1, Hhip) is further increased in activin-treated EBs. (D): Differential localization of Shh in control EBs (outer visceral endoderm layer) versus EBs treated with activin A for endoderm induction (mainly inner cell layers). (E): Increased immunoreactivity for Hedgehog target Gli1 on endoderm induction. (F): Though widely expressed in both conditions, Foxa2 immunoreactivity was more uniform on endoderm induction. Scale bars 100 (D, E) and 200 m (F). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; HNF, hepatocyte nuclear factor; KOSR, knockout serum replacement; SHH, Sonic Hedgehog., p.01;, p.001. Mist1, which are normally expressed by 95% of mature, terminally differentiated pancreatic tissue. The increase in Pdx1 and Ngn3 expressions are probably related to the severe suppression of bulk exocrine tissue differentiation, resulting in a relative increase in progenitor cells. To our surprise, the tendency for pancreas marker repression was not seen with Glucagon (another endocrine marker), the expression of which was fivefold higher in EB-CM, and paralleled by upregulation of TFs required for differentiation of alpha cells in fetal pancreas. It remains to be investigated whether this phenomenon indicates proliferation of the preexisting transient Glucagon-positive cell population present in E12.5 pancreas explants, or the differentiation of endocrine progenitors toward the default alpha cell pathway, as occurs in other systems of perturbed pancreas development [38 40]. Beside this effect on endocrine and exocrine pancreas differentiation, Hedgehog signals are implicated in the transformation of pancreatic mesoderm into intestinal muscle tissue [35, 37]. The induction of smooth muscle marker desmin in explants treated with EB-CM (unpublished observations) holds in this line. Recombinant Shh-N peptide partly mimicked the effect of EB-CM. It is, however, unlikely that the inhibition of pancreas development by EB-CM was attributable to Shh alone. Considering the many cell types that spontaneously differentiate in EBs and their expression profile, previously described, we conceive that other yet unidentified or noninvestigated factors, notably the canonical Wnt pathway and growth factors of the fibroblast growth factor (FGF) and TGF families, play additional or synergistic roles in the observed effects of EB-CM [26, 41]. For instance, Shh in the EB-CM might be inhibiting the terminal differentiation of pancreatic cells, whereas other growth factors such as FGF10 (initially reported to be expressed at high levels in EBs [26], and normally produced by the pancreatic mesenchyme in the embryo to support epithelial cell growth [42 44]) are promoting the proliferation of progenitor cells. Nevertheless, Hedgehog inhibition in diluted EB-CM by use of HIP reduced Gli1 levels, resulting in rescue of beta cell differentiation. These results are concordant with recent findings from human ES cell differentiation where no significant increase in Insulin-positive cells could be observed after definitive endoderm induction performed without Hedgehog pathway inhibition at early stages [27]. Shh concentration in EB-CM, as measured by ELISA, was approximately 200 pg/ml, which is quite low compared to 2.5 g/ml recombinant Shh-N used. The discrepancy in Shh concentrations (EB-CM vs. recombinant Shh-N) and levels of target gene induction is concordant with the 164-fold difference in potency between the natural lipid-modified Shh-N and its recombinant counterpart, though it can also be attributed to the presence of Indian Hedgehog which is expressed but not detectable by our assay [26, 45, 46]. Such differences in potency were shown to be dependent on lipid modifications that normally occur during Hedgehog processing by eukaryotic cells, a phenomenon that is bypassed in the course of recombinant protein production, resulting in a much less active peptide [45, 47 50]. Despite its low level, this concentration remains in the range with potential inhibitory effect on pancreas. Indeed, as little as 50 ng/ml recombinant Shh-N peptide (i.e., 300 pg/ml natural

8 Mfopou, De Groote, Xu et al Figure 6. Effect of definitive endoderm induction and Hedgehog antagonism on pancreas markers expression in EBs. (A): Expression of early pancreas progenitor markers Pdx1 and Ptf1a in EBs exposed to control or endoderm-inducing conditions. Note the Pdx1 decrease in activin-treated EBs by day 10, in contrast to Ptf1a upregulation. (B): Enzyme-linked immunosorbent assay for Shh in the medium from 30 EBs at days 8 and 10. Note the significant increase in Shh production/ secretion after EBs culture in serum-free medium with activin A (endoderm induction), which is inversely correlated to Pdx1 downregulation in (A). (C): Day-10 expression profile of Shh and Gli1 in EBs, showing their downregulation in the presence of the Hedgehog antagonist HIP (10 g/ml). (D): Day-10 expression profile of Pdx1 and Insulin1 in EBs, showing increased levels on HIP supplementation to activin A. Abbreviations: EB, embryoid body; HIP, Hedgehog interacting protein; SHH, Sonic Hedgehog. a, significant versus control; b, significant versus activin A treatment;, p.05;, p.01; ***, p.001. Shh-N equivalent) suppresses pancreas gene expression from ventral endoderm explants [22]. Moreover, it has been shown that 14 pg/ml of Shh secreted by cultured neural progenitor cells is sufficient to induce Islet1/2 and repress Pax7 in chick neural fold explants, a concentration that is very low compared to that estimated from studies using recombinant Shh-N [51]. Since EB-CM elicited Hedgehog responses stronger than recombinant Shh-N, we assume that the lipid-modified Hedgehog ligands in the medium are very potent (as expected) and that this pathway represents a major challenge in the initiation of pancreatic cells types from ES cells. We made use of a fundamental developmental difference between pancreas and liver to show that EB-CM effect on pancreas explants was specific. Endoderm progenitor cells in the posterior foregut are bipotential, capable of giving rise to liver or to pancreas depending on whether they receive Hedgehog signals. Indeed, Shh expression is excluded from pancreas anlagen in contrast to the hepatic bud in which it is induced by cardiac mesoderm and plays a positive role in proliferation or differentiation of early liver progenitors. Thus, Hedgehog is the earliest known signal that diverts the bipotential endoderm progenitor from the default pancreatic fate to a hepatic fate [22, 52, 53]. Inhibition of pancreas explants differentiation that we observed in EB-CM is in line with the requirement for Shh absence during initiation and morphogenesis of this organ. On the contrary, because Shh is induced in the ventral endoderm to favor hepatic fate, its presence in EB-CM is not harmful, but rather beneficial to liver cell differentiation, as we observed. Yet this role for Shh appears permissive because we did not reproduce the increased Alb and Afp expression by treatment with recombinant Shh-N peptide alone, whereas antagonism with the pathway affected only Afp expression, a marker of hepatocyte precursors. These data recall the knowledge that treatment of ventral endoderm with 50 ng/ml Shh-N excluded the default pancreatic fate but was inefficient in eliciting the hepatic program [22], suggesting that additional factors are required, which, in our model, were supplied by the complex biological environment of EB-CM. Furthermore, these observations are in line with the maturation of in vivo implanted definitive endoderm cells derived from ES cells into pneumocytes, hepatocytes, and intestinal cells, but not into pancreatic cells [16, 17]. We further investigated Hedgehog activity during definitive endoderm induction from ES cells and showed higher levels of ligands and targets in activin-treated EBs, which is associated with limited Pdx1 expression, reminiscent of the repressive function of this pathway on pancreas induction and morphogenesis, as described in vivo [23, 30, 35, 41]. This finding is further supported by the contrasting increase in Ptf1a expression after activin treatment, which delineates its independence from Shh activity in contrast to Pdx1. Moreover, it sheds some light on the inability of previous studies to demonstrate pancreatic differentiation from ES cell-derived definitive endoderm both in vitro and in vivo [16 18], and supports the findings that Hedgehog repression at early differentiation stages is mandatory for the generation of Insulin-positive cells from ES cells [27]. The higher Hedgehog activity in this system can also be viewed as a consequence of endoderm formation because activin A-treated EBs are made of at least 60% definitive endoderm cells, and Shh has been identified in the entire gut tube except at the Rathke s pouch and pancreas anlagen [16, 17, 24]. This means that activin-a treatment of ES cells induces definitive endoderm, but not of pancreatic type. Although the tendency for Pdx1 decrease in activin-treated EBs paralleled the increased Hedgehog activity, we could not establish a strong correlation between Pdx1 fold change versus that of Shh, Ptc1, or Gli1, possibly owing to the limited Pdx1 expression in this system, the large variation in Shh fold change, and the observed plateau in Ptc1 and Gli1 activations. In addition, the reported Ptf1a increase in activintreated EBs at 10 days contrasts with its downregulation in pancreas explant models. Interpretation of these data is made difficult by the presence of activin A at high concentrations in ES culture system but not in EB-CM, as well as the use of different references for gene expression analysis (7-day cultured control explant expressing Ptf1a vs. undifferentiated ES cells not expressing Ptf1a). Nevertheless, the rescue of Pdx1 and Insulin1 expression in EBs on Hedgehog inhibition with HIP (10 g/ml) suggests that this pathway represents, as known in vivo, the first barrier to overcome if pancreatic beta cells are to be massively derived from ES cells via the natural definitive endoderm route. In addition, pancreas-inducing signals are needed concomitantly with optimal Hedgehog inhibition to increase the proportion of ES-derived insulin-producing cells [27 29, 54]. Further elucidation of signals that initiate and maintain the pancreas program after Shh repression in the posterior foregut endoderm will represent an asset for future advances in this line. CONCLUSION In summary, this study highlights the main current difficulties associated with beta cell differentiation from ES cells. Although the route to follow should be via the natural definitive endoderm, activation of Hedgehog signaling on endoderm induction represents a major obstacle to derivation of beta cells at

9 1164 EBs Factors Inhibit Pancreas Development a higher frequency. This phenomenon is underscored by Pdx1 downregulation in activin A-treated EBs, as well as the ex vivo demonstration of the deleterious effect of EBs environment (including Shh) on pancreatic growth and beta cell differentiation. Therefore, as occurs in vivo, the Hedgehog pathway blockade represents a mandatory step in the differentiation of beta cells from ES-derived definitive endoderm cells. ACKNOWLEDGMENTS We thank W. Rabiot for assistance in mouse dissection; E. De Blay and M. Baekeland for valuable help in immunocytochemistry; D.G. Pipeleers for logistic support; Dr J. Lardon, S. De Breuck, L. Baeyens, I. Houbracken, and N. De Medts for useful comments and criticisms. This work was supported by the Horizontale Onderzoeksactie of the Vrije Universiteit Brussel (J.K.M.). DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest. REFERENCES 1 Edlund H. 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