Differentiation (2007) 75:1–11 DOI: 10.1111/j.1432-0436.2006.00109.x

r 2006, Copyright the Authors Journal compilation r 2007, International Society of Differentiation

O RI G INA L AR T I C L E

Mio Nakanishi . Tatsuo S. Hamazaki . Shinji Komazaki . Hitoshi Okochi . Makoto Asashima

Pancreatic tissue formation from murine embryonic stem cells in vitro

Received December 15, 2005; accepted in revised form July 6, 2006

Abstract The in vitro formation of organs and/or tissues is a major goal for regenerative medicine that would also provide a powerful tool for analyzing both the mechanisms of development and disease processes for each target organ. Here, we present a method whereby pancreatic tissues can be formed in vitro from mouse embryonic stem (ES) cells. Embryoid body-like spheres (EBSs) induced from ES cell colonies were treated with retinoic acid (RA) and activin, which are candidate regulators of pancreatic development in vivo. These induced tissues had decreased expression of the sonic hedgehog (shh) gene and expressed several pancreatic marker genes. ES cell-derived pancreatic tissue was composed of exocrine cells, endocrine cells, and pancreatic duct-like structures. In addition, the ratio of exocrine to endocrine cells in the induced tissue was found to be sensitive to the concentrations of RA and activin in the present experiment.

Mio Nakanishi
Tatsuo S. Hamazaki
Makoto Asashima . ) (* Department of Life Science (Biology) Graduate School of Arts & Science The University of Tokyo, Meguro Tokyo 153-8902, Japan E-mail: [email protected] . )
Hitoshi Okochi Tatsuo S. Hamazaki (* Department of Tissue Regeneration Research Institute, International Medical Center of Japan Shinjuku, Tokyo 162-8655, Japan E-mail: [email protected] Shinji Komazaki Department of Anatomy Saitama Medical School Iruma, Saitama 350-0495, Japan Makoto Asashima ICORP, Japan Science and Technology Agency (JST) Kawaguchi, Saitama, Japan U.S. Copyright Clearance Center Code Statement:

Key words mouse embryonic stem cells
pancreas
b-cells
sonic hedgehog
pancreatic and duodenal homeobox 1 (Pdx-1)
ptf1a/p48
activin, retinoic acid

Introduction Mouse embryonic stem (ES) cells can differentiate into various cell types in vitro (Loebel et al., 2003), including the insulin-secreting b cells of the pancreas (Soria et al., 2000; Lumelsky et al., 2001; Hori et al., 2002; Shiroi et al., 2002; Blyszczuk et al., 2003; Kim et al., 2003; Leon-Quinto et al., 2004) but not formed any organs or tissues (Le Douarin, 2000). The promise of transplanting such cells into patients with diabetes has driven extensive research into factors that induce the differentiation of endocrine cells. However, the pancreas is a complex organ composed of exocrine cells, endocrine cells, and pancreatic ducts, which are considered to differentiate from a common precursor cell (Slack, 1995; Percival and Slack, 1999), making the in vitro formation of whole pancreas a far more complex undertaking. Retinoic acid (RA) and transforming growth factor-b (TGF-b) play key roles in the regulation of pancreatic organogenesis (Kim and Hebrok, 2001; Kumar and Melton, 2003). RA signaling is involved in patterning along the anteroposterior axis of the endoderm during the late gastrula stage of zebrafish, African clawed frog (Xenopus laevis), and quail. While inhibition of RA signaling in the late stages had little effect on the expression of anterior mesodermic markers, it abrogated pancreatic marker expression. In addition, zebrafish and Xenopus embryos treated with exogenous RA showed enlargement of the pancreas, and in zebrafish also of the liver, in the anterior direction (Stafford and Prince, 2002; Chen et al., 2004; Stafford et al., 2004). Another important mediator of pancreas differentiation is sonic hedgehog (Shh); its down-regulation by signals from the notochord is essential for initiating

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differentiation of the dorsal pancreas in mouse and chicken embryos (Kim et al., 1997). In tissue culture experiments using endoderm isolated from chicken embryos, activin, a member of the TGF-b family, inhibited Shh expression and induced transcription of pancreatic marker genes, thereby mimicking the effects of the notochord signaling (Kim et al., 1997; Hebrok et al., 1998). Some reports have also suggested that differentiation into pancreatic endocrine cells is regulated by TGF-b signal transduction mediated via activin receptors (Sanvito et al., 1994; Ritvos et al., 1995; Miralles et al., 1998; Yamaoka et al., 1998; Shiozaki et al., 1999; Kim et al., 2000). Culture of the mouse embryonic pancreas in vitro in the presence of activin or TGF-b1 led to enhancement of endocrine cell formation, particularly of b cells and pancreatic polypeptide cells (Sanvito et al., 1994), while normal epithelial branching and the development of exocrine cells were disturbed (Ritvos et al., 1995). Furthermore, follistatin, an antagonist for TGF-b signaling (including that via activin), was shown to stimulate the pancreas and differentiate into exocrine cells and reduce the differentiation of endocrine cells (Miralles et al., 1998). In addition, transgenic mice expressing a dominant-negative form of the type II activin receptor showed hypoplasia of the islets (Yamaoka et al., 1998; Shiozaki et al., 1999). Since both RA and activin have considered to regulate pancreas development, we examined whether treatment with both these factors could induce the differentiation of ES cells into pancreatic cells.

Colony formation of ES cells (3 days) on feeder cells in ES medium (15% FBS, +LIF) to isolate the colonies treated with collagenase/dispase

Formation of EBSs (4 days) on non-treated dishes in DMEM (15% KSR, −LIF)

Treatment with RA and activin (2 days) on non-treated dishes in DMEM (15% KSR) containing RA and activin

Expansion of EBSs on gelatin coated tissue culture dishes in DMEM (10% KSR)

Fig. 1 Experimental protocol for the differentiation of embryonic stem (ES) cells into pancreatic tissue with retinoic acid (RA) and activin. Colonies of ES cells were maintained onto feeder cells and incubated in ES medium containing LIF for 3 days. These ES colonies were detached from the feeder cells by collagenase/dispase treatment. The ES cell clusters were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% KnockOut Serum Replacement (KSR) in floating condition. Four days after embryoid body-like spheres (EBSs) began to form, they were transferred in a low cell-binding multi-well plate and incubated for additional 2 days in DMEM supplemented with 15% KSR, containing various concentrations of activin A and all-trans RA. The EBSs were then attached to the gelatin-coated tissue culture dish and continued to culture in DMEM supplemented with 10% KSR until the examination. Immunohistochemistry

Materials and methods Maintenance and differentiation of ES cells Mouse-derived ES cells (E14 and CMTI-1) were seeded onto mouse embryonic fibroblasts pretreated with mitomycin C (10 ng/ml; Sigma, St. Louis, MO) for 2.5 hr. The cells were then incubated in Dulbecco’s modified Eagle’s medium (DMEM; high glucose with Lglutamine and pyruvate; Gibco 11995-065, GIBCO, Invitrogen, Carlsbad, CA) supplemented with 15% ES cell qualified fetal bovine serum (FBS; Gibco), MEM non-essential amino acid solution (Gibco), 0.001% b-mercaptoethanol (Sigma), and LIF ESGRO (1,500 U/ml, Chemicon, Temecula, CA). For the differentiation of ES cells, 3-day ES cell colonies were detached from the feeder cells by the treatment of 1 mg/ml of collagenase/dispase (Roche Diagnostic, Indianapolis, IN) (Fig. 1). The clusters of ES cells were transferred into a low cell-binding dish (Nalge Nunc, Rochester, NY). They were cultured at floating condition in DMEM supplemented with 15% KnockOut Serum Replacement (KSR; Gibco) and the culture medium was renewed 2 days later. Four days after embryoid body-like spheres (EBSs) began to form, they were transferred into a low cell-binding 96-well plate (Nunc) and incubated for additional 2 days in DMEM supplemented with 15% KSR containing activin A (0, 10, 25, 50 ng/ml) and all-trans RA (0, 0.001, 0.01, 0.1, or 1 mM; Sigma). The EBSs were then attached to the wells of tissue culture plates or dishes (TPP) that had been coated overnight with 0.1% gelatin (Sigma). The cells were incubated in DMEM supplemented with 10% KSR, and the medium was renewed every third day.

Thirteen days after the end of treatment (19 days after the beginning of EBS formation), the EBSs were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 40 min at room temperature. The fixed cells were freed from the dish wall and embedded in an LR Gold Resin System (structure probe, Electron Microscopy Sciences, Hatfield, PA), an acrylic resin, and sectioned to yield 600 nm semithin sections. The sections were blocked for 40 min at room temperature in 3% bovine serum albumin (BSA) and phosphate-buffered saline (PBS). They were then exposed to pancreas-specific primary antibodies for 12 hr at 41C. After washing with PBS, the sections were exposed to labeled secondary antibody for 8 hr at 41C. The primary antibodies used were rabbit anti-a amylase antibody (1:1,000 dilution, Sigma), anti-insulin monoclonal antibody (1:400 dilution, Sigma), goat anti-C-peptide antibody (1:800 dilution, Linco Research, Millipore, Billerica, MA), rabbit anti-Pdx-1 antibody (1:200 dilution, Chemicon); the secondary antibodies were Alexa-Fluor 488and Alexa-Fluor 594-conjugated (Molecular Probes, Invitrogen, Carlsbad, CA). The sections were observed under a fluorescence microscope and photographed with AquaCosmos (Hamamatsu Photonics, Hamamatsu, Japan) connected to an ORCA-3CCD camera. For the control, pancreas from 8-week-old mouse was fixed and processed as described above. For smooth muscle detection, EBSs were fixed as described above, blocked with 3% BSA in PBS for 40 min at room temperature, and permeabilized with 0.1% Triton X-100 for 30 min. They were then exposed to fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-a-smooth muscle actin antibody (1:500 dillution; Sigma) for 12 hr at 41C. To calculate the C-peptide-positive cells in the induced EBS, 1 mm serial sections were prepared from each specimen, and examined at every 20 mm thickness by immunostained with anti-C-peptide

3 antibody. Then they were calculated the percentage of the C-peptide-positive cells with image analyzing software (Luminavision). Electron microscopy Eleven days after the end of treatment (17 days after EBS formation), the EBSs were prefixed in a fixative containing 4% paraformaldehyde, 3% glutaraldehyde, and 0.1 M cacodylate buffer (pH 7.4) for 2 hr at room temperature. Then, after washing with 0.1 M cacodylate buffer, they were post-fixed in 1% osmium tetroxide for 30 min at room temperature. The samples were washed again in 0.1 M cacodylate buffer, then dehydrated through an ethanol and acetone series, and embedded in Epoxy resin. Ultrathin (80–90 nm) sections were prepared and stained with uranium acetate and lead citrate for observation under a transmission electron microscope (JEM-1200CX, JEOL, Tokyo, Japan).

7700 Sequence Detector (Perkin Elmer). The forward and reverse primers used and the length of their PCR products were as follows: glucagon, GCA CAT TCA CCA GCG ACT ACA G and GGG AAA GGT CCC TTC AGC ATG TCT (146 bp); somatostatin, CGA GCC CAA CCA GAC AGA GA and CAT TGC TGG GTT CGA GTT GG (115 bp); amylase 2, ATA CTC TGC TTG GGA CTT TAA CGA and CAG AAG GCC AGT CAG ACG A (100 bp); GAPDH, GCT ACA CTG AGG ACC AGG TTG TC and AGC CGT ATT CAT TGT CAT ACC AGG (135 bp); insulin II, AGA AGC GTG GCA TTG TAG ATC AGT and CAG AGG GGT AGG CTG GGT AGT G (102 bp); Pdx-1, GAT GAA ATC CAC CAA AGC TCA CGC and GGG TGT AGG CAG TAC GGG TCC TC (101 bp); ptfla, ATG CAG TCC ATC AAC GAC GCC TTC and GGC TTG CAC CAG CTC GCT GAG (138 bp); Shh, GAC TGC GGG CAT CCA CTG GTA CTC and GTC GGG CTT CAG CTG GAC TTG AC (114 bp). cDNA was amplified by 15 min initial denaturation at 951C, 45 cycles of heating at 941C (30 sec), 551C (30 sec), and 721C (30 sec). This experiment was also carried out in triplicate.

Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from 16 EBSs to normalize the gene expression among the EBSs, using an ISOGEN (Nippon Gene Co., Ltd., Tokyo, Japan). Complementary DNA (cDNA) was synthesized from 1 mg total RNA using a SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA). The forward and reverse primers used and the length of their PCR products were as follows: albumin (Yamada et al., 2002), TGA ACT GGC TGA CTG CTG TG and CAT CCT TGG CCT CAG CAT AG (718 bp); a-phetoprotein (Afp), CCA CCC TTC CAG TTT CCA G and GGG CTT TCC TCG TGT AAC C (609 bp); amylase 2, GCC AAG GAA TGT GAG CGA TAC TTA and CCA GAA GGC CAG TCA GAC GA (418 bp); glucose-6-phosphatase (G6p) (Ishii et al., 2005), TGC ATT CCT GTA TGG TAG TGG and GAA TGA GAG CTC TTG GCT GG; glucagon, AAT GAA GAC AAA CGC CAC T and AAT TCA TAT ACA ATC GTT GGG TTA (554 bp); insulin I, TTA CAC ACC CAA GTC CCG CCG TGA and AGG GGT GGG GCG GGT CGA G (207 bp); insulin II, GGC TTC TTC TAC ACA CCC ATG TCC and TTT ATT CAT TGC AGA GGG GTA GGC (234 bp); Pdx1, AGC AGT CTG AGG GTG AGC GGG TCT and AAC CTC CAA CAG CCG CCT TTC GT (412 bp); somatostatin, CCC AGA CTC CGT CAG TTT CT and TCA ATT TCT AAT GCA GGG TCA AGT (377 bp); Brachuyry (T), GAA TTC GTC CAC CCC CTG TCC TAC and CAA GGG CAG AAC AGT TGA CGG TT (146 bp); b3 tubulin (Tubb3), GTC CTA GAT GTC GTG CGG AAA G and GGA TGT CAC ACA CGG CTA CCT (724 bp); pancreatic polypeptide, GCC CAA CAC TCA CTA GCT CAG and AGA GGA AAG AGC TGG ACC TGT ACT (419 bp); Shh, ACA TGT CCC TTG TCC TGC GTT TCA and CTC GTG GGC TCG CTG CTA GGT (440 bp); Sox17, GGC CAG AAG CAG TGT TAC ACA and TTT GAT AAA AAT CGA TGC GAG AGA (336 bp); tyrosine amino transferase (Tat) TCC AGG AGT TCT GTG AAC AGC and AGT ATA TGG TGC CTG CCT GC; GAPDH, TGA AGG TCG GTG TGA ACG GAT TTG GC and CAT GTA GGC CCA TGA GGT CCA CCA C. cDNA was amplified by a 5 min initial denaturation at 951C, 40 cycles of heating at 941C (20 sec), 541C (20 sec), and 721C (40 sec) and finally, 7 min elongation at 721C. The experiment was carried out in triplicate. To determine the inductive efficiency of pancreatic tissue from the EBSs, we examined the expression of pancreatic marker gene Pdx-1 in each EBS separately (n 5 15 for each treatment). For the examination of gene expression, more than 0.7 mg of total RNA was obtained from each EBS and equal amount (0.7 mg) was used for the RT-PCR. Quantitative PCR Real-time PCR was carried out using a QuantiTectt SYBR Green PCR Master Mix (QIAGEN, Valencia, CA) and ABI PRISMt

Results Differentiation of gut-like and pancreatic tissues from ES cells First, ES cell colonies were isolated after 3 days in culture by collagenase/dispase treatment and were then cultured in DMEM supplemented with 15% KSR to form EBSs, which can initiate differentiation. Four days after the induction of EBS formation, the cells were transferred into a medium containing RA (0, 0.001, 0.01, 0.1, or 1 mM) and activin (10 ng/ml) and incubated for additional 2 days by floating culture, and then seeded on gelatin-coated plates. Six to 8 days after the end of treatment with RA and activin (12–14 days after the commencement of EBS formation), a proportion of the treated EBSs showed tubular or gut-like structures and a motility that resembled smooth-muscle peristalsis. Immunostaining with anti-a-smooth muscle actin antibody demonstrated that abundant smooth muscle cells surrounded these tubular or gut like structures (Figs. 2C–2E). Nine to 12 days after the end of treatment (15–18 days after the commencement of EBS formation), over 20% of the EBSs treated with both RA and activin formed a tissue that contained black spots, which were rarely observed in the untreated EBSs or EBSs treated with activin alone (Figs. 2A,2B). Examination of expression of pancreatic marker genes As an approach to examine differentiation state of the EBSs at the onset of the treatment, we monitored the gene expression patterns of b3-tubulin (Tubb3), Brachyury (T) and Sox17 as early development markers of neuroectoderm, mesoderm, and endoderm, respectively (Fig. 3A). From 3 days after the induction of EBSs formation (a day before the onset of RA and activin treatment), the expressions of Sox17 and T were detected (Fig. 3A and data not shown). In contrast, the

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expression of Tubb3 was not detected until 2 days after the finish of the treatment. In the EBSs treated with RA and activin, the expression of Sox17 was clearly observed. Whereas no differences was observed in the expressions of T and Tubb3 between treated EBSs and untreated EBSs. Sox17 was also detected in the EBSs which were treated with activin alone, but it did not express when they were treated with RA alone. The expression patterns of genes that regulate the development of the pancreas and several pancreatic marker genes were also investigated using RT-PCR (Fig. 3A). The EBSs treated with both RA and activin showed a marked increase in the gene expression of insulin I, insulin II (markers of b cells), glucagons (a cells), pancreatic polypeptide (g cells), somatostatin (d cells), and amylase 2 (a marker of pancreatic exocrine cells), as well as of Pdx-1 and ptf1a/p48, which are genes that regulate pancreatic development. In contrast, the untreated EBSs showed almost no expression of these genes. The expression of amylase and pancreatic polypeptide increased over time from day 6 of treatment (12 days after the commencement of EBS formation), while the gene expression of insulin I, insulin II, glucagon, somatostatin, Pdx-1 and ptf1a/p48 commenced immediately after the end of the treatment. In untreated EBSs, Shh was detected throughout the observation period, while in the EBSs treated with both RA and activin, Shh gene expression was suppressed following the treatment. In the EBSs treated with 0.1 mM RA alone, the expression of glucagon, insulin I, insulin II, and pancreatic polypeptide began to be detected but shh regressed similar to the EBSs treated with the same concentration of RA and 10 ng/ml of activin A. The expression of other

Fig. 2 Phase contrast images of embryoid body-like spheres (EBSs) containing differentiated pancreatic tissue. (A, B) EBS on day 13 after treatment with 0.1 mM of retinoic acid (RA) and 10 ng/ml of activin showed a gut-like structure (g) and pancreatic duct-like structures (d) adjacent to exocrine cells which were seen as many black spots (B, arrowheads). (C–E) Immunostaining of EBSs treated with 0.1 mM RA and 10 ng/ml activin prepared 11 days post-treatment demonstrated that abundant smooth muscle surrounded the gut-like structures (g) in the EBSs. Phase contrast (C), anti-smooth muscle-a-actin antibody staining (D), and merged image of the EBS (E). (Bar 5 50 mm [A and B]).

pancreatic markers, amylase 2 and Pdx1, were also detected but their expression seemed to be weaker than those in the EBSs treated with RA and activin, and the expression of somatostatin was not detected. In the EBSs treated with 10 ng/ml activin A alone, the expression of glucagon and Pdx1 was detected. However, they did not express other pancreatic marker genes (Amylase2, insulin I, insulin II, pancreatic polypeptide, and somatostatin) and shh expression was detected 10 days after the end of the treatment. To examine whether the gene expression level analyzed by RT-PCR were reflected to the mRNA amounts, we performed quantitative PCR using glucagon and somatostatin (Fig. 3B). Expression levels of both genes elevated 2 days after the treatment and increased 4 days later. It was found that the RT-PCR results were almost compatible with that of quantitative PCR. When the concentration of activin kept constant (10 ng/ml) and the RA concentration was changed, the gene expressions of pancreatic marker in EBSs appeared most efficiently when 0.1 mM RA was used with 10 ng/ml activin A (Fig. 3C). Pdx-1 expression was detected in 47.6%  7.3% of the EBSs treated with these conditions. When the concentration of RA were 0.001, 0.01, and 1 mM, the percentage of EBSs expressing Pdx-1 were 4.3%  2.5%, 12.8%  9.6%, and 25.1%  6.6%, respectively. We attempted to determine whether hepatic differentiation was also induced in this system by the examination of expression patterns of Albumin, a-phetoprotein (AFP), glucose-6-phosphatase (G6P), tyrosine amino transferase (TAT) used as hepatic marker genes. As a

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result, no hepatic marker genes were detected by RTPCR in the EBSs treated with both RA and activin, except the AFP expression was faintly detected 4 days after the end of the treatment (data not shown).

Morphological identification of ES cell-derived pancreas tissue Histology of sections of EBSs treated with 0.1 mM RA and 10 ng/ml activin 11 days after the end of treatment (17 days after EBS formation) showed lobular tissue that was intensely stained with toluidine blue in the area adjacent to the tubular lumen (Fig. 4B). Pancreatic duct-like structures were seen in the vicinity of the lobular tissue (Figs. 4C,4D). In contract, neither lobular tissues nor duct-like structures were observed in the sections of untreated EBSs (Fig. 4A). Electron microscopic observation revealed that the cells had a welldeveloped endoplasmic reticulum and contained large amounts of zymogen granules (Figs. 5C,5D), a phenotype consistent with pancreatic exocrine cells (acinar

Fig. 3 The gene expression after induction of differentiation. (A) Expression of pancreatic marker genes examined by reverse transcription-polymerase chain reaction (RT-PCR) in the untreated embryoid body-like spheres (EBSs), and the EBSs treated with either or both retinoic acid (RA) and activin. The expression of insulin II, glucagon, somatostatin, and Pdx1 began to be detected on day 2 post-treatment, while amylase 2 and pancreatic polypeptide expression was on days 6 and 8, respectively. Expression of sonic hedgehog (Shh) diminished after treatment. In the EBSs treated with RA and activin, the expression of Sox17 began to be clearly detected after the treatment, whereas no differences between the treated EBSs and the untreated EBSs were observed in the expression of T and the Tubb3. (B) Quantitative PCR confirmed the RT-PCR results. The gene expression of both glucagon and somatostatin were up-regulated 2–4 days after treatment and kept high level until 10 days of the treated EBSs ( & ) compared with those of the non-treated EBSs (&). (C) The most efficient gene expression of pancreatic marker Pdx1 in EBSs was observed when 0.1 mM RA was used with 10 ng/ml activin A. At these concentrations of RA and activin, 47.6%  7.3% of the treated EBSs expressed Pdx1. Other concentrations did not induce pancreatic gene expressions effectively.

cells). The tissue formed from the treated EBSs resembled pancreatic acini also in that a narrow tubular area was seen abutting the tissue (Fig. 5C). A structure surrounded by low columnar epithelium and resembling a pancreatic duct-like structure was also present (Fig. 4D). Furthermore, cells morphologically resembling pancreatic endocrine cells (Figs. 5E,5F) were observed, although these were smaller in number than the cells resembling pancreatic exocrine cells. These endocrinelike cells contained granules (Fig. 5F), although they were morphologically different from the granules found in the exocrine cells (Fig. 5D) and displayed a clear space between the dark central area and the lateral membrane, considered a feature of typical b cell granules in the pancreas. To confirm whether these granules contained insulin or amylase, the cells were immunostained with anti-amylase and anti-insulin C-peptide (proinsulin) antibodies. The EBSs treated with both RA and activin contained cells with amylase-positive zymogen granules (Fig. 5A) and a relatively small number of cell aggregates with C-peptide-positive granules (Fig. 5B). Immunohistochemical staining also showed that

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Pdx-1-positive cells were localized around the duct-like structures in the EBSs treated with RA and activin A (Fig. 5G). Immunoreactivity of Pdx-1 was localized mainly in cytoplasm and partially in nucleus (Fig. 5H). These morphological findings suggested that pancreatic tissue containing exocrine cells, endocrine cells, and pancreatic ducts were formed from EBSs treated with both RA and activin.

The concentration of activin alters the ratio of exocrine to endocrine cells We further examined whether the inductive effect of activin and RA on the EBSs to form pancreatic tissues was concentration-dependent. To address this, total RNA was extracted from the EBSs 13 days after treatment with RA (0, 0.1, or 1 mM) and activin (0, 10, or 25 ng/ml) and real-time PCR was performed to measure the transcriptional expression of amylase 2, insulin II, ptf1a/p48, and Shh (Figs. 6A–6D) compared with that expressed in untreated EBSs (normalized to a value of 1). Shh expression was reduced by treatment with RA alone, activin alone, as well as by RA plus activin (Fig. 6D). The expression of insulin II and amylase 2 was greater in EBSs treated with RA alone than in the untreated EBSs, and activin alone had no significant effect on the level of insulin II expression but decreased amylase 2 expression compared with the untreated EBSs (Figs. 6A,6B). Interesting results were obtained when the concentration of RA was kept constant (0.1 mM)

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Fig. 4 Morphology of embryoid body-like spheres (EBSs) containing differentiated pancreatic tissue. Sections of EBSs treated with 0.1 mM retinoic acid and 10 ng/ml activin prepared 11 days post-treatment showed lobular tissue intensely stained with toluidine blue adjacent to tubular lumina (B, arrowheads), and pancreatic ductlike structures (d) in the vicinity of the lobular tissue (C, D). A structure resembling a pancreatic duct, surrounded by low columnar epithelium, was also observed (D). In the sections of untreated EBS, neither lobular tissues nor duct-like structures were observed (A). n, nucleus. Scale bars: 50 mm (A–C) and 5 mm (D).

and only the concentration of activin was changed. At relatively low concentrations of activin (10 ng/ml), the levels of amylase 2 and ptf1a/p48 expression (markers of exocrine cells) were much higher in the RA plus activintreated groups than in the untreated EBSs, while the expression of insulin II did not differ between the two groups (Figs. 6A–6C). In contrast, at a higher activin concentration (25 ng/ml), the expression of insulin was markedly increased in the EBSs treated with RA plus activin relative to the untreated group, whereas low the expression levels of amylase 2 were observed in the two groups. The quantitative PCR results were supported by double staining using anti-insulin C-peptide and antiamylase antibodies (Figs. 7A–7D). In EBSs treated with 0.1 mM RA and a low concentration (10 ng/ml) of activin, the number of insulin-positive cells were fewer than amylase-positive cells (Fig. 7C). However, when a high concentration of activin (25 ng/ml) was used with 0.1 mM RA, the percentage of insulin-positive cells markedly increased (Fig. 7D). In the untreated EBSs, insulin-positive cells and amylase-positive cells were not observed (Fig. 7B). These results suggest that the ratio of endocrine cells to exocrine cells in the induced tissue from the EBSs was dependent on the activin concentration. Immunocytochemical analysis also revealed that some of the cells produced the a cell marker protein, glucagon (Fig. 7E). We tried to estimate C-peptidepositive cells in each EBS. Serial sections (1 mm) were cut and examined. As a result, three of 10 EBSs treated

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with 0.1 mM RA and 25 ng/ml activin A were found to contain C-peptide-positive cells. In these C-peptidepositive EBSs, 4.99%  0.59% of total cells contained C-peptide (Table 1). By the evaluation of immunostaining with anti-C-peptide antibody, 30% of EBSs treated with RA and activin showed pancreatic features.

Discussion While a number of studies have isolated pancreatic b cells from ES cells, here we have shown that treatment

Fig. 5 Immunohistochemical and ultrastructural analyses of embryoid body-like spheres (EBSs) containing differentiated pancreatic tissue. (A, B) Immunostaining of EBSs treated with 0.1 mM retinoic acid and 10 ng/ml activin revealed cells containing amylase-positive zymogen granules (A) and a few cells containing insulin C peptide-positive granules (B). Cells were counterstained with DAPI (4 0 ,6-diamidino-2-phenylindole) (A, B). Electron microscopic observation revealed cells containing large amounts of zymogen granules (C, D), well-developed endoplasmic reticulum, and showed dark and homogeneous staining, which are characteristic of acinar cells. A narrow tubular area seen abutting the tissue was also reminiscent of pancreatic acini (C). Cells morphologically resembling pancreatic endocrine cells were also noted (E, F), although they were smaller in number than the cells resembling pancreatic exocrine cells. These cells contained large amounts of endocrine granules (F), which were morphologically different from the granules contained in the exocrine cells (D). A large space between the dark central area and the lateral membrane, consistent with the b cell granules of the pancreas, was also observed (F). (G) Pdx-1-positive cells were observed around the duct-like structures. The localization of Pdx-1 was found mainly in cytoplasm and partially in nucleus (H) (er, endoplasmic reticulum; m, mitochondrion; n, nucleus; z, zymogen granule). Scale bars: 20 mm (A, B, G, H), 5 mm (C–E), 1 mm (F).

of EBSs with RA and activin by floating culture can induce differentiation of complex and functional pancreas that includes all endocrine (a, b, g, and d) cells, acinar cells, and pancreatic duct-like structures. ES cells that spontaneously differentiated into insulin-producing cells in culture have been identified (Loebel et al., 2003; Blyszczuk and Wobus, 2004), and isolated by the gene-trap method (Soria et al., 2000) or by the use of dithizone, a zinc chelator that specifically stains b cells (Shiroi et al., 2002). However, these cells only had minimal expression of a few pancreatic markers and no functional activity, such as glucose concentrationdependent release of insulin (Shiroi et al., 2002).

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Shh

Activin A 10 ng /ml 25 ng /ml

2 1.5 1 0.5

0 ng/ ml

1 Relative mRNA expression

Relative mRNA expression

2.5

Activin A

0 ng / ml

10 ng/ ml 25 ng/ ml

0.8 0.6 0.4 0.2

0 0

0.1

0 1.0 0 Concentration of retinoic acid (µM)

Insulin-secreting cells were generated by selection of cells expressing nestin from spontaneously differentiated ES cells and subsequent treatment of these cells with factors such as nicotinamide (Lumelsky et al., 2001; Hori et al., 2002; Blyszczuk et al., 2003; Shi et al., 2005). Nestin, an intermediate filament protein, is expressed in mesenchymal cells associated with the pancreas, but this protein was not detected in pancreatic endocrine precursor cells (Selander and Edlund, 2002), nor is it expressed in endocrine cells (Delacour et al., 2004). It is therefore unclear whether such induction steps would serve as reliable models for the differentiation of pancreatic cells in vivo. A recent study reported that EBs that formed in the presence of serum could differentiate into b cells by the sequential treatment of activin, RA, and nicotinamide (Shi et al., 2005). In the present study, we first focused on the formation of pancreatic tissues containing all three components: endocrine cells, exocrine

0.1

1.0

Fig. 6 Expression of pancreatic marker genes and sonic hedgehog (shh) in embryoid body-like spheres (EBSs). Total RNA was collected from the EBSs 13 days after the end of treatment with retinoic acid (RA; 0, 0.1, or 1 mM) and activin (0, 10, or 25 ng/ml), and the expression levels of amylase 2 (A), insulin II (B), ptf1a/p48 (C), and Shh (D) were measured using real-time PCR. Expression levels were normalized to those in the untreated EBSs. Shh expression was reduced by all combinations of the treatment with RA alone and activin alone, as well as by RA plus activin (C). Increased amounts of insulin II and amylase 2 were observed in EBSs treated with RA alone, whereas activin alone had no such effect on insulin expression compared with untreated EBSs. Activin alone, however, did induce a decrease in amylase 2 expression (A, B). When the EBSs were treated with a constant concentration of RA (0.1 mM; middle of groups in A, B) and a relatively low concentration of activin (10 ng/ ml), the expression of amylase 2 was much greater in the RA plus activin-treated group than in the untreated group, while the expression of insulin II did not differ between the two groups. In contrast, RA in combination with high activin concentrations (25 ng/ml) induced an approximately 150-fold increase in the expression of insulin II, whereas relatively low levels of amylase 2 expression were observed. The expression patterns of ptf1a/p48 strongly correlated with those of amylase 2.

cells, and duct-like structures from ES cells in vitro by floating cultures of the EBSs treated with both RA and activin. There was a marked increase in the expression of both exocrine and endocrine pancreatic cell marker genes and a decreased expression of the Shh gene, a pattern consistent with the known features of a developing pancreas in vivo. Furthermore, the timing of expression of these marker genes was consistent with previous findings which demonstrated that the increased expression of endocrine marker genes precedes the up-regulation of exocrine marker genes during pancreas development in vivo (Gittes and Rutter, 1992). Ultrastructural analysis identified two morphologically different types of zymogen granule in the treated EBSs: amylase-positive granules that showed features consistent with those found in pancreatic exocrine cells (acinar cells); and the granules that were immunoreactive to anti-C-peptide antibody that are found in pancreatic endocrine cells. Furthermore, a narrow tubular area was observed abutting the

9

A

B

C

D

Amylase/ C-peptide/ DAPI

E

Glucagon/ DAPI

Fig. 7 Effect of activin concentration on the appearance of exocrine and endocrine cells. Pancreas of 8-week-old mouse (A), untreated embryoid body-like spheres (EBSs) (B), and EBSs treated with 0.1 mM retinoic acid (RA) and either 10 ng/ml activin (C) or 25 ng/ ml activin (D, E) were stained with anti-insulin C peptide antibody, anti-amylase antibody and DAPI (4 0 ,6-diamidino-2-phenylindole), 13 days after the end of treatment (19 days after the commencement of EBS formation). No insulin-positive cells or amylase-positive cells were observed in the untreated EBSs (B). In the EBSs treated with 0.1 mM RA and 10 ng/ml activin, there were fewer insulinpositive cells than amylase-positive cells (C). In contrast, EBSs treated with 0.1 mM RA and 25 ng/ml activin markedly increased the insulin-producing cells (D). Moreover, anti-glucagon antibody staining revealed a small but significant number of glucagon-producing cells (E). Scale bar: 50 mm (A–D) and 20 mm (E).

exocrine cells, a structure that resembled the pancreatic ducts surrounded by the interposed area and low columnar epithelium. Immunohistochemical analysis revealed that Pdx-1-positive cells were localized in these duct-like structures. Although Pdx-1 was considered to be localized in nucleus of mature pancreatic cells, it was observed mainly in the cytoplasm and partially in the nucleus of duct-like cells.

This result probably indicates that the immature pancreatic ducts are formed in the present inductive system. RT-PCR and quantitative PCR identified different contributions of RA and activin to the induction of pancreas. Both factors could suppress Shh expression alone or in combination. RA was considered to be essential for the induction of gene expression of pancreatic markers amylase 2, insulin II, glucagon, Pdx-1, and Ppy. In contrast, activin may regulate differentiation and/or the proliferation of pancreatic endocrine and exocrine cells, since both insulin and amylase expression levels changed in an activin concentration-dependent manner in the EBSs treated with RA and activin. Recently, activin A has been reported to induce endoderm differentiation from both human and mouse ES cells (Kubo et al., 2004; D’Amour et al., 2005; Yasunaga et al., 2005). In the present study, the expression level of early endoderm development marker Sox17 was significantly elevated in the EBSs treated with activin A or both activin A and RA than the untreated EBSs or treated with RA alone. Thus, it also showed that activin A has a role for endoderm induction in this experiment. On the other hand, when the concentration of RA was kept constant (0.1 mM) and only the concentration of activin was changed, the low concentrations of activin (10 ng/ml) treatment induced a much higher gene expression level of amylase 2 than that of untreated EBSs, while the expression of insulin II was not different. In contrast, at a higher activin concentration (25 ng/ml), the expression of insulin was markedly increased in the EBSs treated with both activin A and RA than the untreated group, whereas low expression of amylase 2 were observed in the two groups. These results suggested another possible role for activin in the differentiation and/or the proliferation of pancreatic endocrine and exocrine cells, concomitant with RA. It is true that the pancreatic b cells induced by our method occupy a small fraction of total induced tissues compared with other studies (Blyszczuk et al., 2003; Kania et al., 2004; Miyazaki et al., 2004), and that the efficiency of the induction system needs to be improved in this aspect. However, it is important to note that the differentiation system presented here was not designed for the in vitro production of a pure b cell population, but rather for the formation of pancreas-like tissues containing most of all components of intact pancreas. To this end, it was an interesting finding that pancreas-like tissue could be successfully induced from mouse ES cells using a similar treatment regime to that reported by us previously for the induction of pancreas from the presumptive endoderm of frog embryos (Moriya et al., 2000). In conclusion, we have developed a relatively simple approach for inducing the differentiation of ES cells into pancreas-like tissue in vitro, which will provide a good model for analyzing the mechanisms of pancreatic development.

10 Table 1 The percentage of C-peptide-positive areas in the EBSs

RA1activin treated Control

C-peptide-positive area in each EBS (average of positive EBSs, %)

C-peptide-positive area in each EBS (average of total EBSs, %)

Number of EBSs (containing positive signal/total tested)

4.99  0.59 0

1.50  0.78 0

3/10 0/3

To estimate the C-peptide-positive cells in each EBS, serial sections (1 mm thick) of EBSs were made and each section of every 20 mm thickness was stained with anti-C-peptide antibody. Then the C-peptide-positive cells were counted against whole cells in EBs. In the EBSs treated with 0.1 mM RA and 25 ng/ml activin A, three of 10 EBSs contained C-peptide-positive cells. It was found that the C-peptidepositive EBSs, 4.99%  0.59% of total cells were C-peptide positive. RA1activin treated: the EBSs treated with RA (0.1 mM) and activin (25 ng/ml). EBSs, embryoid body-like spheres; RA, retinoic acid.

Acknowledgments We thank Dr. D.A. Melton, Howard Hughes Medical Institute, Harvard University, for his kind comment and suggestions throughout this work. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sport, Culture and Technology, and from the Ministry of Health, Labor and Welfare of Japan.

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