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First published online December 6, 2007
Stem Cells Vol. 26 No. 2 February 2008, pp. 474 -484
doi:10.1634/stemcells.2007-0303; www.StemCells.com
© 2008 AlphaMed Press

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EMBRYONIC STEM CELLS

Activin Alters the Kinetics of Endoderm Induction in Embryonic Stem Cells Cultured on Collagen Gels

Natesh Parashuramaa,b, Yaakov Nahmiasa, Cheul H. Choa, Daan van Polla, Arno W. Tillesa, François Berthiaumea, Martin L. Yarmusha,b

aThe Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA;
bDepartment of Chemical and Biochemical Engineering, Rutgers, the State University of New Jersey, Piscataway, New Jersey, USA

Key Words. Activin • Endoderm • Collagen gel • Embryonic stem cells (mouse) • Follistatin • Epiblast

Correspondence: Correspondence: Martin L. Yarmush, M.D., Ph.D., The Center for Engineering in Medicine, 114 16th Street, Room 1402, Charlestown, Massachusetts 02129-4404, USA. Telephone: 617-371-4882 or 617-726-3474; Fax: 617-573-9471; e-mail: ireis{at}sbi.org

Received on April 24, 2007; accepted for publication on November 23, 2007.

First published online in STEM CELLS EXPRESS  December 6, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic stem cell-derived endoderm is critical for the development of cellular therapies for the treatment of disease such as diabetes, liver cirrhosis, or pulmonary emphysema. Here, we describe a novel approach to induce endoderm from mouse embryonic stem (mES) cells using fibronectin-coated collagen gels. This technique results in a homogeneous endoderm-like cell population, demonstrating endoderm-specific gene and protein expression, which remains committed following in vivo transplantation. In this system, activin, normally an endoderm inducer, caused an 80% decrease in the Foxa2-positive endoderm fraction, whereas follistatin increased the Foxa2-positive endoderm fraction to 78%. Our work suggests that activin delays the induction of endoderm through its transient precursors, the epiblast and mesendoderm. Long-term differentiation displays a twofold reduction in hepatic gene expression and threefold reduction in hepatic protein expression of activin-treated cells compared with follistatin-treated cells. Moreover, subcutaneous transplantation of activin-treated cells in a syngeneic mouse generated a heterogeneous teratoma-like mass, suggesting that these were a more primitive population. In contrast, follistatin-treated cells resulted in an encapsulated epithelial-like mass, suggesting that these cells remained committed to the endoderm lineage. In conclusion, we demonstrate a novel technique to induce the direct differentiation of endoderm from mES cells without cell sorting. In addition, our work suggests a new role for activin in induction of the precursors to endoderm and a new endoderm-enrichment technique using follistatin.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic stem cell-derived endoderm progenitors offer a remarkable potential for the treatment of major diseases affecting the pancreas, liver, lungs, bladder, and prostate [1, 2]. Endoderm progenitors would also be useful for the study of congenital diseases [3], gene discovery, toxicology screening, and drug development [4]. Current knowledge regarding the development of the definitive endoderm, one of three major germ layers, is derived from in vivo fate mapping [5–7], mouse genetics [8], in vitro tissue explants [9], and recent microarray analysis of prospective endoderm [10]. These studies indicate that the endoderm is derived from the inner cell mass through the differentiation of the transient epiblast and mesendoderm (anterior primitive streak) populations [11]. Each of these populations express unique markers or transcription factors. For example, the epiblast expresses Oct4 and Fgf5, mesendoderm expresses Brachyury and Foxa2 [12], and endoderm expresses Foxa2 and Sox 17 [13, 14]. The ability to identify and control these transient precursor populations is a major goal of the field [15, 16]. Although endoderm induction has not been studied extensively, several recent studies have focused on ideal culture conditions for hepatocyte induction, an important product of endoderm differentiation [17–21].

Endoderm induction has been shown to be controlled by soluble factors such as activin-nodal-transforming growth factor (TGF) β, bone morphogenetic protein (BMP), and Wnt [22–27]. Recent studies demonstrated that definitive endoderm can be derived from a mesendoderm precursor using serum-free medium, activin, and serial cell sorting [15, 28]. Activin, which binds to the same receptor as nodal [29], has been shown to generate different tissues as a function of concentration in both Xenopus and mouse embryonic stem (mES) cell studies and has therefore been classified as a potent developmental morphogen [30]. However, recent studies suggest that although activin enhanced endoderm in embryoid body (EB) cultures, it failed to do so in monolayer culture [10]. Furthermore, in human embryonic stem (ES) cells, activin-nodal signaling was shown to inhibit ES cell differentiation, rather than induce endoderm [31–34]. A similar mechanism was shown to inhibit the differentiation of inner cell mass cells in ex vivo mouse blastocyst cultures [32], suggesting a complex role of activin-nodal signaling in ES cell differentiation. The role that follistatin plays in in vitro studies is unclear [35]. However, recent studies suggest that follistatin has an important role during liver regeneration [36] and pancreas differentiation [37].

Studies thus far have relied on multiple cell sorting, complex serum-free formulations, and multiple growth factors for the differentiation of endoderm. Here, we describe a simple cell culture system that induces the differentiation of mES cells toward endoderm in the presence of serum without cell sorting, growth factors, or hormones. The endoderm-like cell population was positive for endoderm-specific markers Foxa2 and Sox17 and negative for major mesodermal and ectodermal markers by day 10 of culture. Surprisingly, activin caused a dose-dependent decrease in the expression endoderm markers while inducing the expression epiblast and mesendoderm markers, such as Brachyury and Fgf5. On the other hand, the activin inhibitor follistatin increased the Foxa2-positive endoderm fraction to 78.4%, without altering the expression kinetics. Long-term gene and protein expression studies indicated that activin-treated cells had reduced hepatic differentiation potential compared with follistatin and controls. In vivo differentiation of activin-treated cells in a syngeneic mouse model generated a heterogeneous, teratoma-like mass, suggesting a primitive population, whereas follistatin-treated cells generated an encapsulated epithelial-like mass, similar to control. In conclusion, these studies demonstrate a novel technique to induce an endodermal cell population in vitro and suggest an intriguing role of activin in endoderm development.


   METHODS
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Reagents
Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, knockout serum, knockout DMEM, bovine gelatin, and dispase were obtained from Invitrogen Corporation (Carlsbad, CA, http://www.invitrogen.com). Human fibronectin was purchased from BD Biosciences (San Jose, CA, http://www.bdbiosciences.com). Hydrocortisone was obtained from Pfizer, Inc (New York, http://www.pfizer.com). Glucagon and insulin were purchased from Eli Lilly (Indianapolis, http://www.lilly.com/). Oncostatin M, human BMP2, hepatocyte growth factor (HGF), human activin, and mouse follistatin were purchased from R&D Systems Inc. (Minneapolis, http://www.rndsystems.com Systems). ESGRO (recombinant leukemia inhibitory growth factor) was purchased from Chemicon (Temecula, CA, http://www.chemicon.com). Immunofluorescence-grade paraformaldehyde was purchased from Electron Microscopy Sciences (Hatfield, PA, http://www.emsdiasum.com/microscopy). Rabbit anti-mouse Foxa2 antibody was purchased from R&D Systems. Goat anti-mouse Sox17 was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com). Goat anti-mouse albumin was purchased from MP Biomedical (Solon, OH, http://www.mpbio.com). For immunofluorescence studies, normal donkey serum and secondary F(ab)2 antibody fragments, multiple-labeling-grade, were obtained from Jackson Immunoresearch Laboratories (Bar Harbor, ME, http://www.jacksonimmuno.com). Unless otherwise noted, all other chemicals, growth factors, and solutions were purchased from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com).

Embryonic Stem Cell Culture
Undifferentiated mouse D3 ES cells (American Type Culture Collection, Manassas, VA, http://www.atcc.org) were cultured on 0.2% gelatin-coated 75 cm2 tissue culture (T75) flasks, at low dilutions of 1:50, such that small colonies were maintained. Medium was changed daily. Experiments were carried out with cells at passage 30. Undifferentiated mES cells were cultured in knockout DMEM supplemented with 15% knockout serum, L-glutamine (4 mM), penicillin (100 U/ml), streptomycin (100 U/ml), gentamicin (10 mg/ml), ESGRO (1,000 U/ml), and 2-mercaptoethanol (0.1 mM). Proliferating ES cell cultures were maintained in a 5% CO2-humidified incubator at 37°C.

Fibroblast Cell Culture
NIH 3T3 mouse fibroblasts (American Type Culture Collection) were cultured at a 1:10 dilution on T75 flasks in high-glucose DMEM containing 10% FBS, penicillin (100 U/ml), streptomycin (10 mg/ml), and gentamicin (1,000 U/ml), and medium was changed every 2 days.

Fibronectin-Coated Collagen Gels
Type I collagen stock solution was prepared from rat tail tendon as described by Dunn et al. [38]. Collagen gelling solution was prepared by mixing nine parts of collagen stock (1.25 mg/ml) with one part of 10x DMEM on ice. The collagen gelling solution was then added at a volume of 250 µl per well (66 µl/cm2), spread evenly on the dish and incubated at 37°C for 30 minutes for gel formation. Fibronectin dissolved in phosphate-buffered saline (PBS) was then added on top of the gel at a concentration of 3.8 µg per well (1 µg/cm2), and the tissue culture plates were incubated for 1 hour at 37°C to ensure fibronectin adsorption on collagen gel.

mES Cell Differentiation
mES cells were directly seeded at a density of 2 x 104 cells per well (5.2 x 103 cells per cm2) onto fibronectin-coated collagen gels. Basal differentiation medium consisted of high-glucose DMEM supplemented with 10% fetal bovine serum, glutamine (100 U/ml), penicillin (100 U/ml), streptomycin (100 U/ml), and gentamicin (10 mg/ml). For growth factor studies, the medium was augmented with activin (20 or 50 ng/ml) or follistatin (100 ng/ml). Medium was changed every 2 days of culture. Differentiating ES cell cultures were maintained in a 10% CO2-humidified incubator at 37°C.

EB cultures were formed by culturing mES cells using the hanging drop method. ES cells were resuspended in differentiation medium and spotted as 30-µl drops on an inverted lid of a 100-mm dish at a concentration of 30,000 cells per milliliter. Dishes were incubated at 37°C for 48 hours in PBS. EBs were recovered and matured in suspension for 48 hours and then transferred to a 12-well plate.

Late Stage In Vitro ES Cell Maturation
Following 10 days of culture, the endoderm-like cells were harvested from gel culture using dispase digestion (1 mg/ml) and filtered using a sterilized 200-µm nylon mesh. Viability was greater than 92%. The harvested endoderm-like cells were then seeded onto fibronectin-coated collagen gels at a concentration of 5 x 104 cells per well (1.3 x 104 cells per cm2) in C+H medium consisting of high-glucose DMEM supplemented with 10% heat-inactivated fetal bovine serum, penicillin/streptomycin (100 U/ml), hydrocortisone (7.5 g/ml), epidermal growth factor (20 ng/ml), glucagon (14 ng/ml), and insulin (0.5 U/ml). C+H medium was augmented with BMP2 (10 ng/ml), HGF (20 ng/ml), and oncostatin M (20 ng/ml).

Late Stage In Vivo Syngeneic Mouse Transplantation
Female 129 mice (Charles River Laboratories, Boston, http://www.criver.com), syngeneic to D3 ES cells and weighing between 25 and 35 g, were used for this study. The animals were treated in accordance with National Research Council guidelines and the Subcommittee on Research Animal Care and Laboratory Animal Resources of Massachusetts General Hospital. Animals had free access to food and water.

ES cells were recovered using dispase digestion from either EB-plated culture, standard collagen culture, activin-treated culture, or follistatin-treated culture and filtered using a sterilized 200-µm nylon mesh. For support of cellular function, 3 x 106 mES cells were mixed with 3 x 104 mouse NIH 3T3 fibroblasts. Mice were anesthetized with intraperitoneal injections of pentobarbital (70 mg/kg). After shaving and cleaning the site of injection with antiseptic solution, cells were injected subcutaneously using a 28-gauge needle such that a subcutaneous wheal could be identified in the paraspinal region between the sixth and eighth ribs. Permanent stitches were placed to mark the injection site. Two weeks after injection, animals were killed by cervical dislocation, and the transplantation site was excised and fixed in formalin. Abdominal and thoracic cavities were inspected for tumor formation.

H&E Staining
Tissue samples were fixed in buffered formalin (1:10), placed in tissue cassettes, dehydrated, embedded in paraffin, and sectioned at 4 µm. Standard hematoxylin and eosin (H&E) staining was performed. Slides were examined using a Nikon Eclipse 800 upright compound microscope (Nikon, Tokyo, http://www.nikon.com).

Reverse Transcriptase Polymerase Chain Reaction
For each experimental condition, cell lysates were generated using a guanidinium isothiocyanate-based solution (Clontech, Palo Alto, CA, http://www.clontech.com) and stored at –80°C for later use. RNA isolation was conducted using the manufacturer's instructions for the Nucleospin II RNA Kit (Clontech). RNA gels were run using 2% agarose (RNase-free) to ensure that RNA was intact prior to reverse transcriptase polymerase chain reaction (RT-PCR). One-step gene-specific RT-PCR (Qiagen, Hilden, Germany, http://www1.qiagen.com) was performed for gene expression analysis and resolved on a 2% agarose gel. Each reaction represents 10 µg of total RNA per condition. Cycle number was 30 for all genes unless otherwise specified. Cycling conditions were 55°C, 30 seconds; 94°C, 30 seconds; and 72°C, 30 seconds; with a 10-minute extension at 72°C. Thermal cycling was done using the Mastercycler Epigradient X (Eppendorf) with 96-well plates. Gels were imaged using a fluorescent gel scanner (Fluor-S Multi-Imager; Bio-Rad, Hercules, CA, http://www.bio-rad.com). Primers were as follows:

B-Actin (forward [F]), 5'-GAGGGAAATCGTGCGTGA-3'; B-Actin (reverse [R]), 5'-CCAAGAAGGAAGGCTGGAA-3';
Oct4 (F), 5'-GGAAAGCCGACAACAATGA-3'; Oct4 (R), 5'-CAAGCTGATTGGCGAATGT-3';
Fgf5 (F), 5'-GTTCAAGCAGTCCGAGCAA-3'; Fgf5 (R), 5'-TAGGCACAGCAGAGGGATG-3';
Otx2 (F), 5'-GGAAGGGAGGGAAGGTCAT-3'; Otx2 (F), 5'-CAGTCGCACAATCCACACA-3';
Brachyury (F), 5'-AAGAACGGCAGGAGGATGT-3'; Brachyury (R), 5'-GCGAGTCTGGGTGGATGTA-3';
Goosecoid (F), 5'-GCACCGCACCATCTTCA-3'; Goosecoid (R), 5'-GTTCCACTTCTCGGCGTTT-3';
Otx2 (F), 5'-GGAAGGGAGGGAAGGTCAT-3'; Otx2 (R), 5'-CAGTCGCACAATCCACACA-3';
Lhx1 (F), 5'-CTGACACGCACACAACCTG-3'; Lhx1 (R), 5'-GCGGCTCTTCTGCTCAAA-3';
Hnf1β (F), 5'-GCCAGTCGGTTTTACAGCA-3'; Hnf1β (R), 5'-TGGGCTTGGGAGGTGTT-3';
Foxa1 (F), 5'-GCCGCCTTACTCCTACATCTC-3'; Foxa1 (R), 5'-TGCCACCTTGACGAAACA-3';
Foxa2 (F), 5'-ACACGCCAAACCTCCCTAC-3'; Foxa2 (R), 5'-GGGCACCTTGAGAAAGCA-3';
Sox17 (F), 5'-ATCCAACCAGCCCACTGA-3'; Sox 17 (R), 5'-TCGGCAACCGTCAAATG-3';
Alb1 (F), 5'-CCCTGTTGCTGAGACTTGC-3'; Alb1 (R), 5'-TGAGGTGCTTTCTGGGTGT-3';
CK8 (F), 5'-AAACCCGAGATGGGAAGC-3'; CK8 (R), 5'-GCCAGAGGATTAGGGCTG-3';
CK18 (F), 5'-CAAGGTGAAGAGCCTGGAAA; CK18 (R), 5'-AAGTCATCGGCGGCAAG-3';
Pdx1 (F), 5'-ACCTCCTCGTGCCCCTAAT-3'; Pdx (R), 5'-CCTGCTCCTCTCTCCATCT-3';
Ins1 (F), 5'-CAGCAAGCAGGTCATTGTTT-3'; Ins1 (R), 5'-AACGCCAAGGTCTGAAGG-3';
IFABP (F), 5'-TGACAATCACACAGGATGGA-3'; IFABP (R), 5'-TCTCGGACAGCAATCAGC-3';
Gata1 (F), 5'-CACCATCAGGTTCCACAGG-3'; Gata1 (R), 5'-TTGAGGCAGGGTAGAGTGC-3';
Foxf1 (F), 5'-CGTGTGTGATGTGAGGTGAG-3'; Foxf1 (R), 5'-CTCCGTGGCTGGTTTCA-3';
Runx2 (F), 5'-TTCCAGACCAGCAGCACTC-3'; Runx2 (R), 5'-GCCGCCAAACAGACTCAT-3';
Lefty2 (F), 5'-CCGTTGTTCCCATTTCTC-3'; Lefty2 (R), 5'-GGACTGTGCTGTGCTGTGTC-3';
Paraxis (F), 5'-TCCAGAAGCCCAAACCAC-3'; Paraxis (R), 5'-TGCTCACATACTACATTCACACAGA-3';
Ascl1 (F), 5'-TGTGACGCTCTTGCTCCA-3'; Ascl1 (R), 5'-GCTGCCCTCGGTCTATTTC-3';
Pax6 (F), 5'-TGCCCTTCCATCTTTGCT-3'; Pax6 (R), 5'-CCATCTTGCGTGGGTTG-3';
Neurog2 (F), 5'-TGAGCCAGTCACAAAGAAGGT-3'; (R), 5' GCAGGCAGTTCGTGTGAA-3'.

Quantitative Real-Time RT-PCR
Reverse transcription and quantitative PCR was performed using the Superscript III Two-Step qRT-PCR kit (catalog no. 11735-032; Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The RT reaction was run using primer containing a mixture of random-hexamers and dT primers, 500 ng of total RNA template, and master mix containing a bioengineered Moloney murine leukemia virus-RT enzyme, nucleotides, and other components. The reaction mix was incubated at 25°C for 10 minutes and 42°C for 50 minutes, followed by termination at 85°C for 5 minutes and RNase H incubation at 37°C for 20 minutes. Real-time quantitative PCR was performed using the Stratagene (La Jolla, CA, http://www.stratagene.com) MX5000P QPCR machine. Triplicates of 10-µl reactions containing 2 µl of primer mixture (0.2 µM), 10 ng of cDNA template, Rox dye, and SYBR Green master mix containing the Platinum Taq Polymerase were used for all reactions. The cycling temperatures were 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds.

Relative Quantitation of Real-Time PCR Data
Real-time data were analyzed using the Stratagene MX-Pro QPCR software using settings with an amplification-based threshold and adaptive baseline. Melting curves for each reaction were obtained and any reactions without a unique PCR product were not analyzed. Threshold cycles (CT) were determined and used to quantify gene expression using the 2–{Delta}{Delta}CT method [39]. Gene expression was measured relative to a normalizer, β-actin, and calibrated using the day 0 condition for the genes of interest. These data, which were then expressed as relative difference of gene expression on day 24 compared with day 0, were normalized to the untreated condition, termed control. Thus, the activin-treated and the follistatin-treated conditions were normalized to the control condition.

Immunofluorescence
Samples were washed three times with PBS and fixed in 4% electron microscopy (EM)-grade paraformaldehyde for 10 minutes at room temperature. Samples were then washed with PBS, permeabilized for 15 minutes with 0.1% Triton X-100, and blocked for 30 minutes with 1% bovine serum albumin, 5% donkey serum at room temperature followed by staining with primary antibodies at overnight at 4°C. The dilutions of primary antibodies used were as follows: Foxa2, 1:100; Sox 17, 1:100; and albumin, 1:500. After additional washes with PBS, samples were stained with fluorescently tagged secondary antibodies at a dilution of 1:500 for 45 minutes at room temperature and washed three times with PBS. Cells were then imaged using phase-contrast and fluorescent microscopy (Carl Zeiss, Jena, Germany, http://www.zeiss.com) and captured using AxioVert software.

Flow Cytometry
Samples were washed three times with cold PBS, kept on ice, and fixed in 1% EM-grade paraformaldehyde for 15 minutes at 4°C. Samples were then washed with ice-cold PBS, permeabilized for 15 minutes with 0.1% Triton X-100 while cells were vortexing, and then washed twice with cold PBS. Cells were blocked for 30 minutes with 1% bovine serum albumin, 5% donkey serum at room temperature for 30 minutes. Primary antibodies diluted in blocking buffer (described above) were then added for 20 minutes at room temperature, at the same dilution as for immunofluorescence. After additional washes with ice-cold PBS, samples were stained with fluorescently tagged secondary antibodies, at the same dilution as for immunofluorescence, for 45 minutes at room temperature and then washed twice more. Flow cytometry was conducted on a Coulter Epics Altra Flow Cytometer (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com), and raw data were captured using Expo 32 Multicomponent software.


   RESULTS
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Culture of ES Cells on Collagen Gels Selectively Induces Endoderm Gene and Protein Expression
The culture of epithelial cells on collagen gels was previously shown to promote epithelial polarity and function [40]. To determine the ability of the collagen gel culture to induce an endodermal phenotype, we cultured mouse ES cells at a seeding density of 5,000 cells per cm2 on fibronectin-coated collagen gels, in the presence of 10% serum. By day 10, the cells acquired a characteristic epithelial morphology, with bright cell borders reminiscent of endoderm-like cells [41–43] (Fig. 1A). In contrast, mouse ES cells cultured on tissue culture plastic coated with a thin layer of collagen and fibronectin formed a morphologically mixed population with numerous spindle-like cells in addition to the epithelial population. Embryoid body culture outgrowths demonstrated a clear heterogeneous morphology by day 10 (Fig. 1A). To clarify serum effects in our system, we compared our results with the current paradigm of serum-free conditions, as well as low serum conditions. Mouse ES cells were seeded on fibronectin-coated collagen gels in serum-free media previously used by Kubo et al. [28]. The cells failed to adhere or proliferate, suggesting that a critical component was missing in the serum-free formulation (supplemental online Fig. 1A). As a result, we were unable to obtain a sufficient sample for analysis of gene expression. Furthermore, gene expression studies on mES cells cultured in low-serum conditions (1% and 5%) demonstrated an increased Fgf5 expression, suggesting a more primitive phenotype (supplemental online Fig. 1B).


Figure 1
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  		Figure 1. Morphology, gene and protein expression of ES cells cultured on collagen gels. (A): Morphologic comparison of day 10 ES cells cultured on fibronectin-coated collagen gels (gel culture), tissue culture-treated 12-well plate coated with collagen and fibronectin (collagen coat), and embryoid bodies plated on tissue culture plastic (embryoid bodies). Scale bar = 100 µm. (B): Germ layer-specific gene expression measured by reverse transcription-polymerase chain reaction (30 cycles) of ES cells cultured on day 10 in gel culture, collagen coat, and embryoid body configurations. Day 0 was used as a negative control, and day 10 mouse embryonic RNA was used as a positive control. Endoderm (Foxa2 and Sox 17), mesoderm (Foxf1, Runx2, GATA1), and neuroect. (Ascl1) were used to assess germ layers. (C): Immunofluorescence images of nuclear staining (DAPI) and either Foxa2 (red) or Sox17 (green) staining of day 10 ES cells cultured on fibronectin-coated collagen gels (gel culture). Scale bar = 100 µm. (D): Flow cytometric analysis of Foxa2-positive cells of gel-cultured ES cells on day 10. Positive fraction was determined by threshold as shown. Abbreviations: d, days; DAPI, 4,6-diamidino-2-phenylindole; ES, embryonic stem; FITC, fluorescein isothiocyanate; NEUROECT., neuroectoderm.

 
To determine whether the observed morphologies correlated with particular genetic phenotypes, we studied the day 10 populations using RT-PCR. Day 10 mouse embryonic RNA was used for a positive control for all primers. We detected endodermal (Foxa2 and Sox17) but not mesodermal (Foxf1, Runx2, and GATA1) [44–46] genes in all three conditions (Fig. 1B). As expected, only the EB condition expressed transcripts for the ectodermal gene Ascl1 [47]. To determine whether the collagen gel-cultured cells with epithelial-like morphology expressed endoderm-specific markers, we performed immunofluorescence staining for the major endoderm transcription factors Foxa2 and Sox17. We found that nearly all cells with epithelial-like morphology expressed both Foxa2 and Sox 17 (Fig. 1C). To determine the relative fraction of these endoderm-like cells, we used flow cytometry, demonstrating that approximately 53% of the cells were positive for Foxa2 by day 10 of culture (Fig. 1D).

Activin Decreases Endoderm and Increases Epiblast-Specific Gene Expression
Activin/Nodal is a member of the TGFβ superfamily, which has been previously shown to be critical in regulating endoderm formation in vitro [28, 43] and in vivo [48]. To test whether endoderm induction in gel cultures responded to stimulation of the activin/nodal axis, we added activin or follistatin (a soluble inhibitor of activin) to the serum-containing culture medium from day 0 to day 10. The addition of 50 ng/ml activin induced the appearance of elongated, spindle-like cells by day 6 of culture (Fig. 2A). In contrast, follistatin-treated cells appeared to have epithelial-like morphology similar to control (Fig. 2A).


Figure 2
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  		Figure 2. Effects of activin and follistatin (activin inhibitor) on morphology, gene and protein expression of embryonic stem (ES) cells cultured on collagen gels. (A): Morphologic comparison of day 10 ES cells cultured in gel culture treated continuously with activin (20 and 50 ng/ml) or follistatin (100 ng/ml). Scale bar = 100 µm. (B): Germ layer-specific gene expression measured by reverse transcription-polymerase chain reaction (30 cycles; 5 ng of RNA) of ES cells cultured on day 10 in gel culture. Primitive (Oct4 and Fgf5), endoderm (Foxa2 and Sox 17), mesoderm (Foxf1, Runx2, and GATA1), early mesoderm patterning (Lefty2 and Paraxis), and neuroectoderm (Ascl1, Pax6, and Ngn2) markers were used. (C, D): Immunofluorescence images of nuclear staining (DAPI) and either Foxa2 (red) or Sox17 (green) staining of day 10 embryonic stem cells cultured in gel culture, treated with 50 ng/ml activin or 100 ng/ml follistatin. Scale bar = 100 µm. (E, F): Flow cytometric analysis of Foxa2-positive cells of gel-cultured embryonic stem cells on day 10. Positive fraction was determined by threshold as shown. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; NEUROECTO., neuroectoderm.

 
We then analyzed these day 10 populations by RT-PCR for epiblast (Oct4 and Fgf5) [49], endoderm (Foxa2 and Sox 17), mesoderm (GATA1, Runx2, and Foxf1) [44, 45], early mesodermal patterning (Paraxis and Lefty2) [50, 51], and neuroectoderm (Pax6, Ascl1 [Mash1], and Ngn2) markers [47, 52] (Fig. 2B). Surprisingly, the addition of activin (20 and 50 ng/ml) decreased endoderm gene expression in a dose-dependent manner (Fig. 2B), whereas the addition of follistatin (100 ng/ml) did not. Interestingly, this decrease in Foxa2 and Sox17 expression in the activin condition correlated with an increase in expression of the primitive epiblast markers Oct4 and Fgf5. As expected, follistatin had the opposite effect, decreasing Fgf5 expression compared with controls. Neuroectodermal and mesodermal markers were absent in all conditions (data not shown). Mesodermal patterning markers, which are activated prior to mesodermal specification in vivo, showed a dose-dependent increase in gene expression with activin, whereas follistatin induced a downregulation of these genes (Fig. 2B).

Immunofluorescence staining for Foxa2 and Sox17 indicated that the activin-treated spindle-like cells did not express Foxa2 or Sox 17 on the protein level, whereas follistatin-treated epithelial cells had strong Foxa2 and Sox 17 expression (Fig. 2C, 2D). To quantify the percentage of endodermal cells, we performed flow cytometry for Foxa2 on day 10 of culture. Activin-treated cells (50 ng/ml) had approximately 10% Foxa2-positive cells, whereas follistatin-treated cells had approximately 78% Foxa2-positive cells (Fig. 2E). Thus, activin decreased the endodermal fraction by 80%, whereas follistatin caused a 47% increase. These results suggest that activin might delay endoderm induction by maintaining upregulation of early genes for epiblast or mesodermal patterning.

Activin Alters the Gene Expression Kinetics of Epiblast, Mesendoderm, and Endoderm
Previous studies have indicated that the and Oct4-positive, Fgf5-positive epiblast population is a transient population that emerges on embryonic day 2 (E2) and is downregulated by E5 as the cells differentiate into the three major germ layers (ectoderm, mesoderm, endoderm) [53]. Our previous results (Fig. 2B) demonstrated that whereas activin downregulated endoderm genes by day 10 of culture, it also upregulated the early epiblast gene Fgf5. To determine whether the kinetics of the epiblast precursor population is altered in the presence of activin, we analyzed epiblast-specific (Oct4, Fgf5, and Otx2) [54], mesendoderm-specific (Brachyury, Goosecoid, and Lim1) [28, 43], and endoderm-specific (Foxa2, Foxa1, Sox17, and HNF1β) gene expression at days 4, 6, and 10 of culture on collagen gel in the presence of activin (20 and 50 ng/ml) or follistatin (100 ng/ml). Oct4 is a specific marker for the inner cell mass as well as the epiblast, Fgf5 is specific for epiblast, and Otx2 is expressed in epiblast in addition to other tissues during development [55]. Whereas control cells have a decreasing expression of Oct4, Fgf5, and Otx2, activin-treated cells appear to have a constant (20 ng/ml) or increasing (50 ng/ml) expression of the same genes, suggesting an earlier phenotype or delayed differentiation. Similarly to the changes in epiblast expression, markers of mesendoderm differentiation (Brachyury, Goosecoid, and Lim1) [28, 43] were downregulated by day 10 of culture in control and follistatin-treated condition following transient expression but remained upregulated by day 10 of culture in activin-treated cells.

The expression kinetics of the endoderm-specific transcription factors Foxa2 and Sox17 appeared to be similar in all conditions, increasing over time (Fig. 3). However, activin-treated cells appeared to express lower levels of the genes in a dose-dependent manner, whereas follistatin-treated cells expressed relatively higher levels of endodermal expression. Furthermore, increasing the dose of activin from 20 to 50 ng/ml delayed the expression of Foxa1 and diminished expression of HNF1β by day 10 of culture. Taken together, the data suggest that increasing activin concentration may shift the kinetics of epiblast and mesendoderm differentiation from a rapid downregulation to a persistent expression of the same markers under our culture conditions.


Figure 3
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  		Figure 3. Kinetics of epiblast, mesendoderm, and endoderm gene expression in control, activin-treated, and follistatin-treated cells. Embryonic stem cells cultured in gel culture were treated with activin (20 and 50 ng/ml) and follistatin (100 ng/ml). Markers for inner cell mass/epiblast (Oct4, Fgf5, and Otx2), mesendoderm (Brachyury, Goosecoid, and Lim1 homeobox) and endoderm (Foxa2, Sox 17, Foxa1, and HN1β) were used. Gene expression was measured by reverse transcription-polymerase chain reaction (30–35 cycles; 10 ng of RNA) on days 4, 6, and 10. Abbreviation: B-Act, β-Actin.

 
Long-Term Endoderm Differentiation Following Activin Stimulation
The patterning of endoderm into its downstream lineages, such as liver, pancreas, and gut, is subject to complex mechanisms in vivo and as a result is poorly understood [1]. To test the differentiation potential of activin- and follistatin-treated cells, we reseeded day 10 cells in hepatic differentiation medium containing BMP2, HGF, and oncostatin [56] but without follistatin or activin. Analysis of cell morphology on days 14, 18, and 22 demonstrated that the cells originating from all three early-differentiation conditions, control, activin (50 ng/ml), and follistatin (100 ng/ml), acquired an epithelial morphology (Fig. 4A). However, activin-treated cells initially formed small clusters reminiscent of EBs formed by primitive mES cells (Fig. 4A).


Figure 4
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  		Figure 4. Endoderm differentiation of control, activin-treated, and follistatin-treated cells. (A): Morphologic comparison of day 10 control, activin-treated, and follistatin-treated embryonic stem cells recultured on fibronectin-coated collagen gels (gel culture) in the presence of hepatic medium (C+H; details are given in Materials and Methods) with bone morphogenic protein 2 (10 ng/ml), hepatocyte growth factor (20 ng/ml), and oncostatin 20 ng/ml). Scale bar = 100 µm. (B): Gene expression was measured by reverse transcription-polymerase chain reaction (RT-PCR) (35 cycles; 10 ng of RNA) for endoderm (Foxa2 and Sox17), foregut (AFP, Alb, Pdx1, and Ins1), midgut (Cdx2), and hindgut (IFABP) markers. (C): Quantitative RT-PCR gene expression for two hepatic differentiation genes, CK8 and CK18. Data for activin- and follistatin-treated cells were normalized to control condition and are expressed as relative gene expression compared with control. (D): Immunofluorescence images (x10) of nuclear staining (DAPI) and either Foxa2 (red) or albumin (green) staining of day 24 control, activin-treated, or follistatin-treated cells recultured on fibronectin-coated collagen gels, in the presence of hepatic differentiation medium. (E): Flow cytometric analysis of albumin-positive cells differentiated in the presence of hepatic differentiation medium for control, activin, and follistatin. Positive fraction determined by threshold as shown. Abbreviations: AFP, {alpha}-fetoprotein; CK, cytokeratin; d, days; DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; IFABP, intestinal fatty acid binding protein.

 
To determine the extent of endoderm induction and patterning, we analyzed gene expression for endoderm patterning markers following 14 days of late-stage differentiation (day 24 of total culture) by RT-PCR. We tested Foxa2 and Sox17, as well as the foregut marker {alpha}-fetoprotein (AFP), albumin, Pdx1, and insulin [57], the midgut marker Cdx2 [58], and the hindgut marker intestinal fatty acid binding protein (IFABP) [58]. Day 10 mouse embryonic RNA was used for a positive control for all primers. Expression of Foxa2, Sox17, and AFP was similar in all three conditions (Fig. 4B). However, the expression of foregut marker albumin and the expression of the hindgut marker IFABP were inhibited by activin but stimulated by follistatin (Fig. 4B), suggesting that activin might delay hepatic differentiation. To quantify these phenomena, we performed quantitative RT-PCR on two genes known to be markers for hepatic differentiation, cytokeratin 8 (CK8) and CK18 (Fig. 4C). Compared with control, activin-treated cells displayed decreased relative expression of CK8 (0.705 ± 0.106) and CK18 (0.79 ± 0.14). On the other hand, follistatin-treated cells displayed increased relative expression of both CK8 (1.43 ± 0.32) and CK18 (1.71 ± 0.84). The ratio of follistatin to activin expression at day 24 was calculated to be 2.02 ± 0.153 for CK8 and 2.09 ± 0.68 for CK18. We extended the analysis of day 24 samples to examine concomitant differences in protein expression. Immunofluorescence staining for intracellular albumin on day 24 showed higher expression in control and follistatin-treated cells than in activin-treated cells (Fig. 4D). To quantify the changes in albumin, we performed flow cytometric analysis of control, activin-treated, and follistatin-treated cells (Fig. 4E). In control and follistatin conditions, 21.8% and 19.3% of cells were positive for albumin, whereas only 5.9% of the activin-treated cells were albumin-positive. The mean intensity values of the albumin-positive population (Table 1) were 25.1% for control, 30.2% for follistatin, and 15.8% for activin. Taken together with the endpoint PCR data, these data strongly suggest that hepatic differentiation was diminished following activin treatment.


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  		Table 1. Albumin positive fraction and mean intensity of albumin for day 24 cultured cells

 
Long-Term In Vivo Differentiation of Activin- and Follistatin-Treated Cells in a Syngeneic Mouse Transplant Model
The in vitro differentiation data on days 10 and 24 of culture suggest that whereas follistatin induces an endoderm-specific differentiation, activin delays differentiation through the induction of primitive lineages. To assess the differences in potency and commitment between these conditions, we transplanted day 10 cultured ES cells subcutaneously in a syngeneic mouse model. The four conditions transplanted were EB, control (no treatment), activin (50 ng/ml), and follistatin (100 ng/ml). Each cell population was mixed with a low ratio of NIH 3T3 fibroblasts (1:100), for mesenchymal support upon transplantation [59]. All implants gave rise to an observable mass (Fig. 5A) by day 14 of transplant (day 24 total culture). The cell mass was significantly larger in the case of activin-treated cells (Fig. 5B).


Figure 5
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  		Figure 5. Long-term in vivo differentiation of day 10 activin or follistatin-treated cells following subcutaneous transplantation in a syngeneic mouse model. Histological evaluation was carried out following 14 days in vivo. Treated cells are compared to no treatment (Control) as well as embryoid body (EB) cultures. (A): Encapsulated superficial mass demonstrated after incision. (B): Day 24 tissue masses after resection were embryoid body (1), gel culture (2), Follist. (100 ng/ml) (3), and activin (50 ng/ml) (4). Note that activin-treated cells resulted in the largest mass. Scale bar = 1 cm. H&E staining of implanted tissue masses. (C): Staining of gel culture demonstrates uniform epithelial-like cells with spindle-like septae. Scale bar = 100 µm. (D): Activin-treated cells demonstrate heterogeneous, thick, tubule-like structures, and mesenchymal elements. Scale bar = 100 µm. (E): Staining of Follist.-treated cells demonstrates uniform cords of epithelial cells separated by septae. Scale bar = 100 µm. (F): EB cells demonstrate heterogeneous tissue with skin, cartilage, and mesenchymal elements. Scale bar = 100 µm. (G): Low magnification image of the encapsulated gel culture mass. Scale bar = 200 µm. (H): Low magnification image of encapsulated Follist.-treated mass. Scale bar = 200 µm. Abbreviation: Follist., follistatin.

 
The excised tissue was analyzed by histological examination and was reviewed by a blinded pathologist. When cells were recovered from an EB and implanted, they generated a teratoma-like cell mass with extensive extracellular matrix, suggesting immature bone or cartilage, evidence of skin, glandular tube-like structures, and immature cells (Fig. 5F). On the other hand, the endoderm-like cell population differentiated on fibronectin-coated collagen (control) generated an encapsulated tissue consisting of immature and mature epithelial cells, reminiscent of intestine, with intervening mesenchymal-like septae (Fig. 5C, 5G). Importantly, activin-treated cells gave rise to a heterogeneous teratoma-like cell mass with both mesodermal components and tubular structures of neural or epithelial origin (Fig. 5D). Furthermore, follistatin-treated cells generated an encapsulated tissue with epithelial-like morphology, similar to control, and demonstrated cords of cells with intervening mesenchymal septae (Fig. 5E). Both embryoid body and activin cases were not encapsulated and invaded into underlying tissue (data not shown).


   DISCUSSION
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
The induction of endoderm in vivo and in vitro is a complex and dynamic process. Therefore, the generation of a simple ES cell culture system that could address fundamental questions, without the need for complex medium formulation or cell sorting, is a major goal in the field. Our work demonstrates that an endoderm-like cell population can be induced by culture on fibronectin-coated collagen gel, without the use of activin, complex serum-free medium, or serial cell sorting, which were previously thought to be essential for endoderm induction [15, 28]. We demonstrated that primitive epiblast and mesendoderm markers were transiently expressed in both control and follistatin cases (Fig. 3). Since these cell populations are the direct precursors to endoderm, this suggests that definitive endoderm was induced by day 10 of culture on collagen gel. Activation of albumin (foregut), IFABP (midgut), and Cdx2 (hindgut) in subsequent late-stage differentiation (day 24) supports this assertion. In vivo differentiation of the day 10 endoderm-like cells with or without follistatin generated a homogenous population of epithelial cells, with intervening fibrous septae, suggesting that our endoderm-like cells remain committed. To our knowledge, this has yet to be reported in literature. Previously, renal capsule injection of endoderm-enriched populations resulted in heterogeneous groups of endoderm and mesoderm derivatives in mouse and human ES cell models of endoderm induction [28, 60].

One of the surprising results in our studies is that activin, a known endoderm inducer, caused a decrease in endoderm-specific gene and an 80% decrease in protein expression by day 10 of culture. Previous work demonstrated that activin-nodal-TGFβ signaling through Smad2/3 is essential for endoderm induction both in vivo and in vitro [15, 28–30]. However, significant evidence suggests that activin-nodal signaling might inhibit the early stages of ES cell differentiation in vitro [31, 33, 34]. Our results demonstrate that in vivo transplantation of activin-treated cells generates a heterogeneous teratoma-like mass with mesodermal, neural, and epithelial components (Fig. 5D), suggesting that the activin-treated cells were still pluripotent. Gene expression kinetics data (Fig. 3) show that day 10 activin-treated cells remain positive to major epiblast (Oct4, Fgf5, and Otx2) and mesendoderm (Brachyury, Goosecoid, and Lhx1) markers, further strengthening the in vivo results. The activin-treated cells also demonstrated delayed hepatic gene and protein expression, as shown in Figure 4. However, activin-treated cells were still competent to generate endoderm in vitro (Fig. 4) and showed a remarkable proliferative potential in vivo (Fig. 5B). Interestingly, the follistatin-treated population showed a 47% increase in the Foxa2-positive ES cell fraction by day 10, suggesting a role for endogenously secreted activin in ES cell self-renewal [33]. Alternatively, follistatin might interact with other pathways, such as Wnt [61] and BMP [62], to induce differentiation. Additional studies will be needed to determine the role of activin in ES cell differentiation using lineage tracing and proliferation studies.

To summarize, in this work, we established a novel approach to induce a homogenous endoderm-like cell population that demonstrates lineage-appropriate gene and protein expression without resorting to cell sorting. Activin, normally an endoderm inducer, caused a dose-dependent decrease in endoderm induction, associated with an increase in its precursor epiblast population. On the other hand, follistatin, a known activin inhibitor, increased the Foxa2-positive endoderm fraction to 78.4%. These studies demonstrate a novel technique to induce the direct differentiation of endoderm from ES cells in vitro without resorting to cell sorting and a new ability to induce critical, transient precursors that will assist in the development of ES-cell-based cellular therapies.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Drs. Monica Casali, Halong Vu, and Heidi Elmoazzen for experimental materials and advice. We also thank Dr. Kamran Badizadegan of the Massachusetts General Hospital Department of Pathology for histologic assessment of specimens and Robert Crowther for processing of tissue specimens. This work was supported by an NIH Biotechnology Fellowship and by a National Research Service Award.


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 Introduction
 Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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