Fetal Bovine Serum (FBS), Dulbecco’s modified Eagle medium (DMEM), penicillin, streptomycin, knockout serum, knockout DMEM, bovine gelatin, and dispase were obtained from Invitrogen Life Technologies (Carlsbad, CA). Human Fibronectin was purchased from BD Biosciences (San Jose, CA, USA). Hydrocortisone was obtained from Pharmacia (Kalamazoo, MI). Glucagon and Insulin were purchased from Eli-Lilly (Indianapolis, IN). Oncostatin M, Human Bone Morphogenic Protein-2 (BMP2), Hepatocyte Growth Factor (HGF), Human Activin, Mouse Follistatin, were purchased from R&D Systems (Minneapolis, MN). ESGRO (Recombinant Leukemia Inhibitory Growth Factor) was purchased Chemicon (Temecula, CA). Immunofluorescence grade paraformaldehyde was purchased from Electron Microscope Sciences (Hatfield, PA). Rabbit anti-mouse Foxa2 antibody was purchased from R&D Systems (Minneapolis, MN). Goat anti-mouse Sox17 was purchased from Santa Cruz (Santa Cruz, CA). Goat anti-mouse Albumin was purchased from ICN Pharmaceuticals (Aurora, OH). For immunofluorescence studies, normal donkey serum and secondary F(ab)2 antibody fragments, ML grade, were obtained from Jackson Immunoresearch (Bar Harbor, ME). Unless otherwise noted, all other chemicals, growth factors, and solutions were purchased from Sigma-Aldrich Chemicals.
Embryonic stem cell culture
Undifferentiated mouse D3-ES cells (ATCC) were cultured on 0.2% gelatin-coated 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 (ATCC) were cultured at 1:10 dilution on tissue culture-treated T75 flasks in high glucose DMEM containing 10% FBS, Penicillin (100 U/ml), Streptomycin (10 mg/ml), 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
]. Collagen gelling solution was prepared by mixing nine parts of collagen stock (1.25 mg/ml) with one part of 10 × DMEM on ice. The collagen gelling solution was then added at a volume of 250 μl/well (66 μl/cm2
), spread evenly on the dish and incubated at 37°C for 30 min for gel formation. Fibronectin dissolved in PBS was then added on top of the gel, at a concentration at 3.8 μg/well (1 μg/cm2
) and the tissue culture plates were incubated for 1h at 37°C to insure ensure fibronectin adsorption on collagen gel.
Mouse embryonic stem (mES) cell differentiation
Mouse embryonic stem (mES) cells were directly seeded at a density of 2 × 104 cells/well (5.2 × 103 cells/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), Gentamycin (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.
Embryoid Body (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/ml. Dishes were incubated at 37°C for 48h in PBS. EB’s were recovered and matured in suspension for 48h 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 × 104 cells/well (1.3 × 104 cells/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 Bone Morphogenic Protein-2 (BMP2) (10 ng/ml), Hepatocyte Growth Factor (HGF) (20 ng/ml) and Oncostatin M (OSM) (20 ng/ml).
Late stage in vivo syngeneic mouse transplantation
Female 129 mice, (Charles River Laboratories, Boston, MA), syngeneic to D3 ES cells, and weighing between 25 and 35 g were used for this study. The animals were cared for in accordance with the guidelines set forth by the Committee on Laboratory Resources, National Institutes of Health, and 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, or follistatin-treated cultures and filtered using a sterilized 200 μm nylon mesh. For support of cellular function, 3×106 mES cells were mixed with 3×104 mouse NIH 3T3 fibroblasts. Mice were anesthetized with intraperitoneal injections of pentobarbitol (70mg/kg). After shaving and cleaning the site of injection with antiseptic solution, cells were injected subcutaneously such that a subcutaneous wheal could be identified in the paraspinal region between the 6th and 8th rib using a 28-guage needle. 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.
Tissue samples were fixed in buffered formalin (1:10) and placed in tissue cassettes, dehydrated, embedded in paraffin, and sectioned at 4μm. Standard H&E (Hematoxylin and Eosin) staining was performed. Slides were examined using a Nikon Eclipse 800 upright compound microscope and analyzed using Advanced Spot 2 software (Molecular Dynamics).
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
For each experimental condition, cell lysates were generated using Gaunidinium Isothiocyanate-based solution (Clonetech) and stored at −80°C for later use. RNA isolation was conducted using the manufacturer’s instructions for the Nucleospin II RNA Kit (Clonetech). RNA gels were run using 2% Agarose (RNAase free) to ensure that RNA was intact prior to RT-PCR. One-step gene-specific RT-PCR (Qiagen) was performed for gene expression analysis and resolved on a 2% agarose gel. Each reaction represents 10 micrograms of total RNA per condition. Cycle number was 30 for all genes unless otherwise specified. Cycling conditions were 55°C, 30 sec, 94°C, 30 sec, and 72°C 30 sec, with a ten minute extension at 72°C. Thermal cycling was done using the Mastercycler Epigradient X (Eppendorf) with 96 well plates Gels were imaged using fluorescent gel scanner, the Fluor-S Multi-Imager (BioRad) and captured using Multi-analyst software. Primers were as follows:
B-Actin (F) 5′-GAGGGAAATCGTGCGTGA-3′; B-Actin (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′-CAG-TCGCACAATCCACACA-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′
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 qPCR was performed using the Superscript III Two-Step qRT-PCR kit (In Vitrogen, Catalog No 11735-032). The RT reaction was run using primer containing a mixture of random-hexamers and dT primers, 500ng of total RNA template, and master mix containing a bioengineered MMLV-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 min and RNAaseH incubation at 37°C for 20 minutes. Real-time quantitative PCR was performed using the Stratagene 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 30s, 57°C for 30s, 72°C for 30s.
Relative Quantitation of Real-Time PCR data
Real time data was 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 cycle (CT) were determined and used to quantify gene expression using the 2−ΔΔCt
]. Gene expression was measured relative to a normalizer, β-Actin, and calibrated using the day 0 condition for the genes of interest. This data, which was then expressed as relative difference of gene expression on day 24 compared to day 0, was normalized to the untreated condition, termed control. Thus the activin-treated and the follistatin treated conditions were normalized to the control condition.
Samples were washed three times with PBS, and fixed in 4% EM-grade paraformaldehyde for 10 min at room temperature. Samples were then washed with PBS and were permeabilized for 15 min with 0.1% Triton X-100, blocked for 30 min 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 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 min at room temperature and washed three times with PBS. Cells were then imaged using phase contrast and fluorescent microscopy (Ziess) and captured using AxioVert software.
Samples were washed three times with cold PBS, kept on ice, and fixed in 1% EM-grade paraformaldehyde for 15 min at 4° C. Samples were then washed with ice cold PBS and were permeabilized for 15 min with 0.1% Triton X-100 while cells were vortexing, and then washed twice with cold PBS. Cells were blocked for 30 min with 1% bovine serum albumin, 5% donkey serum at room temperature for 30 min. Primary antibodies diluted in blocking buffer (above) were then added for 20 min at room temperature, at the same dilution as for immunoflourescence. After additional washes with ice cold PBS, samples were stained with fluorescently tagged secondary antibodies, at the same dilution as immunoflourescence, for 45 min at room temperature, and then washed twice again. Flow cytometry was conducted on a Coulter Epics Altra Flow Cytometer (Beckman Coulter) and raw data were captured using Expo 32 Multicomponent software.