MATERIALS AND METHODS

Cell lines, cell culture and RNA isolation

            The human ES cell lines were derived from the inner cell mass of blastocyststage embryos.  The ES cell lines are all capable of both somatic and extraembryonic (trophoblast) differentiation.  The human EC cell lines, NTERA2, 1777N, 2102Ep, 833KE, GCT27 and TERA1, have all been derived from testicular teratocarcinomas, while NCCIT was derived from an extragonadal germ cell tumor.  NTERA2, GCT27, 1777N, and NCCIT  have all been reported to be capable of significant somatic and/ or extra-embryonic differentiation.  In contrast, 2102Ep, 833KE, and TERA1 no longer show significant somatic or extra-embryonic differentiation.  Of these, 2102Ep and 833KE form xenograft tumors with a pure EC histology in nu/nu athymic mice.  Xenograft tumors of TERA1 have not been reported.  1411H and GCT44 were both derived from human testicular germ cell tumors but exhibit the features of yolk sac carcinoma cells rather than human EC cells.  Both 1411H and GCT44 lack the expression of typical EC marker antigens and form endodermal sinus tumors when grown as xenografts in nu/nu athymic mice.  Primary tissue samples were clinical samples from Stanford University Medical School.  The 17 somatic cell lines are described in the supplementary data (Table 4). 

The five human embryonic stem cell lines were grown as previously described (1).  The human EC cell lines, NTERA2, NCITT, 2102EP, TERA1, 833KE, and 1777N (1777Nrpmet), were maintained by growth in DMEM (Invitrogen) supplemented with 10% fetal calf serum (Gibco, Origin: United States of America) and 2mM L-glutamine (DMEM/FCS) in a 37^oC humidified atmosphere of 10% CO₂ in air. NTERA2 were passaged by scraping with sterile 3mm glass beads (Phillip-Harris Scientific) and were then seeded onto fresh tissue culture plastic (Nunc).  All other cell lines were harvested by treatment with 1ml 0.25% trypsin (w/v):1mM EDTA (Invitrogen) per T75 flask for 5 minutes at 37^oC, harvested with DMEM/FCS, centrifuged, and then resuspended in DMEM/FCS onto fresh tissue culture plastic (Nunc).

GCT44 and GCT27 were grown in 80% DMEM (Invitrogen), 20% defined FBS (Hyclone), with 1mM l-glutamine (Invitrogen), 0.1mM b-mercaptoethanol (Sigma), 1% nonessential amino acids stock (Invitrogen,) in a 37°C humidified incubator with 5%CO₂ in air. Cultures were passaged on mitotically inactivated MEF feeder layers as cells became confluent by incubation at 37°C by treatment with 3ml 0.25% trypsin (w/v):1mM EDTA (Invitrogen) per T75 flask for 5 minutes at 37^oC, harvested with DMEM/FBS, centrifuged, and then resuspended in DMEM/FBS onto fresh tissue culture plastic (Falcon). After several passages, the GCT44 cell line was removed from feeder layers, and several passages later RNA was extracted.

To harvest cells for total RNA extraction, the EC cells were washed once with PBS, harvested by scraping with 3mm glass beads and centrifuged to produce a pellet.   ES cells were separated from the MEF feeder layer by adding 1 ml of 1mg/ml collagenase IV (Invitrogen) to each well of a six-well plate and incubated approximately 10-15 minutes at 37°C in a humidified chamber until colonies began to pull away from the feeder layers. At this time, 0.5ml of 10mg/ml dispase (Invitrogen) in media was added to each culture well and further incubated until colonies became detached and were recovered in the supernatant. Either TRI reagent (Sigma) or RNA-STAT 60 (Tel-Test Inc) was used to isolate the RNA following manufacturers instructions. Subsequently, mRNA was extracted from total RNA with FastTrack mRNA isolation kit (Invitrogen).

Testis tissue samples were flash-frozen on dry ice at the time of radical or simple orchiectomy and stored at –80°C. Histological characterization and purity of all samples was verified by frozen section prior to RNA extraction. Poly-A mRNA extraction and purification were performed according to previously published protocols ((2), The Brown Lab: http://brownlab.stanford.edu).

 

Microarray procedure and data analysis

            Microarray procedures are as described previously (2). Briefly, for each hybridization, 2µg of purified mRNA from each tissue sample was reverse-transcribed, labeled with fluorescence-tagged nucleotides, and hybridized against 1.5µg of a common reference pool of mRNA for 14-18 hours at 65ºC, on cDNA microarrays containing over 44,000 elements, including characterized genes and expressed sequence tags. After several washes, microarrays were scanned with a GenePix 4000 microarray scanner (Axon Instruments, Union City, CA) and were analyzed with Genepix 3.0 software.  Upon visual inspection, spots of insufficient quality were excluded from further analysis.  Data files containing fluorescence ratios were entered into the Stanford Microarray database (http://genome-www4.stanford.edu/Microarray/SMD/index.html).  Hierarchical cluster analysis was performed.  Only those spots with a spot flag of zero were included.  Each spot chosen was required to have a signal over background of at least 1.5 for either the sample or the reference.  We selected genes that had absolute value of log (base 2) normalized R/G greater than or equal to 1.59 from the mean (or median) expression values across the sample set.  Genes with less than 75% well-measured spots were excluded.  Hierarchical cluster analysis was done on both the genes and the arrays using the Pearson correlation.  To demonstrate statistical significance of differences in expression, statistical analysis of microarrays (SAM) was performed on the genes selected for cluster analysis.

 

Reference:

1.         Amit, M., Carpenter, M. K., Inokuma, M. S., Chiu, C. P., Harris, C. P., Waknitz, M. A., Itskovitz-Eldor, J. & Thomson, J. A. (2000) Dev Biol 227, 271-278.

 

2.         Perou, C. M., Sorlie, T., Eisen, M. B., van de Rijn, M., Jeffrey, S. S., Rees, C. A., Pollack, J. R., Ross, D. T., Johnsen, H., Akslen, L. A., et al. (2000) Nature 406, 747-752.