Human embryonic stem cell lines I3, I6, and BG01V used in this study were cultured on mitomycin C-treated mouse embryonic fibroblasts CF-1 (ATCC, SCRC-1040.2; http://www.atcc.org). Cells were cultured at 37°C, in a 5% CO2 atmosphere, in the ES medium of Dulbecco's modified Eagle's medium (D-MEM)/F12 (ATCC 30-2006)80%, supplemented with 2.0 mM L-alanyl-L-glutamine (ATCC 30-2115), 0.1 mM non-essential amino acids (ATCC 30-2116), 0.1 mM 2-mercaptoethanol (Sigma Catalog No. M-7522) and 4 ng/ml basic fibroblast growth factor (bFGF; R & D Systems Catalog No. 233-FB), 5%; Knockout serum replacement (Invitrogen Catalog No. 10828), 15%; fetal bovine serum (ATCC SCRR-30-2020), penicillin (100 I.U./mL) and streptomycin (100 μg/mL) (ATCC 30-2300). An additional 4 ng/ml of bFGF was added in the first 24 hours after thawing the cells. Daily medium changes began after the first 48 hours in culture. The BG01V colony formation was visible within 2–3 days and the other two cell line's colony formation was observed in 3–4 days. Cells were passaged every 4–5 days using collagenase IV (200 Units/mL) (Invitrogen Corporation) for BG01V, I6 was passaged every 6–7 days and I3 was passaged every 7–8 days.
Embryoid body formation
hESCs in culture were removed from feeder cells using collagenase IV (200 Units/mL) (Invitrogen Corporation; http://www.invitrogen.com). hESC clusters were transferred to 10 × 10-cm ultra-low-attachment dishes (corning; http://www.corning.com) and cultured in medium D-MEM/F12 (80%) (ATCC 30-2006) supplemented with ES-Qualified FBS (15%) (ATCC SCRC-30-2020), knockout serum replacement (KSR) (5%) (Invitrogen Corporation), L-alanyl-L-glutamine (2.0 mM) (ATCC 30-2115), non-essential amino acids (1×) (ATCC 30-2116), β-mercaptoethanol (0.1 mM) (Invitrogen Corporation), penicillin (100 I.U./mL)/streptomycin (100 μg/mL) (ATCC 30-2300). The medium was changed every second day. To evaluate the growth rates of EBs, phase-contrast photographs of EBs were taken and the total areas of EBs were measured using Scion Image. The percent increase in total areas of the cell spheroids was compared between different cell lines. Data were calculated as mean ± S.E.M. of at least 3 separate cultures. The statistical significance was determined using the Student's t-test with p < 0.05 considered significant.
Directed neural differentiation of hESCs
The directed neural differentiation method was described previously . Briefly, colonies of the three hESC lines I3, I6, BG01V were removed from MEF feeders and dissociated into small clumps by incubating with collagenase IV (200 Units/ml) (Invitrogen Carlsbad, CA; http://www.invitrogen.com) at 37°C for 35 minutes. The hESC clumps were pelleted and cultured in suspension in low attachment dishes with hESC medium without bFGF for 5 days (the end of this stage is considered as 5 days of differentiation). hESC grew into floating aggregates or embryoid bodies (EBs). The neuroectodermal induction began with EBs transferred into the neural differentiation medium (NDM) that consisted of two parts of a modified Eagle's medium (ATCC 30-2002; http://www.atcc.org), one part F12k- (ATCC 30-2004), 1× N-2 supplement (Gibco Catalog No. 317740; http://www.invitrogen.com), 0.1 mM non-essential amino acids (ATCC 30-2116), penicillin (100 IU/ml)/streptomycin (100 μg/ml) (ATCC 30-2300) and 5 ng/ml bFGF (R& D Systems Catalog No. 233-FB) for 10 days. At days 15–17 of differentiation, EBs were plated on PDL/laminin substrate-coated 35 mm dishes (corning; http://www.corning.com). Although some neural rosettes were formed in floating embryoid bodies (EBs), increased rosettes were visualized after plating of the EBs on substrates. Neuroectodermal cells in rosettes were further differentiated into neural progenitors and their progeny on PDL/laminin substrates.
To compare the growth rate between I3, I6 and BG01V, all three hES cells were plated into 6 well plates containing a feeder layer of mitomycin C-treated fibroblast (MEF). The cells were cultured at 37°C in a 5% CO2 atomosphere. Basic fibroblast growth factor (4 ng/ml) was added to each cell culture after the first 24 hours. The medium was changed daily after 48 hours. The cells from three separate wells were harvested using a 0.25% (w/v) trypsin/0.53 mM EDTA solution (ATCC cat # 30-2101) each day. The cell counts were performed using Cedex Analysis System, Innovatis. Data were calculated as mean ± S.E.M. of at least 3 separate cultures. The statistical significance was determined using the Student's t-test with p < 0.05 considered significant.
Bromodeoxyuridine (BrdU) incorporation and counterstaining with propidium iodide (PI)
To monitor cell proliferation within colonies of hES cells, bromodeoxyuridine (BrdU) incorporation with 5-bromo-2-deoxy-uridine Labeling and Detection Kit I (Roche, Indianapolis, IN; http://www.roche.com) was used as described previously . Briefly, cultures were exposed to 20 μM BrdU for 4 hours and then fixed with 70% alcohol containing 50 mM glycine at PH 2.0. After rinsing with the kit wash buffer, cells were incubated overnight with mouse anti-BrdU (1:1000) followed by incubation with FITC-conjugated donkey anti-mouse IgG (1:50) (Jackson Immunological Research, West Grove, PA) for 45 min. Some cultures that were not exposed to BrdU were used as negative controls which showed no immunoreactivity, demonstrating the specificity of BrdU antibody. In order to quantify the cell proliferation rate, cell nuclei were counterstained by the addition of 5 μg/ml propidium iodide (PI) for 10 min. PI+ and BrdU+ cells were examined and photographed with Nikon eclipse TE 300 microscope. The proliferation index was defined as the percentage of BrdU+ nuclei in the total number of PI+ cells. At least 5 labeled colonies were counted from each dish and three dishes were evaluated. Data were calculated as mean ± S.E.M. which were statistical significance determined by using the Student's t-test with p < 0.05 considered significant.
For the immunostaining of undifferentiated hES colonies for "stemness" markers, undifferentiated hESCs were cultured on mitomycin C-treated feeder cells in 35 mm tissue culture dishes (Corning, Corning, NY, http://www.corning.com). Colonies were rinsed twice before fixation with 4% paraformaldehyde (EMS, Hatfield, PA, http://www.emsdiasum.com) in 1× PBS for 15 min at room temperature. Cells were permeabilized with 0.5% saponin (Sigma, St. Louis, MO, http://www.sigma-aldrich.com) in PBS for 10 min. Primary antibodies against Oct3 (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com, 1:250), NANOG (1:100), and SSEA4 (1:100), and TRA-1-81 (1:50) (all from Millipore, Billerica, MA http://www.millipore.com) were incubated with colonies overnight at 4°C. The secondary antibodies used were either, Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L), (Invitrogen/Molecular Probes, Eugene, OR, http://www.invitrogen.com; 1:50), or FITC-conjugated donkey anti-Mouse IgM (Jackson ImmunoResearch, West Grove, PA, http://www.jacksonimmuno.com, 1:50), or Alexa flour 488-conjugated rabbit anti-goat IgG1 (Invitrogen/Molecular Probes, Eugene, OR, http://www.invitrogen.com). Colonies were incubated with secondary antibodies for 45 min at room temperature.
Immunostaining of hESC-derived neural cells for Nestin and TuJ1 were performed as described previously . Briefly, neural differentiation medium-treated EB were plated on PDL/laminin coated 35 mm Tissue Culture plates (Corning, Corning, NY, http://www.corning.com). Differentiated cells were fixed with 4% paraformaldehyde and permeabilized in 0.5% saponin as described above. Primary antibodies used were rabbit anti-nestin, 1:200, mouse anti-tubulin clone TUJ-1, 1:300, chicken anti-SOX1, 1:200, (all from Millipore, Billerica, MA http://www.millipore.com). Secondary antibodies used were either rhodamine-conjugated donkey anti- rabbit IgG-(H+L) (Jackson Immunoresearch, West Grove, PA; http://www.jacksonimmuno.com), Alexa Fluor 488 conjugated goat anti-mouse- IgG (H+L) (Molecular Probes, Eugene, Oregon; http://www.invitrogen.com) or FITC-conjugated donkey anti-chicken IgG (Millipore, Billerica, MA http://www.millipore.com, 1:50). Cells were counterstained with the 4'-6-diamidino-2-phenylindole (DAPI) with dilution of 1:1000 (Sigma; http://www.sigmaaldrich.com). Immunofluorescence signals were observed and photographed with a Nikon TE 300 epifluorescence microscope (Nikon, Inc. Melville, NY) equipped with a Qicam FAST1394 digital camera (Surrey, BC, Canada) and Openlab vs. 4.0.4 software http://www.improvision.com.
To quantify the percentage of hESC-differentiated neural progenitors, cell counting was performed on cultures immunostained for nestin, together with nuclear DAPI counterstaining, in 35 mm culture dishes coated with different laminin substrates from at least three independent experiments. All data were expressed as mean ± SEM, and Student's t test was used for statistical evaluation. In all instances p < 0.05 was considered statistically significant.
Alkaline Phosphatase staining
Endogenous alkaline phosphatase activity in BG01V, I3 and I6 cells was detected using the ELF® 97 Endogenous Alkaline Phosphatase Detection Kit (ATCC catalog # SCRR-3010) according to the manufacturer's instructions. Cells cultured in 6 well plates (Corning Life Sciences; http://www.corning.com) were treated with 4% paraformaldehyde for 15 minutes at room temperature. The cells were washed with 1× PBS, treated with 0.2% Tween-20 for 10 minutes at room temperature and rinsed with 1× PBS. Fixed cells were then incubated with a filtered 1:20 dilution of the phosphatase substrate in situ, and the reaction was monitored using an epifluorescence microscope. The reaction was terminated using a stop solution consisting of PBS, 25 mM EDTA, 5 mM levamisole, pH 8.0. Cells were rinsed with PBS before mounting on glass microscope slides.
Total RNA was isolated from three hESC lines I3, I6, and BG01V using an RNAeasy Plus Mini kit (Qiagen catalog NO 74134; http://www1.qiagen.com/). The isolated RNAs were quantified using a RNA 6000 Nano Kit (Catalog NO 5067-1511). The integrity of RNA was checked on Agilent 2100 Bioanalyzer (Agilent Technologies; http://www.chem.agilent.com part No G2940CA). Equal amounts of RNA (1 μg) was taken for all samples and reverse transcription was done using RT2 First Strand kit from Superarray Biosciences (SuperArray, catalog No C-03; http://www.superarray.com. The total volume of the reaction was 20 μL and was diluted to 100 μL. PCR reactions were performed using a ABI Fast 7900 using RT2 Real-Time™ SYBR Green PCR master mix PA-012 and qRT-PCR primers from SuperArrray Biosciences. The total volume of the PCR reaction was 10 μL. The qRT-PCR Primers sets catalog numbers are 18srRNA PPH00073A, B-actin PPH05666A, POU5F1, Nanog, UTF1 (undifferentiated) PPH02394A, PPH02391A and PPH17032A, keratin C and NEFL (ectoderm) PPH-21369A and PPH02430A, alpha-globin and Beta-globin (Mesoderm) PPH09054A and PPH12971A, alpha-1AT PPH02413A, nestin and Musashi 1, SOX1 (Neural Progenitor) PPH02388A, PPH13090A and PPH02390A, MAP2, GAD-65 (Neural) PPH02419A, PPHo5950A, S100B and GFAP (astrocytes) PPH02408A and PPH02472A; http://www.superarray.com. The thermocycler parameters were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Each gene for each sample was run in quadruplicate. Relative changes in gene expression were calculated using the ΔΔCt (threshold cycle) method. This method first subtracts the ct (Threshold cycle number) of the gene-avg ct of the two house keeping genes (18srRNA and ACTB) to normalize to the RNA amounts. Finally, the delta delta ct is calculated by subtracting the normalized average ct of the treated cells from the normalized average ct of the undifferentiated cells. Then this delta ct is raised to the negative power of 2 in order to calculate the fold change .
Massively Parallel Signature Sequencing
Massively parallel signature sequencing (MPSS) was performed using 1–2 μg purified total RNA from each of the three human ESC lines (BG01V, I3 and I6) from undifferentiated cells (day 0) and cells at different stages of differentiation (days 7, 14 and 21); the presence and absence of ESC markers and markers of differentiation were evaluated. The quality of total RNAs was evaluated using an Agilent Bioanalyzer. mRNA isolation was processed according to the MPSS protocol as described previously  with some modification. In brief, the mRNA was reverse-transcribed, the cDNA synthesized and digested with Dpn II, then GEX adaptors ligated with Dpn II and amplified by PCR, the cDNA library was ready to sequence. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparison between samples. Only reliable and annotatable signatures against updated human signature database were considered for further analysis.
To generate a complete, annotated human signature database, all the possible signatures from the human genome sequence, the human UniGene sequences, and human mitochondrion were extracted. Each virtual signature was ranked based on its position and orientation in the original sequence. The annotation database is established based on the virtual signatures, their classes and their corresponding genes so that each signature only has one corresponding annotation. The database is then used to annotate the data from the experiment.