Undifferentiated growth of hES cells on MEF feeder and MEF-conditioned medium
Human embryonic stem cell line hES-T3, which is one of the five hES cell lines derived in our laboratory with institutional review board approval and informed consent by couples undergoing IFV treatment in Taiwan , exhibits normal female karyotype (46, XX), and it has been continuously cultured on mitomycin C (10 ug/ml) mitotically inactivated MEF feeder in hES medium under 5% CO2 at 37°C and underwent freezing/thawing processes. The hES culture medium consisted of DMEM/F12 (1:1, GIBCO) supplemented with 20% KSR (Invitrogen), 1% non-essential amino acids, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 4 ng/ml human basic fibroblast growth factor (bFGF; Life Technologies). Routine passages of hES-T3 cells every 5-7 days were done with collagenase (type IV, 1 mg/ml, Invitrogen) treatment and mechanical scrape [4, 5]. The cryopreserved stock of hES-T3 cells (36 passages) were continuously maintained on MEF feeder for additional 14 passages, and these the hES-T3 cells were designated as T3/MEF .
The MEF cells were cultured in MEF medium overnight, and the mitotically inactivated MEF cells were maintained in hES medium containing 4 ng/ml bFGF. After 24 h, the MEF-conditioned medium was collected and filtered through 0.2 um membrane (PN4612, Pall Life Sciences) as previously described . The culture dish was coated with Matrigel diluted with DMEM/F12 (1:30) overnight at 4°C. The cryopreserved stock of hES-T3 cells (36 passages) were continuously maintained on feeder-free Matrigel-coated dish in MEF-conditioned medium (with additional 4 ng/ml bFGF) for 12 passages, and these hES-T3 cells were designated as T3/CMMEF .
Establishment of human hES-T3 differentiated fibroblast-like cells
Autogeneic feeder cells with capacity to support the growth of undifferentiated hES cells were established according to the previously published procedure . hES-T3 (passage 19) cells were transferred into feeder-free and noncoated plate (10 cm) in DMEM supplemented with 10% FBS (GIBCO) under 5% CO2 at 37°C. After 10 days, cells appeared as fibroblast-like morphology, that is, flat cells with elongated nucleus and branching pseudopodia. These hES-T3 differentiated fibroblast-like cells are designated as T3HDF. The expression of transcription factors OCT4, SOX2 and NANOG, which were highly expressed in T3/MEF cells, was shown to be down-regulated in differentiated T3HDF cells. The expression profiles of mRNAs and miRNAs between T3/MEF and T3HDF cells were also found to very different . These T3HDF cells were passaged using trypsin (0.05%, GIBCO) every 4 days or cryopreserved.
Undifferentiated growth of hES cells on T3HDF feeder and T3HDF-conditioned medium
The differentiated fibroblast-like T3HDF cells (passage 8) were inactivated using mitomycin C (10 ug/ml) and used as autogeneic feeder layer in hES medium to maintain the continuously undifferentiated growth of hES-T3 cells (36 passages on MEF) for additional 14 passages . These hES-T3 cells grown on T3HDF feeder were designated as T3/HDF.
The T3HDF cells were cultured in DMEM medium overnight, and the mitotically inactivated T3HDF were maintained in hES medium containing 4 ng/ml bFGF. After 24 h, the T3HDF-conditioned medium was collected and filtered through 0.2 um membrane . The culture dish was coated with Matrigel diluted with DMEM/F12 (1:30) overnight at 4°C. The hES-T3 cells (36 passages on MEF feeder) were first grown on T3HDF feeder for 4 passages and then on Matrigel in T3HDF-conditioned medium for additional 4 passages. The hES-T3 cells grown on feeder-free Matrigel-coated dish in T3HDF-conditioned medium (with additional 4 ng/ml bFGF) were designated as T3/CMHDF
Staining of OCT4 and NANOG
T3/HDF and T3/CMHDF, as well as T3/MEF and T3/CMMEF, colonies were fixed by 4% paraformaldehyde and permeabilized using 0.5% Triton X-100 in the culture dishes. The immunostaining with rabbit polyclonal antibodies against human OCT4 (POU5F1) and NANOG (Santa Cruz Biotechnology) were detected with goat anti-rabbit IgG as described previously [5, 6].
Extraction of total RNAs
Total RNAs from approximately 1 × 106 cells of T3/HDF and T3/CMHDF on 10 cm plate were extracted using TRIZOL reagent, and the same total RNAs from each sample were used for both mRNA microarray analysis and miRNA quantification.
mRNA microarray analysis
The mRNA profilings of T3/HDF and T3/CMHDF cells were analyzed using Affymetrix Human Genome U133 plus 2.0 GeneChip according to the Manufacturer's protocols (Santa Clara, CA, USA, http://www.affymetrix.com) by the Microarray Core Facility of National Research Program for Genomic Medicine of National Science Council in Taiwan as previously described [5, 6]. This Affymetrix GeneChip contains 54,675 probe sets to analyze the expression levels of 47,400 transcripts and variants, including 38,500 well-characterized human genes. GeneChips from the hybridization experiments were read by the Affymetrix GeneChip scanner 3000, and raw data were processed using Affymetrix GeneChip Operating Software MAS5.0 and its default analysis parameters. The raw data were also analyzed by GeneSpring GX software version 7.3.1 (Silicon Genetics, Redwood City, CA, USA, http://www.chem.agilent.com). The correlation coefficients of gene probes expressed between any two samples were calculated from the normalized values by using GeneChip-Robust Multiarray Average (GC-RMA) algorithm. It may be noted that Affymetrix GeneChip expression analysis can be used as a stand-alone quantitative comparison, since the correlation between Affymetrix GeneChip results and TagMan RT-qPCR results was shown in a good linearity of R2 = 0.95 by the MicroArray Quality Control Study, a collaborative effort of 137 scientists led by the US-FDA [7, 8]. A hierarchical clustering and principle component analysis (PCA) of the eight Affymetrix GeneChip data from duplicates of four populations of hES cells were also performed in order to check the quality of microarray results.
Analyses of signaling pathways and GO process networks
The abundantly (more than 3-folds of overall mean) expressed mRNAs of T3/HDF and T3/CMHD, as well as T3/MEF and T3/CMMEF, cells were analyzed for signaling pathways and GO process networks by using MetaCore Analytical Suite (GeneGo Inc., St Joseph, MI, USA) as previously described . The MetaCore includes a curated database of human protein interaction and metabolism, and thus it is useful for analyzing a cluster of genes in the context of regulatory network and signaling pathways.
Quantification of miRNAs
The expression levels of 365 human miRNAs from T3/HDF and T3/CMHD cells were determined using the TaqMan MicroRNA Assays (Applied Biosystems, Foster City, California, USA, http://www.appliedbiosystems.com) [9, 10]. The detailed procedure for miRNA quantification was previously described [5, 6]. In brief, TagMan MicroRNA Assays include two steps: stem loop RT followed by real-time PCR. (90 ng/Rx, with 24-multiplex primers) Each 10 ul RT reaction that includes 90 ng total RNA, 50 nM stem-loop RT primers, 1× RT buffer, 1.25 mM each of dNTPs, 0.25 U/ul RNase inhibitor, and 10 U/ul MultiScribe Reverse Transcriptase was incubated in the PTC-225 Peltier Thermal Cycler (MJ Research, Watertown, Massachusetts, USA) for 30 min each at 16°C and at 42°C, followed by 5 min at 85°C, and then held at 4°C. RT products were diluted twenty times with H2O prior to setting up PCR reaction. Real-time PCR for each miRNA was carried out in triplicates, and each 10 ul reaction mixture included 2 ul of diluted RT product, 5 ul of 2× TagMan Universal PCR Master Mix and 0.2 uM TagMan probe, respectively. The reaction was incubated in an Applied Biosystems 7900HT Sequence Detection System at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The threshold cycle (Ct) is defined as the fraction cycle number at which the fluorescence exceeds the fixed threshold of 0.2. Total RNA input was normalized based on the Ct values of the TagMan U6 snRNA assay as an endogenous control. The fold change was calculated as 2-ΔCT × K, where -ΔCT = -[CTmiRNA-CTU6 snRNA] and K is a constant.
2D-gel analysis of proteins
Approximately 1 × 106 hES cells on 10 cm plate were washed twice each with 1× PBS and cell wash buffer, and then lyzed using NP40 lysis buffer. 1 mL ice-cold acetone/11% w/v trichloroacetic acid (TCA)/20 mM DTT was added per 0.1 mL solubilised sample and incubated for a minimum of 30 min at -20°C. The precipitate was pelleted by centrifugation (12000 rpm for 10 min at 4°C), washed twice with 1 mL cold acetone containing 20 mM DTT, and then air-dried to remove residual acetone. The resulting protein pellet was then resolubilised in the appropriate rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer, 20 mM DTT). The concentration of proteins in the sample was measured by the Bradford method.
Isoelectricfocusing was performed using an Ettan IPGphor II (Amersham Biosciences). 13 cm Immobiline DryStrips (pH 3-10 NL) were rehydrated overnight for 12 h at room temperature in 250 uL rehydration buffer containing 7 M urea, 2 M thiourea, 2% w/v CHAPS, 20 mM DTT, 0.5% IPG buffer and a trace of bromophenol blue. The protein sample (about 100 ug) was mixed in 100 uL sample buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer pH 3-10 NL, 100 mM DeStreak reagent (Amersham biosciences) and a trace of bromophenol blue. Samples were cup-loaded near the anode of the IPG strips using Ettan IPGphor cup-loading (Amersham Biosciences) according to the manufacturer's protocol. Protein focusing was achieved using the following IEF parameters: 300 V, step and hold, 3 h; 600 V, gradient, 1 h; 1000 V, gradient, 1 h; 8000 V, gradient, 1.5 h; 8000 V, step and hold for 3 h, giving a total of 16000 Vh.
After focusing, the strips were removed immediately and equilibrated by gentle shaking for 15 min in 10 mL equilibration buffer (50 mM Tris-base, pH = 8.8, 6 M urea, 30% v/v glycerol, 0.2% w/v SDS and 1% w/v DTT), followed by 10 mL of the same solution containing 2.5% w/v iodoacetamine instead of DTT for 15 min. The second dimension was performed by SDS-polyacrylamide gel electrophoresis (PAGE) on a 12% w/v separation gel using the Hoefer SE 600 vertical chambers. First dimension IPG gel strips were cut and placed on top of the second dimension vertical gels (16 × 18 × 0.01 cm) and sealed in place with boiling 0.5% agarose in running buffer, containing 0.025 M Tris base, 0.192 M glycine, 0.1% w/v SDS, pH 8.3. The second dimension separation was performed sequentially with a constant voltage of 70 V for 0.5 h, and 120 V for 12 h. After SDS-PAGE, the separated gels were visualized by silver staining. The similarities of protein spots on scanned images were analyzed using ImageMaster 2DE platinum software version 5.0 (Amersham Biosciences).