Primary hMSCs were obtained from the Tulane Center for Preparation and Distribution of Adult Stem Cells (http://www.som.tulane.edu/gene_therapy/distribut.shtml). The cells were grown in alpha minimal essential medium ( MEM;GIBCO/BRL, Carlsbad, CA; http://www.invitrogen.com) supplemented with 16.6% fetalbovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2mM L-glutamine (GIBCO/BRL) at 37°C under 5% CO2 atmosphere. The medium was changed twice per week and a subculture was performed after they reached about 80 % confluency.
The hMSC strain (KP) was developed by transfection with the type 16 human papilloma virus proteins E6E7 as described previously (Hung et al., 2004). This strain is grown in DMEM-LG (Gibco, Grand Island, NY; http://www.invitrogen.com) supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2mM L-glutamine. The medium was changed twice per week and a subculture was performed at 1:3 to 1:5 split every week. Using flow cytometry, cells express CD29, CD44, CD73, CD90, CD105, SH2, and SH3, but otherwise, they lack expression of CD34 and CD166.
2. DNA Delivery Methods.
KP cells were transfected with phTERT-IRES2-EGFP, which was generated by inserting a 3.45-kb EcoRI-EcoRI fragment containing the hTERT cDNA into pIRSE2-EGFP (BD company, USA) using Nucleofector technology as recommended by the manufacturer (AMAXA Biosystems, Cologne, Germany). The efficiency of transfection as evaluated by the expression of EGFP was around 70%. The cells were then suspended in an appropriate volume of 20% FBS-supplemented DMEM-LG medium, seeded in 96well plate for selecting single cell clone by neomycin (400μg/ml).
3. Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
Total RNA was extracted using the Tri Reagent (Sigma) according to the manufacturer’s specifications. First strand cDNA synthesis was performed using Superscript III reverse transcriptase (Invitrogen), Random primer (Invitrogen), 10 mM dNTPs (Invitrogen), 5×
First Strand synthesis buffer, 0.1M DTT, and RNaseOUT ribonuclease RNase inhibitor (Invitrogen). PCR was performed using cDNA as the template in a 50 μl reaction mixture containing a specific primer pair of each cDNA according to the published sequences.
The reaction products were resolved by electrophoresis on a 1.5% agarose gel and
visualized with ethidium bromide. Sequences of PCR primers and PCR conditions can be provided onrequest.
4. Real-Time RT PCR.
Real-Time PCR was performed using an ABI PRISM 7700 sequence detection system (Applied Biosystems) and the TaqMan Universal Master Mix (Applied Biosystems).
Analysis of the results was carried out using the software supplied with the machine. The software calculates each gene expression relative to the β-actin housekeeper gene (delta CT) and then relative to controls (delta delta CT) using the fluorescence threshold of the amplification reaction and the comparative CT method. Sequences of PCR primers, probe and PCR conditions can be provided onrequest.
5.Differentiation Protocols
Trophoectoderm differentiation protocol was modified from a previous method (Xu et al., 2002). Cells at 50% of confluence were treated with 100 ng/mL BMP4 (R&D Systems, Minneapolis, MN) in DMEM-LG supplemented with 10% FBS. Medium was changed twice per week. Germline differentiation protocol was performed with a protocol modified from previous report (Nayernia et al., 2006). In brief, cells were plated at a
density of 1~2 x 104 cells/cm2 in DMEM-LG supplemented with 10% FBS and 2μM retinoic acid (RA, Sigma) with medium change twice per week. For osteogenic differentiation, cells were seeded at a density of 104 cells/cm2 and induced in DMEM-LG supplemented with 10% FBS, 50 μg/ml ascorbate-2 phosphate (Nacalai, Kyoto, Japan),
10-8 M dexamethasone (Sigma) and 10 mM β-glycerophosphate (Sigma) with medium
change twice per week. For neurogenic differentiation (Pera et al., 2004), 100ng/ ml recombinant human Noggin (R&D Systems) was added into the serum-free DMEM-LG culture medium.
6. Histochemical Studies
Cells were fixed in 2% paraformaldehyde for 10 min and stained for alkaline phosphatase activity and in vitro mineralization by Alizarin red-S (Hung et al., 2002) to reveal osteogenic differentiation. After washing 5 times with PBS, stained cultures were photographed.
7. DNA Methylation Array
a. DNA preparation
Genomic DNA was extracted from samples using QIAamp® DNA mini kit
(Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol.
b. aPRIMES
1 μg genomic DNA was restricted to completion with 10U MseI at 37oC in a final
volume of 10 μl in the buffer prepared with the 10 × One-Phor-All Buffer PLUS (GE
Healthcare). Heat inactivation was carried out at 65oC for 20 min. MseI fragments were then subjected to ligation with PCR linkers, MseI linker-S (5'-TAA CTA GCA TGC-3’) and MseI linker-L (5'-AGT GGG ATT CCG CAT GCT AGT-3’ ) overnight. Half of the resulting ligated MseI fragments were digested with the restriction enzyme McrBC (New England Biolabs, Beverly, MA, USA) for 3 h following the conditions recommended by the supplier. The other half of the MseI fragments were digested with the three methylation-sensitive endonucleases HpaII (New England Biolabs; recognition site CCGG, 3 h, 37oC), HhaI (New England Biolabs; recognition site CGCG, 3 h, 37oC) and
BstUI (New England Biolabs; recognition site CGCG, 3 h, 60oC) according to the recommendations of the supplier. Digested DNA fragments were then treated with 1 μl
Proteinase K (Invitrogen, Karlsruhe, Germany) for 1 h at 37oC with subsequent heat inactivation at 80oC for 10 min. For the LM-PCR steps, 2X PCR Master Mix (Promega,
Madison, Wisconsin, USA) was added to a final volume of 50 μl. A MJ thermocycler
was programmed to 68oC for 10 min, followed by 27 cycle loops at 94oC (40 s), 57oC (30 s) and 68oC (75 s). Final elongation was carried out at 72oC for 10 min. PCR products were purified by ethanol precipitation. DNA was eluted in 50 μl nuclease free H2O.
c. Labeling and hybridization to microarrays
Both the HpaII/HhaI/BstuI-digested and the McrBC-digested samples were differentially labeled with Cy5- or Cy3-conjugated dUTP by use of an Agilent Genomic DNA Labeling Kit PLUS (Agilent Technologies, USA). Labeled targets were subsequently cleanup by the use of a Centricon YM-30 column (Millipore), pooled and mixed in a 500-μl hybridization mixtures with 50 μg of human Cot-1 DNA (Invitrogen) in 1X
hybridization buffer (Agilent Technologies). Before hybridization to the array, the hybridization mixtures were denatured at 95°C for 3 min and incubated at 37°C for 30 min. To remove any precipitate, the mixture was centrifuged at ≥14,000 ×g for 5min and
the supernatant was transferred to a new tube. The labeled and denatured DNA target was then hybridized to human CpG island microarray (G4492A, Agilent Technologies, USA) at 65°C for 40 h. The arrays were washed with 0.5 × SSC/0.005% Triton X-102 (wash 1)
at room temperature for 5min, and then with 0.1 × SSC/0.005% Triton X-102 (wash 2) at
d. Image and microarray data analysis
After drying by nitrogen gun blowing, microarrays were scanned with an Agilent microarray scanner (Agilent Technologies, USA) at 535 nm and 625 nm for Cy3 and Cy5, respectively. Scanned images were analyzed by Feature extraction 9.1 software (Agilent Technologies, USA) to quantify signal and background intensity for each feature.
Microarray data were firstly normalized with print-tip loess, followed by background-correction, normalization and analysis by the limma package within the R environment (version 2.1.0). The methylation level was determined as the ratio of Cy5/Cy3 in each spot. The raw data from the array experiments is available from the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo) under the series accession number GSE (pending number). For Gene Ontology (GO) analysis of the genes decreased in CpG island methylation, we determined the statistically significant GO terms using the hypergeometric probability distribution. For each GO term, a p-value was calculated representing the probability that the number of genes that are annotated at the term could have been found by chance.
8. Microarray expression data sets and principal component analyses
The expression profile of hTERT-transfected hMSCs was implemented by using the AffymetrixTM HG U133 Plus 2.0. To determine the similarityof the expression profiles between hTERT-transfected hMSCs and various normal human tissues, MSCs, and ESCs, PCA was performed in 31 AffymetrixTM U133 Plus 2.0 array data produced by us or from public accessible array databases using the Partek® Genomics SuiteTM software (Partek
Incorporated, St. Louis, Missouri). All microarray datasets in this paper are available at Gene Expression Ominbus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under the accession no. of GSE7234 and GSE9520.