|
| Status |
Public on Sep 24, 2012 |
| Title |
iP-MSC donor4 cloneA |
| Sample type |
genomic |
| |
|
| Source name |
bone marrow-derived MSC (iP-MSC)
|
| Organism |
Homo sapiens |
| Characteristics |
donor: donor4, cloneA
cell type: iPSC
|
| Treatment protocol |
MSC were isolated from tibia plateau of patients undergoing orthopaedic surgery. All samples were taken after written consent using guidelines approved by the Ethic Committee on the Use of Human Subjects at the University of Aachen (Permit number: EK128/09). MSC were culture-expanded in standard medium consisting of DMEM (PAA; 1g/L glucose) supplemented with glutamine (PAA), penicillin/streptomycin (PAA) and 10% human platelet lysate (hPL).
|
| Growth protocol |
MSC from three donors (passage 3) were infected on two consecutive days with equal amounts of pMXs based retroviruses (Addgene, Cambridge, MA, USA) carrying the OCT3/4, SOX2, KLF4 and c-MYC genes. On day 3, medium was changed to high glucose DMEM (Gibco), 20% FCS, 1% nonessential amino acids (NAA), 1x penicillin/streptomycin, 1x L-glutamine, 0.1 mM β-mercaptoethanol (βME) and 50 ng/ml basic fibroblast growth factor (bFGF) (Peprotech, Hamburg, Germany). On day 6 the cells were plated on gelatin-coated dishes containing irradiated CF1 murine embryonic fibroblasts (MEFs), and on day 7 the culture medium was supplemented with 20 μg/ml vitamin C (Sigma) and 1mM valproic acid (Sigma). Between days 21–30 the ESC-like colonies were observed and mechanically isolated. Established iP-MSC were maintained on MEFs in DMEM/F12 medium supplemented with Glutamax, 20% knockout serum replacer, 1% NAA, 0.1 mM βME and 50 ng/ml bFGF. Cells were passaged by manual dissection of cell clusters every 5-6 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from 10(6) cells using the Qiagen DNA Blood Midi Kit.
|
| Label |
Cy5 and Cy3
|
| Label protocol |
Standard Infinium HD Methylation Assay protocol
|
| |
|
| Hybridization protocol |
Bisulfite converted DNA was amplified, fragmented and hybridized to Illumina Infinium HumanMethylation450 Beadchip using standard Infinium HD Methylation Assay protocol.
|
| Scan protocol |
Standard Infinium HD Methylation Assay protocol
|
| Data processing |
Initial analysis was performed by the Genomestudio 2010.3 (Modul M Version 1.8.5). Data were normalized with internal controls according to Illumina´s standard procedures. Methylation level at each locus was calculated with the GenomeStudio Methylation module as beta-value (ranging from 0 to 1). The number of beads per feature varies between chips and beta-values were calculated as average of at least three technical replica.
|
| |
|
| Submission date |
Dec 23, 2011 |
| Last update date |
Sep 24, 2012 |
| Contact name |
Wolfgang Wagner |
| E-mail(s) |
[email protected]
|
| Phone |
+49 241 8088611
|
| Organization name |
RWTH Aachen University
|
| Department |
Helmholtz Institute for Biomedical Engineering
|
| Lab |
Stem Cell Biology and Cellular Engineering
|
| Street address |
Pauwelsstrasse 20
|
| City |
Aachen |
| ZIP/Postal code |
52074 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13534 |
| Series (3) |
| GSE34688 |
Induced Pluripotent Mesenchymal Stromal Cell Clones Retain Donor-Derived Differences in DNA Methylation Profiles |
| GSE37066 |
Pluripotent Stem Cells Escape From Senescence-Associated DNA Methylation Changes [Illumina] |
| GSE37067 |
Pluripotent Stem Cells Escape From Senescence-Associated DNA Methylation Changes |
|
