|
| Status |
Public on Sep 15, 2010 |
| Title |
hESC_undiff_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
human ES cells (hESC), undifferentiated
|
| Organism |
Homo sapiens |
| Characteristics |
developmental stage: undifferentiated human ES cells
passage number: passage 132
cell line: HES-2
origin of a cell line: ES Cell International, Singapore
|
| Treatment protocol |
Cardiac differentiation of human iPS and ES cells was carried out on the murine visceral endoderm-like cell line END2 as described by Mummery C, et al. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107(21):2733-2740, 2003. To initiate co-cultures, human iPS and ES cell colonies were dissociated into clumps by either using collagenase IV (1mg/ml in DMEM/F-12 at 37°C for 5-10 minutes) or by manual cutting. The differentiation was carried out in Knockout-DMEM having 1mM L-glutamine, 1x non-essential amino acids, 0.1 mmol/L beta-mercaptoethanol and Penicillin-Streptomycin (100 U/ml and 100 ug/ml, respectively) but devoid of serum and serum replacement (all reagents were from Invitrogen). Medium change was performed at day 5, 9, 12 and 15 after beginning of the co-culture. Contracting cardiac outgrowths were first observed on day 11 after the initiation of human iPS and ES cell differentiations.
|
| Growth protocol |
The human iPS and ES cells were maintained on irradiated (2x26 Gray/6 minutes each) mouse embryonic fibroblasts (MEFs). MEFs were derived from outbred CF-1 mice and plated at a density of 19500 cells/sqcm in 60 mm tissue culture dishes (BD Falcon). Both lines were maintained in DMEM/ F12 culture medium supplemented with Glutamax, 20% knockout serum replacer, 1% nonessential amino acids and 0.1 mmol/L beta-mercaptoethanol. Additionally, the medium was supplemented with 100 ng/ml basic fibroblast growth factor (Peprotech) for iPS cells, and 4 ng/ml for human ES cells. Culture media were changed daily and undifferentiated cells were passaged by manual dissection of cell clusters every five to six days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from ES and iPS cell samples was isolated using TRIzol Reagent (Invitrogen). Human fetal and adult RNA samples were purchased from Clontech and were prepared by a guanidinium thiocyanate method.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
|
| |
|
| Hybridization protocol |
Standard Illumina hybridization protocol
|
| Scan protocol |
Standard Illumina scanning protocol
|
| Description |
Illumina HumanHT-12 V3.0 expression beadchip
|
| Data processing |
The data were normalised using quantile normalisation in R
|
| |
|
| Submission date |
Aug 10, 2009 |
| Last update date |
Dec 19, 2012 |
| Contact name |
Joachim Schultze |
| E-mail(s) |
j.schultze@uni-bonn.de
|
| Organization name |
LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)
|
| Department |
Genomics and Immunoregulation
|
| Street address |
Carl-Troll-Strasse 31
|
| City |
Bonn |
| State/province |
NRW |
| ZIP/Postal code |
53115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6947 |
| Series (1) |
| GSE17579 |
Comparative global transcriptomic profiling of human ES and iPSs cells and their derived microdissected cardiac clusters |
|
