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Status |
Public on Oct 22, 2008 |
Title |
Identification of oxygen-sensitive transcriptional programs in human embryonic stem cells. |
Organism |
Homo sapiens |
Experiment type |
Expression profiling by array
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Summary |
To realize the full potential of human embryonic stem cells (hESC), it is important to develop culture conditions that maintain hESC in a pluripotent, undifferentiated state. A low O2 atmosphere (~4% O2), for example, prevents spontaneous differentiation and supports self-renewal of hESC. To identify genes whose expression is sensitive to O2 conditions, microarray analysis was performed on RNA from hESC that had been maintained under either 4% or 20% O2. Of 149 genes differentially expressed, 42 were up-regulated and 107 down-regulated under 20% O2. Several of the down-regulated genes are most likely under the control of hypoxia-inducing factors and include genes encoding enzymes involved in carbohydrate catabolism and cellular redox state. Although genes associated with pluripotency, including OCT4, SOX2 and NANOG were generally unaffected, some genes controlled by these transcription factors, including LEFTY2, showed lowered expression under 20% O2, while a few genes implicated in lineage specification were up-regulated. Although the differences between O2 conditions were generally subtle, they were observed in two different hESC lines and at different passage numbers. The data are consistent with the hypothesis that 4% O2 favors the molecular mechanisms required for the maintenance of pluripotency. This report emphasizes the importance of employing physiological concentrations of O2 when culturing hESC. The transcript profiles of cultures under 20 % O2 suggest that the cells are more poised to differentiate than when they are under the lower 4 % O2 conditions and that the down-regulation of LEFTY2 under 20 % O2 may destabilize the network of genes maintaining ESC pluripotency. Finally, the association of HIF2A with undifferentiated but not differentiating cells is consistent with a particular role for that transcription factor in control of pluripotency. Keywords: different oxygen concentrations in hESC culture condition
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Overall design |
The H1 and H9 hESC lines had been maintained in a physiological O2 (3-5% O2) atmosphere since passage 26 and 23, respectively. The both H1 and H9 cells were propagated under 4% O2 for seven passages on MEF feeder layer, thereafter the both cells were switched to Matrigel coated culture wells for at least three passages prior to RNA collection. RNA extraction was performed within 3 min following removal of the culture plates from respective O2 conditions and well before dissolved O2 in the culture medium showed any detectable changes. Total six RNA samples were collected from H1 cells at two different passage numbers (H1p37 and H1p50) and H9 cells at passage 32 (H9p32) under 4% and 20% O2 conditions, respectively. The cells maintained under 4% O2 were split into both 4% and 20% O2 conditions and cultured for 7 days before of RNA collection.
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Contributor(s) |
Westfall SD, Ezashi T |
Citation(s) |
18811242, 19541600 |
Submission date |
Nov 04, 2007 |
Last update date |
Mar 25, 2019 |
Contact name |
Toshihiko Ezashi |
E-mail(s) |
ezashit@missouri.edu
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Phone |
573 884-9601
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Organization name |
University of Missouri-Columbia
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Department |
Animal Sciences
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Lab |
R.M. Roberts
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Street address |
240a CS Bond Life Sciences Center
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City |
Columbia |
State/province |
MO |
ZIP/Postal code |
65211 |
Country |
USA |
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Platforms (1) |
GPL570 |
[HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array |
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Samples (6)
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Relations |
BioProject |
PRJNA103305 |
Supplementary file |
Size |
Download |
File type/resource |
GSE9510_RAW.tar |
24.9 Mb |
(http)(custom) |
TAR (of CEL, CHP, EXP) |
Processed data included within Sample table |
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