Transcriptome alterations due to physiological normoxic (2% O-2) culture of human embryonic stem cells

Human embryonic stem cells (hESCs) hold great promise therapeutically. In order to deliver on this promise the correct defined conditions for long-term propagation must first be established. Researchers have now provided reports describing the benefits of culturing hESCs in physiologically approximate levels of oxygen. These physiological values fall in the range of 2 to 5% O-2. Benefits include reduced spontaneous differentiation, enhanced chromosomal stability and increased clonality. Aims: The aim of our study was to examine the transcriptional consequences of culturing hESCs in physiological normoxia (2% O-2) using microarray technology. Methods: Three karyoptically normal hESC lines (H1, H9 and RH1) were examined. At the initiation of this experiment, established hESC lines were redesignated as passage (p) 0 in 21% O-2, then bifurcated into 21% O-2 and 2% O-2. and maintained for a further ten passages at which time samples were again collected. RNA was extracted from all sample points and subjected to microarray analysis using the Affymetrix U133 Plus 2.0 platform. Bioinformatic analysis was performed using dChip and GoStat. Results: We performed grouped analyses of gene expression of early (p0) versus late (p10) air-cultured cells. This revealed relative stability with six (air p0 baseline vs p10 experimental) and one (air p10 baseline vs p0 experimental) gene(s) displaying both greater than twofold and statistically significant upregulation. Conversely, we identified 302 gene upregulations and 56 downregulations when comparing 21% 02 (P0P10) with 2% 0 2 (p10). These significantly upregulated changes clustered into 82 over-represented and 9 under-represented ontology terms. These terms were indicative of signaling pathways, developmental potential and metabolism. Hierarchical clustering indicated a trend for 2% 0 2 cultured cells to cluster collectively with reduced heterogeneity when compared with 21% O-2 cultured cells. Conclusions: The gene changes associated with 2% 0 2 culture may be predictive of novel cellular requirements for stable self-renewal, maintenance of pluripotency, and a reduction of hESC-line heterogeneity.