OXYGEN-SUPPLY AND OXYGEN-DEPENDENT GENE-EXPRESSION IN DIFFERENTIATINGEMBRYONIC STEM-CELLS

Blastocyst-derived pluripotent mouse embryonic stem cells can differen tiate in vitro to form so-called embryoid bodies (EBs), which recapitu late several aspects of murine embryogenesis, We used this in vitro mo del to study oxygen supply and consumption as well as the response to reduced oxygenation during the earliest stages of development, EBs wer e found to grow equally well when cultured at 20% (normoxia) or 1% (hy poxia) oxygen during the first 5 days of differentiation. Microelectro de measurements of peri cellular oxygen tension within 13- to 14-day-o ld EBs (diameter 510-890 mu m) done at 20% oxygen revealed efficient o xygenation of the EBs' core region, Confocal laser scanning microscopy analysis of EBs incubated with fluorescent dyes that specifically sta in living cells confirmed that the cells within an EB were viable, To determine the EBs' capability to sense low oxygen tension and to speci fically respond to low ambient oxygen by modulating gene expression we quantified aldolase A and vascular endothelial growth factor (VEGF) m RNAs, since expression of these genes is upregulated by hypoxia in a v ariety of cells, Compared with the normoxic controls, we found increas ed aldolase A and VEGF mRNA levels after exposing 8- to 9-day-old EBs to 1% oxygen, We propose that EBs represent a powerful tool to study o xygen-regulated gene expression during the early steps of embryogenesi s, where the preimplantation conceptus resides In a fluid environment with low oxygen tension until implantation and vascularization allow e fficient oxygenation.