Abstract
The lack of a standardized culture environment for establishment of embryonic stem cell lines has hindered the orchestrated differentiation of cells and the application of this technology. Oxygen concentration has a profound effect on proliferation and differentiation of many cell types. This study tested the hypothesis that establishment dynamics, lactate dehydrogenase (LDH) isoforms, and mRNA expression patterns would be affected by the oxygen tension in the culture environment. Recovered (day 4) murine blastocysts were cultured in a gas environment of 6% CO2 and either 20% or 5% O2 (balance supplemented with N2). More (p < 0.05) blastocysts produced outgrowths in the low (79.3 ± 0.1%) compared to the high (57.1 ± 0.1%) O2 groups, and more (p < 0.05) colonies in the low O2 group (14/15; 93.3 ± 0.1%) stained positive for alkaline phosphatase relative to the high O2 group (9/15; 60.6 ± 0.1%). Oxygen treatment had no effect on the activity of the oxioreductase lactate dehydrogenase. Interestingly, the stem cell lines in both treatments displayed multiple isoforms (III, IV, and V) of LDH, whereas the outgrowths displayed isoforms I and V. In contrast, two-cell embryos and blastocysts displayed only isoform I, and fibroblasts displayed isoforms IV and V. There were no treatment differences in mRNA expression of LDHα in the outgrowths, or established stem cells. LDH transition from the heart (I) to the muscle (V) isoform indicated an increase in glycolytic activity, consistent with the peri-hatching/implantation time period. Reduced O2 environment had significant positive effects on the establishment and maintenance of murine stem cells, supporting the hypothesis, whereas the LDH isozyme transition was consistent among treatments.
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