Effects of oxygen tension on the establishment and lactate dehydrogenase activity of murine embryonic stem cells.
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% CO(2) and either 20% or 5% O(2) (balance supplemented with N(2)). More (p < 0.05) blastocysts produced outgrowths in the low (79.3 +/- 0.1%) compared to the high (57.1 +/- 0.1%) O(2) groups, and more (p < 0.05) colonies in the low O(2) group (14/15; 93.3 +/- 0.1%) stained positive for alkaline phosphatase relative to the high O(2) 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 LDHalpha 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 O(2) 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.
Gibbons
J
J
Colorado Center for Reproductive Medicine, Englewood, CO, USA. jgibbns@clemson.edu
Hewitt
E
E
Gardner
D K
DK
D000818:Animals; D001755:Blastocyst / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology*; D005260:Female; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010100:Oxygen / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000201:enzymology
D012333:RNA, Messenger; D007770:L-Lactate Dehydrogenase; D010100:Oxygen