Normally the majority of mammalian cells, including murine embryonic stem (mES) cells, are immersed in a low oxygen environment (hypoxia); however, mES are generally cultured in an atmosphere containing 21% O2 (normoxia). Such conditions may not be the most appropriate for mES propagation. We have tested the effects of hypoxia and culture on either feeder fibroblasts or gelatin substrate on mES cell growth and spontaneous differentiation. Two ES cell lines (R1 129/Sv from the laboratory of A. Nagy and MAR B6D2 F1 generated in our laboratory) were cultured under two different oxygen tensions (5 and 21%), and on two different substrates, 0.1% gelatin or murine embryonic fibroblasts (mEF). Cell cycle, cell proliferation, mRNA expression of pluripotency and differentiation markers, as well as spontaneous differentiation to cardiomyocytes, were monitored. For cell proliferation measurements, mES after 7 days of culture at the different conditions were labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester, and cultured for up to three more days. Cells were then harvested for flow cytometry analysis every 24 h after labeling (Cell TraceTM CFSE Cell Proliferation Kit; Molecular Probes, Inc., Eugene, OR, USA). For cell cycle analysis, cells grown on mEF under the two different oxygen tensions were fixed after 10 days of culture, and then stained with propidium iodide/Triton-X-100 for flow cytometry analysis (Current Protocols in Cytometry, Chap. 7, 2001). The spontaneous differentiation of embryoid bodies [formed by ES cells in the absence of leukemia inhibitory factor (LIF)] to cardiomyocytes was also monitored. For mRNA expression of pluripotency (Nanog, Oct-3/4, Rex1, GENESIS, FGFR-4, TERF1, Cx43, and GLUT1) and differentiation markers (GATA4, GATA2, AFP, Msx-1, Brachyury, and Myf5), RT-PCR analysis was performed on mES cells from Day 0 to Day 10. Under hypoxia conditions, the proliferation of both types of mES cells was greater than under normoxia, independent of substrate used, and a higher number of apoptotic cells was detected. Moreover, only under normoxia conditions did mES cells lose the expression of pluripotency markers GENESIS and GLUT1. In addition, under lower oxygen tension, the rate of differentiation to beating cardiomyocytes was significantly lower on the feeder layer than that under normoxia (11.9% vs. 28.1%; P = 0.012). The feeder layer supported significantly higher cardiomyocyte formation when compared to 0.1% gelatin at 21% O2 (28.1% vs. 8.3%; P < 0.001). Our results show that normoxia may not be the most appropriate condition for mES cell propagation and that hypoxic culture may be necessary to maintain full pluripotency of mES cells.
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