Combined effects of Matrigel and growth factors on maintaining undifferentiated murine embryonic stem cells for embryotoxicity testing.
Greenlee-AR; Kronenwetter-Koepel-TA; Kaiser-SJ; Ellis-TM; Liu-K
Toxicol In Vitro 2004 Aug; 18(4):543-553
Undifferentiated, murine embryonic stem (mES) cells have shown promise as a substrate for identifying embryotoxic chemicals and for studying mechanisms of early developmental injury. However, long-term maintenance of mES cells in an undifferentiated state is problematic. The present study evaluates the combination of Matrigel matrix and three growth factors for this purpose. Biomarkers of mES cell pluripotency, apoptosis, chromosome number and cardiomyocyte differentiation were monitored over 119 population doublings. D3 mES cells retained undifferentiated characteristics, including sustained expression of alkaline phosphatase and stage specific embryonic antigen-1 (SSEA-1) and continued transcription of Pou5f1 (Oct-4). Cell viability remained at > or=95% and population-doubling times averaged 14.3 h over 10 weeks of observation. Caspase-3 activation, a marker of cellular death by apoptosis, was measured in early- and late-passage mES cells. Early-passage cells showed dose-responsive caspase-3 activation following exposure to sodium arsenite, whereas caspase-3 activation of late-passage cells dropped to background levels at toxicant dosages above 50 ppb. Aneuploidy and impaired differentiation into beating cardiomyocytes were noted for late-passage mES cells. Matrigel, combined with growth factors, may sustain undifferentiated mES cells. However, aneuploidy, reduced caspase-3 activation, and inability to differentiate suggests further modifications to the culture system may be needed for long-term propagation of cells for embryotoxicity endpoints.
Embryotoxicity; Chromosome-disorders; Chromosome-damage; Antigens; Cell-morphology; Toxic-effects; Growth-factors
National Farm Medicine Center, Marshfield Clinic Research Foundation, 1000 North Oak Avenue, Marshfield, WI 54449, USA
Agriculture; Cooperative Agreement
Toxicology in Vitro
Marshfield Medical Research & Education Foundation, Marshfield, Wisconsin