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Combined effects of Matrigel and growth factors on maintaining undifferentiated murine embryonic stem cells for embryotoxicity testing.

Authors
Greenlee-AR; Kronenwetter-Koepel-TA; Kaiser-SJ; Ellis-TM; Liu-K
Source
Toxicol In Vitro 2004 Aug; 18(4):543-553
NIOSHTIC No.
20031990
Abstract
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.
Keywords
Embryotoxicity; Chromosome-disorders; Chromosome-damage; Antigens; Cell-morphology; Toxic-effects; Growth-factors
Contact
National Farm Medicine Center, Marshfield Clinic Research Foundation, 1000 North Oak Avenue, Marshfield, WI 54449, USA
CODEN
TIVIEQ
Publication Date
20040801
Document Type
Journal Article
Email Address
greenlee.anne@mcrf.mfldclin.edu
Funding Amount
1155892
Funding Type
Agriculture; Cooperative Agreement
Fiscal Year
2004
NTIS Accession No.
NTIS Price
Identifying No.
Cooperative-Agreement-Number-U50-CCU-514436
Issue of Publication
4
ISSN
0887-2333
Source Name
Toxicology in Vitro
State
NJ; WI
Performing Organization
Marshfield Medical Research & Education Foundation, Marshfield, Wisconsin
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