THE EFFECT OF ETHANOL ON RHESUS MONKEY EMBRYONIC STEM CELLS

Abstract

Fetal Alcohol Syndrome results from ethanol exposure and can be deleterious to a developing fetus. The earliest stages of fetal development are most sensitive to the effects of ethanol, however that time of development is very difficult to study in vivo. Embryonic stem cells (ESCs) are derived from blastocyst stage embryos making ESCs an appropriate model for early development. Rhesus monkey (Macaca mulatta) ESC lines (ORMES 6 and 7) were treated with 0, 0.01%, 0.1%, or 1.0% ethanol in modified DMEM media for one month. Approximately 30 colonies were cultured per 60mm dish in 5 mL of media. Culture media were changed daily and ESC colonies were passaged twice weekly. Colony number and morphology were observed every 3–4 days and by week 2, morphological differences became apparent. ESCs in the 1.0% ethanol treatment group were either hypotrophic with decreased colony area or showed abnormal growth patterns that may indicate differentiation. No differences in colony numbers or viability were observed. Area measurements indicated a significant decrease (p<0.05) between the control and 1.0% ethanol treatment group for ORMES 6 at fourteen days and for ORMES 7 at twenty-four days and between the control and 0.1% ethanol treatment group at day twenty-eight in ORMES 7. Immunofluorescence staining of key pluripotency markers (OCT4, SSEA1 & 4, TRA-1-60 & 81, and alkaline phosphatase) indicated a loss of pluripotency of the ESC lines used. The morphological changes and immunofluorescence indicates that ethanol affects these early cells of development and may cause uncontrolled differentiation. Further studies are warranted to determine possible mechanisms for the effects observed, especially in the higher ethanol treatment groups. Supported by NIH grants RR00169 and AA014173. (platform)