PARENT SESSION

(T743) WITHDRAWN.

Dyce, Paul¹, Wen, Lihua¹, Li, Julang¹, ¹ University of Guelph, Guelph, ON, Canada

ABSTRACT- We have previously reported the isolation of stem cells from porcine fetal skin. These cells proliferate actively in vitro, express oct4, and can differentiate into neuron, astrocyte and adipocyte. The objective of the current investigation was to study the potential of these stem cells to differentiate into cells that are consistent with granulosa and theca cells in the ovary. Since estrogen synthesis requires the co-action of granulosa and theca cells, the presence of estrogen in the culture medium suggests the presence of these two cell types in the culture. At 40 days of induced differentiation, some follicle-like aggregates formed and suspended in the culture. These aggregates were collected and cultured as a group. Cell medium was assayed using ELISA for the presence of estradiol and progesterone. Both of the steroid hormones were absent in the culture medium at day 0 of culture. At day 10 and 15 of cell aggregate culture, estradiol was detected at the concentration of 86.17±25.88 pg/ml and 186.8±88.42 pg/ml, while progesterone production increased from 836.5±183.9 pg/ml, and 1608.0±292.7 pg/ml, respectively. To confirm that these cell aggregates do have the machinery to produce the steroid hormones, RT-PCR was performed to determine the expression of enzymes that are important for steroid hormone production during induced differentiation. Transcripts of p450 arom, p450c17, and StAR were all detected at day 40 of differentiation. Immunocytochemistry confirmed the expression of aromatase at a subpopulation of the cells and also showed that SCC is expressed at day 40 of differentiation. Immunocytochemistry suggested that the FSH receptor was not expressed before differentiation. The expression was detectable at day 20 of differentiation, and increased during differentiation. When added to the cell aggregate culture, FSH (10 ng/ml) significantly stimulated estradiol production (from 187.3±17.69 pg/ml to 296.3±50.72 pg/ml), indicating that the cell aggregates are functionally responding to the stimulation of the gonadotropin.

KEY WORDS: Hormone, Granulosa, Stem Cell, Differentiation