Ethanol alters the osteogenic differentiation of amniotic fluid-derived stem cells

Abstract

Background: Fetal alcohol spectrum disorder (FASD) is a set of developmental defects caused by prenatal alcohol exposure. Clinical manifestations of FASD are highly variable and include mental retardation and developmental defects of the heart, kidney, muscle, skeleton, and craniofacial structures. Specific effects of ethanol on fetal cells include induction of apoptosis as well as inhibition of proliferation, differentiation, and migration. This complex set of responses suggests that a bioinformatics approach could clarify some of the pathways involved in these responses.

Methods: In this study, the responses of fetal stem cells derived from the amniotic fluid (AFSCs) to treatment with ethanol have been examined. Large-scale transcriptome analysis of ethanol-treated AFSCs indicates that genes involved in skeletal development and ossification are up-regulated in these cells. Therefore, the effect of ethanol on osteogenic differentiation of AFSCs was studied.

Results: Exposure to ethanol during the first 48 hours of an osteogenic differentiation protocol increased in vitro calcium deposition by AFSCs and increased alkaline phosphatase activity. In contrast, ethanol treatment later in the differentiation protocol (day 8) had no significant effect on the activity of alkaline phosphatase.

Conclusions: These results suggest that transient exposure of AFSCs to ethanol during early differentiation enhances osteogenic differentiation of the cells.

Figure 1. Effect of ethanol on cell proliferation and viability

AFSCs were cultured with 0, 25, 50, 75, and 100 mM of ethanol. (A) Cell counts were made after 48 hours of ethanol exposure and compared to counts of cultures that were not treated with ethanol. The results are expressed as percentages relative to cells without ethanol (dark grey bars). The values shown are the mean +/−standard deviation (SD, n=5) of three independent experiments (*, p<0.03 by student T-test). (B) The effect of ethanol on the percentage of non-viable cells was determined by 7-AAD and flow cytometry. Light grey bars indicate the percentage of non-viable cells in the presence of various concentrations of ethanol. The data shown represents the mean number of non-viable cells in 10,000 events from two independent experiments. Error bars represent SDs. Data were not significant. (C) Ethanol concentration in the media was measured by spectrophotometry. Results represent the mean +/− SD from three replicates. (D) Ethanol treatment and osteogenic differentiation began when AFSC reached 60,000 cells. After 24 and 48 hours of 100 mM ethanol, cell counts were performed and showed no significant change in cell number. Ethanol does not have a proliferative effect on AFSC when cultured in osteogenic media.

Figure 1. Effect of ethanol on cell proliferation and viability

AFSCs were cultured with 0, 25, 50, 75, and 100 mM of ethanol. (A) Cell counts were made after 48 hours of ethanol exposure and compared to counts of cultures that were not treated with ethanol. The results are expressed as percentages relative to cells without ethanol (dark grey bars). The values shown are the mean +/−standard deviation (SD, n=5) of three independent experiments (*, p<0.03 by student T-test). (B) The effect of ethanol on the percentage of non-viable cells was determined by 7-AAD and flow cytometry. Light grey bars indicate the percentage of non-viable cells in the presence of various concentrations of ethanol. The data shown represents the mean number of non-viable cells in 10,000 events from two independent experiments. Error bars represent SDs. Data were not significant. (C) Ethanol concentration in the media was measured by spectrophotometry. Results represent the mean +/− SD from three replicates. (D) Ethanol treatment and osteogenic differentiation began when AFSC reached 60,000 cells. After 24 and 48 hours of 100 mM ethanol, cell counts were performed and showed no significant change in cell number. Ethanol does not have a proliferative effect on AFSC when cultured in osteogenic media.

Figure 2. Effect of ethanol on OPN expression in AFSCs

(A) Real-time RT PCR analysis of AFSCs exposed to ethanol for 24 or 48 hours in growth media. CT values were determined from 3 independent experiments from two cell lines and ΔCT values were obtained by subtracting the CT values of β-actin. Mean fold change was determined from four independent experiment-pairs of ethanol-treated AFSCs and non-treated AFSC. Black columns indicate AFSCs without ethanol while grey columns indicate AFSCs exposed to 100 mM ethanol. P-values were determined using a one–tailed paired t test with significance at 24 hours (P < 0.020, n=3) and 48 hours (P < 0.036, n=5). (B) Real-time PCR analysis of AFSCs exposed to ethanol for 24 hours in osteogenic media. CT values were determined from 3 independent experiments using two cell lines and ΔCT values were obtained by subtracting the CT values of β-actin. Mean fold change was determined from 3 independent experiment-pairs of ethanol-exposed AFSCs and non-exposed AFSCs. P-values were determined using a one–tailed paired t test with significance at 24 (P < 0.018).

Figure 3. Effect of ethanol on alkaline phosphatase activity in AFSCs

AFSCs were cultured with or without 100mM ethanol for the first 48 hours of osteogenic differentiation. Ethanol was removed and AFSCs continued to differentiate until days 7–10. At this time, they were assessed for alkaline phosphatase activity. Alkaline phosphatase activity was determined by spectrophotometric measurement of p-nitrophenol conversion. AFSCs exposed to ethanol showed a modest yet significant increase in alkaline phosphatase activity at day 9 and 10 of osteogenic differentiation (*, p < 0.001; ANOVA and two-tail T-test). The values shown are the mean +/− SD from at least ten replicate cultures and similar patterns were observed in a second cell line (data not shown). Differences between treated and non-treated cultures were evaluated by t-tests. Open squares, osteogenic media+ EtOH (48 hours); Black diamonds, osteogenic media; black circles, growth media.

Figure 4. Alkaline phosphatase activity when AFSCs were exposed to ethanol at the midpoint of differentiation

AFSC were treated for 48 hours with 100 mM ethanol beginning at day 8 of osteogenic differentiation. Black columns indicate AFSCs that were not exposed to ethanol while grey bars indicate AFSCs that were exposed to ethanol. Ethanol exposure during the midpoint of differentiation had no significant effect on alkaline phosphatase activity (t test p<0.42). The values shown are the mean +/− SD of twenty cultures.

Figure 5. The effect of ethanol on calcium deposition

AFSCs were exposed to 100 mM ethanol during the first 48 hours of osteogenic differentiation. At day 23 of differentiation, AFSCs were stained for calcium deposition using alizarin red staining. AFSCs exposed for 48 hours to 100 mM ethanol had significantly increased calcium deposition when compared to non-exposed AFSCs (155.1 ± 75.8 μg/mL vs 77.4 ± 26.9 μg/mL). Calcium deposition was detected in non-differentiated AFSCs at a basal level of 53.3 ± 2.2 μg/ml. The values shown are the mean +/− SD (n=3–6) and similar results were confirmed in another cell line (data not shown). * One-tailed t-test of significance of p<0.006.