EFFECTS OF ETHANOL ON MOUSE EMBRYONIC STEM CELL DIFFERENTIATION

Objective: Chronic alcohol consumption during gestation causes fetal malformations, termed fetal alcohol syndrome (FAS). We conducted the present study to clarify the mechanism underlying alcohol consumption-induced malformations.Methods: First, the effects of ethanol on the viability of cell lines, such as EB3 (undifferentiated mouse embryonic stem (ES) cells), 3T3-Swiss albino, Neuro-2a, NCTC Clone 1469, and UBE6T-15, were determined. Furthermore, ethanol-induced cell death patterns were analyzed by the annexin V-Cy3.18 (AnnCy3) immune fluorescent method. Second, the effects of ethanol on ES cell differentiation were assessed by the embryoid body (EB) model. The formation of an EB, accompanied by spontaneous pulsation derived from EB3 cells, was monitored. EB3 cells were cultured in hanging drops of media containing 0-5% ethanol for 8 days. We then analyzed the EB formation grade by counting the EBs accompanied by spontaneous pulsation in four categories and by monitoring the expression of differentiation marker genes: connexin43, GATA4, c-kit, α-SMA, and Oct-3/4.Results: EB3 cells were more sensitive to alcohol than the other four cell lines, and that ethanol-induced death of EB3 cells matched the apoptosis pattern. There were no obvious differences in the formation rates of EBs with pulsation among all ethanol-treated groups. However, c-kit gene expression was significantly decreased in the EBs treated with 3 and 5% ethanol, in comparison to the control EBs.Conclusion: Collectively, the present study suggested that ES cells are more sensitive to ethanol than differentiated cells, and that ethanol-induced down regulation of c-kit expression might be involved in alcohol-induced malformations

Critical <i>in vivo</i> roles of WNT10A in wound healing by regulating collagen expression/synthesis in <i>WNT10A</i>-deficient mice

<div><p>Background</p><p>We have reported that WNT10A plays a critical role in the growth of fibroblasts/myofibroblasts and microvascular endothelial cells, i.e.; wound healing/scarring. To ascertain the <i>in vivo</i> regulatory, central functions of WNT10A, we examined the net effects of WNT10A depletion using <i>WNT10A</i>-deficient mice (<i>WNT10A</i>^–/–).</p><p>Methods and results</p><p>We generated <i>WNT10A</i>^–/–mice, displaying a range of unique phenotypes of morpho/organogenetic failure, such as growth retardation, alopecia, kyphosis and infertility, and then focused on the functions of WNT10A in wound healing. We subjected C57BL/6J wild-type (WT) or <i>WNT10A</i>^–/–mice to skin ulcer formation. The <i>WNT10A</i>^–/–mice had significantly larger injured areas and delayed wound healing, which were associated with (a) a smaller number of fibroblasts/myofibroblasts and microvessels; and (b) more reduced expression and synthesis of collagen, compared with WT mice with intact WNT10A expression, especially in those with activated myofibroblasts.</p><p>Conclusions</p><p>These observations indicate that WNT10A signaling can play a pivotal in vivo role in wound healing by regulating the expression and synthesis of collagen, as one of fibrogenic factors, at least in part, and critical <i>in vivo</i> roles of WNT10A-mediated effective wound healing are extremely closely associated with collagen expression.</p></div

Expression of extracellular matrix associated genes in skin tissue by microarray studies.

<p>Expression of extracellular matrix associated genes in skin tissue by microarray studies.</p

Immunofluorescence study of <i>in vitro</i> murine dermal fibroblasts showing a unique phenotype of little stromagenesis on <i>WNT10A</i>^–/–KO mice.

<p>Immunofluorescence staining shows that a significantly larger number of cultured dermal fibroblasts (<i>blue-stained</i> in nuclei) contain elevated expression of Type I/III collagen (<i>green-stained</i>) in WT mice than in KO mice (n = 3 per group). The basal condition of fibroblastic proliferation is overtly better in WT mice than that in KO mice, and the morphology of fibroblasts from KO skin is relatively poorly preserved. Scale bars = 50 μm.</p

The deficiency of <i>WNT10A</i> suppressed cell proliferation activity in wound healing mice model.

<p>Immunofluorescence staining showed that a significantly larger number of histone H3-positive cells (<i>green-stained</i>) were observed in WT mice wound skin, compared to <i>WNT10A</i>^–/–mice (n = 3 per group). The fluorescence of histone H3 was located in nuclei (inset) (<i>blue-stained</i> in nuclei). Scale bars = 100 μm.</p

<i>WNT10A</i>^–/–KO skin wound showing reduced fibrosis/fibrogenesis associated with the decreased expression of stromagenesis-related genes.

<p><b>A)</b> Representative pictures of Masson’s trichrome staining (Scale bars = 100 μm) show that collagen deposits (<i>blue</i>-stained) were markedly smaller with more reduced fibrosis in the injured incurable skin lesions of KO mice than in those of WT mice (n = 8 mice per group). In addition, an immunofluorescence study (Scale bars = 20 μm) reveals that a large number of WT spindle (myo)fibroblasts (<i>blue-stained</i> in nuclei) have significant expression of Type I/III collagen at day 10 post-skin injury, which is very rarely or not observed in KO (myo)fibroblasts. <b>B)</b> On a quantitative analysis, the <i>blue</i>-stained collagen content of KO injured skin was found to be markedly lower than in WT skin. <b>C)</b> Correspondingly, real-time RT-PCR showed that the mRNA expression of <i>Type I</i> and <i>Type III collagen</i> was significantly lower in the KO skin of the day-3 wound healing model than in the WT skin. Values are means ± SE and were normalized for 18s rRNA expression (real-time RT-PCR). *<i>P</i> < 0.05, ***<i>P <</i> 0.0001.</p