Fig. 6. The p-Erk1, 2/Fst/Fstl1-positive mesenchymal cells were embryonic small intestinal (esi)MSCs. (A) Fluorescence profiles (x-axis) for E18.5 WT iMSCs (passage 5) stained with antigen specific antibodies (red) and isotype-matched negative controls (black) that showed the majority of these cells (y-axis=cell counts) were CD29, CD44, CD54, CD105 and CD106 positive and CD45 negative. (B) Cultured iMSCs expressed genes encoding secreted Tgfβ inhibitors including follistatin (Fst) and follistatin-like 1 (Fstl1). Quantification of relative mRNA amounts of Fst and Fstl1 in multiple cell lines, as determined by qRT-PCR. Expression of Fst and Fstl1 in iMSCs was compared with myofibroblasts (Mic216), macrophages (RAW 264.7) and gut epithelial cells (AGS). (C) Expression of mRNAs encoding Fst (blue), Fstl1 (orange) and Spry2 (a known transcriptional target of Fgf9, red) in iMSCs increased with Fgf9 treatment. A single asterisk indicates that the mean values for the 8 hour time points were statistically significantly different from 0 hours (P<0.05 by Student's t-test). (D) FACS analysis of intracellular -SMA of MIC-216 (intestinal myofibroblast line) cells either cultured alone or in a transwell co-culture with iMSCs. The x-axis is fluorescence intensity for -SMA and the y-axis is forward scatter (an indication of cell size). The box indicates cell population defined as -SMA high. (E) Quantification of the percentage of -SMA low Mic216 cells cultured alone, in the presence of iMSCs, Fst of Fgf9 (n=3 experiments/condition). ^*P<0.05 (F,G) Sections of control E18.5 small intestines stained with p-Erk (green) and (F) CD105 (red) and (G) CD54 (red). The latter two markers colocalize with p-Erk1/2 positive mesenchymal cells (yellow arrows). (H) Section of an E14.5 Dermo1-cre; Rosa26R small intestine stained with anti-β-galactosidase and anti-CD54. Scale bars: 10 µm.