Figure 1.
Evaluation of intestinal lesion after indomethacin administration.
Panels (A) and (B) show that gross appearance of intestines from rats received indomethacin treatment (A) relative to control (B, saline-treated) surviving 4 days, as stained with Evans blue. The small intestines and appendix from the former appear in dark blue (A), whereas the intestine from healthy controls appears pink (B). The intestinal length is shortened in the indomethacin-treated rats relative to controls (C). In H. & E. preparation, the injured intestine show loss and disruption of the villi, the swelling of the intestinal wall, and the ulcerative lesion with fibrinoid plaques, edema, epithelial exfoliation, and granulocyte infiltration of leukocytes (D), relative to normal control (F). Scale bar = 500 µm in (D) applying for (E).
Figure 2.
In vitro expansion and PKH26 labeling of bone marrow mesenchymal stem cells (BMMSCs) harvested from a donor rat, and homing of these labeled cells to the small intestine in recipient rat with indomethacin.
(A) A large number of cells adhering to a culture dish with spindle-shape morphology at 10 days in vitro. (B) Culture cells pre-labeled by PKH26 show bright red fluorescence. Panels (C) and (D) show the distribution of PKH26 positive BMMSCs in the wall of the small intestine 7 days after infusion, with the cells mostly localized to the mucosal region. Scale bar = 500 µm in (A) applying for (C, D), equivalent to 125 µm for (B).
Figure 3.
Fluorescent microscopic images showing potential in situ proliferation and transdifferentiation of transplanted BMMSCs in recipient rat small intestine with indomethacin injury.
The left panels (A, E, I, M, Q) show PKH26 positive BMMSCs in the intestinal mucosa. These cells show partial colocalization with the endogenous proliferative cell nuclear antigen (PCNA) (B–D), and with molecular markers of mucosal stem cells or precursor including Msi-1 (E-H), Lgr5 (I–L) and Ephrin-3 (M–P). Panels (Q–T) show an example of negative assay control, indicating the lack of green fluorescence in the tissue by excluding the DyLight 488-conjugated secondary antibody in immunohistochemical preparation. Scale bar = 250 µm in (A) applying to the 3 left panels, equivalent to 50 µm for the most right panels.
Figure 4.
Effect of BMMSCs and stem cell factor (SCF) therapies on intestinal mRNA expression following indomethacin injury in rats.
Panel (A) shows an example of the traces of real-time quantitative PCR (qPCR) for, with the top reflecting fluorescent signals vs amplification cycles and the ephrin-B3 bottom vs temperature changes. Panel (B) blots the means of fluorescent absorbent signals in the 4 animal groups at 4 surviving time points. Other panels (C–F) show data for the levels of mRNAs of additional markers as indicated. For details regarding statistical analyses, referring to the result section.
Figure 5.
Effect of BMMSCs and stem cell factor (SCF) therapies on intestinal protein expression following indomethacin injury in rats surviving 4 weeks.
Panel (A) shows western blot images of protein levels from one set of comparing groups of animals. Note the increased protein levels of PCNA, Msi-1, Lgr5 and ephrin-B3 relative to the internal control β-actin in the BMMSCs and SCF treated groups in comparison with saline group. The quantitative data and statistics for individual proteins are summarized as panels (B–E) as indicated. All data are expressed as the percent levels of β-actin in the same tissue lysate loads.
Figure 6.
Effect of BMMSCs and stem cell factor (SCF) therapies on intestinal mucosal regeneration following indomethacin injury in rats examined at the 4 week surviving time point.
Image panels (A–H) show representative low and high magnification H. & E. stain images of the small intestines from the 4 animal groups, sampled at the mid-point between beginning of the duodenum and appendix. The relative mucosal area is defined by the measured mucosal area divided by the outside perimeter of small intestine (as illustrated in panel D). There exists a trend of increase in the relative mucosal area in the BMMSCs and SCF treated groups, most evident in the BMMSCs and SCF co-treatment group (I). Relative area of the crypt layer to the total mucosal layer is defined by the ratio in the given intestinal cross-section (as illustrated in panel H). The means of the ration tend to increase in BMMSCs and SCF treated groups, especially in the BMMSCs and SCF co-treatment group (J). Scale bar = 1 mm in (A) applying to (B–D), equivalent to 250 µm for (E–H).