Harvest tissue source does not alter the protective power of stromal cell therapy after intestinal ischemia and reperfusion injury

Abstract

Background: Transplantation of mesenchymal stromal cells (MSCs) may be a novel treatment for intestinal ischemia. The optimal stromal cell source that could yield maximal protection after injury, however, has not been identified. We hypothesized that (1) MSCs would increase survival and mesenteric perfusion, preserve intestinal histologic architecture, and limit inflammation after intestinal ischemia and reperfusion (I/R) injury, and (2) MSCs harvested from different sources of tissue would have equivalent protective properties to the intestine after I/R inury.

Methods: Adult male mice were anesthetized, and a midline laparotomy was performed. The intestines were eviscerated, the small bowel mesenteric root was identified, and baseline intestinal perfusion was determined using laser Doppler imaging. Intestinal ischemia was established by temporarily occluding the superior mesenteric artery for 60 min with a noncrushing clamp. After ischemia, the clamp was removed and the intestines were allowed to recover. Before abdominal closure, 2 × 10(6) human umbilical cord-derived MSCs, bone marrow-derived MSCs, or keratinocytes in 250 μL of phosphate-buffered saline vehicle were injected into the peritoneum. Animals were allowed to recover for 12 or 24 h (perfusion, histology, and inflammatory studies) or 7 d (survival studies). Survival data was analyzed using the log-rank test. Perfusion was expressed as a percentage of the baseline, and 12- and 24-h data was analyzed using one-way analysis of variance and the Student t-test. Nonparametric data was compared using the Mann-Whitney U-test. A P value of <0.05 was considered statistically significant.

Results: All MSCs increased 7-d survival after I/R injury and were superior to vehicle and keratinocytes (P < 0.05). All MSCs increased mesenteric perfusion more than vehicle at 12 and 24 h after injury (P < 0.05). All MSCs provided superior perfusion compared with keratinocytes at 24 h after injury (P < 0.05). Administration of each MSC line improved intestinal histology after I/R injury (P < 0.05). Multiple proinflammatory chemokines were downregulated after the application of MSCs, suggesting a decreased inflammatory response after MSC therapy.

Conclusions: Transplantation of MSCs after intestinal I/R injury, irrespective of a tissue source, significantly increases survival and mesenteric perfusion and at the same time limits intestinal damage and inflammation. Further studies are needed to identify the mechanism that these cells use to promote improved outcomes after injury.

Keywords: Human bone marrow mesenchymal stromal cells; Human umbilical mesenchymal stromal cells; Inflammation; Intestinal ischemia; Mortality; Perfusion; Survival.

Figure 1

Supernatant cytokine analysis following in vitro experiments with keratinocytes, BMSCs and USCs exposed to noxious stimuli (LPS, TNF). Production of trophic growth factors varied with stromal cell source for IL-6 (A), VEGF (B), EGF (C), and IGF-I (D) *=p<0.05 versus keratinocytes, #=p<0.05 versus BMSCs.

Figure 2

Kaplan-Meier plots for 7 day survival after ischemia-reperfusion injury of each MSC treatment group. Both mesenchymal stromal cell lines significantly increased 7 day survival compared to PBS vehicle control and keratinocytes (p<0.05). No survival benefit was seen with the use of keratinocytes (differentiated cell control). No statistically significant survival difference was observed between mesenchymal stromal cell lines. (*=p<0.05 versus PBS, #=p<0.05 versus keratinocytes)

Figure 3

Mesenchymal stromal cells increase mesenteric perfusion following ischemia. A) Representative images of perfusion in all cells lines at baseline, ischemia, 12 hours reperfusion, and 24 hours reperfusion. B) BMSC and USC therapy significantly increased mesenteric perfusion above vehicle and keratinocytes at both 12 and 24 hours of reperfusion. No differences in mesenteric perfusion were observed between MSC treated groups. (*=p<0.05 versus PBS, #=p<0.05 versus keratinocytes)

Figure 3

Mesenchymal stromal cells increase mesenteric perfusion following ischemia. A) Representative images of perfusion in all cells lines at baseline, ischemia, 12 hours reperfusion, and 24 hours reperfusion. B) BMSC and USC therapy significantly increased mesenteric perfusion above vehicle and keratinocytes at both 12 and 24 hours of reperfusion. No differences in mesenteric perfusion were observed between MSC treated groups. (*=p<0.05 versus PBS, #=p<0.05 versus keratinocytes)

Figure 4

Histological examination of small intestine following intestinal ischemia (I/R) and MSC treatment. A) Representative histology slides of each treatment group (hematoxylin and eosin stain, ×40). B) Histological scoring of intestinal specimens: 0, no damage; 1, sub-epithelial space at the villous tip; 2, loss of mucosal lining of the villous tip; 3, loss of less than half of the villous structure; 4, loss of more than half of the villous structure; and 5, transmural necrosis. Statistically significant improvement in histological grade in both MSC lines compared to PBS (*) and keratinocytes (#) respectively (*,# p=<0.05).

Figure 5

Cytokine analysis following I/R injury and treatment at 12 and 24 hours. Following I/R mouse intestines were probed for murine expression of IL-6 (A–B), Mip1α (C–D), Mip2α (E–F), and IP-10 (G–H) *=p<0.05 versus PBS, #=p<0.05 versus Keratinocytes.