Therapeutic effects of human amnion-derived mesenchymal stem cell transplantation and conditioned medium enema in rats with trinitrobenzene sulfonic acid-induced colitis

Abstract

Cell therapy with mesenchymal stem cells (MSCs) is expected to provide a new strategy for the treatment of inflammatory bowel disease (IBD). Large amounts of MSCs can be obtained from human amnion. Therefore, we investigated the effect of transplantation of human amnion-derived MSCs (hAMSCs) or enema of conditioned medium (CM) from hAMSCs into rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. In the first experiment, 10-week-old male Sprague-Dawley rats were intravenously injected with hAMSCs (1 × 10⁶ cells) 3 h after rectal administration of TNBS (45 mg/kg). In the second experiment, rats with TNBS-induced colitis received CM by enema into the colon for 3 days. Colitis was investigated by endoscopy, histology, immunohistochemistry, and by measuring mRNA expression of inflammatory mediators. Administration of hAMSCs or CM enema significantly improved the endoscopic score. In addition, these two interventions resulted in significantly decreased infiltration of neutrophils and monocytes/macrophages and decreased expression levels of TNF-α, CXCL1, and CCL2. In conclusion, transplantation of hAMSCs and CM enema provided significant improvement in rats with TNBS-induced colitis. CM from hAMSCs and hAMSCs may be new strategies for the treatment of IBD.

Keywords: Mesenchymal stem cells; amnion; colitis; conditioned medium; trinitrobenzene sulfonic acid.

Figure 1

Experimental protocol for 2,4,6-trinitrobenzene sulfonic acid (TNBS)-colitis model. Experiment 1: protocol for the human amnion-derived mesenchymal stem cell (hAMSC) transplantation model. Rats received enemas of TNBS on day 0 and 1 × 10⁶ hAMSCs were intravenously infused 3 h later. Experiment 2: protocol for the enema of conditioned medium (CM) model. Rats received enemas of TNBS on day 0 and CM was intrarectally infused 3, 24, and 48 h later.

Figure 2

Characterization of cultured human amnion-derived mesenchymal stem cells (hAMSCs). A. Multipotency of hAMSCs. Differentiation into adipocytes was confirmed by the presence of lipid vesicles stained with oil red O (left panel). Scale bars, 50 µm. Differentiation into osteocytes was confirmed by the existence of mineral nodule deposition stained with alizarin red S (right panel). Scale bars, 200 µm. B. Flow cytometry of hAMSCs. The negative cocktail contained antibodies against CD11b, CD19, CD34, CD45, and HLA-DR. Closed areas indicate staining with a specific antibody, whereas open areas represent staining with isotype control antibodies.

Figure 3

Effect of human amnion-derived mesenchymal stem cell (hAMSC) transplantation in rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. A. Endoscopic imaging. B. Endoscopic score. The following parameters were analyzed and graded as 0 (absent) or 1 (present): changes in the vascular pattern; mucosal granularity; strictures; bleeding; and ulcers (total score ranging therefore from 0 to 5) [20]. Values are expressed as means ± SEM (N = 6-9 animals/group). ††P < 0.01 vs. the control group. **P < 0.01 vs. the TNBS group.

Figure 4

Effect of human amnion-derived mesenchymal stem cell (hAMSC) transplantation on the histological score and infiltration of inflammatory cells. A. Hematoxylin and eosin (H&E) staining and histological score. B. Myeloperoxidase (MPO) staining. C. CD68 staining. D. CD3 staining. The number of positive cells was counted in 10 sections/sample in each low-power field. Scale bars, 200 μm. Values are expressed as means ± SEM (N = 6-9 animals/group). †P < 0.05, ††P < 0.01 vs. the control group. *P < 0.05, **P < 0.01 vs. the 2,4,6-trinitrobenzene sulfonic acid (TNBS) group.

Figure 5

Effect of human amnion-derived mesenchymal stem cell (hAMSC) transplantation on mRNA expression of inflammatory mediators in the colon of rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with tumor necrosis factor (TNF)-α, chemokine (C-X-C motif) ligand (CXCL) 1, chemokine (C-C motif) ligand (CCL) 2, and interleukin (IL)-6. Values are expressed as means ± SEM (N = 6-9 animals/group).

Figure 6

Effect of human amnion-derived mesenchymal stem cell conditioned medium (hAMSC-CM) in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. A. Endoscopic imaging. B. Endoscopic score. The following parameters were analyzed and graded as 0 (absent) or 1 (present): changes in the vascular pattern; mucosal granularity; strictures; bleeding; and ulcers (total score ranging therefore from 0 to 5) [20]. Values are expressed as means ± SEM (N = 6-9 animals/group). ††P < 0.01 vs. the Control group, **P < 0.01 vs. the TNBS+ standard medium (SM) gel group.

Figure 7

Effect of conditioned medium obtained from human amnion-derived mesenchymal stem cell culture (hAMSC-CM) on the histological score and infiltration of inflammatory cells. A. Hematoxylin and eosin (H&E) staining and histological score. B. Myeloperoxidase (MPO) staining. C. CD68 staining. D. CD3 staining. The number of positive cells was counted in 10 sections per sample in each low-power field. Scale bars, 200 µm. Values are expressed as means ± SEM (N = 6-9 animals/group). ††P < 0.01 vs. the control group, **P < 0.01 vs. the 2,4,6-trinitrobenzene sulfonic acid (TNBS) + standard medium (SM) gel group.

Figure 8

Effect of conditioned medium obtained from human amnion-derived mesenchymal stem cell culture (hAMSC-CM) on mRNA expression of inflammatory mediators in the colon of rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with tumor necrosis factor (TNF)-α, chemokine (C-X-C motif) ligand (CXCL) 1, chemokine (C-C motif) ligand (CCL) 2, and interleukin (IL)-6. Values are expressed as means ± SEM (N = 6-9 animals/group).