Endometrial regenerative cells as a novel cell therapy attenuate experimental colitis in mice

Abstract

Background: Endometrial regenerative cells (ERCs) are mesenchymal-like stem cells that can be non-invasively obtained from menstrual blood and are easily grown /generated at a large scale without tumorigenesis. We previously reported that ERCs exhibit unique immunoregulatory properties in vitro, however their immunosuppressive potential in protecting the colon from colitis has not been investigated. The present study was undertaken to determine the efficacy of ERCs in mediating immunomodulatory functions against colitis.

Methods: Colitis was induced by 4% dextran-sulfate-sodium (DSS, in drinking water) in BALB/c mice for 7 days. ERCs were cultured from healthy female menstrual blood, and injected (1 million/mouse/day, i.v.) into mice on days 2, 5, and 8 following colitis induction. Colonic and splenic tissues were collected on day 14 post-DSS-induction. Clinical signs, disease activity index (DAI), pathological and immunohistological changes, cytokine profiles and cell populations were evaluated.

Results: DSS-induced mice in untreated group developed severe colitis, characterized by body-weight loss, bloody stool, diarrhea, mucosal ulceration and colon shortening, as well as pathological changes of intra-colon cell infiltrations of neutrophils and Mac-1 positive cells. Notably, ERCs attenuated colitis with significantly reduced DAI, decreased levels of intra-colon IL-2 and TNF-α, but increased expressions of IL-4 and IL-10. Compared with those of untreated colitis mice, splenic dendritic cells isolated from ERC-treated mice exhibited significantly decreased MHC-II expression. ERC-treated mice also demonstrated much less CD3(+)CD25(+) active T cell and CD3(+)CD8(+) T cell population and significantly higher level of CD4(+)CD25(+)Foxp3(+) Treg cells.

Conclusions: This study demonstrated novel anti-inflammatory and immunosuppressive effects of ERCs in attenuating colitis in mice, and suggested that the unique features of ERCs make them a promising therapeutic tool for the treatment of ulcerative colitis.

Figure 1

ERCs attenuate the development of experimental colitis represented by DAI. A) Representative photo showing bloody stool in untreated colitis mice as compared to ERC-treated colitis mice. B) DAI, scored daily to assess the colitis activity, in different groups (n = 8 mice per group). *p < 0.05, ERC-treated group vs. untreated group on day 14 post-colitis induction.

Figure 2

ERCs attenuate the pathological changes of experimental colitis. A) The length of mouse colon from normal, untreated and ERC-treated groups. *p < 0.05, ERC-treated group vs. untreated group. B) Representative photomicrographs (200×, haemotoxylin and eosin staining) of histological sections of colon from normal, untreated and ERC-treated groups. Overall histology scores in the normal, untreated and ERC-treated groups (n = 8 mice per group). *p < 0.05, ERC-treated group vs. untreated group.

Figure 3

ERCs reduce intra-colon neutrophil and Mac-1 positive cell infiltration in the colitis mice. The levels of intra-colon MPO and Mac-1 positive cell infiltration in normal, untreated and ERC-treated groups. Magnification 400×. (n = 8 mice per group) *p < 0.05, ERC-treated group vs. untreated group.

Figure 4

Effects of ERCs on the regulation of IL-2, TNF- α , IL-4 and IL-10 expressions in mice. The mRNA levels of IL-2, TNF-α, IL-4 and IL-10 were analyzed by real-time PCR. A&B) ERCs reduced IL-2 and TNF-α mRNA expression in colon tissues in DSS-induced colitis mice. (n = 8 mice per group) ^# p < 0.01, untreated group vs. normal mice and the ERC-treated group. C&D) ERCs increased IL-4 and IL-10 mRNA expression in colon tissues in DSS-induced colitis mice. E) ERCs reduced IL-2 and TNF-α mRNA expression in the spleen in DSS-induced colitis mice. (n = 8 mice per group) *p < 0.01, ERC-treated group vs. untreated group.

Figure 5

ERCs modulate the levels of immune cells in the spleen of colitis mice. The levels of immune cells were analyzed by flow cytometry in different groups. A) The level of CD3⁺CD25⁺ cells in the spleen from normal, untreated and ERC-treated groups. B) The level of CD3⁺CD8⁺ cells in the spleen from normal, untreated and ERC-treated groups. C) The level of CD4⁺CD25⁺ Foxp3⁺ Tregs in the spleen from normal, untreated and ERC-treated groups. D) The level of CD11c⁺MHC-II⁺ DCs in the spleen from normal, untreated and ERC-treated groups. (n = 8 mice per group) *p < 0.01, ERC-treated group vs. untreated group.