Trophic and immunomodulatory effects of adipose tissue derived stem cells in a preclinical murine model of endometriosis

Abstract

Endometriosis, which exhibits enigmatic pathological features such as stromal fibrosis and proliferation of ectopic epithelial cells, is known as a refractory disease. Mesenchymal stem cells modulate the fibrosis in stromal tissues through their trophic and immunomodulatory properties. To investigate the potential of stem cells in treating endometriosis, we examined the secondary morphology and molecular alterations in endometriosis-like lesions after the administration of adipose tissue-derived stem cells (ASCs) to an experimental murine model of endometriosis. The infused ASCs were found integrated in the endometriosis-like lesions. Accompanied by the suppression of stromal fibrosis and proliferation of endometriotic epithelial cells, the infusion of ASCs with stemness potential (early passage of ASCs) suppressed the growth of endometriosis-like lesions and inhibited the expression of pro-inflammatory and pro-fibrotic cytokines, whereas no significant attenuation of endometriosis-like lesions occurred after the infusion of ASCs without stemness potential (late passage of ASCs). Accordingly, the trophic and immunomodulatory properties of ASCs may regulate fibrosis in endometriosis-like lesions, suggesting that regenerative medicine could be recognized as an innovative treatment for patients with endometriosis through the accumulation of evidence of preclinical efficacy.

Figure 1

ASC stemness potential in endometriosis-like lesions. (A) Multicolor flow cytometry analysis of cells in the plot of singlet cells based on staining for CD105, CD29, Sca-1, and CD45. The percentages of mesenchymal stem cells, including early passage (3rd passage) adipose tissue-derived stem cells (ASCs), and late passage (20th passage) ASCs, harboring the cell-surface expression markers CD29, Sca-1, and CD105, and negative CD45 are shown. (B) Changes in secondary morphology of endometriosis-like lesion configuration after treatment with ASCs. Control intravenous administration of only PBS on day 1, E-ASCs-D1 intravenous administration of early passage ASCs on day 1, L-ASCs-D1 intravenous administration of late passage ASCs on day 1, E-ASCs-D15 intravenous administration of early passage ASCs on day 15, E-ASCs-D15 intravenous administration of late passage ASCs on day 15. Mice were sacrificed on day 28. n = 10 per group. Data are presented as the mean ± standard error. *P < 0.05 vs. Control.

Figure 2

Macroscopic alterations and histopathology of endometriosis-like lesions after treatment with ASCs. (A) Representative excised implants from the endometriosis-like lesions. The interval at the bottom represents 1 mm. (B–D) Representative images of pathological specimens from excised implants stained with hematoxylin and eosin (B), Masson’s trichrome (C), and Ki67 (D). The red arrow in (B) indicates the maximum stromal thickness from the bottom of the endometrium to the surface of each cyst. The blue and red stains in (C) indicate collagen fibers and cells (mainly: fibroblasts) in the endometriosis-like lesions. (E) Quantification of maximum stromal thickness from the bottom of the endometrium to the surface of each cyst (top) and the percentage of Ki67-positive cells stained in the endometriotic epithelial cells (bottom). Data are presented as the mean ± standard error. *P < 0.01 vs. Control.

Figure 3

Changes in gene expression in endometriosis-like lesions after infusion of ASCs. Quantitative RT-PCR of pro-inflammatory (Il6, Mcp1, Lif), pro-fibrotic (Tgfb1), and anti-inflammatory cytokine (Il4, Il10) as well as angiogenetic factor (Vegfa), matrix metalloprotease (Mmp2, Mmp9), and hormone receptor (Er1, Pgr) gene expression levels in endometriosis-like lesions. Each group included 10 mice. Data are presented as the mean ± standard error. *P < 0.05 vs. Control.

Figure 4

Localization of KuO-ASCs after intravenous administration. (A–C) Phase-contrast and fluorescence microscopy showing the cell morphology of Kusabira Orange (KuO)-expressing ASCs (A); macroscopic localization of KuO-ASCs in the endometriosis-like lesions (B); and microscopic localization of KuO-ASCs in the endometriosis-like lesions (C). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI).