Assessment of multipotency and response of cytokines on ovarian endometriotic stromal clonogenic cells

Abstract

Introduction: Endometriosis, defined as the growth of endometrial tissue outside the uterine cavity is a common gynaecological disorder affecting 10-15% of women in their reproductive years. It is a major clinical problem causing in- flammation, pain and infertility. Despite its common occurrence little is known about its pathogenesis. It has been postulated that endometriosis is developed as a result of inappropriate menstrual shedding of the endometrial tissue consisting of stem/progenitor cells, which reach the peritoneal cavity or ovaries and establish endometriotic lesions. We have demonstrated the existence of stemlike cells based on their clonogenic activity and self-renewal capacity from ovarian endometriosis. However, full characterization of these cells remains to be determined. Endometriosis is often associated with inflammation. The action of inflammatory cytokines on endometriotic stem/progenitor cells is not known. Therefore the aims of this study were to 1) examine the impact of cytokines on the clonogenicity of endometriotic stromal cells and, 2) assess the multipotency of endometriotic stromal stem/progenitor cells. Methods: Ovarian endometriotic cysts were obtained from women undergoing laparoscopic ovarian cystectomy. The tissues were dissociated with collagenase to achieve single cell suspensions. Epithelial and stromal cells were separated using Ber-EP4 Dynabeads and cultured at low seeding density (500 cells/cm2) in serum-containing medium or serum-containing medium supplemented with either: basic fibroblast growth factor (bFGF), interleukin-1B (IL-1B), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-13 (IL-13), tumor necrosis factor-A (TNF-A) and interferon gamma (IFN-G). Cells were cultured for 21 days and colonies were stained with hematoxylin to determine the cloning efficiencies (CE). For in vitro differentiation, isolated endometriotic stromal large clones were trypsinized and pooled together for culture expansion. Medium used for induction into the mesenchymal stem cell (MSC) lineages was adapted from Pittenger et al. Cells were grown in the medium for 4 weeks prior to histological or immunohistochemical differentiation assays. RT-PCR analysis was conducted to confirm the expression of specific differentiation markers. Results: The total CE of stromal endometriotic cells treated with bFGF was 0.14 p 0.03% when compared to untreated samples 0.06 p 0.04% (n = 4). Interestingly, the number of large clones after bFGF treatment (0.11 p 0.03%) was significantly higher (p 0.05) than the untreated samples (0.013 p 0.01%). IL-8, IL-10 and TNF-A slightly supported clonogenicity of endometriotic stromal cells when compared with their corresponding control, though the differences were not yet significantly different (n = 4). In contrast, IL-6, IL-13 and IFN-G did not support clonogenic activity. Cells obtained from large endometriotic stromal clones were able to differentiate into MSC lineages: adipogenesis was indicated by accumulation of neutral lipid vacuoles with the fluorescence lipid stain and detection of the lipid specific marker peroxisome proliferator-activated receptor 2. Chondrogenesis was shown by staining with alcian blue and safranin O, and expression of chondrogenic marker collagen type X and collagen type II. Osteogenesis was indicated by the increase in alkaline phosphatase and osteopontin with expression for core binding factor alpha 1 and parathyroid hormone receptor type 1. Myogenesis was shown by alpha smooth muscle actin and expression with markers for contractile smooth muscle cell, calponin and caldesmon. None of the cells grown in the control medium stained in any of above histological or immunohistchemical differentiation assays. Conclusion: In this study, the addition of angiogenic cytokine (bFGF) promoted the proliferation of endometriotic stromal stem-like cells and 3 cytokines that might support clonogenicity were identified. Endometriotic stromal large clones could differentiate into adipogenic, chondrogenic osteoblastic, and myogenic lineages when cultured under their respective inducing-medium. This finding suggests that in ovarian endometriotic lesions, there is a subpopulation of cells which have the ability to proliferate extensively, and maintain their ability to differentiate into multiple cell types in vitro (multipotency), establishing their stem cell nature.