Posters * Endometriosis, Endometrium and Implantation
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Basic fibroblast growth factor increased endometriotic stromal cell clonogenicity, and these cells differentiated into multiple lineages, confirming their stem cell nature.
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Abstract
Introduction: Endometriosis, defined as the growth of endometrial tissue outside
\nthe uterine cavity is a common gynaecological disorder affecting 10-15%
\nof women in their reproductive years. It is a major clinical problem causing in-
\nflammation, pain and infertility. Despite its common occurrence little is known
\nabout its pathogenesis. It has been postulated that endometriosis is developed as
\na result of inappropriate menstrual shedding of the endometrial tissue consisting
\nof stem/progenitor cells, which reach the peritoneal cavity or ovaries and
\nestablish endometriotic lesions. We have demonstrated the existence of stemlike
\ncells based on their clonogenic activity and self-renewal capacity from
\novarian endometriosis. However, full characterization of these cells remains to
\nbe determined. Endometriosis is often associated with inflammation. The action
\nof inflammatory cytokines on endometriotic stem/progenitor cells is not known.
\nTherefore the aims of this study were to 1) examine the impact of cytokines on
\nthe clonogenicity of endometriotic stromal cells and, 2) assess the multipotency
\nof endometriotic stromal stem/progenitor cells.
\nMethods: Ovarian endometriotic cysts were obtained from women undergoing
\nlaparoscopic ovarian cystectomy. The tissues were dissociated with collagenase
\nto achieve single cell suspensions. Epithelial and stromal cells were separated
\nusing Ber-EP4 Dynabeads and cultured at low seeding density (500 cells/cm2) in serum-containing medium or serum-containing medium supplemented with
\neither: basic fibroblast growth factor (bFGF), interleukin-1B (IL-1B), interleukin-8
\n(IL-8), interleukin-10
\n(IL-10), interleukin-13 (IL-13), tumor necrosis factor-A (TNF-A) and interferon
\ngamma (IFN-G). Cells were cultured for 21 days and colonies were stained
\nwith hematoxylin to determine the cloning efficiencies (CE). For in vitro differentiation,
\nisolated endometriotic stromal large clones were trypsinized and
\npooled together for culture expansion. Medium used for induction into the mesenchymal
\nstem cell (MSC) lineages was adapted from Pittenger et al. Cells
\nwere grown in the medium for 4 weeks prior to histological or immunohistochemical
\ndifferentiation assays. RT-PCR analysis was conducted to confirm
\nthe expression of specific differentiation markers.
\nResults: The total CE of stromal endometriotic cells treated with bFGF was
\n0.14 p 0.03% when compared to untreated samples 0.06 p 0.04% (n = 4). Interestingly,
\nthe number of large clones after bFGF treatment (0.11 p 0.03%)
\nwas significantly higher (p 0.05) than the untreated samples (0.013 p 0.01%).
\nIL-8, IL-10 and TNF-A slightly supported clonogenicity of endometriotic
\nstromal cells when compared with their corresponding control, though the differences
\nwere not yet significantly different (n = 4). In contrast, IL-6, IL-13 and
\nIFN-G did not support clonogenic activity.
\nCells obtained from large endometriotic stromal clones were able to differentiate
\ninto MSC lineages: adipogenesis was indicated by accumulation of
\nneutral lipid vacuoles with the fluorescence lipid stain and detection of the lipid
\nspecific marker peroxisome proliferator-activated receptor 2. Chondrogenesis
\nwas shown by staining with alcian blue and safranin O, and expression of
\nchondrogenic marker collagen type X and collagen type II. Osteogenesis was
\nindicated by the increase in alkaline phosphatase and osteopontin with expression
\nfor core binding factor alpha 1 and parathyroid hormone receptor type
\n1. Myogenesis was shown by alpha smooth muscle actin and expression with
\nmarkers for contractile smooth muscle cell, calponin and caldesmon. None of
\nthe cells grown in the control medium stained in any of above histological or
\nimmunohistchemical differentiation assays.
\nConclusion: In this study, the addition of angiogenic cytokine (bFGF) promoted
\nthe proliferation of endometriotic stromal stem-like cells and 3 cytokines that
\nmight support clonogenicity were identified. Endometriotic stromal large clones
\ncould differentiate into adipogenic, chondrogenic osteoblastic, and myogenic
\nlineages when cultured under their respective inducing-medium. This finding
\nsuggests that in ovarian endometriotic lesions, there is a subpopulation of cells
\nwhich have the ability to proliferate extensively, and maintain their ability to differentiate
\ninto multiple cell types in vitro (multipotency), establishing their stem
\ncell nature.
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- last seen: 2026-06-10T17:14:06.276822+00:00
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