Establishment of a human endometrial cell culture system and characterization of its polarized hormone responsive epithelial cells

Abstract

Uterine epithelial cells from normal human endometrium were cultured as a primary cell culture in a dual-chambered system. The epithelial cells were isolated from endometrial tissue of the proliferative phase obtained by hysterectomy. The epithelial cells were seeded on Millicell CM filters coated with the extracellular matrix Matrigel. Depending on the culture conditions, the epithelial cells formed a polarized cell monolayer on Matrigel or gland-like structures in Matrigel. The epithelial cell polarity was maintained during culture, which could be proved by electron microscopy. The progesterone and estrogen receptors as typical marker molecules for physiologically intact endometrial epithelial cells could be detected immunohistochemically as well as by RT-PCR in vitro and were down-regulated by medroxyprogesterone acetate (MPA) used as progesterone analogue. As this cell culture system exhibits morphological and immunohistochemical characteristics, typical for the in vivo situation, and since it can be modulated by hormone treatment under the in vitro conditions described, it represents a valuable tool for investigating processes that are essential for endometrial differentiation and reproductive functions.

Regulation of functional steroid receptors and ligand-induced responses in telomerase-immortalized human endometrial epithelial cells

“…hTERT-EECs produced a functional ER-alpha that was clearly responsive to estrogen or the synthetic estrogenic compound DES, as demonstrated by a strong induction of luciferase using a ERE-luciferase reporter plasmid. In agreement with observations in primary human EECs (Zhang et al 1995, Classen-Linke et al 1997 and Ishikawa cells (Lessey et al 1996), E2 induced PR expression in hTERT-EECs, which is a classic endometrial ER target gene (Milgrom et al 1973, Classen-Linke et al 1997, Brandenberger et al 1999, Saegusa & Okayasu 2000, Borthwick et al 2003. The dose-dependent decrease of PR transcriptional gene activity in the presence of P4 indicated a functional PR signaling pathway in hTERT-EECs as had been described for primary EECs (Classen-Linke et al 2000, Spencer & Bazer 2002.…”

“…Primary human EECs were reported to retain their polarization for a limited time period only (Classen-Linke et al 1997, Negami & Tominaga 1989 and, when grown on ECM material from an Engelbreth-Holm-Swarm tumor (MatrigelR), showed increased protein secretion (Negami & Tominaga 1989). This Matrigel, however, is supplemented with several growth factors including transforming growth factor-, EGF, insulin-like growth factor (IGF)-I, basic fibroblast growth factor and platelet-derived growth factor, and these growth factors and/or the tumor matrix itself may perturb physiological EEC responses.…”

“…Unique even among primates, studies on the molecular dynamics of the human endometrium require appropriate human cellular in vitro model systems. Primary human endometrial monolayers in culture have limited lifespan and undergo cellular de-differentiation (Mulholland et al 1988, Zhang et al 1995, Classen-Linke et al 1997, Arnold et al 2001, Grümmer et al 2001. Together with problems obtaining normal human endometrial tissue, this restricts the use of isolated human endometrial cells or endometrial tissues for experimental in vitro approaches.…”

Creation of tumorigenic human endometrial epithelial cells with intact chromosomes by introducing defined genetic elements

“…However, in classical tissue culture using plastic or glass dishes, endometrial epithelial cells lose their proliferative capacity during ongoing cultivation over several days, whereas stromal cells are relatively easily cultured long-term. Most investigators in this field have tried to develop endometrial cell cultures with a mixture of stromal and epithelial cells and/or organotipic cultures, but only short-term cultures have been obtained using such methods (Akoum et al, 1996;Classen-Linke et al, 1997;Arnold et al, 2001).…”

Successful Immortalization of Endometrial Glandular Cells with Normal Structural and Functional Characteristics

“…Most investigators in this field have tried to develop endometrial cell cultures with a mixture of stromal cells and/or organotypic cultures, but both for short-term experiments only. [1][2][3] The lack of a stable in vitro culture system of endometrial cells also renders studying the molecular carcinogenesis of the endometrium difficult. Most endometrial cancers arise from endometrial glandular cells via the multistep accumulation of abnormalities in oncogenes and tumor suppressor genes, including PTEN, Ras, and p53.…”