Clonogenicity of Human Endometrial Epithelial and Stromal Cells1

Abstract

The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single- cell suspensions of epithelial and stromal cells were cultured at cloning density (300-500/cm(2)) in serum medium or in serum- free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF- I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGF alpha, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF- I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except alpha(6)-integrin. All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified. This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.

Identification and hormonal regulation of a novel form of NKp30 in human endometrial epithelium

“…5B) as demonstrated previously [13], negative for CD56 by IHC (Fig. 5B) and negative for both CD56 and CD45 by flow cytometry (Fig.…”

“…It is assumed that the colony-initiating cell is an endometrial epithelial progenitor or stem cell, which on continued culture produces large numbers of differentiated epithelial cells [13,30,31]. The Nkp30 gene and protein expression in clonally derived epithelial cells supports this assumption and could possibly serve as a differentiation marker of endometrial epithelial cells.…”

“…Endometrium attached to 5 mm of myometrium was also collected from five hysterectomy samples obtained from ovulating women not taking exogenous hormones for 3 months prior to surgery as described in [13].…”

“…Endometrial tissue was scraped from the underlying myometrium and dissociated with collagenase to produce single-cell suspensions in HEPES-buffered DMED/F-12/5% NCS as described in Chan et al [13]. After filtration through a 40-lm sieve (Becton Dickinson Labware, Franklin Lakes, NJ), red blood cells were removed by centrifugation through FicollPaque (Pharmacia Biotechnology, Uppsala, Sweden).…”

“…Endometrial epithelial cells were obtained by positive selection using BerEP4-coated magnetic Dynabeads (Dynal Biotech, Oslo, Norway) and the supernatant containing stromal cells and leukocytes was also collected. The stromal cells were then negatively selected using anti-CD45 antibody-coated Dynabeads (Dynal Biotech) to remove leukocytes [13]. To establish clones of epithelial and stromal cells initiated by single cells, beaded endometrial epithelial cell and stromal cell suspensions were seeded at very low seeding density, 10-50 cells/cm 2 on six to nine 10-cm culture dishes (Becton Dickinson Labware) coated with gelatin (Sigma-Aldrich, St. Louis, MO) for stromal cells and cultured in serum medium containing bicarbonatebuffered DMEM/F-12 medium, 10% fetal calf serum (FCS; CSL Ltd), 2 mM glutamine (Invitrogen Life Technologies, Australia) and antibiotic-antimycotic solution and cultured at 37 C in 5% CO 2 in air for 20-30 days.…”