Differential expression of Wnt signalling molecules between pre- and post-menopausal endometrial epithelial cells suggests that a population of putative epithelial stem/progenitor cells reside in the basalis layer

The human endometrium is a highly regenerative tissue that undergoes cyclical proliferation, differentiation and shedding each month. The upper functionalis layer of the endometrium is shed in response to circulating levels of estrogen and progesterone, while the lower basalis layer remains. Clonogenic epithelial stem/progenitor cells likely responsible for regenerating endometrial epithelium have been identified in pre-menopausal (Pre-M) and post-menopausal (Post-M) endometrium and may reside in the basalis layer. We undertook a transcriptional profiling of purified epithelial cells from full-thickness Pre-M and Post-M endometrium to identify differentially expressed genes. The hypothesis tested in the present study was that Post-M endometrial epithelial gene profile would be similar to the quiescent basalis epithelium of Pre-M endometrium. We found striking differential gene expression of many Wnt family members between Pre-M and Post-M and other stem cell network genes. Comparative analysis of our endometrial epithelial gene expression profiles to that of endometrial epithelial cells in remodelling endometrium also provides new evidence showing that Post-M endometrial epithelium has a similar gene signature to that of basalis epithelium of menstrual endometrium. Human endometrial tissue was obtained from 8 pre-menopausal and 3 post-menopausal women undergoing hysterectomy for various benign gynaecologic conditions. Endometrial tissue was digested and isolated using combination of DNase and collagenase. Anti-human EpCAM antibody-coated magnetic Dynabeads was used to positively select total epithelial cells from the digested single cell suspensions. Total RNA was extracted from the purified endometrial epithelial cells and hybridised to Illumina Sentrix HT12 beadchip. Resulting data was compared between pre-menopausal and post-menopausal samples.

人类子宫内膜是一种高度再生的组织，每月均会经历周期性的增殖、分化与脱落。子宫内膜的上层功能层会随循环中雌激素与孕激素的水平变化而脱落，而下层基底层则得以保留。现已在绝经前（Pre-M）与绝经后（Post-M）子宫内膜中，鉴定出可能负责再生子宫内膜上皮的克隆源性上皮干细胞/祖细胞，这类细胞大概率定位于基底层中。本研究对取自全层绝经前及绝经后子宫内膜的纯化上皮细胞开展转录组分析，以筛选差异表达基因。本研究验证的假说为：绝经后子宫内膜上皮的基因表达谱，与绝经前子宫内膜处于静息状态的基底层上皮相似。研究发现，绝经前与绝经后样本间，众多Wnt家族成员及其他干细胞网络相关基因均存在显著的差异表达。将本研究获得的子宫内膜上皮基因表达谱，与处于重塑过程中的子宫内膜上皮细胞的表达谱进行对比分析后，本研究还获得了新证据：绝经后子宫内膜上皮的基因特征，与月经周期中子宫内膜基底层上皮的基因特征高度相似。本研究的人类子宫内膜组织取自8名因各类良性妇科疾病接受子宫切除术的绝经前女性，以及3名绝经后女性。研究采用脱氧核糖核酸酶（DNase）与胶原酶联合消化的方法，对子宫内膜组织进行解离与分离；随后使用包被抗人上皮细胞黏附分子（EpCAM）抗体的磁珠（Dynabeads），从消化得到的单细胞悬液中正向分选得到全部上皮细胞。从纯化获得的子宫内膜上皮细胞中提取总RNA，将其与Illumina Sentrix HT12微珠芯片进行杂交，最终对绝经前与绝经后样本的检测数据开展对比分析。