Development and characterization of a polarized human endometrial cell epithelia in an air–liquid interface state
Human endometrial epithelia undergo injury repair and regeneration with the menstrual cycle; however, mechanisms underpinning the roles of endometrial epithelial cells in endometrial lesions and regeneration remain incompletely understood, mainly owing to the difficulty in the isolation and expansion of primary endometrial epithelial cells and the lack of reliable models for in vitro and in vivo studies. In this report, we sought to improve methods for the isolation and expansion of human endometrial epithelial cells with a Rho-associated protein kinase (ROCK) inhibitor–modified medium and subsequently characterize endometrial epithelium generated with primary cells cultured in an air–liquid interface (ALI) state. Immunocytochemistry staining revealed the expression of epithelial cellular adhesion molecule (EpCam) and stage-specific embryonic antigen-1 (SSEA-1) but a lack of CD13 in endometrial epithelial cells. Meanwhile, a large number of proliferative Ki67+ cells were observed in isolated epithelial cells. Importantly, the EpCam+/CD13− cells were capable of forming spheroids, a characteristic of epithelial stem/progenitor cells. Interestingly, these cells also exhibited a capacity to reconstitute epithelial layers in an ALI state. Morphological analysis revealed mucosal secretion of differentiated epithelial cells with cilia and microvilli in ALI epithelial cells as determined by electronic microscopy. Immunoblotting assay further demonstrated the expression of endometrial epithelial cell markers keratin 17/19 and EpCam and stem cell marker OCT3/4 but not stromal cell marker Vimentin protein and CD13 in cell expansions. Furthermore, molecular analysis also showed that the exposure of cells to estrogen elevated the expression of estrogen receptor and progesterone receptors in ALI cultures. Our results shed light on the possibility of expanding sufficient numbers of endometrial epithelial cells for stem cell biology studies, and they provide a feasible and alternative model that can recapitulate the characteristics and physiology of endometrial epithelium in vivo.
KeywordsEndometrium Epithelial cells Stem cells Air–liquid interface Estrogen
Epithelial cellular adhesion molecule
- OCT 3/4
Octamer-binding transcription factor 3/4
Rho-associated protein kinase
Stage-specific embryonic antigen-1
Endometrium is highly regenerative tissue that undergoes a cycle of proliferation, differentiation, shedding, and regeneration 400 times during the menstrual cycle under the control of estrogen or progesterone [1, 2]. It has been demonstrated that endometrial epithelial stem cells play an important role in this repair process and in the integrity and function of endometrium [3, 4]. However, owing to the frequent uterine operation or infection of endometrium, the incidence of endometrial diseases such as intrauterine adhesions has increased in recent years  and this has had a severe impact on quality of life for women .
Nowadays, our understanding of the biology and function of stem cells in endometrial gland and epithelium is limited by the difficulty in endometrial epithelial cell isolation and culturing, and in stem cell identification and the lack of reliable in vitro models. In this report, we described methods for the isolation and culture of human endometrial epithelial cells and characterization of an air–liquid interface (ALI) culture model generated with human endometrial epithelial cells. This study may provide simple and efficient methods for human endometrial epithelial cell isolation and expansion for stem cell biology study, and a reliable and feasible model to recapitulate human endometrium in vivo, which can be employed for investigation into the biology and function of human endometrial epithelial stem cells in vitro.
Materials and methods
Ethnic statement and human endometrial tissue procession
The study and protocol were approved by the ethics committee for conducting human research at the General Hospital of Ningxia Medical University (NXMU-2017-063). All patients analyzed were above 25 years old and were given informed consents. Biopsies of human endometrium samples were obtained from the premenopausal women undergoing hysteroscopy at the General Hospital of Ningxia Medical University. Tissues from 12 donors were analyzed in this study. The endometrium was scraped off and collected into D-Hanks phosphate-buffered saline (PBS) at 4 °C and was subsequently treated for isolation of cells within 2–4 h. Detailed information on materials and methods is provided as supplemental data (Additional file 1), and the antibodies used in this work are listed in Additional file 2.
Isolation and culture of human endometrial glandular epithelial cells
The isolation of human endometrial epithelial cells was conducted as described in a previous study with minimal modification . Briefly, the human endometrial biopsy was minced with scissors into small pieces of less than 1 mm3 before it was washed with PBS containing antibiotics. Subsequently, the minced biopsies were dissociated in a dissociation buffer containing 3.0 mg/mL collagenase type 4 (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 7 min at 37 °C with gentle agitation. Then the same volume of Accumax (Innovative Cell Technologies, San Diego, CA, USA) was added in the dissociated solution and incubated for an additional 7 min at 37 °C with continuous agitation. The digestive reaction was terminated by adding fetal bovine serum to the dissociated fragment/cell suspension in a final concentration of 10%. The dissociated fragment/cell suspension was filtered through a 400-mesh nylon sieve, and residual cell clumps on the sieve were glandular epithelial cells and were washed off with D-Hanks into a tube. The cells were collected by centrifugation (100g for 5 min), resuspended in 2 mL of culture medium containing 10 μM of Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Sigma-Aldrich), and seeded onto a 10-cm culture dish pre-coated with 70 μg/mL collagen type I rat tail (BD Biosciences, Franklin Lakes, NJ, USA). The cells were maintained in the culture medium at 37 °C in a humidified environment with 5% CO2. The adherent cells were dissociated by using Accutase solution (Millipore, Burlington, MA, USA) at 2–3 days after seeding, and the cells were reseeded at a ratio of 1:3–5 for passage. Other materials and methods are provided as supplemental data in Additional files 1 and 2.
Isolation and ALI culture of human endometrial epithelial cells
Morphological analysis of endometrial epithelial cell ALI cultures
Immunological characterization of endometrial glandular epithelial cell ALI cultures
Molecular characterization of endometrial glandular epithelial cell ALI cultures
Discussion and conclusion
In this report, we described the isolation and expansion culture of human endometrial epithelial cells and the characteristics of endometrial epithelial cells cultured in a three-dimensional (3D) ALI state. We showed that a subset of endometrial epithelial cells had potential for spheroid formation, epithelium regeneration, and differentiation into stromal-like cells. This study thus introduces a useful approach for efficient isolation and expansion of human endometrial epithelial cells in stem cell biology research and possibly in studies of autologous cell transplantation therapy for endometrial injury diseases. In addition, the human endometrial epithelial ALI culture may be a feasible and reliable model for investigating the biological characteristics and mechanisms of endometrial epithelial cells or endometrial diseases in vitro. Together with the capacity of epithelial reconstitution and stromal cell differentiation in the ALI state demonstrated by others, our results imply that a subset of endometrial epithelial cells may retain their stem/progenitor cell potency for proliferation and differentiation.
The endometrium is a highly and cyclically regenerating organ by regulating hormones . Estrogen plays an important part in the development and regeneration or repair of injured endometrium [11, 12]. In this context, estrogen could stimulate the proliferation of endometrial epithelial cells at every menstrual cycle in a normal endometrium by binding to ER and PR . In this study, we also demonstrated an induced augmentation of ER and PR along with the increased expression of EpCam, N-cadherin, and CD13 in human ALI endometrial epithelial culture in the presence of progesterone. These data imply that progesterone-promoted proliferation and differentiation of endometrial epithelial cells occur in ALI endometrium, which is similar to the response of endometrial epithelial cells in utero in vivo, suggesting the reliability and feasibility of ALI endometrial epithelium as an in vitro 3D model for mimicking endometrial epithelium in vivo.
In conclusion, this report described methods for the isolation and expansion of human endometrial epithelial cells and generation of human endometrial ALI epithelium. The ALI culture may offer a reliable and feasible model for biomedical research and stem cell biology studies of human endometrium in vitro. However, the limited passages of primary endometrial epithelial cells using ROCK inhibitor–modified medium is a methodological limitation of this study, and further optimizations of culture media or conditions for unlimited culture are required for future autologous endometrial epithelial cell transplantation research in vivo.
This study was supported by the First-Class Discipline Construction Founded Project of Ningxia Medical University and the School of Clinical Medicine (NXYLXK2017A05), a grant from the National Natural Science Foundation of China (no. 31472191) to XL, a grant from the Natural Science Foundation of Ningxia (NZ15172), and internal supporting grants of Ningxia Medical University (XY2017172 and XY201706). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
JY and DL conceived and designed the experiments. DDL, HL, and YW conducted the experiments, analyzed data, and drafted the manuscript. AE, JW, YC, JX, JS, and YJ performed experiments and acquired data. SH, CH, and XL processed biopsies. JY and DL interpreted data and critically revised the manuscript. All authors read and approved the final version of the manuscript.
The study and protocol were approved by the ethics committee for conducting human research at the General Hospital of Ningxia Medical University (NXMU-2017-063).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 5.Hooker AB, de Leeuw R, van de Ven PM, Bakkum EA, Thurkow AL, Vogel NEA, et al. Prevalence of intrauterine adhesions after the application of hyaluronic acid gel after dilatation and curettage in women with at least one previous curettage: short-term outcomes of a multicenter, prospective randomized controlled trial. Fertil Steril. 2017;107(5):1223–31. e1223CrossRefPubMedGoogle Scholar
- 8.Farghaly LM, Atwa KA. Histological changes in the endometrium of women suffering from recurrent pregnancy loss: light and electron microscopic study. Egypt J Histol. 2010;33(2):224–35.Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.