{"paper_id":"e4b225a2-2a8c-4b00-b0ce-ba32192c1259","body_text":"S H O R T R E P O R T Open Access\nDevelopment and characterization of a\npolarized human endometrial cell epithelia\nin an air –liquid interface state\nDandan Li 1,2†, Hui Li 1,3†, Ying Wang 1,3†, Ahmed Eldomany 1,3, Jing Wu 3,4, Chao Yuan 5, Jing Xue 1,3, Juan Shi 1,4,\nYuanyuan Jia 1,3, Chunfang Ha 1,2,6, Shuxia Han 1,2,6, Xiaoming Liu 3,5, Jiali Yang 1,4* and Dan Liu 1,2,3,6*\nAbstract\nHuman endometrial epithelia undergo injury repair and regeneration with the menstrual cycle; however,\nmechanisms underpinning the roles of endometrial epithelial cells in endometrial lesions and regeneration remain\nincompletely understood, mainly owing to the difficulty in the isolation and expansion of primary endometrial\nepithelial cells and the lack of reliable models for in vitro and in vivo studies. In this report, we sought to improve\nmethods for the isolation and expansion of human endometrial epithelial cells with a Rho-associated protein kinase\n(ROCK) inhibitor –modified medium and subsequently characterize endometrial epithelium generated with primary\ncells cultured in an air –liquid interface (ALI) state. Immunocytochemistry staining revealed the expression of\nepithelial cellular adhesion molecule (EpCam) and stage-specific embryonic antigen-1 (SSEA-1) but a lack of CD13 in\nendometrial epithelial cells. Meanwhile, a large number of proliferative Ki67 + cells were observed in isolated\nepithelial cells. Importantly, the EpCam +/CD13− cells were capable of forming spheroids, a characteristic of epithelial\nstem/progenitor cells. Interestingly, these cells also exhibited a capacity to reconstitute epithelial layers in an ALI\nstate. Morphological analysis revealed mucosal secretion of differentiated epithelial cells with cilia and microvilli in\nALI epithelial cells as determined by electronic microscopy. Immunoblotting assay further demonstrated the\nexpression of endometrial epithelial cell markers keratin 17/19 and EpCam and stem cell marker OCT3/4 but not\nstromal cell marker Vimentin protein and CD13 in cell expansions. Furthermore, molecular analysis also showed that\nthe exposure of cells to estrogen elevated the expression of estrogen receptor and progesterone receptors in ALI\ncultures. Our results shed light on the possibility of expanding sufficient numbers of endometrial epithelial cells for\nstem cell biology studies, and they provide a feasible and alternative model that can recapitulate the characteristics\nand physiology of endometrial epithelium in vivo .\nKeywords: Endometrium, Epithelial cells, Stem cells, Air –liquid interface, Estrogen\nIntroduction\nEndometrium is highly regenerative tissue that under-\ngoes a cycle of proliferation, differentiation, shedding,\nand regeneration 400 times during the menstrual\ncycle under the control of estrogen or progesterone\n[1, 2]. It has been demonstrated that endometrial epi-\nthelial stem cells play an important role in this repair\nprocess and in the integrity and function of endomet-\nrium [ 3, 4]. However, owing to the frequent uterine\noperation or infection of endometrium, the incidence\nof endometrial diseases such as intrauterine adhesions\nhas increased in recent years [ 5]a n dt h i sh a sh a da\nsevere impact on quality of life for women [ 6].\nNowadays, our understanding of the biology and\nfunction of stem cells in endometrial gland and epi-\nthelium is limited by the difficulty in endometrial\nepithelial cell isolation and culturing, and in stem cell\nidentification and the lack of reliable in vitro models. In\nthis report, we described methods for the isolation and\n* Correspondence: yangjiali_123@163.com; nxld@163.com\n†Dandan Li, Hui Li and Ying Wang contributed equally to this work.\n1College of Clinical Medicine, Ningxia Medical University, Yinchuan 750004,\nNingxia, China\nFull list of author information is available at the end of the article\n© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0\nInternational License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and\nreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to\nthe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver\n(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.\nLi et al. Stem Cell Research & Therapy  (2018) 9:209 \nhttps://doi.org/10.1186/s13287-018-0962-6\n\nculture of human endometrial epithelial cells and\ncharacterization of an air –liquid interface (ALI) culture\nmodel generated with human endometrial epithelial cells.\nThis study may provide simple and efficient methods\nfor human endometrial epithelial cell isolation and\nexpansion for stem cell biology study, and a reliable\nand feasible model to recapitulate human endometrium\nin vivo , which can be employed for investigation into\nthe biology and function of human endometrial epithe-\nlial stem cells in vitro .\nMaterials and methods\nEthnic statement and human endometrial tissue procession\nThe study and protocol were approved by the ethics\ncommittee for conducting human research at the\nGeneral Hospital of Ningxia Medical University\n(NXMU-2017-063). All patients analyzed were above\n25 years old and were given informed consents. Biop-\nsies of human endometrium samples were obtained\nfrom the premenopausal women undergoing hysteros-\ncopy at the General Hospital of Ningxia Medical\nUniversity. Tissues from 12 donors were analyzed in\nthis study. The endometrium was scraped off and\ncollected into D-Hanks phosphate-buffered saline\n(PBS) at 4 °C and was subsequently treated for isola-\ntion of cells within 2 –4 h. Detailed information on\nm a t e r i a l sa n dm e t h o d si sp r o v i d e da ss u p p l e m e n t a l\ndata (Additional file 1), and the antibodies used in\nthis work are listed in Additional file 2.\nIsolation and culture of human endometrial glandular\nepithelial cells\nThe isolation of human endometrial epithelial cells was\nconducted as described in a previous study with min-\nimal modification [ 7]. Briefly, the human endometrial\nbiopsy was minced with scissors into small pieces of\nless than 1 mm 3 before it was washed with PBS con-\ntaining antibiotics. Subsequently, the minced biopsies\nwere dissociated in a dissociation buffer containing\n3.0 mg/mL collagenase type 4 (Sigma-Aldrich, St. Louis,\nMO, USA) in PBS for 7 min at 37 °C with gentle agitation.\nThen the same volume of Accumax (Innovative Cell\nTechnologies, San Diego, CA, USA) was added in the\ndissociated solution and incubated for an additional\n7 min at 37 °C with continuous agitation. The digest-\nive reaction was terminated by adding fetal bovine\nserum to the dissociated fragment/cell suspension in\na final concentration of 10%. The dissociated frag-\nment/cell suspension was filtered through a 400-mesh\nnylon sieve, and residual cell clumps on the sieve\nwere glandular epithelial cells and were washed off with\nD-Hanks into a tube. The cells were collected by centrifu-\ngation (100 g for 5 min), resuspended in 2 mL of culture\nmedium containing 10 μM of Rho-associated protein\nkinase (ROCK) inhibitor Y-27632 (Sigma-Aldrich), and\nseeded onto a 10-cm culture dish pre-coated with 70\nμg/mL collagen type I rat tail (BD Biosciences, Franklin\nLakes, NJ, USA). The cells were maintained in the\nculture medium at 37 °C in a humidified environment\nwith 5% CO 2. The adherent cells were dissociated by\nusing Accutase solution (Millipore, Burlington, MA,\nUSA) at 2 –3 days after seeding, and the cells were\nreseeded at a ratio of 1:3 –5 for passage. Other materials\nand methods are provided as supplemental data in\nAdditional files 1 and 2.\nResults\nIsolation and ALI culture of human endometrial epithelial\ncells\nIn this study, we initially is olated epithelial cells from\nbiopsies of human endometrial tissue. The workflow of\nisolation and culture of epithelial cells is summarized\nin Fig. 1A. The colonies with morphology of epithelial\ncells were observed when the initially isolated cells\nwere grown on collagen type I rat tail –coated dishes in\nROCK inhibitor –modified medium at 48 h (Fig. 1B).\nThe initially isolated cells (passage 0, P0) were stained\nwith epithelial cell marker epithelial cellular adhesion\nmolecule (EpCam), stroma cell marker CD13, and stem\ncell marker stage-specific embryonic antigen-1 (SSEA-1).\nThe immunocytochemistry assay revealed that the epithe-\nlial cells expressed EpCam but not CD13 (Fig. 1B). Immu-\nnocytofluorescent staining further demonstrated the\nexpression of SSEA-1 (Fig. 1C) and a large number of pro-\nliferation marker Ki67-positive cells in isolated epithelial\ncells (Fig. 1C). In addition, immunoblotting assay revealed\nthe expression of epithelial cell markers Keratin 17/19 and\nEpCam (Fig. 1D) and stem cell markers octamer-binding\ntranscription factor 3/4 (OCT3/4), Sry-box-2 (SOX2),\nP63, c-Myc, and CD117 (c-kit) during the cell expansion\nculture (Fig. 1E). Of interest, the expression of SOX2, P63,\nc-Myc, and CD117 was reduced with the passages of cell\ncultures (Fig. 1E). Equally noteworthy, although the pri-\nmary cells could rapidly proliferate to passage 3 (P3), they\nwere senescent in P4 or slowly grown in P4 in current cul-\nture conditions (data not shown).\nMorphological analysis of endometrial epithelial cell ALI\ncultures\nIn order to characterize endometrial epithelial cells in\nan ALI state, P1 epithelial cells were seeded on colla-\ngen–pre-coated membranes of Millicell inserts and\ncultured in an ALI phase. Scanning electron micros-\ncopy showed anomalous shapes and rough cell sur-\nfaces with abundant secretion and microvilli on the\nsurface of cells cultured in an ALI state (Fig. 2B),\nwhile monolayer cells cultured in the conventionally\nsubmerged condition displayed the morphology of\nLi et al. Stem Cell Research & Therapy  (2018) 9:209 Page 2 of 7\n\ninerratic shape with smooth surfaces (Fig. 2B). Trans-\nmission electron microscopy further unraveled that\nthe structures of bridge, microvilli, and cilia and\nabundant secretory granules and mucus in ALI cul-\ntures (Fig. 2C–E)[ 8, 9].\nImmunological characterization of endometrial glandular\nepithelial cell ALI cultures\nIn order to further characterize endometrial epithelial\ncells in ALI cultures, the expression of cell-specific cell\nmarkers of endometrial epithelial cells was accessed in\nFig. 1 Isolation and identification of endometrial epithelial cells. ( A) The schematic showed the procedure of isolation of endometrial epithelial\ncells and generation of an air –liquid interface (ALI) culture. ( B) Identification of endometrial epithelial cells. Cells grown with Rho-associated\nprotein kinase (ROCK)-modified medium on collagen type I rat tail –coated dishes exhibited a capacity to form colonies, which expressed cell\nsurface antigen epithelial cellular adhesion molecule (EpCam) but not CD13 as determined by an immunocytochemistry assay with hematoxylin\ncounterstaining. (C) Immunofluorescent staining for Ki67 or stage-specific embryonic antigen-1 (SSEA-1) (green) revealed that a subset of primary\nhuman endometrial epithelial cells expressed Ki67 or SSEA-1. ( D) Immunoblotting assay confirmed the expression of indicated proteins of interest\nin native human endometrial biopsy tissues and isolated cell cultures of passage 0 –3. (E) Immunoblotting assay confirmed stem cell marker\nexpression of Nanog, Oct3/4, Sox 2, p63, c-Myc, and CD117 (c-kit) in native human endometrial biopsy tissues and isolated cell cultures of\npassages 0–4. (E′) Semi-quantitative analysis of the fold changes of the expression of proteins in ( E) accessed by a densitometric assay. Compared\nwith passage 0 (P0) cells, * P <0.05; **P <0.01 (analysis of variance). Scale bars = 100 μm( C) and 25 μm( C′)\nLi et al. Stem Cell Research & Therapy  (2018) 9:209 Page 3 of 7\n\nthe whole mount membrane of Millicell by an immuno-\nfluorescent staining assay. This assay showed the epithelial\ncell marker vascular endothelial-cadherin (VE-cadherin)\n(Fig. 3B) and the co-localized expression of epithelial cell\nmarker EpCam and stromal cell marker CD13 in cells\ncultured in an ALI state for 2 weeks (Fig. 3C). Together\nwith aforementioned morphological data, this result sug-\ngested that endometrial epithelial cells held an epithelial\nstem/progenitor potential to differentiate into stromal-like\ncells in an ALI state. In order to investigate the capacity\nof cell differentiation, the P1 cells were resuspended in\n5 0 %B DM a t r i g e la n dc u l t u r e dw i t hm o d i f i e dm e d i u m ,\nand the formation of spheroids was examined. As ex-\npected, the spheroid formation was observed in 14-day\nMatrigel cultures (Fig. 3A). This result further suggests\nthat endometrial epithelial cells can develop spheroids\nwith an epithelial stem/progenitor characteristic.\nMolecular characterization of endometrial glandular\nepithelial cell ALI cultures\nIn normal endometrium, estrogen stimulates the prolif-\neration of endometrial glandular epithelial cells in the\nbasal endometrium, we thus further investigated the\ncharacterization of the effect of estrogen on the expres-\nsion of estrogen receptors (ERs), epithelial and stromal\nmarkers in cells cultured in the ALI state. Interest-\ningly, an exposure of prog esterone exhibited an in-\ncreased expression of ER, progesterone receptor (PR),\nepithelial cell marker N-cadherin, EpCam, and stro-\nmal cell markers CD13, PDGFR-beta, and Vimentin\nin ALI cultured cells (Fig. 4A). However, epithelial\ncell marker Keratin 19 and VE-cadherin were not/19\nand VE-cadherin were not altered in cells cultured in\nthe ALI state. However, the addition of progesterone\ndid not affect or inhibit the expression of ER and PR\nFig. 2 Morphological analysis of electronic microscopy. The passage 1 (P1) endometrial epithelial cells were cultured in an air–liquid interface (ALI) state\nfor 2 weeks, and the ALI epithelial cultures and the P2 submerged monolayer cultures were employed for morphological analysis by scanning electronic\nmicroscopy (SEM) (A, B) and transmission electron microscopy (TEM) (C–E). (A, B) Representative images of SEM for endometrial epithelial cells cultured\nin an ALI state (A) and the logarithmic phase of submerged P2 cell culture (B). A′ and B′ were the higher magnifications of corresponding enlarged fields\nin A and B, respectively. Cells in submerged monolayer cultures showed a morphology of inerratic shapes with smooth surfaces, while ALI cultured cells\nexhibited anomalous shapes and rough cell surfaces with abundant secretions and microvilli on the surface of culture. (C–E) Representative TEM images\nof endometrial epithelial cells grown in ALI culture at magnification of 5,000× (C), 10,000× (D), and 15,000× (E) showed nucleus (n), microvilli (v), cilia (c),\nmitochondria (m), bridge (b), and secretory protein particles (p). Scale bars: 10μm( A and B), 5 μm( A′, B′,a n dC), and 2 μm( D and E)\nLi et al. Stem Cell Research & Therapy  (2018) 9:209 Page 4 of 7\n\nin endometrial epithelial cells cultured in a monolayer\nsubmerged state (Fig. 4B). This result implies that the\ncharacteristics of differentiated ALI human endomet-\nrial cell culture may be a reliable and feasible model\nable to mimic human endometrium in vitro .\nDiscussion and conclusion\nIn this report, we described the isolation and expansion\nculture of human endometrial epithelial cells and the\ncharacteristics of endometrial epithelial cells cultured\nin a three-dimensional (3D) ALI state. We showed that\na subset of endometrial epithelial cells had potential for\nspheroid formation, epithelium regeneration, and dif-\nferentiation into stromal-li ke cells. This study thus in-\ntroduces a useful approach for efficient isolation and\nexpansion of human endometrial epithelial cells in stem\ncell biology research and possibly in studies of autolo-\ngous cell transplantation therapy for endometrial injury\ndiseases. In addition, the human endometrial epithelial\nALI culture may be a feasible and reliable model for in-\nvestigating the biological characteristics and mecha-\nnisms of endometrial epithelial cells or endometrial\ndiseases in vitro . Together with the capacity of epithe-\nlial reconstitution and stromal cell differentiation in the\nALI state demonstrated by others, our results imply\nthat a subset of endometrial epithelial cells may retain\ntheir stem/progenitor cell potency for proliferation and\ndifferentiation.\nThe endometrium is a highly and cyclically regenerating\norgan by regulating hormones [ 10]. Estrogen plays an im-\nportant part in the development and regeneration or repair\nof injured endometrium [ 11, 12]. In this context, estrogen\ncould stimulate the proliferation of endometrial epithelial\ncells at every menstrual cycle in a normal endometrium by\nbinding to ER and PR [ 13]. In this study, we also dem-\nonstrated an induced augmentation of ER and PR along\nwith the increased expression of EpCam, N-cadherin,\nand CD13 in human ALI endometrial epithelial culture\nin the presence of progesterone. These data imply that\nprogesterone-promoted proliferation and differentiation\nof endometrial epithelial cells occur in ALI endometrium,\nwhich is similar to the response of endometrial epithelial\ncells in utero in vivo , suggesting the reliability and feasibil-\nity of ALI endometrial epithelium as an in vitro 3D model\nfor mimicking endometrial epithelium in vivo.\nIn conclusion, this report described methods for the\nisolation and expansion of human endometrial epithelial\ncells and generation of human endometrial ALI epithe-\nlium. The ALI culture may offer a reliable and feasible\nmodel for biomedical research and stem cell biology\nstudies of human endometrium in vitro .H o w e v e r ,t h e\nlimited passages of primary endometrial epithelial cells\nFig. 3 Whole mount immunofluorescent staining of endometrial epithelial cells cultured in an air–liquid interface (ALI) state and spheroid formation.\nThe passage 1 (P1) endometrial epithelial cells were cultured in an ALI state for 2 weeks. ( A) Human endometrial epithelial cells generated the\nspheroids by Matrigel sphere assay. (A′) A higher magnification of image (A). (B) Immunofluorescent staining for endometrial epithelial cell marker\nvascular endothelial-cadherin (VE-cadherin) (green). (B′) A higher magnification of image (B). (C) The co-expression of endometrial epithelial cell\nmarkers epithelial cellular adhesion molecule (EpCam) (green) and CD13 (red) was ascertained by immunofluorescence assay. Cell nuclei were\ncounterstained with DAPI (blue). Arrows denote the co-localization of CD13 and EpCam staining (yellow). Scale bar = 25μm\nLi et al. Stem Cell Research & Therapy  (2018) 9:209 Page 5 of 7\n\nusing ROCK inhibitor –modified medium is a methodo-\nlogical limitation of this study, and further optimiza-\ntions of culture media or conditions for unlimited\nculture are required for fut ure autologous endometrial\nepithelial cell transplantation research in vivo .\nAdditional files\nAdditional file 1: Detail materials and methods. (PDF 359 kb)\nAdditional file 2: Supplementary table of antibodies used for\nimmunostaining in this report. (PDF 128 kb)\nAbbreviations\n3D: Three-dimensional; ALI: Air –liquid interface; EpCam: Epithelial cellular\nadhesion molecule; ER: Estrogen receptor; OCT 3/4: Octamer-binding\ntranscription factor 3/4; P: Passage; PBS: Phosphate-buffered saline;\nPR: Progesterone receptor; ROCK: Rho-associated protein kinase; SOX2: SRY-\nBox 2; SSEA-1: Stage-specific embryonic antigen-1\nFunding\nThis study was supported by the First-Class Discipline Construction Founded\nProject of Ningxia Medical University and the School of Clinical Medicine\n(NXYLXK2017A05), a grant from the National Natural Science Foundation of\nChina (no. 31472191) to XL, a grant from the Natural Science Foundation of\nNingxia (NZ15172), and internal supporting grants of Ningxia Medical University\n(XY2017172 and XY201706). The funders had no role in study design, data\ncollection and analysis, decision to publish, or preparation of the manuscript.\nAvailability of data and materials\nAll data generated or analyzed during this study are included in this\npublished article.\nAuthors’ contributions\nJY and DL conceived and designed the experiments. DDL, HL, and YW\nconducted the experiments, analyzed data, and drafted the manuscript. AE,\nJW, YC, JX, JS, and YJ performed experiments and acquired data. SH, CH, and\nXL processed biopsies. JY and DL interpreted data and critically revised the\nmanuscript. All authors read and approved the final version of the\nmanuscript.\nFig. 4 The expression of endometrial epithelial marker in air–liquid interface (ALI) cultured cells. The passage 1 (P1) endometrial epithelial cells\ncultured in an ALI state for 2 weeks ( A) or in a submerged state ( B) were treated with estrogen for indicated time points, and the cell lysates were\nanalyzed by immunoblotting assay against indicated antibodies against proteins of interest. (A, A′) Representative blots showed a significantly induced\nexpression of indicated endometrial epithelial cell markers of ALI culture cells in response to estrogen (A). (A′) Semi-quantitative analysis of fold\nchanges of the expression of indicated proteins in (A) accessed by a densitometric assay. (B, B′) Representative blots showed the expression of\nestrogen receptor (ER) and progesterone receptor (PR) of submerged cell cultures in response to estrogen (B). (B′) Semi-quantitative analysis of fold\nchanges of the expression of proteins of interest in (B) accessed by a densitometric assay. Compared with cells cultured in the absence of estrogen,\n*P <0.05; **P <0.01; ***P <0.0001 (analysis of variance)\nLi et al. Stem Cell Research & Therapy  (2018) 9:209 Page 6 of 7\n\nEthics approval\nThe study and protocol were approved by the ethics committee for\nconducting human research at the General Hospital of Ningxia Medical\nUniversity (NXMU-2017-063).\nConsent for publication\nNot applicable.\nCompeting interests\nThe authors declare that they have no competing interests.\nPublisher’sN o t e\nSpringer Nature remains neutral with regard to jurisdictional claims in\npublished maps and institutional affiliations.\nAuthor details\n1College of Clinical Medicine, Ningxia Medical University, Yinchuan 750004,\nNingxia, China. 2Department of Gynaecology, General Hospital of Ningxia\nMedical University, Yinchuan 750004, Ningxia, China. 3Institute of Human\nStem Cell Research, General Hospital of Ningxia Medical University, Yinchuan\n750004, Ningxia, China. 4Ningxia Key Laboratory of Clinical and Pathological\nMicrobiology, General Hospital of Ningxia Medical University, Yinchuan\n750004, Ningxia, China. 5College of Life Science, Ningxia University, Yinchuan\n750021, Ningxia, China. 6Key Laboratory of Ministry of Education for Fertility\nPreservation and Maintenance, Ningxia Medical University, Yinchuan 750021,\nNingxia, China.\nReceived: 8 April 2018 Revised: 20 July 2018\nAccepted: 26 July 2018\nReferences\n1. 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