{"paper_id":"ce62ff63-dabe-45d8-b0be-7b5903b21485","body_text":"Abstract\nPurpose\nThis study aimed to establish a protocol for efficiently isolating and expanding adenomyotic lesion-derived endometrial mesenchymal stem cells (A-eMSCs) in vitro.\nMethods\nThree different methods—namely, the enzymatic method, the explant method, and the enzymatic explant method—were employed to isolate A-eMSCs. The isolation and expansion efficiencies of these three methods were subsequently compared. The enzymatic explant method was then used, and the transforming growth factor beta type I receptor (TGF-βR1) inhibitor A83-01 was added to the culture medium to evaluate its impact on the isolation and expansion efficiencies of A-eMSCs.\nResults\nThe enzymatic explant method resulted in improved morphology, shorter cell confluence time, and greater SUSD2 enrichment in the isolation of primary endometrial cells compared to the other two methods. The proliferation and differentiation potential of A-eMSCs obtained by sorting primary endometrial cells via the enzymatic explant method were significantly higher than those obtained via the other two methods in vitro. Using the enzymatic explant method, culture medium containing A83-01 further reduced the confluence time of the cells and increased A-eMSCs enrichment during the primary endometrial cell isolation stage. Furthermore, A83-01 enhanced the proliferation and maintained the differentiation potential of A-eMSCs during the cell expansion stage.\nConclusion\nOur study identified a robust, cost-effective, and efficient protocol for isolating and expanding A-eMSCs and providing an important foundation for further research on the pathogenesis and clinical treatment of AM.\nSimilar content being viewed by others\nData availability\nThe data supporting our findings are available from the corresponding author upon reasonable request.\nAbbreviations\n- AM:\n-\nAdenomyosis\n- eMSCs:\n-\nEndometrial mesenchymal stem cells\n- A-eMSCs:\n-\nAdenomyotic lesion-derived eMSCs\n- TGF-βR1:\n-\nTransforming growth factor beta type I receptor\n- FBS:\n-\nFoetal bovine serum\n- MACS:\n-\nMagnetic-activated cell sorting\n- CCK-8:\n-\nCell Counting Kit-8\n- EdU:\n-\n5-Ethynyl-2-deoxyuridine\nReferences\nMunro MG (2021) Adenomyosis: a riddle, wrapped in mystery, inside an enigma. Fertil Steril 116:89–90. https://doi.org/10.1016/j.fertnstert.2021.04.037\nGarcia-Solares J, Donnez J, Donnez O, Dolmans MM (2018) Pathogenesis of uterine adenomyosis: invagination or metaplasia? Fertil Steril 109:371–379. https://doi.org/10.1016/j.fertnstert.2017.12.030\nVannuccini S, Tosti C, Carmona F, Huang SJ, Chapron C, Guo SW, Petraglia F (2017) Pathogenesis of adenomyosis: an update on molecular mechanisms. Reprod Biomed Online 35:592–601. https://doi.org/10.1016/j.rbmo.2017.06.016\nShilina MA, Domnina AP, Kozhuharova IV, Zenin VV, Anisimov SV, Nikolsky NN, Grinchuk TM (2016) Establishment and characterization of a novel human endometrial mesenchymal stem cell line from a patient with adenomyosis. Cell Tissue Biol 10:10–17. https://doi.org/10.1134/s1990519x16010107\nEl Sabeh M, Afrin S, Singh B, Miyashita-Ishiwata M, Borahay M (2021) Uterine stem cells and benign gynecological disorders: role in pathobiology and therapeutic implications. Stem Cell Rev Rep 17:803–820. https://doi.org/10.1007/s12015-020-10075-w\nSudarikova AV, Shilina MA, Chubinskiy-Nadezhdin VI, Grinchuk TM, Morachevskaya EA, Negulyaev YA (2020) Increased migration ability of adenomyosis-derived endometrial mesenchymal stem cells. Cell Tissue Biol 14:190–195. https://doi.org/10.1134/s1990519x20030062\nChen YJ, Li HY, Chang YL, Yuan CC, Tai LK, Lu KH, Chang CM, Chiou SH (2010) Suppression of migratory/invasive ability and induction of apoptosis in adenomyosis-derived mesenchymal stem cells by cyclooxygenase-2 inhibitors. Fertil Steril 94:1972-1974.e1971-1974. https://doi.org/10.1016/j.fertnstert.2010.01.070\nKao AP, Wang KH, Chang CC, Lee JN, Long CY, Chen HS, Tsai CF, Hsieh TH, Tsai EM (2011) Comparative study of human eutopic and ectopic endometrial mesenchymal stem cells and the development of an in vivo endometriotic invasion model. Fertil Steril 95:1308-1315.e1301. https://doi.org/10.1016/j.fertnstert.2010.09.064\nNiu W, Zhang Y, Liu H, Liang N, Xu L, Li Y, Yao W, Shi W, Liu Z (2023) Single-cell profiling uncovers the roles of endometrial fibrosis and microenvironmental changes in adenomyosis. J Inflamm Res 16:1949–1965. https://doi.org/10.2147/JIR.S402734\nDarzi S, Werkmeister JA, Deane JA, Gargett CE (2016) Identification and characterization of human endometrial mesenchymal stem/stromal cells and their potential for cellular therapy. Stem Cells Transl Med 5:1127–1132. https://doi.org/10.5966/sctm.2015-0190\nLupicka M, Socha B, Szczepanska A, Korzekwa A (2015) Expression of pluripotency markers in the bovine uterus with adenomyosis. Reprod Biol Endocrinol 13:110. https://doi.org/10.1186/s12958-015-0106-0\nSugita N, Moriguchi Y, Sakaue M, Hart DA, Yasui Y, Koizumi K, Chijimatsu R, Shimomura S, Ikeda Y, Yoshikawa H, Nakamura N (2016) Optimization of human mesenchymal stem cell isolation from synovial membrane: implications for subsequent tissue engineering effectiveness. Regen Ther 5:79–85. https://doi.org/10.1016/j.reth.2016.09.002\nWang J, Hu R, Wang Z, Guo Y, Wang S, Zou H, Peng Q, Jiang Y (2022) Establishment of immortalized yak ruminal epithelial cell lines by lentivirus-mediated SV40T and hTERT gene transduction. Oxid Med Cell Longev 2022:8128028. https://doi.org/10.1155/2022/8128028\nBai J, Fu H, Bazinet L, Birsner AE, D’Amato RJ (2020) A method for developing novel 3D cornea-on-a-chip using primary murine corneal epithelial and endothelial cells. Front Pharmacol 11:453. https://doi.org/10.3389/fphar.2020.00453\nKay N, Huang CY, Shiu LY, Yu YC, Chang Y, Suen JL, Tsai EM, Huang SJ (2020) The effects of anti-TGF-beta1 on epithelial-mesenchymal transition in the pathogenesis of adenomyosis. Reprod Sci 27:1698–1706. https://doi.org/10.1007/s43032-020-00139-0\nShen M, Liu X, Zhang H, Guo SW (2016) Transforming growth factor beta1 signaling coincides with epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation in the development of adenomyosis in mice. Hum Reprod 31:355–369. https://doi.org/10.1093/humrep/dev314\nAlmalki SG, Agrawal DK (2016) Key transcription factors in the differentiation of mesenchymal stem cells. Differentiation 92:41–51. https://doi.org/10.1016/j.diff.2016.02.005\nSui BD, Hu CH, Zheng CX, Jin Y (2016) Microenvironmental views on mesenchymal stem cell differentiation in aging. J Dent Res 95:1333–1340. https://doi.org/10.1177/0022034516653589\nUshakov RE, Skvortsova EV, Vitte MA, Vassilieva IO, Shatrova AN, Kotova AV, Kenis VM, Burova EB (2020) Chondrogenic differentiation followed IGFBP3 loss in human endometrial mesenchymal stem cells. Biochem Biophys Res Commun 531:133–139. https://doi.org/10.1016/j.bbrc.2020.07.064\nSu K, Edwards SL, Tan KS, White JF, Kandel S, Ramshaw JAM, Gargett CE, Werkmeister JA (2014) Induction of endometrial mesenchymal stem cells into tissue-forming cells suitable for fascial repair. Acta Biomater 10:5012–5020. https://doi.org/10.1016/j.actbio.2014.08.031\nTojo M, Hamashima Y, Hanyu A, Kajimoto T, Saitoh M, Miyazono K, Node M, Imamura T (2005) The ALK-5 inhibitor A-83–01 inhibits Smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-beta. Cancer Sci 96:791–800. https://doi.org/10.1111/j.1349-7006.2005.00103.x\nGurung S, Werkmeister JA, Gargett CE (2015) Inhibition of transforming growth factor-beta receptor signaling promotes culture expansion of undifferentiated human endometrial mesenchymal stem/stromal cells. Sci Rep 5:15042. https://doi.org/10.1038/srep15042\nGurung S, Williams S, Deane JA, Werkmeister JA, Gargett CE (2018) The transcriptome of human endometrial mesenchymal stem cells under TGFbetaR inhibition reveals improved potential for cell-based therapies. Front Cell Dev Biol 6:164. https://doi.org/10.3389/fcell.2018.00164\nGurung S, Deane JA, Darzi S, Werkmeister JA, Gargett CE (2018) In vivo survival of human endometrial mesenchymal stem cells transplanted under the kidney capsule of immunocompromised mice. Stem Cells Dev 27:35–43. https://doi.org/10.1089/scd.2017.0177\nMurakami K, Lee YH, Lucas ES, Chan YW, Durairaj RP, Takeda S, Moore JD, Tan BK, Quenby S, Chan JK, Gargett CE, Brosens JJ (2014) Decidualization induces a secretome switch in perivascular niche cells of the human endometrium. Endocrinology 155:4542–4553. https://doi.org/10.1210/en.2014-1370\nBozorgmehr M, Gurung S, Darzi S, Nikoo S, Kazemnejad S, Zarnani AH, Gargett CE (2020) Endometrial and menstrual blood mesenchymal stem/stromal cells: biological properties and clinical application. Front Cell Dev Biol 8:497. https://doi.org/10.3389/fcell.2020.00497\nGargett CE (2007) Uterine stem cells: what is the evidence? Hum Reprod Update 13:87–101. https://doi.org/10.1093/humupd/dml045\nHaj Hamoud B, Kasoha M, Sillem M, Solomayer E-F, Sima R-M, Ples L, Schwab R, Olmes GL (2023) Sonographic features of adenomyosis correlated with clinical symptoms and intraoperative findings: a case–control study. Arch Gynecol Obstet 307:1883–1889. https://doi.org/10.1007/s00404-022-06852-2\nWang Z, Cui F, Chen Y, Liu H, Zhang Y, Shi Y, Zhang Y, Wang Y, Liang N, Xu L, Liu Y, Liu Z, Shi W (2024) Establishment of an immortalized cell line derived from human adenomyosis ectopic lesions. Tissue Cell 86:102284. https://doi.org/10.1016/j.tice.2023.102284\nWang YY, Duan H, Wang S, Quan YJ, Huang JH, Guo ZC (2021) Upregulated Talin1 synergistically boosts beta-estradiol-induced proliferation and pro-angiogenesis of eutopic and ectopic endometrial stromal cells in adenomyosis. Reprod Biol Endocrinol 19:70. https://doi.org/10.1186/s12958-021-00756-7\nFang Z, Wang J, Li T, Yin M, Peng Y, Zhang X (2024) A method for isolating and culturing ectopic epithelial and stromal cells to study human adenomyosis. Arch Gynecol Obstet 309:551–563. https://doi.org/10.1007/s00404-023-07254-8\nFakhri O, Tan RS (1975) Short communications: the effect of trypsin on cell surface antigens. Cell Immunol 15:452–456. https://doi.org/10.1016/0008-8749(75)90021-0\nFurcht LT, Wendelschafer-Crabb G (1978) Trypsin-induced coordinate alterations in cell shape, cytoskeleton, and intrinsic membrane structure of contact-inhibited cells. Exp Cell Res 114:1–14. https://doi.org/10.1016/0014-4827(78)90029-0\nAn M, Li D, Yuan M, Li Q, Zhang L, Wang G (2017) Interaction of macrophages and endometrial cells induces epithelial-mesenchymal transition-like processes in adenomyosis. Biol Reprod 96:46–57. https://doi.org/10.1095/biolreprod.116.144071\nLiu L, Jin M, Tao Q, Yu L, Du J, Wang C, Luo Q, Xing T, Xu Y, Shen J, Chu D (2018) Effective amelioration of liver fibrosis through lentiviral vector carrying Toxoplasma gondii gra15(II) in Murine model. Front Immunol 9:1572. https://doi.org/10.3389/fimmu.2018.01572\nLin CH, Lu JH, Hsia K, Lee H, Yao CL, Ma H (2019) The antithrombotic function of sphingosine-1-phosphate on human adipose-stem-cell-recellularized tissue engineered vascular graft in vitro. Int J Mol Sci. https://doi.org/10.3390/ijms20205218\nLiu X, Ding D, Ren Y, Guo SW (2018) Transvaginal elastosonography as an imaging technique for diagnosing adenomyosis. Reprod Sci 25:498–514. https://doi.org/10.1177/1933719117750752\nWang S, Li B, Duan H, Wang Y, Shen X, Dong Q (2021) Abnormal expression of connective tissue growth factor and its correlation with fibrogenesis in adenomyosis. Reprod Biomed Online 42:651–660. https://doi.org/10.1016/j.rbmo.2020.11.002\nShimono M, Clementi F (1977) Intercellular junctions of oral epithelium: II—Ultrastructural changes in rat buccal epithelium induced by trypsin digestion. J Ultrastruct Res 59:101–112. https://doi.org/10.1016/s0022-5320(77)80032-4\nXia W, Chen H, Chen D, Ye Y, Xie C, Hou M (2020) PD-1 inhibitor inducing exosomal miR-34a-5p expression mediates the cross talk between cardiomyocyte and macrophage in immune checkpoint inhibitor-related cardiac dysfunction. J Immunother Cancer. https://doi.org/10.1136/jitc-2020-001293\nZhuang L, Xia W, Chen D, Ye Y, Hu T, Li S, Hou M (2020) Exosomal LncRNA-NEAT1 derived from MIF-treated mesenchymal stem cells protected against doxorubicin-induced cardiac senescence through sponging miR-221–3p. J Nanobiotechnol 18:157. https://doi.org/10.1186/s12951-020-00716-0\nLu Z, Hao C, Qian H, Zhao Y, Bo X, Yao Y, Ma G, Chen L (2022) Tripartite motif 38 attenuates cardiac fibrosis after myocardial infarction by suppressing TAK1 activation via TAB2/3 degradation. iScience 25:104780. https://doi.org/10.1016/j.isci.2022.104780\nYoon JH, Roh EY, Shin S, Jung NH, Song EY, Chang JY, Kim BJ, Jeon HW (2013) Comparison of explant-derived and enzymatic digestion-derived MSCs and the growth factors from Wharton’s jelly. Biomed Res Int 2013:428726. https://doi.org/10.1155/2013/428726\nLee DH, Joo SD, Han SB, Im J, Lee SH, Sonn CH, Lee KM (2011) Isolation and expansion of synovial CD34(-)CD44(+)CD90(+) mesenchymal stem cells: comparison of an enzymatic method and a direct explant technique. Connect Tissue Res 52:226–234. https://doi.org/10.3109/03008207.2010.516850\nBusser H, De Bruyn C, Urbain F, Najar M, Pieters K, Raicevic G, Meuleman N, Bron D, Lagneaux L (2014) Isolation of adipose-derived stromal cells without enzymatic treatment: expansion, phenotypical, and functional characterization. Stem Cells Dev 23:2390–2400. https://doi.org/10.1089/scd.2014.0071\nHendijani F (2017) Explant culture: an advantageous method for isolation of mesenchymal stem cells from human tissues. Cell Prolif. https://doi.org/10.1111/cpr.12334\nRosenberg L, Wang R, Paraskevas S, Maysinger D (1999) Structural and functional changes resulting from islet isolation lead to islet cell death. Surgery 126:393–398\nConconi MT, Burra P, Di Liddo R, Calore C, Turetta M, Bellini S, Bo P, Nussdorfer GG, Parnigotto PP (2006) CD105(+) cells from Wharton’s jelly show in vitro and in vivo myogenic differentiative potential. Int J Mol Med 18:1089–1096\nHong L, Song D, Zeng Y (2015) Comparison and improvement in primary airway fibroblast culture across different mammalian species. Cell Mol Biol (Noisy-le-grand) 61:108–114\nZheng S, Gao Y, Chen K, Liu Y, Xia N, Fang F (2022) A robust and highly efficient approach for isolation of mesenchymal stem cells from Wharton’s jelly for tissue repair. Cell Transpl. https://doi.org/10.1177/09636897221084354\nRajaraman G, White J, Tan KS, Ulrich D, Rosamilia A, Werkmeister J, Gargett CE (2013) Optimization and scale-up culture of human endometrial multipotent mesenchymal stromal cells: potential for clinical application, tissue engineering part C. Methods 19:80–92. https://doi.org/10.1089/ten.tec.2011.0718\nLiu J, Ding Y, Liu Z, Liang X (2020) Senescence in mesenchymal stem cells: functional alterations, molecular mechanisms, and rejuvenation strategies. Front Cell Dev Biol. https://doi.org/10.3389/fcell.2020.00258\nMassague J (2012) TGFbeta signalling in context. Nat Rev Mol Cell Biol 13:616–630. https://doi.org/10.1038/nrm3434\nGurung S, Ulrich D, Sturm M, Rosamilia A, Werkmeister JA, Gargett CE (2020) Comparing the effect of TGF-beta receptor inhibition on human perivascular mesenchymal stromal cells derived from endometrium, bone marrow and adipose tissues. J Pers Med. https://doi.org/10.3390/jpm10040261\nLucciola R, Vrljicak P, Gurung S, Filby C, Darzi S, Muter J, Ott S, Brosens JJ, Gargett CE (2020) Impact of sustained transforming growth factor-β receptor inhibition on chromatin accessibility and gene expression in cultured human endometrial MSC. Front Cell Dev Biol. https://doi.org/10.3389/fcell.2020.567610\nFunding\nThis work was supported by the National Natural Science Foundation of China (81403321), the Traditional Chinese Medicine Science and Technology Development Plan Project of Jiangsu Province (MS2023039 and MS2023079), the Development Fund Project of the Affiliated Hospital of Xuzhou Medical University (XYFY202322), the Science and Technology Project of Xuzhou Municipal Health Commission (XWKYHT20220071), and the Graduate Research and Practice Innovation Plan of Jiangsu Province (SJCX23_0817 and SJCX24_1047).\nAuthor information\nAuthors and Affiliations\nContributions\nXinjun Wei: Conceptualization, Methodology, Software, Funding Acquisition, and Writing (Original Draft); Aiyun Xu and Shuyu Xia: Investigation and Formal Analysis; Jindan Wang and YingYing Qiu: Data Curation, Funding Acquisition, and Visualization; Guiping Wan: Investigation and Visualization; Jian Cao and Zhihui Wang: Resources, Supervision and Writing (Review & Editing); Tao Gui: Conceptualization, Funding Acquisition, Resources, Supervision, and Writing (Review & Editing). All authors read and approved the final manuscript.\nCorresponding authors\nEthics declarations\nConflict of interest\nThe authors declare no competing interests.\nEthical approval\nThis study was approved by the Medical Ethics Committee of Jiangsu Province Hospital with Integration of Chinese and Western Medicine (2023-LWKYZ-050).\nConsent to participate\nAll participants provided written informed consent prior to sample collection.\nAdditional information\nPublisher's Note\nSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\nSupplementary Information\nBelow is the link to the electronic supplementary material.\n404_2024_7854_MOESM1_ESM.tif (download TIF )\nFigure S1 Immunophenotypes of eMSCs isolated via three methods. Flow cytometry analysis revealed that the eMSCs strongly expressed CD73, CD90, and CD105 but barely expressed CD45 or HLA-DR. n = 3. Supplementary file1 (TIF 16204 KB)\n404_2024_7854_MOESM2_ESM.tif (download TIF )\nFigure S2 Effect of A83-01 on the immunophenotype of eMSCs. Flow cytometry analysis revealed that the eMSCs strongly expressed CD73, CD90, and CD105 but barely expressed CD45 or HLA-DR. n = 3. Supplementary file2 (TIF 12810 KB)\n404_2024_7854_MOESM3_ESM.tif (download TIF )\nFigure S3 Morphological observation of primary endometrial cells and flow cytometric analysis. (A) The morphology of primary endometrial cells isolated by enzymatic methods with trypsin, collagenase IV, or a combination of trypsin and collagenase IV (combo) was observed on day 12. Scale bar = 1 mm. (B) Flow cytometry analysis of SUSD2 expression in primary endometrial cells isolated by collagenase IV or the combination. (C) The histogram shows the percentage of SUSD2+ cells among the primary endometrial cells. The data are presented as the mean ± SD, and statistical analysis was performed using an unpaired t test; **p < 0.01. n = 3. Supplementary file3 (TIF 6082 KB)\nRights and permissions\nSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.\nAbout this article\nCite this article\nWei, X., Xu, A., Xia, S. et al. Primary culture of endometrial mesenchymal stem cells derived from ectopic lesions of patients with adenomyosis. Arch Gynecol Obstet 310, 3239–3253 (2024). https://doi.org/10.1007/s00404-024-07854-y\nReceived:\nAccepted:\nPublished:\nVersion of record:\nIssue date:\nDOI: https://doi.org/10.1007/s00404-024-07854-y","source_license":"CC0","license_restricted":false}