Primary culture of endometrial mesenchymal stem cells derived from ectopic lesions of patients with adenomyosis

Purpose This study aimed to establish a protocol for efficiently isolating and expanding adenomyotic lesion-derived endometrial mesenchymal stem cells (A-eMSCs) in vitro.

Three 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.

The 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.

Our 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. Similar content being viewed by others Data availability The data supporting our findings are available from the corresponding author upon reasonable request. Abbreviations - AM: - Adenomyosis - eMSCs: - Endometrial mesenchymal stem cells - A-eMSCs: - Adenomyotic lesion-derived eMSCs - TGF-βR1: - Transforming growth factor beta type I receptor - FBS: - Foetal bovine serum - MACS: - Magnetic-activated cell sorting - CCK-8: - Cell Counting Kit-8 - EdU: - 5-Ethynyl-2-deoxyuridine

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Front Cell Dev Biol. https://doi.org/10.3389/fcell.2020.567610 Funding This 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). Author information Authors and Affiliations Contributions Xinjun 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. Corresponding authors Ethics declarations Conflict of interest The authors declare no competing interests. Ethical approval This study was approved by the Medical Ethics Committee of Jiangsu Province Hospital with Integration of Chinese and Western Medicine (2023-LWKYZ-050). Consent to participate All participants provided written informed consent prior to sample collection. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information Below is the link to the electronic supplementary material. 404_2024_7854_MOESM1_ESM.tif (download TIF ) Figure 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) 404_2024_7854_MOESM2_ESM.tif (download TIF ) Figure 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) 404_2024_7854_MOESM3_ESM.tif (download TIF ) Figure 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) Rights and permissions Springer 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. About this article Cite this article Wei, 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 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s00404-024-07854-y