{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T08:01:13Z","timestamp":1761897673322,"version":"build-2065373602"},"reference-count":41,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2022,2,9]],"date-time":"2022-02-09T00:00:00Z","timestamp":1644364800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003407","name":"Ministry of Education, Universities and Research","doi-asserted-by":"publisher","award":["MARK_RILO_18_02"],"award-info":[{"award-number":["MARK_RILO_18_02"]}],"id":[{"id":"10.13039\/501100003407","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The cyclic regeneration of human endometrium is guaranteed by the proliferative capacity of endometrial mesenchymal stromal cells (E-MSCs). Due to this, the autologous infusion of E-MSCs has been proposed to support endometrial growth in a wide range of gynecological diseases. We aimed to compare two different endometrial sampling methods, surgical curettage and vacuum aspiration biopsy random assay (VABRA), and to validate a novel xeno-free method to culture human E-MSCs. Six E-MSCs cell samples were isolated after mechanical tissue homogenization and cultured using human platelet lysate. E-MSCs were characterized for the colony formation capacity, proliferative potential, and multilineage differentiation. The expression of mesenchymal and stemness markers were tested by FACS analysis and real-time PCR, respectively. Chromosomal alterations were evaluated by karyotype analysis, whereas tumorigenic capacity and invasiveness were tested by soft agar assay. Both endometrial sampling techniques allowed efficient isolation and expansion of E-MSCs using a xeno-free method, preserving their mesenchymal and stemness phenotype, proliferative potential, and limited multi-lineage differentiation ability during the culture. No chromosomal alterations and invasive\/tumorigenic capacity were observed. Herein, we report the first evidence of efficient E-MSCs isolation and culture in Good Manufacturing Practice compliance conditions, suggesting VABRA endometrial sampling as alternative to surgical curettage.<\/jats:p>","DOI":"10.3390\/ijms23041931","type":"journal-article","created":{"date-parts":[[2022,2,9]],"date-time":"2022-02-09T21:22:15Z","timestamp":1644441735000},"page":"1931","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A Novel Xeno-Free Method to Isolate Human Endometrial Mesenchymal Stromal Cells (E-MSCs) in Good Manufacturing Practice (GMP) Conditions"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6402-6750","authenticated-orcid":false,"given":"Stefano","family":"Canosa","sequence":"first","affiliation":[{"name":"Gynecology and Obstetrics 1U, Physiopathology of Reproduction and IVF Unit, S. Anna Hospital, Department of Surgical Sciences, University of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2889-0394","authenticated-orcid":false,"given":"Katia","family":"Mareschi","sequence":"additional","affiliation":[{"name":"Department of Public Health and Paediatrics, University of Torino, 10126 Torino, Italy"},{"name":"Stem Cell Transplantation and Cellular Therapy Laboratory, Paediatric Onco-Haematology Division, Regina Margherita Children\u2019s Hospital, City of Health and Science of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elena","family":"Marini","sequence":"additional","affiliation":[{"name":"Department of Public Health and Paediatrics, University of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7734-4499","authenticated-orcid":false,"given":"Andrea Roberto","family":"Carosso","sequence":"additional","affiliation":[{"name":"Gynecology and Obstetrics 1U, Physiopathology of Reproduction and IVF Unit, S. Anna Hospital, Department of Surgical Sciences, University of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sara","family":"Castiglia","sequence":"additional","affiliation":[{"name":"Stem Cell Transplantation and Cellular Therapy Laboratory, Paediatric Onco-Haematology Division, Regina Margherita Children\u2019s Hospital, City of Health and Science of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Deborah","family":"Rustichelli","sequence":"additional","affiliation":[{"name":"Stem Cell Transplantation and Cellular Therapy Laboratory, Paediatric Onco-Haematology Division, Regina Margherita Children\u2019s Hospital, City of Health and Science of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ivana","family":"Ferrero","sequence":"additional","affiliation":[{"name":"Stem Cell Transplantation and Cellular Therapy Laboratory, Paediatric Onco-Haematology Division, Regina Margherita Children\u2019s Hospital, City of Health and Science of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4390-9428","authenticated-orcid":false,"given":"Gianluca","family":"Gennarelli","sequence":"additional","affiliation":[{"name":"Gynecology and Obstetrics 1U, Physiopathology of Reproduction and IVF Unit, S. Anna Hospital, Department of Surgical Sciences, University of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3663-5134","authenticated-orcid":false,"given":"Benedetta","family":"Bussolati","sequence":"additional","affiliation":[{"name":"Molecular Biotechnology Centre, Department of Molecular Biotechnology and Health Sciences, University of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alberto","family":"Nocifora","sequence":"additional","affiliation":[{"name":"Department of Oncology, Pathology Unit, University of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Valentina","family":"Asnaghi","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Medical Genetics Division, City of Health and Science of Torino, 10124 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Massimiliano","family":"Bergallo","sequence":"additional","affiliation":[{"name":"Department of Public Health and Paediatrics, University of Torino, 10126 Torino, Italy"},{"name":"Paediatric Laboratory Regina Margherita Children\u2019s Hospital, City of Health and Science of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5494-3034","authenticated-orcid":false,"given":"Ciro","family":"Isidoro","sequence":"additional","affiliation":[{"name":"Department of Health Sciences, University of Piemonte Orientale, 13100 Novara, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chiara","family":"Benedetto","sequence":"additional","affiliation":[{"name":"Gynecology and Obstetrics 1U, Physiopathology of Reproduction and IVF Unit, S. Anna Hospital, Department of Surgical Sciences, University of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5797-3722","authenticated-orcid":false,"given":"Alberto","family":"Revelli","sequence":"additional","affiliation":[{"name":"Gynecology and Obstetrics 1U, Physiopathology of Reproduction and IVF Unit, S. Anna Hospital, Department of Surgical Sciences, University of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9257-900X","authenticated-orcid":false,"given":"Franca","family":"Fagioli","sequence":"additional","affiliation":[{"name":"Department of Public Health and Paediatrics, University of Torino, 10126 Torino, Italy"},{"name":"Stem Cell Transplantation and Cellular Therapy Laboratory, Paediatric Onco-Haematology Division, Regina Margherita Children\u2019s Hospital, City of Health and Science of Torino, 10126 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,2,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1738","DOI":"10.1095\/biolreprod.103.024109","article-title":"Clonogenicity of Human Endometrial Epithelial and Stromal Cells","volume":"70","author":"Chan","year":"2004","journal-title":"Biol. Reprod."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1529","DOI":"10.1634\/stemcells.2005-0411","article-title":"Identification of Label-Retaining Cells in Mouse Endometrium","volume":"24","author":"Chan","year":"2006","journal-title":"Stem Cells"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"250","DOI":"10.1111\/j.1479-828X.2006.00582.x","article-title":"Identification and Characterisation of Human Endometrial Stem\/Progenitor Cells","volume":"46","author":"Gargett","year":"2006","journal-title":"Aust. N. Z. J. Obstet. Gynaecol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1136","DOI":"10.1095\/biolreprod.108.075226","article-title":"Isolation and Culture of Epithelial Progenitors and Mesenchymal Stem Cells from Human Endometrium","volume":"80","author":"Gargett","year":"2009","journal-title":"Biol. Reprod."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"672","DOI":"10.1111\/aogs.13598","article-title":"Cellular Therapies for the Endometrium: An Update","volume":"98","author":"Davies","year":"2019","journal-title":"Acta Obstet. Gynecol. Scand."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2903","DOI":"10.1093\/humrep\/dem265","article-title":"Co-Expression of Two Perivascular Cell Markers Isolates Mesenchymal Stem-like Cells from Human Endometrium","volume":"22","author":"Schwab","year":"2007","journal-title":"Hum. Reprod."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1094","DOI":"10.1182\/blood-2004-06-2315","article-title":"Regulator of G-Protein Signaling-5 Induction in Pericytes Coincides with Active Vessel Remodeling during Neovascularization","volume":"105","author":"Berger","year":"2005","journal-title":"Blood"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1095\/biolreprod.111.095885","article-title":"Perivascular Human Endometrial Mesenchymal Stem Cells Express Pathways Relevant to Self-Renewal, Lineage Specification, and Functional Phenotype","volume":"86","author":"Spitzer","year":"2012","journal-title":"Biol. Reprod."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1521","DOI":"10.1016\/j.fertnstert.2012.08.003","article-title":"Sorafenib Inhibits Growth, Migration, and Angiogenic Potential of Ectopic Endometrial Mesenchymal Stem Cells Derived from Patients with Endometriosis","volume":"98","author":"Moggio","year":"2012","journal-title":"Fertil. Steril."},{"key":"ref_10","first-page":"187","article-title":"Angiogenic Properties of Endometrial Mesenchymal Stromal Cells in Endothelial Co-Culture: An in Vitro Model of Endometriosis","volume":"23","author":"Canosa","year":"2017","journal-title":"Mol. Hum. Reprod."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/s13287-019-1496-2","article-title":"Endometrial Stromal Cells Exhibit a Distinct Phenotypic and Immunomodulatory Profile","volume":"11","author":"Davies","year":"2020","journal-title":"Stem Cell Res. Ther."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1186\/s12967-020-02234-x","article-title":"Improved Therapeutics of Modified Mesenchymal Stem Cells: An Update","volume":"18","author":"Ocansey","year":"2020","journal-title":"J. Transl. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e12693","DOI":"10.1111\/aji.12693","article-title":"Physiological and Pathological Angiogenesis in Endometrium at the Time of Embryo Implantation","volume":"78","author":"Chen","year":"2017","journal-title":"Am. J. Reprod. Immunol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"759","DOI":"10.1016\/j.fertnstert.2008.02.096","article-title":"Asherman Syndrome--One Century Later","volume":"89","author":"Yu","year":"2008","journal-title":"Fertil. Steril."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1552","DOI":"10.1016\/j.fertnstert.2015.08.032","article-title":"Human CD133(+) Bone Marrow-Derived Stem Cells Promote Endometrial Proliferation in a Murine Model of Asherman Syndrome","volume":"104","author":"Cabanillas","year":"2015","journal-title":"Fertil. Steril."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1087","DOI":"10.1093\/humrep\/dew042","article-title":"Autologous Cell Therapy with CD133+ Bone Marrow-Derived Stem Cells for Refractory Asherman\u2019s Syndrome and Endometrial Atrophy: A Pilot Cohort Study","volume":"31","author":"Santamaria","year":"2016","journal-title":"Hum. Reprod."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2723","DOI":"10.1093\/humrep\/dew235","article-title":"Autologous Menstrual Blood-Derived Stromal Cells Transplantation for Severe Asherman\u2019s Syndrome","volume":"31","author":"Tan","year":"2016","journal-title":"Hum. Reprod."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1186\/s13287-018-0801-9","article-title":"Human Mesenchymal Stem Cells in Spheroids Improve Fertility in Model Animals with Damaged Endometrium","volume":"9","author":"Domnina","year":"2018","journal-title":"Stem Cell Res. Ther."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1089\/bio.2017.0057","article-title":"The Clinical Applications of Endometrial Mesenchymal Stem Cells","volume":"16","author":"Zuo","year":"2018","journal-title":"Biopreserv. Biobank."},{"key":"ref_20","unstructured":"(2022, January 28). EudraLex\u2014Volume 4. Available online: https:\/\/ec.europa.eu\/health\/medicinal-products\/eudralex\/eudralex-volume-4_en."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"750","DOI":"10.1016\/j.jcyt.2013.12.008","article-title":"Inactivated Human Platelet Lysate with Psoralen: A New Perspective for Mesenchymal Stromal Cell Production in Good Manufacturing Practice Conditions","volume":"16","author":"Castiglia","year":"2014","journal-title":"Cytotherapy"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"8932","DOI":"10.1073\/pnas.132252399","article-title":"Isolated Allogeneic Bone Marrow-Derived Mesenchymal Cells Engraft and Stimulate Growth in Children with Osteogenesis Imperfecta: Implications for Cell Therapy of Bone","volume":"99","author":"Horwitz","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"747","DOI":"10.1016\/j.ymthe.2004.02.012","article-title":"Internalized Antigens Must Be Removed to Prepare Hypoimmunogenic Mesenchymal Stem Cells for Cell and Gene Therapy","volume":"9","author":"Spees","year":"2004","journal-title":"Mol. Ther."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1208","DOI":"10.3324\/haematol.11446","article-title":"No Alloantibodies against Mesenchymal Stromal Cells, but Presence of Anti-Fetal Calf Serum Antibodies, after Transplantation in Allogeneic Hematopoietic Stem Cell Recipients","volume":"92","author":"Sundin","year":"2007","journal-title":"Haematologica"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1080\/14653240600920782","article-title":"Animal Serum-Free Culture Conditions for Isolation and Expansion of Multipotent Mesenchymal Stromal Cells from Human BM","volume":"8","author":"Kordowich","year":"2006","journal-title":"Cytotherapy"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1730","DOI":"10.1056\/NEJMhpr063086","article-title":"FDA Regulation of Stem-Cell-Based Therapies","volume":"355","author":"Halme","year":"2006","journal-title":"N. Engl. J. Med."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"920581","DOI":"10.1155\/2012\/920581","article-title":"Multipotent Mesenchymal Stromal Stem Cell Expansion by Plating Whole Bone Marrow at a Low Cellular Density: A More Advantageous Method for Clinical Use","volume":"2012","author":"Mareschi","year":"2012","journal-title":"Stem Cells Int."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1080\/14653240600855905","article-title":"Minimal Criteria for Defining Multipotent Mesenchymal Stromal Cells. The International Society for Cellular Therapy Position Statement","volume":"8","author":"Dominici","year":"2006","journal-title":"Cytotherapy"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1089\/scd.2014.0299","article-title":"Ex Vivo Expanded Mesenchymal Stromal Cell Minimal Quality Requirements for Clinical Application","volume":"24","author":"Torre","year":"2015","journal-title":"Stem Cells Dev."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2201","DOI":"10.3727\/096368911X637362","article-title":"A Novel Marker of Human Endometrial Mesenchymal Stem-like Cells","volume":"21","author":"Masuda","year":"2012","journal-title":"Cell Transplant."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Cervell\u00f3, I., Gil-Sanchis, C., Mas, A., Delgado-Rosas, F., Mart\u00ednez-Conejero, J.A., Gal\u00e1n, A., Mart\u00ednez-Romero, A., Mart\u00ednez, S., Navarro, I., and Ferro, J. (2010). Human Endometrial Side Population Cells Exhibit Genotypic, Phenotypic and Functional Features of Somatic Stem Cells. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0010964"},{"key":"ref_32","unstructured":"(2022, January 28). EMA Guidelines Relevant for Advanced Therapy Medicinal Products. Available online: https:\/\/www.ema.europa.eu\/en\/human-regulatory\/research-development\/advanced-therapies\/guidelines-relevant-advanced-therapy-medicinal-products."},{"key":"ref_33","unstructured":"(2022, January 28). EU GMP Annex 15: Qualification and Validation\u2014ECA Academy. Available online: https:\/\/www.gmp-compliance.org\/guidelines\/gmp-guideline\/eu-gmp-annex-15-qualification-and-validation."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"530","DOI":"10.1093\/humupd\/dmu011","article-title":"Endometrial Thickness and Pregnancy Rates after IVF: A Systematic Review and Meta-Analysis","volume":"20","author":"Kasius","year":"2014","journal-title":"Hum. Reprod. Update"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1093\/humupd\/dmy044","article-title":"Conventional and Modern Markers of Endometrial Receptivity: A Systematic Review and Meta-Analysis","volume":"25","author":"Craciunas","year":"2019","journal-title":"Hum. Reprod. Update"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1016\/j.rbmo.2016.02.001","article-title":"Strategies to Manage Refractory Endometrium: State of the Art in 2016","volume":"32","author":"Acevedo","year":"2016","journal-title":"Reprod. Biomed. Online"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1097\/GCO.0000000000000378","article-title":"A Comprehensive Review of Asherman\u2019s Syndrome: Causes, Symptoms and Treatment Options","volume":"29","author":"Salazar","year":"2017","journal-title":"Curr. Opin. Obstet. Gynecol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1097\/GCO.0b013e32836024e7","article-title":"Regenerating Endometrium from Stem\/Progenitor Cells: Is It Abnormal in Endometriosis, Asherman\u2019s Syndrome and Infertility?","volume":"25","author":"Deane","year":"2013","journal-title":"Curr. Opin. Obstet. Gynecol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1511","DOI":"10.1007\/s10815-020-01801-x","article-title":"An Update on Stem Cell Therapy for Asherman Syndrome","volume":"37","author":"Benor","year":"2020","journal-title":"J. Assist. Reprod. Genet."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Li, Z., Yan, G., Diao, Q., Yu, F., Li, X., Sheng, X., Liu, Y., Dai, Y., Zhou, H., and Zhen, X. (2019). Transplantation of Human Endometrial Perivascular Cells with Elevated CYR61 Expression Induces Angiogenesis and Promotes Repair of a Full-Thickness Uterine Injury in Rat. Stem Cell Res. Ther., 10.","DOI":"10.1186\/s13287-019-1272-3"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"431","DOI":"10.3389\/fbioe.2019.00431","article-title":"Unraveling the Molecular Signature of Extracellular Vesicles From Endometrial-Derived Mesenchymal Stem Cells: Potential Modulatory Effects and Therapeutic Applications","volume":"7","author":"Marinaro","year":"2019","journal-title":"Front. Bioeng. Biotechnol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/4\/1931\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:16:49Z","timestamp":1760134609000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/4\/1931"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,2,9]]},"references-count":41,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2022,2]]}},"alternative-id":["ijms23041931"],"URL":"https:\/\/doi.org\/10.3390\/ijms23041931","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2022,2,9]]}}}