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The induction of mesenchymal stem cells (MSCs) via neural crest cells (NCCs) from iPSCs offers advantages such as a large-scale cell stock. While research has progressed on the chondrogenic differentiation and regenerative medicine applications of cartilage derived from human iPSC-derived MSCs via a NCCs lineage (iNC), studies on smooth muscle, a critical tracheal component alongside cartilage, remain limited. In this study, we aimed to establish a method for generating airway smooth muscle tissue constructs using human iNCMSCs, assess their contractile function, and evaluate their regenerative potential in tracheal cartilage defects. iNCMSCs were cultured for 28 d in Dulbecco\u2019s Modified Eagle Medium (DMEM) with fetal bovine serum (FBS), with one group receiving transforming growth factor beta 1 (TGF\n                    <jats:italic>\u03b2<\/jats:italic>\n                    1, DMEM-TGF\n                    <jats:italic>\u03b2<\/jats:italic>\n                    1 group) and the other group without TGF\n                    <jats:italic>\u03b2<\/jats:italic>\n                    1 (DMEM group). SMCs markers was assessed using immunofluorescence staining. The tissue constructs were bio-3D printed using spheroids from the DMEM-TGF\n                    <jats:italic>\u03b2<\/jats:italic>\n                    1 group and transplanted as smooth muscle patches into full-thickness defects in the rats\u2019 tracheas. The DMEM-TGF\n                    <jats:italic>\u03b2<\/jats:italic>\n                    1 group showed strong expression of SMCs markers such as\n                    <jats:italic>\u03b1<\/jats:italic>\n                    -smooth muscle actin, calponin, and myosin heavy chain. After 28 d post-transplant, histological evaluation confirmed graft engraftment, adequate blood flow, and epithelial layer extensions from the recipient tissues, along with well-maintained tracheal structures. This study demonstrated the feasibility of using iPSC-derived iNCMSCs to generate bio-3D printed smooth muscle constructs for tracheal regeneration. Our findings support the potential of this strategy as a novel approach for airway reconstruction, offering a scaffold-free cell-based platform for future clinical applications in tissue engineering for airway regeneration.\n                  <\/jats:p>","DOI":"10.1088\/1758-5090\/ae1665","type":"journal-article","created":{"date-parts":[[2025,10,22]],"date-time":"2025-10-22T22:53:06Z","timestamp":1761173586000},"page":"015003","update-policy":"https:\/\/doi.org\/10.1088\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Bio-3D printing with smooth muscle cells derived from human iPSCs via neural crest and its application for the tracheal regeneration"],"prefix":"10.1088","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1849-8754","authenticated-orcid":true,"given":"Shintaro","family":"Hashimoto","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1702-9021","authenticated-orcid":false,"given":"Daisuke","family":"Taniguchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryoichiro","family":"Doi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomohiro","family":"Obata","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6046-2018","authenticated-orcid":false,"given":"Toshio","family":"Shiraishi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-0051-1739","authenticated-orcid":false,"given":"Takamune","family":"Matsumoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-4108-2049","authenticated-orcid":false,"given":"Hiroshi","family":"Maruta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-8466-0383","authenticated-orcid":true,"given":"Keiko","family":"Akao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6099-1702","authenticated-orcid":false,"given":"Satoshi","family":"Mizoguchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0561-4082","authenticated-orcid":true,"given":"Koichiro","family":"Shimoyama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9429-3227","authenticated-orcid":false,"given":"Tetsuro","family":"Tominaga","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2718-2839","authenticated-orcid":false,"given":"Takuro","family":"Miyazaki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5878-4870","authenticated-orcid":false,"given":"Takashi","family":"Nonaka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryota","family":"Otsubo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6506-5070","authenticated-orcid":false,"given":"Katsunori","family":"Takagi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-9803-4358","authenticated-orcid":false,"given":"Mayumi","family":"Iwatake","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7324-8862","authenticated-orcid":false,"given":"Koichi","family":"Nakayama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3930-8032","authenticated-orcid":true,"given":"Makoto","family":"Ikeya","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4344-2322","authenticated-orcid":false,"given":"Keitaro","family":"Matsumoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"266","published-online":{"date-parts":[[2025,11,10]]},"reference":[{"key":"bfae1665bib1","doi-asserted-by":"publisher","first-page":"63","DOI":"10.1016\/j.cell.2006.07.024","type":"journal-article","article-title":"Induction 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