Bio-3D printing with smooth muscle cells derived from human iPSCs via neural crest and its application for the tracheal regeneration

Smooth muscle cells (SMCs) derived from induced pluripotent stem cells (iPSCs) have been used for scaffold-free structures; however, their use in regenerated organs is rare and not well established. 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's Modified Eagle Medium (DMEM) with fetal bovine serum (FBS), with one group receiving transforming growth factor beta 1 (TGF<i>β</i>1, DMEM-TGF<i>β</i>1 group) and the other group without TGF<i>β</i>1 (DMEM group). SMCs markers was assessed using immunofluorescence staining. The tissue constructs were bio-3D printed using spheroids from the DMEM-TGF<i>β</i>1 group and transplanted as smooth muscle patches into full-thickness defects in the rats' tracheas. The DMEM-TGF<i>β</i>1 group showed strong expression of SMCs markers such as<i>α</i>-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.