Stepwise direct reprogramming of adult human dermal fibroblasts into tendon-like cells via transcription factor optimization and 3D culture

Abstract Introduction : Synthetic materials are widely used in pediatric surgery for congenital diaphragmatic hernia repair when primary closure is not feasible but remain associated with complications and lack growth potential. We investigated whether direct reprogramming could promote tenogenic differentiation of adult human dermal fibroblasts (aHDFs) toward patient-derived muscle-tendon constructs for pediatric reconstruction. Methods aHDFs were transduced into directly converted tenoblasts (dTBs) using retroviral transduction of tendon-related transcription factors and Yamanaka factors. Different factor combinations and culture conditions were evaluated. Tenogenic induction was assessed using real-time RT-PCR and immunostaining for tendon markers. In vivo potential was examined by subcutaneous transplantation into NOG/SCID mice. Results Six-factor transduction induced the expression of tendon-related markers tenomodulin (TNMD) and thrombospondin-4 (THBS4), and enabled short-term engraftment in vivo . Optimization identified SCX, MKX, OCT3/4, and MYCL as a sufficient combination for tenogenic conversion. OCT3/4 promoted TNMD expression, whereas SCX affected THBS4 expression. dTBs exhibited elongated tendon-like morphology and positive TNMD and THBS4 staining. Transfer from two-dimensional to three-dimensional culture further increased tenogenic marker expression. Conclusions Direct reprogramming with SCX, MKX, MYCL, and OCT3/4 promoted tenogenic differentiation of aHDFs and induced tendon-like characteristics. This simplified strategy may facilitate development of autologous muscle-tendon constructs for regenerative reconstruction in pediatric surgery. Clinical trial registration Not applicable. This study did not involve a clinical trial