The Effect And Mechanism Study Of Chemical Reprogramming From Fibroblasts Into Articular Chondrocytes

Articular cartilage degeneration can result in serious pain,quality-of-life and labour loss,which is a global problem in the context of growing aged populations.Due to the poor self-renewal capacity and quantity limitation of chondrocytes in adult humans,alternative functional cell sources are urgently needed for articular cartilage regeneration.The advances in cartilage regeneration has allowed fibroblasts to be directly reprogrammed into chondrocytes by viral-induced overexpression of defined transcription factors.However,the methodology brings concerns and risks in clinical application,including tumor formation,genome mutation,and operation complexation.Small-molecule drugs are easy to be stored and transported,and can diffuse though cell membranes without a carrier.Thus cell-fate controlling by small-molecule drugs potentially offers a safe,cost-effective and applicable approach to generate articular chondrocytes.However,the chemical-induced direct reprogramming of fibroblasts into articular chondrocytes,has yet to be investigated.To address this problem,in this study,we established fibroblast-to-chondrocyte chemical reprogramming by combinatory screening of small molecular drugs;we adopted 3-dimentiaonal culture for chemical-induced chondrocytes organoid induction;we conducted single cell RNA-sequencing for the dissection of the dynamical phenotypic changes and molecular mechanisms during the process;we also used animal articular cartilage repair model for the validation of regenerative function of chemical-induced chondrocytes.In this study,we have demonstrated that 1)a combination of 5 small molecule compounds: VPA(a histone deacetylase inhibitor),CHIR-98014(a GSK-3 kinase inhibitor),Repsox(a TGF-β pathway inhibitor),TTNPB(a retinoic acid receptor agonist),and Celecoxib(a cyclooxygenase(COX)2 inhibitor),could reprogram mouse fibroblasts into chondrocytes.2)Using three-dimensional culture,we established a chemical-induced cartilageous organoid induction system,and significantly increased the induction efficiency to ～20%.3)Using single-cell RNA sequencing,we systematically characterized the cellular phenotypes of multiple time points in fibroblast-chondrocyte reprogramming.We also uncovered the phenotypic similarity of induced chondrocytes with articular chondrocytes,and demonstrated that the chondrogenesis induction resembled embryonic cartilage development.4)To explore the molecular mechanisms,we revealed the inhibition of fibrosis features and the activation of cartilage developmental pathways in early reprogramming,which drove fibroblasts toward intermediate chondrogenic progenitors bypassing stem cell state.5)In animal study,chemical-induced cartilageous tissue could promote tissue repair at articular surfaces and rescue over 60% of the mechanical function loss.Our chemical reprogramming approach directly converts fibroblasts into articular chondrocytes with in vivo regenerative function.By using single-cell RNA sequencing,we dissected the cellular phenotypic roadmap and molecular mechanisms.This study provides a potential approach to develop articular chondrocytes resource,a potential target for in situ anti-fibrosis cartilage regeneration,and theoretical basis for chemical reprogramming mechanism study.