Epigenetic programming of human infrapatellar fat pad progenitors by inducible KDM6B to enhance cartilage regeneration and overcome senescence barriers

Articular cartilage injury and osteoarthritis (OA) are leading causes of chronic pain and disability because adult hyaline cartilage has minimal intrinsic repair capacity. Although mesenchymal stem/stromal cell (MSC)–based therapies are promising, clinical and preclinical outcomes remain inconsistent due to donor-to-donor variability, potency loss during ex vivo expansion, and a common failure mode in which engineered cartilage drifts toward hypertrophy, fibrocartilage, or catabolic remodeling. Infrapatellar fat pad–derived progenitor cells (IPFPCs) are an attractive autologous MSC source, harvested from the knee with low morbidity, and retain chondrogenic potential even in OA. However, their performance still declines with expansion and remains constrained by epigenetic repression and senescence-associated inflammatory drift. This project addresses these coupled barriers by coordinating a cell-intrinsic epigenetic program with a pro-regenerative metabolic cue to improve the reliability and stability of IPFPC-derived hyaline cartilage. The central hypothesis is that transient, temporally controlled KDM6B activation will relieve PRC2/H3K27me3-mediated repression at chondrogenic loci to enhance hyaline matrix programs, and that 15-PGDH inhibition (MF-DH-300) will sustain pro-anabolic PGE2 signaling to mitigate senescence-linked dysfunction and restrain hypertrophic drift. In Aim 1, we will establish a KDM6B-conditioned decellularized extracellular matrix (dECM) “niche memory” generated under inducible KDM6B activity, and test whether expansion on KDM6B-dECM with or without MF-DH-300 reduces senescence markers (SA-β-gal, p16INK4A, SASP factors) and produces durable improvements in subsequent 3D pellet chondrogenesis (GAG and type II collagen) without increased hypertrophy/catabolism (type X collagen, COL10A1, MMP13). We will then optimize differentiation-phase schedules of KDM6B induction and MF-DH-300 and define on-target mechanisms using RNA-seq and locus-specific H3K27me3 profiling. In Aim 2, we will translate lead conditioning regimens into an immunodeficient rat focal osteochondral defect model to determine whether preconditioned human IPFPC constructs improve defect fill, integration, and hyaline composition (GAG, COL2, COL10, MMP13, and COL1) over 6-8 weeks. Successful completion will deliver a practical, biomarker-guided conditioning strategy that standardizes potency across donors, establishes in vivo proof of concept, and de-risks a subsequent R01 aimed at durability, scale-up, and a clinically deployable autologous cartilage repair augmented by local small-molecule modulation.