A human organoid drug screen identifies a2-adrenergic receptor signaling as a therapeutic target for cartilage regeneration [scRNA-seq]

Directed differentiation of stem cells toward chondrogenesis in vitro and in situ to regenerate cartilage suffers from off-target differentiation and hypertrophic tendency. Here, we generated a cartilaginous organoid system from human expanded pluripotent stem cells (hEPSCs) carrying a COL2A1mCherry and COL10A1eGFP double reporter, enabling real-time monitoring of chondrogenesis and hypertrophy. After screening 2,040 FDA-approved drugs, we found that a-adrenergic receptor (a-AR) antagonists, especially phentolamine, stimulated chondrogenesis but repressed hypertrophy, while a2-AR agonists reduced chondrogenesis and induced hypertrophy. Phentolamine prevented cartilage degeneration in hEPSC cartilaginous organoid and human cartilage explant models and stimulated microfracture-activated endogenous skeletal stem cells toward hyaline-like cartilage regeneration without fibrotic degeneration in situ. Mechanistically, a2-AR signaling induced hypertrophic degeneration via cyclic guanosine monophosphate (cGMP)-dependent secretory leukocyte protease inhibitor (SLPI) production. SLPI-deleted cartilaginous organoid was degeneration resistant, facilitating large cartilage defect healing. Ultimately, targeting a2-AR/SLPI was a promising and clinically feasible strategy to regenerate cartilage via promoting chondrogenesis and repressing hypertrophy. Overall design: Day 28 hEPSC derived-cartilaginous pellets were treated with hypertrophic induction medium containing 1 µM PM in compared with vehicle. After 14 days of incubation, the D42 pellets with or without PM treatment were digested and analyzed using scRNA-seq.