A human organoid drug screen identifies a2-adrenergic receptor signaling as a therapeutic target for cartilage regeneration [bulk RNA-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: To analyze the overall transcriptome features of cultured cells and dissect subpopulations of hypertrophic cartilaginous organoids, we used FCM-sorted cells to conduct RNA sequencing (RNA-seq) on COL2+COL10- and COL2+COL10+ cell populations generated from day 42 organoids. We next performed RNA sequencing of D42 PM-treated and vehicle-treated pellets to comprehensively understand the effects of targeting a-AR on chondrogenic phenotypes. To identify downstream targets through which SLPI promoted hypertrophic differentiation, we conducted RNA-seq of day 42 SLPI KO and control pellets.