A human organoid drug screen identifies α2-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 α-adrenergic receptor (α-AR) antagonists, especially phentolamine, stimulated chondrogenesis but repressed hypertrophy, while α2-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, α2-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 α2-AR/SLPI was a promising and clinically feasible strategy to regenerate cartilage via promoting chondrogenesis and repressing hypertrophy. 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.