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.

中文翻译：

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人类类器官药物筛选确定 α2-肾上腺素能受体信号转导是软骨再生的治疗靶点


干细胞在体外和原位定向分化至软骨生成 以再生软骨，存在脱靶分化和肥大倾向。在这里，我们从携带 COL2A1mCherry 和 COL10A1eGFP 双报告基因的人扩增多能干细胞 （hEPSC） 中生成了一个软骨类器官系统，能够实时监测软骨形成和肥大。在筛选了 2,040 种 FDA 批准的药物后，我们发现 α-肾上腺素能受体 （α-AR） 拮抗剂，尤其是酚妥拉明，刺激软骨形成但抑制肥大，而 α2-AR 激动剂减少软骨形成并诱导肥大。酚妥拉敏阻止了 hEPSC 软骨类器官和人软骨外植体模型中的软骨变性，并刺激微骨折激活的内源性骨骼干细胞进行透明样软骨再生，而没有原位纤维化变性。从机制上讲，α2-AR 信号传导通过环磷酸鸟苷 （cGMP） 依赖性分泌型白细胞蛋白酶抑制剂 （SLPI） 的产生诱导肥厚性变性。SLPI 缺失的软骨类器官具有抗退性，促进大软骨缺损愈合。最终，靶向 α2-AR/SLPI 是一种有前途且临床可行的策略，通过促进软骨生成和抑制肥大来再生软骨。