Current protocols for the differentiation of human pluripotent stem cells (hPSCs) into chondrocytes do not allow for the expansion of intermediate progenitors so as to prospectively assess their chondrogenic potential. Here we report a protocol that leverages PRRX1–tdTomato reporter hPSCs for the selective induction of expandable and ontogenetically defined PRRX1⁺ limb-bud-like mesenchymal cells under defined xeno-free conditions, and the prospective assessment of the cells’ chondrogenic potential via the cell-surface markers CD90, CD140B and CD82. The cells, which proliferated stably and exhibited the potential to undergo chondrogenic differentiation, formed hyaline cartilaginous-like tissue commensurate to their PRRX1-expression levels. Moreover, we show that limb-bud-like mesenchymal cells derived from patient-derived induced hPSCs can be used to identify therapeutic candidates for type II collagenopathy and we developed a method to generate uniformly sized hyaline cartilaginous-like particles by plating the cells on culture dishes coated with spots of a zwitterionic polymer. PRRX1⁺ limb-bud-like mesenchymal cells could facilitate the mass production of chondrocytes and cartilaginous tissues for applications in drug screening and tissue engineering.

目前将人类多能干细胞 (hPSC) 分化为软骨细胞的方案不允许扩展中间祖细胞，以便前瞻性地评估它们的软骨形成潜力。在这里，我们报告了一种利用 PRRX1–tdTomato 报告基因 hPSC 选择性诱导可扩展和个体遗传学定义的 PRRX1 ^+的方案在确定的无异源条件下的肢芽样间充质细胞，以及通过细胞表面标记 CD90、CD140B 和 CD82 对细胞软骨形成潜力的前瞻性评估。这些细胞稳定增殖并表现出进行软骨分化的潜力，形成与其 PRRX1 表达水平相称的透明软骨样组织。此外，我们表明，源自患者的诱导性 hPSC 的肢芽样间充质细胞可用于鉴定 II 型胶原病的治疗候选者，并且我们开发了一种通过将细胞铺在培养物上来生成大小均匀的透明软骨样颗粒的方法涂有两性离子聚合物斑点的盘子。PRRX1 ⁺肢芽样间充质细胞可以促进软骨细胞和软骨组织的大量生产，用于药物筛选和组织工程。