Mechanistic study comparing cartilage formation from human mesenchymal stromal cells and iPSC-derived multipotent cells [bulk RNA-seq]

Multipotent progenitor cells (iMPCs) created from induced pluripotent stem cells (iPSCs) is a promising cell source for cartilage regeneration. In most studies, bone morphogenetic proteins (BMPs) were shown to significantly enhance transforming growth factor-b (TGFb)-induced iMPC chondrogenesis. In contrast, TGFb alone is sufficient to induce robust chondrogenesis of human primary mesenchymal stromal cells (MSCs). Currently, the mechanism underlying this difference between iMPCs and MSCs has not been fully understood. In this study, we first generated iMPCs from human iPSCs and examined their differentiation capacity at different passages. We then optimized the conditions for chondrogenesis and determined that medium supplemented with TGFb and BMP6 led to robust cartilage formation from iMPCs with minimal hypertrophy. Moreover, the cartilage generated from this new method was resistant to osteogenic transition upon subcutaneous implantation and led to a hyaline cartilage-like regeneration in osteochondral defects in rats. Interestingly, TGFb alone induced phosphorylation of Smad2/3 in iMPCs but not Smad1/5, which led to poor chondrogenesis. In contrast, TGFb resulted in the phosphorylation of both Smad2/3 and Smad1/5 and robust chondrogenesis in human MSCs. We further discovered that the remarkably low level of Activin receptor-like kinase 1 (ACVRL1/ALK1) in iMPCs, when compared to MSCs, partially accounts for this difference. For instance, overexpression of ALK1 in iMPCs activated both Smad1/5 and Smad2/3 upon TGFb only treatment and resulted in a high level of chondrogenesis, which was not observed in control iMPCs. In summary, this study describes a robust method to generate chondrocytes with low hypertrophy for hyaline cartilage repair and elucidates the difference between MSCs and iMPCs in response to TGFb. Overall design: mRNA profiles of two conditions. MSC_B3: MSC under TGFß3 stimulation; iMPC_ B3: iMPC under TGFß3 stimulation. Three repeats per condition

多能祖细胞（multipotent progenitor cells, iMPCs）由诱导多能干细胞（induced pluripotent stem cells, iPSCs）诱导生成，是软骨再生极具潜力的细胞来源。既往多数研究显示，骨形态发生蛋白（bone morphogenetic proteins, BMPs）可显著增强转化生长因子-β（transforming growth factor-β, TGFβ）诱导的iMPC软骨分化。与之相反，单独使用TGFβ即可充分诱导人原代间充质基质细胞（human primary mesenchymal stromal cells, MSCs）发生显著软骨分化。目前，iMPCs与MSCs之间的这种差异机制尚未完全阐明。
本研究首先从人iPSCs中诱导生成iMPCs，并检测了不同传代阶段的分化能力；随后优化了软骨分化培养条件，确定添加TGFβ与BMP6的培养基可使iMPCs生成肥大程度极低的成熟软骨组织。此外，通过该方法生成的软骨在皮下植入后可抵抗成骨转化，且能在大鼠骨软骨缺损模型中实现类似透明软骨的再生。
值得注意的是，单独使用TGFβ可诱导iMPCs中Smad2/3磷酸化，但无法激活Smad1/5，这导致软骨分化效果不佳；而在人MSCs中，TGFβ可同时诱导Smad2/3与Smad1/5磷酸化，进而实现高效软骨分化。本研究进一步发现，与MSCs相比，iMPCs中激活素受体样激酶1（activin receptor-like kinase 1, ACVRL1/ALK1）的表达水平显著偏低，这部分解释了上述差异。例如，在iMPCs中过表达ALK1后，仅用TGFβ处理即可同时激活Smad1/5与Smad2/3，进而实现高水平的软骨分化，而对照组iMPCs则无此现象。
综上，本研究建立了一种可高效生成低肥大程度软骨细胞的方法，用于透明软骨修复，并阐明了MSCs与iMPCs对TGFβ响应的差异机制。
整体实验设计：两种培养条件下的mRNA表达谱。MSC_B3：经TGF-β3刺激的MSCs；iMPC_B3：经TGF-β3刺激的iMPCs。每组设置3次生物学重复。