Human PSC-derived articular cartilage chondrocytes scRNA-seq

Objectives Traditional approaches to study Progressive Pseudorheumatoid Arthropathy of Childhood (PPAC) failed to uncover how loss-of-function mutations in Wnt inducible secreted protein 3 (WISP3) cause premature cartilage failure in children. We developed a human induced pluripotent stem cell (hiPSC)-based model of cartilage development to elucidate pathological changes in WISP3-deficient articular cartilage tissues compared to WISP3-sufficient isogenic cartilage tissues. Methods We generated iPSCs from 2 patients with PPAC, and we corrected the disease-causing mutation in one of these lines using CRISPR/Cas9. We also created a WISP3-knockout human embryonic stem cell line. We generated articular cartilage tissues from these hPSCs by established directed differentiation methods. Bioinformatic analyses were used to identify differentially expressed genes (DEGs) and cell type abundances, which were validated by quantitative RT-qPCR and in situ hybridization. Results WISP3-deficient articular cartilage tissues exhibited significantly different transcriptomic profiles and cellular composition compared with their isogenic controls. WISP3-deficient cartilage transcriptomes exhibited enriched biological processes such as TGFb signaling and epithelial to mesenchymal transition. We validated increased expression of TGFb-related genes in WISP3-deficient cartilage, and determined that the abundance of chondrocyte subtypes was altered compared with isogenic controls. Conclusions We developed a robust and reproducible model of iPSC-derived cartilage development to better understand the role of WISP3 in cartilage biology, and the pathology of cartilage failure associated with loss of WISP3 in patients with PPAC. We identified several altered biological processes and signaling pathways in WISP3-deficient cartilage that can be validated in the future with additional patient-derived cell lines, or large animal models. comparative gene expression profiling of in vitro derived cartilage chrondrocytes from human PSCs Please note: Raw data are not available for the PPAC iPSC samples due to patient privacy concerns.

研究目的：过往研究儿童进行性假性类风湿性关节病（Progressive Pseudorheumatoid Arthropathy of Childhood, PPAC）的传统方法，未能阐明Wnt诱导分泌蛋白3（Wnt inducible secreted protein 3, WISP3）的功能丧失性突变如何导致儿童软骨过早退变。本研究构建了基于人类诱导多能干细胞（human induced pluripotent stem cell, hiPSC）的软骨发育模型，以阐明WISP3缺陷型关节软骨组织与同基因背景下WISP3正常表达的软骨组织之间的病理差异。

研究方法：本研究从2名PPAC患者体内诱导生成了诱导多能干细胞（iPSCs），并利用CRISPR/Cas9基因编辑技术修复了其中一株细胞系中的致病突变。此外，本研究还构建了WISP3基因敲除的人类胚胎干细胞系。通过已建立的定向分化方法，从上述人类多能干细胞（human pluripotent stem cells, hPSCs）中诱导生成关节软骨组织。本研究通过生物信息学分析鉴定差异表达基因（differentially expressed genes, DEGs）与细胞类型丰度，并采用定量RT-qPCR（quantitative RT-qPCR）与原位杂交技术对分析结果进行验证。

研究结果：与同基因对照样本相比，WISP3缺陷型关节软骨组织的转录组谱与细胞组成均存在显著差异。WISP3缺陷型软骨的转录组富集了诸多生物学过程，例如转化生长因子β（transforming growth factor beta, TGF-β）信号通路及上皮间质转化（epithelial to mesenchymal transition, EMT）。本研究验证了WISP3缺陷型软骨中TGF-β相关基因的表达上调，并发现与同基因对照相比，软骨细胞亚型的丰度发生了改变。

研究结论：本研究构建了一套稳定且可重复的iPSC来源软骨发育模型，以更好地阐明WISP3在软骨生物学中的作用，以及PPAC患者中WISP3缺失相关的软骨退变病理机制。本研究在WISP3缺陷型软骨中鉴定出多个发生异常的生物学过程与信号通路，未来可通过更多患者来源的细胞系或大型动物模型对这些结果进行验证。

人类多能干细胞体外诱导软骨细胞的比较基因表达谱分析

请注意：由于患者隐私保护问题，PPAC患者iPSC样本的原始数据暂不对外公开。