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Human PSC-derived articular cartilage chondrocytes scRNA-seq

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE230195 | GEO | 2023/08/24

REPOSITORIES: GEO

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Project description:Background: Meniscus tears are the most common injury in the knee and are associated with an increased risk of osteoarthritis (OA). The molecular profile of knees with meniscus tears is not well-studied. Therefore, to advance our understanding of the early response of the knee to injury, we compared the gene expression profile of meniscus and articular cartilage within the same knees following meniscus injury. Hypothesis/Purpose: To identify differences between the molecular signatures of meniscus and articular cartilage from knees with intact articular cartilage undergoing arthroscopic partial meniscectomy. Study Design: Descriptive laboratory study Methods: Patients (n=12) with a known isolated medial meniscus tear without any knee chondrosis or radiographic OA were consented prior to surgery. During arthroscopic partial meniscectomy, a sample of their injured meniscus and a sample of their articular cartilage off the medial femoral condyle were procured. The transcriptome signatures, as measured through Affymetrix microarray, were compared between the two tissues and underlying biological processes were explored computationally. Results: 3566 gene transcripts were differentially expressed between meniscus and articular cartilage. Gene transcripts down-regulated in articular cartilage were associated with extracellular matrix organization, wound healing, cell adhesion, and chemotaxis. Gene transcripts up-regulated in articular cartilage were associated with blood vessels morphogenesis and angiogenesis. Examples of individual genes with significant differences in expression between the two tissues include IBSP (23.76 fold; P < 0.001), upregulated in meniscus, and TREM1 (3.23 fold; P = 0.006), upregulated in meniscus. Conclusion: The meniscus and articular cartilage have distinct gene expression profiles in knees with meniscus tears and intact articular cartilage. Total RNA obtained from injured meniscus and normal articular cartilage from patients undergoing partial meniscectomy.

Project description:As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that Cbfβ, (subunit of a heterodimeric Cbfβ/Runx1,Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfβ in tamoxifen-induced Cbfβf/fCol2α1-CreERT mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfβ deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/YAP signaling and TGF-β signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfβ and Yap expression and increased active β-catenin expression in articular cartilage, while local AAV-mediated Cbfβ overexpression promoted Yap expression and diminished active β-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfβ overexpression in knee joints of mice with OA showed the significant protective effect of Cbfβ on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfβ may be the cause of OA. Moreover, Local admission of Cbfβ may rescue and protect OA through decreasing Wnt/β-catenin signaling, and increasing Hippo/Yap signaling and TGFβ/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfβ overexpression could be an effective strategy for treatment of OA. Using unbiased genome-wide RNA-seq data from Cbfβf/f;Col2α1-Cre hip joint articular cartilage and Cbfβf/f;Aggrecan-cre knee joint articular cartilage and their controls, we examined Cbfβ-mediated transcriptional targets for articular cartilage regeneration in OA.

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Human PSC-derived articular cartilage chondrocytes scRNA-seq

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