Directed differentiation of human pluripotent stem cells into articular cartilage reveals effects caused by absence ofWISP3, the gene responsible for Progressive Pseudorheumatoid Arthropathy of Childhood
Authors: Li Chaochang , Ribes Mireia Alemany , Raftery Rosanne , Nwoko Uzochi , Warman Matthew L. , Craft April M. 
Published: April 2023

ABSTRACTObjectivesProgressive Pseudorheumatoid Arthropathy of Childhood (PPAC), caused by deficiency ofWNT1 inducible signaling pathway protein 3(WISP3), has been challenging to study because no animal model of the disease exists and cartilage recovered from affected patients is indistinguishable from common end-stage osteoarthritis. Therefore, to gain insights into why precocious articular cartilage failure occurs in this disease, we madein vitroderived articular cartilage using isogenicWISP3-deficient andWISP3-sufficient human pluripotent stem cells (hPSCs).MethodsWe generated articular cartilage-like tissues from induced-(i)PSCs from 2 patients with PPAC and 1 wild-type human embryonic stem cell line in which we knocked out WISP3. We compared these tissues toin vitro-derived articular cartilage tissues from 2 isogenicWISP3-sufficient control lines using histology, bulk RNA sequencing, single cell RNA sequencing, andin situhybridization.ResultsWISP3-deficient andWISP3-sufficient hPSCs both differentiated into articular cartilage-like tissues that appeared histologically similar. However, the transcriptomes ofWISP3-deficient tissues differed significantly fromWISP3-sufficient tissues and pointed to increased TGFβ, TNFα/NFkB, and IL-2/STAT5 signaling and decreased oxidative phosphorylation. Single cell sequencing andin situhybridization revealed thatWISP3-deficient cartilage contained a significantly higher fraction (∼ 4-fold increase,p< 0.001) of superficial zone chondrocytes compared to deeper zone chondrocytes than didWISP3-sufficient cartilage.ConclusionsWISP3-deficient andWISP3-sufficient hPSCs can be differentiated into articular cartilage-like tissues, but these tissues differ in their transcriptomes and in the relative abundances of chondrocyte sub-types they contain. These findings provide important starting points forin vivostudies when an animal model of PPAC or presymptomtic patient-derived articular cartilage becomes available.KEY MESSAGESWhat is already known on this topicLoss-of-function mutations inWISP3cause Progressive Pseudorheumatoid Arthropathy of Childhood (PPAC), yet the precise function ofWISP3in cartilage is unknown due to the absence of cartilage diseaseWisp3knockout mice and the lack of available PPAC patient cartilage that is not end-stage. Thus, most functional studies of WISP3 have been performedin vitrousing WISP3 over-expressing cell lines (i.e., not wild-type) andWISP3-deficient chondrocytes.What this study addsWe describe 3 newWISP3-deficient human pluripotent stem cell (hPSC) lines and show they can be differentiated into articular cartilage-like tissue.We comparein vitro-derived articular cartilage made fromWISP3-deficient and isogenicWISP3- sufficient hPSCs using bulk RNA sequencing, single cell RNA sequencing, andin situhybridization.We observe significant differences in the expression of genes previously associated with cartilage formation and homeostasis in the TGFβ, TNFα/NFkB, and IL-2/STAT5 signaling pathways. We also observe that WISP3-deficient cartilage-like tissues contain significantly higher fractions of chondrocytes that express superficial zone transcripts. These data suggest precocious cartilage failure in PPAC is the result of abnormal articular cartilage formation, dysregulated homeostatic signaling, or both.How this study might affect research, practice or policyThis study usesin vitro-derived articular cartilage to generate hypotheses for why cartilage fails in children with PPAC. This work prioritizes downstream studies to be performed when pre-symptomatic patient-derived cartilage samples or animal model of PPAC becomes available. It is essential to know how WISP3 functions in cartilage to develop therapies that benefit patients with PPAC and other degenerative joint diseases.