Directed differentiation of human pluripotent stem cells into articular cartilage reveals effects caused by absence of WISP3, the gene responsible for progressive pseudorheumatoid arthropathy of childhood.

OBJECTIVES: Progressive pseudorheumatoid arthropathy of childhood (PPAC), caused by deficiency of WNT1 inducible signalling 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 made in vitro derived articular cartilage using isogenic WISP3-deficient and WISP3-sufficient human pluripotent stem cells (hPSCs). METHODS: We generated articular cartilage-like tissues from induced-(i) PSCs from two patients with PPAC and one wild-type human embryonic stem cell line in which we knocked out WISP3. We compared these tissues to in vitro-derived articular cartilage tissues from two isogenic WISP3-sufficient control lines using histology, bulk RNA sequencing, single cell RNA sequencing and in situ hybridisation. RESULTS: WISP3-deficient and WISP3-sufficient hPSCs both differentiated into articular cartilage-like tissues that appeared histologically similar. However, the transcriptomes of WISP3-deficient tissues differed significantly from WISP3-sufficient tissues and pointed to increased TGFß, TNFα/NFκB, and IL-2/STAT5 signalling and decreased oxidative phosphorylation. Single cell sequencing and in situ hybridisation revealed that WISP3-deficient cartilage contained a significantly higher fraction (~4 fold increase, p<0.001) of superficial zone chondrocytes compared with deeper zone chondrocytes than did WISP3-sufficient cartilage. CONCLUSIONS: WISP3-deficient and WISP3-sufficient hPSCs can be differentiated into articular cartilage-like tissues, but these tissues differ in their transcriptomes and in the relative abundances of chondrocyte subtypes they contain. These findings provide important starting points for in vivo studies when an animal model of PPAC or presymptomatic patient-derived articular cartilage becomes available.

Directed differentiation of human pluripotent stem cells into articular cartilage reveals effects caused by absence of WISP3 , the gene responsible for Progressive Pseudorheumatoid Arthropathy of Childhood.

Objectives: Progressive Pseudorheumatoid Arthropathy of Childhood (PPAC), caused by deficiency of WNT1 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 made in vitro derived articular cartilage using isogenic WISP3 -deficient and WISP3 -sufficient human pluripotent stem cells (hPSCs). Methods: We 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 to in vitro -derived articular cartilage tissues from 2 isogenic WISP3 -sufficient control lines using histology, bulk RNA sequencing, single cell RNA sequencing, and in situ hybridization. Results: WISP3 -deficient and WISP3 -sufficient hPSCs both differentiated into articular cartilage-like tissues that appeared histologically similar. However, the transcriptomes of WISP3 -deficient tissues differed significantly from WISP3 -sufficient tissues and pointed to increased TGFß, TNFα/NFkB, and IL-2/STAT5 signaling and decreased oxidative phosphorylation. Single cell sequencing and in situ hybridization revealed that WISP3 -deficient cartilage contained a significantly higher fraction (∼ 4-fold increase, p < 0.001) of superficial zone chondrocytes compared to deeper zone chondrocytes than did WISP3 -sufficient cartilage. Conclusions WISP3 -deficient and WISP3 -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 for in vivo studies when an animal model of PPAC or presymptomtic patient-derived articular cartilage becomes available. KEY MESSAGES: What is already known on this topic: Loss-of-function mutations in WISP3 cause Progressive Pseudorheumatoid Arthropathy of Childhood (PPAC), yet the precise function of WISP3 in cartilage is unknown due to the absence of cartilage disease Wisp3 knockout mice and the lack of available PPAC patient cartilage that is not end-stage. Thus, most functional studies of WISP3 have been performed in vitro using WISP3 over-expressing cell lines (i.e., not wild-type) and WISP3 -deficient chondrocytes. What this study adds: We describe 3 new WISP3 -deficient human pluripotent stem cell (hPSC) lines and show they can be differentiated into articular cartilage-like tissue. We compare in vitro -derived articular cartilage made from WISP3 -deficient and isogenic WISP3 - sufficient hPSCs using bulk RNA sequencing, single cell RNA sequencing, and in situ hybridization. We observe significant differences in the expression of genes previously associated with cartilage formation and homeostasis in the TGFß, 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 policy: This study uses in 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.

Generation and characterization of Six2 conditional mice.

Heterozygous deletion of Six2, which encodes a member of sine oculis homeobox family transcription factors, has recently been associated with the frontonasal dysplasia syndrome FND4. Previous studies showed that Six2 is expressed in multiple tissues during craniofacial development in mice, including embryonic head mesoderm, postmigratory frontonasal neural crest cells, and epithelial and mesenchymal cells of the developing palate and nasal structures. Whereas Six2 -/- mice exhibited cranial base defects but did not recapitulate frontonasal phenotypes of FND4 patients, Six1 -/- Six2 -/- double mutant mice showed severe craniofacial defects including midline facial clefting. The complex phenotypes of FND4 patients and of Six1 -/- Six2 -/- mutant mice indicate that Six2 plays crucial roles in distinct cell types at multiple stages of craniofacial morphogenesis. Here we report generation of mice carrying insertions of a pair of loxP sites flanking exon-1 of the Six2 gene (Six2 f allele) using CRISPR/Cas9-mediated genome editing. We show that the Six2 f allele functions normally and is effectively inactivated by Cre-mediated recombination in vivo. Furthermore, we show that Six2 f/f ;Wnt1-Cre mice recapitulated cranial base defects but not neonatal lethality of Six2 -/- mice. These results indicate that Six2 f/f mice enable systematic investigation of cell type- and stage-specific Six2 function in development and disease.