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• W3154201908 abstract "Purpose: By applying exome sequencing to an extended early-onset osteoarthritis (OA) family followed by linkage analysis, a high impact, likely causal mutation (C518F) was identified in the fibronectin (FN1) gene. Founded by the hypothesis that the underlying pathway in which this high impact mutation is causing OA may likely be extrapolated to common, age related, OA disease pathways we here studied the effects of the identified high impact mutation on neo-cartilage formation. Hereto the identified FN1 mutation was introduced in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome engineering technology while employing these cells to an established 3D in vitro chondrogenesis model. Methods: hiPSCs were electroporated with gRNA/Cas9 complex targeting FN1 and single-strand oligodeoxynucleotide template containing the specific FN1 mutation, after which the hiPSCs were plated at low seeding density to obtain clonal colonies. After 14 days, the multicellular colonies were picked and transferred into a 96-wells plate for clonal screening and expansion. Positive clones were identified by PCR followed by HincIII digestion and confirmed by Sanger sequencing. Mesodermal lineage differentiation towards induced chondroprogenitor cells (iCPCs) was performed by stepwise changes of the culture medium. After 14 days, iCPC aggregates were manually picked and chondrogenesis was initiated in 3D using chondrogenic differentiation medium. Pellets were refreshed twice per week and medium and pellets were collected following 5 weeks of chondrogenesis. Simultaneously, conditioned medium of the wildtype and mutant FN1 pellets was collected. After concentrating the medium, a collagen type II solid-phase binding assay was performed, to compare binding affinity of wildtype and mutant FN1 to collagen type II. Results: Sequencing confirmed that out of 274 hiPSC clones 14 (5.1%) were heterozygous and 3 (1.1%) were homozygous for the C518F FN1 mutation. To investigate the effect of the mutation on chondrogenesis, in vitro derived neo-cartilage constructs of wildtype, heterozygous, and homozygous FN1 mutant hiPSCs were generated. As can be seen in Figure 1, we observed a different morphology in the 3D neo-cartilage pellets formed by homo- and heterozygous FN1 mutated cells as compared to those of wildtype, being that the mutant chondrogenic pellets are more protrusive and frail. Alcian blue and collagen type II staining revealed that the frail matrix was not cartilage. On the other hand, neo-cartilage produced by the iCPCs showed comparable staining intensities between the mutant and wildtype groups. To study whether the C518F mutation, located in the gelatin-binding domain of fibronectin, affected binding to collagen type II, we next determined binding affinity between collagen type II and wildtype, heterozygous and homozygous mutated fibronectin. As shown in Figure 2, we observed a significant (P = 0.002) dose response reduction in binding as function of C518F mutated fibronectin. To study the effect of the mutation on integrity of neo-cartilage formed, we next explored chondrocyte signaling as result of the mutation by performing RT-qPCR. To adjust for differences in efficiency between neo-cartilage formation we used COL2A1 and COL1A1 as housekeeping genes of cartilage tissue. Notably, ADAMTS-5 and RUNX2 showed a dose response significant upregulation, while ACAN, and FN1 expression were downregulated with the C518F mutated as compared to wildtype neo-cartilage chondrocytes. Conclusions: By introducing a high impact OA mutation in human iPSCs while utilizing them in an established 3D in vitro chondrogenesis model we showed that the binding capacity between fibronectin and collagen type II is essential for proper cartilage formation. Moreover, as reflected by the aberrant chondrocyte signaling towards a hypertrophic state, such improper binding makes the cartilage prone to an OA state. Together our work merits further exploration of fibronectin as potential target for therapeutic interventions. We advocate that restoring or maintaining proper binding between fibronectin and collagen type 2 should be the focus of such a quest." @default.
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• W3154201908 date "2021-04-01" @default.
• W3154201908 modified "2023-09-27" @default.
• W3154201908 title "High impact mutation FN1 affects cartilage integrity via aberrant binding to collagen type II" @default.
• W3154201908 doi "https://doi.org/10.1016/j.joca.2021.02.525" @default.
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