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• W2967655775 abstract "Abstract Risk factors for the development of osteoarthritis (OA) include genetic background and focal cartilage injury. The search for disease-modifying OA therapies would benefit from a more comprehensive knowledge of the genetic variants that contribute to chondrocyte dysfunction and the barriers to cartilage regeneration. One goal of this study was to establish a system for producing engineered cartilage tissue from genetically-defined primary human chondrocytes through genome editing and single-cell expansion. This process was utilized to investigate the functional effect of bi-allelic knockout of the cell cycle inhibitor p21. The use of ribonucleoprotein (RNP) CRISPR/Cas9 complexes targeting two sites in the coding region of p21 resulted in a high frequency (16%) of colonies with homozygous p21 knockout. Chondrogenic pellet cultures from expanded chondrocytes with complete loss of p21 produced more glycosaminoglycans (GAG) and maintained a higher cell number. Single-cell derived colonies retained the potential for robust matrix production after expansion, allowing for analysis of colony variability from the same population of targeted cells. The effect of enhanced cartilage matrix production in p21 knockout chondrocytes persisted when matrix production from individual colonies was analyzed. Chondrocytes had lower levels of p21 protein with further expansion, and the difference in GAG production with p21 knockout was strongest at early passages. These results support previous findings that implicate p21 as a barrier to cartilage matrix production and regenerative capacity. Further, this work establishes the use of genome-edited human chondrocytes as a promising approach for engineered tissue models containing user-defined gene knockouts and other genetic variants for investigation of OA pathogenesis." @default.
• W2967655775 created "2019-08-22" @default.
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• W2967655775 date "2019-08-09" @default.
• W2967655775 modified "2023-09-23" @default.
• W2967655775 title "Engineered cartilage from human chondrocytes with homozygous knockout of cell cycle inhibitor p21" @default.
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• W2967655775 doi "https://doi.org/10.1101/731216" @default.
• W2967655775 hasPublicationYear "2019" @default.
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