Engineered cartilage from human chondrocytes with homozygous knockout of cell cycle inhibitor p21

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.

Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes

“…CRISPR-Cas9 mediated gene editing of primary human chondrocytes is challenging and previous reported transfection efficiencies have varied between 16% and 70% 28 , 29 . To explore the effects of gene editing on chondrocyte biology it has been necessary to examine either edited chondrocyte cell lines 30 , chondrocytes derived from edited induced pluripotent stem cells (iPSCs) 31 or using clonally expanded edited chondrocytes that are likely to have lost their chondrocytic phenotype.…”

An Injectable Hydrogel Scaffold With Kartogenin-Encapsulated Nanoparticles for Porcine Cartilage Regeneration: A 12-Month Follow-up Study

“…19 However, most previous studies followed traditional tissue engineering techniques: Namely, chondrocytes or MSCs were seeded into tissue-engineered scaffolds in vitro, induced into cartilage by growth factors, and finally transplanted into cartilage defects. 6,13,38 However, many drawbacks remain in these traditional techniques: Most tissue engineering materials facilitate extreme cell extension, thus inhibiting chondrogenesis 8,25 ; growth factor substances may lead to immunoreaction in vivo 3 ; and the seeded chondrocytes or MSCs can limit proliferation capacity in vitro. Recently, an innovative cartilage tissue engineering technique (ie, in situ injectable hydrogel) has emerged as a promising method to facilitate cartilage repair.…”

3D Chromatin Structure in Chondrocytes Identifies Putative Osteoarthritis Risk Genes

“…To assess the functional role of SOCS2 , we used CRIS-PR-Cas9 to knock out SOCS2 in primary human chondrocytes isolated from three individual donors. After targeting the SOCS2 gene with two guide RNAs that flank exon 2 (a constitutive exon that contains the translational start site), we used our previously developed method that employs PCR to screen single-cell-derived colonies 53 . The screening primers generated a 1068 bp product if the region was intact and a novel 240 bp amplicon if the two guides successfully deleted the intended 828 bp region ( Fig 4A ).…”

“…Chondrocytes were trypsinized, washed with PBS and transfected with the RNP complex as previously described with modifications; volumes were scaled up for transfection of more cells in larger cuvettes 53 . Two million cells were resuspended in 100 μl of P3 Primary Cell Nucle-ofector™ solution (V4XP-3024, Lonza).…”