title: Knockdown of the cell cycle inhibitor p21 enhances cartilage formation by induced pluripotent stem cells.
abstract: The limited regenerative capacity of articular cartilage contributes to progressive joint dysfunction associated with cartilage injury or osteoarthritis. Cartilage tissue engineering seeks to provide a biological substitute for repairing damaged or diseased cartilage, but requires a cell source with the capacity for extensive expansion without loss of chondrogenic potential. In this study, we hypothesized that decreased expression of the cell cycle inhibitor p21 would enhance the proliferative and chondrogenic potential of differentiated induced pluripotent stem cells (iPSCs). Murine iPSCs were directed to differentiate toward the chondrogenic lineage with an established protocol and then engineered to express a short hairpin RNA (shRNA) to reduce the expression of p21. Cells expressing the p21 shRNA demonstrated higher proliferative potential during monolayer expansion and increased synthesis of glycosaminoglycans (GAGs) in pellet cultures. Furthermore, these cells could be expanded ∼150-fold over three additional passages without a reduction in the subsequent production of GAGs, while control cells showed reduced potential for GAG synthesis with three additional passages. In pellets from extensively passaged cells, knockdown of p21 attenuated the sharp decrease in cell number that occurred in control cells, and immunohistochemical analysis showed that p21 knockdown limited the production of type I and type X collagen while maintaining synthesis of cartilage-specific type II collagen. These findings suggest that manipulating the cell cycle can augment the monolayer expansion and preserve the chondrogenic capacity of differentiated iPSCs, providing a strategy for enhancing iPSC-based cartilage tissue engineering.
authors.affiliation: 1 Department of Orthopaedic Surgery, Duke University Medical Center , Durham, North Carolina.
authors.forename: Brian O
authors.initials: BO
authors.lastname: Diekman
authors.forename: Pratiksha I
authors.initials: PI
authors.lastname: Thakore
authors.forename: Shannon K
authors.initials: SK
authors.lastname: O'Connor
authors.forename: Vincent P
authors.initials: VP
authors.lastname: Willard
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Brunger
authors.forename: Nicolas
authors.initials: N
authors.lastname: Christoforou
authors.forename: Kam W
authors.initials: KW
authors.lastname: Leong
authors.forename: Charles A
authors.initials: CA
authors.lastname: Gersbach
authors.forename: Farshid
authors.initials: F
authors.lastname: Guilak
mesh_terms: D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D002356:Cartilage / Q000254:growth & development* / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D020219:Chondrogenesis; D003094:Collagen / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism; D024121:Collagen Type X / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006025:Glycosaminoglycans / Q000378:metabolism; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice
chemical_list: D024042:Collagen Type I; D024121:Collagen Type X; D050759:Cyclin-Dependent Kinase Inhibitor p21; D006025:Glycosaminoglycans; D003094:Collagen; D004247:DNA; D001973:Bromodeoxyuridine