Stem cell therapies have shown promise for regenerative treatment for musculoskeletal conditions, but their success is mixed. To enhance regenerative effects, growth factors are utilized to induce differentiation into native cell types, but uncontrollable in vivo conditions inhibit differentiation, and precise control of expressed matrix proteins is difficult to achieve. To address these issues, we investigated a novel method of enhancing regenerative phenotype through direct upregulation of major cartilaginous tissue proteins, aggrecan (ACAN), and collagen II (COL2A1) using dCas9-VPR CRISPR gene activation systems. We demonstrated increased expression and deposition of targeted proteins independent of exogenous growth factors in pellet culture. Singular upregulation of COL2A1/ACAN interestingly indicates that COL2A1 upregulation mediates the highest sulfated glycosaminoglycan (sGAG) deposition, in addition to collagen II deposition. Through RNA-seq analysis, this was shown to occur by COL2A1 upregulation mediating broader chondrogenic gene expression changes. Multiplex upregulation of COL2A1 and ACAN together resulted in the highest sGAG, and collagen II deposition, with levels comparable to those in chondrogenic growth factor-differentiated pellets. Overall, this work indicates dCas9-VPR systems can robustly upregulate COL2A1 and ACAN deposition without growth factors, to provide a novel, precise method of controlling stem cell phenotype for cartilage and intervertebral disc cell therapies and tissue engineering. Impact statement Stem cell therapies have come about as a potential regenerative treatment for musculoskeletal disease, but clinically, they have mixed results. To improve stem cell therapies, growth factors are used to aid a regenerative cell phenotype, but their effects are inhibited by in vivo musculoskeletal disease environments. This article describes CRISPR gene activation-based cell engineering methods that provide a growth factor-free method of inducing chondrogenic extracellular matrix deposition. This method is demonstrated to be as/more potent as growth factors in inducing a chondrogenic phenotype in pellet culture, indicating potential utility as a method of enhancing stem cell therapies for musculoskeletal disease.

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