Synergistic CRISPRa-Regulated Chondrogenic ECM Deposition Without Exogenous Growth Factors.

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 indicated that COL2A1 upregulation mediates highest sGAG deposition in addition to collagen II deposition. Through RNA-seq analysis this was indicated 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.