Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes

For decades, gene amplification was described as an attribute of tumor cells and as a physiological mechanism to increase gene copy numbers for the higher protein demand during the development of amphibians and flies. Notably, gene amplification has since been detected in normal mammalian cells during differentiation. Numerous amplified genes detected in tumor cells overlap with amplified genes detected during stem cell differentiation. Since stem cells have a valuable potency in regenerative therapies and since cartilage regeneration is a highly demanded therapeutic strategy, the current study investigated gene amplification dynamics during the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). Using quantitative PCR, copy number changes were analyzed for genes previously implicated in differentiation, as well as genes amplified in chondrosarcoma, including CDK4, MDM2, AGAP2, CPT1B, SHANK3, TRIB1 and MYC. Amplifications were transient and stage-specific: CDK4, CPT1B and SHANK3 exhibited the highest copy number increases at day 2, followed by a gradual decline by day 7, whereas AGAP2 and MDM2 increased later in differentiation. Laser microdissection of toluidine blue-stained areas revealed heterogeneity in amplification patterns: CDK4 amplification was prominent in areas lacking or showing moderate proteoglycan deposition; CPT1B amplification occurred in regions with absent, moderate or intense proteoglycan deposition; and SHANK3 amplification was restricted to areas with intense proteoglycan deposition. Notably, regions with the strongest proteoglycan staining exhibited no gene amplification, suggesting that gene amplification is an early, transient event that diminishes as differentiation progresses. These findings highlight gene amplification as a mechanism during chondrogenesis, potentially critical for early differentiation stages and genome stability in mature cells.