Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes

Abstract

For decades gene amplifications were described as an attribute of tumor cells and as a physiological mechanism to increase gene copy numbers for the higher protein demand during development of amphibians and flies. An increasing number of publications describe gene amplifications in normal mammalian cells during differentiation. Many 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, we investigated gene amplification dynamics during chondrogenic differentiation of human mesenchymal stem cells (hMSCs). Using quantitative PCR, we analyzed copy number changes 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, while 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.