Human adipose-derived mesenchymal stromal cells in vitro expansion for regenerative applications: tracking small genetic variants reveals dynamic changes within a genetically stable framework

BACKGROUND: The extended use of mesenchymal stromal cells (MSC) for cellular therapies raises safety concerns, and preliminary controls are needed before clinical application to guarantee patient safety. In vitro expansion is frequently required to obtain sufficient material, with an increased risk of appearance and selection of genetically altered or senescent cells, which could impair not only safety but also the biological potential and therapeutic efficacy of these cells. In vitro manipulated MSCs must adhere to the guidelines of Good Manufacturing Practices including the verification of genomic stability, transformation potential and senescence. Several tests are currently used, mainly karyotyping, which enables the detection of large alterations. Small alterations, albeit potentially important, are usually not evaluated, except for selected known mutations in a few crucial genes. METHODS: Here, the potential of Next Generation Sequencing (NGS) to detect small DNA alterations was exploited as a reliable tool to monitor the genetic stability of cells manipulated prior to therapeutic application. The acquisition of small somatic mutations during in vitro culture of human adipose-derived mesenchymal stromal cells from 23 osteoarthritis patients was investigated at different times from passage 1 to passage 10 using targeted NGS of a panel of human cancer-related genes. Moreover, copy number variations in selected genes were analyzed using both the 'Coverage Analysis' plugin and digital PCR. RESULTS: Overall genetic stability was maintained, despite the emergence of sporadic variants, some with pathogenic potential or of uncertain significance, reflecting dynamic changes in culture. No copy number variations occurred during in vitro culture. CONCLUSIONS: Time in culture is confirmed as a key variable influencing the acquisition of genetic alterations, underscoring the importance of minimizing expansion times to preserve genetic stability. Given the potential impact of small genetic alterations on cellular function and the high next generation sequencing processivity and throughput, exploring incoming mutations in MSC cells via NGS turns out to be not only a perspective research pursuit, but also a robust strategy for surveillance of genetic stability. Integrating this approach into quality control guidelines would support standardized monitoring of the genomic integrity of manipulated MSCs prior to clinical application.