Genetic stability assessment in bone tissue-engineered products

December 18, 2019

CĂ©line Pierard, Nicolas Theys

Bone tissue engineering using osteoblastic differentiated adipose mesenchymal stem cells (AMSCs) is tested in clinical trials to treat critical size bone defects and bone non-unions. Manufacturing of cellbased product may be associated with accumulation of genetic instability. Our study was designed to better understand the physiology of the AMSCs during expansion and differentiation steps to improve understanding of genetic stability data. After induction of osteogenic differentiation, the structural composition of our product prevents use of conventional karyotype technique. Evidence of potential abnormalities and the tumorigenicity were therefore evaluated with a risk-based approach that included combination of selected conventional cytogenetics assays during early cellular proliferation phase and molecular assays performed on biopsies collected at later stages. We performed extensive cytogenetic analysis on several cellular preparations of AMSCs. Several of them showed genetic subclonal and clonal abnormalities or autosomal genetic disorders in tumor suppressor gene. Our study showed that, independently of the initial genetic status and the age of the donor, the senescent rate increased for all independent preparation reaching a senescent phenotype after numerous passages. The population doubling time was also significantly increased with proliferation time. For each donor, the expression levels of tumor suppressor genes CDKN2A and p21 were gradually increasing reaching highest levels at the latest passage. Moreover, none of the cells expressed telomerase subunit. The ability of cells to differentiate and form a 3-dimensional osteogenic graft was not altered by genetic abnormalities observed in initial ASCs preparation. A combined genetic molecular analysis (CGH array, Q-PCR and exomic sequencing) was performed on differentiated cells. Tumors suppressors genes were expressed significantly more than in control cancer cells and expression of telomerase was low. Minor genetic abnormalities observed by CGH were never associated to mutation in oncogenes and tumor suppressor genes. In conclusion, a combined approach is effective to increase the likelihood of detecting genetic abnormality in cell-based products for human use.