Assessment of senescence in adipose mesenchymal stem cells for optimized ex vivo expansion and therapeutic potential

Abstract

Background & Aim Bone tissue engineering using osteoblastic differentiated adipose mesenchymal stem cells (AMSCs) is tested in clinical trials to heal critical size bone defects such as bone non-unions. For such therapies, a high number of cells is desirable for bioactivity, requiring extensive ex vivo cell expansion. However, adult MSC proliferation is limited as long-term culture causes continuous changes resulting in induction of replicative senescence. Replicative senescence is a complex process whereby stem cells grow old and is related to dysfunction of telomeres, the end part of eukaryotic chromosomes consisting of nucleotide repeats that are shortened after each replication cycle. The changes in stem cell functions related to aging of the donor, can be attributed to a decline in the effectiveness of regenerative capability. Methods, Results & Conclusion We present the results of a study where specific markers in the physiology of the AMSCs from 6 adults subjects of different ages were monitored during long-term ex vivo expansion as surrogates of their proliferation, differentiation and therapeutic potential. Telomere dysfunction, defects of Lamin-A and expression levels of the senescence markers p16, p21 genes and several Senescence-Associated Secretory Phenotype (SASP) markers (IL6, IL8, MCP1) were evaluated during early, intermediate and late passages. We show that, independent of the age of the donor, the senescence rate increases steadily for all cell samples reaching a senescent phenotype after numerous passages. The population doubling time is also significantly increased with proliferation time. Interestingly, higher senescence rate is observed at early passages in cells derived from older donors paralleling the slower proliferation potential of their cells which may have an impact on the potency and efficacy or product derived therefrom. For each donor, the expression levels of specific tested senescence markers correlates with to the senescence status of the corresponding cell samples. In conclusion, our study provides new insights to understand the AMSC senescence mechanisms for the improvement of cellular therapies and quality control processes.