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Human mesenchymal stem cell (MSC) therapy holds promise for treating degenerative and immune system diseases and repair of damaged tissues. Human tissues are cryopreserved and isolated MSCs are expanded in various culture media when prepared for clinical applications. These manipulations can significantly alter the MSC genetic characteristics. Such MSCs enter senescence state, resulting in impairment of their therapeutic properties. Moreover, such MSCs can undergo spontaneous neoplastic transformation with karyotype changes. To date no molecular markers of MSC senescence are available and the tumorigenic potential of MSCs has become the most important concern for safe clinical use of MSCs. This study revealed a normal karyotype of MSCs isolated from human bone marrow and expanded in vitro to clinically relevant amounts under a novel protocol. MSCs became senescent during in vitro long-term expansion, paralleling the significant difference in expression of 13 genes and 33 miRNAs. Umbilical cord tissue controlled freezing-thawing process did not affect the genomic stability of MSCs. MSC expansion in culture medium enriched with recombinant proteins had significant impact on the expression of genes related to the cell cycle. Novel MSC isolation and expansion technique developed during this study could be applied at institutions producing an advanced therapy medicinal products. The cellular therapy quality control could be developed on the basis of the methods used in this study.

Human mesenchymal stem cell (MSC) therapy holds promise for treating degenerative and immune system diseases and repair of damaged tissues. Human tissues are cryopreserved and isolated MSCs are expanded in various culture media when prepared for clinical applications. These manipulations can significantly alter the MSC genetic characteristics. Such MSCs enter senescence state, resulting in impairment of their therapeutic properties. Moreover, such MSCs can undergo spontaneous neoplastic transformation with karyotype changes. To date no molecular markers of MSC senescence are available and the tumorigenic potential of MSCs has become the most important concern for safe clinical use of MSCs. This study revealed a normal karyotype of MSCs isolated from human bone marrow and expanded in vitro to clinically relevant amounts under a novel protocol. MSCs became senescent during in vitro long-term expansion, paralleling the significant difference in expression of 13 genes and 33 miRNAs. Umbilical cord tissue controlled freezing-thawing process did not affect the genomic stability of MSCs. MSC expansion in culture medium enriched with recombinant proteins had significant impact on the expression of genes related to the cell cycle. Novel MSC isolation and expansion technique developed during this study could be applied at institutions producing an advanced therapy medicinal products. The cellular therapy quality control could be developed on the basis of the methods used in this study.