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Mesenchymal stem cells (MSCs) represent one of the most promising stem cells for a number of degenerative conditions due to their multipotency, immunoprivileged properties, and easy expansion in vitro. However, the limited life span of primary MSCs during in vitro expansion greatly hampers their use in clinical applications and basic research. Immortalization of MSCs will overcome this problem and may provide a very useful tool with which to study MSC biology. Here we showed that silencing p53 expression with lentivirus-mediated small interfering RNA delayed the senescence by extended passage number, but was not sufficient to immortalize primary MSCs. However, combination of p53 knockdown and human telomerase reverse transcriptase (hTERT) overexpression was sufficient to immortalize MSCs. The effects of p53 knockdown and hTERT overexpression on MSCs, including proliferation, colony formation, and differentiation, were determined. The resultant immortal MSCs displayed similar surface antigen profile to primary MSCs and retained MSC differentiation potential. Gene expression profile showed high similarity between immortalized MSCs and primary MSCs. In addition, immortalization-associated genes were also identified. Our data suggested immortalization of MSCs related to upregulation of cell cycle regulator and DNA repair genes enabling them to bypass cell crisis and complete mitosis. This study provides a new cellular model for basic studies of MSCs and understanding of the molecular basis of MSC immortalization.

stem cells and development

Molecular Basis of Immortalization of Human Mesenchymal Stem Cells by Combination of p53 Knockdown and Human Telomerase Reverse Transcriptase Overexpression