Extended in vitro culture of primary human mesenchymal stem cells downregulates Brca1-related genes and impairs DNA double-strand break recognition.

Mesenchymal stem cells (MSCs) are multi-lineage adult stem cells with considerable potential for cell based regenerative therapies. In vitro expansion changes their epigenetic and cellular properties, with a poorly understood impact on DNA damage response and genome stability. We report here results of a transcriptome-based pathway analysis of in vitro expanded hBM-MSCs, supplemented with cellular assays focusing on DNA double-strand break (DSB) repair. Gene pathways affected by in vitro aging were mapped using gene ontology, KEGG, GSEA, and were found to involve DNA repair, homologous recombination, cell cycle control and chromosomal replication. Assays for the recognition (gamma-H2AX+53BP1 foci) and repair (pBRCA1+gamma-H2AX foci) of X-ray induced DNA DSBs in hBM-MSCs show that over a period of 8 weeks of in vitro aging (i.e. about 10 doubling times), cells exhibit a reduced DNA damage response and a higher fraction of residual DNA damage. Furthermore, a distinct sub-population of cells with impaired DNA DSB recognition was observed. Several genes which participate in DNA repair by homologous recombination (e.g., Rad51, Rad54, BRCA1) show a 2.3 to 4 fold reduction of their mRNA expression by qRT-PCR. We conclude that the in vitro expansion of hMSCs can lead to aging-related impairment of the recognition and repair of DNA breaks.