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

Abstract

Mesenchymal stem cells (MSCs) are multilineage 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 (DDR) and genome stability. We report here results of a transcriptome-based pathway analysis of in vitro-expanded human bone marrow-derived mesenchymal stem cell (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, and GSEA, and were found to involve DNA repair, homologous recombination (HR), cell cycle control, and chromosomal replication. Assays for the recognition (c-H2AX + 53BP1 foci) and repair (pBRCA1 + c-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 DDR and a higher fraction of residual DNA damage. Furthermore, a distinct subpopulation of cells with impaired DNA DSB recognition was observed. Several genes that participate in DNA repair by HR (e.g., Rad51, Rad54, BRCA1) show a 2.3-to fourfold 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.Mesenchymal stem or mesenchymal stromal cells (MSCs) are adult stem cells that reside in bone marrow stroma and other connective tissues. Their lifelong potential to generate committed precursor cells for various lineages is essential for both the continuous replacement of cellular losses and the recovery of connective tissue damage. In addition to their role as a stem cell pool, MSCs are also a source for immunomodulatory paracrine factors, thereby serving as a regulator of inflammation and immune response

Comparison of different gene addition strategies to modify placental derived-mesenchymal stromal cells to produce FVIII

“…The PLCs employed in the present study exhibit a phenotype and biological properties that closely resemble that of MSC from other tissues ( 3 , 22 , 68 ). It is noteworthy that prior studies have reported the downregulation of components of the DNA damage response (DDR) and HRR pathways in MSC with time in culture ( 94 – 96 ). As such, it is possible that the PLCs used in the present study, having been first cultured by explant, then selected for c-kit, and finally expanded for gene modification, no longer expressed sufficient levels of some of the key players in the HRR pathway required for efficient CRISPR/Cas9 gene-editing to occur.…”

Alopecia treatment using minimally manipulated human umbilical cord-derived mesenchymal stem cells: Three case reports and review of literature

“…MM-UC-MSCs are expected to be a safer agent than cultured MSCs, embryonic stem cells, and induced pluripotent stem cells in vivo because minimally manipulated MSCs have little tumorigenicity, genetic mutations, and aging-related modifications[ 33 , 36 , 37 ]. As expected, we did not observe any immune rejection despite the allogeneic transplantation because MSCs have immunomodulatory properties[ 21 , 22 ].…”

Role of ex vivo Expanded Mesenchymal Stromal Cells in Determining Hematopoietic Stem Cell Transplantation Outcome

“…Extensive in vitro culture induces the activation of a DNA damage response altering the functional properties of MSCs. This is mainly due to the accumulation of ROS associated with the oxidative metabolism of cultured cells and to the downregulation of genes involved in DNA repairing ( Bao et al, 2020 ). Indeed, a decrease in the number of repairing γH2AX/53BP1 DSB repair foci was observed in long-term culture of MSCs, in addition to a slower DNA repair kinetics, and an increased number of residual DNA double-strand breaks 7 h post irradiation.…”