Human Bone Marrow-Derived Mesenchymal Stem Cells Do Not Undergo Transformation after Long-Term In Vitro Culture and Do Not Exhibit Telomere Maintenance Mechanisms.

Mesenchymal sem cells (MSCs) are multipotent progenitors with the ability to differentiate along multiple cell lineages and are today considered a useful tool for cell therapy and tissue engineering approaches. Concerns that adult human MSCs may undergo malignant transformation have been recently raised. In fact, human adipose tissue-derived MSCs have been shown to spontaneously transform after long-term in vitro culture. Aim of this study was to investigate the susceptibility to transformation of human bone marrow (BM)-derived MSCs at different time points of in vitro culture. MSCs were isolated from BM of 10 healthy donors and propagated continuously in vitro until reaching a senescence phase or passage (P) 25. MSCs in the senescence phase were closely monitored for 8–12 weeks, to look for the appearance of a crisis phase. MSCs were characterized by morphology, differentiation capacity and immunophenotype at different time-points in culture. The genetic characterization of MSCs was investigated through array comparative genomic hybridization (array-CGH), classical cytogenetics and subtelomeric FISH analysis both before and after prolonged in vitro culture. MSCs were tested for the expression of telomerase activity (TA), hTERT transcripts and alternative mechanisms of telomere lengthening (ALT) at different time-points in culture. MSCs from 5 donors were also analyzed for telomere length at P3 and P15. p53 gene status was also analyzed in MSCs from donors #1 and #2 that rapidly reached senescence in culture. A huge variability between donors was noted in terms of proliferative capacity and in vitro life-span of MSCs. All MSCs maintained the typical spindle-shaped morphology, phenotype and ability to differentiate into osteoblasts and adipocytes throughout the culture period. All MSCs displayed a progressive decrease in the proliferative capacity until reaching senescence, not followed by any further growth. Array-CGH, karyotype and subtelomeric FISH analyses demonstrated that MSCs expanded in vitro did not show chromosomal rearrangements, also after long term culture. TA was not evidenced in the samples tested, including a post-senescence culture. hTERT transcripts (full-length and the additional splice variants a⁻, b⁻, a⁻b⁻) were found not to be expressed in any of the examined cultures. Telomeres shortened during the culture period. ALT were not evidenced in the MSCs tested, as indicated by the lack of ALT-associated promyelocytic leukemia bodies. Direct DNA sequencing of exons 2–11 in pre-senescence cultures from donors #1 and #2 did not evidence the presence of mutation in the p53 gene. Human BM-derived MSCs do not display an aptitude for spontaneous transformation and can be safely expanded in vitro without any sign of immortalization or development of chromosomal abnormalities. Our results provide support to the concept that the biological properties of human BM-derived MSCs after ex vivo expansion remain suitable for use in cell-therapy approaches; however, it is strongly recommended that phenotype, functional and genetic characteristics of MSCs after in vitro culture and before in vivo infusion are tested, to further guarantee safety for the patient.