Human bone marrow mesenchymal stromal cells (BM-MSC) represent one of the most investigated "advanced therapeutic medicinal products".1 Recent safety concerns have focused attention on the possible malignant transformation due to mutations acquired during their large-scale in vitro expansion.2 Indeed, spontaneous oncogenic transformation has been described for murine MSC 3 although not for human cells, 4,5 with the exception of a few studies,
6which were subsequently retracted when it was realized that this was due to cross-contamination by a tumor cell line. 7,8 One single report has described the in vitro outgrowth of a transformed subpopulation from a normal BM sample.9 Furthermore, genetic aberrations of MSC have been very occasionally observed after long-term cultures 4,10,11 but interpreted to be related to senescence.
5In order to investigate the frequency of cytogenetic alterations in a broad "collection" of clinical-grade BM-MSC products, we performed cytogenetic analysis of 92 preparations expanded under Good Manufacturing Practice conditions.12 More precisely, 67 expansions were performed from 33 healthy donors, 4 β−thalassemia patients and 21 multiple sclerosis patients (Table 1). MSC were expanded from BM washouts or aspirates using human platelet lysate as previously described.12,13 Metaphases were prepared according to standard procedures 12 and analyzed by QFQ-banding. At least 20 metaphases per sample were analyzed. Karyotype was described according to the International System for Human Cytogenetic Nomenclature. Furthermore, p53 gene mutations were analyzed by deep sequencing of exons 5 to 11.Chromosomal abnormalities were detected in 17 of 86 expansions (19.8%). In all cases, the genetic lesions were spontaneous abnormalities.2 In 14 cases they were nonclonal: in 8 they involved one metaphase (MSC46, MSC70, MSC74, MSC79, MSC82, MSC87, MSC121, MSC126); in 5 two different chromosome abnormalities in two metaphases (MSC52, MSC55, MSC66 MSC100, MSC116). Only in one case were three different alterations in three metaphases found (MSC80). "Clonal chromosome changes" 2 were detected in 3 cases: in MSC114 monosomy of chromosome X was found in three metaphases, while in MSC119 and MSC122 inversion of chromosome 1 and a translocation involving chromosomes 9 and 4, respectively, were found in two metaphases. We also examined the results of multiple expansions from the same donors. Chromosomal anomalies were observed for 7 out of 13 donors (ns. 18, 20, 23, 32, 37, 50, 63), but these lesions were not recurrent and present only in some of the expansions performed. This suggests that chromosome aberrations do not associate with specific donors. In 6 cases, cytogenetic evaluation could not be performed on the final fresh P2 products due to lack of metaphases (Table 1) but in 5 of these the analyses could be repeated using a frozen P2 aliquot and in 4 cases karyotypes were normal. Similarly, when spontaneous and non-clonal abnormalities were detected, the karyotype analysis was repeated using a frozen P2 aliqu...
