Absence of Oncogenic Transformation in Long-Term Culture Mesenchymal Cells from Bone Marrow of Children.

Mesenchymal stromal cells (MSC) have attracted considerable interest in both the scientific and clinical fields because of their potential to regenerate tissues and support hematopoiesis. Although laboratory isolation of MSC is established, the lack of an MSC specific marker and their low frequency in bone marrow necessitates their in vitro expansion in order to obtain a sufficient cell number for applications. During this process their characteristics may be altered and cells may acquire oncogenic properties or abnormal proliferative capacity. We investigated the effect of in vitro expansion on MSC, isolated from BM of children with idiopathic thrombocytopenic purpura (ITP, n=10) and autoimmune neutropenia (AIN, n=6). MSC were maintained in culture for a number of serial passages (P1–P6). We studied the expression profile of tumor suppressor genes (p53, p16, Rb) and the oncogene (H-Ras) along with hTERT, involved in malignant transformation at different passages. The cell line HUVEC was used as normal control for adherent cells and the M5 as positive control for hTERT expression. P2-MSC and P6-MSC (n=9) were grown on soft agar to assess their anchorage dependence. P2-MSC showed normal, relative to GAPDH, expression of p53, p16, Rb and H-Ras (ITP: 0.37, 0.2, 0.53, 0.64 respectively and AIN: 0.41, 0.27, 0.62, 0.75 respectively) compared to HUVEC cells (0.5, 0.23, 0.73, 0.93, respectively) which remained stable upto P6 (ITP P2vsP6: p= 0.63, p= 0.63, p= 0.82, p=0.33, AIN P2vsP6: p= 0.85, p= 0.07, p= 0.14, p=0.07). In all samples, hTERT expression was undetectable both at P2 and P6, and moreover none of the samples showed anchorage independent growth. These findings suggest that expression profile of oncogenes and tumor suppressor genes does not change during in vitro expansion of MSC. Importantly, the similar transcript levels between MSC and HUVEC, the absence of hTERT expression and anchorage independent growth suggest that MSC may not be transformed for a number of passages. In conclusion, although in vivo models are required, such properties encourage MSC use in clinical practice and should be considered as candidates for therapeutic application.