Abstract
Mesenchymal Stem Cells (MSC) are multipotent adult stem cells harboring a wide range of differentiation capacities as well as non-HLA restricted immunosuppressive properties, leading to an increase in their uses for immunomodulation and regenerative medicine. Even if processes using recombinant growth factors (e.g. FGF2) or platelet lysate have demonstrated their interest, definitive standards to produce clinical –grade MSC are still lacking notably due to the lack of fully validated controls of the final cell product efficacy and safety.. In France, MSC are generally cultivated from bone marrow (BM) using media supplemented with either FCS + FGF2 or platelet lysate. According to the process of the SFGM-TC including FCS + FGF2 (1 ng/mL), a randomized multicentric doubled blinded clinical trial for preventing aGVHD started in 2007. At the same time, for treating tissue lesion in irradiated patients, MSC were cultivated using platelet lysate. Between March and august 2007, 15 MSC productions were done with FGF2 in three independent cell therapy units and 5 with platelet lysate at the CTSA. The expected CD73posCD105posCD90posCD45neg MSC phenotype was obtained for all the 20 productions on more than 98% of the cells at the end of passage 1 (P1). For FGF2-derived cultures, the total cell increase reached 64 (±61) fold after 21 (±2) population doublings.,. Surprisingly, HLA-DR was heterogeneously expressed by MSC (HLADRpos cells at P1: 17.4±15.3%; rMFI: 1.8±1) independently of the production process. However, even HLA-DRpos clinical-grade MSC remained poorly immunogenic and suppressed efficiently T-cell proliferation in agreement with a strong IFNg-dependent production of the immunosuppressive enzyme IDO. A karyotype was systematically done on the final products (P1). For cultures with FCS and FGF2, 4karyotypes out of 11 showed the presence of aneuploidy with extra chromosomes 5 and 8. For cultures in platelet lysate, 1 karyotype out of 5 showed aneuploidy features with an extra chromosome 5. To analyze the significance of this aneuploidy, 3 abnormal productions as well as 4 normal productions were cultured until 2 months after growth arrest. At each passage, a karyotype, a FISH, and a quantitative analysis of expression of genes related to transformation and senescence (hTERT, c-myc, p53, p21 and p16ink4a) were performed. All the MSC stopped to growth after 4 to 7 passages. For the 3 abnormal productions, as early the second passage, the aneuploidic clone disappeared. For normal productions, karyotype remained normal till growth arrest. In both groups, gene expression showed a normal and stable expression of c-myc, p53, p21, whereas hTERT was never expressed. Finally, when reaching growth arrest, MSC expressed p16ink4a and anchorage growth independence was never obtained in soft agar independently of the previous detection of an aneuploidic clone. These results demonstrated that whatever the culture method, aneuploidy could occur during clinical-grade production of BM-MSC but does not lead to cell transformation.
Senescent Growth Arrest in Mesenchymal Stem Cells Is Bypassed by Wip1-Mediated Downregulation of Intrinsic Stress Signaling Pathways
