Platelet-Lysate for In Vitro Expansion of Human Multipotent Mesenchymal Stromal Cells in Approaches of Cell-Therapy.

There is large interest in the use of mesenchymal stromal cells (MSCs) in approaches of cell-therapy and tissue engineering. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns in the case of clinical grade cellular preparations because of the theoretical risk of transmission of zoonoses and triggering immune reactions in the host. Therefore, the identification of a serum-free medium appropriate for both the extensive expansion necessary to reach the large numbers of MSCs required for clinical application, and the exclusion of risks connected with the use of animal products, is warranted. Aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL) are endowed with biological properties appropriate for cell-therapy approaches. MSCs were generated from bone-marrow of 8 healthy hematopoietic stem cell donor; 4 different culture conditions were tested:

• 10 % FCS;

• 5% PL;

• 2,5% PL;

• 1% PL.

MSCs were harvested when reaching ≥ 80% confluence and replated for expansion at 4.000 cells/cm² until passage 5. CFU-F frequency, proliferative capacity, morphology, surface phenotype and differentiation capacity were evaluated. In particular, the immune regulatory effect on alloantigen-specific immune response, the kinetics of cytokine production and the resistance to spontaneous transformation into tumor cells of MSC expanded in the presence of either PL or FCS were investigated. Our results demonstrate that MSCs expanded in either FCS or PL display comparable morphology, phenotype and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. Immune-regulatory effect of MSCs was investigated on alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to:

• decrease alloantigen-induced cytotoxic activity;

• favor differentiation of CD4⁺ T-cell subsets expressing Treg phenotype;

• increase early secretion of IL-10 in MLC supernatant, as well as to induce a striking augmentation of IL-6 production.

As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and in reducing early IFNg-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by both molecular karyotyping (array-comparative genomic hybridization) and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the hypothesis of a remarkable immune functional plasticity of human MSCs and suggest that the use of MSCs-PL, which seem to be endowed with a relatively low immune suppressive activity, could be more appropriate in approaches of reparative/regenerative cell-therapy or in strategies aimed at improving hematopoietic/immune recovery after hematopoietic stem cell transplantation (HSCT). On the contrary, as MSCs-FCS seem to display a more pronounced immune suppressive function, they might be more suitable for preventing or treating alloreactive-related immune complications, such as severe Graft-versus-host disease (GvHD) in HSCT and graft rejection in HSCT and solid organ transplantation.