GMP compliant isolation and expansion of MSCs with inactivated human platelet lysate

Mensenchymal stem cells (MSCs) are used for a very wide range of therapeutic applications for their multipotent, bystander and immunomodulant proprieties. For this reason, the increasing use of MSCs as Advanced Therapy Medicinal Products (ATMP) has led to production processes that need to meet Good Manufacturing Practice (GMP). The use of xenogeneic protein free GMP-compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for foetal bovine serum (FBS). As the use of human-derived blood materials, as growth supplement for MSC expansion, alleviates immunologic risks, but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL). In this study we compared three different MSC culture conditions (alpha+MEM + 10% of FBS or HPL or iHPL) and we firstly analized the immunophenotype, differentiative potential and embryonic markers, than deepen the MSC immunomodulant properties in the three experimental conditions. Since T-cells are the primary cells in adoptive immune response, inhibitory effects of MSCs on total activated T-cells with Phytoemoagglutin (PHA-PBMC) were evaluated and compared. Cellular and molecular methods were used: in vitro cell separations, proliferative assay, cytofluorimetric analysis, Elisa assay and real time PCR. The MSCs cultivated in the three different culture conditions showed no significant differences in terms of CFU-F number, immunophenotype, or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics which may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or katyotype modifications were observed. In all co-culture conditions with PHA-PBMC and MSCs (independently from the growth media which was used for their expansion) data showed: 1) T proliferation inhibition; 2) Naïve T Increase and Memory T decrease; 3) Treg increase; 4) strong Th2 polarization associated to increase IL-10 and IL-4; Th1 inhibition (IL-2, TNF-α, IFN-γ and IL-12 significantly decrease) and Th17 induction confirmed from the production of high concentration of IL-6 and IL-17; 5) IDO mRNA induction in MSCs co-cultured with PHA-PBMC. No significant differences emerged from co-culture with FBS-MSC and HPL/iHPL-MSC. We demonstrated that iHPL is safer than HPL and represents a good, GMP-compliant alternative to FBS for MSC clinical production, which is even more advantageous in terms of cellular growth and stemness and preserve the MSC immunodulant proprieties.