Quality controls in Advanced Therapy Medicinal Products:safety assessment of Mesenchymal Stromal Cells to treat human bone defects

Over the last 25 years, there has been a growing interest towards regenerative medicine and this has led to the development of a new category of medicinal products based on the use of cells, grouped under the name of “Advanced Therapies”. The European Regulation N°1394/2007 classified three kinds of Advanced Therapy Medicinal Products: “gene therapy medicinal products”, “somatic cell therapy medicinal products” and “tissue engineered products”. Aim of cellular therapies in regenerative medicine is to treat or prevent human diseases through the pharmacological, immunological or metabolic action of the cells. Mesenchymal Stromal Cells (MSC) are key candidates for cellular therapies for different reasons: they are multipotent cells easy to obtain from different tissues; they expand rapidly in culture and produce trophic factors which modulate inflammation, tissue remodeling and apoptosis; they possess immune regulatory properties. The employment of these cells in clinical trials requires in vivo studies to test their efficacy, and their manufacturing, like any kind of injectable drug, must follow GMP (Good Manufacturing Practice) rules, which regulate precisely processes, final product characterization and quality controls. All steps in MSC manufacturing need to be standardized to ensure a reproducible cellular quality and potency assessment. Aim of this work is the demonstration of the safety of MSCs for bone regeneration and is focused on the standardization of quality controls necessary for their use in patients. Starting from pre-clinical data obtained in collaboration with the group of Burastero G., who performed in vivo experiments in animals demonstrating the ability of MSCs to induce bone regeneration, we developed a phase I clinical protocol for the treatment of scaphoid bone defects with autologous MSCs ex-vivo expanded, associated with Orthoss® ( an inorganic bone matrix of bovine origin) and BMP-7 (Osigraft, osteoinductive human recombinant protein). The production of clinical-grade MSCs will be carried out in the Cell Factory of the AOUI of Verona, located at “U.O.C. di Medicina Trasfusionale” of the Policlinico G.B. Rossi, Verona. We set up a panel of assays using five different bone marrow samples from healthy donors expanded in Cell Factory and we obtained the following results: 1) the analyses of viability and phenotype showed the preservation of the identity of MSC after the expansion; 2) the analysis on the presence of endotoxin, viruses, fungi or microbial species demonstrated no contamination and a sterile cellular product; 3) after the addiction of specific stimuli, the MSCs were able to differentiate into osteoblasts and adipocytes; 4) the clonogenicity of MSCs after expansion was maintained; 5) the immunomodulatory properties towards the immune effector cells were confirmed; 6) the gene expression analysis and the mutagenesis test demonstrated the absence of neoplastic transformation after the expansion process; 7) the karyotype analysis showed no chromosomal aberrations in the cellular product. All these validated tests have become the definitive quality controls of the clinical protocol “Ricostruzione di deficit dell’osso scafoide mediante l’uso di cellule staminali mesenchimali autologhe espanse ex-vivo”(Eudract N°2012-002424-33), which has been submitted to the approval of the responsible organs, i.e. ISS (Istituto Superiore di Sanità) and AIFA (Agenzia Italiana del Farmaco).