Human Bone Marrow MSC Transformation in Different Culture Conditions.
Authors: Saccardi Riccardo , Urbani Serena , Mazzanti Benedetta , Pozzo Simone Dal , Benvenuti Susanna , Peri Alessandro , Frontera Salvatore , Genuardi Maurizio , Bosi Alberto 
Published: April 2020

Abstract
Human mesenchymal stem cells (MSC) have been isolated from different sources, expanded and characterized extensively. Their plasticity and immunomodulatory properties made these cells extremely promising in the fields of immunotherapy and regenerative medicine. Both safety and modality of preparation in a clinical setting are still be defined.
We generated human MSC from normal donors bone marrow cells following 2 different isolation methods and culture conditions:
white blood cells (buffy-coat) were plated in IMDM with gentamycin, 10% FBS and 2% Ultroser® G. Half of the complete medium was changed after one week and then the whole medium was added every 3–4 days. When approximately 80% of the flask surface was covered, the adherent cells were trypsinized and re-suspended in complete medium. mononucleated cells (MNC) purification by Ficoll density gradient separation and cultivation in DMEM-Low Glucose supplemented with 10% FBS and 1% antibiotic-antimycotic solution. Complete medium was changed after 3 days. When the cultures were near confluence, the cells were detached and replated.
In both protocols, the isolated MSC (P0) were characterized by the number of Colony Forming Units-Fibroblasts (CFU-F), osteogenic and adipogenic differentiation, and immunophenotype based on the following monoclonal antibodies: CD34, CD45, CD14 (in order to quantify hematopoietic and monocytic contamination); CD29, CD44, CD166, CD90, CD73, HLA-DP DQ DR, HLA-ABC and CD105.
We observed a spontaneous transformation of MSC in 6/29 cases (20%), irrespective of the isolation and expansion protocols and the cells showed a different morphologic, immunophenotypic, proliferative and cytogenetic pattern. In particular, cells assumed uniform cuboidal and round morphology and lost their spindle shaped aspect.
MSC transformation usually occurred after the 4th passage in culture; only once it occurred after only one passage in culture (P1). Flow cytometric analysis showed a complete down regulation of MSC surface markers such as CD73, CD105, CD166, CD90, CD44, CD29, HLA ABC. The typical biparametric FSC/SSC distribution of RS cells and mMSC was lost as well. Transformed MSC showed very low expression of KDR, Ulex and vWF. Differentiation capability of the transformed cells was also lost and only small areas stained positive for classic differentiation tests (Oil Red O for adipogenic and Alizarin S for osteogenic differentiation). Cytogenetic analysis showed aneuploidy and a wide pattern of chromosomal aberrations. These cells also showed a high level of telomerase activity when compared with non-transformed MSC. Finally, their proliferation rate was greatly increased.
CONCLUSIONS: we observed spontaneous transformation of MSC generated under various conditions. We speculate that this phenomenon might be related to the high proliferation rate in culture of cells still retaining a rather undifferentiated pattern. Further investigations about the transformation modality and its biological pathways are needed. The use of these cells in a clinical setting should be adequately evaluated for safety reasons.