Robust, Efficient and Pure Induced Mesenchymal Stem Cells Generation frommRNA Induced Pluripotent Stem Cells in Suspension

Mesenchymal Stem Cells (MSCs) are involved in many promising clinical trials tackling vastly complicated diseases. Many factors are determining the safety in these clinical trials such as the purity of tissue-derived MSCs cell population used in therapies. Also, the efficacy of the injected MSCs must be tested in-vitro, before application, through proliferation capacity and reproducibility over continuous passages. In addition to, the importance of choosing the right source of MSCs derivation for successful cellular therapy and transplantation. This study demonstrates robust generation of iMSCs from induced Pluripotent Stem Cells (iPSC) of healthy human donor (with full genetic test done prior) using non-integrative (mRNA) method. This conversion method comprises (i) differentiating a population of iPSCs in suspension without iMatrix, (ii) passaging the cells differentiated in step (i) in the presence of a conditioned MSC medium for a time and under conditions sufficient to produce iMSC in culture for long term with no sign of epigenetic memory. Analysis of Pluripotent markers expression (Oct-4, SSEA-4, Sox-2, Tra-1-60) was confirmed by flow cytometry and Immunocytochemistry through Fluorescence microscope visual assessment. No teratoma was developed by in-vivo injection of the iMSC population in male hamsters, confirming the transformed purity of iMSCs and the immunemodulating property in culture without iPSC respectively. For cell cycle and senescence studies, pure in-vitro iMSCs were tested using flow cytometry using CD73, 90 and 105 expression analysis and compared with UC-MSC. Later, iMSCs demonstrated tri-differentiation of chondrocytes, osteocytes and adipocytes relative to UC-MSCs, which could make it possible to address the drawbacks of using adult MSCs and thus provide a valuable tool for future use in various clinical settings.