Mesenchymal stem cells (MSCs) can be derived from several human and animal sources. According to this systematic review, the immortalization of these cells, by viral or gene transfer techniques (plasmid) and non-viral methods, are useful to ensure the reproducibility of the experiments and the prospect of using these cells in clinical studies. The resultant immortalized MSCs cells must undergo through different validation assays in order to prove their safety and phenotypic and genotypic stability; these assays include flow cytometry for specific MSC markers, trilineage differentiation, RT-PCR and qRT-PCR expression analysis for pluripotency genes, karyotype and telomere length and in vivo tumorigenicity assays.