SENESCENCE STATE OF MESENCHYMAL STEM CELLS IN LOW CULTURE PASSAGES: IMPLICATIONS FOR CLINICAL USE

Abstract

<h3>Background</h3> Mesenchymal stem cells (MSCs) are multipotent cells found in various tissues and are easily isolated and cultivated. These cells are plastic-adherent and have the expression of CD90, CD73 and CD105 and lack of expression of CD45, CD34, CD14, CD19 and HLA-DR; they must differentiate to osteoblasts, adipocytes and chondroblasts ‘in vitro'. In order to use these cells in clinical protocols, MSCs must be expanded to obtain enough number of cells. However, MSCs expansion is limited and a senescence state could happen after some passages, reducing cellular replicative potential. Under such conditions, MSCs may change some properties and the age of donors is a major factor determining MSC replicative potential. In this study, we present a case where MSCs derived from an aged donor enter a senescence state after 3 passages in culture. <h3>Methods</h3> Bone marrow was aspirated from a female patient submitted to incontinency urinary protocol; MSCs was cultivated with DMEM, supplemented with 10% autologous serum (AS) plus 1% L-Glutamin and 1% Antibiotic/Antimycotic. Senescence analysis was performed by β-Galactosidase staining after 24h and 48h. Controls are established using BM-MSC from healthy donors and used in senescence and gene expression assays. Gene expression was performed by RT-PCR for pluripotency genes, such as SOX2, POU5F1, NANOG, and KLF4. Both control and the patient's MSCs were cultivated with AS or fetal bovine serum (FBS). <h3>Results</h3> Patient's MSCs expansion with AS presented early senescence state at passage 3. MSCs were then submitted to two different culture conditions: 1) Cells with AS or 2) FBS supplementation and cultivated up to passage 3. Senescence state was assessed after 24h and no statistical difference were observed; however, patient's cells cultured with AS presented more senescent cells when compared to FBS media supplementation after 48h (<i>p</i> = 0.0018). Gene expression was performed in both conditions to check activity status of pluripotency genes. Increased expression of KLF4 was observed in the patient's cells in comparison to healthy controls (<i>p</i> = 0.0016). Reduced gene expression was observed for NANOG (<i>p</i> = 0.0016) and SOX2 (<i>p</i> = 0.0014) genes. <h3>Conclusion</h3> MSCs expansion could enter a senescence state in low passage number and it could impact MSC quality in clinical application, reducing its efficacy when administered.