Introduction: Bioactive factors contained in Platelet Lysate (PL) promote the isolation and proliferation of Mesenchymal Stem Cells (MSCs) and the growth of cell lines. It is well known that PL not only enhances cell proliferation compared to Fetal Bovine Serum (FBS) without affecting the cell differentiation capability, but it is also able to activate resting quiescent cells to re-enter cell cycle. The main proliferation-relate pathway, involving Cyclin, Akt and ERK1/2, is activated after a short exposure of the cells to PL but the effect on proliferation is higher in primary cell cultures than in cell lines.
To better understand this effect, we focused our attention on a family of highly conserved proteins involved in different cellular mechanisms: C-MYC's family. This protein has three different isoforms: C-MYC1 particularly expressed in those cells that are in suffering condition; C-MYC2 expressed in growing cells and C-MYCS, a short isoform whose expression is transient during cell growth.
Methodology: MSCs from different tissues, Articular Chondrocytes, Osteoblasts, Amniotic Fluid Stem Cells from human samples and HeLa cell line, were cultivated in 10% FBS or 5% PL-supplemented medium. The proliferation capability was evaluated in terms of cell doublings and Ki67 expression by immunofluorescence and the possible apoptotic effect evaluating Annexin V by FACS analysis and C-MYC protein expression by Western Blot.
Results: All primary cell cultures treated with PL showed a high proliferation rate in comparison with FBS treated cultures. C-MYC1 expression was absent in FBS cultured cells but was observed in presence of PL also after a short time of exposure. This pattern of expression was different for the cell line where C-MYC1 was present also in the FBS-cultured cells. C-MYC2 isoform was expressed in all the cells independently from the culture conditions. Immunofluorescence analysis indicated that MSC cultures showing C-MYC1 protein expression were in a proliferative stage.
We also find that the cells treated with PL was not in an apoptotic stage, showing only a 15% of Annexin V positive cells.
Conclusions: PL induces cell proliferation increasing C-MYC1 isoform expression in primary cell cultures and much more slightly in the cell line, modifying the described apoptotic function of C-MYC1 isoform. These results pave the way to a deeper molecular study in order to understand peculiar differences of PL stimulation on various types of cells.