Determination of calcium-calmodulin dependent protein kinase 2 (CAMkII) enzyme in mesenchymal stem cell viability and proliferation under the stress conditions

Introduction and aim: Calcium/Calmodulin Dependent Protein Kinase 2 (CAMKII) is a member of serine threonin kinase family and it is found intensively in neuronal cells. It has been shown that CaMKII has a key role on the learning process, on the memory formation and on the synaptic plasticity. It has been suggested that nonspecific inhibition of calcium / calmodulin dependent protein kinase 2 can prevent apoptosis in neuronal cells, and also inhibition of induced and autonomic CAMKII activity decreases glutamate-induced neuronal cell death in primary cultures. In addition in recent years it has been suggested that change in the amount of Calcium/calmodulin-dependent protein kinase kinase (CaMKK) enzyme which belongs to the same family as CaMKII controls hemopoietic stem cell proliferation. Although the effects of CaMKII enzyme on cellular response in stem cell proliferation and under the stress conditions are still unclear. In this project, changes in the levels of calcium / calmodulin dependent protein kinase 2 on mesenchymal stem cells survival and proliferation changes and on the changes of CaMKII levels under the Hydrogen peroxide (H2O2) induced stress conditions were aimed to investigate. Besides changes on the amount of gene expressions of CaMKII isoforms (alpha, beta, gama, delta) were aimed to investigate under the stress conditions. Methods: In our project, mesenchymal stem cells were obtained from human umbilical cord by explant method. Gene and protein expressions of CD44, CD90, CD105 surface antigens were determined by RT-qPCR, immünohistochemistry and flow cytometry, thus MSCs were characterized. CaMKII enzyme levels and activity levels were determined photometrically and CaMKII enzyme isoforms levels were determined by RT-qPCR under the hydrogen peoxide induced stress conditions. Effects of application of KN-93 which is a CaMKII inhibitor on stem cell viability and proliferation were determined byy MTT and MTS tests. Results: In this thesis project, the minimal criterias which are the capacity of differentiation into osteogenic, chondrogenic and adipogenic cells on the characterization of Mesenchymal Stem Cell's (MSC) according to International Society for Cellular Therapy (ISCT) were shown histochemically by using the stem cells which were isolated from Wharton Jelly. It was shown that the application of 1mM H2O2 for 30 minutes to generate oxidative stress decreased the cell viability ~40 – 50% in proportion to control group. It was determined that CaMKII enzyme inhibition by the application of KN-93 with the H202 application decreased the cell viability more than the H202 application alone and this effect was not exist in the application of KN-92 negative inhibitor. It was identified that Wharton's Jelly Mesenchymal Stem Cells (WJ-MSC) express mostly Beta isoform of CaMKII enzyme under the H202 induced stress conditions. Conclusion: As a result, according to our literature review the effects of CaMKII on the cell viability and H2O2 induced stress conditions were shown for the first time. It is thought that the data which were obtained from our research will provide a fundamental ground to understand response mechanism of MSCs to the stress conditions and to develop targeted stem cell treatment strategies. Keywords: Mesenchymal Stem Cell, Wharton's Jelly, Oxidative Stress, CaMKII, KN-93, KN-92