Studies on metabolomic analysis with immortalized mesenchymal stem cells by SV40 T-antigen

Abstract

This study was carried out by transfection of SV40 T antigen into mesenchymal stem cell hMSCs collected from human bone marrow. Immortalized hMSCs-T cell is a mesenchymal stem cell capable of multiple cell division. The cell cycle of hMSCs was relatively short, but hMSCs-T cell was able to divide more than 80 passages. The amount of amino acids (AAs) in the cell was measured by gas chromatography in order to examine the metabolism of the cell according to the elongated cell cycle. Amino acids are the key component of protein synthesis and intracellular nitrogen. In addition, we aimed to study the amino acid alteration and find a biomarker that can support the continuous growth of cells that are capable of continuously production for the industrial use of immortalized stem cells. The alterations of amino acids in mesenchymal stem cells and immortalized stem cells are written in chapter 1. Beside hMSCs-T cell, HEK293 transfected with synphilin-1 also altered amino acid composition. Synphilin-1 is an α-synuclein interacting protein in Lewy body, which is a characteristic of Parkinson's disease. When HEK293 and S293, HEK293 stably overexpressed synphilin-1, were starved with KRBB buffer, the morphological changes and cell aggregations were different between cell types. The difference might be from intracellular metabolites and protein compositions. Amino acid sensing pathway is controlled by intracellular and extracellular amount of amino acid and changes of amino acids are important factors in controlling protein synthesis and degradation. The mammalian target of rapamycin (mTOR) is important in amino acid sensing that regulates cell growth and cell survival. This study is to estimate the amount of amino acid and detect the phosphorylation of S6K1 and Akt influenced by mTORC1 and mTORC2. HEK293 and S293 were starved and challenged with glutamine, glutamic acid, and proline to confirm the amino acid alteration induced from synphilin-1. The results of amino acid and biological data were recorded in chapter 2.