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.
