Abstract
BackgroundMesenchymal stem cells (MSCs) are multipotent cells holding much promise for applications in regenerative medicine. However, with problems such as aging, increases in heteroploid cells, genomic instability, and reduced maintenance of stemness, more stable culturing methods and the production of MSCs with an improved therapeutic effect are desired. Ascorbic acid (AsA), which is a cofactor for a variety of enzymes and has an antioxidant effect, cannot be synthesized by certain animals, including humans. Nevertheless, little attention has been paid to AsA when culturing MSCs.MethodsWe analyzed the effect of adding AsA to the culture medium on the proliferation and metabolism of human MSCs by serial analysis of gene expression and metabolome analysis.ResultsWe found that AsA promotes MSC proliferation, and is particularly useful when expanding MSCs isolated from bone marrow. Serial analysis of gene expression and metabolome analysis suggested that, due to HIF1α accumulation caused by decreased activity of the enzymes that use AsA as a coenzyme in cultures without AsA, genes downstream of HIF1α are expressed and there is a conversion to a hypoxia-mimetic metabolism. AsA promotes HIF1α breakdown and activates mitochondria, affecting cell proliferation and metabolism. Comprehensive evaluation of the effects of AsA on various metabolic products in MSCs revealed that AsA increases HIF1α hydroxylase activity, suppressing HIF1a transcription and leading to mitochondrial activation.ConclusionsAdding AsA during MSC expansion leads to more efficient preparation of cells. These are expected to be important findings for the future application of MSCs in regenerative medicine.
Highlights
Mesenchymal stem cells (MSCs) are multipotent cells holding much promise for applications in regenerative medicine
Ascorbic acid (AsA) promotes MSC proliferation and promotes MSC expansion from Mononuclear cells (MNC) To investigate the effect of AsA on MSC proliferation, we evaluated proliferation after adding 0, 0.1, 0. 3, 1, or 3 mM of ascorbic acid 2-phosphate (AAP) to the culture medium, using MSCs at P4
When MSCs are expanded from bone marrow-derived MNCs (BMMNCs) from which hematocytes have been eliminated, proliferation takes a long time because of the very low percentage of MSCs
Summary
Mesenchymal stem cells (MSCs) are multipotent cells holding much promise for applications in regenerative medicine. With problems such as aging, increases in heteroploid cells, genomic instability, and reduced maintenance of stemness, more stable culturing methods and the production of MSCs with an improved therapeutic effect are desired. Little attention has been paid to AsA when culturing MSCs. Mesenchymal stem cells (MSCs) have the ability to differentiate into mesenchymal cells such as fat cells, bone cells, and cartilage cells, and they hold promise for applications in regenerative medicine. Ascorbic acid (AsA), an organic compound with a lactone structure, has long been widely studied and is known to have multiple physiological functions: it eliminates reactive oxygen species caused by oxidative stress, is involved in cell division, and suppresses aging. AsA is considered to be involved in collagen synthesis promotion, the MEKERK1/2 pathway, NADPH oxidase (NOX), and endothelial NO synthase (eNOS) [10]
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