Intracellular pH monitoring in stem cells during differentiation using fluorescence microscopy and pH-sensor SypHer-2

Abstract

Among the functional changes accompanying the process of differentiation of stem cells, alterations to the intracellular pH are some of the most important parameters. The concentration of protons in the cytoplasm plays an important role in switching metabolic pathways from oxidative phosphorylation to aerobic glycolysis. Modern fluorescence methods of investigation are currently preferable for the study of pH dynamics. Fluorescence microscopy, in combination with genetically-coded sensors and exogenous markers, allows non-invasive investigation of the functional changes underlying the dynamics of differentiation. The aim of this study was to investigate the dynamic changes in intracellular pH in mesenchymal stem cells (MSCs) undergoing differentiation in three directions: adipogenic, osteogenic, and chondrogenic using fluorescence microscopy and pH-sensor SypHer-2. Materials and Methods. Differentiation was induced by incubating the human MSCs in commercial adipogenic or osteogenic or chondrogenic mediums. We used the genetically-coded pH-sensor SypHer-2 as a fluorescent probe. To obtain a temporarily transfected line, MSC-SypHer-2, electroporation of cells was performed with this protein. To convert relative units of pH into absolute units we conducted a calibration of the SypHer-2 pH-sensor. We determined the correlation between the intensities of fluorescence caused in the pH-sensor by illumination at 488 nm and that from illumination at 405 nm (I488/I405) for each value of pH using fluorescence microscopy. In accordance with the calibration curve we defined the absolute pH values in non-differentiated MSCs and in the MSCs undergoing adipogenic, osteogenic or chondrogenic differentiation. The data representing changes of intracellular pH were obtained on days 7, 14, and 21 of differentiation. Results. In our work, we showed acidification of the intracellular pH during adipogenic, chondrogenic, and osteogenic MSC differentiation. Moreover, this work identified a correlation between changes in the dynamics of intracellular pH and the metabolic status changes of the MSCs that had not been previously described. As the pH in cells undergoing adipogenic differentiation is lower than that in other types of MSC differentiation, this is probably connected with the transfer into the cytosol of the citrate necessary for the biosynthesis of fatty acids and with the conversion of malate into pyruvate. The relatively high pH levels found during osteogenic and chondrogenic differentiation enhance the activity of enzymes needed for catalyzing the oxidation of proline and lysine in collagen, with the participation of ascorbic acid, in particular, proline and lysine hydroxylases. Presented results provide the basis for further development of effective evaluation methods for characterizing the potential of stem cells, particularly by assessing intracellular pH as an indicator of the status of cell differentiation. In addition, these results can serve the development of an integrated approach to assessing functional changes in the stem cells, reflecting the peculiarities of a particular differentiation in the early stages.