Intracellular pH Monitoring in Stem Cells During Differentiation Using Fluorescence Microscopy and pH-Sensor SypHer-2

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.

1. Folmes C.D.L., Dzeja P.P., Nelson T.J., Terzic A. Metabolic plasticity in stem cell homeostasis and differentiation. Cell Stem Cell 2012; 11(5): 596–606, https://doi.org/10.1016/j.stem.2012.10.002.
2. Narae K., Minami N., Yamada M., Imai H. Immobilized pH in culture reveals an optimal condition for somatic cell reprogramming and differentiation of pluripotent stem cells. Reprod Med Biol 2017; 16(1): 58–66, https://doi.org/10.1002/rmb2.12011.
3. McBrian M.A., Behbahan I.S., Ferrari R., Su T., Huang T-W., Li K., Hong C.S., Christofk H.R., Vogelauer M., Seligson D.B., Kurdistani S.K. Histone acetylation regulates intracellular pH. Mol Cell 2013; 49(2): 310–321, https://doi.org/10.1016/j.molcel.2012.10.025.
4. Occhipinti R., Boron W.F. Mathematical modeling of acid-base physiology. Prog Biophys Mol Biol 2015; 117(1): 43–58, https://doi.org/10.1016/j.pbiomolbio.2015.01.003.
5. Damaghi M., Wojtkowiak J.W., Gillies R.J. pH sensing and regulation in cancer. Front Physiol 2013; 4: 370, https://doi.org/10.3389/fphys.2013.00370.
6. Gao W., Zhang H., Chang G., Xie Z., Wang H., Ma L., Han Z., Li Q., Pang T. Decreased intracellular pH induced by cariporide differentially contributes to uman umbilical cord-derived mesenchymal stem cells differentiation. Cell Physiol Biochem 2014; 33(1): 185–194, https://doi.org/10.1159/000356661.
7. Gerweck L.E., Seetharaman K. Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer. Cancer Res 1996; 56(6): 1194–1198.
8. Ailing T., Mantalaris A., Lim M. Influence of culture pH on proliferation and cardiac differentiation of murine embryonic stem cells. Biochemical Engineering Journal 2014; 90: 8–15, https://doi.org/10.1016/j.bej.2014.05.005.
9. Bryne U., Grillo-Hill B.K., Benitez M., Azimova D.R., Barber D.L., Nystul T.G. Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation. J Cell Biol 2016; 215(3): 345–355, https://doi.org/10.1083/jcb.201606042.
10. Boussouf A., Gaillard S. Intracellular pH changes during oligodendrocyte differentiation in primary culture. J Neurosci Res 2000; 59(6): 731–739, https://doi.org/10.1002/(sici)1097-4547(20000315)59:6731::aid-jnr53.0.co;2-g.
11. Kohn D.H., Sarmadi M., Helman J.I., Krebsbach P.H. Effects of pH on human bone marrow stromal cells in vitro: implications for tissue engineering of bone. J Biomed Mater Res 2002; 60(2): 292–299, https://doi.org/10.1002/jbm.10050.
12. Leem Y.H., Kim J.H., Lee K.S., Lee D.H., Yun J., Chang J.S. The effects of extracellular pH on proliferation and differentiation of human bone marrow stem cells. Korean Journal of Bone Metabolism 2012; 19(1): 35, https://doi.org/10.11005/kjbm.2012.19.1.35.
13. Calderón Montaño J.M., Burgos Morón E., Pérez Guerrero C., Salvador Bofill F.J., Robles Frías A., López Lázaro M. Role of the Intracellular pH in the metabolic switch between oxidative phosphorylation and aerobic glycolysis — relevance to cancer. WebmedCentral 2011; 2(3): 1–10.
14. Shum L.C., White N.S., Mills B.N., Bentley K.L., Eliseev R.A. Energy metabolism in mesenchymal stem cells during osteogenic differentiation. Stem Cells Dev 2016; 25(2): 114–122, https://doi.org/10.1089/scd.2015.0193.
15. Pattappa G., Heywood H.K., de Bruijn J.D., Lee D.A. The metabolism of human mesenchymal stem cells during proliferation and differentiation. J Cell Physiol 2010; 226(10): 2562–2570, https://doi.org/10.1002/jcp.22605.
16. Han J., Burgess K. Fluorescent indicators for intracellular pH. Chem Rev 2010; 110(5): 2709–2728, https://doi.org/10.1021/cr900249z.
17. Battisti A., Digman M.A., Gratton E., Storti B., Beltram F., Bizzarri R. Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging. Hem Commun (Camb) 2012; 48(42): 5127–5129, https://doi.org/10.1039/c2cc30373f.
18. Zuk P.A., Zhu M., Mizuno H., Huang J., Futrell J.W., Katz A.J., Benhaim P., Lorenz H.P., Hedrick M.H. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001; 7(2): 211–228, https://doi.org/10.1089/107632701300062859.
19. Belousov V.V., Fradkov A.F., Lukyanov K.A., Staroverov D.B., Shakhbazov K.S., Terskikh A.V., Lukyanov S. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 2006; 3(4): 281–286, https://doi.org/10.1038/nmeth866.
20. Lemasters J.J., Holmuhamedov E. Voltage-dependent anion channel (VDAC) as mitochondrial governator — thinking outside the box. Biochim Biophys Acta 2006; 1762(2): 181–190, https://doi.org/10.1016/j.bbadis.2005.10.006.
21. Mannella C.A., Kinnally K.W. Reflections on VDAC as a voltage-gated channel and a mitochondrial regulator. J Bioenerg Biomembr 2008; 40(3): 149–155, https://doi.org/10.1007/s10863-008-9143-0.
22. Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P. Molecular biology of the cell. New York: Garland Science; 2002.
23. Trivedi B., Danforth W.H. Effect of pH on the kinetics of frog muscle phosphofructokinase. J Biol Chem 1966; 241(17): 4110–4112.
24. Erecińska M., Deas J., Silver I.A. The effect of pH on glycolysis and phosphofructokinase activity in cultured cells and synaptosomes. J Neurochem 1995; 65(6): 2765–2772, https://doi.org/10.1046/j.1471-4159.1995.65062765.x.
25. Fantin V.R., St-Pierre J., Leder P. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell 2006; 9: 425–434, https://doi.org/10.1016/j.ccr.2006.04.023.
26. Huber V., De Milito A., Harguindey S., Reshkin S.J., Wahl M.L., Rauch C., Chiesi A., Pouysségur J., Gatenby R.A., Rivoltini L., Fais S. Proton dynamics in cancer. J Transl Med 2010; 8: 57, https://doi.org/10.1186/1479-5876-8-57.
27. Varum S., Rodrigues A.S., Moura M.B., Momcilovic O., Easley C.A. 4th, Ramalho-Santos J., Van Houten B., Schatten G. Energy metabolism in human pluripotent stem cells and their differentiated counterparts. PLoS One 2011; 6(6): e20914, https://doi.org/10.1371/journal.pone.0020914.
28. Meleshina A.V., Dudenkova V.V., Shirmanova M.V., Bystrova A.S., Zagaynova E.V. Multiphoton fluorescence lifetime imaging of metabolic status in mesenchymal stem cell during adipogenic differentiation. Proc. SPIE 9712, Multiphoton Microscopy in the Biomedical Sciences XVI 2016, https://doi.org/10.1117/12.2209056.
29. Meleshina A.V., Dudenkova V.V., Shirmanova M.V., Shcheslavskiy V.I., Becker W., Bystrova A.S., Cherkasova E.I., Zagaynova E.V. Probing metabolic states of differentiating stem cells using two-photon FLIM. Sci Rep 2016; 6: 21853, https://doi.org/10.1038/srep21853.
30. Meleshina A.V., Dudenkova V.V., Bystrova A.S., Kuznetsova D.S., Shirmanova M.V., Zagaynova E.V. Two-photon FLIM of NAD(P)H and FAD in mesenchymal stem cells undergoing either osteogenic or chondrogenic differentiation. Stem Cell Res Ther 2017; 8(1): 15, https://doi.org/10.1186/s13287-017-0484-7.

Meleshina A.V., Kashirina A.S., Dudenkova V.V., Vdovina N.V., Cherkasova E.I., Zagaynova E.V. Intracellular pH Monitoring in Stem Cells During Differentiation Using Fluorescence Microscopy and pH-Sensor SypHer-2. Sovremennye tehnologii v medicine 2018; 10(1): 93, https://doi.org/10.17691/stm2018.10.1.12