The homeostasis of intracellular pH (pHi ) affects many cellular functions. Our previous study has established a functional and molecular model of the active pHi regulators in human induced pluripotent stem cells (hiPSCs). The aims of the present study were to further quantify passive pHi buffering power (β) and to investigate the effects of extracellular pH and Na+ -H+ exchanger 1 (NHE1) activity on pluripotency in hiPSCs. pHi was detected by microspectrofluorimetry with pH-sensitive dye-BCECF. Western blot, immunofluorescence staining, and flow cytometry were used to detect protein expression and pluripotency. Our study in hiPSCs showed that (a) the value of total (βtot ), intrinsic (βi ), and CO2 -dependent ( ) buffering power all increased while pHi increased; (b) during the spontaneous differentiation for 4 days, the β values of βtot and changed in a tendency of decrease, despite the absence of statistical significance; (c) an acidic cultured environment retained pluripotency and further upregulated expression and activity of NHE1 during spontaneous differentiation; (d) inhibition on NHE1 activity promoted the loss of pluripotency. In conclusion, we, for the first time, established a quantitative model of passive β during differentiation and demonstrated that maintenance of NHE1 at a higher level was of critical importance for pluripotency retention in hiPSCs.
Keywords: BCECF; Na+-H+ exchanger 1; human induced pluripotent stem cells; passive buffering power; pluripotency; reversed pH gradient.
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