Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells

AIM: To establish a functional and molecular model of the intracellular pH (pH(i)) regulatory mechanism in human induced pluripotent stem cells (hiPSCs). METHODS: hiPSCs (HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital (IRB No. B-106-09). Changes in the pH(i) were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K(+)/nigericin method. NH(4)Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power (β) was calculated from the ΔpH(i) induced by perfusing different concentrations of (NH(4))(2)SO(4). Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pH(i) regulators and pluripotency markers. RESULTS: In this study, our results indicated that (1) the steady-state pH(i) value was found to be 7.5 ± 0.01 (n = 20) and 7.68 ± 0.01 (n =20) in HEPES and 5% CO(2)/HCO(3)(-)-buffered systems, respectively, which were much greater than that in normal adult cells (7.2); (2) in a CO(2)/HCO(3)(-)-buffered system, the values of total intracellular buffering power (β) can be described by the following equation: β(tot) = 107.79 (pH(i))(2) - 1522.2 (pH(i)) + 5396.9 (correlation coefficient R(2) = 0.85), in the estimated pH(i) range of 7.1-8.0; (3) the Na(+)/H(+) exchanger (NHE) and the Na(+)/HCO(3)(-) cotransporter (NBC) were found to be functionally activated for acid extrusion for pH(i) values less than 7.5 and 7.68, respectively; (4) V-ATPase and some other unknown Na(+)-independent acid extruder(s) could only be functionally detected for pH(i) values less than 7.1; (5) the Cl(-)/ OH(-) exchanger (CHE) and the Cl(-)/HCO(3)(-) anion exchanger (AE) were found to be responsible for the weakening of intracellular proton loading; (6) besides the CHE and the AE, a Cl(-)-independent acid loading mechanism was functionally identified; and (7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pH(i) value and diminished the functional activity and protein expression of the NHE and the NBC. CONCLUSION: For the first time, we established a functional and molecular model of a pH(i) regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.