AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(HPS0077) were kindly provided by Dr. Dai from the Tr...AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(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_4Cl 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)_2SO_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 steadystate 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 pHi 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 pHi 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 pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.展开更多
基金Supported by Ministry of Science and Technology Grants of Taiwan,No.MOST 106-2320-B-016-003-MY2(to Loh SH)and No.MOST 106-2314-B-016-037-MY3(to Tsai YT)National Defense Medical Center Grants of Taiwan,No.MAB-106-033(to Loh SH),No.MAB-105-043 and No.MAB-106-034(to Dai NZ)Teh-Tzer Study Group for Human Medical Research Foundation of Taiwan,No.A1061037 and No.A1061054(to Loh SH)
文摘AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(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_4Cl 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)_2SO_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 steadystate 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 pHi 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 pHi 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 pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.
