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

Figure 1 Functional characterization of acid extruders in the HEPES-buffered system. A-D: The top bar shows the buffer system used in perfusion experiments. The application of NH₄Cl and different conditions were respectively shown with the solid and dotted lines above the trace. The trace shown in A showed a typical pH_i recovery slope after NH₄Cl prepulse-induced intracellular acidosis in HEPES-buffered solution as a control. The traces shown in B-D showed the effect of the removal of extracellular Na⁺ (Na⁺-free), addition of 30 μmol/L HOE 694 (H₃₀) and Na⁺-free + 30 μmol/L bafilomycin A1 (Ba₃₀) on the pH_i recovery slope. E: The curve of the pH_i recovery rates for Na⁺-free, H₃₀ and Na⁺-free with Ba₃₀ were collected from 3-6 similar experiments shown in A-D. F: After pre-treatment with NH₄Cl for 5 min, HPS0077 cells were treated with Na⁺-free + Ba₃₀, Na⁺-free + Ba₃₀ + 40 μmol/L SCH-28080 (SCH₄₀) and Na⁺/Cl^--free + Na⁺-free + Ba₃₀ in HEPES-buffered solution, and the change in pH_i was detected by a multimode reader. Error bars represent the mean ± SE.

Figure 2 Calibration of the BCECF fluorescence ratio and pluripotency characterization. A: The trace showed the protocol of BCECF fluorescence ratio (510 nm emission at 490 nm and 440 nm excitations) calibration in HPS0077 cells. The top bars represent the application of different conditions; B: The plots of pH_ivs the BCECF fluorescence ratio were collected from 6 similar experiments shown in A; C: Immunofluorescence analysis showed the expression of pluripotency markers, OCT4, SSEA4, TRA-1-60 and SOX2, in HPS0077 cells.

Figure 3 Functional characterization of acid extruders in the 5% CO₂/HCO₃^--buffered system. A-E: The trace shown in A showed a typical pH_i recovery slope after NH₄Cl prepulse-induced intracellular acidosis in HEPES-buffered solution as a control. The traces shown in B-E showed the effect of the removal of extracellular Na⁺ (Na⁺-free), addition of 90 μmol/L S0859 (S₉₀), addition of 30 μmol/L HOE 694 (H₃₀) and addition of S₉₀ + H₃₀ on the pH_i recovery slope; F: The curve of the pH_i recovery rates after the addition of Na⁺-free, S₉₀, H₃₀ and S₉₀ + H₃₀ were collected from 2-10 similar experiments shown in A-E. Error bars represent the mean ± SE.

Figure 4 Functional characterization of the acid loader. A-D: The traces shown in A and C showed typical pH_i recovery slopes after Na-acetate prepulse-induced intracellular alkalization in HEPES and 5% CO₂/HCO₃^--buffered solution as a control. The traces shown in B and D showed the effect of the removal of extracellular Cl^- (Cl^--free) on the pH_i recovery slope in HEPES and 5% CO₂/HCO₃^--buffered solution; E: The curve of the pH_i recovery rates in HEPES and 5% CO₂/HCO₃^--buffered solution were collected from 3-4 similar experiments shown in A and C. Error bars represent the mean ± SE.

Figure 5 Steady-state pH_i in HEPES and 5% CO₂/HCO₃^--buffered solution and the change in the expression of pH_i regulators during the loss of pluripotency in human induced pluripotent stem cells. A: The resting pH_i was a steady-state taken from the completely recovered pH_i after intracellular acidification or alkalization. The dotted line indicates the value of the resting pH_i; B: The max/min chart of the resting pH_i in hiPSCs was collected from A (n = 20) and Figures 4C and D (n = 5). The means of the resting pH_i in HEPES and 5% CO₂/HCO₃^--buffered solution were found to be 7.50 ± 0.01 and 7.68 ± 0.01, respectively. After treatment with H₃₀ and S₉₀ in 5% CO₂/HCO₃^--buffered solution, the resting pH_i shifted to 7.66 ± 0.02 and 7.46 ± 0.02, respectively; C: Immunoblot analysis of the expression of NHE1, NHE3, V-ATPase, NBCe1, NBCe2, NBCn1 and OCT4 in hiPSCs in different culture media for different days (E8 and E6-1d to E6-4d). The histograms in B display the mean and the min to max values. hiPSCs: Human induced pluripotent stem cells; NHE: The Na⁺/H⁺ exchanger; NBC: The Na⁺/HCO₃^- cotransporter; V-ATPase: Vacuolar-ATPase.

Figure 6 The change in the activity of the Na⁺/H⁺ exchanger and the Na⁺/HCO₃^- cotransporter and the resting pH_i during the loss of pluripotency in human induced pluripotent stem cells. A: The traces showed the changes in pH_i recovery after NH₄Cl prepulse-induced intracellular acidification in E8 medium (containing fibroblast growth factor 2, FGF2, and transforming growth factor β1, TGFβ1) and E6 medium (without FGF2 and TGFβ1) for 1 to 4 d (E6-1d to E6-4d) in HEPES-buffered solution; B: The charts showed the pH_i recovery rate in E6-1d to -4d normalized to the rate in E8 (% of E8) in HEPES-buffered solution, which was estimated at pH_i = 6.9 and 7.2, respectively, and averaged for 3 experiments similar to that shown in A; C: The max/min plots showed the resting pH_i in E8, E6-1d, E6-2d, E6-3d and E6-4d media that were averaged from similar experiments shown in A (n = 5-20); D: The traces showed the changes in pH_i recovery after NH₄Cl prepulse-induced intracellular acidification in E8 and E6-1d to E6-4d media in 5% CO₂/HCO₃^--buffered solution; E: The graphs show the pH_i recovery rate in E6-1d to E6-4d normalized to the rate in E8 (control) in 5% CO₂/HCO₃^--buffered solution, which was estimated at pH_i = 6.9, 7.2 and 7.5, respectively, and averaged for 3 experiments similar to that shown in D; F: The max/min plots showed the resting pH_i in E8 E6-1d, E6-2d, E6-3d and E6-4d media, averaged from similar experiments shown in D (n = 5-20). Error bars represent the mean ± SE. The histograms in C and F show the mean and min to max values. NS: No significant difference; hiPSCs: Human induced pluripotent stem cells.

Figure 7 Kinetic model of the pHi regulatory mechanism in human induced pluripotent stem cells. A: A kinetic model illustrating the pH_i regulatory mechanism in HPS0077 cell, including acid extrusion, acid loading and passive buffering power. For the first time, we demonstrated that the active membrane pH_i regulators NHE1, NHE3, V-ATPase, NBCe1, NBCe2, NBCn1, AE and CHE functionally coexisted in hiPSCs, and in addition, unknown Na⁺-independent acid extruder(s) and Cl^--independent acid loader(s) were also observed. The length of the triangle indicates the pH_i range of pH_i regulator activation, and the height indicates the magnitude of the pH_i regulatory activity. For example, the NHE, NBC, AE and CHE were activated at pH_i ≤ 7.5, between 6.9 and 7.68, ≥ 7.4 and between 7.6 and 8.1, respectively. The non-NHE acid extruders [V-ATPase, unknown Na⁺-independent acid extruder(s)] and unknown Cl^--independent acid loader(s) were activated during extreme intracellular acidification, i.e., pH_i < 7.1, and alkalization, i.e., pH_i > 7.9, respectively. Moreover, the intracellular passive buffering capacity (β) increased as the pH_i shifted to the alkalization direction; B: In the process of the loss of pluripotency, the activity of the acid extrusion mechanism gradually decreased, including the participation of at least the NHE, the NBC and V-ATPase, and resulted in the resting pH_i shifting from 7.68 to 7.48. hiPSCs: Human induced pluripotent stem cells; NHE: The Na⁺/H⁺ exchanger; NBC: The Na⁺/HCO₃^- cotransporter; V-ATPase: Vacuolar-ATPase; AE: Anion exchanger; CHE: Cl^--OH^- exchanger.