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©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells
Shih-Chi Chao, Gwo-Jang Wu, Shu-Fu Huang, Niann-Tzyy Dai, Hsu-Kai Huang, Mei-Fang Chou, Yi-Ting Tsai, Shiao-Pieng Lee, Shih-Hurng Loh
Shih-Chi Chao, Shih-Hurng Loh, Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 11490, Taiwan
Gwo-Jang Wu, Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
Shu-Fu Huang, Mei-Fang Chou, Shih-Hurng Loh, Department of Pharmacy Practice, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
Niann-Tzyy Dai, Division of Plastic and Reconstructive Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
Hsu-Kai Huang, Division of Chest Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
Yi-Ting Tsai, Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei 11490, Taiwan
Yi-Ting Tsai, Division of Cardiovascular Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
Shiao-Pieng Lee, Division of Oral and Maxillofacial Surgery, Department of Dentistry, School of Dentistry, Tri-Service General Hospital and National Defense Medical Center, Taipei 11490, Taiwan
Shih-Hurng Loh, Department of Pharmacology, National Defense Medical Center, Taipei 11490, Taiwan
Author contributions: Chao SC and Loh SH performed the majority of experiments and analyzed the data; Chao SC, Wu GJ, Dai NT and Loh SH performed conception and design; Wu GJ, Dai NT and Loh SH participated equally in iPSC treatment and providing; Chao SC, Wu GJ, Huang SF, Dai NT, Huang SK, Tsai YT, Lee SP and Loh SH coordinated and interpreted the research; Cha SC and Loh SH wrote the paper; Cha SC, Wu GJ, Huang SF and Loh SH drafted the article or making critical revisions related to important intellectual content of the manuscript; Loh SH final approval of the version of the article to be published.
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).
Institutional review board statement: The manuscript is approved by Institutional Review Board of Tri-Service General Hospital.
Conflict-of-interest statement: All authors declare no potential conflict of interest.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author to: Shih-Hurng Loh, DPhil, Director, Full Professor, Department of Pharmacology, and Department of Pharmacy Practice, Tri-Service General Hospital, National Defense Medical Center, No. 161, Sec. 6, Minquan E. Rd., Taipei 11490, Taiwan.
shloh@ndmctsgh.edu.tw
Telephone: +886-2-87924861 Fax: +886-2-87924861
Received: October 17, 2018
Peer-review started: October 17, 2018
First decision: October 26, 2018
Revised: November 14, 2018
Accepted: December 4, 2018
Article in press: December 5, 2018
Published online: December 26, 2018
Processing time: 69 Days and 1.1 Hours
AIM
To establish a functional and molecular model of the intracellular pH (pHi) 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 pHi 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. NH4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power (β) was calculated from the ΔpHi induced by perfusing different concentrations of (NH4)2SO4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pHi regulators and pluripotency markers.
RESULTS
In this study, our results indicated that (1) the steady-state pHi value was found to be 7.5 ± 0.01 (n = 20) and 7.68 ± 0.01 (n =20) in HEPES and 5% CO2/HCO3--buffered systems, respectively, which were much greater than that in normal adult cells (7.2); (2) in a CO2/HCO3--buffered system, the values of total intracellular buffering power (β) can be described by the following equation: βtot = 107.79 (pHi)2 - 1522.2 (pHi) + 5396.9 (correlation coefficient R2 = 0.85), in the estimated pHi range of 7.1-8.0; (3) the Na+/H+ exchanger (NHE) and the Na+/HCO3- 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-/HCO3- 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 pHi 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.
Core tip: For the first time, we established a model of the intracellular pH (pHi) regulation mechanism in human induced pluripotent stem cells (hiPSCs). The steady-state pHi value of hiPSCs was 7.50-7.68, which greater than that of normal adult cells. The Na+-H+ exchanger, the Na+-HCO3- cotransporter and vacuolar-ATPase were the main acid extruders, while the Cl--HCO3- anion exchanger and the Cl--OH- exchanger were the main acid loaders. Moreover, the pHi and acid-extruding mechanism were decreased during the loss of pluripotency in hiPSCs. pHi regulators represent an attractive target for differentiation efficiency or culture quality.
