Lowering the increased intracellular pH of human-induced pluripotent stem cell-derived endothelial cells induces formation of mature Weibel-Palade bodies
Authors: Tiemeier Gesa L. , Koning Rozemarijn , Wang Gangqi , Kostidis Sarantos , Rietjens Rosalie G. J. , Sol Wendy M. P. J. , Dumas Sébastien J. , Giera Martin , Berg Cathelijne W. , Eikenboom Jeroen C. J. , Berg Bernard M. , Carmeliet Peter , Rabelink Ton J. 
Published: March 2020

Abstract
Differentiation of human-induced pluripotent stem cells (hiPSCs) into vascular endothelium is of great importance to tissue engineering, disease modeling, and use in regenerative medicine. Although differentiation of hiPSCs into endothelial-like cells (hiPSC-derived endothelial cells [hiPSC-ECs]) has been demonstrated before, controversy exists as to what extent these cells faithfully reflect mature endothelium. To address this issue, we investigate hiPSC-ECs maturation by their ability to express von Willebrand factor (VWF) and formation of Weibel-Palade bodies (WPBs). Using multiple hiPSCs lines, hiPSC-ECs failed to form proper VWF and WPBs, essential for angiogenesis, primary and secondary homeostasis. Lowering the increased intracellular pH (pHi) of hiPSC-ECs with acetic acid did result in the formation of elongated WPBs. Nuclear magnetic resonance data showed that the higher pHi in hiPSC-ECs occurred in association with decreased intracellular lactate concentrations. This was explained by decreased glycolytic flux toward pyruvate and lactate in hiPSC-ECs. In addition, decreased expression of monocarboxylate transporter member 1, a member of the solute carrier family (SLC16A1), which regulates lactate and H+ uptake, contributed to the high pHi of hiPSC-EC. Mechanistically, pro-VWF dimers require the lower pH environment of the trans-Golgi network for maturation and tubulation. These data show that while hiPSC-ECs may share many features with mature EC, they are characterized by metabolic immaturity hampering proper EC function.

Significance statement The formation of functional and mature Weibel-Palade bodies (WPBs), essential for endothelial cell (EC) function, in human induced pluripotent stem cell (hiPSC)-ECs is a crucial step in the development of the full potential of hiPSC-EC for tissue regeneration, organ vascularization, and disease modeling. Current differentiation protocols fail to create hiPSC-EC with mature WPBs in vitro. To the best of the authors' knowledge, this is the first study with detailed characterization of von Willebrand factor (VWF) and WPBs in hiPSC-ECs and 13C-labeled glucose flux metabolomics of hiPSC-ECs. The results of this study show that hiPSC-ECs have a higher intracellular pH (pHi) than mature EC, where pro-VWF dimers require the lower pH environment for maturation and tubulation. Metabolic experiments showed that higher pHi in hiPSC-ECs occurred in association with decreased glycolysis and reduced intracellular lactate concentrations. In addition, decreased expression of MCT1, a pHi-sensitive member of the solute carrier family (SLC16A1), which regulates lactate and H+ uptake was observed. By lowering pHi with acetic acid, formation of mature WPBs in hiPSC-ECs could be induced.