Lowering the increased intracellular pH of human-induced pluripotent stem cell-derived endothelial cells induces formation of mature Weibel-Palade bodies

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.

HPS6 Regulates the Biogenesis of Weibel–Palade Body in Endothelial Cells Through Trafficking v-ATPase to Its Limiting Membrane

“…The existence of regular tubules of ultra-large vWF is an indicator of WPB maturation that is a driving force to form rod-shaped WPBs ( McCormack et al, 2017 ; Tiemeier et al, 2020 ). Studies have shown that vWF is released by regulated secretion and unfurls into long strings that are highly efficient in recruiting platelets under flow in neutral plasma ( Michaux et al, 2006 ; Ferraro et al, 2014 ; El-Mansi and Nightingale, 2021 ).…”

Approaches to induce the maturation process of human induced pluripotent stem cell derived-endothelial cells to generate a robust model

“…Parallel to this study, our collaborating group published a paper in which the intracellular pH of hiPSC-ECs was lowered with acetic acid [18]. They showed that, in combination with the (adjusted) Orlova protocol, elongated WPBs appeared in the hiPSC-ECs upon lowering the pH,…”

Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening

“…Interestingly, our data show that 3PO inhibits the angiogenic sprouting of iPSC-ECs, which suggests that, like HUVECs, they undergo the same metabolic switch to use glycolysis as the main energy source during angiogenic sprouting [ 21 ]. While it has been shown that some endothelial cells differentiated from iPSCs have lower activity of glycolysis [ 27 ], the question remains how the culture conditions dictate the cells phenotype, and whether this effect could be attributed to the specific cues added by our culture platform. For example, it has been shown that both tip cells and non-tip cells use glycolysis as well as mitochondrial respiration for energy production, and that this balance depends on microenvironmental circumstances [ 28 ].…”