hiPSC-ECs differentiation using the Aoki protocol.

A) Timeline and corresponding IF images from day 8 onwards of the expansion of hiPSC-ECs. During this time the hiPSC-ECs were expanded for up to 28 days (passage 3) before they show deterioration in cell morphology. B) IF images showing hiPSC-ECs at day 23 (passage 2) and 28 (passage 3). Some hiPSC-ECs showed elongated WPBs. However, these were very few, surrounded by cells showing the immature rounded WBPs.</p

hiPSC-ECs cultured with HDAC inhibitor sodium butyrate.

A) Confocal microscopy showing VWF and VE-cadherin after the addition of different concentrations of sodium butyrate. Bottom panel 2x zoomed in. B) ELISA showing VWF secretion (medium). C) Gene expression heatmaps of VWF and VWF related transcription factors. SB, sodium butyrate; NT, no treatment.</p

Reprogramming characterization of PBMCs into hiPSC.

A) Donor information and FACS data of pluripotency markers. B) IF images of markers showing differentiation potential into the three germlines. (TIF)</p

ECFCs cultured with acetic acid.

A) Confocal microscopy showing VWF (green) and VE-cadherin (red) after the addition of different concentrations of acetic acid at days 8 (top panel) and 12 (bottom panel). B) ELISA showing VWF production (lysates). C) ELISA showing VWF secretion (medium). HAc, acetic acid.</p

hiPSC-ECs cultured under variable conditions.

hiPSC-ECs differentiated with low or high concentrations of VEGF. A) VWF and VE-Cadherin levels by confocal microscopy. B) VWF and VE-Cadherin levels by confocal microscopy when cultured under static (left) or flow (right) conditions. C) FACS analysis showing endothelial and hematopoietic cell marker levels in hiPSC-ECs. D) ELISA showing VWF production (Lysates) and secretion (medium).</p

Morphology and endothelial markers of hiPSC-ECs.

A) VWF and VE-Cadherin in hiPSC-ECs (left) and ECFCs (right). B) FACS analysis showing endothelial and hematopoietic cell marker expression in the three hiPSC-ECs and ECFCs.</p

Additional optimization strategies.

A) hiPSC-EC cocultures with hiPSC-PCs. B) Varying concentration of endothelial differentiation factor CHIR99021. C) Addition of different HDAC inhibitors. (TIF)</p

hiPSC-ECs differentiation using the ETV2 protocol.

Timeline and corresponding IF images from day 4 onwards of the expansion of hiPSC-ECs. During this time the hiPSC-ECs were expanded for up to 40 days (passage 3) before they show deterioration in cell morphology A limited number of iPSC-ECs showed elongated WPBs, but again, these were very few, surrounded by cells showing the immature rounded WBPs.</p

Medium and supplements used in this study.

BackgroundEndothelial cells generated from induced pluripotent stem cells (hiPSC-ECs) show the majority of endothelial cell characteristics and markers, such as cobblestone morphology and the expression of VEGF and VE-cadherin. However, these cells are failing to show a mature endothelial cell phenotype, which is represented by the low expression and production of von Willebrand Factor (VWF) leading to the round morphology of the Weibel Palade Bodies (WPBs). The aim of this study was to improve the maturation process of hiPSC-ECs and to increase the levels of VWF.MethodshiPSC-ECs were differentiated by a standard differentiation protocol from hiPSCs generated from healthy control donors. To induce maturation, the main focus was to increase the expression and/or production of VWF by the adjustment of potential parameters influencing differentiation and maturation. We also compared alternative differentiation protocols. Cells were analyzed for the expression of endothelial cell markers, WPB structure, and the production and secretion of VWF by flow cytometry, confocal microscopy and ELISA.ResultsThe generated hiPSC-ECs have typical endothelial cell surface expression profiles, with low expression levels of non-endothelial markers as expected. Co-culture with pericytes, varying concentrations and timing of differentiation factors, applying some level of flow, and the addition of HDAC inhibitors did not substantially improve maturation of hiPSC-ECs. Transfection with the transcription factor ETV2 to induce a faster hiPSC-EC differentiation process resulted in a limited increase in VWF production, secretion, and elongation of WPB structure. Alternative differentiation protocols had limited effect.ConclusionhiPSCs-ECs have the potential to show a more mature endothelial phenotype with elongated WPBs after >30 days in culture. However, this comes with limitations as there are very few cells detected, and cells are deteriorating after being in culture for extended periods of time.</div

Schematic overviews of the (adjusted) three endothelial differentiation protocols used in this study.

A) Orlova protocol. B) Aoki protocol. C) ETV2 protocol. (TIF)</p