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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering Undergraduate Fire Talks

Directed Differentiation of Induced Pluripotent Stem Cells into Arterial Endothelial Cells for Vascular Disease Modeling

Saturday, October 11, 2025

8:55 AM - 9:00 AM PDT

Location: Room 33B

Presenting Author(s)

Lisa Garnier

Student
Duke Univeristy
Durham, North Carolina, United States

Co-Author(s)

Jake J. Klimek

PhD Student
Duke University, United States
Claudia Wong

PhD Candidate
Duke University
Durham, North Carolina, United States
SG

Sharon Gerecht

Paul M. Gross Distinguished Professor and Chair, Biomedical Engineering
Duke University
Durham, North Carolina, United States

Last Author(s)

George Truskey

R. Eugene and Susie E. Goodson Distinguished Professor of Biomedical Engineering
Duke University
Durham, North Carolina, United States

Primary Investigator(s)

George Truskey

R. Eugene and Susie E. Goodson Distinguished Professor of Biomedical Engineering
Duke University
Durham, North Carolina, United States

Introduction: : Endothelial cells (ECs) are regulators of vascular function, responsible for maintaining vascular tone, barrier function, and immune homeostasis. Dysfunctional ECs are involved in diseases such as Hutchinson-Gilford Progeria Syndrome (HGPS), where vascular pathology leads to premature cardiovascular mortality. While most of the research has focused on vascular smooth muscle cells (SMCs) as the primary site of progerin toxicity, recent work by Atchison et al. [1] demonstrated that induced pluripotent stem cell (iPSC) derived ECs from HGPS patients impair vasodilation and exhibit upregulated inflammatory markers when incorporated into tissue-engineered blood vessels (TEBVs). These findings highlight the importance of endothelial health and identity in modeling disease-relevant vascular function.
Importantly, EC subtypes differ molecularly and functionally. Arterial ECs express markers such as SOX17, while venous ECs express NR2F2. Our study aimed to generate arterial endothelial cells (aiECs) from iPSCs using stepwise differentiation protocols. We tested combinations of Activin A (TGF-β agonist), SB505124 (TGF-β inhibitor), and GDC-0941 (PI3K inhibitor), based on the hypothesis that TGF-β promotes arterial fate while PI3K promotes venous identity. These small molecule manipulations were tested exclusively on the 168CL2 iPSC line following the Loh protocol [2]. In a subsequent experiment, we examined both the Loh and Shen (unpublished) protocols on iPSCs from two different donors to evaluate if each protocol effectively produced high fidelity aiECs. Our overarching goal is to produce high-purity aiECs for use in vascular tissue engineering and disease modeling platforms, particularly TEBVs, where arterial identity may enhance physiological relevance and application in diseases like HGPS.

Materials and

Methods: : Arterial endothelial cell differentiation was performed using two protocols: Loh and Shen. Both followed a stepwise induction of mesoderm and endothelial lineages over 6–7 days using combinations of different small molecules such as BMP4, VEGF, and XAV939, with adjustments to TGF-β signaling. SB505124 and SB431542 (TGF-β inhibitors) were included in select stages to guide lineage commitment. Protocols differed in the maintenance phase: the Loh protocol omitted small molecules post-differentiation, while the Shen protocol included SB505124 and VEGF. MACS purification was performed on Shen-derived cells to enhance aiEC purity.
Two experiments were conducted. In Experiment 1, we used the Loh protocol on iPSC line 168CL2 from the Progeria Research Foundation and evaluated post-differentiation treatment with combinations of SB505124 (SB), GDC-0941 (GDC; PI3K inhibitor), and ascorbic acid (AA; TGF-β agonist), resulting in eight conditions. The -SB/-GDC/-AA condition served as the control. In Experiment 2, we directly compared the Loh and Shen protocols using iPSC lines 168CL3 and DU11, with vascular endothelial cells (viECs) derived from iPSCs as the reference control. In this experiment, the viECs were maintained in endothelial cell media, which may have influenced their gene expression profile.
Seeding density was optimized at 250,000 cells per well to maximize confluency and arterial marker expression. RT-qPCR was used to assess gene expression of eleven targets across arterial (e.g., SOX17), venous (e.g., NR2F2), and pan-endothelial (e.g., VE-cadherin) categories, normalized to GAPDH. Immunofluorescence was used to visualize SOX17, NR2F2, and VE-cadherin expression.

Results, Conclusions, and Discussions:: In Experiment 1, we assessed the impact of continued small molecule treatment during the maintenance phase of the Loh protocol. Among the tested conditions, the -++ combination (no SB, with GDC and AA) increased arterial marker expression (Figure 1) but also led to undesired upregulation of venous markers. In contrast, the ++- condition (with SB and GDC, without AA) produced the most favorable profile, characterized by moderate arterial marker expression with unchanged or slightly elevated venous marker levels. This challenges the original assumption that TGF-β inhibition would suppress arterial identity, suggesting instead that SB505124 may enhance arterial specification while limiting venous gene expression. These findings reveal limitations in the Loh protocol’s maintenance strategy and support extending TGF-β inhibition beyond initial differentiation.
In Experiment 2, we directly compared the Loh and Shen protocols using two iPSC lines (168CL3 and DU11) to assess reproducibility and robustness. A key limitation was that only the Shen-derived cells were MACS-purified, which may have influenced the observed differences. When compared to viECs, neither protocol resulted in uniform upregulation of arterial markers across both lines. However, the Shen protocol consistently downregulated venous genes FLRT2 and APLNR in both cell lines. This finding aligns with the observation (from Figure 3) that arterial cells may express similar arterial markers to viECs but reduced venous markers, providing an alternative metric for assessing arterial identity.
It is important to note that viECs are a heterogeneous population of endothelial cells that line blood vessels and express both arterial and venous markers depending on their anatomical location. Therefore, additional validation using primary cells, including human aortic endothelial cells (HAOECs) and human umbilical vein endothelial cells (HUVECs), as gene expression controls will be conducted.
Future work will involve immunostaining across all cell lines and conditions using isotype controls to visualize VE-cadherin, SOX17, and NR2F2.
In conclusion, while neither protocol achieves perfect arterial specification, results suggest that with further optimization, such as post-differentiation small molecule supplementation and consistent purification steps, improved and more reproducible generation of aiECs may be feasible. These findings provide valuable insight for refining directed differentiation protocols toward effective arterial endothelial fates.

Acknowledgements and/or References (Optional): : We would like to thank Mengcheng Shen for providing the unpublished differentiation protocol and the Progeria Research Foundation for providing cells.

References
[1]L. Atchison et al., “iPSC-Derived Endothelial Cells Affect Vascular Function in a Tissue-Engineered Blood Vessel Model of Hutchinson-Gilford Progeria Syndrome,” Stem Cell Reports, vol. 14, no. 2, pp. 325–337, Feb. 2020, doi: https://doi.org/10.1016/j.stemcr.2020.01.005.
[2]L. T. Ang et al., “Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses,” Cell, vol. 185, no. 14, pp. 2523-2541.e30, Jul. 2022, doi: https://doi.org/10.1016/j.cell.2022.05.024.