Professor of Bioengineering Northeastern University Boston, Massachusetts, United States
Introduction: Endothelial differentiation of human pluripotent stem cells (hPSCs) holds great promise for vascular tissue engineering and disease modeling. Previously, several protocols have been developed to derive endothelial cells (ECs) from hPSCs using exogenously added growth factors and/or small molecules. However, there are still significant limitations in current methods such as EC phenotypical drifting, loss of EC commitment and functionality, and growth arrest during extended culture and passaging [1-3]. To overcome these limitations, we explore the alternative approach of EC differentiation via transcription factor ETV2 induction. As a master regulator of EC specification, ETV2 plays a crucial role in vascular development [4]. Current research shows promising results for the involvement of ETV2 in vascular differentiation, yet its precise role in directing hPSCs towards a functional endothelial fate remains incompletely understood. The goal of this work is to create hPSC-ECs via ETV2 induction that retain their endothelial morphology, expression, and function through prolonged culture.
Materials and
Methods: The stem cell line used in this project contains a vascular endothelial cadherin-promoter-mOrange-reporter and a Tet-On lentiviral vector containing ETV2 that is inducible by doxycycline. The mOrange reporter allows for the visualization of vascular endothelial cadherin (VECAD) expression in real-time during the differentiation process. The ETV2 vector allows for modulation of ETV2 expression with the addition of doxycycline in culture media. By modulating ETV2, we can tune the downstream expression of VECAD and platelet endothelial cell adhesion molecule 1 (PECAM) to produce functional hPSC-derived ECs. The vascular differentiation protocol used in this project was modified from a previously published protocol utilizing small molecules and growth factors alone [5]. This published condition acted as a control and consisted of a 6-day protocol in which hPSCs were first pushed to the mesoderm lineage with the addition of small molecule CHIR99021 and then to a vascular fate with the addition of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2). The ETV2 conditions were induced with doxycycline from days 2-6 of differentiation. One of the ETV2 conditions was differentiated with doxycycline alone, without any small molecules or growth factors (ETV2-EC), while the other ETV2 condition was differentiated with a combination of all factors in addition to doxycycline (ETV2-CVF-EC). After differentiation, each condition was sorted via magnetically activated cell sorting (MACS) using PECAM beads. The resulting positive populations were kept in culture for over two passages and analyzed through immunofluorescent staining, quantitative PCR, and vessel formation within hydrogel.
Results, Conclusions, and Discussions: When compared to the small molecule and growth factor-only control condition (CVF-EC), the ETV2 conditions (ETV2-EC and ETV2-CVF-EC) varied both in morphology and mO-VECAD expression during differentiation. However, after sorting, both condition types displayed a ‘cobblestone’ endothelial morphology and VECAD expression similar to what is normally seen in human umbilical vein endothelial cells (HUVECs) (Figure 1A). A preliminary quantitative PCR screen showed increased expression of both VECAD and PECAM in passage 2 ETV2-ECs when compared to CVF-ECs and HUVECs (Figure 1B). In addition, ETV2-ECs expressed higher levels of Krüppel-like Factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) than CVF-ECs, approaching the level seen in HUVECs, while Von Willebrand factor (vWF) remained low in both hPSC-EC conditions (Figure 1B). When seeded into fibrin gel alongside human astrocytes and pericytes, both the control CVF-EC condition and combined ETV2-CVF-EC condition formed vascular networks via EC-pericyte interactions within hydrogel (Figure 1C). These results suggest that ETV2-based differentiation methods could be an encouraging next step in deriving functional ECs over prolonged culture. Additionally, such methods could give further insights into creating more accurate, timely, and cost-effective vascularized tissue models.
Acknowledgements (Optional):
References: [1] Wang, K. et al. Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA. Sci Adv 6, (2020). [2] Kim, T. M., Lee, R. H., Kim, M. S., Lewis, C. A. & Park, C. ETV2/ER71, the key factor leading the paths to vascular regeneration and angiogenic reprogramming. Stem Cell Research & Therapy 2023 14:1 14, 1–16 (2023). [3] Grath, A. & Dai, G. SOX17/ETV2 improves the direct reprogramming of adult fibroblasts to endothelial cells. Cell Reports Methods 4, 100732 (2024). [4] Garry, D. J. Etv2 IS A MASTER REGULATOR OF HEMATOENDOTHELIAL LINEAGES. Trans Am Clin Climatol Assoc 127, 212 (2016). [5] Bertucci, T., Kakarla, S., Kim, D. & Dai, G. Differentiating Human Pluripotent Stem Cells to Vascular Endothelial Cells for Regenerative Medicine, Tissue Engineering, and Disease Modeling. Methods in Molecular Biology 2375, 1–12 (2022).
