The inside layer of blood vessels consists of endothelial cells, which are significant in the progression of cardiovascular diseases. To explore the causes of these diseases through “in vitro” studies, researchers need human endothelial cells. A team from the University Hospital Bonn (UKB) and the University of Bonn has developed an efficient, affordable, and reproducible technique to produce functional endothelial cells from human induced pluripotent stem cells (hiPSCs) for use in laboratory tests. Their findings have been published in the journal Cardiovascular Research.
Endothelial cells are the cells that line blood vessels, playing crucial roles in functions like regulating blood pressure and clotting. They are also integral to understanding cardiovascular diseases. For studying the mechanisms of these diseases outside the human body (“in vitro”), human endothelial cells are essential. “Human induced pluripotent stem cells (hiPSCs) offer a promising avenue for this research because they are adaptable and can develop into various cell types, including endothelial cells,” explains Prof. Bernd K. Fleischmann, senior author and Director of the Institute of Physiology I at the UKB, part of the Cluster of Excellence ImmunoSensation2 and the Transdisciplinary Research Area (TRA) “Life & Health” at the University of Bonn.
Numerous methods have been explored to differentiate hiPSCs into endothelial cells. One of the most effective strategies involves using a variety of growth factors along with a purification stage to enhance the yield of correctly formed endothelial cells. Another method involves the targeted activation of specific transcription factors to guide the transformation of hiPSCs into endothelial cells. Recently, a global research team led by George Church from Harvard Medical School and Volker Busskamp from the UKB identified the transcription factor “ETS variant transcription factor 2,” or ETV2, as a crucial component in this process. They also created a hiPSC line that allows for the specific activation of ETV2 through the addition of doxycycline, known as “PGP1 ETV2 iso2.”
Efficient, economical, and consistent method for creating human endothelial cells
Dr. Sarah Rieck’s research team at the Institute of Physiology I, in collaboration with Kritika Sharma from Prof. Volker Busskamp’s Ophthalmology group at the UKB, has refined the differentiation protocol for the PGP1-ETV2-iso2 line (referred to as the ETV2 protocol) and compared it against the growth factor-based method. “Our findings indicate that the ETV2 protocol we optimized is more efficient and economical than the growth factor protocol,” states Dr. Sarah Rieck, the co-corresponding and leading author, who also conducts research at the University of Bonn. This method produces endothelial cells faster, requires fewer supplements in the culture medium, and eliminates the need for an extra purification step. Additionally, the process is highly reproducible and can be adapted to other hiPSC lines. The endothelial cells produced are not mixed with other cell types and maintain stability over extended culture times. They express proteins that are characteristic of endothelial cells and display typical functional traits as well. By adjusting the differentiation protocol, it’s also possible to specifically obtain endothelial cells resembling either arterial or venous types.
Although similar to endothelial cells produced via the growth factor method, there is some indication that ETV2 protocol-derived endothelial cells are slightly more mature. “However, compared to human endothelial cells from the umbilical vein, both types of hiPSC-derived endothelial cells are still not completely mature, likely due to the lack of external factors, such as blood flow,” adds Prof. Busskamp, who heads the “Neurodegenerative Retinal Diseases” research group at the UKB and is part of the Cluster of Excellence ImmunoSensation2 and the TRA “Life & Health” at the University of Bonn.
Looking ahead, the researchers in Bonn believe that the PGP1 ETV2 iso2 line and the endothelial cells generated from it will serve as models for studying human vascular diseases linked to the endothelium within laboratory cultures. This research focus is being pursued by Dr. Rieck and Prof. Fleischmann in project C01 of the DFG Collaborative Research Center Transregio (TRR) 259 “Aortic Diseases.” Additionally, these endothelial cells could aid in organoid research for creating organoids with a vascular system. “Moreover, we aim to explore cultivation techniques that enhance the ‘maturity degree’ of endothelial cells after differentiation, to better mimic the profile of adult endothelial cells,” remarks Dr. Rieck.
