Production of functional endothelial cells

The researchers proposed an efficient and cost-effective way to produce endothelial cells from stem cells.
The innermost layer of blood vessels is formed by endothelial cells, which in turn are involved in the development of diseases of the cardiovascular system.

Human endothelial cells are therefore needed to investigate “in vitro” the causes of these diseases.

Researchers at Bonn University Hospital have developed a highly efficient, cost-effective and reproducible way to produce functional endothelial cells from human induced pluripotent stem cells for testing in cell culture containers.

Importance of endothelial cells

Endothelial cells line the inside of the blood vessels. They perform a number of tasks, such as regulating blood pressure and blood clotting in the human body.

They are also involved in the development of diseases of the cardiovascular system. Human endothelial cells are needed to study the basis of these diseases in vitro, that is, outside the human body.

In this regard, human induced pluripotent stem cells (hiPSCs) are a promising approach for this.

Endothelial (EC) cell dysfunction plays a key role in the onset and progression of cardiovascular disease. However, studying these disorders in ECs from patients is challenging. Hence, the use of human induced pluripotent stem cells (hiPSCs) and their laboratory differentiation into ECs shows a very promising approach.

Therefore, we investigated the application of a forward programming approach using transgenic hiPSC cell lines.

Production of endothelial cells from ESC and iPSC differentiation

It is from the mesoderm subset that both hematopoietic and EC emerge. EC is organized into vascular structures in their EB. The addition of a variety of growth factors increases the differentiation of EC in EB.

In the second method, ESC and iPSC differentiation are cultured with a type of stromal cells to promote the differentiation of the EC lineage from the mesoderm.

Mouse cranial mesenchymal cells called OP9 cells are widely used to promote differentiation and facilitate the emergence of cardiac, hematopoietic, endothelial and other lineages.

Some researchers prefer the distinction of ESC and iPSC in two-dimensional culture on tissue culture plates coated with protein substrates (matrixel, fibronectin, gelatin, or other proteins) using specific culture media with sequential recombinant growth factors.

Different differentiation strategies for the production of endothelial cells

Various differentiation strategies for hiPSCs into endothelial cells have already been developed in the past.

The so-called transcription factors are specifically activated to control the conversion of hiPSCs into endothelial cells.

Fast, Cost-Effective and Repeatable Pathway in Human Endothelial Cell Production

The research group of Dr. Sarah Rick from the Institute of Physiology, together with Kritika Sharma from Professor Volker Bascamp’s team at the UKB Ophthalmology Clinic, improved the PGP1-ETV2-iso2 cell line differentiation protocol and combined it with a strategy using new growth factors.

We were able to show that the ETV2 protocol we have improved is more efficient and cost-effective than the protocol with growth factors alone.

It produces endothelial cells faster, requires fewer additives for the culture medium, and does not require an additional purification step.

In addition, this process is highly repeatable and can be easily transferred to other hiPSC cell lines.

They produce the characteristic proteins of endothelial cells and also exhibit the typical functional properties of endothelial cells.

By modifying the differentiation protocol, it is possible to preferably obtain endothelial cells with arterial or venous characteristics.

Although they are similar to growth factor differentiated endothelial cells, there is some evidence that ETV2 protocol endothelial cells have a slightly higher degree of maturity.

Compared to human endothelial cells produced from the umbilical cord vein, both types of hiPSC-derived endothelial cells are not fully developed, which is likely due to the lack of external influences such as the absence of blood flow.

Next step of the study

Now we have developed an efficient, cost-effective, and repeatable differentiation protocol for generating functional hECs through forward programming.

Transgenic hECs can be genetically modified and are powerful tools for disease modeling, tissue engineering, and translation purposes.

The Bonn researchers hypothesize that the PGP1 ETV2 iso2 cell line and the endothelial cells produced from it are used to model and study human vascular diseases in which the endothelium is involved in the cell culture container.

This scientific question has been investigated by Dr. Rieck and Professor Fleischmann in the C01 project at the DFG Transregio Joint Research Center (TRR) 259 “Aortic Diseases”.

Endothelial cells can also be used in organoid research to produce organoids with the vascular system. “In addition, we are also interested in which culture methods increase the ‘degree of maturity’ of endothelial cells after differentiation, so that their gene expression characteristics are more consistent with mature endothelial cells,” says Dr. Rieck.

Source: Stem Cell Information Database