Endothelial cells are responsible for communication between blood and tissues and are found throughout the body. In terms of disease, their dysfunction is associated with an assortment of disorders, including hypertension and thrombosis. Several researchers have prepared endothelial cells from pluripotent stem cells with the goal of new therapeutics, but the yield and purity of the endothelial cells is not satisfactory for clinical purpose.

A collaboration between the Megumu Saito lab at CiRA at the Kiyotoshi Sekiguchi lab at Osaka University has shown that changing the environment in which the stem cells are cultured midway during the differentiation process to endothelial cells reaches unprecedented yield and purity. The study can be read in Scientific Reports.

"When preparing endothelial cells, we culture the stem cells on laminin-coated extracellular matrix," explains Associate Professor Megumu Saito, lead researcher of the study. It is not uncommon for one laminin to be selected throughout the whole culture process. However, Saito observed, "We show switching the type of laminin increases the number of endothelial cells." There are at least 15 different types of human laminin (LM). LM511 is normally used to culture stem cells, but Saito and his team found that using LM511 for the first three days of culture and then LM411 for the next four significantly improved the differentiation to endothelial cells.

"Almost all of our cells are attached to matrices, so matrices must be a very important factor in cell fate," said Assistant Professor Akira Niwa, another scientist at CiRA involved in the project. "But we tend to focus more on their function as a scaffold." Niwa noted that LM511 reacts well with stem cells, but as the stem cells differentiate, LM411 is more effective at specifying the lineage towards endothelial cells. "We noticed LM411 has very specific distribution in the body. LM411 is located around blood vessels and bone marrow," he remarked.

As such, LM511 was appropriate to promote the early stages of differentiation. On the other hand, LM411 could steer the later stages of differentiation to endothelial cells. In fact, using only LM511 in their protocol resulted in 80% of the stem cells initiating differentiation, but only 10% reaching the stage of endothelial cells, with the remaining becoming an assortment of other cell types that compromised both yield and purity. On the other hand, using a LM411 derivative, LM411-E8, mid-stage, the scientists achieved a purity of endothelial cells that was over 95%, more than twice the purity of any other report, and a yield of 15 endothelial cells per stem cell, again better than any previous value reported.

According to Niwa, "LM411-E8 binds to specific types of integrin, which promotes endothelial cell differentiation." Further study revealed the molecule that bridged pathways downstream of LM411-E8, VEGF. "VEGF is a common hormone used to promote endothelial growth," said Niwa. Accordingly, the new protocol uses includes VEGF during the LM411-E8 stage. It also uses another compound, CHIR99021, during the LM511 stage to promote the early stages of differentiation. Gene analysis confirmed that the resulting endothelial cells were consistent with those seen in the body.

"These are very exciting findings," said Saito. "Since many diseases are linked to poor endothelial function, the derivation of highly purified functional PSC-derived endothelial cells provides a platform for future cell therapy and disease modeling."

• doi:10.1038/srep35680