Human pluripotent stem cells (hPSCs) typically take between 9 and 15 days to differentiate into vascular endothelial cells. At their most productive, only about 60% actually differentiate. A new method reduces that time to only five days, delivers 99% differentiation, and, according to the researchers, is easy and scalable.
This two-stage approach was developed by Sean Palecek, PhD, and Eric Shusta, PhD, both professors of chemical and biological engineering, and colleagues at the University of Wisconsin-Madison. It focuses on overexpressing ETV2, a transcription factor to reprogram the hPSCs to endothelial cells. Then, they expand the inducible ETV2 (iETV2) endothelial cells in an endothelial medium.
“We were somewhat surprised that transient expression of a single transcription factor, ETV2, was sufficient to generate a homogeneous population of endothelial cells,” Palecek tells GEN. “Stem cell populations are often heterogeneous, so it was a pleasant surprise to see that this process was so efficient.”
Writing in a recent paper, senior authors Palecek and Shusta report that, “By optimizing seeding density and medium composition, we achieved 99% pure CD31+ CD144+ iETV2-ECs without cell sorting in five days.” The differentiated cells were comparable to those of the same cell line that were differentiated using usual mesoderm progenitor methods. Gene expression profiles, angiogenesis potential, acetylated low-density lipoprotein uptake, and cytokine response were all similar.
“The ability to generate pure populations of endothelial cells without sorting increases scaling and reduces time and cost,” Palecek says. This streamlines the process of using them for developing in vitro models or cell therapies that involve vascularization.
The first differentiation stage, which induced ETV2 expression in genetically engineered hPSCs, occurred in a basal medium that did not contain growth factors or serum. It lasted three days.
In the second stage, ETV2 induction was removed and the endothelial cells continued differentiation and were “expanded in a serum-free hECSR medium on collagen IV matrix for two or more days.”
The scientists confirmed the expression and morphology of important endothelial markers in the differentiated cells using flow cytometry and ICC.
In further study, they found that the endothelial cells formed through their method responded to factors that induce endothelial cell specialization in the brain the same as native endothelial cells.
As they explained, in the brain, endothelial cells and Wnt ligands secreted from the central nervous system (CNS) are important in forming the blood-brain barrier (BBB). The BBB, in turn, is pivotal in regulating drug transport to the brain to treat neurodegenerative diseases.
This new technique, notes Palecek, can be used “to identify factors involved in the specialization of naïve endothelial cells to tissue-specific endothelial cells. Then we will devise protocols to advance manufacturing of human in vitro BBB models to enable drug discovery and disease modeling.”
The post Cutting hPSC Production Time By 66% appeared first on GEN - Genetic Engineering and Biotechnology News.
This two-stage approach was developed by Sean Palecek, PhD, and Eric Shusta, PhD, both professors of chemical and biological engineering, and colleagues at the University of Wisconsin-Madison. It focuses on overexpressing ETV2, a transcription factor to reprogram the hPSCs to endothelial cells. Then, they expand the inducible ETV2 (iETV2) endothelial cells in an endothelial medium.
“We were somewhat surprised that transient expression of a single transcription factor, ETV2, was sufficient to generate a homogeneous population of endothelial cells,” Palecek tells GEN. “Stem cell populations are often heterogeneous, so it was a pleasant surprise to see that this process was so efficient.”
Streamlining the process
Writing in a recent paper, senior authors Palecek and Shusta report that, “By optimizing seeding density and medium composition, we achieved 99% pure CD31+ CD144+ iETV2-ECs without cell sorting in five days.” The differentiated cells were comparable to those of the same cell line that were differentiated using usual mesoderm progenitor methods. Gene expression profiles, angiogenesis potential, acetylated low-density lipoprotein uptake, and cytokine response were all similar.
“The ability to generate pure populations of endothelial cells without sorting increases scaling and reduces time and cost,” Palecek says. This streamlines the process of using them for developing in vitro models or cell therapies that involve vascularization.
The first differentiation stage, which induced ETV2 expression in genetically engineered hPSCs, occurred in a basal medium that did not contain growth factors or serum. It lasted three days.
In the second stage, ETV2 induction was removed and the endothelial cells continued differentiation and were “expanded in a serum-free hECSR medium on collagen IV matrix for two or more days.”
The scientists confirmed the expression and morphology of important endothelial markers in the differentiated cells using flow cytometry and ICC.
In further study, they found that the endothelial cells formed through their method responded to factors that induce endothelial cell specialization in the brain the same as native endothelial cells.
As they explained, in the brain, endothelial cells and Wnt ligands secreted from the central nervous system (CNS) are important in forming the blood-brain barrier (BBB). The BBB, in turn, is pivotal in regulating drug transport to the brain to treat neurodegenerative diseases.
This new technique, notes Palecek, can be used “to identify factors involved in the specialization of naïve endothelial cells to tissue-specific endothelial cells. Then we will devise protocols to advance manufacturing of human in vitro BBB models to enable drug discovery and disease modeling.”
The post Cutting hPSC Production Time By 66% appeared first on GEN - Genetic Engineering and Biotechnology News.