Effective isolation and culture of endothelial cells in embryoid body differentiated from human embryonic stem cells.
We describe the isolation of endothelial cells from the center region of the attached embryoid body (EB) by a two-step enzyme treatment. The isolated cells from the center and outgrowth region of the EB were characterized separately. As human embryonic stem (hES) cells differentiated in EB, they lost expression of the undifferentiated marker Oct-4, whereas expression levels of endothelial-specific markers were increased. Using RT-PCR, fluorescence-activated cell sorting (FACS) analysis, and immunofluorescence, we have shown that various endothelial cell markers, including platelet/endothelial cell adhesion molecule (PECAM), von Willebrand factor (vWF), Flk-1, and Tie2, were expressed on the attached EB. Compared with the outgrowth region of EB, the center region had a higher population of cells with these endothelial cell markers. Once isolated by the twostep enzyme treatment, cells from the center region continued to differentiate into endothelial cell lineage while expression level of endothelial cell markers in cells from the outgrowth region decreased in subcultures. This study has demonstrated that the isolation of EB by a two-step enzyme treatment is a useful technique to obtain endothelial cell marker-positive cells with high yield. Furthermore, a similar approach can be taken to identify the location and distribution of specific cell types in EB and thereby allow us to isolate and expand specific cell types.
Kim
Jumi
J
Stem Cell Research Laboratory, CHA Stem Cell Institute, Pochon CHA University and CHA Biotech Co., Ltd., Seoul 135-081, Korea.
Moon
Sung-Hwan
SH
Lee
Soo-Hong
SH
Lee
Dong-Ryul
DR
Koh
Gou-Young
GY
Chung
Hyung-Min
HM
D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D042783:Endothelial Cells / Q000166:cytology; D006801:Humans; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism
D015415:Biomarkers; D050814:Octamer Transcription Factor-3; D019408:Platelet Endothelial Cell Adhesion Molecule-1