Isolation and expansion of endothelial progenitor cells derived from mouse embryonic stem cells.
The unlimited differentiation and proliferation capacity of embryonic stem cells represents a great resource for regenerative medicine. Here, we describe a method for differentiating, isolating, and expanding endothelial cells (ECs) from mouse embryonic stem cells (mESCs). First, mESCs are expanded on a mouse embryonic fibroblast (mEF) feeder layer and partially differentiated into embryoid bodies (EBs) by growing the cells in an ultra-low attachment plate for up to 5 days. The EBs are then differentiated along the endothelial lineage using endothelial growth medium supplemented with 40 ng/mL vascular endothelial growth factor (VEGF). The differentiated endothelial population expresses both Fetal Liver Kinase 1 (Flk-1) and VE-Cadherin on the cell surface which can be further purified using a fluorescence-activated cell sorting (FACS) system and subsequently expanded on 0.1 % gelatin-coated plates. The differentiated cells can be analyzed by real-time PCR and flow cytometry to confirm enrichment of EC-specific genes and proteins.
Department of Surgery, University of California San Francisco, San Francisco, CA, USA.
S Bahram
SB
Bahrami
Mandana
M
Veiseh
Nancy J
NJ
Boudreau
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D058732:Embryoid Bodies / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D005786:Gene Expression Regulation; D051379:Mice; D008810:Mice, Inbred C57BL; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D000099152:Cadherin 5
D015703:Antigens, CD; D015415:Biomarkers; D015820:Cadherins; D040301:Vascular Endothelial Growth Factor Receptor-2; D000099152:Cadherin 5