Endothelial progenitor populations in differentiating embryonic stem cells. II. Drug selection and functional characterization.
Despite the enormous potential of embryonic stem cells (ESCs) for use in tissue engineering and cell therapies, clinical use of ESCs has been deterred by difficulties in obtaining desired cell populations without undifferentiated or other undesired cell phenotypes. Previously we demonstrated the time-dependent variation of endothelial specific promoter activities in differentiating mouse ESCs. Here, we evaluated the functional behavior of endothelial cells (ECs) selected using a drug resistance gene (Puro(R)) under the control of different EC promoters (Flk1, Tie1, and platelet endothelial cell adhesion molecule (PECAM), as well as vascular endothelial (VE)-cadherin). All four promoters tested yielded cells with EC-specific protein expression and acetylated low density lipoprotein, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL) uptake after puromycin treatment. However, the percentage of puromycin-surviving cells varied depending on EC promoters, indicating different levels of promoter activity. Continuous selection of ECs in puromycin reduced the presence of smooth muscle cells or undifferentiated cells. Puro-selected cells under all the EC promoters were positive for capillary-like network formation in Matrigel. Prostacyclin secretion by ESC-derived ECs showed improvement with orbital shear stress exposure corresponding to 0-16 dynes/cm(2); however, prostacyclin levels were lower than those seen in immortalized mouse EC lines. Tissue plasminogen activator (tPA) secretion by Puro-selected cells was comparable to that seen in cell lines, but tPA secretion after shear stress exposure showed mixed results, with tPA increasing upon shear under PECAM and Tie1 promoters and tPA decreasing upon shear under Flk1 and VE-cadherin promoters.
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA.
Saejeong
S
Kim
Horst A
HA
von Recum
D000818:Animals; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D011464:Epoprostenol / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006487:Hemostasis / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008858:Microscopy, Phase-Contrast; D018919:Neovascularization, Physiologic / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D011691:Puromycin / Q000494:pharmacology*; D015203:Reproducibility of Results; D010959:Tissue Plasminogen Activator / Q000378:metabolism
D049452:Green Fluorescent Proteins; D011691:Puromycin; D011464:Epoprostenol; D010959:Tissue Plasminogen Activator