title: Multifactorial Optimizations for Directing Endothelial Fate from Stem Cells.
abstract: Embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells are attractive in vitro models of vascular development, therapeutic angiogenesis, and tissue engineering. However, distinct ESC and iPS cell lines respond differentially to the same microenvironmental factors. Developing improved/optimized differentiation methodologies tailored/applicable in a number of distinct iPS and ESC lines remains a challenge in the field. Currently published methods for deriving endothelial cells (EC) robustly generate high numbers of endothlelial progenitor cells (EPC) within a week, but their maturation to definitive EC is much more difficult, taking up to 2 months and requiring additional purification. Therefore, we set out to examine combinations/levels of putative EC induction factors-utilizing our stage-specific chemically-defined derivation methodology in 4 ESC lines including: kinetics, cell seeding density, matrix signaling, as well as medium treatment with vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The results indicate that temporal development in both early and late stages is the most significant factor generating the desired cells. The generation of early Flk-1+/KDR+ vascular progenitor cells (VPC) from pluripotent ESC is directed predominantly by high cell seeding density and matrix signaling from fibronectin, while VEGF supplementation was NOT statistically significant in more than one cell line, especially with fibronectin matrix which sequesters autocrine VEGF production by the differentiating stem cells. Although some groups have shown that the GSK3-kinase inhibitor (CHIR) can facilitate EPC fate, it hindered the generation of KDR+ cells in our preoptimized medium formulations. The methods summarized here significantly increased the production of mature vascular endothelial (VE)-cadherin+ EC, with up to 93% and 57% purity from mouse and human ESC, respectively, before VE-cadherin+ EC purification.
authors.affiliation: School of Engineering, University of California, Merced, United States of America.|Graduate Program in Biological Engineering and Small-scale Technologies, University of California, Merced, United States of America.
authors.forename: Drew E
authors.initials: DE
authors.lastname: Glaser
authors.affiliation: School of Engineering, University of California, Merced, United States of America.
authors.forename: William S
authors.initials: WS
authors.lastname: Turner
authors.affiliation: Graduate Program in Quantitative and Systems Biology, University of California, Merced, United States of America.
authors.forename: Nicole
authors.initials: N
authors.lastname: Madfis
authors.affiliation: School of Engineering, University of California, Merced, United States of America.|Graduate Program in Biological Engineering and Small-scale Technologies, University of California, Merced, United States of America.
authors.forename: Lian
authors.initials: L
authors.lastname: Wong
authors.affiliation: Department of Physics, University of California, Merced, United States of America.|Department of Molecular and Cellular Biology, University of California, Merced, United States of America.
authors.forename: Jose
authors.initials: J
authors.lastname: Zamora
authors.affiliation: School of Engineering, University of California, Merced, United States of America.
authors.forename: Nicholas
authors.initials: N
authors.lastname: White
authors.affiliation: School of Engineering, University of California, Merced, United States of America.
authors.forename: Samuel
authors.initials: S
authors.lastname: Reyes
authors.affiliation: Department of Molecular and Cellular Biology, University of California, Merced, United States of America.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Burns
authors.affiliation: Department of Physics, University of California, Merced, United States of America.
authors.forename: Ajay
authors.initials: A
authors.lastname: Gopinathan
authors.affiliation: School of Engineering, University of California, Merced, United States of America.|Graduate Program in Biological Engineering and Small-scale Technologies, University of California, Merced, United States of America.
authors.forename: Kara E
authors.identifier: http://orcid.org/0000-0002-4674-3219
authors.initials: KE
authors.lastname: McCloskey
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D015820:Cadherins / Q000096:biosynthesis; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D060833:Cellular Microenvironment / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000008:administration & dosage; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects; D051379:Mice; D042461:Vascular Endothelial Growth Factor A / Q000008:administration & dosage; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D016222:Fibroblast Growth Factor 2; D042461:Vascular Endothelial Growth Factor A; D000099152:Cadherin 5