Endothelial cells (EC) generated in vitro from stem cells are desirable for their potential in a variety of in vitro models and cell-based therapeutic approaches; however, EC can take on a number of functionally and phenotypically distinct specializations. Here, we show the generation of functionally distinct EC subpopulations, including (1) the pro-angiogenic migrating tip-like and proliferative stalk-like EC, and (2) the less migratory cobblestone-shaped phalanx-like EC. Both embryonic stem cell (ESC)-derived EC subpopulations are generated from outgrowths of Flk-1+ vascular progenitor cells with high levels of vascular endothelial growth factor treatment, while the phalanx-like ESC-derived EC (ESC-EC) are subsequently isolated by selecting for cobblestone shape. Compared with the ESC-derived angiogenic endothelial cells (named ESC-AEC) that contain only 14% Flt-1+ and 25% Tie-1+ cells, the selected phalanx-like ESC-EC express higher numbers of cells expressing the phalanx markers Flt-1+ and Tie-1+, 89% and 90%, respectively. The ESC-AEC also contain 35% CXCR4+ tip cells, higher expression levels of stalk marker Notch-1, and lower expression levels of Tie-2 compared with the phalanx-type ESC-EC that do not contain discernible numbers of CXCR4+ tip cells. Perhaps most notably, the ESC-AEC display increased cell migration, proliferation, and 3 times more vessel-like structures after 48 h on Matrigel compared with the phalanx-like ESC-EC. This work analyzes, for the first time, the presence of distinct EC subtypes (tip/stalk, and phalanx) generated in vitro from ESC, and shows that phalanx-like EC can be purified and maintained in culture separate from the tip/stalk-like containing EC.
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References
1.
AsaharaT, MuroharaT, SullivanA, SilverM, van der ZeeR, LiT, WitzenbichlerB, SchattemanG, IsnerJM. 1997. Isolation of putative progenitor endothelial cells for angiogenesis. Science, 275:964–967.
IngramDA, MeadLE, TanakaH, MeadeV, FenoglioA, MortellK, PollokK, FerkowiczMJ, GilleyD, YoderMC. 2004. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood, 104:2752–2760.
4.
LinY, WeisdorfDJ, SoloveyA, HebbelRP. 2000. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest, 105:71–77.
5.
PeichevM, NaiyerAJ, PereiraD, ZhuZ, LaneWJ, WilliamsM, OzMC, HicklinDJ, WitteLet al.2000. Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood, 95:952–958.
6.
Planat-BenardV, SilvestreJS, CousinB, AndréM, NibbelinkM, TamaratR, ClergueM, MannevilleC, Saillan-BarreauC, DuriezM. 2004. Plasticity of human adipose lineage cells toward endothelial cells physiological and therapeutic perspectives. Circulation, 109:656–663.
7.
LevenbergS, FerreiraLS, Chen-KonakL, KraehenbuehlTP, LangerR. 2010. Isolation, differentiation and characterization of vascular cells derived from human embryonic stem cells. Nat Protoc, 5:1115–1126.
8.
LevenbergS, GolubJS, AmitM, Itskovitz-EldorJ, LangerR. 2002. Endothelial cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A, 99:4391–4396.
9.
McCloskeyKE, LyonsI, RaoRR, SticeSL, NeremRM. 2003. Purified and proliferating endothelial cells derived and expanded in vitro from embryonic stem cells. Endothelium, 10:329–336.
10.
McCloskeyKE, SticeSL, NeremRM. 2006. In vitro derivation and expansion of endothelial cells from embryonic stem cells. Methods Mol Biol, 330:287–301.
11.
NishikawaSI, HirashimaM, NishikawaS, OgawaM. 2001. Cell biology of vascular endothelial cells. Ann N Y Acad Sci, 947:35–40; discussion 41.
12.
NishikawaSI, NishikawaS, HirashimaM, MatsuyoshiN, KodamaH. 1998. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development, 125:1747–1757.
13.
SoneM, ItohH, YamaharaK, YamashitaJK, Yurugi-KobayashiT, NonoguchiA, SuzukiY, ChaoTH, SawadaNet al.2007. Pathway for differentiation of human embryonic stem cells to vascular cell components and their potential for vascular regeneration. Arterioscler Thromb Vasc Biol, 27:2127–2134.
YamashitaJK. 2007. Differentiation of arterial, venous, and lymphatic endothelial cells from vascular progenitors. Trends Cardiovasc Med, 17:59–63.
16.
BlancasA, ShihAJ, LauerNE, McCloskeyKE. 2011. Endothelial cells from embryonic stem cells in a chemically defined medium. Stem Cells Dev, 20:2153–2161.
17.
KaneNM, MeloniM, SpencerHL, CraigMA, StrehlR, MilliganG, HouslayMD, MountfordJC, EmanueliC, BakerAH. 2010. Derivation of endothelial cells from human embryonic stem cells by directed differentiation: analysis of microRNA and angiogenesis in vitro and in vivo. Arterioscler Thromb Vasc Biol, 30:1389–1397.
De SmetF, SeguraI, De BockK, HohensinnerPJ, CarmelietP. 2009. Mechanisms of vessel branching. Arterioscler Thromb Vasc Biol, 29:639–649.
20.
HellstromM, PhngLK, HofmannJJ, WallgardE, CoultasL, LindblomP, AlvaJ, NilssonAK, KarlssonL, GaianoN. 2007. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature, 445:776–780.
21.
del ToroR, PrahstC, MathivetT, SiegfriedG, KaminkerJS, LarriveeB, BreantC, DuarteA, TakakuraN, FukamizuAet al.2010. Identification and functional analysis of endothelial tip cell-enriched genes. Blood, 116:4025–4033.
22.
StrasserGA, KaminkerJS, Tessier-LavigneM. 2010. Microarray analysis of retinal endothelial tip cells identifies CXCR4 as a mediator of tip cell morphology and branching. Blood, 115:5102.
23.
LarriveeB, FreitasC, SuchtingS, BrunetI, EichmannA. 2009. Guidance of vascular development: lessons from the nervous system. Circ Res, 104:428–441.
24.
GerhardtH, RuhrbergC, AbramssonA, FujisawaH, ShimaD, BetsholtzC. 2004. Neuropilin 1 is required for endothelial tip cell guidance in the developing central nervous system. Dev Dyn, 231:503–509.
25.
HuberAB, KolodkinAL, GintyDD, CloutierJF. 2003. Signaling at the growth cone: ligand-receptor complexes and the control of axon growth and guidance. Annu Rev Neurosci, 26:509–563.
26.
CarmelietP, De SmetF, LogesS, MazzoneM. 2009. Branching morphogenesis and antiangiogenesis candidates: tip cells lead the way. Nat Rev Clin Oncol, 6:315–326.
MazzoneM, DettoriD, Leite de OliveiraR, LogesS, SchmidtT, JonckxB, TianYM, LanahanAA, PollardP, Ruiz de AlmodovarC. 2009. Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization. Cell, 136:839–851.
29.
KrutzikPO, NolanGP. 2003. Intracellular phospho protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry Part A, 55:61–70.
30.
McCloskeyKE, GilroyME, NeremRM. 2005. Use of embryonic stem cell-derived endothelial cells as a cell source to generate vessel structures in vitro. Tissue Eng, 11:497–505.
NehlsV, SchuchardtE, DrenckhahnD. 1994. The effect of fibroblasts, vascular smooth muscle cells, and pericytes on sprout formation of endothelial cells in a fibrin gel angiogenesis system. Microvasc Res, 48:349–363.
33.
UngerRE, GhanaatiS, OrthC, SartorisA, BarbeckM, HalstenbergS, MottaA, MigliaresiC, KirkpatrickCJ. 2010. The rapid anastomosis between prevascularized networks on silk fibroin scaffolds generated in vitro with cocultures of human microvascular endothelial and osteoblast cells and the host vasculature. Biomaterials, 31:6959–6967.
34.
EvansIM, BrittonG, ZacharyIC. 2008. Vascular endothelial growth factor induces heat shock protein (HSP) 27 serine 82 phosphorylation and endothelial tubulogenesis via protein kinase D and independent of p38 kinase. Cell Signal, 20:1375–1384.
35.
PiotrowiczRS, HickeyE, LevinEG. 1998. Heat shock protein 27 kDa expression and phosphorylation regulates endothelial cell migration. FASEB J, 12:1481.
36.
ChangE, HeoKS, WooCH, LeeH, LeNT, ThomasTN, FujiwaraK, AbeJ. 2011. MK2 SUMOylation regulates actin filament remodeling and subsequent migration in endothelial cells by inhibiting MK2 kinase and HSP27 phosphorylation. Blood, 117:2527.
37.
DopplerH, StorzP, LiJ, CombMJ, TokerA. 2005. A phosphorylation state-specific antibody recognizes Hsp27, a novel substrate of protein kinase D. J Biol Chem, 280:15013.
38.
EvansIM, ZacharyIC. 2011. Protein kinase D in vascular biology and angiogenesis. IUBMB Life, 63:258–263.
39.
KruegerJ, LiuD, ScholzK, ZimmerA, ShiY, KleinC, SiekmannA, Schulte-MerkerS, CudmoreMet al.2011. Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo. Development, 138:2111–2120.
40.
GilleH, KowalskiJ, LiB, LeCouterJ, MoffatB, ZioncheckTF, PelletierN, FerraraN. 2001. Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants. J Biol Chem, 276:3222–3230.
41.
PoratRM, GrunewaldM, GlobermanA, ItinA, BarshteinG, AlhonenL, AlitaloK, KeshetE. 2004. Specific induction of tie1 promoter by disturbed flow in atherosclerosis-prone vascular niches and flow-obstructing pathologies. Circ Res, 94:394–401.
LibbyP. 2002. Inflammation in atherosclerosis. Nature, 420:868–874.
47.
O'BrienKD, McDonaldTO, ChaitA, AllenMD, AlpersCE. 1996. Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation, 93:672–682.
48.
MurakamiM, NguyenLT, ZhuangZW, MoodieKL, CarmelietP, StanRV, SimonsM. 2008. The FGF system has a key role in regulating vascular integrity. J Clin Invest, 118:3355–3366.
49.
DavidL, MalletC, KeramidasM, LamandeN, GascJM, Dupuis-GirodS, PlauchuH, FeigeJJ, BaillyS. 2008. Bone morphogenetic protein-9 is a circulating vascular quiescence factor. Circ Res, 102:914–922.
50.
BlumA, BalkanW, HareJM. 2012. Advances in cell-based therapy for peripheral vascular disease. Atherosclerosis, 223:269–277.
51.
TeebkenOE, HaverichA. 2002. Tissue engineering of small diameter vascular grafts. Eur J Vasc Endovasc Surg, 23:475–485.
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