Ontology engineering

lettersα-CD31e10 510 410 3ahVPrGFPhVPrGFP1.31%(+/− 0.3)VEGFR214.6%(+/− 2.6)fbα-VEGFR2hVPrGFPhVPrGFP0.56%(+/− 0.2)VEGFR26.2%(+/− 1.4)10 2 −140 0 10 2 10 3 10 4 10 5 −170 0 10 2 10 3 10 4 10 5Relative Id1 expressionc20015010050gScrId1ScrId1d75k50k25khScrTotal cellsCD31 + cellsHUVEC hVprGFP −SB +SBd14+8 (+SB) VE-cadherin Nuc hVPr-GFP hVPr-GFP GIB4−Cell numberId1ScrId1i© 2010 Nature America, Inc. All rights reserved.Figure 4 TGFβ inhibition upregulates Id1 expression and is necessary for the increased yield of functional endothelial cells capable of in vivo neoangiogenesis.(a,b) Human VPr-GFP hESCs that were stably transduced with control (a) or Id1-specific (b) shRNAs were differentiated according to theprotocol shown in Figure 1d and assessed at day 14 for the prevalence of VEGFR2 + (blue) and hVPr-GFP + (green) cells. The insets show plots of sidescatter on the y axis and hVPr-GFP on the x axis. (c) Control and Id1-specific shRNAs were added to HUVEC or freshly isolated (at day 14) hVPr-GFP +cells, and the relative Id1 transcript levels were measured after 3 d. *, P < 0.05. Error bars, s.d. of experimental values performed in triplicate.(d) Control and Id1-specific shRNAs were added to freshly isolated hVPr-GFP + cells, which were cultured in the absence or presence of SB431542.After 5 d, the total cell number and proportion of CD31 + cells was measured by flow cytometry. Error bars, s.d. of experimental values performed intriplicate. Scr, scrambled control shRNA. (e–g) Human VPr-GFP + cells were isolated by FACS at day 14 and expanded in monolayer culture (e) for8 d while retaining expression of both the endogenous VE-cadherin (f) and the hVPr-GFP transgene (g). Panel e shows a mosaic view of one well ofa 24-well dish. A magnified view of the boxes in e and f are shown in f and g, respectively. (h,i) Expanded cells were injected in Matrigel plugs intoimmunodeficient mice and excised after 10 d following intravital labeling of functional vasculature with lectin (GIB4, blue). h, View of hVPr-GFP + cellsalone; i, view of hVPr-GFP + cells merged with GIB4 + cells. Scale bars, 100 µM.a stable BAC transgenic hESC line 20 containing yellow fluorescent proteindriven by the Id1 promoter (Id1-YFP) (Fig. 3b–f) (Nam, H.S. and Benezra,R., unpublished data). Differentiated endothelial cells were isolated at day14 from Id1-YFP cultures (Fig. 1d), sub-fractionating the CD31 + populationinto Id1-YFP high-expressing (Fig. 3c) and low-expressing (Fig. 3d)cells, and these populations were serially expanded for 7 d with or withoutthe TGFβ inhibitor (Fig. 3e,f). Flow cytometric analysis of these cellsrevealed a direct relationship between upregulation of Id1 expression andTGFβ inhibition. Notably, although SB431542 increased the percentage ofthe CD31 + population, the mean fluorescence intensity of CD31 on thesecells was lower than that of unstimulated cells. These data suggested thatTGFβ inhibition increased expansion of hESC-derived endothelial cellsby maintaining high levels of Id1 expression and preserving an immatureproliferative phenotype.To determine the requirement for Id1 in mediating endothelial cellcommitment, we transduced hVPr-GFP + cells with lentiviral short hairpin(sh)RNA targeted against the Id1 transcript (Fig. 4a,b). In the presence ofSB431542, knockdown of Id1 reduced the numbers of both VEGFR2 + vascularprogenitors and hVPr-GFP + cells at day 14. When the Id1 shRNA constructwas introduced after isolation of the hVPr-GFP + fraction (Fig. 4c),it elicited a marked decrease in CD31 + endothelial cells after 5 d ofSB431542 treatment (Fig. 4d). These results identified TGFβ inhibition–mediated Id1 upregulation as a primary effector in promoting endothelialcell expansion and maintaining long-term vascular identity.To demonstrate that our cultured endothelial cells could form functionalvessels, we grew purified hVPr-GFP + cells from day 14 differentiationcultures for an additional 8 d in the presence of SB431542. Theseendothelial cells showed high proliferative potential (up to ten cell divisions)and generated homogenous hVPr-GFP + VE-cadherin + monolayers(Fig. 4e–g) with retention of hVPr-GFP fluorescence at the single-celllevel (arrowheads in Fig. 4g). These cells were subcutaneously injected inMatrigel plugs into nonobese (NOD)/severe combined immunodeficient(SCID) mice and 10 d later extracted after intravenous injection of lectininto live animals. In Matrigel plugs, hVPr-GFP + cells co-localized withlectin + cells, forming chimeric vessels along with host cells (Fig. 4h–i andSupplementary Videos 8 and 9). These data indicated that the endothelialcells generated by our methods could function in vivo.A prerequisite to therapeutic vascularization using hESC-derived cellsis generation of abundant durable endothelial cells that upon expansionmaintain their angiogenic profile without differentiating into nonendothelialcell types. Here, we show that differentiation of hESCs into alarge number of stable and proliferative endothelial cells can be achievedby early-stage TGFβ-mediated mesoderm induction followed by TGFβinhibition beginning at day 7 (phase 1) and after isolation at day 14 (phase2). Using this approach, we achieved a 36-fold net expansion of committedendothelial cells. The increased yield allowed transcriptional analysis,which revealed a molecular signature that sheds light on the regulatoryinfluences that govern embryonic vasculogenesis. Indeed, genes encodingnature biotechnology volume 28 number 2 february 2010 165