US20150329822A1 - Differentiated cell population of endothelial cells derived from human pluripotent stem cells, composition, system, kit and uses thereof - Google Patents

Differentiated cell population of endothelial cells derived from human pluripotent stem cells, composition, system, kit and uses thereof Download PDF

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US20150329822A1
US20150329822A1 US14/651,548 US201314651548A US2015329822A1 US 20150329822 A1 US20150329822 A1 US 20150329822A1 US 201314651548 A US201314651548 A US 201314651548A US 2015329822 A1 US2015329822 A1 US 2015329822A1
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Helena Sofia ESMERALDO DE CAMPOS VAZÃO
Lino Da Silva Ferreira
Hugo Agostinho MACHADO FERNANDES
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Biocant Associacao de Transferencia de Tecnologia
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Definitions

  • the present disclosure relates to a differentiated cell population of endothelial cells derived from human pluripotent stem cells.
  • the present invention also relates to a composition, a system and a kit comprising those cells and uses thereof.
  • Vascular cells control the permeability of blood vessels, inflammation and immunity, cell growth, among other key functions, which have an important impact in the homeostasis of the human being.
  • vascular cells derived from human pluripotent stem cells represent a potential cell source for vascular kits 1, 2 .
  • the use of “embryonic” ECs may represent an opportunity to screen toxicity of compounds that affect embryonic vasculature.
  • ECs have been derived from human embryonic stem cells (hESCs) using several methodologies such as embryoid bodies (EBs) which recapitulates in vivo embryogenesis 3, 4 , a mixture of EBs with 2D or 3D culture systems 1, 5-7 and co-culture with cell lines 8-10 .
  • EBs embryoid bodies
  • hemodynamic forces found in arteries and veins can be a major driver in the specification and maturation of the ECs 11, 12 .
  • hemodynamic forces as shear stress have the capacity to program or redirect the specification of blood vessel type during development 11,12 .
  • vascular kits require the development of microfluidic platforms to screen multiple compounds in a high-throughput while the cells are exposed to shear stress forces typically found in vivo. Only recently, researchers have replicated the circular cross-section of blood vessels in microfluidic devices 14-16 . However, so far, these tools have not been used in the context of drug screening/toxicology assessment.
  • the present disclosure relates to a differentiated cell population of endothelial cells derived from human pluripotent stem cells wherein a portion of the said endothelial cells express ephrin B2. These differentiated cell population is particular useful in the screening embryonic vascular toxicity or therapeutic compounds.
  • the pluripotent stem cells used in the present disclosure are obtained without having to recur to a method necessarily involving the destruction of human embryos, namely with the use of iPS cells.
  • the portion of differentiated cell population of endothelial cells derived from human pluripotent stem cells has at least 20% of ephrin B2, preferably at least 25%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 100%, more preferably 50% to 75% of the cells express ephrin B2.
  • These cells may have the ability to form cord-like structures when cultured in the basement membrane Martrigel.
  • the cell population of endothelial cells may further express the following marker Ac-LDL.
  • the cell population of endothelial cells may further express one of the following markers: vWF, CD31, CD34, vascular endothelial cadherin, Flk-1/KDR.
  • the cell population of endothelial cells may has a high expression of one of the following arterial endothelial cell genes: jagged 1—JAG1, jagged 2—JAG2, ephrin B1, Hey-2.
  • the cell population of endothelial cells may has a high expression of one of the following arterial endothelial cell genes: receptor protein tyrosine phosphatase, T-cell acute lymphocyte leukemia, N-cadherin, angiopoietin 1, DNA-binding protein inhibitor ID-1.
  • the cell population of endothelial cells may has a low expression of venous genes such as EphB4, lefty-A and lefty-B.
  • Another aspect of the present disclosure is related with a differentiated cell population of endothelial cells derived from human pluripotent stem cells wherein a portion of the said endothelial cells express: receptor protein tyrosine phosphatase, T-cell acute lymphocyte leukemia, N-cadherin, angiopoietin 1, DNA-binding protein inhibitor ID-1.
  • receptor protein tyrosine phosphatase T-cell acute lymphocyte leukemia
  • N-cadherin angiopoietin 1
  • DNA-binding protein inhibitor ID-1 DNA-binding protein inhibitor
  • Another aspect of the present disclosure is differentiated cell population of endothelial cells derived from human pluripotent stem cells wherein a portion of the said endothelial cells express at least one of the following markers: vWF, CD31, CD34, vascular endothelial cadherin (VE-CAD), Flk-1/KDR for the use in the screening embryonic vascular toxicity or therapeutic compounds.
  • vWF vascular endothelial cadherin
  • Flk-1/KDR Flk-1/KDR
  • the portion of differentiated cell population of endothelial cells derived from human pluripotent stem cells has at least 20% of the said cells express at least one of the following markers: vWF, CD31, CD34, vascular endothelial cadherin (VE-CAD), Flk-1/KDR, preferably at least 25%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 100%.
  • VE-CAD vascular endothelial cadherin
  • Flk-1/KDR preferably at least 25%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 100%.
  • These cells may have the ability to form cord-like structures when cultured in the basement membrane Martrigel.
  • Another aspect is the use in medicine of the differentiated cell population of endothelial cells derived from human pluripotent stem cells. Namely, using the cell population in the screening embryonic vascular toxicity or therapeutic compounds.
  • composition comprising the cell population above described, namely a pharmaceutical composition.
  • kits for use in screening vascular toxicity or therapeutic compounds comprising the cell population above described.
  • a fluidic system for use in screening therapeutic drugs or embryonic vascular toxicity comprising a channel with a millimeter or micrometer dimension and a differentiated cell population of endothelial cells as described in the present disclosure.
  • the cells cultured under physiologic shear stress are cells seeded and exposed to the media flow, and may produce glycocalyx.
  • the media flow of the system may be above 1 dyne/cm2, preferably is above 4 dyne/cm2, preferably 20 dyne/cm2.
  • the channel may comprises poly(dimethylsiloxane).
  • system may further comprises plasma such as argon.
  • plasma such as argon.
  • system may further comprises gelatin, collagen, or fibronectin, or fibrin, or matrigel or mixtures thereof.
  • the media flow of the system is above 1 dyne/cm2, preferably is above 4 dyne/cm2, preferably 20 dyne/cm2.
  • the cells of the system above described may be the cell population above described.
  • a device for use in screening vascular toxicity or therapeutic compounds comprising: endothelial cells derived from human pluripotent stem cells as above descrided; and a fluidic system as above described.
  • the device could be use in screening embryonic vascular toxicity or therapeutic compounds, or in screening embryonic arterial endothelial cell toxicity or therapeutic compounds or in screening embryonic arterial endothelial cell toxicity or therapeutic compounds in conditions that mimic the in vivo conditions, or use in the screening of antitumor or anticancer drugs.
  • the compounds of the device may be selected from the following group: danazol, chlorpromazine hydrochloride, ellipticine, 3′,4′-dichlorobenzamil, fluphenazine dihydrochloride, 7-cyclopentyl-5-(4-phenoxyl)phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine, among others.
  • Another aspect is a method of promoting the viability of arterial endothelial cells comprising differentiation from human pluripotent stem cells with the following steps:
  • the reported methodology applies where the pluripotent stem cells are induced pluripotent cells that do not require the death of the embryo.
  • the human pluripotent stem cells were differentiated in conditions that Shh and Notch signaling is activated.
  • the human pluripotent stem cells were differentiated in the presence of DLL4 and purmorphamine preferably, 100 ng/mL of DLL4, and 1 ⁇ M of purmorphamine.
  • the predetermined conditions comprise a seeding density of 15,000 cells per cm2 in a gelatin-coated dish.
  • the method further comprising adding to the cell population of CD31+, SB431542 with a concentration higher than 1 ⁇ M, preferably 5 ⁇ M-10 ⁇ M.
  • the method further comprising adding to the cell population of CD31+ miRNAs by a transfection agent.
  • a transfection agent Preferably the said agent may be a nanoparticle.
  • the said endothelial cell medium for culturing CD31+ cells has at least one of the following growth factors: VEGF, PDGF, angiopoietin (Ang), ephrin (Eph), fibroblast growth factor (FGF), placental growth factor (PIGF), transforming growth factor ⁇ -1 [(TGF)- ⁇ -1], cytokines, erythropoietin, thrombopoietin, transferring, insulin, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF) and their mixtures.
  • SCF stem cell factor
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • the method further comprising co-culturing CD31+ cells with arterial endothelial cells.
  • the present disclosure also described the combination of arterial ECs derived from human pluripotent stem cells with a microfluidic system to create a vascular kit for high-throughput drug screening and/or toxicology analysis.
  • This technology may find particular use for the identification of drugs that may have a fetal cytotoxic effect.
  • FIG. 1 Specific of hPSC-derived ECs into arterial phenotype.
  • A EC marker expression and functionality of hESC-derived ECs. In all figures bar corresponds to 50 ⁇ m.
  • B Microarray analysis showing that hESC-derived ECs share many arterial genes as shown in hUAECs and hAECs. The list of genes correlates with the heat map.
  • G qRT-PCR analysis for embryonic endothelial markers in hESC-derived ECs at passage 4, hUAECs, mouse embryonic ECs at day 12.5 (mAEC E12.5) and postnatal day 1 (p1).
  • H Microarray analysis showing that hESC-derived ECs express embryonic genes not present in fetal hUAECs or adult hAECs. The list of genes correlates with the heat map.
  • II Variation of intracellular Ca 2+ in FURA-2-loaded cultured hESC-derived ECs, HUAECs or HUVECs in response to several agonists. Traces are representative of 6 independent experiments for each condition. In C, D, E and F, gene expression was normalized by the expression of GAPDH. In all figures, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • FIG. 2 Effect of fluidic shear stress on hPSC-derived ECs.
  • A Cell alignment and expression of endothelial (VECad) and arterial (EphB2) markers in hESC-derived ECs. Bar corresponds to 50 ⁇ m.
  • B Flow cytometry analysis of hESC-derived ECs cultured for 7 days in static and flow (20 dyne/cm2) conditions. Percentages of positive cells were calculated based in the isotype controls (grey plot) and are shown in each histogram plot. EphB2 expression on HUAECs cultured under static or flow conditions are also shown for reference.
  • FIG. 3 hPSC-derived ECs as a model to evaluate vascular toxicity.
  • A Expression and localization of VECad in hESC-derived cells and HUAECs cultured in medium supplemented with terbinafine. Cells were cultured under flow (20 dyne/cm2), in medium without terbinafine, for 7 days, after which the cells were cultured in medium supplemented with terbinafine (0.1 or 1 ⁇ M) for 24 h, under flow. Bar corresponds to 50 ⁇ m.
  • (B) Expression of genes involved in inflammation (ICAM-1; E-selectin), oxidative stress (HO-1), vascular modulation (eNOS) and vascular injury (DDAH1 and DDAH2; genes that encode for enzymes that degrade ADMA) in hESC-derived ECs and HUAECs cultured in medium supplemented with terbinafine. Results are mean ⁇ SEM (n 4). *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • C Quantification of ADMA and vWFpp:vWF by ELISA in hESC-derived ECs and HUAECs.
  • FIG. 4 High-throughput screening (HTS) to identify compounds with embryonic vascular toxicity.
  • A Schematic representation of the HTS assay.
  • B Small molecules identified after the analysis of the primary screen. The hits have higher cytotoxicity against hESC-derived ECs than HUAECs.
  • FIG. 5 Value of the hit 7-Cyclo in static and flow conditions.
  • A Macroscopic view of the PDMS microfluidic system (the microchannels have a diameter of 900 ⁇ m and an average length of 0.5 cm) and fluorescent images of microchannel cross-sections showing that ECs can populate the inner surface of the microfluidic channel after 48 h and be stable for at least 7 days at 20 dyne/cm2. Scale bars are 50 ⁇ m.
  • FIG. 6 Mechanism of 7-Cyclo.
  • B Microarray analysis showing the expression of tyrosine kinases in hESC-derived ECs, hUAECs and hAECs.
  • tyrosine kinases are higher expressed in hESC-derived ECs than in hUAECs or hAECs (zoom of the microarray).
  • FIG. 7 Induction of endothelial differentiation on hPSCs.
  • A Scheme showing the differentiation protocol.
  • B Expression of CD31 marker, as quantified by FACS, in hESCs differentiated for 18 days in media supplemented with VEGF165, T ⁇ 4 and SB431542 at different times, or only media without supplements. Values indicate mean ⁇ SEM from 3 independent experiments.
  • hESC-derived ECs were obtained from CD31+ cells isolated by MACS and differentiated for 3 passages (approximately 22 days after cell seeding). Gene expression was normalized as in C.
  • E Flow cytometry analysis of hESCderived ECs. Percentages of positive cells were calculated based in the isotype controls (grey plot) and are shown in each histogram plot.
  • FIG. 8 Induction of vascular differentiation on hESCs.
  • A Scheme summarizing the differentiation protocols (Prot1-Prot7).
  • B Expression of CD31 marker in hESCs differentiated for 18 days. Values indicate mean ⁇ SEM from 3 independent experiments.
  • FIG. 9 Charge of hESC-derived ECs, HUVECs and HUAECs.
  • A Time-course proliferation of hESC-derived ECs.
  • B hESC-derived ECs (Prot7, passage 3, approximately 22 days after CD31+ cell seeding) have cobblestone morphology and do not express ⁇ -SMA, a smooth muscle cell marker.
  • C Expression of arterial (EphB2) and venous (Lefty) markers in HUVECs and HUAECs. Bar corresponds to 50 ⁇ m.
  • FIG. 10 Charge of hIPS-derived ECs (hIPSC—Human Induced Pluripotent Stem Cell).
  • hIPSC Human Induced Pluripotent Stem Cell
  • CD31+ cells isolated by MACS were plated and differentiated for 4 passages (approximately 17 days after cell seeding) using the differentiation protocol previously used for hESCs.
  • A Flow cytometry analysis of hIPS-derived ECs. Percentages of positive cells were calculated based in the isotype controls (grey plot) and are shown in each histogram plot.
  • B EC marker expression and functionality of hESC-derived ECs. In all figures bar corresponds to 50 ⁇ m.
  • C qRT-PCR analysis for EC markers.
  • Vascular gene expression in each experimental group was normalized by the corresponding gene expression observed in HUVECs, with the exception of Oct-4, which was normalized by the corresponding gene expression in undifferentiated hIPS.
  • FIG. 11 Expression of arterial and venous genes during the inductive and spontaneous differentiation protocol.
  • A Scheme summarizing the inductive and spontaneous differentiation protocols.
  • D Effect of activation and inhibition of Shh and Notch signaling pathways in the expression of EphB2 in CD31+ cells.
  • Shh inhibitor is cyclopamine (5 ⁇ M) and Shh activator is purmorphamine (1 ⁇ M).
  • Notch inhibitor is L685458 (1 ⁇ M) and Notch activator is DLL4 (100 ng/mL).
  • FIG. 12 Example of arterial and venous EC markers in HUAECs cultured on static and flow conditions (7 days), as evaluated by immunocytochemistry (A) and qRT-PCR analysis (B).
  • A bar corresponds to 50 ⁇ m.
  • FIG. 13 Effect of physiological shear stress in the subphenotype of hESC-derived ECs.
  • A Cells cultured under static or flow conditions for 7 days do not express the venous EC marker Lefty. Bar corresponds to 50 ⁇ m.
  • B hESC-derived ECs and HUAECs cultured under static conditions do not express heparan sulfate proteoglycan (HSPG), while both cells express HSPG under flow conditions. Bar corresponds to 50 ⁇ m.
  • HSPG heparan sulfate proteoglycan
  • FIG. 14 Hait validation. After being selected from the primary screen, the identified compounds (hits) were subjected to a half-logaritimic dilutions in order confirm the positive hits and to assess the optimal concentration inducing cytotoxicity.
  • the present disclosure relates to differentiated cell population of endothelial cells derived from human pluripotent stem cells.
  • the said pluripotent stem cells used in the present invention are obtained without having to recur to a method necessarily involving the destruction of human embryos.
  • hPSCs human plurpotent stem cells
  • EphB2 the arterial ephrin receptor B2
  • the cells align in the direction of the flow and further up-regulate the expression of arterial genes.
  • the process is likely mediated by heparan sulfate proteoglycan (HSPG), a component of glycocalyx, which is activated by fluidic shear stress.
  • HSPG heparan sulfate proteoglycan
  • the utility of embryonic arterial ECs cultured under flow conditions for toxicological assessment was then demonstrated.
  • the higher sensitivity to cytotoxic compounds such as terbinafine of hESC-derived ECs cultured under physiologic shear stress than cells cultured in static conditions was shown.
  • the disclosed platform is a powerful platform for drug screening and to study embryonic vascular biology under physiologic conditions.
  • hPSCs can differentiate into arterial ECs.
  • the ECs were characterized at protein level by the expression of EphB2 and the absence of venous Lefty 1/2 and lymphatic podoplanin markers. At gene level, the cells express most of arterial markers shown by HUAECs and HAECs.
  • the arterial specification is mediated in part by Shh and Notch signaling pathways. Results show that the activation of both pathways is required to enhance arterial specification, as previously shown 47,48,12.
  • the activation of both Shh and Notch signaling pathways from the very beginning of the differentiation procedure (from day 0 up to day 18; before the isolation of CD31 + cells) increased significantly the percentage of cells (from 18% to 36%) already committed into an arterial sub-phenotype.
  • hPSCs into arterial ECs largely occurs after the isolation and differentiation of CD31 + cells.
  • the arterial ECs differentiated in this study have an “embryonic” phenotype.
  • a defined set of embryonic EC markers have not been identified so far, in the present disclosure was also identified receptor protein tyrosine phosphatase ⁇ (PTPRu) 24 , T-cell acute lymphocyte leukemia 1 (Tall) 25, 26 , and some cadherins 27, 28 , among others, as putative markers of embryonic ECs, based in gene microarray analyses on human ECs as well as gene expression results in mouse embryonic ECs.
  • PTPRu receptor protein tyrosine phosphatase ⁇
  • Tall T-cell acute lymphocyte leukemia 1
  • cadherins 27, 28 cadherins 27, 28
  • the present disclosure also show that fluidic shear stress enhanced the maturation of arterial ECs, and preferably in the maturation of arterial ECs and in the induction of HSPG (shown by the up regulation in the expression of ephrins (1 and 2), notch receptors (1 to 4), and notch ligands (Jagged1 and delta-like ligand 3) and the induction of HSPG.
  • HSPG shown by the up regulation in the expression of ephrins (1 and 2), notch receptors (1 to 4), and notch ligands (Jagged1 and delta-like ligand 3) and the induction of HSPG.
  • Previous studies have supported the idea that hemodynamic forces have the capacity to program or redirect the specification of blood vessel type during development 11,12 .
  • ECs not only have the ability to sense hemodynamic forces, but they have the ability to discriminate between different types of biomechanical stimuli.
  • HSPGs may mediate this maturation effect.
  • HSPG is a mechanosensor mediating shear stress-induced EC differentiation from mouse embryonic stem cell-derived ECs49.
  • HSPG is a part of the endothelial glycocalyx, which is only expressed in flow conditions and absent in static conditions 33 . It is conceivable that HSPGs are physically displaced when exposed to shear and the displacement transmitted to the intracellular machinery. It has been suggested that HSPGs are physically activated (direct or not) to actin and nitric oxide synthase mediating the mechanotransduction process 31 . These intracellular processes may contribute for the maturation of the cells under shear stress.
  • terbinafine an antifungal drug with anti-angiogenesis and anti-tumoral activity 34, 50 .
  • Terbinafine inhibits endothelial cell migration by inhibiting kinases in the Rho-kinase pathway 34 .
  • Results show that hESC-derived ECs cultured under flow respond to very low concentrations of terbinafine (0.1 ⁇ M). This was correlated with an up-regulation of oxidative-sensing and inflammatory genes, down-regulation of genes (DDAH1 and DDAH2) encoding enzymes that degrade an inhibitor (ADMA) of nitric oxide synthase, an increase in the secretion of ADMA and vWF pro-peptide, markers of EC injury.
  • ADMA oxidative-sensing and inflammatory genes
  • 7-Cyclo an embryonic arterial EC inhibitor, 7-Cyclo, by high-throughput screening, which was further validated by a dose-response study and cell culture under flow conditions.
  • 7-Cyclo is a Src family tyrosine kinase inhibitor.
  • 7-Cyclo (20 ⁇ M) has been reported to interfere with angiogenic sprouting and disrupt blood vessel formation in Xenopus embryos, although it was unclear whether such effect was related to the embryonic stage of the vasculature or if any vasculature could have the same consequences 51 .
  • 7-Cyclo (1 ⁇ M) inhibits HUVECs and lymphatic EC tube formation 51 , and it is an inhibitor of lymphangiogenesis 52 .
  • Results indicate that hESC-derived ECs exposed to medium supplemented with 7-Cyclo (1 ⁇ M) for 24 h under flow conditions show significant alterations in cell morphology, up-regulation of inflammatory genes, and secretion of vascular injury markers. This effect is higher on hESC-derived ECs than HUAECs.
  • Similar results were obtained for mouse embryonic ECs and post-natal ECs, i.e., mouse embryonic ECs were sensitive to the toxicity of 7-Cyclo while post-natal ECs show no measurable effect against the same compound. Therefore, the microfluidic system formed by hESC-derived arterial ECs is a sensitive platform for embryonic vascular toxicological assessment.
  • the inhibitory mechanism of 7-Cyclo against embryonic arterial cells involves the inhibition of tyrosine kinases highly expressed in embryonic ECs than in fetal or adult ECs.
  • Results show that embryonic ECs (both human or mouse) express higher levels of tyrosine kinase genes that are susceptible to the inhibitory effect of 7-Cyclo. This might explain the enhanced susceptibility of hESC-derived ECs to the effect of 7-Cyclo.
  • the platform described here is promising for the identification of compounds with embryonic toxicity as well as to study embryonic vascular biology under physiologic conditions.
  • Undifferentiated hESCs (passages 33-36; H9, WiCell, Wisconsin) or hiPSCs K2 (passages 32-35; cord blood derived iPSCs kindly donated by Ulrich Martin) were grown on an inactivated mouse embryonic fibroblast (MEF) feeder layer, as previously described 1, 2.
  • MEF mouse embryonic fibroblast
  • the undifferentiated hESCs were treated with 2 mg/mL type IV collagenase (Invitrogen) for 2 h and then transferred (2:1) to low attachment plates (Corning) containing 10 mL of differentiation medium[80% KO-DMEM, 20% fetal bovine serum (FBS, Invitrogen), 0.5% L-glutamine, 0.2% ⁇ -mercaptoethanol, 1% nonessential amino acids].
  • differentiation medium 80% KO-DMEM, 20% fetal bovine serum (FBS, Invitrogen), 0.5% L-glutamine, 0.2% ⁇ -mercaptoethanol, 1% nonessential amino acids.
  • the differentiation medium was supplemented with VEGF165 (50 ng/mL, Prepotech), T ⁇ 4 (100 ng/mL, Caslo) and SB431542 (10 ⁇ M, Tocris) according to the following timeline: [(VEGF165)days0-18+(T ⁇ 4)days4-18+(SB431542)days7-18].
  • VEGF165 50 ng/mL, Prepotech
  • T ⁇ 4 100 ng/mL, Caslo
  • SB431542 10 ⁇ M, Tocris
  • the differentiation medium was supplemented with one or two of the following agents: 1 ⁇ M purmorphamine (Shh activator; Cayman Chemical), 5 ⁇ M cyclopamine (Shh inhibitor; Sigma), 100 ng/ml DLL-4 (Notch activator; Prepotech), or 1 ⁇ M Y-secretase inhibitor L685458 (Notch inhibitor; Tocris Biosciences) 47.
  • CD31+ cells were isolated from differentiated hESCs at day 18 using MACS (Miltenyi Biotec). Isolated cells were grown on petri dishes (1.5 ⁇ 104 cells/cm2) coated with 0.1% gelatin and containing EGM-2 (Lonza) supplemented with SB431542 (10 ⁇ M). Cell characterization at gene, protein and functional levels can be found in Supplementary Information. HUVECS and HUAECs (both from Lonza) were used as controls for the differentiation studies. Cells were cultured in EGM-2 media or EGM-MV media (both from Lonza; until passage 5) and the medium changed every 2 days.
  • Dil-labeled acetylated low-density lipoprotein Dil-Ac-LDL
  • Dil-Ac-LDL Dil-labeled acetylated low-density lipoprotein
  • HUVEC, HUAEC or hESC-derived ECs were loaded with Fura-2 calcium fluorescent indicator by incubation with 5 ⁇ M of the membrane permeable acetoxymethyl (AM) derivative FURA-2/AM (1 mM in DMSO, Molecular Probes) and 0.06% (w/v) Pluronic F-127 (Sigma), using basal medium (M199, Sigma) as a vehicle (35 ⁇ L/well, not supplemented with serum nor antibiotics), for 1 h at 37° C. in 5% CO 2 and 90% humidity.
  • AM membrane permeable acetoxymethyl
  • FURA-2/AM FURA-2/AM
  • Pluronic F-127 Pluronic F-127
  • HESC-derived ECs (2.6 ⁇ 10 6 cells/mL), HUAECs, HUVECs were seeded on flow chamber untreated-slides (p-slide 0.4, luer, IBIDI) and grown until confluence.
  • IBIDI flow chamber untreated-slides
  • the cell monolayer was perfused with EGM-2 medium for 7 days, at flow rate of 15.1 mL/min (corresponding to shear stress of 20 dyne/cm2), or 3.02 mL/min (corresponding to shear stress of 4 dyne/cm2). After 7 days the flow was stopped, the cells imaged, and then stained or collected for posterior gene analysis. Medium was collected for ELISA assays.
  • cells were cultured in medium supplemented with terbinafine (0.1 ⁇ M or 1 ⁇ M) (Sigma) for a maximum of 24 h, under shear stress. Static controls were performed in the chamber coated-slides but without flow. The IBIDI slides allow a laminar perfusion in rectangular flow geometry.
  • the flow experiments were carried out in a microfluidic system developed by us.
  • the microfluidic device was obtained using a cylindrical mold of 20 Gauge.
  • Polydimethylsiloxane (PDMS) (Sylgard 184 Silicone elastomere base) was mixed at a 10:1 ratio (w/w) with curing agent (Sylgard 184 Silicon Elastomer Curing Agent) and the solution was poured onto a mold (20 gauge needle laid on a plastic dish) and cured at 80° C. for about 3 h.
  • PDMS Polydimethylsiloxane
  • curing agent Sylgard 184 Silicon Elastomer Curing Agent
  • the microchannel was cut to 0.5 cm length (900 ⁇ m inner diameter) and treated with Plasma Clean (Electronic Diener Femto Plasma Surface Technology version 5) for 2 min with argon gas (2 mBar) before immersing it in a solution of 0.1% gelatin.
  • Plasma Clean Electro Diener Femto Plasma Surface Technology version 5
  • HESC-derived ECs (20 ⁇ 10 6 cells/mL) or HUAECs were seeded on PDMS microchannels (3.18 ⁇ L cell suspension per channel) devices and grown until confluence.
  • IBIDI pump with positive pressure, the cell monolayer was perfused with EGM-2 medium for 7 days; shear stress of 20 dyne/cm2 (After 7 days the flow was stopped, the cells imaged, and then stained or collected for posterior gene analysis.
  • ELISA kits analyzed supernatants collected from the shear stress experiments for vWF and vWFpp (Gen-Probe GTI Diagnostic) and ADMA (Enzo Life Sciences), according to manufacturer's recommendations.
  • High-Throughput Screening (HTS).
  • HUAECS HUAECS
  • hESC-derived ECs were cultured in EGM-2 medium while human anterior cruciate ligament cells (ACL cells) were cultured in Dulbecco's modified Eagle's medium (DMEM; PAA) supplemented with 10% FBS, 0.2 mM ascorbic acid 2-phosphate magnesium salt (Sigma Aldrich), 100 ⁇ M/mL streptomycin and 100 U/mL penicillin (Life Technologies).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS 0.2 mM ascorbic acid 2-phosphate magnesium salt
  • streptomycin 100 ⁇ M/mL streptomycin and 100 U/mL penicillin (Life Technologies).
  • ACL cells were isolated from patients that have signed an informed consent form, in compliance with the Dutch legislation. The ethical committee of Medisch Spectrum Twente Hospital approved the collection.
  • the LOPAC library (Sigma-Aldrich) was used to screen embryonic endothelial-specific cytotoxic compounds. The compounds were solubilized in DMSO. The final compound concentration used in the screen was 4.5 ⁇ M in a final volume of 200 ⁇ L per well (96-well plate). HUAECs, hESC-derived EC and ACL cells were seeded at 16,000 cells/well (HUAECs and hESC-derived ECs) and 5,000 cells/well (ACL cells) and allowed to reach near confluence (approximately two days). After two days, medium was exchanged and test compounds and controls were added to the 96-well plates (all wells contained 0.25% (v/v) DMSO).
  • PrestoBlue Invitrogen
  • This assay is based on a resazurin-based solution that indicates the reducing power of living cells and therefore measures indirectly their number.
  • cell medium was removed and the PrestoBlue solution (10%) was added for 3 h at 37° C. upon which the absorbance was measured at 560-590 nm.
  • PrestoBlue solution 10%) was added for 3 h at 37° C. upon which the absorbance was measured at 560-590 nm.
  • a list of compounds were selected based on their higher cytotoxic to HUAECs (more than 50%) than ACL cells.
  • a list of compounds was selected based on their higher cytotoxicity to hESC-derived ECs (more than 20%) than HUAECs.
  • the hit compounds obtained from the primary screen were then re-evaluated at eight different concentrations in order to find a dose-response curve.
  • hESC-derived ECs and HUAECs were seeded at 16,000 cells/well (96 well plate), in EGM-2, and allowed to reach sub-confluency.
  • the compounds were serially diluted in DMSO in logarithmic steps, ranging from 0.01 ⁇ M to 100 ⁇ M and added to the cell culture medium (200 ⁇ L per well; EGM-2 medium). Untreated cells were used as control. Cell viability was assessed as described before for the LOPAC library using the PrestoBlue assay.
  • CD31 + cells were isolated by magnetic activated cell sorting (MACS) and cultured in EGM-2 medium supplemented with SB431542 (10 ⁇ M) ( FIG. 9A ).
  • MCS magnetic activated cell sorting
  • FIG. 9A Gene expression analysis in cells differentiated for 3 passages (between 18 and 22 days after cell seeding) indicate that they express CD34, VE-Cad and Flk-1/KDR at the same or higher level as the one found in HUVECs, albeit have lower expression of vWF and CD31 which may indicate different levels of maturation ( FIG. 7D ).
  • the differentiated cells had low expression of Oct-4 confirming their differentiated state.
  • FIG. 7E and FIG. 7A Flow cytometry and immunocytochemistry analyses show that CD31+ cells cultured for 3 passages expressed high levels of EC markers ( FIG. 7E and FIG. 7A ). Differentiated CD31 + cells stained positively for VE-Cad at cell-cell adherent junctions and produced vWF ( FIG. 1A ). In addition, they do not express markers of other mesoderm-derived cell lineages such as the smooth muscle cell marker ⁇ -SMA ( FIG. 9B ). Overall, the results indicate that CD31 + cells give rise to ECs.
  • hESC-derived ECs were determined, i.e., whether the ECs have been committed to arterial, venous or lymphatic lineages.
  • podoplanin podocyte membrane mucroprotein21
  • EphB2 a transmembrane ligand13
  • Lefty 1/222 as lymphatic, arterial and venous markers, respectively.
  • human umbilical arterial ECs (HUAECs) express EphB2 but do not express Lefty 1/2
  • HUVECs express Lefty 1/2 but do not express EphB2 ( FIG. 9C ).
  • HESC-derived ECs stain positive for arterial marker EphB2 but not for the venous marker Lefty 1/2 or the lymphatic marker podoplanin ( FIG. 1A ).
  • HESC-derived ECs express most of arterial markers shown by HUAECs or human aortic arterial endothelial cells (HAECs) such as JAG1, JAG2, EFNB1, EFNB2, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL3, DLL4, ALDHA1, HEY2, LIPG, CD44, KRT78, FSTI) 22 ( FIG. 1B and Table 1). These results were further confirmed by qRT-PCR.
  • hESC-derived ECs have high expression of arterial genes such as JAG1, EFNB1 and Hey-2 and low expression of venous genes such as EphB4, Lefty-1 and Lefty-2 ( FIG. 1C ).
  • HUAECs fetal phenotype
  • HAECs adult phenotype
  • the receptor protein tyrosine phosphatase ⁇ (PTPRu), the T-cell acute lymphocyte leukemia 1 (TAL1), N-cadherin (CDH2), angiopoietin 1 (ANGPT1) and DNA-binding protein inhibitor ID-1 (ID1), are up-regulated in mouse ECs E12.5 as compared to mouse ECs P1 ( FIG. 1G ). Similarly, those genes are up-regulated in hESC-derived ECs as compared to HUAECs.
  • the receptor protein tyrosine phosphatase ⁇ (PTPRu) has been reported as present in the aorta of mouse embryos, but not in the adult mice 24 .
  • T-cell acute lymphocyte leukemia 1 also known as ScI
  • ScI T-cell acute lymphocyte leukemia 1
  • major cadherins expressed in early embryos of Xenopus laevis are E-cadherin, N-cadherin and a maternal cadherin known as either C-cadherin or EP-cadherin 27,28 .
  • Microarray analyses showed also that the previous embryonic genes as well as others are higher expressed in hESC-derived ECs than in hUAECs or hAECs ( FIG. 1H and Table 2). Overall, results indicate that the hESC-derived ECs have an embryonic arterial phenotype.
  • FIG. 1A confirm that hESC-derived ECs are able to take up Dil-Ac-LDL and to form cord-like structures when cultured in the basement membrane Matrigel. Furthermore, the hESC-derived ECs respond to the vasoactive agonists as normal ECs by increasing the intracellular levels of Ca 2+ ( FIG. 11 ).
  • HESC-derived ECs share a similar response profile to thrombin as HUAECs but different response profiles to VEGF 165 , prostaglandin H2-analogue and histamine. No similarity was found in the response profiles of hESC-derived ECs and HUVECs.
  • hESC-derived ECs are functional however showing different response profiles to vasoactive agonists as compared to HUAECs and HUVECs.
  • the differences found between hESC-derived ECs and somatic arterial ECs (i.e. HUAECs) are likely ascribed to their embryonic and adult phenotypes.
  • VEGFR-2 tyrosine kinases extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), p38 mitogen-activated protein kinase and AKT serine/thronine kinases and transcription factors such as NF-kB.
  • ERKs extracellular signal-regulated kinases
  • JNKs c-Jun amino-terminal kinases
  • p38 mitogen-activated protein kinase and AKT serine/thronine kinases and transcription factors such as NF-kB.
  • NF-kB transcription factors
  • HESC-derived ECs cultured in arterial flow (20 dyne/cm 2 ) conditions align morphologically in the direction of the flow and show alignment of the proteins VE-Cad and actin (stained with phalloidin) ( FIG. 2A ). Similar results have been obtained for HUAECs ( FIG. 12 ).
  • the alignment of the cells is dependent on the magnitude of the shear stress since cells cultured in low (4 dyne/cm 2 ) or no flow (static conditions) show low or no alignment, respectively.
  • cells cultured in flow or static conditions express EphB2 but not lefty A/B ( FIG. 2A and FIG. 12 ).
  • EphB2 The expression of EphB2 is higher in flow than in static conditions, since 75% and 50% of the cells express EphB2 in flow and in static conditions, respectively ( FIG. 2B ).
  • the expression of CD31, VE-Cad and VEGFR-2 (KDR/FIK-1) is also up-regulated in flow conditions ( FIG. 2B ).
  • the up-regulation of these proteins is in agreement with previous data showing that their involvement on a mechanosensory complex that mediates the EC response to fluid shear stress 30 .
  • the maturation of hESC-derived ECs into arterial cells was also observed at gene level ( FIG. 2C ).
  • Cells cultured under arterial or venous flow conditions express higher levels of arterial genes such as Notch receptors (1, 2, 3 and 4), Notch ligands (DLL3 and Jagged-1), Notch transcription factor Hey-2, aldehyde dehydrogenase 1 (ALDH1A1), and ephrin-B1 (EFNB1) and ephrin-B2 (EFNB2), which are ligands of ephrin receptors.
  • Gene expression increased with an increase with flow.
  • An upregulation of venous genes such as EphB4 and Lefty was also observed when the cells were cultured under flow conditions; however, no lefty protein was observed under these conditions ( FIG. 2C and FIG. 12A ).
  • HUAECs cultured under static or flow conditions have the same gene expression profile ( FIG. 12B ), and therefore it suggests that hESC-derived ECs are more prone to respond to shear stress than mature somatic cells.
  • the results indicate that hESC-derived ECs respond to arterial flow by the alignment of VECAD and actin fibers, up-regulation of the mechanosensory complex VECad, CD31 and VEGFR2, and by the up-regulation of arterial marker EphB2.
  • Heparan sulfate proteoglycan a component of glycocalyx layer of ECs, has been reported to be a fluid stress sensor on ECs 31, 32 .
  • a class of HSPG, syndecans is known to associate with cytoskeletal elements including actin, either directly or through associated actin-binding proteins 31 .
  • HSPG is absent on ECs grown and maintained under standard cell culture conditions in vitro, i.e. without flow 33 . Therefore was investigated whether hESC-derived ECs express HSPG under flow culture conditions (20 dyne/cm 2 ).
  • Terbinafine is an antifungal agent (inhibitor of ergosterol synthesis) that inhibits angiogenesis by suppressing endothelial cell proliferation, inhibits DNA synthesis and activates EC apoptosis 34, 35 .
  • Terbinafine is cytotoxic for HUVECs for concentrations above 120 ⁇ M 36 .
  • terbinafine 0.1 and 1 ⁇ M
  • ADMA dimethylarginine-dimethyl-amino-hydrolases
  • ADMA ADMA
  • DDAH1 and DDAH2 are two isoforms of DDAH.
  • DDAH1 and DDAH2 have been found in mammals.
  • the decrease of DDAH1 and DDAH2 in hESC-derived ECs was only observed when cells were culture under flow conditions.
  • vWFpp von Willebrand factor pro-peptide
  • vWF von Willebrand factor
  • hESC-derived ECs or HUAECs cultured in static conditions in the presence of the drug have no significant higher secretion of ADMA or vWFpp:vWF than in control conditions (i.e., without the drug).
  • the sensitivity of HUAECs to terbinafine was lower than hESC-derived ECs.
  • the concentration of ADMA secreted by hESC-derived cells at 24 h is 3.5 times higher than in basal conditions. Importantly, this shift in concentration is observed in human patients with cardiovascular diseases.
  • microvessels of hESC-derived ECs, HUAECs and HAECs were formed on top of Matrigel and then exposed to the drug for 20 h. Results show that there is a statistically significant reduction in the network length and number of sprouts in microvessels formed by hESC-derived ECs after incubation with 1 ⁇ M of 7-Cyclo ( FIG. 15 ) while negligible effect was observed in microvessels formed by HUAECs and HAECs.
  • hESC-derived ECs were cultured in a poly(dimethylsiloxane) (PDMS) microfluidic system with cylindrical channels for 7 days at 20 dyne/cm2 ( FIG. 5A ). ECs were able to form a confluent monolayer in the entire inner surface of the channel after 48 h. At day 7, cells were exposed to EGM-2 medium supplemented with 1 ⁇ M of 7-Cyclo for 24 h, and finally analyzed at morphological, genetic and secretion levels. Results show that hESC-derived ECs show significant alterations in cell morphology in contrast to HUAECs cultured under the same conditions ( FIG. 5B ).
  • PDMS poly(dimethylsiloxane)
  • ICAM-1 ICAM-1
  • E-selectin E-selectin
  • HO-1 and eNOS HO-1 and eNOS
  • hESC-derived ECs or HUAECs cultured in static conditions in the presence of the drug have similar secretion of ADMA or vWFpp:vWF as in control conditions (i.e., without the drug).
  • results indicate that hESC-derived ECs are more sensitive to the effect of 7-Cyclo than HUAECs showing high levels of vascular dysfunction.
  • mouse embryonic ECs were incubated at day 12.5 (mAEC E12.5) and postnatal day 1 (p1) with 7-Cyclo (1 ⁇ M) for 24 h.
  • Inflammation, oxidative stress sensing, vascular modulation and vascular injury sensing genes are statistically up-regulated in mouse aortic endothelial cells (mAEC) at E12.5 as compared to cells without treatment ( FIG. 6A ).
  • mAEC mouse aortic endothelial cells
  • 7-Cyclo has no effect in p1 ECs.
  • the degree of action of 7-Cyclo in mAEC E12.5 is similar to the one identified in hESC-derived ECs ( FIG. 5C ).
  • 7-Cyclo is a cell-permeable pyrrolopyrimidine that acts as a potent inhibitor of tyrosine kinases such as SRC, KDR, TIE-2, BLK, FYN, LYN, CSK, EGFR, PKC, CDC2/B and ZAP-7045.
  • tyrosine kinases such as SRC, KDR, TIE-2, BLK, FYN, LYN, CSK, EGFR, PKC, CDC2/B and ZAP-7045.
  • the expression of the most significant genes was further characterized and confirmed by qRT-PCR ( FIG. 6C ).
  • the qRT-PCR included the analysis of mouse embryonic and postnatal ECs, to validate the results obtained for hESC-derived ECs. The same trend was observed, i.e, tyrosine kinases were found to be more expressed in embryonic ECs (both in human and mouse) than in fetal/adult tissues (exception for EGFR in mouse).
  • Undifferentiated hESCs (passages 33-36; H9, WiCell, Wisconsin) or hiPSCs K2 (passages 32-35; cord blood derived iPSCs kindly donated by Ulrich Martin) were grown on an inactivated mouse embryonic fibroblast (MEF) feeder layer, as previously described 1, 2.
  • MEF mouse embryonic fibroblast
  • the undifferentiated hESCs were treated with 2 mg/mL type IV collagenase (Invitrogen) for 2 h and then transferred (2:1) to low attachment plates (Corning) containing 10 mL of differentiation medium[80% KO-DMEM, 20% fetal bovine serum (FBS, Invitrogen), 0.5% L-glutamine, 0.2% ⁇ -mercaptoethanol, 1% nonessential amino acids].
  • the differentiation medium was supplemented with VEGF165 (50 ng/mL, Prepotech), T ⁇ 4 (100 ng/mL, Caslo) and SB431542 (10 ⁇ M, Tocris) according to different timelines (see below).
  • HUAEC human umbilical arterial endothelial cells
  • HUVEC human umbilical venous endothelial cells
  • Lonza http://www.lonza.com/
  • Total RNA of human Aortic Endothelial cells, mouse aortic endothelial cells (E 12.5 and p1) were bought from ScienceCell (http://www.sciencellonline.com/).
  • Cells were tripsinized, aliquoted (1.25-2.5 ⁇ 10 5 cells per condition), washed in PBS, centrifuged at 1200 g and then resuspended in PBS containing 5% FBS.
  • Cells were labeled with human CD31 monoclonal antibody (eBioscience, clone: WM59; 1.25:100), CD34 (Miltenyi Biotec, clone: AC136; 5:100), vwF (Dako; clone F8/86; 1:50), Flk-1/KDR (R&D, clone: 89106; 5:100), VeCad (R&D, clone: 123413; 5:100) or EphB2 (R&D, clone: 512012; 5:100).
  • Cells were characterized on a FACS Calibur (BD) and the data analyzed by Cell Quest software. Twenty thousand events were collected in each run.
  • RNA from experimental groups was isolated using a protocol with TRIzol (Invitrogen) and RNeasy Minikit (Qiagen, Valencia).
  • a 24-well plate was coated with Matrigel (0.4 mL, BD Biosciences) per well and incubated at 37° C. for 30 min. Cells were seeded on top of the polymerized Matrigel at a concentration of 1 ⁇ 10 5 cells per 300 ⁇ L of EGM-2 medium. After 1 h of incubation at 37° C., an extra 1 mL of EGM-2 was added. After 12 h, medium supplemented with 7-Cyclo (0, 0.1 or 1 ⁇ M) was added. Cord formation was evaluated by phase contrast microscopy (Carl Zeiss International, Germany), at time 0, 3 h and 20 h after 7-Cyclo addition.
  • phase contrast microscopy Carl Zeiss International, Germany
  • HUVECs, HUAECs or hESC-derived ECs were loaded with Fura-2 calcium fluorescent indicator by incubation with 5 ⁇ M of the membrane permeable acetoxymethyl (AM) derivative FURA-2/AM (1 mM in DMSO, Molecular Probes, http://www.invitrogen.com) and 0.06% (w/v) Pluronic F-127 (Sigma, http://www.sigmaaldrich.com), using basal medium (M199, Sigma) as vehicle (35 ⁇ l/well, not supplemented with serum nor antibiotics), for 1 h at 37° C. in 5% CO 2 and 90% humidity.
  • AM membrane permeable acetoxymethyl
  • the medium was then replaced by the respective basal medium and cells were incubated in the same conditions for 30 min to allow hydrolysis of the acetoxymethyl (AM) esters by cellular esterases, resulting in intracellular capture of the membrane impermeant Fura-2. Afterwards cells were washed twice with 100 ⁇ L sodium salt solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Glucose, 10 mM HEPES-Na + pH 7.4). The buffer was replaced again (100 ⁇ l/well) immediately prior to incubating or not with test compounds.
  • sodium salt solution 140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Glucose, 10 mM HEPES-Na + pH 7.4
  • Cells located in wells on a plate row were incubated at 25° C. (inside the microplate reader, during basal reading). Cells were then stimulated with 100 ⁇ M Histamine 53,54 (Sigma), 100 ng VEGF 165 (Prepotech, www.peprotech.com) 55,56 , 10 ⁇ M Prostaglandin U46619 (Cayman, http://www.caymanchem.com) 57,58 , 50 mM KCl 53,54 (Merck, http://www.merck.com) or 2U Thrombin (Sigma) 59 by adding 1 ⁇ l of a stock solution.
  • REU relative fluorescence units
  • each experiment consisted of three or four wells plus three or four wells containing cells incubated with inhibitors and all cells were stimulated and read simultaneously.
  • the dose-response curves for the effect of stimuli on the intracellular Ca 2+ variation were determined using the software GraphPad PrismTM.
  • the kinase activity of cells was measured by a Kinase-Glo® Luminescence Kinase Assay (Promega). Cells incubated with 7-Cyclo for 24 h were treated with the kinase reagent from the kit and luminescence acquired using a Spectra Max Gemini luminometer. The luminescence signal is correlated with the amount of ATP present and its inversely correlated with the amount of kinase activity.
  • hESC-derived EC, HUAEC or AEC adult aortic endothelial cells
  • Trizol reagent Invitrogen
  • total RNA was extracted by using the RNeasy Mini Kit (Qiagen, Valencia, USA), according to manufacturer's instructions.
  • the quality of the RNA was assessed in the Agilent 2100 Bioanalyser (G2943CA) using the RNA 6000 Pico Kit (5067-1513).
  • Agilent 2100 Bioanalyser G2943CA
  • RNA 6000 Pico Kit 5067-1513
  • Labeled cRNA was hybridized to the Whole Human Genome (4 ⁇ 44K) Microarray (G4112F from Agilent Technologies). From each sample, 1.65 ⁇ g cyanine 3-labeled cRNA was adjusted to 41.8 ⁇ l with DNAse-free Water, mixed with 11 ⁇ l Agilent 10 ⁇ blocking Agent and 2.2 ⁇ l Fragmentation Buffer and incubated at 60° C. for exactly 30 minutes to fragment RNA. To stop de fragmentation reaction 55 ⁇ l of 2 ⁇ GEx Hybridization Buffer was added.
  • the labelled cRNA mixture was applied to a microarray slide, assembled in a SureHyb Hybridization Chamber fitted with a gasket slide (Agilent), and incubated for 18H at 65° C. in a hybridization oven (G2545A, SHEL LAB—Agilent), with 10 rpm rotation speed. Slides were washed as described in the Agilent One-Color Microarray-Based Gene Expression Analysis protocol.
  • the microarray and gasket slide were briefly disassembled inside a staining dish containing 250 ml of GE Wash Buffer 1 and the slides (up to 4 slides) were washed in fresh 250 ml of GE Wash Buffer 1 solution at room temperature, during 1 minute, with gentle agitation from a magnetic stirrer.
  • a second wash step was carried out by immersing the slides in GE Wash Buffer 2 solution, previously warmed to 37° C., during 1 minute, also with gentle magnetic stirring. Finally, slides were dried by centrifugation at 800 rpm for 3 minutes.
  • Microarrays were scanned in the Agilent B Scanner (G2565BA) using specific scanning protocols for gene expression microarrays and the format 4 ⁇ 44K.
  • Agilent Feature Extraction Image Analysis Software (Version 10.7.3.1) was used to obtain fluorescence intensities from raw microarray image files.
  • BRB-ArrayTools v3.4.0 developed by Dr. Richard Simon and BRB-ArrayTools Development Team 54 .
  • BRB-Array Tools incorporates the Bioconductor R functions and the R programming language required for raw data normalization within arrays 55 .
  • Each gene's measured intensity was median normalized to correct for differences in the labelling efficiency between samples. This analyzes provided a median normalized dataset that was subjected to statistical analysis and clustering using MeV software 56 .
  • the previous step provided a differentially expressed genes (DEGs) list for each strain that was used to calculate the M-value and Fold-change variation. It was considered as differentially expressed a variation equal or higher than 2 ⁇ between conditions. Only genes with significance level below an alpha corrected p-value of 10 ⁇ 3 were considered as differentially expressed.
  • the down- and up-regulated genes were analyzed using DAVID 6.7 (Database for Annotation, Visualization and Integrated Discovery, http://david.abcc.ncifcrf.gov/) web-accessible program to identify the altered cellular processes and functions.
  • the microarray data has been deposited in NCBI's Gene Expression Omnibus database and is accessible through the GEO series accession number GSE51642.
  • FIG. 1-H Probe NM pubmed Symbol Complete name A_23_P63371 NM_003189 TAL1 T-cell acute lymphocytic leukemia 1 A_23_P149064 NM_005704 PTPRU protein tyrosine phosphatase, receptor type, U A_23_P137381 NM_002167 ID3 inhibitor of DNA binding 3, dominant negative helix- loop-helix protein A_23_P252306 NM_002165 ID1 inhibitor of DNA binding 1, dominant negative helix- loop-helix protein A_23_P216023 NM_001146 ANGPT1 angiopoietin 1 A_23_P60079 NM_001147 ANGPT2 angiopoietin 2 A_23_P132378 NM_014246 CELSR1 cadherin, EGF LAG seven- pass G-type receptor 1 (flamingo homolog, Drosophila ) A_23_P1446
  • FIG. 6-B Probe NM pubmed Symbol Complete name A_23_P10559 NM_001080395 AATK apoptosis-associated tyrosine kinase A_23_P60180 NM_005157 ABL1 c-abl oncogene 1, non-receptor tyrosine kinase A_23_P208389 NM_021913 AXL AXL receptor tyrosine kinase A_23_P253602 NM_001721 BMX BMX non-receptor tyrosine kinase A_23_P152024 NM_004383 CSK c-src tyrosine kinase A_23_P400716 NM_001010846 SHE Src homology 2 domain containing E A_23_P93311 NM_013993 DDR1 discoidin domain receptor tyrosine kinase

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110159522A1 (en) * 2008-04-08 2011-06-30 Kamm Roger D Three-Dimensional Microfluidic Platforms and Methods of Use Thereof
WO2011090684A2 (en) * 2009-12-29 2011-07-28 Cornell University Methods for developing endothelial cells from pluripotent cells and endothelial cells derived

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CN101680018B (zh) * 2007-01-10 2017-03-15 海莫希尔有限责任公司 体外血液动力学的内皮/平滑肌细胞共培养模型在鉴定血管疾病的新型治疗靶标中的应用
US20120211373A1 (en) * 2011-02-22 2012-08-23 The Regents Of The University Of Michigan Microfluidic system for measuring cell barrier function
EP2782866A4 (en) * 2011-11-23 2015-08-05 Univ Toronto DEVICES AND METHODS FOR PRODUCING FLAT POLYMERIC MATERIALS USING MICROFLUIDIC DEVICES

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110159522A1 (en) * 2008-04-08 2011-06-30 Kamm Roger D Three-Dimensional Microfluidic Platforms and Methods of Use Thereof
WO2011090684A2 (en) * 2009-12-29 2011-07-28 Cornell University Methods for developing endothelial cells from pluripotent cells and endothelial cells derived

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Endothelial Cell Markers," from Millipore, pages 1-5, accessed from https://www.emdmillipore.com/US/en/product/Endothelial-Cell-Markers,MM_NF-C133595, August 4, 2016. *
Bochenek et al., J. of Cell Science, 123: 1235-1246, 2010. *
Pahakis et al., Biochem. Biophys. Res. Commun., 355(1): 228-233, 2007. *

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