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March 26, 2018

Co-Emergence of Specialized Endothelial Cells from Embryonic Stem Cells

Identification of Cell Subphenotypes Could Aid the Development of In Vitro Disease Models

Co-Emergence of Specialized Endothelial Cells from Embryonic Stem Cells

Figure. 1. Derivation of tip- and stalk-like EC and phalanx EC. Mouse ESCs were first induced into Flk-1+ VPCs on fibronectin matrix in medium supplemented with BMP-4 and VEGF. The purified Flk-1+ cells were then replated, again on fibronectin matrix in medium supplemented with bFGF and VEGF for EC specification. The Flk-1+ outgrowths from the Flk-1+ cells contained tip-, stalk-, and phalanx-like EC. These populations are then purified further with the cobblestone-shaped EC replated in a separate dish. These two EC subcultures maintain distinct subphenotypic expansion for ~10 passages. EC, endothelial cell; ESC, embryonic stem cell; VPC, vascular progenitor cell; VEGF, vascular endothelial growth factor.

  • Introduction

    The field of vascular biology has firmly rejected the antiquated belief that blood vessels are merely “plumbing” for the distribution of blood. We now know that endothelial cells (ECs) play a dynamic role in regulating immune cell responses1, leukocyte trafficking2, vascular tone3, blood coagulation and clotting4, vascular permeability5, tissue repair6,7, and tumor growth8. In addition to the range of EC functions, EC specialization has been observed aligning with the specific needs of the tissue in which they reside9–12, and these are potentially designated before blood vessel maturation13–15. For example, arterial ECs are largely quiescent ECs that exhibit antithrombotic activity and release vasoactive molecules that control vessel relaxation. Conversely, postcapillary venular ECs are the primary site of trafficking for white blood cells16,17. EC morphology of the smallest arterioles is longer and narrower compared with arterial ECs. The microvascular ECs are involved in initiation of inflammatory signals following injury and infection, as well as angiogenesis and vascular pruning16. These ECs also exhibit distinct functions correlating with their anatomical location13 and respond differentially to a variety of angiogenic stimuli18,19. The heterogeneity of EC genes expressed between tissue specific capillary beds reflects the importance of extracellular surface expression in function of EC subphenotypes12.

    Morphologically distinct vascular EC subphenotypes are also found within a sprouting blood vessel. Positioned at the leading edge of a sprouting vessel, “tip” ECs have been shown to upregulate delta-like ligand 4 (Dll4)20,21, CXCR422, Flt-420,23, Nrp120,24, and Unc5B20, exhibit more organized stress fibers with numerous probing filopodia, and readily migrate toward an angiogenic stimulus20. However, tip ECs do not proliferate significantly or form lumens20,25,26.

    The “stalk” ECs are found trailing behind the tip ECs forming the stalk of the sprouting vessel. Unlike tip cells, stalk cells exhibit greater cell proliferation, lumen formation, increased extracellular matrix (ECM) production, and shorter filopodia21. Moreover, Notch signaling from the tip cells dampens the vascular endothelial growth factor (VEGF)-induced expression of Dll4 on stalk cells20,21 allowing the tip cells to maintain their position at the leading edge of the sprouting vessel. It is thought that the downregulation of VEGFR2 (KDR/Flk-1) and Dll4 in the stalk cells also helps maintain balanced numbers of tip cells for more efficient sprouting and network formation21.

    There potentially exists a distinct nonsprouting, less proliferative, and less migratory EC subphenotype, named a “phalanx” EC20,27. These cells are recognized by their “cobblestone” morphology and high levels of soluble and membrane bound Flt-1 that mitigate VEGF signaling, as well as potentially increased extracellular VE-cadherin expression20,27. Although phalanx-type ECs are capable of responding to VEGF signaling, VEGF signaling in phalanx ECs acts as an apoptosis rescue from serum-deprived conditions, rather than as the migratory and proliferative responses seen in tip and stalk ECs27.

    The current dogma views the specification of an EC as a tip, stalk, or phalanx EC to be a stochastic process with ample plasticity and reversibility between these phenotypes during sprouting25. Our earlier studies challenged this dogma33. Using staged differentiation and chemically defined media to generate ECs from human and mouse ESCs28–32, we discovered the co-emergence of sprouting tip and stalk-containing ECs and nonsprouting phalanx-containing ECs within our two-dimensional derivations33. Specifically, the phalanx ECs were purified from the tip/stalk-containing cultures, and both cultures sequentially expanded for up to 9–10 passages using the identical cell culture medium formulations. The expanded tip/stalk ECs stably exhibited increased levels of cell migration, proliferation, and increased vasculogenic- and angiogenic-like sprouting on Matrigel™ compared with the phalanx ECs33. The tip/stalk ECs also exhibited extensive and complex actin networks and phosphorylation of HSP27—required to release the cap ends from actin filaments and allow the generation of new polymerization required for cell migration34,35. Conversely, the phalanx-like ECs contained greater numbers of cells expressing Flt-1, Tie-1, and Tie-233.

    This was the first report of stable distinct subphenotypes emerging together in vitro from stem cells33. Subsequently, a second group found that in vitro cultured sprouting ECs expressing low levels of CD143 exhibit enhanced angiogenic potential in alleviating local ischemia36. In this paper, we further examined these distinct phenotypes for unique surface markers, gene expression profiles, gel degradation/remodeling, and positional affinities during sprouting. The results show that the tip-specific EC is a distinct and relatively stable EC subphenotype, even in the absence of its morphological association within the sprouting vessel.

  • A well-formed and robust vasculature is critical to the health of most organ systems in the body. However, the endothelial cells (ECs) forming the vasculature can exhibit a number of distinct functional subphenotypes like arterial or venous ECs, as well as angiogenic tip and stalk ECs. In this study, we investigate the in vitro differentiation of EC subphenotypes from embryonic stem cells (ESCs). Using our staged induction methods and chemically defined mediums, highly angiogenic EC subpopulations, as well as less proliferative and less migratory EC subpopulations, are derived. Furthermore, the EC subphenotypes exhibit distinct surface markers, gene expression profiles, and positional affinities during sprouting. While both subpopulations contained greater than 80% VE-cad+/CD31+ cells, the tip/stalk-like EC contained predominantly Flt4+/Dll4+/CXCR4+/Flt-1 cells, while the phalanx-like EC was composed of higher numbers of Flt-1+ cells. These studies suggest that the tip-specific EC can be derived in vitro from stem cells as a distinct and relatively stable EC subphenotype without the benefit of its morphological positioning in the sprouting vessel.

  • Methods

    Generation of tip/stalk-like and phalanx-like EC

    The formulations and stage-specific derivation methodology (Figure 1) using chemically defined mediums were conducted as previously reported28,29,33. Briefly, R1 murine embryonic stem cells (mESCs) were maintained on 0.5% gelatin coated plates in serum-free medium containing Knockout Dulbecco's modified Eagle's medium (KO-DMEM; Invitrogen), 15% Knockout Serum Replacer (KSR; Invitrogen), 1× Penicillin-Streptomycin (Invitrogen), 1× nonessential amino acids (Invitrogen), 2 mM l-glutamine (Invitrogen), 0.1 mM 2-mercaptoethanol (Calbiochem), 2,000 U/mL of leukemia inhibitory factor (LIF-ESGRO; Chemicon), and 10 ng/mL of bone morphogenetic protein-4 (BMP-4; R&D Systems). The initial induction that was induced using a medium optimized by our laboratory was named “NS1D2b.” This consists of alpha-MEM (Cellgro), 20% KSR (Invitrogen), 1 × penicillin-streptomycin (Invitrogen), 1 × nonessential amino acids (NEAA; Invitrogen), 2 mM l-glutamine (Invitrogen), 0.05 mM 2-mercaptoethanol (Calbiochem), 30 ng/mL of VEGF (R&D Systems), and 5 ng/mL BMP-4 (R&D Systems). After 2 days, the Flk-1+ cells were stained with APC-conjugated anti-mouse CD309 (1:200, BioLegend) and viability fixative efluor760 (eBioscience) and enriched using Fluorescence Activated Cell Sorting (FACS, Aria II).

    The 10,000 Flk-1+ cells/cm2 were then seeded onto 50 μg/mL fibronectin-coated dishes in “LDSk” medium containing 70% alpha-MEM (Mediatech) and 30% DMEM (Invitrogen) plus 100 ng/mL VEGF (R&D Systems), 1% Nutridoma CS (Roche), 50 ng/mL bFGF (Sigma), 2 mM l-glutamine (Invitrogen), 1 × penicillin-streptomycin (Invitrogen), 1 × nonessential amino acids (Invitrogen), and 0.1 mM 2-mercaptoethanol (Calbiochem)28. After approximately 10 days, the cobblestone-shaped Flk-1+outgrowths were purified by manual selection (Figure 1). These ECs have been shown to be consistent with phalanx EC subphenotype33. Both the selected phalanx-like and remaining nonselected tip/stalk-containing EC were subsequently maintained in a medium composed of 50% LDSk and 50% serum-free EGM-2™ supplemented with the EGM-2 BulletKit™ containing hydrocortisone, bFGF, VEGF, IGF, ascorbic acid, hEGF, heparin, and GA-1000 (Lonza), mixture called “LDSF.”

    Control EC

    The isolation of primary mouse aortic endothelial cells (MAECs) from adult mice was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, Merced. Briefly, adult 129/Sv+c/+p mice (Jackson Laboratories) were anesthetized using isoflurane before cervical dislocation. The abdominal aorta was excised, stripped of the tunica adventitia, cut into small pieces, and sandwiched on Matrigel drops with 0.1–0.2 mL of EBM-2 media (with EGM-2 BulletKit Supplements; Lonza) with 50 ng/mL VEGF. MAEC was allowed to migrate out of the aortas for 7 days before aortas were removed to prevent smooth muscle cell migration. MAEC outgrowths were purified using a combination of manual selection from the aorta outgrowths and then purified by FACS for CD31/CD144 positive cells. A commercially available immortalized mouse cardiac endothelial cell (MCEC; CELLutions Biosystems) was also used, cultured in the same medium as described above.

    Staining for FACS analysis

    EC cultures were collected by incubating the cells with Cell Dissociation Buffer (Life Technologies) for 10 min and resuspended in PBS with 1% bovine serum albumin (BSA), mouse Fc Block (1:1,000; BD Biosciences), and fixable viability dye e780 (BD Biosciences). Live cells were stained for the following surface markers with corresponding IgG controls: anti-mouse CD31 PECy7 (1:200; BioLegend), anti-mouse Dll4 APC (1:400; BioLegend), anti-mouse Notch1 PE (1:200; eBioscience), anti-mouse VE-cad BV421 (1:400; BioLegend), anti-mouse Flt-4 Alexa Fluor® 488 (1:200; R&D), and anti-mouse Flt-1 Alexa Fluor 488 (1:100; RND systems). Titration optimizations were performed on all antibodies before analysis using fluorescence activated cell scanning (FACS; BD LSR II). Live cell populations were positively selected according to nonfluorescence and FSC/SSC dot plots. Data analysis and gating statistics were obtained using FlowJo software.

    Staining for fluorescence imaging

    Samples of purified cultures at passage 9 were fixed using 4% paraformaldehyde (PFA) for 15 min and permeabilized with 1% Triton (Sigma) for 5 min, then blocked in 5% donkey serum. Samples were incubated overnight in the following antibodies and compared against corresponding IgG controls: goat DDR2 (1:200; Santa Cruz), goat calponin-1 (1:200; Santa Cruz), and rabbit alpha-smooth muscle actin FITC (1:400; Santa Cruz). The secondary antibodies for anti-goat FITC (1:200; Santa Cruz) and anti-rabbit PE (1:200; Santa Cruz) were incubated for 1 h and counterstained with DAPI. Images were obtained using a Nikon Zeiss Microscope.

    Fibrin bead sprouting assay

    The tip/stalk-like EC was stained with 4 μm of CellTracker™ Green CMFDA Dye (Molecular Probes), while the phalanx-like EC was stained with 4 μm of CellTracker Red CMFDA Dye (Molecular Probes) for 45 min. EC cultures were filtered through a 70 μm nylon mesh filter and incubated with Cytodex® three microcarrier beads (200 cells per bead; Sigma) precoated with 50 μg/mL fibronectin. To facilitate attachment, the cell was placed on a nutator and incubated with microcarrier beads for 3 h. After confirming cell attachment, the cell-coated beads were resuspended in fresh media and incubated overnight. Fibrin gels were formed by resuspending cell-coated beads in 0.15 U/mL aprotinin and 2 mg/mL bovine plasma fibrinogen (Sigma). Bovine thrombin solution (0.625 U/mL; Sigma) was placed in the bottom of each well of a 24-well plate and the fibrinogen/bead-cell mixture was gently mixed into each well for 5 min, then allowed to polymerize at 37°C. After 15 min, 1 mL of ESC-EC maintenance media (LDSF) containing ∼20,000 normal lung human fibroblasts (Lonza) was added to the top of each gel. The media was replaced every other day, and images were taken (Nikon Zeiss Microscope) every 24 h for up to 7 days (or until the fibrin gel degraded). Sprouting was quantified (sprout number and average length) on 100–150 beads per experimental condition.


    Total RNAs were isolated with TRIzol reagent (Life Technologies) and RNAeasy Kit (Qiagen) according to the manufacturer's instructions. Ribo-Zero Gold rRNA Removal Kit (Illumina) was used to remove ribosomal RNA before preparation of sequencing libraries using the ScriptSeq RNA-Seq Library Prep Kit (Illumina). Sequencing was performed with Illumina HiSeq 4000 systems, and raw sequence reads were examined for quality using FastQC37. The reads were subsequently trimmed to remove adaptors and filtered for bad quality bases using Trim Galore38,39. Clean sequence reads were aligned to mouse genome, mm10, using STAR aligner40. Gene counts were called using HTSeq (5), and differentially expressed genes were identified using DESeq2 R package41. Gene ontology (GO) analysis was carried out using DAVID42,43 to identify enriched biological functional groups and processes.

    Cathepsin and MMP zymography

    Total protein concentration was determined from the tip/stalk- and phalanx-containing EC lysates. Equal amounts were loaded for cathepsin or matrix metalloprotease (MMP) zymography, as previously described44,45. Zymogram gels were imaged with an ImageQuant LAS 4000 (GE Healthcare), and densitometry was performed with ImageJ (NIH) to quantify cleared white bands, indicative of proteolytic activity.

    Statistical analysis

    Data are representative of at least three independent assays (N = 3). Student's unpaired t-test was used to establish significance.

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