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This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.
Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.
In the past three decades, target-based drug discovery (TDD) has been widely employed in drug discovery by the pharmaceutical industry. TDD incorporates a defined molecular target playing an important role in a disease and relies on the development of relatively simple cell culture systems and readouts for drug screening1. Most typical disease models used in TDD programs include traditional cell culture methods such as cancer cells or immortalized cell lines grown within artificial environments and non-physiological substrates. Although many of these models have provided viable tools for identifying successful drug candidates, the use of such s....
1. Media preparation and culture of mESC
The protocol overview of the blood vessel sprouting assay is shown in Figure 1. Nine-day-old EBs derived from three independent 129/Ola mESC lines (Z/Red, R1, and E14) were enzymatically dissociated into single cells using collagenase A. Cells were stained for PECAM-1 and analyzed by Fluorescence-activated cell sorting (FACS) as described. All the cell lines exhibited robust endothelial differentiation, and no differences in their ability to differentiate into endothelial cells were observed.......
This protocol describes an unbiased, robust, and reproducible 3D EB-based vascular sprouting assay that is amenable to screening for drugs and genes modulating angiogenesis. This method offers advantages over many widely used two dimensional (2D) assays using endothelial cell cultures such as Human Umbilical Vein Endothelial Cells (HUVECs) to monitor migration (lateral scratch assay or the Boyden chamber assay)22,23 or proliferation (counting cell number, de.......
This work was supported by grants from the Nederlandse organisatie voor gezondheidsonderzoek en zorginnovatie (ZonMW 446002501), Health Holland (LSHM19057-H040), Leading Fellows Programme Marie Skłodowska-Curie COFUND, and by the Association Maladie de Rendu-Osler (AMRO).
....Name | Company | Catalog Number | Comments |
2-mercaptoethanol | Milipore, Merck | 805740 | Biohazard: adequate safety instructions should be taken when handling |
Agar Noble | Difco, BD Pharmigen | 214220 | |
Alexa Fluo 555 goat anti rat IgG | Life technologies | A21434 | |
APC conjugated rat anti-mouse PECAM-1 antibody (clone MEC13.3) | BD Biosciences | 551262 | |
APC Rat IgG2a κ Isotype Control (Clone R35-95) | BD Biosciences | 553932 | |
Axiovert 25 inverted phase contrast tissue culture microscope | ZEISS | ||
Basic Fibroblast Growth Factor-2 (bFGF) | Peprotech | 450-33 | |
Benchtop Centrifuge, Allegra X-15R | Beckman Coulter | 392932 | |
Biosafety cabinet BioVanguard (Green Line) | Telstar | 133H401001 | |
Bovine Serum Albumin (BSA) | Sigma-Aldrich | A9418 | |
Cell counting chamber, Buerker, 0.100mm | Marienfeld | 640211 | |
Cell culture dishes 60 x 15mm | Corning | 353802 | |
Cell culture dishes, 35 x 10 mm | Corning | 353801 | |
Cell culture plates 12-well | Corning | 3512 | |
CFX96 Touch Real-Time PCR Detection System | Biorad | 1855196 | |
Chicken serum | Sigma-Aldrich | C5405 | |
CHIR-99021 (CT99021) HCl | Selleckchem | S2924 | |
Collagen I, High Concentration, Rat Tail, 100mg | Corning | 354249 | |
Collagenase A | Roche | 10103586001 | |
Confocal Laser Scanning Microscope, TCS SP5 | Leica | ||
Cover glasses, 24 × 50 mm | Vwr | 631-0146 | |
DAPT γ‑secretase inhibitor | Sigma Aldrich | D5942 | |
DC101 anti mouse VEGFR-2 Clone | BioXcell | BP0060 | |
DC101 isotype rat IgG1 | BioXcell | BP0290 | |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2438-5X | Biohazard: adequate safety instructions should be taken when handling |
DPBS (10x), no calcium, no magnesium | Gibco, Thermofisher scientific | 14200067 | |
EDTA 40 mM | Gibco, Thermofisher scientific | 15575-038 | |
Embryonic stem-cell Fetal Bovine Serum | Gibco, Thermofisher scientific | 16141-079 | Should be lot-tested for maximum ES cell viability and growth. Heat inactivate at 60°C and store at −20 °C for up to 1 year |
Eppendorf Microcentrifuge 5415R | Eppendorf AG | Z605212 | |
Erythropoietin, human (hEPO), 250 U (2.5 µg) (1 mL) | Roche | 11120166001 | |
ESGRO Recombinant Mouse LIF Protein (10⁷ units 1 mL) | Milipore, Merck | ESG1107 | |
Falcon tubes 15 mL | Greiner Bio-One | 188271 | |
Falcon tubes 50 mL | Greiner Bio-0ne | 227270 | |
Filter tip ,clear ,sterile F.Gilson, P-200 | Greiner Bio-One | 739288 | |
Filter tip ,clear ,sterile F.Gilson, P10 | Greiner Bio-One | 771288 | |
Filter tip ,clear ,sterile F.Gilson, P1000 | Greiner Bio-One | 740288 | |
FITC conjugated anti-α Smooth Muscle Actin (SMA) (clone 1A4) | Sigma Aldrich | F3777 | |
FITC conjugated rat anti-mouse CD45 (clone 30-F11) | Biolegend | 103107 | |
FITC Rat IgG2b, κ Isotype Ctrl Antibody (clone RTK4530) | Biolegend | 400605 | |
Fluorscent mounting media | DAKO | S3023 | |
Gascompress | Cutisoft | 45846 | |
Gauze Cutisoft 10 x 10 cm | Bsn Medical | 45844_00 | |
Gel blotting paper, Grade GB003 | Whatman | WHA10547922 | |
Gelatin solution, type B | Sigma-Aldrich | G1393-100 ml | |
Glasgow's MEM (GMEM) | Gibco, Thermofisher scientific | 21710082 | |
IHC Zinc Fixative | BD Pharmigen | 550523 | |
IncuSafe CO2 Incubator | PHCBi | MCO-170AICUV-PE | |
Interleukin-6, human (hIL-6) | Roche | 11138600001 | |
L-Glutamine 200 mM | Gibco, Thermofisher scientific | 25030-024 | |
MEM Non-Essential Amino Acids Solution (100x) | Gibco, Thermofisher scientific | 11140035 | |
Microscope slide box | Kartell Labware | 278 | |
Microscope slide, Starfrost | Knittel glass | VS113711FKB.0 | |
Mm_Cdh5_1_SG QuantiTect Primer Assay | Qiagen | QT00110467 | |
Mm_Eng_1_SG QuantiTect Primer Assay | Qiagen | QT00148981 | |
Mm_Epha4_1_SG QuantiTect Primer Assay | Qiagen | QT00093576 | |
Mm_Ephb2_1_SG QuantiTect Primer Assay | Qiagen | QT00154014 | |
Mm_Flt1_1_SG QuantiTect Primer Assay | Qiagen | QT00096292 | |
Mm_Flt4_1_SG QuantiTect Primer Assay | Qiagen | QT00099064 | |
Mm_Gapdh_3_SG QuantiTect Primer Assay | Qiagen | QT01658692 | |
Mm_Kdr_1_SG QuantiTect Primer Assay | Qiagen | QT00097020 | |
Mm_Notch1_1_SG QuantiTect Primer | Qiagen | QT00156982 | |
Mm_Nr2f2_1_SG QuantiTect Primer Assay | Qiagen | QT00153104 | |
Mm_Pecam1_1_SG QuantiTect Primer | Qiagen | QT01052044 | |
Mm_Tek_1_SG QuantiTect Primer Assay | Qiagen | QT00114576 | |
Mouse (ICR) Inactivated Embryonic Fibroblasts (2 M) | Gibco, Thermofisher scientific | A24903 | Store vials in liquid nitrogen (195.79 °C) indefinitely |
Mouse embryonic stem cell line 7AC5/EYFP (ATCC SCRC-1033) | ATCC | SCRC-1033 | Generated by Dr A Nagy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave, Toronto, Ontario, M5G 1X5, Canada. [Hadjantonakis, A. K., et al. Mechanisms of Development. 76 (1–2), 79–90 (1998)]. |
Mouse embryonic stem cell lines Acvrl1 +/- and Acvrl1 +/+ | Generated at Leiden University Medical Centre [Thalgott, J.H. et al. Circulation. 138 (23), 2698–2712 (2018)]. | ||
Mouse embryonic stem cells line E14 | Provided by M Letarte laboratory and generated according to Cho, S. K., et al. Blood. 98 (13), 3635–3642 (2001). | ||
Mouse embryonic stem cells line R1 (ATCC SCRC-1011) | ATCC | SCRC-1011 | Generated by Dr A Nagy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave, Toronto, Ontario, M5G 1X5, Canada. [Nagy, A., et al. Procedings of the National Academy of Sciences of the United States of America. 90 (18), 8424–8428 (1993)]. |
Mouse embryonic stem cells line Z/Red (strain 129/Ola) | Generated by Dr A Nagy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave, Toronto, Ontario, M5G 1X5, Canada [Vintersten, K., et al. Genesis. 40 (4), 241–246 (2004)]. | ||
NanoDrop 1000 UV/VIS Spectrophotometer | Thermo Fischer Scientific | ND-1000 | |
PD0325901 | Selleckchem | S1036 | |
PDGF-BB, Recombinant Human | Peprotech | 100-14B | |
Pecam-1 antibody, Rat Anti-Mouse | BD Biosciences | 550274 | |
Penicillin-streptomycin (10,000 U/mL) | Gibco, Thermofisher scientific | 15140122 | |
Petri dish, PS, 94/16 mm, standard ,with vents, sterile | Greiner Bio-One | 633181 | |
Pipetboy acu 2 | Integra-Biosciences | 155 019 | |
Pipetman G Multichannel P8 x 200G | Gilson | F144072 | |
Pipetman G Starter Kit, 4 Pipette Kit, P2G, P20G, P200G, P1000G | Gilson | F167360 | |
Recombinant Human BMP-4 Protein | R&D Systems | 314-BP | |
RNeasy Plus mini Kit | QIAGEN | 74134 | |
Serological pipettes, 10 mL | Greiner Bio-One | 607 180 | |
Serological pipettes, 25 mL | Greiner Bio-One | 760 180 | |
Serological pipettes, 5 mL | Greiner Bio-One | 606 180 | |
Sodium hydroxide (NaOH) | Merck | 106498 | |
Sodium pyruvate 100 mM | Gibco, Thermofisher scientific | 11360039 | |
Test tubes 5ml round-bottom with cell-strainer cap | Corning | 352235 | |
Thermal cycler, T100 | Biorad | 1861096 | |
Triton X-100 (BioXtra) | Sigma Aldrich | T9284 | |
Trypan Blue Solution, 0.4% | Gibco, Thermofisher scientific | 15250061 | |
Trypsin (2.5%) | Gibco, Thermofisher scientific | 15090046 | |
Vacuum Filter/Storage Bottle System, 500 mL | Corning | 430758 | |
VEGFA165 , recombinant murine | Peprotech | 450-32 | |
Water, Sterile | Fresenius-Kabi | B230531 | |
Waterbath, Lab-Line Digital | Thermo Fischer Scientific | 18052A |
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