In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Media preparation and culture of mESC

1. Prepare conditioned medium +/- (CM+/-) using the supplement 1x Glasgow MEM (G-MEM BHK-21) medium with 10% (vol/vol) heat-inactivated Fetal Bovine Serum (FBS), 0.05 mM β-mercaptoethanol, 1x non-essential amino acids (NEAA 1x), 2 mM L-glutamine, and 1 mM sodium pyruvate.
2. Prepare conditioned medium +/+ (CM+/+) using the supplement CM+/- medium with Leukemia Inhibitory Factor (LIF) (1,500 U/mL) and 0.1 mM β-mercaptoethanol.
3. Prepare conditioned.......

Discussion

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.......

Materials

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