体外 使用小鼠胚胎干细胞进行血管疾病建模和药物测试的血管生成的三维发芽测定
Summary
该测定利用分化成在3D胶原蛋白凝胶中培养的拟胚体的小鼠胚胎干细胞来分析体 外控制发芽血管生成的生物学过程。该技术可用于测试药物，模拟疾病以及在胚胎致命的缺失背景下研究特定基因。
Abstract
诱导多能干细胞（iPSC）和基因编辑技术的最新进展使得为表型药物发现（PDD）程序开发了基于人类细胞的新型疾病模型。尽管这些新型设备可以更准确地预测研究药物在人类中的安全性和有效性，但它们的开发仍然强烈依赖于哺乳动物数据，特别是小鼠疾病模型的使用。因此，与人类类器官或器官芯片疾病模型并行，相关 体 外小鼠模型的开发对于评估物种与 体内 和 体外 条件之间的直接药物疗效和安全性比较的需求尚未得到满足。在这里，描述了利用分化成拟胚体（EB）的小鼠胚胎干细胞的血管发芽测定。培养到3D胶原凝胶上的血管化EB可形成扩张的新血管，这一过程称为发芽血管生成。该模型概括了 体内 萌芽血管生成的关键特征 – 从预先存在的血管网络形成血管 – 包括内皮尖端细胞选择，内皮细胞迁移和增殖，细胞引导，管形成和壁细胞募集。它适用于筛选调节血管生成的药物和基因，并与最近描述的基于人类 iPSC 技术的三维 （3D） 血管测定有相似之处。
Introduction
在过去的三十年中，基于靶点的药物发现（TDD）已被制药行业广泛用于药物发现。TDD包含明确的分子靶标，在疾病中起重要作用，并依赖于相对简单的细胞培养系统的开发以及用于药物筛选的读数1。TDD项目中使用的大多数典型疾病模型包括传统的细胞培养方法，例如癌细胞或在人工环境中生长的永生化细胞系和非生理基质。尽管其中许多模型为识别成功的候选药物提供了可行的工具，但由于其疾病相关性差，使用此类系统可能值得怀疑2。
对于大多数疾病，潜在的机制确实是复杂的，并且经常发现各种细胞类型，独立的信号通路和多组基因有助于特定的疾病表型。对于遗传性疾病也是如此，其中主要原因是单个基因的突变。随着最近人类诱导多能干细胞（iPSC）技术和基因编辑工具的出现，现在可以生成3D类器官和器官芯片疾病模型，可以更好地概括体内人类的复杂性3，4。这些技术的发展与对表型药物发现（PDD）计划的兴趣重新抬头有关1。PDD可以与经验性筛查进行比较，因为它们不依赖于对特定药物靶标身份的了解或关于其在疾病中的作用的假设。PDD方法现在越来越被公认为对发现一流药物做出了巨大贡献5。由于人类类器官和器官芯片技术的发展仍处于起步阶段，预计iPSC模型（辅以创新的成像和机器学习工具6，7）将在不久的将来提供多种新型的基于复杂细胞的疾病模型，用于药物筛选和相关PDD程序，以克服TDD方法的低生产率8，9.
虽然人类类器官和器官芯片模型可以为疾病复杂性和新药的鉴定提供重要的见解，但将药物带入新的临床实践也强烈依赖动物模型的数据来评估其疗效和安全性。其中，转基因小鼠无疑是哺乳动物最青睐的模型。它们具有许多优点，因为它们对哺乳动物的生成时间相对较短，具有许多与人类疾病相似的表型，并且可以很容易地进行遗传操作。因此，它们被广泛用于药物发现计划10。然而，弥合小鼠和人类之间的差距仍然是一个重要的挑战11。开发相当于人类类器官和器官芯片模型的 体 外小鼠模型可以至少部分填补这一空白，因为它将允许在 体内 小鼠和 体外 人类数据之间进行直接的药物疗效和安全性比较。
在这里，描述了小鼠拟胚体（EB）中的血管萌芽测定。血管由内皮细胞（血管壁内壁）、壁细胞（血管平滑肌细胞和周细胞）组成12。该协议基于小鼠胚胎干细胞（mESCs）的分化为血管化EB，使用悬挂液滴概括 从头 内皮细胞和壁细胞分化13，14。小鼠ESC可以很容易地在具有不同遗传背景的小鼠囊胚的分离第3.5天培养物中建立15。它们还为克隆分析、谱系追踪提供了可能性，并且可以很容易地进行基因操作以生成疾病模型13，16。
由于血管滋养所有器官，因此许多疾病（如果不是全部）与微脉管系统的变化有关也就不足为奇了。在病理条件下，内皮细胞可以采用活化状态或功能障碍，导致壁细胞死亡或从血管迁移。这些可导致血管生成过多或血管稀疏，可诱发异常血流和血管屏障缺陷，导致免疫细胞外渗和炎症12，17，18，19。因此，开发调节血管的药物的研究很高，并且已经确定了用于治疗靶向的多个分子参与者和概念。在这种情况下，所描述的方案特别适用于构建疾病模型和药物测试，因为它概括了体内发芽血管生成的关键特征，包括内皮尖端和茎细胞选择、内皮细胞迁移和增殖、内皮细胞引导、管形成和壁细胞募集。它还显示出与最近描述的基于人类iPSC技术的3D血管测定的相似之处20。
Protocol
1. mESC的培养基制备与培养 使用补充剂 1x 格拉斯哥 MEM （G-MEM BHK-21） 培养基与 10%（体积/体积）热灭活胎牛血清 （FBS）、0.05 mM β-巯基乙醇、1x 非必需氨基酸 （NEAA 1x）、2 mM L-谷氨酰胺和 1 mM 丙酮酸钠制备条件培养基 +/- （CM+/-）。 使用含有白血病抑制因子 （LIF） （1，500 U/mL） 和 0.1 mM β-巯基乙醇的补充剂 CM+/+ （CM+/+） 制备条件培养基 +/+ （CM+/+）。 使用含有1μM PD0325901和3?…
Representative Results
血管发芽测定的方案概述如图1所示。使用胶原酶A将来自三个独立的129 / Ola mESC系（Z / Red，R1和E14）的9天大的EB酶解散成单个细胞.对细胞进行PECAM-1染色并通过荧光激活细胞分选（FACS）进行分析。所有细胞系均表现出强大的内皮分化，并且未观察到其分化为内皮细胞的能力存在差异。所有细胞系产生约10.5%±1.3%的内皮细胞（图2A）。还定量了PECAM-1（+）细…
Discussion
该协议描述了一种无偏倚，稳健且可重复的基于3D EB的血管发芽测定，该测定适用于筛选调节血管生成的药物和基因。与许多广泛使用的二维 （2D） 测定相比，该方法具有优势，使用内皮细胞培养物（例如人脐静脉内皮细胞 （HUVEC）来监测迁移（横向划痕测定或 Boyden 室测定）22，23 或增殖（计数细胞数量、DNA 合成检测、增殖标志物检测或代谢测定）<su…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了荷兰组织（ZonMW 446002501），荷兰卫生（LSHM19057-H040），主要研究员计划Marie Skłodowska-Curie联合基金和Maladie de Rendu-Osler协会（AMRO）的资助。
Materials
| 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 |
References
- Moffat, J. G., Vincent, F., Lee, J. A., Eder, J., Prunotto, M. Opportunities and challenges in phenotypic drug discovery: an industry perspective. Nature Reviews Drug Discovery. 16 (8), 531-543 (2017).
- Horvath, P., et al. Screening out irrelevant cell-based models of disease. Nature Reviews. Drug Discovery. 15 (11), 751-769 (2016).
- Low, L. A., Mummery, C., Berridge, B. R., Austin, C. P., Tagle, D. A. Organs-on-chips: into the next decade. Nature Reviews. Drug Discovery. , (2020).
- Ma, C., Peng, Y., Li, H., Chen, W. Organ-on-a-Chip: A new paradigm for drug development. Trends in Pharmacological Sciences. 42 (2), 119-133 (2021).
- Swinney, D. C., Anthony, J. How were new medicines discovered. Nature Reviews Drug Discovery. 10 (7), 507-519 (2011).
- Hussain, S., et al. High-content image generation for drug discovery using generative adversarial networks. Neural Networks: The Official Journal of the INternational Neural Network Society. 132, 353-363 (2020).
- Scheeder, C., Heigwer, F., Boutros, M. Machine learning and image-based profiling in drug discovery. Current Opinion in Systems Biology. 10, 43-52 (2018).
- Wagner, B. K., Schreiber, S. L. The power of sophisticated phenotypic screening and modern mechanism-of-action methods. Cell Chemical Biology. 23 (1), 3-9 (2016).
- Scannell, J. W., Bosley, J. When quality beats quantity: Decision theory, drug discovery, and the reproducibility crisis. PLoS One. 11 (2), 0147215 (2016).
- Webster, J. D., Santagostino, S. F., Foreman, O. Applications and considerations for the use of genetically engineered mouse models in drug development. Cell and Tissue Research. 380 (2), 325-340 (2020).
- Howland, D. S., Munoz-Sanjuan, I. Mind the gap: models in multiple species needed for therapeutic development in Huntington’s disease. Movement Disorders: Official Journal of the Movement Disorder Scoiety. 29 (11), 1397-1403 (2014).
- Galaris, G., Thalgott, J. H., Lebrin, F. P. G. Pericytes in hereditary hemorrhagic telangiectasia. Advances in Experimental Medicine and Biology. 1147, 215-246 (2019).
- Thalgott, J. H., et al. Decreased expression of vascular endothelial growth factor receptor 1 contributes to the pathogenesis of hereditary hemorrhagic telangiectasia type 2. Circulation. 138 (23), 2698-2712 (2018).
- Lebrin, F., et al. Thalidomide stimulates vessel maturation and reduces epistaxis in individuals with hereditary hemorrhagic telangiectasia. Nature Medicine. 16 (4), 420-428 (2010).
- Czechanski, A., et al. Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains. Nature Protocols. 9 (3), 559-574 (2014).
- Elling, U., et al. A reversible haploid mouse embryonic stem cell biobank resource for functional genomics. Nature. 550 (7674), 114-118 (2017).
- Cheng, J., et al. Targeting pericytes for therapeutic approaches to neurological disorders. Acta Neuropathologica. 136 (4), 507-523 (2018).
- Chade, A. R. Small vessels, big role: Renal microcirculation and progression of renal injury. Hypertension. 69 (4), 551-563 (2017).
- Jourde-Chiche, N., et al. Endothelium structure and function in kidney health and disease. Nature Reviews. Nephrology. 15 (2), 87-108 (2019).
- van Duinen, V., et al. Standardized and scalable assay to study perfused 3D angiogenic sprouting of iPSC-derived endothelial cells in vitro. Journal of Visualized Experiment: JoVE. (153), e59678 (2019).
- Chappell, J. C., Taylor, S. M., Ferrara, N., Bautch, V. L. Local guidance of emerging vessel sprouts requires soluble Flt-1. Developmental Cell. 17 (3), 377-386 (2009).
- Sato, Y., Rifkin, D. B. Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture. Journal of Cell Biology. 109 (1), 309-315 (1989).
- Tchaikovski, V., Olieslagers, S., Bohmer, F. D., Waltenberger, J. Diabetes mellitus activates signal transduction pathways resulting in vascular endothelial growth factor resistance of human monocytes. Circulation. 120 (2), 150-159 (2009).
- Staton, C. A., Reed, M. W., Brown, N. J. A critical analysis of current in vitro and in vivo angiogenesis assays. International Journal of Experimental Pathology. 90 (3), 195-221 (2009).
- Herbert, S. P., Stainier, D. Y. Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nature Reviews Molecular Cell Biology. 12 (9), 551-564 (2011).
- Nakatsu, M. N., Hughes, C. C. An optimized three-dimensional in vitro model for the analysis of angiogenesis. Methods in Enzymology. 443, 65-82 (2008).
- Nakatsu, M. N., Davis, J., Hughes, C. C. Optimized fibrin gel bead assay for the study of angiogenesis. Journal of Visualized Experiments: JoVE. (186), (2007).
- Gau, D., et al. Pharmacological intervention of MKL/SRF signaling by CCG-1423 impedes endothelial cell migration and angiogenesis. Angiogenesis. 20 (4), 663-672 (2017).
- Torres-Estay, V., et al. Androgens modulate male-derived endothelial cell homeostasis using androgen receptor-dependent and receptor-independent mechanisms. Angiogenesis. 20 (1), 25-38 (2017).
- Merjaneh, M., et al. Pro-angiogenic capacities of microvesicles produced by skin wound myofibroblasts. Angiogenesis. 20 (3), 385-398 (2017).
- Rezzola, S., et al. In vitro and ex vivo retina angiogenesis assays. Angiogenesis. 17 (3), 429-442 (2014).
- Wang, X., Phan, D. T. T., George, S. C., Hughes, C. C. W., Lee, A. P. 3D Anastomosed microvascular network model with living capillary networks and endothelial cell-lined microfluidic channels. Methods in Molecular Biology. 1612, 325-344 (2017).
- Nicosia, R. F., Ottinetti, A. Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro. Laboratory Investigation; A Journal of Technical Methods and Pathology. 63 (1), 115-122 (1990).
- Nicosia, R. F. The aortic ring model of angiogenesis: a quarter century of search and discovery. Journal of Cellular and Molecular Medicine. 13 (10), 4113-4136 (2009).
- Nowak-Sliwinska, P., et al. Consensus guidelines for the use and interpretation of angiogenesis assays. Angiogenesis. 21 (3), 425 (2018).
- Belair, D. G., Schwartz, M. P., Knudsen, T., Murphy, W. L. Human iPSC-derived endothelial cell sprouting assay in synthetic hydrogel arrays. Acta Biomaterialia. 39, 12-24 (2016).
- Bezenah, J. R., Kong, Y. P., Putnam, A. J. Evaluating the potential of endothelial cells derived from human induced pluripotent stem cells to form microvascular networks in 3D cultures. Scientific Reports. 8 (1), 2671 (2018).
- Henderson, A. R., Choi, H., Lee, E. Blood and lymphatic vasculatures on-chip platforms and their applications for organ-specific in vitro modeling. Micromachines (Basel). 11 (2), 147 (2020).
- Lin, D. S. Y., Guo, F., Zhang, B. Modeling organ-specific vasculature with organ-on-a-chip devices. Nanotechnology. 30 (2), 024002 (2019).
- Pollet, A., den Toonder, J. M. J. Recapitulating the vasculature using organ-on-chip technology. Bioengineering. 7 (1), 17 (2020).
- Cochrane, A., et al. Advanced in vitro models of vascular biology: Human induced pluripotent stem cells and organ-on-chip technology. Advanced Drug Delivery Reviews. 140, 68-77 (2019).
Cite This Article
Galaris, G., Thalgott, J. H., Teston, E., Lebrin, F. P. In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing. J. Vis. Exp. (171), e62554, doi:10.3791/62554 (2021).