通过鸡胚胎的腹内移植对肾脏类器官进行有效的血管化
1Department of Internal Medicine – Nephrology,Leiden University Medical Center, 2Einthoven Laboratory of Vascular and Regenerative Medicine,Leiden University Medical Center, 3IBPS, CNRS UMR7622, Developmental Biology Laboratory,Sorbonne Université, 4The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW),Leiden University Medical Center
Summary
在这里，我们提出了在鸡胚胎的天体腔中移植肾脏类器官的详细方案。该方法在8天内诱导类器官的血管形成和增强成熟，可用于以有效的方式研究这些过程。
Abstract
源自人类诱导的多能干细胞的肾脏类器官含有肾单位样结构，在一定程度上类似于成人肾脏中的结构。不幸的是，由于缺乏功能性脉管系统，因此体 外成熟有限，它们的临床适用性受到阻碍。在鸡胚胎的天体腔中移植肾脏类器官通过灌注血管诱导血管形成，包括肾小球毛细血管的形成，并增强其成熟。这种技术非常有效，可以移植和分析大量的类器官。本文描述了鸡胚胎中肾类器官的细胞内移植的详细方案，然后注射荧光标记的凝集素以染色灌注的脉管系统，并收集移植的类器官进行成像分析。该方法可用于诱导和研究类器官血管化和成熟，以寻找在 体外 增强这些过程和改善疾病建模的线索。
Introduction
人类诱导的多能干细胞（hiPSC）来源的肾脏类器官已被证明具有发育研究的潜力1，2，3，4，毒性筛查5，6和疾病建模5，7，8，9，10，11，12，13.然而，由于缺乏血管网络，它们对这些和最终临床移植目的的适用性受到限制。在胚胎肾脏发育过程中，足细胞、系膜细胞和血管内皮细胞 （EC） 相互作用形成肾小球的复杂结构。没有这种相互作用，由足细胞、肾小球基底膜（GBM）和EC组成的肾小球滤过屏障就无法正常发育14，15，16。尽管体外的肾脏类器官确实含有一些EC，但这些EC无法形成适当的血管网络并随着时间的推移而减少17。因此，类器官仍然不成熟也就不足为奇了。小鼠移植诱导肾脏类器官18，19，20，21的血管化和成熟。不幸的是，这是一个劳动密集型过程，不适合分析大量类器官。
一个多世纪以来，鸡胚一直被用来研究血管形成和发育22。它们易于接近，需要低维护，缺乏功能齐全的免疫系统，并且在打开蛋壳23，24，25，26后可以正常发育。类器官在其脉络膜尿囊膜（CAM）上的移植已被证明可导致血管形成27。然而，CAM上的移植持续时间以及移植物的成熟水平受到CAM形成的限制，CAM形成需要直到胚胎第7天才能完成。因此，最近开发了一种通过鸡胚胎中鞘内移植来有效血管化和成熟肾脏类器官的方法28。自 1930 年代以来，众所周知，鸡胚胎的天体腔是胚胎组织分化的有利环境29，30。它可以在胚胎发育的早期进入，并允许移植物在各个方向上相对无限地扩张。
本文概述了在第 4 天鸡胚胎的天体腔中移植 hiPSC 衍生的肾类器官的方案。该方法在8天内诱导类器官的血管形成和增强成熟。在牺牲胚胎之前注射荧光标记的晶状体凝集素（LCA），可以通过共聚焦显微镜可视化类器官内的灌注血管。
Protocol
根据荷兰法律，这项研究不需要动物福利委员会的批准。 1. 制备用于移植的hiPSC来源的肾类器官 使用高里等人开发的协议将hiPSCs与肾脏类器官分化4，18，31。按照该协议在具有0.4μm孔的聚酯细胞培养插入物（细胞培养插入物）上培养类器官，直到分化的第7 + 12天。每个细胞培养插入片?…
Representative Results
图1A总结了hiPSCs分化为肾类器官、受精鸡蛋孵化、肾类器官移植、LCA注射和类器官收集的方法和时间表。协调类器官分化和鸡蛋孵化的时间很重要，在孵化前 15 天开始分化。孵化第 0、3、4 和 12 天的操作由时间线下方的照片说明。类器官在分化的第7 + 12天移植到第4天（HH 23-24）鸡胚中。移植后8天将LCA注射到胚胎的静脉系统中，以染色灌注的脉管系统，然后牺牲胚胎并取回类…
Discussion
在这份手稿中，展示了鸡胚胎中hiPSC衍生的肾类器官的细胞内移植方案。移植后，类器官通过灌注的血管进行血管化，这些血管由人类器官来源和鸡来源的EC的组合组成。它们遍布整个类器官并侵入肾小球结构，使EC和足细胞之间的相互作用成为可能。先前表明，这导致类器官肾小球和管状结构的成熟增强28。移植非常有效，每个胚胎需要~5分钟，胚胎唯一需要的维护是定期补充培…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢George Galaris（LUMC，莱顿，荷兰）在鸡胚注射方面的帮助。我们感谢Saskia van der Wal-Maas（荷兰莱顿LUMC解剖与胚胎学系），Conny van Munsteren（荷兰莱顿LUMC解剖与胚胎学系），Manon Zuurmond（LUMC，莱顿，荷兰）和Annemarie de Graaf（LUMC，莱顿，荷兰）的支持。M. Koning由’Nephrosearch Stichting tot steun van het wetenschappelijk onderzoek van de afdeling Nierziekten van het LUMC’提供支持。这项工作部分得到了莱顿大学基金“Jaap de Graeff-Lingling Wiyadhanrma Fund”GWF2019-02 的支持。这项工作得到了再生医学跨境（RegMedXB）和Health Holland，Top Sector Life Sciences & Health的合作伙伴的支持。C.W. van den Berg和T.J. Rabelink由诺和诺德基金会干细胞医学中心（reNEW）支持，诺和诺德基金会干细胞医学中心由诺和诺德基金会资助（NNF21CC0073729）支持。
Materials
| 0.2 µm filter: Whatman Puradisc 30 syringe filter 0.2 µm | Whatman | 10462200 | |
| 35 mm glass bottom dishes | MatTek Corporation | P35G-1.5-14-C | |
| Aspirator tube assemblies for calibrated microcapillary pipettes | Sigma-Aldrich | A5177-5EA | Contains silicone tubes, mouth piece and connector |
| Confocal microscope: Leica White Light Laser Confocal Microscope | Leica | TCS SP8 | |
| Dissecting forceps, simple type. Titanium, curved, with fine sharp tips | Hammacher Karl | HAMMHTC091-10 | |
| Dissecting forceps, simple type. Titanium, straight, with fine sharp tips | Hammacher Karl | HAMMHTC090-11 | |
| Dissecting microscope | Wild Heerbrugg | 355110 | |
| Dissecting scissors, curved, OP-special, extra sharp/sharp | Hammacher Karl | HAMMHSB391-10 | |
| Donkey serum | Sigma-Aldrich | D9663 | |
| Donkey-α-mouse Alexa Fluor 488 | ThermoFisher Scientific | A-212-02 | dilution 1:500 |
| Donkey-α-sheep Alexa Fluor 647 | ThermoFisher Scientific | A-21448 | dilution 1:500 |
| Double edged stainless steel razor blades | Electron Microsopy Sciences | 72000 | |
| DPBS, calcium, magnesium (DPBS-/-) | ThermoFisher Scientific | 14040133 | |
| DPBS, no calcium, no magnesium (DPBS+/+) | ThermoFisher Scientific | 14190094 | |
| Egg cartons or custom made egg holders | NA | NA | |
| Fertilized white leghorn eggs (Gallus Gallus Domesticus) | Drost Loosdrecht B.V. | NA | |
| Incubator | Elbanton BV | ET-3 combi | |
| Lotus Tetragonolobus lectin (LTL) Biotinylated | Vector Laboratories | B-1325 | dilution 1:300 |
| Micro scissors, straight, sharp/sharp, cutting length 10 mm | Hammacher Karl | HAMMHSB500-09 | |
| Microcapillaries: Thin wall glass capillaries 1.5 mm, filament | World Precision Instruments | TW150F-3 | |
| Micropipette puller | Sutter Instrument Company | Model P-97 | We use the following settings: Heat 533, Pull 60, Velocity 150, Time 200 |
| Microscalpel holder: Castroviejo blade and pins holder, 12 cm, round handle, conical 10 mm jaws. | Euronexia | L-120 | |
| Mounting medium: Prolong Gold Antifade Mountant | ThermoFisher Scientific | P36930 | |
| Olivecrona dura dissector 18 cm | Reda | 41146-18 | |
| Parafilm | Heathrow Scientific | HS234526B | |
| Penicillin-streptomycin 5,000 U/mL | ThermoFisher Scientific | 15070063 | |
| Perforated spoon | Euronexia | S-20-P | |
| Petri dish 60 x 15 mm | CELLSTAR | 628160 | |
| Plastic transfer pipettes | ThermoFisher Scientific | PP89SB | |
| Purified mouse anti-human CD31 antibody | BD Biosciences | 555444 | dilution 1:100 |
| Rhodamine labeled Lens Culinaris Agglutinin (LCA) | Vector Laboratories | RL-1042 | This product has recently been discontinued. Vectorlabs does still produce Dylight 649 labeled LCA (DL-1048-1) and fluorescein labeled LCA (FL-1041-5) |
| Sheep anti-human NPHS1 antibody | R&D systems | AF4269 | dilution 1:100 |
| Sterile hypodermic needles, 19 G | BD microlance | 301500 | |
| Streptavidin Alexa Fluor 405 | ThermoFisher Scientific | S32351 | dilution 1:200 |
| Syringe 5 mL | BD Emerald | 307731 | |
| Transparent tape | Tesa | 4124 | Available at most hardware stores |
| Triton X | Sigma-Aldrich | T9284 | |
| Tungsten wire, 0.25 mm dia | ThermoFisher Scientific | 010404.H2 |
Riferimenti
- Taguchi, A., et al. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells. Cell Stem Cell. 14 (1), 53-67 (2014).
- Morizane, R., et al. Nephron organoids derived from human pluripotent stem cells model kidney development and injury. Nature Biotechnology. 33 (11), 1193-1200 (2015).
- Kim, Y. K., et al. Gene-edited human kidney organoids reveal mechanisms of disease in podocyte development. Stem Cells. 35 (12), 2366-2378 (2017).
- Takasato, M., et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature. 526 (7574), 564-568 (2015).
- Hale, L. J., et al. 3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening. Nature Communications. 9 (1), 5167 (2018).
- Vanslambrouck, J. M., et al. Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids. Nature Communications. 13 (1), 5943 (2022).
- Tanigawa, S., et al. Organoids from nephrotic disease-derived iPSCs identify impaired NEPHRIN localization and slit diaphragm formation in kidney podocytes. Stem Cell Reports. 11 (3), 727-740 (2018).
- Forbes, T. A., et al. Patient-iPSC-derived kidney organoids show functional validation of a ciliopathic renal phenotype and reveal underlying pathogenetic mechanisms. American Journal of Human Genetics. 102 (5), 816-831 (2018).
- Freedman, B. S., et al. Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids. Nature Communications. 6, 8715 (2015).
- Cruz, N. M., et al. Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease. Nature Materials. 16 (11), 1112-1119 (2017).
- Gupta, N., et al. Modeling injury and repair in kidney organoids reveals that homologous recombination governs tubular intrinsic repair. Science Translational Medicine. 14 (634), eabj4772 (2022).
- Hiratsuka, K., et al. Organoid-on-a-chip model of human ARPKD reveals mechanosensing pathomechanisms for drug discovery. Scencei Advances. 8 (38), eabq0866 (2022).
- Dorison, A., et al. Kidney organoids generated using an allelic series of NPHS2 point variants reveal distinct intracellular podocin mistrafficking. Journal of the American Society of Nephrology. , (2022).
- Eremina, V., et al. Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. The Journal of Clinical Investigation. 111 (5), 707-716 (2003).
- Kitamoto, Y., Tokunaga, H., Tomita, K. Vascular endothelial growth factor is an essential molecule for mouse kidney development: glomerulogenesis and nephrogenesis. The Journal of Clinical Investigation. 99 (10), 2351-2357 (1997).
- Sison, K., et al. Glomerular structure and function require paracrine, not autocrine, VEGF-VEGFR-2 signaling. Journal of the American Society of Nephrology. 21 (10), 1691-1701 (2010).
- Ryan, A. R., et al. Vascular deficiencies in renal organoids and ex vivo kidney organogenesis. Biologia dello sviluppo. 477, 98-116 (2021).
- vanden Berg, C. W., et al. Renal subcapsular transplantation of PSC-derived kidney organoids induces neo-vasculogenesis and significant glomerular and tubular maturation in vivo. Stem Cell Reports. 10 (3), 751-765 (2018).
- Sharmin, S., et al. Human induced pluripotent stem cell-derived podocytes mature into vascularized glomeruli upon experimental transplantation. Journal of the American Society of Nephrology. 27 (6), 1778-1791 (2016).
- Bantounas, I., et al. Generation of functioning nephrons by implanting human pluripotent stem cell-derived kidney progenitors. Stem Cell Reports. 10 (3), 766-779 (2018).
- vanden Berg, C. W., Koudijs, A., Ritsma, L., Rabelink, T. J. In vivo assessment of size-selective glomerular sieving in transplanted human induced pluripotent stem cell-derived kidney organoids. Journal of the American Society of Nephrology. 31 (5), 921-929 (2020).
- Asai, R., Bressan, M., Mikawa, T. Avians as a model system of vascular development. Methods in Molecular Biology. 2206, 103-127 (2021).
- Jankovic, B. D., et al. Immunological capacity of the chicken embryo. I. Relationship between the maturation of lymphoid tissues and the occurrence of cell-mediated immunity in the developing chicken embryo. Immunology. 29 (3), 497-508 (1975).
- Alkie, T. N., et al. Development of innate immunity in chicken embryos and newly hatched chicks: a disease control perspective. Avian Pathology. 48 (4), 288-310 (2019).
- Rawles, M. E. Transplantation of normal embryonic tissues. Annalls of the New York Academy of Sciences. 55 (2), 302-312 (1952).
- Rawles, M. E. The development of melanophores from embryonic mouse tissues grown in the coelom of chick embryos. Proceedings of the National Academy of Sciences. 26 (12), 673-680 (1940).
- Garreta, E., et al. Fine tuning the extracellular environment accelerates the derivation of kidney organoids from human pluripotent stem cells. Nature Materials. 18 (4), 397-405 (2019).
- Koning, M., et al. Vasculogenesis in kidney organoids upon transplantation. NPJ Regenerative Medicine. 7 (1), 40 (2022).
- Hamburger, V. Morphogenetic and axial self-differentiation of transplanted limb primordia of 2-day chick embryos. Journal of Experimental Zoology. 77 (3), 379-399 (1938).
- Dossel, W. E. New method of intracelomic grafting. Science. 120 (3111), 262-263 (1954).
- Takasato, M., Er, P. X., Chiu, H. S., Little, M. H. Generation of kidney organoids from human pluripotent stem cells. Nature Protocols. 11 (9), 1681-1692 (2016).
- Hamburger, V., Hamilton, H. L. A series of normal stages in the development of the chick embryo. Developmental Dynamics. 195 (4), 231-272 (1992).
- Aleksandrowicz, E., Herr, I. Ethical euthanasia and short-term anesthesia of the chick embryo. ALTEX. 32 (2), 143-147 (2015).
- . ACUC. Guideline: The Use and Euthanasia Procedures of Chicken/Avian Embryos. , (2012).
Citazione di questo articolo
Koning, M., Lievers, E., Jaffredo, T., van den Berg, C. W., Rabelink, T. J. Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos. J. Vis. Exp. (192), e65090, doi:10.3791/65090 (2023).