建立人肺类器官和近端分化以产生成熟的气道类器官
Summary
该协议提出了一种从原代肺组织衍生人肺类器官的方法，扩大肺类器官并诱导近端分化以产生3D和2D气道类器官，从而忠实地对人气道上皮进行表型检查。
Abstract
缺乏强大的人体呼吸 道上皮体外 模型阻碍了对呼吸系统生物学和病理学的理解。我们描述了一种明确的方案，从肺组织中的成体干细胞中提取人肺类器官，并诱导近端分化以产生成熟的气道类器官。然后以高稳定性连续扩张肺类器官超过1年，而分化的气道类器官用于在形态学和功能上模拟人气道上皮至接近生理水平。因此，我们建立了人类气道上皮的鲁棒类器官模型。肺类器官和分化气道类器官的长期扩张产生了稳定和可再生的来源，使科学家能够在培养皿中重建和扩增人气道上皮细胞。人肺类器官系统为各种应用提供了独特且具有生理活性 的体外 模型，包括研究病毒 – 宿主相互作用，药物测试和疾病建模。
Introduction
类器官已成为器官发育 体外 建模和研究生物学和疾病的强大而通用的工具。当在生长因子定义的培养基中培养时，来自各种器官的成体干细胞（ASC）可以在3维（3D）中扩增并自组装成由多种细胞类型组成的器官样细胞簇，称为类器官。Clevers的实验室在2009年报告了第一个ASC衍生的类器官，人类肠道类器官的衍生1，2。之后，ASC衍生的类器官已被确定用于各种人体器官和组织，包括前列腺3，4，肝脏5，6，胃7，8，9，胰腺10，乳腺11和肺 12，13.这些ASC衍生的类器官保留了天然器官的关键细胞，结构和功能特性，并在长期扩增培养物中保持了遗传和表型稳定性14，15。
类器官也可以来源于多能干细胞（PSC），包括胚胎干（ES）细胞和诱导多能干细胞（iPS）细胞16。虽然PSC衍生的类器官利用器官发育的机制来建立它们，但ASC可以通过重建在生理组织自我更新或组织修复期间模仿干细胞位的条件来强制形成类器官。PSC衍生的类器官是探索发育和器官发生的有利模型，尽管无法达到ASC衍生类器官的可比成熟水平。PSC衍生类器官的胎儿样成熟状态，以及建立这些类器官的复杂性，大大阻碍了它们在研究成熟组织中的生物学和病理学的广泛应用。
从鼻子到末端细支气管的人体呼吸道内衬气道上皮，也称为假分层纤毛上皮，由四种主要细胞类型组成，即纤毛细胞，杯状细胞，基底细胞和俱乐部细胞。我们与Clevers的实验室12，13合作，从人类肺组织中建立了ASC衍生的人类肺类器官。这些肺类器官在扩张介质中连续扩张超过一年;确切的持续时间因从不同供体获得的不同类器官系而异。然而，与天然气道上皮相比，这些长期可扩张的肺类器官还不够成熟，因为纤毛细胞（人体气道中的主要细胞群）在这些肺类器官中代表性不足。因此，我们开发了一种近端分化方案，并产生了3D和2D气道类器官，其形态学和功能上将气道上皮进行表观镜检查至接近生理水平。
在这里，我们提供一种视频方案，从原代肺组织中衍生出人肺类器官，扩大肺类器官并诱导近端分化以产生3D和2D气道类器官。
Protocol
本文所述的所有使用人体组织的实验均已获得香港大学/医院管理局香港西区（UW13-364和UW21-695）机构审查委员会的批准。在组织收集之前获得患者的知情同意。 1. 人肺类器官的衍生 实验材料的制备 通过用2mM谷氨酰胺，10mM HEPES，100 U / mL青霉素和100μg / mL链霉素补充高级DMEM / F12培养基来制备基础培养基。 通过用10%R-spondin 1条件培养基，10%诺金?…
Representative Results
该方案能够以高成功率衍生出人肺类器官。将新鲜的人体肺组织切成小块，然后用胶原酶分解。将所得的单细胞嵌入基底基质中，并在肺类器官扩增培养基中孵育，并补充一系列用于上皮干细胞生长的利基因子（步骤1.1.2）。 图1 显示了嵌入在2型（BME; 图 1A（左）。囊性类器官出现并随着时间的推移而扩大（图1A，右）。同时，不…
Discussion
人气道内衬气道上皮，也称为假分层纤毛上皮。上气道上皮的主要细胞类型是纤毛细胞，其能够协调其顶端纤毛运动以从气道中排出粘液和吸入颗粒，产生和分泌粘液的杯状细胞以及排列在基底膜上并参与再生的基础细胞。在小气道（如细支气管）中，长方体气道上皮包含分泌性俱乐部细胞和比上气道区域更少的纤毛细胞。我们描述了一种从人类肺组织中的上皮干细胞中提取人体肺类器官的强大?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢香港大学李嘉诚医学院泛有机科学与电子显微镜中心在共聚焦成像和流式细胞术方面的协助。这项工作部分得到了食物及卫生局卫生及医学研究基金（HMRF，17161272及19180392）的资助;研究资助局一般研究基金（GRF，17105420）;及香港特别行政区政府创新科技署Health@InnoHK。
Materials
| Reagents for lung organoid culture | |||
| Advanced DMEM/F12 | Invitrogen | 12634010 | – |
| A8301 | Tocris | 2939 | 500nM |
| B27 supplement | Invitrogen | 17504-044 | 1x |
| Cultrex Reduced Growth Factor Basement Membrane Matrix, Type 2 (BME 2) | Trevigen | 3533-010-0 | 70-80% |
| FGF-10 | Peprotech | 100-26 | 20 ng/mL |
| FGF-7 | Peprotech | 100-19 | 5 ng/mL |
| GlutaMAX (glutamine) | Invitrogen | 35050061 | 1x |
| HEPES 1M | Invitrogen | 15630-056 | 10 mM |
| Heregulin β-1 | Peprotech | 100-03 | 5 nM |
| N-Acetylcysteine | Sigma-Aldrich | A9165 | 1.25 mM |
| Nicotinamide | Sigma-Aldrich | N0636 | 10 mM |
| Noggin (conditional medium) | home made | – | 10x |
| Penicillin-Streptomycin (10,000 U/mL) | Invitrogen | 15140-122 | 1x |
| Primocin | Invivogen | ant-pm-1 | 100 µg/mL |
| Rspondin1 (conditional medium) | home made | – | 10x |
| SB202190 | Sigma-Aldrich | S7067 | 1 µM |
| Y-27632 | Tocris | 1254 | 5 µM |
| Proximal differentiation medium | |||
| DAPT | Tocris | 2634 | 10 µM |
| Heparin Solution | StemCell Technology | 7980 | 4 µg/mL |
| Hydrocortisone Stock Solution | StemCell Technology | 7925 | 1 µM |
| PneumaCult-ALI 10X Supplement | air liquid interface supplement | ||
| PneumaCult-ALI Basal Medium | StemCell Technology | 05001 | air liquid interface basal medium |
| PneumaCult-ALI Maintenance Supplement | air liquid interface maintenance supplement | ||
| Y-27632 | Tocris | 1254 | 10 µM |
| Equipment | |||
| Biological safety cabinet | Baker | 1-800-992-2537 | |
| Carl Zeiss LSM 780 or 800 | Zeiss | confocal microscope | |
| CO2 Incubator | Thermo Fisher Scientific | 42093483 | |
| Stereo-microscope | Olympus Corporation | CKX31SF | |
| Centrifuge | Eppendorf | 5418BG040397 | |
| Serological pipettor | Eppendorf | ||
| Micropipette | Eppendorf | ||
| ZEN black or ZEN blue software | Zeiss | analysis software | |
| Consumables | |||
| 12mm Trans-well | StemCell Technology | #38023 | |
| 12-well cell culture plate | Cellstar | 665970 | |
| 15- and 50 ml conical tubes | Thermo Fisher Scientific | L6BF5Z8118 | |
| 24-well cell culture plate | Cellstar | 662160 | |
| 6.5mm Trans-well | StemCell Technology | #38024 | |
| Medical Syringe Filter Unit, 0.22 µm | Sigma-Aldrich | SLGPR33RB | |
| Microfuge tubes | Eppendorf | ||
| Micropipette tips | Thermo Fisher Scientific | TFLR140-200-Q21190531 | |
| Pasteur pipette glass | Thermo Fisher Scientific | 22-378893 | |
| Serological pipettes(5ml, 10ml, 25ml) | Thermo Fisher Scientific | BA08003, 08004, 08005 | |
| Antibodies | |||
| Goat Anti-Mouse Alexa Fluor 594 | Invitrogen | A11005 | |
| Goat Anti-Mouse, Alexa Fluor 488 | Invitrogen | A11001 | |
| Goat Anti-Rabbit Alexa Fluor 488 | Invitrogen | A11034 | |
| Goat Anti-Rabbit Alexa Fluor 594 | Invitrogen | A11037 | |
| Goat Anti-Rat Alexa Fluor 594 | Invitrogen | A11007 | |
| Mouse Anti-Cytokeratin 5 | Abcam | ab128190 | |
| Mouse Anti-FOX J1 | Invitrogen | 14-9965-82 | |
| Mouse Anti-Mucin 5AC | Abcam | ab3649 | |
| Mouse Anti-β-tubulin 4 | Sigma | T7941 | |
| Rabbit Anti-p63 | Abcam | ab124762 | |
| Rat Anti-Uteroglobin/CC-10 | R&D Systems | MAB4218-SP | |
| Other reagent | |||
| TrypLE Select Enzyme (10X) | Thermo Fisher Scientific | A1217701 | dissociation enzyme |
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Citer Cet Article
Li, C., Chiu, M. C., Yu, Y., Liu, X., Xiao, D., Huang, J., Wan, Z., Zhou, J. Establishing Human Lung Organoids and Proximal Differentiation to Generate Mature Airway Organoids. J. Vis. Exp. (181), e63684, doi:10.3791/63684 (2022).