分离和富集人肺上皮祖细胞用于类器官培养
Summary
本文提供了组织解离和细胞分馏方法的详细方法，允许从人肺的近端和远端区域富集活的上皮细胞。本文将这些方法应用于通过使用3D类器官培养模型对肺上皮祖细胞进行功能分析。
Abstract
上皮类器官模型是研究器官系统基础生物学和疾病建模的宝贵工具。当作为类器官生长时，上皮祖细胞可以自我更新并产生分化的后代，其表现出与 体内 对应物相似的细胞功能。在这里，我们描述了一种从人肺中分离区域特异性祖细胞并生成3D类器官培养物作为实验和验证工具的分步方案。我们定义肺的近端和远端区域，目的是分离区域特异性祖细胞。我们利用酶解离和机械解离的组合从肺和气管中分离出总细胞。然后使用基于细胞类型特异性表面标志物的荧光相关细胞分选（FACS）从近端或远端起源细胞中分离出特异性祖细胞，例如用于分选基底细胞的NGFR和用于分选II型肺泡细胞的HTII-280。分离的基础或肺泡II型祖细胞用于生成3D类器官培养物。远端和近端祖细胞均形成类器官，当在第30天接种5000个细胞/孔时，远端区域的集落形成效率为9-13%，近端区域的集落形成效率为7-10%。远端类器官在培养物中维持HTII-280 + 肺泡II型细胞，而近端类器官在第30天分化为纤毛和分泌细胞。这些3D类器官培养物可用作研究肺上皮细胞生物学和上皮间充质相互作用的实验工具，以及开发和验证针对疾病中上皮功能障碍的治疗策略。
Introduction
人体呼吸系统的空气空间大致可分为传导区和呼吸区，分别介导气体的运输及其随后穿过上皮- 微血管屏障的交换。传导气道包括气管、细支气管、细支气管和终末细支气管，而呼吸空气空间包括呼吸细支气管、肺泡管和肺泡。这些空域的上皮衬里沿近端-远端轴改变组成，以适应每个功能独特区域的独特要求。气管支气管气道的假分层上皮由基底、分泌和纤毛三种主要细胞类型组成，此外还有较少的细胞类型，包括刷子、神经内分泌和离子细胞1，2，3。支气管气道具有形态上相似的上皮细胞类型，尽管它们的丰度和功能特性有区别。例如，基底细胞在支气管气道内的丰度较低，分泌细胞包括更大比例的俱乐部细胞，而不是气管支气管气道中占主导地位的浆液细胞和杯状细胞。 呼吸区域的上皮细胞包括呼吸细支气管中定义不明确的立方体细胞类型，以及肺泡导管和肺泡的I型肺泡（ATI）和II型（ATII）细胞1，4。
上皮茎和祖细胞类型的特性有助于每个区域上皮的维持和更新，其特征描述不完整，并且主要从动物模型5，6，7，8中的研究中推断出来。对小鼠的研究表明，假分层气道的基础细胞，或细支气管气道的俱乐部细胞或肺泡上皮的ATII细胞，都可以作为基于无限自我更新和多能分化能力的上皮干细胞7，9，10，11，12.尽管无法进行遗传谱系追踪研究来评估人肺上皮细胞类型的干性，但基于类器官的培养模型的可用性，以评估上皮干细胞和祖细胞的功能潜力，为小鼠和人类13，14，15，16，17之间的比较研究提供了工具。
我们描述了从人肺不同区域分离上皮细胞类型的方法，并使用3D类器官系统来概括区域细胞类型。已经开发了类似的方法，用于来自其他器官系统的上皮细胞的功能分析和疾病建模18，19，20，21。这些方法为鉴定区域上皮祖细胞，进行机械研究以研究其调节和微环境以及实现疾病建模和药物发现提供了平台。尽管在动物模型中进行的肺上皮祖细胞研究可以从 体内 或 体外的分析中受益，但对人类肺上皮祖细胞身份的见解在很大程度上取决于模型生物的外推。因此，这些方法提供了一个桥梁，将人肺上皮细胞类型的特性和行为与他们研究的干细胞/祖细胞的调节联系起来。
Protocol
人体肺组织是从已故组织捐赠者那里获得的，符合国际医学促进会（IIAM）制定的同意程序，并得到锡达斯 – 西奈医学中心内部审查委员会的批准。 1. 用于从气管支气管或小气道/实质（小气道和肺泡）区域分离肺细胞的组织处理 在细胞分离前一天准备并高压灭菌所有解剖仪器，玻璃器皿和适当的溶液。 接收肺组织后，识别并分离近端和远端区域。气管和支气?…
Representative Results
源肺组织气管和肺外支气管（图1A）被用作分离近端气道上皮细胞和随后产生近端类器官的源组织。包括实质和直径小于2mm的小气道（图1A）的远端肺组织用于分离小气道和肺泡上皮细胞（远端肺上皮）和生成小气道或肺泡类器官。由假分层上皮衬里的近端气道包括丰富的基底祖细胞，这些细胞对膜蛋白NGFR具有免疫反应性（<strong class="xf…
Discussion
我们描述了一种可靠的方法，用于从人体肺组织中分离出确定的肺细胞亚群，以进行分子或功能分析和疾病建模。方法的关键要素包括实现组织解离和表面表位保存的能力，这允许抗体介导的新鲜分离细胞的富集，以及用于有效生成区域特异性上皮类器官的培养方法的优化。我们专注于恢复和富集上皮祖细胞，当在三维培养中与基质支持细胞重组时能够形成类器官。尽管我们没有定义这些培养物?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢美津野贵子对IFC和H和E染色的支持，凡妮莎·加西亚对组织切片的支持，以及阿尼卡·钱德拉塞卡兰对手稿准备的帮助。这项工作得到了美国国立卫生研究院（5RO1HL135163-04，PO1HL108793-08）和新基IDEAL联盟的支持。
Materials
| Cell Isolation | |||
| 10 mL Sterile syringes, Luer-Lok Tip | Fisher scientific | BD 309646 | |
| 30 mL Sterile syringes, Luer-Lok Tip | VWR | BD302832 | |
| Biohazard bags | VWR | 89495-440 | |
| Biohazard bags | VWR | 89495-440 | |
| connecting ring | Pluriselect | 41-50000-03 | |
| Deoxyribonuclease (lot#SLBF7798V) | sigma Aldrich | DN25-1G | |
| Disposable Petri dishes | Corning/Falcon | 25373-187 | |
| Funnel | Pluriselect | 42-50000 | |
| HBSS | Corning | 21-023 | |
| Liberase TM Research Grade | sigma Aldrich | 5401127001 | |
| needle 16G | VWR | 305198 | |
| needle 18G | VWR | 305199 | |
| PluriStrainer 100 µm (Cell Strainer) | Pluriselect | 43-50100-51 | |
| PluriStrainer 300 µm (Cell Strainer) | Pluriselect | 43-50300-03 | |
| PluriStrainer 40 µm (Cell Strainer) | Pluriselect | 43-50040-51 | |
| PluriStrainer 500 µm (Cell Strainer) | Pluriselect | 43-50500-03 | |
| PluriStrainer 70 µm (Cell Strainer) | Pluriselect | 43-50070-51 | |
| Razor blades | VWR | 55411-050 | |
| Red Blood Cell lysis buffer | eBioscience | 00-4333-57 | |
| Equipment’s | |||
| GentleMACS C Tubes | MACS Miltenyi Biotec | 130-096-334 | |
| GentleMACS Octo Dissociator | MACS Miltenyi Biotec | 130-095-937 | |
| Leica ASP 300s Tissue processor | |||
| LS Columns | MACS Miltenyi Biotec | 130-042-401 | |
| MACS MultiStand** | Miltenyi Biotech | 130-042-303 | |
| Thermomixer | Eppendorf | 05-412-503 | |
| Thermomixer | Eppendorf | 05-412-503 | |
| HBSS+ Buffer | |||
| Amphotericin B | Thermo fisher scientific | 15290018 | 2ml |
| EDTA (0.5 M), pH 8.0, RNase-free | Thermo fisher scientific | AM9260G | 500µl |
| Fetal Bovine Serum | Gemini Bio-Products | 100-106 | 10ml |
| HBSS Hank's Balanced Salt Solution 1X 500 ml | VWR | 45000-456 | 500ml bottle |
| HEPES (1 M) | Thermo fisher scientific | 15630080 | 5ml |
| Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture | Thermo fisher scientific | 15640055 | 5ml |
| List of antibodies for FACS | |||
| Alexa Fluor 647 anti-human CD326 (EpCAM) Antibody | BioLegend | 369820 | 1:50 |
| BD CompBead Anti-Mouse Ig, K/ Negative control particles set | Fisher Scientific | BDB552843 | |
| CD31 MicroBead Kit, human | Miltenyi Biotec | 130-091-935 | 20µl/ 107 total cells |
| CD45 MicroBeads, human | Miltenyi Biotec | 130-045-801 | 20µl/ 107 total cells |
| DAPI | Sigma Aldrich | D9542-10MG | 1:10000 |
| FITC anti-human CD235a | BioLegend | 349104 | 1:100 |
| FITC anti-human CD31 | BioLegend | 303104 | 1:100 |
| FITC anti-human CD45 | BioLegend | 304054 | 1:100 |
| FITC anti-mouse IgM Antibody | BioLegend | 406506 | 1:500 |
| Mouse IgM anti human HT2-280 | Terrace Biotech | TB-27AHT2-280 | 1:300 |
| PE anti-human CD271(NGFR) | BioLegend | 345106 | 1:50 |
| Composition of Organoid Culture mediums | |||
| MRC-5 | ATCC | CCL-171 | |
| PneumaCult -ALI Medium | Stemcell Technologies | 5001 | |
| Small Airway Epithelial Cell Growth Medium | PromoCell | C-21170 | |
| ThinCert Tissue Culture Inserts, Sterile | Greiner Bio-One | 662641 | |
| Y-27632 (ROCK inhibitor) 100mM stock (1000x) | Stemcell Technologies | 72302 | |
| Mouse Basal medium: | |||
| Amphotericin B | Thermo fisher scientific | 15290018 | 50 µl |
| DMEM/F-12, HEPES | ThermoFisher scientific | 11330032 | 50 ml |
| Fetal Bovine Serum | Gemini Bio-Products | 100-106 | 5 ml |
| Insulin-Transferrin-Selenium (ITS -G) (100X) | ThermoFisher scientific | 41400045 | 500 µl |
| Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture | Thermo fisher scientific | 15640055 | 500 µl |
| SB431542 TGF-β pathway inhibitor (stock 100 mM) | Stem cell | 72234 | 5 µl |
| List of antibodies for Immunohistochemistry | |||
| Antigen unmasking solution, citric acid based | Vector | H-3300 | 937 µl in 100ml water |
| Histogel | Thermo Scientific | HG-4000-012 | |
| Primary Antibodies | |||
| Anti HT2-280 | Terracebiotech | TB-27AHT2-280 | 1:500 |
| FOXJ1 Monoclonal Antibody (2A5) | Thermo Fisher Scientific | 14-9965-82 | 1:300 |
| Human Uteroglobin/SCGB1A1 Antibody | R and D systems | MAB4218 | 1:300 |
| Keratin 5 Polyclonal Chicken Antibody, Purified [Poly9059] | Biolegend | 905901 | 1:500 |
| MUC5AC Monoclonal Antibody (45M1) | Thermo Fisher Scientific | MA5-12178 | 1:300 |
| PDPN / Podoplanin Antibody (clone 8.1.1) | LifeSpan Biosciences | LS-C143022-100 | 1:300 |
| Purified Mouse Anti-E-Cadherin | BD biosciences | 610182 | 1:1000 |
| Sox-2 Antibody | Santa Cruz biotechnologies | sc-365964 | 1:300 |
| Secondary Antibodies | |||
| Donkey anti-rabbit lgG, 488 | Thermo Fisher Scientific | A-21206 | 1:500 |
| FITC anti-mouse IgM Antibody | BioLegend | 406506 | 1:500 |
| Goat anti-Hamster IgG (H+L), Alexa Fluor 594 | Thermo Fisher Scientific | A-21113 | 1:500 |
| Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | A-21121 | 1:500 |
| Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | A-21131 | 1:500 |
| Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 | Thermo Fisher Scientific | A-21134 | 1:500 |
| Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 | Thermo Fisher Scientific | A-21144 | 1:500 |
| Buffers | |||
| Immunohistochemistry Blocking Solution | 3% BSA, o.4% Triton-x100 in TBS (Tris based saline) | ||
| Immunohistochemistry Incubation Solution | 3% BSA, ).1% Triton-X100 in TBS | ||
| Immunohistochemistry Washing Solution | TBS with 0.1% Tween 20 | ||
Referências
- Rackley, C. R., Stripp, B. R. Building and maintaining the epithelium of the lung. Journal of Clinical Investigation. 122 (8), 2724-2730 (2012).
- Montoro, D. T., et al. A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature. 560 (7718), 319-324 (2018).
- Plasschaert, L. W., et al. A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature. 560 (7718), 377-381 (2018).
- Barkauskas, C. E., et al. Type 2 alveolar cells are stem cells in adult lung. Journal of Clinical Investigation. 123 (7), 3025-3036 (2013).
- Barkauskas, C. E., et al. Lung organoids: current uses and future promise. Development. 144 (6), 986-997 (2017).
- Leeman, K. T., Fillmore, C. M., Kim, C. F. Lung Stem and Progenitor Cells in Tissue Homeostasis and Disease. Stem Cells in Development and Disease. 107, 207-233 (2014).
- Rawlins, E. L., et al. The Role of Scgb1a1(+) Clara Cells in the Long-Term Maintenance and Repair of Lung Airway but Not Alveolar, Epithelium. Cell Stem Cell. 4 (6), 525-534 (2009).
- Rock, J. R., et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proceedings of the National Academy of Sciences of the United States of America. 106 (31), 12771-12775 (2009).
- Chang, W. I., et al. Bmp4 is essential for the formation of the vestibular apparatus that detects angular head movements. Plos Genetics. 4 (4), 1000050 (2008).
- McQualter, J. L., Bertoncello, I. Concise Review: Deconstructing the Lung to Reveal Its Regenerative Potential. Stem Cells. 30 (5), 811-816 (2012).
- Gonzalez, R. F., Allen, L., Gonzales, L., Ballard, P. L., Dobbs, L. G. HTII-280, a Biomarker Specific to the Apical Plasma Membrane of Human Lung Alveolar Type II Cells. Journal of Histochemistry & Cytochemistry. 58 (10), 891-901 (2010).
- Rock, J. R., et al. Notch-Dependent Differentiation of Adult Airway Basal Stem Cells. Cell Stem Cell. 8 (6), 639-648 (2011).
- Page, H., Flood, P., Reynaud, E. G. Three-dimensional tissue cultures: current trends and beyond. Cell and Tissue Research. 352 (1), 123-131 (2013).
- Hynds, R. E., Giangreco, A. Concise Review: The Relevance of Human Stem Cell-Derived Organoid Models for Epithelial Translational Medicine. Stem Cells. 31 (3), 417-422 (2013).
- Lancaster, M. A., Knoblich, J. A. Organogenesis in a dish: Modeling development and disease using organoid technologies. Science. 345 (6194), (2014).
- Weber, C. Organoids test drug response. Nature Cell Biology. 20 (6), 634 (2018).
- Fatehullah, A., Tan, S. H., Barker, N. Organoids as an in vitro model of human development and disease. Nature Cell Biology. 18 (3), 246-254 (2016).
- Nikolic, M. Z., Rawlins, E. L. Lung Organoids and Their Use To Study Cell-Cell Interaction. Current Pathobiology Reports. 5 (2), 223-231 (2017).
- Sato, T., et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology. 141 (5), 1762-1772 (2011).
- Reynolds, B. A., Rietze, R. L. Neural stem cells and neurospheres–re-evaluating the relationship. Nature Methods. 2 (5), 333-336 (2005).
- Chua, C. W., et al. Single luminal epithelial progenitors can generate prostate organoids in culture. Nature Cell Biology. 16 (10), 951-961 (2014).
- Teisanu, R. M., et al. Functional analysis of two distinct bronchiolar progenitors during lung injury and repair. American Journal of Respiratory and Cellular Molecular Biology. 44 (6), 794-803 (2011).
- Chen, H., et al. Airway epithelial progenitors are region specific and show differential responses to bleomycin-induced lung injury. Stem Cells. 30 (9), 1948-1960 (2012).
- Benam, K. H., et al. Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro. Nature Methods. 13 (2), 151-157 (2016).
- Huh, D., et al. Reconstituting organ-level lung functions on a chip. Science. 328 (5986), 1662-1668 (2010).
- Jain, A., et al. Primary Human Lung Alveolus-on-a-chip Model of Intravascular Thrombosis for Assessment of Therapeutics. Clinical Pharmacology & Therapeutics. 103 (2), 332-340 (2018).
- Mulay, A., et al. SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery. bioRxiv. , (2020).
Citar este artigo
Konda, B., Mulay, A., Yao, C., Beil, S., Israely, E., Stripp, B. R. Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture. J. Vis. Exp. (161), e61541, doi:10.3791/61541 (2020).