从诱导多能干细胞生成3D全肺类器官，用于模拟肺发育生物学和疾病
Summary
本文描述了诱导多能干细胞与含有近端和远端上皮肺细胞以及间充质的三维全肺类器官的逐步定向分化。
Abstract
由于缺乏生物学相关的 体外 模型系统，人类肺部发育和疾病一直难以研究。人类诱导多能干细胞（hiPSCs）可以逐步分化成3D多细胞肺类器官，由上皮细胞群和间充质细胞群组成。我们通过暂时引入各种生长因子和小分子来概括胚胎发育线索，以有效地产生明确的内胚层，前颈内胚层，以及随后的肺祖细胞。然后将这些细胞嵌入生长因子减少（GFR）基底膜基质培养基中，使它们能够响应外部生长因子自发发育成3D肺类器官。这些全肺类器官（WLO）在暴露于地塞米松，环AMP和异丁基黄嘌呤后经历早期肺部发育阶段，包括分支形态发生和成熟。WLO具有表达标志物KRT5（基底），SCGB3A2（俱乐部）和MUC5AC（杯）的气道上皮细胞以及表达HOPX（肺泡I型）和SP-C（肺泡II型）的肺泡上皮细胞。还存在间充质细胞，包括平滑肌肌动蛋白（SMA）和血小板衍生的生长因子受体A（PDGFRα）。iPSC衍生的WLO可以在3D培养条件下维持数月，并且可以分类为表面标记物以纯化特定的细胞群。iPSC衍生的WLO也可用于研究人肺发育，包括肺上皮和间充质之间的信号传导，模拟人肺细胞功能和发育的基因突变，并确定感染因子的细胞毒性。
Introduction
肺是一个复杂的、异质的、动态的器官，在六个不同的阶段发育 – 胚胎期、假性腺体、小管性、囊状、肺泡和微血管成熟1，2。后两个阶段发生在人类发育的前后阶段，而前四个阶段只发生在胎儿发育过程中，除非发生早产3。胚胎期从内胚层开始，以气管和肺芽的萌芽结束。肺部发育部分通过上皮细胞和间充质细胞之间的信号传导发生4。这些相互作用导致肺分支，增殖，细胞命运决定和发育中肺的细胞分化。肺分为传导区（气管到末端细支气管）和呼吸区（呼吸道细支气管到肺泡）。每个区域包含独特的上皮细胞类型;包括传导气道中的基底细胞、分泌细胞、纤毛细胞、刷状细胞、神经内分泌细胞和离子细胞5，其次是呼吸道上皮中的肺泡 I 型和 II 型细胞6。关于各种细胞类型的发育和对损伤的反应，还有很多未知数。iPSC衍生的肺类器官模型能够研究驱动人类肺部发育的机制，基因突变对肺功能的影响，以及上皮和间充质对感染因子的反应，而无需原发性人体肺组织。
与胚胎分化各个阶段相对应的标志物包括用于确定性内胚层 （DE）7 的 CXCR4、cKit、FOXA2 和 SOX17、用于前前肠内胚层 （AFE）8 的 FOXA2、TBX1 和 SOX2，以及用于早期肺祖细胞的 NKX2-19。在胚胎肺发育中，前肠分为背食道和腹侧气管。右肺和左肺的芽表现为气管芽周围的两个独立的外垂10。在分支形态发生过程中，上皮周围的间充质产生弹性组织、平滑肌、软骨和脉管系统11。上皮和间充质之间的相互作用对于正常的肺部发育至关重要。这包括间充质分泌的FGF1012和上皮产生的SHH13。
在这里，我们描述了一种将hiPSCs定向分化为三维（3D）全肺类器官（WLO）的方案。虽然有类似的方法通过LPC阶段的分选分离肺祖细胞，以制造肺泡样14，15 （远端）类器官或气道16 （近端）类器官，或产生腹侧前肠球状体，使人肺类器官表达肺泡细胞和间充质标志物以及芽尖祖器官17，这种方法的优势在于包括肺上皮细胞和间充质细胞类型，以在 体外模式和协调肺分支形态发生，成熟和扩增。
该协议使用小分子和生长因子来指导多能干细胞通过确定性内胚层，前叶内胚层和肺祖细胞的分化。然后，这些细胞通过重要的发育步骤（包括分支和成熟）被诱导成3D全肺类器官。分化方案的摘要如图 1a 所示，内胚层和类器官分化的代表性明场图像如图 1b所示。 图1c、d 显示了完成分化后内胚层分化的基因表达细节以及肺上皮细胞近端和远端群体的基因表达。
Protocol
该研究方案由UCSD人类研究保护计划机构审查委员会批准（181180）。 1. 诱导多能干细胞的确定性内胚层诱导（第1-3天） 使用前30分钟在冰上缓慢解冻生长因子减少（GFR）基底膜（BM）基质培养基。在冷的DMEM/F12混合物中，以1∶1的比例稀释GFR BM基质培养基，使其构成该培养基的50%。使用前将 P1000 移液器吸头放入冰箱中冷藏。 用在冰冷的DMEM / F12中制备的500μL50%GFR?…
Representative Results
电镀后24小时，第1天，iPSCs应为50%-90%汇合。在第 2 天，DE 应为 90%-95% 汇合。在DE诱导期间，通常在第4天观察到显着的细胞死亡，但附着的细胞将保留紧凑的鹅卵石形态（图2b）。如果大多数贴壁细胞分离，则停止分化，并考虑将活性素A的DE培养基暴露时间缩短6-12小时。在AFE诱导期间，细胞死亡是最小的，细胞保持贴壁，但会显得更小，更异质。只有当双阳性SOX2和FOXA2的产量>…
Discussion
3D全肺类器官（WLO）的成功分化依赖于多步骤，6周的方案，并注重细节，包括暴露于生长因子和小分子的时间，传代后的细胞密度以及hiPSCs的质量。故障排除请参见 表2。hiPSCs 应约为 70%-80% 汇合，解离前自发分化小于 5%。该协议要求”mTeSR+”介质;然而，普通的”mTeSR”培养基也被使用，结果相当，而且价格更便宜。对于细胞外基质，我们使用GFR基底膜基质培养基质。我们使用ReLesR（见 <stron…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了加州再生医学研究所（CIRM）（DISC2-COVID19-12022）的支持。
Materials
| Cell Culture | |||
| 12 well plates | Corning | 3512 | |
| 12-well inserts, 0.4um, translucent | VWR | 10769-208 | |
| 2-mercaptoethanol | Sigma-Aldrich | M3148 | |
| Accutase | Innovative Cell Tech | AT104 | |
| ascorbic acid | Sigma | A4544 | |
| B27 without retinoic acid | ThermoFisher | 12587010 | |
| Bovine serum albumin (BSA) Fraction V, 7.5% solution | Gibco | 15260-037 | |
| Dispase | StemCellTech | 7913 | |
| DMEM/F12 | Gibco | 10565042 | |
| FBS | Gibco | 10082139 | |
| Glutamax | Life Technologies | 35050061 | |
| Ham’s F12 | Invitrogen | 11765-054 | |
| HEPES | Gibco | 15630-080 | |
| Iscove’s Modified Dulbecco’s Medium (IMDM) + Glutamax | Invitrogen | 31980030 | |
| Knockout Serum Replacement (KSR) | Life Technologies | 10828028 | |
| Matrigel | Corning | 354230 | |
| Monothioglycerol | Sigma | M6145 | |
| mTeSR plus Kit (10/case) | Stem Cell Tech | 5825 | |
| N2 | ThermoFisher | 17502048 | |
| NEAA | Life Technologies | 11140050 | |
| Pen/strep | Lonza | 17-602F | |
| ReleSR | Stem Cell Tech | 5872 | |
| RPMI1640 + Glutamax | Life Technologies | 12633012 | |
| TrypLE | Gibco | 12605-028 | |
| Y-27632 (Rock Inhibitor) | R&D Systems | 1254/1 | |
| Growth Factors/Small Molecules | |||
| Activin A | R&D Systems | 338-AC | |
| All-trans retinoic acid (RA) | Sigma-Aldrich | R2625 | |
| BMP4 | R&D Systems | 314-BP/CF | |
| Br-cAMP | Sigma-Aldrich | B5386 | |
| CHIR99021 | Abcam | ab120890 | |
| Dexamethasone | Sigma-Aldrich | D4902 | |
| Dorsomorphin | R&D Systems | 3093 | |
| EGF | R&D Systems | 236-EG | |
| FGF10 | R&D Systems | 345-FG/CF | |
| FGF7 | R&D Systems | 251-KG/CF | |
| IBMX (3-Isobtyl-1-methylxanthine) | Sigma-Aldrich | I5879 | |
| SB431542 | R&D Systems | 1614 | |
| VEGF/PIGF | R&D Systems | 297-VP/CF | |
| Primary antibodies | Dilution rate | ||
| CXCR4-PE | R&D Systems | FAB170P | 1:200 (F) |
| HOPX | Santa Cruz Biotech | sc-398703 | 0.180555556 |
| HTII-280 | Terrace Biotech | TB-27AHT2-280 | 0.145833333 |
| KRT5 | Abcam | ab52635 | 0.180555556 |
| NKX2-1 | Abcam | ab76013 | 0.25 |
| NKX2-1-APC | LS-BIO | LS-C264437 | 1:1000 (F) |
| proSPC | Abcam | ab40871 | 0.215277778 |
| SCGB3A2 | Abcam | ab181853 | 0.25 |
| SOX2 | Invitrogen | MA1-014 | 0.180555556 |
| SOX9 | R&D Systems | AF3075 | 0.180555556 |
| SPB (mature) | 7 Hills | 48604 | 1: 1500 (F) 1:500 (W)a |
| SPC (mature) | LS Bio | LS-B9161 | 1:100 (F); 1:500 (W) a |
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Cite This Article
Leibel, S. L., McVicar, R. N., Winquist, A. M., Snyder, E. Y. Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease. J. Vis. Exp. (170), e62456, doi:10.3791/62456 (2021).