Name: generation of 3d whole lung organoids from induced pluripotent stem cells for modeling lung developmental biology and disease
Uploaded: 2021-04-12T15:00:00
Description: Watch this Scientific Journal Video about Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease at JoVE.com

这项研究得到了加州再生医学研究所（CIRM）（DISC2-COVID19-12022）的支持。

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）
<ol><li>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。</li>
	<li>用在冰冷的DMEM / F12中制备的500μL50%GFR?...

<ol>
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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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12 well plates</td>
			<td>Corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well inserts, 0.4um, translucent</td>
			<td>VWR</td>
			<td>10769-208</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Tech</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 without retinoic acid</td>
			<td>ThermoFisher</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) Fraction V, 7.5% solution</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>StemCellTech</td>
			<td>7913</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>10565042</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10082139</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;s F12</td>
			<td>Invitrogen</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#8217;s Modified Dulbecco&#8217;s Medium (IMDM) + Glutamax</td>
			<td>Invitrogen</td>
			<td>31980030</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement (KSR)</td>
			<td>Life Technologies</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Monothioglycerol</td>
			<td>Sigma</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR plus Kit (10/case)</td>
			<td>Stem Cell Tech</td>
			<td>5825</td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>ThermoFisher</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/strep</td>
			<td>Lonza</td>
			<td>17-602F</td>
			<td></td>
		</tr>
		<tr>
			<td>ReleSR</td>
			<td>Stem Cell Tech</td>
			<td>5872</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 + Glutamax</td>
			<td>Life Technologies</td>
			<td>12633012</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Gibco</td>
			<td>12605-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 (Rock Inhibitor)</td>
			<td>R&#38;D Systems</td>
			<td>1254/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth Factors/Small Molecules</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&#38;D Systems</td>
			<td>338-AC</td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans retinoic acid (RA)</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-BP/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Br-cAMP</td>
			<td>Sigma-Aldrich</td>
			<td>B5386</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Abcam</td>
			<td>ab120890</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>R&#38;D Systems</td>
			<td>3093</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>R&#38;D Systems</td>
			<td>236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>R&#38;D Systems</td>
			<td>345-FG/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF7</td>
			<td>R&#38;D Systems</td>
			<td>251-KG/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX (3-Isobtyl-1-methylxanthine)</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>R&#38;D Systems</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF/PIGF</td>
			<td>R&#38;D Systems</td>
			<td>297-VP/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td>Dilution rate</td>
		</tr>
		<tr>
			<td>CXCR4-PE</td>
			<td>R&#38;D Systems</td>
			<td>FAB170P</td>
			<td>1:200 (F)</td>
		</tr>
		<tr>
			<td>HOPX</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-398703</td>
			<td>0.180555556</td>
		</tr>
		<tr>
			<td>HTII-280</td>
			<td>Terrace Biotech</td>
			<td>TB-27AHT2-280</td>
			<td>0.145833333</td>
		</tr>
		<tr>
			<td>KRT5</td>
			<td>Abcam</td>
			<td>ab52635</td>
			<td>0.180555556</td>
		</tr>
		<tr>
			<td>NKX2-1</td>
			<td>Abcam</td>
			<td>ab76013</td>
			<td>0.25</td>
		</tr>
		<tr>
			<td>NKX2-1-APC</td>
			<td>LS-BIO</td>
			<td>LS-C264437</td>
			<td>1:1000 (F)</td>
		</tr>
		<tr>
			<td>proSPC</td>
			<td>Abcam</td>
			<td>ab40871</td>
			<td>0.215277778</td>
		</tr>
		<tr>
			<td>SCGB3A2</td>
			<td>Abcam</td>
			<td>ab181853</td>
			<td>0.25</td>
		</tr>
		<tr>
			<td>SOX2</td>
			<td>Invitrogen</td>
			<td>MA1-014</td>
			<td>0.180555556</td>
		</tr>
		<tr>
			<td>SOX9</td>
			<td>R&#38;D Systems</td>
			<td>AF3075</td>
			<td>0.180555556</td>
		</tr>
		<tr>
			<td>SPB (mature)</td>
			<td>7 Hills</td>
			<td>48604</td>
			<td>1: 1500 (F) 1:500 (W)a</td>
		</tr>
		<tr>
			<td>SPC (mature)</td>
			<td>LS Bio</td>
			<td>LS-B9161</td>
			<td>1:100 (F); 1:500 (W) a</td>
		</tr>
	</tbody>
</table>

generation of 3d whole lung organoids from induced pluripotent stem cells for modeling lung developmental biology and disease

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。

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>...

Watch this Scientific Journal Video about Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease at JoVE.com

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

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。

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

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

该协议使用诱导多能干细胞将它们分化为肺细胞，以便在培养皿中模拟人类肺部疾病和发育。该协议意义重大，因为它很难获得和培养原代人肺组织。将肺细胞与多能干细胞区分开来的主要优点是具有恒定的特定肺细胞供应，以研究人的肺部发育和疾病。这些细胞可以在细胞培养物中长时间维持，可以冷冻保存以备将来应用，并且可以进行遗传操纵以研究基因突变。演示该过程的将是Rachel McVicar，我实验室的博士候选人。在开始实验之前，在冰上缓慢解冻生长因子还原或GFR基底膜基质培养基，并将其稀释在等体积的冷DMEM F12中。使用前将 P1000 吸头放入冰箱中冷藏。接下来，用500微升的50%GFR基底膜基质培养基涂12孔板的每个孔，并从孔中除去任何多余的介质混合物和气泡。将孔板放在湿冰或冰箱上，以4摄氏度的温度凝固20分钟，然后将孔板移至37摄氏度的培养箱过夜。当高PSE达到70%汇合度时，向人诱导多能干细胞中加入10微摩尔ROCK抑制剂Y27632，并在解离前等待一小时。吸出培养基，并用PVS洗涤细胞一次。通过每孔添加500微升细胞分离培养基来解离IPSC，并在5%二氧化碳培养箱中以37摄氏度孵育20分钟。孵育后，每孔加入500微升干细胞传代培养基。使用P1000吸头轻轻移液细胞以获得单细胞悬浮液。然后将解离的细胞转移到15毫升管中，并在300倍G下离心5分钟，离心后，吸出培养基，并用一毫升mTeSR Plus培养基重悬细胞沉淀，并补充10微摩尔ROCK抑制剂。计数细胞后，向12孔GFR介质包衣板的每个孔中加入两次10到第五IPSC，并在37摄氏度下孵育过夜。第二天，在添加确定性内胚层或DE诱导培养基之前，先吸走mTeSR Plus。在第二天和第三天，更换DE感应培养基。第 4 天，通过用无血清基础培养基替代 DE 诱导培养基开始 AFE 诱导。每天更换AFE培养基三天，然后在第六天结束时分析AFE效率。在第七天，在冰上解冻GFR基底膜基质培养基体供以后使用。同时，通过吸入AFE培养基并用PVS洗涤孔来进行肺祖细胞分化。向孔中加入一毫升细胞分离溶液，并在37摄氏度下孵育10分钟。孵育后，向含有细胞分离溶液的孔中加入一毫升淬灭培养基。通过轻轻地上下移液来保持细胞为聚集体。确保所有细胞都被移位，但将它们转移到15毫升的锥形管中，以300倍G离心五分钟。除去上清液并将细胞沉淀重悬于LPC诱导培养基中。计数细胞，并向第五个细胞加入2.5次10到100微升冷的GFR基底膜基质培养基中，并充分混合。将液滴放入12孔板的孔中，并在37摄氏度下孵育30至60分钟。接下来，每孔添加一毫升LPC培养基，确保培养基液滴完全浸没并在37摄氏度下孵育过夜。在第八天，更换LPC培养基以除去ROCK抑制剂Y27632。每隔一天更换一次介质，并在第16天结束时分析LPC效率。在第17天，洗孔并加入500微升，每毫升分散酶2微克。用P1000移液器移取分散酶混合物并孵育15分钟，然后移取混合物并再孵育15分钟。孵育后，将一毫升淬火培养基加入含有孔的解酶中，并将细胞转移到锥形管中。离心并将沉淀重悬于一毫升冷却的PVS中，然后重复离心。然后将沉淀重悬于一毫升细胞分离培养基中。将细胞在37摄氏度下孵育12分钟，然后加入等体积的淬灭培养基并再次离心。将沉淀重悬于一毫升淬火培养基和10微摩尔ROCK抑制剂Y27632中。执行细胞计数以计算获得每孔八次10至第四个细胞所需的体积。然后将所需体积的LPC细胞聚集体等分到1.5毫升微量离心管中，并以300倍G离心5分钟，除去多余的上清液而不搅动细胞沉淀，留下10微升残留介质。将细胞沉淀重悬于200微升的冷GFR基底膜基质培养基中，并将细胞转移到细胞培养膜插入物中。在37摄氏度下孵育30至60分钟。孵育后，将一毫升3D类器官诱导培养基加入插入物的基底外侧腔室，并每隔一天用新鲜培养基替换一次，持续六天。在第 23 天，将培养基切换到 3D 分支培养基，然后每隔一天更换一次培养基，持续六天。在第29天，更换为3D成熟培养基，并在接下来的六天内每隔一天继续更换培养基。在DE诱导的第四天，附着的细胞显示出紧凑的鹅卵石形态。表达了覆盖有细胞核的明确内胚层标志物 CXCR4 和 SOX17，证实了内胚层分化。第七天确认了前肠前部诱导，形态显示细胞更紧密地压实，并且标记FOXA2和SOX2覆盖有细胞核。用3D球体证实了3D肺祖细胞的产生，以及NKX2-1 GFP在报告细胞系中的内源性表达。经过三周的全肺类器官分化后，通过免疫细胞化学分析肺标志物。在类器官中观察到由细胞核覆盖的分支形态发生SOX2和SOX9的标志物。近端肺标志物P63和KRT5，两者都是基底细胞的标志物，与SCGB3A2一起被成功检测，SCGB3A2是俱乐部细胞的标志物。远端肺标志物诱导肺泡II型细胞的Pro SPC和SPB标志物。和肺泡I型细胞的HOPX标志物。此外，NKX2-1和ZO1与原子核叠加表达。间充质肺细胞标志物PGFR Alpha是成纤维细胞的标志物，与SOX9共同表达，代表远端间充质。Vimentin也被表达并分散在整个肺部。在此程序之后，肺类器官可以与诱导多能干细胞衍生的内皮和免疫细胞一起投资。肺发育和疾病通过上皮细胞群和间充质细胞群之间的信号传导发生，但也来自内皮细胞和巨噬细胞。3D肺组织模型系统与研究人类疾病和治疗方法具有临床相关性。

Research

JoVE Journal

Developmental Biology

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

诱导多能干细胞使用成纤维样滑膜生成类风湿关节炎患者的关节分离

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

科研

发育生物学

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

从血细胞使用仙台病毒和离心诱导多能干细胞代

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

脐带血诱导的多能干细胞的软骨形成颗粒形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

部分小叶切除术: 形态学肝再生的外科模型

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...