Name: generation of scaffold-free, three-dimensional insulin expressing pancreatoids from mouse pancreatic progenitors in vitro
Uploaded: 2018-06-02T14:00:00
Description: Watch this Scientific Journal Video about Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro at JoVE.com

我们感谢 Jolanta Chmielowiec 就该协议和手稿提供了有益的讨论。我们还感谢本杰明 Arenkiel 获得共聚焦显微镜。这项工作得到了 NIH (P30-DK079638 到手机和 T32HL092332-13 到 M.A.S. 和手机)、捷尔医学基金会 (手机) 以及在医疗知识和发展残疾研究中心的共焦核心 (nih U54 HD083092 的支持)。肯尼迪国家儿童健康和人类发展研究所)。

该方法所描述的所有动物实验均经贝勒医学院机构动物护理和使用委员会批准。
1. 小鼠胚胎日10.5 胰祖细胞的制备
注: 本协议不需要在无菌条件下遵循, 直到步骤 2, 但它是最佳的消毒解剖工具和喷雾与70% 乙醇之前使用。
<ol><li>为解剖, 填充冰桶和放置一个容器磷酸盐缓冲盐水 (PBS) 在冰。清洁两个细尖钳和解剖剪刀与70% 乙醇。设置在解剖区附近, 一个容?...

<ol>
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细胞培养模型的进展对于正确的模型开发, 产生临床相关的细胞类型, 测试药物疗效, 甚至移植到病人来说是至关重要的。然而, 人工综述在菜肴中的发展是有挑战性的, 因为我们还远未了解器官和生理的机制在体内.因此,体外细胞的生成效率低下, 不能充分发挥作用, 不能长时间维持, 或使体内可比细胞的其他异常发生。这是因为许多不同的细胞类型相互作用, 而复杂的形态发生变化影?...

界定正常发展和生理学的机制, 对于了解疾病病因, 最终培养治疗方法至关重要。虽然培养和区分干细胞能够快速和高通量地分析发展, 但它受现有的知识机构的限制, 这些机制调控细胞命运和人为概括发展在一个相对同构, 二维状态1,2。不仅是受外部影响影响的 "体内" 开发, 在利基和环境中提供分泌信号和组织支持的不同单元格类型也可用于引导器官, 但这些细胞的功能也依赖于其用于指导的环境3,4,5。鉴于这些外部线索的重要性, 差异化协议的局限性, 以及体内老鼠模型的费力性质, 需要新的系统来实验研究基本的发育过程和生理学。
生成三维复杂 organoids 的协议的出现, 提供了一个方便和一致的系统来研究器官发生、生理学、药物功效, 甚至发病机制.建立小鼠 organoids 为不同的系统, 如胃6和肠道7已经扩大了我们对器官的理解, 提供了一个工具来研究发展复杂性较少的限制比在体内和体外模型。由于这些进展在小鼠 organoid 形成和人类多潜能干细胞的出现, 人类肠道8, 视网膜9, 肾脏10,11, 和大脑12 organoids 已经产生, 这《汇辑》仅限于现有关于发展机制的知识。
特别感兴趣的是胰腺 organoids 的产生, 因为无数的疾病瘟疫不同的胰腺细胞类型, 包括腺泡细胞和导管在外分泌的胰腺功能不全13, 胰腺炎的腺泡细胞14, 并β细胞在糖尿病15。获得有关这些不同细胞类型发展的知识有助于理解其病理学, 也可以作为个性化药物筛选或移植的平台。以前, Greggio et开发了一种方法来创建小鼠胰腺 organoids, 它概括了体内形态发生并发展了由所有主要的胰腺上皮细胞组成的三维复杂结构。类型16,17。这是胰腺领域向前迈出的重要一步, 特别是使细胞体外能够使β细胞的生物研究得以发展。然而, 在本议定书中形成的内分泌细胞的匮乏, 除非 organoids 移植到组织, 在那里利基可以互动和提供指导提示17。间质构成利基的最大部分, 严重包裹发育上皮从器官的早期阶段到以后阶段, 包括内分泌分层和分化 3, 4, 18。间质与发展中的胰腺的相互作用又是外部信号的另一个例子, 也是维持体内细胞复杂性来研究器官的重要性. 
在这里, 我们描述如何产生三维胰腺 organoids, 称为 pancreatoids, 从分离 e10.5 小鼠胰腺祖细胞。这些 pancreatoids 保留本机间质, 自组装在自由漂浮条件, 并产生所有主要的胰腺细胞类型, 包括强健的数量的内分泌β细胞 19.这种方法最适合分析内分泌的发展, 因为以前的协议缺乏强健的内分泌分化。然而, 使用 Greggio et所描述的胰 organoids 的协议, 更适合于分析胰腺上皮分支和形态发生, 因为 pancreatoids 中分支更受限制。

<table border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">2-Mercaptoethanol</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">M6250</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Aspirator Tube Assemblies for Calibrated Microcapillary Pipettes</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">A5177</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BarnStead NanoPure Nuclease-free water</td>
			<td>ThermoFisher</td>
			<td>D119</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Borosilicate Capillary Tubes</td>
			<td style="width:123px;">Sutter Instruments</td>
			<td style="width:344px;">GB1007515</td>
			<td style="width:167px;">O.D. 1mm, I.D. 0.75mm, 1.5cm length</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">CaCl2</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">C5080</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Cell-Repellent 96-Well Microplate</td>
			<td style="width:123px;">Greiner Bio-One</td>
			<td style="width:344px;">650970</td>
			<td style="width:167px;">U-bottom</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Centrifuge 5424 R</td>
			<td>Eppendorf</td>
			<td>5401000013</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Chloroform</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">233306</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chromogranin-A antibody</td>
			<td>Abcam</td>
			<td>ab15160</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Compact, Modular Stereo Microscope M60</td>
			<td style="width:123px;">Leica</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Countess Automated Cell Counter</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">C10310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Countess Cell Counter Slides</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">C10312</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CryoStar NX70</td>
			<td>ThermoFisher</td>
			<td>957000L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">D-(+)-Glucose</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">G7528</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DAPI (4&#39;,6-Diamidine-2&#39;-phenylindole-dihydrochloride)</td>
			<td style="width:123px;">Roche</td>
			<td style="width:344px;">10 236 276 001</td>
			<td style="width:167px;">Powder</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DBA antibody</td>
			<td>Vector Lab</td>
			<td>RL-1032</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dispase II, Powder</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:344px;">17105041</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DMEM/F-12, HEPES</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:344px;">11330032</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dnase I</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">18068-015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dumont #5 Forceps</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:344px;">11251-10</td>
			<td style="width:167px;">0.05 x 0.02 mm; Titanium; Biology tip</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">EGF (Epidermal growth factor)</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">E9644</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Ethanol, 200 Proof</td>
			<td style="width:123px;">Decon Laboratories</td>
			<td style="width:344px;">2716</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Forma Steri Cycle CO2 Incubators</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:344px;">370</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Fluoromount-G</td>
			<td style="width:123px;">Southern Biotech</td>
			<td style="width:344px;">OB10001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Heparin sodium salt from porcine intestinal mucosa</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">H3149-10KU</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">INSM1 Antibody</td>
			<td style="width:123px;">Santa Cruz BioTechnology</td>
			<td style="width:344px;">sc-271408</td>
			<td style="width:167px;">Polyclonal Mouse IgG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Isopropanol</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:344px;">a4164</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isothesia Isoflurane, USP</td>
			<td>Henry Schein</td>
			<td>11695-6776-2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Insulin Antibody</td>
			<td style="width:123px;">Dako</td>
			<td style="width:344px;">A056401</td>
			<td style="width:167px;">Polyclonal Guinea Pig</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">KAPA SYBR FAST Universal</td>
			<td style="width:123px;">KAPA Biosystems</td>
			<td style="width:344px;">KK4618</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">KCl</td>
			<td style="width:123px;">KaryoMax</td>
			<td>10575090</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">KnockOut Serum Replacement</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">10828028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Leica TCS SPE High-Resolution Spectral Confocal</td>
			<td style="width:123px;">Leica</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">MgCl2</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">442615</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Mouse C-Peptide ELISA</td>
			<td style="width:123px;">ALPCO</td>
			<td style="width:344px;">80-CPTMS-E01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Mouse Ultrasensitive Insulin ELISA</td>
			<td style="width:123px;">ALPCO</td>
			<td style="width:344px;">80-INSMSU-E01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">MX35 Microtome Blades</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:344px;">3052835</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NaCl</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">S7653</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NaHCO3</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">S3817</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NaH2PO4</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Normal Donkey Serum</td>
			<td style="width:123px;">Jackson Immuno Research</td>
			<td style="width:344px;">017-000-121</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Paraformaldehyde</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">158127</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">PBS 1X</td>
			<td style="width:123px;">Corning</td>
			<td style="width:344px;">21-040-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pdx1 antibody</td>
			<td>DSHB</td>
			<td>F6A11</td>
			<td>Monoclonal Mouse MIgG1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Peel-A-Way Disposable Embedding Molds</td>
			<td style="width:123px;">VWR</td>
			<td style="width:344px;">15160-157</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Penicillin-Streptomycin Solution</td>
			<td style="width:123px;">Corning</td>
			<td style="width:344px;">MT30002CI</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">PMA (Phorbol 12-Myristate 13-Acetate)</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">P1585</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Protein LoBind Microcentrifuge Tubes</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:344px;">22431081</td>
			<td style="width:167px;">1.5mL Capacity</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Recombinant Human FGF-10 Protein</td>
			<td style="width:123px;">R&#38;D Systems</td>
			<td style="width:344px;">345-FG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Recombinant Human FGF-Acidic</td>
			<td style="width:123px;">Peprotech</td>
			<td style="width:344px;">100-17A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Recombinant Human R-Spondin I Protein</td>
			<td style="width:123px;">R&#38;D Systems</td>
			<td style="width:344px;">4546-RS</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">BenchRocker 2D</td>
			<td style="width:123px;">Benchmark</td>
			<td style="width:344px;">BR2000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sucrose 500g</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">S0389</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">SuperFrost Plus Microscope Slides</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:344px;">12-550-15</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Super Pap Pen</td>
			<td style="width:123px;">Electron Microscopy Sciences</td>
			<td style="width:344px;">71310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Thermomixer R</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:344px;">05-412-401</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Tissue Tek O.C.T. Compound</td>
			<td style="width:123px;">Sakura</td>
			<td style="width:344px;">4583</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Triton X-100</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">T8787</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">TRIzol Reagent</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">15596018</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">TrypLE Express</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">12604039</td>
			<td style="width:167px;">(1x), no Phenol Red</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Trypan Blue Stain</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">15250061</td>
			<td style="width:167px;">For cell counting slides</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Trypsin-EDTA (0.05%)</td>
			<td style="width:123px;">Corning</td>
			<td style="width:344px;">25-052-CI</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:123px;">Gibco</td>
			<td>25200072</td>
			<td style="width:167px;">Phenol Red</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Ultra-Low Attachment 24-Well Plate</td>
			<td style="width:123px;">Corning</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Ultra-Low Attachment Spheroid Plate 96-Well</td>
			<td style="width:123px;">Corning</td>
			<td>4520</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Vimentin Antibody</td>
			<td style="width:123px;">EMD Millipore</td>
			<td style="width:344px;">AB5733</td>
			<td style="width:167px;">Polyclonal Chicken IgY</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Vortex Genie</td>
			<td style="width:123px;">BioExpres</td>
			<td style="width:344px;">S-7350-1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Y-27632 Dihydrochloride</td>
			<td style="width:123px;">R&#38;D Systems</td>
			<td>1254</td>
			<td style="width:167px;">Also known as ROCK inhibitor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Zeiss 710 Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of scaffold-free, three-dimensional insulin expressing pancreatoids from mouse pancreatic progenitors in vitro

胰腺是由许多不同细胞类型组成的复杂器官, 它们协同工作来调节血糖的稳态和消化。这些细胞类型包括分泌酶的腺泡细胞, 一种 arborized 导管系统, 负责将酶输送到肠道, 以及产生激素的内分泌细胞。
内分泌β细胞是体内唯一的细胞类型, 它能产生胰岛素以降低血糖水平。糖尿病是一种以丧失或β细胞功能障碍为特征的疾病, 正达到流行的程度。因此, 必须建立一些协议来调查β细胞的发展, 它可以用于筛选目的, 以获得药物和细胞的治疗方法。虽然实验性研究老鼠的发展是必不可少的,在体内研究是费力和耗时的。培养细胞为筛查提供了更方便的平台;然而, 他们无法维持细胞多样性, 建筑组织, 和细胞之间的相互作用发现在体内.因此, 有必要开发新的工具来调查胰腺器官和生理。
胰腺上皮细胞在与间质的密切联系中发展, 从器官发生开始, 细胞组织并分化成复杂的、生理上有能力的成体器官。胰腺间质为内分泌的发展提供了重要信号, 其中许多还没有得到很好的理解, 因此在体外文化中很难重述。在这里, 我们描述一个协议, 文化三维, 细胞复杂的老鼠 organoids 保留间质, 称为 pancreatoids。e10.5 小鼠胰芽被解剖, 分离, 并培养在一个无支架的环境。这些漂浮细胞自组装与间质包络的发展 pancreatoid 和强健的数量的内分泌β细胞与腺泡和导管细胞一起发展。该系统可用于研究细胞的命运决定, 结构组织, 和形态发生, 细胞间的相互作用, 在器官发生, 或为药物, 小分子, 或基因筛选。

仔细解剖小鼠胚胎在 e10.5 从子宫角应该产生未损坏的胚胎在 PBS 进一步解剖 (图 1A)。胃肠道可以有效地从胚胎中移除 (图 1B), 允许辨别肠道和胃交界处的背胰芽 (图 1C-F)。e10.5 的胰芽以前有特征;祖祖先应表达Pdx1、Sox9、Ptf1a、和Hes120?...

Watch this Scientific Journal Video about Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro at JoVE.com

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

在这里, 我们提出了一种生成胰岛素表达3D 小鼠 pancreatoids 从游离漂浮 e10.5 分离的胰祖细胞和相关间质的协议。

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

This method could help answer key questions in the pancreatic field by using these pancreatoids to understand how endocrine cells form in an architecturally and cellularly complex environment. The main advantage of this technique is that the pancreatoids develop endocrine cells due to the inclusion of the surrounding mesenchyme. Generally, individuals new to this method will struggle because the pancreas is very small at e10.5.And the dissection is crucial to the formation of pancreatoids to get enough non-contaminating tissue. To set up for dissection, fill an ice bucket and place a container of phosphate buffered saline in the ice. Clean two fine-tip forceps and dissection scissors with 70%ethanol.Near the dissection area, set a container with at least three borosilicate capillary tubes, a lighter, and filtered 1000 microliter pipet tips. Next, pipet at least one milliliter of 1.25 milligram per milliliter cold dispase diluted in sterilized water in the second well of a 12-well plate on ice. Pipet PBS in the first, third, and fourth wells of the 12-well plate.Also, pipet one milliliter of 0.05%trypsin in a 1.5 milliliter tube on ice. After euthanizing e10.5 timed pregnant mice in accordance with IACUC guidelines, spray the abdomen with 70%ethanol to clean the region. Then, make a V-shaped incision at the genital area of the mouse using scissors and forceps and continue the incision up to the diaphragm.Carefully excise the uterus by making an incision at the uppermost lateral portions of the uterus, pulling the organ upwards away from the mouse and following this incision down to the genital region before repeating on the opposite side. Place the excised uterus in a 10 centimeter petri dish with the cold PBS. Under a light dissection microscope, place the petri dish and carefully open the uterine tissue by placing two forceps between each embryo and peeling tissue away from the yolk sac.Transfer the embryos by grasping the yolk sac gently with forceps and placing them in a 10 centimeter petri dish with fresh, cold PBS. Place the petri dish on ice. Next, place multiple PBS drops on the 10 centimeter petri dish lid.Use these droplets to transfer the embryo during dissection as extra-embryonic tissues are removed to ensure visibility. Transfer an embryo to a PBS drop and gently remove it from the yolk sac using forceps while the remaining embryos stay in PBS on ice. Remove the head before moving the embryo to a new PBS drop using forceps to lightly scoop the embryo without damaging the tissue.In a new PBS drop, remove the four limb buds using forceps. Place the forceps into the opening where limb bud was present and gently tear only the most external tissue anteriorly. Rotate the embryo and repeat in the posterior direction, stopping at the hind limb bud.At this point, the gastrointestinal tract should be visible. The posterior region of the gastrointestinal tract has a slight bend;insert forceps behind this bend in the opening between the gastrointestinal tract and the spinal region at the body wall. Detach the gastrointestinal tract, working slowly upwards until the most anterior region is reached, where the cardiac region connects.In the following steps, it is critical to avoid the surrounding tissue in the culture, so as to not contaminate the pancreatoids with non-pancreatic tissue. However, if you do not grab the whole pancreatic bud, then the number and size of the pancreatoids will be impaired. Transfer the gastrointestinal tract to the fresh PBS droplet.Continue to remove the outer tissue surrounding the gastrointestinal tract. Once this outer tissue is removed, the pancreatic bud can be visualized posterior to the liver buds, in between the stomach and intestine. Then, take forceps and gently pinch the tissue beneath the protruding bud in the intestine and slightly lift the pancreatic bud off.After pinching the region where the bud connects to the intestine, the bud is separated. Wash one time in a new PBS bubble before transferring the bud into the first well of the 12-well plate and cold PBS on ice. Repeat until all of the buds are collected from the embryos and placed in the first well of the 12-well plate.Next, place the 12 well dish under the light dissection microscope. Count the number of pancreatic buds successfully dissected to later calculate the split ratio. Place a filtered 1000 microliter pipet tip into the pipet with the capillary tube attached.Flame sterilize the capillary tube and create a bend in the tube for the ease of use. Use the capillary to transfer the buds to the second well containing cold dispase solution for two minutes before transferring to the third well containing clean PBS. Pipet the buds up and down and transfer them to the fourth well containing clean PBS.Finally, using the capillary device, transfer the buds to the 1.5 milliliter tube with 0.05%trypsin. Place the tube in the pre-warmed 37 degrees celsius shaker and shake at 1, 500 RPM for four to five minutes. After vortexing for approximately 10 seconds, immediately place the tube in the centrifuge and spin for five minutes at 200g.Cautiously remove all but approximately 50 microliters of solution as to not disturb the centrifuged cells. For every bud collected at 400 microliters of organogenesis media to the centrifuged cells, pulse vortex three times and plate 100 microliters per well of a low-attachment 96 well plate, splitting buds at a one to four ratio. Check under the microscope to visualize dissociated cells freely floating.Then place the culture dish in a 37 degrees celsius incubator with 5%carbon dioxide on a rocker at medium speed. Careful dissection of mouse embryos at e10.5 from the uterine horn should yield undamaged embryos in PBS for further dissection. The gastrointestinal tract can be efficiently removed from the embryo, permitting discernment of the dorsal pancreatic bud at the junction of the intestine and stomach.In organogenesis media, these free-floating scaffold-free cells self-assemble and organize into three-dimensional pancreatoids that grow and persist for at least 10 days in culture. The application of a protein kinase C activator at high concentrations alters the morphology of developing pancreatoids, leading to loosely associated epithelial cells and increased branching. Additionally, the use of transgenic animals allows visualization of cells as they differentiate in real time.To assess the association of pancreatic mesenchyme tissue with pancreatic epithelial cells, immunostaining can be performed for markers of each tissue. Immunostaining of a duct marker with an endocrine marker and nuclei, reveal multi-lineage formation in pancreatoids. A mesenchyme marker co-stained with a pancreatic progenitor marker shows that the mesenchyme envelops the pancreatoid.Beta cell development is revealed by staining for the epithelial marker and the beta cell marker. Using quantitative PCR analysis, transcripts of progenitors and differentiating cells can be assessed, such as progenitor genes and differentiated markers. Once mastered, this technique can be done in approximately one hour, including prep time, if it is performed properly.After watching this video, you should have a good understanding of how to perform the dissection to obtain pancreatic tissue from 10.5 mice to generate pancreatoids. While attempting this procedure, it's important to remember to carefully remove the GI tract from the embryos, otherwise it will be very difficult to find the pancreatic bud. Following this procedure, other methods like GSIS and ELISA can be performed in order to answer additional questions, like how much insulin or C-peptide is produced after glucose stimulation.After its development, this technique paved the way for us to explore the morphological consequences of PKC activation using small molecules. A way of further implementing this system to study the effect of transcription factor of expression and even to perform important epistasis experiments.

Research

JoVE Journal

Developmental Biology

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

科研

发育生物学

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

牙龈三维炎症人体组织当量

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1+后后福格内胰腺祖细胞

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

人多潜能干细胞第一心场样心脏祖细胞和心室样细胞的生成

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...