Name: a simplified method for generating kidney organoids from human pluripotent stem cells
Uploaded: 2021-04-13T14:00:00
Description: Watch this Scientific Journal Video about A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells at JoVE.com

这项研究由美国国立卫生研究院R01 DK069403，UC2 DK126122和P30-DK079307以及ASN肾脏研究基金会Ben J. Lipps研究奖学金计划资助。

所有使用hPSC的实验均按照机构指南进行，并在II类生物安全罩中进行，并配有适当的个人防护装备。除非另有说明，否则所有试剂均为细胞培养级。将所有培养物在37°C，5%CO2 空气气氛中孵育。在测定的所有阶段，可以收集胚体或肾类器官，并固定或准备用于分析。用于生成此数据的hPSC线已经过完全表征并已发布18。
1. 制备培养板
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<ol>
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先前的研究表明，初始方案步骤对于中间中胚层分化5，19，20 至关重要，因此，在此阶段实施严格的培养基组成至关重要。从方案的第一阶段去除未定义的组分，例如血清，白蛋白，无蛋白杂交瘤培养基II，可能有助于提高测定21之间的一致分化效率。
肾细胞的代谢状态对其功能至关重要...

近年来，已经开发了许多将人类多能干细胞分化为肾类器官的方案1，2，3，4，5。类肾类化合物为研究新的再生医学方法，模拟肾脏相关疾病，进行毒性研究和治疗药物开发提供了重要工具。尽管它们具有广泛的适用性，但肾脏类器官具有局限性，例如缺乏成熟，体外长期培养能力有限，以及缺乏在人肾中发现的几种细胞类型6，7，8。最近的工作重点是通过修改现有方案9，10，11，12来提高类器官成熟水平，延长培养期并扩大肾细胞群的复杂性。在我们已建立的方案5，13的当前迭代中，我们将方案第一阶段的培养基组分修改为补充胰岛素 - 转铁蛋白 - 硒 - 乙醇胺（ITSE），脂质，聚乙烯醇（E5-ILP）和CHIR99021的无血清基础培养基（图1）。这些变化提供了一种完全定义的、无血清的低蛋白培养基，其成分比我们以前的培养基组成5，13少，并且没有额外的生长因子。因此，与我们之前发布的版本相比，第一阶段培养基的制备劳动强度较低，并且可以减少批次之间的差异5。先前的研究表明，胰岛素和转铁蛋白在无血清培养14，15中都很重要，然而，高水平的胰岛素可以抑制中胚层分化16。我们保持了原始方案中规定的低胰岛素水平，并在测定的第二阶段进一步降低了KOSR（含胰岛素）的水平。根据肾类器官形成的其他方案，较低水平的KOSR有利于维持肾组织增殖和分化之间的平衡17。此外，我们还降低了II期培养基13中的葡萄糖浓度。
我们的方法描述了一种用于肾类器官悬浮测定的设置，从原始出版物5，13中所述的初始〜60%融合hPSC 100mm培养板中产生多达约1，000个类器官。该协议可以很容易地扩展到从多个100 mm或150 mm板开始，以进一步增加类器官的数量。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo Fisher</td>
			<td>21-985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-adherence rinsing solution</td>
			<td>STEMCELL Technologies</td>
			<td>7010</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>STEMCELL Technologies</td>
			<td>72054</td>
			<td>10 mM stock in DMSO</td>
		</tr>
		<tr>
			<td>Corning&#160;disposable spinner flasks</td>
			<td>Fisher Scientific</td>
			<td>07-201-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Ultra-Low Attachment 6-well plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-601</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Slow-Speed Stirrers</td>
			<td>Fisher Scientific</td>
			<td>11-495-03</td>
			<td>Multi plate magnetic stirrer for spinner flask culture</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>STEMCELL Technologies</td>
			<td>7923</td>
			<td>Aliquot and freeze</td>
		</tr>
		<tr>
			<td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td>
			<td>Thermo Fisher</td>
			<td>11054020</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 1x, no calcium, no magnesium</td>
			<td>Thermo Fisher</td>
			<td>14-190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg / Oval Stirring Bars</td>
			<td>2mag</td>
			<td>PI20106</td>
			<td></td>
		</tr>
		<tr>
			<td>Excelta General-Purpose Tweezers</td>
			<td>Fisher Scientific</td>
			<td>17-456-103</td>
			<td>Keep sterile in the cell culture hood</td>
		</tr>
		<tr>
			<td>EZBio Single Use Media Bottle, 250mL</td>
			<td>Foxx Life Sciences</td>
			<td>138-3211-FLS</td>
			<td>Used to make PVA 10%</td>
		</tr>
		<tr>
			<td>Falcon Standard Tissue Culture Dishes (100 mm)</td>
			<td>Thermo Fisher</td>
			<td>08-772E</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Sterile Aspirating Pipet 2mL</td>
			<td>Fisher Scientific</td>
			<td>14-955-135</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160; Cell Lifters - Cell lifter</td>
			<td>Fisher Scientific</td>
			<td>08-100-240</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Multi Function 3D Rotators</td>
			<td>Fisher Scientific</td>
			<td>88-861-047</td>
			<td>Orbital shaker</td>
		</tr>
		<tr>
			<td>Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
			<td>Thermo Fisher</td>
			<td>A1413302</td>
			<td>BME. Aliquot on ice and freeze. Another suitable matrix alternative is Matrigel or Cultrex.</td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>STEMCELL Technologies</td>
			<td>7174</td>
			<td>GCDR</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher</td>
			<td>35-050-061</td>
			<td>L-glutamine supplement.</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Thermo Fisher</td>
			<td>15-630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-Ethanolamine</td>
			<td>Thermo Fisher</td>
			<td>51-500-056</td>
			<td>ITSE</td>
		</tr>
		<tr>
			<td>KnockOut&#160; Serum Replacement - Multi-Species</td>
			<td>Thermo Fisher</td>
			<td>A3181502</td>
			<td>KOSR. Aliquot and freeze</td>
		</tr>
		<tr>
			<td>Lipid Mixture 1, Chemically Defined</td>
			<td>Millipore-Sigma</td>
			<td>L0288-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution</td>
			<td>Thermo Fisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliporeSigma Stericup Quick Release-GP Sterile Vacuum Filtration System 500mL</td>
			<td>Fisher Scientific</td>
			<td>S2GPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliporeSigma&#160; Stericup Quick Release-GP Sterile Vacuum Filtration System 250mL</td>
			<td>Fisher Scientific</td>
			<td>S2GPU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>MIXcontrol MTP / Variomag TELEcontrol MTP Control Unit</td>
			<td>2mag</td>
			<td>VMF 90250 U</td>
			<td></td>
		</tr>
		<tr>
			<td>MIXdrive 6 MTP / Variomag TELEdrive 6 MTP Microplate Stirring Drive</td>
			<td>2mag</td>
			<td>VMF 40600</td>
			<td>6MSP</td>
		</tr>
		<tr>
			<td>MP Biomedicals&#160; 7X Cleaning Solution</td>
			<td>Fisher Scientific</td>
			<td>MP0976670A4</td>
			<td>Tissue culture suitable detergent. Make a 5% solution in water</td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>STEMCELL Technologies</td>
			<td>85850</td>
			<td>hPSC medium.TeSR-E8, NutriStem XF, and mTeSR Plus medium have also been tested and are suitable alternatives.&#160;</td>
		</tr>
		<tr>
			<td>Nunc 50 mL Conical, Sterile Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>12-565-270</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 15mL Conical Sterile Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>12-565-268</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15-140-122</td>
			<td>Aliquot and freeze</td>
		</tr>
		<tr>
			<td>Plasmocin</td>
			<td>Invivogen</td>
			<td>ant-mpt</td>
			<td>Anti-mycoplasma reagent. Aliquot and freeze</td>
		</tr>
		<tr>
			<td>pluriStrainer&#174; 200 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>NC0776417</td>
			<td>Cell strainer</td>
		</tr>
		<tr>
			<td>pluriStrainer&#174; 500 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>NC0822591</td>
			<td>Cell strainer</td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol) 87-90% hydrolyzed&#160; (PVA)</td>
			<td>Millipore-Sigma</td>
			<td>P8136-250G</td>
			<td>10% in DPBS stirring at 98 degrees C until disolves, make in 138-3211-FLS</td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y-27632 (ROCKi)</td>
			<td>STEMCELL Technologies</td>
			<td>72304</td>
			<td>10 mM stock in DPBS</td>
		</tr>
		<tr>
			<td>Sterile Disposable Serological Pipets&#160; - 10mL</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Disposable Serological Pipets - 25mL</td>
			<td>Fisher Scientific</td>
			<td>13-678-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Disposable Serological pipette - 5 mL</td>
			<td>Fisher Scientific</td>
			<td>13-678-12D</td>
			<td></td>
		</tr>
		<tr>
			<td>TeSR-E5</td>
			<td>STEMCELL Technologies</td>
			<td>5916</td>
			<td>Serum-free, low protein base medium for E5-ILP</td>
		</tr>
		<tr>
			<td>Variomag distriBOX 2 Distributor</td>
			<td>2mag</td>
			<td>VMF 90512</td>
			<td>If you use more than one MIXdrive</td>
		</tr>
	</tbody>
</table>

a simplified method for generating kidney organoids from human pluripotent stem cells

由hPSCs产生的肾类器官提供了无限的肾组织来源。人肾类器官是研究肾脏疾病和损伤，开发基于细胞的疗法以及测试新疗法的宝贵工具。对于此类应用，需要大量均匀的类器官和高度可重复的测定。我们以之前发布的类肾类器官方案为基础，以改善类器官的整体健康状况。这种简单，强大的3D方案涉及在含有脂质，胰岛素 - 转铁蛋白 - 硒 - 乙醇胺补充剂和聚乙烯醇的最小组分培养基中形成均匀的胚体，使用GSK3抑制剂（CHIR99021）3天，然后在含有敲除血清置换（KOSR）的培养基中培养。此外，搅拌测定可以减少胚体的结块并保持均匀的大小，这对于减少类器官之间的变异性非常重要。总体而言，该协议为产生大量肾类器官提供了一种快速，高效且具有成本效益的方法。

在我们方案的最新版本中，肾脏类器官分化是在定义的低蛋白培养基中启动的。该测定完全在悬浮液中进行，并依赖于hPSCs分化和组织启动微管生成的先天能力。来自100mm〜60%融合hPSC培养板的单次测定通常产生500-1，000个肾脏类器官，如我们之前的出版物5所示。由于产生如此大量的类器官，该方案非常适合化合物测试。我们通常使用6孔格式进行化合物测试，但是，该协议可以在?...

Watch this Scientific Journal Video about A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells at JoVE.com

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

在这里，我们描述了一种从人类多能干细胞（hPSCs）生成肾类器官的方案。该方案在两周内产生肾脏类器官。所得的类肾类器官可以在大型旋转瓶或多孔磁力搅拌板中培养，以进行平行的药物测试方法。

一种从人类多能干细胞中生成肾类器官的简化方法

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

该方法可以在任何配备细胞培养实验的实验室中进行。使用这种方法，我们可以进一步了解肾脏发育和疾病通路。与其他用于产生肾脏类器官的方法相比，这是一种相对简单且便宜的方法。该技术的最大挑战是维持高质量的人类多能干细胞培养。至关重要的是，细胞保持在小于80%的汇合度并定期分裂。刚接触人类多能干细胞培养的研究人员在进行分化之前，应熟悉干细胞系。通过将两毫升完整的E5-ILP培养基加入六孔超低附着板的每个孔中来开始肾类器官测定。在100毫米组织培养处理的平板中培养人多能干细胞或hPSCs，直到它们达到60%汇合度。用大约8毫升DPBS洗涤hPSCs两次。吸出DPBS，然后滴加两毫升分散酶以覆盖细胞，并在37摄氏度下孵育六分钟。用大约10毫升DPBS洗涤细胞三次。吸出DPBS，然后将板倾斜45度，并用细胞提升器将细胞刮下。使用10毫升血清移液管用六毫升完整的E5-ILP培养基从顶部冲洗菌落。然后，垂直握住移液器，通过轻轻上下移液来分解大的菌落，注意不要接触培养板的侧面。通过将每孔一毫升加入六孔板来均匀分布菌落簇，然后将板置于37摄氏度培养箱内的轨道振荡器上。或者，如果没有轨道振荡器，则可以静态培养细胞。在第二天，让胚体沉降在板的底部后，将板倾斜45度，然后从顶部缓慢吸出培养基，每孔留下一毫升。每孔加入两毫升制备的完整培养基，并将板放回摇摇杯中。在第三天，如前所述吸取培养基后，使用10毫升血清学移液管从每个孔中仔细收集所有胚体，并将其转移到50毫升锥形管中。要收集任何剩余的胚体，请用一毫升低葡萄糖DMEM冲洗每个孔，并将它们添加到相同的50毫升锥形管中。在等待胚体沉降在管的底部时，向六孔板的每个孔中加入两毫升的II期培养基。接下来，通过将200微米的细胞过滤器放在新的50毫升锥形管的顶部，并将所有胚体小心地移液到细胞过滤器上，筛选出大的胚状体。用另外五毫升DMEM冲洗细胞过滤器，以收集卡在过滤器中的任何胚状体。在后期阶段，首先使用500微米的过滤器筛出非常大的胚状体，然后将滤液通过200微米的过滤器筛出非常小的胚状体，没有小管。通过将200微米过滤器倒置到新的50毫升管中并用DPBS洗涤过滤器，收集200至500微米之间正确尺寸的胚体。过滤后，让胚体沉降到锥形管的底部，然后吸出上清液，并用约10毫升DMEM洗涤。然后，将胚体重悬于六毫升的II期培养基中。将胚体移回六孔超低附着板中，将它们均匀地分布在孔中。由于大多数类器官聚集在移液器的顶部，因此在末端留下约半毫升，然后轻轻地将尖端触摸到每个孔中的培养基，使类器官流入孔中。将胚体转移到带有45毫升II期培养基的125毫升旋转瓶中。将磁力搅拌速度设置为120 rpm，并将烧瓶放入培养箱中。为了喂养胚体或肾类器官，让它们短暂地沉降到旋转瓶的底部，然后从烧瓶的一侧臂上提起盖子，并将吸气移液器放在里面，尖端接触对面的内壁。慢慢地将移液器向下倾斜，并吸出大约一半的培养基。通过同一开口移液，用20毫升新鲜的II期培养基补充。使用长无菌镊子小心地将一个磁力搅拌棒放入具有胚体或肾类器官的六孔板的每个孔中。将板放在六轮磁力搅拌板上，并将速度设置为120 rpm。要使磁力搅拌棒卡入到位并开始旋转，请首先将功率水平设置为100。一旦所有棒材开始旋转，将功率水平降低到25。如前所述，通过改变一半的培养基来维持肾脏类器官。第 14 天肾类器官石蜡切片的免疫荧光显示存在肾单位节段，例如表达肝细胞核因子-1 β 和莲花四蕈洛布斯凝集素的肾小管，以及表达 V-maf 肌肉腱鞘神经纤维肉瘤同源物 B 和肾素的足细胞簇。该方案中的修饰可以支持内皮细胞的扩增，正如在培养的第26天用血小板和内皮细胞粘附分子-1染色所证实的那样。人类多能干细胞需要用Dispase处理适当的时间，然后彻底洗涤以去除所有痕迹。如果分散酶没有被完全去除，它可能导致细胞死亡和胚胎体聚集。这种方法帮助我们研究了先天性肾脏疾病的发展，以及各种肾损伤途径和体外治疗干预的潜力。

Research

JoVE Journal

Developmental Biology

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

科研

发育生物学

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

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. ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

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 ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

一种从人类胚胎干细胞中生成脑器官的静态自导方法

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

从人类诱导多能干细胞诱导的体外神经肌肉结点

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

血源性内皮细胞与人多能干细胞的定向分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...