Name: guided differentiation of mature kidney podocytes from human induced pluripotent stem cells under chemically defined conditions
Uploaded: 2020-07-02T13:00:00
Description: Watch this Scientific Journal Video about Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions at JoVE.com

这项工作得到了杜克大学普拉特工程学院、杜克医学院肾脏病学系、杜克大学医学系主席研究奖和伯劳斯·威康基金PDEP职业过渡 特别 奖的支持，该奖授予S.M。M.B得到了国家科学基金会研究生研究金计划的支持。我们感谢Bursac实验室慷慨地为我们提供了DU11干细胞系，以及杜克大学的Varghese实验室暂时与我们的团队共享他们的组织培养设施。本出版物是献给麻省理工学院诺华化学教授劳拉·基斯林教授的，以庆祝她的60岁生日 。

1. 试剂的制备
<ol><li>稀释解冻5倍高PS细胞培养介质（CCM）补充在HIPS细胞培养基质介质获得1倍溶液的HIPS CCM。 注： 冷冻 5 倍高音响 CCM 补充剂需要一个缓慢的解冻过程， 理想情况下在 4 °C 过夜.1x hiPS CCM 的 Aliquot 可在 -20 °C 下存储长达 6 个月。</li>
	<li>准备地下室膜 （BM） 矩阵 1 涂层板用于 hiPS 细胞培养：解冻 BM 矩阵 1 在 4 °C 的冰上过夜。解冻后，根据制造商的建议，准备具有适当稀释因子?...

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiopatch+platform+enables+maturation+and+scale-up+of+human+pluripotent+stem+cell-derived+engineered+heart+tissues.">Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues.</a> Nature Communications. 8, 1825 (2017).</li><li>Satoh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=aPKC&#955;+maintains+the+integrity+of+the+glomerular+slit+diaphragm+through+trafficking+of+nephrin+to+the+cell+surface.">aPKC&#955; maintains the integrity of the glomerular slit diaphragm through trafficking of nephrin to the cell surface.</a> The Journal of Biochemistry. 156, 115-128 (2014).</li><li>Mae, S. 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在这份报告中，我们描述了从HIPS细胞中生成肾球状细胞的一个协议。HIPS细胞衍生的足细胞表现出形态和分子特征与成熟的肾多细胞表型13相关。在以前的出版物中，我们表明，HIPS细胞衍生的足细胞可以模仿肾球菌的结构和选择性过滤功能，当与球状微血管内皮细胞在一个敷衍的微流体器官片上设备8，11共同培养。
<p class="jove_content...

人类多能干细胞培养的进步，准备通过为研究人员提供可再生的、可扩展的生物材料来源，从而彻底改变再生医学、疾病建模和药物筛选。此策略对于衍生出专业和功能性细胞类型特别有用，否则将难以获得。人类诱导的多能干细胞（hiPS）细胞2，3，4，5特别有吸引力，因为它们的体细胞起源和它们代表个性化医学的潜力。然而，由于频繁使用定义不明确的培养条件，导致效率低下，非特定生成异质细胞群6，7，开发方法从HIPS细胞中获取其他细胞血统仍然具有挑战性。
这里介绍的是一种在化学定义条件下从具有特异性和高效率的HIPS细胞中提取成熟肾细胞的方法。通过考虑细胞微环境中多种因素的作用，制定了干细胞分化策略，包括优化细胞培养介质中呈现的可溶性因子以及不溶性因素，如细胞外基质组件或粘合基板。鉴于特格林信号在细胞发育和功能中的重要性，初步检查了细胞表面的特格林受体的表达。β1集成物不仅在hiPS细胞中，而且在它们的衍生物中，包括中皮细胞8，9，10中表达。随后的实验证实，与 β1 集成物（包括层压素 511 或层压素 511-E8 片段）结合使用时，与下面描述的可溶性感应介质结合使用时，支持 hiPS 细胞的粘附和分化到 podocyt 中。
细胞谱系承诺的诱导首先确认，在具有Activin A、CHIR99021和Y27632岩石抑制剂的介质存在的情况下，在层压涂层表面培养了两天的HIPS细胞可以分化成表达早期间皮标记HAND1、鹅科和胸针8、11的细胞。用骨形态遗传蛋白7（BMP-7）和CHIR99021补充的中等体质细胞治疗14天，使表达肾病的中间间皮细胞得以衍生 原体细胞标记威尔姆的肿瘤1（WT1），奇跳过相关蛋白质1（OSR1）8，11，和配对盒基因2蛋白质（PAX2）12。为了获得成熟的肾球状细胞，中间间皮细胞经过45天的治疗，其新介质包括BMP-7、Activin A、血管内皮生长因子（VEGF）、全跨视网膜酸和CHIR99021。流细胞学和免疫染色用于确认>90%的细胞表现出成熟肾细胞8、11、13的分子、形态和功能特征。这些特点包括发展初级和二级足部工艺：足细胞谱系特异性基因的表达，包括SYNPO、PODXL、MAF、EFNB28和蛋白质的表达，包括波多辛、肾素和WT1 14、15、16。此外，还发现，通过使用商用介质8，11，在培养中可保持长达四周的体外培养物，从而在下游实验的时机上提供额外的灵活性。有关用于确定 hiPS-podo 细胞纯度的流细胞学面板的更多信息，请参阅我们之前的出版物11。

<table>
	<tbody>
		<tr>
			<td>Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DU11 human iPS cells</td>
			<td></td>
			<td></td>
			<td>The DU11(Duke University clone #11) iPS cell line was generated at the Duke University iPSC Core Facility and provided to us by the Bursac Lab at Duke University. This line has been tested for mycoplasma and was last karyotyped in July 2019 by our lab, and found to be karyotypically normal.</td>
		</tr>
		<tr>
			<td>Growth Factors and Media Supplements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans retinoic acid (500 mg)</td>
			<td>72262</td>
			<td>Stem Cell Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 serum-free supplement</td>
			<td>17504044</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>04-0004</td>
			<td>Stemgent</td>
			<td>May show lot-to-lot variation</td>
		</tr>
		<tr>
			<td>Complete Medium Kit with CultureBoost-R</td>
			<td>4Z0-500-R</td>
			<td>Cell Systems</td>
			<td>Podocyte maintenance media</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>12634028</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with GlutaMAX supplement</td>
			<td>10565042</td>
			<td>Thermo/Life Technologies</td>
			<td>DMEM/F12 with glutamine supplement</td>
		</tr>
		<tr>
			<td>Heat-inactivated FBS</td>
			<td>10082147</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human activin A</td>
			<td>PHC9564</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BMP7</td>
			<td>PHC9544</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF</td>
			<td>PHC9394</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 medium</td>
			<td>05850</td>
			<td>Stem Cell Technologies</td>
			<td>hiPS cell culture media (CCM)</td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin, liquid (100&#215;)</td>
			<td>15140-163</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632 ROCK inhibitor</td>
			<td>1254</td>
			<td>Tocris</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488&#8211; and Alexa Fluor 594&#8211;conjugated secondary antibodies</td>
			<td>A32744; A32754; A-11076; A32790</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Brachyury(T&#65289;</td>
			<td>ab20680</td>
			<td>Abcam</td>
			<td></td>
		</tr>
		<tr>
			<td>Nephrin</td>
			<td>GP-N2</td>
			<td>Progen</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT4</td>
			<td>AF1759</td>
			<td>R&#38;D Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>PAX2</td>
			<td>71-6000</td>
			<td>Invitrogen</td>
			<td></td>
		</tr>
		<tr>
			<td>WT1</td>
			<td>MAB4234</td>
			<td>Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM Molecules</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iMatrix-511 Laminin-E8 (LM-E8) fragment</td>
			<td>N-892012</td>
			<td>Iwai North America</td>
			<td>Basement membrane (BM) matrix 2</td>
		</tr>
		<tr>
			<td>Matrigel hESC-qualified matrix, 5-mL vial</td>
			<td>354277</td>
			<td>BD Biosciences</td>
			<td>Basement membrane (BM) matrix 1. May show lot-to-lot variation</td>
		</tr>
		<tr>
			<td>Enzymes and Other Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>A1110501</td>
			<td>Thermo/Life Technologies</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>A9418</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>D2438</td>
			<td>Sigma-Aldrich</td>
			<td>DMSO is toxic. Should be handled in chemical safety hood</td>
		</tr>
		<tr>
			<td>Enzyme-free cell dissociation buffer, Hank&#8217;s balanced salt</td>
			<td>13150016</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol solution, 70% (vol/vol), biotechnology grade</td>
			<td>97065-058</td>
			<td>VWR</td>
			<td>Ethanol is flammable and toxic</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>431097</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>28906</td>
			<td>Thermo/Life Technologies</td>
			<td>PFA should be handled in a chemical fume hood with proper personal protection equipment, including gloves, lab coat, and safety eye glasses. Avoid inhalation and contact with skin.</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>14190-250</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Distilled Water</td>
			<td>15230162</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>97062-208</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05%</td>
			<td>25300-120</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirating pipettes, individually wrapped</td>
			<td>29442-462</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-15R Centrifuge</td>
			<td>B99516</td>
			<td>Beckman Coulter</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tube, 15 mL</td>
			<td>352097</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tube, 50 mL</td>
			<td>352098</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryoboxes</td>
			<td>3395465</td>
			<td>Thermo/Life Technologies</td>
			<td>For storing frozen aliquots</td>
		</tr>
		<tr>
			<td>EVOS M7000</td>
			<td>AMF7000</td>
			<td>Thermo/Life Technologies</td>
			<td>Flourescent microscope used to acquire images of fixed and stained iPS cells and their derivatives</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>100503-092</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell VIOS 160i CO2 incubator</td>
			<td>51030403</td>
			<td>Thermo/Life Technologies</td>
			<td>For the routine culture and maintenace of iPS cells and their derivatives</td>
		</tr>
		<tr>
			<td>Inverted Zeiss Axio Observer equipped with AxioCam 503 camera</td>
			<td>491916-0001-000(microscope) ; 426558-0000-000(camera)</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Used to acquire phase contrast images of live iPS cells and their derivatives at each stage of podocyte differentiation</td>
		</tr>
		<tr>
			<td>Kimberly-Clark nitrile gloves</td>
			<td>40101-346</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, large</td>
			<td>21905-049</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, small</td>
			<td>21905-026</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 precision barrier pipette tips</td>
			<td>P1096-FR</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P100 barrier pipette tips</td>
			<td>P1125</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 barrier pipette tips</td>
			<td>P1126</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 barrier pipette tips</td>
			<td>P1121</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 barrier pipette tips</td>
			<td>P1122</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 10 mL, individually wrapped</td>
			<td>356551</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 25 mL, individually wrapped</td>
			<td>356525</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 5 mL, individually wrapped</td>
			<td>356543</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip, 0.22 &#956;m, PES</td>
			<td>SCGP00525</td>
			<td>EMD Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Microcentrifuge Tubes</td>
			<td>1138W14</td>
			<td>Thomas Scientific</td>
			<td>For aliquoting growth factors</td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;treated 12-well plates</td>
			<td>353043</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;treated six-well plates</td>
			<td>353046</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR white techuni lab coat</td>
			<td>10141-342</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-beveled cell lifter</td>
			<td>3008</td>
			<td>Corning</td>
			<td></td>
		</tr>
	</tbody>
</table>

guided differentiation of mature kidney podocytes from human induced pluripotent stem cells under chemically defined conditions

肾脏疾病影响全球10%以上的人口，并花费数十亿美元的联邦开支。最严重的肾脏疾病和最终的末期肾衰竭通常是由球状细胞的损伤引起的，球状细胞是高度专业化的上皮细胞，与内皮细胞和球状基底膜一起发挥作用，形成肾脏的过滤屏障。肾医学的进步因初级组织供应有限和缺乏强大的人类功能性肾细胞（如足细胞）的衍生方法而受到阻碍。从干细胞等可再生能源中提取足细胞的能力有助于增进目前对人类肾脏发育和疾病机制的理解，并为治疗发现提供新的工具。本协议的目标是开发一种方法，从具有高效率和特异性的人类诱导多能茎（hiPS）细胞中提取成熟、后粒细胞，并在化学定义条件下。这种方法产生的HIPS细胞衍生的足细胞表达血统特异性标记（包括肾上腺素、波多辛和威尔姆肿瘤1），并表现出与成熟和功能性足细胞相关的特殊形态特征（包括初级和二次足部过程）。有趣的是，这些特殊特征在该领域广泛使用的不朽的足细胞系中明显缺乏，这表明此处描述的协议产生的人类肾细胞比通常用于研究人类肾脏生物学的现有足细胞系具有更成熟的表型。

本协议的目的是证明成熟的人类卵母细胞可以在化学定义的条件下从HIPS细胞中提取。这份手稿中提供的数据是使用DU11 hiPS细胞线17生成的，该线首先被测试，并发现没有支原体。还进行了染色体分析，发现这些细胞在肌体上是正常的。从未分化的 DU11 HIPS 细胞开始， 本报告概述的分化策略（图1）首先将干细胞（图2A）分化为表达Brachyu...

Watch this Scientific Journal Video about Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions at JoVE.com

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

这里介绍了一个化学定义的协议，用于从诱导多能干细胞中提取具有高效率（>90%）的人类肾细胞，并且独立于遗传操作或亚人口选择。此协议在 26 天内生成所需的细胞类型，可用于肾毒性测试和疾病建模。

在化学定义条件下，成熟肾细胞与人类诱导多能干细胞的引导分化

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Research

JoVE Journal

Bioengineering

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

科研

生物工程

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...