一种从人类多能干细胞中生成肾类器官的简化方法
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 <p class="jove_content…
Representative Results
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?…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 2-Mercaptoethanol | Thermo Fisher | 21-985-023 | |
| Anti-adherence rinsing solution | STEMCELL Technologies | 7010 | |
| CHIR99021 | STEMCELL Technologies | 72054 | 10 mM stock in DMSO |
| Corning disposable spinner flasks | Fisher Scientific | 07-201-152 | |
| Corning Ultra-Low Attachment 6-well plates | Fisher Scientific | 07-200-601 | |
| Corning Slow-Speed Stirrers | Fisher Scientific | 11-495-03 | Multi plate magnetic stirrer for spinner flask culture |
| Dispase | STEMCELL Technologies | 7923 | Aliquot and freeze |
| DMEM, low glucose, pyruvate, no glutamine, no phenol red | Thermo Fisher | 11054020 | |
| DPBS 1x, no calcium, no magnesium | Thermo Fisher | 14-190-250 | |
| Egg / Oval Stirring Bars | 2mag | PI20106 | |
| Excelta General-Purpose Tweezers | Fisher Scientific | 17-456-103 | Keep sterile in the cell culture hood |
| EZBio Single Use Media Bottle, 250mL | Foxx Life Sciences | 138-3211-FLS | Used to make PVA 10% |
| Falcon Standard Tissue Culture Dishes (100 mm) | Thermo Fisher | 08-772E | |
| Fisherbrand Sterile Aspirating Pipet 2mL | Fisher Scientific | 14-955-135 | |
| Fisherbrand Cell Lifters – Cell lifter | Fisher Scientific | 08-100-240 | |
| Fisherbrand Multi Function 3D Rotators | Fisher Scientific | 88-861-047 | Orbital shaker |
| Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix | Thermo Fisher | A1413302 | BME. Aliquot on ice and freeze. Another suitable matrix alternative is Matrigel or Cultrex. |
| Gentle Cell Dissociation Reagent | STEMCELL Technologies | 7174 | GCDR |
| GlutaMAX Supplement | Thermo Fisher | 35-050-061 | L-glutamine supplement. |
| HEPES (1M) | Thermo Fisher | 15-630-080 | |
| Insulin-Transferrin-Selenium-Ethanolamine | Thermo Fisher | 51-500-056 | ITSE |
| KnockOut Serum Replacement – Multi-Species | Thermo Fisher | A3181502 | KOSR. Aliquot and freeze |
| Lipid Mixture 1, Chemically Defined | Millipore-Sigma | L0288-100ML | |
| MEM Non-Essential Amino Acids Solution | Thermo Fisher | 11140-050 | |
| MilliporeSigma Stericup Quick Release-GP Sterile Vacuum Filtration System 500mL | Fisher Scientific | S2GPU05RE | |
| MilliporeSigma Stericup Quick Release-GP Sterile Vacuum Filtration System 250mL | Fisher Scientific | S2GPU02RE | |
| MIXcontrol MTP / Variomag TELEcontrol MTP Control Unit | 2mag | VMF 90250 U | |
| MIXdrive 6 MTP / Variomag TELEdrive 6 MTP Microplate Stirring Drive | 2mag | VMF 40600 | 6MSP |
| MP Biomedicals 7X Cleaning Solution | Fisher Scientific | MP0976670A4 | Tissue culture suitable detergent. Make a 5% solution in water |
| mTeSR1 | STEMCELL Technologies | 85850 | hPSC medium.TeSR-E8, NutriStem XF, and mTeSR Plus medium have also been tested and are suitable alternatives. |
| Nunc 50 mL Conical, Sterile Centrifuge Tubes | Fisher Scientific | 12-565-270 | |
| Nunc 15mL Conical Sterile Centrifuge Tubes | Fisher Scientific | 12-565-268 | |
| Penicillin-Streptomycin | Thermo Fisher | 15-140-122 | Aliquot and freeze |
| Plasmocin | Invivogen | ant-mpt | Anti-mycoplasma reagent. Aliquot and freeze |
| pluriStrainer® 200 µm | Fisher Scientific | NC0776417 | Cell strainer |
| pluriStrainer® 500 µm | Fisher Scientific | NC0822591 | Cell strainer |
| Poly(vinyl alcohol) 87-90% hydrolyzed (PVA) | Millipore-Sigma | P8136-250G | 10% in DPBS stirring at 98 degrees C until disolves, make in 138-3211-FLS |
| ROCK inhibitor Y-27632 (ROCKi) | STEMCELL Technologies | 72304 | 10 mM stock in DPBS |
| Sterile Disposable Serological Pipets – 10mL | Fisher Scientific | 13-678-11E | |
| Sterile Disposable Serological Pipets – 25mL | Fisher Scientific | 13-678-11 | |
| Sterile Disposable Serological pipette – 5 mL | Fisher Scientific | 13-678-12D | |
| TeSR-E5 | STEMCELL Technologies | 5916 | Serum-free, low protein base medium for E5-ILP |
| Variomag distriBOX 2 Distributor | 2mag | VMF 90512 | If you use more than one MIXdrive |
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Citazione di questo articolo
Przepiorski, A., Crunk, A. E., Holm, T. M., Sander, V., Davidson, A. J., Hukriede, N. A. A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells. J. Vis. Exp. (170), e62452, doi:10.3791/62452 (2021).