灵长类动物诱导的多能干细胞来源于尿液的产生和维持
Summary
本协议描述了一种将人和非人灵长类动物尿液来源细胞分离、扩增和重新编程为诱导多能干细胞 （iPSC） 的方法，以及新生成的 iPSC 的无饲养层维护说明。
Abstract
研究灵长类多能干细胞及其衍生物的跨物种方法对于更好地了解疾病、发育和进化的分子和细胞机制至关重要。为了使灵长类动物诱导的多能干细胞（iPSCs）更容易获得，本文提出了一种从尿源性细胞中产生人和非人灵长类动物iPSCs的非侵入性方法，并使用无饲养层培养方法维持它们。
尿液可以从非无菌环境（例如动物的笼子）中取样，并在原代细胞培养过程中用广谱抗生素混合物处理，以有效减少污染。在尿源性细胞繁殖后，iPSCs通过市售仙台病毒载体系统的改良转导方法产生。第一个iPSC菌落可能在5天后已经可见，最早可以在10天后采摘。使用无酶解离缓冲液进行常规团块传代，支持生成的 iPSC 的多能性超过 50 次传代。
Introduction
人类和非人类灵长类动物（NHP）的基因组比较对于了解我们的进化历史和人类特异性状的进化至关重要1。此外，这些比较允许通过识别保守的DNA序列2来推断功能，例如，优先考虑与疾病相关的变异3。分子表型（如基因表达水平）的比较对于更好地解释基因组比较和发现例如细胞表型差异至关重要。此外，它们具有 – 类似于DNA水平的比较 – 推断功能相关性的潜力，从而更好地解释人类内部的医学相关变异4。将全面的分子表型数据纳入这些比较研究需要适当的生物资源（即跨物种的直系同源细胞）。然而，伦理和实践原因使得难以或不可能获得这种可比细胞，特别是在发育过程中。诱导多能干细胞（iPSCs）允许在体外产生这种难以接近的细胞类型5，6，在实验上是可接近的，并且已用于灵长类动物比较6，7，8，9，10，11，12，13，14。
为了产生iPSC，需要获得要重新编程的原代细胞。从尿液中分离的细胞的优点是可以从灵长类动物中非侵入性地采样，并且可以很容易地重新编程，这可能是由于它们的干细胞样分子谱15。维持灵长类动物iPSC的培养条件与重编程一样重要;传统上，人多能干细胞的培养需要一种非定义的、基于血清的培养基和小鼠胚胎成纤维细胞（所谓的饲养层细胞）的共培养，为胚胎干细胞（ESC）提供必需的营养物质和支架16。自从化学成分明确且无饲养层的培养系统开发以来17，18，现在有多种市售iPSC培养基和基质可供选择。然而，这些培养条件中的大多数已针对人类ESC和iPSC进行了优化，因此在NHP iPSC培养中可能效果较差。在此视频协议中，我们提供了生成和维持源自尿细胞培养的人类和NHP iPSC的说明。
自2006年首次报道通过成纤维细胞中特定因子的强制表达产生iPSC以来，该方法已应用于各种来源的许多不同细胞类型19，20，21，22，23，24，25，26，27，28，29，30，31，32.其中，只能以完全无创的方式获得尿源性细胞。基于Zhou等人先前描述的方案33，通过补充广谱抗生素，甚至可以从非无菌样品中分离和扩增灵长类动物尿液中的细胞15。值得注意的是，通过该方案采样的尿液来源细胞表现出产生iPSCs的高潜力，在比传统的成纤维细胞重编程（20-30天，根据我们的经验）更短的时间内（集落在5-15天内可见），并且具有足够高的成功率。这些尿源性细胞被归类为间充质干细胞样细胞和膀胱上皮细胞的混合群体，导致重编程效率高15。
除了原代细胞的变化外，产生iPSCs的重编程方法也根据使用目的而有所不同。人体细胞的常规重编程程序是通过用逆转录病毒或慢病毒载体过表达重编程因子来进行的，这允许外源性DNA整合到基因组中5，34，35。为了保持生成的iPSCs基因组完整，研究人员开发了多种非整合系统-可切除的PiggyBac载体36，37，游离体载体38，39，仙台病毒40和腺病毒41等非整合病毒载体，mRNA转染42，蛋白质转染43，44和化合物处理45.由于高效且易于处理，该协议中使用了基于仙台病毒的重编程载体。原代细胞的感染在接种前以5的感染多重性（MOI）在细胞和病毒的1小时悬浮培养中进行。与将病毒直接添加到贴壁细胞培养物中的常规方法相比，这种修改的步骤可以增加细胞表面与病毒接触的可能性，从而产生更多的iPSC集落15。
人类和NHP多能干细胞的传代可以通过团块传代和单细胞传代来完成。乙二胺四乙酸（EDTA）是一种具有成本效益的螯合剂，可结合钙和镁离子，从而防止钙粘蛋白和整合素的贴壁活性。EDTA也被用作温和的选择性解离试剂，因为未分化的细胞由于其不同的粘附分子而在分化的细胞之前分离。完全解离通过含有蛋白激酶（Rho/Rock）介导的肌球蛋白过度激活的Rho/Rho相关线圈 诱导 灵长类iPSCs的大量细胞死亡。因此，对于需要悬浮液中的单细胞的实验，补充Rho/Rock抑制剂培养基至关重要46，47。在该协议中，我们建议将团块传代作为常规传代方法，并建议仅在必要时进行单细胞传代，例如，当需要接种定义的细胞数量时或在亚克隆期间。
Protocol
该实验程序得到了负责人体实验伦理委员会（20-122，Ethikkommission LMU München）的批准。所有实验均按照相关指南和规定进行。注意：在开始处理人类和非人灵长类样品的实验之前，必须获得相应伦理委员会的批准。所有实验程序必须按照相关指南和规定进行。以下每个步骤都应在生物安全柜中使用无菌技术进行。所有缓冲液和培养基组成均可在 补充表S1中找到。确保在将所有培养基…
Representative Results
从人和非人灵长类尿液中分离细胞时，可以在分离后直接鉴定不同类型的细胞。鳞状细胞以及各种较小的圆形细胞随尿液排出体外;女性尿液含有比男性尿液多得多的鳞状细胞（图1B – 第0天; 补充图S1）。在原代尿培养基中培养5天后，可以看到第一个贴壁增殖细胞（图1A，B – 第5天）。此时，每天用REMC增殖培养基替换一半的培养?…
Discussion
iPSCs是有价值的细胞类型，因为它们允许 在体外产生原本无法接近的细胞类型。作为重编程的起始材料，例如，并非所有灵长类动物都能获得成纤维细胞，本文提出了一种从尿液来源的细胞中生成iPSCs的方案。这些细胞可以通过在培养基中补充广谱抗生素以非侵入性方式获得，甚至可以从非无菌灵长类动物尿液样本中获得。
协议中的几个关键步骤值得进一步讨论。首先…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了DFG EN 1093/5-1（项目编号458247426）的支持。M.O.得到了JSPS海外研究奖学金的支持。所有图形都是用 BioRender.com 创建的。流式细胞术是在慕尼黑生物医学中心的核心设施流式细胞术的帮助下进行的。我们要感谢京都大学ASHBi的Makoto Shida和Tomoyo Muto对摄像的支持。
Materials
| Accumax™ cell detachment solution (Detachment solution) | Sigma-Aldrich | SCR006 | |
| Amphotericin B-Solution | Merck | A2941-100ML | |
| Anti-Human TRA-1-60 Mouse Antibody | Stem Cell Technologies | 60064 | Dilution: 1/200 |
| Anti-Human TRA-1-60 PE-conjugated Antibody | Miltenyi Biotec | 130-122-965 | Dilution: 1/50 |
| Bambanker™ (Cell freezing medium) | Nippon Genetics | BB01 | |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A3059-100G | |
| Cell culture multiwell plate, 12-well CELLSTAR | Greiner BIO-ONE | 665180 | |
| Countess™ II automated cell counter | Thermo Fisher Scientific | AMQAX1000 | |
| CryoKing® 1.5 mL Tubes with 2D Barcode (Cryotubes) | Sued-Laborbedarf | 52 95-0213 | Different types of Cryotubes can be used for freezing. The 2D barcode tubes have the advantage that the sample info can be stored in a database with unique tube information. |
| CytoTune™ EmGFP Sendai Fluorencence Reporter (GFP Sendai virus) | Thermo Fisher Scientific | A16519 | |
| CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Sendai virus reprogramming kit) | Thermo Fisher Scientific | A16518 | |
| DAPI 4',6-Diamidine-2'-phenylindole dihydrochloride | Sigma-Aldrich | 10236276001 | |
| DMEM High Glucose | TH.Geyer | L0102 | |
| DMEM/F12 w L-glutamine | Fisher Scientific | 15373541 | |
| Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 | Thermo Fisher Scientific | A-21202 | Dilution: 1/500 |
| Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 | Thermo Fisher Scientific | A-21207 | Dilution: 1/500 |
| DPBS w/o Calcium w/o Magnesium | TH.Geyer | L0615-500 | |
| EpCAM Recombinant Polyclonal Rabbit Antibody (22 HCLC) | Thermo Fisher Scientific | 710524 | Dilution: 1/500 |
| Ethylenediamine tetraacetic acid (EDTA) | Carl Roth | CN06.3 | |
| Falcon Tube 15 mL conical bottom | Greiner BIO-ONE | 188271-N | |
| Falcon Tube 50 mL conical bottom | Greiner BIO-ONE | 227261 | |
| Fetal Bovine Serum, qualified, heat inactivated, Brazil (FBS) | Thermo Fisher Scientific | 10500064 | |
| FlowJo V10.8.2 | FlowJo | 663441 | |
| Gelatin from porcine skin | Sigma-Aldrich | G1890-1KG | |
| Geltrex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix | Thermo Fisher Scientific | A1413301 | |
| GlutaMAX™ Supplement | Thermo Fisher Scientific | 35050038 | |
| Heracell™ 240i CO2 incubator | Fisher Scientific | 16416639 | |
| Heraeus HeraSafe safety cabinet | Kendro | 51017905 | |
| Human EGF, premium grade | Miltenyi Biotec | 130-097-749 | |
| ImageJ | Fiji | Version 2.9.0 | |
| MEM Non-Essential Amino Acids Solution (100X) | Thermo Fisher Scientific | 11140035 | |
| Microcentrifugation tube PP, 1.5 mL | Nerbe Plus | 04-212-1000 | |
| Microscope Nikon eclipse TE2000-S | Nikon | TE2000-S | |
| Mouse anti-alpha-Fetoprotein antibody | R&D Systems | MAB1368 | Dilution: 1/100 |
| Mouse anti-alpha-Smooth Muscle Actin antibody | R&D Systems | MAB1420 | Dilution: 1/100 |
| Mouse anti-beta-III Tubulin antibody | R&D Systems | MAB1195 | Dilution: 1/100 |
| mTeSR™ 1 | STEMCELL Technolgies | 85850 | |
| Nanog (D73G4) XP Rabbit mAb | Cell Signaling Technology | 4903S | Dilution: 1/400 |
| Normocure™ (Antimicrobial Reagent) | Invivogen | ant-noc | |
| Oct-4 Rabbit Antibody | Cell Signaling Technology | 2750S | Dilution: 1/400 |
| Paraformaldehyde (PFA) | Sigma-Aldrich | 441244-1KG | |
| Penicillin-Streptomycin (10.000 U/ml) (PS) | Thermo Fisher Scientific | 15140122 | Penicillin-Streptomycin mix contains 100 U/mL Penicillin and 100 µg/mL Streptomycin. |
| Recombinant Human FGF-basic | PeproTech | 100-18B | |
| Recombinant Human PDGF-AB | PeproTech | 100-00AB | |
| Refrigerated benchtop centrifuge | SIGMA | 4-16KS | |
| Renal Epithelial Cell Basal Medium | ATCC | PCS-400-030 | |
| Renal Epithelial Cell Growth Kit | ATCC | PCS-400-040 | |
| Sox2 (L1D6A2) Mouse mAb #4900 | Cell Signaling Technology | 4900S | Dilution: 1/400 |
| SSEA4 (MC813) Mouse mAb | NEB | 4755S | Dilution: 1/500 |
| StemFit® Basic02 | Nippon Genetics | 3821.00 | The production of this medium was discontinued, use StemFit Basic04CT for human cell lines or StemFit Basic03 for non-human primates instead. |
| Triton X-100 | Sigma-Aldrich | T8787-50ML | |
| TrypLE™ Select Enzyme (1x), no phenol red (Dissociation enzyme) | Thermo Fisher Scientific | 12563011 | |
| Waterbath Precision GP 05 | Thermo Fisher Scientific | TSGP05 | |
| Y-27632, Dihydrochloride Salt (Rock Inhibitor) | Biozol | BYT-ORB153635 | |
| Antibody dilution buffer | For composition see the supplementary table S1 | ||
| Blocking buffer | For composition see the supplementary table S1 | ||
| REMC medium | For composition see the supplementary table S1 | ||
| Primary urine medium | For composition see the supplementary table S1 | ||
| PSC culture medium | For composition see the supplementary table S1 | ||
| PSC generation medium | For composition see the supplementary table S1 | ||
| Urine wash buffer | For composition see the supplementary table S1 |
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Cite This Article
Radmer, J., Geuder, J., Edenhofer, F. C., Enard, W., Ohnuki, M. Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine. J. Vis. Exp. (197), e64922, doi:10.3791/64922 (2023).