结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞
1Laboratory of Biomedical Embryology, Department of Health, Animal Science and Food Safety and Center for Stem Cell Research,Università degli Studi di Milano, 2Department of Veterinary Medicine,University of Sassari, 3Department of Agricultural and Environmental Sciences – Production, Landscape, Agroenergy and Center for Stem Cell Research,Università degli Studi di Milano
Summary
我们在这里提出了一种将化学表观遗传擦除与机械检测相关线索相结合的方法，以有效地产生哺乳动物多能细胞，而无需基因转染或逆转录病毒载体。因此，这种策略对转化医学很有希望，并且代表了干细胞类器官技术的显着进步。
Abstract
细胞表型可以用不同的方法逆转或修饰，具有每种技术特有的优点和局限性。在这里，我们描述了一种新策略，该策略将化学表观遗传擦除与机械检测相关线索相结合，以产生哺乳动物多能细胞。需要两个主要步骤。在第一步中，成体成熟（终末分化）细胞暴露于表观遗传橡皮擦5-氮杂胞苷以驱动它们进入多能状态。该方案的这一部分是基于对控制细胞命运和分化的表观遗传机制的日益了解而开发的，并且涉及使用表观遗传修饰剂来消除细胞分化状态，然后驱动进入瞬时高可塑性窗口。
在第二步中，将擦除的细胞封装在聚四氟乙烯（PTFE）微生物反应器（也称为Liquid Marbles）中，以促进3D细胞重排以扩展并稳定地保持获得的高可塑性。PTFE是一种非反应性疏水性合成化合物，其使用允许产生细胞微环境，这在传统的2D培养系统中无法实现。该系统通过生物机械检测相关线索鼓励和促进多能性的维持。
这里描述的技术程序是允许在成人体细胞中诱导和维持高可塑性状态的简单策略。该协议允许在所有测试的哺乳动物物种中衍生出高可塑性细胞。由于它不涉及基因转染的使用，并且没有病毒载体，因此它可能代表了转化医学应用的显着技术进步。此外，微生物反应器系统通过体外重新创建特定的微环境，允许长期培养高可塑性细胞，即ESC，iPSCs，表观遗传擦除细胞和MSC，从而为干细胞类器官技术提供了显着的进步。
Introduction
在过去的几十年里，被广泛接受的单向进展到细胞承诺和分化的概念被完全修改。已经证明，细胞规格可以颠倒过来，并且使用不同的方法可以将终末分化的细胞推向不那么投入和更高的宽容状态。
在提出的几种方法中，最有前途的方法之一涉及使用化合物诱导细胞进入所谓的化学诱导多能性。这种方法中使用的小分子能够相互作用并修饰成年成熟细胞的表观遗传特征，从而避免了对任何转基因和/或病毒载体1，2，3，4，5，6，7，8，9，10的需求。.最近的许多研究表明，通过提供诱导高甲基化基因11，12，13，14，15再激活的特定生化和生物刺激，可以将细胞从一种表型切换到另一种表型。这些去甲基化事件允许将终末分化的细胞转化为原始祖细胞，多能或高可塑性/多能细胞1，2，3，4，5，6，7，8，9，10。
同时，许多研究最近都集中在理解机械检测相关线索上，更具体地说，是使用机械力直接影响细胞可塑性和/或分化的可能性16，17，18，19。事实上，已经清楚地表明，细胞外基质（ECM）在控制细胞命运中起着关键作用。特别是，ECM产生的生物力学和生物物理信号直接调节分子机制和信号通路，影响细胞行为和功能20，21。这些最近的数据为开发新型3D培养系统铺平了道路，这些系统更接近于模仿体内细胞微环境，复制驱动细胞行为的机械和物理刺激。
我们在这里描述了一个两步方案，它将化学表观遗传擦除与机械衰老相关线索相结合，以产生哺乳动物多能细胞。在第一步中，将细胞与去甲基化分子5-氮杂胞苷（5-氮杂-CR）一起孵育。该试剂能够通过直接的10-11易位2（TET2）介导的作用8，10 和间接抑制DNA甲基转移酶（DNMT）22，23的组合作用来诱导显着的全局DNA去甲基化。该步骤诱导表观遗传阻滞的去除，随后重新激活多能性相关基因表达，因此产生高可塑性细胞1，2，3，8，10，以下称为“表观遗传擦除细胞”。在第二步中，将细胞封装在3D培养系统中。为此，将非反应性疏水性合成化合物聚四氟乙烯（PTFE;粒径为1μm）用作微生物反应器，其允许创建通过使用传统2D培养系统无法实现的细胞微环境10。PTFE粉末颗粒粘附在液滴的表面，其中细胞被重新悬浮并将液核与支撑表面隔离开来，同时允许内部液体和周围环境24之间的气体交换。由此获得的“PTFE微生物反应器”，也称为“液体大理石”，鼓励细胞彼此自由相互作用，促进3D细胞重排25，26，27，并通过生物机械测量相关线索10延伸并稳定地维持获得的高可塑性状态。
Protocol
所有研究均由米兰大学伦理委员会审查和批准。所有动物实验均按照美国国立卫生研究院（NIH）发布的《实验动物护理和使用指南》进行。从健康的成年个体中分离人类细胞已获得米兰Ospedale Maggiore Policlinico伦理委员会的批准。我们研究中的所有方法都是根据批准的指南进行的。 1. 皮肤成纤维细胞分离 注意：下面描述的所有程序都可以应用于从不同哺乳动…
Representative Results
本方案描述了从成体细胞生成和稳定维持哺乳动物多能细胞的所有步骤。这种方法已经成功地从不同的哺乳动物物种（即小鼠，猪和人类）中分离出成纤维细胞。这里报道的代表性结果是从所有细胞系获得的，无论来源物种如何。 形态学分析表明，在与去甲基化剂5-氮杂-CR孵育18小时后，成纤维细胞封装在PTFE微生物反应器（3D Post 5-aza-CR）聚集体中并形成3D球形结构，显示出均…
Discussion
在过去的几十年里，一些研究的重点是制定策略，使终末分化的细胞恢复到不那么投入和更高的宽容状态。这里描述的方案允许从成体成熟的终末分化细胞开始产生和长期维持多能细胞。该方法结合了两个独立的步骤，涉及诱导高允许状态，这是通过化学表观遗传擦除实现的，并使用3D培养系统确保其后续维护。
在PTFE包封的细胞中观察到的3D球体结构的形成（…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作由Carraresi基金会和MiND FoodS Hub ID：1176436资助。所有作者都是COST Action CA16119体外3-D全细胞指导和健身（CellFit）的成员。
Materials
| 2-Mercaptoethanol | Sigma-Aldrich | M7522 | Component of ESC medium |
| 5-Azacytidine | Sigma-Aldrich | A2385 | 5-aza-CR, for fibroblast epigenetic erasing |
| Adenosine | Sigma-Aldrich | A4036 | Component of nucleoside mix for ESC medium |
| Antibiotic Antimycotic Solution (100×) | Sigma-Aldrich | A5955 | Component of fibroblast and ESC media |
| CFX96 Real-Time PCR | Bio-Rad Laboratories | NA | Thermal cycler for quantitative PCR |
| Cytidine | Sigma-Aldrich | C4654 | Component of nucleoside mix for ESC medium |
| DMEM, high glucose, pyruvate | Thermo Fisher Scientific | 41966052 | For fibroblast isolation and culture medium |
| DMEM, low glucose, pyruvate | Thermo Fisher Scientific | 31885023 | For ESC medium |
| Dulbecco’s Phosphate Buffered Saline | Sigma-Aldrich | D5652 | PBS; for biopsy and cell wash and for solution preparation |
| Dynabeads mRNA DIRECT Micro Purification Kit | Thermo Fisher Scientific | 61021 | mRNA estraction |
| ESGRO Recombinant Mouse LIF Protein | Sigma-Aldrich | ESG1106 | Component of ESC medium |
| Fetal Bovine Serum, qualified, heat inactivated | Thermo Fisher Scientific | 10500064 | Component of fibroblast and ESC media |
| FGF-Basic (AA10-155) Recombinant Human Protein | Thermo Fisher Scientific | PHG0024 | Component of ESC medium |
| Gelatin from porcine skin | Sigma-Aldrich | G1890 | For dish coating |
| GeneAmp PCR System 2700 | Applied Biosystems | NA | Thermal cycler for qualitative PCR |
| Global DNA Methylation ELISA Kit | CELL BIOLABS | STA-380 | Methylation study |
| GoTaq G2 Flexi DNA Polymerase | Promega | M7801 | Qualitative PCR |
| Guanosine | Sigma-Aldrich | G6264 | Component of nucleoside mix for ESC medium |
| Ham's F-10 Nutrient Mix | Thermo Fisher Scientific | 31550031 | For ESC medium |
| KnockOut Serum Replacement | Thermo Fisher Scientific | 10828028 | Component of ESC medium |
| KOVA glasstic slide 10 with grids | Hycor Biomedical | 87144 | For cell counting |
| Leica MZ APO Stereo Microscope | Leica | NA | For organoid observation |
| L-Glutamine solution | Sigma-Aldrich | G7513 | Component of fibroblast and ESC media |
| MEM Non-Essential Amino Acids Solution (100X) | Thermo Fisher Scientific | 11140035 | Component of ESC medium |
| Millex-GS 0.22 µm pore filters | Millipore | SLGS033SB | For solution sterilization |
| M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant | Promega | M3681 | mRNA reverse transcription |
| Multiskan FC Microplate Photometer | Thermo Fisher Scientific | 51119000 | For ELISA plate reading |
| Nikon Eclipse TE300 Inverted Phase Contrast Microscope | Nikon | NA | For cell observation |
| Perkin Elmer Thermal Cycler 480 | Perkin Elmer | NA | Thermal cycler for reverse transcription |
| Poly(tetrafluoroethylene) 1 μm particle size | Sigma-Aldrich | 430935 | For generating micro-bioreactor |
| PureLink Genomic DNA Mini Kit | Thermo Fisher Scientific | K182001 | Genomic DNA estraction |
| TaqMan Gene Expression Cells-to-CT Kit | Thermo Fisher Scientific | AM1728 | Quantitative PCR |
| Thymidine | Sigma-Aldrich | T1895 | Component of nucleoside mix for ESC medium |
| Tissue Culture Dish 100X20 mm, Standard | Sarstedt | 833902 | For fibroblast isolation |
| Tissue Culture Dish 35X10 mm, Standard | Sarstedt | 833900 | For Fibroblast isolation |
| Tissue Culture Dish 35X10 mm, Suspension | Sarstedt | 833900500 | Bacteriology petri dish for liquid marble culture |
| Tissue Culture Plate 96 Well,Standard,F | Sarstedt | 833924005 | For liquid marble culture |
| Trypsin-EDTA solution | Sigma-Aldrich | T3924 | For fibroblast dissociation |
| Tube 15ml, 120x17mm, PS | Sarstedt | 62553041 | For cell suspension centrifugation |
| Uridine | Sigma-Aldrich | U3003 | Component of nucleoside mix for ESC medium |
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Citazione di questo articolo
Pennarossa, G., Ledda, S., Arcuri, S., Gandolfi, F., Brevini, T. A. L. A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells. J. Vis. Exp. (162), e61655, doi:10.3791/61655 (2020).