Pennarossa Georgia, Ledda Sergio, Arcuri Sharon, Gandolfi Fulvio, Brevini Tiziana A L
Laboratory of Biomedical Embryology, Department of Health, Animal Science and Food Safety and Center for Stem Cell Research, Università degli Studi di Milano;
Department of Veterinary Medicine, University of Sassari.
J Vis Exp. 2020 Aug 29(162). doi: 10.3791/61655.
Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window. In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues. The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.
细胞表型可以通过不同方法逆转或改变，每种技术都有其特定的优点和局限性。在此，我们描述一种新策略，该策略将化学表观遗传擦除与机械传感相关线索相结合，以生成哺乳动物多能细胞。这需要两个主要步骤。第一步，将成年成熟（终末分化）细胞暴露于表观遗传擦除剂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深入理解而开发的，涉及使用表观遗传修饰剂来消除细胞分化状态，然后驱动其进入短暂的高可塑性窗口。第二步，将擦除后的细胞封装在聚四氟乙烯（PTFE）微生物反应器中，也称为液体弹珠，以促进3D细胞重排，从而扩展并稳定维持所获得的高可塑性。PTFE是一种非反应性疏水性合成化合物，其使用允许创建一种细胞微环境，这在传统的二维培养系统中是无法实现的。该系统通过与生物机械传感相关的线索来促进和增强多能性的维持。这里描述的技术程序是在成年体细胞中诱导和维持高可塑性状态的简单策略。该方案允许在所有测试的哺乳动物物种中获得高可塑性细胞。由于它不涉及基因转染的使用且不含病毒载体，它可能代表了转化医学应用中的一项显著技术进步。此外，微生物反应器系统通过在体外重新创建一个特定的微环境，为干细胞类器官技术带来了显著进步，该微环境允许长期培养高可塑性细胞，即胚胎干细胞（ESCs）、诱导多能干细胞（iPSCs）、表观遗传擦除细胞和间充质干细胞（MSCs）。
