Department of Biology, University of Padua, Padua, Italy.
Department of Industrial Engineering, University of Padua, Padua, Italy.
Methods Mol Biol. 2022;2416:53-71. doi: 10.1007/978-1-0716-1908-7_5.
Human induced pluripotent stem cells (iPSCs) are generated from somatic cells by the expression of a cocktail of transcription factors, and iPSCs have the capacity to generate in vitro all cell types of the human body. In addition to primed (conventional) iPSCs, several groups recently reported the generation of human naïve iPSCs, which are in a more primitive developmental state and have a broader developmental potential, as shown by their ability to form cells of the placenta. Human iPSCs have broad medical potential but their generation is often time-consuming, not scalable and requires viral vectors or stable genetic manipulations. To overcome such limitations, we developed protocols for high-efficiency generation of either conventional or naïve iPSCs by delivery of messenger RNAs (mRNAs) using a microfluidic system. In this protocol we describe how to produce microfluidic devices, and how to reprogram human somatic cells into naïve and primed iPSCs using these devices. We also describe how to transfer the iPSC colonies from the microfluidic devices over to standard multiwell plates for subsequent expansion of the cultures. Our approach does not require stable genetic modifications, is reproducible and cost-effective, allowing to produce patient-specific iPSCs for cell therapy, disease modeling, and in vitro developmental studies.
人类诱导多能干细胞（iPSCs）是通过表达转录因子混合物从体细胞中产生的，iPSCs 具有体外生成人体所有细胞类型的能力。除了已被激活（常规）的 iPSCs 外，最近有几个研究小组还报告了人类原始 iPSCs 的产生，这些细胞处于更原始的发育状态，具有更广泛的发育潜力，这可从它们形成胎盘细胞的能力中得到证明。人类 iPSCs 具有广泛的医学潜力，但它们的产生通常耗时较长，无法规模化，且需要病毒载体或稳定的遗传操作。为了克服这些限制，我们使用微流控系统通过传递信使 RNA（mRNA）开发了高效生成常规或原始 iPSCs 的方案。在本方案中，我们将描述如何制作微流控设备，以及如何使用这些设备将人类体细胞重编程为原始和已被激活的 iPSCs。我们还将描述如何将 iPSC 集落从微流控设备转移到标准的多孔板上，以便随后对培养物进行扩增。我们的方法不需要稳定的遗传修饰，可重复性强，成本效益高，允许为细胞治疗、疾病建模和体外发育研究生产患者特异性 iPSCs。
