Lopez-Yrigoyen Martha, May Alisha, Ventura Telma, Taylor Helen, Fidanza Antonella, Cassetta Luca, Pollard Jeffrey W, Forrester Lesley M
Centre for Regenerative Medicine, Scottish Centre for Regenerative Medicine, University of Edinburgh; Centre for Reproductive Health, The Queen's Medical Research Institute, University of Edinburgh;
Centre for Regenerative Medicine, Scottish Centre for Regenerative Medicine, University of Edinburgh.
J Vis Exp. 2020 Apr 16(158). doi: 10.3791/61038.
Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.
巨噬细胞存在于大多数脊椎动物组织中，由广泛分散且功能各异的异质细胞群体组成。它们是健康与疾病中的关键角色，在免疫防御过程中作为吞噬细胞发挥作用，并介导营养、维持和修复功能。尽管已经能够研究一些与人类巨噬细胞功能相关的分子过程，但事实证明，将基因工程技术应用于原代人类巨噬细胞非常困难。这严重阻碍了我们探究巨噬细胞生物学中复杂遗传途径以及生成特定疾病状态模型的能力。因此，一种适合大量基因操作技术的现成人类巨噬细胞来源，将为该领域提供一种有价值的工具。我们提出了一种优化方案，可在体外从人类诱导多能干细胞（iPSC）生成巨噬细胞。这些iPSC衍生的巨噬细胞（iPSC-DM）表达人类巨噬细胞表面标志物，包括CD45、25F9、CD163和CD169，我们的活细胞成像功能分析表明它们具有强大的吞噬活性。通过添加LPS和IFNg、IL4或IL10，培养的iPSC-DM可被激活至不同的巨噬细胞状态，这些状态表现出改变的基因表达和吞噬活性。因此，该系统提供了一个平台，可生成携带模拟特定人类疾病的基因改变的人类巨噬细胞，以及用于药物筛选或细胞治疗以治疗这些疾病的细胞来源。
