Sue and Bill Gross Stem Cell Research Center, University of California, Irvine, California, USA.
Department of Anatomy and Neurobiology, University of California, Irvine, California, USA.
J Neurochem. 2024 Sep;168(9):3095-3107. doi: 10.1111/jnc.16173. Epub 2024 Jul 17.
Microglia, the immune cells of the central nervous system, are dynamic and heterogenous cells. While single cell RNA sequencing has become the conventional methodology for evaluating microglial state, transcriptomics do not provide insight into functional changes, identifying a critical gap in the field. Here, we propose a novel organelle phenotyping approach in which we treat live human induced pluripotent stem cell-derived microglia (iMGL) with organelle dyes staining mitochondria, lipids, lysosomes and acquire data by live-cell spectral microscopy. Dimensionality reduction techniques and unbiased cluster identification allow for recognition of microglial subpopulations with single-cell resolution based on organelle function. We validated this methodology using lipopolysaccharide and IL-10 treatment to polarize iMGL to an "inflammatory" and "anti-inflammatory" state, respectively, and then applied it to identify a novel regulator of iMGL function, complement protein C1q. While C1q is traditionally known as the initiator of the complement cascade, here we use organelle phenotyping to identify a role for C1q in regulating iMGL polarization via fatty acid storage and mitochondria membrane potential. Follow up evaluation of microglia using traditional read outs of activation state confirm that C1q drives an increase in microglia pro-inflammatory gene production and migration, while suppressing microglial proliferation. These data together validate the use of a novel organelle phenotyping approach and enable better mechanistic investigation of molecular regulators of microglial state.
小胶质细胞是中枢神经系统的免疫细胞，是具有动态和异质性的细胞。虽然单细胞 RNA 测序已成为评估小胶质细胞状态的常规方法，但转录组学并不能洞察功能变化，这是该领域的一个关键空白。在这里，我们提出了一种新的细胞器表型分析方法，我们用细胞器染料对活的人诱导多能干细胞衍生的小胶质细胞（iMGL）进行染色，以线粒体、脂质、溶酶体为研究对象，并通过活细胞光谱显微镜获取数据。降维技术和无偏聚类识别允许根据细胞器功能以单细胞分辨率识别小胶质细胞亚群。我们使用脂多糖和白细胞介素 10 处理来分别将 iMGL 极化为“炎症”和“抗炎”状态，验证了这种方法的有效性，然后应用它来鉴定 iMGL 功能的一种新型调节剂，补体蛋白 C1q。虽然 C1q 传统上被认为是补体级联反应的启动子，但在这里，我们使用细胞器表型分析来确定 C1q 通过脂肪酸储存和线粒体膜电位调节 iMGL 极化的作用。使用激活状态的传统读出值对小胶质细胞进行后续评估，证实 C1q 可促进小胶质细胞促炎基因的产生和迁移，同时抑制小胶质细胞增殖。这些数据共同验证了一种新型细胞器表型分析方法的使用，并能够更好地研究小胶质细胞状态的分子调节剂的机制。
