人类ipsc衍生小胶质细胞大规模生成和高通量功能表征的改进方法

Manisha Padmakumar, Steven Biesmans, Jorge S. Valadas, Jan R. Detrez, Gaëlle Gillet, Priscillia Bresler, Marie-Laure Clénet, Irena Kadiu
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引用次数: 0

摘要

由于相关人类细胞模型的获取受限,以及现有临床前研究结果对人类病理生理学的可翻译性有限,神经科学药物的发现面临着重大挑战。诱导多能干细胞(iPSCs)已成为一种有前景的解决方案,提供了产生患者特异性细胞类型的潜力,包括近年来ipsc衍生的小胶质细胞(iMGL)。目前的方法依赖于复杂且耗时的区分程序,导致批次之间存在相当大的可变性,从而阻碍了标准化和可重复的高通量功能筛选方法的建立。解决这些挑战对于确保生成具有一致功能特性的同质iMGL种群至关重要。在这项研究中,我们描述了一种改进的高产量协议,用于生成iMGL,它允许在执行高通量功能屏幕时增加再现性和灵活性。我们介绍了胚胎样体(EB)维持的两步过程和允许在造血祖细胞(iHPC)阶段冷冻保存的停止点。此外,我们在一系列高通量功能分析中证明了这种修改后的方案在操作者之间的稳健性,包括吞噬作用、溶酶体酸化、趋化性和细胞因子释放。我们的研究强调了在ipsc分化和功能分析设置的各个阶段进行质量控制检查的重要性,强调了针对现有挑战的新解决方案,如有限的产量,灵活性和可重复性,这些都是药物发现的关键。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
An improved method for large scale generation and high-throughput functional characterization of human iPSC-derived microglia
Neuroscience drug discovery has faced significant challenges due to restricted access to relevant human cell models and limited translatability of existing preclinical findings to human pathophysiology. Induced pluripotent stem cells (iPSCs) have emerged as a promising solution, offering the potential to generate patient-specific cell types, including in the recent years, iPSC-derived microglia (iMGL). Current methods rely on complex and time-consuming differentiation procedures, leading to considerable batch-to-batch variability consequently hindering the establishment of standardized and reproducible high-throughput functional screening approaches. Addressing these challenges is critical in ensuring the generation of homogenous iMGL populations with consistent functional properties. In this study we describe an improved high-yield protocol for generating iMGL, which allows for increased reproducibility and flexibility in the execution of high-throughput functional screens. We introduce a two-step process in embryoid bodie (EB) maintenance and a stop point allowing for cryopreservation at the hematopoietic progenitor cell (iHPC) stages. Furthermore, we demonstrate inter-operator robustness of this modified protocol in a range of high-throughput functional assays including phagocytosis, lysosomal acidification, chemotaxis, and cytokine release. Our study underscores the importance of quality control checks at various stages of iPSC-differentiation and functional assay set up, highlighting novel workarounds to the existing challenges such as limited yield, flexibility, and reproducibility, all critical in drug discovery.
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