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Abstract
Macrophage polarization critically contributes to a multitude of human pathologies. Hence, modulating macrophage polarization is a promising approach with enormous therapeutic potential. Macrophages are characterized by a remarkable functional and phenotypic plasticity, with pro-inflammatory (M1) and anti-inflammatory (M2) states at the extremes of a multidimensional polarization spectrum. Cell morphology is a major indicator for macrophage activation, describing M1(-like) (rounded) and M2(-like) (elongated) states by different cell shapes. Here, we introduced cell painting of macrophages to better reflect their multifaceted plasticity and associated phenotypes beyond the rigid dichotomous M1/M2 classification. Using high-content imaging, we established deep learning- and feature-based cell painting image analysis tools to elucidate cellular fingerprints that inform about subtle phenotypes of human blood monocyte-derived and iPSC-derived macrophages that are characterized as screening surrogate. Moreover, we show that cell painting feature profiling is suitable for identifying inter-donor variance to describe the relevance of the morphology feature 'cell roundness' and dissect distinct macrophage polarization signatures after stimulation with known biological or small-molecule modulators of macrophage (re-)polarization. Our novel established AI-fueled cell painting analysis tools provide a resource for high-content-based drug screening and candidate profiling, which set the stage for identifying novel modulators for macrophage (re-)polarization in health and disease.
Highlights
Macrophages (MΦs) are innate immune cells characterized by high functional and phenotypic plasticity with the classically activated pro-inflammatory (M1) and the alternatively activated anti-inflammatory (M2) states at the extreme ends of a multidimensional polarization spectrum [1–5]
Frozen induced pluripotent stem cells (iPSC)-Derived MΦs Are Suitable as a Screening Surrogate of Monocyte-Derived MΦs Generated from Frozen peripheral blood mononuclear cells (PBMCs)
iPSC-derived MΦs (IDMs) generated from frozen CD34+ progenitors showed no obvious difference regarding surface marker expression compared with CD14+ progenitors and Monocyte-derived MΦs (MDMs) from several donors differentiated from blood monocytes that were purified from frozen PBMCs (Figures S1C and 1B)
Summary
Macrophages (MΦs) are innate immune cells characterized by high functional and phenotypic plasticity with the classically activated pro-inflammatory (M1) and the alternatively activated anti-inflammatory (M2) states at the extreme ends of a multidimensional polarization spectrum [1–5]. Owing to their critical function in pathogen defense, immune regulation, tissue homeostasis, and repair [6], MΦ polarization has been implicated in critically contributing to many human diseases including cancer, fibrosis, and obesity, as well as cardiovascular, inflammatory, and neurodegenerative diseases [7–10]. The limited availability of monocytes obtained from blood donations as well as their functional and phenotypic heterogeneity due to inter-donor variance [16] severely hampers their utilization for large-scale high-throughput cpd screening and drug candidate profiling [17]. We and others have recently shown that human-induced pluripotent stem cells (iPSC) can be differentiated towards iPSC-derived MΦs (IDMs) that functionally resemble MDMs [18–26]
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