Abstract
<title>Abstract</title> <p> Human microglia play central roles in brain homeostasis, immune surveillance and neurodegeneration, yet <italic>in vitro</italic> human microglial models differ widely in cellular origin and culture context, complicating interpretation and translational application. Here, we systematically compare transcriptional and functional states across primary monocytes, monocyte-derived macrophages (MDMa), two-dimensional (2D) and three-dimensional (3D) monocyte-derived microglia-like cells (MDMi) and induced pluripotent stem cell-derived microglia (iPSC-iMG). Using targeted NanoString profiling, we interrogated key microglial biology categories including homeostatic microglial identity, purinergic signalling, TGF-β signalling, interferon responses, complement activation, antigen processing and presentation, phagocytosis and neurodegeneration-associated microglial neurodegenerative phenotype (MGnD)/ disease-associated microglia (DAM) programmes. We find that cellular origin is a dominant determinant of microglial transcriptional identity. iPSC-iMG preferentially express homeostatic and CNS-patterned genes ( <italic>P2RY12</italic> , <italic>CX3CR1</italic> , <italic>ADORA1</italic> ) and TGF-β-associated regulators ( <italic>SMAD3</italic> ), consistent with a homeostatic microglial state. In contrast, MDMi retain immune-alert programmes characterised by interferon, complement and phagocytic gene modules ( <italic>IFIT1</italic> , <italic>STAT1</italic> , <italic>C1QA, C1QC</italic> , <italic>TREM2</italic> , <italic>APOE</italic> ), with partial overlap with MGnD/DAM-associated signatures. Despite these transcriptional differences, both models display comparable phagocytic uptake, indicating that distinct gene networks can support shared core functions. Compared with 2D MDMi, 3D differentiation shifted toward a less immune-alert and more tissue-adapted microglia-like transcriptional state, characterised by increased expression of selected microglial identity-associated genes ( <italic>AIF1</italic> and <italic>TMEM119</italic> ) and reduced inflammatory and antigen-processing signatures ( <italic>IL18</italic> and <italic>CD74)</italic> . However, 3D MDMi remained distinct from iPSC-iMG. Together, these data define model-specific baseline microglial states and provide a practical framework for selecting human microglial platforms for disease modelling and translational drug testing. </p>