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<p>Brain organoids require the integration of microglia to faithfully recapitulate​ physiological brain functions, as these immune cells are critical for neurodevelopmental processes​ and homeostatic regulation​. Here, we present a protocol for generating cortical organoids integrated with microglial progenitors derived from human embryonic stem cells (hESCs) by differentiating hESCs into Yolk Sac Embryoid Bodies (YS-EBs). Within the cultured organoids, the integrated microglia progenitors undergo differentiation, migration, and maturation. Key optimizations include enhanced microglial progenitor yield and organoid maturation, optimized integration timing, and implementation of standardized cryosectioning and dual tissue-clearing methods (iDISCO, CUBIC) to enable comprehensive 2D and 3D validation of microglial integration and their interactions with neural cells. This protocol supports long-term co-culture (up to 46&#xa0;days) with minimal necrotic core formation, enabling studies of neuroimmune interactions in a physiologically relevant 3D human brain model.</p> Graphical Abstract <p></p>

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Generating Microglia-Integrated Cortical Organoids from Human Embryonic Stem Cells

  • 期刊论文

  • 发表日期：

    2026年4月16日

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  • Hongxin Quan,
  • Xianglin Liu,
  • Min Yan,
  • Zongyong Ai,
  • Baohua Niu,
  • Jing Jiang,
  • Hong Chang,
  • Wenxiang Fu,
  • Runrui Zhang

摘要

Brain organoids require the integration of microglia to faithfully recapitulate​ physiological brain functions, as these immune cells are critical for neurodevelopmental processes​ and homeostatic regulation​. Here, we present a protocol for generating cortical organoids integrated with microglial progenitors derived from human embryonic stem cells (hESCs) by differentiating hESCs into Yolk Sac Embryoid Bodies (YS-EBs). Within the cultured organoids, the integrated microglia progenitors undergo differentiation, migration, and maturation. Key optimizations include enhanced microglial progenitor yield and organoid maturation, optimized integration timing, and implementation of standardized cryosectioning and dual tissue-clearing methods (iDISCO, CUBIC) to enable comprehensive 2D and 3D validation of microglial integration and their interactions with neural cells. This protocol supports long-term co-culture (up to 46 days) with minimal necrotic core formation, enabling studies of neuroimmune interactions in a physiologically relevant 3D human brain model.

Graphical Abstract