Engineering a Perfusion Bioreactor System for hiPSC‐Derived Progenitor Co‐Culture Capturing Microglial Features in CNS Development

Microglia are critical regulators of brain homeostasis and immune responses in the central nervous system (CNS). However, existing human-based models fail to reproduce the early and complex microglia-neural cell interactions. The differentiation of human induced pluripotent stem cells (hiPSCs) into specialized cell types offers promising avenues for understanding human development and disease modeling. Herein, a methodology for the differentiation of hiPSC-derived erythromyeloid progenitors (iEMPs) and their 3D co-culture with hiPSC-derived neurospheres were explored, utilizing the Ambr 250 Modular stirred-tank bioreactor (STB) system. The aim of this study was to build a complex co-culture model between iEMP and neurospheres in a scalable and controlled environment. Our results demonstrate that the STB effectively supports the co-culture process, with iEMP integration into the neurospheres, exhibiting cell density, aggregate morphology, and concentration similar to the neurosphere cultures. The co-culture environment induced the upregulation of transcription factors critical for microglial lineage commitment. iEMP-neurospheres displayed a unique secretory profile, releasing proteins involved in extracellular matrix remodeling and neuronal differentiation, essential for microenvironment remodeling. In conclusion, this study underscores the role of iEMPs in CNS development and presents a robust platform for preclinical research.