Midbrain microglia-integrated organoids as a next-generation tool for chronic morphine withdrawal research: A pilot study

In the midst of an opioid epidemic, opioid use disorder (OUD) can lead to significant clinical impairment or distress (such as opioid dependence and/or addiction) and overdose deaths. Therefore, it is significant to elucidate the exact molecular mechanisms of OUD and identify more effective therapies. Human induced pluripotent stem cell (hiPSC)-derived organoids are self-organized 3D tissues and mimic in vivo human organs. Increasingly, human-specific features of opioid abuse are being investigated using emerging brain organoids, which faithfully model the key functional, structural and biological complexities of neural tissues. However, a significant limitation of brain organoids is the absence of microglia which plays a fundamental role in OUD-related remodeling of neural circuits. In this pilot study, using hiPSC KOLF2.1J cell line, we developed our approach for generating both midbrain organoid and microglia and verified that our midbrain organoid was successfully integrated with microglia. We found that chronic exposure to morphine triggers key elements of immune responses including increased TNFα signaling and downregulation of sirtuin3 (Sirt3, a mitochondrial protein, preventing oxidative stress) and manganese superoxide dismutase (MnSOD, located in the mitochondrial matrix) in microglia-organoid co-cultures. Our findings suggest that immunocompetent midbrain organoids provide excellent models with which to study human-specific mechanisms of neuroinflammation and neurodegeneration. By combining disease relevance with scalability, the model system can be utilized as an effective tool for drug screening and toxicity testing.