Host circuit engagement of human cortical organoids transplanted in rodents

Human neural organoids represent promising models for studying neural function; however, organoids grown in vitro lack certain microenvironments and sensory inputs that are thought to be essential for maturation. The transplantation of patient-derived neural organoids into animal hosts helps overcome some of these limitations and offers an approach for neural organoid maturation and circuit integration. Here, we describe a method for transplanting human stem cell–derived cortical organoids (hCOs) into the somatosensory cortex of newborn rats. The differentiation of human induced pluripotent stem cells into hCOs occurs over 30–60 days, and the transplantation procedure itself requires ~0.5–1 hours per animal. The use of neonatal hosts provides a developmentally appropriate stage for circuit integration and allows the generation and experimental manipulation of a unit of human neural tissue within the cortex of a living animal host. After transplantation, animals can be maintained for hundreds of days, and transplanted hCO growth can be monitored by using brain magnetic resonance imaging. We describe the assessment of human neural circuit function in vivo by monitoring genetically encoded calcium responses and extracellular activity. To demonstrate human neuron–host functional integration, we also describe a procedure for engaging host neural circuits and for modulating animal behavior by using an optogenetic behavioral training paradigm. The transplanted human neurons can then undergo ex vivo characterization across modalities including dendritic morphology reconstruction, single-nucleus transcriptomics, optogenetic manipulation and electrophysiology. This approach may enable the discovery of cellular phenotypes from patient-derived cells and uncover mechanisms that contribute to human brain evolution from previously inaccessible developmental stages. Key points The protocol involves surgical implantation of human cortical organoids in the cerebral cortex of rat pups. Organoid growth is monitored by using MRI, whereas their functional integration in the host neural circuitry is carried out by using behavioral, electrophysiological and optogenetic approaches. Transplanted organoids enable multimodal genomic measurements from millions of cells, facilitating characterization of human–human and human–rodent cellular interactions, including neural circuit activity patterns and relationships between glia and neurons. The transplantation of human cortical organoid