Bioengineered microtissue model of the human nigrostriatal pathway featuring centimeter-scale axon tracts and structural maturation over 6 months in vitro

The basal ganglia (BG) comprise a critical neural system involved in voluntary motor coordination and balance. While small animal models have been useful to improve our basic understanding of the development and function of neural systems, they are relatively low-throughput, lack human-specific genomic and target specificity, and fail to recapitulate critical anatomical features such as centimeter-scale axon tracts. Accordingly, bioengineered models replicating human systems-level features such as neuronal constituents, neurophysiology, and long-distance connectivity would be valuable to study BG circuit development, maturation, and function, as well as responses to neurotrauma and neurodegenerative diseases. To address this unmet need, we report the fabrication of a three-dimensional tissue engineered nigrostriatal pathway (TE-NSP) featuring human induced pluripotent stem cell (iPSC) derived dopaminergic (DA) neurons and centimeter-scale axon tracts that were maintained as a long-term in vitro model of key components of BG circuitry. TE-NSPs were fabricated using hyaluronic acid hydrogel microcolumns filled with collagen-laminin solution that were used to seed two types of neuronal aggregates, namely, human iPSC-derived DA and striatal neurons. These TE-NSPs featuring the co-culture of DA and striatal neurons were characterized for phenotypic stability, axonal growth, synaptic integration, and viability over 6 months in vitro. Our results demonstrate anatomically-relevant 3 cm long TE-NSPs with discrete 3D compartmentalized structures connected via > 2 cm long dopaminergic axon tracts. The TE-NSPs exhibited survival up to 6 months in vitro with dense TH + axon tracts and high cell viability. Immunohistochemistry revealed the widespread expression of mature synaptic markers within the TE-NSPs at 180 days in vitro featuring similarity with native in vivo rat striatal tissue. These long-lived TE-NSPs showed robust axonal outgrowth and innervation within the striatal compartment of the engineered tissue. We have established a mature living human iPSC-derived neural microtissue that recapitulates human-relevant anatomical connections and models a key component of the BG circuitry. This bioengineered human microtissue model could serve as a powerful platform for studying neural systems development, function, and neurodegenerative sequelae such as Parkinson’s disease, while also providing the basis for an implantable microtissue for circuit-level reconstruction following neurotrauma or neurodegenerative disease.