Three-dimensional cell-cell interactions promote direct reprogramming of patient fibroblasts into functional and transplantable neurons

Direct reprogramming of somatic cells into induced neurons (iNs) has become an attractive strategy for the generation of patient-specific neurons for disease modeling and regenerative neuroscience. To this end, adult human dermal fibroblasts (hDFs) present one of the most relevant cell sources. However, iNs generated from adult hDFs using traditional two-dimensional (2D) cultures are difficult to maintain long-term in vitro and face challenges in survival upon transplantation into the adult brain. These limitations impose considerable constraints on the biomedical applications and translational potential of iN technology. Here, we present a platform for direct in vitro reprogramming of adult hDFs inside three-dimensional suspension microcultures (3D-iNs) that eliminates some of the major bottlenecks in the direct neuronal reprogramming field. We show that the 3D environment favors neuronal over fibroblast cellular identity to yield more robust conversion into functional neurons with extended culturing span. The 3D reprogramming approach also provides a platform for fusion of different neuronal populations into induced assembloids. Importantly, unlike conventional iNs, 3D-iNs can be gently harvested and transplanted into the adult rodent brain to reproducibly generate functionally integrated neuron-rich grafts. Due to its simplicity, versatility, and robustness, our approach could readily be adapted as a culturing platform used for a broad range of in vitro and in vivo studies to improve disease modeling, drug screening, and other biomedical applications.