Three-dimensional brain organoids have emerged as a valuable model system for studies of human brain development and pathology. Here we establish a midbrain organoid culture system to study the developmental trajectory from pluripotent stem cells to mature dopamine neurons. Using single cell RNA sequencing, we identify the presence of three molecularly distinct subtypes of human dopamine neurons with high similarity to those in developing and adult human midbrain. However, despite significant advancements in the field, the use of brain organoids can be limited by issues of reproducibility and incomplete maturation which was also observed in this study. We therefore designed bioengineered ventral midbrain organoids supported by recombinant spider-silk microfibers functionalized with full-length human laminin. We show that silk organoids reproduce key molecular aspects of dopamine neurogenesis and reduce inter-organoid variability in terms of cell type composition and dopamine neuron formation.

Brain organoid technology has transformed both basic and applied biomedical research and paved the way for novel insights into developmental processes and disease states of the human brain. While the use of brain organoids has been rapidly growing in the past decade, the accompanying bioengineering and biofabrication solutions have remained scarce. As a result, most brain organoid protocols still rely on commercially available tools and culturing platforms that had previously been established for different purposes, thus entailing suboptimal culturing conditions and excessive use of plasticware. To address these issues, we developed a 3D printing pipeline for the fabrication of tailor-made culturing platforms for fluidically connected but spatially separated brain organoid array culture. This all-in-one platform allows all culturing steps—from cellular aggregation, spheroid growth, hydrogel embedding, and organoid maturation—to be performed in a single well plate without the need for organoid manipulation or transfer. Importantly, the approach relies on accessible materials and widely available 3D printing equipment. Furthermore, the developed design principles are modular and highly customizable. As such, we believe that the presented technology can be easily adapted by other research groups and fuel further development of culturing tools and platforms for brain organoids and other 3D cellular systems.

Two-dimensional neuronal cultures have a limited ability to recapitulate the in vivo environment of the brain. Here, we introduce a three-dimensional in vitro model for human glia-to-neuron conversion, surpassing the spatial and temporal constrains of two-dimensional cultures. Focused on direct conversion to induced dopamine neurons (iDANs) relevant to Parkinson disease, the model generates functionally mature iDANs in 2 weeks and allows long-term survival. As proof of concept, we use single-nucleus RNA sequencing and molecular lineage tracing during iDAN generation and find that all glial subtypes generate neurons and that conversion relies on the coordinated expression of three neural conversion factors. We also show the formation of mature and functional iDANs over time. The model facilitates molecular investigations of the conversion process to enhance understanding of conversion outcomes and offers a system for in vitro reprogramming studies aimed at advancing alternative therapeutic strategies in the diseased brain.

Direct lineage reprogramming of glial cells to induced neurons has the potential for restoring brain circuits and function in neuronal disorders and states. We introduce three-dimensional (3D) human glia reprogramming into neurons with a GABAergic interneuron phenotype using stem cell–derived human glia. Single-nucleus RNA sequencing of the converted cells demonstrates distinct neuronal clusters within 2 weeks, including a parvalbumin (PV) cluster with high neuronal maturity and features of chandelier interneurons. A lineage trajectory analysis of the glia-to-neuron conversion reveals a distinct lineage pathway to PV chandelier fate, including various neuronal developmental stages and the establishment of synaptic machinery. This analysis reveals PV fate-important genes that are previously unknown to neural reprogramming with promising functional importance for future derivations. Our data demonstrate successful human glia conversion into interneurons with features of bona fide PV subtype and highlight the reprogramming trajectory with key transitional genes. This advancement holds promise for future human brain cell engineering and repair.

Direct reprogramming of brain-resident glial progenitor cells (GPCs) into induced neurons (iN) shows great promise for future regenerative therapies for brain diseases. This type of cell conversion is facilitated by introducing lineage-specific transcription factors or small molecules to somatic cells, and can even be achieve in vivo , targeting resident glia cells in the brain for in vivo conversion. While this have shown promising results in animal models, a crucial step is to show the long-term survival and maturation of human reprogrammed cells in vivo. However, this remains largely unexplored partly due to the inaccessible cell source of GPC and difficulty in transplanting converted neurons. In this study, we assessed human GPCs derived iNs for their long-term structural and functional neuronal maturation after transplantation to the immunodeficient mouse brain. GPCs were transduced with established transcription factor cocktail and transplanted to the medial prefrontal cortex as three-dimensional cultures to improve survival and integration upon transplantation. Our results reveal survival of hiNs, for up to 10 months post-transplantation (MPT) and wide distribution across local and proximal functionally connected brain regions. Morphological analysis revealed a gradual neuronal maturation over time, characterized by increase in cellular complexity and expression of neuronal markers. Supporting these findings, electrophysiological recordings demonstrated progressive functional maturation up to 5 MPT with increase in sodium and potassium currents, and the ability to generate multiple evoked and spontaneous action potentials, validating a neuronal phenotype. Importantly, these changes were absent in control grafts of non-converted hGPCs that retained their glia identity. Our findings indicate that hGPCs derived iNs maintain a stable neuronal phenotype that persist in the mouse brain for extended periods. These cells exhibit survival and remarkable adaptability to the host environment, enabling both structural and functional maturation over time. These results highlight their potential as a promising strategy for neural replacement therapies aimed at restoring damaged circuits and promoting brain repair. Highlights: • Human neurons reprogrammed from glia progenitor cells survive long term upon transplantation in the immunodeficient mouse brain. • The human induced neurons migrate to local and distal connected brain regions within prefrontal cortex circuitry. • Grafted neurons gradually develop neuronal complex morphology and electrophysiological functional properties in the mouse brain.