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.

Glial progenitor cells are widely distributed in brain parenchyma and represent a suitable target for future therapeutic interventions that generate new neurons via in situ reprogramming. Previous studies have shown successful reprogramming of mouse glia into neurons whereas the conversion of human glial cells remains challenging due to the limited accessibility of human brain tissue. Here, we have used a recently developed stem cell-based model of human glia progenitor cells (hGPCs) for direct neural reprogramming by overexpressing a set of transcription factors involved in GABAergic interneuron fate specification. GABAergic interneurons play a key role in balancing excitatory and inhibitory neural circuitry in the brain and loss or dysfunction of these have been implicated in several neurological disorders such as epilepsy, schizophrenia, and autism. Our results demonstrate that hGPCs successfully convert into functional induced neurons with postsynaptic activity within a month. The induced neurons have properties of GABAergic neurons, express subtype-specific interneuron markers (e.g. parvalbumin) and exhibit a complex neuronal morphology with extensive dendritic trees. The possibility of inducing GABAergic interneurons from a renewable in vitro hGPC system could provide a foundation for the development of therapies for interneuron pathologies.

Direct reprogramming is an appealing strategy to generate neurons from a somatic cell by forced expression of transcription factors. The generated neurons can be used for both cell replacement strategies and disease modelling. Using this technique, previous studies have shown that γ-aminobutyric acid (GABA) expressing interneurons can be generated from different cell sources, such as glia cells or fetal fibroblasts. Nevertheless, the generation of neurons from adult human fibroblasts, an easily accessible cell source to obtain patient-derived neurons, has proved to be challenging due to the intrinsic blockade of neuronal commitment. In this paper, we used an optimized protocol for adult skin fibroblast reprogramming based on RE1 Silencing Transcription Factor (REST) inhibition together with a combination of GABAergic fate determinants to convert human adult skin fibroblasts into GABAergic neurons. Our results show a successful conversion in 25 days with upregulation of neuronal gene and protein expression levels. Moreover, we identified specific gene combinations that converted fibroblasts into neurons of a GABAergic interneuronal fate. Despite the well-known difficulty in converting adult fibroblasts into functional neurons in vitro, we could detect functional maturation in the induced neurons. GABAergic interneurons have relevance for cognitive impairments and brain disorders, such as Alzheimer’s and Parkinson’s diseases, epilepsy, schizophrenia and autism spectrum disorders.

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.

Native tissues are characterized by high cellular density and continuous reciprocal interactions between cells and the extracellular matrix (ECM), yet recreating these features in vitro remains a major challenge. Current three-dimensional (3D) culture systems typically capture either dense cell-cell interactions or a defined ECM environment, but rarely both. Here, we introduce a strategy that exploits direct fibroblast-to-neuron reprogramming to decouple microenvironment formation from final cell identity. Human dermal fibroblasts were embedded in collagen microgels, where their intrinsic matrix-remodeling capacity drove rapid ECM compaction. Concurrently, the contractile fibroblasts were converted into induced neurons (iN), generating densely packed neuronal microcultures within a remodeled collagen matrix. This approach transforms a defining function of the starting cell type into a mechanism for engineering the microenvironment of the target cell type. The resulting iN-microgels combine high cell density, defined ECM composition, and tunable biophysical properties in a scalable microscale format. By coupling matrix remodeling with cell-fate conversion, our platform establishes a general framework for engineering tissue-like microenvironments and reveals how dynamic 3D extracellular contexts can be integrated into direct cell reprogramming.