SummaryConverting resident glia into functional and subtype-specific neurons in vivo by delivering reprogramming genes directly to the brain provides a step forward toward the possibility of treating brain injuries or diseases. To date, it has been possible to obtain GABAergic and glutamatergic neurons via in vivo conversion, but the precise phenotype of these cells has not yet been analyzed in detail. Here, we show that neurons reprogrammed using Ascl1, Lmx1a, and Nurr1 functionally mature and integrate into existing brain circuitry and that the majority of the reprogrammed neurons have properties of fast-spiking, parvalbumin-containing interneurons. When testing different combinations of genes for neural conversion with a focus on pro-neural genes and dopamine fate determinants, we found that functional neurons can be generated using different gene combinations and in different brain regions and that most of the reprogrammed neurons become interneurons, independently of the combination of reprogramming factors used.

Cell replacement is a long-standing and realistic goal for the treatment of Parkinsonʼs disease (PD). Cells for transplantation can be obtained from fetal brain tissue or from stem cells. However, after transplantation, dopamine (DA) neurons are seen to be a minor component of grafts, and it has remained difficult to determine the identity of other cell types. Here, we report analysis by single-cell RNA sequencing (scRNA-seq) combined with comprehensive histological analyses to characterize intracerebral grafts from human embryonic stem cells (hESCs) and fetal tissue after functional maturation in a pre-clinical rat PD model. We show that neurons and astrocytes are major components in both fetal and stem cell-derived grafts. Additionally, we identify a cell type closely resembling a class of recently identified perivascular-like cells in stem cell-derived grafts. Thus, this study uncovers previously unknown cellular diversity in a clinically relevant cell replacement PD model.

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.

Human in vitro models of neural tissue with tunable microenvironment and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing inside granular gels holds great promise as it allows precise patterning of extremely soft tissue constructs. However, granular printing support formulations are restricted to only a handful of materials. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high‐fidelity support for embedded printing akin to granular gels. To address this need, Authors present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self‐Healing Annealable Particle‐Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular‐matrix solution to enable precise and programmable patterning of human stem cells and consequent generation mature subtype‐specific neurons that extend projections into the volume of the annealed support. The developed approach further allows multi‐ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular‐like channels. Due to its modularity and versatility, SHAPE biomanufacturing toolbox has potential to be used in applications beyond functional modeling of mechanically sensitive neural constructs.

Compartmentalized microfluidic platforms are an invaluable tool in neuroscience research. However, harnessing the full potential of this technology remains hindered by the lack of a simple fabrication approach for the creation of intricate device architectures with high‐aspect ratio features. Here, a hybrid additive manufacturing approach is presented for the fabrication of open‐well compartmentalized neural devices that provides larger freedom of device design, removes the need for manual postprocessing, and allows an increase in the biocompatibility of the system. Suitability of the method for multimaterial integration allows to tailor the device architecture for the long‐term maintenance of healthy human stem‐cell derived neurons and astrocytes, spanning at least 40 days. Leveraging fast‐prototyping capabilities at both micro and macroscale, a proof‐of‐principle human in vitro model of the nigrostriatal pathway is created. By presenting a route for novel materials and unique architectures in microfluidic systems, the method provides new possibilities in biological research beyond neuroscience applications.