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.

Ventral midbrain dopaminergic neurons project to the striatum as well as the cortex and are involved in movement control and reward-related cognition. In Parkinson’s disease, nigrostriatal midbrain dopaminergic neurons degenerate and cause typical Parkinson’s disease motor-related impairments, while the dysfunction of mesocorticolimbic midbrain dopaminergic neurons is implicated in addiction and neuropsychiatric disorders. Study of the development and selective neurodegeneration of the human dopaminergic system, however, has been limited due to the lack of an appropriate model and access to human material. Here, we have developed a human in vitro model that recapitulates key aspects of dopaminergic innervation of the striatum and cortex. These spatially arranged ventral midbrain–striatum–cortical organoids (MISCOs) can be used to study dopaminergic neuron maturation, innervation and function with implications for cell therapy and addiction research. We detail protocols for growing ventral midbrain, striatal and cortical organoids and describe how they fuse in a linear manner when placed in custom embedding molds. We report the formation of functional long-range dopaminergic connections to striatal and cortical tissues in MISCOs, and show that injected, ventral midbrain-patterned progenitors can mature and innervate the tissue. Using these assembloids, we examine dopaminergic circuit perturbations and show that chronic cocaine treatment causes long-lasting morphological, functional and transcriptional changes that persist upon drug withdrawal. Thus, our method opens new avenues to investigate human dopaminergic cell transplantation and circuitry reconstruction as well as the effect of drugs on the human dopaminergic system.

Human pluripotent stem cells (hPSCs) are intrinsically able to self-organize into cerebral organoids that mimic features of developing human brain tissue. These three-dimensional structures provide a unique opportunity to generate cytoarchitecture and cell-cell interactions reminiscent of human brain complexity in a dish. However, current in vitro brain organoid methodologies often result in intra-organoid variability, limiting their use in recapitulating later developmental stages as well as in disease modeling and drug discovery. In addition, cell stress and hypoxia resulting from long-term culture lead to incomplete maturation and cell death within the inner core. Here, we used a recombinant silk microfiber network as a scaffold to drive hPSCs to self-arrange into engineered cerebral organoids. Silk scaffolding promoted neuroectoderm formation and reduced heterogeneity of cellular organization within individual organoids. Bulk and single cell transcriptomics confirmed that silk cerebral organoids display more homogeneous and functionally mature neuronal properties than organoids grown in the absence of silk scaffold. Furthermore, oxygen sensing analysis showed that silk scaffolds create more favorable growth and differentiation conditions by facilitating the delivery of oxygen and nutrients. The silk scaffolding strategy appears to reduce intra-organoid variability and enhances self-organization into functionally mature human brain organoids.

Dopaminergic (DA) neurons exhibit significant diversity characterized by differences in morphology, anatomical location, axonal projection pattern, and selective vulnerability to disease. More recently, scRNAseq has been used to map DA neuron diversity at the level of gene expression. These studies have revealed a higher than expected molecular diversity in both mouse and human DA neurons. However, whether different molecular expression profiles correlate with specific functions of different DA neurons or with their classical division into mesolimbic (A10) and nigrostriatal (A9) neurons, remains to be determined. To address this, we have developed an approach termed TARGET-seq (Tagging projections by AAV-mediated RetroGrade Enrichment of Transcriptomes) that links the transcriptional profile of the DA neurons with their innervation of specific target structures in the forebrain. Leveraging this technology, we identify molecularly distinct subclusters of human DA neurons with a clear link between transcriptome and axonal target-specificity, offering the possibility to infer neuroanatomical-based classification to molecular identity and target-specific connectivity. We subsequently used this dataset to identify candidate transcription factors along DA developmental trajectories that may control subtype identity, thus providing broad avenues that can be further explored in the design of next-generation A9 and A10 enriched DA-neurons for drug screening or A9 enriched DA cells for clinical stem cell-based therapies.

Human midbrain dopamine (DA) neurons are a heterogeneous group of cells that share a common neurotransmitter phenotype and are in close anatomical proximity but display different functions, sensitivity to degeneration, and axonal innervation targets. The A9 DA neuron subtype controls motor function and is primarily degenerated in Parkinson’s disease (PD), whereas A10 neurons are largely unaffected by the condition, and their dysfunction is associated with neuropsychiatric disorders. Currently, DA neurons can only be reliably classified on the basis of topographical features, including anatomical location in the midbrain and projection targets in the forebrain. No systematic molecular classification at the genome-wide level has been proposed to date. Although many years of scientific efforts in embryonic and adult mouse brain have positioned us to better understand the complexity of DA neuron biology, many biological phenomena specific to humans are not amenable to being reproduced in animal models. The establishment of human cell-based systems combined with advanced computational single-cell transcriptomics holds great promise for decoding the mechanisms underlying maturation and diversification of human DA neurons, and linking their molecular heterogeneity to functions in the midbrain. Human pluripotent stem cells have emerged as a useful tool to recapitulate key molecular features of mature DA neuron subtypes. Here, we review some of the most recent advances and discuss the current challenges in using stem cells, to model human DA biology. We also describe how single cell RNA sequencing may provide key insights into the molecular programs driving DA progenitor specification into mature DA neuron subtypes. Exploiting the state-of-the-art approaches will lead to a better understanding of stem cell-derived DA neurons and their use in disease modeling and regenerative medicine.

Dopaminergic (DA) neurons derived from human pluripotent stem cells (hPSCs) represent a renewable and available source of cells useful for understanding development, developing disease models, and stem-cell therapies for Parkinson’s disease (PD). To assess the utility of stem cell cultures as an in vitro model system of human DA neurogenesis, we performed high-throughput transcriptional profiling of ~20,000 ventral midbrain (VM)-patterned stem cells at different stages of maturation using droplet-based single-cell RNA sequencing (scRNAseq). Using this dataset, we defined the cellular composition of human VM cultures at different timepoints and found high purity DA progenitor formation at an early stage of differentiation. DA neurons sharing similar molecular identities to those found in authentic DA neurons derived from human fetal VM were the major cell type after two months in culture. We also developed a bioinformatic pipeline that provided a comprehensive long noncoding RNA landscape based on temporal and cell-type specificity, which may contribute to unraveling the intricate regulatory network of coding and noncoding genes in DA neuron differentiation. Our findings serve as a valuable resource to elucidate the molecular steps of development, maturation, and function of human DA neurons, and to identify novel candidate coding and noncoding genes driving specification of progenitors into functionally mature DA neurons.