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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&rsquo, 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.

International audience; Neural stem cell populations generate a wide spectrum of neuronal and glial cell types in a highly ordered fashion. MicroRNAs are essential regulators of this process. T-UCstem1 is a long non-coding RNA containing an ultraconserved element, and in vitro analyses in plurip-otent stem cells provided evidence that it regulates the balance between proliferation and differentiation. Here we investigate the in vivo function of T-UCstem1. We show that T-UCstem1 is expressed in the forebrain neurogenic lineage that generates interneurons for the postnatal olfactory bulb. Gain of function in neural stem cells increased progenitor proliferation at the expense of neuron production, whereas knockdown had the opposite effect. This regulatory function is mediated by its interaction with miR-9-3p and miR-9-5p. Based thereon, we propose a mechanistic model for the role of T-UCstem1 in the dynamic regulation of neural progenitor proliferation during neurogenesis.

Parkinson disease (PD) is characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The selective loss of this cell population makes PD a good candidate for cell-based therapies. Clinical trials using cells derived from human fetal ventral midbrain (hVM) have shown that dopamine release was restored to normal levels and in some PD patients produced substantial long-term clinical improvement. Although new sources of cells such as human embryonic stem cells (hESCs) and pluripotent stem cells (IPCs) are now been the focus of studies and new clinical trials, the hVM tissue represent the gold standard for cell based therapies and serve as important comparator for new cell sources. Therefore there is a need of an increased understanding and characterization of this tissue with new technologies such as organoid culture conditions and single cell sequencing. This study is designed to give a detailed characterization of human fetal VM tissue in standard culture conditions (2D) versus organoids (3D). We used immunocytochemistry to study the presence of ventral midbrain markers in 2D versus 3D condition together with single cell RNA sequencing to transcriptionally compare fetal VM tissue in standard or organoid cultures. Our analysis confirmed that the 3D condition preserve the neuronal composition of the tissue more than standard culture where instead, over a long period of time, the majority of the cells are non-neuronal. This suggest that 3D culture represent a better condition to maintain and study dopaminergic neurons in culture.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson's disease (PD) and they provide the option of using the patient's own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD.OBJECTIVE: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control.METHODS: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo.RESULTS: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line.CONCLUSION: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

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.

Parkinson disease (PD) is characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The selective loss of this cell population makes PD a good candidate for cell-based therapies. Clinical trials using cells derived from human fetal ventral midbrain (hVM) have shown that dopamine release was restored to normal levels and in some PD patients produced substantial long-term clinical improvement. Although new sources of cells such as human embryonic stem cells (hESCs) and pluripotent stem cells (IPCs) are now been the focus of studies and new clinical trials, the hVM tissue represent the gold standard for cell based therapies and serve as important comparator for new cell sources. Therefore there is a need of an increased understanding and characterization of this tissue with new technologies such as organoid culture conditions and single cell sequencing. This study is designed to give a detailed characterization of human fetal VM tissue in standard culture conditions (2D) versus organoids (3D). We used immunocytochemistry to study the presence of ventral midbrain markers in 2D versus 3D condition together with single cell RNA sequencing to transcriptionally compare fetal VM tissue in standard or organoid cultures. Our analysis confirmed that the 3D condition preserve the neuronal composition of the tissue more than standard culture where instead, over a long period of time, the majority of the cells are non-neuronal. This suggest that 3D culture represent a better condition to maintain and study dopaminergic neurons in culture.

Parkinson disease (PD) is characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The selective loss of this cell population makes PD a good candidate for cell-based therapies. Clinical trials using cells derived from human fetal ventral midbrain (hVM) have shown that dopamine release could be restored to normal levels and in some PD patients produced substantial long-term clinical improvement. The ethical issues related to the use of human fetal material as well as the low number of hVM tissue available have limited the spread of this approach and have lead researchers to find new sources of cells such as human embryonic stem cells (hESCs) and pluripotent stem cells (IPCs). This study was designed to provide a detailed molecular and functional of the fetal VM tissue in order to give a deeper understanding of its composition and characteristics. The characterization is based on the comparison of standard culture conditions (2D) versus organoids of hVM (3D). We used unbiased single cell RNA sequencing to transcriptionally compare fetal VM tissue in 2D and 3D conditions together with a functional analysis performed using whole cell patch clamp technique. Our analysis determined the molecular and functional properties of the tissue that originates dopaminergic neurons and also confirmed important differences between the culture conditions as well as it . The data can be used for quality assessment of new cell sources and provide new molecular targets for improved subtype specificity during stem cell differentiation.