Research on human brain development and neurological diseases is limited by the lack of advanced experimental in vitro models that truly recapitulate the complexity of the human brain. Here, we describe a robust human brain organoid system that is highly specific to the midbrain derived from regionally patterned neuroepithelial stem cells. These human midbrain organoids contain spatially organized groups of dopaminergic neurons, which make them an attractive model for the study of Parkinson's disease. Midbrain organoids are characterized in detail for neuronal, astroglial, and oligodendrocyte differentiation. Furthermore, we show the presence of synaptic connections and electrophysiological activity. The complexity of this model is further highlighted by the myelination of neurites. The present midbrain organoid system has the potential to be used for advanced in vitro disease modeling and therapy development.

The human brain is a complex, three‐dimensional structure. To better recapitulate brain complexity, recent efforts have focused on the development of human‐specific midbrain organoids. Human iPSC‐derived midbrain organoids consist of differentiated and functional neurons, which contain active synapses, as well as astrocytes and oligodendrocytes. However, the absence of microglia, with their ability to remodel neuronal networks and phagocytose apoptotic cells and debris, represents a major disadvantage for the current midbrain organoid systems. Additionally, neuroinflammation‐related disease modeling is not possible in the absence of microglia. So far, no studies about the effects of human iPSC‐derived microglia on midbrain organoid neural cells have been published. Here we describe an approach to derive microglia from human iPSCs and integrate them into iPSC‐derived midbrain organoids. Using single nuclear RNA Sequencing, we provide a detailed characterization of microglia in midbrain organoids as well as the influence of their presence on the other cells of the organoids. Furthermore, we describe the effects that microglia have on cell death and oxidative stress‐related gene expression. Finally, we show that microglia in midbrain organoids affect synaptic remodeling and increase neuronal excitability. Altogether, we show a more suitable system to further investigate brain development, as well as neurodegenerative diseases and neuroinflammation.

Parkinson’s disease is a slowly progressive neurodegenerative disease characterised by dysfunction and death of selectively vulnerable midbrain dopaminergic neurons and the development of human in vitro cellular models of the disease is a major challenge in Parkinson’s disease research. We constructed an automated cell culture platform optimised for long-term maintenance and monitoring of different cells in three dimensional microfluidic cell culture devices. The system can be flexibly adapted to various experimental protocols and features time-lapse imaging microscopy for quality control and electrophysiology monitoring to assess cellular activity. Using this system, we continuously monitored the differentiation of Parkinson’s disease patient derived human neuroepithelial stem cells into midbrain specific dopaminergic neurons. Calcium imaging confirmed the electrophysiological activity of differentiated neurons and immunostaining confirmed the efficiency of the differentiation protocol. This system is the first example of an automated Organ-on-a-Chip culture and has the potential to enable a versatile array of in vitro experiments for patient-specific disease modelling.

Background: The etiology of Parkinson's disease (PD) is only partially understood despite the fact that environmental causes, risk factors, and specific gene mutations are contributors to the disease. Biallelic mutations in the phosphatase and tensin homolog (PTEN)‐induced putative kinase 1 (PINK1) gene involved in mitochondrial homeostasis, vesicle trafficking, and autophagy are sufficient to cause PD.Objectives: We sought to evaluate the difference between controls' and PINK1 patients' derived neurons in their transition from neuroepithelial stem cells to neurons, allowing us to identify potential pathways to target with repurposed compounds.Methods: Using two‐dimensional and three‐dimensional models of patients' derived neurons we recapitulated PD‐related phenotypes. We introduced the usage of midbrain organoids for testing compounds. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9), we corrected the point mutations of three patients' derived cells. We evaluated the effect of the selected compound in a mouse model.Results: PD patient‐derived cells presented differences in their energetic profile, imbalanced proliferation, apoptosis, mitophagy, and a reduced differentiation efficiency to tyrosine hydroxylase positive (TH+) neurons compared to controls' cells. Correction of a patient's point mutation ameliorated the metabolic properties and neuronal firing rates as well as reversing the differentiation phenotype, and reducing the increased astrocytic levels. Treatment with 2‐hydroxypropyl‐β‐cyclodextrin increased the autophagy and mitophagy capacity of neurons concomitant with an improved dopaminergic differentiation of patient‐specific neurons in midbrain organoids and ameliorated neurotoxicity in a mouse model.Conclusion: We show that treatment with a repurposed compound is sufficient for restoring the impaired dopaminergic differentiation of PD patient‐derived cells. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Genome editing and human induced pluripotent stem cells hold great promise for the development of isogenic disease models and the correction of disease-associated mutations for isogenic tissue therapy. CRISPR-Cas9 has emerged as a versatile and simple tool for engineering human cells for such purposes. However, the current protocols to derive genome-edited lines require the screening of a great number of clones to obtain one free of random integration or on-locus non-homologous end joining (NHEJ)-containing alleles. Here, we describe an efficient method to derive biallelic genome-edited populations by the use of fluorescent markers. We call this technique FACS-assisted CRISPR-Cas9 editing (FACE). FACE allows the derivation of correctly edited polyclones carrying a positive selection fluorescent module and the exclusion of non-edited, random integrations and on-target allele NHEJ-containing cells. We derived a set of isogenic lines containing Parkinson's-disease-associated mutations in α-synuclein and present their comparative phenotypes.

The p.G2019S mutation of the leucine-rich repeat kinase 2 (LRRK2) has been identified as the most prevalent genetic cause of familial and sporadic Parkinson's disease (PD). The Cre-LoxP recombination system has been used to correct the LRRK2-G2019S mutation in patient derived human induced pluripotent stem cells (hiPSCs) in order to generate isogenic controls. However, the remaining LoxP site can influence gene expression. In this study, we report the generation of a footprint-free LRRK2-G2019S isogenic hiPS cell line edited with the CRISPR/Cas9 and piggyBac technologies. We observed that the percentage of Tyrosine Hydroxylase (TH) positive neurons with a total neurite length of >2000μm was significantly reduced in LRRK2-G2019S dopaminergic (DA) neurons. The average branch number in LRRK2-G2019S DA neurons was also decreased. In addition, we have shown that in vitro TH positive neurons with a total neurite length of >2000μm were positive for Serine 129 phosphorylated (S129P) alpha-Synuclein (αS) and we hypothesize that S129P-αS plays a role in the maintenance or formation of long neurites. In summary, our footprint-free LRRK2-G2019S isogenic cell lines allow standardized, genetic background independent, in vitro PD modeling and provide new insights into the role of LRRK2-G2019S and S129P-αS in the pathogenesis of PD.