Abstract
Although brain organoids are an innovative technique for studying human brain development and disease by replicating the structural and functional properties of the developing human brain, some limitations such as heterogeneity and long-term differentiation (over 2 months) impede their application in disease modeling and drug discovery. In this study, we established simplified brain organoids (simBOs), composed of mature neurons and astroglial cells from expandable hPSC-derived primitive neural stem cells (pNSCs). simBOs can be rapidly generated in 2 weeks and have more homogeneous properties. Transcriptome analysis revealed that three-dimensional (3D) environment of simBOs facilitates the conversion of pNSCs to mature neuronal systems compared to a two-dimensional environment in the context of neurotransmitter release, synaptic vesicle formation, ion channels, calcium signaling, axonal guidance, extracellular matrix organization, and cell cycle. This result was correlated with the translocation of YAP1 into the cytoplasm by sensing matrix stiffness on the 3D models. Furthermore, we demonstrated that simBOs could easily be specified into midbrain-like simBOs by treatment with Shh and FGF8. Midbrain-like simBOs from a Parkinson’s disease patient (LRRK2G2019S)-derived pNSCs and gene-corrected (LRRK2WT) control pNSCs represented disease-associated phenotypes in terms of increased LRRK2 activity, decreased dopaminergic neurons, and increased autophagy. Treatment with the LRRK2 inhibitor, PFE-360, relieved the phenotype of Parkinson’s disease in midbrain-like simBOs. Taken together, these approaches could be applied to large-scale disease models and alternative drug-testing platforms.
Highlights
Neurological disorders, an immense threat to human health, are diseases with psychological and/or physical symptoms characterized by an abnormality in the development, patterning, and maintenance of homeostasis of the human brain or nervous system (Thakur et al, 2016)
Given that primitive neural stem cells (pNSCs) differentiated from Human pluripotent stem cells (hPSCs) are highly proliferative, cryopreservable, and capable of producing various cell types present in the brain and nervous system (Li et al, 2011; Liu et al, 2012), we hypothesized that self-organization of pNSCs and subsequent differentiation in a stirred bioreactor could rapidly generate simplified brain organoids (simBOs)
We differentiated pNSCs from human induced pluripotent stem cells (hiPSCs) derived from healthy human somatic cells following a previously reported procedure (Supplementary Figures 1A,B) (Liu et al, 2012). pNSCs showed high expression of neuronal stem cell markers (PAX6, NESTIN, and SOX2), marginal expression of pan-neuronal marker (TUJ1), and no expression of mature neuronal marker (MAP2), implying that pNSCs harboring the characteristics of neural stem cells (Supplementary Figure 1C)
Summary
Neurological disorders, an immense threat to human health, are diseases with psychological and/or physical symptoms characterized by an abnormality in the development, patterning, and maintenance of homeostasis of the human brain or nervous system (Thakur et al, 2016). Generation of Simplified Brain Organoids obtaining human brain tissues impedes understanding of pathogenesis and drug discovery. Human neuronal tissues can only be obtained from postmortem or surgically removed brain tissues. The experimental animal models have provided considerable insights into the molecular basis of normal brain development, disease pathogenesis, and therapeutic options for neurological disorders, but the inter-species differences in development (Liu et al, 2012; Homem et al, 2015) and pathogenesis (Burbulla et al, 2017; Gitler et al, 2017) make understanding the human brain and disease challenging. New experimental models are required for replicating the characteristics of the human brain and pathological conditions, in order to better understand the human brain development and disease
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