Characterization of simplified brain organoids (simBOs) derived from primitive neural stem cells (pNSCs)
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PROVIDER: PRJNA657816 | ENA |
REPOSITORIES: ENA
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Project description:We established simplified brain organoids (simBOs), composed of mature neurons and astrocytes from expandable hPSC-derived primitive neural stem cells (pNSCs) that can be rapidly generated in 2 weeks and have more homogeneous properties. Transcriptome analysis revealed that three-dimensional (3D) differentiation 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 was correlated with the translocation of YAP1 into the cytoplasm by sensing matrix stiffness on the 3D models.
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Project description:Microglia are specialised brain-resident macrophages that arise from primitive macrophages colonising the embryonic brain. Microglia contribute to multiple aspects of brain development, but their precise roles in early human brain remain poorly understood due to limited access to relevant tissues. The generation of brain organoids from induced human pluripotent stem cells (iPSC) recapitulates some key features of human embryonic brain development, but current approaches do not incorporate microglia and thus are lacking. Here, we generated microglia-sufficient brain organoids by co-culturing brain organoids with primitive-like macrophages generated from the same human iPSC (iMac). In organoid co-cultures, iMac differentiated into cells with microglia-like phenotypes and functions (iMicro), and modulated neuronal progenitor cell (NPC) differentiation, limiting NPC proliferation and promoting axonogenesis. Mechanistically, iMicro contained high levels of PLIN2+ lipid droplets that exported cholesterol and its esters which were taken up by NPC in the organoids. We also detected PLIN2+ lipid droplet-loaded microglia in mouse and human embryonic brain. Overall, our approach significantly advances current human brain organoid approaches by incorporating microglial cells, illustrated by the discovery of a key pathway of lipid-mediated crosstalk between microglia and NPC leading to improved neurogenesis.
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Project description:Cerebral organoids generated according to unguided protocols produce neural tissue with exceptional cell diversity and fidelity to in vivo. However, with only minimal extrinsic intervention, the importance of high quality starting material becomes paramount. Better understanding of what constitutes a high quality stem cell line and how to maintain those properties throughout prolonged culture is therefore a crucial foundation for successful organoid differentiation. In this study, we investigate the proteome and phospho-proteome of human pluripotent stem cells to uncover the mechanisms that drive neural organoid competence. We identify aberrant cell-extracellular matrix interaction and increased oxidative metabolism as hallmarks of poor neural differentiators. Drawing on the proteomic data and published literature, we optimise culturing conditions by using improved coating matrix, sustained supply of the key growth factor FGF2 and reducing oxidative stress. These adjustments improve brain organoid generation across all tested cell lines, though with varying degrees of efficiency. This work highlights the importance of optimal culture conditions to best support stem cells, ultimately enhancing the quality of brain organoids produced.
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Project description:In order to systematically investigate, to which extent cell lines and experimental batches affect the consistency of organoid patterning, we conducted a multiplexed morphogen patterning experiment to study the reproducibility of the effects induced by retinoic acid, FGF-8, SHH and CHIR. We have tested influence of 2 neural induction methods, 2 experimental batches and 4 cell lines: H1 (XY), H9 (XX), WTC (XY) and WIBJ (XX) on cell type composition consistency after morphogen patterning using single-cell transcriptomic readout (Parse Bioscience) and pooling 3 organoids for each condition.
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Project description:During embryogenesis, optic vesicles develop from the diencephalon via a complex process of organogenesis. Using iPSC-derived human brain organoids, we attempted to simplify the complexities and demonstrate the formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day thirty, brain organoids could assemble optic vesicles, which progressively develop as visible structures within sixty days. These optic vesicle-containing brain organoids (OVB-Organoids) constitute a developing optic vesicle’s cellular components, including the primitive cornea and lens-like cells, developing photoreceptors, retinal pigment epithelia, axon-like projections, and electrically active neuronal networks. Besides, OVB-Organoids also display synapsin-1, CTIP-positive, myelinated cortical neurons, and microglia. Interestingly, various light intensities could trigger photoreceptor activity of OVB-Organoids, and light sensitivities could be reset after a transient photo bleach blinding. Thus, brain organoids have the intrinsic ability to self-organize forebrain-associated primitive sensory structures in a topographically restricted manner and can allow conducting interorgan interaction studies within a single organoid.
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Project description:In our study, we define neural network activities in human cortex+ganglionic eminence (GE) fusion orgaonids using a combination of calcium incator imaging and electrophysiology. We futher define abnormal network activities using fusion organoids generated from isogenic induced pluripotent stem cells containing or lacking MECP2 function derived from Rett syndrome patients. Single cell sequencing (10X genomics) was used to characterize the compositon of the fusion organoids used in our studies, and to define differentially expressed genes associated with MECP2 mutation.
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Project description:Here we have provided scRNAseq datasets of unguided human brain organoids. The organoids were sequenced as a 1) Timecourse, 2) With different extracellular matrix treatments, 3) chemical treatment and 4) from organoids ere generated with a gene knockout cell line. Brain organoids enable the mechanistic study of human brain development, and provide opportunities to explore self-organization in unconstrained developmental systems. We have established long-term, live light sheet microscopy on unguided brain organoids generated from fluorescently labeled human induced pluripotent stem cells, which enables tracking of tissue morphology, cell behaviors, and subcellular features over weeks of organoid development. Based on imaging and single-cell transcriptome modalities, we find that lumenal expansion and cell morphotype composition within the developing neuroepithelium are associated with modulation of gene expression programs involving extracellular matrix (ECM) pathway regulators and mechanosensing. We show that an extrinsically provided matrix enhances lumen expansion as well as telencephalon formation, and unguided organoids grown in the absence of an extrinsic matrix have altered morphologies with increased neural crest and caudalized tissue identity. Matrix induced regional guidance and lumen morphogenesis are linked to the WNT and Hippo (YAP1) signaling pathways, including spatially restricted induction of the Wnt Ligand Secretion Mediator (WLS) that marks the earliest emergence of non-telencephalic brain regions. Altogether, our work provides a new inroad into studying human brain morphodynamics, and supports a view that matrix-linked mechanosensing dynamics play a central role during brain regionalization.
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