Reliability of human cortical organoid generation

Yoon, Se-Jin; Elahi, Lubayna S.; Pașca, Anca M.; Marton, Rebecca M.; Gordon, Aaron; Revah, Omer; Miura, Yuki; Walczak, Elisabeth M.; Holdgate, Gwendolyn M.; Fan, H. Christina; Huguenard, John R.; Geschwind, Daniel H.; Paşca, Sergiu P.

doi:10.1038/s41592-018-0255-0

Cited by 373 publications

(391 citation statements)

References 29 publications

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“…Our TLS already display remarkable reproducibility both at 166 the morphological and molecular level. Nevertheless, we realize the importance of further 167 integrated comparative analysis of single TLS at single-cell resolution, such as recently 168 published for brain and kidney organoids 37,38 . Finally, in vivo somites are formed in a rhythmic 169 process involving an oscillator -the segmentation clock 39 .…”

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confidence: 98%

Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites

Veenvliet

Bolondi

Kretzmer

et al. 2020

Preprint

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One sentence summary: A platform for generating trunk-like-structures with precursors of spinal cord, bone and muscle from stem cells in a dish Abstract: Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes inaccessible to deep analysis in vivo. Mouse embryonic stem cells (mESCs) can form aggregates reflecting the post-occipital embryo (gastruloids), but lacking proper morphogenesis. Here we show that embedding of aggregates derived from mESCs in an extracellular matrix compound results in Trunk-Like-Structures (TLS) with a high level of organization comprising a neural tube and somites. Comparative single-cell RNA-seq analysis demonstrates that TLS execute gene-regulatory programs in an embryo-like order, and generate primordial germ cell like cells (PGCLCs). TLS lacking Tbx6 form ectopic neural tubes, mirroring the embryonic mutant phenotype. ESC-derived trunk-like structures thus constitute a novel powerful in vitro platform for investigating lineage decisions and morphogenetic processes shaping the post-implantation embryo.2 Vertebrate post-implantation development comprises a multitude of complex morphogenetic 1 processes, which result from self-organization of stem cells and their descendants shaping the 2 embryonic body plan 1 . Recently developed stem cell models represent powerful platforms for 3 deconstructing the dynamics of these processes that are inaccessible in the embryo 1, 2 . The 4 most advanced models in terms of developmental stage accomplished so far are gastruloids 2,3 , 5 aggregates of mESCs able to self-organize into the three body axes 3 . However, gastruloids lack 6 proper morphogenesis, such as a failure of neural tube formation from neural cells or somite 7 condensation from presomitic cells 3 . In vivo, the extracellular matrix (ECM) has a critical role in 8 tissue morphogenesis 4 . In vitro, matrigel can serve as ECM surrogate, and culture media 9 supplemented with a low percentage of matrigel have been shown to induce complex 10 morphogenesis in organoids 5 . 11We therefore explored if embryo-like morphogenetic features could be induced by 12 embedding 96h post aggregation gastruloids in 5% matrigel for an additional 24h period ( Fig. 13 1a). We also tested a combination of matrigel together with the WNT signaling activator 14 CHIR99021 (CHIR) and the BMP signaling inhibitor LDN193189 (LDN), that have been reported 15 to induce a (pre-)somitic mesoderm ((P)SM) fate in 2D and 3D differentiation protocols 6-8 . To 16 facilitate high-throughput characterization and quantification of our conditions, we generated 17 mESCs with T::H2B-mCherry (hereafter T mCH ) and Sox2::H2B-Venus (hereafter Sox2 VE ) reporters, 18 marking the mesodermal (ME) or neural (NE) lineage respectively (Extended Data Fig. 1a). 19 Strikingly, embedding in 5% matrigel alone was sufficient for segmentation in the T mCH+ ME 20 domain and formation of a Sox2 VE+ neural tube like structure ( Fig. 1b,d; Extended Data Fig. 21 1b,c). The vast majorit...

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mentioning

confidence: 98%

Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites

Veenvliet

Bolondi

Kretzmer

et al. 2020

Preprint

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“…Our optimized self-directed protocol that generates highly reproducible hCOs, complements those reported in other recent studies that use a directed patterning approach involving small molecule inhibitors and growth factors (Kadoshima, Sakaguchi et al, 2013, Velasco et al, 2019, Yoon et al, 2019. The availability of several robust platforms to produce highly similar hCOs, including the one described herein, thus allows for systematic molecular and cellular characterization of the development of the human cortex and how disease-causing mutations alter development and perhaps also homeostatic events.…”

Section: Discussionmentioning

confidence: 74%

“…Several protocols have been developed to generate cerebral organoids including both directed and self-directed (Kadoshima, Sakaguchi et al, 2013, Velasco et al, 2019, Yoon et al, 2019 and here we selected a previously established method of selfpatterned organoids (Lancaster & Knoblich, 2014a, Lancaster et al, 2013 as a starting point. Our work to standardize production revealed that optimization of the early steps of organoid generation including the embryoid body formation and neural induction phases were key in ensuring the production of similar COs.…”

Section: Discussionmentioning

confidence: 99%

“…To establish a method to produce highly similar brain organoids (Fig. 1a), we explored modifications to a previously established protocol for generating selfpatterned whole-brain organoids (Lancaster & Knoblich, 2014a, Lancaster et al, 2013, which yields organoids with variable morphology and cell type composition (Quadrato, Nguyen et al, 2017, Velasco, Kedaigle et al, 2019, Yoon, Elahi et al, 2019. We primarily used female H9 human Embryonic Stem Cells (hESCs), and also validated results in a male hESC model (H1; see below).…”

Section: Optimization Of Cerebral Organoid Productionmentioning

confidence: 99%

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Robust Production of Uniform Human Cerebral Organoids from Pluripotent Stem Cells

Sivitilli

Gosio

Ghoshal

et al. 2020

Preprint

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Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human specific cortical development and pathophysiology in an experimentally tractable system. While diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent (ie uniform) hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single cell profiling, that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.

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“…How the mutations identified in FEZF2 allele-linked families contribute to layer organisation of the human cortex can be studied using induced Pluripotent Stem Cells (iPSCs). These pluripotent stem cells show a remarkable ability to self-organize and differentiate in vitro in three-dimensional aggregates, known as organoids or organ spheroids, and to recapitulate aspects of human brain development and function (62,63). Analysis of deep layer organization enables one to define the involvement of mutated genes discovered in patients of FEZF2 allele-linked families.…”

Section: Discussionmentioning

confidence: 99%

Multi-hit autism genomic architecture evidenced from consanguineous families with involvement of FEZF2 and mutations in high-risk genes

Bensaid

Loe-Mie

Lepagnol-Bestel

et al. 2019

Preprint

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words)Autism Spectrum Disorders (ASDs) are a heterogeneous collection of neurodevelopmental disorders with a strong genetic basis. Recent studies identified that a single hit of either a de novo or transmitted gene-disrupting, or likely gene-disrupting, mutation in a subset of 65 strongly associated genes can be sufficient to generate an ASD phenotype. We took advantage of consanguineous families with an ASD proband to evaluate this model. By a genome-wide homozygosity mapping of ten families with eleven children displaying ASD, we identified a linkage region of 133 kb in five families at the 3p14.2 locus that includes FEZF2 with a LOD score of 5.8 suggesting a founder effect. Sequencing FEZF2 revealed a common deletion of four codons. However, the damaging FEZF2 mutation did not appear to be sufficient to induce the disease as non-affected parents also carry the mutation and, similarly, Fezf2 knockout mouse embryos electroporated with the mutant human FEZF2 construct did not display any obvious defects in the corticospinal tract, a pathway whose development depends on FEZF2. We extended the genetic analysis of these five FEZF2-linked families versus five FEZF2 non-linked families by studying de novo and transmitted copy number variation (CNV) and performing Whole Exome Sequencing (WES). We identified damaging mutations in the subset of 65 genes strongly associated with ASD whose co-expression analysis suggests an impact on the prefrontal cortex during the mid-fetal periods. From these results, we propose that both FEZF2 deletion and multiple hits in the repertoire of these 65 genes are necessary to generate an ASD phenotype. Significance Statement (120 words)The human neocortex is a highly organized laminar structure with neuron positioning and identity of deep-layer cortical neurons that depend on key transcription factors, such as FEZF2, SATB2, TSHZ3 and TBR1. These genes have a specific spatio-temporal pattern of expression in human midfetal deep cortical projection neurons and display mutations in patients with Autism Spectrum Disorder (ASD). Here, we identified a linkage region involving FEZF2 gene in five consanguineous families with an ASD proband. For these FEZF2-allele linked probands, we identified a four-codon deletion in FEZF2 and damaging mutations in other high-risk ASD genes, that exhibit regional and cell type-specific convergence in neocortical deep-layer excitatory neurons, suggesting a multi-hit genomic architecture of ASD in these consanguineous families.

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Reliability of human cortical organoid generation

Cited by 373 publications

References 29 publications

Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites

Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites

Robust Production of Uniform Human Cerebral Organoids from Pluripotent Stem Cells

Multi-hit autism genomic architecture evidenced from consanguineous families with involvement of FEZF2 and mutations in high-risk genes

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