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      RosetteArray ® Platform for Quantitative High-Throughput Screening of Human Neurodevelopmental Risk

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          SUMMARY

          Neural organoids have revolutionized how human neurodevelopmental disorders (NDDs) are studied. Yet, their utility for screening complex NDD etiologies and in drug discovery is limited by a lack of scalable and quantifiable derivation formats. Here, we describe the RosetteArray ® platform’s ability to be used as an off-the-shelf, 96-well plate assay that standardizes incipient forebrain and spinal cord organoid morphogenesis as micropatterned, 3-D, singularly polarized neural rosette tissues (>9000 per plate). RosetteArrays are seeded from cryopreserved human pluripotent stem cells, cultured over 6–8 days, and immunostained images can be quantified using artificial intelligence-based software. We demonstrate the platform’s suitability for screening developmental neurotoxicity and genetic and environmental factors known to cause neural tube defect risk. Given the presence of rosette morphogenesis perturbation in neural organoid models of NDDs and neurodegenerative disorders, the RosetteArray platform could enable quantitative high-throughput screening (qHTS) of human neurodevelopmental risk across regulatory and precision medicine applications.

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          Cerebral organoids model human brain development and microcephaly

          Madeline Lancaster, Magdalena Renner, Carol-Anne Martin … (2013)
          The complexity of the human brain has made it difficult to study many brain disorders in model organisms, and highlights the need for an in vitro model of human brain development. We have developed a human pluripotent stem cell-derived 3D organoid culture system, termed cerebral organoid, which develops various discrete though interdependent brain regions. These include cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes. Furthermore, cerebral organoids recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells. Finally, we use RNAi and patient-specific iPS cells to model microcephaly, a disorder that has been difficult to recapitulate in mice. We demonstrate premature neuronal differentiation in patient organoids, a defect that could explain the disease phenotype. Our data demonstrate that 3D organoids can recapitulate development and disease of even this most complex human tissue.
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            Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR

            Maximilian Haeussler, Kai Schönig, Hélène Eckert … (2016)
            Background The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements. Results We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening. Conclusions To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website (http://crispor.org) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1012-2) contains supplementary material, which is available to authorized users.
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              Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals.

              Mototsugu Eiraku, Kiichi Watanabe, Mami Matsuo-Takasaki … (2008)
              Here, we demonstrate self-organized formation of apico-basally polarized cortical tissues from ESCs using an efficient three-dimensional aggregation culture (SFEBq culture). The generated cortical neurons are functional, transplantable, and capable of forming proper long-range connections in vivo and in vitro. The regional identity of the generated pallial tissues can be selectively controlled (into olfactory bulb, rostral and caudal cortices, hem, and choroid plexus) by secreted patterning factors such as Fgf, Wnt, and BMP. In addition, the in vivo-mimicking birth order of distinct cortical neurons permits the selective generation of particular layer-specific neurons by timed induction of cell-cycle exit. Importantly, cortical tissues generated from mouse and human ESCs form a self-organized structure that includes four distinct zones (ventricular, early and late cortical-plate, and Cajal-Retzius cell zones) along the apico-basal direction. Thus, spatial and temporal aspects of early corticogenesis are recapitulated and can be manipulated in this ESC culture.
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                Author and article information

                Journal
                Journal ID (nlm-ta): bioRxiv
                Journal ID (publisher-id): BIORXIV
                Title: bioRxiv
                Publisher: Cold Spring Harbor Laboratory
                ISSN (Electronic): 2692-8205
                Publication date (Electronic): 20 June 2024
                Electronic Location Identifier: 2024.04.01.587605
                Affiliations
                [1 ]Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53705, USA
                [2 ]Medical Scientist Training Program, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI, 53705 USA
                [3 ]Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
                [4 ]Neurosetta LLC, 330 N. Orchard Street Rm 4140A, Madison, WI 53715 USA
                [5 ]Center of Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143, USA
                [6 ]Department of Neurological Surgery, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI 53705, USA
                [7 ]Departments of Statistics and Computer Science, University of Chicago, Chicago, IL 60637, USA
                Author notes
                [*]

                Equal contribution

                AUTHOR CONTRIBUTIONS

                B.F.L, G.T.K., and R.S.A. took part in conception and design of all RosetteArray experiments and jointly interpreted all data. K.K. and R.W. conceived and designed all RosetteDetect experiments and jointly interpreted all related data. B.F.L. and G.T.K. performed all RosetteArray experimentation unless otherwise noted. N.J.K. performed all RosetteArray assays whose control data was included in Z-factor analysis. N.R.I. aided in derivation of WA09 and SCRIB mutant cell banks. B.F.L., G.T.K., N.J.F., J.E.M., and M.R.C. performed data acquisition and data analysis. N.R.Iz. and J.E.M. assisted with platform manufacturing, immunostaining, and imaging. J.F.R. and B.J.I. assisted with design and interpretation of DNT and NTD experiments. B.F.L, G.T.K., and RSA drafted the manuscript for publication with comments from all authors.

                [8 ]Lead Contact, correspondence: rashton2@ 123456wisc.edu
                Author information
                http://orcid.org/0000-0002-9597-0936
                http://orcid.org/0000-0002-6842-7022
                Article
                DOI: 10.1101/2024.04.01.587605
                PMC ID: 11118315
                PubMed ID: 38798648
                SO-VID: 041b76d0-0c3c-486b-8afc-b8027150a00c
                License:

                This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.

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