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      Cerebral organoids model human brain development and microcephaly

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          Abstract

          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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          Most cited references 61

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          Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.

          Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.
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            Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.

            The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling Lgr5(+) stem cells at the bottoms of small-intestinal crypts. Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5(+) stem cells can also initiate these cryptvillus organoids. Tracing experiments indicate that the Lgr5(+) stem-cell hierarchy is maintained in organoids. We conclude that intestinal cryptvillus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.
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              Generation of germline-competent induced pluripotent stem cells.

              We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression. These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras. Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells. The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells. We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation. Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene. Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                18 October 2013
                28 August 2013
                19 September 2013
                19 March 2014
                : 501
                : 7467
                Affiliations
                [1 ]IMBA - Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna 1030, Austria
                [2 ]MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK
                [3 ]Wellcome Trust Sanger Institute, Cambridge, UK
                [4 ]Department of Clinical Genetics, St. George’s University, London, UK
                Author notes
                Correspondence and requests for materials should be addressed to J.A.K. ( juergen.knoblich@ 123456imba.oeaw.ac.at ).

                Author Contributions: M.A.L and J.A.K. conceived of the project and experimental design and wrote the manuscript. M.A.L. performed experiments and analyzed data. M.R., C.-A.M., and D.W. performed experiments and analyzed data under supervision of J.A.K., J.M.P., and A.P.J. L.S.B., M.H., and T.H. performed patient diagnosis and provided MRIs coordinated by A.P.J. J.A.K. directed and supervised the project.

                EMS54265
                10.1038/nature12517
                3817409
                23995685

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                Funding
                Funded by: Austrian Science Fund FWF :
                Award ID: Z 153-B09 || FWF_
                Funded by: Austrian Science Fund FWF :
                Award ID: I 552-B19 || FWF_
                Funded by: European Research Council :
                Award ID: 250342 || ERC_
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