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.

Classical experiments performed half a century ago demonstrated the immense self-organizing capacity of vertebrate cells. Even after complete dissociation, cells can reaggregate and reconstruct the original architecture of an organ. More recently, this outstanding feature was used to rebuild organ parts or even complete organs from tissue or embryonic stem cells. Such stem cell-derived three-dimensional cultures are called organoids. Because organoids can be grown from human stem cells and from patient-derived induced pluripotent stem cells, they have the potential to model human development and disease. Furthermore, they have potential for drug testing and even future organ replacement strategies. Here, we summarize this rapidly evolving field and outline the potential of organoid technology for future biomedical research.

protocol INtroDUctIoNIn vitro methods to model human development and disease are part of a rapidly expanding field of stem cell biology with major therapeutic implications 1 . Organoid protocols stand at the forefront of these technologies, as these 3D approaches more accurately reproduce in vivo developmental events leading to more precise in vitro models 2,3 . Organoids have already been developed for several organ systems, including retina 4 , intestine 5 , thyroid 6 , liver 7 , pituitary 8 , inner ear 9 , kidney 10-12 and brain 13 . However, only a handful of methods exist for generating such tissues from human pluripotent stem cells (hPSCs) [13][14][15][16][17] . In this protocol, we describe the methodology that was recently published for generating brain organoid tissues from hPSCs 13 . Development of the protocolThe method described here builds on an extensive foundation of protocols for neural differentiation, 3D tissue culture and tissue engineering. Cerebral organoids develop through intrinsic selforganizing processes upon timely application of components and culture environments that had previously been described individually. Thus, the method is an amalgamation of previous methods, combined in a specific manner to address two main objectives: (i) the establishment of neural identity and differentiation and (ii) the recapitulation of 3D structural organization. Establishment of brain identityThe first goal of the protocol is induction and differentiation of neural tissue. This involves identification of media formulations and additives to drive neural identity and to stimulate brain development in vitro. To achieve this, a number of medium formulations were tested at various time points. Rather than describing the multitude of tested combinations, we focus here on the successful outcome.Neural tissue develops in vivo from a germ layer called the ectoderm 18 . Similarly, PSCs in vitro can be stimulated to develop germ layers, including ectoderm, within aggregates called embryoid bodies (EBs) 19 . A number of previous studies have described successful differentiation of EBs in embryonic stem cell (ESC) medium with decreased basic fibroblast growth factor (bFGF) 20 and high-dose rho-associated protein kinase (ROCK) inhibitor to limit cell death 21 . Similarly, cerebral organoids develop from EBs grown initially in ESC medium with low bFGF and ROCK inhibitor.Subsequent neural induction of EBs follows a minimal medium formulation very similar to that established by Zhang et al. 22,23 for the induction of neural rosettes, a 2D polarized organization of neuroepithelial cells. However, for the generation of cerebral organoids, EBs are kept in suspension, leading to uniform neural ectoderm formation along the outer surface of EBs, whereas inner non-neural mesendodermal tissues do not develop.Neural ectoderm in vivo establishes radially organized neuroepithelia that expand to form various brain structures. Similarly, organoids placed in a differentiation medium that supports both neural progenitors and their p...

Cerebral organoids-3D cultures of human cerebral tissue derived from pluripotent stem cells-have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear.Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.