Building three-dimensional human brain organoids Sergiu P. Paşca The organogenesis of the human central nervous system is an intricately culture methods, to self-organize into brain spheroids or organoids. orchestrated series of events that occurs over several months and These organoid cultures can be derived from any individual, can be ultimately gives rise to the circuits underlying cognition and behavior. guided to resemble specific brain regions, and can be employed to model There is a pressing need for developing reliable, realistic, and complex cell-cell interactions in assembloids and to build human circuits. personalized in vitro models of the human brain to advance our This emerging technology, in combination with bioengineering and other understanding of neural development, evolution, and disease. Pluripotent state-of-the-art methods for probing and manipulating neural tissue, has stem cells have the remarkable ability to dierentiate in vitro into any of the potential to bring insights into human brain organogenesis and the the germ layers and, with the advent of three-dimensional (3D) cell pathogenesis of neurological and psychiatric disorders. Brain organogenesis in vitro and in vivo Brain organogenesis in vitro and in vivo Brain assembloids Methods for generating neural cells in vitro aim to recapitulate Spinal Forebrain Forebrain Blastocyst Cerebral Dorsal Pallial-subpallial assembloid key stages of in vivo brain organogenesis. Folding of the Neural Neural cord Paired recordingOptogenetics Pharmacology fold crest cortex Glutamate uncaging ectoderm-derived neural plate gives rise to the neural tube, Embryonic Yo lk sac Region 1 Region 2Region 3 which becomes enlarged on the anterior side to form the stem cell Neural groove Ventral Inner cell forebrain in the central nervous system (CNS). Corticogenesis forebrain In vitro mass Neural Forebrain modeling involves the sequential generation and positioning of layer- tube Spinal cord specific glutamatergic neurons from progenitors that line the Neural crest Cortico-thalamic assembloid MZ Ventral Emission Thalamus ventricles in the dorsal forebrain, the migration of GABAergic Gut tube interneurons that are born in the ventral forebrain, and waves of Live imaging gliogenesis to form astrocytes and oligodendrocytes, which Outer Neural crest derivatives continue postnatally. CP Upper + Pluripotent stem cells, derived from the inner mass of the layer Detection Single cells neurons blastocyst (embryonic stem cells) or from reprogrammed Fibroblasts Pluripotent Neural progenitors Neurons (2D) Neural Intestinal Study gut-neural somatic cells (induced pluripotent stem cells), can be stem cells Rosettes (2D) crest cells organoid interactions dierentiated into neural cells in bi-dimensional (2D) cultures Disease models using 3D brain cultures — where early on neuroepithelial cells position themselves into Inner Deep CP + Disease EtiologyCell types Phenotype structures called rosettes — or in self-organizing three- layer Genetic macrocephaly9 Homozygous deletion of PTEN Neural progenitors Cell cycle re-entry and increased proliferation with morphological changes dimensional (3D) brain spheroids or organoids1. Intermediate neurons Microglia Cortical Study neuroimmune Timothy syndrome10 Gain of function mutation in Cortical GABAergic neurons Defects in migration, which can be rescued by pharmacological manipulation of ('2.5D') cultures can be obtained when neural cells CACNA1C (Ca 1.2 voltage in forebrain assembloids the Ca 1.2 channel Neural aggregates (3D) Neurons (2.5D) Astrocyte organoid interactions V V dierentiated in 2D are lifted and cultured in 3D conditions to SP gated calcium channel) 2 form cellular aggregates or when dierentiated 3D aggregates UndirectedDirected Genetic microcephaly CDK5RAP2 loss of functionNeural progenitorsPremature neuronal dierentiation Organoid Brain-region-specific organoids IZ Blood Autism spectrum disorder IdiopathicForebrain progenitors and neurons Increased production of GABAergic neurons, which is modulated by FOXG1 derived from pluripotent stem cells are subsequently plated for + vessel with macrocephaly6 culture in 2D. Aicardi-Goutieres syndrome11 Loss of function of TREX1 Cortical neurons and astrocytes Apoptosis and reduced size of cortical organoids. Neurotoxic eects of astrocytes. Endothelial Cortical Study BBB Brain organoids can be generated from aggregates of 12, 13 Intermediate cells organoid development Miller-Dieker syndrome 17p13.3 deletion (LIS1, YWHAE) Radial glia and outer radia glia Defects in migration and cell division of radial glia that can be restored progenitors genetically or pharmacologically pluripotent stem cells through undirected dierentiation Cerebral cortex 5, 14, 15 2,3 (dorsal forebrain) SVZ/ Viral microcephaly Infection with the Zika virus Cortical progenitors, neurons, astrocytes Increased cell death methods that lack inductive signals , or by patterning through Outer oSVZ directed dierentiation methods to resemble specific brain radial glia regions4-8 (e.g., forebrain, midbrain, retina). Challenges and future directions Optic cup Midbrain • Improving reliability, anatomical accuracy (cortex psychiatric disease. monogenic, and polygenic causes of CNS disease Brain assembloids • + expansion, folding, white matter), predictability, and Modeling advanced stages, including postnatal, of to explore questions about convergent and divergent To model interactions between brain regions, organoids can be Radial VZ scalability of brain organoids. human brain development and incorporating missing pathogenesis in psychiatric disorders (e.g., autism glia patterned to resemble specific regions of the nervous system Ventral Dorsal Brain • Developing methods for transplantation of organoids cell types, e.g. glia, endothelial cells and pericytes, to spectrum disorders, schizophrenia). forebrain forebrain assembloid and then can be fused to generate brain assembloids1. Another into rodents or other species to obtain circuit-wide study neuroimmune and neurovascular interactions • Building large-scale platforms for drug discovery way to generate assembloids is by spatio-temporally controlling integration and oscillatory activity, to study sensory or cancer cells (e.g., oncogenesis and metastasis). and genetic screens. patterning within one 3D aggregate, by embedding organizer- input, and to develop more accurate models of • Developing reliable models of environmental, like structures (i.e., cells, coated beads) that release or block Applications of brain organoids developmental signals. A third method involves combining other Culturing cells in 3D Methods for culturing single cells into brain organoids: for instance, by embedding cells in 3D include Evolution Development and maturation Hanging drop Aggregation in Embedding Detachment from plates yolk-sac-derived microglia to study neuroimmune interactions, I I hanging drop cultures II U- or V-bottomed wells by embedding mesoderm-derived vascular cells to study the III II Graft hiPSCs attached to a slide, IV 300+ days Matrigel blood-brain barrier, or by embedding tumor cells to study brain III cell aggregation by V IV Human metastasis. These 3D cultures can be probed using genetic, brain centrifugation in U- Brain VI V organoid anatomical and functional read-outs. organoid Fetal Astrocyte Postnatal or V- bottom wells, state state Chimpanzee VI Human Applications of brain organoids embedding into Drug screening Disease extracellular matrices, Brain organoids and assembloids can be used to ask questions Maintaining 3D cell cultures and detachment of intact about evolutionary innovation in human and non-human Genetic Environmental pluripotent colonies that are then Bioreactor Oxygen chamber Shaker Low-attachment plates primates and to understand the developmental program and CRISPR/Cas9 moved to ultra-low-attachment maturation of the nervous system (e.g., the programs underlying Add Mutation Zika virus Organoid dishes. These 3D cultures can be astrocyte transition from a fetal to a postnatal state). Brain candidate deterioration drugs subsequently maintained in low- organoids derived from patients or that have been genetically or high-oxygen conditions, shaken engineered to carry genetic variants associated with disease (i.e., Healthy or spun in a bioreactor, or Patient vs. Control Control Control organoids isogenic lines) can be used to investigate disease pathogenesis in hiPSC/hESC Organoids maintained in low-attachment the nervous system. Lastly, as these 3D cultures become more Potential leads plates without shaking. scalable and assays probing 3D tissue improve, drug and CRISPR- Cas9-based screens can be used to identify therapeutic targets. Abbreviations photosensitive human brain organoids. Nature 545, 48–53, stratified neural retina from human ESCs. Cell Stem Cell 10, 771–785, 13 Iefremova, V. et al. An organoid-based model of cortical development Contact information and acknowledgements https://doi.org/10.1038/nature22047 (2017). https://doi.org/10.1016/j.stem.2012.05.009 (2012). identifies non-cell-autonomous defects in Wnt signaling contributing STEMCELL Technologies STEMdi™ Cerebral Organoid Kit (#08570) At STEMCELL, science is our foundation. We BBB: blood–brain barrier; CP: cortical plate; ENS: enteric 4Paşca, A. M. et al. Functional cortical neurons and astrocytes from 9Li, Y. et al. Induction of expansion and folding in human cerebral to Miller-Dieker syndrome. Cell Reports 19, 50–59, https://doi. Sergiu P. Paşca is at the Department of Psychiatry and are Scientists