Abstract
Induction of Expansion and Folding in Human Cerebral Organoids
Li Y, Muffat J, Omer A, Bosch I, Lancaster MA, Sur M, Gehrke L, Knoblich JA, Jaenisch R. Cell Stem Cell 2017;20:385–396.e3.
An expansion of the cerebral neocortex is thought to be the foundation for the unique intellectual abilities of humans. It has been suggested that an increase in the proliferative potential of neural progenitors (NPs) underlies the expansion of the cortex and its convoluted appearance. Here we show that increasing NP proliferation induces expansion and folding in an in vitro model of human corticogenesis. Deletion of PTEN stimulates proliferation and generates significantly larger and substantially folded cerebral organoids. This genetic modification allows sustained cell cycle reentry, expansion of the progenitor population, and delayed neuronal differentiation, all key features of the developing human cortex. In contrast, Pten deletion in mouse organoids does not lead to folding. Finally, we utilized the expanded cerebral organoids to show that infection with Zika virus impairs cortical growth and folding. Our study provides new insights into the mechanisms regulating the structure and organization of the human cortex.
Human iPSC-Derived Cerebral Organoids Model Cellular Features of Lissencephaly and Reveal Prolonged Mitosis of Outer Radial Glia
Bershteyn M, Nowakowski TJ, Pollen AA, Di Lullo E, Nene A, Wynshaw-Boris A, Kriegstein AR. Cell Stem Cell 2017;20:435–449.e4.
Classical lissencephaly is a genetic neurological disorder associated with mental retardation and intractable epilepsy, and Miller-Dieker syndrome (MDS) is the most severe form of the disease. In this study, to investigate the effects of MDS on human progenitor subtypes that control neuronal output and influence brain topology, we analyzed cerebral organoids derived from control and MDS-induced pluripotent stem cells (iPSCs) using time-lapse imaging, immunostaining, and single-cell RNA sequencing. We saw a cell migration defect that was rescued when we corrected the MDS causative chromosomal deletion and severe apoptosis of the founder neuroepithelial stem cells, accompanied by increased horizontal cell divisions. We also identified a mitotic defect in outer radial glia, a progenitor subtype that is largely absent from lissencephalic rodents but critical for human neocortical expansion. Our study, therefore, deepens our understanding of MDS cellular pathogenesis and highlights the broad utility of cerebral organoids for modeling humanneurodevelopmental disorders.
Assembly of Functionally Integrated Human Forebrain Spheroids
Birey F, Andersen J, Makinson CD, Islam S, Wei W, Huber N, Fan HC, Metzler KRC, Panagiotakos G, Thom N, O'Rourke NA, Steinmetz LM, Bernstein JA, Hallmayer J, Huguenard JR, Pa ca SP. Nature 2017;545:54–59.
The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.
Commentary
Experimental models involving rodents and other animals have begun to elucidate the pathophysiology of mesial temporal lobe epilepsy and the histologic changes seen in the hippocampus after status epilepticus (1). Emerging technologies involving human embryonic or induced pluripotent stem cells (hESCs or iPSCs) and three-dimensional (3D) cerebral organ-oids provide a novel approach to examining neurologic disease. Early models of monogenic diseases will pave the way for further understanding epileptogenesis in human neurons by providing scientific tools to tease apart the complex neurobiology of epilepsy and neurodevelopmental disorders. However, one key question is whether these 3D cerebral organoids possess the regional specificity, organization, and function of the intact human brain.
Bershteyn et al., used 3D cerebral organoids to investigate a severe cortical malformation syndrome, Miller-Dieker syndrome (MDS). The authors sought to expand upon the prevailing model that MDS and other forms of lissencephaly are due to defective neuronal migration (2) by generating iPSCs from three patients with MDS due to 17p13.3 deletion. For the first time, the experiments of Bershteyn et al. were able to visualize the saltatory migration of human lissencephalic neurons using live imaging techniques. Mutant neurons initiated migration but could not maintain typical migration compared with wild type (WT) neurons – they became stuck leading to a significant and early phenotypic difference in vitro. They found that this migratory defect was cell autonomous by using 5-week-old virally modified organoids cocultured into a scaffolding of human brain tissue. In this experiment, the majority of WT neural progenitors migrated radially to the pial surface, while mutant neurons showed impaired migration patterns with relatively few of the neural progenitors making it to the pial surface. To understand the mechanistic basis for these migration defects, the group performed single-cell RNA sequencing, immunohistochemistry analysis, and time-lapse imaging of MDS organoids. The authors identified specific mitotic defects in the outer radial glial (oRG) progenitors, which are believed to be a special population of cells to promote the developmental and evolutionary expansion of the human neocortex (3). This specific cell population is not robustly proliferative in rodent models, thus human-derived iPSC organoid models provide an emerging technique to evaluate oRG dysfunction, migration abnormalities, and other cellular features found in human cortical malformation syndromes.
Li and colleagues take human cerebral organoids a step further by showing that neural progenitor cells contribute to migration abnormalities and contain expanded ventricular and outer progenitors that lead to surface folding with homozygous PTEN deletion. Many human mutations, which activate the PI3K-AKT-mTOR pathway (PTEN is an upstream phosphatase inhibitor) have been implicated in megalencephaly, hemimegalencephaly, and polymicrogyria (4). Genetic rodent models have provided limited insight to the pathogenesis of cortical migration disorders as rodents have a naturally lissencephalic cortex and many cerebral overgrowth models have mild phenotypes in rodents while causing severe disease in humans. Typically, PTEN gene mutations are heterozygous in the human condition, but to maximize the experimental model, they used homozygous knock out (KO) models in both human and rodent organoids. Ultimately, the authors showed a species-specific difference with human PTEN KO organoids being larger than WT and curiously developing complex folding while mouse Pten KO organoids were larger, but did not show the complex folding seen in the human-derived organoids. The authors demonstrate that the expansion of the neural progenitor pool, a unique feature of the human organoids, contributed to the organoid folding phenotype. The identification of the complex role of neural progenitor cells in human organoid development further emphasizes the need to develop species-specific models when investigating neurodevelopmental disorders, including human cortical malformation syndromes.
Finally, Birey and colleagues address a key limitation to organoid technology, especially with respect to modeling epilepsy, by investigating whether organoids can model the assembly of circuits composed of both glutamatergic neurons and GABAergic interneurons. To accomplish this, the authors combine two types of cerebral organoid technology – glutamatergic enriched organoids named human cortical spheroids (hCS), and GABAergic enriched organoids named human sub-pallium spheroids (hSS). Sixty-day-old hCS and hSS organoids were fused and viral labeling of Dlxi1/2b, a marker of medial ganglionic eminence derivatives and source of GABAergic interneurons, allowed for specific investigation of the migration of GABAergic neural progenitors. Over multiple weeks, the position of GABAergic cells was monitored using time-lapse cellular imaging. In WT organoid fusion, GABAergic neural progenitors migrate in a cell autonomous fashion, seemingly to functionally integrate with the glutamatergic hCS. In a patient-derived iPSC model of Timothy syndrome, a rare genetic cause of autism, they demonstrate interneuron migration abnormalities elucidating an important pathophysiologic feature of this rare developmental disorder.
Collectively, these papers highlight the utility of cerebral organoids as an emerging in vitro model to study epilepsy and neurodevelopmental diseases. These articles demonstrate that the model can give robust phenotypes for a range of CNS disorders including lissencephaly, megalencephaly, and autism. By combining technologies that allow for direct visualization of neuron migration, single-cell RNA sequencing, and other functional assays, work on optimizing and standardizing this model has potential to make important discoveries in the biology of neurological and developmental disorders. However, current organoid technologies are limited by the lack of behavioral component (shared by all in vitro experimental models of epilepsy), lack of a blood supply, and unclear axial and anterior-posterior patterning. Functional neuronal activity has been demonstrated with patch clamp recordings of sliced cerebral organoids (5). Additionally, spontaneous neuronal network activity is beginning to be reported in cerebral organoids using high density silicon microelectrodes (6). Further possibilities of high density multielectrode array analysis may allow for additional characterization of cerebral organoid neural networks in both WT and experimental conditions. However, it is unclear if organoids have the capacity to create functional networks that can recapitulate the complicated networks seen in the human brain such as cerebral oscillations or electrographic seizure activity. A final concern is the differences between emerging organoid protocols, each one slightly changed from the last, making cross-study comparisons difficult. While researchers continue to optimize protocols to generate the best “human brain-in-a-dish” model, scientists have begun to use the organ-oid platform to screen drug candidates to address the Zika virus epidemic (7). In spite of 3D organoid model limitations, the technology provides an exciting way to study human-specific diseases using human-derived tissue that otherwise is only available postmortem or after surgical resection. This technology has the potential to advance neurologic research to focus on precision medicine that would otherwise be unattainable without using species-specific experimental models.