Abstract
Brain-Region-Specific Organoids Using Mini-Bioreactors for Modeling ZIKV Exposure
Qian X, Nguyen HN, Song MM, Hadiono C, Ogden SC, Hammack C, Yao B, Hamersky GR, Jacob F, Zhong C, Yoon KJ, Jeang W, Lin L, Li Y, Thakor J, Berg DA, Zhang C, Kang E, Chickering M, Nauen D, Ho CY, Wen Z, Christian KM, Shi PY, Maher BJ, Wu H, Jin P, Tang H, Song H, Ming GL. Cell 2016;165:1238–1254.
Cerebral organoids, three-dimensional cultures that model organogenesis, provide a new platform to investigate human brain development. High cost, variability, and tissue heterogeneity limit their broad applications. Here, we developed a miniaturized spinning bioreactor (SpinΩ) to generate forebrain-specific organoids from human iPSCs. These organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a distinct human-specific outer radial glia cell layer. We also developed protocols for midbrain and hypothalamic organoids. Finally, we employed the forebrain organoid platform to model Zika virus (ZIKV) exposure. Quantitative analyses revealed preferential, productive infection of neural progenitors with either African or Asian ZIKV strains. ZIKV infection leads to increased cell death and reduced proliferation, resulting in decreased neuronal cell-layer volume resembling microcephaly. Together, our brain-region-specific organoids and SpinΩ provide an accessible and versatile platform for modeling human brain development and disease and for compound testing, including potential ZIKV antiviral drugs.
Commentary
Modeling neural network disorders such as epilepsy can be difficult in vitro due to the limitations of monolayer cultures. To overcome this hurdle with human embryonic or induced pluripotent stem cells (hESCs or iPSCs), the first three-dimensional (3D) neuronal culturing techniques took advantage of the self-aggregating properties of PSCs to generate polarized neural tissue (1–3). However, these cultures lacked the organization and complexity of the human neocortex. The advent of cerebral organoid technology (4, 5) transformed our ability to probe disease mechanisms and understand human neural development by allowing for studies in a more physiological 3D environment. Since then, the technology has been honed by several groups to create brain region-specific organoids, particularly cortical organoids. Pa ca et al. developed laminar cortical spheroids that were electrophysiologically active (6), while Mariani et al. reported an alternative cortical organoid strategy that led them to uncover a putative glutamatergic/GABAergic imbalance in autism spectrum disorder (7). However, the feasibility of using these organoids in biological applications, particularly disease modeling and drug discovery, is limited by factors such as the inability to generate all six cortical layers, disorganization of structures, and concerns regarding reproducibility and heterogeneity.
Qian et al. recently pioneered a miniature bioreactor (SpinΩ) accompanied by an innovative protocol utilizing Wnt agonists and SMAD inhibitors to generate highly organized cortical tissue that assembles into six cortical layers with a distinct outer radial glia layer—both critical components in the developing human cortex. Furthermore, the group established a set of protocols that takes advantage of SpinΩ to generate organoids that model other human brain regions, including midbrain and hypothalamus. Using their system, the group explored the mechanism of Zika virus (ZIKV) exposure and found that infected forebrain organoids have increased death of neural progenitors, leading to microcephaly.
So, how did they accomplish all of this? The authors were looking for an approach that allowed them to generate fore-brain organoids in a highly organized, reproducible fashion while simultaneously being scalable for high throughput applications without exponential increases in cost. They were able to engineer SpinΩ, a miniature bioreactor that features an individual spinning shaft in each well of a 12-well cell culture plate. Each well uses a minimal number of media (2 mL), and each plate can harbor a variety of culture conditions that make the system highly suitable for drug testing along with disease modeling applications.
Forebrain organoids are initiated by prepatterning iPSC-derived embryoid bodies into forebrain lineage and subsequently embedding the primitive organoids in Matrigel. A key feature of this new protocol is the utilization of CHIR99021, a downstream Wnt agonist, which helps reduce the significant cell death seen with other organoid protocols to date. After embedding in Matrigel for 7 days, the organoids are placed into SpinΩ in standard neuronal culture media (N2/B27). They are spun under these conditions for 8 weeks, after which the various growth factors are added to the culture to facilitate maturation. As the organoids are spun, they begin to develop distinctly organized cortical-like tissue composed of a SOX2+ ventricular zone (VZ) surrounded by TBR2+ intermediate progenitor cells (IPCs) and CTIP2+ neurons. Compared with organoids generated with previous protocols, the SpinΩ organoids are much more homogeneous in their development in terms of pureness of the ventricular zone and neuronal populations, as well as the growth and development of the cortical layers over time.
Qian and colleagues go on to systematically validate their organoids at different time points during development. At day 28, organoids have VZs that are surrounded by IPCs, followed by deep layer cortical neurons as well as an outer layer of Reelin+ Cajal-Retzius cells mimicking the marginal zone/layer 1 of the developing neocortex. By day 56, the organoids begin to develop late-born cortical neurons, characteristic of the inside-out development of the cerebral cortex. After 84 days, the upper layer neurons increase, and the cells in the outer organoid regions express markers for all six neocortical layers. Remarkably, at this time point, the organoids also develop a distinct outer VZ cell layer that is separate from the inner VZ. The presence of an outer radial glia layer is a feature unique to primate cortical development and largely absent in rodent corticogenesis (8); also, while outer radial glia cells have been observed sparsely in previous organoid protocols, it is a robust feature in the current study.
To further validate the recapitulation of human cortical development by the SpinΩ organoids, the authors perform RNA-seq of global transcriptomes from their organoids at different time points. When they compare the transcription profiles with those of developing fetal brain, the authors found that the forebrain organoids at day 100 closely correlate with fetal brains at 17 to 24 postconception weeks (PCW); however, correlation with some regions, such as the orbital frontal cortex, is as late as 35 PCW. The authors also show that the organoids are functionally active by generating acute slices and performing electrophysiology. Neurons within the forebrain organoids fire action potentials trains and exhibit spontaneous excitatory postsynaptic currents that are blocked by an AMPA/kainate receptor antagonist.
Finally, the study employs the forebrain organoids to examine a recent public health crisis: the ZIKV outbreak that can lead to microcephaly and other neural malformations in developing fetuses. The authors infect forebrain organoids with ZIKV at different time points to model the effects of ZIKV infection at different stages of human corticogenesis. They find that ZIKV mainly infects the neural progenitor cells (NPCs) in both the inner and outer VZs, with the number of ZIKV-infected cells increasing over time. This infection increases NPC apoptosis and decreases VZ thickness, mimicking the features of microcephaly in human disease. Interestingly, non-cell autonomous effects are seen, as many NPCs without evidence of ZIKV infection still undergo apoptosis. The authors conclude that the increased death of NPCs and decreased proliferation of the VZ, especially in the early organoids, mimic the effects of ZIKV on the developing fetal brain.
Qian et al. have developed an innovative strategy for generating brain-region-specific organoids using the SpinΩ. The ability to consistently generate functional, homogeneous forebrain organoids with six cortical layers and a distinct outer VZ makes this tool particularly powerful for investigating epilepsies that arise from altered brain development. For instance, generating iPSC-derived cortical spheroids from patients with gene mutations that cause megalencephaly or focal malformations of cortical development (e.g., mechanistic target of rapamycin pathway disorders) may offer a unique opportunity to assess their impact on different stages of human embryonic brain development such as NPC proliferation, migration, differentiation or synaptogenesis, and thereby suggest specific epilepsy mechanisms. In particular, the ability to identify specific time points and transitional periods in development that may lead to epilepsy susceptibility could offer insight into early interventions for disease prevention. The brain organoid approach should be useful not only for exploring human brain developmental changes underlying epileptogenesis, but also as a drug-screening platform for targeted personalized therapies.
It is important to note that there are some key limitations to the technology. First, as the organoids grow in size, diffusion deep to their centers diminishes, and the core ultimately becomes deprived of oxygen and nutrients. The authors recognized that this is a major reason that the forebrain organoids are unable to develop past what is comparable to second or early third trimester in human fetal development. In addition, while the forebrain organoids indeed contain some astrocytes and inhibitory interneurons, they are incomplete insofar as they do not have medial ganglionic eminence-derived inhibitory cell types. In addition, the authors have acknowledged that the forebrain organoids lack the intermediate zone and subplate regions, important areas for cortical development. A last concern is that, while the SpinΩ organoids contain cells that express markers of all six neocortical layers, their separation is indistinct. This poor lamination may reflect a lack of radial glial processes reaching the cortical surface. Nonetheless, this study paves the way for future organoid technologies and should serve as a powerful strategy for understanding the many epilepsy syndromes related to abnormal brain development and for identifying therapeutic targets.