26 febbraio 2018

Self-organized cerebral organoids predict effective drugs to combat Zika virus infection

The human cerebral cortex shows distinct structural and functional features: accordingly, great efforts have been concentrated on the development of human brain-like structures termed organoids. Watanabe and colleagues from David Geffen School of Medicine at UCLA have reported on Oct 2017 an optimized organoid culture methods that efficiently and reliably produce cortical and basal ganglia structures similar to those in the human fetal brain in vivo. Scientists further demonstrate the utility of this organoid system for modeling the teratogenic effects of Zika virus on the developing brain and identifying more susceptibility receptors and therapeutic compounds.


The study of early human brain development is challenging due to ethical and practical considerations. Consequently, attention has been placed on the generation of in vitro models using human embryonic and induced pluripotent stem cells (Watanabe et al., 2017). Recently, several protocols for cerebral organotypic cultures derived from hPSC, often referred to as “organoids” have been established, with improvements in neuronal organization and generation of basal progenitors (Kadoshima et al., 2013; Lancaster et al., 2013; Pasca et al., 2015). Organoid techniques have thus opened the door for studies of human specific developmental features and disease modeling (Bershteyn et al., 2017; Lancaster et al., 2013; Mariani et al., 2015; Qian et al., 2016). Watanabe and colleagues from David Geffen School of Medicine at UCLA established a simple, yet efficient and reproducible cerebral organoid differentiation method where 80-90% of structures expressed forebrain markers (FOXG1 and LHX2) and displayed characteristic neuroepithelial organization. Hystochemical analysis confirms that these cerebral organoids efficiently recapitulates the histological organization and patterned expression of developmental regulators within the human fetal cortex in vivo. In addition, unbiased transcriptomic analyses prove that this 3D human brain model closely match fetal brain and developmental transitions in vivo up to the second trimester.

Scientists further have found that augmented stimulation of the STAT3 pathway improves the production of basal progenitors, the formation and separation of neuronal layers, and promotes astrogliogenesis, while neurons exhibited action potentials and spontaneous ensemble network activities. Finally, this 3D organoid platform was used to model Zika virus (ZIKV)-associated microcephaly and teratogenic effects on the developing brain, Watanabe et al. suggested that the susceptibility of neural progenitors to ZIKV infection is due to the expression of 4 entry receptors tyrosine kinases: AXL tyrosine kinase receptor, TYRO3 and MER (collectively termed TAM receptors), T-cell immunoglobulin and mucin domain 1 (TIM1) family receptors. ZIKV infection causes widespread cortical progenitor apoptosis and overall growth restriction since it activates innate immune responses leading to programmed cell death. Moreover, scientists from UCLA have identified two drugs that mitigate ZIKV-induced cytopathy: Duramycin and Ivermectin.

Since this organoid method do not require any special apparatus such as spinning bioreactors, the platform is easily scaled up or down. After aggregates are formed in 96-well plates, they can be transferred to bacterial dishes or multi-well plates for testing different media conditions or therapeutic compounds (Watanabe et al., 2017). Microfluidic nature of CellViewer technology (CellDynamics) reduces mechanical damage to the organoids, often seen with growth in spinning bioreactors, and represents a powerful platform for culturing and analyzing this 3D cerebral organoid model.

Figure 5. Infection of cerebral organoids with ZIKV leads to widespread progenitor apoptosis and overall growth restriction (A-B) Cerebral organoids immunostained for the ZIKV E protein, candidate ZIKV receptor (AXL), and PAX6+/SOX2+ progenitors, TBR2+ IP, CTIP2+ excitatory neurons, or AQP1+ choroid plexus markers. (C) VZ/SVZ regions of W8 cerebral organoids immunostained for the additional ZIKV candidate receptors TYRO3, MER, TIM1, and CD209. (D) Top, percentage of all ZIKV+ cells also labeled with indicated markers. Bottom, percentage of each cell type also infected with ZIKV. n=3 independent experiments. (E) Cerebral organoids stained with propidium iodide (PI) to label dead cells and the general nuclear stain. (F) Cell death was measured as PI intensity relative to HOECHST, area in mm2, and perimeter in mm. Mock n=23, ZIKV n=26. (G) Cerebral organoids collected 14 dpi immunostained for ZIKV, SOX2, TBR2, CTIP2, and clCASP3. (H) ZIKV infection was scored as the percentage of each cell type displaying ZIKV envelope staining. Cell death was scored as the percentage of each cell type exhibiting clCASP3 staining. n=3. Data in all charts are represented as mean ± SEM. Scale bars: A, 50 μm; B, 500 μm; E top, 200 μm; E brightfield, 1 mm; G, 100 μm. (Watanabe M. et al., Self-organized cerebral organoids with human specific features predict effective drugs to combat Zika virus infection. Cell Reports. 2017;21(2):517-532).





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Watanabe M., Jessie E. Buth, Neda Vishlaghi, Luis de la Torre-Ubieta, Jiannis Taxidis, Baljit Khakh, Giovanni Coppola, Caroline A. Pearson, Ken Yamauchi, Danyang Gong, Xinghong Dai, Robert Damoiseaux, Roghiyh Aliyari, Simone Liebscher, Katja Schenke-Layland, Christine Caneda, Eric J. Huang, Ye Zhang, Genhong Cheng, Daniel H. Geschwind, Peyman Golshani, Ren Sun, and Bennett G. Novitch. Self-organized cerebral organoids with human specific features predict effective drugs to combat Zika virus infection. Cell Reports. 2017;21(2):517-532.

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