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1. Assembly of Human Stem Cell-Derived Cortical Spheroids and Vascular Spheroids to Model 3-D Brain-like Tissues

   https://doi.org/10.1038/s41598-019-42439-9  

   Song, Liqing; Yuan, Xuegang; Jones, Zachary; Griffin, Kyle; Zhou, Yi; Ma, Teng; Li, Yan (April 2019, Scientific Reports)

   Abstract Human cerebral organoids derived from induced pluripotent stem cells (iPSCs) provide novel tools for recapitulating the cytoarchitecture of human brain and for studying biological mechanisms of neurological disorders. However, the heterotypic interactions of neurovascular units, composed of neurons, pericytes, astrocytes, and brain microvascular endothelial cells, in brain-like tissues are less investigated. The objective of this study is to investigate the impacts of neural spheroids and vascular spheroids interactions on the regional brain-like tissue patterning in cortical spheroids derived from human iPSCs. Hybrid neurovascular spheroids were constructed by fusion of human iPSC-derived cortical neural progenitor cell (iNPC) spheroids, endothelial cell (iEC) spheroids, and the supporting human mesenchymal stem cells (MSCs). Single hybrid spheroids were constructed at different iNPC: iEC: MSC ratios of 4:2:0, 3:2:1 2:2:2, and 1:2:3 in low-attachment 96-well plates. The incorporation of MSCs upregulated the secretion levels of cytokines VEGF-A, PGE2, and TGF-β1 in hybrid spheroid system. In addition, tri-cultured spheroids had high levels of TBR1 (deep cortical layer VI) and Nkx2.1 (ventral cells), and matrix remodeling genes, MMP2 and MMP3, as well as Notch-1, indicating the crucial role of matrix remodeling and cell-cell communications on cortical spheroid and organoid patterning. Moreover, tri-culture system elevated blood-brain barrier gene expression (e.g., GLUT-1), CD31, and tight junction protein ZO1 expression. Treatment with AMD3100, a CXCR4 antagonist, showed the immobilization of MSCs during spheroid fusion, indicating a CXCR4-dependent manner of hMSC migration and homing. This forebrain-like model has potential applications in understanding heterotypic cell-cell interactions and novel drug screening in diseased human brain. 

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2. The Use of Pluripotent Stem Cell-Derived Organoids to Study Extracellular Matrix Development during Neural Degeneration

   https://doi.org/10.3390/cells8030242  

   Yan, Yuanwei; Bejoy, Julie; Marzano, Mark; Li, Yan (March 2019, Cells)

   The mechanism that causes the Alzheimer’s disease (AD) pathologies, including amyloid plaque, neurofibrillary tangles, and neuron death, is not well understood due to the lack of robust study models for human brain. Three-dimensional organoid systems based on human pluripotent stem cells (hPSCs) have shown a promising potential to model neurodegenerative diseases, including AD. These systems, in combination with engineering tools, allow in vitro generation of brain-like tissues that recapitulate complex cell-cell and cell-extracellular matrix (ECM) interactions. Brain ECMs play important roles in neural differentiation, proliferation, neuronal network, and AD progression. In this contribution related to brain ECMs, recent advances in modeling AD pathology and progression based on hPSC-derived neural cells, tissues, and brain organoids were reviewed and summarized. In addition, the roles of ECMs in neural differentiation of hPSCs and the influences of heparan sulfate proteoglycans, chondroitin sulfate proteoglycans, and hyaluronic acid on the progression of neurodegeneration were discussed. The advantages that use stem cell-based organoids to study neural degeneration and to investigate the effects of ECM development on the disease progression were highlighted. The contents of this article are significant for understanding cell-matrix interactions in stem cell microenvironment for treating neural degeneration. 

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3. Multicellular 3D Neurovascular Unit Model for Assessing Hypoxia and Neuroinflammation Induced Blood-Brain Barrier Dysfunction

   https://doi.org/10.1038/s41598-020-66487-8  

   Nzou, G; Wicks, R. T.; N. R., Mekky; G. A., Seale; S. A., Aya; ... & Atala, A. J (June 2020, Scientific reports)

   The blood-brain barrier (BBB) is a dynamic component of the brain-vascular interface that maintains brain homeostasis and regulates solute permeability into brain tissue. The expression of tight junction proteins between adjacent endothelial cells and the presence of efflux proteins prevents entry of foreign substances into the brain parenchyma. BBB dysfunction, however, is evident in many neurological disorders including ischemic stroke, trauma, and chronic neurodegenerative diseases. Currently, major contributors to BBB dysfunction are not well understood. Here, we employed a multicellular 3D neurovascular unit organoid containing human brain microvascular endothelial cells, pericytes, astrocytes, microglia, oligodendrocytes and neurons to model the effects of hypoxia and neuroinflammation on BBB function. Organoids were cultured in hypoxic chamber with 0.1% O2 for 24 hours. Organoids cultured under this hypoxic condition showed increased permeability, pro-inflammatory cytokine production, and increased oxidative stress. The anti-inflammatory agents, secoisolariciresinol diglucoside and 2-arachidonoyl glycerol, demonstrated protection by reducing inflammatory cytokine levels in the organoids under hypoxic conditions. Through the assessment of a free radical scavenger and an anti-inflammatory endocannabinoid, we hereby report the utility of the model in drug development for drug candidates that may reduce the effects of ROS and inflammation under disease conditions. This 3D organoid model recapitulates characteristics of BBB dysfunction under hypoxic physiological conditions and when exposed to exogenous neuroinflammatory mediators and hence may have potential in disease modeling and therapeutic development. 

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4. Genomics Analysis of Metabolic Pathways of Human Stem Cell-Derived Microglia-Like Cells and the Integrated Cortical Spheroids

   https://doi.org/10.1155/2019/2382534  

   Bejoy, Julie; Yuan, Xuegang; Song, Liqing; Hua, Thien; Jeske, Richard; Sart, Sébastien; Sang, Qing-Xiang Amy; Li, Yan (November 2019, Stem Cells International)

   Brain spheroids or organoids derived from human pluripotent stem cells (hiPSCs) are still not capable of completely recapitulating in vivo human brain tissue, and one of the limitations is lack of microglia. To add built-in immune function, coculture of the dorsal forebrain spheroids with isogenic microglia-like cells (D-MG) was performed in our study. The three-dimensional D-MG spheroids were analyzed for their transcriptome and compared with isogenic microglia-like cells (MG). Cortical spheroids containing microglia-like cells displayed different metabolic programming, which may affect the associated phenotype. The expression of genes related to glycolysis and hypoxia signaling was increased in cocultured D-MG spheroids, indicating the metabolic shift to aerobic glycolysis, which is in favor of M1 polarization of microglia-like cells. In addition, the metabolic pathways and the signaling pathways involved in cell proliferation, cell death, PIK3/AKT/mTOR signaling, eukaryotic initiation factor 2 pathway, and Wnt and Notch pathways were analyzed. The results demonstrate the activation of mTOR and p53 signaling, increased expression of Notch ligands, and the repression of NF- κ B and canonical Wnt pathways, as well as the lower expression of cell cycle genes in the cocultured D-MG spheroids. This analysis indicates that physiological 3-D microenvironment may reshape the immunity of in vitro cortical spheroids and better recapitulate in vivo brain tissue function for disease modeling and drug screening. 

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5. Microglia-targeting nanotherapeutics for neurodegenerative diseases

   https://doi.org/10.1063/5.0013178  

   Zhao, Nanxia; Francis, Nicola_L; Calvelli, Hannah_R; Moghe, Prabhas_V (September 2020, APL Bioengineering)

   Advances in nanotechnology have enabled the design of nanotherapeutic platforms that could address the challenges of targeted delivery of active therapeutic agents to the central nervous system (CNS). While the majority of previous research studies on CNS nanotherapeutics have focused on neurons and endothelial cells, the predominant resident immune cells of the CNS, microglia, are also emerging as a promising cellular target for neurodegeneration considering their prominent role in neuroinflammation. Under normal physiological conditions, microglia protect neurons by removing pathological agents. However, long-term exposure of microglia to stimulants will cause sustained activation and lead to neuronal damage due to the release of pro-inflammatory agents, resulting in neuroinflammation and neurodegeneration. This Perspective highlights criteria to be considered when designing microglia-targeting nanotherapeutics for the treatment of neurodegenerative disorders. These criteria include conjugating specific microglial receptor-targeting ligands or peptides to the nanoparticle surface to achieve targeted delivery, leveraging microglial phagocytic properties, and utilizing biocompatible and biodegradable nanomaterials with low immune reactivity and neurotoxicity. In addition, certain therapeutic agents for the controlled inhibition of toxic protein aggregation and for modulation of microglial activation pathways can also be incorporated within the nanoparticle structure without compromising stability. Overall, considering the multifaceted disease mechanisms of neurodegeneration, microglia-targeted nanodrugs and nanotherapeutic particles may have the potential to resolve multiple pathological determinants of the disease and to guide a shift in the microglial phenotype spectrum toward a more neuroprotective state. 

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