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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)

   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. Synthetic amyloid beta does not induce a robust transcriptional response in innate immune cell culture systems

   https://doi.org/10.1186/s12974-022-02459-1  

   Quiroga, I. Y. ; Cruikshank, A. E. ; Bond, M. L. ; Reed, K. S. M. ; Evangelista, B. A. ; Tseng, J. H. ; Ragusa, J. V. ; Meeker, R. B. ; Won, H. ; Cohen, S. ; et al ( April 2022 , Journal of Neuroinflammation)

   Alzheimer’s disease (AD) is a progressive neurodegenerative disease that impacts nearly 400 million people worldwide. The accumulation of amyloid beta (Aβ) in the brain has historically been associated with AD, and recent evidence suggests that neuroinflammation plays a central role in its origin and progression. These observations have given rise to the theory that Aβ is the primary trigger of AD, and induces proinflammatory activation of immune brain cells (i.e., microglia), which culminates in neuronal damage and cognitive decline. To test this hypothesis, many in vitro systems have been established to study Aβ-mediated activation of innate immune cells. Nevertheless, the transcriptional resemblance of these models to the microglia in the AD brain has never been comprehensively studied on a genome-wide scale.

   We used bulk RNA-seq to assess the transcriptional differences between in vitro cell types used to model neuroinflammation in AD, including several established, primary and iPSC-derived immune cell lines (macrophages, microglia and astrocytes) and their similarities to primary cells in the AD brain. We then analyzed the transcriptional response of these innate immune cells to synthetic Aβ or LPS and INFγ.

   We found that human induced pluripotent stem cell (hIPSC)-derived microglia (IMGL) are the in vitro cell model that best resembles primary microglia. Surprisingly, synthetic Aβ does not trigger a robust transcriptional response in any of the cellular models analyzed, despite testing a wide variety of Aβ formulations, concentrations, and treatment conditions. Finally, we found that bacterial LPS and INFγ activate microglia and induce transcriptional changes that resemble many, but not all, aspects of the transcriptomic profiles of disease associated microglia (DAM) present in the AD brain.

   These results suggest that synthetic Aβ treatment of innate immune cell cultures does not recapitulate transcriptional profiles observed in microglia from AD brains. In contrast, treating IMGL with LPS and INFγ induces transcriptional changes similar to those observed in microglia detected in AD brains.

    

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5. Differentiation of Brain Pericyte‐Like Cells from Human Pluripotent Stem Cell−Derived Neural Crest

   https://doi.org/10.1002/cpz1.21  

   Gastfriend, Benjamin D. ; Stebbins, Matthew J. ; Du, Feifan ; Shusta, Eric V. ; Palecek, Sean P. ( January 2021 , Current Protocols)

   Brain pericytes regulate diverse aspects of neurovascular development and function, including blood‐brain barrier (BBB) induction and maintenance. Primary brain pericytes have been widely employed in coculture‐based in vitro models of the BBB, and a method to generate brain pericytes from human pluripotent stem cells (hPSCs) could provide a renewable, genetically tractable source of cells for BBB modeling and studying pericyte roles in development and disease. Here, we describe a protocol to differentiate hPSCs to NG2⁺PDGFRβ⁺αSMA^lowbrain pericyte‐like cells in 22‐25 days through a p75‐NGFR⁺HNK‐1⁺neural crest intermediate, which mimics the developmental origin of forebrain pericytes. The resulting brain pericyte‐like cells have molecular and functional attributes of brain pericytes. We also provide protocols for maintenance, cryopreservation, and recovery of the neural crest intermediate, and for molecular and functional characterization of the resulting cells. © 2021 Wiley Periodicals LLC.

   This article was corrected on 18 July 2022. See the end of the full text for details.

   Basic Protocol 1: Differentiation of hPSCs to neural crest

   Basic Protocol 2: Differentiation of neural crest to brain pericyte‐like cells

   Support Protocol 1: Flow cytometry analysis of neural crest cells

   Support Protocol 2: Maintenance, cryopreservation, and recovery of neural crest cells

   Support Protocol 3: Molecular characterization of brain pericyte‐like cells

   Support Protocol 4: Cord formation assay with endothelial cells and brain pericyte‐like cells

    

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