. Fully defined human pluripotent stem cell-derived microglia and tri-culture system model C3 production in Alzheimer's disease. Nat Neurosci. 2021 Feb 8; PubMed.

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  1. This interesting study from the Studer lab establishes a defined and modular triculture model of hPSC-derived neurons, astrocytes, and microglia that is useful to study cell-cell interactions and cell-type-specific disease contributions in the context of Alzheimer’s disease. Using APPSwe/Swe knock-in stem-cell line and isogenic control (Paquet et al., 2016), they show that triculture of the three cell types is required to recapitulate inflammatory phenotypes, including increased C3 secretion and C1q deposition.

    Guttikonda et al. started out by developing and carefully characterizing a novel protocol to differentiate microglia from human pluripotent stem cells (hPSCs). The protocol yields highly pure populations of cells that are similar to fetal human microglia on a transcriptomic level and show typical functions such as surveillance of the environment and phagocytosis of synaptic material. The new protocol is based on primitive hematopoiesis followed by patterning of precursors to mature microglia, which is conceptually similar to existing protocols (Pandya et al., 2017; Douvaras et al., 2017; Abud et al., 2017), and it is encouraging to see that the gene-expression signatures of the resulting human microglia-like cells also largely overlap with microglia obtained from those protocols, as well as primary human microglia.

    They next established a triculture system of their microglia with hPSC-derived neurons and astrocytes using protocols published by their lab (Qi et al., 2017; Tchieu et al., 2019), and investigated neuroinflammatory “cross talk” between the different cell types. After challenging the cultures with LPS, they found increased levels of secreted C3, a known marker for neuroinflammation that is also increased in AD brains. Remarkably, the effect was strongest in the tricultures of neurons, astrocytes, and microglia, also compared to neuron-microglia cultures, indicating a cross talk of microglia and astrocytes that potentiates C3 expression. Indeed, further analyses suggested a reciprocal signaling between the two cell types, where microglia activate astrocytes, which in turn (further) activate microglia, leading to synergistic increases in C3 secretion.

    Previous studies had already implicated C3 in AD pathogenesis (Lian et al., 2015; Shi et al., 2017; Wu et al., 2019), but the underlying mechanisms are poorly understood and have not been studied in fully human co-culture models. The authors therefore applied their triculture platform to study neuroinflammation in an AD state using homozygous APPSwe knock-in iPSCs. Interestingly, C3 secretion was increased in tricultures with AD neurons compared to tricultures with isogenic WT neurons (astrocytes and microglia were WT in all cases), while it was not increased in cultures lacking astrocytes and/or microglia. This again corroborated the requirement for all three cell types. Finally, they analyzed C1q, an upstream regulator of C3 that is also known to accumulate in AD, and found increased C1q secretion and accumulation in APPSwe tricultures, with microglia being the main source.

    Taken together their data imply that increased Aβ secretion by neurons causes C1q and C3 secretion in microglia, which in turn stimulates a cycle of increased C3 secretion in astrocytes and microglia that may trigger neuroinflammation. Pro-inflammatory cytokines such as TNFα may also be involved.

    Their findings emphasize the importance of studying not only iPSC-derived human neurons in vitro, but more physiological combinations of brain-cell types to increase the relevance and translatability of the models. The experiments also nicely illustrate how the modularity of the triculture approach can be exploited to get mechanistic insights. Down the line, their simple and modular platform may as well offer potential for genetic and drug-screening approaches to find factors and compounds that break this vicious cycle of reciprocal activation.

    As with every intriguing study, important questions remain that may be studied with the established model:

    Is the activation of microglia directly mediated by increased Aβ production or could other APP-processing products such a β-CFTs play a role (Kwart et al., 2019)? In this context, it would be interesting to directly block Aβ production with inhibitors. How is Aβ detected by microglia and what are the players downstream? Is C1q directly involved as an inductive factor and how does this relate to C3 activation by NFκB (Lian et al., 2015)? Future studies could also investigate if glial activation and subsequent C3 and C1q deposition lead to downstream pathologies in the model, such as synaptic degeneration, for example by aberrant phagocytosis of synapses by microglia for which C1q and C3 are important factors (Hong et al., 2016), and eventually neurodegeneration, if the cultures are kept long-term.

    Finally, while defined and modular tricultures can be very powerful to study cellular crosstalk, as nicely demonstrated by Guttikonda et al., they may not be sufficient to study more complex, three-dimensional physiological cellular interactions, such as myelin involvement and microglia-synapse cross talk, or pathologic effects such as protein aggregation. This would require brain tissue models obtained by three-dimensional co-culture or organoids containing all relevant cell types, in which both Aβ and complement factors cannot easily diffuse away into the media but accumulate and act locally on surrounding cell types (discussed in Klimmt et al., 2020). 

    References:

    . Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature. 2016 May 5;533(7601):125-9. Epub 2016 Apr 27 PubMed.

    . Differentiation of human and murine induced pluripotent stem cells to microglia-like cells. Nat Neurosci. 2017 May;20(5):753-759. Epub 2017 Mar 2 PubMed.

    . Directed Differentiation of Human Pluripotent Stem Cells to Microglia. Stem Cell Reports. 2017 Jun 6;8(6):1516-1524. Epub 2017 May 18 PubMed.

    . iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases. Neuron. 2017 Apr 19;94(2):278-293.e9. PubMed.

    . Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells. Nat Biotechnol. 2017 Feb;35(2):154-163. Epub 2017 Jan 23 PubMed.

    . NFIA is a gliogenic switch enabling rapid derivation of functional human astrocytes from pluripotent stem cells. Nat Biotechnol. 2019 Mar;37(3):267-275. Epub 2019 Feb 25 PubMed.

    . NFκB-activated astroglial release of complement C3 compromises neuronal morphology and function associated with Alzheimer's disease. Neuron. 2015 Jan 7;85(1):101-15. Epub 2014 Dec 18 PubMed.

    . Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP/PS1 mice. Sci Transl Med. 2017 May 31;9(392) PubMed.

    . Complement C3 Is Activated in Human AD Brain and Is Required for Neurodegeneration in Mouse Models of Amyloidosis and Tauopathy. Cell Rep. 2019 Aug 20;28(8):2111-2123.e6. PubMed.

    . A Large Panel of Isogenic APP and PSEN1 Mutant Human iPSC Neurons Reveals Shared Endosomal Abnormalities Mediated by APP β-CTFs, Not Aβ. Neuron. 2019 Oct 23;104(2):256-270.e5. Epub 2019 Aug 12 PubMed.

    . Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science. 2016 May 6;352(6286):712-6. Epub 2016 Mar 31 PubMed.

    . Neurodegeneration in a dish: advancing human stem-cell-based models of Alzheimer's disease. Curr Opin Neurobiol. 2020 Apr;61:96-104. Epub 2020 Feb 26 PubMed.

    View all comments by Dominik Paquet
  2. Emerging evidence strongly supports that neural-glial interactions play a pivotal role in Alzheimer's disease pathogenesis. However, there are few human cellular models for studying these interactions. Lorenz Struder's team described a fully defined human neuron-astrocyte-microglia triculture model that recapitulates the elevated C3 complement cascade in AD.

    Previously, our lab, in collaboration with Hansang Cho, developed a three-dimensional human triculture model of AD, which recapitulates robust neurodegeneration and neuroinflammation, as well as Aβ and phospho-tau accumulation/aggregation (Choi et al., 2014; Park et al., 2018). We used immortalized transgenic human neural progenitor cells to generate AD neurons and astrocytes while using SV40-immortalized human microglia, as well as the microfluidic devices to facilitate robust Aβ accumulation, Aβ-induced tau pathology, and microglial migration.

    In this study, Guttikonda and colleagues used an elegant approach to generate a human triculture model using human pluripotent stem cell (hPSC)-derived neurons, astrocytes, and microglia. This full hPSC-derived triculture model will provide a useful tool for studying physiological and pathological neural-glial interactions in human cells, like their previous groundbreaking human cellular models. We congratulate Struder's team for developing a triculture model of AD with more physiologically relevant cell components.

    However, we would like to caution that the hPSC-derived triculture model might be limited in recapitulating full-blown AD pathology without robust Aβ and Aβ-driven tau hallmarks of AD pathology. We previously found that it is not easy to induce sufficient extracellular Aβ accumulation/aggregation and Aβ-driven tau using the conventional two-dimensional cell-culture conditions without the transgenic approaches (Choi et al., 2014). This iPSC-derived triculture can still provide a valuable model to study neural-glial interactions at the early stage of AD pathogenesis. However, without robust Aβ and tau pathology, the current model might be limited in recapitulating full-blown neuroinflammation and neuronal death, as well as microglial regulation of Aβ clearance in AD brains.

    We wonder if synaptic pruning (via C3 complement pathway activation) and neuronal death (by C1q activating A1 cytotoxic astrocytes) are also elevated in this hPSC-derived triculture model with the APP Swedish mutation. It is also exciting to see the selective elevation of the C3 complement cascade in this model. Still, it has not yet been fully characterized if this is through Aβ-dependent or independent pathways. 

    Despite these concerns, we believe that the improved triculture system described in this study holds great promise for understanding neural-glial cross-talks in AD. We hope this study triggers more sophisticated and physiologically relevant human brain cellular models for basic mechanistic studies and drug testing.  

    References:

    . A three-dimensional human neural cell culture model of Alzheimer's disease. Nature. 2014 Nov 13;515(7526):274-8. Epub 2014 Oct 12 PubMed.

    . A 3D human triculture system modeling neurodegeneration and neuroinflammation in Alzheimer's disease. Nat Neurosci. 2018 Jul;21(7):941-951. Epub 2018 Jun 27 PubMed.

    View all comments by Rudy Tanzi
  3. This is a great study and another proof of concept that human IPSC-derived culture/organoid systems, although not perfect since not recapitulating fully what is happening in vivo, are providing better models of human physiology and will be useful to gain new insights in the pathophysiology of AD. This study really exemplifies the importance of studying cell type interaction to identify circuits of regulation/reciprocal signaling pathways that could be now targeted on both cell types to potentiate the effect of an anti-inflammatory intervention.

    View all comments by Florent Ginhoux

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