In the brain, complex interactions between different cell types can drive inflammation and disease. This cross talk is hard to study in vitro, because culture systems are often too simple to recapitulate the intricacies of the brain, or the mix of cells is too poorly defined to give reliable results. In the February 8 Nature Neuroscience, researchers led by Lorenz Studer at the Sloan Kettering Institute for Cancer Research in New York City describe a triculture system of neurons, microglia, and astrocytes that combines pure populations of each cell type in a defined ratio. Generated from human pluripotent stem cells, each cell type can be genetically manipulated. Because the cells can be frozen in large quantities, the cultures can be readily scaled up as well.
- A new triculture system combines pure populations of neurons, astrocytes, and microglia.
- The model revealed inflammatory cross talk between microglia and astrocytes.
- Mutant APP exacerbates this cross talk, implicating it in AD.
Studer noted that the iPSC field has struggled with reliability, with the same protocol often producing different data from one experiment to the next due to impurities. He believes his methodology offers a better option. “The system combines flexibility and reliability with sufficient complexity,” Studer told Alzforum. “The precision is unprecedented.”
Other researchers agreed. “This methodology is a huge step forward for the field,” Cynthia Lemere at Brigham and Women’s Hospital, Boston, told Alzforum. Doo Yeon Kim, Joseph Park, Matthias Hebisch, and Rudolph Tanzi at Massachusetts General Hospital, Boston, called the approach elegant. “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,” they wrote (full comment below).
The authors used this system to investigate inflammation. They found that microglia and astrocytes both had to be present to generate a strong immune response to an inflammatory challenge. Microglia were the instigators, pumping out inflammatory factors that triggered astrocytes to release complement protein C3. C3 then fed back on microglia, spurring them to pour out C3 as well.
The same inflammatory loop engaged when neurons carried the APP Swedish familial Alzheimer’s mutation, with both microglia and astrocytes participating. “To understand the neuroinflammatory response in AD, we think it is essential to have these three cell types together,” Studer told Alzforum. That is not always done in cell culture experiments.
Inflammatory Cross Talk. Under inflammatory conditions, microglia release factors that stimulate astrocytes to produce C3, which feeds back on microglia and triggers them to generate their own supply. [Courtesy of Guttikonda et al., Nature Neuroscience, 2021.]
Studer’s group previously developed protocols for producing pure neuronal and astrocyte populations from embryonic or induced pluripotent stem cells (Chambers et al., 2009; Qi et al., 2017; Tchieu et al., 2019). They wanted a similar method for microglia, which originate, together with macrophages, from a hematopoietic, not neural precursor lineage. The researchers wanted to mimic embryonic hematopoietic development, without embryoid bodies or cumbersome cell-sorting steps, which might contaminate the culture with other cells.
Starting with human pluripotent stem cells (PSCs), first author Sudha Guttikonda pharmacologically activated a burst of WNT signaling for exactly 18 hours before quenching it. This was followed by two days of Nodal signaling, which normally occurs early in embryonic development, to produce hemangioblasts, the precursors to all blood and vascular cells. A week of treatment with hematopoietic growth factors VEGF and FGF2 coaxed these cells into becoming erythromyeloid progenitors, which gave rise to premacrophages. Culturing premacrophages for 10 days with IL-34 and M-CSF generated a nearly pure population of macrophage-like cells. Lorenz noted that macrophages and microglia have the same developmental lineage; macrophages that migrate to the brain become microglia, suggesting that interactions with neurons may complete their patterning.
To make tricultures, the authors combined these macrophage-like cells with human PSC-derived astrocytes and neurons in a ratio of 2:1:8. Astrocytes proliferated, while some macrophages failed to attach; this led to a final ratio of 1:11:20, similar to the proportions of these cells in the human brain. As expected, once mixed with neurons, macrophages extended cellular processes and behaved like microglia, probing their environments and engulfing synapses (see image above). Their gene-expression profile matched those of microglia generated by other differentiation protocols, and those isolated from human brain (Muffat et al., 2016; Douvaras et al., 2017; Galatro et al., 2017).
This complement protein rises in inflammatory conditions such as aging and AD. When only neurons and astrocytes were present in the culture, C3 was almost undetectable. Neuron and microglia co-cultures made only a little more. With all three cell types, however, C3 shot up sevenfold. The same pattern occurred when cultures were challenged with lipopolysaccharide (LPS) to produce a vigorous immune response—both glial cell types were required for robust C3 secretion. The amount of C3 doubled under LPS.
How did each glia contribute? To dissect this, the authors used CRISPR to knock down C3 in the human PSCs before differentiating them. When they made tricultures with C3 knockout microglia but wild-type astrocytes and neurons, the media contained almost no C3. This was notable because in the brain, astrocytes, not microglia, are the main source of the protein. The data suggested microglial signaling was necessary to trigger astrocyte C3 production. Confirming this, media from wild-type, but not C3-knockout, microglial cultures stimulated C3 secretion from astrocytes.
The authors profiled C3 knockout microglia, and found they did not produce the pro-inflammatory cytokine TNF-α. Adding TNF-α to astrocyte-neuron cultures triggered C3 release, confirming this cytokine as a mediator of glial cross talk. As with wild-type tricultures, the authors saw the same results with and without LPS challenge, with the sole difference that maximum C3 levels were somewhat higher in the LPS condition.
Meanwhile, tricultures made with C3 knockout astrocytes but wild-type microglia produced about half as much C3 as did control tricultures. This showed that microglia also make C3. Adding C3 to microglia-neuron cultures stimulated additional C3 secretion, indicating the presence of an autocrine feed-forward loop in microglia. Conversely, adding C3 to astroglia-neuron cultures had no effect on astrocyte C3 production.
Altogether, the data suggested that microglia containing C3 kick off an inflammatory loop by releasing TNF-α. This provokes astrocytes to gush C3, which stimulates microglia to produce even more of the complement protein (see model above). “If you take away C3, nearly the whole neuroinflammatory system crashes,” Studer told Alzforum.
What happens in Alzheimer’s disease? When the authors used human neurons carrying the APP Swedish mutation in their tricultures, C3 levels were 64 percent higher than in isogenic wild-type tricultures without adding LPS. As before, both microglia and astrocytes fueled the cycle. It is unclear how mutant APP triggers this glial response. Overproduction of Aβ by itself has no effect on C3, Studer told Alzforum. He is trying to identify the neuronal factor responsible by way of transcriptomic and proteomic profiling.
Mutant APP tricultures also made more of another complement factor, C1Q, than did wild-type cultures, again without adding LPS. C1Q interacts with C3 to stimulate inflammation and synaptic pruning. It is made by microglia and acts on astrocytes. Curiously, when tricultures contained C3 knockout astrocytes or microglia, they accumulated more C1Q than did their wild-type counterparts, hinting that C3 may help trigger phagocytosis and cleanup of C1Q.
Lemere noted that these cell-culture findings resemble data from C3 conditional knockout mice made in her lab. In those otherwise wild-type animals, C1Q rises in the brain after C3 is shut off by inactivating the gene. “This [triculture] model seems to reflect processes going on in human disease,” Lemere noted. “It’s a great system and will be able to quickly define cellular pathways.” However, she cautioned that the findings need to be validated in vivo, because the triculture does not capture age- and time-dependent effects. For example, André Batista in Lemere’s group found that in mice, the increase in C1Q lasts about three months after C3 knockdown before falling back to baseline, perhaps due to compensatory mechanisms kicking in.
Similarly, Dominik Paquet and Julien Klimmt at the University of Munich noted a spatial limitation to these tricultures, in that they are two-dimensional. They believe three-dimensional culture systems will be necessary to fully model processes such as protein aggregation and microglial-synapse interactions. Nonetheless, they said the tricultures could be a powerful tool. “Their simple and modular platform may offer potential for genetic and drug-screening approaches,” they wrote (full comment below). Florent Ginhoux at the Agency for Science, Technology and Research in Singapore agreed the cultures could be useful for identifying therapeutic targets and testing anti-inflammatory interventions (comment below).
This triculture approach is not limited to AD. By replacing cortical neurons with dopaminergic or motor neurons, Studer already uses the same system to study Parkinson’s disease and amyotrophic lateral sclerosis.—Madolyn Bowman Rogers
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