An international research team headed by scientists at the Salk Institute for Biological Sciences has developed an organoid model—a three-dimensional collection of cells that mimics features of human tissues—that makes it possible to study the development and function of human microglia (hMG) for the first time in living human-derived tissue. In a newly published paper describing the organoid technology, the team reported on a study to examine patient-derived microglia from children with macrocephalic autism spectrum disorder (a condition where infant head circumference is greater than 97% of other infants) to determine whether brain environment influences the development of more reactive microglia.

Their findings highlight the importance of immune cell and brain interaction and offer up new insights to aid our understanding of neurodegenerative and developmental diseases, such as autism spectrum disorder (ASD) and Alzheimer’s disease.

“Outside of the brain environment, microglia lose almost all function and meaning,” said Rusty Gage, PhD, holder of the Vi and John Alder chair for research on age-related neurodegenerative disease. “We knew that if we found a way to replicate the human brain environment in an organoid in order to study human microglia, then we would finally have a tool for examining how the healthy and diseased brain influence microglia and, reciprocally, how healthy and diseased microglia influence the brain.”

Left: An immune brain cell (microglia) in a human brain. Right: Microglia in the novel organoid model with a human-brain-like environment. The cells are almost indistinguishable. [Salk Institute]

Gage is senior author of the team’s published paper in Cell, which is titled, “An in vivo neuroimmune organoid model to study human microglia phenotypes.”

Situated at the intersection of the human immune system and the brain, microglia are specialized brain immune cells that play a crucial role in development and disease. “Microglia are specialized brain-resident macrophages that play crucial roles in brain development, homeostasis, and disease,” the authors noted. “Mounting evidence from human and animal studies suggests that microglia may be implicated in various brain disorders, including neurodevelopmental conditions such as autism spectrum disorder (ASD).”

But while the importance of microglia is undisputed, modeling and studying them has remained a difficult task. Unlike some human cells that can be studied outside of the body or in nonhuman models, human microglia are difficult to study when removed from the human brain-like environment. “… until now, the ability to model interactions between the human brain environment and microglia has been severely limited,” the scientists continued.

Simon Schafer, Rusty Gage, and Axel Nimmerjahn
Simon Schafer, PhD, Rusty Gage, PhD, and Axel Nimmerjahn, PhD [Salk Institute]

Over the last decade, organoids have become a prevalent tool to bridge the gap between cell and human studies. Organoids can mimic human development and organ generation better than other laboratory systems, allowing researchers to study how drugs or diseases affect human cells in a more realistic setting. Brain organoids are typically grown in culture dishes, but the organoids are structurally and functionally limited by the lack of blood vessels, short survival time, and inability to sustain diverse cell types (such as microglia).

So, as the scientists commented, “Apart from phenomenological observations in postmortem tissue, the role and function of microglia during human brain development remains an almost entirely uncharted territory due to the lack of suitable model systems that allow the study of subject-specific neuron-microglia interactions.”

Unlike previous models, the researchers created a human brain organoid that had microglia and a human-brain-like environment, and this finally allowed them to look at environmental influences on microglia throughout brain development. “To create a brain organoid model that contains mature microglia and enables us to research them, we used a novel transplantation technique to create a human-brain-like environment,” said co-first author Abed Mansour, PhD, a former postdoctoral researcher in Gage’s lab and now an assistant professor at the Hebrew University of Jerusalem. “So we could finally make a human brain organoid that had all the features necessary to orchestrate human microglia growth, behavior, and function.”

Abed Mansour, PhD [Salk Institute]

The authors further explained, “To model microglia identity and to assess the interaction and response of hMGs to the human brain environment, we harnessed our recently developed xenotransplantation approach to develop a transplanted immunocompetent human brain organoid (iHBO) model that allows the investigation of hPSC [human pluripotent stem cell]-derived human microglia within vascularized human brain organoids under physiological conditions in vivo. One advantage that our transplanted iHBO model offers is the ability to study how systemic and local perturbations in the human-brain-like environment affect hMG crosstalk over several months of in vivo development.”

Using their new technology the team found that a characteristic protein called SALL1 appeared as early as eleven weeks into development and served to confirm microglial identity and promote mature function. Additionally, they found that brain environment-specific factors, like the proteins TMEM119 and P2RY12, were necessary for microglia to function.

“Creating a human brain model that can effectively replicate the human brain environment is very exciting,” said co-author associate professor Axel Nimmerjahn, PhD. “With this model, we can finally investigate how human microglia function within the human brain environment.”

As the team learned more about microglia, the importance of the relationship between brain environment and microglia became clear, especially in disease scenarios. The lab had previously examined neurons derived from people with ASD and found that their neurons grew faster and had more complex branches than neurotypical counterparts. With the new organoid model, the team could ask whether those neuronal differences altered the brain environment and influenced microglia development.

To do so, they compared microglia derived from skin samples from three individuals with macrocephalic ASD versus three neurotypical individuals with macrocephaly. The researchers found that individuals with ASD exhibited the neuronal differences the team had previously noted, and that the microglia were influenced by those differences in their growth environment. Because of this neuron-dependent environmental change, the microglia became more reactive to damage or intruders—a finding that may explain the brain inflammation observed in some individuals with ASD.

“We provide the first experimental evidence for an environment-induced cell-non-autonomous shift in microglial phenotype that may indicate chronic and early emerging responses to aberrant neurodevelopmental processes,” they commented. “The morphological changes observed have been associated with a primed and more reactive microglial state and have previously been reported in post-mortem tissue of patients with ASD. Importantly, our experiments revealed that these changes in microglia reactivity were induced by the environment and not by the microglia themselves.”

Since this was a preliminary study with a small sample size, the team plans to examine more microglia from additional people in the future to verify their findings. They also aim to expand their research to study other developmental and neurodegenerative diseases to see how microglia are contributing to disease onset.

“Rather than deconstruct the brain, we decided to construct it ourselves,” commented co-first author Simon Schafer, PhD, a former postdoctoral researcher in Gage’s lab and now an assistant professor at Technical University of Munich. “By building our own brain model we can work from the bottom up and see solutions that may be impossible to see from the top down. We are eager to continue improving on our model and unraveling the relationship between the brain and immune system.”

Noting limitations of their study, the authors concluded, “The novel platform developed here will provide the unique opportunity to model the functional interaction of hMGs with their human neuronal environment, thus rendering it a highly suitable technology for unmasking the role of context-dependent hMG phenotypes during development and disease.”