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Brain Illustration

Article by Anushka Srivastava & Ilaria Baldassarri

Choreographing Early Brain Development

Organoids are essentially miniature versions of our organs that scientists have discovered how to develop in vitro. There is a growing body of literature showing how this has been accomplished following a few prevalent paths. Toshiro Sato in the laboratory of Hans Clevers was the first to show that adult stem cells from the intestine could recreate the key units of the gut in a petri dish. A decade later, researchers now use patient tumor biopsies to establish cancer organoid lines that preserve the architecture of the original malignancy. Others instead follow the stages of human development using well-defined growth factor cocktails to push pluripotent stem cells (PSCs) of either embryonic (hPSCs) or induced (iPSCs) origin to transform into brains, livers, hearts, lungs and many more organs in an effort to study health and disease. While these models effectively replicate the intricate cellular structure of the specific organ under study, they currently lack crucial supporting cell types. Specifically, most of these models do not include vasculature nor immune cells. As a result, they not only overlook essential contributors to the organ's development and homeostasis (i.e., to maintain conditions within steady and safe limits), but also fall short in performing vital functions, such as response to infection (learn more about our body’s lines of defense here). In this study, the focus is on the early-stage, or embryonic, brain, and its foremost resident immune cells, microglia. Specifically, the goal of the team was to uncover whether and how microglia interact with other brain cells during the creation of neurons (or neurogenesis).

What did these researchers do?

In this study, cerebral organoids and primitive macrophages (iMac) were separately differentiated from the same iPSC source. These cells are termed primitive because they are rather plastic and can adapt to the surrounding microenvironment taking on specific traits. In particular, by combining the two cultures at a specific time, the scientists tried to recreate the moment microglial precursors from the yolk sac reach the embryonic brain to become microglia. Proving their hypothesis, iMac differentiated into microglia-like cells (iMicro) with consistent phenotypes (i.e., distinct and defining characteristics) and functions, including response to injury and clearance of toxic molecules. Critically, the researchers showed that iMicro were controlling the balance between neuronal progenitor cell (NPC) proliferation and axon development during early brain development, favoring the latter. Diving further into the mechanistic explanation of this process, they unveiled that at the origin of these observations was the transfer of lipids (i.e., fatty molecules) from iMicro to NPCs in the form of droplets. 


Why is this important?

Currently, our knowledge on brain development is hindered by limited access to relevant human tissues. Organoids capture development in a dish and therefore represent an ideal tool for its investigation in health and disease. Here, the researchers were able to co-culture macrophages with brain organoids driving the emergence of microglial traits and elucidating how these cells orchestrate neurogenesis in the embryo. On one hand, given broad efforts to establish more complex in vitro models, this study demonstrates that it is feasible to co-culture immune cells with organoids causing the acquisition of tissue-resident phenotypes. In addition, the authors gather evidence that the presence of microglia in the brain might promote the formation of blood vessels (or vasculogenesis), thus hinting at the possibility to incorporate in the model a further layer of complexity in the form of vasculature. On the other hand, the implications of the study go beyond bioengineering considerations. The authors show that microglia support NPCs with lipids during embryogenesis to modulate their differentiation, covering for astrocytes that are not yet present in the early brain (but would usually play this role). As discussed in this work, aberrations in microglia have already been correlated to increased likelihood of developing Alzheimer’s, and more could be uncovered on their role at the onset of neurodegenerative diseases using analogous models. Finally, validating their in vitro findings with in vivo data, the researchers corroborated the fidelity of their model as a proxy of the developing human brain. Such developments enhance the confidence of scientists in using organoid models for future brain research, making it an important stepping stone in brain research.

How did the researchers do this?

The team generated a co-culture of brain organoids and iMac derived from the same iPSCs and compared it to brain organoids and iMac in isolation. They found that iMac penetrated the organoids and took on a microglia-like phenotype. They confirmed this by monitoring the spatial distribution of brain and immune cells, noting increased microglial markers on iMac, and using single-cell RNA sequencing (scRNA-seq) to show the upregulation of genes indicating the transformation of macrophages into iMicro. Additionally, they demonstrated improved neuronal function in the co-culture by studying the electrical activity of neurons using a technique known as patch-clamping. They also registered a reduction in size of the co-cultured brain organoids associated (i) to lower total cell numbers, and (ii) absolute and relative reduction in NPCs, which were less proliferative and were forming more frequent and long neurites (i.e., protrusions from the cell body that later become dendrites or axons) compared to the brain organoid-only controls. In other words, they gathered evidence that iMicro were limiting the proliferation of NPCs and favoring their differentiation into more mature neuronal cells. The researchers then asked themselves how iMicro were accomplishing this. Their scRNA-seq database had already hinted at an enhancement in lipid transport and storage in iMicro compared to iMac and a higher lipid content in co-cultured NPCs. Yet, NPCs were not synthesizing these lipids themselves. By tagging cholesterols with a tracker, researchers discovered that iMicro package them into droplets, which are then transferred to NPCs. Interestingly, when they used a drug to inhibit cholesterol transport, brain organoids not only grew larger but also showed an increase in NPC proliferation. Importantly, the team ultimately corroborated their findings by comparing them to publicly available datasets of mouse and human microglia at various developmental stages, in fact observing compatible trends. 

Screenshot 2024-01-09 at 11.12.24.png

3D images of brain organoids cocultured with (top) or without (bottom) iMacs

What comes next?

The scientists have hereby developed a robust method for generating microglia-sufficient brain organoids, proving them as a valuable tool for understanding how microglia contribute to human brain development. This research enhances brain organoid technology, which can be further refined to mimic more aspects of the human brain by integrating other cell types. In the future, it would be interesting to assess whether macrophages derived from iPSCs following protocols from other labs are able to undergo analogous transformations. More broad accessibility to similar models would likely accelerate advances in the field and, if successfully applied to drug testing platforms, reduce the reliance on animal models for brain studies. 

The discovery of microglia's role in cholesterol transfer and neurogenesis holds promise for the future of complex brain organoids. Researchers anticipate exciting developments as they delve into specific molecular pathways and explore how microglial dysfunction may contribute to developmental brain disorders. This newfound understanding may revolutionize our approach to neurodevelopmental disorders like autism, ADHD, and intellectual disabilities, as well as neurodegenerative diseases such as Alzheimer's or Parkinson's, providing for more understanding of the intricacies of the brain. By unraveling the interactions of microglia with other brain cells, scientists aim to pave the way for innovative treatments, offering a thrilling outlook towards the treatment of a wide range of neurological conditions.

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