Scientists headed by teams at the Princess Máxima Center for pediatric oncology and the Hubrecht Institute have developed 3D mini-organs from human fetal brain tissue that self-organize in vitro. The researchers say the lab-grown human fetal brain organoids (FeBOs) enable new ways of studying how the brain develops, and also investigating the development and treatment of diseases related to brain development, including brain tumors.

Commenting on the work, research co-lead Benedetta Artegiani, PhD, at the Princess Máxima Center for pediatric oncology, said, “Brain organoids from fetal tissue are an invaluable new tool to study human brain development. We can now more easily study how the developing brain expands, and look at the role of different cell types and their environment. Our new, tissue-derived brain model allows us to gain a better understanding of how the developing brain regulates the identity of cells. It could also help understand how mistakes in that process can lead to neurodevelopmental diseases such as microcephaly, as well as other diseases that can stem from derailed development, including childhood brain cancer.”

Artegiani and colleagues reported on their studies in Cell, in a paper titled “Human fetal brain self-organizes into long-term expanding organoids.”

Scientists use different ways to model the biology of healthy tissue and disease in the lab. These include cell lines, laboratory animals, and more recently 3D organoids, or mini-organs. Organoids have characteristics and a level of complexity that allows scientists to closely model the functions of an organ in the lab.

“Human organoids are stem cell-derived three-dimensional (3D) structures that mimic features of the pertinent tissue, including molecular specification, cellular composition and architecture, and functionality,” the authors further explained. Organoids can be formed directly from cells of a tissue. Scientists can also guide embryonic stem cells, or stem cells from some adult tissue or embryo-derived stem cells, to develop into the organ they aim to study. “Two sources of stem cells can be used to derive organoids: pluripotent stem cells (PSCs) and tissue stem cells (TSCs),” the authors continued.

Until now, brain organoids were grown in the lab by coaxing embryonic or pluripotent stem cells to grow into structures representing different areas of the brain.  “… to date, brain organoids can solely be established from pluripotent stem cells,” the investigators noted. Using a specific cocktail of molecules, researchers attempted to mimic the natural development of the brain.

Artegiani and colleagues have now reported on the development of brain organoids directly from human fetal brain tissue. The team demonstrated that using small pieces of fetal brain tissue, rather than individual cells was vital in growing the brain organoids. To grow other mini-organs, such as gut, scientists normally break down the original tissue to single cells. Instead, the team found that small pieces of fetal brain tissue could self-organize into the brain organoids.

These structures, each roughly the size of a grain of rice, demonstrate a complex 3D make-up that includes a number of different types of brain cells. Importantly, the brain organoids contained many outer radial glia—a cell type found in humans and our evolutionary ancestors. This underlines the organoids’ close similarity to—and utility for studying—the human brain.

An image of a whole human fetal brain organoid. Stem cells are marked by SOX2 (grey) and neuronal cells (TUJ1) are color coded from pink to yellow based on depth.
An image of a whole human fetal brain organoid. Stem cells are marked by SOX2 (grey) and neuronal cells (TUJ1) are color coded from pink to yellow based on depth. [Princess Máxima Center, Hubrecht Institute/B Artegiani, D Hendriks, H Clevers]
The brain tissue pieces also produced proteins that make up extracellular matrix (ECM)—the scaffolding around cells. The team believes these proteins could be the reason why the pieces of brain tissue were able to self-organize into 3D brain structures. “Notably, a key finding of our study is that the preservation of tissue integrity and thus of native cell-cell interactions appear instrumental to produce a proper tissue-like ECM niche, which we hypothesize might be important to maintain long-term expansion,” the investigators noted. The presence of extracellular matrix in the organoids will make it possible to study the environment of brain cells, and what happens when something goes awry.

The researchers also found that the tissue-derived organoids kept various characteristics of the specific region of the brain from which they were derived. “FeBOs can be derived from distinct parts of the CNS while broadly capturing the original regional identity over prolonged periods of time,” they wrote. “FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity.” The resulting organoids were shown to respond to signaling molecules known to play an important role in brain development. This finding suggests that the tissue-derived organoids could be used to further explore and potentially untangle the complex network of molecules involved in directing brain development.

Given the ability of the tissue-derived organoids to quickly expand, the researchers also investigated their potential utility in modeling brain cancer. They used CRISPR-Cas9 gene editing technology to introduce faults in the cancer gene TP53, in a small number of cells in the organoids. They found that after three months the cells with defective TP53 had completely overtaken the healthy cells in the organoid. These cells had effectively acquired a growth advantage, a typical feature of cancer cells.

Four zoom-in images of parts of different human fetal brain organoids. Different neural markers are stained, depicting their cellular heterogeneity and architecture.
Four zoom-in images of parts of different human fetal brain organoids. Different neural markers are stained, depicting their cellular heterogeneity and architecture. [Princess Máxima Center, Hubrecht Institute/B Artegiani, D Hendriks, H Clevers]
The investigators also used CRISPR-Cas9 to switch off three genes—TP53, PTEN, and NF1—that are linked to the brain tumor glioblastoma. The responses of these mutant organoids to existing cancer drugs could then be evaluated. These results showed the potential to use the organoids for cancer drug research, to link certain drugs to specific gene mutations. “We further demonstrate the use of the FeBO culture system to address disease-related questions,” the authors stated. “In particular, expandable CRISPR-mutated FeBO lines with defined genetic make-ups can be generated as bottom-up cancer models. Such mutant FeBO lines represent scalable and reproducible systems amenable to a plethora of functional screenings, including mutation-drug sensitivity assays.”

The tissue-derived organoids continued to grow in a dish for more than six months. Importantly, the scientists could multiply them, allowing them to grow many similar organoids from one tissue sample. “FeBOs could be reliably passaged by cutting of a whole organoid, and each of these single FeBO pieces consistently reformed entire organoids, yielding stably expanding FeBO lines within 20–30 days of culture,” the researchers noted. “Altogether, FeBO lines possess an active stem/progenitor cell pool, accounting for their long-term expansion upon repeated splitting while at the same time capturing broad cellular heterogeneity and organization by the generation of neuronal cells, providing a “snapshot” of the tissue of origin.”

The mini-tumors with the glioblastoma gene changes—were also capable of multiplying, keeping the same mix of mutations. This feature indicates that scientists will be able to carry out repeat experiments with the tissue-derived organoids, increasing the reliability of their findings.

Next, the researchers aim to further explore the potential of the new tissue-derived brain organoids, and plan to continue their work with bioethicists—who were already involved in shaping this research—to guide the future development and applications of the new brain organoids.

Research co-lead Delilah Hendriks, PhD, affiliated group leader at the Princess Máxima Center for pediatric oncology, postdoctoral researcher at the Hubrecht Institute and oncode investigator, commented.” ‘These new fetal tissue-derived organoids can offer novel insights into what shapes the different regions of the brain, and what creates cellular diversity. Our organoids are an important addition to the brain organoid field, that can complement the existing organoids made from pluripotent stem cells. We hope to learn from both models to decode the complexity of the human brain. Being able to keep growing and using the brain organoids from fetal tissue also means that we can learn as much as possible from such precious material. We’re excited to explore the use of these novel tissue organoids for new discoveries about the human brain.”

Added research co-lead Hans Clevers, PhD, pioneer in organoid research and former research group leader at the Hubrecht Institute and the Princess Máxima Center for pediatric oncology and Oncode Investigator, “‘With our study, we’re making an important contribution to the organoid and brain research fields. Since we developed the first human gut organoids in 2011, it’s been great to see that the technology has really taken off. Organoids have since been developed for almost all tissues in the human body, both healthy and diseased—including an increasing number of childhood tumors. ‘Until now, we were able to derive organoids from most human organs, but not from the brain—it’s really exciting that we’ve now been able to jump that hurdle as well.’”

The study was performed in collaboration with Leiden University Medical Center, Utrecht University, Maastricht University, Erasmus University Rotterdam, and National University of Singapore.

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