Jürgen Knoblich: “Cerebral organoids are great for studying processes that only happen in human brains”

Cross-section of a human cerebral organoid (Credit: Madeline Lancaster/IMBA)

For the last few years, scientists have been able to grow 3D nervous structures in the lab – called “cerebral organoids” – which mimic the brain of a few-week-old human embryo. In 2016, they allowed scientists to prove that the infamous Zika virus causes microcephaly in the human fetus. More generally, they are paving new ways to study brain development, in sickness and in health, like never before.

Recipe for “cerebral organoids”: Grow human embryonic cells in a dish – cells that have the potential to give rise to all tissues in the body. Given the right conditions, they will form little three-dimensional balls of cells and start developing into an embryonic nervous system.

Place small portions of this tissue into droplets of a special gel, which mimics these embryonic cells’ natural environment. Introduce the droplets, together with the appropriate nutrients, into a rotating bioreactor (so that the movement fosters rapid growth).

If you did everything right, ten days later, you’ll see that neurons start to develop – and in 20 more days, regions similar to certain brain structures will appear! Congratulations! You have just cooked a bunch of 3D tissue cultures called “cerebral organoids”, each around four millimeters in diameter, which can last for at least a year. 

It’s exciting, but you did not manufacture an artificial brain. Although some ethical concerns have been raised, cerebral organoids are nowhere close to being real brains, as it is highly unlikely that they have any sensory or cognitive capabilities. 

On the other hand, they do have many advantages over classical, bi-dimensional cell cultures in a dish, including the fact that they allow the study of structured tissues – not just cells. Also, scientists can now study processes that only happen in human brains. 

It was the late Yoshiki Sasai, at the RIKEN Center for Developmental Biology in Kobe, Japan, who pioneered the use of pluripotent stem cells for growing nervous system tissue in three dimensions. Some six years later, building on Sasai’s and others’ work, Jürgen Knoblich’s lab, at the Institute of Molecular Biology at the Austrian Academy of Sciences, developed a culture method to generate three-dimensional organ cultures that actually resemble the human brain.

Knoblich came to the Champalimaud Centre for the Unknown, where he was invited to speak at the Lisbon 2019 Champalimaud Symposium under the motto “Tissue Environment in Health and Disease”. We took advantage of the opportunity to interview him.

In 2013, you started growing cerebral organoids. What are they?

They are organ cultures that very much resemble and have morphological features of human fetal brains. It was Madeline Lancaster from my lab who developed a three-dimensional culture method that allowed us to generate them. Other labs before ours had cultured neurons of all types and variations. But I think what makes our work special was that we showed that you can do something with these organ cultures.

Around the same time, other labs showed that the human brain is actually fundamentally different from that of a mouse, or any other animal. Some of the cell types that generate our neurons simply don’t exist in a mouse. 

One thing we showed is that those human-specific cell types are present in the human cerebral organoids, – whereas if you generate the same kind of culture from mouse cells, they are not. This opens up great possibilities to recapitulate [reproduce] the human-specific features of brain development.

The second thing we did was to actually use cells from a patient to make cerebral organoids. In collaboration with a clinician, we took cells from a patient and made IPS cells from them. [IPS cells or “induced pluripotent stem” cells, are cells that can be generated directly from adult cells by making these adult cells “go back in time” and return to an embryonic-like state.] 

The patient had microcephaly, which means his brain was much smaller than normal, and its structure much simpler. And what we did was to use those patient-derived IPS cells to grow an organoid that reproduced those features. 

So you obtained, in vitro, a 3D cellular structure with hallmarks of the disease the patient was afflicted with?

That’s right. We just took a skin sample from the patient, and used the groundbreaking method that was developed in 2007 by Shinya Yamanaka [currently at Kyoto University] to reprogram those cells into their embryonic state. Then we applied our culture method, and we saw that the patient-derived organoids were very, very different from the ones we got from a patient who did not have microcephaly. They were smaller, so we now knew that this feature, the smaller brain size, could be recapitulated.

Once we had a cellular lab model for this feature, we could make hundreds and thousands of this patient’s organoids, and we could ask why they were so much smaller. And what we found is that there is actually a very specific defect that is responsible for their reduced size, which has to do with the initial proliferation of stem cells [IPS cells in the organoid]. 

What is the specific defect?

In a normal brain, stem cells give rise to neurons, but not right away. They start out by making many more of themselves. They amplify their population; one makes two, four, eight, sixteen and so on. It’s only when a lot of these stem cells have been made that they start making neurons.

But that didn’t happen in the microcephalic patient’s brain, where the stem cells only made a few other stem cells, then started making neurons almost right away. They did not expand properly, and there just weren’t enough of them. Moreover, when the patient’s organoids started making neurons, they did it at a much slower pace than organoids derived from a person without microcephaly. That’s the reason why these patients have less neurons and their brains are much smaller than healthy brains.

We even found the gene that is responsible for this. It has this horrible name, CDK5RAP2, and is responsible for how the cells divide. And the way the cells divide determines whether they make more stem cells or whether they make neurons right away. This gene is deficient in the microcephalic patient’s brain, and that is why the patient suffers from this terrible disease. 

We can even go one step further: we can now take the so-called CRISPR-Cas [a recent and powerful genome-editing] technology and repair the mutation of the patient’s gene in the organoid. And when we do that, the organoid’s development becomes normal again.

In a disease like microcephaly, this may seem trivial to do. But imagine people who suffer from schizophrenia, or from autism. There, the relationship between the disease and their genome is often much less clear. We don’t know if it has to do with the environment, or with nutrition, or if it’s in the genes. And I believe this combination of genome editing and organoid technology will allow us to answer those questions.

In 2015, there was a very serious outbreak of a virus called Zika in Brazil, and thousands of cases of microcephaly in newborns were reported. Was it thanks to brain organoids that scientists obtained the first direct proof of a causal link between this virus and microcephaly in humans?

Yes. Proving that the Zika virus causes microcephaly is one of the biggest success stories of cerebral organoid models. The causal link was established by Guo-li Ming and Hongjun Song [from Emory and Johns Hopkins University], and also by Patricia Garcez [from the Federal University] in Rio de Janeiro and her group. They had prime access to the virus. 

Just imagine: here is a human-specific virus, that you cannot model in the mouse, and then there is a country with an epidemic of that virus. At the same time, the number of cases of microcephaly increases. But how do you actually show that there’s a causal link between the two?

It was really the experiments using the cerebral organoid model which showed that, yes, if you put the Zika virus on the organoid, the virus will amplify in the organoid and cause microcephaly. 

Why were cerebral organoids crucial for that proof? Couldn’t it have been done with classical cell cultures?

In a classical 2D cell culture, the cells are “miles” away from each other at least at the neuronal scale, so it’s not so easy to study the propagation of a virus. You generate tons of virus and you put it onto those cells, but then you study every cell in isolation.

One key advantage of the organoid model is that we are looking at a tissue, not just a set of cells, and we can study the actual spread of a virus through the tissue to see what would actually happen in a real fetal brain.

More recently, you have been trying to study brain cancers in cerebral organoids. How?

We thought maybe we could recapitulate brain tumors in cerebral organoids. And to do that, we did something very simple: we developed an organoid system where we can introduce DNA mutations.

The second thing we made use of is the fact that in a [malignant] brain tumor, the mutations which lead to the brain tumor are the ones which make cells divide the fastest. So we put those mutations in, and we looked for the combinations of mutations that made the cells grow the fastest. And they turned out to be precisely the combinations of mutations that are most commonly found in human [malignant] brain tumors.

We then tried a number of different combinations and we made one model that resembles a rare childhood tumor, called “primitive neuro-ectodermal tumor” and three models that resemble various subtypes of glioblastoma, one of the most common types of brain cancers. 

You proposed that the Zika virus itself might be used as a therapeutic weapon against brain cancer. In what way? By “microcephalizing” it, so to speak?

We are doing a number of studies in my lab with this in mind. First, we are trying to put the Zika virus onto the brain tumors to see whether it has some effect on them. The rationale behind this  being that if a virus infects progenitor cells, then a tumor that is full of progenitor cells will have a higher chance of being infected [and its number of cells reduced] by the virus. 

I think we stand to gain from these model systems. Animal models such as the mouse are already being used to the limit of their possibilities – and still, things often fail to work at the level of clinical trials. I have the feeling that organoid models can really help us to fill in that last bit of information we need in order to translate research results to humans.

All the same, before considering any human trials, won’t you have, at some point, to transplant the cerebral organoids into real organisms, with a blood circulation for example, to really see how things work?

That depends. Maybe someone will make an organoid that has a blood circulation – that would be great. In any case, for microcephaly, for example, I don’t think it matters much that there were no blood vessels in our organoids. So it depends on the particular disease. 

What I’m saying is that we can already do a lot with organoid models. With every technological problem we solve – something that is now happening on a weekly basis – we expand the number of biological processes we can simulate in organoid models and the number of disorders we can potentially look at. 

Just to give you an example, the cortex of a mouse is complete when it’s born. However, in a human cortex, work that was done in the lab of Arturo Álvarez-Buylla [at the University of California] has shown that the vast majority of the interneurons [neurons connecting motor to sensory neurons] is just not there. So there’s this gigantic stream of cells that goes into the human cortex after birth. 

All of this work was done in post-mortem material, a situation that is only sustainable for a while. You can’t do functional experiments on post-mortem material. That is why I think cerebral organoids are really good for studying processes that only happen in human brains.

Apart from studying disease, could brain organoids also be useful to study the normal development of the brain?

This is a very good question. There’s even a debate going on within my lab. We are asking ourselves, can we actually use them to study human-specific development? I’m a bit hesitant, I feel that for that, we would probably need to get a bit better at mimicking many other aspects of the brain, such as vascularization and all that.

But there is something we can do right now. Each disease, teaches us something about the healthy process. Take the example of microcephaly; it showed us that a defect in the way cells divide can cause a defect in brain size. That’s very important – it’s not trivial, right? So, by studying brain diseases, we will get more and more information about the healthy brain.

Edited by: Liad Hollender, CR SciCom Office.

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