Meet Pineal Gland Organoids, a New Tool for Studying Sleep

Organoids are miniature, simplified versions of an organ. Over the past two decades, scientists have developed them for the gut, lung, liver, mammary gland, brain, and more. Now, researchers at Yale School of Medicine (YSM) have organoid-ized the pineal gland, a small structure in the brain that regulates sleep patterns through its production of the hormone melatonin.

In a study published in Cell Stem Cell, the researchers demonstrated how pineal gland organoids can be used to study sleep dysfunction in conditions like Angelman syndrome, autism, and depression.

“In a number of neuropsychiatric conditions, severe sleep problems are a major symptom,” says In-Hyun Park, PhD, associate professor of genetics at YSM and senior author of the study. “With pineal gland organoids, we may be able to uncover the causes of those sleep disturbances and possibly identify treatments.”

Organoids are made from stem cells. When given the right chemical environment, stem cells can develop into nearly any type of mature cell, such as neurons or germ cells (sperm and eggs). To get cells to self-organize into the 3D structures that organoids are, researchers have to figure out what chemicals to provide the stem cells, when to provide them, and for how long.

“It took almost a year to find the conditions that allowed us to generate a pineal gland organoid from stem cells,” says Park.

Once the organoids were generated, the researchers had to confirm they were representative of the real thing. Ferdi Ridvan Kiral, PhD, and Woo Sub Yang, PhD, associate research scientists in Park’s lab and lead authors of the study, found that the organoids indeed had the machinery to produce melatonin.

Then they confirmed that the organoids could be stimulated to release melatonin. In mammals, the hormone is released when a cluster of nerve cells called the superior cervical ganglion stimulates the pineal gland.

To reproduce this, the researchers created what’s known as an assembloid—two or more types of organoids linked together. They generated a second organoid to stand in for the nerve cells and connected it to the pineal gland organoid. When the nerve cell organoid was stimulated, it, in turn, successfully stimulated the pineal gland organoid to release melatonin.

As a final test, the researchers replaced pineal glands in mice with organoid versions.

“We found that the mice with pineal gland organoids still had melatonin circulating in their blood, suggesting the organoids were functional in vivo,” says Park.

Sleep dysfunction is a symptom of a number of neurodevelopmental and psychiatric conditions, including Angelman syndrome, autism, depression, and bipolar disorder. It’s also a common issue among older adults and linked to neurodegenerative diseases like Alzheimer’s. Pineal gland organoids could be a powerful tool for studying sleep problems and testing treatments, say the researchers.

To show how organoids can be used to study disease, the researchers developed a pineal gland organoid for Angelman syndrome, in partnership with Yong-Hui Jiang, MD, PhD, Dorys McConnell Duberg Professor of Neuroscience, professor of genetics, and co-author of the study.

Angelman syndrome is a rare genetic disorder characterized by developmental delay, intellectual disability, speech impairment, and problems with balance and coordination. Most people with the syndrome also experience severe and persistent sleep challenges, such as difficulty falling or staying asleep and reduced melatonin secretion.

To develop the disease model pineal gland organoid, the researchers used stem cells derived from patients with Angelman syndrome.

During the early stages of development, Angelman syndrome pineal gland organoids appeared smaller than their typical counterparts, and that size difference increased throughout development.

More surprisingly, the Angelman syndrome organoids began to develop structures not seen in typical pineal gland organoids.

“We found that the cells arising in the Angelman syndrome pineal gland organoid looked like choroid plexus—the network of tissue in the brain’s ventricles that produces cerebrospinal fluid,” says Park.

When they looked to see which genes were active in the cells, those involved in producing choroid plexus were upregulated in the Angelman syndrome pineal gland organoid. But genes necessary for synthesizing melatonin were almost completely inactive. The findings indicate a sort of swap in cell activity, with Angelman syndrome pineal gland cells being unable to produce melatonin and instead assuming choroid plexus functions.

The researchers then looked at Jiang’s Angelman syndrome animal models and found that choroid plexus tissue was invading their pineal glands.

“Now we’re trying to see if people with Angelman syndrome have similar changes to their pineal glands,” says Park.

Looking forward, the researchers are exploring the idea of whether transplants of pineal glands or their main cell types might be a treatment for the sleep challenges that come with Angelman syndrome, autism, or even old age.

“I’m getting old and lots of old people have some type of sleep problem!” says Park. “Transplantation might be a big step for mild or intermediate sleep challenges, but for those with really severe issues, we could see a future where we create pineal gland organoids, isolate their cells, and transplant them into a person.”

Currently, the researchers are trying to use pineal gland organoids to replicate circadian rhythms and sleep cycles in the lab to further investigate the causes of and novel treatments for sleep problems.

“If we can develop those types of models,” says Park, “we could help a lot of people who struggle with sleep.”