man talking to crying woman illustration

cry me a river

Lab-grown body parts could cry just like you — unless they explode

A watershed moment.

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Whether they are tears of pain or tears of joy, the truth about tears is that these tiny, salty droplets are essential for the nutrition, lubrication, and protection of our eyes.

But the precise biological mechanism behind how tears accomplish these feats was hazy. Part of the problem is there are few good ways to study crying in the lab, but a new study might offer a pathway to greater clarity. In a new paper published this week in the journal Cell Stem Cell, scientists describe how they grew cultured cell analogs of human tear glands in the lab, known as organoids, and, perhaps more incredibly, how they managed to make these tear glands cry.

Why it matters — Aside from the visual poetry of having artificial lacrimal glands bawling away in a petri dish in a lab, the fact is this may represent a watershed moment in treating dry-eye disease.

Hans Clevers is group leader at the Hubrecht Institute for Developmental Biology and Stem Cell Research in the Netherlands and lead author on the new study. He tells Inverse that the organoids open new avenues for treatment previously denied people who experience chronic dry eyes. An estimated 5 percent of the population experience dry eyes.

“The biggest promise is the fact that we are now in a position to start thinking about treatment of dry eyes with cells,” Clevers tells Inverse.

“And I guess the nicest takeaway for the non-scientists is the fact that you can actually grow tear glands in a dish and make them cry,” he adds.

The lab-grown organoids, which are essentially a collection of human stem cells cultured in a dish and coaxed into taking on certain traits analogous with actual human cells, could potentially parlay into a totally new treatment themselves. Essentially, it may be possible to one day transplant organoid-based replacement glands into a human eye: something the research community has been working towards for years.

An image of one of the organoids, in this case derived from mouse stem cells.Clevers et al. (2021)

Here’s the background — Researchers at the Hubrecht Institute were among the first scientists to start working with organoids — that was more than a decade ago.

“At the time it was a surprising finding — that you can take one gut cell and grow it to many guts. We called them ‘mini-guts.’ After that it turned out we can do this for other organs and we’ve published about it for the past 10 years,” Clevers says.

But despite all the work done to refine organoids, there is still a chasm between the basic science and their potential applications. Scientists are still sorting through how to best grow organoids and make them function well — either as a model to study biology in the lab, as a way to test drugs or other treatments, or as a potential treatment in and of themselves.

For example, in 2020 Clevers and his team published a paper explaining how to grow venom-gland organoids to model the poisonous capabilities of snakes in dish, and how to induce the organoids to secrete large amounts of venom. It was this experiment which inspired the new study, Clevers explains.

How they did it — In this study, Clevers and his team selected stem cells from both a mouse and a human, and then cultured them in the lab by stimulating their growth using an injected growth factor.

Over time, the stem cells grew and proliferated, eventually forming miniature, 3D-structures that mimicked the function and structure of tear glands. Once they reached a point analogous in development with real, mature, and fully-functioning lacrimal glands, the team injected the organoids with various chemical stimuli, including a neurotransmitter called noradrenaline, to induce tears.

What they discovered —  After the hit of noradrenaline, the tear glands appear to cry, Clevers explains. However, these organoids shed their tears on the inside — they have no eye to cry into, after all — and some swelled up like miniature balloons. The “fatter” the tears, the more the organoids swole up — to the point that some of them even exploded from the simulated sadness.

This video shows the human stem-cell organoids swelling up with tears.Clevers at al. (2021)

Once the organoids started to “cry,” the researchers were also surprised to learn not all tears were created equal.

“We learned there are three types of tears and they all have different compositions,” Clevers says.

  1. Maintenance tears: The researchers describe these tears as standard tears of the kind that keep your eyes wet, moist, and function as a protective layer.
  2. Pain tears: These tears are essentially a reflex reaction to feeling pain — like when you get a fly stuck in your eye, or a chili flake. In this kind of crying, your tear glands produce large amounts of liquid to try flush your eye out.
  3. Emotional tears: These are the tears of sadness, joy, or laughter, and are rooted in a deeper part of the human brain.

“In both the latter cases, when the neurotransmitters are signaling [something is wrong], the molecule that the nerve gives off to the gland is adrenaline. And our organoid glands were very responsive to adrenaline,” Clevers says.

Clevers and his colleagues also report that their organoids’ tears very close in composition to real human tears — of the different cell types known to make up the different components of tears in the real world, the researchers saw almost all of the same cell types in the organoids.

Importantly, Clevers and his team specifically created ductal cells — the cells that release tears, but not the other, complementary cells which make up a full human eye’s tear gland. But the team says they have a roadmap for how it may be done and what it would be like, by creating an atlas of the cells in the tear gland using single-cell sequencing.

Due to the complexity and the variability of tear composition according to what the eye needs and why it is crying, there currently aren’t any artificial tear substitutes comparable to those the eye naturally cries itself. This discovery might change everything.

“The possibility of regenerating active parts of the lacrimal gland is of utmost interest and opens incredible perspectives for dry eye patients,” Maurizio Rolando tells Inverse. Rolando is associate professor at the University of Genoa, Italy, and Vice President of the European Dry Eye Society. He was not involved in this study.

“A working organoid model of glands will also be a formidable instrument for testing the ability of secretagogues to induce tearing and how to stimulate these tissues,” he says. The paper does not show that organoid engraftment could restore lacrimal gland function just yet, but it is promising.

What’s next — Dry-eye disease is a common condition that causes your eyes to produce either an insufficient amount of tears, poor quality tears, or no tears at all. Aside from the eye-watering idea of a bunch of stem-cell models sobbing into their agar agar culture, these miniature organ models could offer the opportunity for new therapies to improve eye health and cure tear-gland disorders. These organoids could help identify new drugs, and test out artificial tears to help people see more clearly.

Most people who have dry-eye disease do tear up in response to pain or emotional stimuli, although they are less able to produce the day-to-day, maintenance-level tears, Rolando says. Eye drops may be enough to help get them through the day. But for people with Sjogren’s Syndrome, an immune disorder which causes chronic dry eyes and dry mouth, it is a struggle to produce any tears at all — for them, these organoids may one day offer promise as a therapy.

“It is clear that the possibility of regeneration of the gland will be an outstanding new perspective and a game changer for this group of patients,” Rolando says.

Replacing non-functioning tear glands with ones grown in a lab is very much a future prospect, but it speaks to the greater promise — as yet unrealized — of organoid technology.

“We still have to optimize it, modify it, to make it safe, and find out exactly how to transplant,” Clevers says. “But that’s the plan.”

A recent experiment on salivary glands — which resemble the tear glands, according to Clevers — offers some hope. Led by Rob Coppes at the University of Groningen, the research team behind that experiment plan to start a preliminary, phase one clinical trial to test transplanting salivary gland organoids into humans, Clevers says. In Coppes’ protocol, they simply inject stem cells directly into human saliva glands, right under the skin, Clevers explains, and these cells eventually replace the deficient gland with a new, functioning gland.

“We would follow the same procedure,” Clevers says. Ultimately, he believes transplanting organoids to replace small, malfunctioning glands in humans may be a lot closer to reality than we think.

“That would be the promise,” he says.

Abstract: The lacrimal gland is essential for lubrication and protection of the eye. Disruption of lacrimal fluid production, composition, or release results in dry eye, causing discomfort and damage to the ocular surface. Here, we describe the establishment of long-term 3D organoid culture conditions for mouse and human lacrimal gland. Organoids can be expanded over multiple months and recapitulate morphological and transcriptional features of lacrimal ducts. CRISPR-Cas9-mediated genome editing reveals the master regulator for eye development Pax6 to be required for differentiation of adult lacrimal gland cells. We address cellular heterogeneity of the lacrimal gland by providing a single-cell atlas of human lacrimal gland tissue and organoids. Finally, human lacrimal gland organoids phenocopy the process of tear secretion in response to neurotransmitters and can engraft and produce mature tear products upon orthotopic transplantation in mouse. Together, this study provides an experimental platform to study the (patho-)physiology of the lacrimal gland.

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