Research Area: Stem Cell and Developmental Biology

Lab-grown fat cells help scientists understand type 2 diabetes
Eva Frederick | Whitehead Institute
June 16, 2022

In research published June 17 in the journal Science Advances, researchers in the lab of Whitehead Institute Founding Member Rudolf Jaenisch present a way to create fat cells that can be modified to display different levels of insulin sensitivity.

The cells accurately model healthy insulin metabolism, as well as insulin resistance, one of the key hallmarks of type 2 diabetes. “This system, I think, will be really useful for studying the mechanisms of this disease,” said Jaenisch, who is also a professor of biology at the Massachusetts Institute of Technology (MIT).

“It’s really exciting,” said Max Friesen, a postdoctoral researcher in Jaenisch’s lab and a first author of the study. “This is the first time that you can actually use a human stem cell-derived [fat cell] to show a real insulin response.”

Body fat — also known as adipose tissue — is essential for regulating your body’s metabolism and plays an important role in the storage and release of energy. When fat cells called adipocytes encounter the hormone insulin, they suck up sugar from the blood and store it for future use.

But over many years, factors such as genetics, stress, certain diets, or polluted air or water can cause this process to go awry, leading to type 2 diabetes. In this disease, adipocytes, as well as cells in the muscles and liver, become resistant to insulin and therefore unable to regulate the levels of sugar in the blood.

Tools to model diabetes in the lab generally rely on mice or on cells in a petri dish or test tube. Both these systems have their own problems. Mice, although they are comparable with humans in some respects, have a completely different metabolism and do not experience human diabetes comorbidities like heart attacks. And cell culture has, in the past, failed to replicate key markers of diabetes in a way that is comparable to human tissues.

That’s why Friesen and Andrew Khalil, another postdoc in Jaenisch’s lab, set out to create a new model. The researchers started with human pluripotent stem cells. These cells are the shapeshifters of the body — given the right conditions, they can assume the specific characteristics of almost any human cell type. The Jaenisch Lab has used them in the past to replicate liver cells, brain cells, and even cancerous tumors.

They decided to try to optimize an existing method for differentiating pluripotent cells into fat cells. The protocol created cells that looked like adipocytes, but these cells did not recreate the conditions of healthy insulin signaling or insulin resistance seen in the human body in type 2 diabetes. When healthy adipocytes encounter insulin in the human body, they respond by taking up glucose out of the bloodstream. These lab-made fat cells weren’t doing that, unless the researchers cranked up insulin levels to a thousand times higher than levels ever seen in humans. “Taking up glucose [in response to normal levels on insulin] is really the main function of an adipocyte, so if the model fails to do that, anything downstream in terms of disease research is not going to work either,” Friesen said.

Friesen and Khalil wondered if the lab-grown adipocytes’ low sensitivity to insulin could be a product of the conditions in which they grew. “We thought that maybe this happens because we’re feeding them an artificial culture medium, with all kinds of extra supplements that might be inhibiting their metabolic response,” Friesen said.

Friesen and Khalil decided to use a method called the Design of Experiments approach, which allows researchers to tease out the contributions of different factors to a specific outcome. Informed by this approach, they created nearly 30 different media compositions, each with slightly different levels of key ingredients such as glucose, insulin, the growth factor IGF-1, and albumin, a protein found in blood serum.

The medium that worked best had concentrations of insulin and glucose that were similar to the levels in the human body. When grown in this new medium, the cells responded to much lower concentrations of insulin, just like cells in the body. “So this is our healthy adipocyte,” Friesen said. “Next we wanted to see if we could make a disease model out of this — to make it an insulin-resistant adipocyte like you would see in the progression to type 2 diabetes.”

To desensitize the cells, they flooded the media with insulin for a short period of time. This caused the cells to become less sensitive to the hormone, and respond similarly to diabetic or pre-diabetic fat cells in a living person.

The researchers could then study how the cells responded to the change — such as what genes the insulin resistant cells expressed that healthy cells did not — in order to tease out the underlying genetics of insulin resistance. “We saw small changes in a lot of genes that are metabolism regulated, so that seems to be pointing to a deficiency of the metabolism or mitochondria of the insulin-resistant cells,” Friesen said. “That’s one thing we want to pursue in the future — figure out what is wrong with their metabolism, and then hopefully how to fix it.”

Now that they have created this new model for studying insulin resistance in fat cells, the researchers hope to develop similar procedures for other cells affected in diabetes.  “It seems that with some modifications, we can apply this method to other tissues as well,” Friesen said. “In the future, this will hopefully lead to a unified system for all stem cell-derived tissues, including liver, skeletal muscle, and other cell types, to get a really robust insulin response.”

A heart-racing deadline for a heartfelt collaboration

In a whirlwind team project, undergraduates Aniket Dehadrai SB ’22 and Brindha Rathinasabapathi SB ’24 of the Boyer lab pioneered a new method to study how hearts are built.

Celina Zhao
May 23, 2022

Can’t miss a beat

The lab was bustling with activity, with everyone working together on a team project comprised of many moving parts. Once one person finished a step of the experiment, it was whisked off to the next person. There was no time to lose.

During MIT’s Independent Activities Period (IAP) in January of 2022, several members of the Boyer Lab were hard at work — among them, Aniket Dehadrai, a junior studying Course 5-7 (Chemistry and Biology), and Brindha Rathinasabapathi, a sophomore studying Course 7 (Biology). Fueled with coffee every morning from the lab’s handy Keurig, the team was on a time crunch.

Working alongside Dehadrai and Rathinasabapathi were research scientist Vera Koledova, lab manager Kirsten Schneider, and fellow undergraduate researcher Caroline Zhang. They had a hard deadline at the end of the month to finish the project: studying how the absence of a certain protein affects the growth of cardiomyocytes, the cells responsible for pumping blood around the heart.

The Boyer lab — headed by Professor Laurie Boyer, the “Queen of Hearts” — specializes in heart cells. The lab is particularly interested in one intriguing question: Is it possible to heal the heart? Injuries like heart attacks often cause permanent damage that can eventually lead to heart failure. Scientists have found that at birth, injured heart cells are able to repair or replace themselves after such an event. However, that ability shuts off just a few days post-birth. Afterwards, heart cells, once damaged, are unfixable.

But what if adult cardiomyocytes could regain the ability to repair themselves, and thus repair trauma in heart tissue? The Boyer lab is intrigued by this possibility. But in order to answer that question, they must start from ground zero: learning how cardiomyocytes themselves develop.

The operation

Dehadrai, Rathinasabapathi, and the rest of the team were studying one part of that puzzle — the role histones play in cardiomyocyte growth. Histones are proteins that act as spools for DNA to wind around. DNA is extremely long, so histones help fit all this genetic information into the tiny space of a nucleus.

There are many types of histones (called “variants”), each of which has a unique effect on how DNA is wrapped. The tighter the DNA is packed, the more difficult it is for proteins to access the DNA — all of which affects how genes are expressed. As a result, each variant has a unique effect on how certain genes are regulated.

For the IAP project, the Boyer lab’s team focused on one histone variant called H2AZ.1. Prior studies have shown that H2AZ.1 is essential in most organisms, particularly when it comes to gene expression in stem cells. Stem cells are cells that essentially begin as blank slates, with the ability to form the many different cell types in the body. But through a differentiation process, they develop specific identities: skin, brain, or heart, to name a few.

By the end of the four weeks, the team planned to create and streamline a completely new process to “knock out,” or entirely remove, H2AZ.1 by degrading it during cardiomyocyte differentiation — the process where stem cells become specialized heart cells. Building this procedure to remove H2AZ.1 could later help identify what role H2AZ.1 plays in cardiomyocyte differentiation, a key step in both heart development and regeneration.

Microscopy image of heart muscle cells
The histone variant H2A.Z.1 (red) is located in the nucleus (blue) of cardiac muscle cells. Actin, a component of the sarcomere, is shown in green. The striated structure of the muscle cells gives them strength to beat throughout our entire lives. Credit: Boyer lab

To begin creating the knockout procedure, the team started by culturing stem cells from a cell line specifically developed by the Boyer lab to study the H2AZ.1 histone. The goal was to see if removing H2AZ.1 would have a visible effect on how stem cells eventually become mature cardiomyocytes.

The amount of careful planning and execution to do in just one month — simply running through one full differentiation cycle took 15 days at a time — meant working together as a team was critical. “There was one late night with all five people in the lab, doing this giant experiment as well as we could without mixing up the different variables in play,” Rathinasabapathi says. “It was really critical for us to look over each other’s shoulders and double check each other.”

In all, the team tested out 10 different variations of a method to optimize the experimental procedure. Despite the time crunch, they succeeded in pioneering a procedure to efficiently remove H2AZ.1 during cardiac differentiation. It turns out that H2AZ.1 does, in fact, have a functional impact on heart cells.

Without H2AZ.1, the beating rate of mature cardiomyocytes was notably different, changing from rhythmic to arrhythmic. The research team also found varying levels of maturity in the cells, suggesting that the progression through the differentiation process was also changed.

All of this suggests that H2AZ.1 has a significant influence in gene regulation, which they plan to continue studying in greater detail in the future.

“We’re breaking new ground,” Dehadrai says. “And importantly, it’s a great framework for future work in this field.”

With the procedure the team developed, the lab is now able to ask and answer more questions. For one, they can zoom in on certain parts of cardiomyocyte differentiation to see when H2AZ.1 has the greatest impact on gene expression. They can also use this procedure as a model to study how other histone variants affect heart cell growth. Ultimately, they can begin piecing together how histones, their effect on gene regulation, and cardiomyocyte development unite to build the heart.

“The better we can understand how heart cell development works, the better we can understand heart development, injury, and response — all of which have a lot of different implications in the medical field,” Rathinasabapathi says.

Following their hearts

The two credit the cohesiveness of the team as a big part of their success. “Brindha is really responsible, helpful, and willing to put in the hours,” Dehadrai says . “You can’t take stuff like that for granted.”

“Ani is just as dependable, and I’ve learned a lot from him as a senior with a lot of experience in the lab,” Rathinasabapathi says.

Another strength of the team was their ability to draw upon many different academic areas: a hallmark of the Boyer lab, which is known for its interdisciplinary approach to heart research. Members come from all sorts of backgrounds: biology, chemistry, biological engineering, mechanical engineering, and more. Research in the lab also spans a wide expanse, from uncovering the secrets of heart regeneration to building better microscopy techniques to study the heart. In fact, that was one of the reasons why Dehadrai initially chose to join the lab. “Here, there’s people who pretty much know how to do everything,” he says.

Although the IAP project has concluded, both Dehadrai and Rathinasabapathi are committed to continuing their passion for medical research. Dehadrai, who is graduating in the spring, is planning to take a gap year to work on clinical research projects before applying to medical school.

Rathinasabapathi, on the other hand, still has two years at MIT. She plans to stay in the Boyer Lab and is eager to take more advanced courses in the Department of Biology. “I’m impatient — I wish I already had the solid foundation to attack the research at different angles and come up with more cool new things,” she says. “There’s just so much more that I want to know.”

When equinox appears, repair transitions into regrowth
Greta Friar | Whitehead Institute
May 18, 2022

When animals experience a large injury, such as the loss of a limb, the body immediately begins a wound healing response that includes sealing the wound site and repairing local damage. In many animals, including humans, when the local wound site is taken care of, this response ends. However, in some animals, the initial wound response soon transitions into another stage of healing: regeneration, regrowing the parts that were lost.

Whitehead Institute Member Peter Reddien, also a professor of biology at MIT and a Howard Hughes Medical Investigator (HHMI), has long studied a flatworm known as the planarian (Schmidtea mediterranea), capable of regrowing any part of its body, to understand the mechanisms underlying regeneration. New research from staff scientist M. Lucila Scimone, graduate students Jennifer Cloutier and Chloe Maybrun, and Reddien identifies a previously undescribed gene, equinox, as playing a key role in initiating the transition from the initial wound healing stage into the regeneration stage in planarians. The work, published in Nature Communications on May 18, also reveals an important role for the wound epidermis, the skin that grows to cover a wound site, in initiating regeneration. Discovering what enables animals like planarians to regrow lost body parts can inform the field of regenerative medicine, which seeks to understand the limits of wound healing in humans and to improve our capacity for recovery and regeneration.

“The more we understand about the genes and mechanisms that play key roles in regeneration in animals that are capable of it, the better we may understand why humans lack that ability and, perhaps, the feasibility of future approaches to improve human wound healing,” says co-first author Scimone.

The case of the mystery gene

When the researchers began this project, they had no idea that it would lead them to identify a new gene that was crucial for regeneration. They originally set out to learn more about bmp4, a gene they had previously studied. BMP signaling, which includes bmp4, is involved in dorsal-ventral patterning, or the formation of the body around an axis between its top (dorsal) and bottom (ventral) sides. Previously, Reddien had found that bmp4 was necessary for regeneration after injuries to an animal’s side. Using new technologies that had not been around when they first studied the gene, the researchers now found that planarians without bmp4 failed to regenerate after large injuries anywhere on the body. This suggested a much more fundamental role for bmp4 in regeneration than the researchers expected, given that its main function relates to only one body axis. The researchers hypothesized that along with its role in dorsal-ventral patterning, bmp4 might help to activate an unknown gene that played some important, as yet unidentified role in regeneration. Bmp4 would therefore be necessary for regeneration because of its connection to this mystery gene.

The researchers started looking at genes regulated by bmp4 and found a promising candidate. They learned that bmp4 was needed to activate their mystery gene during the initial wound healing response, and that the mystery gene was crucial for wound healing to progress into regeneration after large injuries. When the gene was not activated, the steps that usually follow the initial wound healing response to prepare the body for regeneration would not occur. The wound would heal but the missing parts would never regrow, much like what would happen in a human. The researchers named the mystery gene equinox in honor of its appearance during a key transition period to move the body towards renewal.

“We know of a few genes that, when they are inactivated, the hallmarks of regeneration do not occur,” says co-first author Cloutier. “When equinox is not activated, we see an even more powerful inhibition of regeneration at an early phase. It appears to be required early on to allow for the other steps to proceed.”

Skin gets a starring role

The researchers found that equinox is expressed, or active in, wound epidermis, a skin tissue that is integral to regeneration after large injuries in a number of animals and yet had not been known to play a role in the signaling that initiates regeneration in planarians. After an injury, the wound epidermis covers and protects the wound site. As animals begin regeneration, the wound epidermis facilitates the formation of an outgrowth of cells called a blastema, in which the body produces the cell types it needs to replace the parts lost in the injury. Correspondingly, the researchers found that equinox is needed for regeneration in any injury that requires a blastema—essentially any large external injury where the replacement tissues grow out from the body.

Previously, the Reddien lab had found key genes required for regeneration expressed largely in muscle. Muscle in planarians maintains an active blueprint of the body, a network of positional genes that lets cells and tissues know where they are supposed to be. After an injury requiring regeneration, these positional genes rescale their body map near the wound site and guide new cells in building replacement tissues in the correct places. However, if equinox is not expressed, then the muscle tissue does not rescale its map. The body also fails to ramp up production of planarian stem cells or to begin differentiating stem cells into the cell types that were lost. Together, these findings flesh out the researchers’ understanding of the complete steps needed for regeneration to occur, revealing an early key role for wound epidermis, through its expression of equinox, in the signaling sequence that enables regrowth after an injury.

“There’s a cascade of events in which wound signaling activates, among other genes, equinox; equinox promotes wound-induced gene expression in muscle; and that promotes positional information resetting that can then lead to regeneration,” Reddien says. “What’s exciting about filling in this picture is that we’re identifying the key regulatory logic that can bring about regeneration.”

The promise of regeneration

HMS grad Jennifer Cloutier has a habit of pushing limits

Christine Paul | Harvard Medical School News
May 17, 2022

When Jennifer Cloutier receives her MD from Harvard Medical School in May, it will be 12 years since she won a Canadian national waterskiing championship.

Although that feat alone is impressive, it’s even more extraordinary because the competition was designed for individuals with disabilities, and because of her lower-body paralysis, Cloutier, now 30, performed tricky slalom turns and acrobatics from a special seat bolted to her skis.

But then, pushing limits has been Cloutier’s signature style.

“In the 20-second period allowed for trick skiing, if you fall off the seat, your performance is over,” she said. “So, my goal was to always perform the hardest trick I could do without falling.”

Skiing triumphs were just the beginning of many of Cloutier’s achievements, demonstrating her refusal to be deterred by the spinal-cord injury she experienced at age 6 in a car accident, which also left her younger brother paralyzed.

Pushing limits

Cloutier was encouraged by her parents not to let her injury impede her future ambitions, and during the six months she was initially hospitalized after the accident, she gained firsthand appreciation of the marvels of rehabilitative medicine, which she says helped inspire her to become a doctor.

But childhood came first. At age 10, the Ottawa, Ontario, native also embraced alpine skiing, becoming a ski instructor during high school.

Then, turning to watersports, she competed internationally and became a volunteer administrator for SkiAbility Ottawa, a waterskiing organization for people with chronic illnesses and disabilities.

Winning medal after medal, Cloutier’s athletic successes and volunteer work with disabled people culminated in her being selected in 2011 to Canada’s Top 20 Under 20, a prestigious list published by Youth in Motion.

At the time, Cloutier was already at Harvard College, graduating with a bachelor’s in human developmental and regenerative biology in 2013, and serving as president of Women in Science at Harvard-Radcliffe from 2011 to 2013.

She says her early traumatic injury was pivotal in defining her research goal—to understand how tissues regenerate after they are damaged. HMS and the Massachusetts Institute of Technology (MIT) have given her a unique opportunity to pursue this goal.

Enrolled in the joint Harvard-MIT Program in Health Sciences and Technology (HST), which immerses students in rigorous interdisciplinary studies on both campuses, Cloutier will receive an MD in 2022 from HMS, complementing the PhD in biology she received from MIT in 2020.

Compressed into overlapping years, HST students on the MD track receive training to become physician-scientists. In addition to classroom studies on the HMS campus and clinical rotations at HMS-affiliated hospitals, they spend long hours in HMS or MIT laboratories, working with leading scientists on critical questions.

“As a physician-scientist, I am very interested in how organs and tissues re-form in adult organisms that are attempting to regenerate from injury,” Cloutier said.

She has studied the regenerative ability of a tiny planarian, or flatworm, named Schmidtea mediterranea.

For two centuries, this freshwater planarian has been a model organism for studying development and regeneration, because of its distinct anatomical features—eyes, gut, brain, central nervous system, and more—and its capacity to regenerate any missing body region, even the whole body, from minuscule body parts.

Working in the lab of Peter Reddien, professor and associate head of the MIT Department of Biology, Cloutier’s research has focused on planarian signaling pathways that recruit stem cells for regenerating tissues.

“Our research team is seeking to identify the genes and signals involved in initiating regeneration,” Cloutier said. “We are converging on a promising regulator that is expressed within hours of injury in the planarian wound epidermis. Such a discovery would offer key insights to the cellular signals that drive regeneration and could potentially lead someday to therapeutic strategies for better repair after injury,” she said.

Precursor cells

While at Harvard College, she worked as an undergraduate in the labs of Konrad Hochedlinger, HMS professor of genetics at Massachusetts General Hospital, and Guillermo Garcia-Cardeña, HMS associate professor of pathology at Brigham and Women’s Hospital, again focusing on questions of cell fate.

Before beginning her research at MIT, Cloutier gained experience as a doctoral student in the laboratory of HMS Dean George Q. Daley, well known for his research on hematopoietic stem cells, precursor cells in the blood and bone marrow that give rise to mature blood cells which are often used in therapies designed to replace or rebuild a patient’s hematopoietic system.

Then, in 2018, something besides regenerative biology caught her eye. Crossing the MIT campus one day, Cloutier noticed a group of mechanical engineering students designing their capstone project—a wheelchair with an adjustable seat that could be raised 12 inches.

Recognizing the potential benefit of this device for physicians with disabilities when they are performing patient procedures and physical exams, she provided valuable feedback to the group of 20 students.

Feeling at home

On-campus mobility was essential to her studies, and Cloutier credits Harvard for striving to make its facilities accessible. One of her favorite aspects of Harvard life was living as an undergrad at Quincy House in Cambridge, where she later returned during her time as an HST student to serve as resident tutor and mentor.

“Harvard’s house system is incredible and unique,” she said. “It feels like home.”

Even after Cloutier graduates from HMS, she’s not planning on going far. Her residency assignment is at Mass General, an HMS affiliate, where she’ll train in internal medicine.

Again, her primary interest is tissue regeneration, particularly the liver, which has the greatest regenerative capacity of any organ in the body. But if the liver is injured beyond its ability to regenerate itself, a transplant is often called for.

“During my principal clinical experience at HMS, I realized how much liver disease affects a patient,” Cloutier said, which led to more questions. “With the current shortage of livers available for transplant, is there a way to prolong the liver’s regeneration capacity and extend the bridge to transplant?”

Harkening back to the accident that changed her life, Cloutier’s also concerned about people who cope daily with chronic illnesses and pain, like her father, whose leg injury from that car accident that injured her still causes him significant discomfort.

“As physicians, we can’t fix all problems,” she said. “But we can focus on what matters to our patients and try to improve their quality of life.”

With her future before  her, Cloutier is reminded of the powerful life lesson she learned at an early age.

“One day you may wake up, and things might be quite different,” she said. “But you still can do really cool things.”

Image: Gretchen Ertl
Thyroid hormone found to be a missing ingredient in lab-made liver cells
Greta Friar | Whitehead Institute
April 20, 2022

Stem cells are the versatile building blocks from which every cell type in the body, from neurons, to skin cells, to blood cells, is ultimately descended. Researchers have also figured out how to turn stem cells into different cell types in the lab, which has been helpful for studying health and disease in their normal cellular contexts, and could be used to generate cells for medical transplants. Whitehead Institute Founding Member Rudolf Jaenisch not only uses these cells in his research, but has spent much of his career discovering and improving the methods for making accurate laboratory models out of stem cell-derived cells.

One challenge that Jaenisch’s lab is focusing on is how to eliminate the differences between cell types as they are found in the body and their stem cell-derived equivalents. In particular, they have found that stem-cell derived cells are often immature, more closely resembling the cells found in fetuses rather than in adults. These differences can make the cells less accurate research models and prevent them from being medically useful as functional transplant cells. Stem cells in the body receive complex cocktails of molecular signals as they transform into different cell types. The challenge for researchers lies in figuring out which of the many molecular signals in the body are relevant and then get the recipe exactly right in their recreations.

Postdoc Haiting Ma in the Jaenisch lab decided to tackle this problem for hepatocytes, the main type of cell in the liver. In work published in Cell Stem Cell on April 21, Jaenisch and Ma share their findings on why stem cell-derived liver cells resemble fetal liver cells, and what’s needed to make them mature—including an important role for a thyroid hormone.

The liver filters everything that enters the body through the digestive system. It helps to store and modify nutrients, safely break down toxins and waste, process medications, and more. There is still a lot to learn about how the liver functions, and what goes wrong in a number of liver-associated diseases, and accurate stem cell-derived models will help with that research. Liver cells are also needed to treat end-stage liver disease, and if researchers could mass produce stem cell-derived liver cells that can function safely in an adult liver, this could help to meet the demand for liver cell transfusions.

For this study, Jaenisch and Ma grew liver cells from stem cells in two setups: a typical 2D culture, in which the cells were grown in a dish, and a 3D spheroid, in which cells that started out in the normal culture were then allowed to grow into three-dimensional balls of cells. The spheroids can be designed to mimic some aspects of the cells’ natural environment in ways that a 2D culture cannot. In each case, the researchers exposed the cells to a carefully timed mixture of signals to prompt them to develop into liver cells. The researchers then analyzed cells from both the 2D and 3D cultures and compared them to primary liver cells, or cells from a body, using a variety of techniques to look for differences related to DNA and gene expression. They found that the cells cultivated in the 3D system were closer to cells from the adult body than those in the 2D system.

“The 3D culture not only contributes to maturation of the liver cells, but it can also be used to scale up production of the cells, which could be very useful for cell therapies in the future,” Ma says.

However, both sets of lab-derived cells lacked important features of adult liver cells. The analyses pointed to one important missing factor in particular: in the adult liver cells, a hormone receptor called Thyroid Hormone Receptor Beta (THRB) binds to a number of places in the DNA. THRB then senses the presence or absence of thyroid hormones, and regulates a variety of gene expression processes accordingly. However, the researchers found that while the stem cell-derived liver cells made the right amount of THRB, something was preventing it from binding where it should and performing its function.

Normally, THRB has a partner that helps it bind to DNA, the thyroid hormone T3. When the researchers added T3 to their 2D and 3D cultures, this led to more typical binding of THRB, which in turn made the cells—especially the cells from the 3D culture—more closely resemble adult liver cells in a number of ways. Improved THRB binding increased the expression of key liver genes, restored the activity of regulatory elements in the DNA that modify gene expression, and reduced the expression of a fetal liver gene. The researchers also gained insights into the molecules that THRB interacts with and the mechanisms by which it affects liver maturation, painting a more complete picture of its key roles in liver cells.

Altogether, this work led to a better recipe for making adult liver cells from stem cells in the lab–using the 3D spheroid culture and adding T3. When cells developed with this approach were incorporated into the livers of mice, the cells integrated successfully and the liver maintained normal function long term.

The new and improved stem cell-derived liver cells are still not a perfect match for adult liver cells—the researchers have ideas about which missing characteristics they could tackle next—but the current cells’ ability to seamlessly integrate into the liver, as well as indicators from the analyses that they would be good models for liver-associated diseases, suggest that they will be useful in a variety of projects.

“As we improve the authenticity of our stem cell-derived cell types, we open up new opportunities for research,” Jaenisch says. “We can build more accurate models in which to study high-impact diseases, such as liver diseases, diabetes, and chronic viral infections, and using those models we can develop strategies for treatment and prevention.”

Yukiko Yamashita, unraveler of stem cells’ secrets

The MIT biologist’s research has shed light on the immortality of germline cells and the function of “junk DNA.”

Anne Trafton | MIT News Office
March 22, 2022

When cells divide, they usually generate two identical daughter cells. However, there are some important exceptions to this rule: When stem cells divide, they often produce one differentiated cell along with another stem cell, to maintain the pool of stem cells.

Yukiko Yamashita has spent much of her career exploring how these “asymmetrical” cell divisions occur. These processes are critically important not only for cells to develop into different types of tissue, but also for germline cells such as eggs and sperm to maintain their viability from generation to generation.

“We came from our parents’ germ cells, who used to be also single cells who came from the germ cells of their parents, who used to be single cells that came from their parents, and so on. That means our existence can be tracked through the history of multicellular life,” Yamashita says. “How germ cells manage to not go extinct, while our somatic cells cannot last that long, is a fascinating question.”

Yamashita, who began her faculty career at the University of Michigan, joined MIT and the Whitehead Institute in 2020, as the inaugural holder of the Susan Lindquist Chair for Women in Science and a professor in the Department of Biology. She was drawn to MIT, she says, by the eagerness to explore new ideas that she found among other scientists.

“When I visited MIT, I really enjoyed talking to people here,” she says. “They are very curious, and they are very open to unconventional ideas. I realized I would have a lot of fun if I came here.”

Exploring paradoxes

Before she even knew what a scientist was, Yamashita knew that she wanted to be one.

“My father was an admirer of Albert Einstein, so because of that, I grew up thinking that the pursuit of the truth is the best thing you could do with your life,” she recalls. “At the age of 2 or 3, I didn’t know there was such a thing as a professor, or such a thing as a scientist, but I thought doing science was probably the coolest thing I could do.”

Yamashita majored in biology at Kyoto University and then stayed to pursue her PhD, studying how cells make exact copies of themselves when they divide. As a postdoc at Stanford University, she became interested in the exceptions to that carefully orchestrated process, and began to study how cells undergo divisions that produce daughter cells that are not identical. This kind of asymmetric division is critical for multicellular organisms, which begin life as a single cell that eventually differentiates into many types of tissue.

Those studies led to a discovery that helped to overturn previous theories about the role of so-called junk DNA. These sequences, which make up most of the genome, were thought to be essentially useless because they don’t code for any proteins. To Yamashita, it seemed paradoxical that cells would carry so much DNA that wasn’t serving any purpose.

“I couldn’t really believe that huge amount of our DNA is junk, because every time a cell divides, it still has the burden of replicating that junk,” she says. “So, my lab started studying the function of that junk, and then we realized it is a really important part of the chromosome.”

In human cells, the genome is stored on 23 pairs of chromosomes. Keeping all of those chromosomes together is critical to cells’ ability to copy genes when they are needed. Over several years, Yamashita and her colleagues at the University of Michigan, and then at MIT, discovered that stretches of junk DNA act like bar codes, labeling each chromosome and helping them bind to proteins that bundle chromosomes together within the cell nucleus.

Without those barcodes, chromosomes scatter and start to leak out of the cell’s nucleus. Another intriguing observation regarding these stretches of junk DNA was that they have much greater variability between different species than protein-coding regions of DNA. By crossing two different species of fruit flies, Yamashita showed that in cells of the hybrid offspring flies, chromosomes leak out just as they would if they lost their barcodes, suggesting that the codes are specific to each species.

“We think that might be one of the big reasons why different species become incompatible, because they don’t have the right information to bundle all of their chromosomes together into one place,” Yamashita says.

Stem cell longevity

Yamashita’s interest in stem cells also led her to study how germline cells (the cells that give rise to eggs and sperm cells) maintain their viability so much longer than regular body cells across generations. In typical animal cells, one factor that contributes to age-related decline is loss of genetic sequences that encode genes that cells use continuously, such as genes for ribosomal RNAs.

A typical human cell may have hundreds of copies of these critical genes, but as cells age, they lose some of them. For germline cells, this can be detrimental because if the numbers get too low, the cells can no longer form viable daughter cells.

Yamashita and her colleagues found that germline cells overcome this by tearing sections of DNA out of one daughter cell during cell division and transferring them to the other daughter cell. That way, one daughter cell has the full complement of those genes restored, while the other cell is sacrificed.

That wasteful strategy would likely be too extravagant to work for all cells in the body, but for the small population of germline cells, the tradeoff is worthwhile, Yamashita says.

“If skin cells did that kind of thing, where every time you make one cell, you are essentially trashing the other one, you couldn’t afford it. You would be wasting too many resources,” she says. “Germ cells are not critical for viability of an organism. You have the luxury to put many resources into them but then let only half of the cells recover.”

Uncovering the mysteries of methylation in plants
Eva Frederick | Whitehead Institute
January 11, 2022

Growing up is a complex process for multi-celled organisms — plants included. In the days or weeks it takes to go from a seed to a sprout to a full plant, plants express hundreds of genes in different places at different times.

In order to conduct this symphony of genes, plants rely in part on an elegant regulatory method called DNA methylation. By adding or removing small molecules called methyl groups to the DNA strand, the plant can silence or activate different regions of its genetic code without changing the underlying sequence.

In a new paper from the lab of Whitehead Institute Member Mary Gehring, researchers led by former Gehring lab postdoc Ben Williams (now an assistant professor at the University of California, Berkeley) tease apart the role of proteins governing this system of genetic control, and reveal how enzymes that regulate methylation can affect essential decisions for plants such as when to produce flowers. “We’re starting to see that there is actually a broader role for  demethylation [in plant development] than we thought,” Gehring said.

In the model plant Arabidopsis thaliana, methylation is regulated in part by enzymes encoded by  a family of four genes called the DEMETER genes. The protein products of these genes are in charge of demethylating, or removing those methyl groups from the DNA, allowing different parts of the strand to be expressed. “You have these enzymes that can come in and completely change the way the DNA is read in different cells, which I find super interesting,” Williams said.

But teasing apart the role of each DEMETER gene has proved difficult in the past, because one member of this gene family in particular, called DME, is essential for seed development. Knock out DME, and the seed is aborted. “We had to design a synthetic gene to get around that,” Williams said. “We had to create plants that would rescue the reproductive failure, but then still be mutated throughout the rest of the life cycle.”

The researchers accomplished this by putting the DME gene under the control of a genetic element called a promoter that allowed it to be expressed in a cell that only existed in the plant during seed development. Once the plant was past the critical point where DME was needed for development, the gene would no longer be expressed, allowing the plant to grow up as a dme knockout. “It was an exciting thing, finally being able to create this knockout,” Gehring said.

Now, for the first time, the researchers could create plants with any combination of the DEMETER family genes knocked out, and then compare them to try and understand what the enzymes produced by each of the four genes was doing.

As expected, plants missing any of the DEMETER demethylases ended up with areas of their genomes with too many methyl groups (this is called hypermethylation). These areas were often overlapping, suggesting that the four DEMETER genes shared responsibility for demethylating certain areas of the genome.

“When one of these enzymes is gone, the others are surprisingly good at knowing that they need to step forward and do the job instead,” Williams said. “So the system has flexibility built in, which makes sense if it’s going to be involved in making important decisions like when to make flowers. You’d want there to be multiple layers of responsibility, right? It’s like in an organization, you don’t want to load all responsibility on one person — you’d want a few people who can take on that responsibility.”

Williams hypothesizes that while the DEMETER enzymes could step in for each other when needed, each specialized in demethylating DNA in particular types of plant tissue. “If you look at the protein sequences,they are actually really similar,” he said. “What’s different about them is they’re expressed in different cell types.”

A crucial finding of the study came about when the researchers knocked out all four genes in the DEMETER family at the same time. “All flowering plants have this really important decision of when to make flowers,” Williams said. “For plants out in the wild, that decision is usually dependent on temperature and pollinators. What we found really strange is that these mutants just flowered straight away. It’s almost like they weren’t even putting any effort into the decision. They made a few leaves, then boom, flower.”

When the researchers dove deeper, they saw that one area of the genome in particular that controls flowering time is under very careful and continuous regulation by methylating and demethylating enzymes. “We don’t really know why they’re doing that,” he said. “But when you knock out the demethylases, that gene just becomes methylated, and it’s then switched off. And that just sends plants into an automatic flowering state.”

In the future, the researchers plan to investigate other outcomes associated with their quadruple knockout of the DEMETER genes. “When we knocked out all four of the enzymes, it led to a lot of interesting phenotypes and tons of stuff to study,” Williams said. “We’ve learned through doing this that with DEMETER, like many gene families, we had to knock out all the players to find out the importance of what they are doing.”

Gehring will continue the research at Whitehead Institute. Williams recently started his own lab at the University of California, Berkeley. “I feel very lucky because this project has given me two or three different avenues that I can pursue in my new lab,” Williams said. “It has opened a lot of doors, which is very rewarding.”

3 Questions: Kristin Knouse on the liver’s regenerative capabilities

The clinically-trained cell biologist exploits the liver’s unique capacities in search of new medical applications.

Grace van Deelen | Department of Biology
December 15, 2021

Why is the liver the only human organ that can regenerate? How does it know when it’s been injured? What can our understanding of the liver contribute to regenerative medicine? These are just some of the questions that new assistant professor of biology Kristin Knouse and her lab members are asking in their research at the Koch Institute for Integrative Cancer Research. Knouse sat down to discuss why the liver is so unique, what lessons we might learn from the organ, and what its regeneration might teach us about cancer.

Q: Your lab is interested in questions about how body tissues sense and respond to damage. What is it about the liver that makes it a good tool to model those questions?

A: I’ve always felt that we, as scientists, have so much to gain from treasuring nature’s exceptions, because those exceptions can shine light onto a completely unknown area of biology and provide building blocks to confer such novelty to other systems. When it comes to organ regeneration in mammals, the liver is that exception. It is the only solid organ that can completely regenerate itself. You can damage or remove over 75 percent of the liver and the organ will completely regenerate in a matter of weeks. The liver therefore contains the instructions for how to regenerate a solid organ; however, we have yet to access and interpret those instructions. If we could fully understand how the liver is able to regenerate itself, perhaps one day we could coax other solid organs to do the same.

There are some things we already know about liver regeneration, such as when it begins, what genes are expressed, and how long it takes. However, we still don’t understand why the liver can regenerate but other organs cannot. Why is it that these fully differentiated liver cells — cells that have already assumed specialized roles in the liver — can re-enter the cell cycle and regenerate the organ? We don’t have a molecular explanation for this. Our lab is working to answer this fundamental question of cell and organ biology and apply our discoveries to unlock new approaches for regenerative medicine. In this regard, I don’t necessarily consider myself exclusively a liver biologist, but rather someone who is leveraging the liver to address this much broader biological problem.

Q: As an MD/PhD student, you conducted your graduate research in the lab of the late Professor Angelika Amon here at MIT. How did your work in her lab lead to an interest in studying the liver’s regenerative capacities?

A: What was incredible about being in Angelika’s lab was that she had an interest in almost everything and gave me tremendous independence in what I pursued. I began my graduate research in her lab with an interest in cell division, and I was doing experiments to observe how cells from different mammalian tissues divide. I was isolating cells from different mouse tissues and then studying them in culture. In doing that, I found that when the cells were isolated and grown in a dish they could not segregate their chromosomes properly, suggesting that the tissue environment was essential for accurate cell division. In order to further study and compare these two different contexts — cells in a tissue versus cells in culture — I was keen to study a tissue in which I could observe a lot of cells undergoing cell division at the same time.

So I thought back to my time in medical school, and I remembered that the liver has the ability to completely regenerate itself. With a single surgery to remove part of the liver, I could stimulate millions of cells to divide. I therefore began exploiting liver regeneration as a means of studying chromosome segregation in tissue. But as I continued to perform surgeries on mice and watch the liver rapidly regenerate itself, I couldn’t help but become absolutely fascinated by this exceptional biological process. It was that fascination with this incredibly unique but poorly understood phenomenon — alongside the realization that there was a huge, unmet medical need in the area of regeneration — that convinced me to dedicate my career to studying this.

Q: What kinds of clinical applications might a better understanding of organ regeneration lead to, and what role do you see your lab playing in that research?

A: The most proximal medical application for our work is to confer regenerative capacity to organs that are currently non-regenerative. As we begin to achieve a molecular understanding of how and why the liver can regenerate, we put ourselves in a powerful position to identify and surmount the barriers to regeneration in non-regenerative tissues, such as the heart and nervous system. By answering these complementary questions, we bring ourselves closer to the possibility that, one day, if someone has a heart attack or a spinal cord injury, we could deliver a therapy that stimulates the tissue to regenerate itself. I realize that may sound like a moonshot now, but I don’t think any problem is insurmountable so long as it can be broken down into a series of tractable questions.

Beyond regenerative medicine, I believe our work studying liver regeneration also has implications for cancer. At first glance this may seem counterintuitive, as rapid regrowth is the exact opposite of what we want cancer cells to do. However, the reality is that the majority of cancer-related deaths are attributable not to the rapidly proliferating cells that constitute primary tumors, but rather to the cells that disperse from the primary tumor and lie dormant for years before manifesting as metastatic disease and creating another tumor. These dormant cells evade most of the cancer therapies designed to target rapidly proliferating cells. If you think about it, these dormant cells are not unlike the liver: they are quiet for months, maybe years, and then suddenly awaken. I hope that as we start to understand more about the liver, we might learn how to target these dormant cancer cells, prevent metastatic disease, and thereby offer lasting cancer cures.

How sea stars get their symmetry
Greta Friar | Whitehead Institute
November 4, 2021

In a paper published Nov. 4 in the journal Current Biology, Zak Swartz, a postdoctoral researcher at Whitehead Institute, along with researchers in the lab of Whitehead Institute Member Iain Cheeseman and collaborators at the Massachusetts Institute of Technology (MIT), the University of Miami, and the Marine Biological Laboratory Embryology Course delve into the origins of the initial polarity in an animal’s first cell, which establishes an axis of symmetry for the developing organism and underlies the first steps of development. Their research reveals how a specific protein, called Dishevelled, localizes in a cell to help create this polarity.

All multicellular organisms begin as a single cell — the oocyte, precursor cell to the egg — which carries within it a “plan” for the fully developed, complex creature it will become. “How that multifunctional body plan is created is one of the deepest questions in developmental biology,” said Swartz.

“Sea stars, and a huge diversity of other animals, have an incredibly complex body plan, none of which is possible without the polarity of the initial cell,” said Cheeseman. “This work shows how the polarity originates as early as the meiotic divisions in the developing oocyte through an unexpected strategy to break its symmetry and achieve the asymmetric distribution of developmental factors.”

To study the intricate process of body patterning, Cheeseman Lab researchers used a type of sea star called the bat star, or Patiria miniata. These colorful animals are radially symmetric as adults — they usually have five arms, sometimes more — but as larvae they are bilaterally symmetric like humans.

The sea star larvae’s mirror-image symmetry is established when they are egg cells, called oocytes. A key step in the development of this organization involves a protein called Dishevelled, which localizes to the vegetal, or “bottom” end of the oocyte (which will define the posterior end of the embryo) as the cell gets ready to divide into two daughter cells.

Dishevelled — so named because a mutation in the homologous protein in fruit flies lends their tiny hairs a messy, tousled look  — is a component of a common signaling pathway called the Wnt pathway, which is found in many creatures throughout the animal kingdom. The pathway serves various purposes in the cells, from body patterning to cell proliferation. “The Wnt pathway is evolutionarily ancient,” Swartz said. “Jellyfish use it, sea stars use it, people use it, and I think that’s really quite profound.”

In the sea stars, the pathway provides a link between the initial asymmetry of the oocyte and the polarity of the resulting embryo. Dishevelled serves as a messenger on the inside of the sea star’s cells, relaying external signals that are then transmitted through a molecular pathway to the cells’ nuclei.

The researchers used time-lapse imaging to visualize how Dishevelled moved around the oocyte as the cell went through different phases of its development. When the sea star oocyte was in a non-dividing phase, Dishevelled could be found distributed uniformly in small aggregations throughout the cytoplasm.

As the oocyte got ready to divide, however, Dishevelled aggregations dissolved and then reformed at the bottom of the cell at the furthest point from the nucleus.  This provided a clear difference between the two ends of the oocyte.

Swartz was curious about how exactly the protein was localizing to the bottom of the oocyte. There were a number of protein transport options to investigate, so he began systematically ruling them out; the protein was not transported by the cell’s cytoskeleton (“You can think of these like little railroad tracks,” Swartz said), nor was it buoyed along on cytoplasmic currents, nor repelled by some factor at the “top” of the oocyte.

At this interval, Swartz reached out to two collaborators in MIT’s physics department, who helped design experiments to further probe the behavior of Dishevelled in the oocytes. “That’s when we started to consider the idea of dissolution and reassembly, which is kind of the punchline of the paper,” Swartz said. “You can think of it like salt crystals dissolving in water — rather than taking a pre-assembled thing and physically transporting it down [to the bottom of the oocyte], the idea is that these Dishevelled assemblies start out everywhere, get dissolved into their individual components, and then selectively reform in the vegetal region.”

The exact mechanism of this dissolution and reformation is not yet clear. Swartz was able to show that the reformation could not take place in the absence of another Wnt pathway protein called Frizzled, but because Frizzled is not exclusive to the bottom of the oocyte, it is not the only thing driving the reassembly.

In the future, Swartz plans to investigate whether the Dishevelled aggregates are formed in precise structures, or whether they group together as phase-separated droplets such as the RNA molecules studied in Whitehead Institute Member Ankur Jain’s lab or the protein molecules involved in transcription from Whitehead Institute Member Richard Young’s lab. “I’m interested in the broader composition of these structures,” he added. “Do they only contain Dishevelled, or are there other ingredients?”

Regardless of how the assemblies form, the new information on how Dishevelled localizes shines a light on a previously mysterious step in how the Wnt pathway plays a role in early body patterning in sea stars.

“It’s quite striking that Dishevelled localization seems to be an important feature in the Wnt pathway in sea stars, but also in distantly related vertebrates,” Swartz said. “My feeling is that the ability to activate this pathway in selective parts of the early embryo by interpreting polarity built into the oocyte may be a really critical feature of the evolution of the animal body plan.”