Researchers from Tel Aviv University have discovered a species of ascidian, a marine animal commonly found in the Gulf of Eilat, capable of regenerating all of its organs – even if it is dissected into three fragments. The study was led by Prof. Noa Shenkar, Prof. Dorothee Huchon-Pupko, and Tal Gordon of Tel Aviv University’s School of Zoology at the George S. Wise Faculty of Life Sciences and the Steinhardt Museum of Natural History. The findings of this surprising discovery were published in the leading journal Frontiers in Cell and Developmental Biology.
“It is an astounding discovery, as this is an animal that belongs to the Phylum Chordata – animals with a dorsal cord – which also includes us humans,” explains Prof. Noa Shenkar. “The ability to regenerate organs is common in the animal kingdom, and even among chordates you can find animals that regenerate organs, like the gecko who is able to grow a new tail. But not entire body systems. Here we found a chordate that can regenerate all of its organs even if it is separated into three pieces, with each piece knowing exactly how to regain functioning of all its missing body systems within a short period of time.”
There are hundreds of species of ascidians, and they are found in all of the world’s oceans and seas. Anyone who has ever opened their eyes underwater has seen ascidians without knowing it, as they often camouflage themselves as lumps on rocks and are therefore difficult to discern. The animal that is the subject of this new study is an ascidian from the species Polycarpa mytiligera, which is very common in the coral reefs of Eilat.
“By all accounts, the ascidian is a simple organism, with two openings in its body: an entry and an exit,” says Tal Gordon, whose doctoral dissertation included this new research. “Inside the body there is a central organ that resembles a pasta strainer. The ascidian sucks in water through the body’s entry point, the strainer filters the food particles that remain in the body, and the clean water exits through the exit point. Among invertebrates, they are considered to be the closest to humans from an evolutionary point of view.”
Ascidians are famous for their regenerative ability, but until now these abilities have been identified mainly in asexual reproduction. Never before has such a high regenerative capacity been detected in a chordate animal that reproduces only by sexual reproduction.
“There are species of ascidians that perform simple regeneration in order to reproduce,” Gordon says. “These are species with a colonial lifestyle, with many identical individuals connected to one another. They replicate themselves in order to grow. In contrast, the ascidian from Eilat, Polycarpa mytiligera, is an organism with a solitary lifestyle, without the capacity for asexual reproduction, similar to humans. In previous studies we showed that this species is able to regenerate its digestive system and its points of entrance and exit within a few days. But then we wanted to see if it is capable of renewing all of its body systems. We took a few individual ascidians from Eilat and dissected them into two parts, which were able to replenish the removed sections without any problem. In a subsequent experiment, we dissected several dozen ascidians into three fragments, leaving a part of the body without a nerve center, heart, and part of the digestive system. And contrary to our expectations, not only did each part survive the dissection on its own, all of the organs were regenerated in each of the three sections. Instead of one ascidian, there were now three. This is very astonishing. Never before has such regenerative capacity been discovered among a solitary species that reproduces sexually, anywhere in the world.”
Prof. Shenkar concludes: “Since the dawn of humanity, humans have been fascinated by the ability to regenerate damaged or missing organs. Regeneration is a wonderful ability that we have, to a very limited extent, and we would like to understand how it works in order to try and apply it within our own bodies. Anyone snorkeling in the Gulf of Eilat can find this intriguing ascidian, who may be able to help us comprehend processes of tissue renewal that can help the human race.”
Reference: Tal Gordon, Arnav Kumar Upadhyay et al., “And Then There Were Three…: Extreme Regeneration Ability of the Solitary Chordate Polycarpa mytiligera”, Front. Cell Dev. Biol., 15 April 2021 | https://doi.org/10.3389/fcell.2021.652466
Capturing highly motile and invasive neuromast-associated ionocytes
One of the evolutionary disadvantages for mammals, relative to other vertebrates like fish and chickens, is the inability to regenerate sensory hair cells. The inner hair cells in our ears are responsible for transforming sound vibrations and gravitational forces into electrical signals, which we need to detect sound and maintain balance and spatial orientation. Certain insults, such as exposure to noise, antibiotics, or age, cause inner ear hair cells to die off, which leads to hearing loss and vestibular defects, a condition reported by 15% of the US adult population. In addition, the ion composition of the fluid surrounding the hair cells needs to be tightly controlled, otherwise hair cell function is compromised as observed in Ménière’s disease.
While prosthetics like cochlear implants can restore some level of hearing, it may be possible to develop medical therapies to restore hearing through the regeneration of hair cells. Investigator Tatjana Piotrowski, PhD, at the Stowers Institute for Medical Research is part of the Hearing Restoration Project of the Hearing Health Foundation, which is a consortium of laboratories that do foundational and translational science using fish, chicken, mouse, and cell culture systems.
“To gain a detailed understanding of the molecular mechanisms and genes that enable fish to regenerate hair cells, we need to understand which cells give rise to regenerating hair cells and related to that question, how many cell types exist in the sensory organs,” says Piotrowski.
The Piotrowski Lab studies regeneration of sensory hair cells in the zebrafish lateral line. Located superficially on the fish’s skin, these cells are easy to visualize and to access for experimentation. The sensory organs of the lateral line, known as neuromasts, contain support cells which can readily differentiate into new hair cells. Others had shown, using techniques to label cells of the same embryonic origin in a particular color, that cells within the neuromasts derive from ectodermal thickenings called placodes.
It turns out that while most cells of the zebrafish neuromast do originate from placodes, this isn’t true for all of them.
In a paper published online April 19, 2021, in Developmental Cell, researchers from the Piotrowski Lab describe their discovery of the occasional occurrence of a pair of cells within post-embryonic and adult neuromasts that are not labeled by lateral line markers. When using a technique called Zebrabow to track embryonic cells through development, these cells are labeled a different color than the rest of the neuromast.
“I initially thought it was an artifact of the research method,” says Julia Peloggia, a predoctoral researcher at The Graduate School of the Stowers Institute for Medical Research, co-first author of this work along with another predoctoral researcher, Daniela Münch. “Especially when we are looking just at the nuclei of cells, it’s pretty common in transgenic animal lines that the labels don’t mark all of the cells,” adds Münch.
Peloggia and Münch agreed that it was difficult to discern a pattern at first. “Although these cells have a stereotypical location in the neuromast, they’re not always there. Some neuromasts have them, some don’t, and that threw us off,” says Peloggia.
By applying an experimental method called single-cell RNA sequencing to cells isolated by fluorescence-activated cell sorting, the researchers identified these cells as ionocytes—a specialized type of cell that can regulate the ionic composition of nearby fluid. Using lineage tracing, they determined that the ionocytes derived from skin cells surrounding the neuromast. They named these cells neuromast-associated ionocytes.
Next, they sought to capture the phenomenon using time-lapse and high-resolution live imaging of young larvae.
“In the beginning, we didn’t have a way to trigger invasion by these cells. We were imaging whenever the microscope was available, taking as many time-lapses as possible—over days or weekends—and hoping that we would see the cells invading the neuromasts just by chance,” says Münch.
Ultimately, the researchers observed that the ionocyte progenitor cells migrated into neuromasts as pairs of cells, rearranging between other support cells and hair cells while remaining associated as a pair. They found that this phenomenon occurred all throughout early larval, later larval, and well into the adult stages in zebrafish. The frequency of neuromast-associated ionocytes correlated with developmental stages, including transfers when larvae were moved from ion-rich embryo medium to ion-poor water.
From each pair, they determined that only one cell was labeled by a Notch pathway reporter tagged with fluorescent red or green protein. To visualize the morphology of both cells, they used serial block face scanning electron microscopy to generate high-resolution three-dimensional images. They found that both cells had extensions reaching the apical or top surface of the neuromast, and both often contained thin projections. The Notch-negative cell displayed unique “toothbrush-like” microvilli projecting into the neuromast lumen or interior, reminiscent of that seen in gill and skin ionocytes.
“Once we were able to see the morphology of these cells—how they were really protrusive and interacting with other cells—we realized they might have a complex function in the neuromast,” says Münch.
“Our studies are the first to show that ionocytes invade sensory organs even in adult animals and that they only do so in response to changes in the environment that the animal lives in,” says Peloggia. “These cells therefore likely play an important role allowing the animal to adapt to changing environmental conditions.”
Ionocytes are known to exist in other organ systems. “The inner ear of mammals also contains cells that regulate the ion composition of the fluid that surrounds the hair cells, and dysregulation of this equilibrium leads to hearing and vestibular defects,” says Piotrowski. While ionocyte-like cells exist in other systems, it’s not known whether they exhibit such adaptive and invasive behavior.
“We don’t know if ear ionocytes share the same transcriptome, or collection of gene messages, but they have similar morphology to an extent and may possibly have a similar function, so we think they might be analogous cells,” says Münch. Our discovery of neuromast ionocytes will let us test this hypothesis, as well as test how ionocytes modulate hair cell function at the molecular level,“ says Peloggia.
Next, the researchers will focus on two related questions—what causes these ionocytes to migrate and invade the neuromast, and what is their specific function?
“Even though we made this astounding observation that ionocytes are highly motile, we still don’t know how the invasion is triggered,” says Peloggia. “Identifying the signals that attract ionocytes and allow them to squeeze into the sensory organs might also teach us how cancer cells invade organs during disease.” While Peloggia plans to investigate what triggers the cells to differentiate, migrate, and invade, Münch will focus on characterizing the function of the neuromast-associated ionocytes. “The adaptive part is really interesting,” explains Münch. “That there is a process involving ionocytes extending into adult stages that could modulate and change the function of an organ—that’s exciting.”
Other coauthors of the study include Paloma Meneses-Giles, Andrés Romero-Carvajal, PhD, Mark E. Lush, PhD, and Melainia McClain from Stowers; Nathan D. Lawson from the University of Massachusetts Medical School; and Y. Albert Pan, PhD, from Virginia Tech Carilion.
The work was funded by the Stowers Institute for Medical Research and the National Institute of Child Health and Human Development of the National Institutes of Health (award 1R01DC015488-01A1). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Lay Summary of Findings
Humans cannot regenerate inner ear hair cells, which are responsible for detecting sound, but non-mammalian vertebrates can readily regenerate sensory hair cells that are similar in function. During the quest to understand zebrafish hair cell regeneration, researchers from the lab of Investigator Tatjana Piotrowski, PhD, at the Stowers Institute for Medical Research discovered the existence of a cell type not previously described in the process.
The research team found newly differentiated, migratory, and invasive ionocytes located in the sensory organs that house the cells giving rise to new hair cells in larval and adult fish. The researchers published their findings online April 19, 2021, in Developmental Cell. Normal invasive (that is, non-metastatic) behavior of cells after embryonic development is not often observed. Future research by the team will focus on identifying triggers for such behavior and the function of such cells, including how this process may relate to hair cell regeneration.
Founded in 1994 through the generosity of Jim Stowers, founder of American Century Investments, and his wife, Virginia, the Stowers Institute for Medical Research is a non-profit, biomedical research organization with a focus on foundational research. Its mission is to expand our understanding of the secrets of life and improve life’s quality through innovative approaches to the causes, treatment, and prevention of diseases.
The Institute consists of twenty independent research programs. Of the approximately 500 members, over 370 are scientific staff that includes principal investigators, technology center directors, postdoctoral scientists, graduate students, and technical support staff. Learn more about the Institute at www.stowers.org and about its graduate program at www.stowers.org/gradschool.
Research into 3D bioprinting has grown rapidly in recent years as scientists seek to re-create the structure and function of complex biological systems from human tissues to entire organs.
The most popular 3D printing approach uses a solution of biological material or bioink that is loaded into a syringe pump extruder and deposited in a layer-by-layer fashion to build the 3D object. Gravity, however, can distort the soft and liquid bioinks used in this method.
In APL Bioengineering, by AIP Publishing, researchers from Carnegie Mellon University provide perspective on the Freefrom Reversible Embedding of Suspended Hydrogels (FRESH) 3D bioprinting approach, which solves this problem by printing within a yield-stress support bath that holds the bioinks in place until they are cured.
Until now, the distortion of bioinks, which results in a loss of fidelity, had presented a challenge to fabricating functional adult-sized tissues and organs and is a barrier to the long-term goal to supplement the limited donor supply for transplant. Consequently, most 3D-bioprinted tissue constructs to date have been relatively small when compared to the tissues or organs they are intended to replace.
“Our goal is to be able to FRESH 3D-print complex 3D tissue and organ models out of a wide range of biocompatible hydrogel and cell-laden bioinks,” said author Adam Feinberg.
The FRESH technique embodies several unique aspects. First, a support bath enables the printing of cells and bioinks that maintain their position as they cure, while still allowing for the movement of the extrusion needle. The FRESH support bath also provides an environment during the printing process that maintains high cell viability.
FRESH provides the ability to work with the widest range of bioinks of any 3D-bioprinting method. Finally, it uses a nondestructive print release by warming up the ink to 37 degrees Celsius to gently melt the support bath at body temperature.
Since it was developed in 2015, FRESH has been adopted by many research labs, for projects such as the FRESH printing of nanocellulose, conductive hydrogels, scaffolds for stem cell grown, and ventriclelike heart chambers composed of beating heart muscle cells.
The researchers have recently initiated a number of studies to FRESH 3D-print skeletal muscle, including controlling muscle architecture and regenerating muscle tissue after volumetric muscle loss.
Featured image: Customizability of the FRESH bioprinting platform CREDIT: Adam Feinberg and Andrew Hudson, Carnegie Mellon University
Reference: Daniel J. Shiwarski, Andrew R. Hudson, Joshua W. Tashman, and Adam W. Feinberg, “Emergence of FRESH 3D printing as a platform for advanced tissue biofabrication”, APL Bioengineering, 2021. https://aip.scitation.org/doi/10.1063/5.0032777
The synthetic ‘PIC’ hydrogel discovered in Nijmegen proves highly suitable as in vitro culture platform for (human) cells, as is demonstrated by Radboud researchers in a number of recent publications. The gel is a valuable tool for research into the development of diseases as well as potential treatments, including stem cell therapies for personalised medicine.
Chemists at Radboud University discovered a unique material in 2013. From a synthetic polymer named polyisocyanopeptide (PIC), they made a gel that behaves similar to the matrix that surrounds the cells in your body. The material exhibits a special property that is not found in other materials: it gels on heating and returns to a liquid solution as it is cooled. With this special combination of properties, there is a vast number of potential clinical applications for the gel.
In a series of recent publications researchers at Radboud University and Radboud university medical center demonstrate that the PIC gel is extremely suitable as a cell culture platform outside of the body (in vitro). By modifying the properties of the gel, they can tune cell growth and development. In this way it is possible to control the growth of organoids – tiny organs that are produced from human cells in a test tube – through the gel. With the gel they are also able to direct stem cells growth and development and which substances they secrete.
Standard model organs
“Scientists normally use mouse tumour extract as a culture medium for organoids”, says Paul Kouwer, molecular chemist at Radboud University. “Due to the unstable composition of this extract, however, the results are highly variable.”
“The advantage of the PIC gel is that each time the growth medium has exactly the same composition, which means that the organoids are more suitable for use as standard model organs for medical research, for example into cancer.” In a subsequent publication, the researchers demonstrated that the PIC gel can be used to control the cellular development, which we can use for the growth of tissues.
No stem cells needed in the body
In stem cell therapy, stem cells from the patient or a donor are introduced into the body to replace absent or defective cells in blood, tissues or organs. After transplantation the stem cells generally die. Kouwer: “Even though the stem cells typically do not survive very long, stem cell therapy is nevertheless successful. One theory suggests that the important factor is not the stem cells themselves, but what they secrete in the body.”
“In one of our publications we have shown that the PIC gel can be tuned to control which substances stem cells secrete. These bioactive compounds can then be used to treat illnesses, which means that it is no longer necessary to introduce the stem cells into the body.”
The results underline the versatility of the PIC gel. “We aim to further develop materials such they can ultimately be applied in a clinical setting.” Simultaneously, the chemists in Kouwer’s group are further advancing research into other possible applications of the gel.
The studies are the result of collaborations between researchers at Radboud University and researchers from the departments Radiotherapy & Oncoimmunology (Paul Span, Marleen Ansems, Gosse Adema), Urology (Egbert Oosterwijk), Cell biology (Mirjam Zegers) and Dentistry (John Jansen, Frank Walboomers, Fang Yang) of the Radboud university medical center.
List of publications:
Liu, et al. ‘Synthetic Extracellular Matrices as a Toolbox to Tune Stem Cell Secretome” ACS Appl. Mater. Interfaces2020. DOI: 10.1021/acsami.0c16208.
Zhang, et al. ‘Tunable interpenetrating network matrices drive epithelial morphogenesis and YAP translocation’ Adv. Sci.2020. DOI: 10.1002/advs.202003380.
Zhang, et al. ‘Polyisocyanide Hydrogels as a Tunable Platform for Mammary Gland Organoid Formation’ Adv. Sci.2020. 7, 202001797. DOI: 10.1002/advs.202001797.
Wang, et al. ‘Antimicrobial and anti-inflammatory thermo-reversible hydrogel for periodontal delivery’ Acta Biomater.2020, 116, 259. DOI: 10.1016/j.actbio.2020.09.018.
Immune cells called “killer T cells,” also known as cytotoxic or cytolytic CD8 T cells, normally stay in the bloodstream and do not enter organs and other tissues, according to a new study from scientists in the Perelman School of Medicine at the University of Pennsylvania.
The discovery, published in Cell, may help resolve many conundrums in immunology, including mysteries with medical relevance—for example, why recently developed cancer therapies using modified killer T cells fail to work well against solid tumors, and why the AIDS-causing virus HIV, which is considered highly vulnerable to killer T cells, seems able to evade these immune cells indefinitely by hiding outside the bloodstream.
“This finding tells us that killer T cells normally do not migrate out of the bloodstream,” said Michael Betts, Ph.D., Professor of Microbiology at Penn Medicine. “Now that we know this, we can, for example, start to engineer better solutions that employ these powerful cells.”
Killer T cells have long been considered the main battle tanks of the immune system. Each killer T cell has a receptor that, like an antibody receptor, can recognize a specific target. Killer T cells are called “cytotoxic” or “cytolytic” because they possess special molecular weapons that enable them to directly attack and destroy other cells displaying targets they recognize, for example, a virus-infected cell or even a cancerous cell.
Traditionally immunologists have believed that killer T cells circulate more or less continuously from the bloodstream into tissues and then back again, ever-ready to destroy targets they recognize anywhere in the body. But this view is based mainly on studies in animals. Human studies of T cells have mostly been confined to sampling these cells from the bloodstream. In this study, Betts and his team were able to take a broader look at T-cell movement in the body by analyzing samples—from people as well as macaque monkeys—of both blood and lymph.
Lymph is a whitish, watery fluid that flows from various tissues and organs in the body into the bloodstream via a network of vessels and nodes called the lymphatic system. T cells and other immune cells that move from the bloodstream into tissues flow back to the bloodstream via this lymphatic route. The scientists sampled from a part of the lymphatic network called the thoracic duct, through which most lymph flows.
In this way, the researchers for the first time were able to catalogue the detailed molecular characteristics of T cells sampled from thoracic duct lymph, comparing them to T cells collected from the bloodstream in the same subjects.
Of the many findings in the study, the most striking was that the CD8 T cells present in lymph—the CD8 T cells that had moved through organs and other tissues outside the bloodstream—generally were not the classic killer T cells that are abundantly present in blood. Virtually all of the CD8 T cells in lymph did not have a direct cell-killing capability; instead, they seemed equipped for producing chemicals called immune cytokines that summon other elements of the immune system. These non-cytotoxic CD8 T cells also seemed to recognize the same targets as their killer T cell counterparts in blood, hinting that these two sets of CD8 T cells develop from the same progenitor cells to have distinct but complementary roles in fighting the same pathogens.
The discovery is significant for basic immunology, the researchers say, because it extends the understanding of how these important immune cells work, and overturns the traditional assumption that killer T cells circulate from the bloodstream into tissues and back again. And while much remains to be learned, for example, about the role of the non-cytotoxic CD8 T cells that migrate through tissues beyond the bloodstream, the findings appear to have important implications for medicine.
One implication concerns CAR T-cell therapy for cancer, which uses engineered killer T cells from patients to home in on and kill their cancerous cells. CAR T-cell therapies have had significant successes against leukemias and other cancers accessible via the bloodstream, but so far little success against solid tumors in organs and tissues outside the bloodstream. A possibility suggested by the new findings is that CAR-T cells could be further engineered to venture beyond the bloodstream and attack solid tumors effectively.
“It may be that cytotoxic T cells put into the blood cannot access tumors in the lungs or intestines or breast, for example, because they don’t have the right properties to do so,” Betts said.
Similarly, according to Betts, the new findings may help explain why some viruses, such as HIV, can elude the immune system indefinitely while infecting organs and tissues outside the bloodstream.
At the same time, the findings could lead to better ways of stopping killer T cells from inappropriately migrating outside the bloodstream and doing harm to the body, for example in the immune rejection of transplanted organs, and in autoimmune disorders that are caused in part by inappropriate T cell activity, such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis.
Betts and his colleagues are now following up with further research in several directions. In one project they will examine how to re-engineer CAR-T cells to migrate better to solid tumors. In another they will try to discover how maturing CD8 T cells become either bloodborne cytotoxic CD8 cells or tissue-transiting, non-cytotoxic CD8 T cells.
Reference: Marcus Buggert et al. The Identity of Human Tissue-Emigrant CD8+ T Cells, Cell (2020). DOI: 10.1016/j.cell.2020.11.019 http://www.upenn.edu/
Heidelberg researchers gain groundbreaking new insights into the regulation and evolution of gene expression in mammalian organs.
A large-scale study conducted by molecular biologists from Heidelberg University has yielded groundbreaking new insights into the evolution and regulation of gene expression in mammalian organs. The scientists investigated RNA synthesis and subsequent protein synthesis in the organs of humans and other representative mammals, and with the aid of sequencing technologies, they analysed more than 100 billion gene expression fragments from various organs. They were able to demonstrate that the finely tuned interplay of the two synthesis processes during evolution was crucial for shaping organ functions.
A complex interplay of activity between a large number of genes – known as gene expression – underlies organ functions. “Until now, our understanding of these essential genetic programmes in mammals was limited to the first layer of gene expression – the production of messenger RNAs,” explains Prof. Dr Henrik Kaessmann, group leader of the “Functional evolution of mammalian genomes” research team at the Center for Molecular Biology of Heidelberg University (ZMBH). “The next layer – the actual synthesis of proteins at the ribosome through the translation of the messenger RNAs – remained largely unknown.”
It is this second synthesis process that the Heidelberg researchers have now studied more closely. Using so-called next-generation sequencing technologies, they analysed the gene expression of various organs on both layers. They studied the brain, liver and testes from humans and other selected mammals, including rhesus monkeys, mice, opossum and platypus. “On the basis of these data, we could jointly investigate both gene expression layers and compare them across mammalian organs using state-of-the-art bioinformatics approaches,” explains Dr Evgeny Leushkin of the ZMBH.
In their large-scale study, the ZMBH researchers showed that the finely tuned interplay of the two synthesis processes during evolution was critical for shaping organ functions. For the first time, they were able to show that – in addition to regulation of messenger RNA production – other regulatory mechanisms at the layer of translation are crucial for optimising the amount of protein produced in all organs. This is especially true in the testes, where translational regulation is key for sperm development. Another important finding concerns mutational changes in gene expression regulation that arose during evolution. These changes were often balanced between the two layers. Changes that offset one another were primarily maintained to ensure the production of consistent amounts of protein.
Researchers from France and Switzerland contributed to the study. Funding was provided by the German Research Foundation and European Research Council. The data are available in a public access database. Their research results were published in Nature.
References: Zhong-Yi Wang, Evgeny Leushkin, Angélica Liechti, Svetlana Ovchinnikova, Katharina Mößinger, Thoomke Brüning, Coralie Rummel, Frank Grützner, Margarida Cardoso-Moreira, Peggy Janich, David Gatfield, Boubou Diagouraga, Bernard de Massy, Mark E. Gill, Antoine H. F. M. Peters, Simon Anders, Henrik Kaessmann. Transcriptome and translatome co-evolution in mammals. Nature, 2020; DOI: 10.1038/s41586-020-2899-z
The closer people are physically to one another, the higher the chance for exchange, of things like ideas, information, and even infection. Now researchers at MIT and Boston Children’s Hospital have found that, even in the microscopic environment within a single cell, physical crowding increases the chance for interactions, in a way that can significantly alter a cell’s health and development.
In a paper published today in the journal Cell Stem Cell, the researchers have shown that physically squeezing cells, and crowding their contents, can trigger cells to grow and divide faster than they normally would.
While squeezing something to make it grow may sound counterintuitive, the team has an explanation: Squeezing acts to wring water out of a cell. With less water to swim in, proteins and other cell constituents are packed closer together. And when certain proteins are brought in close proximity, they can trigger cell signaling and activate genes within the cell.
In their new study, the scientists found that squeezing intestinal cells triggered proteins to cluster along a specific signaling pathway, which can help cells maintain their stem-cell state, an undifferentiated state in which can quickly grow and divide into more specialized cells. Ming Guo, associate professor of mechanical engineering at MIT, says that if cells can simply be squeezed to promote their “stemness,” they can then be directed to quickly build up miniature organs, such as artificial intestines or colons, which could then be used as platforms to understand organ function and test drug candidates for various diseases, and even as transplants for regenerative medicine.
Guo’s co-authors are lead author Yiwei Li, Jiliang Hu, and Qirong Lin from MIT, and Maorong Chen, Ren Sheng, and Xi He of Boston Children’s Hospital.
To study squeezing’s effect on cells, the researchers mixed various cell types in solutions that solidified as rubbery slabs of hydrogel. To squeeze the cells, they placed weights on the hydrogel’s surface, in the form of either a quarter or a dime.
“We wanted to achieve a significant amount of cell size change, and those two weights can compress the cell by something like 10 to 30 percent of their total volume,” Guo explains.
The team used a confocal microscope to measure in 3D how individual cells’ shapes changed as each sample was compressed. As they expected, the cells shrank with pressure. But did squeezing also affect the cell’s contents? To answer this, the researchers first looked to see whether a cell’s water content changed. If squeezing acts to wring water out of a cell, the researchers reasoned that the cells should be less hydrated, and stiffer as a result.
They measured the stiffness of cells before and after weights were applied, using optical tweezers, a laser-based technique that Guo’s lab has employed for years to study interactions within cells, and found that indeed, cells stiffened with pressure. They also saw that there was less movement within cells that were squeezed, suggesting that their contents were more packed than usual.
Next, they looked at whether there were changes in the interactions between certain proteins in the cells, in response to cells being squeezed. They focused on several proteins that are known to trigger Wnt/β-catenin signaling, which is involved in cell growth and maintenance of “stemness.”
“In general, this pathway is known to make a cell more like a stem cell,” Guo says. “If you change this pathway’s activity, how cancer progresses and how embryos develop have been shown to be very different. So we thought we could use this pathway to demonstrate how cell crowding is important.”
A “refreshing” path
To see whether cell squeezing affects the Wnt pathway, and how fast a cell grows, the researchers grew small organoids — miniature organs, and in this case, clusters of cells that were collected from the intestines of mice.
“The Wnt pathway is particularly important in the colon,” Guo says, pointing out that the cells that line the human intestine are constantly being replenished. The Wnt pathway, he says, is essential for maintaining intestinal stem cells, generating new cells, and “refreshing” the intestinal lining.
He and his colleagues grew intestinal organoids, each measuring about half a millimeter, in several Petri dishes, then “squeezed” the organoids by infusing the dishes with polymers. This influx of polymers increased the osmotic pressure surrounding each organoid and forced water out of their cells. The team observed that as a result, specific proteins involved in activating the Wnt pathway were packed closer together, and were more likely to cluster to turn on the pathway and its growth-regulating genes.
The upshot: Those organoids that were squeezed actually grew larger and more quickly, with more stem cells on their surface than those that were not squeezed.
“The difference was very obvious,” Guo says. “Whenever you apply pressure, the organoids grow even bigger, with a lot more stem cells.”
He says the results demonstrate how squeezing can affect a organoid’s growth. The findings also show that a cell’s behavior can change depending on the amount of water that it contains.
“This is very general and broad, and the potential impact is profound, that cells can simply tune how much water they have to tune their biological consequences,” Guo says.
Going forward, he and his colleagues plan to explore cell squeezing as a way to speed up the growth of artificial organs that scientists may use to test new, personalized drugs.
“I could take my own cells and transfect them to make stem cells that can then be developed into a lung or intestinal organoid that would mimic my own organs,” Guo says. “I could then apply different pressures to make organoids of different size, then try different drugs. I imagine there would be a lot of possibilities.”
References: Yiwei Li, Ming Guo et al., “Volumetric Compression Induces Intracellular Crowding to Control Intestinal Organoid Growth via Wnt/β-Catenin Signaling”, Cell Stem Cell, 2020 https://doi.org/10.1016/j.stem.2020.09.012
The number of proteins in the human body, collectively known as the proteome, is vast. Somewhere between 80,000 and 400,000 proteins circulate in our cells, tissues and organs, carrying out a broad range of duties essential for life. When proteins go awry, they are responsible for a myriad of serious diseases.
Now, researchers at the Biodesign Center for Applied Structural Discovery and ASU’s School of Molecular Sciences, along with their colleagues, investigate a critically important class of proteins, which adorn the outer membranes of cells. Such membrane proteins often act as receptors for binding molecules, initiating signals that can alter cell behavior in a variety of ways.
A new approach to acquiring structural data of membrane proteins in startling detail is described in the new study. Cryogenic electron microscopy (or cryo-EM) methods, a groundbreaking suite of tools, is used. Further, use of so-called LCP crystallization and Microcrystal electron diffraction (MicroED) help unveil structural details of proteins that have been largely inaccessible through conventional approaches like X-ray crystallography.
The findings describe the first use of LCP-embedded microcrystals to reveal high-resolution protein structural details using MicroED. The new research graces the cover of the current issue of the Cell Press journal Structure.
“LCP was a great success in membrane protein crystallization, according to Wei Liu, a corresponding author of the new study. “The new extensive application of LCP-MicroED offers promise for improved approaches for structural determination from challenging protein targets. These structural blueprints can be used to facilitate new therapeutic drug design from more precise insights.”
One class of membrane proteins of particular interest are the G-protein-coupled receptors (GPCRs), which form the largest and most varied group of membrane receptors found in eukaryotic organisms, including humans.
The physiological activities of GPCRs are so important that they are a major target for a wide range of therapeutic drugs. This is where problems arise however, as determining the detailed structure of membrane proteins–an essential precursor to accurate drug design– often poses enormous challenges.
The technique of X-ray crystallography has been used to investigate the atomic-scale structures and even dynamic behavior of many proteins. Here, crystallized samples of the protein under study are struck with an X-ray beam, causing diffraction patterns, which appear on a screen. Assembling thousands of diffraction snapshots allows a high-resolution 3D structural image to be assembled with the aid of computers.
Yet many membrane proteins, including GPCRs, don’t form large, well-ordered crystals appropriate for X-ray crystallography. Further, such proteins are delicate and easily damaged by X-radiation. Getting around the problem has required the use of special devices known as X-ray free electron lasers or XFELS, which can deliver a brilliant burst of X-ray light lasting mere femtoseconds, (a femtosecond is equal to one quadrillionth of a second or about the time it takes a light ray to traverse the diamere of a virus). The technique of serial femtosecond X-ray crystallography allows researchers to obtain a refraction image before the crystalized sample is destroyed.
Nevertheless, crystallization of many membrane proteins remains an extremely difficult and imprecise art and only a handful of these gargantuan XFEL machines exist in the world.
Enter cryogenic electron microscopy and MicroED. This ground-breaking technique involves flash-freezing protein crystals in a thin veneer of ice, then subjecting them to a beam of electrons. As in the case of X-ray crystallography, the method uses diffraction patterns, this time from electrons rather than X-rays, to assemble final detailed structures.
MicroED excels in collecting data from crystals too small and irregular to be used for conventional X-ray crystallography. In the new study, researchers used two advanced techniques in tandem in order to produce high-resolution diffraction images of two important model proteins: Proteinase K and the A2A adenosine receptor, whose functions include modulation of neurotransmitters in the brain, cardiac vasodilation and T-cell immune response.
The proteins were embedded in a special type of crystal known as a lipidic cubic phase or LCP crystal, which mimics the native environment such proteins naturally occur in. The LCP samples were then subjected to electron microscopy, using the MicroED method, which permits the imaging of extremely thin, sub-micron-sized crystals. Further, continuous rotation of LCP crystals under the electron microscope allows multiple diffraction patterns to be acquired from a single crystal with an extremely low, damage-free electron dose.
The ability to examine proteins that can only form micro- or nanocrystals opens the door to the structural determination of many vitally important membrane proteins that have eluded conventional means of investigation, particularly GPCRs.
References: Lan Zhu, Guanhong Bu, Liang Jing, Tamir Gonen, Wei Liu, Brent L. Nannenga, “Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED”, 28(10), pp. 1149-59, 2020, DOI: https://doi.org/10.1016/j.str.2020.07.006
Key control mechanism allows cells to self-organize and build tissues, organs, and anatomical structures.
Under a microscope, the first few hours of every multicellular organism’s life seem incongruously chaotic. After fertilization, a once tranquil single-celled egg divides again and again, quickly becoming a visually tumultuous mosh pit of cells jockeying for position inside the rapidly growing embryo.
Yet, amid this apparent pandemonium, cells begin to self-organize. Soon, spatial patterns emerge, serving as the foundation for the construction of tissues, organs, and elaborate anatomical structures from brains to toes and everything in between. For decades, scientists have intensively studied this process, called morphogenesis, but it remains in many ways enigmatic.
Now, researchers at Harvard Medical School and the Institute of Science and Technology (IST) Austria have discovered a key control mechanism that cells use to self-organize in early embryonic development. The findings, published in Science on Oct. 2, shed light on a process fundamental to multicellular life and open new avenues for improved tissue and organ engineering strategies.
Studying spinal cord formation in zebrafish embryos, a team co-led by Sean Megason, professor of systems biology in the Blavatnik Institute at Harvard Medical School (HMS), revealed that different cell types express unique combinations of adhesion molecules in order to self-sort during morphogenesis. These “adhesion codes” determine which cells prefer to stay connected, and how strongly they do so, even as widespread cellular rearrangements occur in the developing embryo.
Insights into how cells self-organize in early development could also aid efforts to engineer tissues and organs for clinical uses such as transplantation.
The researchers found that adhesion codes are regulated by morphogens, master signaling molecules long known to govern cell fate and pattern formation in development. The results suggest that the interplay of morphogens and adhesion properties allows cells to organize with the precision and consistency required to construct an organism.
“My lab’s goal is to understand the basic design principles of biological form,” said Megason, co-corresponding author on the study. “Our findings represent a new way of approaching the question of morphogenesis, which is one of the oldest and most important in embryology. We see this as the tip of the iceberg for such efforts.”
Insights into how cells self-organize in early development could also aid efforts to engineer tissues and organs for clinical uses such as transplantation, the authors said.
“Constructing artificial tissues for research or medical applications is a critically important goal, but currently one of the biggest problems is inconsistency,” said lead study author Tony Tsai, research fellow in systems biology in the Blavatnik Institute at Harvard Medical School. “There is a clear lesson to learn from understanding and reverse engineering how cells in a developing embryo are able to build the components of an organism in such a robust and reproducible way.”
Tug of war
Spearheaded by Tsai and in collaboration with Carl-Philipp Heisenberg and colleagues at IST Austria, the research team first looked at one of the most well-established frameworks for morphogenesis, the French flag model.
In this model, morphogens are released from localized sources in the embryo, exposing nearby cells to higher levels of the signaling molecule than cells farther away. The amount of morphogen a cell is exposed to activates different cellular programs, particularly those that determine cell fate. Concentration gradients of morphogens therefore “paint” patterns onto groups of cells, evocative of the distinct color bands of the French flag.
This model has limitations, however. Previous studies from the Megason lab used live-cell imaging and single-cell tracking in whole zebrafish embryos to show that morphogen signals can be noisy and imprecise, particularly at the boundaries of the “flag.” In addition, cells in a developing embryo are constantly dividing and in motion, which can scramble the morphogen signal. This results in an initial mixed patterning of cell types.
Nevertheless, cells self-sort into precise patterns, even with a noisy start, and in the current study, the team set out to understand how. They focused on a hypothesis proposed over 50 years ago, known as differential adhesion. This model suggests that cells adhere to certain other cell types, self-sorting in a way similar to how oil and vinegar separates over time. But there was little evidence that this plays a role in patterning.
To investigate, Megason, Tsai and colleagues developed a method to measure the force by which cells adhere to one another. They placed two individual cells together and then pulled on each cell with precisely controlled suction pressure from two micropipettes. This allowed the researchers to measure the precise amount of force needed to pull the cells apart. By analyzing three cells at once, they could also establish adhesion preferences.
The team used this technique to study the patterning of three different types of neural progenitor cells involved in building the nascent spinal cord in zebrafish embryos.
The experiments revealed that cells of a similar type strongly and preferentially adhered to one another. To identify the relevant adhesion molecule-encoding genes, the researchers analyzed the gene expression profile of each cell type using RNA sequencing. They then used CRISPR-Cas9 to block the expression of candidate genes, one at a time. If pattern formation became disrupted, they applied the pulling assay to see how much the molecule contributed to adhesion.
Three genes — N-cadherin, cadherin 11 and protocadherin 19 — emerged as essential for normal patterning. The expression of different combinations and different levels of these genes was responsible for differences in adhesion preference, representing what the team dubbed an adhesion code. This code was unique to each of the cell types and determined which other cells each cell type stays connected to during morphogenesis.
“All three adhesion molecules we looked at are expressed in different amounts in each cell type,” Tsai said. “Cells use this code to preferentially adhere to cells of their own type, which is what allows different cell types to separate during pattern formation. But cells also maintain some level of adhesion with other cell types since they have to collaborate to form tissues. By piecing together these local interaction rules, we can illuminate the global picture.”
Because the adhesion code is cell-type specific, the researchers hypothesized that it is likely controlled by the same processes that determine cell fate — namely, morphogen signaling. They looked at how perturbations to one the most well-known morphogens, Sonic hedgehog (Shh), affected cell type and corresponding adhesion-molecule gene expression.
“The issue with tissue engineering right now is that we just don’t know what the underlying science is. … Our goal is to figure out what those rules are for the embryo.” Sean Megason, Blavatnik Institute at HMS.
The analyses revealed that both cell type and adhesion-molecule gene expression were highly correlated, both in level and spatial position. This held true across the entire nascent spinal cord, where patterns of gene expression for cell type and adhesion molecule changed together in response to differences in Shh activity.
“What we found is that this morphogen not only controls cell fate, it controls cell adhesion,” Megason said. “The French flag model gives a rough sketch, and differential adhesion then forms the precise pattern. Combining these different strategies appears to be how cells build patterns in 3D space and time as the embryo is forming.”
The researchers are now further investigating the interplay between morphogen signaling and adhesion in developing embryos. The current study looked at only three different cell types, and there are many other adhesion-molecule candidates and morphogens that remain to be analyzed, the authors said. In addition, the details of how morphogens control both cell type and adhesion molecule expression remain unclear.
Better understanding these processes could help scientists uncover and reverse engineer the fundamental mechanisms by which a single-celled egg constructs a whole organism, the authors said. This could have profound implications in biotechnology, particularly for efforts to build artificial tissues and organs for transplantation or for testing new drug candidates.
“The issue with tissue engineering right now is that we just don’t know what the underlying science is,” Megason said. “If you want to build a little bridge over a stream, maybe you could do that without understanding physics. But if you wanted to build a big suspension bridge, you need to know a lot about the underlying physics. Our goal is to figure out what those rules are for the embryo.”
References: Tony Y.-C. Tsai, Mateusz Sikora, Peng Xia, Tugba Colak-Champollion, Holger Knaut, Carl-Philipp Heisenberg, Sean G. Megason, “An adhesion code ensures robust pattern formation during tissue morphogenesis”, Science, 2020, Vol. 370, Issue 6512, pp. 113-116 DOI: 10.1126/science.aba6637 link: https://science.sciencemag.org/content/370/6512/113/tab-article-info