What makes stem cells so attractive for research is that they start off in an undifferentiated state — they're capable of becoming anything in the human body. Working with them, scientists can construct organoids: limited-function, synthetic versions of organs or other biological structures for study. (Organoids can't grow into independent living organisms.) However, the natural mechanism that causes embryonic stem cells to become different cell types remains an area about which not a lot is known.
Now a study identifies a particular growth factor — bone morphogenetic protein 4, or "BMP4"— that appears to be the trigger for a critical event, "symmetry breaking." It occurs about two weeks after conception via a process called gastrulation, taking place just after an embryo attaches to its mother's uterus. It's a pivotal moment at which a clump of cells first begins to separate into upper and lower body regions. "Symmetry breaking drives almost everything that happens during embryonic development," says Mijo Simunovic, a junior Fellow in the lab that conducted the research. "Our heads don't look like our feet, and that's because, at some point, the embryo breaks into two parts, anterior and posterior."
The researchers' conclusion regarding BMP4 is just part of what they've achieved — the study also validates the unique 3D model, or "embryoid," they developed as being accurate enough to use in laboratory investigations of other developmental processes.
The research is published in Nature Cell Biology.
Breaking symmetry
Artist's rendering of stem cells
Image source: Giovanni Cancemi/Shutterstock
Earlier research using mouse embryoids demonstrated symmetry breaking, and it was observed in human embryonic stem cells a few years ago, leading to the hope that symmetry breaking might also occur in an experimental embryoid if the model emulated the real thing well enough.
By combining bioengineering, physics, and developmental biology, the researchers — Simunovic, Ali H. Brivanlou and Eric D. Siggia — were able to create a new type of 3D model from human embryonic stem cells. It mimics the genetics, shape, and size of a roughly 10-day-old human embryo. (Researchers are prohibited from creating embryoids whose development goes beyond that of a natural 14-day-old.)
To test their embryoid, the researchers exposed it to a range of chemical signals the placenta releases during pregnancy until they got to bone morphogenetic protein. Simunovic recalls, "We added BMP4, and two days later one part of the three-dimensional culture became the future posterior, and the opposite part became the future anterior."
A model embryoid
Image source: ValentinaKru/Shutterstock
The teams' work results in the introduction of a new type of 3D model that may lead to better understanding of pregnancy complications, such as why some embryos fail to attach to the uterus successfully. "About 50 to 75 percent of embryos do not attach, creating a huge bottleneck to pregnancy," says Simunovic. "We don't know why that is, but using this model we may be able to find out."
Simunovic says that the model may also be useful in exploring developmentally incurred diseases. He says, "We can create 3D embryonic models of genetic conditions, and follow the developmental process in real time. These models can finally advance the understanding of a wide range of diseases for which we currently have no idea where and when things begin to go wrong."
The resemblance is uncanny: choanocytes, single-celled organisms found in sea sponges, look an awful lot like choanoflagellates. Since the former are believed to be the closest single-celled relatives of animals, scientists have surmised a connection that would make choanocytes the earliest predecessors to animals, including us.
"Most biologists for decades have believed this theory to be true," says Associate Professor Sandie Degnan from the University of Queensland (UQ), Australia. "But their transcriptome signatures simply don't match, meaning that these aren't the core building blocks of animal life that we originally thought they were."
Her assertion unsettles nearly settled evolutionary science. "We're taking a core theory of evolutionary biology and turning it on its head," says Degnan, co-senior author of a paper published in Nature on June 12 with UQ's Professor Bernard Degnan.
New technology reveals an old history
The Degnans and their colleagues sequenced the genes expressed by sponge somatic cell types, which are believed to be similar to those present in the last single-celled common ancestor prior to the emergence of multicellular metazoans and ultimately, animals. Using the CEL-Seq2 gene-sequencing platform, they analyzed:
- choanocytes — described in the study as "internal epithelial feeding cells that capture food by pumping water through the sponge."
- pinacocytes — epithelial cells that line sponges' internal canals and their outer surface.
- archaeocytes — mesenchymal cells from sponges' middle collagenous layer that perform a range of functions.
Image source: QU
Choanocytes are not your great, great, great, etc. grandmother
The researchers found "no support for the long-standing hypothesis that multicellular animals evolved from an ancestor that was an undifferentiated ball of cells resembling extant choanocytes and choanoflagellates." Instead, the truth seemed to lay more in the flexible behavior of archeocytes. The researchers concluded that the "ancestral metazoan cell type had the capacity to exist in and transition between multiple cell states."
In other words, says Professor Degnan, "We've found that the first multicellular animals probably weren't like the modern-day sponge cells, but were more like a collection of convertible cells. And so, the "great-great-great-grandmother of all cells in the animal kingdom, so to speak, was probably quite similar to a stem cell."
Associate Professor Degnan notes that modern genetic sequencing tools "helped us finally address an age-old question, discovering something completely contrary to what anyone had ever proposed." As for the exact identity of our earliest single-celled ancestor, she adds, "Now we have an opportunity to re-imagine the steps that gave rise to the first animals, the underlying rules that turned single cells into multicellular animal life."
Amphimedon queenslandica, from which the study's were taken (left), and a choanocyte chamber containing multiple choanocytes (right)
Image sources: Karin Taylor, Rebecca Fieth, QU
It should come as no surprise that microbiology is a difficult discipline. The sheer amount of work that it takes to be knowledgeable about the current state of the field is staggering, and advancing the field as a whole is even more challenging. A recent study out of Cincinnati Children's Hospital has made a significant advancement, with researchers growing a human esophagus using stem cells for the first time.
The esophagus in question wasn't very large — just 800 micrometers long, which works out to be about 0.03 inches. (We're still a far way off from growing whole human organs in a laboratory.) However, this research represents an important step in that direction, and the ability to grow small models of organs (called organoids) makes us better at developing treatments for common diseases affecting those organs. What's more, the new research also means that it will be possible to regenerate damaged tissue in existing esophagi.
Growing an esophagus
Developing this small esophagus organoid took a lot of precision. The 800-micrometer organoid was grown over the period of two months, but it started out as a slurry of pluripotent stem cells (PSCs). Unlike adult stem cells, which can only grow into specific, specialized types of tissues, PSCs can develop into any type of cell in the body. Essentially, they are our original components — every human started off as a similar slurry of PSCs.
The researchers exposed these cells to precise amounts of different chemicals that recreated the kind of events a PSC would undergo in order to develop into an esophagus in a normal developing fetus. These chemicals manipulated cellular signaling pathways — essentially, a chain of reactions that occur when a cell is exposed to a certain molecule. In the cell, a cascading series of reactions occurs that triggers some kind of event in a cell, such as cell death, replication, or, in this case, differentiation into esophagus cells.
Previous studies had tried to develop human esophagus organoids, but these usually ended up as a mixture of different tissues, including those found in the pharynx, the esophagus, and the respiratory tract. To develop esophageal tissues, the researchers needed to precisely time similarly precise quantities of chemicals to trigger the right signaling pathways for the right amount of time.
As an example, exposing the cells to retinoic acid for four days caused them to develop into tissues found lower down in the foregut, below the esophagus. Treating the cells in retinoic acid for just one day, however, seemed to be the right amount of exposure to encourage esophageal tissues to develop. In addition, treating the cells with Noggin — a curiously named protein — encouraged the tissues to develop into esophageal tissues rather than respiratory tissues.
A diagram depicting the various possible tissues the stem cells could have developed into. Exposing the cells to different molecules, such as retinoic acid (RA) and Noggin (NOG) encourages the stem cells to develop into different tissues.
Trisno et al., 2018
What's useful about this?
Growing a model of the human esophagus is an interesting project, but science like this isn't done out of sheer curiosity. Regarding its utility, Jim Wells, a researcher working on the project, said, "In addition to being a new model to study birth defects like esophageal atresia, the organoids can be used to study diseases like eosinophilic esophagitis and Barrett's metaplasia, or to bioengineer genetically matched esophageal tissue for individual patients." There are other potential applications of this research on esophageal cancer, gastroesophageal reflux disease (GERD), and achalasia, which affects the esophagus's lower muscles, preventing food from passing through. The researchers noted that all of these conditions need better treatments.
To demonstrate the usefulness of this model organ, the researchers examined the impact of the SOX2 gene on the development of the esophagus. In both humans and mice, when SOX2 is repressed or inactivated, the esophagus peters out and fails to connect to the stomach. For babies born with esophageal atresia, this condition can be life-threatening and requires surgery to correct.
Researchers have known that SOX2 was associated with this condition, but the exact mechanism was unknown. By studying the growth of the esophageal organoid and comparing it with the esophagi of mice whose Sox2 genes had been inactivated, the researchers discovered that a molecule called Wnt was the likely reason behind esophageal atresia. Remember how growing this organoid require precisely timed applications of various chemicals? Wnt works like that to — only in a developing body, the SOX2 gene inhibits the amount of Wnt the cells are exposed to. When SOX2 doesn't work correctly, Wnt encourages developing cells to become part of the respiratory tract rather than the esophagus, resulting in esophageal atresia.
This kind of work is very much in the early stages. Before 1998, scientists had no idea how to harvest human stem cells, and now we're building model organs with them. While growing an entire organ is still very much a thing of the future, it's important to remember that every step along the way to that goal will make use better at fighting disease, saving lives, and understanding how the human body functions.
