Unlocking the secrets of how stem cells decide what kind of cell they’re going to be

Laszlo Nagy, Ph.D., M.D.

Laszlo Nagy, Ph.D., M.D.: Sanford Burnham Prebys Medical Discovery Institute

Before joining CIRM I thought OCT4 was a date on the calendar. But a new study says it may be a lot closer to a date with destiny, because this study says OCT4 helps determine what kinds of cell a stem cell will become.

Now, before we go any further I should explain for people who have as strong a science background as I do – namely none – that OCT4 is a transcription factor, this is a protein that helps regulate gene activity by turning certain genes on at certain points, and off at others.

The new study, by researches at Sanford Burnham Prebys Medical Discovery Institute (SBP), found that OCT4 plays a critical role in priming genes that cause stem cells to differentiate or change into other kinds of cells.

Why is this important? Well, as we search for new ways of treating a wide variety of different diseases we need to find the most efficient and effective way of turning stem cells into the kind of cells we need to regenerate or replace damaged tissue. By understanding the mechanisms that determine how a stem cell differentiates, we can better understand what we need to do in the lab to generate the specific kinds of cells needed to replace those damaged by, say, heart disease or cancer.

The study, published in the journal Molecular Cell, shows how OCT4 works with other transcription factors, sometimes directing a cell to go in one direction, sometimes in another. For example, it collaborates with a vitamin A (aka retinoic acid) receptor (RAR) to convert a stem cell into a neuronal precursor, a kind of early stage brain cell. However, if OCT4 interacts with another transcription factor called beta-catenin then the stem cell goes in another regulatory direction altogether.

In an interview with PhysOrg News, senior author Laszlo Nagy said this finding could help develop more effective methods for producing specific cell types to be used in therapies:

“Our findings suggest a general principle for how the same differentiation signal induces distinct transitions in various types of cells. Whereas in stem cells, OCT4 recruits the RAR to neuronal genes, in bone marrow cells, another transcription factor would recruit RAR to genes for the granulocyte program. Which factors determine the effects of differentiation signals in bone marrow cells – and other cell types – remains to be determined.”

In a way it’s like programming all the different devices that are attached to your TV at home. If you hit a certain combination of buttons you get to one set of stations, hit another combination and you get to Netflix. Same basic set up, but completely different destinations.

“In a sense, we’ve found the code for stem cells that links the input—signals like vitamin A and Wnt—to the output—cell type. Now we plan to explore whether other transcription factors behave similarly to OCT4—that is, to find the code in more mature cell types.”



Filling the Holes in our Understanding of Stem Cell Fate

How does a single-celled human embryo transform into a human body with intricate organ systems containing trillions of specialized cells? Step into any college lecture discussing this question and I bet “transcription factors” is a phrase you’ll often hear.

Transcription factors are DNA-binding proteins that act as cell fate control switches during development. For cells destined to become, say muscle tissue, transcription factors bind DNA and help activate muscle-specific genes or keep non-muscle-specific genes silent. And so it goes for all other cell types as they form inside the growing embryo.

But file this blog entry under “Hold up a moment” because in research published today in Stem Cell Reports, CIRM-funded scientists at U.C. San Diego (UCSD) pinpoint another cellular process that appears equally as important as transcription factors in cell fate decisions. The process they studied is called nonsense-mediated mRNA decay (NMD). To go into details about NMD we need to first delve a bit more into transcription factors.

A bit of molecular biology 101
When a gene is said to be activated, or “turned on”, that’s just shorthand for describing the process of transcription in which a stretch of DNA corresponding to a gene is “read” by cell machinery and transcribed into messenger RNA (mRNA). The mRNA is then translated into a string of amino acids which forms a particular protein. By binding to the DNA in the vicinity of a gene, the transcription factors provide a physical platform for the transcribing machinery to form the mRNA. Frequent transcribing leads to more mRNA and more protein.


Transcription and translation: turning genes on to produce proteins (image: tokresource.org)


The nonsense mRNA decay (NMD) pathway (Wikipedia)

The NMD pathway regulates transcription from the opposite end of the process by promoting the degradation of mRNAs. It was once thought to only be involved in getting rid of mRNAs that contain transcribing errors but more recent studies have shown that NMD has roles in normal cell functions. For instance, the UCSD team had shown that neural stem cells contain high levels of NMD which must be reduced to allow those stem cells to specialize into neurons. In the current paper, the team sought to better understand these underlying mechanisms that enable the NMD pathway to regulate development.

To accomplish this goal, NMD function was examined during the very early stages of human development using human embryonic stem cells (hESC). All adult tissues are derived from the three germ layers that form during embryogenesis: endoderm (which gives rise to lung and gut), mesoderm (which gives rise to muscle, bone, blood) and ectoderm (which gives rise to skin and neurons). When the researchers grew the hESCs under conditions that favored endoderm formation, they observed dramatically reduced levels of NMD activity. But growing hESC toward mesoderm and ectoderm fates, led to increased levels of NMD. So these very early forks in the road of cell fate decisions led to diverging levels of NMD.

NMD activities: cell fate bystander or participant?
But does this change in NMD activity play a direct role during the specialization of hESCs? To answer this question, the team focused on the manipulating NMD activity as hESCs formed into endoderm. Instead of the natural decrease in NMD proteins during endodermal formation, NMD levels were artificially maintained at high levels. Sure enough, this hampered the ability of the hESCs to take on properties of endodermal cells and instead they kept hallmarks of stem cells. Approaching this analysis from the opposite angle, NMD factors were removed from the hESCs under conditions that should block endoderm formation. In support of the previous experiment, this artificial drop in NMD activity led to the initiation of endodermal differentiation.

Further experiments determined that NMD activity specifically inhibits TGF-b, a protein that signals cells toward an endoderm fate. Conversely, the team’s results also suggest that NMD stimulates BMP which is an important signal for a mesoderm fate. So just like transcription factors, the activity of NMD modulates the balance of other proteins which ultimately direct the fate of a cell’s identity. In fact, the TGF- b and BMP pathways themselves stimulate the actions of transcription factors so there’s likely some cooperation going with these factors and NMD. We reached out to UCSD professor Miles Wilkinson, the principal investigator for this study, to get his team’s reaction to their results:

“Most of what we know about human embryonic stem cell fate revolves around the role of factors that regulate RNA synthesis – transcription factors.  In our study, we examined the other side of the coin – the role of factors required for a selective RNA decay pathway.  We were surprised to find that not only did the NMD RNA decay pathway influence embryonic cell differentiation, but it is critical for cell fate decisions through its effect on signaling pathways.  Thus, we envisage that RNA decay pathways and transcriptional pathways converge on signaling pathways to control embryonic stem cell fate.”


And a better understanding of how embryonic stem cell fate is controlled could help optimize stem cell-based therapies for a given tissue or organ. Whatever the case, it shouldn’t be long before future college students in a developmental biology class will hear “NMD” in the same breath as “transcription factors”.

A cardiac love triangle: how transcription factors interact to make a heart

 Here’s a heartfelt science story for all those Valentine’s day fans out there. Scientists from the Gladstone Institutes have identified how a group of transcription factors interact during embryonic development to make a healthy heart. Their work will increase our biological understanding of how the heart is formed and could produce new methods for treating cardiovascular disease.

The study, published today in the journal Cell, describes a tumultuous love story between cardiac transcription factors. Transcription factors are proteins that orchestrate gene expression. They have the power to turn genes on or off by binding to specific DNA sequences and recruiting other proteins that will eventually turn the information encoded in that gene into a functional protein.

Every organ has its own special group of transcription factors that coordinate the gene expression required for that organ’s development. Often times, transcription factors within a group directly interact with each other and work together to conduct a specific sequence of events. These interactions are essential for making healthy tissues and organs, but scientists don’t always understand how these interactions work.

For the heart, scientists have already identified a group of transcription factors essential for cardiac development, and genetic mutations in any of these factors can impair heart formation and cause heart defects in newborns. What’s not known, however, are the details on how some of these cardiac transcription factors interact to get their job done.

A cardiac love triangle

In the Gladstone study, the scientists focused on how three key cardiac transcription factors – NKX2.5, TBX5, and GATA4 – interact during heart development. They first proved that these transcription factors are essential for the formation of the heart in mouse embryos. When they eliminated the presence of one of the three factors from the developing mouse embryo, they observed abnormal heart development and heart defects. When they removed two factors (NKX2.5 and TBX5), the results were even worse – the heart wasn’t able to form and none of the embryos survived.

Normal heart muscle cells, courtesy Kyoto University

Normal cardiomyocytes or heart cells, courtesy Kyoto University

Next, they studied how these transcription factors interact to coordinate gene expression in heart cells called cardiomyocytes made from mouse embryonic stem cells that lacked either NKX2.5, TBX5, or both of these factors. Compared to normal heart cells, cardiomyocytes that lacked one or both of these two transcription factors started beating at inappropriate times – either earlier or later than the normal heart cells.

Taking a closer look, the scientists discovered that TBX5, NKX2.5 and GATA4 all hangout in the same areas of the genome in embryonic stem cells that are transitioning into cardiomyocytes. In fact, each individual transcription factor required the presence of the others to bind their DNA targets. If one of these factors was missing and the love triangle was broken, the remaining transcription factors became confused and bound random DNA sequences in the genome, causing a mess by turning on genes that shouldn’t be on.

First author on the study, Luis Luna-Zurita, explained the importance of maintaining this cardiac love triangle in a Gladstone Press Release:

Luis Luna-Zurita, Gladstone Institute

Luis Luna-Zurita, Gladstone Institute

“Transcription factors have to stick together, or else the other one goes and gets into trouble. Not only are these transcription factors vital for turning on certain genes, but their interaction is important to keep each other from going to the wrong place and turning on a set of genes that doesn’t belong in a heart cell.”

Crystal structure tells all

Protein crystal structure of NKX2.5 and TBX5 bound to DNA.

Protein crystal structure of NKX2.5 and TBX5 bound to DNA. (Luna-Zurita et al. 2016)

The last part of the study proved that two of these factors, NKX2.5 and TBX5, directly interact and physically touch each other when they bind their DNA targets. In collaboration with a group from the European Molecular Biology Laboratory (EMBL) in Germany, they developed protein crystal structures to model the molecular structure of these transcription factors when they bind DNA.

Co-author and EMBL scientist Christoph Muller explained his findings:

“The crystal structure critically shows the interaction between two of the transcription factors and how they influence one another’s binding to a specific stretch of DNA. Our detailed structural analysis revealed a direct physical connection between TBX5 and NKX2-5 and demonstrated that DNA plays an active role in mediating the interaction between the two proteins.”

Big picture

While this study falls in the discovery research category, its findings increase our understanding of the steps required to make a healthy heart and sheds light on what goes wrong in patients or newborns with heart disease.

Senior author on the paper and Gladstone Professor Benoit Bruneau explained the biomedical applications of their study for treating human disease:


Benoit Bruneau, Gladstone Institute

“Gene mutations that cause congenital heart disease lower the levels of these transcription factors by half, and we’ve shown that the dosage of these factors determines which genes are turned on or off in a cell. Other genetic variants that cause heart defects like arrhythmias also affect the function of these factors. Therefore, the better we understand these transcription factors, the closer we’ll come to a treatment for heart disease. Our colleagues at Gladstone are using this knowledge to search for small molecules that can affect gene regulation and reverse some of the problems caused by the loss of these transcription factors.”


I think it’s worth mentioning that these studies were done using mouse embryos and mouse embryonic stem cells. Future work should be done to determine whether this cardiac love triangle and the same transcription factor interactions exist in human heart cells.

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CIRM-Funded Scientists Build a Better Neuron; Gain New Insight into Motor Neuron Disease

Each individual muscle in our body—no matter how large or how small—is controlled by several types of motor neurons. Damage to one or more types of these neurons can give rise to some of the most devastating motor neuron diseases, many of which have no cure. But now, stem cell scientists at UCLA have manufactured a way to efficiently generate motor neuron subtypes from stem cells, thus providing an improved system with which to study these crucial cells.

“Motor neuron diseases are complex and have no cure; currently we can only provide limited treatments that help patients with some symptoms,” said senior author Bennett Novitch, in a news release. “The results from our study present an effective approach for generating specific motor neuron populations from embryonic stem cells to enhance our understanding of motor neuron development and disease.”

Normally, motor neurons work by transmitting signals between the brain and the body’s muscles. When that connection is severed, the individual loses the ability to control individual muscle movement. This is what happens in the case of amyotrophic lateral sclerosis, or ALS, also known as Lou Gehrig’s disease.

These images reveal the significance of the 'Foxp1 effect.' The Foxp1 transcription factor is crucial to the normal growth and function of motor neurons involved in limb-movement.

These images reveal the significance of the ‘Foxp1 effect.’ The Foxp1 transcription factor is crucial to the normal growth and function of motor neurons involved in limb-movement.

Recent efforts had focused on ways to use stem cell biology to grow motor neurons in the lab. However, such efforts to generate a specific type of motor neuron, called limb-innervating motor neurons, have not been successful. This motor-neuron subtype is particularly affected in ALS, as it supplies nerves to the arms and legs—the regions usually most affected by this deadly disease.

In this study, published this week in Nature Communications, Novitch and his team, including first author Katrina Adams, worked to develop a better method to produce limb-innervating motor neurons. Previous efforts had only had a success rate of about 3 percent. But Novitch and Adams were able to boost that number five-fold, to 20 percent.

Specifically, the UCLA team—using both mouse and human embryonic stem cells—used a technique called ‘transcriptional programming,’ in order to coax these stem cells into become fully functional, limb-innervating motor neurons.

In this approach, which was funded in part by a grant from CIRM, the team added a single transcription factor (a type of protein that regulates gene function), which would then guide the stem cell towards becoming the right type of motor neuron. Here, Novitch, Adams and the team used the Foxp1 transcription factor to do the job.

Normally, Foxp1 is found in healthy, functioning limb-innervating motor neurons. But in stem cell-derived motor neurons, Foxp1 was missing. So the team reasoned that Foxp1 might actually be the key factor to successfully growing these neurons.

Their initial tests of this theory, in which they inserted Foxp1 into a developing chicken spinal cord (a widely used model for neurological research), were far more successful. This result, which is not usually seen with most unmodified stem-cell-derived motor neurons, illustrates the important role played by Foxp1.

The most immediate implications of this research is that now scientists can now use this method to conduct more robust studies that enhance the understanding of how these cells work and, importantly, what happens when things go awry.