gene expression

Using stem cells to take an inside approach to fixing damaged livers

/ Kevin McCormack / Leave a comment

Often on the Stem Cellar we write about work that is in a clinical trial. But getting research to that stage takes years and years of dedicated work. Over the next few months we are going to profile some of the scientists we fund who are doing Discovery, or early stage research, to highlight the importance of this work in developing the treatments that could ultimately save lives.

 This first profile is by Pat Olson, Ph.D., CIRM’s Vice President of Discovery & Translation

liver

Most of us take our liver for granted.  We don’t think about the fact that our liver carries out more than 500 functions in our bodies such as modifying and removing toxins, contributing to digestion and energy production, and making substances that help our blood to clot.  Without a liver we probably wouldn’t live more than a few days.

Our liver typically functions well but certain toxins, viral infections, long-term excess alcohol consumption and metabolic diseases such as obesity and type 2 diabetes can have devastating effects on it.  Under these conditions, functional liver cells, called hepatocytes, die and are replaced with cells called myofibroblasts.  Myofibroblasts are cells that secrete excess collagen leading to fibrosis, a form of scarring, throughout the liver.  Eventually, a liver transplant is required but the number of donor livers available for transplant is small and the number of persons needing a functional liver is large.  Every year in the United States,  around 6,000 patients receive a new liver and more than 35,000 patients die of liver disease.

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Dr. Holger Willenbring

Dr. Holger Willenbring, a physician scientist at UCSF, is one of the CIRM-funded researchers pursuing a stem cell/regenerative medicine approach to discover a treatment for patients with severe liver disease.  There are significant challenges to treating liver disease including getting fully multi-functional hepatocytes and getting them to engraft and/or grow sufficiently to achieve adequate mass for necessary liver functions.

In previous CIRM–funded discovery research, Dr. Willenbring and his team showed that they could partially reprogram human fibroblasts (the most common cell found in connective tissue) and then turn them into immature hepatocytes.  (see our Spotlight on Liver Disease video from 2012 featuring Dr. Willenbring.) These immature hepatocytes, when transplanted into an immune-deficient mouse model of human liver failure, were shown to mature over time into hepatocytes that were comparable to normal human hepatocytes both in their gene expression and their function.

This was an important finding in that it suggested that the liver environment in a living animal (in vivo), rather than in a test tube (in vitro) in the laboratory, is important for full multi-functional maturation of hepatocytes.  The study also showed that these transplanted immature human hepatocytes could proliferate and improve the survival of this mouse model of chronic human liver disease.  But, even though this model was designed to emphasizes the growth of functional human hepatocytes, the number of cells generated was not great enough to suggest that transplantation could be avoided

A new approach

Dr. Willenbring and his team are now taking the novel approach of direct reprogramming inside the mouse.  With this approach, he seeks to avoid the challenge of low engraftment and proliferation of transplanted hepatocytes generated in the lab and transplanted. Instead, they aim to take advantage of the large number of myofibroblasts in the patient’s scarred liver by turning them directly into hepatocytes.

Recently, he and his team have shown proof-of principle that they can deliver genes to myofibroblasts and turn them into hepatocytes in a mouse. In addition these in vivo myofibroblasts-derived hepatocytes are multi-functional, and can multiply in number, and can even reverse fibrosis in a mouse with liver fibrosis.

From mice to men (women too)

Our latest round of funding for Dr. Willenbring has the goal of moving and extending these studies into human cells by improving the specificity and effectiveness of reprogramming of human myofibroblasts into hepatocytes inside the animal, rather than the lab.

He and his team will then conduct studies to test the therapeutic effectiveness and initial safety of this approach in preclinical models. The ultimate goal is to generate a potential therapy that could eventually provide hope for the 35,000 patients who die of liver disease each year in the US.

 

 

‘STARS’ Help Scientists Control Genetic On/Off Switch

/ cirmweb / Leave a comment

All life on Earth relies, ultimately, on the delicate coordination of switches. During development, these switches turn genes on—or keep them off—at precise intervals, controlling the complex processes that guide the growth of the embryo, cell by cell, as it matures from a collection of stem cells into a living, breathing organism.

Scientists have found a new way to control genetic switches.

Scientists have found a new way to control genetic switches.

If you control the switch, you could theoretically control some of life’s most fundamental processes.

Which is precisely what scientists at Cornell University are attempting to do.

Reporting in today’s issue of Nature Chemical Biology, synthetic biologists have developed a new method of directing these switches—a feat that could revolutionize the field of genetic engineering.

At the heart of the team’s discovery is a tiny molecule called RNA. A more simplified version of its cousin, DNA, RNA normally serves as a liaison—translating the genetic information housed in DNA into the proteins that together make up each and every cell in the body.

In nature, RNA does not have the ability to ‘turn on’ a gene at will. So the Cornell team, led by Julius Lucks, made a new kind of RNA that did.

They engineered a new type of RNA that they are calling Small Transcription Activating RNAs, or STARS, that can serve as a kind of artificial switch. In laboratory experiments, Lucks and his team showed that they could control how and when a gene was switched on by physically placing the STARS system in front of it. As Lucks explained in a news release:

“RNA is like a molecular puzzle, a crazy Rubik’s cube that has to be unlocked in order to do different things. We’ve figured out how to design another RNA that unlocks part of that puzzle. The STAR is the key to that lock.”

RNA is an attractive molecule to manipulate because it is so simple, says Lucks, much simpler than proteins. Many efforts aimed at protein manipulation have failed, due to the sheer complexity of these molecules. But by downshifting into the simpler, more manageable RNA molecules, Lucks argues that greater strides can be made in the field of synthetic biology and genetic engineering.

“This is going to open up a whole set of possibilities for us, because RNA molecules make decisions and compute information really well, and they detect things really well,” said Lucks.

In the future, Lucks envisions a system based solely on RNA that has the capability to manipulate genetic switches to better understand fundamental processes that guide the healthy development of a cell—and provide clues to what happens when those processes go awry.

Scientists identify gene that causes good protein to turn bad

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There exists a protein that, most of the time, helps keep the growth of cancer cells in check. But every so often it does the opposite—with potentially deadly consequences.

But now, researchers have discovered precisely why this protein, known as TGF-beta, can perform such an abrupt about-face. The results, published today in the journal Science Signaling, shed light on potential therapies that can succeed where others have failed—and attack the most aggressive forms of cancer.

TGF-beta is a type of tumor suppressor, a protein that normally keeps cells from growing, dividing and multiplying too quickly, which is how most tumors originate. But scientists have long observed that in many forms of cancer, TGF-beta has switched sides: it becomes a tumor promoter fostering the out-of-control growth of cells.

In this study, scientists at the University of Michigan Comprehensive Cancer Center have figured out that a gene called Bub1 seems to be pulling the strings—essentially flipping the switch on TGF-beta. The finding that Bub1 played such an important role in regulating TGF-beta caught the team completely off guard. According to the paper’s senior author Alnawaz Rehemtulla:

“Bub1 is well-known for its role in cell division. But this is the first study that links it to TGF-beta. We think this may explain the paradox of TGF-beta as a tumor promoter and a tumor suppressor.”

The team reached this conclusion by screening gene candidates against lung cancer and breast cancer cells. After screening over 700 genes, they narrowed down the potential gene to Bub1.

Further experiments revealed that Bub1 physically binds to TGF-beta, turning it to a tumor promoter in the process. And when the team prevented Bub1 from binding to TFG-beta, essentially blocking it, TGF-beta never turned sides.

These initial results have left the research team optimistic, in large part because Bub1 is known to be active, or ‘expressed,’ in so many forms of cancer. So, if they can find a way to block Bub1 in one type of cancer, they may be able to do so with other types.

Even at this early stage, the team has developed a compound that could block Bub1. Initial lab tests show that this so-called Bub1 ‘inhibitor’ could shut off the gene without affecting surrounding regions. Said Rehemtulla:

“When you look at gene expression in cancer, Bub1 is in the top five…. But we never knew why. Now that we have that link, we’re a step closer to shutting down this cycle.”

Key stem cell gene controlled from afar, Canadian scientists discover

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Embryonic stem cells can, by definition, mature into any cell type in the body. They are able to maintain this state of so-called pluripotency with the help of a gene called Sox2. And now, researchers at the University of Toronto (U of T) have discovered the unseen force that controls it. These findings, reported in the latest issue of Genes & Development, offer much-needed understanding of the steps a cell must take as it grows up.

Mouse embryonic stem cells grown in a round colony of cells (A) and express Sox2 (B), shown in red. Sox2 control region (SCR)-deleted cells have lost the typical appearance of embryonic stem cells (C) and do not express Sox2 (D). [Credit: Jennifer Mitchell/University of Toronto]

Mouse embryonic stem cells grown in a round colony of cells (A) and express Sox2 (B), shown in red. Sox2 control region (SCR)-deleted cells have lost the typical appearance of embryonic stem cells (C) and do not express Sox2 (D). [Credit: Jennifer Mitchell/University of Toronto]

Led by U of T Professor Jennifer Mitchell, the research team were, for the first time, able to identify the specific molecular regulator that switched the Sox2 gene on and off at specific times during an embryonic cell’s lifetime. As Mitchell explained:

“We studied how the Sox2 gene is turned on in mice, and found the region of the genome that is needed to turn the gene on in embryonic stem cells. Like the gene itself, this region of the genome enables these stem cells to maintain their ability to become any type of cell.”

The team named this region the Sox2 control region, or SCR.

For the last decade scientists have been using knowledge gleaned from the Human Genome Project to map how and when genes are switched on and off. Interestingly, the regions that control the gene in question aren’t always located close by.

This was the case with Sox2, said Mitchell. Early on, researchers had argued that Sox2 was regulated from nearby. But in this study, the team found the SCR, which controls Sox2, to be located more than 100,000 DNA base pairs away. According to Mitchell, the process by which the SCR activates Sox2 is fascinating:

“To contact the gene, the DNA makes a loop that brings the SCR close to the gene itself only in embryonic stem cells… It is possible that the formation of the loop needed to make contact with the Sox2 gene is an important final step in the process by which researchers practicing regenerative medicine can generate pluripotent cells from adult cells.”

Indeed, despite a flurry of research breakthroughs and a promising number of clinical trials moving forward, there are still some fundamental aspects of stem cell biology that remain unknown. This discovery, argues Mitchell, is an important step towards reaching toward improving the way in which scientists manipulate stem cells to treat disease.

These Are the Cells You’re Looking for: Scientists Devise New Way to Extract Bone-Making Stem Cells from Fat

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Buried within our fat tissue are stashes of stem cells—a hidden reservoir of cells that, if given the right cues, can transform into cells that make up bone, cartilage or fat. These cells therefore represent a much-needed store for regenerative therapies that rebuild bone or cartilage lost to disease or injury.

Finding cells that have bone-making potential is more efficiently done by looking at the genes they express (in this case, ALPL) than at proteins on their surface. The bone matrix being produced by cells is stained red in samples of cells that do not express ALPL (left), those that do express ALPL (right). [Credit: Darling lab/Brown University]

Finding cells that have bone-making potential is more efficiently done by looking at the genes they express (in this case, ALPL) than at proteins on their surface. The bone matrix being produced by cells is stained red in samples of cells that do not express ALPL (left), those that do express ALPL (right). The center image shows both types of cells prior to sorting [Credit: Darling lab/Brown University]

The only problem with these tucked-away cellular reservoirs, however, is identifying them and getting them out.

But now, researchers at Brown University have devised a unique method of identifying, extracting and then cultivating these bone-producing stem cells. Their results, published today in the journal Stem Cell Research & Therapy, seem to offer a much-needed alternative resource for growing bone.

Traditional methods attempting to locate and extract these stem cells focused on proteins that reside on the surface of the cells. Find the proteins, scientists reasoned, and you’ve found the cell.

Unfortunately, that method was not fool proof, and many argued that it wasn’t finding all the cells that reside in the fat tissue. So Brown scientists, led by Dr. Eric Darling found an alternative.

They knew that a gene called ALPL is an indicator of bone-making cells. If the gene is switched on, the cell has the potential to make bone. If it’s switched off, it does not. So Darling and his team devised a fluorescent marker, or tag, that stuck to the cells with activated ALPL. They then used a special machine to sort the cells: those that glowed went into one bucket, those that did not went into the other.

To prove that these ALPL-activated cells were indeed capable of becoming bone and cartilage, they then cultivated them for several weeks in a petri dish. Not only did they transform into the right cell types—they did so in greater numbers than cells extracted using traditional methods.

Hetal Marble, a graduate student in Darling’s lab and the paper’s first author, argues that tagging genes—rather than surface proteins—in order to distinguish and weed out cell types represents an important paradigm shift in the field. As he stated in a press release:

“Approaches like this allow us to isolate all the cells that are capable of doing what we want, whether they fit the archetype of what a stem cell is or is not. The paradigm shift is thinking about isolating populations that are able to achieve an end point rather than isolating populations that fit a strictly defined archetype.”

While their method is both precise and accurate, there is one drawback: it is slow.

Currently, it takes four days to tag, extract and cultivate the bone-making cells. In the future, the team hopes they can shorten this time frame so that they could perform the required steps within a single surgical session. As Darling stated:

“If you can take a patient into the OR, isolate a bunch of their cells, sort them and put them back in—that’s ideally where we’d like to go with this.”