Live streaming genes in living cells coming to a computer near you!

Christmas has come early to scientists at the University of Virginia School of Medicine. They’ve developed a technology that allows you to watch how individual genes move and interact in living cells. You can think of it as Facebook’s live streaming meets the adventurous Ms. Frizzle and her Magic School Bus.

Using a gene editing system called CRISPR/Cas9, the team tagged genes of interest with fluorescent proteins that light up under a microscope – allowing them to watch in real time where these genes are in a cell’s nucleus and how they interact with other genes in the genome. This research, which was funded in part by a CIRM Research Leadership award, was published in the journal Nature Communications.

Watching genes in living cells

Traditional methods for observing the locations of genes within cells, such as fluorescent in situ hybridization (FISH), kill the cells – giving scientists only a snapshot of the complex interactions between genes. With this new technology, scientists can track genes in living cells and generate a 3D map of where genes are located within chromatin (the DNA/protein complex that makes up our chromosomes) during the different stages of a cell’s existence. They can also use these maps to understand changes in gene interactions caused by diseases like cancer.

Senior author on the study, Dr. Mazhar Adli, explained in a news release:

Mazhar Adli (Josh Barney, UVA Health System)

“This has been a dream for a long time. We are able to image basically any region in the genome that we want, in real time, in living cells. It works beautifully. With the traditional method, which is the gold standard, basically you will never be able to get this kind of data, because you have to kill the cells to get the imaging. But here we are doing it in live cells and in real time.”

Additionally, this new technique helps scientists conceptualize the position of genes in a 3D rather than in a linear fashion.

“We have two meters of DNA folded into a nucleus that is so tiny that 10,000 of them will fit onto the tip of a needle,” Adli explained. “We know that DNA is not linear but forms these loops, these large, three-dimensional loops. We want to basically image those kind of interactions and get an idea of how the genome is organized in three-dimensional space, because that’s functionally important.”

Not only can this CRISPR technology light up specific genes of interest, but it can also turn their activity on or off, allowing the scientists to observe the effects of one gene’s activity on others. The flexibility of this approach for visualizing genes in live cells is something that the research world currently lacks.

“We were told we would never be able to do this. There are some approaches that let you look at three-dimensional organization. But you do that experiment on hundreds of millions of cells, and you have to kill them to do it. Here, we can look at the single-cell level, and the cell is still alive, and we can take movies of what’s happening inside.”

This is a pretty nifty imaging tool for scientists that allows them to watch where genes are located and how they move as a cell develops and matures. Live-streaming the components of the genetic engine that keeps a cell running could also provide new insights into why certain genetic diseases occur and potentially open doors for developing better treatments.

Scientists tracked specific genomic locations in a living cell over time using their CRISPR/Cas9 technology. (Nature communications)

Why is a cell therapy that restores sight to the blind against the law?


A lot of people are frustrated with the US Food and Drug Administration (FDA) and its woefully slow process for approving stem cell therapies. That’s one of the reasons why we started the CIRM Stem Cell Champions campaign, to gather as many like-minded supporters of stem cell research as possible and help to change the way the FDA works, to create a more efficient approval process.

You can read more about that campaign and watch a short video on what being a Stem Cell Champion involves (hint: not very much).

Now Randy Mills, our President and CEO, has teamed up with former US Senator Bill Frist to explain precisely why the FDA needs to change the way it regulates stem cells, and to offer a simple way to create the system that will best serve the needs of patients.

This Op Ed appeared on Fox News’ online Opinion section on Friday, May 20th.

Cell therapy reversed blindness for 47,000 patients in 2015. So why is it against the law?

By C. Randal Mills Ph.D., Sen. Bill Frist M.D.

As medical miracles go, restoring sight to the blind is right up there. A mother seeing her baby for the first time, or a child being able to count the stars is a beautiful gift, and its value cannot be overstated. Last year 47,000 Americans received that gift and had their blindness reversed through the transplantation of cells from a corneal donor’s final selfless act.

It is safe, it is effective, and because it is curative, it is a relatively cost effective procedure. It is medicine at its most beautiful. And according to FDA regulations, the distribution of this cell therapy is in violation of federal law.

That’s right. The regulation says that no matter how competent the surgeon, the FDA must first approve cells from donated corneas as if they were a drug—a process that takes over a decade and can costs billions of dollars — all for a practice that has been successfully restoring sight for more than 50 years. And this is only one example.

The good news: the FDA doesn’t always adhere to its regulations and has not in this case.

The bad news: inconsistent enforcement creates uncertainty, deterring innovation for other unmet medical needs such as arthritis, back pain, and diabetic ulcers.

How did a country known for pioneering medical breakthroughs get here?

Appropriate regulation of living cells that treat disease is inherently complex. Some therapies, like corneal cell transplants, are well-understood. Others are far more sophisticated and can involve forcing cells to change from one type to another, cutting out defective genes, and growing cells in culture to expand their numbers into the billions. Although this may sound like science fiction, it’s the type of very real science that will revolutionize the practice of medicine. And it is a challenging spectrum to regulate.

Unfortunately, what we have today amounts to a regulatory light switch for cell therapy; one that is either OFF or ON. For some cell therapies there is essentially no pre-market regulation. But at some point of added complexity, often arbitrarily decided by the FDA, the switch flips to ON and the cell becomes a drug in the minds of the Agency. And the consequences could not be more profound.

A product can be introduced through the OFF pathway in days with no FDA review and at very little cost. The ON pathway on the other hand, takes 10-20 years and can cost over a billion dollars. For cell therapy, there is no in between.

It is not possible to regulate the continuum of cell therapies fairly and effectively by using this binary approach. The system is broken and is impeding the hunt for safe and effective treatments for suffering patients.

Why? Because sensible people don’t invest significant capital gambling that the FDA will give them a pass out of its rules. They evaluate the time and cost of development assuming they will be forced down the ON pathway. They also assume that this arbitrary approach to regulation will (and often does) work against them by allowing a competitor to enter the market through the OFF pathway, placing them at a prohibitive disadvantage. The results speak for themselves. After 15 years under this paradigm we have had only a few cell therapies approved, all commercial disasters.

This is because the ON-OFF approach fails to adequately account for the difference in cell therapy complexity. To better understand, imagine this methodology applied to the regulation of automobiles. The government might permit low tech cars, say the Model T, to be sold without pre-market regulation. But if a manufacturer wanted to improve the vehicle by adding air conditioning, a radio or other such feature, the car would be subject to massive pre-market regulation. And not just on the new feature. Instead, the addition of the new feature would trigger a bumper-to-bumper evaluation of the entire car, increasing its development cost from basically nothing to that of a Lamborghini. The result would be streets full of hot, radio-less go-karts, except for a few ultra-high-end sports cars whose manufacturers are now defunct because they were never able to recoup the disproportionate costs of satisfying the regulatory system. This is what we see with cell therapies today: progress that is sluggish at best.

How can we move forward?

Ironically, the FDA identified a solution to the problem. In order to account for the broad spectrum inherent to cell therapy, in the late 90’s the FDA proposed a progressive, risk-based approach. The higher the risk, the greater the regulation. This guards against under regulation that might put patients at risk and prevents overregulation that can disincentivize the development of new or improved products.

In the FDA’s own words, the regulation they proposed would abide by a few basic principles:

  • “Under this tiered, risk-based approach, we propose to exert only the type of government regulation necessary to protect the public health.”
  • “The regulation of different types of human cells… will be commensurate with the public health risks…”
  • “These planned improvements will increase the safety of human cells… while encouraging the development of new products.”

It was a remarkably common sense approach that would have balanced safety with the need for innovation over an exceptionally broad range of technological complexity and risk.

It would have.

Unfortunately, the regulatory framework that was promised was never delivered, and it is time to resuscitate it. The burden placed on the development of cell therapies must accurately reflect the risks; must be balanced against the very real consequences of doing nothing (patients continuing to suffer); and must be consistently and fairly applied. In short, the FDA had it right and we need to give them the tools to deliver the regulatory paradigm they originally envisioned.

If we fix this highly fixable problem, we can create a system that will drive new innovations and better outcomes. Europe and Japan have already acted and are seeing the benefits. People with great ideas are coming off the bench, and game changing therapies are entering practice. While challenging the status quo does not sit well with some, particularly those who stand to prosper from the built-in barriers to entry the current structure provides, in the United States we have a responsibility to do better for patients and fix this broken system.

Randal Mills, Ph.D., is the President and CEO of the California Institute for Regenerative Medicine

William “Bill” H. Frist, M.D. is a nationally-acclaimed heart and lung transplant surgeon, former U.S. Senate Majority Leader, and chairman of the Executive Board of the health service private equity firm Cressey & Company.

UCLA Scientists Find 3000 New Genes in “Junk DNA” of Immune Stem Cells

Genes and Junk

Do you remember learning about Junk DNA when you took Biology in high school? The term was used to described 98% of the human genome that doesn’t make up its approximately 22,000 genes. We used to think that Junk DNA didn’t serve a purpose, but that was before we discovered special elements called non-coding RNAs that call Junk DNA their home. But we’re getting ahead of ourselves, so let’s take a step back.

Genes are sequences of DNA that contain the blueprints for the proteins that make your cells and organs function. Before a gene can become a protein, its transformed into a molecule called an RNA. RNAs contain messages that tell a cell’s machinery what types of protein to make and how many.

Not Junk After All

Now back to “Junk DNA”… scientists thought that because this mass of DNA sequences was never turned into protein, it served no purpose. It turns out that they couldn’t be farther from the facts.

There are actually sequences of DNA in our genomes that are blueprints for RNAs that never become proteins. Scientists call them “non-coding” RNAs, and they play very important roles in the body such as replicating DNA and regulating gene expression – deciding which genes are turned on and which are turned off.

Another important function that non-coding RNAs control is cell differentiation, or the maturation of immature cells into adult cells. Differentiation is a complicated process, and because non-coding RNAs are relatively new to the scientific world, we haven’t figured out their exact roles in the differentiation of stem cells into adult cells.

Understanding Immune Cell Development

In a study published this week in Nature Immunology, UCLA scientists reported the discovery of 3000 new genes that make a type of non-coding RNA called a long non-coding RNA (lncRNA) that regulates the differentiation of stem cells into mature immune cells like B and T cells, which play a key role in fighting infection. This important study was funded in part by CIRM.

UCLA scientists David Casero and Gay Crooks with the sequencing machine that separated the genetic information within the bone marrow and thymus gland tissue stem cells. (Image credit: Mirabai Vogt-James, UCLA Broad Stem Cell Research Center)

UCLA scientists David Casero and Gay Crooks with the sequencing machine used to identify the 3000 new genes. (Image credit: Mirabai Vogt-James, UCLA Broad Stem Cell Research Center)

Using sequencing technology and bioinformatics, they mapped the RNA landscape (known as the transcriptome) of rare stem cells isolated from human bone marrow (hematopoietic stem cells) and the thymus (lymphoid progenitor cells). They identified over 9000 genes that produced lncRNAs that were important for moderating various stages of immune cell development. Of this number, over 3000 were genes whose lncRNAs hadn’t been found before.

First author, David Casero explained the importance of their discovery in a UCLA press release:

Our findings are exciting because they provide a huge and unique resource for the whole immunology community. We will now be able to drill down on the specific LncRNA genes that seem to be most important at each stage of immune cell development and understand how they function individually and together to control the process.


Co-senior author and UCLA professor Gay Crooks explained that the goal of their work was to gain a better understanding of how the immune system develops in order to battle serious diseases that affect it and open up avenues for generating better cell therapies.

If we can understand how the immune system is generated and maintained during life, we can find ways to improve production of immune cells for potential therapies after chemotherapy, radiation and bone marrow transplant, or for patients with HIV and inherited immune deficiencies. In addition, by understanding the genes that control this process we can better understand how they are changed in cancers like leukemia and lymphoma.


Final Words

While this study focused on the role of lncRNAs in the development of the immune system and the differentiation of immune stem cells, the technology in this study can be used to understand the development of other systems and organs.

Scientists are already publishing papers on the role of lncRNAs in the differentiation of stem cells in the brain and heart, and further work in this field will undoubtedly uncover many new and important lncRNA genes. If the pace keeps up, the term “Junk DNA” will need to be retired to the junk yard.


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Specialized Embryonic Stem Cells Yield Insights into X Chromosome Inactivation

Please don’t be intimidated by the title of this post! By the end of this blog, you’ll be well versed in X chromosome inactivation, and you’ll understand why you should care about this topic.

Males and females are different in countless ways, but the underlying cause of these differences originates with chromosomes. Women have two X chromosomes while men have an X and a Y. The X chromosome is much larger than the Y chromosome, and consequently it harbors a larger number of genes (there are about 1000) with very important functions. Female cells have evolved to inactivate or silence one of their X chromosomes so that both male and female cells receive the same the same “dosage” of X chromosome genes.

Calico Cat.

Calico cats are a result of X-inactivation.

A great example of X-inactivation in nature is a cat with a calico coat. Did you notice that most calico cats are female? This is because there are two different versions of the fur color gene (orange and black) located on different X chromosomes. In calico cats, some patches of fur turn off the X-chromosome with the black gene while others turn off the one with the orange gene. The result is the beautiful and crazy patchwork of orange and black.

The process of X chromosome inactivation is extremely important for many reasons other than feline coat color. Think about that time you ate an extra-large pizza by yourself. That was pushing your limits right? Well imagine if you actually ate two of those pizzas. Your stomach would likely explode, and you would meet an untimely end. Apply this somewhat disturbing analogy to female cells with two active X chromosomes. You can now imagine that having double the dosage of X chromosome genes could be toxic and result in dead or very unhappy cells.

How X-inactivation works
The jury is still out on the full answer to how X-inactivation works; however, some pieces of the puzzle are known.

The major player in X-inactivation is a molecule called Xist. Xist is produced in cells with two X chromosomes, and its job is to inactivate one of these X’s. During X-inactivation, hundreds of Xist molecules swarm and attach to one of the two X chromosomes. Xist then recruits other molecule buddies to join the silencing party. These other molecules are thought to modify the X chromosome in a way that inactivates it.

This theory is where the field is at right now. However, a study published recently in Cell Reports by Dr. Anton Wutz’s group at ETH Zurich found another piece to this puzzle: a new molecule that’s critical to X-inactivation.

New Study Sheds Light on X-inactivation

Specialized haploid embryonic stem cells engineered to produce the X-inactivator Xist upon drug treatment. (Cell Reports)

Specialized haploid embryonic stem cells engineered to produce the X-inactivator Xist upon drug treatment were used to identify genes important to X-inactivation. (Cell Reports, Montfort et al. 2015)

The Wutz lab used a novel and powerful mouse embryonic stem cell (ESC) model that was engineered to have only one of each chromosome, and therefore only one X instead of two. These “haploid” ESCs were also manipulated to produce copious amounts of the X chromosome silencer Xist when treated with a specific drug. Thus, when these haploid ESCs received the drug, Xist was turned on and inactivated the only X chromosome in these cells, causing them to die.

In an example of brilliant science, Wutz and colleagues used this haploid ESC model to conduct a large-scale screen for genes that work with Xist to cause X-inactivation. Wutz and his colleagues identified genes whose loss of function (caused by mutations made in the lab) saved the lives of haploid ESCs treated with the Xist-inducing drug.

In total, the group identified seven genes that they think are important to Xist function. Their most promising candidate was a gene called Spen. When they mutated the Spen gene in their specialized ESC model, the ESCs survived treatment with the Xist-inducing drug. Further studies revealed that Spen directly interacts with Xist and recruits the other molecules that cause X-inactivation.

Big Picture
But why does this research matter? From a scientific standpoint, it highlights the power of embryonic stem cells as a model for understanding fundamental human processes. In terms of human health, it’s important because X-inactivation is actually a defense mechanism against diseases caused by mutations in genes on the X chromosome (X-linked genes).

In women with that have a disease-causing mutation in only one copy of an X-linked gene, X-inactivation of the chromosome with the mutation will prevent that woman from getting the disease. However, sometimes X-inactivation can be incomplete or biased (favoring the inactivation of one X chromosome over the other), both of which could cause activation of X chromosomes with X-linked disease mutations.

These events are hypothesized to be the cause of some cancers (although this hypothesis is still under speculation), mental impairment, and X-linked diseases such as Rett’s syndrome and autoimmune disorders. Therefore, a better understanding of X-inactivation may one day lead to treatments that prevent these diseases.