CIRM invests $1.3 million to study stem cells in metabolic liver disease

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Dr. Tracy Grikscheit. Image courtesy of Children’s Hospital LA.

Metabolic liver disease, is an emerging public health concern in Western countries, but has largely been overshadowed by health issues such as cancer and diabetes. Chronic liver disease (of which metabolic liver disease is a significant contributor) however, is a significant public health concern, evidenced by its contribution to nearly 2 million deaths per year worldwide.

The primary treatment option for metabolic liver disease is a liver transplant. In fact, of the liver transplants performed every year, 14% are due to damage associated with metabolic disorders. With any organ transplant, however, such a procedure comes with drawbacks, the most frustrating of which is the need for patients to wait for an organ donor.

As transplants are not a reasonable or feasible option for many people, alternative treatment options are necessary.  Enter Dr. Tracy Grikscheit, a doctor-scientist at the Children’s Hospital Los Angeles, who hopes to make liver transplant a thing of the past for the millions of people who live with metabolic liver disease.

Dr. Grikscheit was awarded a $1.3 million grant to study how stem cells can be used to treat liver disease caused by metabolic disorders. In a press release, Dr. Grikscheit details the importance and practicality of using stem cells to treat liver disease:

“Liver-based metabolic diseases are the perfect starting point to apply cellular therapy to liver disorders. The only current therapy — a liver transplant — is costly and in short supply. Plus, it requires suppressing the patient’s immune system, which has long-term consequences.”

The project, termed UPLiFT for Universal Pluripotent Stem Cell Therapy, aims to use pluripotent stem cells (cells that can turn into any cell in the body) to correct liver associated disorders like Crigler-Najjar Syndrome. A genetic mutation in liver cells of these patients makes them unable to covert bilirubin (a byproduct of red blood cell degradation) to its non-toxic form. Dr. Grikscheit hopes to bypass the need for a liver transplant by giving these patients pluripotent stem cells that can become liver cells without the genetic mutation, and are able to convert bilirubin to its non-toxic form. The use of pluripotent stem cells would also potentially eliminate the need for lifelong immunosuppressive therapy

Dr. Grikscheit will use the CIRM grant to test safety and efficacy of the stem cell treatment in pre-clinical trials to determine the optimal cell dosage that will be both safe and relieve disease symptoms, as well as assessing any off-target effects of the treatment. She has previously received a grant from CIRM to study stem cell therapy options for digestive neuromuscular condition, which you can read about here.

 

Mechanical forces are the key to speedy recovery after blood cancer treatment

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Mesenchymal stem cells grown on a surface with specialized mechanical properties. Image courtesy of Krystyn Van Vliet at MIT.

Blood cancers, such as leukemia and lymphoma, are projected to be responsible for 10% of all new cancer diagnoses this year. These types of cancers are often treated by killing the patient’s bone marrow (the site of blood cell manufacturing), with a treatment called irradiation. While effective for ridding the body of cancerous cells, this treatment also kills healthy blood cells. Therefore, for a time after the treatment, patients are particularly vulnerable to infections, because the cellular components of the immune system are down for the count.

Now scientists at MIT have devised a method to make blood cells regenerate faster and  minimize the window for opportunistic infections.

Using multipotent stem cells (stem cells that are able to become multiple cell types) grown on a new and specialized surface that mimics bone marrow, the investigators changed the stem cells into different types of blood cells. When transplanted into mice that had undergone irradiation, they found that the mice recovered much more quickly compared to mice given stem cells grown on a more traditional plastic surface that does not resemble bone marrow as well.

This finding, published in the journal Stem Cell Research and Therapy, is particularly revolutionary, because it is the first time researchers have observed that mechanical properties can affect how the cells differentiate and behave.

The lead author of the study attributes the decreased recovery time to the type of stem cell that was given to mice compared to what humans are normally given after irradiation. Humans are given a stem cell that is only able to become different types of blood cells. The mice in this study, however, were give a stem cell that can become many different types of cells such as muscle, bone and cartilage, suggesting that these cells somehow changed the bone marrow environment to promote a more efficient recovery. They attributed a large part of this phenomenon to a secreted protein call ostepontin, which has previously been describe in activating the cells of the immune system.

In a press release, Dr. Viola Vogel, a scientist not related to study, puts the significance of these findings in a larger context:

“Illustrating how mechanopriming of mesenchymal stem cells can be exploited to improve on hematopoietic recovery is of huge medical significance. It also sheds light onto how to utilize their approach to perhaps take advantage of other cell subpopulations for therapeutic applications in the future.”

Dr. Krystyn Van Vliet, explains the potential to expand these findings beyond the scope of just blood cancer treatment:

“You could imagine that by changing their culture environment, including their mechanical environment, MSCs could be used for administration to target several other diseases such as Parkinson’s disease, rheumatoid arthritis, and others.”

 

A 3-D model of heart tissue gives scientists a leg up in studying heart disease

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3-D printed cardiac microtissue generated from human stem cells. Image credit to Zhen Ma PhD, courtesy of the Gladstone Institutes

Hank Gathers was a 23-year-old basketball player at Loyola Marymount University with numerous accolades to his name and a promising future in the sport. This all came to an end when he suddenly collapsed in the middle of one of his games and subsequently passed away.  It was the beginning of numerous such sudden death instances in the sporting world, that have been traced to hypertrophic cardiomyopathy (HCM)

HCM affects approximately 1 in 200 people and belongs to a family of diseases called  cardiomyopathies, all of which impair the ability of heart muscle to effectively pump blood throughout your body. While there are several environmental factors that can contribute to the onset of HCM, one of the most common factors is a genetic mutation in a particular myosin binding protein called MYBPC3. This protein is responsible for both proper development of heart tissue, as well as controlling heart muscle contraction. Mutation in this gene is also associated with the most common type of cardiomyopathy, called dilated cardiomyopathy.

In HCM, mutation of MYBPC3 results in enlarged heart muscle with irregular contraction patterns. There is no cure, but medications to alleviate symptoms are available as well as surgical options. Surgery, however, can lead to serious complications and is not an option for everyone. Better understanding of the disease is necessary to develop treatment options that are effective for all patients.

A CIRM funded scientific collaboration between labs at UC Berkeley and the Gladstone Institutes has found a better way to study heart diseases such as HCM by generating a 3-D model heart tissue. While the genetic mutations that lead to cardiomyopathies are fairly well studied, how exactly the mutation leads to disease symptoms is not well understood, partially because many of those scientific studies relied on insights derived from two-dimensional culture systems. While informative in many ways, these types of cultures do not mimic the 3-D interactions that occur in nature. To overcome this challenge, these scientists used laser-guided 3-D printing system along with human cardiac stem cells to assemble the heart microtissue.

To confirm that the microtissue functioned like a real heart, they changed the structure of the printing scaffold to mimic the stress that hearts undergo during development and in different environmental conditions. They found that the microtissue derived from healthy human cardiac stem cells was able to adapt and contract normally in these changing environmental conditions.

When comparing the structure of WT and MYBPC3 generated microtissues, they surprisingly saw no different in microtissue architecture derived from WT or mutant cells. They did, however, observe functional differences between the normal and mutant tissues: mutant tissues displayed an increased contraction rate in response to stress and dysregulated contraction, both of which are hallmarks of HCM. Thus, this microtissue can mimic both normal and disease states.

Zhen Ma, lead author of the study explains the importance of this technology in a press release:

“With these microtissues we were able to observe how the human heart can develop this syndrome. Even though this is a microscopically tiny part of the heart, we could measure its contraction, the mechanical forces generated, and the calcium flow associated with the electrical signaling that triggers contraction of heart muscle. This advance gives us an opportunity to study cardiac disease in a much more precise manner.”

The most exciting aspect of this bioengineering success is its applicability beyond even heart disease. Bruce Conklin, one of the lead authors of the study explains:

“Some of the worst drug safety issues are due to problems with side effects on the heart, so we need better ways to test drugs for potential cardiac effects. It’s possible that in the future microtissues might become the preferred choice for their capability to capture a fuller range of cardiac physiology.

A cancer therapy developed at a CIRM Alpha Stem Cell Clinic tests its legs against breast cancer

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Three-dimensional culture of human breast cancer cells, with DNA stained blue and a protein on the cell surface membrane stained green. Image courtesy The National Institutes of Health

A Phase 1 clinical trial co-sponsored by CIRM and Oncternal Therapeutics, has started treating patients at UC San Diego (UCSD). The goal of the trial is to test the safety and anti-tumor activity of the Oncternal-developed drug, cirmtuzumab, in treating breast cancer.

Breast cancer is the second most common cancer to occur in women, regardless of race or ethnicity. More than 260,000 new cases are expected to be diagnosed this year in the United States alone. Typically, breast cancer cases are treated by a combination of surgery to remove the tumor locally, followed by some kind of systemic treatment, like chemotherapy, which can eliminate cancer cells in other parts of the body. In certain cases, however, surgery might not be a feasible option. Cirmtuzumab may be a viable option for these patients.

The drug acts by binding to a protein called ROR1, which is highly abundant on the surface of cancer cells. By blocking the protein Cirmtuzumab is able to promote cell death, stopping the cancer from spreading around the body.

Because ROR1 is also found on the surface of healthy cells there were concerns using cirmtuzumab could lead to damage to healthy tissue. However, a previous study revealed that using this kind of approach, at least in a healthy non-human primate model did not lead to any adverse clinical symptoms. Therefore, this protein is a viable target for cancer treatment and is particularly promising because it is a marker of many different types of cancers including leukemia, lung cancer and breast cancer.

Phase 1 clinical trials generally enroll a small number of patients who have do not have other treatment options. The primary goals are to determine if this approach is safe, if it causes any serious side-effects, what is the best dosage of the drug and how the drug works in the body. This clinical trial will enroll up to 15 patients who will receive cirmtuzumab in combination with paclitaxel (Taxol), a vetted chemotherapy drug, for six months.

Earlier this year, a similar clinical trial at UCSD began to test the effectiveness a of cirmtuzumab-based combination therapy to treat patients with B-cell cancers such as chronic lymphocytic leukemia. This trial was also partially funded by CIRM.

In a press release, Dr. Barbara Parker, the co-lead on this study states:

“Our primary objective, of course, is to determine whether the drug combination is safe and tolerable and to measure its anti-tumor activity. If it proves safe and shows effectiveness against breast cancer, we can progress to subsequent trials to determine how best to use the drug combination.”

Support cells have different roles in blood stem cell maintenance before and after stress

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Expression of pleiotrophin (green) in bone marrow blood vessels (red) and stromal cells (white) in normal mice (left), and in mice 24 hours after irradiation (right). UCLA Broad Stem Cell Research Center/Cell Stem Cell

A new study published in the journal Cell Stem Cell, reveals how different types of cells in the bone marrow are responsible for supporting blood stem cell maintenance before and after injury.

It was already well known in the field that two different cell types, namely endothelial cells (which line blood vessels) and stromal cells (which make up connective tissue, or tissue that provides structural support for any organ), are responsible for maintaining the population of blood stem cells in the bone marrow. However, how these cells and the molecules they secrete impact blood stem cell development and maintenance is not well understood.

Hematopoietic stem cells are responsible for generating the multiple different types of cells found in blood, from our oxygen carrying red blood cells to the many different types of white blood cells that make up our immune system.

Dr. John Chute’s group at UCLA had previously discovered that a molecule called pleiotrophin, or PTN, is important for promoting self-renewal of the blood stem cell population. They did not, however, understand which cells secrete this molecule and when.

To answer this question, the scientists developed mouse models that did not produce PTN in different types of bone marrow cells, such as endothelial cells and stromal cells. Surprisingly, they saw that the inability of stromal cells to produce PTN decreased the blood stem cell population, but deletion of PTN in endothelial cells did not affect the blood stem cell niche.

Even more interestingly, the researchers found that in animals that were subjected to an environmental stressor, in this case, radiation, the result was reversed: endothelial cell PTN was necessary for blood stem cell renewal, whereas stromal cell PTN was not. While an important part of the knowledge base for blood stem cell biology, the reason for this switch in PTN secretion at times of homeostasis and disease is still unknown.

As Dr. Chute states in a press release, this result could have important implications for cancer treatments such as radiation:

“It may be possible to administer modified, recombinant versions of pleiotrophin to patients to accelerate blood cell regeneration. This strategy also may apply to patients undergoing bone marrow transplants.”

Another important consideration to take away from this work is that animal models developed in the laboratory should take into account the possibility that blood stem cell maintenance and regeneration is distinctly controlled under healthy and disease state. In other words, cellular function in one state is not always indicative of its role in another state.

This work was partially funded by a CIRM Leadership Award.

 

 

For the first time, scientists entirely reprogram human skin cells to iPSCs using CRISPR

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CRISPR iPSC colony of human skin cells showing expression of SOX2 and TRA-1-60, markers of human embryonic pluripotent stem cells

Back in 2012, Shinya Yamanaka was awarded the Nobel Prize in Physiology or Medicine for his group’s identification of “Yamanaka Factors,” a group of genes that are capable of turning ordinary skin cells into induced pluripotentent stem cells (iPSCs) which have the ability to become any type of cell within the body. Discovery of iPSCs was, and has been, groundbreaking because it not only allows for unprecedented avenues to study human disease, but also has implications for using a patient’s own cells to treat a wide variety of diseases.

Recently, Timo Otonkoski’s group at the University of Helsinki along with Juha Kere’s group at the Karolinska Institutet and King’s College, London have found a way to program iPSCs from skin cells using CRISPR, a gene editing technology. Their approach allows for the induction, or turning on of iPSCs using the cells own DNA, instead of introducing the previously identified Yamanka Factors into cells of interest.

As detailed in their study, published in the journal Nature Communications, this is the first instance of mature human cells being completely reprogrammed into pluripotent cells using only CRISPR. Instead of using the canonical CRISPR system that allows the CAS9 protein (an enzyme that is able to cut DNA, thus rendering a gene of interest dysfunctional) to mutate any gene of interest, this group used a modified version of the CAS9 protein, which allows them to turn on or off the gene that CAS9 is targeted to.

The robustness of their approach lies in the researcher’s identification of a DNA sequence that is commonly found near genes involved in embryonic development. As CAS9 needs to be guided to genes of interest to do its job, identification of this common motif allows multiple genes associated with pluripotency to be activated in mature human skin cells, and greatly increased the efficiency and effectiveness of this approach.

In a press release, Dr. Otonkoski further highlights the novelty and viability of this approach:

“…Reprogramming based on activation of endogenous genes rather than overexpression of transgenes is…theoretically a more physiological way of controlling cell fate and may result in more normal cells…”

 

“Junk” DNA is development gold for the dividing embryo

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Single two-cell mouse embryos with nuclear LINE1 RNA labeled magenta – Credit Ramalho-Santos lab

The DNA in our cells provide the instructions to make proteins, the workhorses of our body. Yet less than 2% of the 3 billion base pairs (the structural units of DNA) in each of our cells are actually involved in protein production. The rest, termed non-coding DNA for not being involved in protein production, has roles in regulating genetic activity, but, largely, these genetic regions have remained a mystery causing some to mis-characterize it as “junk” DNA.

One of the largest components of these “junk” DNA regions are transposons, which make up 50% of the genome. Transposons are variable length DNA segments that are able to duplicate and re-insert themselves into different locations of the genome which is why they’re often called “jumping genes”.

Transposons have been implicated in diseases like cancer because of their ability to disrupt normal gene function depending on where the transposon inserts itself. Now, a CIRM-funded study in Miguel Ramalho-Santos’ laboratory at UCSF has found a developmental function for transposons in the dividing embryo. The report was published today in the Journal Cell.

Of the transposons identified in humans, LINE1 is the most common, composing 24% of the entire human genome. Many investigators in the field had observed that LINE1 is highly expressed in embryonic stem cells, which seemed paradoxical given that these pieces of DNA were previously thought to be either inert or harmful. Because this DNA was present at such high levels, the investigators decided to eliminate it from fertilized mouse embryos at the two-cell stage and observe how this affected development.

To their surprise, they found that the embryo was not able to progress beyond this stage. Further investigation revealed that LINE1, along with other proteins, is responsible for turning off the genetic program that maintains the two-cell state, thus allowing the embryo to further divide and develop.

Dr. Ramalho-Santos believes that this is a fine-tuned mechanism to ensure that the early stages of develop progress successfully. Because there are so many copies of LINE1 in the genome, even if one is not functional, it is likely that there will be functional back up, an important factor in ensuring early mistakes in embryo development do not occur.

In a press release, Dr. Ramalho-Santos states:

“We now think these early embryos are playing with fire but in a very calculated way. This could be a very robust mechanism for regulating development…I’m personally excited to continue exploring novel functions of these elements in development and disease.”

Timing is a critical factor in kidney development

Through countless studies, it’s clear that genes and environmental factors are important for determining cellular identity. Now, a research team at the University of Southern California  (USC) have found that timing is another critical factor in determining cell fate during organ development.

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Developing human nephron, the filtering unit of the kidney. Image by Nils O. Lindstrom and Tracy Tran/McMahon Lab USC Stem Cell

In findings published in Development Cell, Dr. Andy McMahon’s group shows that development of the nephron, the filtering structure of the kidney, is acutely dependent on when cells arrive in that developmental region. Cells that arrive in the developing nephron early become part of the tubule, which is responsible for reabsorption of water and salt, whereas cells that arrive late become part of the glomerulus, the structure that is responsible for filtering blood.

The scientists verified that timing influenced cell identity with a combination of microscopy, which allowed them to follow particular cell types as they developed, and single-cell RNA sequencing, which allowed them to determine how gene expression changes in a population of cells.

In a press release, Dr. McMahon details the importance of these findings:

“By studying normal human nephron development, we’re gaining important information about how to replicate this intricate process in the laboratory. The hope is that laboratory-grown nephrons can be used to further study the process of development, screen potential therapies to treat disease, and eventually provide the building blocks to assemble functional kidneys for transplantation into patients.”

Understanding kidney development is crucial because approximately 30 million people suffer from chronic kidney disease and it is the ninth leading cause of death in the United States alone. Insights into the basics of kidney biology can provide important advances to develop novel therapeutics for this devastating condition.

CIRM funded study results in the first ever in utero stem cell transplant to treat alpha thalassemia

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Dr. Tippi MacKenzie (left) of UCSF Benioff Children’s Hospital San Francisco, visits with newborn Elianna and parents Nichelle Obar and Chris Constantino. Photo by Noah Berger

Imagine being able to cure a genetic disorder before a baby is even born. Thanks to a CIRM funded study, what would have been a mere dream a couple of years ago has become a reality.

Drs. Tippi MacKenzie and Juan Gonzalez Velez of the University of California San Francisco (UCSF) have successfully treated alpha thalassemia in Elianna Constantino, using stem cells from her mother’s bone marrow. Alpha thalassemia is part of a group of blood disorders that impairs the body’s ability to produce hemoglobin, the molecule that is responsible for transporting oxygen throughout the body on red blood cells. Present in approximately 5% of the population, alpha thalassemia is particularly prevalent among individuals of Asian heritage. Treatment options for this disease are severely limited, generally requiring multiple rounds of blood transfusions or a bone marrow transplant which requires immunosuppressive therapy. Normally, fetuses die in the womb or the pregnancy is aborted because of the poor prognosis.

The revolutionary treatment pioneered at UCSF involved isolating blood stem cells (cells that are capable of turning into all blood cell types) from the mother’s bone marrow and injecting these cells into Elianna’s bloodstream via the umbilical vein. The doctors were able to observe the development of healthy blood cells in the baby’s blood stream, allowing for efficient oxygen transport throughout the baby’s body. Because the cells were transplanted at the fetal stage, a time when the immune system is not fully developed, there was low risk of rejection and the transplant occurred without aggressive immunosuppressive therapy.

The baby was born healthy earlier this year and has been allowed to return home. While it is still too early to tell how effective this treatment will be in the long term, it is very encouraging that both the mother and baby have endured the treatment thus far.

In a press release, Dr. MacKenzie states:

“Her healthy birth suggests that fetal therapy is a viable option to offer to families with this diagnosis.”

The in utero stem cell transplant was performed as part of a clinical trial conducted at the UCSF Benioff Children’s Hospitals in San Francisco and Oakland. The trial is currently enrolling 10 pregnant women to test the safety and effectiveness of this treatment over a wider population.

If successful, this type of treatment is particularly exciting because it could be expanded to other types of hereditary blood disorders such as sickle cell anemia and hemophilia.

 

 

 

Making stem cell-derived liver cells to study fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) affects approximately 30% of the population, with that number increasing to 75% in obese individuals. Shockingly, the number of cases is expected to increase 21% by the year 2030 in the United States alone.

liver_fattyliverNAFLD refers to a broad range of liver conditions, which are all characterized by abnormally high levels of fat deposits in the livers of people who do not drink excessive amounts of alcohol. While not always fatal, NAFLD can lead to liver cirrhosis, or extensive scaring of the liver tissue. Cirrhosis, in turn, can cause life-threatening conditions such as liver cancer or liver failure. Whether or not N

AFLD will lead to extensive liver damage is not well understood and the primary therapeutic option is weight loss with no FDA-approved drug options. The projected increase in NALD cases combined with the poor treatment options makes this disease a significant public health burden.

Studying NALD can be quite complicated because the liver is complex organ made up of multiple different cell types. Investigators at the University of Edinburgh have simplified some of this complexity by figuring out a way to generate liver cells in a dish.

In studies published in the Philosophical Transactions of the Royal Society B, these scientists used human embryonic stem cells to generate hepatocyte-like cells (HLCs), or cells that are highly similar to liver cells isolated from humans. When exposed to fatty acids, they saw that the HLCs exhibited hallmarks of NAFLD, such as fat accumulation in liver cells, and changes in gene expression that are indicative of NAFLD.

In a press release, Dr. David Hay, one of the two senior investigators of this study, states:

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Dr. David Hay

“Our ability to generate human hepatocytes from stem cells, using semi-automated procedures, allows us to study the mechanisms of human liver disease in a dish and at scale.”

 

This approach is particularly valuable because it would replace the need to use cancer cell lines for this type of work. While valuable for many reasons, research done in cancer cells lines can be difficult to draw therapeutic conclusions from, because cell lines have significant genetic alternations from normal cells. Generating liver cells from human stem cells provides an important tool for high throughput screening of medically relevant therapies for NALD.