Nusse Laboratory at Sanford Identifies Elusive Mouse Liver Stem Cell

Stanford University life science researchers have managed to successfully identify a stem cell population that has eluded many laboratories for some time. Essentially, the Stanford team has discovered a previously unknown population of liver cells in mice that function as liver stem cells. Such a find could aid drug testing and increase our present understanding of liver biology and disease.

Researchers in the laboratory of Roel Nusse at the Stanford University School of Medicine have identified a cell type in the liver of laboratory mice that can both self-renew and make new liver cells. This discovery by Nusse and others settles a long-standing conundrum of how the liver maintains itself when liver cells grow old and die.

“There’s always been a question as to how the liver replaces dying hepatocytes,” said professor of developmental biology Roel Nusse, PhD. “Most other tissues have a dedicated population of cells that can divide to make a copy of themselves, which we call self-renewal, and can also give rise to the more-specialized cells that make up that tissue. But there never was any evidence for a stem cell in the liver.”

It was assumed for some time that mature hepatocytes (liver cells) would themselves divide to replace a dying neighbor. However, hepatocytes have an abnormal amount of DNA, which would make cell division extremely difficult.

Nusse and his team published this research in the Aug. 5 edition of the journal Nature. The first author of this paper, Bruce Wang, MD, an assistant professor of gastroenterology and hepatology at the University of California-San Francisco, led the research while serving as a visiting scholar in Nusse’s lab.

The liver is a large, multi-lobed organ that filters toxins from the blood, synthesizes blood proteins, and makes digestive enzymes and bile. It is involved in many important metabolic processes. The liver contains a central vein that carries blood through it. The stem cells identified by Wang and Nusse are found adjacent to this vein.

Classically, hepatocytes were largely thought to be all alike. Most mature hepatocytes are “polyploid,” which means that they have more than the normal two copies of each chromosome. With all this extra DNA, it makes it difficult or even impossible, for these cells to divide normally, but this extra DNA might confer other benefits.

“If it’s not necessary for a cell to maintain the capacity to divide, it can do whatever it wants with its genome,” said Nusse. “Red blood cells, for instance, have no DNA. Muscle cells have many copies of each chromosome.” Having extra copies of chromosomes might allow these cells to make large amounts of particular proteins quickly, for example.

However, the cell population identified by Wang and Nusse in the livers of laboratory mice is diploid, and have only two copies of each chromosome. These cells can divide to make carbon copies of themselves, or to make cells that begin their lives as diploid but then acquire additional copies of their chromosomes as they move away from the central vein into the main body of the liver.

“People in the field have always thought of hepatocytes as a single cell type,” said Wang. “And yet the cell we identified is clearly different from others in the liver. Maybe we should accept that there may be several subtypes of hepatocytes, potentially with different functions.”

To identify liver-specific stem cells, Wang and Nusse identified cells that express the protein Axin2. They found these axin2-expressing cells surrounding the central vein. Axin2 is produced by cells in response to the presence of members of the Wnt signaling protein family, and decades of research by Nusse and other laboratories have shown that the Wnt proteins play a critical role in embryonic development, and in the growth and maintenance of stem cells throughout the body.

Interestingly, Wang and Nusse, and others showed that the endothelial cells that line the interior surface of the central vein in the liver produce Wnt2 and Wnt9b. These secreted Wnt proteins confer stem cell properties on the neighboring hepatocytes that surround the central vein.

Finally, Nusse’s team discovered that a portion of the descendants of the Axin2-expressing cells move outward from the central vein over time, become polyploid and begin to express other, hepatocyte-specific genes. About one year after being born, these descendant cells had effectively replaced about 30 percent of the entire mouse liver, and made up about 40 percent of all hepatocytes in the liver.

These newly identified liver stem cells also express genes associated with very early embryonic development, which may give a clue as to when and where they arise.

“Perhaps these stem cells in the adult liver actually arise very early in development,” said Nusse, “when the embryo sets aside a certain population of cells to maintain the organ during adult life.”

Although the current research was conducted in mice, it is possible that there are more than just one kind of hepatocyte in humans as well, and this realization could transform the study of liver biology. For example, hepatocytes have proven notoriously difficult to grow in laboratory culture for study or for use in drug testing.

“The most common reason that promising new drugs for any type of condition fail is that they are found to be toxic to liver,” said Wang. “Researchers have been trying for decades to find a way to maintain hepatocytes in the laboratory on which to test the effects of potential medications before trying them in humans. Perhaps we haven’t been culturing the right subtype. These stem cells might be more likely to fare well in culture.”

There’s also an opportunity to better understand human disease.

“Does liver cancer arise from a specific subtype of cells?” said Wang. “This model also gives us a way to understand how chromosome number is controlled. Does the presence of the Wnt proteins keep the stem cells in a diploid state? These are fundamental biological questions we can now begin to address.”

Liver-Based Stem Cells Regenerate Animal Livers

Biologists from the MRC Center for Regenerative Medicine at the University of Edinburgh have managed to restore liver function in mice by using stem cell transplants to regenerate them. This is the first time such a procedure has succeeded in a living animal.

If liver stem cells from human livers behave the same way as did the mouse cells in this study, then this procedure could potentially be used in place of liver transplants in human patients. This work was published by Professor Stuart Forbes and his colleagues in the journal Nature Cell Biology.

According to Forbes: “Revealing the therapeutic potential of these liver stem cells brings us a step closer to developing stem cell based treatments for patients with liver disease. It will be some time before we can turn this into reality as we will first need to test our approach using human cells. This is much needed as liver disease is a very common cause of death and disability for patients in the UK and the rest of the world.”

Liver cells are also called “hepatocytes” and even though such cells are used for liver transplants, the technology does not yet exist to easily propagate human hepatocytes in the laboratory. In this study, Forbes and his group designed a protocol that could wipe out close to 98% of the cells in the liver of laboratory mice. They genetically engineered mice whose liver cells would delete the MDM2 gene. The MDM2 gene encodes a protein called “E3 ubiquitin ligase,” which is an enzyme that tags junk proteins so that they are properly degrades and recycled. Without a functional E3 ubiquitin ligase, the vast majority of the liver cells underwent programmed cell death. Under these conditions, a group of liver-specific stem cells called hepatic progenitor cells or HPCs were transplanted from healthy mice into the adult mice with severely damaged livers. The transplanted HPCs significantly restored the structure of the liver, regenerating hepatocytes and the cells of the “biliary epithelia,” which compose the ducts that move bile into the gall bladder. This highlights the potency of these transplanted HPCs as liver regenerators. Essentially, after several months, Forbes and his coworkers discovered that major areas of the liver had regrown and these new cells significantly improved the liver’s physiological performance.

Transplanted hepatic progenitor cells can self-renew (yellow, left image) and differentiate into hepatocytes (green) to repair the damaged liver. Image credit: Dr Wei-Yu Lu.
Transplanted hepatic progenitor cells can self-renew (yellow, left image) and differentiate into hepatocytes (green) to repair the damaged liver. Image credit: Dr Wei-Yu Lu.

This is the first time that biologists have succeeded in regenerating an organ in a living animal by using stem cells. Even human cells have significant differences from mouse cells, if these human cells can be manipulated so that they behave in a similar manner to these mouse stem cells, transplanting stem cells or, perhaps administering drugs that activate a patient’s own liver to produce stem cells and regenerate itself, could replace liver transplants.

In a press release, Dr. Rob Buckle, director of science programs for the U.K.’s Medical Research Council, said: “This research has the potential to revolutionize patient care by finding ways of co-opting the body’s own resources to repair or replace damaged or diseased tissue. Work like this, building upon a precise understanding of the underlying human biology and supported by the UK Regenerative Medicine Platform, will give doctors powerful new tools to treat a range of diseases that have no cure, like liver failure, blindness, Parkinson’s disease and arthritis.”

Putting Peps in Your Heps

The liver is a special organ that performs a whole host of essential functions. The liver stores iron, vitamins and minerals; it detoxifies alcohol, drugs, and other chemicals that accumulate in our bloodstreams, and it produces bile (used to dissolve fats so that they can be degraded), and blood-based proteins like clotting factors and albumin. The liver also stores sugar in the form of glycogen. All of these tasks are undertaken by a single cell type, the hepatocyte (otherwise known as a liver cell).


When your liver fails, you get really sick. This was greatly illustrated to me by one of my colleagues where I teach whose wife suffered extensive liver damage as a result of her battle with lupus (short for systemic lupus erythematosus, an autoimmune disease). Now that this dear lady has had a liver transplant, she is a new person. What a difference a healthy liver makes.

What can regenerative medicine do for patients with failing livers? Human pluripotent stem cells, either embryonic stem cells or induced pluripotent stem cells, can be directed to differentiate into liver cells in culture, but the liver cells made by these cells are very immature. They express proteins commonly found in fetal liver cells (for example, alpha-fetoprotein) and they also lack key enzymes associated with adult cells (such as cytochrome P450s). Rashid and others in the Journal of Clinical Investigation (2010; 120: 3127-3136) showed this. The development of three-dimensional culture systems have increased the maturity of such cells, but there is still a long way to go (see T Takebe and others, Nature 2013; 499:481-484 and J Shan and others, Nature Chemical Biology 2013; 9: 514-520).

Two papers from the journal Cell Stem Cell might show a way forward to making mature liver cells for regenerative liver treatments without destroying embryos or even using and pluripotent stem cell lines. These papers utilize the procedure known as “direct reprogramming,” otherwise known as “direct lineage conversion.” Direct reprogramming requires the forced overexpression of particular genes that causes the cells to switch their cell types.

In the first of these papers, Pengyu Huang and his colleagues from the Chinese Academy of Sciences in Shanghai, China overexpressed a three-gene combination in mouse embryonic fibroblasts that converted the cells into hepatocytes at an efficiency of 20% after 14 days in culture. This gene combination, known as 3TF (HNF4/HNF1A/FOXA3), converted the mouse embryonic skin cells into mature liver cells that made blood proteins and drug-processing enzymes. The only problem was that these mature cells could not grow in culture because they were mature. Therefore, Huang and others infected these cells with a virus called SV40, which drove the cells to divide. Now these cells could be grow in culture and expanded for further experiments.

When transplanted into the livers of mice with failing livers, the induced liver cells made by Huang and others restored proper liver function and allowed the mice to survive.

A second paper by Yuanyuan Du and others from the Peking-Tsinghua Center for Life Sciences at Peking University in Beijing, China, used a large gene combination to make mature liver cells from human skin fibroblasts. This gene combination included eight genes (HNF1A/HNF4A/HNF6/ATF5/PROX1/CEBPA/p53 ShRNA/C-MYC) that converted the human skin cells into liver cells after 30 days in culture at an efficiency of nearly 80%. Again, these cells metabolized drugs as they should, made blood proteins, took up cholesterol, and stored glycogen. Du and others compared the gene expression profile of these human induced hepatocytes or “hiHeps” to the gene expression profile of liver cells taken from liver biopsies. While there were differences in gene expression, there was also significant overlap and a large overall similarity. In fact the authors state, “these results indicate that hiHeps show a similar expression profile to primary human hepatocytes.”

Next, Du and others used three different mouse models of liver failure in all three cases, the hiHeps were capable of colonizing the damaged liver of the mouse and regenerating it. Mind you, the hiHeps did not do as good a job as human primary hepatocytes, but they still worked pretty well. This shows that this direct reprogramming protocol, as good as it is, can still be optimized and improved.

These studies show that the production of highly functional human hepatocyte-like cells using direct reprogramming is feasible and represents an exciting step towards the production of a supply source of cells for drug development, and therapies for liver disease.

Repopulation of Damaged Livers With Skin-Derived Stem Cells

Patients with severe liver disease must receive a liver transplant. This major procedure requires that the patient survives major surgery and then takes anti-rejection drugs for the rest of their lives. In general, liver transplant patients tend to fair pretty well. The one-year survival rate of liver transplant patients approaches 90% (see O’Mahony and Goss, Texas Heart Institute Journal 2012 39(6): 874-875).

A potentially better way to treat liver failure patients would be to take their own liver cells, convert them into induced pluripotent stem cells (iPSCs), differentiate them into liver cells, and use these liver cells to regenerate the patient’s liver. Such a treatment would contain a patient’s own liver cells and would not require anti-rejection drugs.

Induced pluripotent stem cells or iPSCs are made from genetically-engineered adult cells that have had four specific genes (Oct4, Klf4, Sox2, and c-Myc) introduced into them. As a result of the heightened expression of these genes, some of the adult cells dedifferentiate and are reprogrammed into cells that resemble embryonic stem cells. Normally, this procedure is relatively inefficient, slow, and induces new mutations into the engineered cells. Also, when iPSCs are differentiated into liver cells (hepatocytes), they do not adequately proliferate after differentiation, and they also fail to properly function the way adult hepatocytes do.

New work from laboratories at the University of California, San Francisco (UCSF), has differentiated human hepatocytes by means of a modified technique that bypasses the pluripotency stage. These cells were then used to repopulate mouse livers.

“I really like this paper. It’s a step forward in the field,” said Alejandro Soto-Gutiérrez, assistant professor of pathology at the University of Pittsburgh, who was not involved in the work. “The concept is reprogramming, but with a shortcut, which is really cool.”

Research teams led by Holger Willenbring and Sheng Ding isolated human skin cells called fibroblasts and infected them with engineered viruses that forced the expression of three genes: OCT4, SOX2, and KLF4. These transduced cells were grown in culture in the presence of proteins called growth factors and small molecules in order to induce reprogramming of the cells into the primary embryonic germ layer known as endoderm. In the embryo, the endoderm is the inner-most layer of cells that forms the gastrointestinal tract and its associated structures (liver, pancreas, and so on). Therefore, the differentiation of adult cells into endodermal progenitor cells provides a handy way to form a cell type that readily divides and can differentiate into liver cells.

“We divert the cells on their path to pluripotency,” explained coauthor Holger Willenbring, associate professor of surgery at UCSF. “We still take advantage of what is intrinsic to reprogramming, that the cells are becoming very plastic; they’ve become flexible in what kind of cell type they can be directed towards.”

The authors called these cells induced multipotent progenitor cells (iMPCs). The iMPCs were easily differentiated into endodermal progenitor cells (iMPC-EPCs). These iMPC-EPCs were grown in culture with a cocktail of small molecules and growth factors to increase iMPC-EPC colony size while concomitantly maintain them in an endodermal state. Afterwards, Willenbring and others cultured these cells with factors and small molecules known to promote liver cell differentiation. When these iMPC-Hepatocytes (Heps) were transplanted into mice with damaged livers, the iMPC-Hep cells continued to divide at least nine months after transplantation. Furthermore, the transplanted cells matured and displayed gene expression profiles very similar to that of typical adult hepatocytes. Transplantation of iMPC-Heps also improved the survival of a mouse model of chronic liver failure about as well as did transplantation of adult hepatocytes.

“It is a breakthrough for us because it’s the first time that we’ve seen a cell that can actually repopulate a mouse’s liver,” said Willenbring. Willenbring strongly suspects that iMPCs are better able to repopulate the liver because the derivation of iMPC—rather than an iPSC—eliminates some steps along the path to generating hepatocytes. These iMPCs also possess the ability to proliferate in culture to generate sufficient quantities of cells for therapeutic purposes and, additionally, can functionally mature while retaining that proliferative ability to proliferate. Both of these features are important prerequisites for therapeutic applications, according to Willenbring.

Before this technique can enter clinical trials, more work must be done. For example: “The key to all of this is trying to generate cells that are identical to adult liver cells,” said Stephen Duncan, a professor of cell biology at Medical College of Wisconsin, who was not involved in the study. “You really need these cells to take on all of the functions of a normal liver cell.” Duncan explained that liver cells taken directly from a human adult might be able to repopulate the liver in this same mouse model at levels close to 90 percent.

Willenbring and his colleagues observed repopulation levels of 2 percent by iMPC-Heps, which is substantially better than the 0.05 percent repopulation typically accomplished by hepatocytes derived from iPSCs or embryonic stem cells. However: “As good as this is, the field will need greater levels of expansion,” said Ken Zaret of the Institute for Regenerative Medicine at the University of Pennsylvania, who did not participate in the work. “But the question is: What is limiting the proliferative capacity of the cells?”

Zaret explained that it is not yet clear whether some aspect of how the cells were programmed that differed from how they normally develop could have an impact on how well the population expands after transplantation. “There still is a ways to go [sic],” he said, “but [the authors] were able to show much better long-term repopulation with human cells in the mouse model than other groups have.”

See S. Zhu et al., “Mouse liver repopulation with hepatocytes generated from human fibroblasts,” Nature, doi:10.1038/nature13020, 2014.

Transplanted Liver Cells do Better When Co-Cultured with Mesenchymal Stem Cells

Implanting frozen liver cells is a relatively new procedure that has, reportedly, been used to treat very young patients with liver problems. Thawing frozen liver cells, however, tends to cause a fraction of the cells to die off and other damaged cells show poor function.

To ameliorate this problem, researchers at Kings College Hospital, London have used mesenchymal stem cells from fat or umbilical cord to improve the viability and function of frozen liver cells.

Emer Fitzpatrick and her colleagues at Kings College Hospital reasoned that mesenchymal stem cells and the multitudes of healing molecules that these cells secrete should be able to “lend proregenerative characteristics to liver cells.”

Thus by co-culturing thawed liver cells with mesenchymal stem cells from fat or umbilical cord, Fitzpatrick and others demonstrated that the rate of cell survival of the liver cells and their functionality increased in comparison with liver cells grown on their own.

Fitzpatrick hopes that such a co-culture technique might improve the clinical usefulness of frozen liver cells for transplantation.

An Efficient Method for Converting Fat Cells to Liver Cells

I have a friend whose wife has systemic lupus erythematosis, and her liver has taken a beating as a result of this disease. She has never had a drop of alcohol for decades and yet she has a liver that looks like the liver of a 70-year-old alcoholic. The scarring of the liver as result of repeated damage and healing has seriously compromised her liver function. She is now a candidate for a liver transplant. Wouldn’t it be nice to simply give her liver cells to heal her liver?

This dream came a little closer to becoming reality in October of this year when scientists at Stanford University developed a fast and efficient way to convert fat cells isolated from routine liposuction into liver cells. Even though these experiments used mice, the stem cells were isolated from human liposuction procedures.

This experiment did not use embryonic stem cells or induced pluripotent stem cells to generate liver cells. Instead it used adult stem cells from fat.

Fat-based stem cells

The liver builds complex molecules, filters and breaks down waste products and toxic substances that might otherwise accumulate to dangerous concentrations.

The liver, unlike other organs, has a capacity to regenerate itself to a significant extent, but the liver’s regenerative abilities cannot overcome the consequences of acute liver poisoning, or chronic damage to the liver, as a result of hepatitis, alcoholism, or drug abuse.

For example, acetaminophen (Tylenol) is a popular pain-reliever, but abusing acetaminophen can badly damage the liver. About 500 people die each year from abuse of acetaminophen, and some 60,000 emergency-room visits and more than 25,000 hospitalizations annually are due to acetaminophen abuse. Other environmental toxins, such as poisonous mushrooms, contribute more cases of liver damage.

Fortunately, the fat-to-liver protocol is readily adaptable to human patients, according to Gary Peltz, professor of anesthesia and senior author of this study. The procedure takes about nine days, which is easily fast enough to treat someone suffering from acute liver poisoning, who might die within a few weeks without a liver transplant.

Some 6,300 liver transplants are performed annually in he United States, and approximately 16,000 patients are on the waiting list for a liver. Every year more than 1,400 people die before a suitable liver can be found for them.

Even though liver transplantations save the lives of patients, the procedure is complicated, not without risks, and even when successful, is fraught with after effects. The largest problem is the immunosuppressant drugs that live patients must take in order to prevent their immune system from rejecting the transplanted liver. Acute rejection is an ongoing risk in any solid organ transplant, and improvements in immunosuppressive therapy have reduced rejection rates and improved graft survival. However, acute rejection still develops in 25% to 50% of liver transplant patients treated with immunosuppressants. Chronic rejection is somewhat less frequent and is declining and occurs in approximately 4% of adult liver transplant patients.

Peltz said, “We believe our method will be transferable to the clinic, and because the new liver tissue is derived from a person’s own cells, we do not expect that immunosuppressants will be needed.”

Peltz also noted that fat-based stem cells do not normally differentiate into liver cells. However, in 2006, a Japanese laboratory developed a technique for converting fat-based stem cells into induced liver cells (called “i-Heps” for short). This method, however, is inefficient, takes 30 days, and relies on chemical stimulation. In short, this technique would not provide enough material to regenerate a liver.

The Stanford University group built upon the Japanese work and improved it. Peltz’s group used a spherical culture and were able to convert fat-bases stem cells into i-Heps in nine days and with 37% efficiency (the Japanese group only saw a 12% rate). Since the publication of their paper, Peltz said that workers in his laboratory have increased the efficiency to 50%.

Dan Xu, a postdoctoral scholar and the lead author of this study, adapted the spherical culture methodology from early embryonic-stem-cell literature. However, instead of growing on flat surfaces in a laboratory dish, the harvested fat cells are cultured in a liquid suspension in which they form spheroids. Peltz noted that the cells were much happier when they were grown in small spheres.

Once they had enough cells, Peltz and his co-workers injected them into immune-deficient laboratory mice that accept human grafts. These mice were bioengineered in 2007 as a result of a collaboration between Peltz and Toshihiko Nishimura from the Tokyo-based Central Institute for Experimental Animals. These mice had a viral thymidine kinase gene inserted into their genomes and when treated with the drug gancyclovir, the mice experienced extensive liver damage.

After gancyclovir treatment, Peltz and his coworkers injected 5 million i-Heps into the livers of these mice, using ultrasound-guided injection procedures, which is typically used for biopsies.

Four weeks later, the mice expressed human blood proteins and 10-20 percent of the mouse livers were repopulated with human liver cells. Blood tests also showed that the mouse livers, which were greatly damaged previous to the transplantation, were processing nitrogenous wastes properly. Structurally, the mouse livers contained human cells that made human bile ducts, and expressed mature human liver cells.

Other tests established that the i-Heps made from fat-based stem cells were more liver-like than i-Heps made from induced pluripotent stem cells.

Two months are injection of the i-Heps, there was no evidence of tumor formation.

Peltz said, “To be successful, we must regenerate about half of the damaged liver’s original cell count.” With the spherical culture, Peltz is able to produce close to one billion injectable i-Heps from 1 liter of liposuction aspirate. The cell replication that occurs after injection expands that number further to over 100 billion i-Heps.

If this is possible, then this procedure could potentially replace liver transplants. Stanford University’s Office of Technology Licensing has filed a patent on the use of spherical culture for hepatocyte (liver cell) induction. Peltz’s group is optimizing this culture and injection techniques,talking to the US Food and Drug Administration, and gearing up for safety tests on large animals. Barring setbacks, the new method could be ready for clinical trials within two to three years, according the estimations by Peltz.

Artificial Liver Replaces Liver Function in Mice

Once again, I apologize to my readers for the week-long hiatus, but I was at Roberts Wesleyan College for the 60th Free Methodist National Bible Quiz. My teams had some successes and failure, but I am exceedingly proud of them.

Now on to our news for the day.

Takanori Takebe, a stem-cell biologist at Yokohama City University in Japan, and his colleagues have transplanted small liver buds constructed from human stem cells into mice that restored liver function in mice. Even though this is a preliminary study, these results offer a potential path towards developing treatments for the thousands of patients who are awaiting liver transplants every year.

Takebe and others reported their data in the journal Nature recently. The liver buds constructed by Takebe and co-workers are about 4 milliliters in diameter and were able to prevent death in mice suffering from liver failure. The transplanted liver buds also differentiated into cells that assumed a range of liver functions that ranged from secreting liver-specific proteins and producing human-specific metabolites. Most notably, these buds quickly made connections with nearby blood vessels and continued to grow after transplantation.

Valerie Gouon-Evans, who studies liver development and regeneration at Mount Sinai Hospital in New York, noted that even these results are preliminary but promising. “This is a very novel thing,” she said. Because these liver buds are supported by the host’s blood system, transplanted cells continue to proliferate and perform liver functions.

However, Gouon-Evans cautioned, the transplanted animals must be placed under observation for several more months in order to determine if the transplanted cells begin to degenerate or form tumors.

Globally and even in the United States, there is a dismal scarcity of human livers for transplant. In 2011, 5,805 adult liver transplants were done in the United States. That same year, 2,938 people died waiting for new livers or became too sick to remain on waiting lists.

However, attempts to create complex organs in the laboratory have been challenging. Takebe believes that his study represents the first time that people have made a solid organ using induced pluripotent stem cells (iPSCs), which are created by reprogramming mature adult cells into an embryonic stem cell-like state.

Unfortunately, determining whether or not these liver buds could help sick patients is years away, according to Takebe. Apart from the need for longer-term experiments in animals, it is not yet possible to make liver buds in quantities sufficient for human transplantation.

In the current work, Takebe surgically transplanted liver buds at sites in the cranium or the abdomen, but in future work, Takebe hopes to generate liver buds small enough to be delivered intravenously in mice and, eventually, in humans. Takebe also hopes to transplant the buds to the liver itself, where he hopes they will form bile ducts, which are crucial important for proper digestion and were not observed in the latest study.

The researchers make the liver buds from three types of human cells. First, they induce iPSCs to differentiate into a cell type that expresses liver genes. To these cells, they add endothelial progenitor cells (EPCs; endothelial line blood vessels) from umbilical cord blood, and mesenchymal stem cells, which can make bone, cartilage and fat. These cell types also come together as the liver begins to form in the developing embryo.

“It’s a great day for developmental biology,” says Kenneth Zaret, who studies regenerative medicine and liver development at the University of Pennsylvania in Philadelphia. “By reconstituting cell interactions that we know are important for natural liver progression, they get what appears to be robust, mature tissue.”

The project began with an unexpected phenomenon, says Takebe. He initially hoped to design ways of to make vascularized liver tissues. Therefore, he tried culturing multiple cell types together and noticed that they began to self-organize into three-dimensional structures. After this, the process for making liver buds took hundreds of trials to adjust various experimental parameters (e.g., the maturity and ratios of the different cell types).

This strategy takes a middle path between two common strategies in regenerative medicine. For simple, hollow organs such as the bladder and trachea, researchers seed scaffolds with living cells and then transplant the entire organ into patients. Researchers have also worked to create pure cultures of functional cells in the laboratory, hoping that cells could be infused into patients, where they would establish themselves. But even if the cells work perfectly in the laboratory, says Gouon-Evans, the process of harvesting cells can damage them and destroy their function.

Zaret thinks that the liver buds work might encourage an intermediate approach. “Basically, put the cells in a room together and let them talk to each other and make the organ.”

Self-organizing structures from stem cells have also been observed for other organ systems, such as the optic cup, an early structure in eye development. And “mini-guts” have been grown in culture from single human stem cells.

Takebe believes that the self-organizing approach might also be applicable to other organs, such as lung, pancreas and kidney.

The Use of Stem Cells in Drug Development

Why is it that one person can have surgery and wake up, eat a full lunch and show no ill effects while others are sick for several days after receiving general anesthesia?

The fact is that we all process drugs differently, and these differences are a function of the genetic diversity between all of us. These differences stem from 1) different targets; 2) different liver enzyme activities; and 3) different levels of absorption, excretion and distribution.

A few examples might be illustrative. It is fairly well established that a particular type of blood pressure medicines called “ACE inhibitors” do not work terribly well in African-Americans (see Park IU, Taylor AL. Ann Fam Med. 2007 Sep-Oct;5(5):444-52). The reason for this is that the target of ACE inhibitors, the enzyme angiotensin converting enzyme, which is mercifully abbreviated ACE, works on a substrate that already exists at low concentrations in most African-American patients. Thus a target difference causes differential responses to particular blood pressure medicines.

As a second example, two liver enzymes that degrade drugs, Cyp2C19 and Cyp2D6 are encoded by genes that are subject to genetic variation. In 3-10% of whites, the Cyp2D6 enzyme does not completely function and the drugs processed by this enzyme, a blood pressure medicine called debrisoquine and a heart medicine called sparteine, show impaired degradation. Thus these patients are in danger of overdosing on these drugs at normal dosages, since they are degraded and excreted at such low rates. Other people, however, have a version of Cyp2D6 that is hyperactive. This variant is most commonly found in Ethiopians and Saudi Arabians that consequently, drugs degraded by this enzyme, such as tricyclic antidepressants (e.g., nortriptyline) must be dosed at two the three times the normal concentration. Also, some drugs are given as prodrugs, which are inactive until the liver activates them. In individuals with the overactive Cyp2D6 enzyme variant, a prodrug, such as codeine is overactivated and at normal doses, causes severe side effects (stomach pains). Thus a distinct enzyme difference causes different clinical outcomes with the same drugs (see JK Hicks, et al., Clin Pharmacol Ther. 2013 May;93(5):402-8).

So then, how do we test for drug safety and efficacy given these variations in drug metabolism?

Stem cell technology has the ability to improve drug testing in a multitude of ways. Drug safety can be tested with stem cells as can drug efficacy without feeding them to human volunteers.

Now scientists from the University of Edinburgh have shown that stem cell-based drug tests are almost ready for the prime time. David Hay from the Medical Research Centre for Regenerative Medicine at the University at the University of Edinburgh and his colleagues have generated cell in the laboratory that reach the gold standard required by the pharmaceutical industry to test drug safety.

In this study, the Hay laboratory made liver cells from H9 human embryonic stem cells and from 33D6 human induced pluripotent stem cells. Since is the liver is the main organ that biochemically processes drugs in our bodies (a phenomenon known as biotransformation), testing drug safety in cultured liver cells makes good sense.

Next, Hay and his colleagues found that these pluripotent-derived liver cells were equally effective in drug safety tests as frozen human liver tissue extracted from cadavers. Such livers are in short supply and the results researchers derive from them varies wildly according to the genetic make-up of the donor. Thus frozen liver tissue is not optimal for such drug testing protocols.

However, these drug-testing protocols that use stem cell-based protocols can provide reproducible drug safety results and can also be adapted for individuals with particular genetic compositions who process drugs differently from other people.

David Hay explained it this way: “Differing genetic information plays a key role in how patients’ livers process drugs. We are now able to efficiently produce human liver cells in the laboratory from different people model the functional differences in human genetics.”

Hay and others hope to generate liver cells that contain distinct DNA sequences that will reflect the genetic variations in metabolism found in the population. These cultured liver cells from human pluripotent stem cells can be used to identify differences in drug biotransformation.

These laboratory-generation liver cells could also be used to screen certain drugs that need close monitoring in order to optimize the efficacy of patient treatment, and the safety of these treatment regimes.

Hay and his colleagues are working with Edinburgh BioQuarter in order to form a spin-off company that will commercialize this research and its clinical ramifications.

New Liver Stem Cell Might Aid in Liver Regeneration

For patients with end-stage liver disease, a liver transplant is the only viable option to stave off death. Liver failure is the 12th leading cause of death in the United States, and finding a way to regenerate failing livers is one of the Holy Grails of liver research. New research suggests that one it will be feasible to use a patient’s own cells to regenerate their liver.

Researchers at the Icahn School of Medicine at Mount Sinai have discovered that a particular human embryonic stem cell line can be differentiated into a previously unknown liver progenitor cell that can differentiate into mature liver cells.

“The discovery of the novel progenitor represents a fundamental advance in this field and potentially to the liver regeneration field using cell therapy,” said Valerie Gouon-Evans, the senior author of this study and assistant professor of medicine at the Icahn School of Medicine. “Until now, liver transplantation has been the most successful treatment for people with liver failure, but we have a drastic shortage of organs. This discovery may help circumvent that problem.”

Gouon-Evans collaborated with the laboratory of Matthew J. Evans and showed that the liver cells that were made from the differentiating liver progenitor cells could be infected with hepatitis C virus. Since this is a property that is exclusive to liver cells, this result shows that these are bona fide liver cells that are formed from the progenitor cells.

One critical step in this study was the identification of a new cell surface protein called KDR, which is the vascular endothelial growth factor 2. KDR was thought to be restricted to blood vessels, blood vessels progenitor cells (EPCs), and blood cells.  However, the Evans / Gouon-Evans study showed that activation of KDR in liver progenitor cells caused them to differentiate into mature liver cells (hepatocytes).  KDR is one of the two receptors for VEGF or vascular endothelial growth factor.  Mutations of this gene are implicated in infantile capillary hemangiomas.

KDR Protein Crystal Structure
KDR Protein Crystal Structure

The next step in this work is to determine if liver cells formed from these embryonic stem cells could potentially facilitate the repair of injured livers in animal models of liver disease.