Functional, Though Not Completely Structurally Normal Tissue-Engineered Livers Made from Adult Liver Cells

Tracy C. Grikscheit and her research team from the Saban Research Institute at the Children’s Hospital Los Angeles have produced functional, tissue-engineered human and mouse liver from adult stem and progenitor cells.

The largest organ in our bodies, the liver executes many vital functions. It is located in the upper right portion of the abdomen protected by the rib cage. The liver has two main lobes that are divided into many tiny lobules.

Liver cells are supplied by two different sources of blood. The hepatic artery provides oxygen-rich blood from the heart and the portal vein supplies nutrients from the intestine and the spleen. Normally, veins return blood from the body to the heart, but the portal vein allows chemicals from the digestive tract to enter the liver for “detoxification” and filtering prior to entering the general circulation. The portal vein also delivers the precursors liver cells need to produce the proteins, cholesterol, and glycogen required for normal body activities.

The liver also makes bile. Bile is a mixture of water, bile acids (made from stored cholesterol in the liver), and other sundry chemicals. Bile made by the liver is then stored in the gallbladder. When food enters the duodenum (the uppermost part of the small intestine), the gallbladder contracts and secretes bile is secreted into the duodenum, to aid in the digestion of fats in food.

The liver also stores extra sugar in the form of glycogen, which is converted back into glucose when the body needs it for energy. It also produces blood clotting factors, processes and stores iron for red blood cell production, converts toxic nitrogenous wastes (usually in the form of ammonium) into urea, which is excreted in urine. Finally, the liver also metabolizes foreign substances, like drugs into substances that can effectively excreted by the kidneys.

Both adults and children are affected by various types of liver disease. Liver can be caused by infectious hepatitis, which is caused by a variety of viruses, chronic alcoholism, inherited liver abnormalities (e.g., Wilson’s disease, hemochromatosis, Gilbert’s disease) or various types of liver cancer. One in ten people in the United States suffer from liver cancer and need a liver transplant. Liver transplantation is the only effective treatment for end-stage liver disease, but the scarcity of liver donors and the necessity of life-long immunosuppressive therapy limit treatment options. In some cases (such as inborn errors of metabolism or acute bouts of liver insufficiency), patients may be effectively treated by transplanting small quantities of functional liver tissue.

Alternate approaches that have been investigated, but these protocols have significant limitations. For example, “hepatocyte transplantation” involves the infusion of liver cells from a donated liver. This protocol, however, wastes many cells that do not integrate into the existing liver and such a treatment is usually little more than a stop-gap solution, since most patients require a liver transplant within a year of this treatment.

Human-induced pluripotent stem (iPS) cells are another possibility but, so far, iPS cells differentiate into immature rather than mature, functional, proliferative hepatocytes.

A need remains for a robust treatment that can eliminate the need for immunosuppressive theory. “We hypothesized that by modifying the protocol used to generate intestine, we would be able to develop liver organoid units that could generate functional tissue-engineered liver when transplanted,” said Dr. Grikscheit.

Grikscheit and her co-workers extracted hardy, multicellular clusters of liver cells known as liver organoid units (LOUs) from resected human and mouse livers. These LOUs were loaded onto scaffolds made from nonwoven polyglycolic acid fibers. These scaffolds are completely biodegradable and they provide a structure upon which the LOUs can grow, fuse, and form a structure that resembles a liver.

After transplantation of the LOU/scaffold combinations, they generated tissue-engineered livers or TELis. Tissue-engineered livers developed from the human and mouse LOUs and possessed a variety of key liver-specific cell types that are required for normal hepatic function. However, the cellular organization of these TELis did differ from native liver tissue.

The tissue-engineered livers (TELis) made by Grikscheit’s laboratory contained normal liver components such as hepatocytes that properly expressed the liver-specific protein albumin, CK19-expressing bile ducts, vascular structures surrounded by smooth muscles that expressed smooth muscle-specific actin, desmin-expressing stellate cells, and CD31-expressing endothelial cells. The production of albumin by the TELi hepatocytes indicated that these cells were executing their normal secretory function. In a mouse model of liver failure, their tissue-engineered liver provided some hepatic function. In addition, the hepatocytes proliferated in the tissue-engineered liver.

A cellular therapy for liver disease that utilizes technologies like this would completely change the treatment options for many patients. In particular, children with metabolic disorders and require a new liver to survive might see particular benefits if such a treatment can come to the clinic. By generating functional hepatocytes comparable to those in native liver, establishing that these cells are functional and proliferative, Grikscheit and her colleagues have moved one step closer to that goal.

To access this paper, please see: Nirmala Mavila et al., “Functional Human and Murine Tissue-Engineered Liver Is Generated From Adult Stem/Progenitor Cells,” Stem Cells Translational Medicine, August 2016 DOI: 10.5966/sctm.2016-0205.

German Group Uses Induced Pluripotent Stem Cells to Model Nonalcoholic Fatty Liver Disease

A German research group has used pluripotent stem cells to design a new in vitro model system for investigating nonalcoholic fatty liver disease (NAFLD).  NAFLD, or steatosis, is a liver disease whose prevalence is probably much higher than estimated, and the new cases of it are increasing every year throughout the world.  NAFLD is typically associated with obesity and type-2 diabetes.  An estimated one-third of the general population of Western countries is thought to be affected with NAFLD, with or without symptoms.  It usually results from a high caloric diet in combination with a lack of exercise.  The liver begins to accumulate fat as lipid droplets.  Initially, this is a benign state, but it can develop into nonalcoholic steatohepatitis (also known as NASH), an inflammatory disease of the liver.  Then many patients develop fibrosis, cirrhosis or even liver cancer.  However, in many cases patients die of heart failure before they develop severe liver damage.

A major obstacle that dogged NAFLD research was that biopsies of patients and healthy individuals were required.  Researchers from the Institute for Stem Cell Research and Regenerative Medicine at the University Clinic of Düsseldorf, Germany solved this problem by reprogramming skin cells into induced pluripotent stem cells (iPSCs) that they differentiated into hepatocyte-like cells.

“Although our hepatocyte-like cells are not fully mature, they are already an excellent model system for the analysis of such a complex disease”, said Nina Graffmann, first author of the paper that appeared in the journal Stem Cells and Development.

The researchers recapitulated important steps of the disease in cultured cells.  They demonstrated up-regulation of PLIN2, a protein called perilipin that surrounds lipid droplets. Mice without PLIN2 do not become obese, even when overfed with a high fat diet.  Also the key role of PPARα, a transcription factor involved in controlling glucose and lipid metabolism, was reproduced in the tissue culture system.  “In our system, we can efficiently induce lipid storage in hepatocyte-like cells and manipulate associated proteins or microRNAs by adding various factors into the culture.  Thus, our in vitro model offers the opportunity to analyse drugs which might reduce the stored fat in hepatocytes,” Graffmann said.

Senior author James Adjaye and his colleagues hope to expand their model by deriving iPSCs from NAFLD patients.  They hope to discover differences that might explain the course of NAFLD.

“Using as reference the data and biomarkers obtained from our initial analyses on patient liver biopsies and matching serum samples, we hope to better understand the etiology of NAFLD and the development of NASH at the level of the individual, with the ultimate aim of developing targeted therapy options,” said Adjayer.

This paper can be found at Nina Graffmann et al., “Modeling NAFLD with human pluripotent stem cell derived immature hepatocyte like cells reveals activation of PLIN2 and confirms regulatory functions of PPARα,”Stem Cells and Development, 2016; DOI: 10.1089/scd.2015.0383.

Mesenchymal Stem Cells from Bone Marrow Improve Liver Function and Reduce Liver Scarring in Patients with Alcoholic Cirrhosis

Dr Soon Koo Baik from the Yonsei University Wonju College of Medicine, and Dr. Si Hyun Bae from The Catholic University of Korea and their colleagues have conducted an important phase 2 clinical trial that tests the ability of mesenchymal stem cells from bone marrow to treat cirrhosis of the liver. In this trial, seventy-two patients who had established cirrhosis of the liver participated in a multicenter, randomized, open-label, phase 2 trial (published in the journal Hepatology, DOI:10.1002/hep.28693).

The liver is a hugely important organ. Not only is it the largest internal organ in our bodies, but it serves as the main metabolic factory of the body because of the outsized role it plays in metabolism. The liver takes up and stores and processes nutrients from food. Once it processes fats, sugars, and amino acids, the liver delivers them to the rest of the body. The liver also makes new proteins, such as clotting and immune factors, produces bile, which helps the body absorb fats, cholesterol, and fat-soluble vitamins, and removes waste products the kidneys cannot remove, such as fats, cholesterol, toxins, and medications.

The condition known as cirrhosis is a condition in which the liver gradually deteriorates and becomes unable to function normally due to chronic, or long-lasting, injury. The accumulation of scar tissue in the liver is typically slow and gradual and as scar tissue replaces more healthy liver tissue, the liver begins to fail. Scar tissue also partially blocks the flow of blood through the liver. Chronic liver failure (also known as end-stage liver disease) culminates in the inability of the liver to perform important functions. Since the liver is an organ that have a good deal of regenerative ability, end-stage liver disease essentially becomes so damaged that it cannot effectively replace damaged cells.

Cirrhosis is most commonly called by chronic alcoholism, but so can chronic viral infections by viruses like hepatitis B virus and hepatitis C virus.  Additionally, particular genetic diseases can also cause cirrhosis in children or young adults.

Mesenchymal stem cells have the ability to secrete cocktails of pro-healing molecules that might be able to support the growth and survival of liver cells. A variety of experiments in animals have established that the administration of mesenchymal stem cells (MSCs) from bone marrow (Truong, NH, et al., Stem Cells Int. 2016;2016:5720413. doi: 10.1155/2016/5720413; Almeida-Porada G, et al., Exp Hematol. 2010;38:574–580; Berardis S, et al., World J Gastroenterol. 2015;21:742–758), and other sources (De Ugarte DA, et al., Cells Tissues Organs. 2003;174:101–109; in ‘t Anker PS, etr al., Haematologica. 2003;88:845–852; Lee OK, et al., Blood. 2004;103:1669–1675) can decrease inflammation within the liver, inhibit the death of liver cells and promote their survival, and promote the regeneration of residential liver cells.

In clinical trials, administration of MSCs to cirrhosis patients has established the safety of MSC-based treatments (Amin MA, et al., Clin Transplant. 2013;27:607–612; El-Ansary M, et al., Stem Cell Rev. 2012;8:972–981; Jang YO, et al., Liver Int. 2014;34:33–41; Kharaziha P, et al., Eur J Gastroenterol Hepatol. 2009;21:1199–1205; Mohamadnejad M, et al., Arch Iran Med. 2007;10:459–466). Unfortunately, the design of these trials involved the mixing of patients with alcohol-based cirrhosis, viral-based cirrhosis, and other types of cirrhosis. Therefore, it is impossible to draw any conclusions about the efficacy of MSC transplantations on the basis of these trials. However, one trial, by Jang, et al, examined the effect of MSCs from bone marrow in patients with alcoholic cirrhosis. After 11 patients received MSC implantations, improvements in liver tissue architecture were observed in 6/11 patients, and 10 patients showed recovery of liver function. These 10 patients had decreased expression of molecules that induce scarring in the liver (i.e. TGF-β1, collagen type I, and α-smooth muscle actin). Significantly, Jang and others observed these improvements in the absence of significant complications or side effects during the study period. On the strength of these results, a larger phase 2 study is certainly warranted (see F. Ezquer, et al., World J Gastroenterol. 2016 Jan 7; 22(1): 24–36).

In this Bak and Bae clinical trials, 72 patients were randomly assigned to three groups that consisted of a control group and two autologous bone marrow-based MSC groups that underwent either one-time or two-time hepatic arterial injections of 5 × 10[7] MSCs, 30 days after bone marrow aspiration. All patients also underwent a follow-up biopsy 6 months after enrollment and adverse events were monitored for 12 months.

The primary endpoint in this study was the improvement in the amount of scar tissue in biopsies (as assayed by Picrosirius-red staining). The secondary endpoints included liver function tests, a measure of the severity of cirrhosis called the Child-Pugh score, and another score called the Model for End-stage Liver Disease (MELD) score. The outcomes were analyzed by per-protocol analysis.

When it comes to the amount of scar tissue in the patient’s livers, patients that received one-time and two-time bone marrow-based MSC administrations, showed 25% (19.5±9.5% vs. 14.5±7.1%) and 37% (21.1±8.9% vs. 13.2±6.7%) reductions in the amount of liver scar after MSC administration, respectively (P0.05). The Child-Pugh scores of both BM-MSC groups (one-time: 7.6±1.0 vs. 6.3±1.3 and two-time: 7.8±1.2 vs. 6.8±1.6) were also significantly improved following BM-MSC transplantation (P<0.05) compared to the control group that did not receive MSCs. Most significantly, perhaps, is that the proportion of patients with adverse events did not differ among the three groups.

From this larger phase 2 study, it seems that transplantation of a patient’s own bone marrow-based MSCs can safely improve the degree of scarring in the liver of cirrhosis patients and also improve liver function in patients with alcoholic cirrhosis. This study seems to confirm what was observed in preclinical studies in laboratory animals and extends what was observed in the phase 1 studies. While more work is certainly required, these results are certainly hopeful.

Treating a Damaged Liver: Bone Marrow CD45 Cells are Superior to Mesenchymal Stem Cells

Scarring of the liver, otherwise known as “liver fibrosis” usually results when the liver is constantly assaulted by inflammation. Conventional treatments for liver fibrosis are usually not very effective. Therefore, mesenchymal stem cells (MSCs) is an attractive alternative due to their ability to suppress inflammation. Unfortunately, transplanted MSCs tend to show poor survival in the scarred liver, and they have an additional tendency to stimulate the formation of new scar tissue. These characteristics have bred skepticism among many investigators.

New work by Asok Mukhopadhyay and his colleagues from New Delhi, India has compared bone marrow (BM)-derived cells with MSCs as a treatment for liver fibrosis. They used CCl4 to induce liver fibrosis in laboratory mice. Then they treated liver-damaged mice with either BM-CD45 cells or fat-based MSCs.

Liver tests and tissue samples of both sets of mice clearly showed that the BM-CD45 cells did a much job attenuating liver scarring than did the fat derived MSCs. Interestingly, the anti-scarring capacity of the BM-CD45 cells was compromised by the presence of MSCs.

Why did the BM-CD45 cells do a better job? The bone marrow cells expressed rather high level expressions of matrix metalloproteinases. These enzymes chopped through scar tissue and also suppressed the hepatic stellate cells, which are responsible for making the scar tissue in liver. Apparently, the BM-CD45 cells induced the die off of the stellate cells. MSCs, however, released two growth factors (TGFβ and IGF-1) that are known to activate hepatic stellate cells, and promote the formation of scar tissue. As an added bonus, transplantation of CD45 cells led to functional improvement of the damaged liver, and this functional improvement seems to the result of improved liver repair and regeneration.  Thus transplanted MSCs were pro-scarring while transplanted BM-CD45 cells were pro-regeneration, at least in the liver.

To summarize the results of these experiments, BM-derived CD45 cells appear to be a superior candidate for the treatment of liver fibrosis. The structural and functional improvement of CCl4-damaged livers was substantially better in animals that received transplants of BM-CD45 than those who received fat-derived MSCs.

Newly Discovered Liver Cells Regenerate Liver Without Forming Tumors

The means by which the liver repairs itself is still a matter of debate. Now a new study from the University of San Diego has discovered a population of liver cells that do a better job at regenerating liver tissue than ordinary liver cells, or hepatocytes. This study has identified a cell population called “hybrid hepatocytes” that are able to regenerate liver tissue without giving rise to cancer.

This latest study was led by Michael Karin, PhD, Distinguished Professor of Pharmacology and Pathology at University of San Diego. Karin and his colleagues published their study in the August 13th edition of the journal Cell, and their paper is the first to identify these so-called “hybrid hepatocytes.” Karin and his coworkers also showed that hybrid hepatocytes are able to regenerate liver tissue without giving rise to cancer. Although the majority of the work described in this study was done in mouse models, Karin and his group also found similar cells in human livers.

Of all major organs, the liver has the highest capacity to regenerate. This is the main reason some liver diseases, including cirrhosis and hepatitis, can often be cured by transplanting a piece of liver from a healthy donor. The liver’s regenerative properties were previously credited to a population of adult stem cells known as oval cells. However, recent studies concluded that oval cells do not give rise to hepatocytes, since oval cells tend to make bile duct cells. These discoveries prompted researchers to begin looking for other cell populations in the liver that serve as the primary source of new hepatocytes in liver regeneration.

To find this new cell population, Karin and others traced the cells responsible for replenishing hepatocytes following chronic liver injury after laboratory animals were fed the liver toxin carbon tetrachloride. The liver regeneration that was stimulated by carbon tetrachloride-induced liver damage was traced to a unique population of hepatocytes in one specific area of the liver, called the “portal triad.” The portal triad is a region of the liver named after its triangular shape and its three major components: the hepatic artery, the hepatic portal vein, and the hepatic ducts, or bile ducts. The portal triad is also known by its clinical term, portal hepatis, transverse fissure and portal fissure. The portal triad serves as a blood-vessel gateway or entrance of the liver’s hepatic lobule. These special liver cells that reside in the portal triad hepatocytes undergo extensive proliferation and replenish liver mass after chronic liver injuries. Since these cells are similar to normal hepatocytes, but express low levels of bile duct cell-specific genes, Karin and his team dubbed these cells “hybrid hepatocytes.”

Portal Triad

Many other research labs around the world are attempting to use induced pluripotent stem cells (iPSCs) to repopulate diseased livers and prevent liver failure. “Although hybrid hepatocytes are not stem cells, thus far they seem to be the most effective in rescuing a diseased liver from complete failure,” said Joan Font-Burgada, PhD, postdoctoral researcher in Karin’s lab and first author of the study.

While iPSCs hold a lot of promise for regenerative medicine, it might be theoretically difficult to ensure that they stop proliferating once their therapeutic job is done. As a result, iPSC-derived cells might pose a significant risk for tumor formation.  To test the safety of hybrid hepatocytes, Karin’s team examined three different mouse models of liver cancer. They found no signs of hybrid hepatocytes in any of the tumors, leading the researchers to conclude that these cells do not contribute to liver cancer caused by obesity-induced hepatitis or chemical carcinogens.

“Hybrid hepatocytes represent not only the most effective way to repair a diseased liver, but also the safest way to prevent fatal liver failure by cell transplantation,” Karin said.

Liver Cells from Circulating Blood Cells Under Clinically Safe Conditions

Can we convert circulating blood cells into working liver cells? Think of what this would mean for people who have liver problems. While is sounds like science fiction, the laboratory of James Ross at the University of Edinburgh, in collaboration with other scientists, has managed to do exactly that.

Ross and his colleagues developed an efficient method for converting circulating white blood cells into induced pluripotent stem cells (iPSCs). As previously mentioned on this blog, iPSCs are made from mature, adult cells by genetically engineering those cells with a cocktail of genes (in this case Oct4, Sox2, Klf4, L-Myc, and Lin28), and then culturing the cells in a special culture system that allows them to grow and become pluripotent stem cells that can theoretically differentiate into any of the 210 adult cell types in the human body.

Since the production of iPSCs from mature cells requires the insertion of particular genes into those cells, scientists typically use viruses or other vehicles to do this, which can introduce mutations into the genomes of the cells. Ross and his coworkers, however, used a non-integration method for reprogramming fresh or frozen white blood cells. They inserted small circles of DNA called “episomes” into these cells using a technique called “electroporation,” which binds the DNA to the surfaces of the cells and then subjects them to an electrical pulse that quickly moves the DNA into the cells without harming them. The genes on the episome are then expressed, but only transiently, which is all that is required to reprogram the adult cells into iPSCs. The cells were also cultured in a feeder-free system, which means that no animal products were involved in the production of these iPSC lines.  This constitutes, so-called “Good Manufacturing Practice” or GMP, which is required is a product is to be used for human patients.

Ross and others achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×105 seeded MNC), and when they used the same protocol to cord blood or fetal liver-derived blood-making (CD34+) cells, they achieved a reprogramming rate of 0.148% (148 iPSC colonies from 105 seeding cells). These iPSC lines were then used to make differentiated liver cells. This procedure tends to produce quasi-liver cells that do not have the characteristics of mature liver cells, but in this case, Ross and others derived cells that have proper drug metabolic function. This suggests that the iPSC-derived liver cells were at least mature enough to express many of the enzymes necessary to properly metabolize drugs. While these cells were probably not fully mature, they were a good deal further along than those derived in other experiments.

These experiments show that it is feasible to make liver cells for drug screen from circulating blood cells in a manner that is clinically safe. It is presently unclear if these cells can serve as material to heal a damaged liver, and that will take more work. Also, this procedure almost certainly would cost a good deal of money, and for that reason, banked iPSCs from white blood cells that have been fully tissue typed might be a better way to use cells made in this manner.

See Jing Liu, and others, Experimental Cell Research, 6 August 2015, Article ECR15383.

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.”