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.”

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.”

REALISTIC Trial to Test Efficacy of Bone Marrow Stem Cells on Liver Disease


Chronic liver disease is the fifth leading cause of death in the United Kingdom. With the long-standing shortage of donated, transplantable livers, the prognosis of such patients seems grim.

Several preclinical studies in animals have established that mobilization of bone marrow stem cells or direct injection of bone marrow stems into a damaged liver can augment healing and improve survival (Sukaida I, and others, Hepatology 2004;40:1304–11; and Yannaki E, and others, Exp Hematol 2005;33:108–19). Some small clinical trials have examined the use of a patient’s own bone marrow stem cells to prime the liver and stimulate its own internal healing mechanisms. These studies were small and varied in the manner in which the stem cells were delivered, but they di show that the stem cell treatments were safe and even improved the health of the liver significantly (Gordon MY, and others, Stem Cells 2006;24:1822–30; Terai S, and others, Stem Cells 2006;24:2292–8). Also, in patients with liver cancer who had to have portions of their livers removed, bone marrow stem cell treatments accelerated liver healing (am Esch JS, and others, Ann Surg 2012;255:79–85; am Esch JS, and others, Stem Cells 2005;23:463–70; and Furst G, and others, Radiology 2007;243:171–9).

Clearly there is a need for a larger, more systematic study of the efficacy of bone marrow stem cells as a therapeutic agent in patients with liver failure. To that end, Philip Newsome and his colleagues at the University of Birmingham, in collaboration with colleagues from Scotland, Newcastle, and Nottingham have initiated the REALISTIC trial, which stands for REpeated AutoLogous Infusions of STem cells In Cirrhosis.

This is a multi-center clinical trial and it will examine patients with Cirrhosis (fatty liver disease), regardless of the cause of that liver disease. Patients whose livers were damaged by excessive alcohol use, hepatitis B or C infections, or genetic conditions are all eligible for this study, but anyone who liver is too far-gone to be helped by a treatment like this or has had a liver transplant is not eligible.

Patients will receive injections of a drug called lenograstim (G-CSF) to mobilize bone marrow stem cells into the blood. These blood-based stem cells will then be collected and concentrated, and then implanted into the liver. Patients will be assessed at 3 months after the treatment and then followed-up for 1 year. Liver health will be assessed by means of medical imaging of the liver and various blood tests.

Patients will be evaluated using the Model for End-Liver Disease or MELD scoring system. Secondary tests will measure the degree of liver scarring, the degree of liver stiffness, blood tests, survival, and liver function.

Patients will also be placed into three groups. One group will only have the bone marrow stem cells mobilized from bone marrow without being collected. Another group will have the cells collected and implanted into the liver. The third group will receive standard care with not stem cells treatments.

There is a need for a study like this. I only hope that Newsome and his group can recruit the patients and get started collecting data as soon as they can.

Tonsil-Based Stem Cells To Repair the Liver


Byeongmoon Jeong and colleagues report in the journal ACS Applied Materials & Interfaces that injections of stem cells from tonsils, a body part we don’t need, can repair damaged livers without the need for surgery. The liver rids the body of toxins, makes blood proteins, and metabolizes a goodly number of molecules from our food. Liver failure is a deadly condition and a liver transplant is often the only option to restore the patient to health. Unfortunately there is a need for available organs for transplantation, Also, liver transplantation presents certain risks and also is extremely expensive.

A promising alternative to liver transplantation is the implantation of liver cells. Adult stem cells can be used to make new liver cells, and bone marrow-based stem cells have been used, but they these cells have inherent limitations. Recently, scientists have identified another stem cell source that can be used for this purpose from tonsils. Every year, thousands of tonsillectomies are performed to remove tonsils, and the extirpated tonsils are discarded. Now, however, these throw-away tissues could have a new purpose. Scientists have devised ways to grow tonsil-based stem cells on a three-dimensional scaffold that simulates living liver tissue.

Jeong’s team encapsulated tonsil-derived stem cells in a heat-sensitive liquid that solidifies into a gel at body temperature. To these cells ensconced in this gel, they added protein growth factors to stimulate the stem cells to differentiate into liver cells. The stem cells differentiated into liver cells, degraded the scaffold, and formed functioning liver cells. Jeong and others think that with a little tweaking, this procedure could potentially provide an injectable tissue engineering technique to treat liver disease without surgery.

See Seung-Jin Kim, Min Hee Park, Hyo Jung Moon, Jin Hye Park, Du Young Ko, Byeongmoon Jeong. Polypeptide Thermogels As a 3D Culture Scaffold for Hepatogenic Differentiation of Human Tonsil-derived Mesenchymal Stem Cells. ACS Applied Materials & Interfaces, 2014; 140905122318006 DOI:10.1021/am504652y.

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).

human-liver-diagram

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.

Human Umbilical Cord Stem Cells Prevent Liver Failure in Mice


Acute liver failure results from massive liver damage over a short period of time. Viral infections (hepatitis B virus), drugs (acetaminophen, halothane), sepsis, Wilson’s disease, or autoimmune hepatitis can all cause acute liver failure, but acute liver failure can be life-threatening. Remember, the liver makes the vast majority of blood proteins such as clotting factors or albumin, and without a functioning liver, multi-organ failure ensues.

Liver transplantation can offer effective treatment of acute liver failure, except that there is a global shortage of available livers. The wife of my colleague at Spring Arbor University waited years and years for a liver until a liver was given to her as the result of a dying declaration. THe need is substantial and the supply is miniscule.

Several experiments have demonstrated that the transplantation of mesenchymal stem cells (MSCs) can treat acute liver failure. Human umbilical cord MSCs (hUCMSCs) can be differentiated into cells that closely resemble liver cells (known as hepatocytes) and these i-Heps, as they are called, display many liver-specific functions (secretion of albumin, storage of glycogen, see Campard et al., Gastroenterology 134 2008: 833-848). Likewise, UBMSCs secrete a host of interesting pro-regenerative molecules that seem to aid in liver recovery, regeneration, and healing when implanted into a damaged liver (see Banas et al., J Gastroenterol Hepatol 24 2009: 70-77; van Poll, et al., Hepatology 47 2008: 1634-1643; Moslem, et al., Cell Transplant 22(10) 2013: 1785-99).

To this end, scientists from the Chinese Academy of Sciences in Shenzhen, China have done an interesting side-by-side comparison of the ability of i-Heps and undifferentiated UCMSCs to mitigate acute liver damage in a mouse model.

Ruiping Zhou and Zhuokun Li in the laboratory of Zhi-Ying Chen and their colleagues injected a mixture of D-Galactosamine and a bacterial compound called LPS (lipopolysaccharide) into the bellies of NOD/SCID mice (non-obese diabetic, severe combined immunodeficiency) to induce acute liver damage. Half of the mice injected with this concoction died of acute liver failure, and autopsies of the mice in these experiments showed that half of the liver cells in their livers had been burned out. A control group was injected with salt solutions and showed no such liver damage.

Of these mice, some of the were injected with either two million UBMSCs or two million i-Heps, six hours after the induction of acute liver damage. The cells were given intravenously, in the tail vein.

Interestingly, both groups of mice – those that had received the UBMSCs and those that had received the i-Heps – showed improved survival and improved liver function as ascertained by several liver function tests. Liver biopsies revealed lower levels of cell death within the liver in both cases. Also when the liver is damaged, there are several blood tests that can reveal the presence of liver damage and indicate the degree of liver damage. In all cases where the D-Galactosamine and LPS were administered, the levels of these liver enzymes increased the first after their administration, but in those animals that received either UBMSCs or i-Heps, the markers of liver damage neither climbed as high, nor did they stay high as long, indicating the damage to the liver was mitigated by the infused stem cells.

Liver biopsies of the laboratory animals further confirmed the decreased levels of liver scarring in those animals that had received the stem cells with the D-galactosamine and LPS. Also the levels of cell division, indicative of healing, were increased in the stem cell-treated animals. Two weeks after the initial liver damage, large areas of the liver were observed that showed the signs of cell division, which indicates the presence of active liver repair activities at work in the stem cell-treated animals. Mice not treated with stem cells showed extensive liver damage with little signs of healing if they survived at all.

This interesting study shows that both hUCMSCs and hUCMSC-derived -i-Heps exhibited similar therapeutic effects for mouse acute liver failure. Also, when injected into the tail vein, the stem cells were able to home to the damaged liver and set up shop there. The liver regeneration in both cases seemed to be due to the stimulation of resident liver cells rather substantial contributions from the infused stem cells.

What does this mean for human regenerative medicine? Umbilical cord MSCs are probably a good source of material to treat liver failure. However, such cells will need to be matched to the tissue type of the patient. Secondly, a point emphasized in this paper is that MSCs should not be overly manipulated before they are used because some experiments with MSCs have shown that if these cells are grown in long-term culture, they can undergo malignant transformation (see Rosland, et al., Cancer Res 69 2009: 5331-5339).

Thus beefing the number of cells up for therapeutic purposes to treat a human, which is larger than a mouse, might represent a challenge. However, it is possible to expand MSCs in culture without transforming them into cancer cells, as long as it is done for a short period of time. Finally, MSCs represent an excellent alternative for the shortage of livers, since they can stimulate the liver’s internal healing systems to heal themselves on a short-term basis without the need for a liver transplantation. This sounds like a win-win situation. Of course more work must be done first. Preclinical studies like this must be expanded and then larger animals will need to be used as well before human clinical trials can be planned.

Prostaglandin E Switches Endoderm Cells From Pancreas to Liver


The gastrointestinal tract initially forms as a tube inside the embryo. Accessory digestive organs sprout from this tube in response to inductive signals from the surrounding mesoderm. Both the pancreas and the liver form at about the same time (4th week after fertilization) and at about the same place in the embryonic gut (the junction between the foregut and the midgut).

Pancreatic development

The pancreas forms as ventral and dorsal outgrowths that eventually fuse together when the gut rotates. The liver forms from the “hepatic diverticulum” that grows from the gut about 23-26 days after fertilization. These liver bud cells work with surrounding tissues to form the liver.

Liver development

What determines whether an endodermal cell becomes a liver or pancreatic precursor cell?

Wolfram Goessling and Trista North from the Harvard Stem Cell Institute (HSCI) have identified a gradient of the molecule prostaglandin E (PGE) in zebrafish embryos that acts as a liver/pancreas switch.

Postdoctoral researcher Sahar Nissim in the Goessling laboratory has uncovered how PGE toggles endodermal cells between the liver-pancreas fate. Nissim has shown that endodermal cells exposed to more PGE become liver cells and those exposed to less PGE become pancreas. This is the first time that prostaglandins have been reported as the factor that can switch cell identities from one fate to another.

After completing these experiments, HSCI scientists collaborated with colleague Richard Mass to determine if their PGE-mediated cell fate switch also occurred in mammals. Here again, Richard Sherwood from the Mass established that mouse endodermal cells became liver if exposed to PGE and pancreas if exposed to less PGE.  Sherwood also demonstrated that PGE enhanced liver growth and regeneration.

Goessling become interested in PGE in 2005, when a chemical screen identified PGE as an agent that amplified blood stem cell populations in zebrafish embryos. Goessling that transitioned this work to human patients, and a phase 1b clinical trial that uses PGE to increase umbilical cord blood transplants has just been completed.

PGE might be useful for instructing pluripotent human stem cells that have been differentiated into endodermal cells to form completely functional, mature liver cells that can be used to treatment patients with 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.

Safe and Efficient Cell Reprogramming Inside a Living Animal


Research groups at the University of Manchester, and University College, London, UK, have developed a new technique for reprogramming adult cells into induced pluripotent stem cells that greatly reduces the risk of tumor formation.

Kostas Kostarelos, who is the principal investigator of the Nanomedicine Lab at the University of Manchester said that he and his colleagues have discovered a safe protocol for reprogramming adult cells into induced pluripotent stem cells (iPSCs). Because of their similarities to embryonic stem cells, many scientist hope that iPSCs are a viable to embryonic stem cells.

How did they do it? According to Kostarelos, “We have induced somatic cells within the liver of adult mice to transient behave as pluripotent stem cells,” said Kostarelos. “This was done by transfer for four specific gene, previously described by the Nobel-prize winning Shinya Yamanaka, without the use of viruses but simply plasmid DNA, a small circular, double-stranded piece of DNA used for manipulating gene expression in a cell.”

This technique does not use viruses, which was the technique of choice in Yamanaka’s research to get genes into cells. Viruses like the kind used by Yamanaka, can cause mutations in the cells. Kostarelos’ technique uses no viruses, and therefore, the mutagenic properties of viruses are not an issue.

Kostarelos continued, “One of the central dogmas of this emerging field is that in vivo implantation of (these stem) cells will lead to their uncontrolled differentiation and the formation of a tumor-like mass.”

However, Kostarelos and his team have determined that the technique they designed does not show this risk, unlike the virus-based methods.

“[This is the ] only experimental technique to report the in vivo reprogramming of adult somatic cells to plurpotentcy using nonviral, transient, rapid and safe methods,” said Kostarelos.

Since this approach uses circular plasmid DNA, the tumor risk is quite low, since plasmid DNA is rather short-lived under these conditions. Therefore, the risk of uncontrolled growth is rather low. While large volumes of plasmid DNA are required to reprogram these cells, the technique appears to be rather safe in laboratory animals.

Also, after a burst of expression of the reprogramming factors, the expression of these genes decreased after several days. Furthermore, the cells that were reprogrammed differentiated into the surrounding tissues (in this case, liver cells). There were no signs in any of the laboratory animals of tumors or liver dysfunction.

This is a remarkable proof-of-principle experiment that shows that reprogramming cells in a living body is fast and efficient and safe.

A great deal more work is necessary in order to show that such a technique can use useful for regenerative medicine, but it is certainly a glorious start.

 

Also involved in this paper were r, , and .

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.

Radio Interview About my New Book


I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

New Liver Drug Gets Fast Tracked by the FDA


Liver scarring (fibrosis) and cirrhosis (deposition and build up of fatty deposits in the liver) are life-threatening events. We normally associate cirrhosis in our thinking with chronic alcoholism, but there are many other conditions that can cause liver fibrosis and cirrhosis. For example, chronic systemic lupus erythematosis, which is normally just known s lupus, can wreak havoc upon the patient’s liver. Likewise Crohn’s disease, chronic hepatitis infections, or even certain genetic can cause liver disease. Once a patient’s liver scars over to a certain point. The last stop for them is either a liver transplant, or Hospice.

Until now? A biotechnology company called Galectin Therapeutics has announced that the U.S. Food and Drug Administration (FDA) has granted their new drug, GR-MD-O2 (galactoarabino-rhamnogalacturonate – say that five times fast) so-called “Fast Track designation” as an experimental treatment for non-alcoholic steatohepatitis (NASH) with hepatic fibrosis, which is also commonly known as fatty liver disease with advanced fibrosis.

GR-MD-02 is an experimental name for a complex carbohydrate drug that targets galectin-3. Galectin-3 is a cell surface protein found on liver cells and it plays a critical protein in the pathogenesis of fatty liver disease and fibrosis. Galectin proteins are central players in those diseases that involve scaring of organs such as cancer, and inflammatory and fibrotic disorders. The drug binds to galectin proteins and disrupts their function. Preclinical data have shown that GR-MD-02 can reverse fibrosis and cirrhosis in kidney, lung, and liver.

Galectin-3
Galectin-3

What is “fast track” designation? Here how this works: Fast Track is a process designed by the FDA to speed up the development and review of drugs to treat serious conditions and fill an unmet medical need. The goal is to get important new drugs to the patient earlier. Determining whether a condition is serious is a matter of judgment, but generally is based on whether the drug will have an impact on such factors as survival, day-to-day functioning, or the likelihood that the condition, if left untreated, will progress from a less severe condition to a more serious one. The kinds of conditions that have qualified for fast tracking in the past include AIDS, Alzheimer’s, heart failure and cancer. .

To qualify for fast tracking, the drug must either treat or prevent a condition with no currently available, or if there are available therapies, a fast track drug must show some advantage over available therapy. These advantages would come in the following forms:

1. Show superior effectiveness (outcomes or improved effect on serious outcomes);
2. Avoid serious side effects of an available therapy;
3. Improve the diagnosis of a serious condition (in those cases where early diagnosis results in an improved outcome);
4. Decreases clinically significant toxicity of an available therapy
5. Ability to address emerging or anticipated public health need

If a drug is fast tracked, then is will receive more frequent meetings with FDA, more frequent written correspondence from FDA, or eligibility for Accelerated Approval and Priority Review, or some combination of these.

Galectin Therapeutics is currently conducting a phase 1 clinical trial to evaluate the safety, tolerability and efficacy for single and multiple doses of GR-MD-02 over four weekly doses of GR-MD-02 treatment in patients with fatty liver disease with advanced fibrosis. In this study, Galectin will enroll eight patients in each dose escalation cohort and there will be at least three cohorts and potentially up to five cohorts, with a maximum of 40 patients at six clinical sites in the US, which each have extensive experience in clinical trials in liver disease.

“Our preclinical data has shown that GR-MD-02 has robust treatment effects in reversing fibrosis and cirrhosis. Fast Track designation enables us to expedite the compound’s development and review process, with the ultimate goal of bringing a first-in-class treatment to the millions of Americans suffering from fatty liver disease with advanced fibrosis,” said Dr. Peter Traber, president, chief executive officer and chief medical officer of Galectin Therapeutics Inc. “We are very pleased that the FDA sees the clinical value of GR-MD-02 and seriousness of fatty liver disease, and we look forward to working closely with the FDA throughout this process.”

Induced Pluripotent Stem Cells Replace Liver Function in Mice


Liver transplants save lives and in the United States there is a shortage of livers for transplantation. Between July 1, 2008 and June 30, 2011, well over 14,601 adult donor livers were recovered and transplanted. Of these livers that were transplanted, many other patients died from liver failure. If there was a way to restore liver function in patients with liver failure without dependence on a liver from a liver donor, then we might be able to extend their lives.

A paper from the laboratory of Hossein Baharvand at the University of Science and Culture in Tehran, Iran provides a step towards doing just that. In this paper, Baharvand and his colleagues used human induced pluripotent stem cells to make hepatocyte-like cells or HLCs. Hepatocyte is a fancy word for a liver cell. These HLCs were then transplanted into the spleen of mice that have damaged livers, and they rescued liver function in these mice.

The liver is a vital organ. It processes molecules absorbed by the digestive system, processes foreign chemicals to make them more easily excreted. It also produces bile, which helps dispose of fat-soluble waste and solubilize fats for degradation in the small intestine during digestion. It also produces blood plasma proteins, cholesterol and special proteins to cholesterol and fat transport, converts excess glucose into glycogen for storage, regulates blood levels of amino acids (the building blocks of proteins), processes used hemoglobin to recycle its iron content, converts poisonous ammonia to urea, regulates blood clotting, and helps the body resist infections by producing immune factors and removing bacteria from the bloodstream. Thus without a functioning liver, you are in deep weeds.

Induced pluripotent stem cells or iPSCs are made from adult cells that have been genetically engineered to de-differentiate into embryonic-like stem cells. They can be grown in culture to large numbers, and can also be differentiated into, potentially, any cell type in the adult body.

In this paper, Baharvand and his colleagues grew human iPSCs in “matrigel,” and then grew them in suspension. Matrigel is gooey and the cells stick to it and grow, and they were grown in matrigel culture for 1 week. After one week, the cells were grown in liquid suspension for 1-2 weeks. The cells have better access to soluble growth factors in liquid culture and tend to grow faster. After this they were grown in a stirred culture (known as a spinner).  This expanded the cells into large numbers for further use.

 Expansion and characterization of human induced pluripotent stem cells (hiPSCs) in a dynamic suspension culture. (A) Schematic representation of the suspension culture and expansion of human pluripotent stem cells (hPSCs) from adherent to stirred bioreactor. The cells were transferred to bacterial dishes to adapt to three-dimensional (3D) environment and then transferred into the dynamic phase, stirred flask. (B) Morphology of a tested hiPSC line (hiPSC1) after passage in a stirred suspension bioreactor as monitored by dark-field microscopy. (C) Immunostaining of cross sections of spheroids for OCT4 and TRA-1-81. Scale bar: 50 μm. (D) Flow cytometry analysis and (E) normal karyotype of suspended cells in the bioreactor.
Expansion and characterization of human induced pluripotent stem cells (hiPSCs) in a dynamic suspension culture. (A) Schematic representation of the suspension culture and expansion of human pluripotent stem cells (hPSCs) from adherent to stirred bioreactor. The cells were transferred to bacterial dishes to adapt to three-dimensional (3D) environment and then transferred into the dynamic phase, stirred flask. (B) Morphology of a tested hiPSC line (hiPSC1) after passage in a stirred suspension bioreactor as monitored by dark-field microscopy. (C) Immunostaining of cross sections of spheroids for OCT4 and TRA-1-81. Scale bar: 50 μm. (D) Flow cytometry analysis and (E) normal karyotype of suspended cells in the bioreactor.

Getting cells to grow in liquid suspension tends to be a bit of an art form, but these iPSCs grew rather well. Also, the iPSCs were differentiated into definitive endoderm, which is the first step in bringing cells to the liver cell stage. The drug Rapamycin and activin (50 ng / L for those who are interested) were used to bring the growing iPSCs to the definitive endoderm.  The cells expressed all kinds of endoderm-specific genes.  Endoderm is the embryonic germ layer from which the digestive system and its accessory organs forms.

 Induction of hiPSCs into definitive endoderm. (A) Diagrammatic representation of the experimental groups for endoderm induction of hiPSCs in the bacterial dish static suspension, which include rapamycin (Rapa) “priming” and activin A “inducing” phases, and positive control groups of hiPSCs cultured in the absence of Rapa in suspension or adherent cultures in the presence of activin A. (B) Gene expression analysis of hiPSCs induced into endodermal cells. Quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) showed no significant differences in SOX17 and FOXA2 [definitive endoderm (DE) markers], and BRA (mesoendoderm marker) in all groups. SOX7 (visceral endoderm marker) was expressed at a low level. Influence of the refreshment strategy of the induction medium on DE formation in suspension cultures by qRT-PCR (C), immunostaining (D), and flow cytometry (E). We compared single (SR), double (DR), and triple (TR) refreshment of induction medium per 24 h for 4 days after Rapa administration in the static suspension of hiPSCs. Scale bar: 100 μm. The target gene expression level in qRT-PCR was normalized to GAPDH and calibrated with (presented relative to) hiPSCs. Data are presented as mean±SD. Statistical analysis as determined by one-way ANOVA with Tukey's post hoc test, n=3 for B, C, and E. *P<0.05, **P<0.01.
Induction of hiPSCs into definitive endoderm. (A) Diagrammatic representation of the experimental groups for endoderm induction of hiPSCs in the bacterial dish static suspension, which include rapamycin (Rapa) “priming” and activin A “inducing” phases, and positive control groups of hiPSCs cultured in the absence of Rapa in suspension or adherent cultures in the presence of activin A. (B) Gene expression analysis of hiPSCs induced into endodermal cells. Quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) showed no significant differences in SOX17 and FOXA2 [definitive endoderm (DE) markers], and BRA (mesoendoderm marker) in all groups. SOX7 (visceral endoderm marker) was expressed at a low level. Influence of the refreshment strategy of the induction medium on DE formation in suspension cultures by qRT-PCR (C), immunostaining (D), and flow cytometry (E). We compared single (SR), double (DR), and triple (TR) refreshment of induction medium per 24 h for 4 days after Rapa administration in the static suspension of hiPSCs. Scale bar: 100 μm. The target gene expression level in qRT-PCR was normalized to GAPDH and calibrated with (presented relative to) hiPSCs. Data are presented as mean±SD. Statistical analysis as determined by one-way ANOVA with Tukey’s post hoc test, n=3 for B, C, and E. *P<0.05, **P<0.01.
After the cells went through this culture protocol, they were grown in a stirred liquid culture called a “spinner.” The culture system contain a cocktail of growth factors that differentiated the definitive endoderm cells into HLCs.  The cells formed little spheres that expressed a host of liver-specific genes.

Differentiation of hepatocyte-like cells (HLCs) from hiPSCs in the stirred bioreactor. (A) Stepwise protocol for differentiation of hiPSCs into HLCs. (B) The morphology and cross section of spheroids at day 21. The spheroids in this step were observed as cystic and dense spheroids. Hematoxylin and eosin (H&E) staining of spheroid cross sections indicated cystic and dense epithelioid appearances in the transplant and dense spheroids, respectively. Scale bar: 100 μm. (C) Comparative relative mRNA expression in cystic and dense spheroids normalized to GAPDH and calibrated to undifferentiated hiPSCs. Transplant and dense spheroids expressed more early and late hepatic lineage markers, respectively. Data are presented as mean. n=3. (D) Transmission electron microscopy (TEM) of differentiated cells in dense spheroids at day 21. Nucleus (N), nucleoli (n), mitochondria (M), Golgi apparatus (G), lysosomes (Ly), rough endoplasmic reticuli (arrowhead), glycogen granules (GR), intermediate filaments (CK), tight junctions (TJ), gap junctions (GJ), fascia adherens (FA), junctional complex (JC), microvilli (MV), and bile-like canaliculus (BLC). Scale bar: 1 μm.
Differentiation of hepatocyte-like cells (HLCs) from hiPSCs in the stirred bioreactor. (A) Stepwise protocol for differentiation of hiPSCs into HLCs. (B) The morphology and cross section of spheroids at day 21. The spheroids in this step were observed as cystic and dense spheroids. Hematoxylin and eosin (H&E) staining of spheroid cross sections indicated cystic and dense epithelioid appearances in the transplant and dense spheroids, respectively. Scale bar: 100 μm. (C) Comparative relative mRNA expression in cystic and dense spheroids normalized to GAPDH and calibrated to undifferentiated hiPSCs. Transplant and dense spheroids expressed more early and late hepatic lineage markers, respectively. Data are presented as mean. n=3. (D) Transmission electron microscopy (TEM) of differentiated cells in dense spheroids at day 21. Nucleus (N), nucleoli (n), mitochondria (M), Golgi apparatus (G), lysosomes (Ly), rough endoplasmic reticuli (arrowhead), glycogen granules (GR), intermediate filaments (CK), tight junctions (TJ), gap junctions (GJ), fascia adherens (FA), junctional complex (JC), microvilli (MV), and bile-like canaliculus (BLC). Scale bar: 1 μm.

From the figure above, we can see that these HLCs, not only express liver-specific genes, but when they are examined in the electron microscope they look, for all intents and purposes, like liver cells.  Functional tests of these spheres of HLCs showed that they 1) took up low-density lipoprotein; 2) produced albumin (a major blood plasma protein); 3) expressed cytochrome P450s, which are the major enzymes used to process drugs; 4) produced urea from amino acids, just like real liver cells; 5) accumulated glycogen; 6) and made liver proteins (HNF4a, ALB, etc).

So it looks like liver, quacks like liver, but can it replace liver?  These HLCs were transplanted into the spleen of mice whose livers had been treated with carbon tetrachloride.  Carbon tetrachloride tends to make mincemeat of the liver, and these mice are in trouble, since their livers are toast.  Transplantation of the iPSC-derived HLCs into the spleens of these mice increased their survival rate and decreased the blood levels of liver enzymes that are usually present when there is liver damage.

This paper is significant because the procedure used provides an example of a “scalable” protocol for making large quantities of iPSCs, and their mass differentiation into definitive endoderm and then liver cells,  Because this can potentially provide enough cells to replace a nonfunctional liver, it represents a major step forward in regenerative medicine.