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).


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.