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.

New Liver Stem Cell Might Aid in Liver Regeneration

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

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

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

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

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

KDR Protein Crystal Structure
KDR Protein Crystal Structure

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

Mature Liver Cells Seem to be Better Than Stem Cells for Liver Therapy

Japanese research team has compared the ability of liver progenitor cells (liver stem cells) and mature liver cells to effectively repopulate a damaged liver. They have concluded that mature liver cells (hepatocytes) are better than stem cells for liver repopulation.

Workers in the laboratory of Toshihiro Mitaka of the Cancer Research Institute of the Sapporo Medical University School of Medicine, Sapporo, Japan used a rat model to test these hypotheses. They injured the livers of these rats with surgery and chemicals and then used transplanted cells to repopulate these livers. Up to two weeks after transplantation, the growth rate of the stem cells was significantly higher than that of the mature liver cells, but after two weeks the majority of the stem cells died before they could confer any significant benefit on the liver. The mature cells, however, grew slower, but survived much better.

Toshihiro Mitaka made this comment: “Cell-based therapies as an alternative to liver transplantation to treat liver disease have shown promise. However, the repopulation efficiency of hepatic progenitor/stem cells and mature hepatocytes (liver cells) had not been comprehensively assessed and questions concerning the efficiency of each needed to be resolved.”

Mitaka’s team noticed that the shortage of liver cell sources and the difficulties of preserving the available liver cell sources by freezing (cryopreservation) have limited the available material, and therefore, the clinical applications of cell-based therapies for liver disease. Liver stem cells (liver progenitor cells) have been considered to be the best option to treat liver disease, since they can be expanded in culture and preserved by freezing for long periods of time.

However, once the liver progenitor cells were transplanted into the damaged livers of rats, the stem cells failed to survive terribly well. After two months, the vast majority of the transplanted stem cells had disappeared. In contrast, the mature liver cells gradually repopulated the rat livers and the even continued growing and repopulating the damaged livers for one year after transplantation.

Transplanted liver cells did not make uniform cells. Mitaka noted that “the small hepatocytes repopulated significantly less well than the larger ones. We also found that serial transplantations did not enhance nor diminish the repopulation capacity of the cells to any significant degree.”

In this paper, Mitaka and his colleagues argue that because the stem cells died much earlier than the mature hepatocytes, the stem cells were eliminated from the host livers and this reduced their potential regenerative capacity. They conclude in the paper that further “experiments are required to clarify the mechanism by which this might occur.”

My take on this is that damaged livers probably contain a respectable amount of inflammation. Therefore, they are probably a rather hostile environment for any transplanted cells. We have seen in previous posts that stem cells have a mechanism to resist stressful conditions, but also, that this pathway for resisting stress must be activated in the stem cells before they are capable of resisting stress. Therefore, I would suggest that the next experiment Mitaka and his co-workers should do, is to either precondition his stem cells with oxygen and glucose deprivation or pre-treat them with insulin-like growth factor-1 (IGF-1). Both of these treatments have been shown to activate the stress resistant pathway in stem cells transplanted into the heart. Therefore, if this pathway could be induced in liver progenitor cells, then perhaps the stem cells can be stress-adapted and tolerate the stressful conditions in the damaged livers.

High Blood Pressure Medicine Improves Mesenchymal Stem Cell Treatments of Heart Attacks

Mesenchymal stem cells (MSCs) exist in a variety of places throughout the body. They are found in bone marrow, the lower levels of the skin, umbilical cord and umbilical cord blood, placenta, amniotic membrane, muscle, blood vessels, liver, synovial membranes that surround joints, endometrial glands, fat, tendons, and other locations as well. MSCs have the ability to differentiate into cartilage-making cells, fat-making cells, muscle-making cells or bone-making cells  Other protocols exist to differentiate MSCs into heart muscle (Williams AR, Hare JM. Mesenchymal stem cells: biology, pathophysiology, translational findings, and therapeutic implications for cardiac disease.Circ Res. 2011 Sep 30;109(8):923-40; Song YH, Pinkernell K, Alt E.Stem cell induced cardiac regeneration: fusion/mitochondrial exchange and/or transdifferentiation? Cell Cycle. 2011 Jul 15;10(14):2281-6.), neurons (Scuteri A, Miloso M, Foudah D, Orciani M, Cavaletti G, Tredici G.Mesenchymal stem cells neuronal differentiation ability: a real perspective for nervous system repair? Curr Stem Cell Res Ther. 2011 Jun;6(2):82-92), and liver cells (Al Battah F, De Kock J, Vanhaecke T, Rogiers V. Current status of human adipose-derived stem cells: differentiation into hepatocyte-like cells. ScientificWorldJournal. 2011;11:1568-81).  The therapeutic possibilities of MSCs has been widely recognized by stem cell scientists and MSCs have been the subject of many past and ongoing clinical trials.

The use of MSCs to treat heart attack patients has been the subject of several clinical trials (Mazo M, Araña M, Pelacho B, Prosper F. Mesenchymal stem cells and cardiovascular disease: a bench to bedside roadmap. Stem Cells Int. 2012;2012:175979).  While MSCs do provide a modicum of healing to damaged hearts, the ability of MSCs to differentiate into heart muscle is low.  Many experiments have focused upon increasing the percentage of implanted MSCs  that differentiate into heart muscle cells.  However, a recent paper from a research group at the Keio University School of Medicine and the National Institute for Child Health and Development in Tokyo, Japan has taken a different approach to this problem.

Drugs that treat blood pressure include the “angiotensin II receptor blockers” or ARBs.  ARBs prevent a small polypeptide called angiotensin II from binding its receptor.  WHen it binds to its receptor, angiotensin II causes rather substantial constriction of blood vessels throughout the body, and this raises blood pressure.  By preventing blood vessel constriction, ARBs can lower blood pressure.  Also, many heart attack patients are on blood pressure medicines, and ARBs are one of the those normally given to heart attack patients.

One particular ARB is called candesartan, and the commercial names are Atacand, Amias, Blopress, and Ratacand.  In this paper by Yohei Namasawa and colleagues in the laboratories of Kaoru Segawa, Satoshi Ogawa, and Akihiro Umezawa, determined if treating human MSCs from bone marrow could increase the ability of these cells to form heart muscle cells.  To induce heart muscle cells, they used a popular technique from the literature that grows MSCs in culture with mouse heart muscle cells.  The interaction between the MSCs and the heart muscle cells in culture drives the MSCs to form heart muscle-like cells at a somewhat low-frequency.  This group determined if MSCs became heart muscle cells by testing for the presence of heart muscle-specific proteins (cardiac-specific troponin-I).  To prevent them from confusing MSCs with the mouse heart muscle cells, the MSCs were pre-labeled with a fluorescent protein.

Candesartan treatment of MSCs more than doubled the ability of MSCs to form heart muscle cells in culture.  When these same cells were transplanted into the hearts of rats that had suffered heart attacks, the results were even more interesting.  MSC transplantation into the hearts of rats that had recently suffered a heart attack.  Those animals that had undergone surgery but were not given any heart attacks, showed an average reduction of about 3% in their ejection fraction (percentage of blood that pumped from the heart during each heart beat).  Given that the standard deviation was close to this number, this change is not significant.  The control animals that were not given MSC treatments showed an average decrease of just over 10% in their ejection fraction.  Animals treated with MSCs that had suffered heart attacks showed a decrease of about 6-7%.  This is significantly less of a decrease than in the control, but it is still a decrease.  When the rat hearts were treated with MSCs that had been pretreated with candesartan, they showed an average 3-4% increase in ejection fraction.  If the rats were given candesartan after the heart attack, it raised the ejection fraction 1-2%.  If the rats were given candesartan, and treated with bone marrow cells after the heart attack, their ejection fractions decreased by the same as the sham group.  However, if the rats were given candesartan and MSCs that had been pretreated with candesartan after the heart attack, their ejection fractions increased by 10-12%.  Other heart function indicators improved too, since transplantation of the candesartan-treated bone marrow cells improved the “end systolic dimension,” which is an indication of how well the heart contracts.

When hearts were examined after the animals died, those animals that had received transplantations of the candesartan-pretreated bone marrow cells had 2-3 times more heart muscle cells derived from the implanted MSCs than did the controls transplanted with non-treated bone marrow.  Also, post-mortem examination of hearts from the treated rats showed that the rats treated with candesartan-pretreated bone marrow cells had much small heart scars than the other groups (5%-7% smaller).

These experiments, though pre-clinical, suggest that pre-treatment of MSCs with compounds like candesartan can increase their ability to differentiate into heart muscle cells.  This would certainly augment their ability of heal the hearts of patients after a heart attack.  While further work is certainly warranted, a clinical study should be proposed to test if this efficacy applies to human hearts as well.