Multipotent Adult Progenitor Cells for Immunomodulation after Liver Transplantation

Mesenchymal stem cells and multipotent adult progenitor cells (MAPCs) have received a good deal of discussion by scientists as agent for solid organ transplant recipients. Why? Because these cells, with their ability to suppress unwanted immune responses might be able to reduce the need for drugs that suppress the immune system, which have extensive side effects.

The study under discussion today is the clinical course of the first patient of the phase I, dose-escalation safety and feasibility study, MiSOT-I (Mesenchymal Stem Cells in Solid Organ Transplantation Phase I).

The patient received a living-related liver graft, each patient was given one intraportal injection (injection into the portal vein) and one intravenous infusion of third-party MAPC in combination with a low-dose of an anti-tissue-rejection drug.

The results so far are still coming in, but it seems that the administration of the cells is easy and is technically feasible. How well did the patients tolerate them? Quite well it turns out. There was no evidence of acute toxicity associated with infusions of the MAPCs. Also, there was some indication that the patient’s white blood cells were less reactive to foreign substances. However, it is difficult to make definitive statements about the efficacy of this treatment at this time.

Recruitment and follow-up of participants in the MiSOT-I trial continue, and completion of the study is currently projected for autumn 2016.

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.

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.

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.