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.

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.