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.

Mesenchymal Stem Cells Found Around Blood Vessels in the Liver

Mesenchymal stem cells (MSCs) are found throughout the body and it is possible that every organ in our body has a MSC population. MSCs have the ability to differentiate into three main tissues: bone, fat and cartilage. However, the efficiency of this differentiation differs from one MSC population to another. Also, some MSCs can form smooth muscle for blood vessels and there is even evidence that MSCs can form blood vessels under certain conditions (for example, see Wingate K, Bonani W, Tan Y, Bryant SJ, Tan W. Acta Biomater. 2012 8(4):1440-9. doi: 10.1016/j.actbio.2011.12.032).

One of the places MSCs are usually found is around blood vessels. MSCs like to hang out on the outside of blood vessels in some tissues, and for this reason, MSCs are sometimes called “perivascular” stem cells.

One organ that has a stem cell population is the liver, but there is disagreement as to where they reside. Now a new publication has established that cells that hang out near blood vessels in liver are the MSC population in liver.

Eva Schmelzer from the McGowan Institute for Regenerative Medicine at the University of Pittsburgh has published a fine paper in the journal Stem Cells and Development detailing, with the help of her trusty laboratory colleagues, the characterization of liver MSCs.

Briefly, Schmelzer and her colleagues obtained fetal and adult lover tissue from tissue suppliers and minced them up, digested them with the appropriate enzymes, pushed them through cell strainers and then destroyed all the contaminating red blood cells. The remaining cells were grown in a cell culture medium. The stem cells would outgrow all the other cells, which would make their isolation and purification easy.

To purify the cells, Schmelzer’s co-workers used a technique called “flow cytometry.” When they had purified the liver MSCs, they set about characterizing them.

The liver MSCs grew quite well in culture and also grew quickly. They also expressed lots of surface proteins normally found on MSCs, confirming that they are MSCs. When gene expression experiments examined what genes these MSCs expressed, they expressed some smooth muscle genes and a several other genes enriched in cells near blood vessels. When Schmelzer examined cross sections of liver to determine where these cells are located, she found them curled up next to blood vessels.

In culture, the liver MSCs did not make very good cartilage or fat. However, they did make very good smooth muscle and bone. The efficiency of MSC differentiation tends to depend on where they were isolated. The rule of thumb is that MSCs most easily differentiate into those tissues that are closest to their own tissue of origin. Therefore, we would expect bone marrow MSCs to make better bone and cartilage than fat-based MSCs, and we would expect fat-based MSCs to make better fat than bone or liver-based MSCs. The ability of liver MSCs to be so good and making bone might be a little surprising, but when we consider that bone marrow stem cells begin their lives in the liver before they migrate to the bone marrow, perhaps this finding makes more sense.

In short, the adult and fetal liver contain a MSC population that is found on the outside of the blood vessels and these cells have an excellent capacity to make bone and smooth muscle for blood vessels. Thus liver biopsies might provide do more than provide material for diagnostic purposes – they might secure cells for regenerative purposes.

Umbilical Mesenchymal Stem Cells Improve the Symptoms of Patients With Decompensated Liver Cirrhosis

One of the most central organs for the body’s metabolism is the liver. When the gastrointestinal tract absorbs food molecules, the first stop for most of these molecules is the liver. The liver makes many blood-specific proteins, detoxifies foreign molecules to make them more water-soluble so that the body can excrete them, and stores energy reserves in the form of glycogen. Consequently, damage to the liver from chronic liver infections (e.g., hepatitis B & C, bilharzia or schistosomiasis, illegal drug use, etc.), alcoholism, or exposure to liver-damaging chemicals (carbon tetrachloride, chloroform, etc.) seriously compromises the capacity of the body to store energy, process food molecules, make blood specific proteins (which include clotting factors), and process and synthesize metabolic wastes. Repeated damage to the liver causes extensive scarring and deposition of fatty tissues, and such a condition is called “cirrhosis.”

Cirrhosis ultimately leads to liver failure, and tough scar tissue with nodules replaces once healthy liver tissue. There are two main types of cirrhosis. Compensated cirrhosis of the liver refers to early liver damage in which the body functions well despite the damaged liver tissue. Even though liver function is decreases, the body still operates within normal parameters, and the patient often shows no symptoms of disease. Even though people with compensated liver cirrhosis are often asymptomatic, they may display symptoms of weakness, fatigue, loss of appetite, vomiting, weight loss and easy bruising. Liver function tests may reveal increased levels of certain liver enzymes. Liver damage is not reversible, but treating the underlying cause can prevent further damage. Additionally, constant monitoring is required for the early detection of loss of liver function that leads to life-threatening complications.

Decompensated liver cirrhosis is a life-threatening complication of chronic liver disease, and it is also one of the major indications for liver transplantation. The symptoms of decompensated cirrhosis are internal bleeding from the esophagus (bleeding varices), fluid in the belly (ascites), confusion (encephalopathy), yellowing of the eyes and skin (jaundice). When someone becomes this sick, there is little to be done, but receive a liver transplant.

Can stem cells help patients with decompensated liver cirrhosis? Perhaps they can. A paper from the Journal of Gastroenterology and Hepatology (2012; 27 Suppl 2:112-20) has examined the ability of human umbilical cord mesenchymal stem cells to improve symptoms in patients with decompensated liver cirrhosis (DLC). The paper’s first author is Z. Zhang and the title of the paper is “Human Umbilical Cord Mesenchymal Stem Cells Improve Liver Function and Ascites in Decompensated Liver Cirrhosis Patients.” These authors are from the Research Center for Biological Therapy at the Beijing 302 Hospital, in Beijing, China.

In this study, the safety and efficacy of umbilical cord-derived MSCs (UC-MSC) were infused into in patients with DCL. They used a total of 45 chronic hepatitis B patients, all of whom were diagnosed with DCL. 30 patients received transfusions of UC-MSCs, and another 15 patients were given saline as the control. After transfusions, all 45 patients were followed for a 1-year follow-up period.

In none of the 45 patients who were infused, were any significant side-effects observed. Also, there were no significant complications were observed in either group. As to the symptoms suffered by the patients, those who had received the UC-MSC transfusion showed a significant reduction in the volume of ascites in comparison to those patients who had received the control saline transfusions. When liver function parameters were examined, UC-MSC therapy also significantly increased of serum albumin levels (albumin is made by the liver), decreased in total serum bilirubin levels (bilirubin is a waste that is processed by the liver), and stabilized the sodium levels for patients (patients with cirrhosis have low blood sodium levels).

Further follow-up of these patients is clearly warranted, but for the year follow-up. It seems clear that UC-MSC transfusions are clinically safe. Furthermore, when compared to controls, they also seem to improve liver function and reduce the volume of belly fluid in patients with DCL. UC-MSC transfusions might represent a novel therapeutic approach for patients with DCL.