Liver cirrhosis is scarring of the liver, resulting from injury or long-term disease, and is primarily characterized by abnormal liver structure and function. The main causes of cirrhosis are alcoholism, viral hepatitis and genetic disorders. The liver continues to regenerate and create new cells; however, intracellular relationships within the tissue are deranged and extracellular matrix is deposited. Liver cirrhosis induces edema and ascites, esophageal varices bleeding, and complications in the kidney, lungs, spleen and cognition and may progress to hepatocellular carcinoma. The therapeutic goals in treating cirrhosis patients include arrest of disease progression and restoration of liver function. These aims may be achieved by diet, viral treatment, immunomudolatory drugs and liver transplantation. However, transplantation is limited by donor shortage, high cost, risk and immune rejection. Therefore, recent works have been focusing on the ability to use cellular resources to bridge patients until transplantation or to restore liver mass and function via less invasive procedures (i.e. cellular intraportal administration).
Cell therapy for liver cirrhosis patients is directed toward: 1) improved regeneration and reduced scarring by modulating the liver's own regenerative processes and stimulating hepatocytes and oval cells, (2) down-regulation of immune-mediated liver damage, (3) supply of stem cells-derived hepatocyte-like cells (HLCs) for use in extracorporeal bio-artificial liver (BAL) machines, and (4) use of HLCs derived from different sources, such as stem or progenitor cells, for cell transplantation to supplement or replace hepatocyte function.
Biological support systems such as BAL machines have shown great promise and demonstrate the importance of both restoration of failing detoxification, and restoration of hepatic metabolic function. The development of a humanized BAL has, to date, been limited by the inability to generate efficient numbers of metabolically competent primary human hepatocytes.
Without a mitotic challenge and a regenerative advantage, such as in a genetic metabolic defect, to the donor hepatocytes, repopulation is rather low (approximately 1%). There is doubt whether this is sufficient to supply the metabolic capacity necessary to overcome the malfunction of the damaged liver. Modern cell sources include primary hepatocytes, immortalized cell lines, and an array of stem cells, including liver stem cells, bone marrow stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells. Many of these treatments are autologous requiring no immunosuppression, which is a great advantage.
Some investigators have developed immortalized human hepatocytes via spontaneous transformation, introduction of telomerase constructs, or retroviral transfection. C3A, a subclone of the HepG2 hepatoblastoma cell line, is the only human-based cell line that has been clinically tested in a BAL device with no evidence of C3A cell transmission ever reported. Because of the risk of tumor transmission, the clinical use of immortalized cell lines such as C3A has been limited to extracorporeal devices which possess a membrane to block cell spread to the patient. In an attempt to bypass the limitations associated with tumor cells, researchers have attempted to immortalize hepatocytes from non-tumor-derived hepatocytes, which may be useful as a potential source of liver support in BAL systems.
Many protocols have been developed for the differentiation of hepatic and extra-hepatic stem cells into hepatocytes. However, they are all at the preclinical stages, inducing improvement in the clinical status of assorted animal models.
Various studies have reported that cells from the bone marrow are released into the circulation, migrate to the liver and differentiate into hepatocytes. However, the extent to which this occurs (< 0.01% to 40%) and the mechanism(s) involved remain highly controversial. Stem cells derived from the peripheral blood or the bone marrow are administered to animal models, with or without culturing, via the hepatic artery or a peripheral vein. This protocol has been studied in variable animal models leading to improvement in liver function however, there is great controversy whether the cells transdifferentiate or fuse to other cells.
The role of mesenchymal stem cells (MSCs) in liver cirrhosis is also arguable, as demonstrated in various animal models. Some claim that MSCs play a role in the pathology of liver cirrhosis, constituting a source for myofibroblasts that produce fibrosis, while others claim MSCs stimulate liver regeneration and restore liver function during chronic liver injury by enhancing the degradation of liver fibrosis. MSCs have been shown to ameliorate liver fibrosis in mice and rats, which is likely due to the reduction of collagen synthesis and the induction of expression of metalloproteinases, the major players in matrix degradation and remodeling. Futhermore, MSCs have been reported to contribute to the direct production of new hepatocytes, as well as to stimulate proliferation of endogenous hepatocytes. However, irrespective of the site of application, i.e., systemic infusion, intrahepatic injection, intrasplenic delivery, or portal vein infusion, MSCs were found in the host liver forming clusters of donor cells.
Embryonic stem (ES) cells can be induced along the endodermal and hepatocytic lineages in culture and then transplanted into the liver differentiating into both mature hepatocytes and bile duct epithelial cells. However, lineage-specified ES cells are not yet therapeutically effective as a result of low yield and may also be tumorigenic.
Induced pluripotent stem cells (iPS) are generated from somatic cells complemented with pluripotency factors, introduced via viral, chemical, and DNA-mediated delivery techniques. All of these methods raise safety concerns, which, in addition to the tendency of the iPS to form teratomas, restrict the clinical use of these cells so far. However, first liver repopulation experiments in mice demonstrated the high regenerative potential of iPS, demonstrating their clinical relevance.
Clinical and pre-clinical trials
Phase I/II clinical trials have involved transplantion of primary hepatocytes to treat inherited metabolic diseases (e.g., Crigler-Najjar Disease, familial hypercholesterolemia, urea cycle disorders) or liver failure, in patients who are unlikely to survive without extensive medical therapy or transplantation as a 'bridge' to whole-organ transplantation or improved liver function. Clinical hepatocyte transplantation efficacy ranged from 0% to a maximum of 12%, and the graft was often lost within several months or years. However, during this limited time normalization of liver functions was achieved. Phase I trials employing a BAL device with xenogenic material (porcine hepatocytes) improved both the patients' neurological state and hemodynamics.
Results of clinical studies strongly suggest that liver function-improving effects can be achieved by infusion of stem cells. CD34-positive cells, derived from the peripheral blood following G-CSF induction, administered via the hepatic artery, were reported to improve serum levels of bilirubin and albumin, increasing proliferation of hepatic progenitor cells. However, caution must be exercised when administering G-CSF, as it may induce rupture of the spleen.
Infusion of autologous bone marrow cells via a peripheral vein yielded significantly improved serum albumin levels, total protein levels, and Child–Pugh score in treated patients. There was also an improvement in quality of life, with no serious adverse events. Bone marrow-derived cells injected via the hepatic artery improved hepatic function in the early period. Long-term observations showed no change in the incidence of hepatocellular carcinoma after the administration of bone marrow cells, suggesting the possibility of an improved survival rate. Unselected autologous bone marrow cells infused via the portal vein improved serum albumin levels, total protein levels, and CTP score at 6 months in liver cirrhosis patients.
In patients suffering from decompensated liver cirrhosis, treatment with umbilical cord-derived MSCs reduced ascites volume and improved liver function in the short-term range. Patients with end-stage liver failure demonstrated reduced ascites volume and improvements in Child–Pugh score after autologous bone marrow-derived MSC transplantation. These phase I/II clinical trials demonstrated safety of hepatic MSC transplantation, but efficacy still awaits confirmation. Even if some clinical parameters improve, the fate and long-term survival of the transplanted cells in the host liver, their mode of action, and long-term safety remain to be demonstrated. In other trials, bone marrow-derived MSCs were isolated and cultured for 2 weeks before infusing to the patients. This treatment resulted in improved liver function.
Cultured autologous bone marrow-derived cells (stimulated to hepatic lineage using hepatic growth factor) were transmitted into the spleen or the liver, inducing improvement in liver function. Hepatic integration and function of human adipose tissue-derived MSCs pre-differentiated into hepatocyte-like cells prior to transplantation, were shown in CCl4-treated mice. A significant increase of liver function post-liver resection has been documented in patients with cirrhosis pre-treated with autologous MSCs transplantation.
Mouse ES cell-derived hepatocytes have improved survival and liver function in a number of injury models including carbon tetrachloride (CCl4), dimethylnitrosamine-induced cirrhosis without teratoma formation, though low percentages of donor-derived cells were present. Human ES cell-derived hepatocytes have been demonstrated to integrate into the CCl4-severely injured SCID mouse liver, without evidence of teratoma formation.
Bieke Biotech is evaluating the safety and efficacy of NU215-01, human umbilical cord mesenchymal stem cells, for treatment of liver cirrhosis. Stempeutics are studying StempeucelLC™, autologous ex vivo-expanded bone marrow-derived mesenchymal stem cells, for the treatment of cirrhosis.