Mesenchymal Stem Cells for Liver Regeneration in Liver Failure: From Experimental Models to Clinical Trials.

The liver centralizes the systemic metabolism and thus controls and modulates the functions of the central and peripheral nervous systems, the immune system, and the endocrine system. In addition, the liver intervenes between the splanchnic and systemic venous circulation, determining an abdominal portal circulatory system. The liver displays a powerful regenerative potential that rebuilds the parenchyma after an injury. This regenerative mission is mainly carried out by resident liver cells. However, in many cases this regenerative capacity is insufficient and organ failure occurs. In normal livers, if the size of the liver is at least 30% of the original volume, hepatectomy can be performed safely. In cirrhotic livers, the threshold is 50% based on current practice and available data. Typically, portal vein embolization of the part of the liver that is going to be resected is employed to allow liver regeneration in two-stage liver resection after portal vein occlusion (PVO). However, hepatic resection often cannot be performed due to advanced disease progression or because it is not indicated in patients with cirrhosis. In such cases, liver transplantation is the only treatment possibility, and the need for transplantation is the common outcome of progressive liver disease. It is the only effective treatment and has high survival rates of 83% after the first year. However, donated organs are becoming less available, and mortality and the waiting lists have increased, leading to the initiation of living donor liver transplantations. This type of transplant has overall complications of 38%. In order to improve the treatment of hepatic injury, much research has been devoted to stem cells, in particular mesenchymal stem cells (MSCs), to promote liver regeneration. In this review, we will focus on the advances made using MSCs in animal models, human patients, ongoing clinical trials, and new strategies using 3D organoids.

Immunosuppressive properties of mesenchymal stem cells: advances and applications.

Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, such as bone marrow, skeletal muscle, dental pulp, bone, umbilical cord and adipose tissue. MSCs are used in regenerative medicine mainly based on their capacity to differentiate into specific cell types and also as bioreactors of soluble factors that will promote tissue regeneration from the damaged tissue cellular progenitors. In addition to these regenerative properties, MSCs hold an immunoregulatory capacity, and elicit immunosuppressive effects in a number of situations. Not only are they immunoprivileged cells, due to the low expression of class II Major Histocompatibilty Complex (MHC-II) and costimulatory molecules in their cell surface, but they also interfere with different pathways of the immune response by means of direct cell-to-cell interactions and soluble factor secretion. In vitro, MSCs inhibit cell proliferation of T cells, B-cells, natural killer cells (NK) and dendritic cells (DC), producing what is known as division arrest anergy. Moreover, MSCs can stop a variety of immune cell functions: cytokine secretion and cytotoxicity of T and NK cells; B cell maturation and antibody secretion; DC maturation and activation; as well as antigen presentation. It is thought that MSCs need to be activated to exert their immunomodulation skills. In this scenario, an inflammatory environment seems to be necessary to promote their effect and some inflammation-related molecules such as tumor necrosis factor-α and interferon-γ might be implicated. It has been observed that MSCs recruit T-regulatory lymphocytes (Tregs) to both lymphoid organs and graft. There is great controversy concerning the mechanisms and molecules involved in the immunosuppressive effect of MSCs. Prostaglandin E2, transforming growth factor-ß, interleukins- 6 and 10, human leukocyte antigen-G5, matrix metalloproteinases, indoleamine-2,3-dioxygenase and nitric oxide are all candidates under investigation. In vivo studies have shown many discrepancies regarding the immunomodulatory properties of MSCs. These studies have been designed to test the efficacy of MSC therapy in two different immune settings: the prevention or treatment of allograft rejection episodes, and the ability to suppress abnormal immune response in autoimmune and inflammatory diseases. Preclinical studies have been conducted in rodents, rabbits and baboon monkeys among others for bone marrow, skin, heart, and corneal transplantation, graft versus host disease, hepatic and renal failure, lung injury, multiple sclerosis, rheumatoid arthritis, diabetes and lupus diseases. Preliminary results from some of these studies have led to human clinical trials that are currently being carried out. These include treatment of autoimmune diseases such as Crohn's disease, ulcerative colitis, multiple sclerosis and type 1 diabetes mellitus; prevention of allograft rejection and enhancement of the survival of bone marrow and kidney grafts; and treatment of resistant graft versus host disease. We will try to shed light on all these studies, and analyze why the results are so contradictory.

Portosystemic hepatic encephalopathy model shows reversal learning impairment and dysfunction of neural activity in the prefrontal cortex and regions involved in motivated behavior.

Hepatic encephalopathy (HE) is a neurological complication that affects attention and memory. Experimental animal models have been used to study HE, the most frequent being the portacaval shunt (PCS). In order to investigate learning impairment and brain functional alterations in this model, we assessed reversal learning and neural metabolic activity in a PCS rat model. PCS and sham-operated rats were tested for reversal learning in the Morris water maze. Brains were then processed for cytochrome oxidase (CO) histochemistry. The PCS group had reversal learning impairment and a reduction in CO activity in the prefrontal cortex, ventral tegmental area and accumbens shell nucleus. These results suggest that this model of portosystemic HE shows learning impairments that could be linked to dysfunction in neural activity in the prefrontal cortex and regions involved in motivated behavior.

The interstitial lymphatic peritoneal mesothelium axis in portal hypertensive ascites: when in danger, go back to the sea.

Portal hypertension induces a splanchnic and systemic low-grade inflammatory response that could induce the expression of three phenotypes, named ischemia-reperfusion, leukocytic, and angiogenic phenotypes.During the splanchnic expression of these phenotypes, interstitial edema, increased lymph flow, and lymphangiogenesis are produced in the gastrointestinal tract. Associated liver disease increases intestinal bacterial translocation, splanchnic lymph flow, and induces ascites and hepatorenal syndrome. Extrahepatic cholestasis in the rat allows to study the worsening of the portal hypertensive syndrome when associated with chronic liver disease. The splanchnic interstitium, the mesenteric lymphatics, and the peritoneal mesothelium seem to create an inflammatory pathway that could have a key pathophysiological relevance in the production of the portal hypertension syndrome complications. The hypothetical comparison between the ascitic and the amniotic fluids allows for translational investigation. From a phylogenetic point of view, the ancestral mechanisms for amniotic fluid production were essential for animal survival out of the aquatic environment. However, their hypothetical appearance in the cirrhotic patient is considered pathological since ultimately they lead to ascites development. But, the adult human being would take advantage of the potential beneficial effects of this "amniotic-like fluid" to manage the interstitial fluids without adverse effects when chronic liver disease aggravates.

Hepatectomies in the rat: A look at the caudate process through microsurgery.

Hepatectomies in the rat can be improved using microsurgical techniques. The distribution variations of the vascular and biliar lobular branches of the liver are observed under magnification with an operative microscope and, therefore their dissection, ligation and section are more accurate. The vascularization and bile drainage of the caudate process, a liver sector located between the right lateral and the caudate lobes, can be identified using microsurgery. The viability of the animal's evolution after different types (90%, 95%, 97%) of subtotal hepatectomies depends on an effective identification of these vascular and biliary branches.

Prehepatic portal hypertension worsens the enterohepatic redox balance in thioacetamide-cirrhotic rats.

BACKGROUND: Oxidative stress has been reported as a key pathogenic factor in many human liver diseases and in experimental models of cirrhosis related to hepatotoxin administration. The aim of this study was to verify the hypothesis that prehepatic portal hypertension aggravates the enterohepatic redox imbalance in thioacetamide-cirrhotic rats. MATERIALS AND METHODS: Wistar male rats were used: Control (n=9); rats with prehepatic portal hypertension by triple partial portal vein ligation (TPVL; n=9); thioacetamide-cirrhotic rats (TAA; n=9) and TPVL-rats associated to TAA administration (TPVL+TAA; n=9). Three months after the operation, portal pressure (PP), mesenteric venous vasculopathy (MVV) and portosystemic collateral circulation were studied. Liver and ileal levels of malondialdehyde (MDA), as a lipid peroxidation marker, and catalase (CAT), glutathione peroxidase (GSH-Px), glutathione transferase (GSH-t) and cytosolic and mitochondrial superoxide dismutases (cSOD and mSOD), as antioxidative enzymatic mechanisms, were measured. RESULTS: Liver and ileal MDA increased in all the experimental groups, although the higher increase occurred in the ileum of rats with portal hypertension. CAT levels decreased in the liver and the ileum in the three experimental groups. The decrease in liver and ileal GSH-Px and GSH-t was greater in rats with portal hypertension, alone or associated with TAA. mSOD activation was demonstrated in the liver when portal hypertension was added to TAA. On the contrary, this compensatory response was not activated in the ileum, where mSOD was significantly decreased. CONCLUSION: Prehepatic portal hypertension by triple partial portal vein ligation impaired the enterohepatic antioxidative activity and aggravated the intestinal oxidative stress in thioacetamide-cirrhotic rats.

The inflammatory response recapitulates phylogeny through trophic mechanisms to the injured tissue.

The post-traumatic local acute inflammatory response is described as a succession of three functional phases of possible trophic significance: 1. Nervous or immediate (ischemia-reperfusion); 2. Immune or intermediate (infiltration by inflammatory and bacterial cells) and 3. Endocrine or late (angiogenesis with regeneration and/or cicatrization). Each of these phases emphasizes the trophic role of the mechanisms in the damaged tissue. Hence, the nervous phase is predominated by nutrition by diffusion; in the immune phase trophism is mediated by inflammatory cells and bacteria and, finally, in the endocrine phase, the blood circulation and oxidative metabolism play the most significant nutritive role. Since these trophic mechanisms are of increasing complexity, progressing from anoxia to total specialization in the use of oxygen to obtain usable energy, it could be speculated that they represent the successive reappearance of the stages that take place during the evolution of life on Earth, from ancient times without oxygen. In this sense, the inflammatory response could recapitulate phylogeny through the successive expression of pathophysiologic mechanisms that have a trophic meaning to the injured tissue.

Progressive hepatocytic fatty infiltration in rats with prehepatic portal hypertension.

BACKGROUND/AIMS: Homogenous evolution, with a narrow range of portal hypertension, degree of portosystemic shunt and hepatic atrophy has been described in the experimental model of prehepatic portal hypertension in the rat. However the great differences observed in the rats' liver weight could be attributed to a pathological alteration of the liver. Based on this, we performed an evolutive histological study of the liver. This study shows the existence of a progressive hepatocytic fatty infiltration. METHODOLOGY: Male Wistar rats with portal hypertension induced by triple stenosing ligation of the portal vein at 1 month (group II, n=4) and at 1 year (group IV, n=10) of postoperative evolution were used. The portal pressure, body, liver and splenic weights, types of collateral circulation and degree of mesenteric venous congestion were studied. The intracytoplasmatic lipid microvacuoles were quantified in hepatocytes with an image analyzer (software MIP/CID, Spain). The results were compared with those obtained in control rats with the same evolutive periods (Groups I and III). RESULTS: The hepatic fatty infiltration in Group II (TPVS 1 month) (30.12+/-53.92 micron2) is similar to that presented by Group III (Control 1 year) (16.52+/-45.20 micron2), while there is an increase (p<0.001) in Group IV (triple portal vein stenosis 1 year) (182.03+/-371.42 micron2) in relation to the other groups studied. The progressive hepatic fatty infiltration in triple portal vein stenosis rats is associated with a decrease of portal pressure and of the incidence of liver hepatic atrophy, portosystemic collateral circulation and mesenteric venous congestion. CONCLUSIONS: TPVS produces progressive hepatocytic fatty infiltration in the rat so that this prehepatic portal hypertension experimental model could also be considered as a hepatic steatosis model.

Evaluation of two experimental models of hepatic encephalopathy in rats.

The serious neuropsychological repercussions of hepatic encephalopathy have led to the creation of several experimental models in order to better understand the pathogenesis of the disease. In the present investigation, two possible causes of hepatic encephalopathy, cholestasis and portal hypertension, were chosen to study the behavioral impairments caused by the disease using an object recognition task. This working memory test is based on a paradigm of spontaneous delayed non-matching to sample and was performed 60 days after surgery. Male Wistar rats (225-250 g) were divided into three groups: two experimental groups, microsurgical cholestasis (N = 20) and extrahepatic portal hypertension (N = 20), and a control group (N = 20). A mild alteration of the recognition memory occurred in rats with cholestasis compared to control rats and portal hypertensive rats. The latter group showed the poorest performance on the basis of the behavioral indexes tested. In particular, only the control group spent significantly more time exploring novel objects compared to familiar ones (P < 0.001). In addition, the portal hypertension group spent the shortest time exploring both the novel and familiar objects (P < 0.001). These results suggest that the existence of portosystemic collateral circulation per se may be responsible for subclinical encephalopathy.

Evaluation of two experimental models of hepatic encephalopathy in rats

The serious neuropsychological repercussions of hepatic encephalopathy have led to the creation of several experimental models in order to better understand the pathogenesis of the disease. In the present investigation, two possible causes of hepatic encephalopathy, cholestasis and portal hypertension, were chosen to study the behavioral impairments caused by the disease using an object recognition task. This working memory test is based on a paradigm of spontaneous delayed non-matching to sample and was performed 60 days after surgery. Male Wistar rats (225-250 g) were divided into three groups: two experimental groups, microsurgical cholestasis (N = 20) and extrahepatic portal hypertension (N = 20), and a control group (N = 20). A mild alteration of the recognition memory occurred in rats with cholestasis compared to control rats and portal hypertensive rats. The latter group showed the poorest performance on the basis of the behavioral indexes tested. In particular, only the control group spent significantly more time exploring novel objects compared to familiar ones (P < 0.001). In addition, the portal hypertension group spent the shortest time exploring both the novel and familiar objects (P < 0.001). These results suggest that the existence of portosystemic collateral circulation per se may be responsible for subclinical encephalopathy.

Microsurgical extrahepatic cholestasis in the rat: a long-term study.

An experimental model of microsurgical cholestasis is studied as an alternative to the most frequently used surgical techniques, based on the section of the common bile duct. This microsurgical technique consists of the resection of the extrahepatic biliary tract, that is, of the common bile duct in continuity with the bile ducts that drain the four lobes of the rat liver. At 30 days of evolution, rats with microsurgical cholestasis do not develop biliary pseudocysts or intraperitoneal hilar hepatopulmonary abscesses and show an increase (p < 0.001) in total bilirubin (9.50 +/- 1.50 mg/dL vs. 1.60 +/- 0.35 mg/dL), bile acids (225 +/- 87 micromol/L vs. 12.5 +/- 14.50 micromol/L), gamma-glutamyltranspeptidase (375 +/- 143 U/L vs. 8 +/- 11 U/L), and alkaline phosphatase (73 +/- 25 U/L vs. 23 +/- 4 U/L) levels. The histological study shows fibrosis with biliary proliferation. The microsurgical cholestasis technique is a valid alternative to other techniques and can be an adequate experimental model for the study of etiopathogenic mechanisms of obstructive jaundice and especially to study extrahepatic biliary atresia.

Posttraumatic inflammation is a complex response based on the pathological expression of the nervous, immune, and endocrine functional systems.

The successive phases that make up both the local and systemic posttraumatic acute inflammatory response could represent the expression of three concatenated pathological or "primitive" functional systems with trophic properties: the nervous, immune, and endocrine ones. The nervous functional system would play an important role in the phenomenon of ischemia-reperfusion, which would be represented by nutrition by diffusion that is either anaerobic (ischemia) or with defective use of oxygen (reperfusion) and, thus, with a limited energy requirement. The immune functional system would be represented by the infiltration of the tissues by inflammatory cells and bacteria, which would become mediators in providing nutrition to the injured tissues. Although the use of oxygen would still be defective, hypermetabolism and fever would occur. In these inflammatory response phases, the lymphatic is the most important circulation. The endocrine functional system would be the most specialized and would have high energy requirements because it would be represented by the blood capillary-mediated nutrition. Highly specialized epithelial cells would already possess a perfected oxidative metabolism. The successive expression of these three functional systems during embryonic development and also during the evolutionary development of our species could explain why the inflammatory response is a ubiquitous mechanism that is common to multiple diseases, because it is an integrator of the ontogeny and phylogeny.

Portal hypertension: return to fetal life to re-attempt differentiation?

We speculate on the final meaning of the alterations that characterize portal hypertensive enteropathy. The similarity of these alterations with certain morphofunctional characteristics of prenatal splanchnic development makes it possible to hypothesize that the dedifferentiation with return to early stages of development could constitute a portal hypertension induced pathogenic mechanism.

Budesonide ameliorates early portal hypertension in the rat: possible antiexudative splanchnic action.

Major portal pressure increase occurs on the second day post-stenosing-ligation of the portal vein in the rat and it is associated with pancreatic edema, intraperitoneal free exudate, hypoalbuminemia and hypoproteinemia. All this suggests the development of a regional exudative inflammatory response. In order to verify this hypothesis the steroid budesonide, whose antiinflammatory activity could prevent these alterations, was administered to rats with prehepatic portal hypertension. Wistar male rats were divided into the following groups: Control rats that were administered saline solution (CS; n = 10), Control rats that were administered budesonide (36 mg/kg per day; CB; n = 10), triple stenosing ligation of portal vein (TSLP) with saline solution (n = 10) and triple stenosing ligation of portal vein with budesonide (36 mg/kg per day; n = 10). In rats with prehepatic portal hypertension at 48 h of postoperative evolution, budesonide decreases the incidence of pancreatic edema, of peritoneal free exudate, of mesenteric adenopathies and prevents hypoproteinemia, hypoalbuminemia and hyper-beta-globulinemia. Some of the macroscopic intra-abdominal alterations and some of the changes in the electrophoretic pattern found in portal hypertensive rats could have an inflammatory etiopathogeny because budesonide shows an effective prophylaxis.

Chronic portal hypertension in the rat by triple-portal stenosing ligation.

A surgical technique based on the development of a triple stenosing ligation is used to worsen the complications inherent to the prehepatic chronic portal hypertension. The results have been compared with those obtained in rats with a single-portal stenosing ligation. An increase (p <.05) in the body, liver, spleen, and kidney weights as well as a decrease (p <.001) in the testes weight to body weight ratio were produced in both groups of animals. In addition, the variability in the obtained weights, particularly in the liver weight, stands out. The incidence of portosystemic and portohepatic collateral circulation and of the mesenteric venous vasculopathy increases in the animals with triple-portal stenosing ligation. The new proposed technique is a valid alternative to the classic one that used single portal stenosing ligation.

Altered proteinogram in short term portal vein stenosed rats.

The electrophoretic pattern of serum proteins has been studied in short-term prehepatic portal hypertensive rats since atrophy is produced in the liver, which is the main origin of most of these proteins, during this postoperative period. After 28 days of evolution, rats (n = 9) with triple stenosing ligated portal vein showed hypoalbuminemia, hypo-alpha-globulinemia, hyper-alpha2-globulinemia and hyper-gamma-globulinemia, the albumin/globulin ratio decreased with respect to the control animals (n = 8). These alterations are associated with hepatic atrophy, portosystemic and portohepatic (44.4%) collateral circulation. The proteinogram alterations found in rats with short-term prehepatic portal hypertension suggest that hepatic failure exists in spite of potential portohepatic revascularization which is frequently originated by the development of portohepatic collateral circulation.

Neuro-immune-endocrine functional system and vascular pathology.

A new interpretation of the response to injury by the nervous, immune and endocrine system is proposed, in order to integrate biochemical knowledge into the respective clinical areas. The discovery that the signaling molecules of the classical nervous, immune and endocrine systems, that is, the neurotransmitters, cytokines and hormones, respectively, are expressed and perceived by the three systems, has enabled us to establish a functional concept of these systems. The hypothetical integration of different pathological processes in a functional response made up by three phases, the immediate or nervous, intermediate or immune and late or endocrine ones, makes it possible to consider that all of them represent different forms of expression of a functional response whose meaning is always the same, that is, inflammation. If the functions that characterize each one of these three phases represent the activity of the nervous, immune and endocrine systems, the biochemical knowledge could be integrated into the functional meaning of each system.