Optimization of methods for intrasplenic administration of human amniotic epithelial cells in order to perform safe and effective cell-based therapy for liver diseases.

In animal experimental models the administration of stem cells into the spleen should ensure high effectiveness of their implantation in the liver due to a direct vascular connection between the two organs. The aim of this study was to update the methods of experimental intrasplenic cell transplantation using human amniotic epithelial cells (hAECs) which are promising cells in the treatment of liver diseases. BALB/c mice were administered intrasplenically with 0.5, 1, and 2 million hAECs by direct bolus injection (400 µl/min) and via a subcutaneous splenic port by fast (20 µl/min) and slow (10 µl/min) infusion. The port was prepared by translocating the spleen to the skin pocket. The spleen, liver, and lungs were collected at 3 h, 6 h, and 24 h after the administration of cells. The distribution of hAECs, histopathological changes in the organs, complete blood count, and biochemical markers of liver damage were assessed. It has been shown that the method of intrasplenic cell administration affects the degree of liver damage. The largest number of mice showing significant liver damage was observed after direct administration and the lowest after slow administration through a port. Liver damage increased with the number of administered cells, which, paradoxically, resulted in increased liver colonization efficiency. It was concluded that the administration of 1 × 106 hAECs by slow infusion via a subcutaneous splenic port reduces the incidence of complications at the expense of a slight decrease in the effectiveness of implantation of the transplanted cells in the liver.

Dynamics of Chronic Liver Injury in Experimental Models of Hepatotoxicity.

BACKGROUND: In humans, chronic liver disease (CLD) is a serious clinical condition with many life-threatening complications. Currently, there is no therapy to stop or slow down the progression of liver fibrosis. Experimental mouse models of CLD, induced by repeated intraperitoneal injections of carbon tetrachloride (CCL4) and D-galactosamine (D-GalN), can be used to evaluate therapies that cannot be performed in humans. A major drawback of these animal models is the different dynamics of liver fibrosis progression depending on the animal strain, administered hepatotoxin, its dose, duration of intoxication, and frequency of injections. The aim of this study was to describe and compare the dynamics of progression of pathological changes in the BALB/c mouse and Sprague Dawley rat models of CLD induced by CCl4 and D-GalN. We defined the onset and duration of these changes and suggested the optimal time for therapeutic intervention in the analyzed CLD models. METHODS: CLD was induced by repeated intraperitoneal injection of CCl4 in mice (12.5 µL/100 g bw every 5 days) and rats (25-100 µL/100 g bw twice a week) and D-GalN in mice (75 mg/100 g bw twice a week) and rats (25 mg/100 g bw twice a week). Blood and liver samples were collected at weeks 2, 4, 6, 8, 10, and 12 of intoxication. Liver injury and its progression were assessed by using complete blood count and liver function blood tests as well as by analyzing histopathological changes, including fibrosis, proliferation activity, apoptosis, stellate cell activation, and gene expression. RESULTS: In mice and rats treated with CCl4, early fibrosis was observed in most pericentral areas from week 2 to 4 of intoxication. Established fibrosis developed in both rats and mice at week 6 of intoxication. Incomplete cirrhosis, defined as the presence of occasional cirrhotic nodules, was observed in rats at week 12 of intoxication. The dynamics of liver fibrosis in CCl4-treated animals were greater than in the D-GalN groups. In D-GalN-intoxicated rats and mice, the first signs of liver fibrosis were observed at weeks 4 and 10 of intoxication, respectively. The rats developed early fibrosis after 8 weeks of D-GalN intoxication. The progression of collagen deposition was accompanied by histological changes and alteration of certain genes and blood liver parameters. CONCLUSIONS: The dynamics of liver fibrosis in CCl4 treated rodents is greater than in the D-GalN treated ones. In the CCl4 models, two appropriate times for therapeutic intervention are indicated, which to varying degrees reflect the real clinical situation and may potentially differ in the obtained results: early intervention before week 4 of intoxication (early fibrosis) and late intervention after week 8 of intoxication (when signs of established fibrosis are present). Rodent models of D-GalN-induced fibrosis are not recommended due to the long incubation period and weak toxic effect.

Dynamics of Acute Liver Injury in Experimental Models of Hepatotoxicity in the Context of Their Implementation in Preclinical Studies on Stem Cell Therapy.

BACKGROUND AND AIMS: Experimental models using carbon tetrachloride (CCl4) and D-galactosamine (D-GalN) can be used in preclinical assessment of acute liver failure (ALF) therapies. Unfortunately, these models are characterized by different dynamics of liver injury depending on the animal strain, administered hepatotoxin, and its dose. The aim of this study was to compare known rat and mouse models of ALF with a view to their future introduction into preclinical cell therapy experiments. In particular, based on histopathological and molecular changes, we suggested experimental time cut-off points for an effective stem cell therapeutic intervention. METHODS: ALF was induced by a single intraperitoneal injection of CCl4 in mice (50 µL/100 g b.w.) and rats (200 µL/100 g b.w.) and D-GalN in mice (150 mg/100 g b.w.) and rats (50 mg/100 g b.w.). Blood and liver samples were collected 12 h, 24 h, 48 h and 7 days after intoxication. Blood morphology, liver function blood tests, histopathological changes, proliferation activity, apoptosis, fibrosis, and gene expression were analysed to assess liver damage. RESULTS: At 12 h, 24 h, and 48 h after CCl4 injection, mouse livers showed moderate inflammatory infiltration and massive pericentral necrosis. In rats treated with CCl4, minor lymphocytic infiltration in the liver parenchyma was seen at 12 h, followed by necrosis that appeared around central veins at 24 h and persisted to 48 h. In D-GalN-injected mice, the first histopathological signs of liver injury appeared at 48 h. In the livers of D-GalN-treated rats, moderate pericentral inflammatory infiltration occurred after 12 h, 24 h, and 48 h, accompanied by increased proliferation and apoptosis. All histological changes were accompanied by decreasing expression of certain genes. In most experimental groups of rats and mice, both histological and molecular parameters returned to the baseline values between 48 h and 7 days after intoxication. CONCLUSIONS: In mice and rats with CCl4-induced ALF, signs of liver failure can be seen as early as 12 h and develop to 48 h. In the D-GalN-induced model, mice are more resistant to the hepatotoxic effect than rats (after 12 h), and the early hepatitis phase can be observed much later, after 48 h. These cut-off points seem to be optimal for suppressing inflammation and applying effective stem cell therapy for acute liver injury.

Paracrine activity of adipose derived stem cells on limbal epithelial stem cells.

Limbal stem cells deficiency (LSCD) is an eye disease caused by the loss of stem cells in the corneal limbus as a succession of an injury due physical, biological, or chemical agents. Current therapies of LSCD are focused on the transplantation of donor corneas or tissue equivalents produced from autologous limbal stem cells. Every year there are waiting millions of patients for the cornea transplantation all over the world and the list is growing due to the relatively low number of cornea donors. On the other hand, the transplantation of tissue or cells into the recipient's body is associated with the higher risk of possible side effects. The possibility of the application of an indirect treatment using the properties of the paracrine activity of stem cells, would be beneficial for the patients with transplant failures. This study was to evaluate the paracrine effect of mesenchymal stem cells derived from adipose tissue (ADSC) on the viability of limbal epithelial stem cells (LESC). The paracrine effect was assessed by treating LESC with conditioned medium collected from ADSC culture. Cell viability, cytotoxicity, apoptosis and proliferation were evaluated using in vitro assays in standard conditions and induced inflammation. After the exposure to the examined conditions, the expression of genes related to pro- and anti- inflammatory factors was evaluated and compared to the secretion of selected cytokines by ELISA test. Moreover, the changes in LESC phenotype were assessed using of phenotype microarrays. Our findings suggest that paracrine activity of ADSC on LESC promotes its proliferation and has a potential role in mitigation of the adverse impact of inflammation induced by lipopolysaccharide.