Approaching Thrombospondin-1 as a Potential Target for Mesenchymal Stromal Cells to Support Liver Regeneration after Partial Hepatectomy in Mouse and Humans.

Extended liver resection carries the risk of post-surgery liver failure involving thrombospondin-1-mediated aggravation of hepatic epithelial plasticity and function. Mesenchymal stromal cells (MSCs), by interfering with thrombospondin-1 (THBS1), counteract hepatic dysfunction, though the mechanisms involved remain unknown. Herein, two-thirds partial hepatectomy in mice increased hepatic THBS1, downstream transforming growth factor-ß3, and perturbation of liver tissue homeostasis. All these events were ameliorated by hepatic transfusion of human bone marrow-derived MSCs. Treatment attenuated platelet and macrophage recruitment to the liver, both major sources of THBS1. By mitigating THBS1, MSCs muted surgery-induced tissue deterioration and dysfunction, and thus supported post-hepatectomy regeneration. After liver surgery, patients displayed increased tissue THBS1, which is associated with functional impairment and may indicate a higher risk of post-surgery complications. Since liver dysfunction involving THBS1 improves with MSC treatment in various animal models, it seems feasible to also modulate THBS1 in humans to impede post-surgery acute liver failure.

Organotypic 3D Co-Culture of Human Pleura as a Novel In Vitro Model of Staphylococcus aureus Infection and Biofilm Development.

Bacterial pleural infections are associated with high mortality. Treatment is complicated due to biofilm formation. A common causative pathogen is Staphylococcus aureus (S. aureus). Since it is distinctly human-specific, rodent models do not provide adequate conditions for research. The purpose of this study was to examine the effects of S. aureus infection on human pleural mesothelial cells using a recently established 3D organotypic co-culture model of pleura derived from human specimens. After infection of our model with S. aureus, samples were harvested at defined time points. Histological analysis and immunostaining for tight junction proteins (c-Jun, VE-cadherin, and ZO-1) were performed, demonstrating changes comparable to in vivo empyema. The measurement of secreted cytokine levels (TNF-α, MCP-1, and IL-1ß) proved host-pathogen interactions in our model. Similarly, mesothelial cells produced VEGF on in vivo levels. These findings were contrasted by vital, unimpaired cells in a sterile control model. We were able to establish a 3D organotypic in vitro co-culture model of human pleura infected with S. aureus resulting in the formation of biofilm, including host-pathogen interactions. This novel model could be a useful microenvironment tool for in vitro studies on biofilm in pleural empyema.

Novel 3D organotypic co-culture model of pleura.

Pleural mesothelial cells are the predominant cell type in the pleural cavity, but their role in the pathogenesis of pleural diseases needs to be further elucidated. 3D organotypic models are an encouraging approach for an in vivo understanding of molecular disease development. The aim of the present study was to develop a 3D organotypic model of the pleural mesothelium. Specimens of human pleura parietalis were obtained from patients undergoing surgery at the University Hospital Leipzig, Germany. 3D co-culture model of pleura was established from human pleural mesothelial cells and fibroblasts. The model was compared to human pleura tissue by phase-contrast and light microscopy, immunochemistry and -fluorescence as well as solute permeation test. Histological assessment of the 3D co-culture model displayed the presence of both cell types mimicking the morphology of the human pleura. Vimentin and Cytokeratin, PHD1 showed a similar expression pattern in pleural biopsies and 3D model. Expression of Ki-67 indicates the presence of proliferating cells. Tight junctional marker ZO-1 was found localized at contact zones between mesothelial cells. Each of these markers were expressed in both the 3D co-culture model and human biopsies. Permeability of 3D organotypic co-culture model of pleura was found to be higher for 70 kDa-Dextran and no significant difference was seen in the permeability for small dextran (4 kDa). In summary, the presented 3D organoid of pleura functions as a robust assay for pleural research serving as a precise reproduction of the in vivo morphology and microenvironment.

Human Mesenchymal Stromal Cells Resolve Lipid Load in High Fat Diet-Induced Non-Alcoholic Steatohepatitis in Mice by Mitochondria Donation.

Mesenchymal stromal cells (MSC) increasingly emerge as an option to ameliorate non-alcoholic steatohepatitis (NASH), a serious disease, which untreated may progress to liver cirrhosis and cancer. Before clinical translation, the mode of action of MSC needs to be established. Here, we established NASH in an immune-deficient mouse model by feeding a high fat diet. Human bone-marrow-derived MSC were delivered to the liver via intrasplenic transplantation. As verified by biochemical and image analyses, human mesenchymal stromal cells improved high-fat-diet-induced NASH in the mouse liver by decreasing hepatic lipid content and inflammation, as well as by restoring tissue homeostasis. MSC-mediated changes in gene expression indicated the switch from lipid storage to lipid utilization. It was obvious that host mouse hepatocytes harbored human mitochondria. Thus, it is feasible that resolution of NASH in mouse livers involved the donation of human mitochondria to the mouse hepatocytes. Therefore, human MSC might provide oxidative capacity for lipid breakdown followed by restoration of metabolic and tissue homeostasis.

Mesenchymal stromal cells mitigate liver damage after extended resection in the pig by modulating thrombospondin-1/TGF-ß.

Post-surgery liver failure is a serious complication for patients after extended partial hepatectomies (ePHx). Previously, we demonstrated in the pig model that transplantation of mesenchymal stromal cells (MSC) improved circulatory maintenance and supported multi-organ functions after 70% liver resection. Mechanisms behind the beneficial MSC effects remained unknown. Here we performed 70% liver resection in pigs with and without MSC treatment, and animals were monitored for 24 h post surgery. Gene expression profiles were determined in the lung and liver. Bioinformatics analysis predicted organ-independent MSC targets, importantly a role for thrombospondin-1 linked to transforming growth factor-ß (TGF-ß) and downstream signaling towards providing epithelial plasticity and epithelial-mesenchymal transition (EMT). This prediction was supported histologically and mechanistically, the latter with primary hepatocyte cell cultures. MSC attenuated the surgery-induced increase of tissue damage, of thrombospondin-1 and TGF-ß, as well as of epithelial plasticity in both the liver and lung. This suggests that MSC ameliorated surgery-induced hepatocellular stress and EMT, thus supporting epithelial integrity and facilitating regeneration. MSC-derived soluble factor(s) did not directly interfere with intracellular TGF-ß signaling, but inhibited thrombospondin-1 secretion from thrombocytes and non-parenchymal liver cells, therewith obviously reducing the availability of active TGF-ß.

In Vitro Tool: 3D Cell Culture of Human Adipose Tissue-Derived Mesenchymal Stromal Cells on Low Stiffness Silicone Scaffolds.

Conventional two-dimensional as well as established three-dimensional cell culture systems exhibit a high modulus of elasticity, which largely do not represent the stiffness of human organs. This chapter describes a protocol to establish 3D cell cultures of human adipose tissue-derived mesenchymal stromal cells (hAT-MSC) on low stiffness silicon scaffolds. These feature a defined growing environment with a stiffness close to physiology, thus being useful for in vitro investigations for multiple applications.

Impairment of Host Liver Repopulation by Transplanted Hepatocytes in Aged Rats and the Release by Short-Term Growth Hormone Treatment.

Hepatocyte transplantation is an alternative to whole liver transplantation. Yet, efficient liver repopulation by transplanted hepatocytes is low in livers of old animals. This restraint might be because of the poor proliferative capacity of aged donor hepatocytes or the regenerative impairment of the recipient livers. The age-dependent liver repopulation by transplanted wild-type hepatocytes was investigated in juvenile and senescent rats deficient in dipeptidyl-peptidase IV. Repopulation was quantified by flow cytometry and histochemical estimation of dipeptidyl-peptidase IV enzyme activity of donor cells in the negative host liver. As a potential pathway involved, expression of cell cycle proteins was assessed. Irrespective of the age of the donor hepatocytes, large cell clusters appeared in juvenile, but only small clusters in senescent host livers. Because juvenile and senescent donor hepatocytes were likewise functional, host-derived factor(s) impaired senescent host liver repopulation. Growth hormone levels were significantly higher in juvenile than in senescent rats, suggesting that growth hormone might promote host liver repopulation. Indeed, short-term treatment with growth hormone augmented senescent host liver repopulation involving the growth hormone-mediated release of the transcriptional blockade of genes associated with cell cycle progression. Short-term growth hormone substitution might improve liver repopulation by transplanted hepatocytes, thus augmenting the therapeutic benefit of clinical hepatocyte transplantation in older patients.

Deep Digging: Far Red Imaging for the Monitoring of Transplanted Hepatocytes in Rats.

Technologies for in vivo imaging of the distribution and integration of cell transplants gain significance for the use of novel cell therapy approaches in regenerative medicine. Applied to adequate animal models, they provide information on the spatio-temporal engraftment and functional performance of the cells transplanted. This chapter includes a detailed description of the in vivo tracking of transplanted hepatocytes in rat liver including the conjugation of antibodies to fluorochromes for far red imaging using a multispectral optical imager.

Age-dependent hepatocyte transplantation for functional liver tissue reconstitution.

The transplantation of hepatocytes could be an alternative therapeutic option to the whole organ transplantation for the treatment of end-stage liver diseases. However, this cell-based therapy needs the understanding of the molecular mechanisms to improve efficacy. This chapter includes a detailed method of a rat model for liver regeneration studies after age-dependent hepatocyte transplantation.

Human mesenchymal stem cells towards non-alcoholic steatohepatitis in an immunodeficient mouse model.

Non-alcoholic steatohepatitis (NASH) is a frequent clinical picture characterised by hepatic inflammation, lipid accumulation and fibrosis. When untreated, NASH bears a high risk of developing liver cirrhosis and consecutive hepatocellular carcinoma requiring liver transplantation in its end-stage. However, donor organ scarcity has prompted the search for alternatives, of which hepatocyte or stem cell-derived hepatocyte transplantation are regarded auspicious options of treatment. Mesenchymal stem cells (MSC) are able to differentiate into hepatocyte-like cells and thus may represent an alternative cell source to primary hepatocytes. In addition these cells feature anti-inflammatory and pro-regenerative characteristics, which might favour liver recovery from NASH. The aim of this study was to investigate the potential benefit of hepatocyte-like cells derived from human bone marrow MSC in a mouse model of diet-induced NASH. Seven days post-transplant, human hepatocyte-like cells were found in the mouse liver parenchyma. Triglyceride depositions were lowered in the liver but restored to normal in the blood. Hepatic inflammation was attenuated as verified by decreased expression of the acute phase protein serum amyloid A, inflammation-associated markers (e.g. lipocalin 2), as well as the pro-inflammatory cytokine TNFα. Moreover, the proliferation of host hepatocytes that indicate the regenerative capacity in livers receiving cell transplants was enhanced. Transplantation of MSC-derived human hepatocyte-like cells corrects NASH in mice by restoring triglyceride depositions, reducing inflammation and augmenting the regenerative capacity of the liver.

Human bone marrow mesenchymal stem cell-derived hepatocytes improve the mouse liver after acute acetaminophen intoxication by preventing progress of injury.

Mesenchymal stem cells from human bone marrow (hMSC) have the potential to differentiate into hepatocyte-like cells in vitro and continue to maintain important hepatocyte functions in vivo after transplantation into host mouse livers. Here, hMSC were differentiated into hepatocyte-like cells in vitro (hMSC-HC) and transplanted into livers of immunodeficient Pfp/Rag2⁻/⁻ mice treated with a sublethal dose of acetaminophen (APAP) to induce acute liver injury. APAP induced a time- and dose-dependent damage of perivenous areas of the liver lobule. Serum levels of aspartate aminotransferase (AST) increased to similar levels irrespective of hMSC-HC transplantation. Yet, hMSC-HC resided in the damaged perivenous areas of the liver lobules short-term preventing apoptosis and thus progress of organ destruction. Disturbance of metabolic protein expression was lower in the livers receiving hMSC-HC. Seven weeks after APAP treatment, hepatic injury had completely recovered in groups both with and without hMSC-HC. Clusters of transplanted cells appeared predominantly in the periportal portion of the liver lobule and secreted human albumin featuring a prominent quality of differentiated hepatocytes. Thus, hMSC-HC attenuated the inflammatory response and supported liver regeneration after acute injury induced by acetaminophen. They hence may serve as a novel source of hepatocyte-like cells suitable for cell therapy of acute liver diseases.

A fat option for the pig: hepatocytic differentiated mesenchymal stem cells for translational research.

STUDY BACKGROUND: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention in the pig model. METHODS: Mesenchymal surface antigens and multi-lineage differentiation potential of porcine MSC isolated by collagenase digestion either from bone marrow or adipose tissue (subcutaneous/visceral) were assessed by flow cytometry. Morphology and functional properties (urea-, glycogen synthesis and cytochrome P450 activity) were determined during culture under differentiation conditions and compared with primary porcine hepatocytes. RESULTS: MSC from porcine adipose tissue and from bone marrow express the typical mesenchymal markers CD44, CD29, CD90 and CD105 but not haematopoietic markers. MSC from both sources displayed differentiation into the osteogenic as well as adipogenic lineage. After hepatocyte differentiation, expression of CD105 decreased significantly and cells adopted the typical polygonal morphology of hepatocytes. Glycogen storage was comparable in adipose tissue- and bone marrow-derived cells. Urea synthesis was about 35% lower in visceral than in subcutaneous adipose tissue-derived MSC. Cytochrome P450 activity increased significantly during differentiation and was twice as high in hepatocyte-like cells generated from bone marrow as from adipose tissue. CONCLUSION: The hepatocyte differentiation of porcine adipose tissue-derived MSC was shown for the first time yielding hepatocyte-like cells with specific functions similar in bone marrow and subcutaneous adipose tissue-derived MSC. That makes them good pre-clinical candidates for supportive approaches after liver resection in the pig.

Phenotype selection reveals coevolution of muscle glycogen and protein and PTEN as a gate keeper for the accretion of muscle mass in adult female mice.

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.

Mesenchymal stem cell-derived hepatocytes for functional liver replacement.

Mesenchymal stem cells represent an alternate cell source to substitute for primary hepatocytes in hepatocyte transplantation because of their multiple differentiation potential and nearly unlimited availability. They may differentiate into hepatocyte-like cells in vitro and maintain specific hepatocyte functions also after transplantation into the regenerating livers of mice or rats both under injury and non-injury conditions. Depending on the underlying liver disease their mode of action is either to replace the diseased liver tissue or to support liver regeneration through their anti-inflammatory and anti-apoptotic as well as their pro-proliferative action.

A methionine-choline-deficient diet elicits NASH in the immunodeficient mouse featuring a model for hepatic cell transplantation.

Non-alcoholic staetohepatitis (NASH) is associated with fat deposition in the liver favoring inflammatory processes and development of fibrosis, cirrhosis and finally hepatocellular cancer. In Western lifestyle countries, NASH has reached a 20% prevalence in the obese population with escalating tendency in the future. Very often, liver transplantation is the only therapeutic option. Recently, transplantation of hepatocyte-like cells differentiated from mesenchymal stem cells was suggested a feasible alternative to whole organ transplantation to ameliorate donor organ shortage. Hence, in the present work an animal model of NASH was established in immunodeficient mice to investigate the feasibility of human stem cell-derived hepatocyte-like cell transplantation. NASH was induced by feeding a methionine/choline-deficient diet (MCD-diet) for up to 5 weeks. Animals developed a fatty liver featuring fibrosis and elevation of the proinflammatory markers serum amyloid A (SAA) and tumor necrosis factor alpha (TNFα). Hepatic triglycerides were significantly increased as well as alanine aminotransferase demonstrating inflammation-linked hepatocyte damage. Elevation of αSMA mRNA and collagen I as well as liver architecture deterioation indicated massive fibrosis. Both short- and long-term post-transplantation human hepatocyte-like cells resided in the mouse host liver indicating parenchymal penetration and most likely functional engraftment. Hence, the NASH model in the immunodeficient mouse is the first to allow for the assessment of the therapeutic impact of human stem cell-derived hepatocyte transplantation.

CD13: Waving the flag for a novel cancer stem cell target.

Cancer stem cells (CSCs) are generally dormant or slowly cycling tumor cells that have the ability to reconstitute tumors. They are thought to be involved in tumor resistance to chemo/radiation therapy and tumor relapse and progression. However, neither their existence nor their identity within many cancers has been well defined. Here, we have demonstrated that CD13 is a marker for semiquiescent CSCs in human liver cancer cell lines and clinical samples and that targeting these cells might provide a way to treat this disease. CD13+ cells predominated in the G0 phase of the cell cycle and typically formed cellular clusters in cancer foci. Following treatment, these cells survived and were enriched along the fibrous capsule where liver cancers usually relapse. Mechanistically, CD13 reduced ROS-induced DNA damage after genotoxic chemo/radiation stress and protected cells from apoptosis. In mouse xenograft models, combination of a CD13 inhibitor and the genotoxic chemotherapeutic fluorouracil (5-FU) drastically reduced tumor volume compared with either agent alone. 5-FU inhibited CD90+ proliferating CSCs, some of which produce CD13+ semiquiescent CSCs, while CD13 inhibition suppressed the self-renewing and tumor-initiating ability of dormant CSCs. Therefore, combining a CD13 inhibitor with a ROS-inducing chemo/radiation therapy may improve the treatment of liver cancer.

Hepatic transplantation of mesenchymal stem cells in rodent animal models.

The hepatocyte is the smallest functional entity of the liver and executes the majority of this organ's -metabolic functions. Hence, hepatocyte transplantation has become a versatile alternative to whole organ liver transplantation. This novel treatment option is based on the assumption that transplanted -hepatocytes integrate into the host liver, proliferate at the site of tissue damage, take over the long-term hepatic -synthetic capacity, and thus substitute for the diseased host tissue. However, clinical success is still waiting for a breakthrough, likely because of two major reasons including (1) the scarcity of cadaveric donor livers and (2) the largely poor quality of cells isolated from marginal quality donor organs. Therefore, alternative cell sources have to be established to further prompt the clinical success of hepatocyte transplantation. Due to their multiple differentiation potential and nearly unlimited availability, stem cells are an attractive -alternate resource. Because of both clinical and ethical objections, adult stem cells are often preferred over embryonic stem cells as a starting material. Recent studies have demonstrated the ability of mesenchymal stem cells derived from various tissues to differentiate into hepatocyte-like cells in vitro as well as showing specific hepatocyte functions in vivo after transplantation into the livers of mice or rats.

Lack of dietary carbohydrates induces hepatic growth hormone (GH) resistance in rats.

GH is a well established regulator of growth, lipid, and glucose metabolism and therefore important for fuel utilization. However, little is known about the effects of macronutrients on the GH/IGF system. We used low-carbohydrate/high-fat diets (LC-HFD) as a model to study the impact of fat, protein, and carbohydrates on the GH/IGF-axis; 12-wk-old Wistar rats were fed either regular chow, a moderate, protein-matched LC-HFD, or a ketogenic LC-HFD (percentage of fat/protein/carbohydrates: chow, 16.7/19/64.3; LC-HF-1, 78.7/19.1/2.2; LC-HF-2, 92.8/5.5/1.7). After 4 wk, body and tibia length, lean body mass, and fat pad weights were measured. Furthermore, we investigated the effects of LC-HFD on 1) secretion of GH and GH-dependent factors, 2) expression and signaling of components of the GH/IGF system in liver and muscle, and 3) hypothalamic and pituitary regulation of GH release. Serum concentrations of IGF-I, IGF binding protein-1, and IGF binding protein-3 were lower with LC-HF-1 and LC-HF-2 (P < 0.01). Both LC-HFD-reduced hepatic GH receptor mRNA and protein expression, decreased basal levels of total and phosphorylated Janus kinase/signal transducers and activators of transcription signaling proteins and reduced hepatic IGF-I gene expression. Hypothalamic somatostatin expression was reduced only with LC-HF-1, leading to increased pituitary GH secretion, higher IGF-I gene expression, and activation of IGF-dependent signaling pathways in skeletal muscle. In contrast, despite severely reduced IGF-I concentrations, GH secretion did not increase with LC-HF-2 diet. In conclusion, lack of carbohydrates in LC-HFD induces hepatic GH resistance. Furthermore, central feedback mechanisms of the GH/IGF system are impaired with extreme, ketogenic LC-HFD.

Induction of ketosis in rats fed low-carbohydrate, high-fat diets depends on the relative abundance of dietary fat and protein.

Low-carbohydrate/high-fat diets (LC-HFDs) in rodent models have been implicated with both weight loss and as a therapeutic approach to treat neurological diseases. LC-HFDs are known to induce ketosis; however, systematic studies analyzing the impact of the macronutrient composition on ketosis induction and weight loss success are lacking. Male Wistar rats were pair-fed for 4 wk either a standard chow diet or one of three different LC-HFDs, which only differed in the relative abundance of fat and protein (percentages of fat/protein in dry matter: LC-75/10; LC-65/20; LC-55/30). We subsequently measured body composition by nuclear magnetic resonance (NMR), analyzed blood chemistry and urine acetone content, evaluated gene expression changes of key ketogenic and gluconeogenic genes, and measured energy expenditure (EE) and locomotor activity (LA) during the first 4 days and after 3 wk on the respective diets. Compared with chow, rats fed with LC-75/10, LC-65/20, and LC-55/30 gained significantly less body weight. Reductions in body weight were mainly due to lower lean body mass and paralleled by significantly increased fat mass. Levels of ß-hydroxybutyate were significantly elevated feeding LC-75/10 and LC-65/20 but decreased in parallel to reductions in dietary fat. Acetone was about 16-fold higher with LC-75/10 only (P < 0.001). In contrast, rats fed with LC-55/30 were not ketotic. Serum fibroblast growth factor-21, hepatic mRNA expression of hydroxymethylglutaryl-CoA-lyase, peroxisome proliferator-activated receptor-γ coactivator-1α, and peroxisome proliferator-activated receptor-γ coactivator-1ß were increased with LC-75/10 only. Expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase was downregulated by 50-70% in LC-HF groups. Furthermore, EE and LA were significantly decreased in all groups fed with LC-HFDs after 3 wk on the diets. In rats, the absence of dietary carbohydrates per se does not induce ketosis. LC-HFDs must be high in fat, but also low in protein contents to be clearly ketogenic. Independent of the macronutrient composition, LC-HFD-induced weight loss is not due to increased EE and LA.

The generation of hepatocytes from mesenchymal stem cells and engraftment into murine liver.

Donor organ shortage is still the major obstacle for the clinical application of hepatocyte transplantation in the treatment of liver diseases. However, generation of hepatocyte-like cells from mesenchymal stem cells (MSCs) has become a real alternative to the isolation of primary hepatocytes. MSCs are extracted from the tissue by collagenase digestion and enriched by their capacity to grow on plastic surfaces. Enriched cells display distinct mesenchymal surface markers and are capable of multiple lineage differentiation. In the presence of specific growth conditions, the cells adopt functional features of differentiated hepatocytes. After orthotopic transplantation, differentiated human stem cells engraft in the host liver parenchyma of immunocompromised mice. This protocol describes the in vitro differentiation of stem cells from human bone marrow and their transplantation into livers of immunodeficient mice. The cell culture procedures take about 4-5 weeks, and cells engrafted in the mouse liver may be detected 2-3 months after transplantation.