Cell Therapy For Liver Diseases Research Articles

Cellular Therapy for Liver Disease

Regenerative medicine is energizing and empowering basic science and has the potential to dramatically transform health care in the future. Given the remarkable intrinsic regenerative properties of the liver, as well as widespread adoption of regenerative strategies for liver disease (eg, liver transplant, partial hepatectomy, living donor transplant), hepatology has always been at the forefront of clinical regenerative medicine. However, an expanding pool of patients awaiting liver transplant, a limited pool of donor organs, and finite applicability of the current surgical approaches have created a need for more refined and widely available regenerative medicine strategies. Although cell-based therapies have been used extensively for hematologic malignant diseases and other conditions, the potential application of cellular therapy for acute and chronic liver diseases has only more recently been explored. New understanding of the mechanisms of liver regeneration and repair, including activation of local stem/progenitor cells and contributions from circulating bone marrow-derived stem cells, provide the theoretical underpinnings for the rational use of cell-based therapies in clinical trials. In this review, we dissect the scientific rationale for various modalities of cell therapy for liver diseases being explored in animal models and review those tested in human clinical trials. We also attempt to clarify some of the important ongoing questions that need to be addressed in order to bring these powerful therapies to clinical translation. Discussions will cover transplant of hepatocytes and liver stem/progenitor cells as well as infusion or stimulation of bone marrow-derived stem cells. We also highlight tremendous scientific advances on the horizon, including the potential use of induced pluripotent stem cells and their derivatives as individualized regenerative therapy for liver disease.

Isolation and Hepatocyte Differentiation of Mesenchymal Stem Cells From Porcine Bone Marrow—“Surgical Waste” as a Novel MSC Source

Mesenchymal stem cells (MSC) isolated from bone marrow and differentiated into hepatocyte-like cells have increasingly gained attention for clinical cell therapy of liver diseases because of their high regenerative capacity. They are available from bone marrow aspirates of the os coxae after puncture of the crista iliaca or from bone marrow “surgical waste” gained from amputations or knee and hip operations. Thus, the aim of the study was to demonstrate whether these pBM-MSC (porcine bone marrow-derived mesenchymal stem cells) displayed mesenchymal features and hepatocyte differentiation potential. MSC were isolated either from crista iliaca punctures or after sampling and collagenase digestion of bone marrow from the os femoris. Mesenchymal features were assessed by flow cytometry for specific surface antigens and their ability to differentiate into at least 3 lineages. Functional properties, such as urea or glycogen synthesis and cytochrome P450 activity, as well as the cell morphology were examined during hepatocyte differentiation. pBM-MSC from both sources lacked the hematopoietic markers CD14 and CD45 but expressed the typical mesenchymal markers CD44, CD29, CD90, and CD105. Both cell types could differentiate into adipocyte, osteocyte, and hepatocyte lineages. After hepatocyte differentiation, CD105 expression decreased significantly and cells changed morphology from fibroblastoid into polygonal, displaying significantly increased glycogen storage, urea synthesis, and cytochrome activity. pBM-MSC from various sources were identical in respect to their mesenchymal features and their hepatocyte differentiation potential. Hence, long bones might be a particularly useful resource to isolate bone marrow mesenchymal stem cells for transplantation.

Mesenchymal stem cells (MSC) from porcine bone marrow (pBM) of the Os femoris and the Crista iliaca

MSC increasingly gain attention for clinical cell therapy of liver diseases. Before translation into the clinics feasibility must be proven preferably in large animal models.The aim of the study was to demonstrate in the large animal model of the pig whether MSC from bone marrow (pBM‐MSC) of the Crista iliaca and from BM of the femur/shank display differences in regard to their hepatogenic differentiation potential.MSC were isolated either from BM of the Crista iliaca or Os femoris. Mesenchymal features were analysed by flow cytometry. Functional properties like the synthesis of urea and glycogen were compared during hepatogenic differentiation.pBM‐MSC from both sources were negative for hematopoietic markers CD14, CD45 but positive for mesenchymal markers CD44, CD29, CD90, CD105. pBM‐MSC from both sources could be differentiated along the adipocyte, osteocyte and hepatocyte lineage. With differentiation progressing glycogen and urea synthesis increased featuring typical hepatocyte functions. After 21 days of differentiation urea synthesis amounted to 3 nmol/10.000 cells × 24h comparable to the rate in primary pig hepatocytes after 8 days of culture.pBM‐MSC from various sources are not different in respect to their mesenchymal features and their hepatogenic differentiation potential. Hence, hollow bones might be particularly useful to isolate allogeneic bone marrow cell transplants.

Efficient generation of functional hepatocyte-like cells from menstrual blood-derived stem cells

In recent years, the advantages of menstrual blood-derived stem cells (MenSCs), such as minimal ethical considerations, easy access and high proliferative ability, have inspired scientists to investigate the potential of MenSCs in cell therapy of different diseases. In order to characterize the potency of these cells for future cell therapy of liver diseases, we examined the potential of MenSCs to differentiate into hepatocytes, using different protocols. First, the immunophenotyping properties and potential of MenSCs to differentiate into osteoblasts, adipocytes and chondrocytes were evaluated. Thereafter, the differentiation protocols developed by two concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM), in combination with other components in serum-supplemented or serum-free culture media, were also investigated. The sequential differentiation was monitored by real-time PCR, immunostaining and functional assays. Our primary data revealed that the isolated MenSCs exhibited mesenchymal stem cell markers in parallel to OCT-4 as an embryonic marker. Regardless of differentiation procedures, the developed cells expressed mature hepatocyte markers, such as albumin, tyrosine aminotransferase and cytokeratin-18 at the mRNA and protein levels. They also showed functional properties of hepatocytes, including albumin secretion, glycogen storage and cytochrome P450 7A1 expression. However, the degree of differentiation was dependent on the concentrations of HGF and OSM. Indeed, omission of serum during the differentiation process caused typical improvement in hepatocyte-specific functions. This study is a novel report demonstrating the differentiation potential of MenSCs into hepatocyte-like cells. We recommend a complementary serum-free differentiation protocol for enrichment of in vitro production of functional MenSC-derived hepatocyte-like cells that could lead to a major step toward applied stem cell therapy of chronic liver diseases.

The fetal liver as cell source for the regenerative medicine of liver and pancreas.

Patients affected by liver diseases and diabetes mellitus are in need for sources of new cells to enable a better transition into clinic programs of cell therapy and regenerative medicine. In this setting, fetal liver is becoming the most promising and available source of cells. Fetal liver displays unique characteristics given the possibility to isolate cell populations with a wide spectrum of endodermal differentiation and, the co-existence of endodermal and mesenchymal-derived cells. Thus, the fetal liver is a unique and highly available cell source contemporarily candidate for the regenerative medicine of both liver and pancreas. The purpose of this review is to revise the recent literature on the different stem cells populations isolable from fetal liver and candidate to cell therapy of liver diseases and diabetes and to discuss advantages and limitation with respect to other cell sources.

Possibility of the Enhanced Progression of Fetal Liver Stem/Progenitor Cells Therapy for Treating End-stage Liver Diseases by Regulating the Notch Signaling Pathway

Cell therapy is the most promising therapy for end-stage liver diseases (ESLDs). Fetal liver stem/progenitor cells (FLSPCs) have the advantages of a high survival rate, high proliferation, small volume, and high safety, which make them one of the ideal cells for stem cell therapy for liver diseases. During the early phase of our study, we applied a three-step separation method to enrich FLSPCs and obtained a separation efficiency that was similar to the flow cell-sorting method. Additionally, using a fulminant hepatic failure rat model, we demonstrated that FLSPCs can contribute to the recovery of hepatic morphogenesis and function. However, two problems remain to be resolved to explore the therapeutic potential of FLSPCs. First, how can FLSPCs be induced to accurately differentiate into hepatocytes and cholangiocytes? Second, how do FLSPCs maintain self-renewal? The Notch signaling plays a critical role in regulating the differentiation and self-renewal of many types of stem cells. Additionally, our previous findings have shown that the Notch signaling plays an important role in FLSPC differentiation into hepatocytes. Therefore, we hypothesized that the Notch signaling may be involved in the differentiation and self-renewal of FLSPCs. We began a study on the regulatory effects and relative molecular mechanisms of the Notch signaling on FLSPCs and found the corresponding interfering target, which may become an index for the clinical application of FLSPCs.

Comparison of in vitro hepatogenic differentiation potential between various placenta-derived stem cells and other adult stem cells as an alternative source of functional hepatocytes

Mesenchymal stem cells (MSCs) are powerful sources for cell therapy in regenerative medicine. The capability to obtain effective stem cell-derived hepatocytes would improve cell therapy for liver diseases. Recently, various placenta-derived stem cells (PDSCs) depending on the localization of placenta have been suggested as alternative sources of stem cells are similar to bone marrow-derived MSC (BM-MSCs) and adipose-derived MSC (AD-MSCs). However, comparative studies for the potentials of the hepatogenic differentiation among various MSCs largely lacking. Therefore, we investigated to compare the potentials for hepatogenic differentiation of PDSCs with BM-MSCs, AD-MSCs, and UCB-MSCs. Several MSCs were isolated from human term placenta, adipose tissue, and umbilical cord blood and characterized isolated MSCs and BM-MSCs was performed by quantitative reverse transcription-PCR (RT-PCR) and special stains after mesodermal differentiation. The hepatogenic potential of PDSCs was compared with AD-MSCs, UCB-MSCs, and BM-MSCs using RT-PCR, PAS stain, ICG up-take assays, albumin expression, urea production, and cytokine assays. MSCs isolated from different tissues all presented similar characteristics of MSCs. However, the proliferative potential of PDSCs and the expression of hepatogenic markers in differentiated PDSCs were higher than other MSCs. Interestingly, the expression of hepatocyte growth factor (HGF) increased in PDSCs after hepatogenic differentiation. Interestingly, stem cell factor (SCF) expression in chorionic plate-derived MSCs, one of the PDSCs, was significantly higher than in the other PDSCs. Taken together, the results of the present study suggest that MSCs isolated from various adult tissues can be induced to undergo hepatogenic differentiation in vitro, and that PDSCs may have the greatest potential for hepatogenic differentiation and proliferation. Therefore, PDSCs could be used as a stem cell source for cell therapy in liver diseases.

Cell therapies for liver diseases

Cell therapies, which include bioartificial liver support and hepatocyte transplantation, have emerged as potential treatments for a variety of liver diseases. Acute liver failure, acute-on-chronic liver failure, and inherited metabolic liver diseases are examples of liver diseases that have been successfully treated with cell therapies at centers around the world. Cell therapies also have the potential to be widely applied to other liver diseases, including noninherited liver diseases and liver cancer, and to improve the success of liver transplantation. Here we briefly summarize current concepts of cell therapy for liver diseases.

New horizons for stem cell therapy in liver disease

There is an increasing range of potential applications of stem cells in liver diseases, with many clinical studies already undertaken. We identify four of the main areas which we propose stem cell therapy could be a realistic aim for in the future: (1) to improve regeneration and reduce scarring in liver cirrhosis by modulating the liver's own regenerative processes, (2) to down-regulate immune mediated liver damage, (3) supplying hepatocyte-like cells (HLCs) derived from stem cells for use in extracorporeal bio-artificial liver machines, and (4) to use stem cell derived HLCs for cell transplantation to supplement or replace hepatocyte function.

584. Regenerative Cell Therapy for Liver Diseases with Skin Adipose Tissues-Derived Stem Cells

584. Regenerative Cell Therapy for Liver Diseases with Skin Adipose Tissues-Derived Stem Cells

137P In vitro differentiation of human umbilical vein mesenchymal stem cells into hepatocyte-like cells

Human umbilical vein, as a readily available source of stem cells, is one of the good alternative source for mesenchymal stem cells. Human umbilical cord vein mesenchymal stem cells (UVMSCs) have been recently isolated and demonstrated the ability to differentiate into various cell types such as fat, bone, cartilage and neuronal cells. In this study, we have investigated whether human UVMSC are also able to differentiate into hepatocyte-like cells. Hepatic differentiation was done with a 2-step protocol and with the use of hepatocyte growth factor and oncostatin M. The cells were cultured with hepatocyte growth factor (HGF) and oncostatin M (OSM). During 4 weeks of induction most of the cells displayed a cuboidal morphology. Immunological analysis indicated that UVMSCs-derived hepatocyte-like cells expressed liver-specific protein markers such as albumin (ALB) and cytokeratin-18 (CK-18). The hepatocyte-like cells also displayed several characteristics of hepatocytes, including expression of transthyretin, glucose-6-phosphatase (G6P), cytokeratin-8,18 (CK-8,18), alpha-fetoprotein (AFP), hepatocyte nuclear factor-3β (AFP) and ALB. The result of indocyanine green (ICG) cell uptake as a test substance to evaluate hepatocyte-like cells function was positive for differentiated cells. Glycogen storage was examined by periodic acid–Schiff (PAS) staining and accumulation of intracellular glycogen were detected in hepatocyte-like cells. Based on these observations it can be concluded that UVMSCs encompass suitable hepatogenic potential which may provide a source of stem cells for cell therapy in liver diseases.

Transplant of Primary Human Hepatocytes Cocultured With Bone Marrow Stromal Cells to SCID Alb-uPA Mice.

Hepatocytes are vulnerable to loss of function and viability in culture. Modified culture methods have been applied to maintain their functional status. Heterotypic interactions between hepatocytes and nonparenchymal neighbors in liver milieu are thought to modulate cell differentiation. Cocultivation of hepatocyte with various cell types has been applied to mimic the hepatic environment. Bone marrow stromal cells (BMSC) are plastic cell lines capable of transforming to other cell types. In this study hepatocyte coculture with BMSCs achieved long-term function of human hepatocytes in culture for 4 weeks. In vitro functional status of human hepatocytes in BMSC coculture was compared with fibroblast coculture and collagen culture by measuring albumin, human-α-1-antitrypsin (hAAT), urea secretion, CYP450 activity, and staining for intracellular albumin and glycogen. After 2 weeks in culture hepatocytes were retrieved and transplanted to severe combined immunodeficiency/albumin linked-urokinase type plasminogen activator (SCID Alb-uPA) mice and engraft-ment capacity was analyzed by human hepatic-specific function measured by hAAT levels in mouse serum, and Alu staining of mouse liver for human hepatocytes. Hepatocytes from BMSC coculture had significantly higher albumin, hAAT secretion, urea production, and cytochrome P450 (CYP450) activity than other culture groups. Staining confirmed the higher functional status in BMSC coculture. Transplantation of hepatocytes detached from BMSC cocultures showed significantly higher engraftment function than hepatocytes from other culture groups measured by hAAT levels in mouse serum. In conclusion, BMSC coculture has excellent potential for hepatocyte function preservation in vitro and in vivo after transplant. It is possible to use BMSC hepatocyte coculture as a supply of cell therapy in liver disease.

Postnatally Induced Over-Expression of FGF10 Promotes Murine Hepatic Progenitor Cell Expansion Similar to the DDC Diet Model

Introduction: Liver cell replacement therapy is an area of active investigation given the lack of organ donors. Greater understanding of what regulates HPC expansion during hepatogenesis may lead to effective stem cell therapies for liver diseases as well as other system pathologies. Fibroblast Growth Factors (FGFs), a family of 23 ligands and 4 FGFR tyrosine kinases, regulate many aspects of development including hepatogenesis. We previously showed that FGF10 promotes the proliferation and survival of fetal hepatic progenitor cells (HPC) called hepatoblasts during early hepatogenesis in mice (Berg et al, Hepatology, 2007).

Current Status of Stem Cell Therapy for Liver Diseases

Liver failure is one of the main causes of death worldwide and is a growing health problem. Since the discovery of stem cell populations capable of differentiating into specialized cell types, including hepatocytes, the possibility of their utilization in the regeneration of the damaged liver has been a focus of intense investigation. A variety of cell types were tested both in vitro and in vivo, but the definition of a more suitable cell preparation for therapeutic use in each type of liver lesions is yet to be determined. Here we review the protocols described for differentiation of stem cells into hepatocytes, the results of cell therapy in animal models of liver diseases, as well as the available data of the clinical trials in patients with advanced chronic liver disease.

Serum from Radiofrequency-Injured Livers Induces Differentiation of Bone Marrow Stem Cells into Hepatocyte-Like Cells

Roles that bone marrow stem cells (BMSCs) play in liver repair after liver injury and the cell therapy for liver diseases are widely accepted. However, the availability of hepatocyte-like cells from BMSCs and possible animal diseases association with culturing in fetal calf serum (FCS) are the major limitations of clinical therapy. Therefore, this study was designed to search for a new cell source for the treatment of liver injuries through investigating whether serum from radiofrequency ablation-injured rabbit livers can induce the differentiation of BMSCs into hepatocyte-like cells. Serum was collected from rabbits 36 h after radiofrequency ablation (RFA) treatment of the liver. BMSCs were isolated from rabbit bone marrow and were cultured in the collected serum. Cellular morphology and cell cycle were observed. Hepatocyte markers of the differentiated cells were detected by immunohistochemistry. After induction for 7 d, spindle-shaped BMSCs turned into round cells that resembled the morphology of hepatocyte-like cells. Flow cytometry showed that the percentage of cells in the S/G2/M phase was higher in the RFA group than that in the FCS group and HGF groups. This result suggests that BMSC can transform into mature cells from stem cell phase. Albumin and CK18 were considered as typical marker of hepatocytes. Following induction for 14 d, the differentiated cells expressed immunofluorescence of FITC-labeled albumin and TRITC-labeled CK18. BMSCs treated with serum collected from radiofrequency ablation-injured livers can differentiate into hepatocyte-like cells providing a cell source to cell therapy.

Differentiation of human umbilical cord mesenchymal stromal cells into low immunogenic hepatocyte-like cells

Mesenchymal stromal cells (MSC) isolated from several human tissues have been known to differentiate into the hepatic lineage in vitro, but the immunogenicity of the differentiated hepatocyte-like cells (DHC) has not been reported. Umbilical cord (UC) MSC are thought to be an attractive cell source for cell therapy because of their young age and low infection rate compared with adult tissue MSC. Hepatic differentiation of UC-MSC was induced with a 2-step protocol. The expressions of hepatic markers were detected by RT-PCR and immunofluorescence staining. Albumin production and urea secretion were measured by ELISA and colorimetric assay respectively. The immunosuppressive properties of DHC was detected by mixed lymphocyte culture. After incubation with specific growth factors, including hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF), UC MSC exhibited a high hepatic differentiation ability in an adherent culture condition. The differentiated UC MSC showed hepatocyte-like morphology and expressed several liver-specific markers at gene and protein levels. Furthermore, the DHC exhibited hepatocyte-specific functions, including albumin secretion, low-density lipoprotein uptake and urea production. More importantly, DHC did not express major histocompatibility complex (MHC) II antigen and were not able to induce lymphocyte proliferation in mixed lymphocyte culture, as undifferentiated UC MSC did. Our results indicate that UC MSC are able to differentiate into functional hepatocyte-like cells that still retain their low immunogenicity in vitro. More importantly, DHC incorporated into the parenchyma of liver when transplanted into mice with CCl(4)-induced liver injury. Therefore, DHC may be an ideal source for cell therapy of liver diseases.

Stem and progenitor cells for liver repopulation: can we standardise the process from bench to bedside?

There has been recent progress in the isolation and characterisation of stem/progenitor cells that may differentiate towards the hepatic lineage. This has raised expectations that therapy of genetic or acquired...

Analysis of gene expression patterns of hepatocyte‐like cells derived from human bone marrow mesenchymal stem cells

The aim of the study was to determine the degree of differentiation of hepatocyte‐like cells derived from human mesenchymal stem cells (hMSC) by comparing global gene expression patterns of differentiated and undiffentiated hMSC from bone marrow (hBM‐MSC), adult liver and fetal hepatocytes.hMSC were isolated from bone marrow and differentiated into hepatocyte‐like cells. Microarray analysis was performed using the HG‐U133A array from Affymetrix. For comparative analysis of expression patterns published datasets and results obtained from this study were combined. All datasets were analysed by Microarray Suite 5.0 and Genesis software.In comparison to undifferentiated hBM‐MSC, hepatocyte differentiated hBM‐MSC showed a dramatic change of the gene expression profile. We found approximately 1000 genes upregulated and 1400 genes were downregulated in differentiated MSC with a fold change ≥3. Among these, around 60% and 40% showed similar expression levels as in adult liver, respectively. Expression of glutamine synthetase and ribonuclease 4 is significantly upregulated in hepatocyte differentiated MSC, typical for adult hepatocytes in the perivenous area of the liver lobule. Thus, hepatocyte‐like cells derived from hBM‐MSC feature human hepatocyte gene expression patterns, which encourages to step forward to use hBM‐MSC as a novel source for cell therapy of liver diseases.

Bone marrow transplantation into hemochromatotic mice decreases hepatic and duodenal iron overload

Human hereditary hemochromatosis is a disorder of iron homeostasis characterized by increased absorption of iron and its deposition in parenchymal organs. The maintenance of iron homeostasis is regulated by molecules involved in the absorption, transport, storage and redox of iron. The potential of hematopoietic stem cell therapy for liver diseases has been studied in some experimental animal models. Our objective was to evaluate the effect of bone marrow transplantation from wild type mice on the status of iron overload in Hfe knockout hemochromatotic mice ( Hfe −/−). The transplanted cells were detected in the liver (11% of the total cells) and characterized as hepatocytes and myofibroblasts. They were also detected in the duodenum and characterized as myofibroblasts. The iron content in the Hfe −/− mice descended 2.9-fold in the liver and 2.4-fold in the duodenum 6 months after transplantation. Non-significant changes of relative mRNA abundance of genes of iron metabolism were observed in the liver and duodenum of Hfe −/− transplanted mice. At 6 months after transplantation the proportion of Hfe mRNA in Hfe −/− mice reached 3.8% of the levels in wild type mice in the liver and 1.6% in the duodenum. In conclusion, adult stem cells from bone marrow transplant were able to differentiate into hepatocytes and myofibroblasts in hemochromatotic mice. Bone marrow transplantation assisted in reducing the iron overload in this murine model of hemochromatosis. These findings might contribute to the knowledge of pathways involved in the regulatory system of iron homeostasis.

Regenerative Medicine and Liver Injury: What Role for Bone Marrow Derived Stem Cells?

In recent years, great interest has been aroused by the discovery of the ability of adult stem cells to contribute to regeneration processes and repair of damaged tissues. In particular, bone marrow derived stem cells (BMSCs), the most well known population of multipotent stem cells in adults, have been shown to be able to generate many different committed cellular types. In this review, we systematically organize the numerous hypotheses emerging from the most recent studies, in animal and humans, which evaluated the potentiality of BMSCs to contribute to tissue repair in different types of liver damage. Our aim is to give scientists and clinicians who are interested in regenerative medicine the rational basis for planning future studies on stem cell therapy for liver diseases.