Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes

Recent observations indicate that several stem cells can differentiate into hepatocytes; thus, cell-based therapy is a potential alternative to liver transplantation. The goal of the present study was to examine the in vitro hepatic differentiation potential of adipose tissue-derived mesenchymal stem cells (AT-MSCs). We used AT-MSCs from different age patients and found that, after incubation with specific growth factors (hepatocyte growth factor [HGF], fibroblast growth factor [FGF1], FGF4) the CD105(+) fraction of AT-MSCs exhibited high hepatic differentiation ability in an adherent monoculture condition. CD105(+) AT-MSC-derived hepatocyte-like cells revealed several liver-specific markers and functions, such as albumin production, low-density lipoprotein uptake, and ammonia detoxification. More importantly, CD105(+) AT-MSC-derived hepatocyte-like cells, after transplantation into mice incorporated into the parenchyma of the liver. CONCLUSION: Adipose tissue is a source of multipotent stem cells that can be easily isolated, selected, and induced into mature, transplantable hepatocytes. The fact that they are easy to procure ex vivo in large numbers makes them an attractive tool for clinical studies in the context of establishing an alternative therapy for liver dysfunction.     

Biochemical and molecular characterization of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel three-dimensional biocompatible nanofibrous scaffold

Background:  There is significant interest in using nanofibers in tissue engineering from stem cells. The transdifferentiation of mesenchymal stem cells into the hepatic lineage in a nanofibrous structure has not been reported. In this study, a three dimensional nanofibrous scaffold is introduced for differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs) into hepatocytes.
Methods:  A scaffold composed of Poly (ε-caprolactone), collagen and polyethersulfone was fabricated by the electrospinning technique. After characterization of isolated hBMSCs, the performance of the cells on the scaffold was evaluated by Scanning Electron Microscopy (SEM) and MTT assay. Cytological, molecular and biochemical markers were measured to confirm differentiation potential of hBMSCs into hepatocytes.
Results:  The isolated cells possessed the basic properties of mesenchymal stem cells (MSCs). Based on scanning electron microscope (SEM) analysis and MTT assay, it was shown that the cells adhere, penetrate and proliferate on the nanofibers. Cultured cells on the nanofibers differentiated into hepatocyte-like cells and expressed hepatocyte specific markers such as albumin, α-fetoprotein, cytokeratin-18, cytokeratin-19 and cytochrome P450 3A4 at mRNA levels. Appearance of a considerable number of albumin-positive cells cultivated on the scaffold (47 ± 4%) as compared to the two-dimensional culture system (28 ± 6%) indicates the supporting role of the scaffold. The efficiency of the cells to produce albumin, urea, transferrin, serum glutamic pyruvic transaminase and serum oxaloacetate aminotransferase in hepatocytes on the scaffold further attest to the functionality of the cells.
Conclusion:  The data presented in this study show that the engineered nanofibrous scaffold is a conductive matrix which supports and enhances MSC development into functional hepatocyte-like cells.     

Role of stem cells in repair of liver injury:Experimental and clinical benefit of transferred stem cells on liver failure

Although the liver has a high regenerative capacity,as a result of massive hepatocyte death,liver failure occurs. In addition to liver failure,for acute,chronic and hereditary diseases of the liver,cell transplantation therapies can stimulate regeneration or at least ensure sufficient function until liver transplantation can be performed. The lack of donor organs and the risks of rejection have prompted extensive experimental and clinical research in the field of cellular transplantation. Transplantation of cell lineages involved in liver regeneration,including mature hepatocytes,fetal hepatocytes,fetal liver progenitor cells,fetal stem cells,hepatic progenitor cells,hepatic stem cells,mesenchymal stem cells,hematopoietic stem cells,and peripheral blood and umbilical cord blood stem cells,have been found to be beneficial in the treatment of liver failure.In this article,the results of experimental and clinical cell transplantation trials for liver failure are reviewed,with an emphasis on regeneration.     

Therapeutic potential and related signal pathway of adipose-derived stem cell transplantation for rat liver injury

Aim:  Liver transplantation is the only currently effective therapy for end-stage chronic liver disease and severe acute liver failure, but its use is limited by high cost and a shortage of allografts. Here we explored the effectiveness of transplanting adipose-derived stem cells (ADSCs) into rats with experimentally induced liver injury.
Methods:  ADSCs obtained from rats were hepatogenic induced in vitro with MAPK pathways inhibitors preconditioning. In vivo , ADSCs were transplanted into rats via different routes and serum liver function markers from post-operative rats were tested.
Results:  When grown in adipogenic induction medium, ADSCs were able to differentiate into adipocytes. In hepatogenic induction medium, ADSCs were able to differentiate into hepatocyte-like cells, with appropriate changes in morphology and appropriately elevated expression of hepatocyte-specific markers. ERK1/2 phosphorylation activity was also significantly upregulated during the hepatogenic differentiation process, and was blocked by the ERK/MAPK pathway-specific inhibitor PD98059. In a rat liver injury model, intravenously injected ADSCs successfully engrafted into recipient livers. We found that injection via the hepatic portal vein was more efficient than via the dorsal vein of the penis. ADSC transplantation into damaged livers significantly decreased the level of serum liver enzymes such as alanine aminotransferase and aspartate aminotransferase, and improved serum albumin level. Both the number of engrafted cells and the improvement of liver function reached a peak two weeks after transplantation.
Conclusion:  Transplanted ADSCs appear to be therapeutically effective in the rat liver injury model, which may ultimately provide a therapeutic alternative to liver transplantation in human patients.     

Efficacy and limitation of bone marrow transplantation in the treatment of acute and subacute liver failure in rats

Aim:  Recent reports have shown that bone marrow cells (BMC) retain the potential to differentiate into hepatocytes. Thus, the BMC have been recognized as an attractive source for liver regenerative medicine. However, it has not been clarified whether BMC transplantation can be used to treat liver damage in vivo . In the present study, we explored whether BMC possess therapeutic potential to treat acute and/or subacute liver failure.
Methods:  Fulminant hepatic failure (FHF) was induced by 70% hepatectomy with ligation of the right lobe pedicle (24% liver mass), followed by transplantation of BMC into the spleen. Dipeptidyl peptidase IV-positive (DPPIV⁺) BMC were then transplanted into DPPIV-negative (DPPIV^-) recipients following hepatic irradiation (HIR) in which 70% of the liver was resected and the remnant liver irradiated.
Results:  There was no benefit of BMC transplantation towards survival in the FHF model. DPPIV⁺ hepatocytes appeared in the liver tissues of the DPPIV^- HIR model rats, but DPPIV⁺ hepatocytes replaced less than 13% of the recipient liver.
Conclusion:  BMC transplantation may have limitations in the treatment of fulminant or acute liver failure because they do not have sufficient time to develop into functional hepatocytes. Preparative HIR may be beneficial in help to convert the transplanted BMC into host hepatocytes, and provide a survival benefit. Although, However, the precise mechanism warrants further studies.     

Hepatoma-derived growth factor is induced in liver regeneration

Aim:  Hepatoma-derived growth factor (HDGF) is a heparin-binding protein, which has been suggested to be involved in the development of kidneys, the cardiovascular system and the liver. We have shown that HDGF is highly expressed in parenchymal hepatocytes in the developing liver and promotes fetal hepatocyte proliferation. In the present study, we asked whether HDGF expression was related to liver regeneration.
Methods:  We examined the mRNA and protein expressions of HDGF in two liver regeneration models. In addition, cellular distribution of HDGF in the regenerating liver was investigated by immunohistochemistry.
Results:  In the carbon tetrachloride (CCl₄)-treated liver, HDGF expression was induced and the peak was detected at 24 h after the CCl₄ injection. HDGF expression was also enhanced in the hepatectomy model and the peak was detected at 12 h after surgery. The increased expression of HDGF protein was also confirmed by western blotting. Expression of the HDGF gene in the regenerating liver was dominantly detected in parenchymal hepatocytes.
Conclusion:  These findings showed that HDGF expression was induced in parenchymal hepatocytes before the DNA synthesis in the regenerating liver, suggesting the possible involvement of HDGF in liver regeneration as an autocrine factor.     

Vascular endothelial growth factor reduces Fas-mediated acute liver injury in mice

Background and Aim:  Fulminant hepatitis is still a fatal liver disease, and no specific treatment for it has been available. Vascular endothelial growth factor (VEGF) is the focus of attention because of its various actions. We investigated the effect of vascular endothelial growth factor (VEGF) on Fas-induced fulminant hepatic failure (FHF).
Method:  Male Balb/c mice were treated with an intraperitoneal injection of an anti-Fas antibody (Jo-2 Ab) with or without premedication with intraperitoneally administered human recombinant VEGF.
Results:  The serum level of alanine aminotransferase (ALT) was up to 300 times higher that of normal mice following the Jo-2 Ab injection, and histological analysis revealed hepatic injury and massive hepatocyte apoptosis. The VEGF significantly suppressed an elevation in serum ALT levels and hepatocyte apoptosis. Immunohistochemically, VEGF-treated mice showed that Bcl-xL in hepatocytes was strongly expressed.
Conclusions:  Since hepatocytes do not express VEGF receptors, we speculated that VEGF acts on sinusoidal endothelial cells (SECs) and promotes production of cytokines such as hepatocyte growth factor in SECs, resulting in reducing apoptosis through an increase expression of Bcl-xL in hepatocytes. We suggest that VEGF has a potent antiapoptotic effect on hepatocytes through cell–cell interaction between SECs and hepatocytes.     

Alternative sources of hepatocytes for cell therapy

There is an urgent need to search for alternatives to whole organ transplantation. Several methods have been proposed. Among these strategies, cell transplantation is currently one of the most promising. To achieve this aim, in addition to highly differentiated adult hepatocytes, the use of stem cells is considered a highly attractive therapeutic method for the treatment of liver disease and for temporary support of hepatic function until a liver becomes available for organ transplantation. This strategy is based on the ability of stem cells to differentiate into different cellular types according to their environment. Therefore, stem cells could be an unlimited source of hepatic cells for transplantation and gene therapy. Bone marrow is considered the most promising source of adult stem cells, partly due to the versatility of the cells obtained in repairing damaged tissues of several lineages. Several different types of stem cells have been described in bone marrow: hematopoietic, mesenchymal, side population and multipotent adult stem cells. Bone marrow cells have been hypothesized as a third recruitment source in liver regeneration in addition to hepatocytes and endogenous liver stem cells. Consequently, attempts have been made to differentiate them into hepatic lineage for their subsequent use in hepatic cell therapy. The present article reviews the progress made in this field or research.     

Use of mesenchymal stem cells to treat liver fibrosis:Current situation and future prospects

Progressive liver fibrosis is a major health issue for which no effective treatment is available,leading to cirrhosis and orthotopic liver transplantation.However,organ shortage is a reality.Hence,there is an urgent need to find alternative therapeutic strategies.Cellbased therapy using mesenchymal stem cells(MSCs) may represent an attractive therapeutic option,based ontheir immunomodulatory properties,their potential to differentiate into hepatocytes,allowing the replacement of damaged hepatocytes,their potential to promote residual hepatocytes regeneration and their capacity to inhibit hepatic stellate cell activation or induce their apoptosis,particularly via paracrine mechanisms.The current review will highlight recent findings regarding the input of MSC-based therapy for the treatment of liver fibrosis,from in vitro studies to pre-clinical and clinical trials.Several studies have shown the ability of MSCs to reduce liver fibrosis and improve liver function.However,despite these promising results,some limitations need to be considered.Future prospects will also be discussed in this review.     

Hepatic differentiation of amniotic epithelial cells

Hepatocyte transplantation to treat liver disease is largely limited by the availability of useful cells. Human amniotic epithelial cells (hAECs) from term placenta express surface markers and gene characteristics of embryonic stem cells and have the ability to differentiate into all three germ layers, including tissues of endodermal origin (i.e., liver). Thus, hAECs could provide a source of stem cell-derived hepatocytes for transplantation. We investigated the differentiation of hAECs in vitro and after transplantation into the livers of severe combined immunodeficient (SCID)/beige mice. Moreover, we tested the ability of rat amniotic epithelial cells (rAECs) to replicate and differentiate upon transplantation into a syngenic model of liver repopulation. In vitro results indicate that the presence of extracellular matrix proteins together with a mixture of growth factors, cytokines, and hormones are required for differentiation of hAECs into hepatocyte-like cells. Differentiated hAECs expressed hepatocyte markers at levels comparable to those of fetal hepatocytes. They were able to metabolize ammonia, testosterone, and 17α-hydroxyprogesterone caproate, and expressed inducible fetal cytochromes. After transplantation into the liver of retrorsine (RS)-treated SCID/beige mice, na?ve hAECs differentiated into hepatocyte-like cells that expressed mature liver genes such as cytochromes, plasma proteins, transporters, and other hepatic enzymes at levels equal to adult liver tissue. When transplanted in a syngenic animal pretreated with RS, rAECs were able to engraft and generate a progeny of cells with morphology and protein expression typical of mature hepatocytes. CONCLUSION: Amniotic epithelial cells possess the ability to differentiate into cells with characteristics of functional hepatocytes both in vitro and in vivo, thus representing a useful and noncontroversial source of cells for transplantation.     

Autologous Bone Marrow–Derived Cells in the Treatment of Liver Disease Patients

Liver transplantation is universally accepted as a “cure” procedure, and yet is not universally applicable for the treatment of end-stage liver diseases (ESLD) because of the shortage of donors, operative complications, risk of rejection, and high cost. Bioartificial liver device is an option to temporarily improve the liver function and to bridge the patients to liver transplantation. However, bioartificial liver device has many problems in clinical application, such as hepatocyte allograft rejection and maintenance of hepatocyte viability and function. Another therapeutic option is stem cell transplantation. There are two broad types of stem cells: Embryonic stem cells and adult stem cells. The latter are sourced from bone marrow (BM), adipose tissue, and blood. This review will concentrate on BM-derived cells. BM-derived cell transplantation, although not ideal, is theoretically an optimal modality for the treatment of ESLD. Autologous BM-derived cells have no graft rejection, have the capability of regeneration and self-renewal, and are multipotent stem cells that can differentiate into a variety of cell types which include hepatocytes. The pathway from BM-derived cell to hepatocyte is well documented. The present review summarizes the delivery routes of BM-derived cells to the liver, the evidences of engraftment of BM-derived cells in the liver, and the possible mechanisms of BM-derived cells in liver repair and regeneration, and finally, updates the clinical applications.     

Multipotent mesenchymal stromal cells: A promising strategy to manage alcoholic liver disease

Chronic alcohol consumption is a major cause of liver disease. The term alcoholic liver disease (ALD) refers to a spectrum of mild to severe disorders including steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. With limited therapeutic options, stem cell therapy offers significant potential for these patients. In this article, we review the pathophysiologic features of ALD and the therapeutic mechanisms of multipotent mesenchymal stromal cells, also referred to as mesenchymal stem cells (MSCs), based on their potential to differentiate into hepatocytes, their immunomodulatory properties, their potential to promote residual hepatocyte regeneration, and their capacity to inhibit hepatic stellate cells. The perfect match between ALD pathogenesis and MSC therapeutic mechanisms, together with encouraging, available preclinical data, allow us to support the notion that MSC transplantation is a promising therapeutic strategy to manage ALD onset and progression.     

Differentiation of hepatocytoid cell induced from whole-bone-marrow method isolated rat myeloid mesenchymal stem cells

AIM: To explore the expansion and differentiation of hepatocytoid cell induced from myeloid mesenchymal stem cell (MSC) in vitro, in order to find suitable resource of hepatocytes for bioartificial liver or liver transplantation. METHODS: The rat myeloid MSC was isolated and divided into three groups which were cultured by Frieden-steion method, and then were induced by culture fluid, culture fluid plus cholestatic serum and culture fluid plus hepatocyte growth factor (HGF), respectively. Hepatocytoid cell as well as expression of CK18 and AFP was observed by immunohistochemistry. RESULTS: After the induction for 21 d, hepatocytoid cell was observed, and its expression of CK18 and AFP was detected by immunohistochemistry in MSC cultured with cholestatic serum. Furthermore, on the 35th d, albumin mRNA was expressed in the cell, suggesting the inducing effect was similar to that by HGF. CONCLUSION: Rat myeloid MSC can differentiate into hepatocyte lineage under appropriate condition. This method is easy to operate.     

Differentiation of hepatocytoid cell induced from whole-bone-marrow method isolated rat myeloid mesenchymal stem cells

AIM: To explore the expansion and differentiation of hepatocytoid cell induced from myeloid mesenchymal stem cell (MSC)in vitro, in order to find suitable resource of hepatocytes for bioartificial liver or liver transplantation. METHODS: The rat myeloid MSC was isolated and divided into three groups which were cultured by Frieden steion method, and then were induced by culture fluid, culture fluid plus cholestatic serum and culture fluid plus hepatocyte growth factor (HGF), respectively. Hepatocytoid cell as well as expression of CK18 and AFP was observed by immunohistochemistry. RESULTS: After the induction for 21 d, hepatocytoid cell was observed, and its expression of CK18 and AFP was detected by immunohistochemistry in MSC cultured withcholestatic serum. Furthermore, on the 35th d, albumin mRNA was expressed in the cell, suggesting the inducing effect was similar to that by HGF. CONCLUSION: Rat myeloid MSC can differentiate into hepatocyte lineage under appropriate condition. This method is easy to operate.     

Liver regeneration is impaired by FK778 in partially hepatectomized rats, while supplemental uridine restores both liver growth and hepatocyte proliferation

Aim:  The impact of mandatory immunosuppression on liver regeneration after segmental liver transplantation is of clinical importance. FK778, a novel immunosuppressant, inhibits pyrimidine biosynthesis and prevents rejection after organ transplantation in a dose-dependent manner. We investigated the effect of FK778 at a highly effective dose on liver regeneration in a small animal model.
Methods:  Inbred Lewis rats were subjected to 70% partial hepatectomy (PH) and treated with saline ( n  = 28), uridine ( n  = 16), FK778 alone ( n  = 28) or in combination with uridine ( n  = 16). FK778 was given intravenously daily at a dose of 25 mg/kg bodyweight (bw) and uridine was given daily intraperitoneally at a dose of 250 mg/kg bw. Liver bodyweight ratio (LBR), hepatocyte proliferation index (PI), blood chemistry and morphological analysis were incorporated. PI was determined by Ki-67 immunostaining. De Ritis ratio was calculated to assess the extent of liver damage.
Results:  In FK778-treated animals PI was decreased at 24 h and 72 h and LBR was lower at 48 h and 72 h ( P  < 0.05) after the PH. In addition, morphological analysis showed confluent central lobular necrosis at 72 h in four of seven animals. Uridine supplementation restored PI, LBR and the de Ritis ratio in FK778-treated animals and no confluent necroses were observed.
Conclusion:  FK778 is antihepatotrophic as well as antiproliferative during rat liver regeneration. Both liver growth and hepatocyte proliferation are completely restored by supplementation with uridine. In addition, supplemental uridine markedly reduces the severity of morphological abnormalities consistent with FK778 toxicity.     

Update on hepatic stem cells

Abstract: The liver, like most organs in an adult healthy body, maintains a perfect balance between cell gain and cell loss. Though normally proliferatively quiescent, simple hepatocyte loss such as that caused by partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response to restore liver mass. This restoration of moderate cell loss and ‘wear and tear’ renewal is largely achieved by hepatocyte self‐replication. Furthermore, cell transplant models have shown that hepatocytes can undergo significant clonal expansion. Such observations indicate that hepatocytes are the functional stem cells of the liver. More severe liver injury activates a facultative stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of biliary epithelia within the lobules before these cells differentiate into hepatocytes. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells can contribute to the albeit low renewal rate of hepatocytes, make a more significant contribution to regeneration, and even completely restore normal function in a murine model of hereditary tyrosinaemia. How these three stem cell populations integrate to achieve a homeostatic balance is not understood. This review focuses on three aspects of liver stem cell biology: 1) the hepatic stem cell candidates; 2) models of cell transplantation into the liver; and 3) the therapeutic potential of hepatic stem cells.     

Role of peroxisome proliferator-activated receptor-gamma (PPARgamma) during liver regeneration in rats

Background and Aim:  Peroxisome proliferator-activated receptor-gamma (PPARγ), a member of the nuclear receptor superfamily, is widely expressed in adipocytes and other tissues, including the liver. Several reports have shown that PPARγ activation induced cell-cycle arrest and apoptosis in tumor cells. We investigated the role of the PPARγ/ligand system and the effect of the PPARγ agonist during liver regeneration.
Methods:  Expression of PPARγ and serum levels of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) by enzyme immunoassay were evaluated in rats following partial hepatectomy (PH group). Further, the effect of the PPARγ agonist, pioglitazone, on liver regeneration (PH + PGZ group) was evaluated by proliferating cell nuclear antigen labeling index, relative liver weight, and expression of cell-cycle regulators.
Results:  The number of PPARγ-stained hepatocytes decreased at 24 h (PH, 15.8 ± 2.2%; sham, 35.5 ± 2.4%; P  < 0.001) and increased in the late phase of liver regeneration compared to the sham-operated group ( P  < 0.001 at 48–120 h). The peaks of serum 15d-PGJ2 (627.0 ± 91.1 pg/ml) and PPARγ expression (90.6 ± 3.1%) coincided in the late phase of liver regeneration. Also, oral administration of pioglitazone inhibited hepatocyte proliferation, in terms of the proliferating cell nuclear antigen (PCNA) labeling index and p27 expression during the late phase of liver regeneration, and caused a transient reduction in liver mass when compared to the PH group.
Conclusions:  These results indicate that the PPARγ/ligand system may be one of the key negative regulators of hepatocyte proliferation and may be responsible for the inhibition of liver growth in the late phase of liver regeneration.     

Update on hepatic stem cells

The liver, like most organs in an adult healthy body, maintains a perfect balance between cell gain and cell loss. Though normally proliferatively quiescent, simple hepatocyte loss such as that caused by partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response to restore liver mass. This restoration of moderate cell loss and 'wear and tear' renewal is largely achieved by hepatocyte self-replication. Furthermore, cell transplant models have shown that hepatocytes can undergo significant clonal expansion. Such observations indicate that hepatocytes are the functional stem cells of the liver. More severe liver injury activates a facultative stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of biliary epithelia within the lobules before these cells differentiate into hepatocytes. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells can contribute to the albeit low renewal rate of hepatocytes, make a more significant contribution to regeneration, and even completely restore normal function in a murine model of hereditary tyrosinaemia. How these three stem cell populations integrate to achieve a homeostatic balance is not understood. This review focuses on three aspects of liver stem cell biology: 1) the hepatic stem cell candidates; 2) models of cell transplantation into the liver; and 3) the therapeutic potential of hepatic stem cells.     

骨髓间充质干细胞对大鼠减体积肝移植术后肝细胞再生与修复的作用

目的探讨骨髓间充质干细胞（BMSCs）对大鼠减体积肝移植术后肝脏再生与修复的影响。方法采用生化、免疫组化等方法检测术后不同时间的血清和肝脏标本。（1）取同周龄Wistar大鼠骨髓体外培养BMSCs,标记CSFE荧光后制备悬液;（2）建立大鼠50%减体积肝移植模型（供体鼠为SD大鼠,受体鼠为Wistar大鼠）,分为实验组（取BMSCs悬液经门静脉植入）和对照组（取等量生理盐水经门静脉注射）,观察大鼠生存状态,测量肝再生的相关指标。结果 （1）成功分离培养BMSCs;（2）术后2h,大鼠均自由活动饮水;（3）实验组肝组织中存在大量BMSCs,术后第2、3 d实验组肝体比显著高于对照组。病理显示：实验组肝细胞损伤较对照组轻微。结论植入BMSCs后,可以促进大鼠减体积肝移植术后肝细胞增殖和修复。