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. Cell-based therapy using mesenchymal stem cells (MSCs) may represent an attractive therapeutic option, based on their 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.
Liver fibrosis; Cirrhosis; Mesenchymal stem cells; Cell therapy; Hepatic stellate cells
AIM: To test the ability of adult-derived human liver stem/progenitor cells (ADHLSC) from large scale cultures to conjugate bilirubin in vitro and in bilirubin conjugation deficient rat.
METHODS: ADHLSC from large scale cultures were tested for their phenotype and for their capacity to conjugate bilirubin in vitro after hepatogenic differentiation. In vivo, Gunn rats [uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1) deficient animal] were injected with ADHLSC and cryopreserved hepatocytes (positive control). Two, 4, 13 and 27 wk post-transplantation, transplanted Gunn rat bilirubin serum levels were determined by high performance liquid chromatography. Human transplanted cell engraftment was assessed 27 wk post-transplantation using immunohistochemistry and RTqPCR.
RESULTS: Large scale culture conditions do not modify ADHLSC phenotype, ADHLSC were able to specifically conjugate bilirubin. ADHLSC were intraportally injected into Gunn rats and blood UCB was measured at different times post-transplantation, infused-Gunn rats exhibited a metabolic effect 3 mo post-transplantation and maintained over a 6 mo period. ADHLSC engraftment into Gunn rat’s liver was demonstrated by RTqPCR and immunohistochemistry against albumin and UGT1A1.
CONCLUSION: ADHLSC from large scale cultures are efficient in conjugating bilirubin in vitro and in restoring a deficient metabolic function (reducing bilirubin level) in hyperbilirubinemic rats.
Liver stem/progenitor cells; Gunn rat; Hepatocyte; in vitro and in situ differentiation; Uridine diphosphate-glucuronosyltransferase 1A1
Pancreatic and duodenal homeobox 1 (PDX1), a member of the homedomain-containing transcription factor family, is a key transcription factor important for both pancreas development and mature beta-cell function. The ectopic overexpression of Pdx1, Neurog3, and MafA in mice reprograms acinar cells to insulin-producing cells. We developed a qPCR-based gene-expression assay to screen >60,000 compounds for expression of each of these genes in the human PANC-1 ductal carcinoma cell line. We identified BRD7552, which up-regulated PDX expression in both primary human islets and ductal cells, and induced epigenetic changes in the PDX1 promoter consistent with transcriptional activation. Prolonged compound treatment induced insulin mRNA and protein, and enhanced insulin expression induced by the three-gene combination. These results provide a proof of principle for identifying small molecules that induce expression of transcription factors to control cellular reprogramming.
Advanced therapies including stem cells are currently a major biotechnological development. Adult liver stem cells can differentiate into hepatocyte like cells and be infused in the recipient’s liver to bring a missing metabolic function. These cells can be produced in large quantities in vitro. Allogeneic stem cells are required to treat genetic diseases, and this approach allows to use one single source of tissue to treat different diseases and many recipients. Mesenchymal stem cells can in addition play an immunomodulatory and anti-inflammatory role and possibly prevent the accumulation of fibrous tissue in the liver. From a regulatory point of view, stem cells are considered as medicinal products, and must undergo a pharmaceutical development that goes beyond the research and proof-of-concept phases. Here, we review the track followed from the first hepatocyte transplantation in 2000 to the next generation product issued from stem cell technology, and the start of EMA approved clinical trials to evaluate the safety and potency of liver stem cells for the treatment of inborn errors of the liver metabolism.
AIM: To investigate the activity and expression of EAAT2 glutamate transporter in both in vitro and in vivo models of cholestasis.
METHODS: This study was conducted on human hepatoblastoma HepG2 cell cultures, the liver of bile duct ligated rats and human specimens from cholestatic patients. EAAT2 glutamate transporter activity and expression were analyzed using a substrate uptake assay, immunofluorescence, reverse transcription-polymerase chain reaction, and immunohistochemistry, respectively.
RESULTS: In HepG2 cells, cholestasis was mimicked by treating cells with the protein kinase C activator, phorbol 12-myristate 13-acetate. Under such conditions, EAAT2 transporter activity was decreased both at the level of substrate affinity and maximal transport velocity. The decreased uptake was correlated with intracellular translocation of EAAT2 molecules as demonstrated using immunofluorescence. In the liver of bile duct ligated rats, an increase in EAAT2 transporter protein expression in hepatocytes was demonstrated using immunohistochemistry. The same findings were observed in human liver specimens of cholestasis in which high levels of γ-glutamyl transpeptidase were documented in patients with biliary atresia and progressive familial intrahepatic cholestasis type 3.
CONCLUSION: This study demonstrates the alteration in glutamate handling by hepatocytes in liver cholestasis and suggests a potential cross-talk between glutamatergic and bile systems.
Glutamate transport; Hepatocyte; Bile duct ligation; Cholestasis; Biliary atresia
Adult-derived human liver stem/progenitor cells (ADHLSC) are obtained after primary culture of the liver parenchymal fraction. The cells are of fibroblastic morphology and exhibit a hepato-mesenchymal phenotype. Hepatic stellate cells (HSC) derived from the liver non-parenchymal fraction, present a comparable morphology as ADHLSC. Because both ADHLSC and HSC are described as liver stem/progenitor cells, we strived to extensively compare both cell populations at different levels and to propose tools demonstrating their singularity.
ADHLSC and HSC were isolated from the liver of four different donors, expanded in vitro and followed from passage 5 until passage 11. Cell characterization was performed using immunocytochemistry, western blotting, flow cytometry, and gene microarray analyses. The secretion profile of the cells was evaluated using Elisa and multiplex Luminex assays.
Both cell types expressed α-smooth muscle actin, vimentin, fibronectin, CD73 and CD90 in accordance with their mesenchymal origin. Microarray analysis revealed significant differences in gene expression profiles. HSC present high expression levels of neuronal markers as well as cytokeratins. Such differences were confirmed using immunocytochemistry and western blotting assays. Furthermore, both cell types displayed distinct secretion profiles as ADHLSC highly secreted cytokines of therapeutic and immuno-modulatory importance, like HGF, interferon-γ and IL-10.
Our study demonstrates that ADHLSC and HSC are distinct liver fibroblastic cell populations exhibiting significant different expression and secretion profiles.
A 3-year-old girl suffering from ornithine carbamoyltransferase (OTC) deficiency was poorly equilibrated under conventional diet and scavenger treatment. Following unsuccessful cryopreserved hepatocyte transplantation, she received two infusions of Adult Derived Human Liver Stem/Progenitor Cells (ADHLSCs) expanded in vitro under GMP settings, the quantity being equivalent to 0.75% of her calculated liver mass. Using FISH immunostaining for the Y chromosome, the initial biopsy did not detect any male nuclei in the recipient liver. Two liver biopsies taken 100 days after ADHLSC transplantation showed 3% and 5% of male donor cells in the recipient liver, thus suggesting repopulation by donor cells. Although limited follow-up did not allow us to draw conclusions on long-term improvement, these results provide a promising proof of concept that this therapy is feasible in an OTC patient.
Umbilical cord matrix stem cells (UCMSC) have generated great interest in various therapeutic approaches, including liver regeneration. This article aims to analyze the specific characteristics and the potential occurrence of premalignant alterations of UCMSC during long-term expansion, which are important issues for clinical applications. UCMSC were isolated from the umbilical cord of 14 full-term newborns and expanded in vitro until senescence. We examined the long-term growth potential, senescence characteristics, immunophenotype and multilineage differentiation capacity of these cells. In addition, their genetic stability was assessed through karyotyping, telomerase maintenance mechanisms and analysis of expression and functionality of cell cycle regulation genes. The tumorigenic potential was also studied in immunocompromised mice. In vitro, UCMSC reached up to 33.7±2.1 cumulative population doublings before entering replicative senescence. Their immunophenotype and differentiation potential, notably into hepatocyte-like cells, remained stable over time. Cytogenetic analyses did not reveal any chromosomal abnormality and the expression of oncogenes was not induced. Telomere maintenance mechanisms were not activated. Just as UCMSC lacked transformed features in vitro, they could not give rise to tumors in vivo. UCMSC could be expanded in long-term cultures while maintaining stable genetic features and endodermal differentiation potential. UCMSC therefore represent safe candidates for liver regenerative medicine.
Islet and hepatocyte transplantation are associated with tissue factor-dependent activation of coagulation which elicits instant blood mediated inflammatory reaction, thereby contributing to a low rate of engraftment. The aim of this study was i) to evaluate the procoagulant activity of human adult liver-derived mesenchymal progenitor cells (hALPCs), ii) to compare it to other mesenchymal cells of extra-hepatic (bone marrow mesenchymal stem cells and skin fibroblasts) or liver origin (liver myofibroblasts), and iii) to determine the ways this activity could be modulated. Using a whole blood coagulation test (thromboelastometry), we demonstrated that all analyzed cell types exhibit procoagulant activity. The hALPCs pronounced procoagulant activity was associated with an increased tissue factor and a decreased tissue factor pathway inhibitor expression as compared with hepatocytes. At therapeutic doses, the procoagulant effect of hALPCs was inhibited by neither antithrombin activators nor direct factor Xa inhibitor or direct thrombin inhibitors individually. However, concomitant administration of an antithrombin activator or direct factor Xa inhibitor and direct thrombin inhibitor proved to be a particularly effective combination for controlling the procoagulant effects of hALPCs both in vitro and in vivo. The results suggest that this dual antithrombotic therapy should also improve the efficacy of cell transplantation in humans.
AIM: To evaluate the presence of progenitor cells in healthy adult rat liver displaying the equivalent advanced hepatogenic profile as that obtained in human.
METHODS: Rat fibroblastic-like liver derived cells (rFLDC) were obtained from collagenase-isolated liver cell suspensions and characterized and their phenotype profile determined using flow cytometry, immunocytochemistry, reverse transcription polymerase chain reaction and functional assays.
RESULTS: rFLDC exhibit fibroblastoid morphology, express mesenchymal (CD73, CD90, vimentin, α-smooth muscle actin), hepatocyte (UGT1A1, CK8) and biliary (CK19) markers. Moreover, these cells are able to store glycogen, and have glucose 6 phosphatase activity, but not UGT1A1 activity. Under the hepatogenic differentiation protocol, rFLDC display an up-regulation of hepatocyte markers expression (albumin, tryptophan 2,3-dioxygenase, G6Pase) correlated to a down-regulation of the expression of the biliary marker CK19.
CONCLUSION: Advanced hepatic features observed in human liver progenitor cells could not be demonstrated in rFLDC. However, we demonstrated the presence of an original rodent hepato-biliary cell type.
Hepato biliary profile; Hepatogenic differentiation; Liver; Progenitor cell; Rat
Mitochondrial dysfunction participates in the progression of several pathologies. Although there is increasing evidence for a mitochondrial role in liver disease, little is known about its contribution to hepatic stellate cell (HSC) activation. In this study we investigated the role of mitochondrial activity through mild uncoupling during in vitro activation of HSCs.
Cultured primary human and mouse HSCs were treated with the chemical uncouplers FCCP and Valinomycin. ATP levels were measured by luciferase assay and production of reactive oxygen species was determined using the fluorescent probe DCFH-DA. Possible cytotoxicity by uncoupler treatment was evaluated by caspase 3/7 activity and cytoplasmic protease leakage. Activation of HSCs and their response to the pro-fibrogenic cytokine TGF-β was evaluated by gene expression of activation markers and signal mediators using RT-qPCR. Proliferation was measured by incorporation of EdU and protein expression of α-smooth muscle actin was analyzed by immunocytochemistry and western blot.
FCCP and Valinomycin treatment mildly decreased ATP and reactive oxygen species levels. Both uncouplers increased the expression of mitochondrial genes such as Tfam and COXIV while inducing morphological features of quiescent mouse HSCs and abrogating TGF-β signal transduction. Mild uncoupling reduced HSC proliferation and expression of pro-fibrogenic markers of mouse and human HSCs.
Mild mitochondrial uncoupling inhibits culture-induced HSC activation and their response to pro-fibrogenic cytokines like TGF-β. These results therefore suggest mitochondrial uncoupling of HSCs as a strategy to reduce progression of liver fibrosis.
Hepatic stellate cell; Mitochondria; Uncoupler; Fibrosis
Background & Aims
Progressive familial intrahepatic cholestasis (PFIC) with normal serum levels of gamma-glutamyltranspeptidase can result from mutations in ATP8B1 (encoding familial intrahepatic cholestasis 1 [FIC1]) or ABCB11 (encoding bile salt export pump [BSEP]). We evaluated clinical and laboratory features of disease in patients diagnosed with PFIC, who carried mutations in ATP8B1 (FIC1 deficiency) or ABCB11 (BSEP deficiency). Our goal was to identify features that distinguish presentation and course of these 2 disorders, thus facilitating diagnosis and elucidating the differing consequences of ATP8B1 and ABCB11 mutations.
A retrospective multi-center study was conducted, using questionnaires and chart review. Available clinical and biochemical data from 145 PFIC patients with mutations in either ATP8B1 (61 “FIC1 patients”) or ABCB11 (84 “BSEP patients”) were evaluated.
At presentation, serum aminotransferase and bile salt levels were higher in BSEP patients; serum alkaline phosphatase values were higher, and serum albumin values were lower, in FIC1 patients. Elevated white blood cell counts, and giant or multinucleate cells at liver biopsy, were more common in BSEP patients. BSEP patients more often had gallstones and portal hypertension. Diarrhea, pancreatic disease, rickets, pneumonia, abnormal sweat tests, hearing impairment, and poor growth were more common in FIC1 patients. Among BSEP patients, the course of disease was less rapidly progressive in patients bearing the D482G mutation.
Severe forms of FIC1 and BSEP deficiency differed. BSEP patients manifested more severe hepatobiliary disease, while FIC1 patients showed greater evidence of extrahepatic disease.
cholestasis; genetics; transport protein; pediatrics; P-type ATPase; ATP binding cassette protein; ATP8B1; FIC1; ABCB11; BSEP
Liver cell transplantation presents clinical benefit in patients with inborn errors of metabolism as an alternative, or at least as a bridge, to orthotopic liver transplantation. The success of such a therapeutic approach remains limited by the quality of the transplanted cells. Cryopreservation remains the best option for long-term storage of hepatocytes, providing a permanent and sufficient cell supply. However, isolated adult hepatocytes are poorly resistant to such a process, with a significant alteration both at the morphological and functional levels. Hence, the aim of the current review is to discuss the state of the art regarding widely-used hepatocyte cryopreservation protocols, as well as the assays performed to analyse the post-thawing cell quality both in vitro and in vivo. The majority of studies agree upon the poor quality and efficiency of cryopreserved/thawed hepatocytes as compared to freshly isolated hepatocytes. Intracellular ice formation or exposure to hyperosmotic solutions remains the main phenomenon of cryopreservation process, and its effects on cell quality and cell death induction will be discussed. The increased knowledge and understanding of the cryopreservation process will lead to research strategies to improve the viability and the quality of the cell suspensions after thawing. Such strategies, such as vitrification, will be discussed with respect to their potential to significantly improve the quality of cell suspensions dedicated to liver cell-based therapies.
Hepatocyte; Cryopreservation; Quality; Mitochondria; Intracellular ice formation
Liver cell transplantation is an attractive technique to treat liver-based inborn errors of metabolism. The feasibility and efficacy of the procedure has been demonstrated, leading to medium term partial metabolic control of various diseases. Crigler-Najjar is the paradigm of such diseases in that the host liver is lacking one function with an otherwise normal parenchyma. The patient is at permanent risk for irreversible brain damage. The goal of liver cell transplantation is to reduce serum bilirubin levels within safe limits and to alleviate phototherapy requirements to improve quality of life. Preliminary data on Gunn rats, the rodent model of the disease, were encouraging and have led to successful clinical trials. Herein we report on two additional patients and describe the current limits of the technique in terms of durability of the response as compared to alternative therapeutic procedures. We discuss the future developments of the technique and new emerging perspectives.
Hepatocyte transplantation; Cell therapy; Inborn error of metabolism; Crigler-Najjar; Liver regeneration; Animal models
Stem cells from extra- or intrahepatic sources have been recently characterized and their usefulness for the generation of hepatocyte-like lineages has been demonstrated. Therefore, they are being increasingly considered for future applications in liver cell therapy. In that field, liver cell transplantation is currently regarded as a possible alternative to whole organ transplantation, while stem cells possess theoretical advantages on hepatocytes as they display higher in vitro culture performances and could be used in autologous transplant procedures. However, the current research on the hepatic fate of stem cells is still facing difficulties to demonstrate the acquisition of a full mature hepatocyte phenotype, both in vitro and in vivo. Furthermore, the lack of obvious demonstration of in vivo hepatocyte-like cell functionality remains associated to low repopulation rates obtained after current transplantation procedures. The present review focuses on the current knowledge of the stem cell potential for liver therapy. We discuss the characteristics of the principal cell candidates and the methods to demonstrate their hepatic potential in vitro and in vivo. We finally address the question of the future clinical applications of stem cells for liver tissue repair and the technical aspects that remain to be investigated.
Stem cells; Hepatocyte differentiation; Liver regeneration; Cell therapy
AIM: To investigate the presence and role of liver epithelial cells in the healthy human adult liver.
METHODS: Fifteen days after human hepatocyte primary culture, epithelial like cells emerged and started proliferating. Cell colonies were isolated and sub-cultured for more than 160 d under specific culture conditions. Cells were analyzed for each passage using immunofluorescence, flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR).
RESULTS: Flow cytometry analysis demonstrated that liver epithelial cells expressed common markers for hepatic and stem cells such as CD90, CD44 and CD29 but were negative for CD34 and CD117. Using immunofluorescence we demonstrated that liver epithelial cells expressed not only immature (α-fetoprotein) but also differentiated hepatocyte (albumin and CK-18) and biliary markers (CK-7 and 19), whereas they were negative for OV-6. RT-PCR analysis confirmed immunofluorescence data and revealed that liver epithelial cells did not express mature hepatocyte markers such as CYP2B6, CYP3A4 and tyrosine amino-transferase. Purified liver epithelial cells were transplanted into SCID mice. One month after transplantation, albumin positive cell foci were detected in the recipient mouse parenchyma.
CONCLUSION: According to their immature and bipotential phenotype, liver epithelial cells might represent a pool of precursors in the healthy human adult liver other than oval cells.
Epithelial-like cells; Liver stem cell; Hepatocyte; Differentiation; Cell therapy
Fifty-three patients with chronic hepatitis B and active viral replication were studied for 4 weeks while on treatment and for 12 weeks after treatment with the oral nucleoside analogue lamivudine. Children aged 2 to 12 years were randomized to receive twice-daily doses of 0.35, 1.5, or 4 mg of lamivudine solution per kg of body weight or once-daily doses of 3 mg of lamivudine solution per kg. Adolescents aged 13 to 17 years received lamivudine at 100 mg (as tablets). Blood samples for pharmacokinetic assay were taken on days 1 and 28. Lamivudine was rapidly absorbed following oral administration, with the maximum concentration in serum being reached 0.5 to 1 h postdosing. Apparent oral clearance (CL/F) was higher in younger children and decreased with age, with CL/F values for adolescents reaching those seen for adults by the age of 12. All doses produced a dramatic fall in serum hepatitis B virus (HBV) DNA levels, with a median reduction of ≥99.5% after 4 weeks of treatment and with the levels returning to the baseline levels posttreatment. The correlation of dose, area under the concentration-time curve (AUC), and changes in HBV DNA levels, as measured by the Chiron Quantiplex assay, showed maximal antiviral effects (99.9% inhibition and a reduction of the amount of HBV DNA of approximately 3 log10) at 3 mg/kg/day, with no discernible increase in effect seen whether the drug was given at 4 mg/kg twice daily or whether it was given once daily or twice daily. The limit of detection of the assay (2.5 pg/ml) was reached for some but not all patients across the dose ranges, with the smallest number (n = 2) of those having values negative by the Chiron Quantiplex assay being in the lowest-dose group. The 13- to 17-year-olds showed a similar overall response in terms of the HBV DNA level reduction compared to that for patients younger than age 13. Analysis of the same samples by PCR, which has a lower limit of sensitivity than the Chiron Quantiplex assay, also showed average drops in HBV DNA levels of about 3 log10 at 4 weeks for patients for which the AUC was ≥4,000 ng · h/ml, confirming the conclusions given above. Lamivudine treatment was well tolerated at all doses, with no significant adverse events or laboratory data changes. On the basis of pharmacokinetic and pharmacodynamic data, a 3-mg/kg/day dose in children (ages 2 to 12 years) with chronic hepatitis B provides levels of exposure and trough concentrations similar to those seen in adults following the receipt of doses of 100 mg. The 100-mg dose is being evaluated in a large phase III study with HBV-infected pediatric patients.