Publications

Academic publications

Prigent et al. Cell Transplantation; May 20 2014

Human progenitor cell quantification after xenotransplantation in rat and mouse models by a sensitive qPCR assay

Xenotransplantation of human cells in animal models is an essential tool for evaluation of safety and efficacy of cell-based products for therapeutic use. Sensitive and reproducible methods are needed to detect and quantify human cells engrafted into the host tissue either in the targeted organ or in undesired locations. We developed a robust quantitative polymerase chain reaction (qPCR) assay based on amplification of human AluYb8 repeats, to assess the number of human cells present in rat or mouse tissues after transplantation.

Standard curves of mixed human/rodent DNA and mixed human/rodent cells have been performed to determine the limit of detection and linear range of the assay. Standard curves from DNA mixing differed significantly from standard curves from cell mixing. We show here that the AluYb8 qPCR assay is highly reproducible and is able to quantify human cells in a rodent cell matrix over a large linear range that extends from 50 to 0.01% human cells. Short-term in vivo studies showed that human cells could be quantified in mouse liver up to 7 days after intra-splenic transplantation and in rat liver four hours after intra-hepatic transplantation.

Defresne et al. Nucl Med Biol, 2014 Apr;41(4):371-5

Biodistribution of adult derived human liver stem cells following intraportal infusion in a 17-year-old patient with glycogenosis type 1A.

Current treatment of inherited liver inborn errors of metabolism in children consists in appropriate diet and drugs and, for unstable patients, final orthotopic liver transplantation. Unfortunately, liver transplantation remains not easily available because of organ shortage and imposes inherent risks and lifelong immunosuppressive therapy. Therefore alternative treatments are required. Hepatocytes transplantation and its limitations led to consider innovative alternative such as transplantation of adult derived human liver stem cells (ADLHSC). These cells present high proliferative capacity, good resistance to cryopreservation and ability to differentiate into hepatocyte-like cells displaying mature hepatocyte functions.

Biodistribution of ADHLSC had never been assessed after infusion through the portal vein in patients. This information is required to determine the safety of the method.

ADHLSC were efficiently labelled with 111-Indium DTPA radiotracer and SPECT imaging was used for the acquisition of whole body imaging to document short term biodistribution of ADHLSC.

Following infusion through the portal vein, ADHLSC diffused homogenously throughout the liver and remained strictly within the targeted organ. Images were acquired until 5 days after infusion. At that time, no signal was observed in any other organs except the liver. Urinary excretion of 111-Indium DTPA was also monitored.

For the first time, we documented the short term biodistribution of ADHLSC within the liver after infusion through the portal vein

Sokal et al. J Inherit Metab Dis, February 2014

Treating Inborn Errors of Liver Metabolism with Stem Cells: Current Clinical Development

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.

Berardis et al. PLOS ONE, January 2014, Volume 9

Gene Expression Profiling and Secretome Analysis Differentiate Adult-Derived Human Liver Stem/Progenitor Cells and Human Hepatic Stellate Cells

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 a-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-c and IL-10. Our study demonstrates that ADHLSC and HSC are distinct liver fibroblastic cell populations exhibiting significant different expression and secretion profiles.

 

Khuu et al. Cell Transplant. 2012 Dec 4. [Epub ahead of print]

Adult human liver mesenchymal stem/progenitor cells participate to mouse liver regeneration after hepatectomy.

The advances in stem cell science have promoted research on their use in liver regenerative medicine. Beyond the demonstration of their ability to display metabolic functions in vitro, candidate cells should demonstrate achievable in situ differentiation, and ability to participate to liver repopulation. In this work, we studied the in vivo behaviour of adult liver mesenchymal stem/progenitor cells (ADHLSCs) after transplantation into immuno-deficient mice. Kinetic of engraftment and in situ hepatogenic differentiation were analyzed. Response of transplanted ADHLSCs to regenerative stimulus has also been evaluated. Non-differentiated ADHLSCs were intrasplenically transplanted into SCID mice. Efficiency of transplantation was evaluated at the level of engraftment and in situ differentiation using immunohistochemistry, in situ hybridization and RT-PCR. After bromodeoxyuridine (BrdU) implantation, proliferation of transplanted ADHLSCs in response to 20% hepatectomy was assessed using immunohistochemistry. We demonstrated that ADHLSC engraftment in the SCID mouse liver was low, but remained stable up to 60 days post-transplantation, when albumin (ALB) immuno-positive ADHLSCs were still detected and organized as clusters. Co-expression of ornithine transcarbamylase (OTC) demonstrated ADHLSC in situ differentiation mostly near the hepatic portal vein. After 20% hepatectomy on one month transplanted mice, the percentage of BrdU and human ALB immuno-positive ADHLSCs increased from 3 to 28 days post-BrdU implantation to reach 31.3 ± 5.4% of the total analyzed human cells. In the current study, we demonstrate that transplanted ADHLSCs are able to differentiate in the non pre-conditioned SCID mouse liver mainly in the peri-portal area. In response to partial hepatectomy, integrated ADHLSCs proliferate and participate to recipient mouse liver regeneration.

Stephenne et al. PLoS One. 2012;7(8):e42819

Bivalirudin in combination with heparin to control mesenchymal cell procoagulant activity.

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.

In vitro differentiated adult human liver progenitor cells display mature hepatic metabolic functions: a potential tool for in vitro pharmacotoxicological testing.

Khuu et al. Cell Transplant. 2011;20(2):287-302.

The potential use of stem/progenitor cells as alternative cell sources to mature hepatocytes remains basically dependent on their ability to exhibit some, if not all, the metabolic liver functions. In the current study, four major liver functions were investigated in adult derived human liver stem/progenitor cell (ADHLSCs) populations submitted to in vitro hepatogenic differentiation: gluconeogenesis, ammonia detoxification, and activity of phase I and phase II drug-metabolizing enzymes. These acquired hepatic activities were compared to those of primary adult human hepatocytes, the standard reference. Amino acid content was also investigated after hepatogenic differentiation. Differentiated ADHLSCs display higher de novo synthesis of glucose correlated to an increased activity of glucose-6 phosphatase and mRNA expression of key related enzymes. Differentiated ADHLSCs are also able to metabolize ammonium chloride and to produce urea. This was correlated to an increase in the mRNA expression of relevant key enzymes such arginase. With respect to drug metabolism, differentiated ADHLSCs express mRNAs of all the major cytochromes investigated, among which the CYP3A4 isoform (the most important drug-metabolizing enzyme). Such increased expression is correlated to an enhanced phase I activity as independently demonstrated using fluorescence-based assays. Phase II enzyme activity and amino acid levels also show a significant enhancement in differentiated ADHLSCs. The current study, according to data independently obtained in different labs, demonstrates that in vitro differentiated ADHLSCs are able to display advanced liver metabolic functions supporting the possibility to develop them as potential alternatives to primary hepatocytes for in vitro settings.

Najimi et al. Cell Transplant. 2007;16(7):717-28.

Adult-derived human liver mesenchymal-like cells as a potential progenitor reservoir of hepatocytes?

It is currently accepted that adult tissues may develop and maintain their own stem cell pools. Because of their higher safety profile, adult stem cells may represent an ideal candidate cell source to be used for liver cell therapies. We therefore evaluated the differentiation potential of mesenchymal-like cells isolated from adult human livers. Mesenchymal-like cells were isolated from enzymatically digested adult human liver and expanded in vitro. Cell characterization was performed using flow cytometry, RT-PCR, and immunofluorescence, whereas the differentiation potential was evaluated both in vitro after incubation with specific media and in vivo after intrasplenic transplantation of uPA(+/+)-SCID and SCID mice. Adult-derived human liver mesenchymal-like cells expressed both hepatic and mesenchymal markers among which albumin, CYP3A4, vimentin, and alpha-smooth muscle actin. In vitro differentiation studies demonstrated that these mesenchymal-like cells are preferentially determined to differentiate into hepatocyte-like cells. Ten weeks following intrasplenic transplantation into uPA(+/+)-SCID mice, recipient livers showed the presence of human hepatocytic cell nodules positive for human albumin, prealbumin, and alpha-fetoprotein. In SCID transplanted liver mice, human hepatocyte-like cells were mostly found near vascular structures 56 days posttransplantation. In conclusion, the ability of isolated adult-derived liver mesenchymal stem-like cells to proliferate and differentiate into hepatocyte-like cells both in vitro and in vivo leads to propose them as an attractive expandable cell source for stem cell therapy in human liver diseases.

Hepatocytes papers

Darwish et al. Clin Res Hepatol Gastroenterol. 2011 Apr;35(4):271-80.

Paediatric liver transplantation for metabolic disorders. Part 2: Metabolic disorders with liver lesions.

Liver based metabolic disorders account for 10 to 15% of the indications for paediatric liver transplantation. In the last three decades, important progress has been made in the understanding of these diseases, and new therapies have emerged. Concomitantly, medical and surgical innovations have lead to improved results of paediatric liver transplantation, patient survival nowadays exceeding 80% 10 year after surgery with close to normal quality of life in most survivors. This review is a practical update on medical therapy, indications and results of liver transplantation, and potential future therapies, for the main liver based metabolic disorders in which paediatric liver transplantation may be considered. Part 1 focuses on metabolic based liver disorders without liver lesions, and part 2 on metabolic liver diseases with liver lesions.

Darwish et al. Clin Res Hepatol Gastroenterol. 2011 Mar;35(3):194-203.

Paediatric liver transplantation for metabolic disorders. Part 1: Liver-based metabolic disorders without liver lesions.

Liver-based metabolic disorders account for 10 to 15% of the indications for paediatric liver transplantation. In the last three decades, important progress has been made in the understanding of these diseases, and new therapies have emerged. Concomitantly, medical and surgical innovations have lead to improved results of paediatric liver transplantation, patient survival nowadays exceeding 80% 10-year after surgery with close to normal quality of life in most survivors. This review is a practical update on medical therapy, indications and results of liver transplantation, and potential future therapies, for the main liver-based metabolic disorders in which paediatric liver transplantation may be considered. Part 1 focuses on metabolic based liver disorders without liver lesions, and part 2 on metabolic liver diseases with liver lesions.

Stéphenne et al. World J Gastroenterol. 2010 Jan 7;16(1):1-14.

Hepatocyte cryopreservation: is it time to change the strategy?

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.

Najimi et al. Methods Mol Biol. 2009;481:59-74.

Hepatocyte apoptosis.

Apoptosis has been documented as a frequent hurdle phenomenon that occurs in human hepatocytes during isolation, storage, infusion and after engraftment within the recipient liver parenchyma. Apoptosis is an active form of cell death that involves programmed cellular machineries leading to a progressive self-destruction of the cell. In contrary to necrosis, it can affect individual cells within a cell population. It is characterized by chronological alteration of intracellular biochemical signaling pathways followed by cellular morphological changes, DNA fragmentation, perturbation of mitochondrial membrane function and changes in the plasma membrane. These cellular alterations can be analyzed using different methodologies on adherent, suspended and in situ engrafted hepatocytes. This chapter presents a brief overview of these techniques and provides methodology for the evaluation of hepatocyte apoptosis at the structural and biochemical levels.

Stéphenne et al. Liver Transpl. 2007 Apr;13(4):599-606.

Tissue factor-dependent procoagulant activity of isolated human hepatocytes: relevance to liver cell transplantation.

Liver cell transplantation (LCT) aims to correct inborn liver function defects by infusing metabolically active cells into the diseased liver. Further improvement in LCT might depend on the prevention of early loss of transplanted cells. As tissue factor (TF)-dependent activation of coagulation was found to contribute to a low rate of beta cell engraftment in islet transplantation, we investigated the potential procoagulant activity (PCA) of hepatocyte preparations. TF expression on hepatocyte preparations was assessed by flow cytometry, reverse-transcription polymerase chain reaction and immunofluorescence. PCA depending on TF was evaluated in human plasma and in whole blood systems. Coagulation parameters were followed by routine techniques in a LCT recipient Crigler-Najjar patient. We determined that hepatocytes express soluble and membrane-bound forms of TF. We showed that hepatocytes exert a TF-dependent PCA. In parallel, delayed increase in D-dimer levels was observed following the hepatocyte infusions in the Crigler-Najjar patient. Furthermore, in vitro experiments demonstrated that TF-dependent PCA of hepatocytes is inhibited by N-acetyl-L-cysteine. In conclusion, hepatocytes exert TF-dependent PCA, which may contribute to early loss of infused cells. Addition of N-acetyl-L-cysteine to the suspensions of hepatocytes might be beneficial in LCT by inhibiting activation of coagulation.

Stéphenne et al. Gastroenterology. 2006 Apr;130(4):1317-23.

Sustained engraftment and tissue enzyme activity after liver cell transplantation for argininosuccinate lyase deficiency.

Donor cell engraftment with expression of enzyme activity is the goal of liver cell transplantation for inborn errors of liver metabolism with a view to achieving sustained metabolic control.

Sequential hepatic cell transplantations using male and female cells were performed in a 3.5-year-old girl with argininosuccinate lyase deficiency over a period of 5 months. Beside clinical, psychomotor, and metabolic follow-up, engraftment was analyzed in repeated liver biopsies (2.5, 5, 8, and 12 months after first infusion) by fluorescence in situ hybridization for the Y-chromosome and by measurement of tissue enzyme activity.

Metabolic control was achieved together with psychomotor catch-up, changing the clinical phenotype from a severe neonatal one to a moderate late-onset type. The child was no longer hospitalized and was able to attend normal school. Sustained engraftment of male donor liver cells was shown in repeated biopsies, reaching 19% at 8 months and 12.5% at the 12-month follow-up. XXYY tetraploid donor cells were mainly detected during the infusion period (2.5- and 5-month biopsies), whereas in the follow-up 8-month and 1-year biopsies, diploid donor cell subpopulations had become dominant. Moreover, argininosuccinate lyase activity, originally absent, became measurable in 2 different biopsy samples at 8 months, reaching 3% of control activity, indicating in situ metabolic effect and supporting the clinical evolution to a moderate form of the disease.

Liver cell transplantation can achieve donor cell engraftment in humans in a significant proportion, leading to sustained metabolic and clinical control with psychomotor catch-up.

Stéphenne et al. Am J Transplant. 2005 Aug;5(8):2058-61.

Cryopreserved liver cell transplantation controls ornithine transcarbamylase deficient patient while awaiting liver transplantation.

Liver cell transplantation was performed in a child with urea cycle disorder poorly equilibrated by conventional therapy as a bridge to transplantation. A 14-month-old boy with ornithine transcarbamylase (OTC) deficiency received 0.24 billion viable cryopreserved cells/kg over 16 weeks. Tacrolimus and steroids were given as immunosuppressive treatment while the patient was kept on the pre-cell transplant therapy. Mean blood ammonia level decreased significantly following the seven first infusions, while urea levels started to increase from undetectable values. After those seven infusions, an ammonium peak up to 263 microg/dL, clinically well tolerated, was observed. Interestingly, blood urea levels increased continuously to reach 25 mg/dL, after the last three infusions. Eventually, he benefited from elective orthotopic liver transplantation (OLT) and the post-surgical course was uneventful. We conclude that use of cryopreserved cells allowed short- to medium-term metabolic control and urea synthesis in this male OTC-deficient patient while waiting for OLT.

Ambrosino et al. Cell Transplant. 2005;14(2-3):151-7.

Isolated hepatocyte transplantation for Crigler-Najjar syndrome type 1.

Crigler-Najjar syndrome type 1 (CN1) is an inherited disorder characterized by the absence of hepatic uridine diphosphoglucuronate glucuronosyltransferase (UDPGT), the enzyme responsible for the conjugation and excretion of bilirubin. We performed allogenic hepatocyte transplantation (AHT) in a child with CN1, aiming to improve bilirubin glucuronidation in this condition. A 9-year-old boy with CN1 was prepared with plasmapheresis and immunosuppression with prednisolone and tacrolimus. When a graft was made available, 7.5 x 10(9) hepatocytes were isolated and infused into the portal vein percutaneously. After 2 weeks phenobarbitone was added to promote the enzymatic activity of UDPGT of the transplanted hepatocytes. Nocturnal phototherapy was continued throughout the studied period. Total bilirubin was considered a reliable marker of allogenic cell function. There was no significant variation of vital signs nor complications during the infusion. Mean +/- SD bilirubin level was 530 +/- 38 micromol/L before and 359 +/- 46 micromol/L after AHT (t-test, p < 0.001). However, the introduction of phenobarbitone was followed by a drop of tacrolimus level with increase of alanine aminotransferase (ALT) and increase of bilirubin. After standard treatment of cellular rejection bilirubin fell again but from then on it was maintained at a greater level. After discharge the patient experienced a further increase of bilirubin that returned to predischarge levels after readmission to the hospital. This was interpreted as poor compliance with phototherapy. Only partial correction of clinical jaundice and the poor tolerability to nocturnal phototherapy led the parents to refuse further hepatocyte infusions and request an orthotopic liver transplant. After 24 months the child is well, with good liver function on tacrolimus and prednisolone-based immunosuppression. Isolated AHT, though effective and safe, is not sufficient to correct CN1. Maintenance of adequate immunosuppression and family compliance are the main factors hampering the success of this procedure.

Sokal et al. Transplantation. 2003 Aug 27;76(4):735-8.

Hepatocyte transplantation in a 4-year-old girl with peroxisomal biogenesis disease: technique, safety, and metabolic follow-up.

Hepatocyte transplantation is an investigational alternative to orthotopic liver transplantation to treat liver based inborn errors of metabolism. We report successful hepatocyte transplantation in a 4-year-old girl with infantile Refsum disease. Hepatocytes were isolated from the left liver segment of two male donors using a classic two-step perfusion method. Fresh cells were transplanted first and then cryopreserved cells, for a total of 2 billion cells. Total bile acids and abnormal dihydroxycoprostanoïc acid markedly decreased in the patient's serum, indicating resolution of cholestasis and re-population of liver cells. Pipecholic acid decreased by 40% and c26:c22 fatty acid ratio by 36% after 18 months. Donor chromosomes sequences were detected on biopsy posttransplant, indicating engraftment. Hepatocyte transplantation is a safe and promising technique in the treatment of rare inborn errors of metabolism. Future improvements of cell viability and prevention of apoptosis may increase engraftment and subsequent re-population.

Review papers

Jorns et al. Intern Med. 2012 Sep;272(3):201-23.

Hepatocyte transplantation for inherited metabolic diseases of the liver.

Inherited metabolic diseases of the liver are characterized by deficiency of a hepatic enzyme or protein often resulting in life-threatening disease. The remaining liver function is usually normal. For most patients, treatment consists of supportive therapy, and the only curative option is liver transplantation. Hepatocyte transplantation is a promising therapy for patients with inherited metabolic liver diseases, which offers a less invasive and fully reversible approach. Procedure-related complications are rare. Here, we review the experience of hepatocyte transplantation for metabolic liver diseases and discuss the major obstacles that need to be overcome to establish hepatocyte transplantation as a reliable treatment option in the clinic.

Sokal EM. Cell Prolif. 2011 Apr;44 Suppl 1:39-43.

From hepatocytes to stem and progenitor cells for liver regenerative medicine: advances and clinical perspectives.

The parenchymal liver cell is a unique fully functional metabolic unit that can be used for liver regenerative medicine to restore function of the diseased organ; the aim of the procedure is to prevent progression of end-stage disease. The alternative, orthotopic liver transplantation, is highly intrusive, irreversible and limited by general organ shortage. Mature liver cell - hepatocyte - transplantation has been shown to have short- to medium-term efficacy for correction of miscellaneous inborn errors of metabolism. However, although proof of concept has been established, the procedure has not yet achieved full success, due to limited durability of functional benefit. Hepatocyte procurement is also restricted by organ shortage, and their storage is difficult due to poor tolerance of cryopreservation. Alternative cell sources are therefore needed for development and wider accessibility of cell-based liver regenerative medicine. Besides safety, the main challenge for these alternative cells is to acquire similar levels of functionality once implanted into the target organ. In this respect, liver derived progenitor cells may have some advantages over stem cells derived from other tissues.

Dhawan et al. Methods Mol Biol. 2010;640:525-34.

Human hepatocyte transplantation.

Over the last decade the interest in hepatocyte transplantation has been growing continuously and this treatment may represent an alternative clinical approach for patients with acute liver failure and life-threatening liver-based metabolic disorders. The technology also serves as the proof of concept and reference for future development in stem cell technology. This chapter reviews the field of hepatocyte transplantation from bench to bedside.

Sokal EM. Bull Mem Acad R Med Belg. 2009;164(5-6):207-12.

Regeneration of the liver: from hepatocyte cells to deficient hepatic cells.

Regenerative medicine aims to restore the function of a deficient organ without replacing it, i.e. without resection-transplantation, but by the use of healthy cells which will transfer the deficient function inside the diseased organ. Cells can be mature, harvested directly from the source tissue, or be instead produced from stem cells, which can be manipulated in vitro, expanded and/or differentiated to render them functional. Liver cell therapy has brought the proof of concept that a deficient metabolic activity can be transferred via transfusion of heterologous liver cells via the portal vein. The main limitations of the technique include organ shortage, poor renewal capacity of mature cells and poor resistance to cryopreservation. A liver derived progenitor cell has recently been identified in the adult human liver. The cell is selected by a culture process, can be expanded in vitro and differentiated into mature human hepatocytes when transplanted in rodent livers. The cell displays all the essential hepatocyte function, and may replace the mature hepatocyte for regenerative medicine of the liver.

Sancho-Bru P. Gut. 2009 Apr;58(4):594-603.

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 liver disease might be possible by transplanting stem/progenitor cells or their liver-committed progeny. However, it is currently impossible to determine from the many documented studies which of the stem/progenitor cell populations are the best for therapy of a given disease. This is largely because of the great variability in methods used to characterise cells and their differentiation ability, variability in transplantation models and inconsistent methods to determine the effect of cell grafting in vivo. This manuscript represents a first proposal, created by a group of investigators ranging from basic biologists to clinical hepatologists. It aims to define standardised methods to assess stem/progenitor cells or their hepatic lineage-committed progeny that could be used for cell therapy in liver disease. Furthermore standardisation is suggested both for preclinical animal models to evaluate the ability of such cells to repopulate the liver functionally, and for the ongoing clinical trials using mature hepatocytes. Only when these measures have been put in place will the promise of stem/progenitor-derived hepatocyte-based therapies become reality.

Lysy PA et al. World J Gastroenterol. 2008 Feb 14;14(6):864-75.

Stem cells for liver tissue repair: current knowledge and perspectives.

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.