Journal of Hepatology
Volume 45, Issue 1 , Pages 144-159, July 2006

Present status and perspectives of cell-based therapies for liver diseases

  • Andreas Nussler

      Affiliations

    • Fresenius Biotech Bad Homburg, Division of Cell Therapy, Bad Homburg, Germany
    • Corresponding Author InformationCorresponding authors.
    • ,
  • Sarah Konig

      Affiliations

    • University of Goettingen, Department of General Surgery, Goettingen, Germany
    • ,
  • Michael Ott

      Affiliations

    • Medizinische Hochschule Hanover, Department of Gastroenterology, Hanover, Germany
    • ,
  • Etienne Sokal

      Affiliations

    • Cliniques Universitaires St. Luc, Department of Pediatric Hepatology and Liver transplantation, Brussels, Belgium
    • ,
  • Bruno Christ

      Affiliations

    • Martin-Luther-University Halle-Wittenberg, Division of Molecular Hepatology, Halle-Wittenberg, Germany
    • ,
  • Wolfgang Thasler

      Affiliations

    • LMU Munich, Department General Surgery, Munich, Germany
    • ,
  • Marc Brulport

      Affiliations

    • University of Leipzig, Center for Toxicology, University of Leipzig, Leipzig, Germany
    • ,
  • Geredn Gabelein

      Affiliations

    • University Medicine Berlin, Charité, Department of General Surgery and Transplantation, Berlin, Germany
    • ,
  • Wiebke Schormann

      Affiliations

    • University of Leipzig, Center for Toxicology, University of Leipzig, Leipzig, Germany
    • ,
  • Maren Schulze

      Affiliations

    • University Hospital Kiel, Division of Transplantation and Biotechnology, Kiel, Germany
    • ,
  • Ewa Ellis

      Affiliations

    • University of Pittsburgh, Department of Pathology, Pittsburgh, USA
    • ,
  • Matthias Kraemer

      Affiliations

    • Fresenius Biotech Bad Homburg, Division of Cell Therapy, Bad Homburg, Germany
    • ,
  • Frank Nocken

      Affiliations

    • Fresenius Biotech Bad Homburg, Division of Cell Therapy, Bad Homburg, Germany
    • ,
  • Wolfgang Fleig

      Affiliations

    • Martin-Luther-University Halle-Wittenberg, Division of Molecular Hepatology, Halle-Wittenberg, Germany
    • ,
  • Michael Manns

      Affiliations

    • Medizinische Hochschule Hanover, Department of Gastroenterology, Hanover, Germany
    • ,
  • Steven C. Strom

      Affiliations

    • University of Pittsburgh, Department of Pathology, Pittsburgh, USA
    • ,
  • Jan G. Hengstler

      Affiliations

    • University of Leipzig, Center for Toxicology, University of Leipzig, Leipzig, Germany
    • Corresponding Author InformationCorresponding authors.

published online 26 April 2006.

Article Outline

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In recent years the interest in liver cell therapy has been increasing continuously, since the demand for whole liver transplantations in human beings far outweighs the supply. From the clinical point of view, transplantation of hepatocytes or hepatocyte-like cells may represent an alternative to orthotopic liver transplants in acute liver failure, for the correction of genetic disorders resulting in metabolically deficient states, and for late stage liver disease such as cirrhosis. Although the concept of cell therapy for various diseases of the liver is widely accepted, the practical approach in humans often remains difficult. An international expert panel critically discussed the recent published data on clinical and experimental hepatocyte transplantation and the possible role of stem cells in liver tissue repair. This paper aims to summarise the present status of cell based therapies for liver diseases and to identify areas of future preclinical and clinical research.

 

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1. Introduction 

The progress made in the field of liver organ transplantation has revolutionized the treatment of a wide spectrum of liver diseases. Nevertheless, cell-based therapies are emerging as an alternative to whole-organ transplantation. Hepatocyte transplantation has been used to bridge patients to whole-organ transplantation [7], [69], to decrease mortality in acute liver failure [18], [58], and for treatment of metabolic liver disease [3], [16], [19], [22], [29], [30], [47], [48], [66], [67], [69]. Cell transplantation is less invasive than whole-organ transplantation and can be performed repeatedly. However, one major limitation of cell-based therapies for liver disease is the availability of human hepatocytes. A wider use of these techniques will not be possible until adequate numbers of functional cells for transplantation become more readily available. There are at least two possible sources that could meet the needs for transplantation, namely stem and precursor cell derived hepatocyte-like cells or reversibly replicating hepatocyte cell lines [36]. In recent years, numerous articles have reported about the generation of liver cells or ‘hepatocyte-like cells’ from different types of extrahepatic stem or precursor cells. At a first glance, this appears to provide exciting new opportunities for cell therapy, as some types of stem cells proliferate efficiently in vitro and therefore may help to generate a larger supply of human hepatocytes or precursor cells for transplantation. Without doubt, the wide availability of human hepatocytes would be considered a major breakthrough and may open new perspectives for the treatment of liver disease [60], [68]. On the other hand, some studies presenting with far-reaching conclusions with respect to the capacity of stem cell therapy have not yet been reproduced or may have been interpreted in an over-optimistic manner. Here, we review the present status of cell therapies for liver diseases and discuss the most promising future strategies.

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2. Which liver diseases are first line candidates for cell therapy? 

Three categories of liver disease can be distinguished and principally addressed by liver cell therapy: (i) acute liver failure, (ii) inherited metabolic liver disease and (iii) end-stage liver disease (cirrhosis). The conditions and requirements of cell therapy differ for each of these classes.

2.1. Acute liver failure 

Acute liver failure is characterised by rapid deterioration of liver function and a high mortality. Viral hepatitis, idiosyncratic drug reactions, acetaminophen and mushroom ingestion are common causes of acute liver failure. Hepatic encephalopathy, brain edema, coagulopathy, septicemia and multi-organ failure are critical key events [44], [61] during the course of the disease. Cell therapy of acute liver failure should provide rapid support for the failing liver by providing metabolism of liver toxins, the secretion of proteins such as clotting factors or albumin and stabilisation of haemodynamic parameters. In several studies, allogeneic primary hepatocytes isolated from cadaver livers were infused into the splenic artery or the portal vein [53], [69], [7]. Improvements in ammonia levels encephalopathy scores, and prothrombin time levels were reported. Thus, the first studies performed in patients with acute liver failure demonstrated the feasibility of hepatocyte transplantation and presented strong evidence for its therapeutic efficiency.

Due to the large capacity and accessibility of the peritoneal cavity, intraperitoneal transplantation of hepatocytes seems to be a promising alternative strategy to bridge life until the spontaneous regeneration of the liver occurs. In a few patients with acute liver failure and grade III encephalopathy fetal hepatocytes were injected into the peritoneal cavity to bridge patients to organ recovery. Improved survival rates were reported compared to historical controls [26]. Since hepatocytes in the peritoneum survive only short term, alginate-embedded or microcarrier-attached hepatocytes seem to offer a reasonable alternative. For instance, the transplantation of microcarrier-attached hepatocytes into rats subjected to a 90% near total hepatectomy markedly improved long-term survival rates [14]. In contrast, the transplantation of hepatocytes alone did not prolong survival.

Despite the positive results, it should be considered that the evaluation of therapies in acute liver failure may be difficult. This is due to large variations in the course of the disease, multiple aetiologies, complex supportive treatment and a spontaneous recovery rate of approximately 20% by successful hepatic self-regeneration. Thus, the inclusion of adequate controls is difficult. In this respect, studies in patients with inherited metabolic diseases are easier to interpret.

2.2. Inherited metabolic liver disease 

In comparison with acute liver failure, the course of metabolic liver disease usually varies less. In addition, objective parameters such as laboratory data (i.e. bile acid, clotting factors, etc.) can be determined to unequivocally assess the efficacy of the treatment. On the other hand, the situation is rarely immediately life threatening and often acceptable conventional therapies are available. Therefore, the potential benefit must be carefully weighed against any possible complications, such as immunosuppression, hepatocyte embolisation of the pulmonary vascular system, sepsis or haemodynamic instability.

The results of hepatocyte transplantation for many metabolic liver diseases have been encouraging (for review, see: [8]). For instance, therapeutic benefit has been reported in a girl with Crigler–Najjar Syndrome Type I, which is a recessively inherited metabolic disorder characterized by severe unconjugated hyperbilirubinaemia [19]. Isolated hepatocytes were infused through the portal vein and partially corrected plasma bilirubin levels for more than 11 months [19]. Similarly, a 9-year-old boy received 7.5×109 hepatocytes, infused via the portal vein, which resulted in a decrease in bilirubin level from 530±38μmol/L (mean±SD) before to 359±46μmol/L [3]. Hughes et al. [30] also report a 40% reduction in bilirubin levels in a Crigler–Najjar Syndrome Type I patient following transplantation with hepatocytes. Although these data demonstrate efficacy and safety, a single course of cell application seems not sufficient to correct Crigler–Najjar Syndrome Type I completely.

Promising results have also been obtained in a 47-year-old woman suffering from glycogen storage disease type 1a, an inherited disorder of glucose metabolism resulting from mutations in the gene encoding the hepatic enzyme glucose-6-phosphatase [48]. 2×109 ABO-compatible hepatocytes were infused into the portal vein. Nine months after cell transplantation, her metabolic situation had clearly improved. Successful hepatocyte transplantation has also been achieved in a 4-year-old girl with infantile Refsum disease, an inborn error of peroxysome metabolism, leading to increased levels of serum bile acids and the formation of abnormal bile acids [66]. A total of 2×109 hepatocytes from a male donor were given during eight separate intraportal infusions. Abnormal bile acid production (for instance pipecholic acid) had decreased by 40% after 18 months. Recently, hepatocyte transplantation has been used successfully to treat inherited factor VII deficiency [16]. Two brothers (aged 3 months and 3 years) received infusions of 1.1 and 2.2×109 ABO-matched hepatocytes into the inferior mesenteric vein. Transplantation clearly improved the coagulation defect and decreased the necessity for exogenous factor VII to approximately 20% of that prior to cell therapy. As with the other metabolic liver diseases, hepatocyte transplantation has been shown to provide a partial correction of urea cycle defects. Patients showed clinical improvement, reduced ammonia levels and increased production of urea [29], [47], [67], [70]. Some hepatocyte transplantation studies with inherited metabolic liver disease have been summarized in Table 1.

Table 1. Overview over some hepatocyte transplantation studies in patients with inherited metabolic liver disease
RecipientNo. of transplantation hepatocytesRouteOutcomeRef.
DiseaseSexAge
α1-Antitrypsin deficiencyMale18 w PortalOLTa (day 2)[69]
Female52 y2.2×106OLTa (day 4)
Crigler–Najjar IMale10 y7.5×109PortalOLTa (3.5 years)[19]
Ornithine transcarbamylase deficiencyMale5 y1×109PortalNormal ammonia level within 48h; died after 43 days[51]
Ornithine transcarbamylase deficiencyMale10h?4.5×109PortalNormal protein intake possible; OLTa (6 months)[29]
Glycogen storage disease type IaFemale46 y2×109PortalImproved for 3 years[48]
Refsum diseaseFemale4 y2×109PortalImproved for 1 year[66]
Factor VII deficiencyMale3 m1.1×109PortalImproved and decreased requirement for recombinant factor VII[16]
Male2 y, 11 m2.2×109Portal

aOLT, orthotopic liver transplantation.

Relatively little is known about long-term engraftment of the transplanted hepatocytes. An important observation of [16] is an increased requirement for recombinant factor VII six months after cell therapy, suggesting loss or decreased function of the transplanted hepatocytes. Therefore, it will be important to obtain further information whether loss of transplanted allogeneic hepatocytes is inevitable or a consequence of suboptimal immunosuppression.

With respect to long-term engraftment it will be important whether the transplanted hepatocytes will gain a selection advantage over the recipient’s cells. Therefore, it could be reasonable to differentiate between two groups of metabolic liver diseases. A typical representative of group 1 is inherited clotting factor deficiencies. Secretion of clotting factors is important for the organism, but deficiency does not influence survival of hepatocytes. Therefore, a selection advantage of the transplanted hepatocytes cannot be expected. The situation is different for group 2 metabolic liver diseases. A typical example is Wilson’s disease that is caused by a defect in the copper transporting ATPase ATP7B protein. As a consequence of the gene defect copper will accumulate and lead to deterioration of hepatocytes [25]. In this case transplanted wild-type cells may have a selection advantage over the recipient’s hepatocytes. Therefore, higher numbers of transplanted hepatocytes, better cell engraftment and perhaps repeated transplantations may be necessary for the treatment of group 1 metabolic liver diseases. However, clinical data with respect to possible differences in long-term engraft between group 1 and 2 are not yet available.

2.3. End-stage liver disease (cirrhosis) 

Cell therapy of end-stage liver disease is more problematic. Besides loss of functional hepatocytes abnormalities of the hepatic architecture contribute to the decrease in liver function. Intrahepatic portal-to-portal venous shunts may prevent an efficient exchange between hepatocytes and blood plasma. In this situation, the benefit of additionally transplanted hepatocytes into the liver without restoring the normal liver architecture may be questionable. An alternative strategy may be the transplantation of hepatocytes into other sites e.g. the spleen, peritoneum or omentum to support metabolic function and regeneration. To evaluate the efficiency of intrasplenic transplantation several researchers induced liver cirrhosis in rats using phenobarbital and carbon tetrachloride followed by direct injection of cells into the splenic pulp [9], [35], [54]. Only animals with stable liver cirrhosis four weeks after the discontinuation of carbon tetrachloride were subjected to cell therapy. With different cell types applied, namely rat or porcine hepatocytes [54], syngeneic rat hepatocytes [35] or immortalized rat hepatocytes [9] intrasplenic cell therapy clearly improved liver function and prolonged survival.

The response to hepatocyte transplantation in humans with end-stage liver disease, however, has not resulted in the same degree of improvement compared to experimental animal studies [46], [51]. One explanation may be that the hepatocytes in clinical studies were delivered into the splenic artery and not into the splenic pulp. This view is supported by Nagata and colleagues, who have shown that the route of hepatocyte delivery influences hepatocyte engraftment and function [55]. Another open question remains as to whether the human spleen is capable of accommodating a sufficient number of functional hepatocytes to compensate for the cirrhotic liver. From the immunological point of view the spleen represents the ‘lion’s den’, where transplanted cells could possibly cause greater immune response than in most other ectopic transplantation sites. Because of its large capacity and accessibility, the peritoneal cavity is an alternative site for the cell therapy of end-stage liver disease. However, since hepatocyte suspensions do not survive for longer periods only short-term effects may be achieved. Application of cells in patients with decompensated chronic liver disease has resulted in some improvement of laboratory parameters, but was not able to change the natural cause of the disease (Ott M., unpublished). Encapsulation of hepatocytes [4], [62] or their attachment to microcarriers may be an alternative method to improve efficacy of intraperitoneal cell transplantation. Implantable hepatocyte-based devices may represent another alternative for the treatment of end-stage liver disease [10]. However, these approaches are conceptual and still far from clinical application.

2.4. Route of administration 

In metabolic liver disease and acute liver failure the liver architecture is usually intact. In this case the infusion of cells into the portal vein or into the inferior mesenteric vein is adequate. It is known from animal studies that infused hepatocytes disperse with the portal blood flow and finally translocate to the hepatic sinusoids in the periportal region of the liver lobules [37]. Single cells succeed in traversing the endothelial barrier and integrate into the parenchyma (Fig. 1). After re-establishing intercellular contacts with neighbouring host cells, transplanted hepatocytes start to proliferate. Donor cells and their descendents form gradually increasing clusters, thus finally repopulating the recipient liver (Fig. 1). While monitoring the portal pressure in a 4-year-old girl following portal infusion of hepatocytes, a temporary increase was noted, with a return to pre-infusion levels shortly after the infusion [66].

  • View full-size image.
  • Fig. 1. 

    Transplantation of adult hepatocytes and liver repopulation. Primary hepatocytes were injected into the portal vein of dipeptidylpeptidase IV (DPPIV) deficient rats pre-treated with retrorsine and subjected to 30% partial hepatectomy to ensure selective donor growth. Donor cell integration and repopulation was studied by co-localising transplanted cells (DPPIV-positive=red) with hepatic gap junctions (Connexin-32=green) to visualize the intact hepatic architecture, cell nuclei (DAPI=blue). Multilayer immunofluorescence imaging, magnification as indicated). (A) Shortly after transplantation (4h), donor cells form microemboli in the distal branches of the portal vein and translocate further to the sinusoids. (B) From day 3 onwards, donor cells are fully integrated and display re-established gap junctions with neighbouring cells. (C) Donor cells and descendents form clusters and grow out from the periportal areas (21 days). (D) Two months following transplantation, donor cell-derived cell clusters are nearly confluent and range up to 200 cells in number. The integration process is remarkably homogeneous and the architecture of the parenchyma is perfectly maintained without any sign of displacement.

The capacity of the liver to incorporate transplanted hepatocytes can be enhanced for instance by ischaemia. In low-density lipoprotein receptor deficient Watanabe rabbits, transient ischaemia reperfusion injury of the recipient liver has been shown to improve the therapeutic efficacy of allogeneic intraportal hepatocyte transplantation [5]. This technique may offer a perspective to improve the engraftment of transplanted hepatocytes in patients with metabolic liver disease.

When the liver architecture is deranged, cell infusions may cause prolonged portal hypertension and embolization in the lung [71]. Therefore, ectopic sites for hepatocyte engraftment are needed. The best studied ectopic site is the spleen. A study in pigs has shown that direct intrasplenic injection produced engraftment that was far superior to that obtained using splenic artery infusion [55]. Splenic artery infusion caused splenic necrosis due to vascular occlusion with hepatocytes. In contrast, injection into the splenic pulp was tolerated much better and was associated with only clinically insignificant intraabdominal haemorrhage. Therefore, direct intrasplenic injection may be a feasible strategy to support patients with deranged liver architecture. Whether the number of engrafted cells and their metabolic capacity will be sufficient remains an open question.

In several animal studies cells were directly injected into the liver tissue [74], [63], [6] to avoid the requirement of homing. Using this technique, the transplanted cells were observed in the liver tissue, but also in central veins indicating an increased risk of embolization to the lung. Therefore, direct injection of cells into the liver may be used in basic research but not in the clinical setting. The kidney capsule represents a further potential site for ectopic hepatocyte transplantation. Although engraftment was demonstrated the renal subcapsular space will accommodate only relatively small numbers of hepatocytes.

2.5. Which would be the most effective immunosuppression? 

Islet cell transplantation is a well-established clinical option in the therapy of diabetes. Transplantation of islet cells is usually performed transcutaneously into the intrahepatic portal vein system. Therefore, it represents the circumstances of liver cell therapy at least concerning cell administration and may serve as a pioneering example for cell-based therapies. Nevertheless, the clinical outcome of islet cell transplantation with a basal c-peptide >0.5ng/ml, insulin independence of less than 40% after one year (www.med.uni-giessen.de/itr) cannot reach the success of whole-organ transplantation of the pancreas with 70% insulin independence one year after transplantation (www.iptr.umd.edu). However, optimization of immunosuppression substantially improved the clinical outcome of islet cell transplantation [17]. Therefore, it can be expected that the immunosuppressive regimen also plays a major role in cell based therapies for liver diseases. Inhibition of liver resident natural killer cells by specific or local immunosuppression could additionally improve engraftment and proliferation of transplanted cells [20].

The ultimate goal would be an effective immunosuppression with as little side effects as possible. The most common immunosuppressive protocols consist of calcineurin inhibitors (CNI, tacrolimus or cyclosporine) with or without steroids in combination with induction therapy such as IL-2 receptor antibodies (e.g., basiliximab) or anti-thymocyte globulins (e.g., ATG Fresenius or Thymoglobulin). Depending on the underlying disease and existing comorbidities of the patient, immunosuppressive regimens without steroids or reduced doses of CNI are favoured. The reduction or even removal of CNI may be achieved by the addition of other immunosuppressive drugs such as mycophenolate mofetil (MMF) or sirolimus. Since there are few data about immunosuppression after cell transplantation and no specific protocol can be recommended for these patients so far, it is advised to use the standard immunosuppressive protocol of each respective centre. However, it should be taken into account that the immunosuppressive requirements after cell transplantation may be lower than after solid organ transplantation. Encapsulation of the transplanted cells reduces their immunogenicity. This may not only prolong survival of the transplanted cells, but may also allow for less immunosuppression of the recipient.

2.6. Number of cells and frequency of administration 

The human liver consists of approximately 250×109 cells that are organized in about 106 hepatic lobules each containing approximately 250,000 liver cells. Hepatocytes constitute approximately 65–70% of the cell population of the liver. Therefore, the total number of hepatocytes in a human liver is approximately 175×109. As a rule of thumb transplantation of a number of hepatocytes corresponding to 1–5% of total liver mass (1.8–8.8×109 hepatocytes) can be expected to have a positive impact. Although a number as high as 8.8×109 hepatocytes is an ambitious goal it seems desirable to transplant such high numbers of cells. In several published cases lower numbers of hepatocytes were transplanted, which may explain lack or marginal clinical benefit. About 5% of liver mass corresponding to approximately 8.8 billion hepatocytes can safely be transplanted in one transplant event, whereby the transplant may be divided into about 5–6 separate infusions over a number of hours. When portal pressures return to normal, or at least decrease to acceptable levels, it is safe to infuse more cells. It might even be useful to transplant 5% of liver mass as several separate transplants over a prolonged period of time (several weeks to months) so that ultimately one could transplant 15–20% of total liver mass at 4 separate sessions.

2.7. Limited availability of hepatocytes: the major hurdle of liver cell therapy 

Already in 1977 hepatocyte transplantation has been recognized as an attractive option for the management of metabolic liver disease [23]. Groth and colleagues demonstrated that intraportal hepatocyte transplantation in glucuronosyltransferase-deficient rats improved hyperbilirubinaemia. Up to the present, most of the published articles have reported a positive impact of hepatocyte transplantation also in human studies. Despite the positive reports application of hepatocyte transplantation in humans is limited to less than 100 cases. The reason for this discrepancy is the success of orthotopic liver transplantation and limited availability of human hepatocytes. It is reasonable to use all available donor livers for organ transplantation. The numbers and/or quality of hepatocytes isolated from non-transplantable livers will not allow a widespread application of hepatocyte transplantation. This situation will remain unaltered unless an alternative to primary hepatocytes is available. As soon as an easily available cell type equivalent to primary hepatocytes will be on hand, treatment of acute liver failure, acquired or monogenetic metabolic liver diseases and perhaps also of end-stage liver disease will be revolutionized within the near future.

2.8. Are human stem and precursor cells promising candidates for cell therapy of human liver diseases? 

Since 1999, numerous articles have reported the generation of hepatocyte-like cells from different types of extrahepatic stem or precursor cells (review: [27]). At a first glance this seems to open exciting new opportunities for cell therapy. However, after a careful evaluation of published preclinical studies and also considering our own results we concluded that preclinical data are not yet sufficient to justify clinical studies. Further information about the generated hepatocyte-like cells is needed and so are reproducible results in preclinical animal models of human liver disease. This will be illustrated in the next two paragraphs where we will critically discuss recent publications about differentiation of extrahepatic stem or precursor cells to hepatocyte-like cells, either following transplantation into animal livers or in vitro following cytokine and/or liver extract exposures.

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3. Experimental evidence for hepatocyte formation from extrahepatic stem and precursor cells 

3.1. Formation of hepatocyte-like cells from extrahepatic stem cells in vivo 

First publications have shown that haematopoietic stem cells were capable of differentiating into hepatocytes and cholangiocytes yielding in a high degree of engraftment within injured rodent livers. Petersen et al. [59] used three approaches to demonstrate that bone marrow cells contribute to liver cells. First, female rats were lethally irradiated and transplanted with bone marrow from a male rat. Engrafted females were treated with CCl4 and 2-acetylaminofluorene (2-AAF) to simultaneously induce hepatotoxicity and block endogenous hepatocyte proliferation. Under these conditions, Y-chromosome-positive hepatocytes were observed in the female recipients. Second, using the same protocol bone marrow cells from dipeptidylpeptidase IV (DPPIV)+ F-344 male rats were injected into DPPIV− F-344 female rats, resulting in DPPIV expression in bile canalicular sites between hepatocytes of the DPPIV− F-344 recipients. Finally, livers from Lewis rats expressing the major histocompatibility complex class II L21-6 isozyme were transplanted into Brown–Norway rats that do not express L21-6. After the CCl4/2-AAF protocol the recipients (Lewis rats) showed positive L21-6 staining in livers. Similar results were obtained in further studies using also different animal models and purified cell types for transplantation (for review: see [27]). However, marker expression of transplanted extrahepatic cells in combination with hepatocellular factors does not automatically imply that the transplanted cells show transdifferentiation into true hepatocytes, but could more likely be induced by cell fusion. Regardless of the underlying mechanisms, in vivo generation of hepatocytes from bone marrow has one fundamental requirement: the host bone marrow has to be reconstituted by transplanted bone marrow in advance. From a clinical point of view, this requirement may be considered as an impossible option for many patients suffering from severe hepatic disease.

The promising results obtained after transplantation of extrahepatic rodent cells into livers of rodents stimulated a relatively large number of independent groups to study the fate of different types of human stem and precursor cells in livers of experimental animals (Table 2). Without doubt differentiation of human stem cells to genuine hepatocytes or even to liver tissue would be an enormous progress with high clinical relevance. In all experiments published so far a similar strategy was used. Extrahepatic stem cells were introduced into the liver of experimental animals by different routes (Table 2). After periods usually ranging between 3 weeks and 6 months expression of human hepatocyte markers were analysed by immunohistochemistry, RT-PCR or in situ hybridization. Although different human cell types, routes of injection and recipients (mice, sheep, goat) have been tested, similar results were obtained. In 14 of the 15 published studies (Table 2) expression of human albumin was observed in the recipients. In one of the studies [56] listed in Table 2, human albumin was not analysed, but human specific antigen (HepPar1) was detected. In most of these studies the positive immunostaining data have been confirmed by RT-PCR analysis (Table 2). Two studies included analysis of cell fusion and did not find evidence for this mechanism [56], [38]. Other parameters relevant for hepatocytes, including activities of drug metabolizing enzymes, clotting factors and complement, were not yet tested in these experiments (Table 2).

Table 2. Transplantation of human stem and precursor cells into livers of experimental animals: summary of studies
Human cell typeRoute and number of transplanted cellsLiver injuryObservationsRecipient mice (m), sheep (s), goat (g)Ref.
Adherently proliferating cells from cord bloodInjection of 2×105 cells directly into the liver tissueNoneExpression of human albumin 7 and 21 days after transplantation (IHC, RT-PCR), but no expression of α-fetoprotein and GATA4 (RT-PCR)m[6]
Lin−CD38−CD34−ClqRp+ and Lin−CD38−D34+ClqRp+ cells isolated from cord blood and bone marrowTail vein injection of 500 to 7×104 cells3.75 GyExpression of HepPar1 antigen and human c-met 8–10 weeks after transplantation (IHC), human albumin (RT-PCR). Mouse liver suspensions showed a rare population of human HLA- ABC+ and CD45− cells (flow cytometry)m[13]
Unsorted mononuclear cell preparations from human cord bloodTail vein infusion of 50×106 cells2.5 GyExpression of the HepPar1 antigen (IHC) in livers 4, 6 and 16 weeks after transplantation into SCID/NOD mice. No evidence for cell fusion (FISH)m[56]
CD34+ or CD34+, CD38−, CD7− cells isolated by from cord bloodTail vein injection of 2000 CD34+ or 1×105 CD34+, CD38−, CD7− cells3 Gy CCl4Expression of human albumin (WB, RT-PCR, IHC) 5 and 30 days after liver injury. Positive RT-PCR for CK19. Negative results without CCl4 induced liver injurym[76]
CD34+ cells isolated from cord blood and from peripheral bloodTail vein injection of 2×105 cells3.75 Gy CCl4Human albumin positive cells preferentially around bile ducts (IHC, RT-PCR, WB). Neutralization of the SDF-1 receptor CXCR4 abolished homing of human stem cells to the mouse liver, whereas local injection of SDF-1 into the mouse liver increased homingm[39]
Adherently proliferating cells from cord bloodInjection of 10×106 cells into the portal vein2-AAF and one.third hepatectomyExpression of human albumin (RT-PCR, IHC) and HepPar1 antigen” (IHC) in liver, human albumin detection in serum (WB) and detection of human X chromosome centromers (FISH)m[33]
CD34+ or CD45+ cells from cord bloodTail vein injection of 1×105 cells5-Fluorouracil and anti-mouse c-kitExpression of human albumin (RT-PCR), HepPar1 antigen (IHC) and human centromers (FISH)m[31]
Nestin-positive islet-derived adherently proliferating precursor cellsInjection of 0.15, 1.5, and 7.5×105 directly into the liver tissueNoneExpression of human albumin but not of mouse albumin in individual cells (IHC, RT-PCR) 3 and 12 weeks after transplantation. Negative results after injection of 0.15×105 cells, but positive for 1.5 and 7.5×105 injected cellsm[74]
CD34+ cells isolated from cord blood3–5×105 for intra-fetal and 15–20 cells for intra-blastocyst injectionNoneExpression of human albumin, HepPar1 antigen, and human α1-antitrypsin (IHC, RT-PCR) 1 and 4 weeks after birthm[72]
CD34+Lin−CD38− cells from human bone marrow, cord blood and mobilized peripheral bloodIntra-fetal injection of 2×104 cellsNoneExpression of human albumin (IHC, ELISA), “human hepatocyte antigen” (IHC) and detection of Alu-sequences (FISH)s[1]
Adherently proliferating cells from cord bloodIntra-fetal injection of 1500 cells in sheepNoneExpression of human albumin and “human hepatocyte specific antigen” in livers of sheep (IHC, WB)s[38]
Human blood monocyte derived cellsInjection of 7.5×105 cells directly into the liver tissueNoneExpression of human albumin but not of mouse albumin in individual cells (IHC) 3 weeks after transplantationm[63]
Adherently proliferating cells from cord bloodTail vain injection of 5×104 cellsFas-ligand 1.5 GyExpression of human albumin and HepPar1(IHC) as well as albumin, α-fetoprotein, glutamine synthetase and transferrin (RT-PCR)m[57]
CD34+lin− cells derived from cord bloodIntra-fetal injectionNoneExpression off human-specific serum albumin and hHNF-3β mRNAg[78]
Unsorted mononuclear cell preparations from human cord bloodTail vein injection of 1×106 cells2.5 GyExpression of human albumin, but no expression of human CK18 (IHC, RT-PCR) 4 weeks after induction of liver injury and 8 weeks after cell transplantationm[64]

Without doubt, the observation of human albumin- or HepPar1-positive cells in livers of animals after transplantation of human stem cells is intriguing. Nevertheless, it is still questionable if these cells represent genuine human hepatocytes that could take over all hepatocellular functions. Although the data obtained by several groups are comparable (Table 2) the interpretation and discussion remain controversial. Many authors tend to give an optimistic view. For instance [38] transplanted adherently proliferating cells isolated from human cord blood into livers of fetal sheep. The authors observed expression of albumin and human hepatocyte-specific antigen after transplantation and concluded that the human cord blood cells differentiated to human parenchymal hepatic cells. Newsome demonstrated expression of the HepPar1 human hepatocyte-specific antigen, and concluded that cells from human cord blood ‘become mature hepatocytes’ in livers of SCID/NOD mice [56]. Ishikawa detected human albumin and the HepPar1 antigen in livers of immunodeficient mice, postulating that the engrafted cells from human cord blood ‘functioned as hepatocytes’ [31]. Considering these interpretations one might ask why these cells are not yet used in the clinical setting.

However, some challenges may have been underestimated. In addition there is reason to suspect publication bias. At least in recent years a simple interpretation of an albumin- and/or HepPar1-positive stem cell derived cell type as “hepatocyte” could be published easier than a “problematic” study about intermediate cell types expressing some but not all hepatocellular markers. It should also be considered that bone marrow-derived cells as a source for hepatocyte regeneration have also been critically discussed. Cantz et al. [11] investigated the contribution of intrasplenic bone marrow transplants or in vivo mobilized haematopoietic stem cells to the formation of hepatocytes in normal and injured liver. Direct intrasplenic injections of bone marrow mononuclear cells, Scal+/lin− haematopoietic stem cells and highly purified “side population” hematopoietic stem cells derived from enhanced green fluorescent protein (EGFP)-transgenic mice were performed in normal C57Bl/6 mice and in C57Bl/6 mice following two-thirds hepatectomy. The results demonstrate that there is little or no contribution of bone marrow-derived cells to the regeneration of normal and injured liver in the animal models used [11]. Since a realistic assessment is crucial for further progress in this field we will focus on possible limitations and problems in the next paragraphs. It should be considered that albumin expression has been observed in most published studies. However, expression of albumin was not automatically associated with expression of further hepatocyte markers. For instance GATA4, α-fetoprotein and CYP3A4 [6] as well as cytokeratin 18 and DPPIV [64] were negative despite positive staining for human albumin. Thus, mixed or chimeric cell types may occur after stem or precursor cell transplantation. Their impact on liver physiology remains unexplored.

3.1.1. Single cells but no tissue formation 

Typically single cells or small clusters of hepatocyte-like cells were observed after transplantation of the human stem and precursor cells into mouse livers (Fig. 2). However, no human liver tissue formation could be observed in the mouse livers (all mouse studies in Table 2). In contrast ‘more than 20% albumin-producing human parenchymal hepatic cells’ have been reported after transplantation of adherently proliferating cord blood cells into fetal sheep [38]. This is surprising and requires independent confirmation before further conclusions can be drawn. Thus, formation of human liver tissue after transplantation of human stem cells into animal models has not yet been demonstrated (see Fig. 3).

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  • Fig. 2. 

    Morphology of extrahepatic stem cells after transplantation into livers of immunodeficient mice from published studies. (A) Danet et al. [13]: transplantation of purified human Lin−CD38−CD34−or+C1qRp+ cells isolated from human umbilical cord blood. Livers from transplanted mice were recovered 8–10 weeks posttransplant and immunostained for human albumin. Bar: 10μm. (B) Newsome et al. [56]: transplantation of unsorted mononuclear cell preparations of human cord blood and immunostaining for HepPar1. Bar: 20μm. (C) Wang et al. [76]: transplantation of CD34+ cells from human umbilical cord blood and immunostaining for human albumin. Magnification: 100×. (D) Kollet et al. [39]: transplantation of CD34+ cells isolated from human cord blood. Immunostaining for human albumin. Magnification: 100×. (E) Kakinuma et al. [33]: transplantation of adherently proliferating cells isolated from human cord blood. Immunostaining for HepPar1. Bar: 10μm. (F) Von Mach et al. [74]: transplantation of nestin-positive islet-derived adherently proliferating cells. Immunostaining for human albumin. Magnification: 400×. (G) Von Mach et al. [74]: transplantation of nestin-positive islet-derived adherently proliferating cells. Immunostaining for human (red) and mouse (green) albumin. Magnification: 630-fold. (H) Ruhnke et al. [63]: transplantation of primary human hepatocytes (left side) and human blood monocyte derived hepatocyte-like cells (right side). Immunostaining for albumin. Bar: 50μm. (I) Sharma et al. [64]: transplantation of unsorted human cord blood cells. Immunostaining for albumin. Magnification: 40×.

  • View full-size image.
  • Fig. 3. 

    Heterogeneity of human albumin positive cell types. Adherently proliferating cord blood cells derived from a single colony were directly injected into the left liver lobe of SCID/NOD mice followed by immunohistochemistry visualizing human albumin 3 weeks after transplantation. (A) Human albumin positive cells with hepatocyte-like morphology. Magnification: 200×. (B) Human albumin positive cells that do not show a hepatocyte like morphology, but rather resemble monocytes Magnification: 400×. (Hengstler and Brulport, unpublished data).

3.1.2. No functional improvement reported 

Many animal models of liver disease are available and seem to be adequate for preclinical studies (Table 3). Usually clear criteria are available to evaluate the success of therapy, such as liver copper content for the ATP7B-deficient mouse [21] or clotting factor VIII in a factor VIII-deficient mouse [42]. Several of these mouse models have been evaluated by transplantation of primary hepatocytes or precursors. For instance intrasplenic transplantation of embryonic hepatocytes isolated from 14-day fetal mouse livers into a mouse model of Wilson’s disease reduced toxic copper accumulation [65]. However, to our knowledge an improvement of liver function in these mouse models (Table 3) by cell therapy with human extrahepatic stem cells has not yet been reported.

Table 3. Animal models of inherited liver disease adequate for evaluating functionality of transplanted stem and precursor cells
Name/diseaseDefective geneRef.
Toxic milk mouse, Wilson diseaseATP7B[25]
Gunn rat, Crigler–Najjar type IUDP-glucuronosyltransferase 1A1 (UGT1A1)[50]
Nagase analbuminemic ratsMutation of the 5′ splice site of the intron HI leads to skipping of the albumin exon H[52], [28]
Watanabe hyperlipidemic rabbit, familial hypercholesterolemiaDeficiency of the low density lipoprotein receptor gene (in frame deletion in the ligand binding domain)[12], [43]
mdr2-knockout mouse, familial intrahepatic cholestasis (PFIC) type 3mdr2-knockout[15]
FAH mouse, hereditary tyrosinaemia type IFumarylacetoacetate hydrolase (FAH) deficient[75], [73]
Spf(ash) mouse, ornithine transcarbamylase (OTC) deficiencyInactivation of the spf(ash) gene (causing OTC deficiency)[34]
FVII-deficient miceReplacement of exons 2–8 of the FVII gene with a neomycin phosphotransferase (neo) gene[49]
FVIII-deficient mice, haemophilia AFVIII knockout mice[40]
3.1.3. The wrong strategy? 

Many scientists transplanted human stem cells into livers of immunodeficient mice to study the differentiation capacity. An advantage of this strategy is the genuine liver microenvironment which is difficult to imitate in vitro. Many human growth and differentiation factors are known to be efficient also in human hepatocytes (and vice versa). Therefore, it is not implausible to assume that the mouse liver microenvironment may promote differentiation also of human stem cells to hepatocytes. However, when negative results or, for instance, mixed cell types are observed it is not clear, whether this is due to limitations of the specific stem cell type used or to interspecies incompatibilities.

To avoid this dilemma an alternative strategy might be applied. When possible, identical types of stem or precursor cells should be isolated from human and mouse tissue and should be tested in the same mouse model. The worst case scenario is that both, mouse and human cell types, fail to improve liver function. In this case clearly the limited differentiation capacity of the tested stem cell type is responsible for the negative result. However, if the mouse cells improve liver function, whereas the respective human cells remain negative, further, more adequate models must be applied for the human cells. To our knowledge this strategy (comparing human and allogeneic stem cells in the same animal model) has not yet been performed.

On the other hand, studies are available where rodent stem cells have been transplanted into rodents. A well-known example has been published by Jang et al. [79], who used a relatively complex method of isolating haematopoietic stem cells including three steps: (i) isolation of a small-sized cell population from male C57Bl6 mice by counter-flow elutriation of bone marrow cells; (ii) depletion of lineage-positive cells; and (iii) labelling of the resulting cell fraction with the red fluorescence dye PKH26 and isolation after injection into lethally irradiated female C57Bl6 mice. These cells were intravenously injected into CCl4 pretreated mice (100,000 cells per animal). The authors report that liver function was restored already 2–7 days after transplantation. For instance, mean fibrinogen plasma concentrations were reported to be 129mg/dl in mice two days after treatment with CCl4 compared to 252mg/dl in mice two days after treatment with CCl4 plus stem cells. Negative control cells have not been transplanted in this study, which would be important to prove an advantage of the rigorously purified cell fraction. In addition studies in wild-type rodent livers may be difficult, due to the extremely fast spontaneous regeneration. Thus, the data of Jang et al. [79] are promising, but confirmation by independent groups and evaluation in further animal models (for instance, those suggested in Table 3) are needed.

We also noticed that human stem cells have not yet been tested in livers of pigs, which seems to be mandatory. The rodent liver is capable of extremely rapid regeneration, which is much more efficient compared to pig and human. Therefore, the pig liver may represent a more realistic model with respect to human liver regeneration. In addition size and anatomical structure of pig livers more closely resemble the human situation than rodent livers. This will also be important for evaluation of cell application techniques and adverse effects.

3.1.4. Which stem and precursor cell types are most promising? 

Based on the available data it is difficult to compare the different human cell types with respect of their capacity to differentiate to hepatocytes in vivo. Human cell types studied so far are (references in Table 2): (i) adherently proliferating cells from human cord blood. These cells are isolated from the mononuclear cell fraction by adhesion to culture dishes and repeated passaging in culture after trypsinization to remove monocyte contaminations. These cells have a fibroblast-like morphology and proliferate for at least ten passages with 1:5 splitting. (ii) The mononuclear cell fraction from cord blood, either as a crude fraction or after FACS sorting for markers, such as CD34. These cells differ from the adherently proliferating cord blood cells because they do not adhere to cell culture vessels and (usually) do not proliferate in vitro. (iii) Haematopoietic cells from bone marrow, isolated by FACS sorting for well-established markers [32]. As well, these cells do not adhere to culture dishes and cannot be multiplied in vitro by proliferation. (iv) Nestin-positive pancreatic islet cells. These cells are isolated after outgrowth from pancreatic islets in vitro and selected for high nestin expression. These cells have a fibroblast-like morphology and proliferate well in vitro. (v) Hepatocyte-like cells isolated from peripheral blood monocytes by a two-step dedifferentiation/differentiation in vitro protocol. After differentiation the cells stop proliferating and adopt a hepatocyte-like morphology. (vi) Amniotic epithelial cells that develop from the epiblast by 8 days after fertilization and have the capacity to differentiate to hepatocyte-like cells [45]. Of course many more promising extrahepatic stem and precursor cell types are available. To our knowledge these stem and precursor cells have not yet been tested for differentiation into hepatocytes in vivo. As mentioned above it is difficult to compare the capacity of different human cell types. Most scientists have concentrated on a specific cell type. Comparative in vivo studies of several human stem or precursor cell types to evaluate hepatocellular differentiation capacity under standardized conditions are not available. However, concerning the published studies in which human cells have been transplanted into mice, the results appear remarkably similar for the different transplanted cell types. Almost all authors observed albumin and/or HepPar1-positive single cells or small cell clusters, but no tissue formation (Table 2). However, as discussed above it is not yet clear, whether this reflects the true differentiation capacity of the cells or a limitation of the mouse model.

With respect to the implementation of cell therapy, there are clear differences between the above-mentioned cell types. An advantage of the adherently proliferating cells is the availability of large amounts of cells. However, especially for the proliferating cell types it is crucial to exclude malignant transformation after transplantation. An advantage of the monocyte derived hepatocyte-like cells [63] is the opportunity to generate these cells from the recipient’s own blood, thus avoiding immune suppressive medication.

3.1.5. What are the requirements for clinicians to use hepatocyte-like cells in patients? 

Several requirements have to be considered to be met in order to use hepatocyte-like cells in patients. Cells must be produced following GMP criteria in order to ensure safety (transmission of infections such as hepatitis), stability and continuous quality of the graft. Engraftment, survival and functionality of the cells are certainly important factors, since they predict the frequency of recurrent treatments and thereby directly influence therapy success and costs. Cryopreservation of the cells is mandatory in order to ensure permanent availability in regard to the distribution within the clinical centres. Comparable to solid organ transplantation procedures, the transplanted cells should carry the same ABO antigens as the recipient. Therefore, cells of the various blood groups should be kept on stock.

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4. Cell fusion and “fusogenic cell therapy” 

The mechanism underlying the conversion of stem cells has been heavily debated. It is generally accepted that some stem cell types can fuse with the recipient’s cells [2], thus leading to cytoplasmic mixing and reprogramming of cell fate. Alternatively, it seems that stem cells can be instructed by factors of the host’s microenvironment to adopt a hepatocyte fate. To differentiate between cell fusion and transdifferentiation is of fundamental importance and has several practical implications. For instance, fusogenic cells may be used as vehicles that might deliver their own wild-type genes to the deficient genome of the recipient’s hepatocytes.

To our knowledge the efficiency of “fusogenic cell therapy” so far has only been demonstrated in the fumarylacetoacetate hydrolase-deficient mouse model (FAH−/−): an animal model of fatal tyrosinaemia Type 1. FAH−/− mice suffer from severe liver damage as a consequence of accumulation of the hepatotoxic metabolites, fumarylacetoacetate and its precursor maleylacetoacetate. Due to the deterioration of hepatocytes, FAH-deficient mice cannot survive unless they are treated with 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione (NTBC), which prevents production of the toxic metabolites. Due to permanent deterioration of hepatocytes the FAH−/− mice represent an animal model with an extremely high selection pressure for wild-type (i.e., FAH+/− or FAH+/+) hepatocytes. At least three articles (from two independent groups) have convincingly demonstrated that in the FAH−/− mouse model the transplanted stem cells fuse with the host’s hepatocytes, thus leading to liver regeneration [75], [73], [77]. Wang et al. [75] transplanted bone marrow cells from female FAH wild-type LacZ transgenic mice into male FAH−/− recipients. Cytogenetic analysis demonstrated 80 XXXY karyotypes, indicating cell fusion between two diploid cells. Similarly, 120 XXXXYY karyotypes demonstrated cell fusion events between a tetraploid recipient’s hepatocyte to a diploid donor bone marrow cell. Vassilopoulos et al. [73] analysed genomic DNA of FAH-expressing liver nodules after transplantation of FAH+/+ bone marrow cells. Interestingly, the nodules contained more mutant (host) than wild-type (donor) FAH alleles. If donor bone marrow had transdifferentiated into hepatocytes the FAH-expressing liver nodules should have contained mostly donor DNA. Willenbring et al. [77] concentrated on the cell type responsible for therapeutic cell fusion. They demonstrated that differentiated macrophages (obtained from the mononuclear fraction of OSA26+/− [R26R], FAH+/+-mice) could act as fusion partners for FAH−/− hepatocytes. In contrast, hepatocyte–hepatocyte fusion did not occur or was extremely rare, which has been demonstrated by transplantation of FAH wild-type hepatocytes into FAH−/− mice.

Therefore, the experiments with FAH−/− mice clearly provide a proof of principle that a monogenetic liver disease can be cured by “fusogenic cell therapy”. One might argue that the extreme selective pressure in FAH−/− livers may create a situation that facilitates cell fusion. Usually, such an extreme selection pressure cannot be found in human inherited metabolic liver diseases. Therefore, efficiency of gene transfer by cell fusion may be insufficient for a significant improvement of clinical outcome in human metabolic liver disease. At present, cell fusion remains a rare event not considered as the principal mechanism of liver repopulation. Nevertheless, future developments might increase the “fusogenic potential” of cells for “fusogenic therapy”. Good candidates for fusogenic cell therapy are inherited metabolic liver diseases that are caused by defects in a single or a limited number of genes, which have been discussed above (Table 2). However, it should be considered that fusogenic therapy could have serious consequences. Cell fusion leads to aneuploidy and eventually to chromosome instability and loss of chromosomes. Therefore, serious consequences including neoplasia must be carefully excluded before fusogenic therapy can be applied in patients.

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5. Technical approaches related to human cell transplantation 

In order to track the fate of human stem cells following transplantation, the use of suitable animal models is necessary. However, the analysis of human stem cells after their transplantation into the livers of laboratory animals is technically demanding. The experimental transplantation of hepatic cells aiming to repopulate the host liver has two fundamental requirements: Firstly, donor cells need to be identified within the recipient tissue and secondly, selective proliferation stimulus is necessary, so that transplanted cells proliferate in preference to host cells.

Several techniques are available to identify transplanted cells, such as fluorescent dyes, nanoparticles, and genetic markers (Fig. 4A). After identification of the transplanted cells in the recipient tissue by these markers, their human origin should be confirmed, for instance through in situ hybridization with alu- and mouse major satellite probes that allow differentiation between mouse and human nucleic acids (Fig. 4B–D). A next milestone is the demonstration that previously silent hepatocyte markers become expressed in the transplanted cells (Fig. 4E). For this purpose combination of in situ hybridization using alu-probes with immunostaining for human hepatocyte markers may identify cells of human origin that express, for instance, albumin (Fig. 4F). Relying on the species specificity of an antibody alone may be problematic, since it is difficult to establish conditions that guarantee a 100% species specificity. After identification of cells expressing human hepatocyte markers it is recommended to confirm expression by an independent technique, for instance by RT-PCR with species specific primers. If confirmation is positive an important milestone has been achieved. Some examples of cells that fulfil these criteria at least partially have been shown in Fig. 2.

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  • Fig. 4. 

    Milestones in defining hepatocyte-like cells: (A) Marking of cells before transplantation. In this example human fetal hepatocytes have been marked by red fluorescent nanoparticles. (B–D) Identification of cells of human origin in mouse livers by in situ hybridization. (B) SCID/NOD mouse liver tissue after in situ hybridization with mouse major satellite probes (visualized by pink fluorescence). (C) Human liver tissue after in situ hybridization with alu-probes (visualized by green fluorescence). (D) Visualization of human hepatocytes after transplantation into the liver of a SCID/NOD mouse by combined in situ hybridization with alu- and mouse major satellite probes [80]. (E) Double immunohistochemistry for human (red) and mouse (green) albumin, demonstrating that the transplanted cell expresses human but not mouse albumin. (F) Confirmation of the human origin of a human albumin expressing cell. Human albumin is immunohistochemically detected (visualized by red fluorescence), whereas human DNA is identified by in situ hybridization with alu-probes (visualized by green fluorescence). Blue fluorescence: nuclear staining with DAPI. Magnification: A,E,F: 630×; D: 200×.

To allow preferential proliferation of donor cells, the regeneration capacity of the recipient liver can be impaired. In most animal models, chemotoxins or carcinogens targeting the liver (e.g. retrorsine, 2-acetylaminofluorene, CCl4) are widely used for this purpose [41]. However, owing to the severe systemic side effects, these agents are not suitable for human application. Therefore, less harmful stimuli as an alternative to prime the host liver have to be studied. It has been recently shown that liver irradiation may be a promising alternative as a potential preparative regimen for hepatocyte transplantation [24].

A question of relevance is, whether these cells can be regarded as genuine, functional hepatocytes. Some recent studies suggested that expression of albumin and an epithelial morphology alone do not guarantee expression of all functions that make up a hepatocyte [64], [6]; review: [27]. For this purpose functional studies in animal models of liver disease (Table 3) are needed. An important further criterion is whether the hepatocyte-like cells of human origin can form liver tissue when given a selection advantage over the recipient’s hepatocytes. Finally, hepatocyte-like cells of human origin could be isolated after collagenase digestion to be able to characterize enzyme activities and further hepatocellular functions to cultured primary hepatocytes, as recently recommended [27]. In conclusion, many scientists have demonstrated hepatocyte-like cells after transplantation of human stem and precursor cells into livers of experimental animals. However, as a worst case scenario, it cannot yet be excluded that these cells are intermediate cell types expressing only a small number of hepatocyte markers. In terms of clinical application functional analysis in animal models of human liver disease is imperative.

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References 

  1. Almeida-Porada G, Porada CD, Chamberlain J, Torabi A, Zanjani ED. Formation of human hepatocytes by human hematopoietic stem cells in sheep. Blood. 2004;104:2582–2590
  2. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K, et al. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature. 2003;425:968–973
  3. Ambrosino G, Varotto S, Strom SC, Guariso G, Franchin E, Miotto D, et al. Isolated hepatocyte transplantation for Crigler–Najjar syndrome type 1. Cell Transplant. 2005;14:151–157
  4. Aoki T, Jin Z, Nishino N, Kato H, Shimizu Y, Niiya T, et al. Intrasplenic transplantation of encapsulated hepatocytes decreases mortality and improves liver functions in fulminant hepatic failure from 90% partial hepatectomy in rats. Transplantation. 2005;79:783–790
  5. Attaran M, Schneider A, Grote C, Zwiens C, Flemming P, Gratz KF, et al. Regional and transient ischemia/reperfusion injury in the liver improves therapeutic efficacy of allogeneic intraportal hepatocyte transplantation in low-density lipoprotein receptor deficient Watanabe rabbits. J Hepatol. 2004;41:837–844
  6. Beerheide W, von Mach MA, Ringel M, Fleckenstein C, Schumann S, Renzing N, et al. Downregulation of beta2-microglobulin in human cord blood somatic stem cells after transplantation into livers of SCID-mice: an escape mechanism of stem cells?. Biochem Biophys Res Commun. 2002;294:1052–1063
  7. Bilir BM, Guinette D, Karrer F, Kumpe DA, Krysl J, Stephens J, et al. Hepatocyte transplantation in acute liver failure. Liver Transplant. 2000;6:32–40
  8. Burlina AB. Hepatocyte transplantation for inborn errors of metabolism. J Inherit Metab Dis. 2004;27:373–383
  9. Cai J, Ito M, Nagata H, Westerman KA, LaFleur D, Chowdhury JR, et al. Treatment of liver failure in rats with end-stage cirrhosis by transplantation of immortalized hepatocytes. Hepatology. 2002;36:386–394
  10. Chan C, Berthiaume F, Nath BD, Tilles AW, Toner M, Yarmush ML. Hepatic tissue engineering for adjunct and temporary liver support: critical technologies. Liver Transplant. 2004;10:1331–1342
  11. Cantz T, Sharma AD, Jochheim-Richter A, Arseniev L, Klein C, Manns MP, et al. Reevaluation of bone marrow-derived cells as a source for hepatocyte regeneration. Cell Transplant. 2004;13:659–666
  12. Chowdhury JR, Grossman M, Gupta S, Chowdhury NR, Baker JR, Wilson JM. Long-term improvement of hypercholesterolemia after ex vivo gene therapy in LDLR-deficient rabbits. Science. 1991;254:1802–1805
  13. Danet GH, Luongo JL, Butler G, Lu MM, Tenner AJ, Simon MC, et al. C1qRp defines a new human stem cell population with hematopoietic and hepatic potential. Proc Natl Acad Sci USA. 2002;99:10441–10445
  14. Demetriou AA, Reisner A, Sanchez J, Levenson SM, Moscioni AD, Chowdhury JR. Transplantation of microcarrier-attached hepatocytes into 90% partially hepatectomized rats. Hepatology. 1988;8:1006–1009
  15. De Vree JM, Ottenhoff R, Bosma PJ, Smith AJ, Aten J, Oude Elferink RP. Correction of liver disease by hepatocyte transplantation in a mouse model of progressive familial intrahepatic cholestasis. Gastroenterology. 2000;119:1720–1730
  16. Dhawan A, Mitry RR, Hughes RD, Lehec S, Terry C, Bansal S, et al. Hepatocyte transplantation for inherited factor VII deficiency. Transplantation. 2004;78:1812–1814
  17. Ryan Edmond A, Lakey Jonathan RT, Rajotte Ray V, Korbutt Gregory S, Kin2 Tatsuya, Imes Sharleen, et al. Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes. 2001;50:710–719
  18. Fisher RA, Bu D, Thompson M, Tisnado J, Prasad U, Sterling R, et al. Defining hepatocellular chimerism in a liver failure patient bridged with hepatocyte infusion. Transplantation. 2000;69:303–307
  19. Fox IJ, Chowdhury JR, Kaufman SS, Goertzen TC, Chowdhury NR, Warkentin PI, et al. Treatment of the Crigler–Najjar syndrome type I with hepatocyte transplantation. N Engl J Med. 1998;338:1422–1426
  20. Francavilla A, Starzl TE, Barone M, Zeng QH, Porter KA, Zeevi A, et al. Studies on mechanisms of augmentation of liver regeneration by cyclosporine and FK 506. Hepatology. 1991;14:140–143
  21. Fuentealba IC, Aburto EM. Animal models of copper-associated liver disease. Comp Hepatol. 2003;2:5
  22. Goss JA, Stribling R, Martin P. Adult liver transplantation for metabolic liver disease. Clin Liver Dis. 1998;2:187–210
  23. Groth CG, Arborgh B, Bjorken C, Sundberg B, Lundgren G. Correction of hyperbilirubinemia in the glucuronyltransferase-deficient rat by intraportal hepatocyte transplantation. Transplant Proc. 1977;9:313–316
  24. Guha C, Parashar B, Deb NJ, et al. Liver irradiation: a potential preparative regimen for hepatocyte transplantation. Int J Radiat Oncol Biol Phys. 2001;49:451–457
  25. Harada M, Kawaguchi T, Kumemura H, Terada K, Ninomiya H, Taniguchi E, et al. The Wilson disease protein ATP7B resides in the late endosomes with Rab7 and the Niemann-Pick C1 protein. Am J Pathol. 2005;166:499–510
  26. Habibullah CM, Syed IH, Qamar A, Taher-Uz Z. Human fetal hepatocyte transplantation in patients with fulminant hepatic failure. Transplantation. 1994;58:951–952
  27. Hengstler JG, Brulport M, Schormann W, Bauer A, Hermes M, Nussler AK, et al. Generation of human hepatocytes by stem cell technology: definition of the hepatocyte. Expert Opin Drug Metab Toxicol. 2005;1:61–74
  28. Hitomi Y, Sugiyama K, Esumi H. Suppression of the 5′ splice site mutation in the Nagase analbuminemic rat with mutated U1snRNA. Biochem Biophys Res Commun. 1998;251:11–16
  29. Horslen SP, McCowan TC, Goertzen TC, Warkentin PI, Cai HB, Strom SC, et al. Isolated hepatocyte transplantation in an infant with a severe urea cycle disorder. Pediatrics. 2003;111:1262–1267
  30. Hughes RD, Mitry RR, Dhawan A. Hepatocyte transplantation for metabolic liver disease: UK experience. J R Soc Med. 2005;98:341–345
  31. Ishikawa F, Drake CJ, Yang S, Fleming P, Minamiguchi H, Visconti RP, et al. Transplanted human cord blood cells give rise to hepatocytes in engrafted mice. Ann NY Acad Sci. 2003;996:174–185
  32. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;418:41–49
  33. Kakinuma S, Tanaka Y, Chinzei R, Watanabe M, Shimizu-Saito K, Hara Y, et al. Human umbilical cord blood as a source of transplantable hepatic progenitor cells. Stem Cells. 2003;21:217–227
  34. Kiwaki K, Kanegae Y, Saito I, Komaki S, Nakamura K, Miyazaki JI, et al. Correction of ornithine transcarbamylase deficiency in adult spf(ash) mice and in OTC-deficient human hepatocytes with recombinant adenoviruses bearing the CAG promoter. Hum Gene Ther. 1996;7:821–830
  35. Kobayashi N, Ito M, Nakamura J, Cai J, Gao C, Hammel JM, et al. Hepatocyte transplantation in rats with decompensated cirrhosis. Hepatology. 2000;31:851–857
  36. Kobayashi N, Westerman KA, Tanaka N, Fox IJ, Leboulch P. A reversibly immortalized human hepatocyte cell line as a source of hepatocyte-based biological support. Addict Biol. 2001;6:293–300
  37. Koenig S, Stoesser C, Krause P, Becker H, Markus PM. Liver repopulation after hepatocellular transplantation: integration and interaction of transplanted hepatocytes in the host. Cell Transplant. 2005;14:31–40
  38. Kogler G, Sensken S, Airey JA, Trapp T, Muschen M, Feldhahn N, et al. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med. 2004;200:123–135
  39. Kollet O, Shivtiel S, Chen YQ, Suriawinata J, Thung SN, Dabeva MD, et al. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest. 2003;112:160–169
  40. Kumaran V, Benten D, Follenzi A, Joseph B, Sarkar R, Gupta S. Transplantation of endothelial cells corrects the phenotype in hemophilia A mice. J Thromb Haemost. 2005;3:2022–2031
  41. Laconi E, Laconi S. Principles of hepatocyte repopulation. Semin Cell Dev Biol. 2002;13:433–438
  42. Landskroner KA, Olson NC, Jesmok GJ. Thromboelastography measurements of whole blood from factor VIII-deficient mice supplemented with rFVIII. Haemophilia. 2005;11:346–352
  43. Li J, Fang B, Eisensmith RC, Li XH, Nasonkin I, Lin-Lee YC, et al. In vivo gene therapy for hyperlipidemia: phenotypic correction in Watanabe rabbits by hepatic delivery of the rabbit LDL receptor gene. J Clin Invest. 1995;95:768–773
  44. Meier M, Woywodt A, Hoeper MM, Schneider A, Manns MP, Strassburg CP. Acute liver failure: a message found under the skin. Postgrad Med J. 2005;81:269–270
  45. Miki T, Lehmann T, Cai H, Stolz DB, Strom SC. Stem cell characteristics of amniotic epithelial cells. Stem Cells. 2005;[Epub ahead of print]
  46. Mito M, Kusano M, Kawaura Y. Hepatocyte transplantation in man. Transplant Proc. 1992;24:3052–3053
  47. Mitry RR, Dhawan A, Hughes RD, Bansal S, Lehec S, Terry C, et al. One liver, three recipients: segment IV from split-liver procedures as a source of hepatocytes for cell transplantation. Transplantation. 2004;77:1614–1616
  48. Muraca M, Gerunda G, Neri D, Vilei MT, Granato A, Feltracco P, et al. Hepatocyte transplantation as a treatment for glycogen storage disease type 1a. Lancet. 2002;359:317–318
  49. Rosen ED, Chan JC, Idusogie E, Clotman F, Vlasuk G, Luther T, et al. Mice lacking factor VII develop normally but suffer fatal perinatal bleeding. Nature. 1997;390:290–294
  50. Seppen J, van der Rijt R, Looije N, van Til NP, Lamers WH, Oude Elferink RP. Long-term correction of bilirubin UDPglucuronyltransferase deficiency in rats by in utero lentiviral gene transfer. Mol Ther. 2003;8:593–599
  51. Strom SC, Chowdhury JR, Fox IJ. Hepatocyte transplantation for the treatment of human disease. Semin Liver Dis. 1999;19:39–48
  52. Moscioni AD, Rozga J, Chen S, Naim A, Scott HS, Demetriou AA. Long-term correction of albumin levels in the Nagase analbuminemic rat: repopulation of the liver by transplanted normal hepatocytes under a regeneration response. Cell Transplant. 1996;5:499–503
  53. Mito M, Kusano M. Hepatocyte transplantation in man. Cell Transplant. 1993;2:65–74
  54. Nagata H, Ito M, Cai J, Edge AS, Platt JL, Fox IJ. Treatment of cirrhosis and liver failure in rats by hepatocyte xenotransplantation. Gastroenterology. 2003;124:422–431
  55. Nagata H, Ito M, Shirota C, Edge A, McCowan TC, Fox IJ. Route of hepatocyte delivery affects hepatocyte engraftment in the spleen. Transplantation. 2003;76:732–734
  56. Newsome PN, Johannessen I, Boyle S, Dalakas E, McAulay KA, Samuel K, et al. Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion. Gastroenterology. 2003;124:1891–1900
  57. Nonome K, Li XK, Takahara T, Kitazawa Y, Funeshima N, Yata Y, et al. Human umbilical cord blood-derived cells differentiate into hepatocyte-like cells in the Fas-mediated liver injury model. Am J Physiol Gastrointest Liver Physiol. 2005;
  58. Ott M, Schmidt HH, Cichon G, Manns MP. Emerging therapies in hepatology: liver-directed gene transfer and hepatocyte transplantation. Cells Tissues Organs. 2000;167:81–87
  59. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, et al. Bone marrow as a potential source of hepatic oval cells. Science. 1999;284:1168–1170
  60. Petersen J, Ott M, von Weizsacker F. Current status of cell-based therapies in liver diseases. Z Gastroenterol. 2001;39:975–980
  61. Jalan Rajiv. Acute liver failure: current management and future prospects. J Hepatol. 2005;42(Suppl. 1):S115–S123[Epub 2004 Dec 15]
  62. Ringel M, von Mach MA, Santos R, Feilen PJ, Brulport M, Hermes M, et al. Hepatocytes cultured in alginate microspheres: an optimized technique to study enzyme induction. Toxicology. 2005;206:153–167
  63. Ruhnke M, Ungefroren H, Nussler A, Martin F, Brulport M, Schormann W, et al Reprogramming of human peripheral blood monocytes into functional hepatocyte and pancreatic islet-like cells. Gastroenterology. 2005;128:1774–1786
  64. Sharma AD, Cantz T, Richter R, Eckert K, Henschler R, Wilkens L, et al. Human cord blood stem cells generate human cytokeratin 18-negative hepatocyte-like cells in injured mouse liver. Am J Pathol. 2005;167:555–564
  65. Shi Z, Liang XL, Lu BX, Pan SY, Chen X, Tang QQ, et al. Diminution of toxic copper accumulation in toxic milk mice modeling Wilson disease by embryonic hepatocyte intrasplenic transplantation. World J Gastroenterol. 2005;11:3691–3695
  66. Sokal EM, Smets F, Bourgois A, Van Maldergem L, Buts JP, Reding R, et al. Hepatocyte transplantation in a 4-year-old girl with peroxisomal biogenesis disease: technique, safety, and metabolic follow-up. Transplantation. 2003;76:735–738
  67. Stephenne X, Najimi M, Smets F, Reding R, de Ville de Goyet J, Sokal EM. Cryopreserved liver cell transplantation controls ornithine transcarbamylase deficient patient while awaiting liver transplantation. Am J Transplant. 2005;5:2058–2061
  68. Strom S, Fisher R. Hepatocyte transplantation: new possibilities for therapy. Gastroenterology. 2003;124:568–571
  69. Strom SC, Fisher RA, Thompson MT, Sanyal AJ, Cole PE, Ham JM, et al. Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure. Transplantation. 1997;63:559–569
  70. Strom SC, Fisher RA, Rubinstein WS, Barranger JA, Towbin RB, Charron M, et al. Transplantation of human hepatocytes. Transplant Proc. 1997;29:2103–2106
  71. Strom SC, Chowdhury JR, Fox IJ. Hepatocyte transplantation for the treatment of human disease. Semin Liver Dis. 1999;19:39–48
  72. Turrini P, Monego G, Gonzalez J, Cicuzza S, Bonanno G, Zelano G, et al. Human hepatocytes in mice receiving pre-immune injection with human cord blood cells. Biochem Biophys Res Commun. 2005;326:66–73
  73. Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature. 2003;422:901–904
  74. von Mach MA, Hengstler JG, Brulport M, Eberhardt M, Schormann W, Hermes M, et al. In vitro cultured islet-derived progenitor cells of human origin express human albumin in severe combined immunodeficiency mouse liver in vivo. Stem Cells. 2004;22:1134–1141
  75. Wang X, Montini E, Al-Dhalimy M, Lagasse E, Finegold M, Grompe M. Kinetics of liver repopulation after bone marrow transplantation. Am J Pathol. 2002;161:565a–574a
  76. Wang X, Ge S, McNamara G, Hao QL, Crooks GM, Nolta JA. Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells. Blood. 2003;101:4201–4208[Epub 2003 Jan 30]
  77. Willenbring H, Bailey AS, Foster M, Akkari Y, Dorrell C, Olson S, et al. Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med. 2004;10:744–748
  78. Zeng F, Chen M, Katsumata M, Huang W, Gong Z, Hu W, et al. Identification and characterization of engrafted human cells in human/goat xenogeneic transplantation chimerism. DNA Cell Biol. 2005;24:403–409
  79. Jang YY, Collector MI, Baylin SB, Diehl AM, Shatkis SJ. Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol. 2004;6:532–539
  80. Brüstle O, Spiro AC, Karram K, Choudhary K, Okabe S, McKay RD. In vitro-generated newal precursors participate in mammalian brain development. Proc Natl Acad Sci USA. 1997;94:14809–14814

PII: S0168-8278(06)00223-6

doi:10.1016/j.jhep.2006.04.002

Journal of Hepatology
Volume 45, Issue 1 , Pages 144-159, July 2006

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