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European Biopharmaceutical Review

Treatment from Within

Liver cell transplantation is preferred to completely replacing the organ in genetic liver diseases. However, the choice of cells used is crucial to the success of this therapy, with intrahepatic sources having many advantages over cells originating from outside the liver.

The liver is the site of many vital functions. As a result, the impairment of only one protein within its complex metabolic pathway is usually very problematic. This condition is called ‘inborn error of metabolism’, and concerns many genetic diseases linked to a non-functional enzyme in the liver. All these diseases have high mortality rates and, for survivors, affect the quality of life of patients and their families. Both treatments and long-term strategies are currently not efficient or suitable enough and patients would greatly benefit from an innovative therapy that meets this medical need. Currently, orthotopic liver transplantation is the only curative treatment of severe defects and/or end stage diseases, but it is hampered by major drawbacks. Donor livers are rare and the surgery is invasive and irreversible, while still presenting risks of non-functionality of the graft or even long-term graft dysfunction. Also, there are certain cases, such as in young babies, where transplantation is not an option. Cell therapy has therefore been identified as the best alternative tool to overcome the scarcity of organ donation. Several cell types are under investigation and adult liver stem/progenitor cells represent an attractive cell source for liver regenerative medicine.

Inborn Errors of Metabolism

The liver is a key organ in regulating body homeostasis, playing a role in several functions including: blood glucose homeostasis; synthesis of plasma proteins, such as albumin or coagulation factors; and endogenous and xenobiotic detoxification. Hence, impairment of a single liver function may have a dramatic impact on health. According to the World Health Organization, worldwide total incidence of both acute and chronic liver diseases set these pathologies within the top 15 causes of death.

The liver expresses a substantial number of important enzymes, each of which is susceptible for deficiency, with each one being associated to a specific disease. The incidence of these diseases is rare and can be as low as one in a million births. However, altogether, inborn errors of liver metabolism affect one child in every 2,500 live births. This means that, within the European Community, 2,000 new cases are diagnosed every year.

Alongside congenital liver diseases due to a genetic metabolic defect, acquired diseases may also affect the liver due to infectious, toxic or immune-related mechanisms. Acute liver diseases can rapidly lead to severe functional impairments, such as in fulminant hepatitis, while chronic damage can cause progressive fibrosis and cirrhosis.

The spectrum of these genetic diseases range from severe and life-threatening to ones with a milder clinical expression. That said, they all impede the general quality of life of both the patient and his or her family. In particular, patients suffering from urea cycle diseases cannot detoxify free ammonium resulting from protein catabolism. Free ammonium is highly toxic to the central nervous system. These patients are consequently at high risk of metabolic decompensation, leading to irreversible neurological damage. Long-term management of patients suffering from urea cycle diseases is mainly based on a low-protein diet and the use of ammonium scavengers; naso-gastric feeding is also typically required. These efforts however, do not reduce the risk of sudden hyperammonaemia. On top of this, many patients often develop anorexia and intellectual impairment due to chronic ammonium intoxication.

A retrospective study performed in Italy showed that for the 1,935 cases diagnosed with an inborn error of metabolism, only 118 reached adulthood. Currently, the only radical treatment of end stage diseases is orthotopic liver transplantation (OLT). However, this procedure is serious, irreversible and limited by organ shortage.

Liver Cell Transplantation

Liver cell transplantation (LCT) consists of single or repeated infusions of allogeneic mature hepatocytes in the patient’s liver circulation, isolated from healthy adult donors. The procedure has been well described and more than 40 clinical attempts have been reported in the literature.

The proof of concept – for example the feasibility to compensate a missing function within a diseased liver by infusing a suspension of mature hepatocytes – has been established. The best results have been obtained in miscellaneous inborn errors of liver metabolism, such as Crigler Najjar syndrome, urea cycle defects, glycogen storage disease, clotting factor deficiencies and Refsum disease (1).

Clinical reports have demonstrated that LCT is safe and can restore metabolic liver function for up to nine months post-infusion. Repeated doses of mature hepatocyte infusions in a child with urea-cycle disorder and important secondary psychomotor retardation led to the presence of donor cells in liver biopsies up to eight months after the last infusion, thereby restoring de novo activity of arginosuccinate lyase. Infused cells were also shown to have contributed to the restoration of liver metabolism and improvement of psychomotor development (2).

LCT is a fully reversible procedure. It is much less invasive than OLT by avoiding the risks related to native liver transplantation; non-functionality of the graft or even long-term graft rejection. In addition, the possibility to perform OLT at a later stage remains. OLT has, for example, already been performed without complications in children having undergone previous LCT. For treatment of genetic diseases, LCT is based on allogeneic cells and patients receive immunosuppressive treatment following infusion.

There are, however, several limitations in the use of mature hepatocytes that currently limit its wide application:

  • Organ shortage as for classical OLT; only one or two patients can be treated by mature hepatocytes isolated from one donor
  • Weak resistance to cryopreservation. Cryopreservation of mature hepatocytes induces a severe impairment of cell adhesion, mitochondrial respiratory changes, loss of ATP production, alteration of mitochondrial respiratory chain enzymes, increased mitochondrial permeability and loss of membrane potential. Although successful cases have been reported with cryopreserved cells, these are clearly less efficient in the clinical field. It is possible that selecting high-quality cells from young donors may overcome this, but this is rarely achievable in routine transplantation
  • Possible bacterial contamination of fresh hepatocyte preparations, unknown at the time of infusion. Sterility is partially addressed by the use of prophylactic antibiotherapy covering gram-negative strains during the infusion procedure. These storage limitations, in addition to the organ shortage issue, consequently require consideration of other cell sources such as stem/progenitor cells as an alternative for liver cell therapy. Stem/ progenitors cells are undifferentiated cells, displaying a substantial self-renewal and expansion capacity in vitro. Adult tissue derived stem cells are: safer than embryonic, fetal or induced pluripotent cells; closer to clinical applications; and do not raise any ethical issues. Adult stem/ progenitor cells also have a good resistance to cryopreservation
The prospect is that these cells can soon be used for liver cell therapy.

Extrahepatic Sources of Stem/Progenitor Cells

There are two groups of sources of stem cells, either originating from or outside the liver. Two examples of extrahepatic sources are bone marrow and mesenchymal stem cells.

Bone marrow and haematopoietic tissues
Adult bone marrow contains different cell populations including mesenchymal stromal cells, endothelial cells, fibroblastic cells and haematopoietic cells. In different in vivo studies, it has been postulated that these cells are able to improve hepatic functionality. For example, in a mouse model of tyrosinaemia, infusion of bone marrow stem cells from wild type mice was able to restore fumaryl aceto acetase activity in deficient livers.

Besides direct functional activity of the transplanted cells, stem cells may also exert a paracrine effect and act through the release of cytokines or growth factors (secretosomes), thereby modifying the microenvironment and contributing to hepatocyte proliferation/functionality. This mechanism has been suggested as a mode of action in mouse models of toxicity-induced fulminant hepatitis; yet, it cannot explain de novo acquired metabolic functions in models of inborn errors of liver metabolism.

Mesenchymal Stem Cells (MSCs)
MSCs were first described by Freidenstein and colleagues as plastic-adherent fibroblastic cells with high capacity of proliferation and differentiation into osteogenic lineages. MSCs have since then been isolated from various tissues such as skin, Wharton’s Jelly, adipose tissue, amniotic membrane of the placenta, and so on (3). In vitro studies have demonstrated the ability of MSCs from different origins to differentiate into hepatocytes-like cells upon addition of specific growth/differentiation factors to the culture medium.

The adipose tissue is an easily accessible source of MSCs, and can be obtained from plastic surgical waste material (liposuctions and abdominal resections). These cells share similar phenotypic and immunological features with bone marrow-derived MSC, making them an attractive source for allogeneic transplantation. Their hepatic differentiation potential has been confirmed with acquisition of functional activities such as albumin production, glycogen storage and drug metabolising activities. Transplantation into nude mice with acute liver injury can restore liver functions. Pre-differentiation before transplantation may improve engraftment and the thus transplanted cells express human proteins, such as albumin and HepPar.

Besides their substantial differentiation potential, MSCs have been shown to exhibit immunosuppressive and inflammatory-reducing properties.

Intrahepatic Sources of Stem/Progenitor Cells

It is logical to look for a candidate liver progenitor that resides in the adult liver. Mature human hepatocytes themselves play an important role in liver tissue regeneration. They start to proliferate and repopulate the liver following an acute injury, which induce a stimuli from cytokines secreted by non-parenchymal cells. Besides regeneration from mature hepatocytes, different populations of stem/progenitor cells – including oval cells, small hepatocytes, liver epithelial cells and mesenchymal cells – participate in the replacement of liver cell loss due to hepatocyte proliferation impairment.

Oval cells are resident progenitor cells located in the bile ductules (canals of Hering), which are able to generate both hepatocytes and bile duct cells. They constitute the progenitor ‘niche’ or ‘reservoir’ of the liver. In response to injury, these cells proliferate and migrate to the liver parenchyma. However, these cells cannot be isolated, are not deliverable as cell suspensions, and are therefore not suitable in liver regenerative medicine.

Liver epithelial cells are derived from primary cultures of human adult hepatic cells. In vitro, they can be expanded and differentiated into both hepatocytes and biliary cells. They express mesenchymal, haematopoietic, biliary and hepatic markers, and typically differ from oval cells by their polygonal shape. Small hepatocytes are isolated from the non-parenchymal fraction. They display a high proliferation potential, but their in vitro differentiation potential into mature hepatocytes is limited.

Heterologous Human Adult Liver Progenitor Cells

The presence of the mesenchymal cells, also called heterologous human adult liver progenitor cells (HHALPC), were identified in hepatocyte suspensions obtained after collagenase perfusion of normal adult livers (4). These cells were isolated from a primary culture of hepatocytes and demonstrated an important proliferation potential and a more advanced stage to complete morphological and functional differentiation into hepatocytes. In contrary to other MSCs, HHALPC have a preferential differentiation capacity into hepatocytes and not into mature osteogenic and adipogenic cells. The latter feature suggests that these cells are already engaged in the hepatocytic lineage, and can therefore be considered as progenitor cells, rather than stem cells. Khuu and colleagues have demonstrated advanced liver metabolic activity of differentiated HHALPC in vitro and in vivo (5, 6). Indeed, following in vitro differentiation, HHALPC are able to metabolise ammonium, conjugate bilirubin, and express the Phase 1 and 2 enzymes responsible for metabolising both xenogenic (for example, drug), and endogen (for example, bilirubin) compounds.

The HHALPC’s ability to engraft, proliferate and differentiate into hepatocytes have been evaluated in animal models and the results have confirmed their potential use as a liver cell-based therapy for the treatment of many liver diseases. Transplantation of undifferentiated HHALPC into rodents was followed by in vivo differentiation and synthesis of human albumin six weeks later, with the resulting cells showing long-term engraftment potential. HHALPC is a so-called adult ‘progenitor’ cell, being committed to become a mature hepatocytic liver cell. These cells have been well-characterised, patented and are currently being studied for ‘allogeneic’ use. Unlike hepatocytes, HHALPC are not yet mature; therefore, they have greater potential for multiplication, making it possible to treat up to a hundred patients from a single organ. This approach will allow the organ shortage problem to be largely overcome. Furthermore, these cells have a longer lifetime and better resistance towards cryopreservation than hepatocytes. In particular, HHALPC have been successfully isolated from healthy liver samples, multiplied and cryopreserved over long periods. Taken together, differentiated or not, HHALPC is an excellent candidate to liver cell therapy (7).


Stem cell technology opens up opportunities for regenerative treatment of liver diseases, targeting inborn errors of metabolism – so far a high unmet medical need – as well as some acquired diseases of the liver. The proof of concept that cell therapy is able to restore liver function has been obtained with mature hepatocyte transplantation, but efficacy is partial and suitable, alternative sources of cells are needed to replace hepatocytes. HHALPC has shown promising results. The optimal combination of in vitro expansion and hepatic engraftment ability makes this adult progenitor cell a truly competitive tool for liver regenerative medicine. Moreover, the progenitor cell commitment to the hepatic lineage, associated with their ability to be cultured in vitro, can offer the biopharmaceutical industry an interesting tool for pharmacotoxicology screening of new drugs and lead compound optimisation.


  1. Lysy PA, Najimi M, Stephenne X, Bourgois A, Smets F and Sokal EM, Liver cell transplantation for Crigler- Najjar syndrome type I: Update and perspectives, World J Gastroenterol 14: pp3,464-3,470, 2008
  2. Stéphenne X, Najimi M, Sibille C, Nassogne MC, Smets F and Sokal EM, Sustained engraftment and tissue enzyme activity after liver cell transplantation for argininosuccinate lyase deficiency, Gastroenterology 130: pp1,317-1,323, 2006
  3. Campard D, Lysy PA, Najimi M and Sokal EM, Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells, Gastroenterology 134: pp833-848, 2008
  4. Najimi M, Khuu DN, Lysy PA, Jazouli N, Abarca J, Sempoux C and Sokal EM, Adult-derived human liver mesenchymal-like cells as a potential progenitor reservoir of hepatocytes? Cell Transplant 16: pp717-728, 2007
  5. Khuu DN, Scheers I, Ehnert S, Jazouli N, Nyabi O, Buc-Calderon P, Meuleman A, Nussler A, Sokal E and Najimi M, In vitro differentiation Adult human liver progenitor cells display mature hepatic metabolic functions: A potential tool for in vitro pharmacotoxicological testing, Cell Transplant 20: pp287-302, 2011
  6. Khuu DN, Nyabi O, Maerckx C, Sokal E and Najimi M, Adulte human liver mesenchymal stem/progenitor cells participate to mouse liver regeneration after hepatectomy, Cell Transplant (in press)
  7. Scheers I, Maerckx C, Khuu ND, Marecell S, Decottignies A, Najimi M and Sokal E, Adult liver derived progenitor cells in long term culture maintain appropriate gatekeeper mechanisms against transformation, Cell Transplant, Epub ahead of print, 2012

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Eric Halioua is Co-Founder and CEO of the Belgium cell therapy company Promethera Biosciences. Eric holds two Master’s degrees in Pharmacology and Molecular Biology and is a graduate of the ESSEC business school, with an advanced degree from the Health Care ESSEC chair. Eric is Co-Founder of two biotechnology companies called Myosix and Murigenetics. He was principal of the international life sciences practice of Arthur D. Little, based in Paris and Boston, for 11 years.

Etienne Marc Sokal is Founder and CSO of Promethera Biosciences. He is Full Professor and Head of the paediatric research unit at Université Catholique de Louvain, and Head of the paediatric gastroenterology and hepatology service at Cliniques Universitaires St Luc, Brussels. 
Eric Halioua
Etienne Marc Sokal
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