Regenerative Medicine, Stem Cells and the Liver 1st Edition

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 Contents

Preface v
List of Contributors ix
1. Stem Cell Therapy in the Context of Chronic Liver 1
Disease: Promise and Pitfalls
Prakash Ramachandran and John P. Iredale
2. Sources of Human Liver Cells and the Challenge 7
of Working with Primary Tissue
Janet W.C. Kung and James A. Ross
3. Role of Pluripotent Stem Cells in Regenerative Medicine 21
Eriona Hysolli, Xiao-Ling Zhou, Renjing Liu, Ji-Hyun Kim,
Brian Adams, Gareth Sullivan and In-Hyun Park
4. Human Liver Development as a Template to Generate 38
High Fidelity Models
Claire N. Medine, Janet W.C. Kung, Catherine M. Payne,
James R. Black, Richard A. Anderson, James A. Ross,
John P. Iredale and David C. Hay
5. Applying Pluripotent Stem Cell Technology to 49
Modelling Human Liver Disease
Nicholas R.F. Hannan, S. Tamir Rashid and Ludovic Vallier
6. Applying Stem Cell Technology to High Through-put 59
Drug Screening
Mia Emgård and Petter Björquist
7. Stem Cells in the Development of Products for 77
Regenerative Medicine
Paul A. De Sousa, Scott McRae and Glyn Stacey
8. Cells for Bioartificial Liver Support 98
Humphrey Hodgson, Amy Thomas and Clare Selden
9. Stem Cell Technology and Cell Based Therapies 115
Nicholas James Davies and Philip Noel Newsome
viii Regenerative Medicine, Stem Cells and the Liver

10. Advances in Cell Therapy for Liver Disease 138

Luke Boulter
11. Technical and Bioethical Challenges Associated with 154
using Stem Cells for Research and Therapy
Jordi L. Tremoleda, Itziar de Lecuona and Sian E. Harding
Index 189
Color Plate Section 191
 List of Contributors

Brian Adams
Department of Genetics, Yale Stem Cell Center, Yale School of Medicine,
10 Amistad, New Haven, CT. 06520, USA.
Email: [email protected]
Richard A. Anderson
MRC Centre for Regenerative Medicine, University of Edinburgh, 49
Little France Crescent, Edinburgh, EH16 4SB, U.K.
Email: [email protected]
Petter Björquist
Cellectis Stem Cells Cellartis AB, Arvid Wallgrens Backe 20, 41346
Göteborg, Sweden.
Email: [email protected]
James R. Black
MRC Centre for Regenerative Medicine, University of Edinburgh, 49
Little France Crescent, Edinburgh, EH16 4SB, U.K.
Email: [email protected]
Luke Boulter
Research Fellow, Queens Medical Research Institute, 47 Little France
Crescent, Edinburgh, EH16 4TJ, UK.
Email: [email protected]
Nicholas James Davies
Centre for Liver Research and NIHR Biomedical Research Unit,
University of Birmingham,Birmingham, U.K.
Email: [email protected]
Paul A. De Sousa
Scottish Centre for Regenerative Medicine, University of Edinburgh,
Edinburgh, EH16 4SB, UK.
Email: [email protected]
x Regenerative Medicine, Stem Cells and the Liver

Mia Emgård
Cellectis Stem Cells Cellartis AB, Arvid Wallgrens Backe 20, 41346
Göteborg, Sweden.
Email: [email protected]
Nicholas R.F. Hannan
Anne McLAren Laboratories for Regenerative Medicine, Department of
Surgery, University of Cambridge, UK.
Email: [email protected]
Sian E. Harding
Professor of Cardiac Pharmacology, National Heart and Lung Institute,
Faculty of Medicine, Imperial College London, UK, London SW7 2AZ, UK,
Member of the Nuffield Council on Bioethics.
Email: [email protected]
David C. Hay
MRC Centre for Regenerative Medicine, University of Edinburgh,
49 Little France Crescent, Edinburgh, EH16 4SB, U.K.
Email: [email protected]
Humphrey Hodgson
Royal Free Campus, UCL Medical School, London NW3 2PF.
Email: [email protected]
Eriona Hysolli
Department of Genetics, Yale Stem Cell Center, Yale School of Medicine,
10 Amistad, New Haven, CT. 06520, USA.
Email: [email protected]
John P. Iredale
MRC Centre for Regenerative Medicine, University of Edinburgh,
49 Little France Crescent, Edinburgh, EH16 4SB, U.K.
Email: [email protected]
Ji-Hyun Kim
Department of Genetics, Yale Stem Cell Center, Yale School of Medicine,
10 Amistad, New Haven, CT. 06520, USA.
Email: [email protected]
Janet W.C. Kung
MRC Centre for Regenerative Medicine, University of Edinburgh,
49 Little France Crescent, Edinburgh, EH16 4SB, U.K.
Email: [email protected]
 List of Contributors xi

Itziar de Lecuona
Lecturer, Dept. of Public Health, School of Medicine, Bioethics and Law
Observatory, Barcelona Science Park, University of Barcelona, 08028
Barcelona, Spain.
Email: [email protected]
Renjing Liu
Department of Genetics, Yale Stem Cell Center, Yale School of Medicine,
10 Amistad, New Haven, CT. 06520, USA.
Email: [email protected]
Scott McRae
Cell Guidance Systems, Babraham Research and Control-HPA Campus,
Cambridge, UK.
Email: [email protected]
Claire N. Medine
MRC Centre for Regenerative Medicine, University of Edinburgh,
49 Little France Crescent, Edinburgh, EH16 4SB, U.K.
Email: [email protected]
Philip Noel Newsome
Centre for Liver Research and NIHR Biomedical Research Unit,
University of Birmingham, Birmingham, UK.
Email: [email protected]
In-Hyun Park
Department of Genetics, Yale Stem Cell Center, Yale School of Medicine,
10 Amistad, New Haven, CT. 06520, USA.
Email: [email protected]
Catherine M. Payne
MRC Centre for Regenerative Medicine, University of Edinburgh,
49 Little France Crescent, Edinburgh, EH16 4SB, U.K.
Email: [email protected]
Prakash Ramachandran
MRC Centre for Inflammation Research, University of Edinburgh, 47
Little France Crescent, Edinburgh, EH16 4TJ, U.K.
Email: [email protected]
S. Tamir Rashid
Anne McLAren Laboratories for Regenerative Medicine, Department of
Surgery, University of Cambridge, UK.
Email: [email protected]
xii Regenerative Medicine, Stem Cells and the Liver

James A. Ross
MRC Centre for Regenerative Medicine, University of Edinburgh,
49 Little France Crescent, Edinburgh, EH16 4SB, UK.
Email: [email protected]
Clare Selden
Royal Free Campus, UCL Medical School, London NW3 2PF.
Email: [email protected]
Glyn Stacey
National Institute for Biological Standards-HPA, Blanche Lane, South
Mimms, Herts, EN6 3QG.
Email: [email protected]
Gareth Sullivan
MRC Centre for Regenerative Medicine, University of Edinburgh,
49 Little France Crescent, Edinburgh, EH16 4SB, UK.
Email: [email protected]
Amy Thomas
Royal Free Campus, UCL Medical School, London NW3 2PF.
Email: [email protected]
Jordi L.Tremoleda
Investigator, MRC Centre for Clinical Sciences, Faculty of Medicine,
Imperial College London, London W12 0NN, UK.
Email: [email protected]
Ludovic Vallier
Anne McLAren Laboratories for Regenerative Medicine, Department of
Surgery, University of Cambridge, UK.
Email: [email protected]
Xiao-Ling Zhou
Department of Genetics, Yale Stem Cell Center, Yale School of Medicine,
10 Amistad, New Haven, CT., 06520, USA.
Email: [email protected]
 1
Stem Cell Therapy in the
Context of Chronic Liver
Disease: Promise and Pitfalls
Prakash Ramachandran1,a and John P. Iredale1,b,*

Regenerative medicine occupies a unique position in research and translation,

drawing on the disparate but overlapping fields of developmental biology,
cell biology, genetics, epigenetics, inflammation and of course stem cell
biology. Key attributes of each of these fields are critical when organ or
tissue restitution occurs with repair that restores a normal architecture and
replacement of the component cell types that maintain structure and specific
function. Nowhere is this more important or arguably better exemplified
than in liver disease.
To date the major interest in the deployment of stem cells in the context
of liver disease (and numerically the greatest number of publications) has
been focused on the area of parenchymal restitution. Restoration of the liver
parenchyma requires the replacement of diseased or failing hepatocytes
with new hepatocytes, either exogenously derived from stem or progenitor
cell elements or, theoretically, derived from stimuli applied to the liver
progenitor cell component. Is this approach likely to be successful in the
immediate future as a new approach to restoring normal hepatic function
to diseased livers? The single biggest therapeutic challenge clinically in
the context of chronic liver disease is fibrosis and its end stage cirrhosis

1
MRC Centre for Inflammation Research, University of Edinburgh, 47 Little France Crescent,
Edinburgh, EH16 4TJ, U.K.
a
Email: [email protected]
b
Email: [email protected]
*Corresponding author
2 Regenerative Medicine, Stem Cells and the Liver

with attendant hepatocellular failure. Fibrosis and cirrhosis result from

chronic inflammatory injury within the liver and represent the final
common pathway of virtually all chronic liver diseases from paediatric
metabolic disorders to alcohol excess. Our view is that current evidence
suggests that the approach of “adding back” hepatocytes is unlikely to be
uniformly successful unless the underlying inflammatory liver disease and
in particular the consequent fibrosis, which characterizes all chronic liver
diseases, can be brought under control.
Virtually all chronic liver diseases are associated with inflammation to
a greater or lesser extent. This is manifestly obvious in acute viral hepatitis
or aggressive autoimmune disease. The presence of chronic inflammation
in turn leads to the activation of myofibroblasts, the recruitment of key
inflammatory cell elements and the development of fibrosis. Initially
perisinusoidal, fibrosis eventually extends to link the vascular structures
and distort the liver anatomy. At this stage, if there is regenerative activity
from hepatocytes or progenitor cells these in turn form spheres of poorly
functioning liver parenchyma and the resulting structure is termed
cirrhosis.
Thus, in the context of chronic liver disease, hepatocyte dysfunction may
result from direct damage from the causative stimulus, as a result of intense
inflammatory activity or due to changes in specific function mediated by the
abnormal pericellular milieu and altered cell-matrix interactions. The latter
may be important not only for hepatocellular function but also hepatocyte
and hepatic progenitor cell proliferation (Kallis et al. 2011).
The delivery of new hepatocytes derived from progenitor or stem
cells in this context might not be expected to result in a dramatic return
of organ function. Indeed any new hepatocytes would be susceptible to
the injurious agent directly and the consequences of the altered milieu.
Additionally, phenotypic changes in the sinusoidal endothelium following
chronic injury may also impact on the recruitment and engraftment of
exogenous hepatocytes. Put another way, planting the seed in poor quality
soil is unlikely to deliver a significant harvest.
That is not to say, however, that these problems are insurmountable.
It is perhaps not surprising that the greatest successes in hepatocyte
transplantation (in many ways an analogous experiment differing only
in origin of the transplanted cells) have been demonstrated in the context
of orphan paediatric metabolic disorders, such as Crigler–Najar, Primary
Hyperoxaluria or Urea cycle disorders (Meyburg et al. 2009). In such
conditions the metabolic defects are predominantly expressed in the liver
but the organ damage results elsewhere, whilst the liver parenchyma
remains normal, with little by way of liver inflammation and fibrosis. In
these genetic conditions, endogenous regenerative systems will be subject to
the same metabolic defects. Therefore, the transplanted, genetically normal,
 Stem Cell Therapy in the Context of Chronic Liver Disease: Promise and Pitfalls 3

hepatocytes will have a positive selection pressure and can induce a good
functional recovery, acting as either a bridge to liver transplantation or
indeed a medium term therapeutic solution (Meyburg et al. 2009). Similar
findings are seen dramatically in animal models in which transplanted stem
cell derived hepatocytes may have a growth and survival advantage over
the metabolically compromised host cells and in which the engrafted cells
proliferate and deliver a major proportion of the metabolic function of the
liver’s hepatocytes (Shafritz and Oertel 2011, Rhim et al. 1994).
Moreover, the delivery of cells to a heterotypic site, for example the
peritoneum, might overcome some of the problems of the specific liver
milieu (Baldini et al. 2008). Furthermore, it is not inconceivable that an
uninjured peritoneal surface could provide anchorage and cell-cell and
cell matrix signals that may preserve and foster hepatocyte function. An
alternative heterotypic site might, for example be the spleen (Payne et al.
2011). Additionally, it may be possible to engineer cells that are resistant
to the injurious agent thus eliminating direct damage to engrafted cells.
However, assuming that those cells also shared other attributes of the
normal cell phenotype for hepatocytes, the adverse inflammatory and
fibrotic milieu might still result in changes to function.
An alternative approach utilizing stem cells which may come to the
forefront in the immediate future is the development of a bioartifical, or
liver support device. This is likely to be particularly valuable in the clinical
context of acute liver failure, where massive hepatocellular necrosis can
result in previously well patients developing rapidly progressive liver and
multi-organ failure. Often liver transplantation is the only curative option
in this setting, and due to the lack of available organs other treatments are
badly needed. Clearly the liver milieu in this setting would not be conducive
to cellular transplantation. Previous attempts to use liver assisting devices,
akin to renal replacement therapy, as a bridge to transplantation or to
hepatic recovery have not demonstrated a survival benefit (Liu et al. 2004).
Principally, the complexity of the biological functions performed by the liver
mean that standard liver dialysis systems cannot compensate for this loss.
Cellular systems have also been tried to overcome this (Carpentier et al.
2009). However, the sheer volume of metabolically active human hepatocytes
required for an effective liver device has, to date, significantly impacted on
developments in this field. One enormous advantage provided by stem cell
derived hepatocytes is that they can be manufactured to a uniform high
standard, theoretically in infinite numbers, giving exciting opportunities
for their use in “off the shelf” bioartificial liver support devices.
The inflamed and fibrotic liver is, also an excellent model for matrix
remodelling, parenchymal restitution (from hepatocytes and progenitor
cell elements) and a return of normal or more normal architecture, if
the inflammatory and fibrotic processes can be terminated. Previously
4 Regenerative Medicine, Stem Cells and the Liver

considered irreversible, there is now a wealth of evidence from both

animal and human models that with cessation of the injurious stimulus,
liver fibrosis will undergo remodelling with changes in the matrix from a
fibrotic to a more normal sinusoidal pattern (degradations of collagens I
and III and a return to a matrix rich in laminins and collagen IV) (Iredale
2007). This will result not only in return to a more normal architecture, but
is also associated with hepatocyte proliferation and a hepatic progenitor
cell response (Kallis et al. 2011).
During the last 10 years the events underpinning this process of fibrosis
remodelling and resolution have become clear. Whereas in progressive
fibrogenesis myofibroblasts become activated and express high levels
of fibrillar collagens I and III, and concurrently high levels of the tissue
inhibitors of metalloproteinases TIMPs 1 and 2, following withdrawal of the
injurious stimulus this pattern reverses and the expression of TIMPs 1 and 2
and the fibrillar collagens drops rapidly to become undetectable. Moreover,
the myofibroblasts which populate the liver scar undergo apoptosis, thereby
withdrawing both the source of the new fibrillar matrix and the source
of the proteins which protect the matrix from degradation (Iredale et al.
1998). The TIMPs are very potent metalloproteinase inhibitors, binding in
a non-covalent manner and acting against all members of the MMP family.
Thus, during progressive fibrosis, fibrillar matrix accumulates not only
as a result of enhanced synthesis but also as a result of a failure of matrix
degradation. Following withdrawal of the injurious stimulus and the drop
in TIMP levels MMP mediated matrix degrading activity, previously held
in check, can occur and the remodelling process commences (Iredale 2007,
Ramachandran and Iredale 2009).
Central to the process of matrix remodelling is the macrophage. It has
been known for many years that macrophages are necessary to drive the
inflammation and fibrogenic process during progressive fibrosis (Karlmark
et al. 2009). However, an interesting and apparently counterintuitive
observation—that the maximum number of macrophages within a liver
during experimental fibrosis resolution occurs as matrix degradation
commences, suggests a pro resolution function also. In combination with
the observation that the major TIMP expressing cell, the myofibroblast,
undergoes apoptosis during resolution of fibrosis has led to the tantalizing
suggestion that macrophages may be critical for fibrosis resolution. We
and others have now demonstrated unequivocally, using models in which
macrophages can be selectively depleted, that macrophages are necessary
for matrix degradation in the spontaneous resolution of liver fibrosis
(Duffield et al. 2005). Moreover MMPs derived from those macrophages
including a key collagenase (MMP-13) and elastase (MMP-12) in addition
to promiscuous enzymes that will degrade the fibrillar collagens following
initial collagenase mediated cleavage (MMPs 9 and 7) are almost exclusively
 Stem Cell Therapy in the Context of Chronic Liver Disease: Promise and Pitfalls 5

derived from macrophages in experimental rodent models (Fallowfield et al.

2007). Intriguingly, the macrophages that we have observed infiltrating the
liver and fulfilling the matrix degrading functions also express the mitogens
TWEAK and IGF1 both of which have now been identified as important
for hepatic progenitor cell proliferation and hepatocyte proliferation
respectively.
These observations highlight the tantalizing possibility that macrophage
therapy may be a valuable approach to treating the underlying inflammatory
and fibrotic condition within the liver, in addition to promoting regeneration.
Moreover, in ground breaking work, the Sakaida group demonstrated
clear evidence that infusion of bone marrow elements was associated
with enhanced matrix degradation and resolution of fibrosis and that
cells secreting the MMP repertoire of macrophages were likely mediating
this effect (Sakaida et al. 2004). Our group and others have subsequently
demonstrated that myeloid elements, particularly macrophages, are the
cells that are essential to fulfilling this function in experimental engraftment
and in addition can enhance the hepatic progenitor cell response in this
context (Thomas et al. 2011). Furthermore, in work using renal models
of inflammation and fibrosis it has been demonstrated that macrophages
altered to over-express IL-10 or Il-4 will function as a potent anti-
inflammatory agent (Wilson et al. 2002, Kluth et al. 2001). Indeed, because
the macrophages will be home to an area of inflammation they can in some
respect be considered a targetted therapy in these models. Macrophages
are of course in turn derived from myeloid and bone marrow stem cell
and progenitor elements. Thus at an early stage it is likely that we will see
stem cell related trials, in this case of macrophage therapy, being deployed
to treat liver fibrosis.
In conclusion, there remains enormous promise for the use of
regenerative medicine technology in the clinical treatment of human liver
disease. Hepatocyte transplantation and the development of bioartificial
liver devices are likely to find their niche in the management of metabolic
liver disease and acute liver failure respectively. In the context of chronic
disease, the liver remains an extraordinary paradigm for the study of
inflammation, fibrosis resolution and regeneration. Stem cell derived
therapies are likely ultimately to impact on parenchymal reconstitution
but will be more effective if they can be concurrently delivered with an
antifibrotic therapy to improve the milieu into which they will engraft.
Macrophage therapy for fibrosis, in turn is the result of expansion of
progenitor and stem cell elements derived from the bone marrow, shows
enormous promise as a targetted cell therapy of fibrosis in vivo.
6 Regenerative Medicine, Stem Cells and the Liver

References
Baldini E, R Cursio, G De Sousa et al. 2008. Peritoneal implantation of cryopreserved
encapsulated porcine hepatocytes in rats without immunosuppression: viability and
function. Transplant Proc. 40(6): 2049–52.
Carpentier B, A Gautier and C Legallais. 2009. Artificial and bioartificial liver devices: present
and future. Gut. 58(12): 1690–702.
Duffield JS, SJ Forbes, CM Constandinou et al. 2005. Selective depletion of macrophages reveals
distinct, opposing roles during liver injury and repair. J Clin Invest. 115(1): 56–65.
Fallowfield JA, M Mizuno, TJ Kendall et al. 2007. Scar-associated macrophages are a major
source of hepatic matrix metalloproteinase-13 and facilitate the resolution of murine
hepatic fibrosis. J Immunol. 178(8): 5288–95.
Iredale JP. 2007. Models of liver fibrosis: exploring the dynamic nature of inflammation and
repair in a solid organ. J Clin Invest. 117(3): 539–48.
Iredale JP, RC Benyon, J Pickering et al. 1998. Mechanisms of spontaneous resolution of
rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of
metalloproteinase inhibitors. J Clin Invest. 102(3): 538–49.
Kallis YN, AJ Robson, JA Fallowfield et al. 2011. Remodelling of extracellular matrix is a
requirement for the hepatic progenitor cell response. Gut. 60(4): 525–33.
Karlmark KR, R Weiskirchen, HW Zimmermann et al. 2009. Hepatic recruitment of the
inflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis.
Hepatology. 50(1): 261–74.
Kluth DC, CV Ainslie, WP Pearce et al. 2001. Macrophages transfected with adenovirus
to express IL-4 reduce inflammation in experimental glomerulonephritis. J Immunol.
166(7): 4728–36.
Liu JP, LL Gluud, B Als-Nielsen et al. 2004. Artificial and bioartificial support systems for liver
failure. Cochrane Database Syst Rev. (1): CD003628.
Meyburg J, J Schmidt and GF Hoffmann. 2009. Liver cell transplantation in children. Clin
Transplant. Suppl. 21: 75–82.
Payne CM, K Samuel, A Pryde et al. 2011. Persistence of functional hepatocyte-like cells in
immune-compromised mice. Liver Int. 31(2): 254–62.
Ramachandran P and JP Iredale. 2009 Reversibility of liver fibrosis. Ann Hepatol. 8(4):
283–91.
Rhim JA, EP Sandgren, JL Degen et al. 1994. Replacement of diseased mouse liver by hepatic
cell transplantation. Science. 263(5150): 1149–52.
Sakaida I, S Terai, N Yamamoto et al. 2004. Transplantation of bone marrow cells reduces
CCl4-induced liver fibrosis in mice. Hepatology. 40(6): 1304–11.
Shafritz DA and M Oertel. 2011. Model systems and experimental conditions that lead to
effective repopulation of the liver by transplanted cells. Int J Biochem Cell Biol. 43(2):
198–213.
Thomas JA, C Pope, D Wojtacha et al. 2011. Macrophage therapy for murine liver fibrosis
recruits host effector cells improving fibrosis, regeneration, and function. Hepatology.
53(6): 2003–15.
Wilson HM, KN Stewart, PA Brown et al. 2002. Bone-marrow-derived macrophages genetically
modified to produce IL-10 reduce injury in experimental glomerulonephritis. Mol Ther.
6(6): 710–7.
 2
Sources of Human Liver Cells
and the Challenge of Working
with Primary Tissue
Janet W.C. Kung1,a,* and James A. Ross1

Introduction
Liver cirrhosis represents the final common histologic pathway for a wide
variety of chronic liver diseases. With the rising incidence of alcoholic liver
disease, hepatitis C and, more recently, non-alcoholic liver disease associated
with obesity, cirrhosis places an increasing burden on healthcare worldwide.
Currently the only curative treatment is liver transplantation. While liver
transplant has a relatively good 5-yr survival, donor organ shortage costs
many lives every year; and despite improvements in donor schemes, the
use of broader donor criteria, and advances in surgical techniques, the
increase in the number of transplants has been modest (van der Meulen et
al. 2007). New figures from the NHS Blood and Transplant survey reveal
that in the 12-mon period to June 2011, the number of people waiting for a
new liver in the UK rose by 25% when compared with the 12 mon to June
2010 (NHS Blood and Transplant [Internet] 2011). Alternative treatments
are thus urgently needed.
The generation of functional hepatocytes is of great therapeutic
interest with applications in drug screening and disease modelling,
human bioartificial liver construction, and, potentially, in hepatocyte
transplantation. The ability to use human cell types in the pharmaceutical
1
MRC Centre for Regenerative Medicine, University of Edinburgh, 49 Little France Crescent,
Edinburgh, EH16 4SB.
a
Email: [email protected]
*Corresponding author
8 Regenerative Medicine, Stem Cells and the Liver

industry not only allows expedition of novel human drug development,

but also takes into account variability in drug metabolism because of
cytochrome P450 polymorphisms. Phase I trials employing a bio-artificial
liver device have shown some promise as patients exhibited improvement
in neurological state and haemodynamics, but this approach is currently
limited by the use of xenogeneic materials. Such bioartificial devices permit
some restoration of liver metabolic function and are, in the future, likely
to act as a bridge to liver transplantation in patients with advanced or
end-stage liver failure (Gerlach et al. 2008, McKenzie et al. 2008). Attempts
at hepatocyte transplantation to treat some liver-based inborn errors of
metabolism and liver failure (Fisher and Strom 2006, Puppi and Dhawan
2009, Smets et al. 2008) as an alternative to orthotopic liver transplantation
have shown transient clinical improvement and/or partial correction of the
underlying metabolic defect. Whilst significant progress has been made to
develop the technique, there are many obstacles to successful hepatocyte
transplantation and the main constraint remains the difficulty in sourcing
and maintaining viable hepatocytes. This chapter aims to outline the sources
of human hepatocytes and discuss the challenges faced by researchers
working with primary human tissue.

Sources of human hepatocytes

Mature (adult) hepatocytes
The human liver has a remarkable regenerative capacity. Following acute
liver injury such as partial hepatectomy, the tissue mass is restored by mitotic
division of mature hepatocytes (Fausto et al. 2006). However, hepatocytes in
culture rapidly lose their in vivo phenotypic characteristics and functional
abilities with a decline in cytochrome P450 levels occurring within hours of
isolation (Elaut et al. 2006). The compound effect of endotoxin-containing
collagenase digestion, disruption of normal tissue architecture (cell-cell
and cell-matrix interactions), and ischaemic-reperfusion injury during
the isolation process triggers a cascade of signalling pathways leading
to hepatocyte apoptosis and anoikis (Elaut et al. 2006). Despite decades
of research, it remains difficult to maintain liver-specific functions of
primary hepatocytes in culture for more than a week without an adequate
supportive microenvironment either in the form of a three-dimensional
extracellular matrix or the presence of xenogeneic feeder layers.
Nonetheless, de-differentiation of primary hepatocytes appears to be an
inevitable eventuality. Attempts at immortalization of primary hepatocytes
are currently only partially successful as proliferation and hepatocellular
function can appear mutually exclusive (van de Kerkhove et al. 2005). In
addition, hepatocytes do not spontaneously redifferentiate after in vitro