1 - FORBESJHEPATOL2015 Cell Therapy en Enf Heaticas

Review
Cell therapy for liver disease: From liver transplantation to

cell factory
El acceso y uso a este artículo se limita exclusivamente al alumno del "Máster de Hepatología" y para los fines no lucrativos docentes y científicos del propio curso, que incluyen su análisis o comentario. Prohibida la reproducción, en cualquier formato, para otros fines o personas.
Stuart J. Forbes1,⇑, Sanjeev Gupta2, Anil Dhawan3

1
MRC Centre for Regenerative Medicine, Scottish Centre for Regenerative Medicine, 5 Little France Drive, Edinburgh EH16 4UU, United Kingdom;
2
Departments of Medicine and Pathology, Albert Einstein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Avenue, Ullmann
Building, Room 625, Bronx, NY 10461, United States; 3Paediatric Liver GI and Nutrition Center and NIHR/Wellcome Cell Therapy Unit, King’s
College Hospital at King’s College, London SE59RS, United Kingdom
Summary Introduction
Work over several decades has laid solid foundations for the Orthotopic liver transplantation (OLT) is the standard of care for
advancement of liver cell therapy. To date liver cell therapy in peo- people with end-stage liver disease and for certain liver-based
ple has taken the form of hepatocyte transplantation for metabolic metabolic defects [1]. However, successful replacement of defi-
disorders with a hepatic basis, and for acute or chronic liver failure. cient liver functions by transplantation of healthy hepatocytes,
Although clinical trials using various types of autologous cells have e.g., in animal models and people with Crigler-Najjar syndrome
been implemented to promote liver regeneration or reduce liver due to UGT1 enzyme deficiency, familial hypercholesterolemia
fibrosis, clear evidence of therapeutic benefits have so far been due to low-density lipoprotein receptor (LDLR) deficiency, or
lacking. Cell types that have shown efficacy in preclinical models acute and chronic liver failure indicated that OLT could possibly
include hepatocytes, liver sinusoidal endothelial cells, mesenchy- be avoided [2–6]. This general concept has been emphasized by
mal stem cells, endothelial progenitor cells, and macrophages. similar successes with auxiliary partial orthotopic liver trans-
However, positive results in animal models have not always transplantation (APOLT) for enzymatic deficiency states or acute liver
lated through to successful clinical therapies and more realistic failure [7]. In the latter case, discontinuation of immunosuppres-
preclinical models need to be developed. Studies defining the opti- sion when the native liver regenerates after APOLT may lead to
mal repopulation by transplanted cells, including routes of cell spontaneous rejection and atrophy of the allogeneic liver graft
transplantation, superior engraftment and proliferation of trans- [8,9] The clinical experience with APOLT gives credence to the
planted cells, as well as optimal immunosuppression regimens hypothesis that the relevant functional unit of the liver – ‘‘the
are required. Tissue engineering approaches to transplant cells in hepatocyte’’ could be used to correct discrete enzyme defects
extrahepatic locations have also been proposed. The derivation and support metabolic functions for the failing liver after injury
of hepatocytes from pluripotent or reprogramed cells raises hope whilst it regenerates. Similarly, successful correction of haemo-
that donor organ and cell shortages could be overcome in the philia by OLT, indicated that consideration of cell therapy will
future. Critical hurdles to be overcome include the production of be appropriate for other classes of diseases. In principle, cell
hepatocytes from pluripotent cells with equal functional capacity transplantation is far simpler than either OLT or APOLT, because
to primary hepatocytes and long-term phenotypic stability in vivo. 1) cells from a donor liver may be transplanted into multiple
Ó 2015 European Association for the Study of the Liver. Published recipients; 2) cell transplantation is simpler using cell
by Elsevier B.V. All rights reserved. administration via intravascular catheters rather than complex
surgery; 3) if cryopreserved cells are used, therapies could be
undertaken in a prospective non-emergency setting; 4) cells
Keywords: Cell therapy; Stem cells; Liver regeneration; Metabolic liver disease;
may even be transplanted repeatedly, the procedure can be con-
Liver cirrhosis. sidered ‘‘reversible’’ since the native liver is not removed; and 5)
Received 29 December 2014; received in revised form 20 February 2015; accepted 27 the costs of transplanting cells should be considerably less than
February 2015 that of organ transplantation.
⇑ Corresponding author. Tel.: +44 (0) 131 651 9500; fax+44 (0) 131 651 9501.
Subsequent to the early demonstrations of whether trans-
E-mail address: [email protected] (S.J. Forbes).
Abbreviations: ATM, ataxia telangiectasia mutant; APOLT, auxiliary partial planted cells may engraft and function in the liver and in a vari-
orthotopic liver transplantation; ES Cells, embryonic stem cells; FAH/, ety of extrahepatic sites [10], a large body of work in many small
fumarylacetoacetate-hydrolase-deficient; GMP, Good Manufacturing Practice; and large animal models supported studies of the potential of
HPCs, Hepatic progenitor cells; iHeps, induced hepatocytes; iMPCs, induced hepatocyte transplantation [11,12]. More recently, the therapeu-
multipotent progenitor cells; iPSCs, induced pluripotent stem cells; LDLR, low-
density lipoprotein receptor; LSEC, liver sinusoidal endothelial cells; MSCs,
tic value of other liver cell types was elucidated. For instance,
mesenchymal stem cells; NAR, Nagase analbuminemic rats; OLT, Orthotopic liver transplantation of liver sinusoidal endothelial cells (LSECs) cured
transplantation; VEGF, vascular endothelial growth factor; WHHL, Watanabe haemophilia A in mice after LSECs were found to be the major
heritable hyperlipidemic cholesterolemic; HLC, hepatocyte-like cell.
Journal of Hepatology 2015 vol. 62 j S157–S169

Review
Critical components of liver cell therapy and current barriers
Step 1. Step 2. Step 3.

Isolation, characterization Transplanted cell Transplanted cell
and storage of donor cells engraftment proliferation
Hepatocytes Recipient pre- or

post-conditioning
Other cells LSEC
(e.g., stem cell-derived)
± gene transfer
Drug or other modifications
• Inferior donor quality Most transplanted cells • Transplanted cells do not proliferate in
Major the liver
• Low viability or number are cleared rapidly from
barriers
• Freeze-thaw damage the liver • Allografts are rejected
Fig. 1. Depiction of critical components in liver cell therapy and barriers in various steps. The first step in cell therapy requires isolation, characterization and storage of
suitable donor cells. These steps are restricted by donor organ shortages or their inferior quality, procedural limitations in isolating cells of high viability and large numbers,
as well as difficulties in cryopreservation of cells. The second critical step concerns engraftment of transplanted cells in the liver (or extrahepatic sites), which requires
overcoming of early transplanted cell clearances. Cells may be modified by gene transfer vectors, drugs or other ways for improving cell viability, engraftment and
proliferation. In the third and final step, transplanted cells must survive over the long-term and also proliferate to the necessary extents for imparting therapeutic benefits,
which may require conditioning of recipients either before or after cell transplantation, as well as development of suitable regimens for controlling allograft rejection.
source of FVIII [13]. Applications of LSECs may extend to liver donor cells by altering liver growth or cell cycle controls, such that
repair since these cells have been shown to be critical for liver transplanted cells receive survival and/or proliferation advantages
regeneration in mice [14]. Pathophysiological processes that over native cells [18,19]. In this way, the concept of ‘‘liver trans-
could be altered during chronic liver injury and fibrosis by the plantation to cell factory’’ may be gained if one considers that suc-
cell transplantation approach have also gained interest [15]. In cessive generations of daughter cells may emanate in the recipient
some people with acute liver failure, cell transplantation has liver from transplanted hepatocytes, as was elegantly established
been successful for bridging to OLT, whereas in other instances, using serial hepatocyte transplants in the fumarylacetoacetate-
people with liver failure or enzymatic deficiency states had to hydrolase-deficient (FAH/) mouse model [20]. If these
be treated with OLT because cell therapy proved unsuccessful concepts regarding liver repopulation are reduced to drug-based
[6]. In part, this difficulty in achieving superior outcomes of cell approaches then barriers in transplanted cell engraftment and
therapy has been related to immunosuppression following allo- proliferation will be overcome for more effective clinical trials.
geneic cell transplants, since optimal regimens for inducing toler- For many reasons, the clinical application of liver cell therapy
ance to transplanted liver cells are to be established. has proceeded at a gradual pace in people compared to the suc-
In the setting of metabolic liver disease and hepatic injury, e.g., cesses in preclinical animal studies. Some of the obstacles con-
hereditary tyrosinemia type-1 or Wilson’s disease, animal studies cern limited availability of donor livers, difficulties in isolating
established that disease correction can be achieved because even good-quality cells from often suboptimal donor livers, mechanis-
modest numbers of healthy transplanted hepatocytes can pro- tic restrictions in cryopreserving human liver cells without losing
liferate and repopulate the liver [16,17]. This process of liver viability, low levels of engraftment and proliferation in trans-
repopulation has been shown in rodents to be accelerated by planted liver cells, as well as the general lack of therapeutic bene-
recipient organ preconditioning [18]. By contrast, in the setting fits over the long-term due to allograft rejection (Fig. 1). Another
of metabolic diseases where the native liver is unaffected and important point is that the animal models used often translate
remains totally healthy, as in Crigler-Najjar syndrome or familial poorly to the clinic. Liver damage may have accumulated over
hypercholesterolemia, transplanted hepatocytes engraft but do decades in patients with severe distortion of liver architecture
not proliferate in the liver because this is not physiologically and impairment of function. The models of liver injury developed
required. Therefore, in achieving therapeutic levels of repopulation in mice and rats typically occur over days or weeks and are often
further manipulation is required by either: a) preconditioning of milder than the human diseases they seek to model. An impor-
the recipient’s liver using techniques such as DNA-adduct forming tant message is that more realistic models of these liver injuries
chemicals, radiation, oxidative stress or by b) modification of are required.
S158 Journal of Hepatology 2015 vol. 62 j S157–S169

JOURNAL OF HEPATOLOGY
Table 1. Potential clinical indications for liver cell therapy.⁄
Key Points
A. Congenital disorders
• Liver cell therapy has extensive value for paediatric and • Alpha-1 antitrypsin deficiency*
adult populations with many enzyme deficiency states, • Crigler-Najjar syndrome type 1*
metabolic diseases, coagulation disorders, as well as
• Familial hypercholesterolemia*
liver failure
• Congenital coagulation factor VII deficiency*
• Much of the clinical literature is based upon case • Hemophilia A
reports although some controlled studies have also • Glycogen storage disease type I*
been conducted • Infantile Refsum disease
• Maple syrup urine disease
• Widespread applications of liver cell therapy have been
constrained by donor organ shortages and limitations in • Neonatal hemochromatosis
transplanted cell engraftment and proliferation • Progressive familial intrahepatic cholestasis type 2 (PFIC2)
• Urea cycle defects - ornithine transcarbamylase
• Utilization of stem cells offers hope for producing (OTC) deficiency; arginosuccinate lyase deficiency;
suitable liver cells of clinical utility although barriers carbamoylphosphate synthase type 1 deficiency;
include the production of cells with the necessary levels citrullinemia*
of differentiated function and karyotypic stability • Wilson’s disease
B. Acquired disorders
• Approaches to regenerate liver or reducing liver fibrosis
• Acute liver failure (multiple etiologies)*
or promoting endogenous repair and regeneration are
being developed and tested • Fatty liver of pregnancy*
• Acute-on-chronic liver failure (multiple etiologies)*
⁄
Indicates conditions treated by cell transplantation in people.
Major clinical indications and roles of cell therapy
The premise for early studies in people was based on transplant- Table 2. Selected examples of animal models used in cell transplantation
ing healthy cells to replace deficient functions in acquired or studies.
inborn errors of metabolism besides supporting the failing liver
(Table 1). Liver-based metabolic defects are usually secondary Mechanisms in cell engraftment and proliferation
to a missing enzyme with consequences secondary either to the Dpp4-deficient F344 rats and Dpp4 knockout mice
lack of a normally functioning protein or the upstream accumula- Transgenic donor mice (HBV, human alpha-1 antitrypsin, beta-
tion of toxic substances due to impaired metabolism of a protein. galactosidase, alb-uPA, etc.)
These diseases could be further classified into those with no Fumarylacetoacetate hydroxylase (FAH) knockout mice
effects on the liver and those leading to liver injury and fibro- Defects in hepatic detoxification
sis/cirrhosis. The rationale for hepatocyte transplantation in peo- • FAH knockout mice (hereditary tyrosinemia, type-1)
ple with these conditions came from animal studies showing that • Gunn rat and MRP-2 knockout mice (Crigler-Najjar
healthy hepatocytes transplanted into hepatic or extrahepatic syndrome, type-1)
sites possessed properties as follows (Table 2): 1) engraftment • Histidinemia mice (histidinemia)
and proliferation in the liver or other sites; 2) enzymatic activity • MSUD knockout mouse (Maple syrup urine disease)
for detoxification, e.g., bilirubin glucuronidation followed by the • Spf-ash mice (OTC deficiency)
biliary excretion of conjugated bilirubin in Gunn rats modelling
Secretory protein deficiency
Crigler-Najjar syndrome; 3) release of secreted proteins, e.g.,
• Nagase analbuminaemic rat (hypoalbuminaemia)
albumin synthesis and release in Nagase analbuminemic rats
(NAR); 4) receptor-mediated ligand uptake, e.g., LDLR-dependent • Hemophilia A mice
clearance of cholesterol in Watanabe heritable hyperlipidemic Diseases of receptor function
cholesterolemic rabbits (WHHL); 5) ammonia-fixation in rats • Watanabe heritable hyperlipidemic rabbit (familial
with portacaval shunts; and 6) improvements in mortality in rats hypercholesterolaemia)
and rabbits with induced acute liver failure [21–24]. These • Apolipoprotein E knockout mouse (hypercholesterolemia)
demonstrations were better understood after the important dis- Transport defects
covery in rodents that hepatocytes transplanted into liver sinu- • TR- rat and Mdr2 knockout mouse (PFIC1)
soids enter liver parenchyma and integrate with adjacent native • BSEP knockout mouse (PFIC2)
hepatocytes along with the reconstitution of plasma membrane • Long-Evans Cinnamon rat and Atp7b knockout mouse
structures permitting restoration of cell polarity and transport (Wilson’s disease)
or exchange functions (Fig. 2) [25–27]. Acquired disorders
Moreover, studies in rat showed that transplanted cells • Induced acute liver failure (hepatectomy, chemicals, drugs,
retained position-specific and physiologically-regulated patterns viruses, physical methods)
of gene expression, and exhibited lifetime survival with normal • Chronic liver disease (CCl4, thiocetamide, acetyl-
liver growth controls in the absence of allograft rejection aminofluorene, etc.)
Journal of Hepatology 2015 vol. 62 j S157–S169 S159

Review
The experience in single cases or uncontrolled trials of hep-
A B atocyte transplantation via spleen, portal vein or intraperitoneal
administration in several adults and children with acute liver fail-
ure has been mixed but with some notable successes, as defined
by spontaneous recovery or bridging to OLT [6]. Of course, injec-
tion of cells into the portal venous system is made difficult in

these patients by coagulopathy and associated portal hyperten-
sion, which presents the risk of systemic embolization or portal
vein thrombosis. Moreover, liver inflammation and injury could
be aggravated by ischemic injury produced by the arrival of
transplanted cells in liver sinusoids and activation of ischemic
events. Immunosuppression after the transplantation of allo-
geneic cells also adds the risk of sepsis in this vulnerable
population.
In preclinical rat studies, transplantation of healthy hep-
Gap junctions Bile canaliculi atocytes in the liver after acute toxic injury established that the
Fig. 2. Integration of dipeptidyl peptidase IV (Dpp4)-positive adult hep-
transplanted cells required several days before they could pro-
atocytes in liver parenchyma of healthy Dpp4-negative rats. (A) Combined liferate and liver repopulation was very limited [34]. Therefore,
staining for Dpp4 (red color, arrows) and hepatic connexin 32 (brown color, it was of interest when investigators used the peritoneal cavity
arrowheads) showing sharing of gap junctions by adjacent transplanted and for transplanting freshly isolated, allogeneic fetal human hep-
native hepatocytes. (B) Reconstitution of bile canalicular networks in trans-
atocytes in people with acute liver failure [35,36]. Of eight persons
planted hepatocytes (Dpp4 staining, red color, arrows) and native hepatocytes
(ATPase staining, brown color, arrowheads). Modified from Gupta et al. Proc Natl with acute liver failure and hepatocyte transplantation in this
Acad Sci 1995. manner, four people recovered. In these studies hepatocytes were
transplanted without matrix support or anchorage, which would
have led to the rapid loss of transplanted cells, raising a question
[28,29]. These findings prompted numerous studies of liver cell about the mechanisms underlying the rescue. More recently, ani-
biology and stimulated clinical hepatocyte transplantation (listed mal studies established ATM (ataxia telangiectasia mutant) gene
in Table 2). Although hepatocyte transplantation in animal stud- network-related molecular lesions in hepatocytes after drug-
ies has been shown to correct enzymatic, receptor, proteolytic or induced acute liver failure, as another explanation of the failure
transport defects in the setting of no liver injury and early in of liver regeneration [37]. These studies established that rescue
disease processes, in conditions associated with progressive liver did not require reseeding of the liver with transplanted cells since
injury, e.g., alpha-1 antitrypsin deficiency, Wilson’s disease, bil- hepatocytes transplanted into the peritoneal cavity provided
iary phospholipid excretion (Mdr2/3 deficiency) or progressive metabolic support along with paracrine factors to advance liver
familial intrahepatic cholestasis (BSEP deficiency) [18,30], realiz- regeneration. Therefore, transplantation of hepatocytes with
ing such gains in people with these conditions awaits further matrix support offers a viable alternative approach for rescue in
advances as discussed below. acute liver failure as shown in mice [38]. This should be simplified
In an early human trial, autologous hepatocytes isolated from by the possibility of cryopreserving immobilized hepatocytes [39].
resected liver were returned via intrasplenic injection in several In ongoing studies, human hepatocytes encapsulated in alginate
cirrhotic individuals but without clear clinical benefits [31]. The beads (to avoid exchange of immunoglobulins and immunocytes
unique vascular and extracellular matrix architecture of the driving alloresponses but not of desirable proteins) were
spleen permits survival and proliferation of transplanted hep- intraperitoneal administered to several children with acute liver
atocytes although studies in rat show that the majority (90%) failure. In vitro experiments showed that albumin and factor VII
of transplanted cells enter instantaneously into the portal vein were produced for at least 2 weeks or longer when microbeads
and then liver sinusoids [32]. Similarly, after acute injury, the were cultured in ascites fluid obtained from children in the post
liver can be seeded by transplanting cells into the portal vein. liver-transplant period [40]. Of seven children with acute liver fail-
On the other hand, attachment of hepatocytes to extracellular ure, three avoided OLT after intraperitoneal transplantation of
matrix scaffolds can help in their engraftment in extrahepatic alginate-encapsulated hepatocytes (Dhawan A, personal commu-
sites, e.g., subcutaneous fat pads or peritoneal cavity as demon- nication unpublished observations). These results offer mecha-
strated in the rat [24]. Therefore, it was possible to examine nisms for further cell therapy approaches for acute liver failure,
major principles for supporting the failing liver, i.e., to under- including studies with additional cells of therapeutic interest.
stand the role of reseeding the liver with healthy cells vs. For cell therapy in the setting of advanced chronic liver dis-
provision of metabolic support and paracrine factors from trans- ease or cirrhosis due to hepatitis, alcohol or unspecified injury,
planted cells in liver regeneration. Whilst cell transplantation one must take into account the depletion of parenchymal cells,
improved outcomes in animals with induced liver failure, it architectural distortions, vascular reorganization, inflammation
was also found in rat that besides intact cells, fragmented cells, and excessive extracellular matrix deposition, and myofibroblast
culture medium supernatant from cells, and transplantation of activation and proliferation. This distorted liver microenviron-
even allogeneic or xenogeneic hepatocytes that were rapidly ment is naturally challenging for the engraftment and prolifera-
cleared, were also capable of rescue in acute liver failure [33]. tion of transplanted cells. In preclinical studies in rats using
This raised the possibility that paracrine factors could be carbon tetrachloride-induced cirrhosis, transplanted hepatocytes
important for cell therapy in liver failure, which recently gained engrafted but did not proliferate significantly in fibrotic liver after
renewed attention. withdrawal of the injurious agent and no benefits were noted in

outcomes related to hepatic function or liver fibrosis [41]. By con-
trast, transplantation of hepatocytes into the spleen of rats with
end-stage liver failure was found to prolong survival [42]. There
have been a number of clinical studies seeking to transplant adult Intraportal infusion
hepatocytes into patients with liver cirrhosis [15]. These studies (percutaneous)
have attempted various ways to transplant hepatocytes, includ- Hickman line

ing directly into the liver or via spleen as well as in the peritoneal in inferior
mesenteric vein
cavity. Transplantation of cells into the liver in this situation is
obviously difficult because of coagulopathy and portosystemic
collaterals with risks of serious cardiopulmonary complications.
However, despite the possibility that selected parameters may Catheter through
umbilical vein
have improved in some cases, in general these studies have not (in newborn)
met with great success. In the absence of disease processes that
may be modified by transplanted hepatocytes, e.g., mobilization
of copper in Wilson’s disease, removal of toxins in progressive
familial intrahepatic cholestasis, or replenishment of disease-re-
sistant cells in chronic hepatitis, underlying issues of portal
hypertension, excess extracellular matrix deposition and vascular
distortions may not improve. An alternative approach recently Main criteria
suggested, following positive results in mice, the seeding of hep- • Fresh or cryopreserved hepatocytes
atic cells into the lymphatics [43]. Whether this interesting • Cell viability should be >60%
• ABO blood group compatible
approach is translatable in the setting of human cirrhosis remains
• Up to 109 cells per infusion
to be seen. • Portal pressure monitoring
An alternative cell therapy approach to liver cirrhosis is the • Repeated up to 5-10% liver mass
transplantation of cells that may stimulate endogenous regenera- • Immunosuppression-tacrolimus and steroids
tion or decrease fibrosis (see Table 4 for different types of poten-
tial cell therapy depending upon disease scenario). For instance, Fig. 3. Routes for delivering cells into the portal venous system and major
Thomas et al. studied the intraportal injection of bone marrow- criteria for donor cell usage in the clinical setting. The radiographic image in
derived macrophages for liver fibrosis and found significant the box indicates hepatocytes being infused via the umbilical vein into the liver of
a new-born with urea cycle deficiency disorder. From Dhawan, Transplantation.
reduction in liver scarring and improvements in regeneration Used with permission.
and metabolic function in mice [44]. Macrophages directly acti-
vate the liver’s ductular response via Tweak/FN14 signalling
[45] and stimulate such cells towards an hepatocyte phenotype The issues of cell delivery
via Wnt signalling [46]. Injected macrophages have strong para-
crine and chemotactic effects which amplify their benefit but The relatively superficial location of the liver and access through
only survive for a short time in the liver, which may limit efficacy a variety of modalities, including percutaneous, intravascular
without repeated administration. Nakamura et al. used endothe- through either portal vein or hepatic artery, as well as vessels
lial progenitor cells (EPCs) in rat models of liver fibrosis and upstream of the portal vein offer multiple ways to deliver cells
found them to be anti-fibrotic and also capable of stimulating (Fig. 3). However, studies in rat have established that for trans-
liver regeneration [47]. Similarly, DeLeve and colleagues estab- planted cells to integrate in the liver parenchyma, the most effec-
lished that bone marrow-derived liver sinusoidal endothelial pro- tive approach is to deliver cells into hepatic sinusoids [52].
genitor cells could be targeted to the liver with improvement in Injecting cells directly into the liver parenchyma has the risk of
liver inflammation and hepatic injury in rats [48]. inadvertent entry into hepatic venous outflow tract and entry
Terai et al. utilized autologous mononuclear cells from of cells into pulmonary capillaries with embolic consequences.
patients with advanced liver disease by bone marrow aspiration Injection of cells into the hepatic or splenic artery is technically
[49]. The cells were purified prior to re-infusion via the periph- convenient but this may lead to organ infarcts, again due to
eral vein. A pilot study showed encouraging preliminary results embolic processes [53]. Under high-flow conditions of the arterial
where liver function and histological parameters of liver circulation transplanted cells may be destroyed rapidly by shear
regeneration improved following cell therapy. The peripheral forces, which is equally applicable to the pulmonary, hepatic and
administration of autologous mesenchymal stem cells (MSCs) splenic arterial beds. By contrast, transplantation of hepatocytes
have been tested in a randomized, placebo-controlled trial in cir- under low-flow conditions coupled with access to additional cell
rhosis but no beneficial effect was seen, indeed 3 out of 15 attachment factors and extracellular matrix components, as in
patients that received the MSCs died in the first 5 months follow- the liver or spleen sinusoids, has been most effective for hep-
ing cell administration compared to zero from 12 in the control atocyte delivery, persistence and engraftment. In the case of the
group [50]. The clinical studies of various autologous cells for spleen in rat, most (90%) of the cells instantaneously migrate into
liver disease have been systematically reviewed by Moore et al. the portal vein followed by redistribution equally to all liver lobes
and to date no convincing benefit has been noted in adequately in accordance with blood flow and liver volumes [32]. The num-
powered randomized controlled studies [51]. However it is worth ber of cells that may be safely accommodated in the sinusoids is
noting that due to the early state of the ‘‘therapies’’ tested many substantial and approaches 5–10% of the total number of
studies were small uncontrolled studies that did not allow a mea- parenchymal cells in the healthy rat liver [54]. Moreover, studies
sure of efficacy. in rodents have shown that cells may be transplanted repeatedly

Review
without deleterious consequences on hepatic vasculature or liver requires reconstitution over 1–5 days of plasma membrane struc-
sinusoids [55]. tures and physical joining together of transplanted and native
The initial transplanted cell distributions has been examined hepatocytes [56]. Evidence of this process in rats include re-
in rodents using radiolabeled or genetically-marked reporter liver associated gap junctions and bile canalicular networks with com-
cells, which provide additional methods for non-invasive tracking ponents from both native and transplanted hepatocytes [25], as
of the initial fate of transplanted cells [27,32]. A clinically rele- noted above. Despite delays in the integration of transplanted
vant finding is that cell transplantation results in transient portal hepatocytes, it should be noteworthy that transplanted cells con-
hypertension, which is due to transplanted cell size-dependent tinue to express the normal repertoire of genes without interrup-
occlusions in the periportal vascular complex, followed by the tions. However, it has been shown in rodents that hepatic gene
restoration of blood flow through the opening of alternative vas- expression in transplanted cells is better supported in the hepatic
cular channels as well as by the entry of transplanted cells into microenvironment compared with that in the spleen, which in
the liver parenchymal structure over several hours [56]. turn is superior to that in the peritoneal cavity [60]. The process
However, the transplantation of cells in the presence of pre- of transplanted cell integration in liver is aided by the activation
existing portal hypertension and chronic liver disease may lead of stellate cells, which release matrix metalloproteinases and tis-
to excessive translocation of cells into lungs through portosys- sue inhibitors of metalloproteinases to coordinate disruptions
temic collaterals or channels with cardiovascular complications. and restitutions of the extracellular matrix during the entry and
These may be related to the dosages of cells administered. resettling of transplanted cells [61]. After transplanted cells
Therefore, clinical trial design needs to incorporate mechanisms become integrated in the liver parenchyma, gene expression pro-
for the successful initial delivery of cells to liver sinusoids. files are driven by their position in the liver lobule, which is simi-
lar to adjacent native hepatocytes, and is under position-specific
regulation [29]. Moreover, transplanted cells exhibit normal pat-
Engraftment of transplanted cells including tracking and terns of proliferative activity, i.e., no proliferation in the healthy
monitoring of the delivered cells liver, and graded proliferation in response to injury in the liver
lobule that spares them, which is similar to the responses in adja-
After delivery of transplanted cells to liver sinusoids, several cent healthy cells. In these approaches, transplanted cells are
steps follow before cells are fully integrated in the liver parench- noted to life-long survival in rodents [28].
yma (hepatocytes) or in appropriate niches of the liver structure In recent years, novel targets have been defined to improve
(LSEC and other cell types) [13,56,57]. Studies in rats have shown engraftment of transplanted cells at these stages and several
that the initial series of events are driven by the mechanical pro- drug-based strategies have been developed in preclinical models
cess by which cells enter vascular spaces beyond the portal areas, that are potentially clinically relevant for enhancing cell engraft-
which is dependent upon the size of cells and the diameter of ment in the liver (Table 3). The major concepts have concerned
hepatic sinusoids [54]. The larger the size of cells, the more proxi- use of drugs to treat subjects prior to cell transplantation, such
mal will these be to portal areas, and the smaller the size of cells, that vascular or inflammatory changes induced by cell trans-
the more distal in the liver lobule will these be in relationship to plantation are abolished or minimized, the endothelial barrier
portal areas. This essential size-structure relationship drives the interposed between liver sinusoids and parenchyma is disrupted,
nature of cell-cell adhesions, as well as the extent of vaso-occlu- or hepatic stellate cells are induced to release beneficial sub-
sive processes that may be initiated by cell transplantation. stances, e.g., VEGF [58,59,62,63]. Similarly, novel concepts have
Rodent studies have shown that subsequent cell-cell signalling been developed in rodents where donor cells may be modified
events are important. The ischemic injury rapidly activates neu- prior to transplantation, e.g., by addition of extracellular matrix
trophils, Kupffer cells, LSECs and hepatic stellate cells [58,59]. components for better endothelial adhesion or incubation with
The initial engraftment processes where vascular responses drugs to block endothelin (ET)-1 receptors, which otherwise
involve release of locally-acting vasoactive molecules, such as may transduce deleterious intracellular signals to activate
NO and prostacyclins, complement, platelet-related thrombo- NF-jB-mediated cell death [63,64]. Of note, prior treatment of
genic substances, endothelin, cyclooxygenases, cytokines/ recipients with anti-inflammatory drugs, e.g., the TNF-a blocker,
chemokines/receptors, are largely deleterious. However, release etanercept, had remarkable effects in rat by preventing dele-
of some substances, such as vascular endothelial growth factor terious cytokine/chemokine/receptor responses, leading to
(VEGF) from native hepatocytes, Kupffer cells, monocytes and improvements in transplanted cell survival and engraftment in
hepatic stellate cells helps in permeabilization of endothelial the liver [65]. The possibility of combining these approaches
cells. Coupled with the activation of LSEC, transplanted cells are has also been and is being examined for further beneficial effects.
able to penetrate the endothelial barrier and then integrate them- To this effect, the initial losses of transplanted cells may be miti-
selves into the liver parenchyma [56]. This part of the process gated by relatively straightforward approaches using available
requires 16–20 h from cell delivery. However, cumulatively, drugs, which should aid future cell therapy applications.
nearly 80–90% of all transplanted hepatocytes are destroyed The in vivo monitoring of delivered cells in patients is surpris-
due to sinusoidal events, including lack of entry into sinusoids, ingly challenging. Whilst cells can be genetically labelled with
inadequate or no adhesion to sinusoidal endothelium, oxidative fluorescent or other probes in animal models, cell tracking in
stress, cytokine-mediated toxicity, etc. Therefore, considerable humans requires alternative methods. For short-term tracking of
efforts have been devoted to understanding how these dele- transplanted cells, radiolabeling methods have been effective,
terious processes could be harnessed and thus yield drug-based e.g., Indium-111- or 99m-technetium-labelled cells, in animals
approaches for improving cell therapy outcomes. as well as in people [32,66–68]. These methods are particularly
The next process in transplanted cell engraftment concerns suitable for tracking the initial distribution and redistribution of
integration of transplanted cells in the liver parenchyma. This transplanted cells in various vascular beds. An example of this

Table 3. Drug-based approaches for improving transplanted cell engraftment approach is the ability to determine and quantitate the risks of
and proliferation. intrapulmonary shunting of transplanted cells via portosystemic
A. Pre-treatment of recipients prior to cell transplantation collaterals. Moreover, transplanted cells may be localized in extra-
hepatic sites by imaging of hepatic receptor function, e.g., asialo-
Alteration of hepatic vascular responses
glycoprotein receptor as has been shown in mice and rats [69]. For
• ET-1 receptor blockade (bosentan, darusentan)
longer-term tracking of transplanted cells, molecular imaging

• Nitroglycerine
methods have also been developed. One such approach developed
• Prostacyclin in rodents and non-human primates used genetic constructs (her-
Blockade of inflammatory cytokines/chemokines/receptors pes simplex virus thymidine kinase) for metabolizing ganciclovir
• Etanercept substrates for PET imaging [70]. Another approach tested in rats
• Thalidomide utilised the imaging of natural ligands, e.g., radiocopper probes,
Disruption of hepatic endothelial barrier to demonstrate restoration of biliary copper excretion after trans-
• Cyclophosphamide plantation of healthy hepatocytes [71]. Furthermore, genetic
• Doxorubicin reporters capable of identifying unique alleles, including HLA-
specific alleles short tandem repeats, have been proposed as addi-
• Rifampicin/phenytoin
tional molecular methods to establish organ chimerism with
Activation of hepatic stellate cells
transplanted cells in mice [72]. Nonetheless, further clinically
• Naproxen
applicable methods are needed for tracking transplanted cells
• Celecoxib and monitoring their function in humans.
B. Pre-treatment of donor cells prior to transplantation
• Extracellular matrix coating with adhesion factors or Inducing proliferation in transplanted cells for liver
engineered molecules
repopulation
• Dual ET-1 A and B receptor blockade (bosentan)
C. Combined preconditioning of recipients and of donor Despite the integration and indefinite persistence of transplanted
cells prior to or after cell transplantation
cells in the absence of allograft rejection, more needs to be
• Various combinations of drug regimens learned about ways to induce proliferation in transplanted cells.
• Thyroid hormone treatments Although repeated transplantation of cells in large numbers cou-
• Inhibitors of fumarylacetoacetate hydroxylase activity pled with ways to decrease losses of transplanted cells could sub-
• Other drugs promoting liver regeneration or cell survival stantially increase delivery of cells to the liver [65], replacing
under study large amounts of the liver parenchyma with transplanted cells
is of enormous interest for many conditions. The fundamental
principle driving this area was gained from studies in animals
Table 4. Different types of potential cell therapy depending upon disease scenario.
Cell type Potential indication Clinical use? Advantages Disadvantages Reference

Hepatocytes • Metabolic liver Yes • In routine clinical use • Shortage of supply
disease • Engraftment in damaged
• Paediatric liver • Key metabolic and synthetic liver problematic
failure cell • Susceptible to infection with
hepatitis viruses
MSCs • Liver cirrhosis Some clinical • Easy to isolate and expand • Some clinical studies have [50, 111]
• Liver failure reports and • Generally immune tolerising been negative
• Immune mediated small number of • May be used with other cell • Poorly defined cell type
liver disease randomised trials types to reduce inflammation
EPCs • Liver cirrhosis No • Appear anti-fibrotic and pro- • Isolation and clinical use [47]
regenerative unclear
Macrophages • Liver cirrhosis No • Multiple effects on fibrosis • Transient liver engraftment [44, 45]
and regeneration • May be pro-fibrotic in
• Stimulate host macrophages certain circumstances
to amplify the response
Embryonic stem • Metabolic liver No • May be infinitely expandable • Ethics of use has been [93]
cells (ESCs) disease population of cells questioned
• Liver failure • Available from GMP • Long-term stability unclear
compatible sources
Induced • Metabolic liver No • May be infinitely expandable • Question over [102]
pluripotent stem disease population of cells completeness of
cells (IPSCs) • Liver failure • Autologous use is possible, functionality
this would require gene • Long-term stability as yet
modification for genetic unproven
diseases

Review
Obtaining sufficient hepatocytes in the clinic therapy
A B
Human adult hepatocytes
Human hepatocytes are isolated from donor organ livers that are
either unused surplus or are rejected for transplantation in peo-

ple. Cells are typically isolated by using collagenase perfusion
techniques under clinical Good Manufacturing Practice (cGMP)
conditions [76]. The shortage of good-quality donor organs has
led to the use of marginal donors or segment IV/caudate lobes
when the donor liver is split for transplantation into more than
one recipient [77,78]. Progress is being made in understanding
molecular mechanisms underpinning the difficulty in isolating
viable hepatocytes from non-heart-beating cadaveric donors
[79]. Progress is also being made to improve the viability and
No preconditioning Drug preconditioning metabolic function of hepatocytes isolated from steatotic livers,
Fig. 4. Proliferation of transplanted cells in the liver of healthy Dpp4
e.g., by the addition of N-acetylcysteine as an antioxidant to the
knockout mice. (A) Control animal subjected to no hepatic preconditioning perfusion solution [80]. Given the increasing rates of obesity
showing occasional transplanted cells with Dpp4 activity (red color, arrows) after and thus the frequency of fatty organs that are offered for trans-
2 months. (B) Preconditioning of animals with rifampicin, phenytoin and plantation this area of research is of major importance for the
monocrotaline followed by hepatocyte transplantation showed extensive liver
future. Isolated human hepatocytes may be transplanted freshly
repopulation to approximately 50% after 2 months.
after isolation or after cryopreservation even several years later
with progressive liver injury that spared healthy transplanted if stored appropriately, depending on regulatory practices [81].
hepatocytes. For instance, in alb-uPA transgenic mice, trans- However, the cell yield after thawing is low and the freezing pro-
plantation of healthy hepatocytes resulted in progressive liver cess also has a detrimental effect on the metabolic function and
repopulation [18]. Similarly, in FAH/ mice with progressive cell attachment properties [82]. Therefore, an optimised protocol
liver injury, transplanted healthy hepatocytes or hepatocytes may provide significant improvements in cryopreserved cells but
derived as fusion products with components from extrahepatic further refinements in the processes of cryopreservation and
sources, such as donor bone marrow-derived mononuclear cells, mechanisms capable of preserving or enhancing cell viability
proliferated with progressive liver repopulation [73]. In LEC rats are required [83].
modelling Wilson’s disease, extensive liver repopulation was
observed when healthy hepatocytes were transplanted [17].
Furthermore, induced injuries that constituted hepatic pre- Alternative sources of hepatocyte-like cells
conditioning were successful in promoting proliferation of trans-
planted cells in rodents [18]. For instance, DNA-adduct forming Fetal hepatocytes
pyrollizidine alkaloids, retrorsine and monocrotaline, were found
to be effective in combination with additional injuries, such as Clinical studies using fetal hepatocytes have suggested they are a
partial hepatectomy, carbon tetrachloride, or other drugs, potentially useful source of cells for clinical therapy [35,36].
for inducing liver repopulation by transplanted cells. Understanding their behaviour in the host liver will help to define
Preconditioning of the liver with radiation plus partial hep- conditions for their optimal usage. Interestingly, in the rat, fetal
atectomy, ischemia-reperfusion or toxic bile salts has also been hepatocytes have been shown to proliferate within the host liver,
effective for liver repopulation. This included replacement of even in the absence of the usual mitogenic stimulus required for
mouse liver with transplanted hepatocytes as well as LSECs transplanted adult hepatocytes [84]. An interesting feature of this
[13,74]. However, these manipulations are not well-suited to was that the transplanted cells could induce apoptosis in the
clinical applications and more conceptual development is neces- recipient’s nearby host cells and thereby have a form of selective
sary, especially in regards to suitable drugs. Some progress has advantage over the recipient liver cells. Although the proliferative
been made in this area and further progress is anticipated (Fig. 4). capacity of the rat fetal cells is encouraging, other studies utiliz-
An alternative approach is to alter the proliferation of trans- ing human fetal cells have found different results. Haridass et al.
planted cells by inactivation of cell cycle suppressors, which compared human fetal hepatocytes to adult hepatocytes in an
accelerated liver repopulation kinetics with transplanted hep- immunodeficient mouse transplantation model. In this system
atocytes in FAH/ mice [19]. However, this approach is not with- the fetal cells had less repopulation capacity on a cell per cell
out cancer-risk. More recently, regulation of liver growth control basis than the adult hepatocytes [85]. Intriguingly, a recent study
by the Hippo signalling pathway has come to attention [75] has suggested that rat fetal hepatocytes may have anti-fibrotic
which may provide additional targets to promote transplanted properties when transplanted into damaged livers, which would
cell proliferation. have the dual benefit of supporting parenchymal regeneration
It may be that hepatic preconditioning to replace LSECs will be whilst targeting the scarring component of chronic liver disease
simpler than the replacement of hepatocytes because suitable [86]. Whether this finding is a general phenomenon, seen using
endothelial injury could possibly be achieved by ischemia- human fetal hepatocytes, particularly in the setting of chronic
reperfusion injury or other available drugs. Although this needs liver injury where there is significant scarring is unknown.
further study this would be of benefit for haemophilia A targeted Whether fetal cells could be less immunogenic compared with
cell therapy. adult hepatocytes is also unresolved at present. One issue in

transferring these interesting preclinical studies findings into cells have been studied for a considerable time and human ES
clinical therapy is the issue of ethically sourcing sufficient human cells have been derived at GMP level which would be required
fetal hepatocytes for therapy in people. This aspect will likely for clinical use. Protocols have been developed to differentiate
prevent fetal hepatocytes becoming a widely used clinical ES cells sequentially into a hepatocyte-like cell (HLC) phenotype.
resource, however the positive features of the cells may be used These have largely been based upon the signals that arise during
to guide and influence the production of stem cell derived hep- development including Activin A and Wnt 3A to encourage endo-
atocytes in the future. derm differentiation [93,94], bone morphogenic protein (BMP)
and fibroblast growth factor (FGF) to aid hepatocyte differentia-
tion and then oncostatin M and dexamethasone to promote
Hepatic progenitor cells (HPCs) maturity [95]. Further attempts have been made to enhance their
phenotype and stability using synthetic and natural cell matrices
Whilst the normal human liver can regenerate efficiently through [96], however despite this the pluripotent (ES cells and iPSCs-see
hepatocyte division during severe or chronic liver disease the later) derived HLCs have a phenotype more in keeping with a
regenerative capacity of hepatocytes is compromised. Under fetal than an adult hepatocyte phenotype [97]. However, in vivo
these circumstances it has been thought that there are endoge- it is possible that more mature cells may develop. A recent study
nous HPCs that are activated and due to their bipotential nature demonstrated that pluripotent derived human HLCs can support
are able to regenerate both biliary epithelia and hepatocytes [87]. HCV infection and replication in vivo [98]. If the signals and fac-
Recently this understanding has been challenged by studies in tors present in vivo can be recapitulated in vitro it is possible that
mouse which suggest hepatocytes supply all the regenerative more mature HLCs may be produced. 3D culture systems have
capacity of the parenchyma, and the ductular reactions seen in been tested and aid the increased maturity of the HLCs [99].
chronic injury do not regenerate parenchyma. Furthermore some Because of the source and derivation of ES cells it is unlikely that
of the ductular reactions may arise from the de-differentiation of ES cell-derived HLCs would be routinely fully immunologically
mature hepatocytes during injury [88,89]. However recent data matched to the recipient (blood antigen, tissue type and HLA).
using developmental ablation of foxl1 marked HPCs in mice sug- Therefore, like whole liver transplantation, focusing upon blood
gested that HPCs were a significant source of parenchymal antigen matching would be one solution with the resulting need
regeneration and ablation of these foxl1 HPCs had a detrimental for some immunosuppression.
effect upon the recovery from liver injury [90]. Further studies iPSCs are pluripotent cells that can be reprogrammed from
are awaited in this area with conditional ablation of such cell adult cells using so-called pluripotency factors [100]. They have
types to further delineate their regenerative role of such HPCs. the ethical advantage of not requiring embryonic material and
An important unresolved question is whether such studies in have the potential clinical advantage that they can be developed
mice are directly translatable to human liver disease. The injury from autologous starting cells, thereby obviating the requirement
models used are often relatively mild and short lived compared for immunosuppression. Obviously if iPSCs were to be used for
to the severe liver injury that occurs sometimes over decades in the derivation of HLCs for the treatment of a genetic liver disease
humans and results in the significant impairment of hepatocyte then the autologous source would mean some form of ‘‘gene sur-
proliferation. This controversial area is beyond the scope of this gery’’ would be required prior to use. Such an approach has been
review but is relevant for the question of whether an expandable adopted in a preclinical model of alpha-1-antitrypsin deficiency
source of cells with hepatocyte or biliary potential could be [101].
derived for cell therapy [91]. Lgr5 has been shown in mouse to Human HLCs can be efficiently derived from iPSCs [102], how-
identify cells with a HPC characteristic. These Lgr5+ cells could ever using standard differentiation methods they are currently
be grown into organoids with a high clonogenic capacity [46]. more fetal in their phenotype than adult primary hepatocytes
When such organoids were used in the FAH/ mouse they could [97]. The 3D culture of iPSCs has recently been shown to increase
engraft within the parenchyma and provide nodules of function- their maturity closer to that of mature hepatocytes emphasizing
ing parenchyma. Furthermore, organoids have been derived from the need for an appropriate developmental niche for the cells
human livers using EpCAM selection of bipotential hepatic [103]. An exciting development was the demonstration that
epithelial cells that originated in the ductal areas of the liver when human iPSCs were cultured with endothelial and mes-
[92]. Importantly these cells retained chromosomal stability dur- enchymal cells they self-formed in vitro into small liver organoids
ing prolonged culture. In order to translate these interesting find- that could be transplanted and had metabolic and synthetic func-
ings into a clinical therapy a GMP compatible method of organoid tion [104]. For clinical translation of these findings a way to grow
culture will be required; furthermore the engraftment and repop- scalable organoids with appropriate 3D structure, vascularity and
ulation characteristics of the organoids or cells derived from ideally immune cell function would be required. Alternative
these organoids will need to be defined. approaches have been taken to increase the maturity of human
iPSC derived hepatocytes. Kondo et al. used Activin A, dimethyl
sulfoxide, hepatocyte growth factor, oncostatin M, and
Pluripotent sources of hepatocytes: Embryonic stem (ES) cells dexamethasone to induce hepatocyte maturity including drug
and induced pluripotent stem cells (iPSCs) metabolism activity [105].
The use of pluripotent stem cell derived and directly repro-
ES cells are pluripotent stem cells derived from the inner cell grammed HLCs have several factors that would influence their
mass of a blastocyst. In humans this stage is reached at 4–5 days potential clinical use. Cells would need to be phenotypically
post fertilization and consists of approximately 50–150 cells. stable over a long period if they were to be transplanted into
Because the derivation of ES cells requires destruction of the blas- the liver. This is particularly obvious in the setting of paediatric
tocyst there is an inescapable moral dimension to their use. ES cell therapy where decades of safety would be required from

Review
the transplanted cell. The HLC would need to be homeostatic and Conclusions and further perspectives
respond to growth and renewal requirements in an appropriate
manner. Furthermore this capacity for appropriate proliferation There is a clear requirement for an unlimited source of human
would be tested in the setting of chronic liver injury where there HLCs for transplantation with good function, phenotypic stability
are multiple and chronic signals acting to perturb the HLCs phe- and a near zero risk of tumorgenicity. Whilst easily stated this is a
notype and physiological function. A particular concern when demanding goal. Cells with a stem cell like potential that are
contemplating the use of pluripotent cells as a source of HLCs therefore able to give rise in a clonogenic manner to large num-
is the development of tumours although safety measures can bers of progeny do carry a potential risk of unwanted and
be envisaged such as physical encapsulation of the cells or the unregulated cell growth. Such risks can be minimized by screen-
use of an inbuilt suicide gene that could be activated in the situa- ing potential candidate cells in appropriate long-term studies in
tion of unwanted proliferation of the transplanted cells. animal models and by engineering in ‘‘safety devices’’ such as sui-
cide genes which would allow the killing of a cell upon
administration of a drug or small molecule. All risks of therapy
Direct reprogrammed cells are relative to the disease in question and the transplantation
of a cell into an elderly person with a potentially fatal fulminant
A recent technological advance is the direct reprogramming of liver disease is an entirely different risk profile to the use of in a
human fibroblasts into so-called induced hepatocytes (iHeps). baby where the transplanted cell may need to perform for dec-
Sekiya and Suzuki showed that two transcription factors- ades without undergoing oncogenic change.
Hnf4alpha plus Foxa1, Foxa2 or Foxa3 could convert mouse adult Stem cells have frequently been invoked as the future cell to
fibroblasts into iHeps in vitro and could rescue the FAH/ mouse allow an almost limitless source of cells for therapy. However if
[106]. In a similar approach fibroblasts were converted to iHeps hepatocytes are the facultative stem cells of the liver then per-
by the transduction of Gata4, Hnf1alpha and Foxa3 and the inac- haps it is time to re-examine the potential clonogenic capabilities
tivation of p19. Furthermore, the iHeps showed good phenotypic of the humble hepatocyte. Adult hepatocyte exhibits almost
qualities and could repopulate the livers of Fah/Rag2/ mice, unlimited clonogenicity in vivo yet in vitro has been very difficult
rescuing a proportion of recipients [107]. to expand without the cells undergoing de-differentiation.
Human induced hepatocytes (hiHeps) have been developed by Understanding the in vivo cues which allow the homoeostatic
similar techniques to that seen in mouse. Huang et al. found that regulation of hepatocytes has progressed greatly. Hepatocytes
the expression of HNF4, HNF1A, and FOXA3 in fibroblasts allows divide readily when required yet remain quiescent at other times
the production of hiHeps at a conversion rate approaching 20% in the healthy liver. This in vivo understanding still needs to be
[108]. The paper by Du et al. used a more comprehensive set of applied in vitro to human adult hepatocytes and allow division
factors: C-MYC, HNF1A, HNF4A, HNF6, ATF5, PROX1, CEBPA, without de-differentiation. Such knowledge would lead to the
and p53 shRNA to efficiently produce hiHeps [109]. Both sets of ideal scenario of an infinitely expandable cell source for clinical
hiHeps had similar gene expression profiles to mature human cell therapy.
hepatocytes but by no means identical. Encouragingly the
hiHeps also showed good in vivo functionality in a number of
mouse models of liver injury including the FRG (Fah//Rag2/
Conflict of interest
/Il2rg/) mouse.
A further refinement of this technique has been employed in
The authors declared that they do not have anything to disclose
mice whereby fibroblasts are first differentiated into induced
regarding funding or conflict of interest with respect to this
multipotent progenitor cells (iMPCs) [110]. These iMPCs could
manuscript.
be significantly passaged and expanded then differentiated
through an endoderm stage to differentiated HLCs (so-called
iMPC-Heps). These iMPC-Heps could partially repopulate the References
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Review

Cell therapy for liver disease: From liver transplantation to

Stuart J. Forbes1,⇑, Sanjeev Gupta2, Anil Dhawan3

Journal of Hepatology 2015 vol. 62 j S157–S169

Step 1. Step 2. Step 3.

Hepatocytes Recipient pre- or

S158 Journal of Hepatology 2015 vol. 62 j S157–S169

Major clinical indications and roles of cell therapy

Journal of Hepatology 2015 vol. 62 j S157–S169 S159

tion of cells into the portal venous system is made difﬁcult in

S160 Journal of Hepatology 2015 vol. 62 j S157–S169

have attempted various ways to transplant hepatocytes, includ- Hickman line

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S162 Journal of Hepatology 2015 vol. 62 j S157–S169

longer-term tracking of transplanted cells, molecular imaging

Cell type Potential indication Clinical use? Advantages Disadvantages Reference

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either unused surplus or are rejected for transplantation in peo-

S164 Journal of Hepatology 2015 vol. 62 j S157–S169

Journal of Hepatology 2015 vol. 62 j S157–S169 S165

S166 Journal of Hepatology 2015 vol. 62 j S157–S169

niques: large animal models. Methods Mol Biol 2009;481:83–96. 1994;58:951–952.

Journal of Hepatology 2015 vol. 62 j S157–S169 S167

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Journal of Hepatology 2015 vol. 62 j S157–S169 S169

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