Department of Surgery, University of Southern California, USA
Submission: March 29, 2018; Published: May 21, 2018
*Corresponding author: Toshio Miki, Department of Surgery, Keck School of Medicine, University of Southern California 2011 Zonal Avenue, HMR 509A, Los Angeles, CA 90033-9141, USA, Tel: 323-442-7703; Email: firstname.lastname@example.org
How to cite this article: Toshio Miki. Clinical Hepatocyte Transplantation. Adv Res Gastroentero Hepatol 2018; 9(5): 555771.
A severe shortage of suitable allograft is a long-standing and worldwide problem for the patients who are waiting for organ transplantation. Hepatocyte transplantation has been proposed as an alternative therapeutic approach for liver disease patients to address these urgent and unmet medical needs. The cell replacement approach does not replace the orthotopic liver transplantation (OLT), but rather complement OLT especially for the patients who are not required whole liver replacement such as congenital metabolic disorders. This review article summarizes current knowledge and limitations of clinical hepatocyte transplantation and aims to advance our understanding towards the goal of developing novel cell replacement therapies for the patients who are on the OLT waiting list.
Keywords: Hepatocytes; Congenital metabolic disorders; Cell replacement therapy; Regenerative medicine
Hepatocyte transplantation has been proposed as an alternative approach of the orthotropic liver transplantation (OLT). A number of advantages of this cell replacement therapy over the OLT are listed including less invasiveness and cost-effectiveness . The concept of partially replacing damaged or malfunctioned hepatocytes is particularly suitable for the patients who suffer from congenital metabolic disorders and acute liver failure patients who require transient support of their liver function. The keys to the successful hepatocyte transplantation are the indications and the careful evaluation of the eligible recipients, the preconditioning of the recipient’s liver, and the quality of the donor cells. The cell transplantation procedure is technically simple compared to that of OLT. The common interventional technique is required to safely deliver the cells to the liver. Here, we briefly overview the reported clinical cases, suitable target diseases, clinical procedures, limitations, and perspective to overcome the limitations.
The basic studies that led to clinical hepatocyte transplantation were initiated in 1970’s. Rodent models with metabolic disorders and acute liver failure models induced by chemicals, partial hepatectomy, or ischemic-reperfusion liver injury were often used to demonstrate improvements of damaged liver functions by injecting autologous liver cells .
These pre-clinical studies indicated that the allergenic liver cell transplantation had a potential to correct various metabolic defects. The earliest published reports of pre- clinical hepatocyte transplantations were performed in 1976 by a group led by Najarian using UDP-glucuronyl-transferase-deficient rats, the Gunn rat . About a decade later, Mito tested an idea to utilize the spleen as an ectopic liver by transplanting hepatocytes into the spleen with rodent and dog models .
The first attempted human hepatocyte transplantation was performed in 1992 by the same group in Japan . The safety of hepatocyte splenic arterial infusion and the therapeutic efficiency were confirmed by Strom  with chronic end- stage liver disease patients. They demonstrated that transplanted human hepatocytes were viable in splenic nidation and showed typical hepatic cord structures. Three of five treated patients fully recovered and successfully received OLT. Since then, more than 100 clinical hepatocyte transplantations were reported [7-9]. Over the last two decades, more than 15 institutes around world sporadically conducted clinical hepatocyte transplantation, and currently, at least 7 groups are active . However, despite these successes of the clinical studies, hepatocyte transplantation has remained experimental due to the limited supply of donor liver tissue for hepatocyte isolation. Conducting a large-scale randomized clinical trial has been prohibited by the limited supply of sufficient and consistent quality of human hepatocytes.
Orthotropic liver transplantation (OLT) is a significantly
efficient treatment to improve the prognosis in patients with
fulminant hepatic failure, end-stage liver disease, and metabolic
liver diseases. In theory, if we can replace 100% of damaged
patient’s hepatocytes, hepatocyte transplantation can provide
similar therapeutic efficacy in most of these diseases. Therefore,
the indication of hepatocyte transplantation depends on how
much replacement is required to alleviate disease symptoms and
how much donor cells can functionally engraft in the patient’s
liver. In addition, the indications will depend on the risk-benefit
balance. Based on this logic, the disease conditions that inhibit
donor hepatocyte engraftments such as cirrhotic liver and acute
hepatitis will not be suitable.
On the other hand, congenital metabolic disorders (CMDs)
will benefit from hepatocyte transplantation. In general,
metabolic disease patients require only partial replacement of
hepatocytes to compensate for the missing enzyme function.
The required compensation level may vary by case and by
disease. Careful assessments of the impaired enzyme functions
will be required to determine the indication of hepatocyte
transplantation. Approximately 1 in 1,500 children is born
with a CMD. Many of these critical inborn errors of metabolic
or synthetic processes involve principally the liver. Current
therapy for CMDs consists of life -long dietary restriction with
or without supplementation of amino acids. For example, an
infusion of normal hepatocytes that is equivalent to 5% of
the parenchymal mass achieved a medium-term reduction in
serum bilirubin in a patient with Crigler-Najjar syndrome .
An ornithine transcarbamylase deficient child who received
1.9x109 hepatocytes had normalization of plasma ammonia
and glutamine levels on a normal diet without phenylbutyrate/
phenylacetate therapy. These cases clearly demonstrate CMDs
can be effectively treated via cell replacement therapy.
Acute liver failures will also benefit from the cell
replacement strategy. Although the liver is still in the active
inflammation status, injecting hepatocytes into the spleen may
support the patients’ hepatic function temporarily until the
own liver recovers or until the OLT become available. A total
of 68 reported clinical hepatocyte transplantation cases can be
categorized in these two groups, CMD and ALF (Figure 1). Due
to the epidemiologic nature, the patients’ ages are significantly
distinguished (Figure 2). In most of CMD cases except a case
with Progressive familial intrahepatic cholestasis type 2, the
therapeutic efficacies were partial but satisfactory. Recipients
with some conditions may preclude a successful transplant.
Such contraindications may include metastatic cancer outside
of the liver, active drug or alcohol abuse, and active systemic
infections. However, unlike OLT which is a major surgery with
a high incidence of complications, the list of contraindications is
shorter for hepatocyte transplantation.
The source of primary human hepatocyte is the livers unused
for OLT. In result of increasing usage of marginal-suboptimal
donor organs with the extended donor criteria (EDC), the
availability of organs for cell isolation has diminished. The
quality of available donor organ for cell isolation is frequently
poor. The degree of steatosis affects the yield, viability, and
function . Hepatocytes can be obtained from livers removed
from OLT recipients with congenital metabolic disorders. These
hepatocytes can be used to treat different types of congenital
metabolic disorders . The donor criteria can be extended
to advanced-age donors and non-heart-beating donors .
However, the cell quality is highly variable. Therefore, these
cell sources are not reliable and insufficient to overcome the
primary human hepatocyte shortage problem. Hepatocyte
Isolation requires well established a unique enzymatic digestion
technique. The standard protocol was established based on
Seglen’s two-step collagenase perfusion technique for rat
hepatocytes isolation. The protocol was slightly modified for
human hepatocyte isolation [14-16]. Hepatocytes should be
transplanted as soon as possible, preferably within 24hrs of
isolation, as the hepatic function deteriorates when kept at 4 ˚C.
Although 100% replacement of disease hepatocytes with
healthy functional hepatocytes is ideal, the practical goal of
replacement ratio will be 10-15%, which may improve enzyme
functions to the mild phenotype from the most of severe type
congenital metabolic disorders [17-22]. It is assumed that 2×108
cells per kg of body weight may be the upper limit of hepatocytes
that can be safely infused during transplantation. The currently
proposed optimized dose is 30-100×106 cells/kg of body weight
at an infusion rate of 5-10ml/kg per hour, and a concentration of
1-10×106 cells/ml . To achieve the estimated number of cell
transplantation, therefore, multiple infusions are necessary with
certain interval periods .
Cell transplantation performed to target either liver or
spleen. Although cell engraftment in the liver is physiological,
spleen could be a good alternative destination in case the
recipient’s liver suffer from severe fibrosis (cirrhosis). The
route of administration must be intraportal injection regardless
directly inject into the intra hepatic portal vein, inferior
mesenteric vein, umbilical vein, or via spleen. Systemically
injected cells will be trapped in the lung and may cause the
pulmonary thromboembolism. On the other hand, cells injected
into portal vein do not pass through the liver . Although the
mechanism of cell integration in the recipient’s hepatic lobule
structure is not well studied, it is speculated that the intraportal
infusion causes cell embolism at the intra hepatic portal capillary
and increase the portal pressure. The portal hypertension and
mechanical expansion stimulate intercellular signaling exchange
between the non parenchymal cells which increase the vascular
Preconditioning treatments are common strategies used in
preclinical studies to enhance engraftment and proliferation of
donor cells . The aims of the preconditioning treatments can
be classified into four categories:
(1) Decrease recipient’s immune reaction.
(2) Disrupt the native liver structure.
(3) Stimulate liver regeneration signalings and
(4) Supress the native hepatocyte proliferation.
The most common treatment is a partial hepatectomy
combined with radiation or drugs, such as retro sine [29,30].
Although many of these strategies are not clinically acceptable,
Fox  demonstrated a significant increase of cell engraftment
with partial radiation in clinical hepatocyte transplantation .
Irradiation approach may serve all above four aims [32,33].
(1) The irradiation inhibits the phagocytic activity of
(2) Transiently disrupt the sinusoidal endothelial barrier.
(3) Induce apoptosis of native hepatocytes to stimulate
liver regeneration and
(4) Inhibit native hepatocyte proliferation.
In addition, these effects can be controlled by optimizing the
radiation dose. A total dose of 10Gy for patients greater than 3
years of age, and 5Gy for patients less than that age was used
in the clinical trial . The donor cells injected into the portal
vein were guided to the irradiated right lobe by the left branch
occlusion. One of the advantages of hepatocyte transplantation
is the native liver serves as a back-up to the therapy. Unlike OLT, the patient’s condition only returns to the pre-transplantation
state in case of cellular graft failure. A major concern of this
preconditioning treatment is losing this advantage. The eligible
recipients must be evaluated carefully.
There are several obstacles to provide this promising therapy
to the patients as an option of standard therapies. Shortage of
donor organs limits the availability of livers for hepatocyte
isolation. In an endeavour to increase the opportunity to obtain
primary human hepatocyte, the researchers extended the donor
criteria to advanced -age donors and non-heart-beating donors.
Fetal hepatocytes or immortalized hepatocytes were also
considered as alternative cells . However, none of them could
resolve the problem. Recent advancements in stem cell research
have demonstrated that hepatocyte -like cells can be derived
from human stem cells.
Pluripotent stem cells such as embryonic stem cells (ESCs)
and induced pluripotent stem cells (iPSCs) possess tremendous
differentiation potentials, however, the developmental capability
is a double-edged sword, associated with the risk of tumorigenicity
. It is almost impossible to guaranty the safety of injecting
over two billion ESC or iPSC-derived hepatocytes with current
technology. Other stem cells such as mesenchymal stem cells also
proposed that they possess the hepatic differentiation capability.
However, efficiency is still open to debate. One of the placental
stem cells, human amnion epithelial cells (hAEC) has been
getting attention as an alternative for hepatocyte transplantation
[36,37]. hAECs possess multi- lineage differentiation potential
including the ability to differentiate towards the hepatic lineage,
which allows them to express the desired enzymatic functions
Unlike other components of the placenta, the human
amniotic epithelium is derived from pluripotent epiblasts
[39-41]. Studies showed that the embryonic stem cell surface
markers TRA1-60, TRA1-81, SSEA3, and SSEA4 are positive on
most of the fetal amniotic epithelium and some of these stem
cell marker positive cells are retained in term placental amniotic
epithelium . Primary hAECs respond to exogenous stimuli in
vitro and can be induced towards specific differentiation. These
morphological and transcriptional profile changes demonstrate
the developmental plasticity of the hAECs. Under appropriate
culture conditions, hAECs exhibit the capability of differentiating
into endoderm lineage tissues including hepatocytes in vitro and
Transcriptional analysis of hAECs transplanted SCID/
Beige mouse livers indicate that transplanted hAECs terminally
differentiated into mature hepatocytes in mouse liver and
expressed functional marker genes including cytochrome P450
genes at equivalent levels to human primary hepatocytes .
Several preclinical studies have demonstrated that the hAECderived
hepatic cells acquire the desired enzyme function for the
treatment of congenital metabolic disorders using disease model
animals [43,44]. Other preclinical studies also demonstrated
significant therapeutic properties of hAEC for cirrhosis .
Importantly, upon transplantation into the livers of mice,
undifferentiated hAECs have been shown to engraft, display
hepatocyte-like morphology, and express various hepatic
enzymes without tumorigenicity. The lack of direct monitoring/
tracking technology after cell transplantation is another
obstacle that prevents to make the cell therapy as one of the
standard therapies . The biochemical elevations of serum
concentrations of aspartate transaminase (AST) and alanine
transaminase (ALT) are the markers of liver rejection post
OLT, as well as the histological signature of inflammatory cell
infiltration in the graft. However, relatively less number of donor
cells in case of hepatocyte transplantation limit to determine cell
rejection with these assays.
Molecular biological approaches such as detecting
Y chromosome sequences with qRT-PCR may be able to
demonstrate the presence of donor cells, however, it will not
indicate the cell viability. If the target enzyme function is
restored or improved, that could be an indirect evidence of cell
engraftment. However, these parameters may not be sensitive
enough to control immune suppression in a practical manner.
Development of novel methods to label cells will be required
with a detection/tracking technology with single cell level high
resolution. Novel biomarkers that correspond to the rejection
will be helpful to optimize the immune suppression regimen
[47,48]. It is critical to monitor the status of transplanted cells in
order to optimize and design immune suppression regimen for
each patient .
Currently similar immune suppression regimen used for OLT
or islet transplantation is used for hepatocyte transplantation
however, it could be reduced because of the immune privileged
nature of the hepatocytes . In summary, clinical hepatocyte
transplantation studies have clearly demonstrated that this
therapy is a suitable treatment for patients with CMDs. However,
there are some obstacles to provide this promising therapy to
the patients as an option of standard therapies. The obstacles
are the insufficient supply of donor cells, the lack of direct
monitoring/tracking technology after cell transplantation,
which subsequently causes the difficulty to optimize immune
suppression protocols. Further studies on stem cell-derived
hepatic cells and finding novel biomarkers are required to
translate the hepatocyte transplantation into clinic.